United States Department of Commerce

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National Institute of Standards and Technology

NIST Special Publication 874

Methods for Analysis of Cancer

Chemopreventive Agents in

Human Serum

Jeanice Brown Thomas and Katherine E. Sharpless, Editors
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'At Boulder, CO 80303. ^Some elements at Boulder. CO 80303.
NIST Special Publication 874

Methods for Analysis of Cancer

Chemopreventive Agents in

Human Serum

Editors

Jeanice Brown Thomas

and Katherine E. Sharpless

Chemical Science and Technology Laboratory

National Institute of Standards and Technology

Gaithersburg, MD 20899-0001

Research supported in part by:

Division of Cancer Prevention and Control

National Institutes of Health

National Cancer Institute Bethesda, MD 20892

February 1995

Project Officer: Dr. James A. Crowell

Chemoprevention Branch

Division of Cancer Prevention and Control

National Cancer Institute Bethesda, MD 20892

U.S. Department of Commerce

Ronald H. Brown, Secretary

Technology Administration

Mary L. Good, Under Secretary for Technology

National Institute of Standards and Technology

Arati Prabhakar, Director
National Institute of Standards

and Technology

Special Publication 874

Natl. Inst. Stand. Technol.

Spec. Publ. 874

128 pages (Feb. 1995)

CODEN: NSPUE2

U.S. Government Printing Office

Washington: 1995

For sale by the Superintendent

of Documents

U.S. Government Printing Office

Washington, DC 20402-9325
ACKNOWLEDGMENTS

We thank Ms. Donna Sirk for assistance in the preparation of this manual.

The following authors contributed papers:

Jeanice Brown Thomas, National Institute of Standards and Technology

Richard G. Christensen, National Institute of Standards and Technology

Neal E. Craft, Craft Technologies, Inc. Thomas P. Davis, University of Arizona Health Sciences Center

William A. MacCrehan, National Institute of Standards and Technology

Winfred Malone, National Cancer Institute

Sam A. Margolis, National Institute of Standards and Technology

Willie E. May, National Institute of Standards and Technology

Robert C. Paule, National Institute of Standards and Technology

Herbert F. Pierson, Preventive Nutrition Consultants, Inc. Roland Rieger, University of Ulm

Lane C. Sander, National Institute of Standards and Technology

Emil Schonberger, Hebrew University

Katherine E. Sharpless, National Institute of Standards and Technology

Joseph H. Scares, Jr., University of Maryland

Stephen A. Wise, National Institute of Standards and Technology

Regina Ziegler, National Cancer Institute

INTRODUCTION

The Micronutrients Measurement Quality Assurance (QA) Program was organized in 1984 by

the National Cancer Institute (NCI) and the National Institute of Standards and Technology

(NIST, formerly the National Bureau of Standards) to support measurement technology for

selected fat- and water-soluble vitamins, carotenoids, zinc, and selenium in serum and plasma.

This program was initiated as part of investigations supported by the NCI Division of Cancer

Prevention and Control to study the possible role of these analytes as cancer chemoprevention

agents.

Two of the main components of the program have been (1) methods development and

validation, and (2) interlaboratory quality assurance through use of proficiency testing.

Serum-based samples with assigned values for the target analytes and performance-evaluation

standards are distributed by NIST to the laboratories for analysis. NIST staff provide the

laboratories with technical feedback concerning their performance and suggestions for

methods development and refinement. The results from the proficiency testing are used to establish a laboratory performance database for use by NCI to evaluate the quality of the

analytical data from which epidemiological conclusions are to be made.

This report has been compiled as a result of NIST efforts to provide consultation in methods

development and analytical-measurement troubleshooting to laboratories that measure vitamins

A and E, carotenoids, ascorbic acid, 4-hydroxyphenylretinamide, oltipraz, vitamin Kj, and

glycyrrhetinic acid in serum and plasma. A summary of factors that are essential to the

method performance for each procedure is provided as a quick reference. The intended use of

this summary is to help the practitioner focus on the steps needed to implement each

procedure. The guidance offered within these pages should prove suitable for use in clinical

laboratories and for NIST reference methods.

V
DISCLAIMER

Certain commercial equipment, instruments, or materials are identified in this manual to specify adequately the experimental procedure. Such identification does not imply

recommendation or endorsement by the National Cancer Institute or the National Institute of

Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

vi
TABLE OF CONTENTS

Introduction ii Disclaimer iii

The purpose of this manual is to summarize several years of methods development and refinement for the measurement of selected fat- and water-soluble vitamins, carotenoids, oltipraz, and glycyrrhetinic acid in serum and plasma as part of the NIST/NCI Micronutrients Measurement Quality Assurance Program. The manual is divided into six sections, each containing a series ofpublications pertaining to the measurement of the specified

analyte(s). A summary offactors that are important to the method performance for each procedure is provided.

This summary is included to help the analyst focus on the steps needed to implement each procedure.

Sections

1. Measurement of Fat-Soluble Vitamins and Carotenoids in Serum 1-1

Evaluation of Reversed-Phase Liquid Chromatographic Columns

for Recovery and Selectivity of Selected Carotenoids 1-3

K.S. Epler, L.C. Sander, R.G. Ziegler, S.A. Wise, and N.E. Craft

Liquid Chromatographic Method for the Determination of

Carotenoids, Retinoids, and Tocopherols in Human Serum

and in Food 1-16

K.S. Epler, R.G. Ziegler, and N.E. Craft

Determination of Retinol, a-Tocopherol, and ^-Carotene in

Serum by Liquid Chromatography with Absorbance and Electrochemical

Detection 1-28

W.A. MacCrehan and E. Schonberger

Separation and Relative Distribution of All-fra«j-P-Carotene

and its cw-Isomers in ^-Carotene Preparations 1-36

N.E. Craft, L.C. Sander, and H.F. Pierson

Relative Solubility, Stability, and Absorptivity of

Lutein and p-Carotene in Organic Solvents 1-49

N.E. Craft and J.H. Soares, Jr. Optimization of an Isocratic High-Performance Liquid

Chromatographic Separation of Carotenoids 1-53

N.E. Craft, S.A. Wise, and J.H. Soares, Jr. Development of Engineered Stationary Phases for the

Separation of Carotenoid Isomers 1-59

L.C. Sander, K.E. Sharpless, N.E. Craft, and S.A. Wise

Determination of Glycyrrhetinic Acid 2-1

Determination of Glycyrrhetinic Acid in Human Plasma by

High-Performance Liquid Chromatography 2-3

J. Brown Thomas, R.G. Christensen, R. Rieger, W. Malone, and W.E. May

vii
3. Measurement of Oltipraz in Serum 3-1

Determination of Oltipraz in Serum by High-Performance

Liquid Chromatography with Optical Absorbance and

Mass Spectrometric Detection 3-3

R.G. Christensen and W. Malone

4. Ascorbic and Dehydroascorbic Acids Analyses 4-1

Ascorbic and Dehydroascorbic Acids Measured in Plasma Preserved

with Dithiothreitol or Metaphosphoric Acid 4-3

S.A. Margolis, R.C. Paule, and R.G. Ziegler

Stabilization of Ascorbic Acid in Human Plasma, and Its Liquid-Chromatographic Measurement 4-9

S.A. Margolis and T.P. Davis

5. Determination of Vitamin Ki in Serum 5-1

Determination of Vitamin Kj in Serum 5-3

W.A. MacCrehan and E. Schonberger

6. Measurement of (4-Hydroxyphenyl)retinamide in Serum 6-1

NIST Measurements of (4-Hydroxyphenyl)retinamide in Serum 6-2

W.A. MacCrehan

viii
UST OF FIGURES

Page

Section 1 : Measurement of Fat-Soluble Vitamins and Carotenoids in Serum

Structures of Carotenoids Used in Test Mixture 1-7

Separation of Test Mixture on a Monomeric Cig Phase 1-8

Separation of Test Mixture on a Polymeric Cjg Phase 1-9

Separation of Test Mixture on an Intermediate Cjg Phase 1-9

Structures of Retinol, fraAij'-P-Apo-lO'-Carotenal Oxime and the Carotenoids

Measured 1-18

Structures of Retinyl Paknitate, Tocol, and a-, y-, and 5-Tocopherol 1-18

Chromatogram from the Reversed-Phase LC Separation of Retinol,

fra«i'-P-Apo-10'-Carotenal Oxime, Lutein, Zeaxanthin, P-Cryptoxanthin,

rran^-Lycopene, a-Carotene, trans-^-CMoX&nt, and Retinyl Palmitate in

Human Serum Using Absorbance Detection. Chromatogram of Tocol,

6-Tocopherol, y-Tocopherol, and a-Tocopherol Using Ruorescence Detection 1-21

Chromatogram from the Reversed-Phase LC Separation of Retinol, Carotenoids,

and Tocopherols in a Mixture of Apricots, Carrots, Creamed Com, Creamed Peas,

Green Beans, Orange Juice, Peaches, Spinach, Sweet Potatoes, Tomato Paste,

and Winter Squash 1-22

Fast-Isocratic Separation of the Micronutrients in a Serum Extract 1-31

Slow-Gradient-Elution Separation of Micronutrients in a Serum Extract 1-31

Hydrodynamic Voltammograms for All-franj'-Retinol, Tocol, a-Tocopherol,

and All-rrani'-P-Carotene at a Glassy Carbon Electrode 1-32

Comparison of Absorbance and Electrochemical Detection of Carotenes 1-33

Comparison of the Absorbance with the Electrochemical Detection Results for the

Determination of PAl-trans-KtXmol, a-Tocopherol, and P-Carotene 1-34

Comparison of the Separation of an Algal P-Carotene Extract Using Reversed-Phase

Ci8 LC Columns 1-42

ix
List of Figures, Continued

Comparison of the Difference in the Separation Selectivity Using Methanol or

Acetonitrile as the Mobile Phase 1-43

Representative Chromatograms of P-Carotene Preparations Separated Using a Wide-Pore, Polymeric Cjg Column 1-44

Overlay of the Absorption Spectra of the Major P-Carotene Isomers Present

in the Preparations Investigated 1-46

Percent Initial Absorbance at X^^ of Lutein and P-Carotene Monitored in 18

Organic Solvents Over a Period of 10 Days at Ambient Temperature 1-52

The Effect of Nine Solvent Modifiers on the Separation of Seven Carotenoids 1-55

The Effect of Three Concentrations of THE on the Separation of Seven

Carotenoids 1-55

The Effect of Eive Column Temperatures on the Separation of Seven Carotenoids .... 1-56

The Separation of Serum and Mixed Diet Carotenoids Using the Optimized

HPLC Method » 1-56

Structures of Polar and Nonpolar Carotenoids 1-61

Column Selectivity Comparison for Various Cjg Phases Prepared Using

Monomeric and Polymeric Syntheses 1-62

Effect of Endcapping for Polymeric Cjg Phases for Polar and Nonpolar Solutes 1-63

Space Filling Models for P-Carotene and AU-trans Hydrocarbon Chains 1-64

Separations of Carotenoid Standards on Commercial and Monomeric Cjg

Columns and the Engineered C30 "Carotenoid Column" 1-65

Separation of Carotenoids from an Extract of SRM 2383, Vitamins and

Carotenoids in Food 1-66

Separation of a P-Carotene Preparation Containing a High Percentage of

cis-Isomers on Commercial C,g Columns and "Carotenoid Column" 1-66

X
List of Figures, Continued

Section 2: Determination of Glycyrrhetinic Acid

Structures of ISP-Glycyrrhetinic Acid and the Acetate Ester of

Glycyrrhetinic Acid 2-4

HPLC from the Analysis of a Standard Solution of 1 |a.g/mL GRA and

3.4 P-g/mL GRA Acetate Using Methanol-Water-Acetic Acid 2-7

HPLC from the Analysis of Plasma Extract Using Solid-Phase Extraction:

0.38 [ig/mL GRA and 2.9 |ag/mL GRA Acetate 2-7

Section 3: Measurement of Oltipraz in Serum

Schematic of the On-line Extraction System 3-5

Chromatograms of Serum Extracts 3-6

Single-Ion Monitoring of the M' Ion of Oltipraz from the Injection of a Serum Extract Containing About 1 ng of Oltipraz 3-7

Section 4: Ascorbic and Dehydroascorbic Acids Analyses

Chromatograms of Lyophilized and MPA-Preserved Human Plasma and

AA-Supplemented Plasma With and Without DTT Treatment 4-5

Separation of Ascorbic Acid in Human Plasma on an HPX87H Column 4-11

Separation of Ascorbic Acid on an Octadecylsilane Column 4-11

Concentration - Response Curves for Standard Solutions of Ascorbic Acid as a Function of Storage Time 4-12

Effect of DTT and MPA on the Conversion of DHAA to Ascorbic Acid in

Human Plasma 4-13

xi
List of Figures, Continued

Section 5: Determination of Vitamin Kj in Serum

The Catalytic Reduction of Quinone ' 5-3

Block Diagram of Vitamin Ki Catalytic Reduction/LC-Fluorescence System 5-7

Chromatogram of Fractionated Serum Extract 5-8

Section 6: Measurement of (4-Hydroxyphenyl)retinamide in Serum

Structure of Retinamides 6-2

xii
UST OF TABLES

Section 1: Measurement of Fat-Soluble Vitamins and Carotenoids in Serum

List of Columns Used 1-6

Resolution and Selectivity for Zeaxanthin/Lutein and P-Carotene/Lycopene on Selected Cjg Columns Using Methanol and THF-Methanol Compared to the

TBN/BaP a Value on Those Columns 1-9

Average Recovery of Carotenoids on All Columns Tested Using Different

Mobile Phases 1-11

Columns Used in This Study Grouped According to Percent Recoveries 1-12

Average Percent Recovery for Polymeric, Intermediate, and Monomeric

Columns Tested 1-12

Comparison of k' and Recovery for Columns from Different Lots 1-13

Comparison of k' and Recoveries on Columns with Different Frit Materials 1-14

Absorptivities and Maximum Wavelengths Used for Calibration 1-18

Results of the Analysis of SRM 968a, Fat-Soluble Vitamins in Human Serum 1-23

Mean Standard Deviation and Relative Standard Deviation for Two Quality

Control Serum Samples Measured Fifteen Times Over a Five-Week Period 1-24

Recovery of a Carotenoid Mixture on Selected Columns Before and After

Treatment with 0.05 M Ammonium Acetate in Methanol With and Without the

Addition of 0.05% TEA to an Acetonitrile-Based Mobile Phase 1-25

Linear Regression Calibration Curves for the Micronutrients by Each Detector 1-33

Variation of Absorbance Signal for P-Carotene with Mobile Phase

Composition and Frit Type 1-33

Detection Limits for the Analytes 1-33 .

Determination of Micronutrients in a Human Serum Pool 1-34

Source and Description of P-Carotene Products Investigated 1-38

xiii
List of Tables, Continued

LC Column Characteristics 1-39

Spectrophotometric Concentration of P-Carotene Products Relative to the

Gravimetric Concentrations 1-41

LC Determination of the Relative Composition of P-Carotene Isomers in

P-Carotene Preparations 1-45

List of Solvents, Sources, and Lot Numbers 1-50

Relative Solubility and Absorptivity of Lutein and P-Carotene in

Organic Solvents 1-50

Relative Lutein and P-Carotene Degradation in Organic Solvents 1-51

Bonded Phase Properties 1-62

Physical Properties of AJkyl-Modified Silica 1-64

Physical Properties of Intermediate Pore Size SiUcas 1-64

''':-] Section 2: Determination of Glycyrrhetinic Acid

HPLC Measurements of ISP-Glycyrrhetinic Acid in Plasma After Ion-Pairing

Solid-Phase Extraction 2-8

Section 3: Measurement of Oltipraz in Serum • »

-

Comparison of Oltipraz Concentrations Determined by Three Methods 3-7

Section 4: Ascorbic and Dehydroascorbic Acids Analyses

Summary of Analysis of Variance of Plasma Samples, Set 1, Supplemented with

AA at High- and Low-Normal Concentrations 4-5

Summary of Analyses of AA-Supplemented Plasma Samples, Set 2, Preserved

in MPA, 50g/L 4-5

xiv
List of Tables, Continued

Stability of Ascorbic and Dehydroascorbic Acids in MPA, 50 g/L, at 4 °C 4-6

Stability of Ascorbic and Dehydroascorbic Acids in MPA, 50 g/L 4-6

Stability of Ascorbic Acid in Freeze-Dried and Frozen Plasma 4-12

Comparison of Round-Robin AA Values and Values Obtained by NBS (mg/L) 4-12

Conversion of DHAA by DTT 4-12

Ascorbic Acid Concentrations (mg/L) in Plasma of 11 Normal Subjects 4-13

XV

MEASUREMENT OF FAT-SOLUBLE VITAMINS AND CAROTENOIDS IN SERUM

This section of the reference manual contains seven papers pertaining to the analysis of

vitamins A and E and the carotenoids. Most of the essential factors listed below come from

the first three papers (pages 1-3 through 1-35), which compare the recovery and selectivity of

carotenoids on 65 reversed-phase columns and describe two liquid chromatographic methods

employed by NISTfor carotenoid and fat-soluble vitamin analyses. The paper beginning with

page 1-36 compares ^-carotene preparations available from several suppliers. An evaluation

of various properties of lutein (a polar carotenoid) and ^-carotene (a non-polar carotenoid)

in different organic solvents is provided beginning with page 1-49. The separation of

carotenoids using optimized parameters on a polymeric Cjg and a C^q stationary phase

developed at NIST are described in the last two papers (pages 1-53 through 1-66). Listed

below are some of the more important factors related to these analyses.

Important Factors

Calibration and Sample Preparation

• Use a spectrophotometer and Beer's Law to calculate concentrations of calibration

solutions. Correct the concentration for chromatographic purity at the wavelength at which you performed your Beer's Law calculation. We use the wavelength of maximum

absorption.

• Because of its stability, we prefer P-apo-lO'-carotenal oxime as an internal standard for

the retinoids and carotenoids rather than P-apo-8'- or lO'-carotenal. Tocol is used as an

internal standard for the tocopherols.

• To overcome the binding of carotenoids to lipoproteins in serum, the proteins must first be denatured with ethanol. Acetone, acetonitrile, methanol, and perchloric acid yield

lower recoveries of the fat-soluble vitamins and carotenoids than ethanol.

• Use a water-immiscible organic solvent to extract the analytes from the serum matrix.

Hexane has been most frequently used, with a large ratio of volume of extractant to serum.

• Use an antioxidant in the solvent used to reconstitute serum extracts, e.g., 30 |ig/mL 2,6-

di-f-butyl-4-methylphenol (BHT).

1-1
• In our cooled autosampler (15 °C), significant losses of hydrocarbon carotenoids in serum extracts are observed in about 6 hours when extracts are reconstituted in ethanol.

This is presumably a result of coprecipitation with lipids in the extract. The

hydrocarbons (and lipids) may remain in solution longer when extracts are reconstituted

in 50:50 ethyl acetate: ethanol.

• Since carotenoid concentrations are frequently near the detection limit, make sure that

the serum extract is completely dissolved in the reconstituting solvent. Ultrasonic

agitation is helpful.

Chromatography

• Use a column to separate the fat-soluble vitamins and carotenoids in serum.

Polymerically bonded C,8 phases (and some intermediate phases) generally provide better

selectivity for the separation of carotenoids than monomerically bonded Cjg phases. The

C30 column provides better separation of some carotenoid isomers than the Cjg columns.

• Monomeric columns, on average, provide better recovery than polymeric columns.

• Methanol-based solvent systems provide higher carotenoid recoveries than acetonitrilebased systems.

• If you have to use an acetonitrile-based system, add ammonium acetate and

triethylamine to improve carotenoid recoveries.

• To avoid the possible loss of carotenoids, eliminate stainless steel frits.

• Residual acids and metal activity on column packing materials cause carotenoid losses.

• Use Standard Reference Material 968b, Fat-Soluble Vitamins and Cholesterol in Human

Serum, to validate your HPLC method.

For further information, please contact Katherine Sharpless at (301)975-3121 or Jeanice

Brown Thomas at (301)975-3120.

1-2
Journal of Chromatography, 595 (1992) 89-101

Elsevier Science Publishers B.V., Amsterdam

CHROM. 23 924

Evaluation of reversed-phase liquid chromatographic

columns for recovery and selectivity of selected

carotenoids

Katherine S. Epler and Lane C. Sander

Organic Analytical Research Division. Chemical Science and Technology Laboratory, National Institute of Standards and Technology,

Gaithersburg. MD 20899 ( USA

)

Regina G. Ziegler

Environmental Epidemiology Branch, Epidemiology and Bioslatistics Program, Division of Cancer Etiology. National Cancer Institute, Bethesda. MD 20892 (USA)

Stephen A. Wise and Neal E. Craft*

Organic Analytical Research Division. Chemical Science and Technology Laboratory . National Institute of Standards and Technology.

Gaithersburg. MD 20899 ( USA

)

(First received September 17th, 1991; revised manuscript received November 28th, 1991)

ABSTRACT

Sixty commercially available and five experimental liquid chromatography columns were evaluated for the separation and recovery of seven carotenoid compounds. Methanol- and acetonitrile-based solvents (either straight or modified with ethyl acetate or

tetrahydrofuran) were compared to determine which solvent systems and which columns provided better selectivity and recovery. Methanol-based solvents typically provided higher recoveries than did acetonitrile-based solvents. Polymeric C,g phases generally

provided better selectivity for the diflScult separation of lutein and zeaxanthin than did monomeric Cjg phases.

INTRODUCTION

Epidemiologists have observed a lower incidence

of lung cancer in people who have an above-average

intake of fruits and vegetables [1,2]. Studies also

suggest that the intake of fruits and vegetables may

reduce the risk of cancers of the mouth, pharynx,

larynx, esophagus, stomach, colon, rectum, bladder, and cervix [2]. Because low serum levels of

^-carotene are associated with the subsequent development of lung cancer, /3-carotene may be the

protective factor present in the fruits and vegetables

[2]. However, the lowered incidence of cancer may

be due to other carotenoids that are co-ingested

0021-9673/92/S05.00

with the ^-carotene, and serum levels of these other

carotenoids have not been adequately studied.

Liquid chromatography (LC) has been used to sep- arate and measure j9-carotene in serum [3] with concurrent measurement of a-carotene [4-10] and

sometimes lycopene [11-18]. In addtion to numerous cis/trans geometric isomers, human blood se- rum contains at least six structurally distinct carotenoids. To determine which carotenoid compound(s)

may provide anti-cancer effects, it is important to separate and measure the major carotenoids in se- rum and in food. Consequently, a number of workers have reported the measurement of carotenoids

other than lycopene, a-, and j9-carotene in serum © 1992 Elsevier Science Publishers B.V. All rights reserved

1-3
90 K. S. EPLER et al.

[19-31], in skin [32], in human milk [33] and in foods [34^8].

The majority of carotenoid separations reported

in the literature involve the use of reversed-phase

LC, generally on a Cis stationary phase, although a few normal-phase LC separations have been reported [21,24,35]. Most workers using reversed-phase

LC have used one of the following solvent systems: an acetonitrile-based eluent, an acetonitrile-based

eluent to which ammonium acetate has been added,

or a methanol-based eluent.

Acetonitrile-based eluents are used most fre- quently. In 1979, Zakaria et al. [49] separated lycopene, a-carotene, and j9-carotene in tomatoes on a Ci8 column using a mixture of chloroform and acetonitrile. In 1983, Nelis and De Leenheer [19] re- ported LC separations on Cig columns using acetonitrile or acetontrile and 8% methanol with various

organic modifiers added [tetrahydrofuran (THF),

diisopropyl ether, chloroform, dichloromethane,

ethyl acetate]. Most workers have adapted one of

these mixtures so that it provides acceptable results on their particular LC column [4,5,8,9,11-13,16-

18,20,22,25,26,30,31,35,38,40,46,48]. Khachik and

co-worker [37,41,42,47] typically add hexane to a mixture of methanol, acetonitrile, and methylene

chloride. Other workers have used acetonitrile

modified with THF and water [36], with water and

2-propanol [44,45], or in a 1:1 mixture with ethanol

[27].

The second basic method was described by Peng

[6], and employs a mixture of acetonitrile, THF,

methanol, and ammonium acetate. This method

has been adapted by Nierenberg et al. [3,14,29],

Kalman et al. [10], and CuUing-Berglund et al. [32]. Methanol-based separations cited include the tise of straight methanol [15] or methanol that has been

modified with THF or chloroform [35], with water

and THF [7], with water and butanol [23], and with

hexane [28]. Carotenoid separations reported in the literature

employ a wide variety of Cis and other reversedphase columns from different manufacturers. Several workers have compared separations on a small

number of commercially available columns: Nelis

and De Leenheer [19] have described the effects of

five organic modifiers on retention and selectivity of

carotenoids on two different Cis columns. Bushway

[35] has compared the selectivity of eight columns

(two normal-phase and six reversed-phase) using

several different solvent systems. Lauren and

McNaughton [50] have compared ten reversedphase columns with respect to alfalfa carotenoids'

elution order, retention time, peak height and

shape, etc. using acetonitrile and ethyl acetate with

or without 0.1% «-decanol.

Recovery of carotenoids from the LC column is an important factor in carotenoid analysis. Frequently, serum carotenoid concentrations are near

the detection limit so maximum sensitivity is neces- sary for accurate carotenoid measurements. This can only be achieved if carotenoid recovery is essentially 100%. Similarly, since epidemiological studies

continue for extended periods, reproducibility is ex- tremely important to the outcome of studies. Therefore only LC columns offering consistent and maximum recovery can yield the most meaningful results. Finally, there is a need to correlate carotenoid

analysis by LC to older spectrophotometric measurements. If LC columns do not provide complete

and consistent recovery of carotenoids, then LC

measurements cannot be correlated with spectrophotometric measurements of carotenoids. Only

one limited study has been reported that compared

serum carotenoids measured by LC to total carote- noid concentration determined statically in a spectrophotometer [20]. We have compared sixty-five

reversed-phase LC columns (primarily Cis-modified silica) for selectivity and recovery of a mixture

of seven carotenoids, six of which are present in the serum of most American populations. This is the

first study to report absolute recovery of carote- noids from LC columns.

EXPERIMENTAL"

Test mixture

An ethanol solution containing approximately 2

to 10 mg/1 each of lutein (Kemin Industries, Des

Moines, lA, USA); zeaxanthin, ^-cryptoxanthin

(Atomergic Chemetals, Farmingdale, NY, USA);

echinenone (Hoffmann-La Roche, Nutley, NJ,

" Certain commercial equipment, instruments, or materials are

identified in this report to specify adequately the experimental

procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equip- ment identified are necessarily the best available for the purpose.

1-4
EVALUATION OF REVERSED-PHASE LC COLUMNS 91

USA); lycopene (extracted from tomato paste); and

a- and ^-carotene (Sigma, St. Louis, MO, USA)

was prepared, and 2-ml aliquots of this solution were stored in amber glass vials at —20 °C. The

respective concentrations of the compounds in the

mixture were sufficiently different to provide some

indication of identity based on chromatographic

peak area. Prior to injection, 100 ^1 THF were added to a vial of the carotenoid mixture, and the mixture was placed in an ultrasonic bath for 10 min to ensure dissolution. This solution was then trans- ferred to a vial and placed in an autosampler where

it was held at a constant temperature of 15°C; the

autosampler injected a 20-//1 aliquot of the solution

onto the column being tested.

Chromatographic conditions

A liquid chromatograph pumped the mobile

phase at a rate of J. 5 ml/min. Solvents were premixed so as not to depend on reproducible mixing

by the pump; the solvents were sparged with helium.

The column being tested was held at 27°C by the LC

column oven.

Columns

A list of the columns tested, serial number, manufacturer, and classification of the bonded Cis sta- tionary phase (polymeric, monomeric, or intermediate [51]) is provided in Table L Reversed-phase

columns that were potentially useful for the separation of carotenoids were donated by LC column

manufacturers. Columns prepared from different

production lots from the same manufacturer were

also requested in order to assess reproducibility.

Several columns that were currently in use in our

laboratory were tested as well. Most of the columns

tested were Cis, 250 x 4.6 mm LD., with 5-ixm

particles. All columns were made of stainless steel. All columns were alkyl-silane modified silica, except

for the Biotage Unisphere Polybutadiene, which was modified alumina. The polymeric/monomeric/

intermediate classification given in Table I refers to the type of stationary phase modification procedure, and is described in detail in the Results and

Discussion section, as is the separation factor

(axBN-BaP value) to which it is related.

Eluents based in methanol and acetonitrile were

used in this study. Butylated hydroxytoluene-stabilized THF and ethyl acetate modifiers were added

to the mobile phase for those columns that failed to elute all seven compounds with a capacity factor

(k')^\\. Both THF [3,6,7,10,11,14,22,29,32,35,

36,43] and ethyl acetate [19,50] have been used for

carotenoid separations and represent two different

solvent selectivity groups. III and VL respectively.

(Chlorinated solvents such as methylene chloride

and chloroform were not tested because of a previ- ous report of carotenoid losses caused by the hydrochloric acid that may be a trace contaminant in these solvents [52].) Prior to performing a separation, each column was flushed with THF at L5 ml/

min for 4 min to remove any compounds remaining on the column from a previous run. The column

was then equilibrated at 1.5 ml/min for 15 min with

a given eluent. In cases where 100% methanol or

acetonitrile eluted the compounds from the column

with a k' < 7, no attempt was made to increase

retention by the addition of water because of the

minimal solubility of carotenoids in water and the

opportunity for on-column precipitation [19,23].

Recovery determination

The photodiode array detector (Model 990, Waters, Milford, MA, USA) used to monitor the LC

effluent acquired absorbance spectra from 350 to 500 nm with 20 scans/s at 2-s intervals. Data were

acquired for 25 min at 0.5 a.u.f.s. Spectral data were

used for peak identification when necessary. Visible

absorbance at 450 nm was recorded by the data

system for the quantitative comparison discussed

below.

For the recovery study, the column was replaced

with a I95-cm length of PTFE tubing (0.8-mm I.D.)

to provide "peak" dispersion similar to the LC col- umn. Using ethanol as the mobile phase, a 20-fil injection of the test mixture was considered to have

a peak area that represented 100% recovery. Five

such replicate measurements were made at the beginning and at the end of each day's work. With a column in place, the individual areas of the integrated peaks' were totalled, and the total area was nor- malized to the average area of the 10 replicate in- jections when no column was used. Because the test mixture contained small quantities (1 to 7%) of impurities that certain columns may have been capable of resolving, all peaks eluting from a column

were measured, not just the seven main peaks.

The linearity of the detector response was verified

1-5
92 K. S. EPLER et al.

TABLE I LIST OF COLUMNS USED

Columns are Cjg except where noted.

Supplier Column name Serial No. -'TBN/BaP Classification*

1 Beckman Ultrasphere ODS 9UE189S 1.92 Monomeric

2 Beckman Ultrasphere ODS 8UE2757 2.01 Monomeric

3 Beckman Ultrasphere ODS DABS 7UE2306 2.00 Monomeric

4 Analytichem Sepralyte CIS 072851-18 1.82 Monomeric

5 Separations Group Vydac 218TP 890130-16 0.83 Polymeric

6 Separations Group Vydac 201 TP 890130-23 0.78 Polymeric

7 Separations Group Vydac 201 TP 890131-26 0.80 Polymeric

8 Biotage Unisphere-PBD' 2167 3.06 Monomeric

9 ES Industries Gamma Bond CIS 19589-57-17992 1.86 Monomeric

10 ES Industries Chromegabond C22 (C^j) 19589-4-58-17994 1.83 Monomeric . 11 ES Industries Chromegabond MCI

8

195S9-4-57-17991 1.73 Monomeric . . 12 Supelco LC-18 18744F 2.03 Monomeric

13 Supelco LC-18 i8745F 2.02 Monomeric

14 Supelco LC318 110224 2.05 Monomeric

15 Supelco LC318 110232 2.04 Monomeric

16 Supelco XC-PAH 81695 0.70 Polymeric

17 Analytichem Sepralyte CIS 071912-14 1.92 Monomeric

18 AUtech Adsorbosphere CIS 08039GA 2.01 Monomeric

19 EM Science LiChrospher 100 RP-18 86554563 1.45 Intermediate 20 Serva Octadecyl 03118 1.84 Monomeric

21 Serva Triacontyl (Cjg) _d 22 J&W Accusphere ODS 9050825 2.07 Monomeric

23 J&W Accusphere ODS 9072526 1.96 Monomeric

24 MacMod Zorbax RX CS (Cg) AU2642 2.33 Monomeric

25 Perkin Elmer Pecosphere CIS 1119 2.00 Monomeric

26 YMC A303 42511 1.97 Monomeric

27 YMC AMP303 4259 2.01 Monomeric

28 YMC AP303 4250 2.06 Monomeric

29 J.T. Baker exper WP CIS" 1294-43 0.57 Polymeric

30 J.T. Baker exper WPC18" 1294-26 0.93 Polymeric

31 J.T. Baker exper WPC18" 1294-29 0.73 Polymeric

32 J.T. Baker exper WPC18" 1239-64C 1.42 Intermediate 33 J.T. Baker exper WP CIS" 1239-64D 0.22 Polymeric 34 ES industries Chromegabond PFP-'^ 19589-4-58-17993 0.88 Polymeric

35 ES Industries Chromegabond BF-C18 21389-4-60-18079 1.04 Intermediate 36 J.T. Baker* Bakerbond CI

8

A291 13-01 1.25 Intermediate 37 J.T. Baker" Bakerbond WPC18 B36097.25 0.51 Polymeric

38 J.T. Baker« Bakerbond WPC18 none 0.54 Polymeric

39 J.T. Baker* Bakerbond WPCIS B33125.16 1.09 Intermediate >

,

,

40 Keystone Sci. ODS Hypersil 03708 1.95 Monomeric

41 Shiseido Capsell Pak CIS SGI 20 1.99 Monomeric '

"• 42 Nacalai Tesque Cosmosil 5C18-P Waters 391-03 2.04 Monomeric

43 Nacalai Tesque Cosmosil 5C1S Waters 390-47 1.85 Monomeric ^• 44 Waters Nova-Pak CIS T93242 1.97 Monomeric • - 45 YMC ASP303-5 42541 2.05 Monomeric

46 Hewlett-Packard ODS Hypersil 799260D-584 1.98 Monomeric

47 Hewlett-Packard Licnrospner luu Kr-io lyylj\J\J-jo^ ti .jU Intermediate 48 Brownlee Spheri-5 ODS 102454 1.26 Intermediate 49 Bio-Rad Hi-Pore RP31S 890215-11 No. 82 0.59 Polymeric

50 Macherey-Nagel Nucleosil 5 PAH 90702B 0.36 Polymeric

51 Rainin* Microsorb CIS 10681 1.78 Monomeric

52 Phase Separations Spherisorb S5 PAH 23/123 00-1046 0.82 Polymeric

53 Phase Separations Spherisorb S5 ODS 29/69 19-1372 1.68 Intermediate

1-6
EVALUATION OF REVERSED-PHASE LC COLUMNS

TABLE I (continued)

93

Supplier Column name Serial No. a

" TBN/BaP Classification"

54 Phase Separations Spherisorb S5 ODS 30/35 06-10-Fl 1.50 Intermediate

55 Carlo Erba Erbasil 5 C18/L 517250502 1.76 Monomeric

56 Carlo Erba Erbasil 5 C18/M 517250503 1.28 Intermediate 57 Carlo Erba" Erbasil 5 C18/H 517250504 0.91 Polymeric

58 Brownlee Spheri-5 RP-18 109083 1.92 Monomeric

59 MacMod Zorbax RX C18 880967.902 1.50 Intermediate 60 Phenomenex Ultracarb 5 ODS20 PP/4953C 1.95 Monomeric

61 Phenomenex Ultracarb 5 ODS30 PP/4954C 2.01 Monomeric

62 J.T. Baker Bakerbond WP C18 1314-13 0.89 Polymeric

63 J.T. Baker Bakerbond WP C18 DS097 0.67 Polymeric

64 Brownlee Spheri-5 ODS 102402 1.42 Intermediate 65 MacMod* Zorbax ODS F36560 1.80 Monomeric

" Relative retention of 1.2:3,4:5, 6:7, 8-tetrabenzonaphthalene (TBN) to benzo[a]pyrene (BaP); see Results and Discussion section or ref. 51.

' As determined by evaluation of SRM 869 (see refs. 51 and 53).

" PBD = polybutadiene.

Returned to supplier before value could be determined.

' Experimental columns-with varying surface coverage.

/ PFP = pentafluorophenol.

' Used in laboratory prior to evaluation in this study.

* Not tested due to excessive backpressure ( > 200 bar).

by injecting varying amounts of the mixture of sev- en carotenoids. The detector response was linear

from 50 to 300% of the amount injected for evaluation of the columns.

RESULTS AND DISCUSSION

Sixty-five reversed-phase LC columns (Table I) were evaluated to determine selectivity and recovery of selected carotenoids. Lutein, zeaxanthin,

^-cryptoxanthin, echinenone, lycopene, and a- and

^-carotene were selected for use in the test mixture,

and are shown in Fig. 2. Excluding echinenone,

which is present in the human populations that con- sume echinoderms (sea urchins, starfish), these

compounds account for more than 90% of the caro- tenoids present in American serum [20]. Echinenone was included because it has intermediate polarity

and has been used as an internal standard for caro- tenoid measurements [20].

Column classification

A system has been developed for the classification of Ci8 stationary phases based on their sep- aration of a mixture of polycyclic aromatic hydrocarbons (PAHs) [51]. The application of this classification scheme has proven useful for selecting

columns for separating PAH isomers and steroids,

two classes of compounds with rigid molecular

Lutein

OH Ze£ixanthin

p-Cryptoxanthin

Echinenone

Lycopene

a-Carotene

p-Carotene

Fig. 1. Structures of carotenoids used in test mixture.

1-7
94 K. S. EPLER et a!.

Structures [51]. Because carotenoid isomers also

have a rigid structure, it was thought that the classi- fication scheme might provide insight into retention

mechanisms for carotenoids, as well as provide as- sistance in column selection.

The Ci8 columns in this study were classified into

three stationary phase types using Standard Refer- ence Material (SRM) 869, Column Selectivity Test

Mixture for Liquid Chromatography [51,53]. The

SRM is a mixture of three PAHs: benzo[a]pyrene

(BaP), 1 ,2:3,4:5,6:7,8-tetrabenzonaphthalene

(TBN), and phenanthro [3,4-c]phenanthrene. Columns were classified by calculating the relative re- tention of TBN to BaP. The resulting axBN-Bap val- ues were grouped as follows: those columns with an

axBN-BaP less than 1 are classified as polymeric,

those with an aiBN-BaP between 1 and 1.7 are classi- fied as intermediate, and those with an ayeN-BaP

greater than 1.7 are .classified as monomeric. The

ajBN-BaP values for each column are provided in

Table I. These values may not be directly comparable for the five columns whose stationary phases were not Cig. Nonetheless, 35 of the columns tested were classified as monomeric, 17 were polymeric

and 12 were intermediate at 27°C.

This classification scheme is strongly related to the procedures used in the bonded-phase synthesis,

i.e., monomeric phases prepared by the reaction of

silica with monofunctional silanes (usually in the

absence of water) have properties that differ from

polymeric phases prepared by reaction of silica with

trifunctional silanes in the presence of water. (The

third group is an arbitrary classification for which

phase chemistry is less certain; this group has properties that are intermediate to the monomeric and

polymeric classes.)

Selectivity

Optimum carotenoid selectivity of each column

was difficult to assess, since a mobile phase was not

tailored for each. Our design was limited to an isocratic, nonaqueous binary mobile phase separation,

with the capacity factor, k', manipulated through

the addition of THF or ethyl acetate such that 7 ^

k' ^ 11. Under the defined conditions, lutein and

zeaxanthin were the most difficult carotenoids to resolve due to their structural similarity and early

elution.

No monomeric Cig column evaluated in this

study was able to resolve lutein and zeaxanthin us- ing methanol or methanol-based solvents. Using

acetonitrile-based solvents, monomeric Ci8 columns were sometimes able to separate this pair partially. The polymeric Cig columns were usually able

to separate lutein and zeaxanthin. Typical chromatograms for columns classified as monomeric,

polymeric, and intermediate are shown in Figs. 2, 3 and 4, respectively; chromatographic conditions are described in those figures. In Table II, resolution

(R) and a values for zeaxanthin/lutein and jS-carotene/lycopene are shown for a representative onethird of the columns. The actual frequency and in- ability of a class of columns to separate lutein and

zeaxanthin is represented, i.e., if one in every three

monomeric columns failed to separate lutein and

zeaxanthin, then one of every three numbers shown

for the monomeric columns in Table II is for a column that failed to separate this pair. Using monomeric and most intermediate columns, the elution

of lycopene was followed by that of a- and ^-caroACETONITRILE-BASED SOLVENT RECOVERY = 71%

J

METHANOL-BASED SOLVENT RECOVERY = 84%

Ly o

10 15 20

MINUTES

Fig. 2. Separation of test mixture on a monomeric C,g phase. Eluent is THF-methanol (10:90) or THF-acetonitrile (15:85) at a flow-rate of 1.5 ml/min. Legend: L = lutein, Z = zeaxanthin. C = ^-cryptoxanthin. E = echinenone, Ly = lycopene. a =

a-carotene, P = )3-carotene.
EVALUATION OF REVERSED-PHASE LC COLUMNS 95

ACETONITRILE-BASED SOLVENT

P RECOVERY = 50%

I I

METHANOL-BASED SOLVENT RECOVERY = 92%

r

Ly

MINUTES

Fig. 3. Separation of test mixture on a polymeric Cjg phase. Eluent is 100% methanol or 100% acetonitrile at a flow-rate of

1.5 ml/min. For legend, see Fig. 2.

ACETONITRILE-BASED SOLVENT

RECOVERY = 71%

t.X

tij o

z

<

m

DCO

w

CD< •\

METHANOL-BASED SOLVENT

RECOVERY = 86%

10 15 MINUTES20

Fig. 4. Separation of test mixture on an intermediate C,8 phase. Eluent is THF-methanol (7.5:92.5) or THF-acetonitrile

(12.5:87.5) at a flow-rate of 1.5 ml/min. For legend, see Fig. 2.

TABLE II RESOLUTION (R) AND SELECTIVITY (a) FOR ZEAXANTHIN/LUTEIN (Z/L) AND /?-CAROTENE/LYCOPENE (B/Ly) ON

SELECTED C,8 COLUMNS USING METHANOL AND THF-METHANOL COMPARED TO THE TBN/BaP a VALUE ON

THOSE COLUMNS

Column" Character «Z/L "'rBN/BaP

22 Monomeric 0 1.28 13 2.07

14 Monomeric 0 1.20 6.2 2.05

18 Monomeric 0 1.28 14 2.01 25 Monomeric 0 1.18 7.8 2.00 41 Monomeric 0 1.24 7.8 1.99 44 Monomeric 0 1.29 13 1.97 26 Monomeric 0 1.27 12 1.97 40 Monomeric 0 1.29 12 1.95

1 Monomeric 0 1.28 13 1.92 20 Monomeric 0 1.19 7.4 1.84

11 Monomeric 0 1.36 7.9 1.73

53 Intermediate 0 1.15 10 1.68 59 Intermediate 0 1.24 13 1.50

19 Intermediate 0 2.16 19 1.45 35 Intermediate 1.20 2.9 0.91 6.0 1.04

30 Polymeric 1.18 2.9 0.78 8.0 0.93

5 Polymeric 1.21 2.9 0.83 5.8 0.83 6 Polymeric 1.23 4.0 0.74 12 0.78

16 Polymeric 1.22 4.4 0.74 12 0.70 49 Polymeric 1.26 3.9 0.70 16 0.59 37 Polymeric 1.32 4.6 0.56 35 0.51

See Table I for column descriptions.

1-9
96 K. S. EPLER et al.

tene. Using polymeric columns, the elution order

often changed to a-carotene, /^-carotene, lycopene.

thus the a- values for the polymeric columns (and one intermediate column) in Table II are less than

one. In general, if the column could resolve lutein

and zeaxanthin, then lycopene eluted after a- and

j5-carotene. Quackenbush and Smallidge [54] and

Bushway [35] have also observed that Vydac 201 TP

and 218TP columns, which are polymeric, reverse

the order of elution of lycopene and j8-carotene

compared to other (monomeric) columns that they

tested, although they did not recognize the monomeric or polymeric phase synthesis of the columns

as the cause. Polymeric phases have also been re- ported to provide better separation of certain carotenoids and their cis isomers [35,54,55].

