NISTIR 7880-40

NIST Micronutrients Measurement

Quality Assurance Program

Analysis Tutorial

Handout of the

Fat-Soluble Vitamin and Carotenoid Analysis Tutorial

October 27, 1997

Katherine E. Sharpless

Jeanice B. Thomas

David L. Duewer

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NISTIR 7880-40

NIST Micronutrients Measurement

Quality Assurance Program

Analysis Tutorial

Handout of the

Fat-Soluble Vitamin and Carotenoid Analysis Tutorial

October 27, 1997

Katherine E. Sharpless 

Special Programs Office 

Jeanice B. Thomas

David L. Duewer 

Chemical Sciences Division,

Material Measurement Laboratory 

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http://dx.doi.org/10.6028/NIST.IR.7880-40 

September 2016 

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology 

Willie May, Under Secretary of Commerce for Standards and Technology and Director 
Certain commercial entities, equipment, or materials may be identified in this

document in order to describe an experimental procedure or concept adequately.

Such identification is not intended to imply recommendation or endorsement by the

National Institute of Standards and Technology, nor is it intended to imply that the

entities, materials, or equipment are necessarily the best available for the purpose. 
(This page intentionally blank.)
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ABSTRACT

Since 1984, the National Institute of Standards and Technology (NIST) has coordinated what is now 

known as the Micronutrients Measurement Quality Assurance Program (MMQAP) for laboratories 

that measure fat-soluble vitamins and carotenoids in human serum and plasma. The MMQAP has 

provided participants with measurement comparability assessment through use of interlaboratory 

studies, Standard Reference Materials (SRMs) and control materials, methods development and 

validation, workshops, and tutorials. This report reproduces the printed materials provided to 

participants in the 1997 Fat-Soluble Vitamin and Carotenoid Analysis Tutorials, the most recent in a 

series of hands-on training programs for laboratorians involved in fat-soluble vitamin-related 

analytes. Some of the tutorial material has been updated.

KEYWORDS

Analytical methods, Retinol, Tocopherols, Carotenoids, Human serum
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TABLE OF CONTENTS

INTRODUCTION............................................................................................................................... 1

CALIBRATION.................................................................................................................................. 2

Value of Internal Standards........................................................................................................... 3

Internal Standard Characteristics ................................................................................................ 3

Internal Standards Used by Participants in the QA Program ................................................... 4

Synthesis of β-Apo-10'-Carotenal Oxime ..................................................................................... 5

Analyte Ranges from Two Sets of NCI Study Samples............................................................... 6

"Normal" Physiological Concentrations...................................................................................... 6

Preparation of Calibration Solutions............................................................................................ 7

SAMPLE PREPARATION.............................................................................................................. 10

Extraction Procedure ................................................................................................................... 11

Storage and Handling Recommendations .................................................................................. 13

Effect of Light Exposure and Other Extrinsic Factors on Fat-Soluble Vitamins in Serum.. 14

QUALITY CONTROL..................................................................................................................... 15

Standard Reference Material 968c.............................................................................................. 16

CHROMATOGRAPHY................................................................................................................... 17

Percent Recovery of Carotenoid Mixture on Tested Columns................................................. 18

Table 1: Index to the Reversed-Phase Columns Studied................................................................ 19

Average Percent Recovery of Carotenoid Mixture ................................................................... 20

Percent Recovery of Carotenoid Mixture................................................................................... 21

Recovery of a Mixture of Carotenoids from the Bakerbond C18 Column............................... 22

Critical Factors in Chromatographic Analysis of Carotenoids................................................ 23

APPENDIX A: Chemical Structures and Absorptivities............................................................. 27

APPENDIX B: Fat-Soluble Vitamins SRMs................................................................................. 28

Table B1: Production History of the SRM 968 series.................................................................... 28

Table B2: Analyte Information Delivered by the SRM 968 series................................................. 28

Table B3: Other SRMs of Potential Interest................................................................................... 29
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INTRODUCTION

Since 1984, the National Institute of Standards and Technology (NIST) has coordinated what is 

now known as the Micronutrients Measurement Quality Assurance Program (MMQAP) for 

laboratories that measure fat-soluble vitamins and carotenoids in human serum and plasma. The 

MMQAP has provided participants with measurement comparability assessment through use of 

interlaboratory studies, Standard Reference Materials (SRMs) and control materials, methods 

development and validation, workshops, and tutorials. This report reproduces the printed 

materials provided to participants in the 1997 Fat-Soluble Vitamin and Carotenoid Analysis 

Tutorials, the most recent in a series of hands-on training programs for laboratorians involved in 

fat-soluble vitamin-related analytes. Some of the tutorial materials have been updated, and notes 

have been added in italics. Appendix A lists the names and chemical structures, absorptivities, 

and absorption wavelengths of the fat-soluble vitamin-related compounds that have been most 

often reported in the MMQAP interlaboratory studies, and Appendix B provides a history of 

SRM 968 Fat-Soluble Vitamins and Carotenoids in Human Serum.

