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NBS SPECIAL PUBLICATION 260-69

U.S. DEPARTMENT OF COMMERCE / National Bureau of Standards

Standard Reference Materials:

A REFERENCE METHOD FOR THE

DETERMINATION OF LITHIUM IN SERUM

QC

100

U57

260-69

1980
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards' was established by an act of Congress on March 3, 1901. The Bureau's overall goal is to strengthen and advance the Nation's science and technology

and facilitate their effective application for public benefit. To this end, the Bureau conducts

research and provides: (1) a basis for the Nation's physical measurement system, (2) scientific and technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to promote public safety. The Bureau's technical work is per- formed by the National Measurement Laboratory, the National Engineering Laboratory, and

the Institute for Computer Sciences and Technology.

THE NATIONAL MEASUREMENT LABORATORY provides the national system of

physical and chemical and materials measurement; coordinates the system with measurement

systems of other nations and furnishes essential services leading to accurate and uniform

physical and chemical measurement throughout the Nation's scientific community, industry,

and commerce; conducts materials research leading to improved methods of measurement,

standards, and data on the properties of materials needed by industry, commerce, educational

institutions, and Government; provides advisory and research services to other Government

agencies; develops, produces, and distributes Standard Reference Materials; and provides

calibration services. The Laboratory consists of the following centers:

Absolute Physical Quantities2 — Radiation Research — Thermodynamics and

Molecular Science — Analytical Chemistry — Materials Science. THE NATIONAL ENGINEERING LABORATORY provides technology and technical ser- vices to the public and private sectors to address national needs and to solve national problems; conducts research in engineering and applied science in support of these efforts; builds and maintains competence in the necessary disciplines required to carry out this research and technical service; develops engineering data and measurement capabilities;

provides engineering measurement traceability services; develops test methods and proposes

engineering standards and code changes; develops and proposes new engineering practices;

and develops and improves mechanisms to transfer results of its research to the ultimate user. The Laboratory consists of the following centers:

Applied Mathematics — Electronics and Electrical Engineering2 — Mechanical

Engineering and Process Technology2 — Building Technology — Fire Research —

Consumer Product Technology — Field Methods.

THE INSTITUTE FOR COMPUTER SCIENCES AND TECHNOLOGY conducts

research and provides scientific and technical services to aid Federal agencies in the selection, acquisition, application, and use of computer technology to improve effectiveness and

economy in Government operations in accordance with Public Law 89-306 (40 U.S.C. 759), relevant Executive Orders, and other directives; carries out this mission by managing the Federal Information Processing Standards Program, developing Federal ADP standards

guidelines, and managing Federal participation in ADP voluntary standardization activities; provides scientific and technological advisory services and assistance to Federal agencies; and

provides the technical foundation for computer-related policies of the Federal Government.

The Institute consists of the following centers:

Programming Science and Technology — Computer Systems Engineering.

'Headquarters and Laboratories at Gaithersburg, MD, unless otherwise noted; mailing address Washington, DC 20234. 'Some divisions within the center are located at Boulder, CO 80303.
A REFERENCE METHOD FOR THE DETERMINATION OF

NATIOXAt »OTfe»«V OF 8TANOAB&B

UBjUllY ^ JUL 2 l 1980

Standard Reference Materials: no-e -Ore

QCioo

LITHIUM IN SERUM 'no. &o-4>q

I Wo

Ranee A. Velapoldi, Robert C. Paule,

Robert Schaffer, John Mandel,

Lawrence A. Machlan, Ernest L. Garner,

and Theodore C. Rains

National Measurement Laboratory

National Bureau of Standards

Washington, D.C. 20234

With the cooperation of a Committee of Experts:

George N. Bowers, Jr.,

1 Bradley E. Copeland,2 Denis O. Rodgerson,3 and James M. White4

'Hartford Hospital, Hartford, CT

2New England Deaconnes Hospital, Boston, MA

3 Department of Pathology, University of California, Los Angeles, CA

4Center for Disease Control, Atlanta, GA

U.S. DEPARTMENT OF COMMERCE, Philip M. Klutznick, Secretary

Luther H. Hodges, Jr., Deputy Secretary

Jordan J. Baruch, Assistant Secretary for Productivity, Technology and Innovation

£... NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued July 1980
Library of Congress Catalog Card Number: 80-600091

National Bureau of Standards Special Publication 260-69

Nat. Bur. Stand. (U.S.), Spec. Publ. 260-69, 1 14 pages (July 1980) CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1980

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402

Price $4.25

(Add 25 percent for other than U.S. mailing).
PREFACE

Standard Reference Materials (SRM's) as defined by the

National Bureau of Standards are well- characterized materials

produced in quantity and certified for one or more physical

or chemical properties. They are used to assure the accuracy

and compatibility of measurements throughout the Nation.