To investigate whether the selectivity for selected

carotenoids correlates with the selectivities for the

PAHs, aiBN/BaP values for the columns are also

shown in Table II. On the polymeric columns,

aTBN/BaP and azeaxamhin/iutein arc corrclatcd (correlation coefficient, r = — 0.94), as are arBN/BaP and a^.

carotene/iycopene ('' = 0.86). Dcspitc the Small Sample

size (« = 6 polymeric columns), there is more than

95% confidence that a correlation does exist, ac- cording to Pearson Product Moment correlation

coefficient tables [56]. There is no correlation between the axBN/BaP and the a^eaxanthin/iutein ou thc

monomeric columns because all the oCzeaxanthin/iutein values are 1, as is the case for most of the intermediate columns' '^zeaxanthin/lulein ValuCS. ThcrC is alSO nO

correlation between the axBN/BaP and the a^.carotene/iy- copene on both thc monomcHc and intermediate col- umns. Thus, the ajBN/BaP values are useful for predicting whether a column will be able to separate

lutein and zeaxanthin inasmuch as it indicates

whether the column is monomeric, polymeric, or

intermediate, since mainly polymeric columns are capable of resolving this pair. However, it is not

useful for predicting whether an intermediate column will be able to resolve lutein and zeaxanthin, nor can it predict how much separation of lycopene

and ^-carotene a monomeric or intermediate column is likely to provide.

For most columns it was necessary to add a mobile phase modifier to elute compounds with a /c' ^

11. The choice of modifier (THF or ethyl acetate)

did not significantly aff'ect selectivity for either lu- tein/zeaxanthin or lycopene/^-carotene, the two

pairs that were examined. Columns that failed to

resolve lutein and zeaxanthin using methanol-based

solvents failed using each modifier. On a few columns, the use of one modifier in acetonitrile did

provide higher a values than the other, but the dif- ference was not great enough to significantly improve resolution. THF did not consistently provide

higher a values than ethyl acetate or vice versa. For

the separation of lycopene and /9-carotene, selectivity differed slightly using acetonitrile-based mobile phases on some columns. On one or two columns using methanol-based solvents, the a values

for lycopene and ^-carotene may have been more

significantly aff"ected by the choice of modifier.

Again. THF was not consistently better or worse

than ethyl acetate at providing greater resolution.

Pore size of the column can affect the selectivity

for carotenoids of sufficiently diff"erent size {e.g., lycopene and j5-carotene, zeaxanthin and j3-carotene)

but does not affect selectivities for carotenoids of

very similar sizes {e.g., a-carotene and jS-carotene). In a comparison of four monomeric columns from

the same manufacturer (column 45, 26, 27 and 28),

the only difference being pore size of the silica substrate {i.e., 60, 120, 200 and 300 A, respectively),

both (Xp-carotene/lycopene and OC^.carotene/zeaxanthin dcCreaSC

with increasing pore size. This also holds true for a comparison of two other sets of wide-pore columns

and their narrow-pore counterparts (columns 12

and 13, 100 A vs. columns 14 and 15, 300 A; column

36, 120 A vs. column 37, 300 A). In addition, as pore size decreases, more modifier must be added in

all three cases to elute all seven compounds with a

k' ^ 11. This is as expected since the greater surface area of the base silica of the narrow-pore columns

results in a higer carbon load. Although absolute

retention of carotenoids increases with decreasing

pore size (and increasing carbon loading), a values

for similarly sized carotenoids do not change with

column pore size. An example of this trend is the a

values for ^-carotene/a-carotene, which are the same for the sets of columns described above.

Recovery

Sample losses on the column are critical to the

sensitivity and precision of an L<^ method and to quantitative analysis. Carotenoid recoveries ranging from 0 to almost 100% were observed. Recovery was dependent on the mobile phase, stationary

phase type classification, and possibly the column

bed support frit material.

1-10
EVALUATION OF REVERSED-PHASE LC COLUMNS 97

Mobile phase. On almost all the columns tested,

the use of methanol or methanol-based solvents

provided a higher recovery of the carotenoids than

did the use of acetonitrile of acetonitrile-based sol- vents, as shown by the mean recoveries given in Table III. When using acetonitrile-based mobile phases, the THF modifier resulted in a higher percent

recovery on most columns than did the ethyl acetate. This difference in recovery was less noticeable

when the methanol-based eluents were used. Addition of a modifier was not necessary for about half

the columns when methanol was used and about

one-third of the columns when acetonitrile was

used. Typically, it took less THF than ethyl acetate

to elute the compounds with 11, and methanol

required less modifier than acetonitrile. which is contrary to expectations based on solvent strength

parameters, where acetonitrile is classified as a stronger solvent than methanol [57]. However, this observation is consistent with the findings of Nelis

and De Leenheer, who reported that methanol acts as a stronger solvent than acetonitrile for the sep- aration of carotenoids on Cis columns [19]. Since

recoveries were lower and no improvements in se- lectivity were observed, ethyl acetate-modified sol- vents are not discussed further to simplify data

analysis. Henceforth each column has just two sets of results: 100% methanol and 100% acetonitrile, or THF-methanol and THF-acetonitrile if it was

necessary to use a modifier.

TABLE III AVERAGE RECOVERY OF CAROTENOIDS ON ALL

COLUMNS TESTED USING DIFFERENT MOBILE PHASES

THF or ethyl acetate are added to methanol and acetonitrile as modifiers such that 7 ^ k' ^ix ^ ' ^

Mobile phase Recovery ± S.D. (%)

100% methanol 84±8 {n = 29)" Methanol-THF 86± 11 (« = 35) Methanol-ethyl 82± 12 (« = 35) acetate 100% Acetonitrile 56± 19 {n = 21) Acetonitrile-THF 68± 17 (n = 43)

Acetonitrile-ethyl Al±\l (n = 43)

acetate

" « = Number of columns.

Acetonitrile-based solvents are typically used for

the carotenoid separations reported in the literature

[3-12,14.17-20,22,25,26,29-38,40^8]. However,

our studies show that recoveries using acetonitrile

and acetonitrile-based eluents are generally lower

than those obtained using methanol and methanolbased eluents. Nelis and De Leenheer [19] reported

that the incorporation of methanol in acetonitrilebased solvents dramatically enhanced selectivity,

which they speculated was due to hydrogen bonding. Most of the acetonitrile-based methods reported in the literature do involve the use of some methanol [3,6,7,9-11,14,17,19,20,22,26,29-35,37,38,40-

44,46-48]. To determine whether the addition of

methanol improved recovery as well as selectivity, a column that had provided 0% recovery when run

with 100% acetonitrile and 77% recovery with

100% methanol was tested with a methanol-acetonitrile ( 10:90) mixture. Recovery with respect to the

100% acetonitrile run improved only slightly (up

from 0 to 2%). A run using methanol-acetonitrile

(20:80) as the eluent resulted in 4% recovery. Thus

the addition of methanol did improve recovery

marginally. The column tested performed poorly

using 100% acetonitrile; it would be unfair to say

that recovery on all columns using acetonitrilebased solvents would also show only a 2%o increase

in recovery with the addition of methanol.

The poor performance of certain columns appears to be real and not an artifact. When poorly

performing columns were rechecked at a later date,

performance had not changed. In addition, recovery was not affected when various sample sizes were

injected (from 5 /^l to 100 ^I). Lauren and

McNaughton [50] recommend the addition of 0.1%

A7-decanol to the mobile phase to improve performance by minimizing adsorption. We did not find n- decanol to be effective in improving the performance of columns with poor recoveries using acetonitrile-based solvents. However, we have observed

that flushing the column with an ammonium acetate buffer improves recovery on these columns

when an acetonitrile-based mobile phase is used.

We have also found that the addition of ammonium

acetate to the mobile phase improves recoveries.

To determine the reproducibility of recovery results, one column from each of the three classification groups was retested about four months after

the initial test. Columns that had required 100%

1-11
98 K. S. EPLER et al.

methanol were selected to eliminate any eflfect caused by slight variations in the mobile phase composition. Columns were also selected that had provided recoveries greater than 80% in the first test. Relative standard deviations ranged from 2.5 to 5.6%. An insufficient number of runs were made on

individual columns to allow statistical comparisons

of methanol or methanol-based and acetonitrile or

acetonitrile-based recoveries for each column;

however, based on the reproducibility studies, 6%

represents a liberal estimate of the relative standard

deviation. Using this estimate of variability, columns with a 12% difference in recovery between

methanol- and acetonitrile-based eluents would be

significantly different. Columns are grouped ac- cording to their recoveries using methanol or THFmethanol and acetonitrile or THF-acetonitrile in

Table IV.

Selective recovery^ Experiments were performed

to determine whether certain carotenoids contributed a greater share to recovery losses than did the

other carotenoids. Individual carotenoids were in- jected into the system, both with and without a colTABLE IV COLUMNS USED IN THIS STUDY GROUPED ACCORDING TO PERCENT RECOVERIES

Recovery Columns

(%)

Using methanol or THF-methanol:

90-100 3 57 11 15 21 23 24 26 27 28 29 30 32 41 42 43 44 45

52 65 80-89 1 24 68 10 12 13 14 16 17 18 19 22 25 31 35 37 38 39 40 46 47 48 49 50 51 53 54 58 59 60 61 70-79 20 33 36 55 63 64 60-69 42 62 50-59 9 40-49

30-39 56

Using acetonitrile or THF-acetonitrile: 90-100 43 50 51 80-89 2 3 23 27 37 49 60 61 70-79 1 4 8 12 13 18 21 22 26 30 32 41 42 45 47 52 58 59 60-69 5 10 25 28 29 33 36 44 48 65 50-59 9 1114 15 17 20 24 31 38 40 46 40-49 6 7 16 19 35 39 62 64 30-39 63 20-29

10-19 0-9 53 54 55 56

umn, as described. Two monomeric-phase and

three polymeric-phase columns were tested. All columns had previously required 100% methanol for

elution of the carotenoids in less than 25 min. Four

of the columns had required 100% acetonitrile; one

required THF-acetonitrile (5:95). Two trends in re- covery were observed. Losses of zeaxanthin and

/^-carotene, which each contain two ^-rings (Fig. 1), were greater than losses of lutein and a-carotene,

which each contain one fi- and one 8-ring. Within

the group of ^,^-carotenoids used in the mixture

(zeaxanthin, ^-cryptoxanthin, echinenone, and

^-carotene), recovery increased as polarity decreased: less zeaxanthin, a dihydroxy carotenoid, was recovered than ^-cryptoxanthin, a monohydroxy carotenoid. Less ^-cryptoxanthin was reco- vered than echinenone, a mono-keto carotenoid.

And less echinenone was recovered than ^-carotene, a hydrocarbon carotenoid. This trend also

holds true for the two ^,£-carotenoids used in the

mixture. The recovery of the dihydroxy carotenoid,

lutein, was less than that of the hydrocarbon carote- noid, a-carotene. Stationary phase. In these carotenoid studies, as a

rule, polymeric phases required the least modifier (a

mean value of 2% THF in methanol, 3% THF in acetonitrile) and monomeric phases required the

most (a mean value of 6% THF in methanol, 9%

THF in acetonitrile) to elute all seven compounds

from the column within the k' range allowed

(7 ^ A:' ^ 11 ). Intermediate phases required an intermediate amount of modifier (4% THF in methanol,

6% THF in acetonitrile). Percent recovery across

the three groups did not vary consistently. Recovery results for columns divided into the three classi- fication groups are shown in Table V. Thirty per- cent of the columns gave >90% recovery using

TABLE V

AVERAGE PERCENT RECOVERY FOR POLYMERIC,

INTERMEDIATE, AND MONOMERIC COLUMNS TESTED

Eluent Polymeric Intermediate Monomeric

MeOH'^ 83% 79% 88%

ACN* 62% 46% 67%

" MeOH = methanol and methanol-based eluents. ACN = acetonitrile and acetonitrile-based eluents.

1-12
EVALUATION OF REVERSED-PHASE LC COLUMNS 99

methanol or THF-methanol as the solvent; of

these, 70% were classified as monomeric and the

remaining 30% were classified as polymeric.

In several cases, columns were available that contained the "same" packing material from different

lots. Recovery results and k' values for selected

compounds using THF-methanol for columns

from different production lots are presented in Table VT Mean recoveries for many of the columns

appear to be different, but performance of Tukey's

Multiple Comparison test [58] on these data shows

that there is no difference between the mean recoveries at a 95% confidence level for almost all the

column pairs.

Retention times of carotenoids using columns

from different stationary phase lots varied substantially. (On one pair of columns, there was a 15%

difference in ^-carotene's retention time.) This variability can arise from two sources: the base silica from which the stationary phase is prepared and

reproducibility of the bonding procedure. Most LC

column manufacturers obtain silica from outside

suppliers, and differences between column lots is strongly related to differences in the silica—particle

size, pore size, surface area, silanol activity, and

trace metal contamination. Thus, column lot differ- ences can exist for both monomeric and polymeric

phases. The only way to ensure reproducible col- umns is to purchase columns packed with the same

production lot of stationary phase.

Column bed support frits. It has been suggested

that losses occurring on column frit materials may

be partially responsible for low recoveries of carote- noids [3,23]. Data from two sets of columns that were identical except for the presence of different

frits are presented in Table VII. Although recoveries were slightly lower for stainless steel frits, no

significant differences in recoveries between these

sets of columns were detected. Later studies in which frits were placed in series (without a column

in the system) showed no significant difference in recovery using stainless steel, titanium, or "biocompatible" frits. In 1986, Nierenberg and Lester [3] observed difTABLE VI COMPARISON OF k' AND RECOVERY FOR COLUMNS FROM DIFFERENT LOTS

Column" k' Recovery

(%)

Lutein Echinenone ^-carotene

4 0.30 2.91 8.38 87 3 17 0.20 2.75 8.08 82 3

12 0.26 2.60 7.07 92 3 13 0.20 2.66 7.25 89 3

14 0.27 2.21 5.86 88 3 15 0.30 2.31 6.07 90 5

16 0.73 1.83 2.80 86 5 66' 0.66 2.00 2.95 92 5

40 0.43 3.18 8.22 86 3 46 0.49 3.06 7.76 92 3

48 0.35 3.24 8.16 86 3 64 0.38 3.57 9.18 78 3

37 1.03 3.00 4.81 82 3 62 0.74 2.58 3.89 87'' 4

63 0.59 2.60 3.82 73'' 2

" See Table I for column descriptions.

* n = Number of runs.

' Supelco LC-PAH column tested after the completion of the other analyses.

** Statistical difference in recovery as determined by Tukey's Multiple Comparisons.

1-13
100 K. S. EPLER et al.

TABLE VII COMPARISON OF k' AND RECOVERIES ON COLUMNS WITH DIFFERENT FRIT MATERIALS

Column" Frit k'

Lutein Echinenone jS-carotene

Recovery

(%)

n

6 SS" 0.75 2.46 3.30 86 5 7 Ti 0.75 2.53 3.42 88 5 -. .

1 SS 0.28 2.85 8.27 87 5 3 Ti 0.29 3.02 8.93 92 5 2 Hast 0.33 2.98 8.54 93 5

" See Table I for column description.

* SS = Stainless steel; Ti = titanium; Hast = Hastelloy.

ferences in recovery when they switched to a new

column of the same brand from the same manufacturer, but with a different kind of frit, and they

attributed these differences to the use of different

frits (stainless steel vs. Hastelloy). They also observed this phenomenon when two columns were packed with the same lot of packing material,

topped with either stainless steel or Hastelloy frits. During our studies, it was observed that a certain

previously unused column did not provide the same

recovery or retention times as its older, used, counterpart, but that the "new" column gradually at- tained the retention and recovery characteristics of

the "old" column with repeated use. Columns that

had been used in our laboratory prior to this study

have been marked in Table I, in the event that re- sults for these columns have been affected by previ- ous use in the laboratory.

CONCLUSIONS

Several conclusions concerning the selectivity

and recovery of carotenoids in reversed-phase LC

can be drawn from this study: (1) In general, separations using acetonitrile or the frequently employed acetonitrile-based solvents resulted in lower

recoveries than separations using methanol or methanol-based solvents. (2) Monomeric Cig columns generally provided high recoveries, but they were unable to resolve lutein and zeaxanthin using

methanol or methanol-based eluents, which were

the solvents yielding the highest recoveries. (3)

Polymeric Cis columns were usually able to resolve

lutein and zeaxanthin in methanol-based solvents,

but on average had lower recoveries than monomeric Ci8 columns. (4) Using methanol-based sol- vents, the retention time of lycopene is greater than

that of ^-carotene on most polymeric columns. (5)

Columns with the "same" stationary phases from

different lots do not necessarily elute compounds

with the same retention times. (6) When using a new

column, it may be necessary to make several preliminary runs before the separation {i.e., retention

times, recovery, selectivity) becomes reproducible

and comparable to that expected based on the per- formance of the corresponding "old" column. (7)

The most appropriate system for the separation of

carotenoids would probably include the use of a polymeric Cis column (to allow separation of lutein

and zeaxanthin) and a methanol-based mobile

phase (to obtain a high percent recovery).

ACKNOWLEDGEMENTS

This work was supported in part by the National

Cancer Institute, Division of Cancer Etiology,

Agreement No. Y01-CP9-0513. We extend our

thanks to the many companies who supplied us with their columns for this study.

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1-14
EVALUATION OF REVERSED-PHASE LC COLUMNS 101

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1959.

1-15
Journal of Chromatography. 619 (1993) 37^8 '

Biomedical Applications

Elsevier Science Publishers B.V., Amsterdam

CHROMBIO. 7021

Liquid chromatographic method for the determination of

carotenoids, retinoids and tocopherols in human serum

and in food

Katherine S. Epler

Organic Analy tical Research Division. Chemical Science and Technology Lahoratorv . Saiional Institute ul Standards and Technologv.

i S Department of Commerce. Technology .Administration. Oaithershurg. MD 20899 i CSA i

Regina G. Ziegler

Environmental Epidemiology Branch. Epidemiology and Biostatisiics Program. Division of Cancer Etiologv. National Cancer Institute

Bethe.<;da. !WD 20892 'L SA i

Neal E. Craft*

Organic Analytical Research Division. Chemical Science and Technology Laboratory. National Institute of Standards and Technologv.

U.S. Department of Commerce. Technology Administration. Gaithershurg. .\4D 20899 (i'SAi

(First received April 2nd. 1993; revised manuscript received May 31st. 1993)

ABSTRACT

A liquid chromatographic (LC) method has been developed for the quantitative measurement of the six major carotenoids in human

serum (lutein, zeaxanthin. jS-cryptoxanthm. lycopene. 2-carotene, and ^-carotene) as well as retinol, retinyl palmitaie, i-tocopherol.

•/-tocopherol, and ^-tocopherol. Several polar carotenoids. 2'.3'-anhydrolutein. a-cryptoxanthin, and geometric isomers of Kcopene

and ^-carotene are also separated. Retinoids and carotenoids are monitored using a programmable ultraviolet-visible detector, while tocopherols are monitored using a fluorescence detector. The method uses a gradient containing acetonitrile. methanol, and ethyl

acetate. Ammonium acetate is introduced with the methanol to minimize carotenoid losses on the LC column aggravated by the use of

acetonitnle and ethyl acetate. The method is also applicable to the analysis of foods.

INTRODUCTION

Epidemiologists have observed a reduced risk of lung cancer in people who have an elevated

intake of fruits and vegetables [1]. Epidemiologic

studies also suggest that eating fruits and vegetables may reduce the risk of other cancers as well

[1]. Because low serum levels of ^-carotene are associated with the subsequent development of

* Corresponding author.

Present address; Southern Testing and Research Laboratories. Wilson, NC 27893, USA.

0378-4347,93/506.00 t 1993 Elsevier Science Publishers B.V.

lung cancer, j3-carotene may be the protective

factor present in the fruits and vegetables [1]. However, the lowered risk of cancer may be due

to other carotenoids that are ingested along with

the /^-carotene, and the importance of these other

carotenoids has not been studied adequately.

Six carotenoids (lutein, zea.xanthin. ^-cryptoxanthin, lycopene. a-carotene, and j?-carotene)

reportedly account for more than 90% of the ca- rotenoids present in the serum of American populations [2]. The goal of this study was to sep- arate and measure the six major carotenoids in serum (as well as some of the geometric isomers

All rights reserved

1-16
38 A.. .V Hph'r cl til J Clirnmaim;r 1/V /OVJ " J\

of these carotenoids). retinol. tocopherols, and

retinyl palmitate. The reversed-phase liquid chromatographic (LC) method that has been developed allows quantitation of retinol. retinyl palmitate, lutein, zeaxanthin. /J-cryptoxanthin.

^ra/w-lycopene. total lycopene. /ra/?.9-a-carotene. total x-carotene. //-am-Zi-carotene. total [}-Cdvo- tene. - -tocopherol. c)-tocopherol. and a-tocopherol. The method also resolves a-cryptoxanthin.

2'.3'-anhydrolutein. and the cis isomers of lycopene. x-carotene. and /^-carotene, as well as some

unidentified polar carotenoids. Most published

methods use reversed-phase LC to separate major carotenoids but do not resolve geometric iso- mers [2-9]. Some workers do report the separation of geometric isomers, usually those of /^-ca- rotene, when measuring the major carotenoids

[10-12]. Other workers have concentrated on the

separation of just /^-carotene isomers [13.14] or isomers of both a- and /^-carotene [15.16]. To

measure serum levels of each major carotenoid

accurately, it is important that isomers of that

carotenoid not interfere with the measurement of

the isomer of interest, otherwise that measure- ment should be identified as a "total" measurement, i.e.. "total j3-carotene"" as opposed to

"frart5-/J-carotene"' or the somewhat indefinite

"^-carotene." The ideal method would separate

all the carotenoids and geometric isomers of in- terest in the serum as well as the retinoids and

tocopherols. Of course, it is difficult to know

whether all the isomers are separated; if a method

cannot separate two isomers, it is not possible to

tell that there are two isomers present merely by

looking at a chromatogram that has resulted

from the measurement of UV absorbance at a

single wavelength.

In work similar to this study. Hess et al. [17]

separated retinol. the tocopherols. /3-cryptoxanthin. lycopene. a-carotene. /^-carotene, and some

isomers in human serum, and measured them

with both UV absorbance and fluorescence; however, they were unable to resolve lutein and zea- xanthin. Using the method described here, we

have analyzed standard reference material

(SR.M) 968a. fat-soluble vitamins in human se- rum, for the compounds listed above. Although

a-cryptoxanihin. 2'.3'-anhydrolutein. and cis \^o- mers of Kcopene. x-carotene. and /^caro^ene are resolved from the other components using this method, standards were not available to permit

quantitation of these compounds. We have also demonstrated the method's applicability to food

by analyzing a mixture of foods.

EXPERIMENTAL^

Iniernal sumdards

Tocol (Esai. Tokyo. Japan) was used as the internal standard for the tocopherols, tnins-li- .A.po-10'-caroienaroxime. the internal standard

for the retinoids and carotenoids. was prepared

from fra/7.v-/^-apo- lO'-carotenal (HotTman-LaRoche. Basel. Switzerland) [18]. The oxime was

purified by passing a concentrated solution

through a preparative Vydac 201TP column us- ing 50:50 methanol-acetonitrile and collecting

the fraction that contained the truns-li-d^O'\0'

carotenal oxime. The resulting solution was 99.6% pure.

Retinol and carotenoids

Three calibration standards containing known

low. intermediate, and high levels (relative to the physiological levels in serum) of lutein (Kemin

Industries. Des Moines. I A. USA), zeaxanthin

and z^-cryptoxanthin (Atomergic Chemetals.

Farmingdale. NY. USA), and retinol. a-, and

^-carotene (Sigma. St. Louis. MO. USA) were

prepared in ethanol with 30 /zg ml butvlated hydroxytoluene (BHT. an antioxidant) added.

Structures of retinol. these carotenoids. and

//•artj-/^-apo-10'-carotenal oxime are shown in Fig. 1. Stock solutions from which standards were prepared were filtered through 45-//m PTFE

syringe filters. Concentrations of these stock so- lutions were determined spectrophotometncall>.

Certain commercial equipment, instruments, or materials are

identified in this report to specify adequateh the experimental

procedure. Such identification does not impK recommendation or endorsement by the National Institute of Standards and Technology . nor does it imply that the materials or equip- ment identified are necessarily the best available for the purpose.

1-17
K S Epier ei ai V. Chromatogr 619 1 1993) 37-48 39

Fig. I. Structures of retinol, /rarti-i^-apo-lO'-carotenal oxime. and the carotenoids measured using this method.

and LC analysis of the stock solutions was per- formed to determine the purity of each solution.

Absorptivities and maximum wavelengths used

for calibration are shown in Table I. The cali- bration curve was prepared immediately following preparation of the standards. A 1-ml volume

of each calibration solution was combined with

200 ^1 of internal standard solution, and the re- sulting solution was placed in a glass insert m an autosampler vial. The autosampler. which held

the samples at 15°C, injected a lO-^l aliquot of

the solution onto the column.

Due to rapid degradation, three lycopene (Sigma) calibration solutions were prepared separately for immediate use. These solutions were

also prepared m ethanol with 30 /^g ml BHT added, and 1 ml of each solution was mixed with 200

li\ of internal standard solution.

Tocopherols and retinyl palmitate

Solutions containing low, intermediate, and

high levels of a-. 6-, and -/-tocopherol (Eastman

Kodak, Rochester, NY. USA) and retinyl palmitate (Sigma) were prepared as described above.

Structures of retinyl palmitate, tocol. x-tocopherol, d-tocopherol, and y-tocopherol are shown in Fig. 2.

Serum extracts SRM 968a, fat-soluble vitamins in human se- rum (Standard Reference Materials Program.

National Institute of Standards and Technology.

Gaithersburg. MD. USA), was reconstituted

with 1.0 ml of HPLC-grade water. Duplicate

31 Retnyl palmitate

TABLE I ^0,^Y^ ''"s ^ , LJIv^4-CH,-(CH -CH -CH-CH ) -H Tocol ABSORPTIVITIES AND MAXIMUM WAVELENGTHS ° 2 2 2 2

3 USED FOR CALIBRATION

Compound Absorptivity (dl/g • cm) /.^^^ (nm)

Retinol 1850 325

Lutein 2765 445

Zeaxanthin 2416 452

/J-Cryptoxanthin 2486 452

frani- Lycopene 3450 472

//-r7«i-x-carotene 2800 444

^ra«.?-/J-Carotene 2592 452

Retinyl palmitate 975 325

(^-Tocopherol 91.2 297

/-Tocopherol 91.4 298

^-Tocopherol 75.8 292

" PQ-CH2.(CH2.CH2.ch'ch2)3.h ; -tocopherol

"°}^X^Ch^.,ch^.cH2.ch'ch2)3-h a-tocopherol

"°PC>ch2.(Ch^.ch2.ch'ch^,3-h 6-tocopherol

Fig. 2. Structures of retinyl palmitate. tocol. and i-. and (i-to- copherol.

1-18
40 K S- Epltr al J Clirnnnimvr 'j/V /vv.< -V.v

200-,al aliquots of the reconstituted serum were

placed in glass test tubes, and serum protems

were denatured with an equal volume of ethanol

containing the internal standards and BHT. The

samples were mixed on a vortex mixer for 15 s. Hexane ( 1 ml) was added, and the mixtures were

placed on a vortex mixer for 45 s. Then the samples were centrifuged (30 s. ^ ZOOO ,?). and the

supernatants were removed. The hexane extraction process was repeated, and the supernatant

from this second extraction was combined with

that of the first. Extracts were evaporated under a stream of nitrogen and reconstituted in 100 of

ethanol containing 30 /ig ml BHT. The reconstituted extracts were placed in an ultrasonicating

bath for 30-60 s to facilitate dissolution. The extracts were then transferred to glass inserts that were placed in the autosampler vials.

Qualiiy control

Two sets of serum samples containing low and

norma! levels of retinol. carotenoids. and toco- pherols were run fifteen times over a five-week

period to ascertain that the method was in control and to determine the reproducibility of the

method. .A. low and a normal sample were run each day. Peak-area measurements were used for

all nine analytes; retinyl palmitate was not measured in these samples.

Food extracts An extract of a mixture of foods containing

apricots, carrots, creamed corn, creamed peas, green beans, orange juice, peaches, spinach, sweet potatoes, tomato paste, and winter squash was analyzed using the method described below

to demonstrate the method's applicability to food analysis.

Chromatographic conditions

The method involved two linear gradients and

an isocratic step. Solvent .A was acetonitrile. sol- vent B was methanol containing 0.05 .V/ ammonium acetate, and solvent C was ethyl acetate. Each

of the three solvents contained 0.05° o tnethylamine (TE.A) for reasons that are discussed below.

The first linear gradient began with 98° o A-2°'o B

and went to 75% A-18"'o B-7"o Cm M) mm. The

second linear gradient ran from this composition

to 68" 0 A-25'' 0 B-7% C in five min. This composition was held for 10 min longer, then the system was returned to initial conditions (98" o A-2" o B) over 5 min and re-equilibrated for 10 min.

The precolumn ( 10 mm ^ 4.6 mm I.D.. 10 /(m

particle size) containing Vydac 201 TP polymeric

wide-pore C^^ (Separations Group. Hesperia.

CA. USA) stationary phase and the narrow-pore

Bakerbond Cis analytical column (250 mm ^ 4.6 mm I.D.. 5 am. particle size. J. T. Baker. Phil- lipsburg. NJ. USA) were held at 29 = 0.5'C b> a thermostatted water bath. In previous work, col- umn temperature has been shown to influence se- lectivity for carotenoids and other compounds

with molecule shape differences [19]. The stainless-steel frits throughout the chromatographic

system were replaced with biocompatible frits

[20]. A programmable UV-Vis detector (Spectroflow 783. ABI. Foster City. CA. USA) with a tungsten lamp was used for measurement of the

retinoids and the carotenoids. The wavelength was held at 325 nm to measure retinol and then was changed to 450 nm at 4.5 min to measure the internal standard and the carotenoids: the wavelength was changed back to 325 nm at 22 min to measure retinyl palmitate. A fluorescence spec- trometer (LS-4. Perkin-Elmer. Norwalk. CT.

USA) was used to measure the tocopherols and

tocol using an excitation wavelength of 295 nm

and an emission wavelength of 335 nm. Signals

from both detectors were recorded simultaneously by the data system ( Maxima 820. Waters. Div i- sion of Millipore. Milford. M.A. USA).

RESULTS .AND DISCUSSION

Method development

In a previous paper, we reported on a comparison of carotenoid separations on sixt\-fi\e re- versed-phase LC columns [20]. Columns were

classified as monomeric. polymeric, or intermediate based on their selectivity for a test mixture of

polycyclic aromatic hydrocarbons (P.AHs). stan- dard reference material 869 [21]. The selectivity

test of Sander and Wise classifies phases as

1-19
A.' .S. Eptcr ei al J Chromalogr 619 I 1993 1 37-48 41

monomeric. intermediate, or polymeric depending on the elution order of benzo[a]pyrene (BaP)

and tetrabenzonaphthalene (TBN) (axBN BaP < 1 for polymeric C\%\ xtbn saP > 1-7 for monomeric

Ci8 phases) [21]. The selectivity of sixty-five col- umns was evaluated for a mixture of seven carotenoids. using methanol and acetonitrile or these

soK ents modified with tetrahydrofuran (THF) or

ethvl acetate; recovery of the carotenoids from

the columns was measured as well. In that study,

several conclusions were drawn: under the conditions used, polymeric and some intermediate col- umns were able to separate lutein and zeaxanthin. but monomeric columns could not; methanol-based solvents provided higher carotenoid

recoveries from the column than acetonitrilebased solvents; and if a modifier was used. THF

provided slightly better recovery than ethyl acetate and less modifier was required [20].

Column selection

The narrow-pore Bakerbond Cis column that was selected for this method is prepared by a polymeric synthesis [22], but was classified as in- termediate in the earlier study using the PAH test mixture [21]. A second column, while nominally

the same as the column evaluated in the sixtyfive-column comparison study, was classified as polymeric, with an aTSN/saP of 0.9. The earlier in- termediate column (aTBN/BaP = 1-27) was one of

the few intermediate columns able to resolve lu- tein and zeaxanthin in our column comparison

study, but its recovery of the carotenoid mixture

used in that study was low (75% recovery using

100% methanol as the mobile phase, 66% recovery using 5% THF-95% acetonitrile). These re- coveries, which were unacceptable, did not present a problem when recovery was improved by

adding ammonium acetate and TEA to the mobile phase.

Solvent selection

Results of the column comparison study indi- cated that carotenoid recovery was better using

THF rather than ethyl acetate and also better us- ing methanol rather than acetonitrile. However,

to achieve the desired separation with this column, it was necessary to use an acetonitrile-based

solvent despite the observations described above.

We were unable to obtain an acceptable separation using various combinations of THF. methanol, and acetonitrile. and instead developed a method using ethyl acetate, methanol, and acetonitrile. Acid contamination in chlorinated solvents

has been blamed for carotenoid losses [23]; be- cause hydrolysis of ethyl acetate produces acetic

acid, acidity may have caused the losses observed

when ethyl acetate was used in the column comparison study. Ammonium acetate, which was

necessary as a modifier, is poorly soluble in ethyl acetate and in acetonitrile. The addition of 0.05

M ammonium acetate to the methanol alone pro- vided sufficient buffer capacity (assuming that acidity is the factor critical to recovery) so that recoveries were high even though ethyl acetate and acetonitrile were used. Although initial sep- arations in an earlier iteration of the method were

fine after the column was flushed with buffer, ca- rotenoid recovery gradually decreased without

the addition of ammonium acetate to the mobile

phase itself. When a mixture of lutein, zeaxanthin. /J-cryptoxanthin. lycopene. a-carotene.

/^-carotene, and rra«5-/3-apo-8'-carotenal (an earlier internal standard) was chromatographed us- ing the earlier method without the addition of

ammonium acetate, recovery was only 75%. Using the same separation procedure with the addition of ammonium acetate, recovery of that caro- tenoid mixture was 92%.

Kamber and Pfander [24] found it necessary to add N.N-diisopropylethylamine to silica col- umns to obtain good carotenoid recovery. N.NDiisopropylethylamine is a nucleophilic base; in the absence of this compound, we examined

TEA. TEA added to each of the three solvents

(acetonitrile. methanol containing 0.05 ,V/ ammonium acetate, and ethyl acetate) improved recovery. Using the method described in this paper

with 0.1% TEA added to each of the three sol- vents, carotenoid recovery was 101%. When

0.1% TEA was added to the solvents, and the

methanol did not contain ammonium acetate, re- covery was only 89%. TEA behaves as a strong

1-20
K. S Epier fl ai. J Chromaioi^r . 6/V /Wi

modifier, and reduces retention times such that

several peaks were no longer resolved when a TEA concentration ofO-P'o was used. Thus, as a compromise, this method uses 0.05°'o TEA in each of the solvents and 0.05 .V/ ammonium acetate in the methanol; recovery is 94°'o. and the

necessary peaks are resolved.

Chromatograms for the anaKsis of an e.xtract of human serum usins this method are shown in

Fig. 3; all analytes were naturally occurring ex- cept for reiinyl palmitate. which was spiked mto

the serum. Resolution of lutein and zeaxanthin is superior to most published methods. /?-cryptoxanthin is well resolved from lycopene and its iso- mers, and the c is trans isomers of /^-carotene are

resolved, although the cis isomers are not baseline-resolved. The method's applicability to food

separations is demonstrated in Fig. 4. In this figox

n UJ

RP

10 15 20

Tkn« (mkiutaa)

23

IS 23

Vm» IftMm) Re. 3. Chromatogram from the reversed-phase LC separation of retinol (R. 0.734 ml). /rtiw5-/i-dpo- lO'-carotenal oxime (o\i. luisin iL. 0 1 lO.ug ml), zeaxanthin (Z. 0.051 ng ml). /?-cr\ptoxanthin (C. 0.060 ,ug,ml). ?ra/ij-lycopene (Ly. 0.124 ug ml), z-carotene ix. 0 022

^(g ml). f/-u«.?-/i-carotene (//. 0.567 fig ml), and reiinyl palmitate (RP. spiked. 0.297 ng ml) in human serum using absorbance detection, Chromatogram of tocol (T). O-tocopherol (6. 0.084 /ig, ml), /-tocopherol (y. 1.94 fig ml), and i-tocopherol (z-t. 5.69 fig ml) using ^uorescencc detection. -

1-21
K. S. Epler et al. J. Chromaiogr. 619 / 19951 37-4H 43

1^

a-t

VjvJvi

10

TbiM (mfeHJtM)

18 20

Fig. 4. Chromatogram from the reversed-phase LC separation of retinol. carotenoids, and tocopherols in a mixture of apricots, carrots, creamed corn, creamed peas, green beans, orange juice, peaches, spmach. sweet potatoes, tomato paste, and winter squash. Labels are

the same as those for Fig. 3,

ure the chromatograms from the analysis of an

extract of the mixture of foods listed in the Experimental section contain all the major peaks

that are present in serum as well as additional

unidentified peaks. The resolution of the cis/ trans isomers of a- and jS-carotene is more apparent in

this chromatogram because of the increased levels of these isomers relative to those in the serum. (The cis isomers of a- and j5-carotene follow the

trans compounds, which are labelled, in Figs. 3 and 4.)

Internal standard selection

During the analysis of serum samples other

than the SRM, our first choice of internal stan- dard for retinol and the carotenoids, trans-fi- apo-8'-carotenal, co-eluted with polar carote- noids that were present in some of those samples.

Thus, it was necessary to investigate different in- ternal standards. rrfl«5-j5-Apo-10'-carotenal elut- ed with an appropriate retention time, but only

65% of it was recovered from the column. Using an earlier method that did not employ TEA. the

1-22
44 A.' V Epii-r cl ai J Cliriimulo^r 6/V IW< l~ 4.\

TABLE II RESULTS OF THE ANALYSIS OF SRM FAT-SOLL BLE VITAMINS IN HUMAN SERUM

Mean \alues were deiermined from the twelve \alues ihai resulted I'rom anaKsis ot two extracts from each of six bottles at each le\el Uncertainties (values in parenthesesi are one standard deviation I'rom the mean. Uncertainties on certified values represent a 95",i confidence interval.

v. I'l 11 pL' Ul lU R I'v 1 1 1 1 < 1 1 i\j mil I [-*rT 1 ht*i1 rt*t i'*rh»ni 'f* V. ^'mpouriu Results ( ^/g ml

)

Certified reference

^ Ul LICO I lit lllll values i/jg mil

/ /HI li'

Rc*f IX^ iI

1 nt^i 1 1 V ' 1 1*^9 (0 008) 0 1X4 ± {) 016 (' it f 1^1 J.'\. dl UlCIlC L litctn 0.035 (0 002) 0.03 1 i'Ul //"vUlUltllC II y ^ N Ml 1 ) ^A 1

t..^ 7t*;iU.XUlILlllll x:i n r h ! n 0 Ul ; (0 001

1

0.02 0 *~0 1 0 l)JX 1 0 9

li'(^r\ nriixn n t hi n 0 019 (0 DO"*) 0 02 rxwlMlVI R T 1 n v r^' 1 1 m i r i f p jJUllllllclLC N n

Tiii:il I VLTonc^ne 0. 160 (0.019) 0.2 />-Tni"'(inhr'r('»l <' I k.'^VJ [J I ICl I n 1 1 "> 1(1 OM))

1/r/#>» f Ufl.yV - 1 V r^i^npni* L- » f-* L/C lie 0.07 " i ULUpllCl Ul -.DO I U.U J ; _.o

1 t '11 'f—i"\ f pn 11 0'''' ((1 OOil 0.02 2- Tocopherol 10 "^h U\ "» N I

\J.\J I ^ \\J.\J\J \ 1 Ti^ 1 '1 1 rotten** 0 '64 0 079 nAt 1 fJ it 1It \ iJl li£rt It I tr 1 /rc//iv-/^-Ciirotcnc 0.200 (0.013) 0.2 RtX^er1

I

1 nt^l 1 1 V/ 1 1) fi^S lO ()l9l Rctinv! ptilmitiiic ND" () 07'' (0 ()0S» 0 08

(S-Xornnht*rn! 0 054 (0 011) 7p'i v ;i n r h 1 n 0 in

0 801 (0 0^9) 0.8 /i-lrvnlov'infhin fJ \- 1 * L^LVJ.\illlllllll 0 O'^X (0 OO"*^ i-Tocopherol 4.61 (0.38) 4.44 ± 0.22 Total lycopene 0 440 (0.022) 0.4

fc//!,?- Lycopene 0.151 (0.008) 0.2 Medium level Total x-carotene 0-128 (0.007) 0,1 Retinol 0.516 (0.013) 0.495 ± 0.021 rruw.v-i-Carotene 0.080 (0 006) Lutein 0.092 (0.006) 0.09 Total /i-carotene 2 22 (0,11) 2.65 3: 0.22 Zea.xanthin 0.037 (0.003) 0.04 /rt;/7.v-/i-Carotene 1 92 (0.10) 2.4 /{-Crypto.xanthin 0.057 (0.005) 0.06 Retinyl palmiiate 0.022 (0.010) Total lycopene 0.461 (0.022) 0.4 i)-Tocopherol 0.163 (0.036)

^rti/i.v-Lycopene 0.179 (0.01 1) 0.2 : -Tocopherol 3.70 (0.09) 3.9 Total i-carotene 0.066 (0.004) 0.05 a-Tocopherol 16.04 (0.59) 16.19 ± 0.56 ND = not delected; analyte not present or present at levels below the limit of detection.

addition of a small amount of formaldehyde [500

^1 of a 30% (w,v) formaldehyde solution per 1 of

solvent] to each of the three solvents improved

recovery of rra/ti-^-apo-lO'-carotenal ( >90% re- covery). But since recovery of this internal stan- dard might vary over time, we converted the

/ra/i5-^-apo-10'-carotenal to fra«5-/^-apo-10'-carotenal oxime. which has approximately the same

retention time as its aldehyde, is quite stable, and

does not have a recovery problem. Thus, formaldehyde is no longer bemg added to the solvents. Although we did not observe loss of /3-apo-8'-

carotenal in our earlier studies, it is probable that

this aldehyde would be subject to the same losses as /^apo-lO'-carotenal. For this reason the free

carotenals are not recommended for use as in- ternal standards.

Analysis of serum samples

Results for the analysis of SRM 968a are shown in Table II. Certified values are provided

for retinol. total ^-carotene, and a-tocopherol in SRM 968a: reference values are provided for lu- tein, zeaxanthin. /?-cryptoxanthin. /ra/!.v-lycopene, total lycopene, a-carotene. trans-li-cdrotene, and -'-tocopherol. Certified values were determined by two NIST analysts using different

methods and seven collaborating laboratories us- ing different column-mobile phase combinations. Reference values were determined bv a lim1-23
K. S. Epler el al. . J. Chromatogr. 619 i mi) 37-46 45

TABLE III MEAN STANDARD DEVIATION. AND RELATIVE

STANDARD DEVIATION FOR TWO QUALITY CONTROL SERUM SAMPLES MEASURED FIFTEEN TIMES

OVER A FIVE-WEEK. PERIOD

Compound Mean S.D. R.S.D,

(A/g ml (/jg ml) (%)

Sormal qualuy control Retinol 0.474 0.010 2.1 Lutein 0.117 0.005 4.0 Zeaxanthm 0.039 0.002 4.9 ^-Cryptoxanthin 0.058 0.002 3.5 /rani-Lycopene 0.222 0.015 6.8 ^rans-a-Carotene 0.037 0.001 3.5 //•a/;i-/i-Carotene 0.164 0.005 3.0 d-Tocopherol 0.076 0.012 15.2 /-Tocopherol 1.93 0.07 3.7 Dt-Tocopherol 8.58 0.32 3.8

Low quality control Retmol 0.366 0.011 3.1 Lutein 0.091 0.004 4.9 Zeaxanthin 0.030 0.002 7.5 ^-Cryptoxanthin 0.044 0.002 3.8 /ran5-Lycopene 0.167 0.006 3.8 /ran5-a-Carotene 0.028 0.001 5.3 /ran5-^-Carotene 0.128 0.004 3.2 d-Tocopherol 0.059 0.008 14.0 •/-Tocopherol 1.49 0.04 2.4 a-Tocopherol 6.64 0.21 3.2

ited number of analyses and are provided by

NIST for information only. (The method described in this paper was not used to assign values

to the SRM.) Results show good agreement with

reference and certified values for retinol, the to- copherols, and most of the carotenoids. Total

jS-carotene results obtained using this method are somewhat low for the medium- and high-level

SRM when compared to the certified values, but

are comparable to results obtained when the

SRM was distributed to 45 laboratories involved

in a round robin study. As reported in the SRM

Certificate of Analysis, the medium- and highlevel total ^-carotene concentrations determined

in the round robin were 0.89 ±0.12 and 2.34 ±

0.34 mg/1, respectively.