Tutorials for fat-soluble vitamin and carotenoid analysis were held in 1994, 1995, 1996, and 

1997 at NIST. Initially these tutorials were associated with workshops that were also held at 

NIST. When the workshops moved to venues associated with national meetings (AACC and 

Experimental Biology, in particular), the tutorials were eliminated. The tutorials were oriented 

primarily toward new laboratories or lab personnel participating in the MMQAP as well as others 

who were experiencing difficulties with analysis. The tutorials included discussion and 

demonstrations of calibration, sample preparation, and chromatographic techniques for 

measuring fat-soluble vitamins and carotenoids in serum.
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CALIBRATION
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Value of Internal Standards

• Volume correction - corrects for variable injection volume, evaporative 

losses of volatile solvents

• Recovery correction - corrects for spills, incomplete removal of organic 

phase, correction for partial solubility of organic phase in aqueous phase

• Column performance - monitor peak shape, area, and retention time in 

routine analysis

• Quality control - flag possible methodological errors

Internal Standard Characteristics

• Should be stable, of high purity, and readily available

• Should not react with sample components

• Should provide detector response similar to analytes’

• Should elute near analytes

• Should be completely resolved from other sample components
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Internal Standards Used by Participants in the QA Program

retinyl acetate

retinyl butyrate

tocol

α-tocopheryl acetate

echinenone

ethyl-β-apo-8'-carotenoate

trans-β-apo-8'-carotenal

various oximes, including trans-β-apo-8'-carotenal oxime and

trans-β-apo-10'-carotenal oxime
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Synthesis of β-Apo-10'-Carotenal Oxime

• Combine β-apo-10'-carotenal dissolved in ethanol and at least 1000-fold molar 

excess of 1 mol/L hydroxylamine.

• Let stand overnight in the dark at room temperature.

• Extract into hexane by adding water.

• Remove hexane layer and evaporate.

• Reconstitute in ethanol.

• To purify, inject onto Vydac 201TP preparative column, mobile phase of 100% 

acetonitrile at 10 mL/min.

• Extract oxime from mobile phase into hexane with the addition of water.

• Remove hexane layer and evaporate.

• Reconstitute in ethanol containing 30 μg/mL.

• Store at -20 °C.

References:

G.W. T. Groenendijk, W.J. De Grip, and F.J.M. Daemen, Biochim. et Biophys. 

Acta, 617 (1980) 430.

G. Handelman, B. Shen, and N. Krinsky, Meth. Enzymol. 213 (1992) 336.
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Analyte Ranges from Two Sets of NCI Study Samples

(approx. 1400 samples)

Range (μg/mL)

Retinol 0.07 to 1.6

Retinyl palmitate ND* to 1.7

Lutein 0.013 to 0.63

Zeaxanthin 0.002 to 0.20

β-Cryptoxanthin 0.001 to 0.30 (as high as 1.4 in some populations)

Lycopene 0.003 to 0.54

α-Carotene ND* to 0.82

β-Carotene 0.003 to 2.8

δ-Tocopherol ND to 0.90

γ-Tocopherol ND to 11

α-Tocopherol 3.1 to 48

*ND = not detected; not present or present at levels below the limit of detection

"Normal" Physiological Concentrations

Range (μg/mL)

Retinol 0.5 to 0.7

β-Carotene 0.18 to 0.3

α-Tocopherol 8 to 10

Reference: Sharpless, K.S. and Duewer, D.L., “Population Distributions and Intralaboratory 

Variance for Fat-Soluble Vitamin-Related Compounds in Human Serum.” Anal. Chem. 67, 4416 

(1995).
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Preparation of Calibration Solutions

1. Prepare stock solutions at concentrations 5 to 10 times higher than the concentrations 

you'll want in your calibration solutions. The concentrations of your stock solutions do 

not have to be determined during this preparation step; you will be determining the 

concentration spectrophotometrically and correcting for impurities using 

chromatography.

2. Filter stock solutions and determine concentration of stock solutions using 

spectrophotometer.

• blank out the solvent you're using 

(don't forget to add BHT if you used it in your stock solution)

• run spectrum and check the maximum absorbance

• dilute your stock solution if the maximum absorbance is approximately 1 and run the 

spectrum several times

• record the absorbances at λmax and take the average

(some samples will degrade as they sit in the beam of the spectrophotometer)

• calculate concentration using Beer's Law

For a 1+9 dilution of β-cryptoxanthin in ethanol,

A = εbc

0.181 = (2356)(1)(c)

c = 0.0000768 g/dL x 1000 mg/g x 10 dL/L = 0.768 mg/L

(Note that the absorbance here is only 0.181, which means the solution has been diluted 

more than it should have been.)