Results for the analysis of the two quality control sera are shown in Table III. Means are pro- vided to show the levels at which the given rela- tive standard deviations ( R.S.D. s) were attained.

Most of the analytes had R.S.D.s in the 3-5%

range. The R.S.D. for (5-tocopherol is significantly higher due to the very low concentrations pre- sent in the serum (about two orders of magnitude

lower than a-tocopherol). The R.S.D. for the

low-concentration zeaxanthin is also somewhat

high, probably because of the relatively low concentration in this sample.

Dissolution of extract Dissolution of serum extracts is a critical step

in the quantitative analysis of carotenoids in se- rum. The carotenoid concentrations are fre- quently near the detection limit. The carotenoids

and lipid residue are poorly soluble in polar or- ganic solvents, yet the extract must be completely

dissolved in a solvent miscible with the mobile

phase and an adequate volume of the solvent in- jected without creating chromatographic arti- facts. Dissolution can be facilitated by using ul- trasonic agitation. Ideally the extract should be

dissolved in the initial mobile phase or a slightly

weaker solvent to focus the sample at the head of

the column. We compared the effects on quantitation of dissolution in different solvents. After

following the usual hexane extraction procedure,

dissolution of the dried extract was compared in ethanol and in several other solvents. When 1 5:85 acetone-acetonitrile. methanol, a 20:80 methylene chloride-methanol. and 20:80 methylene

chloride-acetonitrile were added to the dried extract, precipitates formed, and low recoveries were observed. Ethyl acetate dissolved the extract with no difference in recovery when compared to ethanol. Because of the increased solubility of lip- ids in ethyl acetate, we are currently reconstituting extracts in 50:50 ethyl acetate-ethanol. with

30 fig/m\ BHT. It is important to note that peak

broadening may become a problem when more

than 20 /il are injected into the predominantly

acetonitrile mobile phase.

Residual acid and metal activity

The improved recovery resulting from the use

1-24
46 ^ S Epii.'r cl at. 7 Chn>mai(i\;r hl'J IWI 4.\

of ammonium acetate and TEA is not limited to the analytical column used in the method described here. During the development of this method, we observed that exposure to minute

quantities of trifiuoroacetic acid caused the col- umn to become unsuitable for carotenoid separations; previous carotenoid separations on the col- umn were acceptable, but no peaks were obtained after the column had been exposed to traces of trifiuoroacetic acid. Treatment with 50:50

methanol-0.01 .V/ ammonium acetate for 45 min

restored the column to its mitial carotenoid-separating state. The column could then be convert- ed back and forth from "bad" to "good" at will by injecting dilute trifiuoroacetic acid and washing the column with solvent containing ammonium acetate. Following this observation, five columns that

had provided poor recovery in our previous six- ty-five-column evaluation were flushed with 0.05

\f ammonium acetate in methanol at 1 ml, min

for 15 mm. then equilibrated for 15 min with

THF-acetonitrile eluent pumped at 1 ml min.

After this buff"er treatment, recovery improved on all five columns. Some columns showed greater improvement than others. Table IV shows the

initial recoveries and recoveries following the

buffer treatment. Mean improvement in recovery

TABLE IV RECOVERY OF A CAROTENOID MIXTURE ON SE- LECTED COLUMNS BEFORE AND AFTER TREATMENT WITH 0.05 V/ AMMONIUM ACET.a.TE IN .METHANOL WITH AND WITHOUT THE ADDITION OF 0.05% TEA TO AN ACETONITRILE-BASED MOBILE PHASE

Column Recovery (%)

Before treatment After treatment: After treatment; no TEA 0.05'"o TEA m mobile phase

1 48 74 102

1 30 99

-7 87 106 4 15 69 104

5 3 i: >87

across this group of five columns was 40" o Thus,

other recoveries m the earlier study may have improved if the columns had first been flushed with an ammonium acetate buffer. After observing that TEA further improved re- covery, these same five columns were again

flushed for 15 min with 0.05 .V/ ammonium acetate in methanol. Columns were equilibrated for

15 min with 5:95 THF-acetonitnle containing

0.05% TEA. and carotenoid recovery was measured using this same solvent mixture. Results of

this study are also included in Table IV. Recovery on all five columns improved dramatically

from initial recoveries. Recovery for the fifth col- umn is ">87°'o" because not all the peaks had

eluted during the 25-min run time using the mobile phase composed of 15:85 THF-acetonitrile

containing 0.05°'o TEA. Reproducibility of re- covery measurements in the column comparison

study was ±6%: recoveries in Table IV that are greater than 100% are within the limits of the method.

These same five columns that had provided

poor recovery in the sixty-five-column study were

also tested for trace metal activity. Acetylacetone

is used as an indicator of metal activity: the sharper the acetylacetone peak, the less metal activity present [25]. Of the five columns tested, the one with the highest initial recoverv had the least metal activity. Columns 4 and 5. two columns

from the same manufacturer, exhibited high metal activity. Columns 2 and 3. also from one manufacturer, exhibited similar metal activity, but

less than columns 4 and 5. Although adding TEA

to the mobile phase improved recovery on these

five columns, it is not likely that TEA is functioning as a chelating agent. However, because of its nucleophilic character. TEA may interact with

positively charged metal species that may be pre- sent in the silica. The use of nucleophilic bases

has been previously reported to improve carote- noid recovery on normal-phase LC columns

[24.26]. Liew e! al. [27]. in a study of the role of

metal ions in bleaching clays, found that Fe-'^. Mg'". and Ca-' play a significant role in the

clay's ability to adsorb ^-carotene. They suggested that the /^-carotene is adsorbed directlv to the

1-25
K S Enter er al. J. Chromaio?r 619 i 1993) }^-4H 47

ferric ion. in particular, and then undergoes fur- ther reaction [27]. At the same time, they believe

that the presence of metal ions causes the dis- sociation of water to form metal hydroxides and

protons, which also cause carotenoid degradation [27]. While there is very little water in our

system ( < 1 % from the ammonium hydroxide

and the acetic acid used to prepare the ammonium acetate), the improvement of carotenoid re- covery with the addition of ammonium acetate (a

buffer) and TEA (a base) in our study supports

their conclusions.

CONCLUSIONS

In this paper, an LC method is described for the measurement of carotenoids, retinol. retinyl

palmitate. and the tocopherols in serum and in food. Retinol. retinyl palmitate. and the carote- noids are separated and measured using a pro- grammable UV-Vis detector. The tocopherols

are measured simultaneously using a fluorescence

detector in series. Apo-carotenals used as inter- nal standards in our initial methods are not ac- ceptable for use in the analysis of all serum samples because of variable recovery and co-elution

with serum carotenoids that are more polar than

lutein. Because of this, we have examined several

other internal standards, and have selected tramj3-apo-10'-carotenal oxime.

Using several different columns, we have also

demonstrated that by flushing with a mixture of

ammonium acetate and methanol, columns that

provide little or no recovery of carotenoids can

be improved. TEA added to the solvents also improves carotenoid recovery. The ammonium acetate and the TEA appear to be performing two

different functions, since the addition of one

without the other did not improve recovery as much as the addition of both in studies with the

analytical column used with this method.

ACKNOWLEDGEMENTS

This work was supported in part by the National Cancer Institute, Division of Cancer Etiol- ogy. Agreement No. Y01-CP9-0513. We thank

Lane Sander (N 1ST) for valuable insights regarding recovery problems, Garry Handelman (University of California at Davis) for providing caro- tenoid compounds to test as internal standards.

Willy Schtiep of Hoffman-LaRoche for providing rra/?5-jS-apo-10'-carotenal for the synthesis of

the /ra«5-j3-apo-10'-carotenal oxime internal

standard, and Shizumasa Kijima of Esai for providing tocol for use as an internal standard.

REFERENCES .

'

1 R. G. Ziegler. i. C///I. -Vi/^r . 53 ( 1991 1 25 IS. 2 J. G. Bien. E. D. Brown and J C Smiih. Jr . J Liq. Chnmuilogr . 8 (1985) 473.

3 H. J, C F, Nelis and A. P. De Leenheer. Anal. Clwni . 55 (1983) 270. 4 M. Stacewicz-Sapuntzakis. P E. Bov.en. J. W Kikendall and M. Burgess. J. Micronutr Anal.. 3 ( 1987) 27.

5 R. Ohmacht. Gy. Toth and G. Voigt. J. Chnmiamsir.. 395 (1987)609

6 C. S. Yang and M,-J. Lee. / Suir Growth Cancer. 4 ( 19X7)

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7 G. Cavina. B. Gallinella. R. Porra. P Pecora and C Suraci.

J. Pharm. Biomed. Anal.. 6 (1988) 259

8 A. B. Barua. R. O. Batres. H C Purr and J A Olson. J. \licronuir. Anal.. 5 (1989) 291. 9 L. R. Cantilena and D. W. Nierenberg. J. Micromiir. Anal.. 6 (1989) 127. 10 W A. MacCrehan and E. Schonberger. Clin Chcm.. 33 (1987) 1585.

11 A. L. Sowell. D. L. Huff. E W Gunter and W J Dnskell. J. Chromamgr.. 431 (1988) 424

12 B. Olmedilla. F. Granado. E. Rojas-Hidalgo and I Blanco. J Liq. Chromaiogr.. 13 (1990) 1455.

13 W. G. Rushin. G. L. Catignani and S. J Schwartz. Clm. Chem.. 36 (1990) 1986.

14 N. E. Craft. L. C. Sander and H. F. Pierson. J. Micrnnuir.

.Anal.. 8 (1990) 209.

15 E. Lesellier. C. Marty, C. Berset and A. Tchapla. J. High

Resolui. Chromaiogr.. 12 (1989)447.

16 A. Pettersson and L. Jonsson. J .\ficronuir. Anal.. 8 (1990)

23. 17 D. Hess. H. E. Keller, B. Oberlin. R. Bonfanti and W.

Schuep. fni. J. Vit. .Vuir. Res.. 61 (1991) 232.

18 G. W. T. Groenendijk. W J De Grip and F J. M Daemen.

Biochim. Biophys. Ada. 617 (1980) 430.

19 L C. Sander and N. E. Craft. Anal. Chem . 62 (1990) 1545. 20 K. S. Epler. L. C. Sander. R. G. Ziegler. S. A. Wise and N. E. Craft. J. Chromaiogr.. 595 ( 1992) 89

21 L. C. Sander and S. A. Wise. LC - GC. 5 (1990) 378. 22 M. Henry. J. T. Baker. Philhpsburg. NJ. personal communication. 23 G. Britton. Meihoch Enzvmol.. Ill (1985) 113.

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'*° K. S. Epier el at. J Chrnmato^r. 619 i IWj ; J

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1-27
[Reprinted from CLINICAL CHEMISTRY, 33, 1585 (1987).] Copyright 1987 by the American Association of Clinical Chemistry and reprinted by permission of the copyright owner.

Determination of Retinol, a-Tocopherol, and /3-Carotene in Serum by Liquid Chromatography

with Absorbance and Electrochemical Detection

William A. MacCrehan' and Emit SchdnbergeH

We describe a method for the determination of retinol, a- tocopherol, and /3-carotene in serum, using a liquid-chromatographic separation with wavelength-programmed ultra- violet/visible absorbance and amperometric electrochemical

detection with a glassy carbon electrode. After protein denaturation and addition of an internal standard, tocol, 250-/u,L samples are twice extracted with hexane. The reversed- phase, gradient-elution chromatographic separation provides

baseline resolution of: the ai\-trans isomer of retinol from the

cis isomers, a- from ytocopherol, and all-/rans-/3-carotene from a-carotene and from c/s-)8-carotene isomers. The lin- earity of response and the detection limits for the two

detectors for the three analytes are measured. A comparison

of the values obtained for serum extracts shows good

agreement between the absorbance and electrochemical

detectors.

Additiorial Keyphrases: micronutrients • chromatography, re- versed-phase • c\s-l3-carotene

In a quality-assurance role, the National Bureau of Stan- dards (NBS) is supporting clinical research funded by the

National Cancer Institute into the cancer-chemopreventive

properties of vit£unin A (all-trarts-retinol), vitamin E (a- tocopherol), and pro-vitamin A (all-fra?is-/3-carotene).3 The

quality-assurance program is for laboratories involved in epidemiological studies, assaying these micronutrients in serum to evaluate the nutritional status of treated and

control populations. For NBS anfdyses, we have developed a method to assay the three analytes by using Uquid chromatography with ultraviolet/visible absorbance detection (LCUV) and oxidative electrochemical detection (LCEC), a new approach for the determination of all three micronutri- ents in serum.

In the past, all three analytes were determined by extraction followed by colorimetry (1—4). That approach is subject

to nonspecific interference fi-om other matrix constituents,

such as carotenoid and xanthophylls as well as unresolved

' National Bureau of Standards, Center for Analytical Chemistry, Organic Analjjtical Research Division, Gaithersburg, MD

20899.

2 National Physical Laboratory, Danzinger Bldg "A," Hebrew

University, Givat Ram, Jerusalem, Israel 91904.

^ Nonstandard abbreviations: NBS, National Bureau of Standards; LCUV, liquid chromatography with ultraviolet/visible absor- bance detection; LCEC, liquid chromatography with electrochemical detection; BHT, 2,6-i-butyl-4-methylphenol. Received March 2, 1987; accepted June 17, 1987.

isomers of the analytes. With the advent of liquid-chromatographic separations, imrecognized interferences present in the earlier techniques became apparent, and those proce- dures are no longer considered reliable. Many liquid-chromatographic methods have been pro- posed for the single (5-11) as well as the simultaneous

multiple determination (12-21) of the three micronutrients

in senmi. Fixed-wavelength ultraviolet/visible absorbfmce

detection can be used to measure the analytes at their expected serum concentrations, but this approach relies heavily on the degree of chromatographic resolution to provide bias-free results. Fluorescence detection (20, 21) has

been used to enhance both selectivity and sensitivity of assays for retinol and tocopherol. Oxidative electrochemical

detection has also been used for the determination of tocopherol (9, 19, 22), but its use for detecting retinol and

carotene has only been suggested (22). The electro-oxidation of retinol (23, 24) and ^-carotene (25) has been studied only

in nonaqueous solvents. Recently, we have studied the

oxidation of retinol at a glassy carbon electrode in methanol/ water solvent such as those used in LC separations (26). The oxidation is a multiple-electron process that forms

several products. The approach has been found suitable for the thin-layer jmiperometric detection of chromatographically separated all-trans as well as the cis isomers of retinol

(26). The objectives of this work were (a) to evaluate the

extraction procedure used to recover the analjrtes from sera,

(6) to develop a reversed-phase separation that would pro- vide baseline resolution of the analj^s of interest from

other senmi constituents and from their isomers, and (c) to investigate the utility of oxidative amperometric detection

for the simultaneous determination of retinol, a-tocopherol,

and /3-carotene.

Materials and Methods^

Reagents/Standards

Standard solutions of the three analytes and the internal standard were prepared by dissolving crysteds (ah-transretinol, all-ira/is-/3-carotene) or oils (a-tocopherol, tocol), in absolute ethanol; the concentrations were determined spec- trophotometrically, corrected for impurities that absorb at

» Certain conunercial equipment, instruments, or materials are

identified in this paper in order to specify adequately the experi- mental procedures. Such identification does not imply reconunendation or endorsement by the National Bureau of Standards, nor does

it imply that the materials or equipment identified are necessarily the best available for the purpose.

1-28

CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1585
the wavelength of measurement (see below). The tocol internal standard was added in equal concentrations to standard mixtures and the serum samples before extraction. AU-fra^-retinol (type IV), retinyl acetate (type I), retinyl

palmitate (tvTJe IV), all-i7-a/ts-/3-carotene (type IV), and all- ^ran5-a-carotene (type V) were obtained from Sigma Chemical Co., St. Louis. MO. dZ-Tocol was a gift from Hoffmann-La

Roche, Basel, Switzerland. ci-a-Tocopherol ("99% minimum"

grade). (i--v^tocopherol, and a-tocopherol acetate came

from Eastman Kodak, Rochester, NY. 2,6-Di-^butyl-4-

methylphenol (BHT) was obtained from Calbiochem, La

JoUa. CA. Lutein, zeaxanthin, cryptoxanthin, and nonaprene were a gift (G. R. Beecher. USDA. Beltsville, MD). All were stored at -4 °C upon receipt and thawed just before preparation of ethanol solutions. The concentrations of the three analyte standards were

determined with a double-beam grating spectrophotometer

vs an absolute ethanol reference. The absorptivity values (L/ cm) in ethanol that we chose for the calibration are: all- fran^-retinol €324 nm = 1850 (27), a-tocopherol ^292 nm = 75.5

(13), and all-^ra/is-/3-carotene £452 nm = 2620 (28). The

solutions were diluted such that the absorbance of the

solution was always between 0.1 and 0.9 A. Once prepared,

the purit>' of the individual standard solutions was evaluat- ed by the "slow-gradient" LCUV separation described below.

If the total impurity at \rnax was between 1% and 5%, as determined by the total area under £l11 chromatographic

peaks (other than that for the desired analyte), then the absorbance of the solution was corrected for the impurities;

otherwise, the solution w£is discarded and fresh material was taken for measurement. From the individual solutions, we prepared dilute standards in ethanol, adding BHT (20

mgL) as a preservative (5). The approximate final concentrations in our working standards for the analyte mixture

(in mg L) were: all-^rarts-retinol 0.5, tocol 7, a-tocopherol 7, and cdl-fraas-/3-carotene 0.2. Aliquots of this mixture were

stored in glass vials at —70 °C eind were stable for at leeist one month. Before use, we warmed the solutions to about

40 °C, agitated them ultrasonically, then cooled them to room temperature.

LC Apparatus For the simultaneous determination of the three analytes, we used a two-pump, computer-controlled solvent-delivery

system. Each pump had two reciprocating pistons with

pressure-feedback compensation. The automated sample

injector was used with Teflon-septa-sealed vials. To separate

the analytes and their isomers, we used a 0.46 x 25 cm

Vydac (The Separations Group, Hesperia, CA) octadecylsilyl-modified silica (Cig) column (201TP) packed with 5-^

particles. This column has a polymeric bonded phase with

large (30-nm) pores, and can separate all of the retinol isomers in 30 min (26). For absorbance detection we used a wavelength-programmable detector (Model 783; Kratos Analytical, Ramsey, NJ) with a noise specification of 1 x 10~°

A (1-s time constant, 236 nm), and a deuterium lamp. The

electrochemical detector was a thin-layer amperometric

detector cell (Model TL-6A; Bioanalytical Systems, West

Lafayette, IN) modified by replacing the original working

electrode block with a custom Teflon holder and a 1.2 mmdiameter glassy carbon (Fluorocarbon Co., Anaheim, CA)

rod. The filling solution for the silver/silver chloride refer- ence electrode contained methanol/water (70/30 by vol before mixing) and 0.1 mol of KCl per liter. All potentieds are

specified with respect to this reference electrode. We used

this reference solution to help address the problems in the commercial electrode of the widely disparate internal (3 mol/L aqueous KCl solution) and external (methemol/water

mobile phase) electrolytes. A 2-s time constant was used for both detectors.

Separation Conditions

Distilled-in-glass solvents (B & J Brand; American Scientific Products, McGaw Park, ID were used in preparing the mobile phase. The electrolyte we used was a combination of Suprapur-grade ammonium hydroxide and acetic acid (Merck, Darmstadt, F.R.G.), which provided a buffer of pH

3.5. This electrolyte was included in all mobile-phase mixtures at a final concentration of 0.02 mol/L. The electrolyte not only was necessary to conduct electrical current for the amperometric detector, but also enhanced the reversed- phase separation of the analytes from their isomers. For

simultaneous determination of the three analytes, we used

the following "slow-gradient" solvent program: initial sol- vent, water/methanol/rt-butanol (15/75/10, by vol); final sol- vent, water/methanol/^-butanol (2/88/10 by vol). The initial solvent composition was used for 3 min, then was changed

linearly to the final solvent composition over a 15-min

period, and was held at the final composition for 17 min

before being returned to the initial solvent composition. The

flow rate was constant at 1.5 mbmin. The "fast-isocratic" mobile phase was water/methanol/ri-butanol (2.6/87.4/10 by

vol), with an initial flow rate of 1.5 mL/min; the flow rate was increased to 3.0 mL/min after 10 min, held constant for 5 min, then returned to 1.5 mL/min.

Sample Preparation

We used three steps to extract the micronutrients from serum for LC analysis: protein denaturation, organic sol- vent extraction, and solvent exchange. Frozen sera, stored

in glass ampules at -70 °C, were thawed thoroughly to room temperature, and vortex-mixed for 5 s before sample

aliquots were removed. We placed 250 ijlL of serum in a 13 mL centrifuge tube, added 25 yu,L of an ethanolic solution of the internal standard, tocol, and vortex-mixed briefly. We

then denatured the proteins with 250 fiL of ethanol containing BHT, 15 mg/L. After vortex-mixing the sample for 1 min

with 1.5 mL of hexane extractant, we centrifuged (=20(K) x

g) it for about 2 min, then removed 1.0 mL of the upf>er

layer. We added a fresh 0.5 mL of hexane to the remaining

lower layer, vortex-mixed for 1 min, then centrifuged 2 min.

We collected 300 /xL of the second extract and combined it with the first extract. To remove the hexane, we placed the sample in a container with a two-holed polyethylene lid and

blew dry nitrogen gas over it, to avoid oxidation by atmospheric oxygen. Finally we dissolved the residue in 250 ^tL of ethanol containing BHT, agitating the sample iiltrasoni- cally for 1 min. Occasionally, the redissolved extract was

cloudy, and an additional centrifugation was necessary

before LC analysis. We placed the redissolved extract in a

glass sample vial with an air-tight Teflon-lined septum, and

injected 25 pL into the chromatograph.

Results and Discussion

Extraction of the Analytes from Serum

Although retinol, tocopherol, and carotene are highly

insoluble in water, they are soluble in serum because they

are transported by lipoproteins. The proteins involved may

be 80% to 90% lipid enclosed in a hydrophilic protein sheath.

1586 CLINICAL CHEMISTRY. Vol. 33, No. 9, 1987 1-29
Specific retinol-binding-proteins are known to selectively

transport retinol in vivo. The presence of lipoprotein

encapsulation imposes several constraints on the available approaches to the determination of these micronutrients in serum. First, it makes impossible meaningful recovery experiments for these analytes in the senim matrix based on a simple addition of the free analyte dissolved in solution. As noted by Bieri et al. (7), the procedure of addition of emalyte solution to the serum prior to extraction fails to adequately simulate the naturally encapsulated analj^tes

because of the slow kinetics of uptake by the lipoproteins.

The slow kinetics of the analyte transfer to and from the

lipoprotein encapsulation is demonstrated by the failure of hexane to directly extract the analytes without the addition

of protein denaturant. In such an experiment we found that

tocopherol is only partially extracted, with little recovery of

retinol and carotene. Although this standard addition ap- proach has been used to determine extraction efficiencies in many studies (12, 14-18, 20-22), it may provide unrealistic "recovery" results for these analytes.

To overcome the serum lipoprotein-binding of the analytes for solvent extraction, one must iirst denature the

proteins, then use a water-immiscible organic solvent to extract the imencapsulated analytes. The final aspect of the

lipoprotein-binding of the serum micronutrients that must

be resolved before complete recovery is possible is the requirement of effective competition of the extraction sol- vent with the serum lipids for solvation of the analytes.

Hexane has been most frequently used for this purpose, with

a large ratio of the volume of organic extractant to serum

providing suitable competition to overcome lipid-binding.

However, even if the distribution ratio of the analytes into the hexane from the precipitated serum were infinite, the recovery would still be <100%, owing to the volume of hexane (about 20% in our method) that does not separate

into the upper layer, presumably because it is bound in lipid

micelles in the aqueous lower layer. The imrecovered vol- ume of extractant graphically demonstrates the importance

of the serum lipids in the procedure and points to the

advisability of the addition of a suitable internal standard to correct for the volume of extractant not recovered. We chose to use tocol (29) as internal standard to correct

for the variations in extractant volume. Tocol is structurally very similar to a-tocopherol but lacks the three methyl

groups on the phenolic ring. It is eluted in an uncrowded

portion of the chromatogram and responded to both absor- bance and electrochemical detectors. We also examined the use of three alternative internal standards: two esters of the vitamins—retinyl acetate (12, 18, 20) and a-tocopherol acetate (7, 12)—and a )8-carotene analog, nonaprene (30, 31). Retinyl acetate was imstable and eluted, with two shoulders, on the LCUV chromatogremi. a-Tocopherol acetate was

not suitable as internal standard for LCEC, because the

esterification of the electro-active phenolic hydroxy group of the tocopherol ring increased the applied potential required

to -1-1100 mV, where detector selectivity and sensitivity are

poor. Nonaprene, a promising internal standard for carot- enoid analysis (30, 31), is an analog of /3-carotene having an

additional isoprene unit; however, in our method, nona- prene co-eluted with serum components that were retained

longer than /3-carotene, making it unsuitable as an internal standard. We evaluated the effect of different protein denaturation

solvents on analyte recovery. Acetone, acetonitrile, methanol, and perchloric acid (100 mL/L), in equivolimie ratio to

the serum sample, yielded less recovery of all three analytes

than ethanol did. Because of the loss of extractant volume, we examined the

utility of a second extraction of the serum denatured with

fresh solvent. We used a pooled serum sample and measured

the analytical recovery of the added internal standard, tocol. After a single extraction, recovery was 81.0 (SD 15.2)% (n =

9). Combining a second extraction with the first recovered

96.8 (SD 3.0)% of the added tocol (n = 72). The recovery of the three analytes was also improved by including the second extraction. Although this additional extraction adds

to the complexity of the procedure, it is necessary for assay

precision.

We evaluated alkaline hydrolysis of the serum prior to the

extraction, as outlined by Thompson et al. (8), for the determination of micronutrients in serum, comparing the

results with those obtained in our method. Results were

identical, within statistical error, for /3-carotene. For all- irans-retinol, however, the alkaline extration method pro- vided higher results than our method did, because of the

additional free retinol recovered by hydrolysis of serum

retinol esters. No a-tocopherol was recovered from the

alkaline hydrolysate because of conversion to the hydrophilic phenolate anion.

We also tested a standardization approach based on the

addition of increments of an ethanol solution of the three analytes to bovine serum albumin, followed by extraction

(15, 16). The rationale for this approach is that this albumin

is commercially available, contains none of the three micronutrients, and provides a matrix similar to human serum. Although matrix-matching for standardization often has

merit, we found serious bias with this particular method:

only about 70% of the added all-^rans-retinol and a-tocopherol and about 40% of /3-carotene were recovered from the bovine serum albumin matrix. Because of the low recovery

of the standards in this matrix, the final determined results

for the three micronutrients in test human serum were

quite high. Thus, this method is not recommended (32).

Separation of the Analytes

Some methods for the determination of all three micronutrients involve an isocratic separation (15, 16, 18). However,

the advantage of rapid repetitive analyses is offset by the

significant compromises that must be made in the selectivity of the chromatographic separation for all-^raras-retinol and a-tocopherol. Because the reversed-phase retention of these two analytes is much less than for /3-carotene, baseline

resolution of the micronutrients from their isomers and

other serum components in a short isocratic chromatographic procedure is not possible with current colvmin technology.

Two types of analytical compromises are illustrated when a

"rapid isocratic" separation is used (Figure 1): (a) peak area

integration of the three analytes is made less precise by the presence of partly co-eluting "shoulders," and (6) the accura- cy is lowered by exactly co-eluting components, which are

revealed only in long-gradient separations. Despite the long

elution and recycle time required for gradient separations, we could not tolerate the attendant losses in the precision

and accuracy resulting from the use of this fast-isocratic separation.

Thus, we used slow-gradient separation (Figure 2), which

provides significantly better resolution of the three micronutrients from their isomers than shown in previously published reversed-phase antdyses (6, 7). We chose this widepore, polymeric, octadecyl stationary phase in conjunction

CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1587 1-30 .
Minutes

Rg. 1 . Fast-isocratic separation of the micronutrients in a serum extract Peaks: 1. retinol; 2, tocol: 3. rtocopherol; 4, a-tocophero); 5. a-carotene; ft all-frans-)3-carotene; 7, os-^^^arotene

UV/visible Absorbance Detection

T

2 mAU

1

325 nm-^ 295 nm

I

1 r- 1 1 1

1 1 r 10

~i——'

'—'—

r

20 30

Electrochemical Detection at *900 mV I'

T

35 nA

x8^

I

' ' ' '

1 ^ 0 10

-l—1

I—I—I—p20 30

Minutes

Fig. 2. Slow-gradient-eiution separation of micronutrients in a serum extract

Peaks: 1. all-tra/is-retirx)l; 2, toco); 3, r-tocopherol; 4, o-tocopherol; 5, lutein; ft zeaxanttiin; 7, cryptoxanttiin; ft a-carotene; 9. ai\-trans-p<ajotene: 10, cis-^orotene

1588 CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1-31
with a methanol/water/n-butanol mobile phase, based on

our previous experience with separation of aW-trans and the mono-CIS isomers of retinol (26). We found, as have others {6,

7, 33), that bonded-phase separations are enhanced by the

addition of a third solvent possessing polar and nonpolar

ends (such as n-butanol). The addition of a supporting

electrolyte was also beneficial to the separation of these

micronutrients. With this gradient separation, aU-transretinol is separated at the baseline from its 13-cis isomer, a- tocopherol is separated from y-tocopherol, and all-trans-ji- carotene is separated, also at the baseline, from the cis isomers and a-carotene. Recently, the 15,15'-cis and 9-cis isomers of /3-carotene have been separated from the all-^ra^

and a-isomer in determinations in foods {34).

Detection of the Analytes in Serum

We evaluated the use of LCEC for the determination of the three micronutrients because of the sensitivity and

selectivity offered by the technique. LCEC has previously

been used for tocopherol determination (9, 19). However, a

recent tocopherol method (9) noted an unexpected pro- nounced pH effect for the hydrodynamic current-voltage curve of the tocopherol isomers, the signal being zero for pH

values <4. We examined the pH dependence of the a- tocopherol electrochemical signal by using rotating disk

voltammetry over the pH range of 1.6 to 10 with a glassy

carbon disk. A single oxidation wave (£12 = +0.55 V) was

found for all pH values <8. Above pH 8, the wave splits, a second wave appearing at lower potential (£, 2 = +0.35 V); the sum of the wave heights remained constant. At low pH,

the oxidation of the protonated form of the phenolic moiety

predominated, whereas the second (easier) oxidation wave

appeared at the higher pH because of the oxidation of the

phenolate anion ipKa ~ 10). The pH dependence for the

electro-oxidation of phenols has been described (55): at pH

values below pKa, the OH moiety is oxidized to a radical cation in the first oxidation step; at pH values near or above

pKa, the oxidation potential decreases because the de- protonated phenolate anion may be more easily oxidized to a

free radical. No change in the protonation level of tocopherol

is expected in the pH range of 2 to 7, and thus no change in the electro-oxidation signal would be expected. We have

concluded that the electro-oxidation of tocopherol shows no pH dependence from pH 2 to 7, and thus we use an ammonium acetate buffer (pH 3.5). Only one preliminary investigation of the use of LCEC for retinol and /3-carotene has been reported (22). Bulk voltammetric studies of the electro-oxidation of /3-carotene {23, 25)

and retinol {23, 24) in nonaqueous solvents have been made.

Recently, we investigated the electro-oxidation reaction and

the use of LCEC for all-^rans-retinol and its cis isomers {26).

The electro-oxidation of retinol is a multi-step process, involving the initial formation of radical cations from the

double bonds, followed by reaction with the protic solvents,

with no involvement of the alcohol moiety. We found little or no selectivity for the electro-oxidation of any one specific

double bond, in the five-bond conjugated system. A charge of

4.7 electrons per retinol molecule occurred rapidly in coulo- metric experiments. A similar sequence of electro-oxidation reactions might be expected for )3-carotene, except the number of electrons transferred may be twice that for retinol (24). As a basis for LCEC determination of the micronutrients, we constructed a hydrodynamic voltammogram (Figure 3). The current response, measured as peak area, was normalApplied Potential (in V)

Fig. 3. Hydrodynamic voltammograms for all-frans-retinol (), focol (+), a-tocopherol (O), and all-frans-/3-carotene (A) at a glassy cartxjn electrode

ized by dividing the response at a given potential by the response at +1000 mV. The optimum potential for the simultaneous determination of all three analytes is + 900

mV, and is limited by the requirement to detect retinol. For

detecting a-tocopherol and /3-carotene, an applied potential of only +0.75 V would be necessary and would provide

additional detector selectivity (see below). For absorbance detection, we used an instrument that allowed rapid (<100 ms) wavelength programming to the absorbance maxima for each analyte. The wavelength

changes and subsequent auto-zero adjustments were timed

to occur at least 3 min before the elution of each micronutri- ent (Figure 2), thereby providing the data system an ade- quate baseline for measurement.

Calibration curves for the three anal3^s were linear through the physiological concentration range for both the

absorbance and electrochemical detectors (Table 1). However, when we used mobile phases containing a large initial proportion of water, the /3-carotene calibration curve dis- played some slight curvature in both detectors. Table 2 shows the effect of the initial proportion of water on the /3- carotene signal. The signal was also dependent on the material used in the column inlet frit. We speculate that the

mechanism of loss of the /3-carotene signal may involve

precipitation of the injected sample onto the oxide surface of the steel frit, with slow redissolution of /3-carotene into mobile phases that contain more water. This specxilation is supported by the "tailing" of the /3-carotene peak in solvents

in which signal loss occurred. We compared the detection limits of the absorbance and

electrochemical detectors for the three analytes (Table 3),

i.e., a signal three times the peak-to-peak baseline noise

levels. The detection limits for the electrochemical detector were somewhat superior to absorbance detection for all of the analytes, especially for a-tocopherol.

The relative selectivity of the two detectors for the deter- mination of the three analytes in serum may be judged by

1-32

CLINICAL CHEMISTRY. Vol. 33, No. 9, 1987 1589
Table 1. Linear Regression Calibration Curves for the Micronutrients by Each Detector

Analyte

AII-fAans-retinol a-Tocopherol

All- frans-^S-carotene

n = 14 each.

Concn range, mg/L

0-1 .3 0-16

0-1.0

SE

0.00841 0.0150 0.0264

LCUV LCEC

Intercept -0.018

0.0032 -0.10

SE

0.0196

0.0109

0.0356

Intercept -0.011

0.0024 -0.084

Table 2. Variation of Absorbance Signal for /3-Carotene

with Mobile Phase Composition and Frit Type

Absorbance (arb. units)

Initial proportion of water

in mobile phase, mLL

565

110

155

Stainless

steel frit 50 100 45 500

39 800

34 600

Teflon frit 76 000

73 400

70 100

69 000

the ability to provide baseline-resolved chromatographic

signals (cf. Figures 1 aind 2). The selectivity of the absor- bance detector was slightly better than the electrochemical

detector for edl-^raris-retinol; however, with the gradientelution solvent program, baseline resolution was possible

with both detectors. The electrochemical detector provides

superior selectivity for tocol and a-tocopherol, as evidenced

by small chromatographic shoulders found on the tocol and

a-tocopherol peaks in some absorbance chromatograms but

not observed with the electrochemical detector. We were

able to alleviate most of this occasional absorbance detection

interference by using cursor-controlled manual integration.

The selectivity of the absorbance detector w£is superior to

that of the electrochemical detector for /3-carotene (Figure

4): interfering serum components, including retinol palmitate and possibly other retinol esters, were detected electro- chemically (middle trace) but not by absorbance measure- ments (lowest trace). The interfering retinol esters could be

detected by their absorbance at the retinol optimum wavelength, 325 nm (upper trace). Although the retinol esters

interfere in the LCEC at the applied potential of +900 mV,

no interference with the )3-carotene signal is observed if a

lower detector potential of +750 mV is used. Perhaps it would be feasible to program a change in potential from

+900 to +750 mV after the elution of retinol and tocol; this would allow the detection of a-tocopherol and /3-carotene with maximum signal and Ijetter selectivity. Again, we

currently rely on cursor-controlled manual integration to

Table 3. Detection Limits for the Analytes

Concn, ftqIL

Analyte Absorbance EI«ctrochemlcal

All-frans-retinol 6.0 (150)* 4.1 (103) a-Tocopherol 96 (2400) 0.65 (16) AII-/rans-^-carotene 29 (730) 2.1 (53)

• Numbers In parentheses irxllcate pg of analyte in 25-fjL injection volume.

UV 325 nm

UV 450 nm

30 35

Minutes

Fig. 4. (Comparison of absorbance and electrochemical detection of carotenes

Lowesf (race; absortance detection at 450 nm; middle (race; amperometric detection at +900 mV; upper fjaoe;absort)ance detection at 325 nm. Peaks: 1. cKarotene; 2, aH-lrans-S-carofene; 3, as-0<arotene; 4, retinol palmitate

1590 CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1-33
[lectrochemical Detection (mg/L) Electrochemical Detection (mg/L) Electrochemical Detection (mg/L)

Fig. 5. Comparison of the absorbance with the electrochemical detection results for the determination of {lefti all-/rans-retinol (n = 72), (middle) a-to- copherol (n = 71), and (righf) /3-carotene (n = 72)

Regression equation parameters are given in the text

minimize this interference with the /?-carotene signal in the

electrochemical detector.

Determination of the Analytes

We evaluated the method by assaying (all in duplicate) a

series of serum samples and one serum pool obtained from

27 Japanese Hiroshima-bomb survivors who have subse- quently developed cancer. The procedure was calibrated with a single external standard, at the beginning and at the

end of the day, to correct for small amounts of drift in the

electrochemical detector response. Correlation plots for the

micronutrient vsdues determined with the absorbance and

electrochemical detectors are presented in Figure 5. Linear

least-squares analysis (n = 72) indicates an excellent corre- lation between the determined values by the two detectors

for all-<rans-retinol, with a slope of 1.01 and standard error

of 0.016, and for a-tocopherol, with a slope of 1.02 and a

standard error of 0.025. However, despite excellent correla- tion between the detectors, apparently some small bias is indicated for the determination of /3-carotene (slope 1.08, standard error 0.02). Overcompensation in the manual

integration of the retinol ester interference with the )3- carotene peak may have produced a negative bias for the

electrochemical detector results. Figure 5 also demonstrates the relative lack of interfer- ence in the micronutrient determinations from unknown

serum components or ingested drugs. Such interferences are not likely to provide response factors simileir to those of the

analj^s in both detectors and thus would appear in the

plots as points far removed from the line of identity.

The results for the anedyses of the pooled senmi are presented in Table 4. The agreement between the two

detectors is good, and the imcertainty (CV) was acceptable

(3% to 4%) for all three analytes.

Table 4. Determination of Micronutrients in a Human

Serum Pool

All-frang-retlnol a>Tocopherol /3-Carotene LCUV LCEC LCUV LCEC LCUV LCEC

X", mg/L 0.563 0.554 13.5 12.9 0.216 0.198 CV, % 4.1 3.0 4.7 3.2 4.1 1.4

n = 18 (nine measurements in duplicate).

Although the absorbance and electrochemical detectors measure the three micronutrients by completely separate

principles with different attendant interferences, the results

for both still depend on the same extraction and separation

technique. Absolute validation of the our method would

require agreement with the analysis of a serum Standard

Reference Material, for which the values of the three micronutrients would have been certified by two totally

independent techniques. Current work at NBS is directed toward the development of such a Standard Reference

Material.

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12. Driskell WJ, Neese JW, Bryant CC, Bashor MM. Measurement

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13. Catignani GL, Bieri JG, Driskell WJ, et al. Proposed Selected Method: simultaneous determination of retinol and a-tocopherol in serum or plasma by liquid chromatography. Clin Chem

1983;29:708-12.

1-34

CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1591
14. Broich CR, Gerber LE, Erdman JW. Determination of lycopene,

a- and ^-carotene and retinol esters in human serum by reverse- phase high-performance liquid chromatography. Lipids

1983;18:253-8.

15. Miller KW, Yang CS. An isocratic high-performance liquid

chromatography method for the simultaneous analysis of plasma

retinol, a-tocopherol and various carotenoids. Anal Biochem

1985;145:21-6.

16. Miller KW, Lorr NA, Yang CS. Simultaneous determination of plasma retinol, o-tocopherol, lycopene, o-carotene and /3-carotene by high-performance liquid chromatography. Anal Biochem

1984;138:340-5.

17. Nieremberg D, Lester DC. Determination of vitamins A £uid E

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18. Milne DB, Botnen J. Retinol, a-tocopherol, lycopene and a- and

^-carotene simultaneously determined in plasma by isocratic liquid

chromatography. Clin Chem 1986;32:874-6.

19. Ueda T, Igarashi O. Evaluation of the electrochemical detector

for the determination of tocopherols in feeds by high-p)erformance

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20. Russell MJ, Thomas BS, Wellock E. Simultaneous assay of serum vitamin A and vitamin E by high performance liquid

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1986;9:281-4.

21. Williams ATR. Simultaneous determination of senmi vitamin A and E by liquid chromatography with fluorescence detection. J Chromatogr 1985;341:198-201.

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23. Park SM. Electrochemical studies of /3-carotene all-trans reti- nal, and ah-tmns retinol in tetrahydrofuran. J Electrochem See

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35. Adams RN. Electrochemistry at solid electrodes. New York: Marcel Dekker, 1969.

1592 CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987 1-35
Journal of Micronutrient Analysis 8 (1990) 209-221

Separation and Relative Distribution of All-^ransjS-Carotene and its cis Isomers in jS-Carotene Preparations

Neal E. Craft,^* Lane C. Sander^ & Herbert F. Pierson^^

"Center for Analytical Chemistry, National Institute of Standards and Technology,

Gaithersburg, Maryland 20899, USA

''Diet and Cancer Branch, National Cancer Institute, Rockville, Maryland 20852, USA

(Received 6 September 1989; revised version received 29 August 1990;

accepted 25 September 1990)

ABSTRACT

The purity and relative distribution of fi-carotene isomers in several

commercially available preparations of ^-carotene was evaluated using

spectrophotometric methods and reversed-phase liquid chromatography

(LC). The purity of five lots of ^-carotene evaluated by comparison of

spectrophotometric to gravimetric determinations wasfound to rangefrom 7-

1

to 82- 9%. Reversed-phase LC was then used to measure the impurities due to other carotenoids and geometric isomers of^-carotene which also absorb in

the 450 nm region. Five different reversed-phase Cjg columns were examined

for their ability to resolve thegeometric isomers of^-carotene which contribute

to the apparent concentration of trans-P-carotene standard solutions. The

columns packed with wide-pore, Cjg polymerically-modified silica exhibited

adequate selectivity to separate the geometric isomers from a//-trans-j9- carotene. Impurities separated by LC accounted for 16-75% of the

absorbance ofcommercial ^-carotenepreparations at 450 nm. Based on these

observations. all-tmns-P-carotene measurements could potentially be only

1/50 of reported values.

Key words: ^-carotene, carotenoids, HPLC, isomers, purity.

*To whom correspondence should be addressed.

209

Journal of Micronutrient Analysis 0266-349X/91/$03.50 © 1991 Elsevier Science

Publishers Ltd, England. Printed in Northern Ireland

1-36
210 Neal E. Craft, Lane C. Sander. Herbert F. Pierson

INTRODUCTION

Although a muhitude of methods have been reported for carotenoid

measurements, only a few address the separation of geometric isomers.

The most complete separations of carotenoid isomers have been

accomplished by liquid chromatography (LC) using lime (Ca(OH)2) and

alumina columns (Vecchi et ai, 1981; Tsukida et al, 1981; Tsukida &

Saiki, 1982; Chandler & Schwartz, 1987; 1988). Tsukida and co-workers

resolved 17 ^-carotene isomers using a column packed in their

laboratory with a hand-selected batch of lime (Tsukida & Saiki, 1982;

Tsukida et aL, 1981). Unfortunately, commercially prepared Ca(OH)2

columns are not available and extensive batch-to-batch variability of

Ca(OH)2 has been reported (Chandler & Schwartz, 1987). Vecchi et al. (1981) separated 12 isomers of )3-carotene using an alumina column;

however, significant modifications of normal LC equipment were

required to regulate the mobile phase temperature and water content.

Successful quantitative analysis requires that the LC column selectivity

must be sufficient to separate the standard of interest from all

interferences contributing absorbance at the detection wavelength,

including geometric isomers. Our goal was to identify a means of

assessing carotene purity for laboratories making routine measurements.

Reversed-phase LC is the method most commonly used to measure

carotenoids. This technique offers the capability to separate a wide range

of compounds and Cjg stationary phases are compatible with most

solvents. The resolution of carotenoid isomers using reversed-phase

columns is less complete than that obtained using lime and alumina

columns; however, the columns are commercially available and the

equipment requires no special modifications.

The investigation ofcis isomers of/^-carotene was further motivated by

the lack of knowledge about the carotenoid isomer content of natural

and commercial ^-carotene products. Since much of the association

between cancer prevention and )3-carotene is based on epidemiological

and food consumption data, it is essential to determine more accurately

which isomers are present naturally in foods and the composition of

commercially available /^-carotene products. The development of

methods that resolve carotenoid isomers is imperative to understanding

which carotenoid(s)/isomer(s) might be beneficial for human cancer

prevention. Therefore, the ability of several reversed-phase C|g columns

to separatee-carotene isomers was investigated. Commercial preparations

of )3-carotene were then analyzed to determine their relative content of

)3-carotene isomers using spectrophotometry and reversed-phase LC

with absorbance detection.

1-37
All-tTans-^-carotene and its cis isomers 111

TABLE 1 Source and Description of ^-Carotene Products Investigated^

Supplier Lot Description

Fluka" 245663582 Synthetic crystalline

Sigma" 83F0218 T" TTT ill* /* * Type III crystalline from carrots Sigma 15F97I5 T^ TX T .11' f Type IV crystalline from carrots Sigma" 1 1 2F0307 Type I synthetic crystalline

MCB 2259 Synthetic crystalline

ICN 14008 C ill* Synthetic crystalline

Kodak E15A Synthetic crystalline

* Betatene 4% algal ^-carotene suspension

* Betatene 30% algal /3-carotene suspension

1 Betatene 10 000 lU algal /3-carotene capsule

* 2 Betatene 10 000 lU algal /3-carotene capsule

* Chinese crystalline

* Vita crystalline

Roche 10% beadlets

"A detailed investigation of lot-to-lot reproducibility has not been

performed. Therefore the lots analyzed may or may not be representative

of the manufacturers products.

^^-Carotene preparations not measured by LC.

* Samples provided by the National Cancer Institute, Division of Cancer

Prevention and Control.

MATERIALS AND METHODS

The descriptions, lot numbers, and suppliers of the ^-carotene products

analyzed are listed in Table 1. All solvents used for the LC analyses were

HPLC grade or equivalent and were used without further treatment.

Gravimetric determinations

Weighed samples (c. 0-35 mg) from five lots of j3-carotene were each

dissolved in 500 jjl of toluene by ultrasonic agitation and diluted to 50 ml

with acetonitrile. The UV/visible spectrum for each sample was recorded

immediately using a spectrophotometer (Hewlett-Packard model 8450A).

Spectrophotometric concentrations were calculated using published

absorptivities for ^-carotene in ethanol (Isler et ai, 1956) since

absorptivities have not been reported in acetonitrile. Using a Cary Model

219 spectrophotometer (Varian Instruments, Walnut Creek, CA), we

found that the absorptivity of/?-carotene in the two solvents varied by less

than 1% and the wavelength maxima in acetonitrile was 1-2 nm higher

than in ethanol. An absorptivity in acetonitrile is not reported here due to

1-38
212 Neal E. Craft, Lane C. Sander Herbert F. Pierson

the presence of impurities in all /3-carotene samples examined. The

relative purity of preparations was determined by comparing the spectrophotometric concentration to the gravimetric concentration.

LC sample preparation

j3-Carotene materials were received as either crystalline material or as an

oily suspension. A representative sample of each product was dissolved

in absolute ethanol. Ethanol was the solvent of choice since it is both

miscible with the LC mobile phase, has not been reported to induce

carotene isomerization, and ethanolic )3-carotene solutions have

demonstrated stability in our laboratory. The Hoffman-La Roche

/^-carotene beadlets are gelatin-coated which necessitated the removal of

the gelatin prior to extraction. This was accomplished either by heating

the beadlets in ethanol to 50°C to dissolve the gelatin, permitting the

extraction of )3-carotene into the ethanol, or denaturing the gelatin with

6 m urea followed by hexane extraction. The hexane extract was

evaporated using a stream of N2 and redissolved in ethanol.

Equipment'"

The liquid chromatographic system consisted of a ternary single-piston

pumping system fitted with a fixed-loop injector. Columns investigated

TABLE 2

LC Column Characteristics"

Column Particle

size (lAtn) & type

Carbon

load (%)

Synthesis

type'' Endcapped

Pore

size (A)

Vydac 201TP C,8 5, spherical 8 Polymeric 300

Bakerbond 5, spherical 10 Polymeric + 300

Widepore Cig

/iBondapak Cig 10, irregular 10 Monomeric 50-300

Ultrasphere Cig 5, spherical 13 Monomeric + 80

Zorbax C|g 5, spherical 20 Monomeric 70

"Nominal data provided by the column manufacturer.

''Monomeric synthesis binds monochlorosilanes to the base silica gel yielding a monolayer.

Polymeric synthesis binds trichlorosilanes to the base silica gel yielding a polymer layer.

* Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply

recommendation or endorsement by NIST, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

1-39
All-trans-IS-carotene and its cis isomers 213

for the separation ofj3-carotene isomers included Bakerbond Wide Pore

C,8 (250 X 4-6 mm, J. T. Baker Chemical Co., Phillipsburg, NJ),

//Bondapak Cig (300 X 3-9 mm. Waters Division of Millipore, Milford,

MA), mtrasphere C-18 (250 x 4-6 mm, Beckman Instruments, San

Ramon, CA), Vydac 201TP (250 X 4-6 mm. The Separations Group,

Hesperia, CA), and Zorbax ODS (250 X 4-6 mm, MacMod Analytical,

Inc., Chadds Ford, PA). Additional physical characteristics of these Cig

stationary phases are listed in Table 2. Column selectivity for carotenoid

isomer separation was tested using methanol or acetonitrile as the

primary mobile phase solvent. Ethyl acetate or water was added as a mobile phase modifier to maintain the capacity factor (/:') for)3-carotene

in the range of 7-5-12-5. The Vydac 201TP column was ultimately used

for the analysis ofthe commercial j3-carotene preparations with a mobile

phase comprised of methanol/water (97:3, v/v) at a flow rate of

l-5mlmin"'. Carotenoids were monitored at 450 nm (absorbance

maximum of trans-p-caTOtene in the mobile phase for the LC detectors)

using both a programmable variable wavelength absorbance detector

(Spectroflow 783, Kratos/ABI, Ramsey, NJ) and a UV/visible photodiode

array detector (H-P 1040A, Hewlett-Packard, Palo Alto, CA) in series.

Data were collected by computer on floppy discs permitting the

reprocessing of chromatograms. The relative standard deviation of the

/ra«5-)3-carotene peak area in a 5 //g ml"' standard solution was 1-5% for

10 injections of 20 //I.

Peak identification

)8-Carotene isomers were tentatively identified by UV/visible absorbance

spectra, Q-ratios and elution order from a Ca(OH)2 column. Q-ratios,

absorbance at the wavelength maximum/absorbance at the cw' peak,

were calculated from spectral data obtained using the photodiode array

detector (PAD) and compared to published values (Tsukida & Saiki,

1982; Quackenbush, 1987). Elution order of the major isomers (9-cis and

13-cw) from Ca(OH)2 was compared to published separations (Tsukida

et aL, 1981; Chandler & Schwartz, 1987). A column (4-6X 250 mm)

packed with Ca(OH)2 (J. T. Baker, Phillipsburg, NJ) at 12 000 psi was

eluted with 1% acetone in pentane. Peaks corresponding to 9-cis and 13-

cis isomers of thermally isomerized )8-carotene were collected and

chromatographed using the reversed-phase system described above.

RESULTS AND DISCUSSION

The purity of five lots of )3-carotene was evaluated by comparing the

concentration calculated using the absorptivity to the gravimetric

1-40
214 Neal E. Craft, Lane C. Sander. Herbert F. Pierson

TABLE 3

Spectrophotometric Concentration

of )3-Carotene Products Relative to the Gravimetric Concentrations

Supplier Percentage'^

1 71

2a 28-4

2c 43-

1

3 72-3

4 82-9

"Spectrophotometric concentration/

gravimetric concentration X 100.

concentration. Estimated purities were low (Table 3), ranging from 7- 1 to

82-9%, in spite of an over-estimation of purity due to the absorbance

contributed by contaminating carotenoids and )3-carotene isomers.

Quackenbush and Smallidge (1986) also observed a similar degree of

variability in )3-carotene preparations from six different suppliers.

Five commercially available Cig columns were investigated for the

separation of a mixture of/3-carotene isomers. The physical characteristics

of the different stationary phases are summarized in Table 2. Wide-pore

polymeric columns were less retentive than monomeric columns. The

mobile phase compositions used to yield a A:' of approximately 9 for

rrfl«5-)3-carotene were as follows: Vydac 201TP and Bakerbond WP,

methanol/water (98:2, v/v); /iBondapak 100 methanol; Ultrasphere,

methanol/ethyl acetate (90:10, v/v); Zorbax, methanol/ethyl acetate

(85:15, v/v). None of the monomeric columns used in this study

possessed adequate selectivity to resolve c/5-)3-carotene isomers from

rra«5-)3-carotene. These observations are in agreement with the reports of

others (Quackenbush & Smallidge, 1986; Quackenbush, 1987; Bushway,

1985) and are illustrated in Fig. 1. The mixture of)3-carotene isomers was

separated by the wide-pore polymeric Cig columns but not by the

monomeric Cig columns. Based on the dimensions of the rigid j8-

carotene molecule (33 A long), the narrow pore size of these monomeric

columns may limit the accessibility of the molecule to the bonded phase

inside the pores. MacCrehan & Schonberger (1987), Ben-Amotz et al.

(1988), Quackenbush (1987) and Bushway (1985) have all reported the

separation of rra«5-j3-carotene from its cis isomers by reversed-phase LC.

In all cases the LC columns used to achieve these separations contained

wide-pore C,8 modified silica synthesized with trichlorosilanes (polymeric synthesis) rather than silica modified with monochlorosilanes

(monomeric synthesis). The unique selectivity of polymeric Cig columns

1-41
All-ir&ns-^-carotene and its cis isomers 215

0 5 10 15 20 25

Time (minutes)

Fig. 1. Comparison of the separation ofan algal /3-carotene extract using reversed-phase

Cig LC columns. For all columns, the flow rate was l-5mlmin~' and eluents were monitored at 450 nm; A Zorbax ODS, methanol/ethyl acetate (85:15, v/v); B Ultrasphere

ODS, methanol/ethyl acetate (90:10, v/v); C/iBondapak, 100 methanol; D Bakerbond

WP ODS, methanol/water (98:2, v/v); E Vydac 201TP, methanol/water (98:2, v/v).

for the separation of structurally similar compounds has been investigated

extensively for polycyclic aromatic hydrocarbons (PAHs) (Wise et ai,

1983; Sander & Wise, 1984, 1986; Wise & Sander, 1985). Wise & Sander

(1985) have demonstrated the ability of polymeric Cig columns to resolve

isomers of PAH that could not be separated by monomeric columns.

These authors have also investigated the influence of pore size, surface

coverage and chain length on the PAH isomer separation and found the

polymeric synthesis to be the most significant determinant. The effects of

specific stationary phase modifications (i.e. pore size, surface coverage,

chain length) on the separation of /3-carotene isomers will be described

in a later publication.

Mobile phase composition was manipulated to maximize the

selectivity for jS-carotene isomers. Although acetonitrile has lower

viscosity than methanol resulting in higher efficiency, methanol yielded

better selectivity and better recovery of^-carotene isomers. The average

recovery of )3-carotene in acetonitrile-based solvents was 74% while

recovery in methanol-based solvents was 89%. The separation factors, a,

for )3-carotene/a-carotene and isomer 2/)3-carotene are larger in the

1-42
216 Neal E. Craft, Lane C. Sander, Herbert F. Pierson

E

coID

O

cm

Sio

V) <

0.06

0.04 -

0.02 -

0.00

methanol mobile phase

acetonitrile mobile phase

n

10 15

Time (minutes)

Fig. 2. Comparison of the difference in the separation selectivity using methanol (solid

line) or acetonitrile (dashed line) as the mobile phase. Algal j3-caroten^ extract injected on a wide-pore polymerically modified reversed-phase Cig LC column and monitored at 450 nm.

methanol mobile phase than the acetonitrile mobile phase. The

difference in selectivity using the two solvents is illustrated in Fig. 2. Therefore, the primary mobile phase constituent was restricted to

methanol. Optimal selectivity and recovery are essential for accurate

standardization and quantification since the isomers of)3-carotene have

different extinction coefficients and absorption maxima which can

influence the value assigned to the '^-carotene peak'.

Representative chromatograms from the reversed-phase separation of

four different /5-carotene products are presented in Fig. 3. Both the

relative purity and isomer content of the commercial ^-carotene reagents

vary significantly among the various sources. The composition of the

products evaluated in this work by reversed-phase LC, expressed as the area per cent of the totalled peak area absorbance at 450 nm, is listed in

Table 4. 7rfl«5-)3-carotene constituted from 22 to 84% and the cis

component comprised at least 10% in all products tested. Quackenbush

& Smallidge (1986) also reported significant variability in the purity and

isomer content of commercial /^-carotene compounds. The presence of

isomers in ^-carotene products coincides with a recent report by Mayne

& Parker (1988) who observed the presence of cis isomers in commercial

preparations of canthaxanthin. However, it refutes their comment that

isomers are not associated with )3-carotene preparations.

1-43
All-tmns-^-carotene and its cis isomers 111

(0 a^ 100 mAUFS

I ^

5 25 45

Time (minutes)

Fig. 3. Representative chromatograms of jS-carotene preparations separated using a wide-pore, polymeric Cig column. Mobile phase methanol/water (97:3, v/v) at a flow rate

1-5 ml min"' and monitored at 450 nm. Cis isomers of ^-carotene are numbered in the

order of elution from the LC column. A = Product 2b, B = Product 9H, C = Product 6b,

D = Product 6a.

The isomer content of product 9 differed significantly with the type of

extraction used (Table 4, 9C vs. 9H). The large cis component observed in

the 'hot extract' (9H) is possibly the result of induced isomerization due

to the elevated extraction temperature. Heating is one method that has

been used to induce isomerization of carotenoids; however, the

temperatures used here are much lower than those typically used to

cause isomerization. When an ethanolic solution ofcrystalline )3-carotene

1-44
218 Neal E. Craft. Lane C. Sander. Herbert F. Pierson

TABLE 4

LC Determination of the Relative Composition of

^-Carotene Isomers in ^-Carotene Preparations

- Product % Composition"

tra 11 Cl n1 1 so isomers Tntnl KJl rl(zr

C/j

1

1 2 3 4

o0

Q A z1I 1 10 6

9C 11 6 7 13 10

2b 12 8 4 12 16

3 68 3 5 1 2 12 20

7 64 2 14 6 2 24 12 4 61 5 5 2 2 14 25

5 61 13 8 20 19

6b 49 9 30 6 1 46 5 9H 48 18 13 1 41 11 6c 33 5 45 4 3 57 10

6d 25 56 8 1 65 10

6a 25 5 49 10 3 67 8

"Based on the % area under the chromatographic peak

relative to the total area of all peaks at 450 nm. Values

rounded to the nearest whole number.

''The 'other category constitutes primarily a-carotene

and other 450 nm absorbing compounds that elute

prior to all-?ra«5-^-carotene.

was submitted to similar conditions, only a 5% change was observed in

the trans isomer.

Of particular note was the very high cis isomer content in some algal

/^-carotene products (6a-d). The proportion of isomer 2 in products 6a, 6c

and 6d is greater than the proportion of rra/75-j3-carotene. The content of

isomer 2 is higher than any isomers reportedly formed by deliberate

isomerization of rra«5-/?-carotene, negating the possibility of it being an

artifact (Zechmeister, 1962). The high ratio of cis/trans along with other

factors influencing the isomer distribution of algal j8-carotene have been

reported previously by Ben-Amotz et al. (1988). Using the UV/visible

absorbance spectra collected by the photodiode array detector during

the LC separation, it was possible to compare the Q-ratio (absorbance at

the wavelength maximum/absorbance at the 'cw' peak) to published

values (Tsukida & Saiki, 1982; Quackenbush, 1987). The primary cis

isomer (peak 2) has been tentatively identified as 9-d5-i3-carotene by its

Q-ratio, absorption spectrum, and elution from Ca(OH)2. This identifi1-45
AH-trans-P-carotene and its cis isomers 219

o

to <

O

C

(0 J3

1 1 r 250 350 450 550

Wavelength (nm)

Fig. 4. Overlay of the absorption spectra of the major /3-carotene isomers present in the

preparations investigated. Cis isomers are identified by order of elution from the LC

column, as illustrated in Fig. 3.

cation is in agreement with a previous report describing the same algal

source of j3-carotene (Ben-Amotz et ai, 1988). Spectra of the major

^-carotene isomers in the carotene products are presented in Fig. 4. The

4 nm downward shift (hypsochromic shift) of the wavelength maxima in

response to the introduction of a cis bond into the conjugated doublebond system is evident along with the characteristic 'cw' peak at 330-

340 nm. Based on Q-ratios and spectral maxima, peaks 1, 3 and 5 have

been tentatively identified as 7-d5-^-carotene, 13-d5-i3-carotene, and 9,

13-di-cw-j8-carotene, respectively. These identifications are termed tentative

since spectral analysis alone is not a definitive technique for the

identification of carotenoid isomers and coelution of isomers may exist.

Studies are in progress to collect sufficient quantities of each isomer for

more definitive identification by nuclear magnetic resonance spectroscopy.

The majority of methods used for carotenoid analysis lack the

selectivity necessary to resolve very similar carotenoids, including the

geometric isomers. In the present study, five LC columns were examined

for their ability to separate the geometric isomers of/3-carotene. Only the

columns packed with wide-pore, polymerically synthesized Cig were able

to separate these isomers. We found that the recovery and selectivity of

these columns was better using methanol-based mobile phases rather

than acetonitrile-based. The cw-component ofall y3-carotene preparations

1-46
220 Neal E. Craft. Lane C. Sander. Herbert F. Pierson

examined ranged from 10 to 67% of the total absorbance due to ^-carotene.

As illustrated here by /3-carotene, accurate carotenoid measurements

require the right selection of column and mobile phase; and due to the

large degree of variability in the purity of commercial ^-carotene

preparations, special precautions must be taken when value assigning

carotenoid calibration standards.

ACKNOWLEDGEMENT

This work was supported in part by research agreement YOl-CN-80655

from the Division of Cancer Prevention and Control, National Cancer

Institute.

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Bushway, R. J. (1985). Separation of carotenoids in fruits and vegetables by high

performance liquid chromatography. 7. Liq. Chromatogr.. 8, 1527-47.

Chandler, L. A. & Schwartz, S. J. (1987). HPLC separation of cis-trans carotene

isomers in fresh and processed fruits and vegetables. /. Food Set, 52, 669-72.

Chandler, L. A. & Schwartz, S. J. (1988). Isomerization and losses of trans- j3-carotene in sweet potatoes as affected by processing treatments. J. Agric.

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Isler, O., Lindlar, H., Montavon, M., Riiegg, R. & Zeller, P. (1956). Synthesen in

der Carotinoid-Reihe. Die technische Synthese von ^-Carotin. Helv. Chim.

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MacCrehan, W. A. & Schonberger, E. (1987). Determination of retinol,

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compounds in canthaxanthin preparations. J. Agric. Food Chem., 36, 478-82.

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Chromatogr., 10, 643-53.

Quackenbush, F. W. & Smallidge, R. L. (1986). Nonaqueous reverse phase liquid

chromatographic system for separation and quantitation of provitamins A.

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Sander, L. C. & Wise, S. A (1984). Synthesis and characterization of polymeric

C18 stationary phases for liquid chromatography. Chem., 56, 504-10.

Sander, L. C. & Wise, S. A. (1986). Investigations of selectivity in RPLC of polycyclic aromatic hydrocarbons. In Advances in Chromatography, Vol. 25 eds

J. C. Giddings, E. Grushka, J. Cazes & P. R Brown. Marcel Dekker, New

York, pp. 139-218.

1-47
All-trans-P-carotene and its cis isomers 221

Tsukida, K. & Saiki, K. (1982). Separation and determination of cis/trans-

)3-carotenes by high-performance liquid chromatography. J. Chromatogr.,

245, 359-64.

Tsukida, K., Saiki, K. & Sugiura, M. (1981). Structural elucidation of the main

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charakterisierung von ^-carotin-isomeren. Helv. Chim. Acta. 64, 2746-58.

Wise, S. A. & Sander, L. C. (1985). Factors affecting the reversed-phase liquid

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Resol. Chromatogr. Chromatogr. Commun., 8, 248-55.

Wise, S. A.. Sander, L. C. & May, W. E. (1983). Modification of selectivity in

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Zechmeister, L. (1962). Cis-Trans Isomeric Carotenoids, Vitamins A. and

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1-48
Copjrright (|) 1992 by the American Chemical Society and reprinted by permission of the copyright owner. Relative Solubility, Stability, and Absorptivity of Lutein and /5-Carotene

in Organic Solvents

Neal E. Craft*

Organic Analytical Research Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899

Joseph H. Scares, Jr.

Nutritional Sciences Program, University of Maryland, College Park, Maryland 20742

The relative solubility, stability, and absorptivity of lutein and ^S-carotene were determined in 18 organic

solvents. The solubility of both carotenoids was greatest in tetrahydrofuran, while hexane exhibited

the least solubility for lutein; methanol and acetonitrile exhibited the least solubility for /9-carotene. Stability was monitored for 10 days at room temperature by measuring absorbance changes at the

wavelength maximum. In the majority of the solvents, initial absorbance decreased by less than 10%

during the 10-day period. Degradation was greatest for both carotenoids in cyclohexanone. The relative absorptivities were determined by calculating the carotenoid concentration in a reference solvent using

a reference absorptivity, and then Beer's Law was applied to the measured absorbance of the same

carotenoid concentration in other organic solvents. Absorbfince maxima and relative absorptivities were in good agreement with available literature values.

Interest in carotenoids has increased during the past

decade. Carotenoids are not only natural pigments and

vitamin A precursors but have been proposed as cancer

prevention agents, ulcer inhibitors, life extenders, and

heart attack inhibitors (Peto et al., 1981; Colditz et al., 1985; Mozsik etal., 1984; Cutler, 1984; Gazianoetal., 1990).

Unfortunately, physical information about these compounds in organic solvents is limited. The wavelength

maxima and absorptivity of carotenoids change with the nature of the solvent in which they are dissolved. For

example, the visible spectrum of /3-carotene in ethanol

has little fine structure with absorbance maxima at 453

and 480 nm, while the visible spectrum in carbon disulfide has more fine structure and exhibits maxima at 484

and 512 nm (Davies, 1976). The molar absorptivities of

)3-carot€ne at Xmai in these two solvents are 140 700 and

107 800 L mol-i cm-\ respectively (Davies, 1976). In the

past, absorbance maxima have been compiled for caro- tenoids in several solvents (Davies, 1976; De Ritter and

Purcell, 1981), but such tables supply limited absorptivities, and frequently the maxima in a given solvent vary by several nanometers depending on the source of infor- mation. These tables also provide no information re- garding the solubility and stability of the carotenoids in the solvents. The lack of information about carotenoid

solubilities and molar absorptivities in a variety of organic

solvents increases the difficulty associated with developing

analytical methods for carotenoid research. Such practical

information is important for the selection of solvents for use in sample preparation and liquid chromatography (LC)

mobile phases and also for the identification and quantification of carotenoids in diverse LC mobile phases.

The two most prominent cyclized carotenoids in human

serum and foods are lutein (/3,«-carotene-3,3'-diol) and 0- carotene (/3,/3-carotene) (Bieri et al., 1985; Khachik et al., 1986). Not only are they the most prominent, but they

also span a wide polarity range and are representative of

the /3,{- and the /3,/3-carotenoids, respectively. Herein we

describe the determination of the relative solubility,

Author to whom correspondence should be addressed.

stability, and absorptivity of these two biologically important carotenoids in various organic solvents. MATERIALS AND METHODS

Reagents. Crystalline /3-carotene (type I, Sigma Chemical

Co., St. Louis, MO) and lutein (provided as a gift by Kemin

Industries, Des Moines, lA) used throughout the study were

assessed by spectrophotometric and liquid chromatographic

techniques to be greater than 90% tran5-/3-carotene and 90%

trans-lutein, respectively. The sources, descriptions, and lot numbers of the solvents used are listed in Table I. Equipment. Spectral measurements were made using a photodioide array scanning spectrometer (H-P 8450, Hewlett-Packard, Palo Alto, CA). The spectrophotometer provided a 1-nm

spectral band pass from 200 to 400 nm and a 2-nm spectral band

pass from 400 to 800 nm. Wavelength accuracy was checked

using a holmium oxide glass filter and found to be correct at the

279-, 361-, 460-, and 536-nm absorption maxima. Solutions were

dispensed with calibrated pipets or gas-tight syringes. Carotenoid purity was determined as previously described (Craft et

al., 1991). Relative Solubility of Lutein and /3-Carotene in Organic

Solvents. Approximately 10 mgoflutein or /3-carotene was added

to 3 mL of each of the solvents listed in Table I. Vials were

ultrasonically agitated for 5 min. If a clear solution with no

residual crystals resulted, additional carotenoid was added until crystalline material remained undissolved. Each solution was

then filtered through a 0.2-iim membrane, and appropriate

dilutions were made until the absorbance at the wavelength

maximum was between 0.5 and 1.0 absorbance unit at ambient

temperature. The background absorbance of each solution was

subtracted using the appropriate solvent containing no caro- tenoid. Carotenoid concentration was calculated using Beer's law and the relative absorptivities determined below (Determination of Relative Absorptivity). Measurements were performed

in triplicate and the calculation used is

(absorbance, at X^)(dilution factoD/molar absorptivity,

where the subscript s is a given solvent. The measured values were rounded to one significant figure since this experiment was

not designed to determine absolute solubility but rather to indicate solubility relative to other solvents. Determination of Relative Absorptivity. Concentrated

solutions (approximately 3 g/L) of lutein and /3-carotene were

prepared in tetrahydrofuran (THF) containing butylated hy0021-8561/92/1440-0431$03.00/'^ 1992 American Chemical Society

1-49
432 J. Agrlc. Food Chem., Vol. 40, No. 3, 1992

Table I. List of Solvents, Sources, and Lot Numbers

Craft and Scares

solvent source, grade lot safety hazards"

acetone Mallinckrodt, SpectAR 2438 1,2,5

acetonitrile J. T. Baker, HPLC C28108 2, 3, 5 benzene J. T. Baker, Photrex C 14603 3, 5 chJoroform EM Science, Omnisolve 5102 3 cyclohexane EM Science, Omnisolve 6041 1, 2,5

cyclohexanone Kasai, GR FAvol 1,2

dichloromethane J. T. Baker, HPLC D25082, D18131 2,3

dimethyiformamide (DMF) Burdick and Jackson, HPLC AK285 1, 2 dimethyl sulfoxide (DMSO) Mallinckrodt, SpectAR KMCD 1, 2, 4

ethanol, absolute Warner-Graham 1, 5

ethyl acetate EM Science, Omnisolve 7278 h 2.5

ethyl ether EM Science, GR 9130 1, 2, 4, 5 hexane J. T. Baker, HPLC D33095 1,2,5

2-propanol Mallinckrodt, AR 3037KDEV, 3035KCAY 1,5 methanol EM Science, Omnisolve 8352 3,5

methyl fert-buty! ether (MTBE) EM Science, reagent 11P23 2,5

tetrahydrofuran (THF) + BHT J. T. Baker, HPLC C24654 2,5

toluene Burdick and Jackson, HPLC AK80 1, 2,5

" 1 , harmful when entering the body; 2, irritant to skin, eyes, and respiratory organs; 3, toxic (harmful if inhaled, ingested, or absorbed through

the skin); 4, explosive; 5, flammable.

Table IL Relative Solubility and Absorptivity of Lutein and ^-Carotene in Organic Solvents

lutein j3-carotene

solubility. absorptivity molar absorptivity," solubility. absorptivity molar absorptivity," solvent mg/L XniM," nm cm-' L mol"' cm'' mg/L Xma," nm £;"'%cm-i L mol"' cm"^

acetone 800 446 2540 144 500 200 452 (452) 2559 137 400

acetonitrile 100 446 2559 145 600 10 452 2540 136 400 benzene 600 456 (458) 2350 133 700 (127 200) 4000 462 (462) 230^, 124 000 (125 500) chloroform 6000 454 (458) 2369 134 800 2000 462 (461) 2330 125 100(128 600) cyclohexane 50 448 2520 143 400 2000 454 (457) 2508 134 700 (134 500) cyclohexanone 4000 454 2359 134 200 2000 462 2359 126 700

dichloromethane 800 452 2320 132 000 6000 460 2369 127 200 DMF 1000 454 2390 136 000 200 460 2389 128 300 DMSO 1000 460 2369 134 800 30 466 2259 121300

ethanol 300 444 (445) 2550 145 100* 30 450 (449) 2529 135 800(140 700)

ethyl acetate 800 446 2529 143 900 500 452 2520 135 300

ethyl ether 2000 444 2629 149 600 1000 448 2659 142 800 hexane 20 444 (445) 2589 147 300 600 448 (453, 450) 2592 139 200^ 2-propanol 400 444 2599 147 900 40 450 2508 134 700 methanol 200 442 (444) 2629 149 600 10 450 2540 136 400 MTBE 2000 444 2589 147 300 1000 450 2588 139 000 THF 8000 450 2469 140 500 10000 456 2399 128 800

toluene 500 456 2290 130 300 4000 462 (463) 2270 121900

" Calculated molar absorptivities and Xn absorptivity for /3-carotene.

droxjrtoluene (BHT) as an antioxidant and filtered through 0.2- lim membranes. To 30 mL of each of the solvents listed in Table

I was added 30 fiL of concentrated lutein or /3-carotene in THF.

We were cautious to work well within the carotenoid solubility

limits (determined above) of the solvents being examined to avoid

precipitation of the carotenoid compounds. Sealed vieds were

ultrasonically agitated for 3 min to assure dissolution. Spectrophotometric scans were performed from 250 to 550 nm, and

absorbance at the wavelength maximum was determined. The

concentrations of the lutein and /3-carotene solutions were

determined using the most widely accepted molar absorptivity

for lutein in ethanol and /3-carotene in hexane (145 100 and 139 200 L mol"' cm"', respectively) (Davies, 1976). Relative absorptivities in the different solvents were determined on the basis of the

calculated concentrations of lutein and /3-carotene determined

in ethanol and hexane, respectively, and the absorbance of the carotenoid solutions at the wavelength maximum in a given

solvent using Beer's law. Absorbance measurements were performed in triplicate, and the combined error (mean standard

deviation of absorbance measurements and estimated limits of

bias) associated with the measurements was approximately 1 % . Lutein and /^-Carotene Stability. The solutions prepared

under Determination of Relative Absorptivity were stored in amber glass vials with Teflon-lined screw caps at room temperature. The UV-vis absorbance spectrum from 250 to 550 nm

was monitored over a 10-day period. Decreases in absorbance and shifts in wavelength maxima were indicators of carotenoid

degradation. Degradation is reported as percent of initial ab- sorbance at Xmax.

in parentheses are taken from Davies (1976). * Reference absorptivity for lutein. ' Reference

RESULTS AND DISCUSSION

Both carotenoids tested were most soluble in THF

(Table II). /3-Carotene was letist soluble in methanol and

acetonitrile, while lutein was least soluble in hexane. Many

existing extraction techniques partition carotenoids into

hexane or petroleum ether from aqueous alcohol or acetone

(Bieri et al., 1985; De Ritter and Purcell, 1981; Simpson

et al., 1985). Given the poor solubility of dihydroxy and more polar carotenoids in hexane, this may lead to losses. Diethyl ether has also been used to partition carotenoids

from aqueous/ polar organic mixtures (Britton, 1985; De

Ritter and Purcell, 1981; Rodriquez-Amaya, 1989); on the

basis of the solubilities listed in Table II, this may present

a more effective approach. One possible disadvantage is the solubility of fatty acid soaps in ether which must be

thoroughly removed with water (Britton, 1985). Although

THF is subject to peroxide formation, it has found

increased use (Bureau and Bushway, 1986; Khachik et al., 1986; Peng et al., 1987) for carotenoid extractions due to the high solubility of a wide polarity range of carotenoids.

Weare unaware ofpublished absorptivities for carotenoids

in THF. This lack of information may hamper its use as a solvent and result in the introduction of errors associated

with evaporation and solvent-tremsfer steps due to the

use of less appropriate solvents with published absorptivities. The information listed in Table II should prove

1-50
wuLoiii ai ivj III wi ^«ii iiw ww)«v>ivis u. f-iyiiK,. I y^iioiii., vol. tyi, no. O, 197^

Table III. Relative Lutein and /3-Carotene Degradation in Organic Solvents

"c of initial absorbance of lutein at '"c of initial absorbance of j3-carotene at X,

solvent

time, days

shift,^ nm

cis peak''

time, days

shift," nm

cis

1 3 peak* 6 10 1 3 g 10 acetone 96 95 yo U

QQyo 96 96 93 0 +

acetonitrile yo 97 94 y4 n ~i QQyy 96 93 92 0 +

benzene 1 AA 99 100 y / r\U 87 77 71 67 0 -

chloroform y ( 96 93 yu —

z

+ 97 91 92 91 -2 +

cyclohexane 100 98 QQyy

A0 98 98 93 91 0 +

cyclohexanone QQ80 70 49 O / o +++ ofa 67 45 32 -4 +++

dichloromethane yo 91 88 oo

AU 11 59 47 34 -30 +

JJMf 100 97 Q7y / U

QCyo 94 93 90 0

—

lUvJ 99 99 y / AU 94 90 86 80 0 —

ethanol yo 96 93 Q1y i A

U + QQyo 94 92 91 -2 +

ethyl acetate QQyo 97 95 Q£yb A0 99 97 96 95 0 —

ethyl ether yo 88 79 bo ++ 94 78 70 69 -2 +

hezane yy 100 100 QQytt A

u 96 95 94 92 0 +

^ (if upelilUl 99 99 99 n Q7y / 94 94 QQoy A0 methanol 97 95 95 90 -2 ++ 97 92 89 88 -4 ++ MTBE 97 90 82 76 0 ++ 96 89 82 74 -2 ++ THF + BHT 100 100 100 99 0 99 98 99 97 0

toluene 99 98 100 97 0 90 80 76 71 0

" Indicates direction and amount of spectral shift at day 10. Indicates presence/absence of cis peak. Number of +'s indicates intensity

of cis peak.

valuable in the selection of solvents employed for caro- tenoid extractions and dissolutions. The wavelen^h maximum for /3-carotene in the various

solvents ranged from 448 to 466 nm. The in hexane

was 448 nm, which is 4-5 nm below the referenced value

(Davies, 1976), although the same reference lists other

citations reporting Xmai in hexane at 449 nm but without an absorptivity. The difference in the reference Xmax aiid the Xmai obtained in this work can be attributed to aromatic

contaminants in some solvents, the presence of cis isomers

in the /3-carotene employed, and differences in the

wavelength calibration or spectral band pass of the spec- trophotometers used for the measurements. For the purposes of this work, the maximum absorbance was used

to determine relative solubilities and absorptivities. The

wavelength maximum for lutein ranged from 442 to 460 nm. The Xmu of lutein in ethanol was within 1 nm of the

reported value of 445 nm (Davies, 1976). It may be that,

in the lutein used, the presence of ~6 % zeaxanthin, which

has a higher Xmu, offset the wavelength lowering due to

cis isomers or differences in wavelength calibration. The

spectrophotometer used for this work was limited to a 2-nm spectral band pass in the visible region. These two

observations may explain why the measured Xmax values were consistently 1-2 nm lower than reported values. Because the absorptivity and Xmax of carotenoids vary

in different solvents and these values are only published

for a few solvents, relative absorptivities and Xmu were determined for lutein and /3-carotene in the solvents listed

in Table I. These values, given in Table II, were used to

calculate the solubility of the carotenoids, also reported

in Table II. Relative absorptivity values listed in Table

II are in good agreement with previously published values

(Davies, 1976; De Ritter and Purcell, 1981). The primary

advantage of the determination of the spectral maxima

and absorptivities is that carotenoid concentrations can

be determined directly in a wide range of solvents. In

addition, data obtained using diode array detectors in conjunction with LC can be better interpreted when the

wavelength shifts that occur in different solvents are known.

Finally, the stability of these two carotenoids in the

various solvents was monitored spectrophotometrically

over a period of 10 days. Carotenoid degradation was

accompanied by decreases in the absorbance and, in some

cases, a downweird shift in the Xmax (Table III). We are

aware that some degradation products (e.g., geometric

isomers and carotenals) contribute to the absorbance in the visible region; however, all degradation products

exhibit lower absorptivity at the wavelength msiximum of

the parent compound. This also implies that changes in absorbance are not necessarily proportional to the concentration of lutein or /3-carotene in the solution. The

definitive measure of degradation would have been to monitor the trans isomer of both carotenoids by HPLC;

however, while attempting to do this, we encountered

technical difficulties. First, it was not possible to make

all of the measurements by HPLC at the appointed times

without staggering the experiments; second, few of the

solvents could be injected directly into the HPLC system;

and third, complete redissolution of carotenoids was

questionable if a solvent evaporation was included. For

these reasons we opted to record the UV-vis spectra to monitor major changes in the andytes. When the ab- sorbance expressed as percent of initial absorbance at \ma. was plotted against time, the degradation function was

similar in all solvents but proceeded at different rates (Figure 1). Stability was poorest for both lutein and /3- carotene in cyclohexanone, retaining only 37% and 32%

,

respectively, of their initial absorbance by day 10. The

degradation of /3-carotene in cyclohexanone was followed

closely by degradation in dichloromethane with ~34%

absorbance remaining at day 10. In general, the rate of

lutein degradation was slower than ^-carotene degradation

as illustrated by the curves shown in Figure 1 representing

the average rate of degradation in all solvents. Even on

day 10, the carotenoid absorbance in most solvents was

clustered above 90% of the initial absorbance. The

conditions incorporated were selected to exacerbate the

degradation process so that stability/ instability would be

evident. In a laboratory setting, greater efforts would be

made to stabilize carotenoid solutions, e.g., by the incor- poration of antioxidants and use of lower storage temperatures.

Little information of this type has been reported

previously. In comprehensive reviews (Davies, 1976; De

Ritter and Purcell, 1981), references to factors important

to solvent selection are mentioned, but specific information

about the influence of solvents on carotenoid stability is lacking. Information presented in Table III should be

used for comparative purposes since stability is also de- pendent on solvent supplier and lot number. The solvents

1-51
434 J. Agrlc. Food Chem.. Vol. 40. No. 3, 1992 Craft and Soares

LUTEIN DEGRADATION IN ORGANIC SOLVENTS

4 6 8 TIME (days)

^CAROTENE DEGRADATION IN ORGANIC SOLVENTS

Figure 1. Percent initial absorbanbe at of lutein and 0- carotene monitored in 18 organic solvents over a period of 10 days at ambient temperature. Solid line represents the average

rate of degradation in all solvents. Actual values for a given

solvent are listed in Table III. A specific symbol is used to

illustrate the degradation in a given solvent; however, duplicates of most symbols were necessary to account for all solvents. As

such, legends are not given for each symbol; however, solvents with less than 85% of initial absorbance at day 10 are indicated by solvent abbreviations. Abbreviations: BZ, benzene; CH, cy- clohexanone; DCM, dichloromethane; EE, ethyl ether; MTBE,

methyl tcrt-butyl ether; TL, toluene.

used may or may not be representative of current lots from a given supplier. No attempt w£is made to sample

various sources of each solvent, and no additional antioxidants were added to the solvents used. However, our

experience with 2-propanol and dichloromethane indicates that the source and lot of solvent used substfmtially

influence the stability of carotenoids in solution. While developing methods for the extraction and de- termination of carotenoids, we found that critical infor- mation was frequently missing from tabulated data and

found it necessary to fill in some gaps. The information

provided in this paper supplements published absorptivities and Xmai values for solvents for which information

is currently unavaulable. This information should also aid in the selection of solvents to be used for carotenoid

research by giving an indication of stability and solubility of two ceirotenoids, which vary greatly in polarity. Finally,

since the chromophore is not strongly influenced by the presence of hydroxyl groups outside the conjugated double

bond system, the molar absorptivity vedues for lutein can be used for estimating concentration values of other /3,e- CMotenoids such as a-tryptoxanthin and a-carotene; the

molar absorptivity values for /3,/3-carotene can be used for /3-cryptoxanthin and zeaxanthin.

ACKNOWLEDGMENT

We are grateful to Chris Nelson and Kemin Industries

for the generous gift of lutein. The work is taken in part

from a dissertation submitted to the Graduate School,

University of Maryland, by N.E.C. in partial fulfillment of the requirement for the Ph.D. degree in Nutritional

Sciences.

LITERATURE CITED

Bieri, J. G.; Brown, E. D.; Smith, J. C. Determination of individual carotenoids in human plasma by high performance liquid chro- matography. J. Liq. Chromatogr. 1985, 8, 473-484.

Britton, G. General Carotenoid Methods. Methods Enzymol.

1985, 111, 113-149. Bureau, J. L.; Bushway, R. J. HPLC determination of carotenoids

in fruits and vegetables in the United States. J. Food Sci. 1986, 51, 128-130.

Colditz, G. A.; Branch, L. G.; Lipnick, R. J.; Willett, W. C; Rosner,

B.; Posner, B. M.; Hennekens, C. H. Increased green and yellow

vegetable intake and lowered cancer deaths in an elderly

population. Am. J. Clin. Nutr. 1985, 41, 32-36.

Craft, N. E.; Sander, L. C; Pierson, H. F. Separation and Relative Distribution of all-trans-|8-Carotene and its cis-Isomers in ff- Carotene Preparations. J. Micronutr. Anal. 1991, 8, 209-221.

Cutler, R. G. Carotenoids and retinol: Their possible importance

in determining longevity of primate species. Proc. Natl. Acad.

Sci. U.S.A. 1984, 87, 7627-7631.

Davies, B. H. Carotenoids. In Chemistry and Biochemistry of Plant Pigments, 2nd ed.; Goodwin, T. W., Ed.; Academic Press: London, 1976; Vol. 2, pp 38-165. De Ritter, E.; Purcell, A. E. Carotenoid Analytical Methods. In Carotenoids as Colorants and Vitamin A Precursors; Bauernfeind, J. C, Ed.; Academic Press: London, 1981; pp 815-923. Gaziano, J. M.; Manson, J. E.; Ridker, P. M.; Buring, J. E.; Hennekens, C. H., /3-Carotene and Heart Disease. Circulation

1990, 82, 796 (Abstract). Khachik, F.; Beecher, G. R.; Whittaker, N. F. Separation,

identification, and quantification of the major carotenoid and

chlorophyll constituents in extracts of several green vegetables

by liquid chromatography. J. Agric. Food Chem. 1986, 34, 603-616. Mozsik, G.; Javor, T.; Toth, G.; Zsoldos, T.; Tigyi, A. Interre- lationships between the Gastric Cytoprotective Effects of Vitamin A and |9-Carotene and the Gastric Mucosal Superoxide Dismutase Activity in Rats. Acta Phys. Hung. 1984, 64, 315-318. Peng, Y.-M.; Xu, M.-J.; Alberts, D. S. Analysis and stability of

retinol in plasma. J. Natl. Cancer Inst. 1987, 78, 95-99.