Multiply by 10 to account for the 1+9 dilution. The concentration in your stock solution 

is 7.68 mg/L, not accounting for impurities.

3. Check filtered stock solution for impurities by running through your usual 

chromatographic method, but with the detector set at the wavelength where you did your

Beer's Law determination (typically the wavelength of maximum absorbance for the 

compound you're measuring).
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Analyte λmax (nm) absorptivity (dL/g·cm)

lutein (EtOH) 445 2550

zeaxanthin (EtOH) 452 2540

β-cryptoxanthin (EtOH) 452 2356

trans-lycopene (hex) 472 3450

trans-α-carotene (hex) 444 2800

trans-β-carotene (hex) 452 2592

9-cis-β-carotene (hex) 445 2090

13-cis-β-carotene (hex) 446 2370

15-cis-β-carotene (hex) 447 1820

δ-tocopherol (EtOH) 297 91.2

γ-tocopherol (EtOH) 298 91.4

α-tocopherol (EtOH) 292 75.8

retinol (EtOH) 325 1843

retinyl palmitate (EtOH) 325 975

(EtOH = ethanol; hex = hexane)

You find that your filtered β-cryptoxanthin solution is 91.4% pure, so correct your 

concentration for this:

7.68 mg/L x 0.914 = 7.02 mg/L

4. Prepare calibration solutions. Say for β-cryptoxanthin, you want three calibration 

solutions in a range of 0.1 to 1 mg/L. To prepare 10 mL of each, you would need:

(10 mL) (0.1 mg/L) = (x mL) (7.02 mg/L): x = 0.142 mL

(10 mL) (0.5 mg/L) = (x mL) (7.02 mg/L): x = 0.712 mL

(10 mL) (1 mg/L) = (x mL) (7.02 mg/L): x = 1.42 mL

When you add your internal standard, you're going to have some dilution, so use 0.2, 0.8, 

and 1.5 mL to prepare your calibration solutions.

The concentrations in the calibration solutions are then:

[(0.20 mL)×(7.02 mg/L)]/10 mL = 0.140 mg/L

[(0.80 mL)×(7.02 mg/L)]/10 mL = 0.562 mg/L

[(1.50 mL)×(7.02 mg/L)]/10 mL = 1.053 mg/L

(The easiest way to do these calculations is on a spreadsheet so that everything is 

calculated for you automatically each time you recalibrate.)
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For simplicity in this example, the calibrants have been prepared from a stock solution 

for which a single determination of concentration has been made. If an error is made in 

the determination of that concentration, that error is propagated into the entire 

measurement system. (This can become apparent if results for quality control materials 

are out of specification.) Thus, it is preferable to determine concentrations individually 

rather than doing serial dilutions.

5. If you don't use an internal standard, you can now inject the calibration solutions (three 

times each) and prepare your calibration curve.

6. If you do use an internal standard, you need to add it to an aliquot of your calibration 

solutions now (or you could have included it when you prepared the calibration solution). 

If you're adding the internal standard to an aliquot of your calibration solution, your 

concentrations must be corrected for the dilution caused by the addition of the internal 

standard. If the concentration of your internal standard is fairly high in its stock solution, 

you have to add less to get a good level in your calibration solutions and thus don't dilute 

your calibration solutions as much.

For example, if you add 200 μL of your internal standard to 1 mL of the low calibration 

solution, the resulting β-cryptoxanthin concentration is:

0.140 μg/mL x (1 mL/1.2 mL) = 0.117 μg/mL.

For an internal standard stock concentration of 5.02 μg/mL, the resultant internal standard 

concentration in your calibrants will be:

5.02 μg/mL x (0.2 mL/1.2 mL) = 0.837 μg/mL.

7. Prepare your calibration curve using the three solutions containing the internal standard.

Calibration time is decreased if you put more than one compound in your calibration 

solutions. When you do this, however, you must be aware that commercial lutein 

preparations contain zeaxanthin as an impurity and vice versa, and α- and β-carotene also 

can be impurities in commercial preparations of each other. This must be taken into 

account when determining the final concentrations. Also, it's best to do lycopene by 

itself since it degrades fairly rapidly.