Peto, R.; Doll, R.; Buckley, J. D.; Sporn, M. B. Can dietary 0- carotene materially reduce human cancer rates? Nature 1 98 1

,

290, 201-208. Rodriquez-Amaya, D. Critical Review of Provitamin A Deter- mination in Plant Foods. J. Micronutr. Anal. 1989, 5, 191-

225. Simpson, K. L.; Tsou, S. C. S.; Chichester, C. 0. Carotenes. In Methods of Vitamin Assay, 4th ed.; Augustin, J., Klein, B. P., Becker, D. A., Venugopal, P. B., Eds.; Wiley: New York, 1985; pp 185-220.

Received for review June 27, 1991. Accepted December 2, 1991. Certain commercial equipment, instruments, or materials are

identified in this paper to specify adequately the experimental

procedure. Such identification does not imply recommendation

of endorsement by NIST, nor does it imply that the equipment

or materials identified are necessarily the best available for the purpose. Registry No. Lutein, 127-40-2; /3-carotene, 7235-40-7; acetone,

67-64-1; acetonitrile, 75-05-8; benzene, 71-43-2; chloroform, 67-

66-3; cyclohexane, 110-82-7; cyclohexanone, 108-94-1; dichlo- romethane, 75-09-2; dimethylformamide, 68-12-2; dimethyl sul- foxide, 67-68-5; ethanol, 64-17-5; ethyl acetate, 141-78-6; ethyl

ether, 60-29-7; hexane, 110-54-3; 2-propanol, 67-63-0; methanol,

67-56-1; methykert-butyl ether, 1634-04-4; tetrahydrofuran, 109-

99-9; toluene, 108-88-3.
Journal of Chromatography, 589 (1992) 171-176

Elsevier Science Publishers B.V.. Amsterdam

CHRO>I. 23 735

Optimization of an isocratic high-performance Uquid

chromatographic separation of carotenoids

Neal E. Craft* and Stephen A. Wise

Organic Analytical Research Division. Chemical Science and Technology Laboratory. National Institute of Standards and Technologv,

Gaithersburg. MD 20899 (USA)

Joseph H. Scares, Jr.

'

Nutritional Sciences Department . University of Maryland. College Park. MD 20742 ( USA

)

(First received June 28th, 1991; revised manuscript received September 10th, 1991)

ABSTRACT

Using a polymeric C,y high-performance liquid chromatographic (HPLC) column, which demonstrated excellent separation selectivity toward carotenoid compounds in an earlier column evaluation, the effects of mobile phase modifier, modifier concentration, and

column temperature were investigated. A seven-component carotenoid mixture was used to monitor changes in separation selectivity in response to variations in HPLC conditions. Both acetonitrile and tetrahydrofuran (THF) improved the resolution of echinenone and

a-carotene; THF was selected for use as a modifier due to its solvating properties. At concentrations greater than 6% THF, the resolution of lutein and zeaxanthin deteriorated significantly. Temperature was varied from 15 to 35°C in 5°C increments. Resolution of

lutein/zeaxanthin and ^-cartone/lycopene were better at lower temperatures while echinenone/a-carotene separation improved as temperature increased. An acceptable separation of all seven carotenoids was achieved at 20°C using 5% THF as a mobile phase

modifier. Method applicability is demonstrated for serum and food carotenoids.

INTRODUCTION

During the past decade carotenoid pigments have

become highly publicized compounds due to their

potential roles in cancer prevention [1-4], as antioxidants [5], and in the reduction of atherogenesis [6]. As a result, several publications have reported highperformance liquid chromatographic (HPLC)

methods for the determination of carotenoids [7-

12]. but no publications have reported the influence

of individual HPLC parameters on the separation

of multiple carotenoids. Few laboratories have the

time or the resources to evaluate a large sample of

HPLC columns during analytical method development. Therefore, most "new" methods of separation are only slight adjustments in mobile phase

composition. Recently, Epler et al. [13] evaluated 65

HPLC columns under standardized conditions with

respect to carotenoid separation and recovery. In

this study they observed, as others have reported

[11,12] that polymeric Cis phases exhibited excellent selectivity for structurally similar carotenoids,

whereas monomeric Cig phases, which represent

the majority of the commercial Cis columns available, exhibited less selectivity for structurally similar carotenoids. Extensive investigations of the sta- tionary phase characteristics responsible for differ- ences in selectivity between monomeric and polymeric Ci8 phases for the separation of polycycHc

aromatic hydrocarbon isomers have been reported

by Sander and Wise [14]. Many of these same sta- tionary phase characteristics influence the separation of carotenoids.

This report describes the optimization of an isocratic carotenoid HPLC separation using one HPLC column that exhibited very good separation

1-53
172 N. E. CRAFT. S. A. WISE. J. H. SCARES. Jr.

of the carotenoid mixture during the column evaluation study. The influence of muUiple solvent

modifiers and temperature on the separation of a carotenoid mixture was examined.

EXPERIMENTAL

Reagents

An ethanolic solution containing between 0.2 and

1.0 //g/ml of lutein [(3i?,3'7?,6'i?)-i?,2-carotene-3,3'-

diol, Kemin Industries, Des Moines, lA. USA],

zeaxanthin[(3i?,3'/?)-^,/J-carotene-3,3'-diol],/?-cryptoxanthin [(3/?)-/?,^-caroten-3-ol, Atomergic Chemetals, Farmingdale, NY, USA], echinenone (fi^fi-c-a.- roten-4-one) (Hoffmann-La Roche, Nutley, NJ,

USA), lycopene ((//,i/'-carotene, extracted from to- mato paste), a-carotene [(6'7?)-^.£-carotene], and

j5-carotene (j?,/S-carotene, Sigma, St. Louis, MO,

USA) was prepared -to monitor column separation

selectivity. The following solvents were used in the

mobile phase mixtures: acetone, acetonitrile, chloroform, diethyl ether, ethyl acetate, hexane, methanol, methylene chloride, tetrahydrofuran (THF)

containing butylated hydroxytoluene (BHT) and

toluene. All solvents were HPLC grade or equivalent and were used without further treatment.

HPLC

The HPLC system consisted of: a dual piston,

quaternary low-pressure gradient solvent delivery

system equipped with helium sparging and column

temperature control; a high sensitivity, programmable, rapid scanning, UV-visible detector fitted with

both deuterium and tungsten lamps and an 8-jul

flow cell; and manual and automatic injection

valves. Carotenoids were monitored at 445 nm and

a computer-controlled data system was used to process data permitting review and manipulation of

peak integration. The HPLC column contained 300

A pore diameter, 5 ^um particle size, silica polymerically modified with Cig (Vydac 201 TP, 25 x 4.6

cm LD. Separations Group, Hesperia, CA, USA).

Other columns with similar chemistries could be

substituted with minor modifications in mobile

phase composition [13]. A 0.2 /im inline filter was

placed between the pump and injection valve to trap particles resulting from piston seal wear. All

stainless steel frits between the injection valve and

the detector were replaced with biocompatible ceramic frits to minimize carotenoid precipitation and

degradation [13,15]. A guard column containing

5-jum Ci8 material similar to the analytical column

was directly attached to the analytical column inlet to remove particulate material and protect the col- umn.A methanol-based mobile phase was modified

with 5 to 10% of the solvents listed under Reagents

to determine the influence of each solvent on the

separation of the carotenoid test mixture. After the

modifier yielding the best selectivity was selected

(THF), incremental portions were added to methanol to achieve the best separation of all seven carotenoids as determined by the shortest analysis time

without compromising the resolution of all carote- noid peaks.

Column temperature was varied in 5°C increments between 15 and 35°C to investigate the influ- ence of temperature on the separation of the carote- noid mixture. Temperature was regulated by the

thermostatically controlled column oven on the

HPLC system. The 15°C column temperature was

achieved by placing a beaker of dry ice at the forced

air inlet to reduce the chamber temperature to below ambient.

Carotenoid recovery

"Total recovery" was determined by flow injection analysis (FIA). To perform the FIA, the HPLC

column was replaced by a 2000 mm x 0.8 mm I.D. PTFE tubing and the individual carotenoid solutions were introduced using a manual injector fitted with a 20-/il loop and recording the peak area at 445 nm. This process was repeated five times for each

carotenoid. Then the HPLC column was replaced

and each individual carotenoid solution was injected onto the column and "peak area" was recorded.

Recovery of carotenoids from the HPLC column

was determined by dividing the mean "peak area"

by mean "total recovery" of the five injections of

each carotenoid solution.

RESULTS AND DISCUSSION :

The carotenoids included in the test mixture span

a wide polarity range and represent j5,)S; j?,£; (/',(/'; monoketo; monohydroxy; and dihydroxy carotenoids. These compounds account for approximately 90% of carotenoids in human blood and the

1-54
HPLC OF CAROTENOIDS 173

f • T » I 0 5 10 15 20 Time (min)

Fig. 1. The effect of nine solvent modifiers on the separation of

seven carotenoids. Chromatographic conditions: Vydac 201 TP

Cig, 5 /im, 250 X 4.6 mm I.D. column; mobile phase as listed, 1.0 ml/min; UV-VIS at 445 nm: column temperature 25°C. Peaks: L = lutein; Z = zeaxanthin; ^-C = /?-cryptoxanthin;

E = echinenone; a = a-carotene; = ^-carotene; Ly = lyco- pene.

three major carotenoids in the US diet [7,16]. Therefore, an HPLC separation developed using this mix

should find wide applicabiHty to both food and se- rum carotenoid analyses.

A wide pore (300 A), polymerically bonded Cig

column with "biocompatible" frits was incorporated based on results of a previous column evaluation

study in which polymerically modified Cis columns

demonstrated greater resolution of the carotenoids

in the ethanolic mixture [13]. Also for duplicate

HPLC columns with different frit materials, recov- ery was improved in columns fitted with "biocompatible" materials (titanium and Hastelloy C) rather than stainless steel [13,15]. In the same study,

total carotenoid recovery was found to be higher

for nearly all columns when using methanol-based

0 5 10 15 20

Time (min)

Fig. 2. The effect of three concentrations of THF on the sep- aration of seven carotenoids. Chromatographic conditions as in Fig. I. Peaks: cis-P = /J-carotene cis isomers; Ly isomers =

lycopene isomers; other peaks as in Fig. 1.

mobile phases rather than acetonitrile-based mobile

phases. In the case of the polymeric Ci8 columns,

the use of methanol actually resulted in improved

carotenoid resolution. For this reason, our initial optimization began with a methanol-based mobile

phase.

The influence of the mobile phase modifiers on

the separation of the seven carotenoids in the test mix is illustrated in Fig. 1. With this particular col- umn and 100% methanol as the mobile phase, echi- nenone coelutes with a-carotene. and lycopene is too far removed from ^-carotene, substantially in- creasing analysis time. The only two modifiers that

facilitated the separation of echinenone and a-carotene were acetonitrile and THF. Both solvents also

selectively reduced the retention time of lycopene.

The solubility of both the xantholphylls and carotenes is greater in THF than acetonitrile [17]. Therefore to improve carotenoid solubility, THF was se- lected as the modifier; although the resolution of the

1-55
174 N. E. CRAFT, S. A. WISE, J. H. SCARES, Jr.

lutein/zeaxanthin pair and echinenone/a-carotene

pair was slightly decreased.

Mobile phases of methods previously developed

using a similar Cis column have been modified with

chloroform. THF, and a mixture of acetonitrileTHF [1 1,12,18]. Each of these reports illustrated the

superior selectivity of the polymeric Cig column to- ward carotenoid compounds and especially geometric isomers, however, none of these reports addressed the separation of the carotenoid pairs discussed above.

The effect of varying the proportion of THF from

4 to 8% on the separation of the carotenoid mixture

is illustrated in Fig. 2. As the THF concentration

increases, resolution decreases between the pairs lu- tein/zeaxanthin and j8-carotene/lycopene. At a column temperature of 25°C, 3 to 5% THF results in

good separation of all the carotenoids in the mixture. Previously, our laboratory has reported that column temperature alters column selectivity, specifically with respect to solute shape recognition

[19,20]. Using 5% THF in methanol, column temperature was varied between 15 and 35°C to determine if the separation of structurally similar carote- noids could be improved (Fig. 3). At 15°C echine- none and lycopene are more strongly retained with

respect to ^-carotene (ae/p 0.80 vs 0.67, and aiy/p =

1.60 vs. 1.24, respectively, at 15 and 35°C). As a

result, at 15°C, echinenone and a-carotene are not

baseline resolved and lycopene elutes distant from

the other carotenoids. Beginning at 25°C and

E

c

in?

o

c

«

p-c Jiiil

35 X

JUUiJ

JUUa)

—l—

10

—1

15 20

Time (min)

Fig. 3. The effect of five column temperatures on the separation of seven carotenoids. Chromatographic conditions: Vydac

20ITP C,8 column; 5% THF in methanol; 1.0 ml/min; UV/VIS

at 445 nm; column temperature as listed. Peak identifications as

in Fig. 2.

E

c

in

(Do

c

(0 G

o

</) < U

Ly isomers

Standards

Vegetable Mix

Ly isomers Serum

ds-p

—

I—

10

I 15 —

r~ 20 —r25 Time (min)

Fig. 4. The separation of serum and mixed diet carotenoids using

the optimized HPLC method. Chromatographic conditions: Vydac 201TP C,8 column; 5% THF in methanol; 1.0 ml/min; UV/

VIS at 445 nm. Peaks: L = lutein; Z = zeaxanthin; a-C =

a-cryptoxanthin; (i-C = ^-crytoxanthin; a = a-carotene; ^ =

/5-carotene; cis-^ = ^-carotene; cis-fi = /3-carotene cis isomers; Ly isomers = lycopene isomers.

1 -56
HPLC OF CAROTENOIDS 175

above, resolution decreases between the lutein/

zeaxanthin pair and /9-carotene/lycopene pair. A

column temperature of 20°C was selected, because

all carotenoids in the mixture are well resolved from

each other, a baseline separation exists between the

trans isomer of ^-carotene and its geometric iso- mers, and there is partial resolution of the geometric isomers of lycopene. Lesellier et al. [21] recently

described the separation of trans/cis-a- and ^-carotenes and observed that temperature was a significant factor in optimizing the separation of these

isomers. Using a narrow pore (80 A) polymeric Cis

column, they identified 21-22°C as the optimum

temperature for the separation of trans-a- and ^-carotene from their cis isomers. However, using the narrow pore column they were unable to resolve the

cis isomers from each other. Previously, we have

demonstrated the separation of individual geometric isomers of ^-carotene using a wide pore polymeric Cis columns [22]. In our recent column evaluation study [13], we

found that total carotenoid recovery from the

HPLC column was influenced by the column packing, mobile phase, and frit material. The percent

recovery and standard deviation of individual caro- tenoids measured by FIA using the optimized

HPLC system described above are: lutein, 96 ±

6%; zeaxanthin, 107 ± 9%; ^-cryptoxanthin, 94 ±

5%; echinenone, 92 ± 6%; a-carotene, 95 ± 3%;

^-carotene, 96 ± 3%; and lycopene, 108 ± 6%.

Recovery of each carotenoid was greater than 92%

and total carotenoid recovery was 98 ± 6%. This

demonstrates that the system not only resolves

these major carotenoids but also elutes them quantitatively from the LC column.

The applicability of the method is demonstrated

in Fig. 4 in which the separations of serum carote- noids and dietary carotenoids are illustrated. Most

isocratic reversed-phase HPLC methods fail to re- solve lutein and zeaxanthin when the capacity fac- tor (k') for the hydrocarbon carotenoids is kept below 10 [7-9]. Previously, polymeric Cis phases have

been reported to exhibit superior ability to recognize subtle differences in molecular structure

[20,22,23]. In this application, the lutein/zeaxanthin

pair, geometric isomers of ^-carotene (trans/9-

cis/\3-cis), and geometric isomers of lycopene

(trans/\3-cis) are separated within 20 min. Although superior to monomeric Cig phases in many

ways, polymeric Ci8 columns are not without problems'. The total carbon load is lower in wide-pore

polymeric phases (due to the lower surface areas of

wide pore silicas) which results in weaker retention

of the carotenoids and limits the amount and type

of modifier used in the mobile phase. The large pore

diameter and thick surface coating slows mass

transfer resulting in slightly broader peaks. Lastly,

column reproducibility from various production

lots tends to be more variable than for monomeric

Ci8 phases [13]. Because of these difficulties, duplication of the results illustrated in this manuscript

with a different polymeric Cig column may require

slight modifications in the mobile phase composition, but similar separations should be attainable. In summary, the isocratic separation of carote- noids was optimized using a polymeric Cis column.

Stainless steel frits were replaced with "biocompatible" frits to minimize oxidative degradation of the

analytes and a methanol-based mobile phase was

used to improve selectivity and recovery. Nine sol- vent modifiers were investigated to determine their

effect on separation selectivity and THF was found

to be the most beneficial modifier. Various combinations of THF composition and column temperature were assessed to achieve the best overall separation. The recovery of individual carotenoids and

a mixture of seven carotenoids from the HPLC column was nearly 100%.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the helpful

discussions with Lane C. Sander regarding stationary phase chemistry and the assistance of Katherine

S. Epler with the initial column evaluations. The

authors are grateful to Chris Nelson of Kemin In- dustries and Hemmige Bhagavan of Hoffmann-La

Roche for their generous contributions of carotenoids.

This work is taken in part from a dissertation

submitted by N.E.C. to the Graduate School of the

University of Maryland in partial fulfillment of the

requirements for the Ph.D. degree in Nutritional

Sciences.

Certain commercial equipment, instruments, or

materials are identified in this paper to specify adequately the experimental procedure. Such identifi- cation does not imply recommendation of endorse1-57
176 N. E. CRAFT, S. A. WISE, J. H. SCARES, Jr.

ment by the National Institute of Standards and

Technology, nor does it imply that the materials or equipment identified are neccesarily the best available for the purpose.

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15 D. W. Nierenberg and D. C. Lester, J. Nutr. Growth Cancer,

3 (1986) 215. 16 N. E. Craft, S. A. Wise, and J. H. Soares. Jr., J. Agric. Food

Chem., submitted for publication. 17 N. E. Craft and J. H. Soares, Jr., J. Agric. Food Chem., in press. 18 F. W. Quackenbush and R. L. Smallidge, J. Assoc. Off. Anal. Chem., 69 (1986) 767. 19 L. C. Sander and N. E. Craft. Anal. Chem., 62 (1990) 1545. 20 L. C. Sander and S. A. Wise. Anal. Chem.. 61 (1989) 1749. 21 E. Lesellier, C. Marty, C. Berset and A. Tchapla. J. High

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1-58
Published in Analytical Chemistry, May 15,1994,pp. 1667-1674 by the American Chemical Society

Development of Engineered Stationary Phases for the

Separation of Carotenoid Isomers

Lane C. Sander,' Katherine Epier Sharpless, Neal E. CraH,^ and Stephen A. Wise

Organic Analytical Research Division, Chemical Science and Technology Laboratory, National Institute of

Standards and Technology, Gaithersburg, Maryland 20899

A variety of bonded phase parameters (endcapping, phase

chemistry, iigand length, and substrate parameters) were studied

for their effect on column retention and selectivity toward

carotenoids. Decisions were made on how each of these

variables should be optimized based on the separation of

carotenoid and polycyclic aromatic hydrocarbon test probes.

A column was designed with the following properties: high

absolute retention, enhanced shape recognition of structured

solutes, and moderate silanol activity. These qualities were

achieved by triacontyl (C30) polymeric surface modification of

a moderate pore size ( 20 nm), moderate surface area ( ~200

mVg) silica, without subsequent endcapping. The effectiveness of this "carotenoid phase" was demonstrated for the separation

of a mixture of structurally similar carotenoid standards, an

extract of a food matrix Standard Reference Material, and a

/3-carotene dietary supplement under consideration as an agent

for cancer intervention/prevention. Carotenoids are highly conjugated polyprenoids found in a variety of natural sources including fruits and vegetables.

Interest in the separation, identification, and quantification

of these compounds is largely motivated by perceived health

benefits; however, carotenoids are also widely utilized as natural pigmenting agents. Carotenoids constitute a class of molecules with diverse properties. Carotenoid hydrocarbons

(carotenes) are nonpolar, whereas oxy-substituted carotenoids

(referred to as xanthophylls) are polar. Subtle molecular

differences exist within these groups, and geometric and

positional isomers of carotenoids are common. Because of

the complexity of carotenoid extracts and the minor shape

differences among carotenoid isomers, separation of individual

species is difficult. In reversed-phase liquid chromatography (LC) systems,

the retention of nonpolar compounds is often attributed to solvophobic interactions, but other properties of the solute may provide an additional basis for separation. For example,

with polycyclic aromatic hydrocarbons (PAHs), separations

of isomers can be achieved on the basis of molecular shape.'"' For solutes with oxygen-containing substituents, dipole or

' Present address: Southern Testing & Research Labs, Inc., Wilson, NC 27893.

(1) Sander, L. C; Wise, S. A. J. Chromawgr. 1993, 656, 335-351.

(2) Sander, L. C; Wise, S. A. LC GC 1990, S, 378-390.

(3) Wise, S. A.; Sander, L. C; Upouyade, R.; Garrigues, P. J. Chromatogr. 1990,

514, 111-122.

(4) Wise, S. A.; Sander, L. C; Chang, H.; Markides, K. E.; Lee, M. L. Chromatographia 1988, 25. 473-480.

(5) Sander, L. C; Wise, S. A. Advances in Chromatography, Giddings, J. C,

Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker; New York, 1986;

Vol. 25, pp 139-218,

(6) Wise, S. A.; Sander, L. C. HRCCC. J. High Resolul. Chromatogr- Chromatogr. Commun. 1985, 8. 248-255.

(7) Cole, S. R.; Dorsey, J. G. J. Chromatogr. 1993, 635, 177-186.

(8) Sentell, K. B.; Dorsey, J. G. J. Chromatogr. 1989, 461, 193-207.

hydrogen bonding interactions may provide another basis for separation. Stationary phases can be designed with properties

engineered for the solution of a given separation problem by

understanding the cause and effect relationship between

bonded phase properties and solute interactions.

The separation of carotene and xanthophyll mixtures has been the subject of considerable research effort spanning

several decades. Most LC separations of carotenes and

xanthophylls have been developed using reversed-phase

systems,^^"^' although normal-phase separations (particularly of geometric isomers of |8-carotene) have also been

reported.^"* In a study of the retention behavior of carotenoid compounds with over 60 commercial Cig columns, better separations of nonpolar carotenoids were usually possible with columns prepared using polymeric surface modification chemistry compared with monomeric surface chemistry.^^ Variations in the retention behavior of polar carotenoids (i.e., xanthophylls) were observed among both monomeric and

polymeric Cig phases.

(9) Sentell, K. B.; Dorsey. J. G. Anal. Chem. 1989, 61, 930-934.

(10) Zechmeister, L. Chem. Rev. 1944, 34, 267-344.

(11) Bickoff. E. M. Anal. Chem. 1948, 20, 51-54.

(12) Williams, R. C; Schmit, J. A.; Henry, R. A. J. Chromatogr. Set. 1972, 10, 494-501.

(13) Weedon, B. C. L. Pure Appl. Chem. 1979, 51, 435-445.

(14) Schwartz, S. J.; von Elbe, J. H. J. Uq. Chromatogr. 1982, 5 (suppl. I), 43-73.

(15) Krinsky, N. I.; Welankiwar. S. Methods Enzymol. 1984, 105, 155-162.

(16) Bushway, R. J. J. Uq. Chromatogr. 1985, S, 1527-1547.

(17) Ruddat, M.; Will, O. H., Ill Methods Enzymol. 1985, ///, 189-200.

(18) Rfledi, P. Pure Appl. Chem. 1985, 57, 793-800.

(19) Schwartz, S. J.; Patroni-KiUam, M. J. Agric. Food Chem. 1985, 33, 1 160-

1163. (20) Taylor, R. F. Advances in Chromatography. Volume 22; Giddings, J. C,

Gnishka, E.; Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1985; pp 157-213.

(21) Chandler, L. A.; Schwartz. S. J. J. Food Sci. 1987, 52, 669-672.

(22) Quackenbush, F. W. J. Uq. Chromatogr. 1987. 10, 643-653.

(23) Craft, N.; Sander, L. C; Pierson, H. F. J. Micronutr. Anal. 1990, 8, 209-22 1

.

(24) Matus, Z.; Ohmacht, R. Chromatographia 1990, 30, 318-322.

(25) Granado, F.; Olmedilla, B.; Blanco, I.; Rojas-Hidalgo, E. J. Uq. Chromatogr.

1991, 14, 2457-2475.

(26) Tsukida, K. Methods Enzymol. 1992, 213, 291-298.

(27) Braumann, T.; Grinune, L. H. Biochim. Biophys. Acta 1981, 637, 8-17.

(28) Nelis, H. J. C. F.; De Leenheer, A. P. Anal. Chem. 1983. 55, 270-275.

(29) Will, O. H., Ill; Ruddat, M. LC Mag 1985, 2, 610-612.

(30) Lcsellier, E.; Marty, C; Berset, C; Tchapla, A. J. High Resolut. Chromatogr.

1989, 12, 447-454.

(31) Craft, N. E. Methods Enzymol. 1992, 213, 185-205.

(32) Berg, W. F.; Sandler, L. Nature 1935. 136, 914-915.

(33) Nelis, H. J. C. F.; van Steenberge, M. M. Z.; Lefevere, M. F.; De Leenheer, A. P. J. Chromatogr. 1986, 353, 295-302.

(34) Hashimoto, H.; Koyama, Y. J. Chromatogr. 1988, 448, 182-187.

(35) Koyama, Y.; Hosomi, M.; Miyata, A.; Hashimoto, H.; Reames, S. A. J. Chromatogr. 1988, 439, 417-422.

(36) Rhodes, S. H.; Netting, A. G.; MUborrow, B. V. J. Chromatogr. 1988. 442, 412-419.

(37) Epler, K. S.; Sander, L. C; Ziegler, R. G.; Wise, S. A.; Craft, N. E. J. Chromatogr. 1992, 595, 89-101.

Analytical Chemistry, Vol. 66, No. 10, May 15. 1994 1087 1-59
In this study the effect of various bonded phase parameters on retention is examined for polar and nonpolar carotenoids. Through the control of appropriate bonded phase variables, the development of a stationary phase tailored for the separation of carotenoids is described. The utility of this column is demonstrated for the separation of standards as

well as complex food extracts and dietary supplements.

EXPERIMENTAL SECTION

Materials and Reagents. Carotene and xanthophyll stan- dards were obtained from a variety of sources, trans-a- and

fra/ts-;8-carotene, and /ra/w-lycopene were obtained from

Sigma Chemical Co. (St. Louis, MO), echinenone, zeaxanthin,

astaxanthin, 5-carotene, capsanthin, canthaxanthin, and 9-cis-, 13-c/j-, and 1 5-a'5-/3-carotene were obtained from HoffmanLaRoche (Nutley, NJ, and Basel, Switzerland), /3-crypto- xanthin was obtained from Atomergic Chemetals (Farmingdale, NY), and lutein was obtained from Kemin Industries (Des Moines, lA). A /S-carotene preparation containing a high percentage of cis isomers was obtained from Betatene

Ltd. (Melbourne, Australia). Octadecyltrichlorosilane, dimethyloctadecylchlorosilane, and triacontyltrichlorosilane were

obtained from Huls America, Inc. (Piscataway, NJ). Four

microparticulate silicas were used in the preparation of bonded

phases: YMC SIL200 5-^m silica (YMC Inc., Wilmington,

NC), IMPAQ 200 5-Mm silica (PQ Corporation, Conshohocken, PA), Kromasil 200 5-jum silica (Eka Nobel, Bohus,

Sweden), and Vydac TP 5-Mm silica (Separations Group,

Hesperia, CA). For comparisons of separations made with commercial columns, a Zorbax (monomeric Cig) column (Mac

Mod Analytical, Inc., Chadds Ford, PA) and a Hypersil Green PAH (polymeric Cis) column (Shandon HPLC, Cheshire,

UK) were employed. HPLC-grade solvents were used in chromatographic separations. Candidate Standard Reference

Material (SRM) 2383, Vitamins and Carotenoids in Food,

and SRM 869, Column Selectivity Test Mixture for Liquid

Chromatography, were obtained from the NIST Standard

Reference Materials Program (NIST, Gaithersburg, MD).

Bonded Phase Synthesis. Monomeric and polymeric Cu

phases were prepared using a previously published procedure.^*'^' Polymeric C30 phases were prepared in a similar

fashion, as follows. Approximately 10 g of triacontyltrichlo- rosilane was added to 100 mL of xylene and heated to boiling. After dissolution, the mixture was filtered while hot to remove

solid impurities. In a separate container ~4 g of 20-nm pore

size, 5-Mm particle size silica was dispersed in 25 mL of xylene. This silica slurry was added to the hot silane reaction mixture. One milliliter of water was added to initiate the polymerization

reaction. The slurry was again heated and refluxed for 3-6

h. The bonded silica was filtered (hot) using a sintered glass

frit funnel and washed with xylene, acetone, methanol, water,

acetone, and pentane. Samples for carbon analysis were dried

at ~100 "C under reduced pressure for 4 h, and shipped to Galbraith Laboratories (Knoxville, TN).

Chromatography. Separations of carotenoid standards and

extracts were carried out using a linear mobile phase gradient

(38) Sander, L. C; Wise. S. A. Anal. Chem. 1984, 56, 504-510.

(39) Sander, L. C; Wise, S. A. Polynuclear Aromatic Hydrocarbons: Eighth

International Symposium on Mechanism. Method and Metabolism, Cooke, M. W., Dennis, A. J., Eds.; Battelle Press: Columbus, OH, 1983; pp 1133-

1144.

from 81:15:4 to 6:90:4 methanol/methyl-/err-butyl ether (MTBE)/water, over 90 min, at a flow rate of 1 mL/min.

Columns were thermostated at 20 °C with a water jacket. Detection for all separations was at 450 nm. Isocratic separations of the high dj-/3-carotene mixture were carried out at 3 "C using an 80:20 acetonitrile/MTBE mobile phase

(90:10 acetonitrile/MTBE for commercial monomeric and

polymeric Ci8 phases).

Extraction of SRM 2383. SRM 2383 Vitamins and

Carotenoids in Food was extracted and saponified in preparation for carotenoid analysis using the modified procedure of

Scott.'"' Approximately 2.0 g of the food mixture was

combined with 0.2 g of calcium carbonate, 5 mL of water, and

100 mL of 50/50 THF/methanol, and the mixture was

homogenized for 1 min. This mixture was vacuum-filtered,

and the filter cake was rinsed with 50-mL portions of 50/50

THF/methanol until the orange color was removed from the

solids. The filtrate was transferred to a separatory funnel, 50 mL of a 10% (w/v) sodium chloride solution was added, and

the analytes were extracted 3 times with 50-mL portions of

diethyl ether. The ether was evaporated to a volume of approximately 50 mL. This solution was shaken twice with petroleum ether to reextract the analytes, and the petroleum

ether phase was evaporated to approximately 500 mL. Ethanol was added to the solution to yield a total volume of 5 mL.

These extracts were further processed to convert xanthophyll esters to free xanthophylls using the following saponification procedure: A 3-mL aliquot of the food extract was

combined with 0.1 mL of 20% (w/v) pyrogallol (1,2,3- benzenetriol) in methanol, 1 .5 mL of 40% potassium hydroxide

in methanol, and 1.5 mL of methanol. This mixture was

allowed to sit at room temperature for 30 min and was then

extracted with three 1 0-mL portions of 50/50 petroleum ether/

diethyl ether. The combined extracts were evaporated under

a stream of nitrogen to a viscous solution. Water and

petroleum ether were added to extract the analytes, and the petroleum ether phase was removed and evaporated. The

resultant waxy substance was redissolved in a 50/50 mixture

of ethyl acetate/ethanol to total 3 mL.

RESULTS AND DISCUSSION

To design a stationary phase with properties tailored for the separation of carotenoid compounds, the effect of various sorbent parameters on retention was examined using repre- sentative carotenoid probes and other test mixtures. The

influence that bonded phase chemistry exhibits on PAHs has been studied in considerable detail.*^-^*-^'"^^ In general,

columns prepared using polymeric surface modification chemistry exhibit enhanced shape selectivity toward isomers and other groups of compounds with similar molecular

structure. This is in comparison to columns prepared using

monomeric surface modification chemistry, which generally

exhibit greater absolute retention, but reduced shape recognition. A trend toward enhanced shape recognition has also been observed for increasing phase length, for both monomeric

and polymeric alkyl bonded phases.** Although silanol activity

(40) Scott K.J. InstituteofFood Research, Norwich, UK. Personal communicatioD,

1993.

(41) Sander, L. C; Parris. R. M.; Wise, S. A.; Garrigues, P. Anal. Chem. 1991,

63, 2589-2597.

(42) Sander, L. C; Wise, S. A. Anal. Chem. 1989, 61, 1749-1754.

(43) Sander, L. C; Wise. S. A. /. Chromatogr. 1984, 316, 163-181.

1668 Analytical Chemistry, Vol. 66. No. 10, May 15, 1994 1-60
Carotenoids

Xanthophylls

Astaxanthin

Capsanthin

Canthaxanthin

Echinenone

Lutein

Zeaxanthin

ji-Cryptoxanthln

Hydrocarbons

FIgur* 1. Structures of polar and nonpolar carotenokds.

a-Carotene

|3-Carotene

5-Carotene

Lycopene

15-cis Carotene

13-cis Carotene

9-cis ^-Carotene

has been shown to have no effect on shape selectivity for nonpolar compounds such as PAHs,' certain carotenoid compounds have oxygen-containing substitutents and exhibit considerable polarity. For this reason, silanol activity has the

potential for influencing carotenoid separations. Finally, substrate properties such as pore diameter and surface area

have been shown to affect the mechanism ofsynthesis ofvarious

alkyl phases and, consequently, the overall retention behavior

of the resulting sorbents.*^ The structures of some of the more important common

carotenoids are shown in Figure 1. Differences in the

structures of these compounds are often minor. For example,

a- and /3-carotene differ in the position of a double bond in one of the terminal rings. Lutein and zeaxanthin are similarly

related and can be thought of as dihydroxy analogs of a- and

j3-carotene. Cis isomers of /3-carotene differ in the location of cis double bonds along the conjugated chain. 5-Carotene and lycopene have one and/or both end rings opened,

respectively. Xanthophylls differ in the extent and position of substitution for hydroxylation or ketonation. It is believed

that geometric isomers exist for many of these compounds

(e.g., cis isomers of a-carotene), and only a small fraction of

the known carotenoids are illustrated in Figure 1. The primary goal of this work was to develop a column

that exhibited significantly enhanced selectivity toward both

polar and nonpolar carotenoids compared to existing commercial columns. In addition, a column with high absolute

(44) Sander, L. C; Wise, S. A. Anal. Chtm. 1987, 59, 2309-2313.

retention was desired to reduce the amount of water required

in the mobile phase and to provide adequate retention for polar carotenoids. The use of mobile phases containing little or no water benefit preparative carotenoid separations since solvent elimination is greatly facilitated by the absence of

water. The low viscosity of such mobile phases also permits

the utilization of longer columns and smaller particle diameter

substrates. A column can be produced with properties

optimized for specific carotenoid separations by understanding

how each parameter affects carotenoid retention. Stationary Phase Chemistry. Three octadecyl phases were

prepared by different approaches. A monomeric Cig phase was prepared on IMPAQ silica using an excess of dimethyloctadecylchlorosilane in the presence of a basic catalyst (2 mL of A^,A^-diethyl-w-toluamide). Another Cig phase was

prepared by reaction of octadecyltrichlorosilane with the silica, under anhydrous conditions. A third Cig phase was synthesized also using octadecyltrichlorosilane, but with the addition of 1 .0 mL of water to initiate polymerization. Carbon loading

and surface coverage values for the stationary phases are listed in Table 1

.

Separations utilizing these columns are shown in Figure

2. The chromatograms at the left of the figure are for SRM

869, Column Selectivity Test Mixture for Liquid Chromatography. The use of this material to characterize stationary

phase type (monomeric vs. polymeric) and shape recognition

properties has been detailed in previous publications.^'^*-''^'' The elution order of the three PAH probes (benzo[a]pyrene

(BaP), phenanthro[3,4-c]phenanthrene (PhPh), and 1,2:3,4:

Analytical Chemistry. Vol. 66, No. 10, May 15, 1994 1869 1-61
Tabto 1. Bonded Phase Properties

stationfiiry phase type silica surf&C6 area** (m'^/g) p6rc6nt Ciirbon coverage (>zm monomeric monofunctioneil Cis IMPAQ 200 240 12.96 3.07 monomeric trifunctional Cig IMPAQ 200 240 12.79 3.03 (anhydrous synthesis) polymeric Cig IMPAQ 200 240 20.01 5.44 polymeric Cts (not endcapped) Vydac TP 7.99 -5.6

polymeric Cis (TMCS endcapped) Vydac TP 7.84 polymeric Cig (HMDS endcapped) Vydac TP ~75* 8.33 polymeric C30 IMPAQ 200 240 23.34 3.93 polymeric C30 YMC SIL200 200* 19.41 3.64 polymeric C30 Kromasil 200 196 14.46 2.54

" Measured values. ' Nominal values reported by the manufacturer.

Bap + PhPh

a^carotene

p-carotene

lycopeiM

0 5 10 IS 20 0 4 8 12 16 20

B ll

a-carotene

p.caroten« iycop«ne

0 5 10 IS 20 0 4 8 12 16 20

aT»»M> = 0.S9 p-carotsiw

0 S 10 15 20 25 30 0 4 8 12 16 20 Figure 2. Ck>lumn selectivity comparison for various C^e phases

prepared using monomeric and polymeric syntheses: (A) monomeric,

monofunctional synthesis: (B) monomeric, trifunctional synthesis

(anhydrous); (C) polymeric synthesis. Separation conditions were as

follows: left (SRM 869). 2 mL/min, 85:15 acetonitrile/water; right (carotenes), 1.5 mL/min, 80:20 methanol/ethyl acetate.

5,6:7,8-tetrabenzonaphthalene (TBN)) is indicative ofcolumn

type. Furthermore, the selectivity factor areN/BaP provides a measure of shape selectivity for structurally similar compounds; low values of axBN/BaP indicate a high degree of shape recognition. The chromatograms in Figure 2 for SRM

869 are typical of those observed for monomeric and polymeric

Ci8 phases. The monomeric Cis phase prepared using the

(45) Sander, L. C; Wise, S. A. SRM 869 Certificateof Analysis, Standard Reference

Materials Program, NIST, Gaithersburg, MD, 1990.

(46) Sander, L. C; Wise, S. A. HRCCC. J. High Resolul. Chromatogr. Chromalogr. Commun. 1988, //, 383-387.

(47) Sander, L. C. J. Chromatogr. Sci. 1988. 26, 380-387.

anhydrous trifunctional synthesis (Figure 2B) exhibits an

elution order PhPh < BaP < TBN (1 < atBN/BaP < 1.7) and

this may indicate slight polymeric character, although it is interesting to note that the carbon loading for this stationary

phase is slightly less than for the monofunctional monomeric

phase.

Of more direct interest is the separation of three repre- sentative nonpolar carotenoids (namely lycopene and a- and

/8-carotene). These carotenes were selected as probes based on their importance in clinical measurements, and their widespread occurrence in fruits and vegetables. The separation of these common carotenes is a prerequisite for any column

designed for carotenoid measurements. With the monofunctional monomeric Ci g phase ( Figure 2A), lycopene was resolved from a- and i8-carotene, but a- and /3-carotene were unresolved. Poorer resolution was observed using the trifunctional monomeric Cig phase (Figure 2B). Nearly baseline separation of

the three carotenoids was achieved with the polymeric Cig

phase (Figure 2C). Thus, at least for the probe compounds

(nonpolar carotenes), better separations were possible using

the polymeric surface modification approach. Several methods

for the analysis of carotenoids have been reported in which

polymeric Cig columns are utilized. The distinction between

monomeric and polymeric Cig phases is not commonly

expressed. Instead, the methods are described with a specific

column brand rather than by bonded phase chemistry or retention behavior. ' A similar situation has existed for the separation of PAHs; however, the realization of the importance

of stationary phase chemistry toward selectivity is becoming more widespread. For example, Lesellier and co-workers

recently studied carotenoid retention mechanisms on commercial monomeric and polymeric Cig columns.^' Endcapping. The actual effect that silanol groups contribute toward retention and selectivity remains controversial even though silanol activity has been the subject of numerous

studies.^**"-''* It is clear, however, that the extent and

magnitude of any silanol effects are dependent on the nature

of the solute molecules under study. Silanols can be expected

to influence the retention behavior of polar solutes to a greater extent than nonpolar hydrocarbons. Traditionally, silanol

(48) Quackenbush, F. W.; Smallidge. R. L. / Assoc. Off. Anal. Chem. 1986, 69, 767-771.

(49) Lesellier, E.; Tchapla, A.; Krstulovic, A. M. Chromatogr. 1993. 645. 29-39.

(50) Nabum, A.; Horvath, C. /. Chromatogr. 1981. 203. 53-63.

(51) Miller, M. L.; Linton, R. W.; Maciel. G. E.; Hawkins, B. L. /. Chromatogr.

1985, 319, 9-21.

(52) Jennings, E. C; Brownlee. R. G. Anal. Chem. 1986. 58. 2895.

(53) Mant, C. T.; Hodges, R. S. Chromatographia 1987, 24, 805-814.

(54) Nawrocki, J. Chromatographia 1988, 25. 404-408.

1870 Analytical Chemistry. Vol. 66. No. 10. May 15. 1994 L-62

-

1
activity is usually considered undesirable, and secondary

silanization reactions using methylsilanes (endcapping) are

carried out to reduce this activity. In previous work, we

investigated the influence of silanol activity and endcapping on retention behavior for a series of polycyclic aromatic

hydrocarbon isomers.''^* A single batch of a polymeric Cis phase was divided into three portions, of which two where

endcapped with trimethylchlorosilane (TMCS) and hexamethyldisilazane (HMDS). The third portion was not end- capped. No difference was apparent for PAH separations

carried out on the three columns, yet a fourth Cig phase

prepared by monomeric surface modification on the same

silica substrate exhibited large differences in retention and

selectivity. In general, column selectivity toward nonpolar

solutes such as PAHs is not affected by silanol activity, but

is strongly affected by column type (i.e., monomeric vs polymeric syntheses).' Because carotenoids span a wide range of polarity and

may potentially have interactions with silanols, the effect of endcapping on retention behavior was studied for polar and

nonpolar carotenoid compounds. Endcapped and nonend- capped phases were prepared (as described above) using a

single batch of a polymeric Cig phase prepared on Vydac TP

silica. Properties of the stationary phases are listed in Table

1. A slight decrease in carbon loading was measured after endcapping with TMCS, whereas endcapping with HMDS

resulted in an increase in carbon loading. This phenomenon

(for endcapping processes utilizing TMCS) was first reported

by Berendsen et al.^' It is thought that HCl formed as a byproduct of endcapping acts to catalyze cleavage of a fraction of the C|8 ligands, resulting in a net loss of carbon. To examine the influence of silanol activity on carotenoid

selectivity, two representative mixtures were used: a- and

/3-carotene and lycopene (nonpolar probes) and lutein and

zeaxanthin (polar probes). Separations of these compounds

are shown in Figure 3 for the endcapped and nonendcapped

stationary phases. Also shown are separations of SRM 869. No appreciable difference exists in the retention behavior of

the three columns toward the PAH probes (SRM 869). This

is to be expected, since SRM 869 indicates differences in shape selectivity based on bonded phase chemistry unrelated

to polarity. Separations of the three nonpolar carotenoids are

also affected little by endcapping for similar reasons. In

contrast, the separation of polar carotenoids lutein and

zeaxanthin is influenced by silanol activity, as indicated in Figure 3. Better separation was observed with the unend- capped stationary phase (Figure 3A) compared to either of

the endcapped phases ( Figure 3 B,C) . Because the separation

of lutein and zeaxanthin is of particular importance in the food science and clinical disciplines, it seems reasonable to use a synthesis approach that facilitates this separation. The

separation of nonpolar carotenoids, as indicated by the three nonpolar probes, is unaffected by the presence or absence of endcapping; therefore, the best synthesis approach would be

to not endcap. Lack of endcapping further eliminates bonded

phase stability problems that have recently been attributed to endcapped phases.^* These conclusions are similar to those

of Matus and Ohmacht, who first reported a detailed

(55) Berendjen, G. E.; Pikaart, K. A.; de Galan, L. J. Uq. Chromalogr. 1980, J, 1437-1464.

lycoperw

0 2 4 6 8 10 0 4 8 12 16 20

J

aTBwsu. = 0.40

BaP 1_J__

a-caroten«

0 2 4 6 8 10 0 4 8 12 16 20

[}-caro1ene

lycopene

0 2 4 6 0 4 8 12 16 20 Figure 3. Effect of endcapping for polymeric Ci8 phases for polar and

nonpolar solutes: (A) polymeric Cis phase, not endcapped; (B) polymeric Ci8 phase, endcapped with trimethylchlorosilane; (C) polymeric Cig

phase, endcapped with hexamethyldlsllazane. Separation conditions were as follows: left (SRM 869) see Figure 2; center (xanthophylls)

1 .5 mL/min methanol; right (carotenoids) 1 .5 mL/mIn, 90: 1 0 methanol/

ethyl acetate.

investigation of phase selectivity for carotenoids indicating

better separations of lutein and zeaxanthin were possible with unendcapped polymeric Cig phases.^^

Stationary Phase Length. In previous studies it has been shown that shape selectivity toward PAHs is closely related

to alkyl phase length.**^ Better separations of isomer sets are

usually achieved with long chain-length phases. This trend

is observed for both monomeric and polymeric alkyl phases. For example, better shape discrimination of PAH isomers is possible with a monomeric C22 phase than with a monomeric

Cg phase. However, for any given alkyl length, polymeric

phases still provide enhanced selectivity for structurally similar compounds.

It is informative to consider the size of carotenoid molecules

for comparison with typical bonded phase dimensions. Thicknesses for various monomeric and polymeric alkyl phases

have been measured using small angle neutron scattering

(SANS),^'-** and a summary of these results is provided in Table 2. These measurements were carried out in a reversed- phase environment (methanol and deuterated methanol),

giving values that have chromatographic relevance. Approximate dimensions of ^S-carotene were determined by

molecular modeling as shown in Figure 4. Structures for

all-trans conformations of octadecane and triacontane are

also shown for comparison, although spectral characterization

(56) Kirkland, J. J.; Dilks, C. H.; Henderson, J. E. 17th International Symposium

on Column Liquid Chromatography, May 9-14, 1993, Hamburg, Germany

1993, Abstract.

(57) Sander, L. C; Glinka, C. J.; Wise, S. A. Anal. Chem. 1990, 62, 1099-1 101. (58) Glinka, C. J.; Sander, L. C; Wise, S. A.; Berk, N. F. Mater. Res. Soc. Symp.

Proc. 1990, 166. 415-420.

1-63

Analytical Chemistry. Vol. 66, No. 10, May 15. 1994 1671
Table 2. Physlcat Properties of Alkyl-Modlfled Silica (from

reference 57)

stationary phase type thickness, nm

Cg monomeric

Ci8 monomeric

Ci8 polymeric

C30 monomeric

1.0 ± 0.2

1.7 ± 0.3

2.1 ± 0.3

2.5 ± 0.4

Table 3. Physical Properties of Intermediate Pore Size Silicas particle pore diameter surface area

silica shape (nm) (mVg)

Kromasil 200 IMPAQ 200 YMC SIL200

spherical

irregular spherical

16.8" 20O

20*

196" 240"

200*

" Measured values. * Nominal values reported by manufacturer.

C18

mono poly mono

C,8 C^g

1 " " 1 " ' M

" "

1 uu, , , ,

1

, , , ,

1 , , , ,

1 0 5 10 IS 20 25 30 35 40 LENGTH, A

Figure 4. Space filling models for ;S-carotene and all-trans hydrocarbon

chains. Dotted lines indicate stationary phase thickness as determined

by snnall angle neutron scattering.

studies indicate that bends and kinks in bonded alkanes reduce

average phase thickness to less than if fully extended.^^"^' Actual phase thicknesses as determined by SANS are indicated by dashed lines. It is clear that carotenoids are significantly

larger than either monomeric or polymeric Cis phases and

closer in dimensions to the monomeric C30 phase. The

thickness of polymeric C30 phases was not determined in the previous SANS investigation (polymeric C30 phases had not

yet been developed), but based on the measurements for monomeric and polymeric C|g phases, it is likely that a polymeric C30 phase would be slightly thicker than a monomeric C30 phase. In light of the relative dimensions of

carotenoids and various alkyl phases, the potential of polymeric C30 phases was indicated for further study. Substrate Properties. Physical and chemical properties of

silica affect the chromatographic properties of the resulting

bonded phase in many ways. Of course, purely mechanical

aspects of the substrate such as particle size and porosity affect

efficiency, backpressure, and mechanical strength, but sta- tionary phase selectivity is also affected by such parameters

as silanol activity (see discussion above) and trace metal

content.*^' To provide adequate retention of polar carotenoids in a reversed-phase system, the column to be developed

should exhibit high absolute retention. Such a column would

(59) Sander, L. C; Callis, J. B.. Field, L. R. Anal. Chem. 1983, 55. 1068-1075.

(60) Verzele, M.; Dewaeie, C. J. Chromatogr. 1981, 217, 399-404.

(61) van den Driest, P. J.; Ritchie, H. J.; Rose, S. LC-GC 1988, 6. 124-132.

(62) Schuddinck, G.; Verzele, M. J. Chromatogr. 1987, ¥07, 159-169.

(63) Nawrocki, J.; Moir, D. L.; Szczepaniak, W. Chromaiographia 1989, 28, 143-

147. (64) Koyama, J.; Nomura, J.; Ohtsu, Y.; Nakata, O.; Takahashi, M. Chem. Lett. 1990, 4, 687-690.

(65) Liew, K. Y.; Tan, S. H.; Morsingh. F.; Khoo, L. E. J. Am. Oil Chem. Soc.

1982, 59, 480-484.

also be advantageous for preparative separations since non- aqueous solvents would facilitate carotenoid reclamation. Yet

another consideration is substrate pore diameter. It has been shown that the enhanced shape recognition exhibited with polymeric Cig phases is compromised when narrow-pore

substrates less than about 15 nm are employed in the

synthesis."*^ This trend is attributed to a size exclusion effect limiting the polymeric phase synthesis, rather than a chro- matographic size exclusion effect. Each of these trends constrains the selection of a substrate with properties appropriate for the development of a "caro- tenoid phase". High absolute retention results from stationary

phases prepared with high surface area substrates, but such

substrates typically have narrow pores. Conversely, wide- pore silicas with pore diameters 30 nm or greater typically have low specific surface areas (<~ 100 m^/g). Three silica substrates were identified with properties intermediate to these extremes (see Table 3). Each of these materials has nominal

pore diameters of 20 nm, with moderately large specific surface areas (200-240 m^/g). This combination of properties was

anticipated to yield a chromatographic sorbent with high

absolute retention and high shape recognition.

Stationary Phase Synthesis and Characterization. Polymeric C30 phases were prepared on each of the 20-nm pore

size substrates using the procedure detailed in the Experimental

Section. A summary of physical characteristics of these materials is listed in Table 1. Bonded phase loadings are higher than most monomeric Cu phases but are typical of polymeric phases.

Columns were evaluated using the carotenoid mixture

described previously. Many of these compounds are isomers, with structures that differ only in the location of a double bond or the position of cis-substitution. The extent of

carotenoid separations varies dramatically among commercial

Cjg columns. As with PAH separations, column selectivity can be categorized broadly into monomeric and polymeric

C18 groups, but differences in silanol activity cause further

variations in selectivity toward xanthophylis in each of these groups. Separations of the carotenoid standards on repre- sentative commercial monomeric and polymeric Cig columns

as well as the jwlymeric C30 column are illustrated in Figure

5. Separation conditions are the same for each column;

however, a weaker mobile phase gradient was also employed

for monomeric and pxjlymeric Cig phases in an attempt to increase retention and improve separation for these columns. The longer retention times did not result in improved separation

and the chromatograms are not shown. Most of the xanthophylis were separated on both the monomeric and polymeric C18 phases (Figure 5A,B); however, lutein and zeaxanthin were not resolved on the monomeric Cig phase. Differences were most apparent for the nonpolar carotenoids, which eluted

1872 Analytical Chemistry, Vol. 66, No. 10, May 15, 1994 1-64
echlnenone

3-cryptoxanthln

canthaxanthin

lutein * zeaxanthin

aataxanthin

capxanthin,

nonpolar carotenoids

—

r10 20 -r30 —

r40 —

r50 60 —I—

70 —

T

80

Carotene and Isomers

B

a-carotene echlnenone.

3-cryptoxanthln canthaxanthin

zeaxanthin

lutein astaxanthin capsanthin

—

r10

5-carotene

/ lycopene

—

r20 —r30 40 —

I—

50 60 —

I—

70 —

T

80

canthaxanthin

zeaxanthin

15-cls ^<arotene 13-cl8 p-carotene

echlnenone

a-carotene / ^ trans 0-carotene 9<l8 ^-carotene

S-carotene lycopene

_J\_

(

' ' ' '

I '

I—'—I—'—'

' '

I

' ' '

I

' ' ' I' ' '

I

' '

'—• I I 0 10 20 30 40 50 60 70 80

Figure S. Separations of carotenoid startdards on commercial (A) monomeric and (B) potymeric Cia columns, as well as the engineered C30

"carotenoid coiumn ". Separation conditions were as follows: 81:15:4 to 6:90:4 methanoi/methyt-ferf-butyl ether (MTBE)/water over 90 min; 1 mi^mln; 20 'C.

as a group with the monomeric phase and were partially

separated with the pwlymeric Cig phase. A better separation

of both polar and nonpolar carotenoid standards was achieved

using the prolymeric C30 column (Figure 5C). Cis-isomers of

/3-carotene were largely resolved from each other and from

other carotenes. Better separations of the xanthophylls were

also observed, including a few unidentified carotenoid impurities.The separations illustrated in Figure 5 are not fully optimized; however, some effort was made to resolve the carotenoid standards with the polymeric C30 column. Separations were carried out at 20 "C, and selectivity was observed

to change significantly with column temperature. The

separation of the xanthophylls was observed to be influenced by the amount of water in the mobile phase whereas nonpolar

carotenoids were relatively insensitive to water content. Good

separation of most of the carotenoid standards was possible

using a nonaqueous mobile phase gradient (methanol and

methyl-rm-butyl ether); however, /3-cryptoxanthin and

echlnenone were not resolved in this environment. The choice of a linear gradient for the separations in Figure 5 was selected

to facilitate column comparison and to give a simple indication

1-65

Analytical Chemlsty, Vol. 66. No. 10, May 15. 1994 1673
0 10 20 30 40 50 60 70 80 Figure 6. Separation of carotenoids from an extract of SRM 2383,

Vitamins and Carotenoids in Food. For separation conditions, see Figure

5.

of overall retention behavior toward carotenoids. In practical

applications separation conditions could probably be modified

to reduce analysis times. Separation of an extract of a food matrix. Standard

Reference Material (candidate SRM 2383, Vitamins and

Carotenoids in Food) is illustrated in Figure 6. Components

were tentatively identified on the basis of retention time. Several peaks did not correspond to any of the carotene or xanthophyll standards, though it is probable that these components are isomers or oxidation products of carotenoid compounds.

To illustrate the potential of the engineered C30 carotenoid column for the separation of carotene isomers, a solution containing various ;8-carotene isomers was chromatographed.

The sample obtained from Betatene Ltd. is prepared from

algae (Dunaliella) and has high levels of cis isomers of

|3-carotene. A separation of this material using the "carotenoid column" is illustrated in Figure 7C. For comparison, separations of the /3-carotene preparation are also shown using conventional commercial monomeric and polymeric Cig

columns (parts A and B of Figure 7, respectively). Mobile

phase conditions were altered in an attempt to improve

separations with the monomeric and polymeric phases by

increasing retention. Better separations were possible with

the "carotenoid column" than with either of the C18 columns. Even so, complete separation of all of the isomers of /3-carotene (and other nonpolar carotenoids) was not achieved.

CONCLUSIONS

An understanding of the cause and effect relationship

between chromatographic variables and solute retention

creates the potential for the development of "designer phases". For carotenoids and related compounds, molecular shape,

polarity, and silanol interactions are possible parameters that can be utilized in the development of such a tailored stationary

phase. Three properties have been identified as desirable for a column intended for carotenoid separations: enhanced shape

recognition of isomers, high absolute retention, and silanol

activity. The preparation of a column with these qualities

requires the choice of an appropriate silica substrate as well

10 20 30 40 50 60 70 BO

10 20 30 40 50 60 70 80

9-ci8 |3-carotene

,

trans Carotene,

trans a-carotene.

13-cis Carotene

IS^Is p-carotene

0 10 20 30 40 50 60 70 80 Figure 7. Separation of a |3-carotene preparation containing a hi^

percentage of cis-isomers on commercial (A) mononr>eric and (B) polymeric ds columns, and (C) "carotenoid column". Separation

conditions were as follows: (A and B) 3 °C, 2 mL7min, 90: 1 0 acetonttrfle/ MTBE; (C) 3 "C. 2 mL/min. 80:20 acetonitrile/t^TBE.

as bonding chemistry. The use of C30 polymeric surface modification chemistry without endcapping in conjunction with a moderate pore-size, moderate surface area silica results

in a bonded phase with properties well suited to the separation of carotenoids. ACKNOWLEDGMENT

The authors thank Robert Cooley ( YMC, Inc., Wilmington,

NC) for gifts of YMC SIL200 silica, Neil Miller (PQ

Corporation, Conshohocken, PA) for gifts of IMPAQ 200

silica. Per Moller (Eka Nobel, Bohus, Sweden) for a gift of Kromasil 200 silica, and Hemmi Bhagavan and Willy Schuep

(Hoffman-LaRoche, Nutley, NJ, and Basel, Switzerland) for providing carotenoid standards. Certain commercial equipment, instruments, or materials are identified in this report

to specify adequately the experimental procedure. Such

identification does not imply recommendation or endorsement

by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are

necessarily the best available for the purpose.

Received for review October 5, 1993. Accepted March 9, 1994. •

• Abstract published in Advance ACS Abstracts. April 15, 1994.

1674 Analytical Chemistry, Vol. 66, No. 10, May 15, 1994 1-66
DETERMINATION OF GLYCYRRHETINIC ACID

This section describes an ion-pairing solid-phase extraction-HPLC protocol for measuring

glycyrrhetinic acid (GRA) in human plasma. This method allows reliable measurements of

GRA in human plasma with a detection limit of 10 ng/mL and a limit of quantification greater

than or equal to 100 ng/mL. The following factors are essential to this procedure.

Important Factors

Calibration and Sample Preparation

• Solutions of glycyrrhetinic acid (GRA) in polar solvents such as methanol, acetonitrile,

and ethanol were found to be stable at room temperature in the dark for up to 3 weeks.

However, we recommend that working standards of GRA be kept for no longer than 1 week at room temperature. We foimd plasma-based calibrants stored at -70°C to be

stable up to 4 months.

• Because of the polar nature of GRA and its affinity for plasma constituents, liquid-liquid

extraction is not effective in removing GRA from the plasma matrix.

• Denaturation of the proteins, followed by ion-pairing, is crucial to obtain GRA

recoveries of >90%. We use urea to denature the proteins. Both acid and base

hydrolyses were tried. GRA appeared to be destroyed by the acid at the required

temperature of 120 °C, while -10% of the GRA was recovered after treatment with base.

• To prevent intrusion of air bubbles into the cartridge, pass the plasma through the

cartridge at a flow rate of 1-2 mL/min.

• Before eluting the GRA from the cartridge with methanol during the ion-pairing solidphase extraction (SPE) process, it is essential to wash the cartridge with 45% methanol

in water to remove unwanted constituents from the plasma matrix. Higher methanol

concentrations (e.g., 50%) will cause significant loss of GRA.

24
Chromatography

In our laboratory we use a polymeric Cig column (0.46 x 25 cm). Monomeric Cjg

columns may also be suitable with adjustments in mobile phase composition.

Isocratic conditions are used for this study. The mobile phase is methanol-water-acetic

acid (75:24.7:0.3, v/v) with UV detection at 248 nm.

For further information, please contact Jeanice Brown Thomas at (301)975-3120.

2-2
Journal of Chromatography, 568 (1991) 232-238

Biomedical Applications

Elsevier Science Publishers B.V., Amsterdam

CHROMBIO. 5906

Short Communication

Determination of glycyrrhetinic acid in liuman plasma by

high-performance liquid chromatography

JEANICE M. BROWN-THOMAS*, RICHARD G. CHRISTENSEN, ROLAND RIEGER",

WINFRED MALONE and WILLIE E. MAY

Organic Analytical Research Division. Center for Analytical Chemistry, National Institute of Standards and

Technology, Gaithersburg, MD 20899 (USA)

(First received September 17th, 1990; revised manuscript received March 19th, 1991)

ABSTRACT

A high-performance liquid chromatographic (HPLC) method has been developed for measuring 18^-

glycyrrhetinic acid (GRA) in human plasma in the range of 0.1-3 ;ig/ml. The acetate ester of GRA is added

to the plasma as an internal standard, plasma proteins are denatured with urea to release GRA, and the GRA and the internal standard are extracted in an ion-pairing solid-phase extraction process. An isocratic,

reversed-phase HPLC separation is used, followed by ultraviolet absorbance detection at 248 nm. The

results from the analysis of five GRA-fortified plasma pools show a mean relative standard deviation of

7% and are accurate to within 10%. With evaporative concentration of the extract, the limit of detection

for GRA in plasma is approximately 10 ng/ml.

INTRODUCTION

Interest in investigating the cancer chemopreventive properties of herb-derived

substances such as hcorice root extracts of 18j5-glycyrrhetinic acid (GRA; see Fig.

1) has created a need for simple, sensitive, and accurate methods for the determination of GRA in human plasma. GRA, the main metabolite of the glucoside

glycyrrhizin, is obtained from licorice root, Glycyrrhiza glabra, and is believed to

act as an anti-tumor agent for humans [1-3].

Only a limited number of methods for quantitative determination of GRA in

human plasma have been published [4-9]. Recently, Yasuda et al. [5] described

the use of ammoniacal ethanol to precipitate the proteins, followed by extraction

of GRA with methylene chloride. This extraction procedure gives good recovery

of GRA, approximately 90%. However, the procedure requires a solvent ex- change on the extract to make it compatible with reversed-phase chromatogra-

' Permanent address: Department of Analytical Chemistry, University of Ulm, Ulm, Germany.

2-3
SHORT COMMUNICATIONS 233

COOH

HO

Glycyrrhetinic acid

Glycyrrhetinic acid acetate

Fig. I. Structures of 18^-glycyrrhetinic acid and the acetate ester of glycyrrhetinic acid.

phy. De Groot et al. [7] have reported results using a multi-dimensional ionpairing-reversed-phase chromatography scheme which requires a sophisticated

valving arrangement for column switching. Most recently, Newman and Welch

[8] have reported the development of a chromatographic method for determining

plasma levels of GRA that uses a mixture of sodium bisulfate and sodium chloride to precipitate proteins, and acetonitrile to extract GRA. They report accurate and precise measurement capabilities, though the limit of detection is about

an order of magnitude greater than the method we report here. Takeda et al. [9]

have developed a reversed phase high-performance liquid chromatographic

(HPLC) method that is useful in the evaluation of the pharmacokinetic profile of

GRA in human serum following the oral administration of Kampo medicines

containing glycyrrhizin. Their method provides a limit of detection and measurement capabilities that are comparable to those reported in our method. Other

reports provided information on only quahtative measurement of GRA [10-13].

In this paper, we describe an HPLC method for the determination of GRA in

human plasma. Denaturation of the plasma proteins with urea renders the GRA

and the internal standard readily extractable from the matrix by ion-pairing solid-phase extraction (SPE). Analysis of the extract is by isocratic reversed-phase

separation with ultraviolet (UV) absorbance detection at 248 nm. Quantitation is accomplished through use of an internal standard, the acetate ester of GRA (see

Fig. 1), which is not commercially available, but can be easily prepared. This

method allows reliable measurements of GRA in human plasma with a detection

hmit of 10 ng/ml and with reliable quantitative measurements being possible at

greater than or equal to 100 ng/ml.

2-4
234 SHORT COMMUNICATIONS

EXPERIMENTAL"

Standards and materials

Standard solutions with concentrations ranging from 0.1 to 10 //g/ml GRA

(Sigma, St. Louis, MO, USA) and from 3 to 7 /ig/ml GRA acetate were prepared

(synthesis described belowj in methanol and in plasma. Working standards were

prepared from the methanol solutions for use in calibration and were run prior to

sample analysis to determine retention times and detector response factors. The

purities of the individual reference compounds were determined by direct injection of the respective reference solutions into the chromatograph, using the

HPLC separation described below.

Tetrabutylammonium phosphate (TBAP; Waters Assoc., Milford, MA, USA)

was used as the ion-pairing reagent. SPE cartridges (Cis Sep-Pak, Waters Assoc.)

were used for extraction. The heparin-treated plasma used for this work was a commercial product that had been tested for hepatitis-B and HIV virus.

Synthesis of GRA acetate

GRA acetate (internal- standard) was prepared by mixing 2 g of GRA, 1 5 ml of

acetic anhydride, and 30 ml of pyridine. The mixture was allowed to react overnight and was then poured into cold water. The GRA acetate precipitated, was

removed by filtration, and was then further purified by recrystallization from

n-propanol.

Sample extraction

To 1 ml of plasma, 0.5 g of urea, 3 /zg of the internal standard in methanol, and

200 /il of 0.5 M TBAP in water were added. The solution was vortex-mixed for 1 min prior to extraction to ensure proper mixing. The pH of the resulting solution

was about 10.

A Ci8 SPE cartridge, conditioned sequentially with 3 ml each of methanol,

water, and 0.5 mM TBAP in water, was used to extract the GRA from the plasma

matrix. The 1-ml plasma sample was passed through the cartridge at a rate of

about 1 ml/min. After washing the cartridge with 3 ml of 5 mM TBAP in water

and 3 ml of 45% methanol in water to remove unwanted constituents, the GRA

and internal standard were eluted with 2 ml of methanol. This eluate was concentrated under a stream of nitrogen to I ml or less, depending on the sensitivity

required.

" Certain commercial products are identified to specify adequately the experimental procedure. Such identification does not imply endorsement or recommendation by the National Institute of Standards and

Technology, nor does it imply that the materials identified are necessarily the best available for the purpose.

2-5
SHORT COMMUNICATIONS 235

HPLC apparatus and conditions

The chromatograph used in this work consisted of a single-piston solvent

delivery system, a variable wavelength UV-VIS detector, a data system, and an

autosampler. The (30-nm-pore) polymerically bonded octadecylsilane column (25 cm X 0.45 cm I.D., Vydac TP) was protected with a direct-connect

pre-column that was packed with 10-^m Cis particles of similar type. Both columns were obtained from The Separations Group (Hesperia, CA, USA). Column

temperature was IS'C. Injection volume was 30 //I. The mobile phase was methanol-water-acetic acid (75:24.7:0.3, v/v), at a flow-rate of 2 ml/min. UV absorbance detection at 248 nm, the absorption maximum of GRA, was used for

monitoring the effluent. Analyte concentrations were calculated using peak-area

ratios of GRA and internal standard.

RESULTS AND DISCUSSION

In the development of this method, preliminary studies were made to identify

an appropriate internal standard, to investigate the stabihty of GRA in various

solvents under feasible storage conditions, to optimize the extraction of GRA

from the plasma matrix, and to develop a chromatographic system for separating

GRA and GRA acetate and matric interferences.

Internal standard

The required properties of an internal standard for this analysis are that it be a

sufficiently close homologue of the analyte so that it would mimic the extractability of the analyte from the matrix and that it would be readily separable from the

analyte by HPLC. The acetate ester of GRA meets these requirements, and,

although not commercially available, is easily prepared with common reagents, as described earlier.

Stability of GRA standard solutions

To verify the stability of GRA in various solvents, solutions at approximate

,

concentrations of 10 /ig/ml were prepared in methanol, acetonitrile, and ethanol,

both with and without the addition of 2,6-di-/er/.-butyl-4- methylphenol (BHT)

as an antioxidant. These were stored at room temperature in glass-stoppered

flasks wrapped in foil to exclude light. The UV absorbance at 248 nm was monitored for three weeks and no significant changes were observed in solutions with

or without BHT. No extraneous peaks appeared upon HPLC analysis of aliquots

of these solutions, further confirming that no degradation occurred. Repeated

analyses of plasma-based calibrants kept frozen at — 70°C showed these materials

to be stable over a four-month period.

Extraction of GRA from plasma

It is diflScult to quantitatively extract GRA from the plasma matrix because of

2-6
236 SHORT COMMUNICATIONS

its polar nature and its affinity for plasma constituents. In evaluating extraction

alternatives, acidic (pH 3) and basic (pH 10) aqueous solutions of GRA were

extracted with butanol, methylene chloride, and hexane. Butanol and methylene

chloride were effective extractants, but hexane was not, suggesting that SPE using

«-alkane bonded phases would require ion-pairing agents to be effective.

We found that ion-pairing with Ci8 SPE quantitatively extracted GRA from

aqueous solutions, while recoveries from plasma were only 60-70%. This indicated that the loss was not due to solubility considerations, but due to the

binding of GRA to plasma constituents. When urea was used to denature the

proteins, recoveries of both GRA and GRA acetate were >90%. Both acid and

base hydrolyses were also investigated as means for denaturing plasma proteins.

GRA appeared to be destroyed by acid at the required temperature ( 1 20°C) and

only approximately 10% of the GRA was recovered after treatment with base.

To obtain quantitative recovery in the SPE extraction, it was necessary to pass

the pre-treated plasma slowly through the cartridge and to prevent intrusion of

air bubbles into the cartridge. A flow-rate of 1-2 ml/min was suitable. An intermediate wash with 45% methanol in water was employed to elute interfering

components from the plasma while retaining GRA and GRA acetate in the cartridge. The use of higher methanol concentrations, e.g. 50%, results in significant

loss of GRA.

5 15 Retention Time (min)

Fig. 2. Lower trace: HPLC from the analysis of a standard solution of (1) 1 ng/m\ GRA and (2) 3.4 fxg/m]

GRA acetate using methanol-water-acetic acid (75:24.7:0.3, v/v) at a flow-rate of 2 ml/min on a C,8

column with UV detection at 248 nm. Responses shown represent 30 ng GRA and 90 ng GRA acetate. Upper trace: HPLC from the analysis of plasma extract using solid-phase extraction: (1) 0.38 i^g/m\ GRA

and (2) 2.9 /ig/ml GRA acetate.

2-7
SHORT COMMUNICATIONS 237

Chromatographic separation

A chromatographic separation that resolves GRA and the internal standard

from one another, and from potentially interfering plasma components, is easily

achieved through use of a mobile phase consisting of methanol-water-acetic acid

(75:24.7:0.3, v/v). Acetic acid is added only to effect ionization suppression.

Though we used a polymerically bonded Ci8 column [14,15], monomerically

bonded Cig columns [15] would probably serve as well with suitable adjustments

in the mobile phase. Chromatograms of a plasma extract and a GRA solution in

methanol using the SPE-HPLC protocol is illustrated in Fig. 2. The elution time

of GRA is 8.5 min and of the internal standard is 16.7 min.

Quantification of GRA

Calibration curves were established from both plasma and methanol solutions over the range 0.1-3 /^g/ml. Linear relationships between concentration and the

ratio of peak areas for GRA and the internal standard were obtained for both

plasma- and solution-based standards. The co-linearity of these calibration curves demonstrates that the method is free of matrix effects. Thus, for convenience, we used solution-based standards for routine calibration.

Plasma samples that contained GRA at concentrations ranging from 0.1 to 3

/ig/ml were analyzed using the SPE-HPLC procedure. At least two aliquots from

each plasma sample were extracted, and duplicate HPLC injections were made

from each extract.

The results of this study are shown in Table L Within-day precision is good,

with relative standard deviations ranging from 4 to 7%. No significant betweenday variability was observed. Agreement with the known values is within 10%.

The limit of detection of GRA in plasma using our method is about 10 ng/ml,

which corresponds to a limit of quantification of approximately 100 ng/ml.

The procedure we have described has been successfully implemented for our

TABLE I HPLC MEASUREMENTS OF 18i?-GLYCYRRHETINIC ACID IN PLASMA AFTER ION-PAIRING SOLID-PHASE EXTRACTION

Two aliquots of each plasma sample were extracted and analyzed in duplicate by HPLC. Solution-based

calibrants were used for these analyses.

Known Mean found Relative

concentration concentration standard deviation

(/ig/mi) (/ig/ml) (%)

0.104 0.104 6

0.520 0.571 10 0.966 1.03 7

1.90 1.74 4

2.74 3.04 7

2-8
238 SHORT COMMUNICATIONS

value assignment of quality control materials distributed to investigators involved in National Cancer Institute-supported pre-clinical trials. It provides a

facile, sensitive, precise, and sufficiently accurate means for laboratory use in

investigative studies to determine subject compliance in licorice root-dosing studies and for evaluating the efficacy of GRA as a chemopreventive agent.

ACKNOWLEDGEMENTS

The authors would hke to acknowledge partial financial support for this re- search from the Division of Cancer Prevention and Control of the National

Cancer Institute (USA) under interagency agreement YOl-CN-80655.

REFERENCES

1 Y. Kiso, M. Tohkin, H. Hikino. M. Hattori. T. Sakamoto and T. Namba, Planta Med.. 50 (1984)

298-302.

2 Y. L. Liu. B. D. Roebuck. J. D. Yager, J. D. Groopman and T. W. Kensler, Toxicol. Appl. Pharmacol.,

93 (1988) 442^51.

3 M. H. Davies. Diss. Abstr. Intr. B, 48 (1988) 2275-2500.

4 T. J. F. Savelkoul and P. van Volten, J. Chromatogr.. 456 (1988) 71-81.

5 K. Yasuda, T. Shibuya, M. Nozaki, K. Tsurumi, H. Fujimura and F. Kaneuchi, Zasshi Yakagaku, 98

(1978) 1545-1547.

6 H. Hughes and R. J. Cowles, N.Z. Med. J.. 85 (1977) 398. 7 G. de Groot, R. Koops, E. A. Hogendoom, C. E. Goewie, T. J. F. Savelkoul and P. van Vloten, J. Chromatogr.. 456 (1988) 71-81.

8 R. A. Newman and M. Welch, J. Liq. Chromatogr.. 13 (1990) 1585-1594.

9 S. Takeda, O. Hiromasa. Y. Wakui, A. Asami, Y. Matsuzaki, H. Sasaki, M. Aburada and E. Hosoya,

J. Chromatog :, 530 (1990) 447^51.

10 L. E. Carlat, H. W. Margraf, H. H. Weathers and T. E. Weichselbaum, Proc. Soc. Biol. Med.. 102

(1959) 245-250.

1 1 J. Killacky, M. S. F. Ross and T. D. Turner, Planta Med. 30 (1976) 310-316.

12 D. V. Parke, S. Pollock and R. T. Williams, J. Pharm. Pharmacol.. 15 (1963) 500-506.

13 D. V. Parke, T. C. Hunt and P. Iveson, Clin. Sci.. 43 (1972) 393-400.

14 L. C. Sander and S A. Wise, Anal. Chem.. 56 (1984) 504-510.

15 L. C. Sander and S. A. Wise, in M. W. Cooke and A. J. Dennis (Editors). Polynuclear Aromatic

Hydrocarbons: Mechanisms, Methods, and Metabolism. Battelle Press. Columbus. OH^ 1983, pp. 1 133-

1144.

2-9

MEASUREMENT OF OLTIPRAZ IN SERUM

This section describes three reversed-phase liquid chromatographic methods for the

determination of Oltipraz in serum Two methods employ offline solid-phase extraction, one

using a labeled internal standard suited to thermospray mass spectrometric detection and the

other using a homologous internal standard for use with optical absorbance detection. The

third method includes an on-line solid-phase extraction step and does not require internal

standards. Detection limits are ca. 1 ng/mL of serum. Listed below are the more important

factors related to this analysis.

Important Factors

Calibration and Sample Preparation

• Oltipraz and related compounds are light-sensitive. Store solutions in the dark when not

in use.

• Aqueous standard solutions are unstable in storage presumably owing to sorption to the

container. Create standards daily by diluting alcoholic stock solutions.

• Recovery in solid-phase extraction is enhanced by diluting serum samples three- or fourfold with water.

• Evaporation of extracts to dryness causes serious losses of Oltipraz. Evaporate to 100-

200 |iL and inject.

• Serum samples containing Oltipraz are not stable in long storage, even at -80 °C. limit

such storage to 1 or 2 months for best results.

Chromatography

• Filtration of extracts can cause losses of Oltipraz owing to sorption on the filter. Use a pre-colimm as a bed filter to retain injected particulate matter.

Buffer in the mobile phase (e.g., ammonium acetate) will often sharpen the peaks and

stabilize the retention times.

3-1
• Several types of Cjg columns were tried and all were suitable once the mobile phase was

adjusted to the proper methanol concentration.

• Oltipraz has absorbance maxima at 232, 305, and 450 nm. The extinction coefficients are 10,800, 13,800, and 8,100 L*mol''*cm \ respectively. Detection at 450 nm is preferred to minimize interferences from serum components.

For further information, please contact Richard Christensen at (301)975-3129.
Journal of Chromatography, 584 ( 1 992) 207-2 1

2

Biomedical Applications

Elsevier Science Publishers B.V., Amsterdam

CHROMBIO. 6606

Determination of Oltipraz in serum by high-performance

Hquid chromatography with optical absorbance and mass

spectrometric detection

Richard G. Christensen

Chemical Science and Technology Laboratory, National Institute of Standards and Technology. Gaithersburg, MD 20899 ( USA

)

Winfred Malone

Chemoprevention Branch. Division of Cancer Prevention and Control, National Cancer Institute. National Institutes of Health, Bethesda. MD 20892 (USA)

(First received July 14th, 1992; revised manuscript received September 29th, 1992)

ABSTRACT

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absor- bance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting

interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass

spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method

detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of

variation were similar for all three methods, being about 5%.

INTRODUCTION

Oltipraz, 4-methyl-5-(2-pyrazinyl)- 1 ,2-dithiole-3-thione, is a synthetic dithiolethione that

has been found to have chemoprevention activity

in breast, skin, lung, and bladder cancer animal

models. Epidemiological studies have also re- ported that consumption of certain vegetables

that are known to contain dithiolethones, namely

Correspondence to: Richard G. Christensen. National Institute of Standards and Technology, Chemical Science and Technol- ogy Laboratory, Organic Analytical Research Division, 222/ 31 58, Gaithersburg, MD 20899, USA.

cauliflower, brussels sprouts, and cabbage, have

been associated with decreased cancer risk in humans [1]. These chemopreventive efficacy studies,

together with relatively low toxicity of Oltipraz

as observed thus far in animals, makes it an important compound for continued investigation as a cancer preventive agent.

This study reports on methods for measuring

Oltipraz in human serum, a necessary initial step

for qualifying a compound for entrance into human clinical trials. The National Institute of

Standards and Technology (NIST) is responsible

for providing assistance to grantee laboratories

of the Division of Cancer Prevention and Con3-3
208 R. G. Christensen and W. Malone / J. CInomatogr. 584 (1992) 207-212

trol, National Cancer Institute, in the areas of

analytical method development and quahty as- surance. The quality assurance approach consists

of circulating performance-evaluation standards

to the laboratories to identify problem areas, and

when problems are discovered, to provide counseling in method refinement and trouble-shooting. The development of several analytical methods for assaying serum levels of Oltipraz was nec- essary in the course of preparing for this program. One or more routine methods suitable for use in the clinical laboratories were needed, as

well as one or more reference methods.

Solid-phase extraction of Oltipraz from serum

samples using silica modified with octadecylsilane (Cis) proved to be quite effective, although

another laboratory [2] reports the successful use of liquid-liquid extraction. Two approaches were

used. The first, an off-line extraction using commercial solid-phase extraction cartridges, re- quires the use of an internal standard for volume

correction and to compensate for non-quantitative recovery. It is desirable that the internal stan- dard used should be chemically similar to Oltipraz, so that the extraction recovery and detector

response will be about the same. This extraction

technique was used with both optical absorbance

detection and mass spectrometric (MS) detection.

Since the internal standards used with the ex- traction technique above are not commercially

available, an on-line solid-phase extraction was

also developed. A method based on this extraction, while somewhat cumbersome to perform

with non-automated equipment, offers the possibility of doing the analysis without reference to an internal standard. It also offers some additional method independence, since the extract concentration steps are eliminated. The use of independent methods is an important means for discovering the presence of (and sources oO analytical bias.

Separation of Oltipraz from other extracted se- rum components was readily effected by re- versed-phase chromatography on silica bonded

with octadecylsilane, using a methanol-water

mobile phase. Oltipraz has strong optical absorbances at 232, 305, and 450 nm. These make it quite amenable to determination by high-performance liquid chromatography with absorbance detection (LC-UV) at the levels likely to be

found in serum from the NCI studies, i.e., 10-500

ng/ml [2]. Analytical determination by LC-MS is also

possible, since Oltipraz provides a good yield of

negatively charged molecular ions, M~, in a thermospray interface with discharge-assisted ionization. We have found that a deuterated internal

standard gives both the desired co-elution and

similarity of ionization mechanism for isotope dilution analysis.

EXPERIMENTAL

LC-UV method with homologous internal stan- dard

The internal standard used in this analysis is 4-ethyl-5-(2-py.razinyl)- 1 ,2-dithiole-3-thione

(I.S.)", a homolog of Oltipraz which was synthesized in this laboratory. It has the desirable attri- butes of being chemically similar to Oltipraz, so

that its extraction recovery should be the same,

and also of having similar optical absorptivity, so

that it can be used at about the same concentration as the Oltipraz.

Standard solutions were prepared by dispensing appropriate aliquots of a stock solution of

Oltipraz (Rhone-Poulenc, Vitry, France) in absolute ethanol (Warner-Graham, Cockeysville,

MD, USA) into 50-ml volumetric flasks. These

were then made up to volume with water. Concentrations ranged between 10 and 500 ng/ml. A

solution of I.S. was also made up in ethanol at about 2 jug/ml. This was used to spike both the

standard solutions and samples. If the same I.S. solution is used to spike both the standards and

the serum samples, the accuracy of its concentration is not critical. Oltipraz in solution is subject to photodegradation, so that flasks should be covered, wrapped

with foil, or kept in the dark when not actually in

" The author will make small quantities of these internal stan- dards available to qualified investigators.

3-4
R. G. Christensen and W. Malone / J. Chromatogr. 584 (1992) 207-212 209

use. For storage, ethanol solutions kept in a re- frigerator have been found to be stable for up to two weeks. The aqueous standard solutions are

subject to possible sorptive losses of Oltipraz,

and therefore should be made up fresh daily.

A calibration curve was constructed by placing

1-ml aliquots of standard solutions in vials and

spiking with 50 fi\ of I.S. solution. The aliquots were then evaporated to 100-200 fi\, either by

blowing down with inert gas or by the use of a

centrifugal vacuum evaporator (SVC-IOOH, Savant Instruments, Farmingdale, NY, USA)". The

concentrates were injected into the chromatograph without further treatment. Subjecting

them to the same extraction-concentration

process that was used with the serum samples

gave identical results. For extraction of Oltipraz from serum, 1-ml

aliquots of serum Were placed in vials and spiked

with 50 fil of I.S. solution. The serum samples were then diluted with about 3 ml of water. This

dilution has been found to increase the recovery.

Sep-Pak solid-phase extraction cartridges (Waters Assoc., Milford, MA, USA) packed with Cis

modified silica were conditioned with several milHliters of methanol followed by 2 ml of water.

The spiked, diluted serum was then passed

through at the rate of about 2 ml/min. It is important to keep the cartridge packing wet and not

allow air bubbles to enter, except as specified later. Each cartridge was then washed with 2 ml of

water and 2 ml of 40% (v/v) methanol in water.

These washings remove many of the unwanted

serum components. The last washing was expelled with 3 ml of air. The Oltipraz was then

eluted with 2 ml of methanol followed by 3 ml of

air. This eluate was concentrated by the same

process used with the standard solutions. The el- uate contained some water and, after evaporative

concentration, the water concentration was high

enough that large volumes could be injected

" Certain commercial products are identified in order to ade- quately specify the experimental procedure. This does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment identified as necessarily the best available for the purpose.

without degradation of chromatographic peak

shapes.

For the LC separations, a mobile phase of 2:1 (v/v) methanol-water containing 0.05 mol/1 am- monium acetate was used. Solvent delivery was

by an ISCO M2350/2360 pumpmg system (ISCO,

Lincoln, NE, USA). Flow-rates of 1.5-2.0

ml/min were satisfactory. The buffer served to sharpen up some of the peaks from serum components which might otherwise interfere with the

Oltipraz or internal standard peaks. The injector was fitted with a 1-ml loop, arranged so that

hquid injected into the loop is back-flushed into

the column. A pre-column of 50 jul capacity (Upchurch Scientific, Oak Harbor, WA, USA), filled with Ci8 Corasil (Waters Assoc.) packing supported on 0.5-/im frits, was used to protect the 25

cm X 4.6 mm Vydac 5-fim Cis column (The Separations Group, Hesperia, CA, USA). This precolumn served not only to remove components

which might irreversibly sorb to the analytical

column, but also acts as a filter bed. The concentrated extracts often contained visible particulate

matter, but off-line pre-filtration caused serious

losses of Oltipraz.

Detection was by optical absorbance at 450 nm

using an ISCO V4 variable-wavelength UV-VIS

detector (ISCO), with sensitivity set at 0.005 or 0.010 absorbance units full scale. If an absorption maximum at 232 or 305 nm is used for detection, the sensitivity is 33 or 70% greater, respectively, but there is a greater risk of interference

from the absorbances of co-eluting serum components.

Peak heights were measured for quantification.

Extraction Pump

Water -Qh— Wash ^— Rinse

Fig. 1. Schematic of the on-line extraction system.

3-5
210 R. G. Christensen and W. Malone / J. Chromatogr. 584 (1992) 207-212

though measurements of peak areas would also

serve. A calibration curve was constructed by

plotting the ratio. R (where R = Oltipraz re- sponse/I. S. response), versus the Oltipraz concentration in the standard solution. The plot was

found to be linear over the range of interest, i.e., 10-500 ng/ml. Oltipraz concentrations in serum

samples could then be inferred by comparing the

value of the ratio, /?, for each sample to the cali- bration curve. LC-UV method with external standard

An additional HPLC pump (M6000, Waters

Assoc.) was used to pump the samples (or stan- dards) and washing solutions through an extraction column which was substituted for the injection loop of the HPLC system, as shown in Fig.

1. A 40 mm x 4.6 mm I.D. extraction column

was constructed and 'packed with 10-//m /^Bondapak Ci8 (Waters Assoc.). This packing is some- what less retentive than the Vydac Ci8 analytical

column, so that some focusing of the extracted

Oltipraz will occur when it is displaced onto the

analytical column.

Serum samples and standard solutions were diluted 1:4 with water prior to injection. With water flowing through the extraction column at 2

ml/min, 1.00 ml of diluted serum or standard so- lution was injected. After the passage of 5 ml of

water, 5 ml of washing solution containing 35%

methanol in water was pumped through. The ex- traction column was then switched in line with

the analytical column, whereupon the extracted

materials were eluted. The extraction column was

conditioned for the next extraction by pumping

through 10 ml of absolute ethanol, followed by

10 ml of water. For quantification, peak heights

from the serum samples were compared to the

peak heights resulting from injections of stan- dard solutions.

LC-MS method

A Vestec Model 201 thermospray LC-MS sys- tem (Vestec, Houston TX, USA) was used as the

detector for isotope dilution determinations. The

LS. used was 4-(methyl-d3)-5-(2-pyrazinyl)-l,2-

dithiole-3-thione (01tipraz-d3), which was synthesized in this laboratory. Standard solutions,

spiking, extraction, and concentration steps were

similar to those used for the LC-UV method.

The mobile phase was the same except that the

ammonium acetate was omitted because it yielded a background ion at m/z 229, interfering with

the ions from the internal standard. The flow-rate

used was 1.00 ml/min. Obviously, the on-line ex- traction described above can also be used in this

method, with the addition of 01tipraz-d3 as LS.