If you have a lot of compounds in each calibration solution, it is often not possible to 

make one solution containing all the low levels, one with all the middle, and one with all 

the high because the volume in the high level would exceed the 10-mL total target 

volume. Therefore, it is sometimes necessary to mix up the levels so that the total 

volume of stock solution is less than 10 mL.
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SAMPLE PREPARATION
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Extraction Procedure

• Precipitate proteins from serum or plasma using ethanol (containing 30 μg/mL 

BHT anti-oxidant and internal standards)

• Vortex.

• Add hexane. Vortex to extract analytes into hexane.

• Centrifuge sample. Remove organic phase.

• Re-extract with hexane.

• Centrifuge. Remove organic phase and combine with first.

• Evaporate organic phase under nitrogen.

• Re-dissolve sample in ethanol containing 30 μg/mL BHT.

References:

Epler, K.S., Ziegler, R.G., and Craft, N.E., “Liquid Chromatographic Method for the 

Determination of Carotenoids, Retinoids, and Tocopherols in Human Serum and in Food.” J. 

Chromatogr. (Biomed. Applications) 619, 37 (1993).

Brown Thomas, J., Kline, M.C., Schiller, S.B., Ellerbe, P.M., Sniegoski, L.T., Duewer, D.L., and 

Sharpless, K.E., “Certification of Fat-Soluble Vitamins and Cholesterol in Human Serum: 

Standard Reference Material 968b.” Fresenius J. Anal. Chem. 356, 1 (1996).

Brown Thomas, J., Kline, M.C., Gill, L.M., Yen, J.H., Duewer, D.L., Sniegoski, L.T., and Sharpless, 

K.E., “Preparation and Value Assignment of Constituents in Standard Reference Material 968c: 

Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum,” Clin. Chim. Acta 305, 

141-155 (2001).
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EXTRACTANT COMMENTS

Ethyl acetate/THF May see sample-to-sample variability since solvent 

partitioning is not well defined; THF is not the ideal solvent 

for extractions since it forms peroxides, thereby leading to 

degradation of vitamin A and carotenoids.

Ethyl acetate/Butanol Gives good recovery of carotenoids and effectively 

overcomes lipid binding.

*Hexane Effectively competes with serum lipids for solvation of the 

analytes and thereby overcomes lipid binding. Hexane is 

not compatible with most common reversed-phase methods' 

mobile phases and must be evaporated under a steam of inert 

gas.

Alkaline hydrolysis Method works for β-carotene analysis. Lower concentration 

(<1 mol/L) of KOH solution does not extract all the 

carotenoids when using volumes of serum greater than 

0.5 mL.

Additional free retinol recovered by hydrolysis of serum 

retinol esters cause erroneous results for all-trans-retinol 

determination.

α-Tocopherol is converted to the hydrophilic phenolate 

anion, thereby making it impossible to recover α-tocopherol 

from the alkaline hydrolysate.
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Storage and Handling Recommendations

• Store samples in amber vials.

• Minimize exposure to light and air.

• Store samples below room temperature (-20 °C or -80 °C). If carotenoids will 

be measured, -80 °C storage is preferable; carotenoids are less stable than 

retinoids and the tocopherols at -20 °C.

• Reconstituted serum should be used immediately or stored at -20 °C for no 

more than 3 days.

• Minimize repeated thawing and refreezing of serum (<5 freeze/thaw cycles).

Reference: Brown Thomas, J., Kline, M.C., Duewer, D.L., and Sharpless, K.E. “The Stability of 

Fat-Soluble Vitamins and Carotenoids in Human Serum,” Clin. Chim. Acta 276, 75 (1998).
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Effect of Light Exposure and Other Extrinsic Factors

on Fat-Soluble Vitamins in Serum

SOURCE EFFECT

Direct sunlight Causes vitamin A and carotenoids to decompose

Artificial light Oxidation of the analytes is minimal but evident during 

short-term (2 hour) exposure. As a safety precaution, direct 

exposure of samples to light should be avoided.

Air, heat, metal ions Vitamin A and carotenoids are readily destroyed by 

oxidation in the presence of air. Heat and the presence of 

metal ions such as iron and copper also add to this 

phenomenon.
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QUALITY CONTROL
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Standard Reference Material 968c

Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum

Certified: Non-certified:

retinol trans-lutein

trans-β-carotene total lutein

total β-carotene total zeaxanthin

δ-tocopherol total β-cryptoxanthin

γ-tocopherol (includes β-tocopherol) trans-lycopene

α-tocopherol total lycopene

cholesterol trans-α-carotene

total α-carotene 25-hydroxyvitamin D

SRM 968c is no longer available. SRM 968e is the currently available (2016) version. Table B1 

in Appendix B describes the production history of the SRM 968 versions. Table B2 identifies the 

analyte information delivered by each version.