The source block was operated at 275°C, and

the vaporizer was operated near the "take-oflT'

temperature, giving a tip temperature of about

210°C, and a vapor temperature of about 240°C.

Discharge-assisted ionization was used, and the

M~ ions of Oltipraz and 01tipraz-d3 were monitored at m/z 226 and 229, respectively. A caUbration curve was constructed by the same method employed for LC-UV quantification. As a check on the integrating algorithm, calculations

have been made using both peak areas and peak

heights, with no significant difference appearing

in the results.

8 r

0'—'—'—I 1 lJ 1 i_

0 10 20

Time, minutes

Fig. 2. Chromatograms of serum extracts. (A) Injection of the

extract from a sample containing 47.7 ng/ml Oltipraz, spiked with 150 ng/ml I.S. (B) Injection of the extract from a sample

containing 20.2 ng/ml Oltipraz, spiked with 150 ng/ml I.S.

3-6
R. G. Chrisiensen and W. Malone I J. Chromaiogr. 584 1 1992) 207-212 211

6 8 10 12 14 Time (min )

Fig. 3. Single-ion monitoring of the M" ion of Oltipraz (miz 226) from the injection of a serum extract containing about 1 ng of

Oltipraz. The upper trace is recorded at 5 x higher sensitivity.

RESULTS AND DISCUSSION

Recoveries observed in the methods using off- Une extraction ranged between 50 and 90%. The

losses appear to occur during the concentration

of the extracts, rather than being due to ineffi- ciency of extraction. Whether the losses are caused by oxidation, photolysis, sorption to the

glass container, or some other process is not

known. However, the good agreement between

the three methods, together with the observation

that the results show no dependence on the level of recovery, leads us to assert that the losses af- fect the analyte and I.S. equally and therefore do

not bias the results. A typical chromatogram obtained by the LCUV method with I.S. is shown in Fig. 2. Other

types of Ci8 columns have been tried and found

to be usable when the methanol concentration in the mobile phase was adjusted to give similar re- tention times. Chromatograms obtained by the

LC-MS method are very clean, typically showing

only peaks from the analyte and I.S. The limits of detection for each method were

calculated from observations of the range of

basehne noise, compared with the sensitivity.

This has been found empirically to be equivalent

to several other criteria [3]. Method detection

limit for the LC-UV method was observed to be

approximately 0.5-1.0 ng/ml, and for the LCMS method 0.1-0.2 ng/ml. The latter can be in- ferred from Fig. 3, which shows an LC-MS peak

from the injection of a serum extract containing

about 1 ng of Oltipraz. The detection limit for the

on-line extraction method is, on one hand, degraded by the smaller sample size. and. on the

other hand, enhanced by the elimination of analyte losses during evaporative concentration. The

resulting method detection limit is about 3.0 ng/

ml.

Comparative data from all three methods are shown in Table L Two samples from each lot of

spiked serum were run in duplicate by each method. The coefficients of variation (C.V.) are typical

of those observed in our analyses of many other serum samples. The agreement between the three

methods is also good.

The methods utilizing internal standards have

been in use in our laboratory for two years to assign Oltipraz concentrations to serum samples

TABLE I COMPARISON OF OLTIPRAZ CONCENTRATIONS DETERMINED BY THREE METHODS

Serum LC-MS. I.S. LC-UV. I.S. LC-UV, external standard No.

Concentration C.V. Concentration C.V. Concentration C.V.

(ng/ml) (%) (ng/ml) (%) (ng/ml) (%)

151 378 4.8 423 3.7 395 6.2 152 210 6.2 '203 3.8 203 4.8 153 58.4 2.3 66.4 1.4 64.0 5.7 154 91.8 7.1 99.5 2.2 95.7 3.8

3-7
212 R. G. Chrislensen and W. Malone / J. Chromato^r. 5H4 (1992) 207-212

circulated in performance evaluation exercises.

The median of the absolute values of relative deviations in the results reported by other laboratories using similar and different methods is about

20%.

The on-line extraction method was developed

to provide a means of Oltipraz determination

where specially synthesized internal standards

are not available. Its lesser sensitivity could presumably be enhanced by extracting a larger volume of sample. It will not prove as well suited to routine use in clinical laboratories, however, unless the number of analyses is high enough to warrant setting up a chromatographic system

with automated valving, dedicated to this analysis.

ACKNOWLEDGEMENT

We would like to acknowledge partial financial

support for this research from the Division of

Cancer Prevention and Control of the National

Cancer Institute (USA) under interagency agreement YOl-CN-80655.

REFERENCES

1 C. W. Boone, G. J. Kelloffand W. E. Malone. Cancer Res., 50

(1990) 2. 2 J. L. Bennett. J. McMillan and N. V. Dimitrov, J. Chromalogr., 573 (1992) 146.

3 R. G. Christensen and W. E. May, J. Liquid Chromalogr., 1 (1978) 385.

3-8
ASCORBIC AND DEHYDROASCORBIC ACIDS ANALYSES

This section contains two papers pertaining to the analysis of ascorbic acid and

dehydroascorbic acid in human plasma. It describes the preparation and stability of quality

control materials and calibration standards. Two HPLC methods for analysis are described

in detail as well as procedures for optimum sample handling and analyte extraction. The

points listed below are some of the more important factors related to this analysis.

Important Factors

Calibration and Sample Preparation

All plasma samples must be preserved immediately after the cells are removed,

preferably in metaphosphoric acid (MPA) at a final concentration of 5% w/v.

Plasma controls may be prepared from ascorbic-acid-free plasma by first adding

dithiothreitol (DTT 1 mg/mL) and then by adjusting the ascorbic acid content to the

desired concentration by adding a solution of ascorbic acid in water: acetonitrile (1:3 v/v)

containing DTT (1 mg/mL). The samples may then be distributed into vials and

lyophilized. If ascorbic acid is added to plasma without the dithiothreitol, it will be

rapidly oxidized.

Frozen plasma samples should be defrosted in a 20 °C water bath for no more than ten

min. A longer duration or a higher temperature will lead to some ascorbic acid

oxidation.

Samples preserved in MPA should be mixed thoroughly before sampling in order to evenly suspend the precipitate.

Sampling should be done with a diluting pump so that a plasma sample is washed out of

the pipette with at least five volumes of distilled water or some other non-precipitating

solvent. The viscosity of the plasma does not permit full delivery of the sample without

a diluting or washout step.

The addition of acetonitrile achieves a more complete precipitation of the proteins,

prevents the accumulation of insoluble material at the top of the column, and extends the

column life.

4-1
The assay is performed using calibration standards prepared with dithiothreitol

(1 mg/mL) in acetonitrile and water 3:1 v/v. These standards are stable for 1 week

when stored at 4 °C. Each analysis run contains duplicate standards at each

concentration, (i.e., six vials, two at each concentration). Two vials are analyzed at the

beginning, the middle, and at the end of the assay to ensure that the conditions are

constant.

Chromatography ^

• The chromatography was performed on a "Fast Acid" analysis column, 100 x 7.8 mm.

• The separation was isocratic using a solvent composed of 0.1 mol/L formic acid and 5%

acetonitrile in water at a flow rate of 1 mL/min. The column temperature was 30 °C.

Detection was electrochemical at +0.7 volts.

For further information, please contact Sam Margolis at (301)975-3137.

4-2
[Reprinted from CLINICAL CHEMISTRY, 36, 1750 (1990).) Copyright 1990 by the American Association of Clinical Chemistry and reprinted by permission of the copyright owner.

Ascorbic and Dehydroascorbic Acids Measured in Plasma Preserved with Dithiothreitol or i\^etaphosphoric Acid

Sam A. Margoiis,^ Robert C. Paule,^ and Regina G. Ziegler'

We describe a rapid metiiod for accurately and precisely

measuring ascorbic acid and dehydroafcorbic acid in plasma. Total analysis time is <10 min, replicate analyses of a single pool provide precisions <2%, and values measured

in supplemented samples agree with known concentrations

of 4.68 and 1 1 .83 mg/L. The stability and homogeneity of lyophilized plasma samples supplemented with ascorbic acid and dithiothreitol are documented. We also describe a pro- cedure in which metaphosphoric acid (50 g/L) is used to prepare a reference material for the measurement of ascorbic acid and dehydroascorbic acid. The procedure for both

acids consists of first measuring the native ascorbic acid, then reducing the dehydroascorbic acid, at neutral pH, with

dithiothreitol, and finally measuring the total ascorbic acid; dehydroascorbic acid is then determined by difference. The

metaphosphoric-acid-treated samples were stable at -70 °C, but stability decreased with temperature over the range examined, 4-50 °C.

Additional Keyphrases: re/isrence mafena/s analytical precautions

Ascorbic acid (AA) and dehydroascorbic acid (DHAA) are normgdly present in both human and animal tissues and

plasma.^ Both the chemical and biochemical destruction of AA occurs solely through the pathway of AA to DHAA to diketogulonic acid—the first reaction being reversible, the

second irreversible (1). Accurate measurement of AA and

DHAA in biological samples such as plasma depends on a

reliable method for stabilizing these materials in the matrix of interest and the availability of a reliable assay for each analyte. Such measurements are necessary to the

design and interpretation of epidemiological studies defining the factors aiffecting the distribution of AA and

DHAA in blood and other tissues. Currently there are three general procedures for quantifying the sum of both analytes (total AA): (a) the dinitro- phenylhydrazine method (2), (6) the o-phenylenediamine

method (3-6), and (c) the reduction of DHAA by sulfhydryl

reagents (7-13).

The first two procedures, both based on the production of a derivative of DHAA with either dinitrophenylhydrazine

or o-phenylenediamine, are used to measure the DHAA

content and, indirectly, to measure total AA after the

^ Organic Analytical Chemistry Division, Center for Analytical

Chemistry, National Measurement Laboratory, National Institute

for Standards and Technology, Gaithersburg, MD 20899.

^ Environmental Epidemiology Branch, Epidemiology and Biostatistics Program, Division of Cancer Etiology, National Cancer

Institute, Bethesda, MD 20892.

^ Nonstandard abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; MPA, metaphosphoric acid; HPLC, high-pressure

liquid chromatography; DTT, dithiothreitol; and EC, electrochemical. Received April 12, 1990; accepted July 27, 1990.

native AA has been oxidized to DHAA (i). However, both of these reagents react with other reductones, including diketogulonic acid, £ind this can lead to measurement errors in a simple chromogenic assay (1). The last two procedures have been used in conjunction

vdth chromatographic methods. Method 6 has been used for both pre- and postcoliunn derivatization of DHAA vdth

o-phenylenediamine and method c for precolumn reduction

of DHAA. The advantage of method b is that it measures DHAA directly. These last two procedures involve either

complex extractions that require the use of internal stan- dards (4), an insensitive ultraviolet method for AA detection (5, 13), or a lengthy separation procedure (6). The procedure we used in this study (a variant of proce- dure c) involves sulfhydryl reagents (7-13) to reduce

DHAA to AA, and requires little additional sample manipulation. Diketogulonic acid is unaffected by dithiothreitol (DTT). This procedure measures only AA directly; DHAA

is measured indirectly after its reduction to AA. The

complete assay can be done by HPLC in 8 min viith use of a very sensitive electrochemical (EC) detector at a constant

electrode potential. To measure DHAA, one must analjrze

each sample twice, once for native AA concentration and

once after the addition of DTT, to meeisure total AA

content. The DHAA content is calculated as the difference between the values for native AA and toted AA.

Accurate results entail both accurate analyses and stable samples. However, there is a paucity of published data on

the long-term stability of AA in acid-stabilized serum or plasma (3S, 14, 15). In one report (5) it was demonstrated

that AA and DHAA were stable when stored at -20 "C for 30 days in 100 mL/L metaphosphoric acid (MPA). In a second (15), AA was found to be stable for five weeks at -70 °C in 50 mlVL MPA. In two studies (3, 14) total AA

was measured in plasma. Bradley et al. (14) demonstrated

that AA was stable in plasma for 21 days at 4, -20, and

-70 °C, in the presence of either MPA or trichloroacetic

acid; Baker et al. (3) foimd AA to be stable for seven days at -70 °C in EDTA-perchloric acid. Using a colorimetric

assay, they (3, 14) observed an initial, imexplained 5% to 13% increase in the concentration of AA viathin the first week of storage, and Baker et al. (3) noted that their

colorimetric results did not agree with results they ob- tained with an HPLC-EC method. Nevertheless, the con- centration of AA determined by the HPLC method corre- sponded to the amount added to AA-free plasma, suggesting a systematic bias in the determination of AA by the

dinitrophenylhydrazine method. We have reported that AA

in plasma can be preserved by adding DTT and have

demonstrated that all of the added AA was accounted for after 88 weeks in samples stored at -70 °C (13). The objectives of the present study were (a) to demonstrate the accuracy and precision (including some of the

sources of systematic error) of the measurements deter- mined by a modified version of our recently reported

HPLC-EC method (13); (h) to demonstrate the feasibility of preparing a plasma pool suitable for use as a reference

1750 CLINICAL CHEMISTRY. Vol. 36, No. 10, 1990 4-3
material in field studies; (c) to show that DHAA formed by

the oxidation of AA in plasma at 22 °C can be quantitatively converted to AA; and id) to demonstrate that no measurable degradation of AA occurs in MPA-treated

plasma at -70 °C, but at 4, 25, and 50 °C AA is more or less slowly oxidized to DHAA and other products.

Materials and Methods'*

Materials AA and DTT were purchased from Sigma Chemical Co.,

St. Louis, MO; metaphosphoric acid from Fisher Scientific,

Fair Lawn, NJ; and the plasma from Western States Plasma Corp., Fallbrook, CA. The acetonitrile was "HPLC"

grade. The AA, examined by proton magnetic resonance, contained <0.1% protonated impurities, including DHAA

and its degradation products. The processed plasma was

stored at -20 °C until used. The method of AA analysis is a modification of that

described in reference 13 as HPLC Method 1. A 100 x 7.8 mm "fast acid analysis" column (Bio-Rad Laboratories,

Richmond, CA) was used at a flow rate of 1 mL/min and the

EC detector was a Model 400 (EG&G, Princeton Applied

Research, Princeton, NJ). Analysis time was shortened

from 35 min to 8 min. A series of three calibration stan- dards (three AA concentrations that bracketed the sample

concentrations) were run at the beginning and end of each

day, to assess the stability of the detector and to provide a

calibration curve for use in calculating the AA concentration of the samples U3). All serum samples were aliquoted

with a calibrated automated positive-displacement pipet in either the dispensing (for reference-material prepeiration) or diluting (for sample analysis) mode.

Preparation of AA-Supplemented Plasma Samples

Set 1. Quality-control sample set. We prepared three

160-sample groups of lyophilized plasma: (a) a group of unsupplemented plasma samples, (6) a group supplemented to give a low-normal concentration of AA, and (c) a group supplemented to give a high-normal concentration of AA. DTT was added to plasma at room temperature to give a concentration of 1 g/L. This plasma (relative density

I. 025) was divided into three lots, two of which were

supplemented by adding a weighed amovmt of an AA

solution (1.184 g of AA per gram of 10 g/L aqueous

DTT:acetonitrile, 1:3 by vol), such that the final AA con- centration of each lot of reference material was 4.68 and

II.83 mg per liter of plasma for the low- and high-normal

samples, respectively. Each lot of reference material was

well mixed, after which 1-mL samples were aliquoted,

lyophilized, and stored at -70 °C as previously described

(13). Each sample was prepared for analysis by equilibrating

it at room temperature and adding 0.97 mL of distilled water. After the solids were completely dissolved, 0.1 mL of reconstituted plasma was diluted with 1 mL of distilled water into a vial containing 160 ixL of a 400 g/L solution of metaphosphoric acid; 400 /u-L of acetonitrile was added, and

the vial was capped and vortex-mixed for 15 s. The result-

* Identification of any commercial product does not imply rec- ommendation or endorsement by the National Institute of Stan- dards and Technology, nor does \\ imply that the material or equipment identified is necessarily the best available for the

purpose.

ing suspension was centrifuged (1000 x 30 min, 4 °C)

and the supernatant fluid was transferred to 1.8-mL au- tosampler vials and analyzed by HPLC.

Set 2. Samples preserved with metaphosphoric acid. The

frozen human plasma was thawed and three 100-sample

lots were prepared: (a) a group of unsupplemented plasma

samples, (6) a group supplemented with a low-normal

concentration of AA, and (c) a group supplemented with a high-normal concentration of AA. Two lots of plasma were

supplemented by adding a weighed amount of an AA

solution (49.56 mg of AA per 53.24 g of aqueous metaphosphoric acid, 100 g/L) such that the final concentration ofAA

in each lot was 4.87 and 11.75 mg per liter of plasma (low

and high normal, respectively). Each plasma lot was thor- oughly mixed. The solutions were exposed to 22 °C for 2 h,

permitting partial oxidation of AA to DHAA. Therefore,

the 6 and c samples could be used to analyze for both AA

and DHAA, and they mimicked real seunples. Of these

plasma samples, 0.5-mL aliquots were then mixed with 0.5 mL of a 100 g/L solution of metaphosphoric acid, in glass

ampules, which were then sealed, their contents frozen,

and stored at -70 °C. At the time of analysis, the test-plasma samples pre- served with metaphosphoric acid were thawed in a water

bath at 20 °C; thawing was complete within 10 min. Two

analyses were performed on each sample, the first to measure the native AA content and the second to measure

the total AA (i.e., AA + DHAA) after reduction with DTT.

Samples for the first analysis were prepared by diluting

0.200 mL of sample with 1 mL of distilled water in a vial containing 160 ixL of metaphosphoric acid (400 g/L); 0.400

mL of acetonitrile was added, the vial was capped, and the

sample was vortex-mixed for 15 s. After centrifuging the

resulting suspension (1000 x g, 30 min, 4 °C), we transferred the supernatant fluid to 1.8-mL autosampler vials and analyzed it by HPLC.

The samples for the second analysis were prepared in the same manner as for the first analysis except that 200 fiL of

test plasma sample was diluted with 1 mL of distilled water

in a vial containing 200 fiL of a solution of 0.5 mol of K2HPO4 and 1 g of DTT per liter. After 1 h at room

temperature, 160 fxL of a 400 g/L MPA solution and 400 fiL of acetonitrile were added, and samples were vortex-mixed

and centrifuged as above. The supernatant fluid was then

transferred to autosampler vials for analysis. All HPLC-EC

measurements were corrected by calibration factors. Statistical analysis. Measurement data on the samples

from set 1 were examined. Separate analyses of variance were made for the low- and the high-concentration plasma

samples. The organization of the data was that of a fourlevel hierarchical design. Analyses were made over 12

diflFerent days, with two different samples for each day, two

aliquots taken from each sample, and two measurements

made on each aliquot. Thus, for each concentration, 96

measurement results were used and four sources of vari- ance were evaluated: days, semiples, aliquots, and replicate

measurements.

Results

The chromatogreuns of the unsupplemented plasma (Fig- ure 1, A and B) indicate that it contained no electrochemically active material with a retention time at or near that

of AA. Furthermore, treatment of this plasma with DTT

did not result in the generation of any material that

4-4

CLINICAL CHEMISTRY, Vol. 36, No. 10, 1990 1751
Table 1 . Summary of Analysis of Variance of Plasma

Samples, Set 1 , Supplemented with AA at High- and

Low-Normal Concentrations

Ascorbic acid, mg/L

2 4 6 Tim* (min)

Rg. 1. Chromatograms of lyophilized and MPA-preserved human

plasma and AA-supplemented plasma with and without DTT treat- ment

A. lyophilized plasma; 6, plasma preserved in MPA (50 g/L) and treated with dipotassium phosphate (0.05 mol/L) and DTT (1 g/L) for 1 h: C, lyophilized plasma containing DTT (1 g/L) and supplemented with 11.83 mg of AA per

liter: D. plasma preserved with MPA (50 g/L) and supplemented with AA, 1 1 .75 mg/l„ upper trace, no DTT, tower (race, treated with DTT (1 g/L) and

dipotassium phosphate (0.05 mol/L) for 1 h. Peak J, MPA; 2, AA; 3, uric acid;

4, DTT

co-chromatographed with AA. Thus the unsupplemented

plasma matrix contained neither AA nor DHAA (which

weis reduced to AA with DTT), and edl of the material

measured in all samples represented either added AA or material converted to AA.

Measurement of AA in Lyophilized Plasma Stored at -70 °C (Set 1) The concentration of AA in the two supplemented lots of plasma (AA, 4.68 and 11.83 mg/L) was meeisured in dupli- cate on each of 12 days over a 21-day period. Twenty-four

samples were selected without conscious bias from each lot and two were analyzed on each day. Analysis of variance of these data (Table 1) gives the various sources of imprecision contributing to the total variability of a single measurement. The total imprecision (i.e., the CV) of a single

measurement for each lot of samples was 2% (11.76 ± 0.26 and 4.70 ±0.11 mg/L). This uncertainty is small and the

measured values vary from the gravimetric value by <1

SD (Table 1). These results demonstrate the stability and

homogeneity of these reference materials relative to the

day of analysis and the order of sample preparation.

Measurement of AA and DHAA in Frozen Plasma

Preserved with MPA (Set 2) The following studies were done to examine the accuracy and precision of the measvirement of existing AA, total AA

after reduction of the DHAA in the senmn sample with

AA added

Grand mean of all 96 measurements

Source of variability Day

Sample

Aliquot

Replicate measurement SD of a single measurement

CV, %

" ns = not significant.

11.83

11.76

4.68

4.70

Component of SD, mg/L

ns^ 0.07

0.23 ns^

0.07 0.06

0.10 0.06

0.26 0.11

~2 ~2

DTT, and DHAA by difference (total AA - existing AA).

Because the reduction of DHAA by DTT is pH dependent

(7), we adjusted the pH of each sample with dipotassium

phosphate. The precipitated material completely dissolved

and the DHAA was completely reduced to AA within 10

min at room temperature (22 °C). The resulting AA con- centration remained constant for at least 90 min after the

sample was prepared. In the absence of the dipotassium

phosphate the reduction of the DHAA was still incomplete

after the mixture had stood for an hour at room temperature. Before being preserved with MPA, the initial decrease in AA (11.75 and 4.87 mg/L) in plasma at 22 °C for 2 h was

1.60 and 0.98 mg/L, respectively (Table 2). After being

preserved with MPA, these samples were analyzed for DHAA content and for anal)rtical recovery of supplemented

AA (i.e., AA + DHAA content) over a period of 98 days. The

total AA remained constant in MPA (50 g/L) when the

sample was stored at -70 °C. These results indicate that

the oxidized AA, namely DHAA, could be quantitatively

converted to AA by DTT, and that no diketogulonic acid was formed. This is demonstrated by the fact that the

values obtained for AA after DTT treatment (11.69 and

5.03 mg/L) were not statistically different (P >0.05, Student's f-test) from the amount gravimetrically added to the

plasma at the beginning of the experiment (11.75 and 4.87 mg/L, Table 2). These results demonstrate the relative homogeneity of the samples preserved with MPA and

indicate that the sample-to-sample variation in total AA

Table 2. Summary of Analyses of AA-Supplemented

Plasma Samples, Set 2, Preserved in MPA, 50 g/L

Concentration, mg/L

AA added 1 1 .75 4.87

(No. of samples) (10) (5) AA^ 10.09 ±0.04 4.05 ±0.13

Total AA" 1 1 .69 ± 0.06 5.03 ± O.OS'' DHAA<^ 1.60 ± 0.07 0.98 ± 0.14

* Mean ± SD of the mean.

" Total ascorbic acid (mean ± SD of mean) was determined as described in the text with use of DTT as a reducing agent.

= This value does not differ significantly (P >0.05 by Student's Mest) from

the gravimetric value.

" Determined by subtracting the mean AA value from the mean total AA

value.

1752 CLINICAL CHEMISTRY, Vol. 36, No. 10, 1990 4-5
content is similar to that observed for those samples pre- pared in DTT and then lyophilized and stored at -70 °C. The CV for the measurement of AA in both sample lots before the reduction of DHAA with DTT, 0.4% and 3%

(10.09 ± 0.04 and 4.05 ± 0.13 mg/L), is similar to the CV

for the total AA value, 0.5% and 1% (11.69 ± 0.06 and 5.03 ± 0.06 mg/L, Table 2). The DHAA content (1.60 ± 0.07 and

0.98 ± 0.14 mg/L)—i.e., the "total AA" minus "AA"—can

be determined to the same general precision as the "total AA" and "AA." The results indicate that <1 mg of DHAA

per liter can be precisely measured.

Stability of AA in Plasma Stabilized with MPA (Set 2) at 4, 25, and 50 °C

In preliminary studies we thawed our samples by putting

them in "warm water" for a variable period of time. Under

these uncontrolled conditions we observed measurable

variation in the initial AA content. Thus at increased

temperatures, plasma AA preserved with MPA does not appear to be stable. We have now examined this instability at 4, 25, and 50 °C, using the plasma samples from set 2. At

4 °C AA degraded slowly. The degradation was detectable

in the high-normal plasma samples after only one day, and

it continued for 14 days (Table 3, experiments 1 and 2). In the low-normal plasma samples (Table 3, experiment 3) about 80% of the AA was lost after 14 days. More importantly, the total AA content was reduced 42%. This indi- cates that some of the DHAA was also degraded along with

the AA. At 25 °C, detectable oxidation of AA occurred

within 5 h, and after 24 h 18% of the AA had been oxidized

Table 3. Stability of Ascorbic and Dehydroascorbic

Acids in MPA, 50 g/L, at 4 X

Concentration, mg/L*

Day AA Total AA" DHAA'= Experiment 1 0 9.9 ± 0.22 11.6 ± 0.08 1.7

1 9.2 ± 0.19 11.6 ± 0.15 2.4 2 9.1 ± 0.10 11.0 ± 0.12 1.9 4 8.6 ± 0.12 11.2 ± 0.06 2.6 7 8.5 ± 0.09 10.6 ± 0.11 2.1 9 7.6 ± 0.06 9.9 ± 0.03 2.3

Total loss 2.3 1.7

Experiment 2

0 10.1 ± 0.14 11.6 ± 0.06 1.5 3 8.8 ± 0.14 10.9 ± 0.12 2.1 7 8.7 ± 0.08 11.2 ± 0.05 2.5 10 7.9 ± 0.13 10.1 ± 0.05 2.2 14 7.2 ± 0.10 9.7 ± 0.12 2.5

Total loss 2.9 1.9

Experiment 3

0 3.9 ± 0.18 5.0 ± 0.06 1.1 3 3.3 ± 0.10 4.6 ± 0.02 1.3 7 2.9 ± 0.07 4.4 ± 0.04 1.5 10 1.9 ± 0.10 3.6 ± 0.07 1.7 14 0.7 ± 0.06 2.9 ± 0.09 2.2

Total loss 3.2 2.1

^ Each numt)er represents the mean (and SD) of four measurements.

" Ascortiic acid content measured after treatment of the sample with DTT.

" The difference between the AA value and the total AA value.

(Table 4). At 50 °C the degradation of AA was even faster,

16% in 3 h (Table 4).

Discussion

Validity of AA and DHAA measurement in clinical plasma samples depends on the availability of a method of demonstrated precision and accuracy for measurement of

each analyte. The modified method described here is characterized by increased speed of analysis (8 min), baseline

peak resolution comparable with that of our previous

method (13), and the elimination of baseline drift. Figure 1 clearly demonstrates the baseline resolution of all peaks.

Furthermore, the chromatograms of the unsupplemented

plasma to which DTT was added indicate that there was no AA, DHAA, or any "contaminating" peaks. However, for patients receiving special therapy, it may be necessary to demonstrate that no interfering materials are present by

measviring the AA content in the presence and absence of

ascorbate oxidase (EC 1.10.3.3). The baseline drift observed

in earlier experiments (13) was reduced by use of the EC

detector such that negligible signal degeneration was ob- served during an 8-h period.

A major source of measurement error is imprecision in sampling the plasma. This is a function of the viscosity of the plasma. We have minimized this error by using an

automated diluting pipet. With this iristrument, an accurate aliquot of plasma is taken and then the sampling pipet

is pvu"ged with at least five volumes of diluent to wash out

the viscous plasma. We tested the dependability of this method by using a single lot of stable reference plasma

samples that had been supplemented vnth AA and pre- served as previously described (13). The results of such an

experiment (Table 1) indicate that this method accurately measures the supplemented concentrations of AA, i.e., measured values of 11.76 ± 0.26 and 4.70 ± 0.11 mg/L as compared with the respective gravimetric values of 11.83

Table 4. Stability of Ascorbic and Dehydroascorbic

Acids in MPA, 50 g/L

AA Total AA" DHAA"

fMinutes' mg/L

Temperature 25 °C

15 10.3" 12.0" 1.7 60 10.1 12.0 1.9 120 10.2 11.8 1.6 180 9.8 11.8 2.0 300 9.7 11.6 1.9 1440 8.4 11.0 2.6

Total loss 1.9 1.0 Temperature 50 °C 0 10.1 11.6 1.5 20 9.8 11.7 1.9 40 9.8 11.8 2.0 60 9.4 11.8 2.4 120 8.9 11.2 2.3 180 8.5 10.7 2.2

Total loss 1.6 0.9

^ Elapsed time after the sample was transferred from -70 °C directly into the controlled-femperature bath.

" Ascorbic acid content measured after treatment of the sample with DTT. The difference between the AA value and the total AA value.

" Each value represents the mean of two determinations.

CLINICAL CHEMISTRY, Vol. 36, No. 10, 1990 1753 4-6
and 4.68 mg/L. The precision of both the low- and highconcentration AA measurements is —2%. The error contri- butions of the different components of the analj^ical

method were assessed by analysis of variance. These components (Table 1) included (a) the variability due to the

handling and processing of samples on different days; (6) the between-sample variability for a given analyte concentration; (c) the variability in the sample preparation pro- cedure, including aliquoting the sample, adding reagents,

and precipitating the proteins; and {d) variability between

dupUcate analyses on the same aliquot that is attributable

to the variation in instrument settings. From a practical

viewpoint, all four sources of variability are small. The

difference between the gravimetric measurement and the

observed values (an index of the accuracy of the meeisxure- ment) falls within the precision of the measurement, the

CV being about 2% of the measured value for a single

measurement. This supports the conclusion that there is no

outstanding source of systematic bias. Fiirthermore these results demonstrate an acceptable

level of stability and homogeneity for both sets of samples

(sets 1 and 2) and clearly indicate that our measurements

are reproducible for both sets of samples. These results are

consistent with our previous observation (i3) on the stability of samples that were similar to set 1. Because these

samples are stable, this product may be a suitable control material for AA measxu-ement by methods used to resolve AA and DTT.

The suitability of a method for preserving AA in biological samples collected for epidemiological studies and the

valid interpretation of data from completed epidemiological

studies depend on demonstrating the stability of plasma

AA preserved by that method. One approach to eveiluating the stability of AA in plasma is to measiu-e, specifically, AA

and its immediate degradation product, DHAA, so that

storage procedures can be effectively evaluated. This is particularly important because some clinical samples may

contain significant concentrations of DHAA (5, 6). For us to evaluate the stability of AA and DHAA in plasma preserved with MPA, it was necessary to prepare a

reference material in which the total AA (AA + DHAA)

was accurately measured. This was achieved by gravimetrically adding a known amount of AA to plasma, and

permitting partial oxidation of the AA to DHAA by letting the supplemented plasma stand at room temperature for 2 h. The DHAA produced this way seemed to us to most

closely approach the conditions under which DHAA is generated when samples are processed in the field and

preserved with MPA.

DHAA was meeisiu-ed after neutralizing the plasma

sample and reducing the DHAA to AA with DTT. The

reduction of the DHAA was complete within 10 min and no

farther change weis observed during the following 80 min.

The nonsupplemented plasma pool contained neither AA

nor DHAA (Figure 1). The results in Table 1 clearly

demonstrate that all of the added AA was accoimted for as AA or DHAA and that both AA and total AA (and by

inference the DHAA) are stable at -70 °C when preserved

in MPA (50 g/L). The small standard deviation indicates that the means for both supplemented lots are precise and

that all of the values from day 6 to day 98 fell within a narrow range. These results, i.e., recovery of all supplemented AA, also indicate that the use of DTT (1 g/L) in the presence of approximately 0.05 mol of dipotassium phosphate per liter (final concentration) effectively reduces all

of the oxidized AA to AA. Thus, this method provides a

suitable technique for measuring the simi of AA plus

DHAA in biological samples after they have been pre- served with MPA (50 g/L).

The identification of the degraded AA as DHAA is based on its reductive conversion to a material chromatographically identical to AA. Because the unsupplemented plasma

does not contain any material that can be converted to a

material that is chromatographically identical to AA or is electrochemically reducible, the material that was detected

by reduction with DTT had to arise from the supplemented

AA. The observation that DHAA in plasma can be reduced

to AA by DTT (13) also supports the identification of the

degraded AA as DHAA.

The studies described above (as opposed to those done on serum samples collected and preserved in field studies) were carried out imder ideal conditions, where the samples were rapidly aliquoted and quickly cooled to -70 °C. At the

time of assay they were carefully thawed and prepared for assay. Close attention must be paid to this procedure

because of the temperature instability of AA and DHAA. A

study of the stability of AA and DHAA at 4, 25, and 50 °C, in the supplemented plasma samples preserved in MPA,

indicates that degradation of AA can be detected at these

temperatures (Tables 3 and 4). At 4 °C, degradation of AA

can be detected between 24 and 48 h. By 14 days, 29% (2.9 mg/L) of the remeiining AA at thi high concentration and

82% (3.2 mg/L) at the low concentration were oxidized. At

25 °C, similar results were observed within 24 h (1.9 mg/L)

and at 50 °C, within 3 h (1.6 mg/L). Furthermore, at all three temperatiu-es a measurable amoimt of the total AA

w£is not accounted for at the end of the experiment; evi- dently some of the DHAA was further degraded. Thus,

under field conditions, MPA-treated clinical samples can be

stored for a day at 4 °C without significant loss of AA or of

its initial degradation product (DHAA), but higher temperatvu-es incur a risk of significant loss. Frozen samples

should be thawed before analysis by weirming the samples

at 20 °C for 10 min to ensure minimal degradation. Furthermore, when MPA-preserved samples are kept at room

temperature for extended periods in the absence of DTT,

the AA can be expected to undergo degradation. These

samples will not yield accurate measurements of native

AA.

Under the conditions described above, we did not observe

AA concentrations significantly exceeding the amount to which the plasma was supplemented. This suggests that

earlier results obtained with a colorimetric method are in

error, the source of which remains undefined (5, 14). We

were also able to account for all of the DHAA that we

generated. This is in contrast to the lesser recovery re- ported by Baker et al. (5), a discrepancy that may be the

result of incomplete conversion of the DHAA dimer to the monomer in the plasma ssmaples supplemented with commercial DHAA. Finally, we have confirmed that aged

plasma can slowly oxidize AA to DHAA at a rate similar to that reported by Baker et al. (3) and that the DHAA

resulting from the oxidation of AA can be determined by

the method described herein.

In conclusion we have described a technique that is suitable for use in the future evaluation of the stability of AA and DHAA in whole blood and for the elucidation of suitable conditions for conserving the physiological ratio of

these compounds so that the epidemiological significance of

1754 CLINICAL CHEMISTRY, Vol. 36, No. 10, 1990 4-7
the natural concentrations of AA and DHAA can be explored.

We thank Dr. Robert SchaflFer for his assistance in the preparation of the ascorbic-acid-supplemented human plasma samples and

Dr. Willie May for coordination of the NIST/NCI Program. This

study was supported in part by National Cancer Institute contract

no. Y01-CP9-0506.

References

1. Pelletier O. Vitamin C. L-Ascorbic acid and dehydro-L-ascorbic

acid [Review]. In: Augustin J, Klein BP, eds. Methods of vitamin

assay. New York: John Wiley and Sons, Inc., 1985:303-^6.

2. Roe JH, Mills MB, Oesteriing MJ, Damron CM. The determi- nation of diketo-L-gulonic, dehydro-L-ascorbic acid and L-ascorbic acid in the same tissue extract by the 2,4-dinitrophenylhydrazine method. J Biol Chem 1948;174:201-8.

3. Baker JK, Kapeghian J, Verlangieri A. Determination of ascorbic acid and dehydroascorbic acid in blood plasma samples. J Liq

Chromatogr 1983;6:1333-^.

4. Huang T, Kissinger PT. Simultaneous LC determination of

ascorbic acid in foods and biological fluids. Curr Separations

1989;9:20-3.

5. Lunec J, Blake DR. The determination of dehydroascorbic acid and ascorbic acid in serum and synovial fluid of patients with rheumatoid arthritis (RA). Free Radical Res Commun 1985;1:31-

9.6. Vanderslice JT, Higgs DJ. Chromatographic separation of

ascorbic acid, isoascorbic acid, dehydroascorbic acid and dehydroisoascorbic acid and their quantitation in food products. J Micro- nutr Anal 1988;4:109-18.

7. Okamura M. An improved method for determination of Lascorbic acid and L-dehydroascorbic in blood plasma. Clin Chim

Acta 1980;103:259-68.

8. Schreiber J, Lohmann W, Unverzagt D, Otter A. Determination

of ascorbic acid, dehydroascorbic acid and isoascorbic acid in blood. Fresenius Z Anal Chem 1986;325:473-5.

9. Otsuka M, Kurata T, Arakawa N. The tissue distribution of

L-ascorbic acid and dehydro-L-ascorbic acid in the guinea pigs

injected intravenously with dehydro-L-ascorbic acid. J Nutr Sci Vitaminol (Tokyo) 1986;32:259-66.

10. Doner LW, Hicks KB. High performance liquid chromatographic separation of ascorbic acid, erythorbic acid, dehydroascorbic acid and diketogulonic acid. Anal Biochem 1981;115:225-30.

11. Dennison DB, Brawley TG, Hunter GLK. Rapid high-perfor- mance liquid chromatographic determination of ascorbic acid and

combined ascorbic acid-dehydroascorbic acid in beverages. J Agric

Food Chem 1981;29:927-9.

12. McGahan MC. Ascorbic acid levels in aqueous and vitreous humors of the rabbit: eflFects of inflammation and ceruloplasmin.

Exp Eye Res 1985;41:291-8.

13. Margolis SA, Davis TP. Stabilization of ascorbic acid in human plasma, and its liquid chromatographic measurement. Clin Chem 1988;34:2217-23.

14. Bradley DW, Emery G, Maynard JD. Vitaunin C in plasma: a

comparative study of the vitamin stabilized with trichloroacetic acid or metaphosphoric acid and the eflFects of storage at —70°,

-20°, 4°, and 25° on the stabilized vitamin. Clin Chim Acta

1973;44:47-52.

15. Kutnink MA, Skala JH, Sauberlich HE, Omaye ST. Simulta- neous determination of ascorbic acid, isoascorbic acid (erythorbic

acid), and uric acid in human plasma by high-performance liquid

chromatography with amperometric detection. J Liq Chromatogr

1985;8:31-^6.

4-8

CLINICAL CHEMISTRY. Vol. 36, No. 10, 1990 1755
[Reprinted from CLINICAL CHEMISTRY, 34, 2217 (1988).] Copyright 1988 by the American Association of Clinical Chemistry and reprinted by permission of the copyright owner.

Stabilization of Ascorbic Acid in Human Plasma, and Its Liquid-Chromatographic Measurement

Sam A. Margolis^ and Thomas P. Davis^

We describe two independent HPLC procedures for the

rapid, accurate analysis for ascorbic acid in human plasma.

No sample extraction or phase separation is required. We

also describe a procedure for preparing a human plasma

reference material for use in clinical laboratory analysis for ascorbic acid. The ascorbic acid in plasma can be deter- mined in 15 min, with as little as 50 /xL of sample. Analytical recoveries are near 1 00% with direct injection of deproteinized plasma. Extensive stability data under several conditions, with dithiothreitol as a preservative (antioxidant), indi- cate that ascorbic acid remains stable in stored plasma for as

long as 57 weeks. CVs for round-robin analysis of 1 1 normal

human blood samples by two independent methods were

between 0.1% and 5.3%. These clinical samples appear to be stable for at least 50 days under the described conditions

of stabilization and sample treatment. Finally, t>ecause

ascorbic acid prepared by the described procedures is stable

at room temperature for at least 18 h, these methods can be

readily adapted to clinical laboratory automation at room

temperature.

Additional Keyphrases: dithiothreitol preservative • reference

materials sample handling electrochemical detection

Currently, the recommended method for preparing plasma for assay of ascorbic acid (AA) consists of drawing the

blood into a suitable anticoagulant, transferring it to the

laboratory, removing the cellular material by centrifugation, and adding metaphosphoric acid (MPA) to precipitate the proteins and prevent oxidation of the AA.^ The resulting

suspension can then be stored at -80 "C for extended

periods il-4) with no decrease in apparent AA content. However, in routine laboratory procedures, 15 min to several hours can elapse between blood sampling and treatment

to stabilize AA, or between the thawing of a frozen specimen

and its analysis. A significant loss of ascorbic acid content, and erroneous measxirements of ascorbic acid in plasma, can

result (6). We confirmed the utility of dithiothreitol (DTD as a preservative for AA in solution (7), and demonstrated that

dilute solutions of AA in acetonitrilerwater (3:1 by vol) containing 1 g of DTT per liter are stable for 60 days when

stored at -20 "C under argon in sealed glass ampules (3). Our objective in this study was to evaluate the degree of

stabilization of AA conferred by DTT at a final concentration of 1 g/L (5.4 mmol/L) under the following conditions: (a) in plasma, when it is added at the same time that the

plasma is supplemented with AA; (6) in blood, when the

blood is drawn into heparinized tubes containing a solution

of DTT such that the final concentration of DTT is 1 g/L; and

' Organic Analytical Chemistry Division, Center for Analytical

Chemistry, National Bureau of Standards, Gaithersburg, MD

20899.

^ University of Arizona Health Sciences Center, Department of Pharmacology, College of Medicine, Tucson, AZ 85724.

^ Nonstandard abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; MPA, metaphosphoric acid; DTT, dithiothreitol; NBS, National Bureau of Standards. Received April 28, 1988; accepted July 7, 1988.

(c) in plasma, when it is added with MPA (17 g/L final concentration). In addition we report two simple, rapid, accurate, and precise HPLC methods for determining AA in plasma that can be easily adapted for routine use by clinical chemistry laboratories.

Materials and Methods*

We used two independent HPLC emalsiiical procedures.

Method 1, performed at the National Bureau of Standards

(NBS), was used to evaluate the stability of AA in DTTsupplemented human plasma samples. This procedure and a

second procedure, method 2 (University of Arizona), were

used to evaluate the stabilizing effects of DTT on patients'

samples.

Method 1 AA, dehydroascorbic acid (DHAA), and DTT were pur- chased from Sigma Chemical Co., St. Louis, MO. The

acetonitrile was "HPLC" grade. The AA, examined by

proton magnetic resonance, contained <0.1% impurities,

including those related to AA or the degradation products of AA. Human plasma was prepared by Alpha ITierapeutic

Corporation and purchased from Western States Plasma

Corp., Fallbrook, CA, in 650-g lots. This plasma was ob- tained by plasmapheresis fit)m human blood to which 50 mL

of a solution of sodiimi citrate per 500 mL of blood (final concentration, 5 g/L) was added. The processed plasma was

stored at -70 "C until used. In the stability studies, supplemented human plasma

extracts containing AA were analjrzed with a Model 5560

chromatograph equipped with a Model 402 data station, a UV-200 variable-wavelength detector, a Model 8085 auto- sampler (all from Varian Associates, Sunnyvale, CA), and a

Model LC-2 electrochemical detector (Bioanalytical Systems

Inc., West Lafayette, EN).

The supplemented plasma (lots 58-61) was prepared as

follows: after slowly thawing a 650-g package of plasma at room temperatvire overnight, we removed any insoluble

material by filtration through cheesecloth. This yielded 595

mL plasma, to which we added 611 mg DTT. The resulting

solution was divided into four 148-mL lots. 'We supplemented three of the lots with AA by adding —0.3, 0.75, or 1.0 mL,

respectively, of a 1.01 g/L AA stock solution. The fourth lot served as a blank for assessing the presence of possible

substances interfering with the measurement methods.