There are also several other SRMs of potential relevance to those interested in measurement of 

fat-soluble vitamin-related analytes. Table B3 in Appendix B lists these materials.

The information on how to order NIST SRMs provided in the original tutorial is no longer valid 

and is not reproduced. To obtain information on, and to order, NIST’s currently available 

SRMs, visit http://www.nist.gov/srm/index.cfm and enter “vitamin” in the “Keyword” search 

box.
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CHROMATOGRAPHY

In the early 1990s we tested 65 reversed-phase columns to evaluate the 

selectivity of various C18 stationary phases with methanol- and 

acetonitrile-based mobile phases and to see whether the carotenoids 

eluted from the columns or were retained. Much of the content of this 

section is drawn from the results of that study, published in:

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

“Evaluation of Reversed-Phase Liquid Chromatography Columns for 

Recovery and Selectivity of Selected Carotenoids.” J. Chromatogr. 595, 

89 (1992).

This publication is included in the freely available compendeum:

Methods for Analysis of Cancer Chemopreventive Agents in Human 

Serum, Brown Thomas, J. and Sharpless, K.E., editors, NIST Special 

Publication 874, U.S. Government Printing Office Washington: 1995
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Percent Recovery of Carotenoid Mixture on Tested Columns

Mobile Phase

Using Methanol or THF/Methanol:

Using Acetonitrile or THF/Acetonitrile:

THF = tetrahydrofuran

0-9%

10-19%

20-29%

30-39% 56

40-49%

50-59% 9

60-69% 46 62

70-79% 20 33 36 55 63 64

80-89% 1 2 4 6 8 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

90-100% 3 5 7 11 15 21 23 24 26 27 28 29 30 32 41 42 43 44 45 52 65

% Carotenoid Recovery

Index of Tested Column

0-9% 53 54 55 56

10-19%

20-29%

30-39% 63

40-49% 6 7 16 19 35 39 62 64

50-59% 9 11 14 15 17 20 24 31 38 40 46

60-69% 5 10 25 28 29 33 36 44 48 65

70-79% 1 4 8 12 13 18 21 22 26 30 32 41 42 45 47 52 58 59

80-89% 2 3 23 27 37 49 60 61

90-100% 43 50 51

% Carotenoid Recovery

Index of Tested Column
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Table 1: Reversed-Phase Columns Tested

Columns are C18 except where noted.

Supplier Column name RRa Classb Supplier Column name RRa Classb

Beckman Ultrasphere ODS 1.92 Monomeric ES Industries Chromegabond PFPf 0.88 Polymeric

Beckman Ultrasphere ODS 2.01 Monomeric ES Industries Chromegabond BF-C18 1.04 Intermediate

Beckman Ultrasphere ODS DABS 2.00 Monomeric J.T. Bakerg Bakerbond C18 1.25 Intermediate

Analytichem Sepralyte C18 1.82 Monomeric J.T. Bakerg Bakerbond WP C18 0.51 Polymeric

Separations Group Vydac 218TP 0.83 Polymeric J.T. Bakerg Bakerbond WP C18 0.54 Polymeric

Separations Group Vydac 201TP 0.78 Polymeric J.T. Bakerg Bakerbond WP C18 1.09 Intermediate

Separations Group Vydac 201TP 0.80 Polymeric Keystone Sci. ODS Hypersil 1.95 Monomeric

Biotage Unisphere-PBDc 3.06 Monomeric Shiseido Capsell Pak C18 1.99 Monomeric

ES Industries Gamma Bond C18 1.86 Monomeric Nacalai Tesque Cosmosil 5C18-P Waters 2.04 Monomeric

ES Industries Chromegabond C22 (C22) 1.83 Monomeric Nacalai Tesque Cosmosil 5C18 Waters 1.85 Monomeric

ES Industries Chromegabond MC18 1.73 Monomeric Waters Nova-Pak C18 1.97 Monomeric

Supelco LC-18 2.03 Monomeric YMC ASP303-S 2.05 Monomeric

Supelco LC-18 2.02 Monomeric Hewlett-Packard ODS Hypersil 1.98 Monomeric

Supelco LC318 2.05 Monomeric Hewlett-Packard LiChrospher 100 RP-18 1.50 Intermediate

Supelco LC318 2.04 Monomeric Brownlee Spheri-S ODS 1.26 Intermediate

Supelco LC-PAH 0.70 Polymeric Bio-Rad Hi-Pore RP318 0.59 Polymeric

Analytichem Sepralyte C18 1.92 Monomeric Macherey-Nagel Nucleosil 5 PAH 0.36 Polymeric

Alltech Adsorbosphere C18 2.01 Monomeric Raining Microsorb C18 1.78 Monomeric

EM Science LiChrospher 100 RP-18 1.45 Intermediate Phase Separations Spherisorb SS PAH 0.82 Polymeric

Serva Octadecyl 1.84 Monomeric Phase Separations Spherisorb SS ODS 1.68 Intermediate

Serva Triacontyl (C30) d Phase Separations Spherisorb SS ODS 1.50 Intermediate

J&W Accusphere ODS 2.07 Monomeric Carlo Erba Erbasil 5 C18/L 1.76 Monomeric

J&W Accusphere ODS 1.96 Monomeric Carlo Erba Erbasil 5 C18/M 1.28 Intermediate

MacMod Zorbax RX C8 (C8) 2.33 Monomeric Carlo Erbah Erbasil 5 C18/H 0.91 Polymeric

Perkin Elmer Pecosphere C18 2.00 Monomeric Brownlee Spheri-5 RP-18 1.92 Monomeric

YMC A303 1.97 Monomeric MacMod Zorbax RX C18 1.50 Intermediate

YMC AMP303 2.01 Monomeric Phenomenex Ultracarb 5 ODS20 1.95 Monomeric

YMC AP303 2.06 Monomeric Phenomenex Ultracarb 5 ODS30 2.01 Monomeric

J.T. Baker exper WP C18e 0.57 Polymeric J.T. Baker Bakerbond WP C18 0.89 Polymeric

J.T. Baker exper WP C18e 0.93 Polymeric J.T. Baker Bakerbond WP C18 0.67 Polymeric

J.T. Baker exper WP C18e 0.73 Polymeric Brownlee Spheri-5 ODS 1.42 Intermediate

J.T. Baker exper WP C18e 1.42 Intermediate MacModg Zorbax ODS 1.80 Monomeric

J.T. Baker exper WP C18e 0.22 Polymeric

a Relative retention of 1,2,3,4,5,6,7,8-tetrabenzonaphthalene (TBN) to benzo[a]pyrene (BaP)

b As determined by evaluation of SRM 869.

c PBD = polybutadiene.

d Returned to supplier before value could be determined.

e Experimental columns-with varying surface coverage.

f PFP = pentafluorophenol.

g Used in laboratory prior to evaluation in this study.

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

Reference: Epler, K.S., Sander, L.C., Wise, S.A., Craft, N.E., and Ziegler, R.G., “Evaluation of ReversedPhase Liquid Chromatography Columns for Recovery and Selectivity of Selected Carotenoids.” J. 

Chromatogr. 595, 89 (1992).
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Average Percent Recovery of Carotenoid Mixture

Stationary Phase

Eluent Polymeric Intermediate Monomeric

MeOH and THF/MeOH 83% 79% 88%

ACN and THF/ACN 62% 46% 67%

ACN = acetonitrile; MeOH = methanol; THF = tetrahydrofuran

Reference: Epler, K.S., Sander, L.C., Wise, S.A., Craft, N.E., and Ziegler, R.G., “Evaluation of ReversedPhase Liquid Chromatography Columns for Recovery and Selectivity of Selected Carotenoids.” J. 

Chromatogr. 595, 89 (1992).
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Percent Recovery of Carotenoid Mixture

Buffer Treatment

Recovery before Recovery after Recovery after buffer,

Column buffer treatment buffer treatment 0.05% TEA in eluent

1 48% 74% 102%

2 1% 30% >99%

3 7% 87% 106%

4 15% 69% 104%

5 3% 12% >87%

TEA = trimethylamine

Reference: Epler, K.S., Sander, L.C., Wise, S.A., Craft, N.E., and Ziegler, R.G., “Evaluation of ReversedPhase Liquid Chromatography Columns for Recovery and Selectivity of Selected Carotenoids.” J. 

Chromatogr. 595, 89 (1992).
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Recovery of a Mixture of Carotenoids from the

Bakerbond C18 Column

In the column comparison study:

100% methanol 75%

5% THF/95% acetonitrile 66%

Using a gradient of acetonitrile, methanol, and ethyl acetate:

No ammonium acetate, no TEA 68%

0.05 mol/L NH4OAc in the methanol 92%

0.1% TEA in all solvents, no NH4OAc in the methanol 89%

0.1% TEA in all solvents, 0.05 mol/L NH4OAc in the methanol 101%

0.05% TEA in all solvents, 0.05 mol/L NH4OAc in the methanol 94%

NH4OAc = ammonium acetate; TEA = trimethylamine; THF = tetrahydrofuran

Reference: Epler, K.S., Sander, L.C., Wise, S.A., Craft, N.E., and Ziegler, R.G., “Evaluation of ReversedPhase Liquid Chromatography Columns for Recovery and Selectivity of Selected Carotenoids.” J. 