Each plasma lot was distributed in 1.3-mL volumes into 5- mL serum vials. The vials were then loosely capped with

split serum stoppers, frt)zen, and freeze-dried. The stoppers,

which were closed automatically after the samples were

completely freeze-dried and the vacuum had been released, were secured with metal caps and stored at either -25 or -80 °C. The process of filtering, supplementing, sampling,

and freezing was completed within 3 h. Each sample could

* Identification of any commercial product does not imply recom- mendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.

CLINICAL CHEMISTRY, Vol. 34, No. 1 1 , 1988 2217 4-9
be reconstituted to its original volume by adding 1.2 mL of

distilled water. To extract the AA from the freeze-dried plasma samples we added 1.5 mL of distilled water to each sample. After the

material completely dissolved, we transferred the entire

solution to a 4-mL screw-cap vial containing 160 /uL of 400

g/L aqueous MPA, capped the vial, and vortex-mixed for 15

s. We then added 400 fih of HPLC-grade acetonitrile, capped

the sample, and vortex-mixed for 15 s. (This step improved

protein aggregation and prevented precipitation of material on the top of the HPLC colvunn.) After centrifuging the

resulting suspension (1000 x g, 30 min, 4 °C), we transferred the supernatant fluid to 1.8-mL vials, placed them in an autosampler, and flushed 300 /iL of sample through a 20-

fjL sampling loop (to eliminate carryover from the previous

sample).

The samples were analj^zed by a modification of the

method of Grun and Loewus (6) with a 300 x 7.8 mm

Aminex HPX87H cation-exchange column (Bio-Rad Laboratories, Richmond, CA). The column was equilibrated at 30 °C with 0.1 mol/L formic acid containing 50 mL of

acetonitrile per Uter, at a flow rate of 0.5 mL/min. We added

the acetonitrile to prevent bacterial growth and improve

peak shape.

The chromatogram was developed isocratically and the

eluted components were detected spectrophotometrically at 265 nm and electrochemically at +0.7 V, in the oxidation

mode. The amount of AA was determined by peak-height

measurement (electrochemical determinations) and by peeik

integration (ultraviolet absorbance and electrochemicjd de- terminations).

Method 2

Ascorbic acid ("Gold Label") was pvuxhased fix)m Aldrich

Chemical Co., Milwaukee, WI. DTT, EDTA, chloroacetic

acid, and MPA were purchased from Sigma Chemical Co. Octyl sodiimi sulfate was purchased from Eastman Kodak

Co., Rochester, NY.

The HPLC instrumentation consisted of a Model 6000A

pump (Millipore, Waters Chromatography Div., Milford,

MA), a Model 7125 sampling valve with a 25-fjL sampling

loop (Rheodyne Inc., Cotati, CA), a Model 3390A integrator

(Hewlett Packard Co., Palo Alto, CA), a Model LC-4A

electrochemical detector (Bioanals^cal Systems Inc.), and a

250 X 4.6 mm Ultreisphere coliomn (Beckman Instruments

Inc., FuUerton, CA) packed with 5-^tm octadecylsilane particles. All chromatographic solvents were filtered through a

0.2-fjm (pore size) Nylon-66 filter (Rainin Instrument Co.

Inc., Wobum, MA) before use. All standard solutions of AA were either refrigerated or

stored on ice until analysis. We prepared the standard

solutions at concentrations that bracketed the expected

range of concentrations in clinical samples by making

appropriate immediate dilutions of a stock solution (1 g/L in an aqueous solution of MPA, 30 g/L). Blank plasma was

supplemented with a similar stock solution. The AA stan- dard curve was determined by transferring 100 /jL of supplemented or unsupplemented plasma to a 1.5-niL "microfiige" tube containing 900 /xL of a cold, freshly prepared

solution of MPA (30 g/L) and DTT (1 g/L). After standing 10

min, the samples were centrifuged (15 min, 11 600 x g) and

a 25-/xL portion of the supemate was analj^zed by HPLC.

Clinical samples of himian plasma were prepared for storage at -70 °C by adding 100 fjL of plasma to 300 ijL of the MPA/DTT solution, mixing, and promptly freezing at

-70 °C. At the time of analysis we added 600 ^iL of a fi^hly

prepared 30 g/L solution of MPA to the sample, thawed the

sample, mixed well, centrifuged (11 600 x g, 15 min), and

analj^zed 25 /uL of the supemate by HPLC.

The analytical colimm and the electrochemical detector were conditioned (passivated) with an aqueous solution of Na4EDTA (2.55 g/L) at a flow rate of 1 mL/min for 30 min

before analyzing for AA. We used isocratic elution at a flow

rate of 0.5 mL/min at ambient temperature with a column

that was eqviilibrated with a mobile phase consisting of 14.1 g of chloroacetic acid, 4.65 g of NaOH, 0.85 g of Na4EDTA,

and 200 mg of octyl sodium sulfate in 1 L of distilled water. The electrochemical detector was set at +0.7 V and the

amount of AA was quantified by peak-height measurement

and electronic peak integration.

The plasma samples for use in the study comparing

methods 1 and 2 were processed within 1 h. We drew 15 mL

of blood into heparinized, foil-wrapped tubes and centri- fuged immediately. Seven milliliters of plasma was promptly removed and 250 ptL of a solution containing 1.2 mg DTT

(final concentration, 0.17 g/L) and 125 mg MPA (final concentration, 17.2 g/L) was added. The samples, divided

into 1.3-mL aliquots, were stored at -80 °C until an£dyzed. For analysis by method 1, we added 1.3 mL of sample to 160 of a 400 g/L solution of MPA and mixed for 15 s, then

added 400 piL of acetonitrile, mixed for 15 s, centrifuged,

and analj'zed the supernatant fluid for AA as described

above. For analysis by method 2 the protocol previously

described was followed. Each of the split samples from each

patient was analyzed in duplicate.

Results

The utilization of DTT as an antioxidant for AA has been

well doctmiented (3, 7, 8). Recent studies at NBS demonstrated that DTT is also an eflFective long-term preservative

for AA (3). In some of the analyses for AA by HPLC (3, 8) an

aminosileme column was used, which resolved AA and DTT.

We foimd it difficult to obtain reproducible chromatographic

separations with different aminosilane columns. We also foimd it diffiailt to prevent degradation of the AA during

the separation process on an aminosilane column without

resorting to elaborate sparging and the use of antioxidants

in the HPLC solvent (3). We circvunvented these problems

by using the HPX87H or an ODS 5-/xm particle column. AA

is not destroyed on these columns and AA is resolved fit)m

MPA, uric acid, and DTT, all of which produce an electro- chemical response and could contribute to errors in measurement if tiiey are not completely separated from AA.

Measurement of AA in plasma with an ultraviolet detector is diffiailt because there are several intense ultraviolet- absorbing peaks that elute very close to AA, and large

amoimts of sample must be used in order to obtain a

sufficient signal for AA quantification. The use of an electro- chemical detector results in increased sensitivity and selec- tivity (9) as compared with the ultraviolet technique. However, the DTT also has a strong electrochemical sipial at the same voltage as AA (3). Because in our experience some ion- pairing methods do not adequately resolve DTT fi:t)m AA, it was necessary to select an HPLC method of analysis that would resolve these compoimds. Methods 1 and 2 both give

baseline resolution between uric acid, DTT, and AA (Fig- ures 1 and 2), and method 1 also resolves two additional

trace materials (Figure 1) that are present in AA-d^leted

plasma.

The range and sensitivity of the electrochemical method

2218 CLINICAL CHEMISTRY, Vol. 34, No. 11, 1988 4-10
are particularly noteworthy: no more than 500 ng of AA can

be quantified under the conditions of these studies. It is also

possible with a favorable signal/noise ratio to measure as

little as 2 ng of AA (Figure 2A). For method 1 the amount of AA routinely analyzed varied from 40 to 400 ng in 20 /xL of sample and for method 2, 5 to 30 ng in 20 /iL of sample

(Figure 2). Furthermore, analysis by method 2 is routinely

completed in <10 min.

Stability of the Ascorbic Acid during Automated Analysis

All of the analyses done by method 1 were performed with an autosampler during ~24 h. Under these conditions, the

stability of AA at room temperature becomes important (1, 2, 10). We evaluated this variable by measuring the area of

the chromatographic peaks absorbing in the ultraviolet seen on chromatographing standard solutions containing DTT.

For periods up to 18 h, little or no deterioration of the standard AA solutions at 21 °C was detected, an observation

supported by the fact that the relation between the concentration of AA and the area of the AA HPLC peak was linear from 2 to 20 mg/L and closely approached the origin (slope =

49124 arb. units, intercept = 817, = 0.9999, n = 6). When

stored at 4 °C, these solutions remained stable for several weeks, after which new standards were prepared. For standards stored at 4 °C (method 2) the AA measured

remained constant for the duration of the experiment. The

concentration curve for these standard solutions was linear from 5 to 20 mg/L, intersecting the origin with a slope of 57.900 and a correlation coefficient of 0.9992. The standards

and samples Einalyzed by this method were never stored at room temperatiu-e.

100

so

5.0 nA

MOB

L JJ

I J L _L

0 10 20 30 0 10 20 30 0 Time (min)

10 20 30 0 10 20 30

Rg. 1 . Separation of ascorbic acid in human plasma on an HPX87H

column (method 1

)

A unsupplemented plasma; S, plasma with acWed AA, 4.80 mg/L; C, plasma of subject 1 (Table 4) assayed on day 52; O, AA standard, 4.99 mg/L, in acetonrtrile/ water (3/1 by vol). Upper traces are electroctiemicaJ ctiromatograms; lower traces are ultraviolet chromatograms at 268 nm. Peak identity: 1, MPA; 2, AA; 3, uric acid; 4, DTT

200

5.0 ng

2

2 cm = 6.6 min.

Time (minutes)

0 3 6 91215

Time (minutes)

Fig. 2. Separation of ascorbic acid on an octadecylsilane column

(method 2) A starxlard solutions; S, human plasma. Peak identity as in Rg. 1

Stability of the Electrochemical Detector Of primary consideration was the time-dependent variation in the sensitivity of the electrochemical (letector. Method 1 was characterized by a time-dependent decrease in the

detector sensitivity, which varied from one analysis to another. This was demonstrated by measuring, in duplicate, a series of standards (three different concentrations) at the

beginning and at the end of an experiment. Each set of measurements was plotted separately. The ultraviolet ab- sorbance of the standards remained constant throughout the

experiment (Figure 3B), whereas the electrochemical re- sponse decreased by a constant vsdue over the entire concentration range, resulting in parallel curves displaced by a

constant value (Figure 3A). Additionally, the standard of intermediate concentration (4.2 mg/L) was assayed after every two plasma samples (three additional times), and the

resiilts decreased steadily throughout the experiment, indi- cating a continuous change in the sensitivity of the electro- chemical detector. In contrast, method 2 was characterized by a timedependent increase in detector sensitivity; that is, the slope of the standard curve increased and the intercept remained

constant over a 4-h period (Figure SO. Thus, for both

methods, fi^uent standards were run, to correct for variations in response of the electrochemical detector.

Stability of Ascorbic Acid in Human Plasma Supplemented

with Dithiothreitol Okamura (7) demonstrated that DTT reduces DHAA, so that total AA in plasma can be measured. To test the utility of using this reagent for reducing plasma DHAA and

preserving plasma AA for extended periods of time, we

modified pools of AA-depleted human plasma by adding

DTT to give a concentration of 1 g/L (5.3 mmol/L) and

subsequently supplemented with AA to give concentrations

between 2 and 16 mg/L in plasma. Both fi^ze-dried and

frozen supplemented 1.3-mL samples of plasma were pre- pared from these pools and stored at -80 °C for as long as

18.5 months. The AA content of these samples, measured at the indicated time intervals, is summarized in Table 1. Randomly selected samples fit)m each of lots 37, 38, 45, and

46 were anal3rzed in duplicate at the time intervals indicat- ed in Table 1. These values were within one standard

CLINICAL CHEMISTRY, Vol. 34. No. 11, 1988 2219 4-11
0.25 0.50 0.75 1.0

Concentration ()jg/ml)

Rg. 3. Concentration-response curves for standard solutions of ascorbic acid as a function of storage time A ultraviolet detection at 268 nm, method 1 ; B, electrochemical detection at 0.700

V, method 1 ; C, electrochemical detection, at 0.700 V, method 2. The symtxDis represent the following time periods: • •, 0 time; 4 h; x-— x, 18 h

deviation of the amount gravimetrically added to the origi- nal plasma pool. This indicates that the AA is not oxidized

in the presence of DTT imder these storage conditions. Lots 58-61 constituted a second group of samples, which was

prepared and freeze-dried. This group included an imsupplemented pool of plasma and three pools of plasma supplemented to contain ~2, 4.8, and 6.7 mg of added AA per liter of plasma. Four samples from each pool were analyzed in duplicate two weeks after the samples were prepared, to confirm the initial AA concentration. The plasma AA con- centration of sfunples randomly selected from these pools was also measured at the time intervals indicated in Table

1. The results complement and support the conclusion that

DTT indeed protects and preserves the integrity of AA in human plasma for at least 66 weeks when stored at -80 °C. Finally, AA was never detected in any of the vmsupplemented plasma samples (lot 59, Figure LA).

The utility of this method for preparing reference materials for the measurement of AA in plasma is further supported by the results obtained in two impublished collaborative studies (lots 37, 45, and 46; and lots 58, 60, and 61) co- sponsored by NBS and The National Cancer Institute (Table

2). The values reported in these collaborative studies, which

were obtained with a variety of methods, show acceptable

agreement with the measurements made at NBS.

Mechanism of Action of DTT

DTT completely reduces DHAA either in plasma or in a

mixture of acetonitrile and water (75:25 by vol). The reacTable 1 . Stability of Ascorbic Acid in Freeze-Dried and

Frozen Piasma

Weeks stored Plasma lot 0' 2 6* 12' 27" 57' 66* 80' 37 6.50« 6.51 6.27 38 15.60 15.63 15.69 15.21 45 8.80 8.80 9.11 46' 8.80 8.54 8.80 58 6.7 6.30 6.41 6.27 6.46 6.30 6.58 60 2.0 1.82 1.85 1.81 1.74 1.83 1.83

61 4.8 4.80 5.12 4.98 4.47 4.94 4.65

"Values for lots 37-46 represent gravimetric additions; values for lots 58-

61 , at 0 time only, represent estimated concentrations. "Means of eight measurements. Means of four measurements.

"Values for lots 37—46 are means of eight measurements and for lots 58-61 means of four measurements. "AA concentration, mg/L. The CV for each value in this table ranges

ttetween 0.25% and 4.43%.

'Lot 46 was frozen only; all other lots were freeze-dried.

Table 2. Comparison of Round-Robin AA Values and

Values Obtained by NBS (mg/L)

Round-robin results* NBS results"

Plasma lot Mean SD« n Mean SO" n 37 6.34 1.09 14 6.33 0.21 13 45 9.34 1.55 14 9.04 0.42 16

46'' 9.25 1.07 14 8.73 0.26 16

58 5.30 1.29 10 6.30 0.21 30

60 1.72 0.46 10 1.81 0.08 30

61 4.22 0.97 10 4.83 0.13 30

"Round-robin measurements on lots 38, 45, and 46 were completed 13 weeks after the samples were prepared and the round-robin measurements on

lots 58, 60, and 61 were completed four weeks after sample preparatnn. Each

round robin represented an independent set of duplicate measurements on

two different samples by each of seven and five latx)ratories, respectively. "Data represent an aggregate mean of all data acquired in the stability studies summarized in Table 1

.

Standard deviation of a single measurement.

"Lot 46 was frozen only; all other lots were freeze-dried.

tion is complete within 60 min (compare the 1- and 4-h

values in Table 3). If MPA is added to the DTT before the

plasma is added, the reduction still occurs (Table 2 and

Figure 4). Thus by measuring the AA both before and after addition of the reducing eigent, DTT, it is possible to measure the AA and DHAA diflFerentially in biological

samples by an electrochemical method. However, it is still impossible to determine by this method whether the DHAA

was initially present in the plasma or was formed during

removal of the blood cells.

Table 3. Conversion of DHAA by DTT

Ascorbic acid, mg/L*

Time of analysis 1 h 4h

Experiment 1 Solvent" 0 0

Solvent + DTT 8.7 8.8 Plasma 0 0 Plasma -i- DTT 9.1 8.8 Experiment 2

Plasma 0 0 Plasma + DTT 6.3 6.8 MPA + DTT -1- plasma 6.1 6.7

* Each value represents the mean of duplicate measurements which differ by <5%.

"Acetonitrilerwater, 3:1 by vol.

2220 CLINICAL CHEMISTRY, Vol. 34. No. 1 1 . 1988 4-12
We evaluated the effect of DTT on AA in blood by

collecting into heparinized tubes two blood samples from a

patient with very low concentrations of AA in his plasma.

One of the blood samples (9.5 mL) was collected into 0.5 mL

of a solution containing 15 mg DTT; the other blood sample

served as a control. AA was added to each blood sample to give a concentration of 7 mg/L, the plasma was prepared,

and the AA was measured by method 2. Five repUcates of each measurement were made. The results indicate that

there was no destruction of AA in either sample [plasma 7.2 (SD 0.6) mg/L, and blood 7.5 (SD 0.5) mg/LJ. Thus one can

collect blood samples directly into DTT solutions without

affecting the ability to remove the cells.

Assessment of the Stability of Patients' Plasma Ascorbic

Acid Preserved in DTT and MPA

The utility of a method for stabilizing AA in human

plasma depends on the demonstration that it is efficient and

that the same results can be obtained by two or more

independent methods. A comparison of the results obtained

by each method (1 vs 2), during the same time period, for the

indicated lots of reference plasma samples showed, for lot 37

(6.00 vs 6.15 mg/L), 45 (8.80 vs 8.95 mg/L), 46 (8.80 vs 9.15 mg/L), 58 (6.27 vs 6.00 mg/L), 60 (1.81 vs 2.05 mg/L), and 61

(4.98 vs 4.90 mg/L). Evidently, the two methods yield

equivalent results. Furthermore, comparison of the experimental values for plasma lots- 37, 45, and 46 with the

gravimetric values in Table 1 also indicates that the methods are accurate. On this basis, a series of 11 blood samples

from normal subjects was collected, centrifuged, and the

pleisma preserved within 1 h as described in the Methods

section. This group consisted of five men and six women, 333 100 2 2

<

- 1 1 1

s ssuodsai

V ^JL i _ » _

4

0

3

A

3

B

2

3 C

2

3

r>0

X

2 ato

1 1

<

1

0 I J. -

4 La

J \ I l/fJ 1 1 L//J \ 1 L

0 10 20 30 0 10 20 30 0 10 20 30 Time (min)

Fig. 4. Effect of DTP and MPA on the conversion of DHAA to ascorbic

acid in human plasma

A DHAA (7 mg/L) in plasma; S, DHAA (7 mg/L) in plasma after exposure to DTT

(1 g/L) for 1 h; C, DHAA (7 mg/L) in plasma after exposure to DTT (1 g/L) and MPA (20 g/L) for 1 h. Upper traces are electrochemical chromatograms; lower

traces are ultraviolet chromatograms at 268 nm. Peak identity as in Fig. 1

Table 4. Ascorbic Acid Concentrations (mg/L) in Plasma of 11 Normal Subjects'

AA concentration, mg/L

Sex of Days stored"

subject 23 32 52 Mean SO'

F 7.8 7.3 7.8 6.9 7.5 0.4 F 19.9 19.2 18.7 18.9 19.2 0.5 F 8.7 8.4 8.6 8.5 8.6 0.1 M 10.8 10.8 10.9 10.8 10.8 0.0 F 12.1 11.8 12.3 12.0 12.1 0.2 F 7.6 8.1 7.7 8.2 7.9 0.3 M 8.7 8.8 8.5 8.6 8.7 0.1 M 9.8 10.0 9.5 10.4 9.9 0.3 M 15.1 15.9 14.9 15.4 15.3 0.4 M 7.9 7.9 7.6 8.0 7.9 0.1 F 16.3 16.5 16.3 16.0 16.3 0.2 "Ago 18 to 40 y. "The samples on days 23 and 51 were analyzed by method 1 and the samples on days 32 and 52 were analyzed by method 2. Each value

represents the mean of duplicate analyses. Standard deviation of a single measurement.

ages 18—40. The plasma samples were prepared at the

University of Arizona, frozen at -80 °C, shipped in solid CO2, and stored at -80 °C imtil the /lA was measured. The

results are summarized in Table 4. The CV for these determinations was between 0.1"^) (subject 1) and 5.3%

(subject 10). The size of the standard deviation does not appear to be concentration dependent (compare subject 1 with 9) or method dependent (same subjects). Furthermore,

the samples appear to be stable for no less than 52 days,

thus establishing the utility of this method for collecting

£Lnd storing patients' plasma for AA measurement.

Discussion

Measuring AA in pleisma is viseful in the diagnosis of

deficiency syndromes, the assessment of human nutritional

status, and in confirming subject adherence (compliance) to experimental protocols such as chemoprevention and che- motherapy studies (13). For large, multilaboratory studies,

it is essential to have suitable stable reference materials to assess the quality control, precision, and accuracy of participating laboratories. The hvunan plasma reference material

that we describe here can be prepared by a central laboratory for the assessment of interlaboratory and intralaboratory performance or can be prepared in the individual

laboratory and used for an extended period of time to assess intralaboratory quality control. The use of reducing agents to convert DHAA to AA is well

docimiented, partioilarly with respect to methods in which

the AA was derivatized before spectrophotometric measure- ment (12) as well as techniques involving HPLC {3, 4, 8, 11). The utility of DTT as a reducing agent for DHAA in plasma was demonstrated by Okamura (7) and used for the measurement of AA in firtiit juices (.4, 8) and urine (8). Okamura

(7) noted that among the reducing agents tested, DTT

exhibited reducing activity over a broader pH range than

the other compounds examined, and in aqueous solution it was more stable than other reducing agents. It was superior

to glutathione, mercaptoethanol, cysteine, homocysteine,

and 2,3-dimercapto-l-propanol. By using DTT to convert DHAA to AA, Okamura was able to measure differentially

DHAA and total plasma AA by assasdng AA in the presence and absence of DTT. Since then, DTT was shown to be an

effective antioxidant for the long-term preservation of AA in

4-13 CLINICAL CHEMISTRY, Vol. 34, No. 11, 1988 2221
aqueous organic solution (3). These observations were ex- tended to include plasma, which can be stored in the presence of DTT for more than one year at -80 °C without

the AA undergoing any oxidation (Table 1). Blood can also be collected into a solution of DTT without causing coagulation. Therefore, DTT can be introduced immediately at the

time of blood sampling, thus preventing any oxidation of AA, either during the process of removal of blood ceils or during subsequent storage. However, this procedure pre- cludes the measurement of any native DHAA.

The differential content of DHAA and AA can be deter- mined by first obtaining the plasma, treating it with MPA

to stabilize it, and then measuring the plasma AA both in the absence and presence of DTT. The difference between

these measurements represents the DHAA content. This

follows from the observation that in the presence of MPA (20

g/L) and DTT (1 g/L), DHAA in plasma is converted to AA

(Table 3 and Figure 4). This type of experiment was done

with fruit juice (4, 8, 11) and urine (8). The measurement of total AA in plasma preserved at -80 °C in DTT (170 mg/L) plus MPA (20 g/L) and the

stability of clinical samples preserved in this manner was

evaluated on split samples from 11 subjects. The samples were stable for 52 days (Table 4) eind detectable amounts of DTT were still present in the sample (Figure lO. We did not

observe any significant consistent variation in the AA

content of these samples, and these observations are consist- ent with the results reported by other investigators (1,4).

Thus DTT prevents oxidation of AA in blood samples

without causing coagulation (at a concentration of 1 g/L),

and in pleisma for extended periods of time in the presence or absence of MPA and in either the frozen or freeze-dried

state. All of the colorimetric methods are based on either the

direct oxidation of AA by a colorimetric reagent or the

oxidation of AA to DHAA and the subsequent derivatization

of DHAA to a material that can be measured colorimetrically (12). The lack of total specificity of these reactions can

lead to significant errors in measurement. Because all of these methods involve the measurement of a reduced form of AA, the presence of relatively large amounts of a reducing

agent (DTT) can introduce a significant error. Okamura (7) solved this problem by reducing his AA samples with

sulfhydryl reagents and then removing the sulfhydryl re- agents by treating his samples with iV-ethylmaleimide.

Thus, reference AA samples treated with DTT can be used

as standards for some colorimetric AA assays if the samples

are first treated with a sulfhydryl reagent such as A^- ethylmaleimide.

Using HPLC methods in conjunction with electrochemical

detectors improves selectivity of AA determinations (8). The

increase in selectivity as well as sensitivity over the ultraviolet detectors can be seen in the dual recording in Figtires

1 and 4. It is apparent that the AA signal is not well

resolved from other ultraviolet peaks, whereas baseline

resolution was observed with electrochemical detection. Furthermore, the electrochemical method permits increases

in sensitivity of at least two orders of magnitude as is demonstrated by method 2 (Figure 2). The three HPLC methods that were used to analyze AA in the presence of DTT were chosen because they resolve DTT

from AA. These include analysis on an aminosilane column

in a previous study (3), an HPX87H colvunn (method 1), and

a reversed-phase column (method 2). Each of these systems

resolves DTT, uric acid, and metaphosphoric acid from AA.

We were unable to obtain consistent results with different aminosilane columns and therefore, have adopted the two methods described herein. Both HPLC methods are capable of measuring accurately the AA concentration in gravimetrically supplemented samples (37, 38, 45, and 46). The accuracy of these two methods was compared by analyzing the reference samples described

in Tables 1 and 2. The results are in good agreement, and

because the unsupplemented plasma contained no electro- chemically detectable material with the retention time of AA, these results indicate that both methods accurately measure the AA in plasma. Furthermore, the use of two

independent methods that are based on different mechanisms of separation (ion exchange vs paired ion chromatography) lends credence to the accuracy of the measurement

and decreases the probability that interfering substances

are present in the analyte peak.

With the knowledge that these methods were accurate within the limits of the two methods and that DTT reduced

DHAA and preserved AA in supplemented plasma samples, we evaluated the plasma AA content of a group of normal

subjects. The results of this study indicate that samples that are preserved with MPA (17 g/L) and DTT (170 mg/L) at -80 °C are stable for no less than 52 days and can be

shipped transcontinentally in solid CO2. The values thus

obtained indicate that in normal subjects the AA concentration in plasma can range from ~7 to 19 mg of plasma per

liter. Thus, a suitable assay procedure should include stan- dards from subnormal concentrations of AA to concentrations comparable to 20 mg of AA per liter of plfisma. Using

either of these separation techniques coupled with electro- chemical detection, a laboratory can easily automate the

analysis of six to eight clinical samples per hour. We thank Ms. Margaret Kline and Dr. Robert Schaffer for their assistance in the preparation of the ascorbic acid-supplemented

human plasma samples; Alison Culling-Berglund and Wendi Morfitt for their technical assistance; Dr. Robert Paule for his advice on

the statistical analysis; and Dr. Willie May for coordination of the NBS/NCI Quality Assurance Program. This study was supported in part by the National Cancer Institute contract nos. YOl-CN-70645

(NBS) and NCI-CA 27502 (University of Arizona).

References

1. Bradley DW, Emery G, Maynard JD. Vitamin C in plasma: a

comp£irative study of the vitamin stabilized with trichloracetic acid

or metaphosphoric acid and the effects of storage at -70°, -20°, 4°, and 25° on the stabilized vitamin. Clin Chim Acta 1973;44:47-52.

2. Baker JK, Kapeghian J, Verlangieri A. Determination of ascorbic acid and dehydroascorbic acid in blood plasma samples. J Liq

Chromatogr 1983;6:1333--44.

3. Margolis SA, Black I. Stabilization of ascorbic acid and its measurement by liquid chromatography in nonfat dry milk. J Assoc

Off Anal Chem 1987;70:806-9.

4. Lloyd LL, McConville JA, Warner FP, Kennedy JF, White CA. HPLC analysis of L-ascorbic acid (vitamin C) using a polymeric

reversed-phase adsorbent. Liq Chromatogr Gas Chromatogr

1987;5:338-41.

5. Sauberlich HE, Green MD, Omaye ST. Determination of ascorbic acid and dehydroascorbic acid. In: Seib PA, Tolbert BM, eds. Ascorbic acid: chemistry, metabolism and uses [Review]. Washington DC: Am Chem Society, 1982:199-221.

6. Grun M, Loewus F. Determination of ascorbic acid in algae by

high-performance liquid chromatography on strong cation-ex- change resin with electrochemical detection. Anal Biochem

1983;130:191-8.

7. Okamura M. An improved method for determination of l-- ascorbic acid and L-dehydroascorbic in blood plasma. Clin Chim

Acta 1980;103:259-68.

2222 CLINICAL CHEMISTRY, Vol. 34, No. 1 1, 1988 4-14
8. Doner LW. Hicks KB. High performance liquid chromatographic

separation of ascorbic acid, erythrobic acid, dehydroascorbic acid and diketogluconic acid. Anal Biochem 1981;115:225-30.

9. Pachla LA, Kissinger PT. Analysis of ascorbic acid by hquid

chromatography with amperometric detection. Methods Enzymol

1979:62:15-24.

10. Rose RC. Nahrwold DL. Ascorbic acid analysis in biological

fluid. Anal Biochem 1982;123:389-93.

11. Dennison DB, Brawley TG, Hunter GLK. Rapid high-performance liquid chromatographic determination of ascorbic acid and

combined ascorbic acid-dehydroascorbic acid in beverages. J Agric Food Chem 1981;29:927-9.

12. Bui-Nguyen MH. Ascorbic acid and related compounds. In: De

Leenheer AP, Lambert WE, De Ruyter MGM, eds. Modem chro- matographic analysis of the vitamins (Chromatographic Sci Ser, Vol 30). New York: Marcel Dekker Inc., 1985:267-301.

13. Jaffe GM. Vitamin C. In: Machlin LJ, ed. Handbook of vita- mins: nutritioned, biochemical and clinical aspects. New York: Marcel Dekker Inc., 1984: 199-244.

4-15

CLINICAL CHEMISTRY, Vol. 34, No. 1 1 , 1988 2223

DETERMINATION OF VITAMIN K, IN SERUM

In this section a new approach to the detection of serum vitamin Kj is described. This

vitamin may have some involvement in the respiration of tumor cells. Details of the

procedure and critical factors are derived from a manuscript accepted for publication in the

Journal of Chromatography, Biomedical Applications. Listed below are some of the factors

important to this analysis.

Important Factors

Calibration and Sample Preparation

• For the liquid-liquid extraction we recommend serum proteins be precipitated with two

volumes of either ethanol or 2-propanol, and that three sequential extractions with four

volumes of fresh hexane be used.

• Care should be taken to minimize losses through the extraction, solvent evaporation,

solid-phase fractionation elution, and final evaporative concentration by careful washing

of container walls and transfer pipets.

• Concentrations of calibration solutions for vitamin Kj should be assigned by

spectrophotometry; we use a molar absorptivity of 41,000 at 242 nm for this

determination.

• Stock solutions were stored refrigerated at a concentration of approximately 50 |lg/mL

and diluted just prior to use. The stock solutions appeared to be stable for at least 2

months.

• Use an internal standard; we used vitamin Kj (which is rare in most sera and is added to the protein precipitant solution).

• Care should be taken to prevent photodecomposition throughout the extraction and

analysis.

Chromatography ^

• We recommend the use of a polymericaUy bonded Cjg column (25 x 0.46 cm) for the

analytical separation.

5-1
• An "on-line" reducer column (5 x 0.2 cm I.D.) and cataljmc oxygen scrubber (10 x 0.46

cm) should be dry-packed using vibration from a column vibrator or an electric

engraving tool.

• Great care should be taken in handling the catalyst during column preparation and in

disposal since contact of organic solvents with even small amounts of the solid causes spontaneous combustion of the liquid.

• Best sensitivity is achieved with a xenon light-source fluorimeter.

For further information, please contact William MacCrehan at (301)975-3122.

5-2
Determination of Vitamin in Serum

William MacCrehan and Emil Schonberger

Vitamin (phylloquinone) is a substituted naphthoquinone, required for proper blood

clotting. Unfortunately, there is very poor agreement in the published literature on the

measured "normal" serum concentration of Kj, with reported values as low as 0.2 ng/mL and

as high as 2 ng/mL. Most of this disagreement is likely to be a result of poor measurement

technology and lack of serum-based Kj standards for the evaluation of method accuracy. The

higher levels that are found in foods (|J.g/g) may be measured with LC using UV detection of

the strong naphthoquinone absorption in the 240 - 260 nm range. Since serum concentrations

of Kj are three orders of magnitude lower, more selective and sensitive detection is required

for the clinical determination.

NIST has developed a new LC method for the determination of Vitamin Kj based on the "online" chemical reduction of the non-fluorescent quinone to the highly fluorescent

hydroquinone. The reduction is done in a post-column catalytic reducer column packed with

a platinum-on-alumina catalyst (10% Pt) that uses an alcohol added to the mobile phase as the

reducing agent. In the reduction reaction, the quinone is reduced by the solvent in the

presence of the catalyst:

R-CHO

0 OH

Quinone Alcohol Hydroquinone Aldehyde

Figure 1 : The Catalytic Reduction of the K Vitamins.

This is the same catalytic reduction approach that we previously developed for the removal of

mobile phase oxygen prior to reductive electrochemical detection of nitropolyaromatic

hydrocarbons {Anal. Chem. 6, 284-286 (1988)}. To apply this concept to the reduction of

Vitamin Ki, we first evaluated the conditions required for quantitative reduction of the

quinone functionality to the hydroquinone. The reductor was inserted ahead of the analytical

column and the disappearance of the parent and product compounds were monitored by UV

absorbance and fluorescence detection.

5-3
The apparatus for the reductive/fluorescence LC detection of Vitamin Kj in serum is shown in

Figure 2. The chemical conversion to a single fluorescent product is nearly quantitative when

either methanol or ethanol are used at a flow rate of 1 mL/min for a 50 x 2 mm catalyst

column. We found that the catalyst column retained full activity for the 10 months of the

study.

A second issue addressed in the method was the isolation of the Vitamin Kj from the other

fluorescent matrix components that are co-extracted from sera. A liquid-liquid extraction and

a column fractionation on a solid-phase extraction (SPE) cartridge were used to selectively

isolate the Vitamin Kj before the final LC determination.

Serum Extraction and Fractionation Procedure for Vitamin Kj Determination

Five hundred microliters of serum (warmed to room temperature and mixed, if necessary) is measured into a conical centrifuge tube. One milliliter of an approximately 1 ng/mL solution

of K2 in 2-propanol is added by vortex mixing for 5 seconds. Three milliliters of hexane are added and the mixture is vortex mixed for 30 seconds. The layers are separated using

centrifugation. The organic layer is drawn off and placed in an amber vial having a twoholed cap. The sample is dried under a current of dry at 30 - 40 °C. The residue is redissolved by careful washing with rotation in 0.5 mL of hexane for sorbing to the SPE

cartridge.

Silica SPE cartridges (Baxter Enviroprep Inert SPE, 500 mg packing) are used for the

isolation of the Vitamins Kj and K2 from matrix fluorescent interferences. New cartridges are washed with 5 mL of hexane prior to sampling. The extract is then sorbed to the column,

and fats and retinol are eluted using 10 mL of hexane, which is discarded. Elution of the

Kj/Kj fraction is done with 5 mL of 3% ether in hexane. The first 1 mL that elutes is discarded and then the subsequent 4 mL are collected for further processing. These volumes

are critical to eliminate the fats and retinol eluted by the hexane (without losing the K

vitamins) and to prevent collection of the interfering tocopherols, which elute following the

collected 4 mL fraction. The eluant is dried under nitrogen, and the residue is redissolved in a volume of ethanol that is determined by the sensitivity of the fluorimetric detector.

Volumes between 100 |J,L and 500 |J.L can be used. Fifty microliters are injected into the

chromatograph for analysis. SPE cartridges may be reused by washing with 10 mL of the 3%

ether followed by 10 mL of hexane.

Rationale for the Procedure

Like other lipid-soluble vitamins, the Vitamins Kj and K2 can be extracted from serum with

hexane following denaturation of the proteins. We found that 2-propanol as protein

precipitant provided better recovery and lower amounts of coextracted interferences than did

ethanol. After a standard liquid-liquid vortex extraction, the hexane/2-propanol layer is drawn

off the top and evaporated under inert gas to dryness. Care must be taken to remove all residual 2-propanol since it causes band broadening and loss of selectivity in the silica

fractionation. The small volume of hexane used to redissolve the extract must be swirled

around the container walls to maximize redissolution.

5-4
An internal standard, added to the serum prior to extraction, is crucial in order to correct for

volume changes and other losses in the procedure. For this senmi work, we chose

menaquinone - 4, Vitamin K2, which is identical to Kj except for saturation of two of the side

chain double bonds. This K vitamin does not generally occur in human serum. The

chromatographic behavior of K2 is nearly identical to for both the fractionation and

reverse-phase separation steps and thus serves as an excellent internal standard.

The crude liquid-liquid extract matrix is too complex for direct Ki determination since it contains excess coextracted fat and compounds that interfere with the LC-fluorescence

determination, such as retinol and the tocopherols. Using the normal-phase SPE fractionation

scheme outlined, retinol and most fats elute before the K vitamins using the hexane solvent.

Tocopherols were found to elute in the fractions following the K vitamins. We found that an

all-glass/fluorocarbon SPE cartridge did not present any interference from background

fluorescence of the dialkyl phthlates found in plastic SPE cartridges. The syringe tube

cartridge type conveniently holds the eluting solvents and provides a gravity flow rate of

about 1 mL/min with hexane. The concentration and volume of the ether in hexane mixture

was found to be crucial for a good separation of the quinones from interfering matrix

compounds.

Chromatographic Determination

For the analytical separation two columns were tested, a Zorbax ODS and a Vydac 201TP C,8

column (25 x 0.46 cm). The Zorbax showed the higher retention and the Vydac showed

much lower k' values. We investigated the retention for both columns using

methanol:ethanol solvent mixtures. The retention time is reduced as the ethanol concentration

is increased; however, some asymmetry at high ethanol concentration is observed, possibly

due to increased solvent viscosity. The best separation of the Vitamin K compounds was

achieved with the Vydac column using 20% ethanol in methanol, with a flow rate of 1 mL/minute. With this separation, the two K vitamins are baseline resolved and well separated

from early eluting interferences. In serum extracts a chromatographic peak with a k' that is twice the value of the K vitamins was observed and was judged to probably be a higher n- value menaquinone. Under these conditions, the chromatographic separation time is about 20

to 25 minutes in length, as shown in Figure 3. The fluorescence detection wavelengths were 242 nm for excitation and 430 nm for emission.

Calibration solutions were prepared by dissolving Ki and K2 in ethanol and value-assigned by

spectrophotometry. The molar absorptivity of Vitamin Kj that was used was 41,000 at 242 nm. Stock solutions of about 40 |J.g/mL were stored in the dark in the refrigerator. The stock

solutions appeared to be stable for at least 2 months. Calibration curves were evaluated in

the range of 50 to 1000 ng/mL and found to be quite linear at the higher concentrations.

There was some non-linearity at the lower concentrations that is similar to what we have

found for P-carotene.

5-5
Performance of the Method

We have carefully evaluated the recovery of both spiked analyte (Kj) and internal standard

(K2) in each of the procedural steps in the analysis. The overall recovery of extraction,

fractionation, evaporation and reconstitution steps was found to be 70 ± 34%. Losses may

occur in several ways. Perhaps most important are losses in the transfer of the small volumes

of extract. Redissolution of the extract following the evaporation of the crude hexane extract

must be done with several rinses and vortex mixing to assure complete dissolution of the fatty

residue. The transfer of the evaporated crude extract to the SPE cartridge must be done with

care to avoid liquid losses. Redissolution of the fractionated extract also requires washing

and mixing since some fatty residue remains which is not rapidly soluble in the ethanol

solvent. Losses by photodegradation are also possible, so all work should be done in amber

or aluminum foil-covered vials. The absolute accuracy of the Vitamin Kj quantitation assumes similarity in the extraction and fractionation behavior (and losses) of the Kj and the

K2 throughout the procedure. We have tested the method on two serum pools from the

NIST/NCI round robin set. A value of 200 pg/mL was measured for Serum #28 and 420

pg/mL for Serum #70.

j

i1

j

1

5-6
5-7

MEASUREMENT OF (4-HYDR0XYPHENYL)RETINAMIDE IN SERUM

This section describes a method used by NISTfor the determination of a retinoic acid

derivative, (4-hydroxyphenyl)retinamide (4-HPR), in human serum. This compound is currently being evaluated for its cancer preventive effects in Phase II clinical trials sponsored

by the NCI. Listed below are important factors related to this analysis.

Important Factors

Calibration and Sample Preparation

• Add the internal standard (4-ethoxyphenyl)retinamide (4-EPR) to the protein

precipitation solvent.

• The liquid-liquid extraction protocol outlined in Clinical Chemistry 33, 1585-1592

(1987) should be used. This paper can be found beginning with page 1-28.

• Sera should be shipped on dry ice and frozen for storage.

• Care should be taken to prevent photodecomposition of the retinamides.

• The extraction volume correction is based on the recovery of the internal standard.

• Use a multiple-point calibration curve.

Chromatography

• The recommended column is a polymerically bonded 5 |Jin Cig column

(0.46 cm X 25 cm).

• Use an isocratic mobile phase: 55% acetonitrile, 10% n-butanol, 35% water, 0.01 mol/L

ammonium acetate.

• Variable wavelength detection should be used: programmed single wavelength at 364 nm for the first 12 minutes, 325 nm for the next 3 minutes, and 364 nm for the last 8 minutes (use wavelength timing to correspond to the elution times of 4-HPR, retinol,

and (4-methoxyphenyl)retinamide and 4-EPR, respectively).

For further information, please contact William MacCrehan at (301)975-3122.

6-1
NIST Measurements of (4-Hydroxyphenyl)retinairade in Serum

William A. MacCrehan

NIST developed a method for the determination of (4-hydroxyphenyl)retinamide (4-HPR, an amide of retinoic acid, see Figure 1), in 1987/88 as part of the NCI/NIST Micronutrients

Measurement Quality Assurance Program. As a candidate cancer chemopreventive agent,

accurate serum measurements are important to determine 4-HPR dosage compliance and

pharmacokinetic behavior in phase 1 and 2 human clinical trials. The NIST method

simultaneously determines the parent compound, the major metabolite (4-

methoxyphenyl)retinamide (4-MPR), and serum retinol. A non-naturally occurring compound,

(4-ethoxyphenyl)retinamide (4-EPR) is used in the procedure as an internal standard.

R = CH2CH3 4-(ethoxyphenyl)retinamide

(internal standard)

Figure 1 : Structure of Retinamides.

The serum preparation procedure follows the liquid-Uquid extraction protocol outlined in

Clinical Chemistry 33, 1585-1592 (1987). Sera were shipped frozen on dry ice and stored

at -70 °C prior to analysis, and thawed thoroughly to room temperature before subsampling.

Extra care was taken to avoid exposure to light as the retinamides are particularly sensitive to photodecomposition (as communicated by McNeil Pharmaceuticals) and the crystalline

standards were stored at -4 °C. Calibration solutions were value-assigned based on

gravimetric measurement of the pure crystalline compounds provided by McNeil, and

corrected for impurities separated by reversed-phase liquid chromatography with absorbance

detection at 364 nm by dividing the peak area of the target analyte by the total area. Calibration was done using a mixture of the three retinamides in ethanol at several levels in

the 0. 1 to 0.5 [ig/mL range. The method detection Hmit was approximately 0.005 jLig/mL on

6-2
a serum basis. The chromatographic separation conditions for the determination were as follows:

* column: Vydac 201TP (5 |im) 0.46 x 25 cm

* isocratic mobile phase: 55% acetonitrile, 10% «-butanol, 35% water, 0.01 mol/L

ammonium acetate * detector: programmed single wavelength at 364 nm for the first 12 minutes, 325 nm

for the next 3 minutes, and 364 nm for the last 8 minutes (wavelength timing to correspond to the elution times of 4-HPR, retinol, and 4-MPR and 4-EPR,

respectively)

* sample size: 10 |J.L

On a set of test sera, we found that 4-HPR values ranged from 0.02 to 0.37, retinol from 0.28

to 0.63 and 4-MPR from less than 0.005 to 0.31 pig/mL.

6-3

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