Chromatogr. 595, 89 (1992).
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Critical Factors in Chromatographic Analysis of Carotenoids

• Use polymeric (or some intermediate) C18 columns if you want to be able to 

separate lutein and zeaxanthin.

• Use a monomeric column, which on average provides better recovery than 

polymeric columns, if you do not need to measure lutein and zeaxanthin.

• Use methanol-based solvent systems rather than acetonitrile-based systems.

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

triethylamine to improve carotenoid recovery.

• Stainless steel frits may cause on-column carotenoid losses.

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

losses.

• Be aware that there are a lot of carotenoid peaks in serum.

Reference: Methods for Analysis of Cancer Chemopreventive Agents in Human Serum, Brown 

Thomas, J. and Sharpless, K.E., editors, NIST Special Publication 874, U.S. Government 

Printing Office Washington: 1995
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Column: Vydac 201TP

Gradient: 67:10:23 / 86.5:10:3.5 Methanol:Butanol:Water with

10 mmol/L HOAc (pH 5.5) over 25 min

Flow rate: 1.5 mL/min

Detection: Retinol - 325 nm

Tocopherols - 292 nm

Carotenoids - 452 nm

R = retinol

T = tocol

g = γ-tocopherol

a-t = α-tocopherol

L = lutein

Z = zeaxanthin

Cr = β-cryptoxanthin

a = α-carotene

b = β-carotene

Reference: Brown Thomas, J., Kline, M.C., Gill, L.M., Yen, J.H., Duewer, D.L., Sniegoski, L.T., 

and Sharpless, K.E., “Preparation and Value Assignment of Constituents in Standard Reference 

Material 968c: Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum,” Clin. 

Chim. Acta 305, 141-155 (2001).

0

1

2

3

4

5

6

0 10 20 30 40 50

Absorbance

Time (minutes)

R

T

g

a-t

LZ Cr a

b

Vydac 201TP column
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Column: Bakerbond C18

Guard column: Vydac 201TP

Temperature: 29 °C

Flow rate: 1.5 mL/min

Solvent A: acetonitrile containing 0.05% triethylamine (TEA)

Solvent B: methanol containing 0.05 mol/L ammonium acetate and 0.05% TEA

Solvent C: ethyl acetate containing 0.05% TEA

Gradient: 98% A/2% B to 75% A/18% B/7% C at 10 min

75% A/18% B/7% C to 68% A/25% B/7% C at 15 min

Hold at 68% A/25% B/7% C for 15 min

Return to initial conditions (98% A/2% B) over 5 min

Re-equilibrate for 5 min

Detection: Retinol and retinyl palmitate - absorbance at 325 nm

Carotenoids - absorbance at 450 nm

Tocopherols - fluorescence - excitation at 295 nm, emission at 335 nm

R = retinol

ox = trans-β-apo-8’-carotenal oxime (internal 

standard)

L = lutein

Z = zeaxanthin

Cr = β-cryptoxanthin

Ly = lycopene

a = α-carotene

b = β-carotene

T = tocol

d = δ-tocopherol

g = γ-tocopherol

a-t = α-tocopherol

Reference: Brown Thomas, J. et al., Clin. Chim. Acta 305, 141-155 (2001).

0.025

0.027

0.029

0.031

0.033

0.035

0 5 10 15 20 25 30 35

Absorbance

R

ox

L Z Cr Ly a

b

Bakerbond C18 column

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 5 10 15 20 25 30 35

Fluorescence

T

d

g

a-t Bakerbond C18 column
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Column: polymeric C30 column (3-μm particles, 15-cm length) prepared in-house

Guard column: in-line titanium frit

Temperature: room temperature

Flow rate: 1.5 mL/min

Solvent A: 8% water/ 92% methanol containing 0.05 M ammonium acetate and

0.05% triethylamine

Solvent B: methyl t-butyl ether

Gradient: 83% A/17% B to 59% A/41% B at 29 min

59% A/41% B to 30% A/70% B at 34 min

Hold at 30% A/70% B for 11 min

Return to initial conditions (83% A/17% B) over 5 min

Re-equilibrate for 10 min

Detection: Carotenoids - absorbance at 450 nm

ox = trans-β-apo-8’-carotenal oxime (internal standard)

L = lutein

Z = zeaxanthin

Cr = β-cryptoxanthin

a = α-carotene

b = β-carotene

Ly = lycopene

Reference: Brown Thomas, J., Kline, M.C., Gill, L.M., Yen, J.H., Duewer, D.L., Sniegoski, L.T., 

and Sharpless, K.E., “Preparation and Value Assignment of Constituents in Standard Reference 

Material 968c: Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum,” Clin. 

Chim. Acta 305, 141-155 (2001).

0.025

0.027

0.029

0.031

0.033

0.035

0.037

0 10 20 30 40 50

Absorbance

ox

L Z Cr

Ly a

b NIST-engineered

polymeric C30 column
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APPENDIX A: Chemical Structures and Absorptivities1

 1Absorptivities were taken from the following references:

Schierle, J.; Härdi, W.; Faccin, N.; Bühler, I.; Schüep, W.; Geometrical Isomers of β,β-Carotene; In: Carotenoids: Isolation and 

Analysis; Britton, G., Liaaen-Jensen, S., and Pfander, H., eds., Basel: Birkhäuser Verlag, Vol. 1A, pp. 265-272 (1995).

Robeson, C.D.; Cawley, J.D.; Weisler, L.; Stern, M.H.; Eddinger, C.C.; Chechak, A.J.; The Synthesis of Geometric Isomers of Vitamin 

A via Methyl β-Methylglutaconate; J. Am. Chem. Soc., Vol. 77, p. 4111 (1955).

Windholz, M.; ed., The Merck Index of Chemicals and Drugs; 9th ed., Rahway, NJ, Merck, p. 1221 (1976).

Schudel, P.; Mayer, H.; Isler, O.; Tocopherols; In: The Vitamins: Chemistry, Physiology, Pathology, Methods; Sebrell, W.H., Jr.; 

Harris, R.S.; eds., New York, Academic Press, pp. 168-218 (1967).

DeRitter, E.; Purcell, A.E.; Carotenoid Analytical Methods; In: Carotenoids as Colorants and Vitamin A Precursors, Bauernfeind, J.C., 

ed., Orlando, FL, Academic Press, pp. 883-923 (1981).

Absorptivity for β-cryptoxanthin in ethanol is calculated from the values for β-cryptoxanthin in petroleum ether and β-carotene in 

ethanol and petroleum ether provided in DeRitter and Purcell.
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APPENDIX B: Fat-Soluble Vitamins SRMs

Table B1: Production History of the SRM 968 series

Version Issued

Number

Levels

Number

Analytes Form Spiked Analytes

968 1989 3 5 Lyophilized Retinol, Tocopherols

968a 1991 3 12 Lyophilized Retinol, Tocopherols

968b 1995 3 15 Lyophilized Retinol, Tocopherols

968c 1999 2 21 Lyophilized Retinol, Tocopherols

968d 2008 1 12 Liquid frozen None

968e 2010 3 17 Liquid frozen None

968f 2017? 2 ?? Liquid frozen None

Table B2: Analyte Information Delivered by the SRM 968 series

Analyte 968 968a 968b 968c 968d 968e

Retinol C C C C C C

Retinyl Palmitate C NC

α-Tocopherol C C C C C C

γ/β-Tocopherol NC NC NC C C C

δ-Tocopherol NC C NC

Total β-Carotene C C C C C C

Trans-β-Carotene NC NC C C NC

Total cis-β-Carotene NC NC

Total α-Carotene C NC NC NC

Trans-α-Carotene NC NC NC

Total Lycopene NC NC NC NC NC

Trans-Lycopene NC NC NC NC NC

Total α-Cryptoxanthin NC NC NC

Total β-Cryptoxanthin NC NC NC C

Total Lutein NC C NC NC C

Trans-Lutein NC

Total Zeaxanthin NC NC NC NC C

Phylloquinone (Vitamin K) NC

Coenzyme Q10 NC NC

25-Hydroxyvitamin D NC

25-Hydroxyvitamin D2 NC

25-Hydroxyvitamin D3 NC

Cholesterol NC C C C C

C: Certified analyte value (traceable to the SI)

NC: Non-certified value (traceable to the material if analyzed with method(s) similar to those used 

to provide the value
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Table B3: Other SRMs of Potential Interest

Number Name

972a Vitamin D Metabolites in Frozen Human Serum

1950 Metabolites in Frozen Human Plasma

2972a 25-Hydroxyvitamin D Calibration Solutions

2973 Vitamin D Metabolites in Frozen Human Serum (High Level)

3950 Vitamin B6 in Frozen Human Serum

These materials are available as of July 2016. For further information on these materials, visit

http://www.nist.gov/srm/index.cfm

and enter the SRM number in the “SRM/RM Number:” search box, click the hyperlink listed under 

the “Description” header, then click the hyperlink next to the “Certificate” bullet. Pricing and 

ordering information is also provided on the webpage containing the “Certificate” bullet.

To obtain information on all currently available vitamin-related SRMs, visit

http://www.nist.gov/srm/index.cfm

and enter “vitamin” in the “Keyword” search box and follow the relevant hyperlinks.