SRM's are widely used as primary standards in many diverse

fields in science, industry, and technology, both within the

United States and throughout the world. They are also used

extensively in the fields of environmental and clinical analysis. In many applications, traceability of quality control

and measurement processes to the national measurement system

are carried out through the mechanism and use of SRM's. For

many of the Nation's scientists and technologists it is there

fore of more than passing interest to know the details of the

SRM certification measurements made at NBS or of recommended

methods for use with SRM's to assure accurate measurements

in the field. 'An NBS series of papers, of which this publica

tion is a member, called the NBS Special Publication - 260

Series , is reserved for this purpose.

This 260 Series is dedicated to the dissemination of

information on different phases of the preparation, measurement, certification and use of NBS-SRM's. In general, much

more detail will be found in these papers than is generally

allowed, or desirable, in scientific journal articles. This

enables the user to assess the validity and accuracy of the

measurement processes employed, to judge the statistical

analysis, and to learn details of techniques and methods

utilized for work entailing the greatest care and accuracy.

These papers also should provide sufficient additional

information not found on the certificate so that new applications in diverse fields not foreseen at the time the SRM was

originally issued will be sought and found.

111
Inquiries concerning the technical content of this

paper should be directed to the author (s). Other questions

concerned with the availability, delivery, price, etc., of

specific SRM's cited in this document will receive prompt

attention from:

Office of Standard Reference Materials

National Bureau of Standards

Washington, D.C. 20234

George A. Uriano, Chief

Office of Standard Reference Materials

iv
FOREWORD

A fundamental requirement for assuring adequate patient

care is the need for the accurate analysis of constituents in

body fluids. Two major functions of the National Bureau of

Standards (NBS) are to provide certified Standard Reference

Materials for the calibration of measurement systems and to

develop new or improved analytical methods. The results presented in this NBS Special Publication provide a methodology

of known accuracy for the determination of lithium in serum.

The evaluation of a reference method by comparison to a definitive method, used for the first time at NBS in the

development of reference methods for calcium, sodium, potassium, and chloride in serum, also was applied to this work.

This hierarchy of analytical procedures has been accepted as

a valid format for developing reference methods by the clinical

community at a recent Conference on an Understanding for a National Reference System in Clinical Chemistry.

In an undertaking of this magnitude, extensive collaboration with a committee of experts, the Center for Disease

Control, the Food and Drug Administration, and a wide

spectrum of participating analytical laboratories that

included Federal, State, hospital, industrial, and academic

laboratories was essential to establish a widely accepted

reference method. It is hoped that this work will provide

an additional basis for the development of future clinical

reference methods through continued collaboration and the

concerted efforts of the individual participants.

Curt W. Reimann, Director

Center for Analytical Chemistry

v
NOTE

Because of concern for the useability of this lithium

reference method, Center for Disease Control management has

declined to endorse the method described in this report.

However, the authors believe the present method should

function as the reference method until the efficacy of a subsequent improved method has been demonstrated, since this

procedure has been shown to satisfy the generally accepted

criteria of a reference method. NBS supports the evolution

of analytical methods and believes it important that the

principles of analytical practice delineated in the present

report be circulated in a timely manner.

NBS will continue to participate in interlaboratory

exercises that are aimed toward establishing the transferability of proposed reference procedures and will maintain

its primary role in supplying SRM's and definitive methods.

vi
OTHER NBS PUBLICATIONS IN THIS SERIES

Catalog of NBS Standard Reference Materials

(1979-80 edition), R. W. Seward, ed., NBS

Spec. Publ. 260 (April 1979) $3.00* SN003-003-

02048-6

Michaelis, R. E., and Wyman, L. L., Standard

Reference Materials: Preparation of White Cast

Iron Spectrochemical Standards, NBS Misc.

Publ. 260-1 (June 1964). COM74-1 1061**

Michaelis, R. E., Wyman, L. L., and Flitsch, R.,

Standard Reference Materials: Preparation of

NBS Copper-Base Spectrochemical Standards,

NBS Misc. Publ. 260-2 (October 1964). COM74-

11063**

Michaelis, R. E., Yakowitz, H., and Moore, G. A.,

Standard Reference Materials: Metallographic

Characterization of an NBS Spectrometric LowAlloy Steel Standard, NBS Misc. Publ. 260-3

(October 1964). COM74-1 1060**

Hague, J. L., Mears, T. W., and Michaelis, R. E., Standard Reference Materials: Sources of

Information, NBS Misc. Publ. 260-4 (February

1965). COM74-11059

Alvarez, R., and Flitsch, R., Standard Reference

Materials: Accuracy of Solution X-Ray Spectrometric Analysis of Copper-Base Alloys, NBS

Misc. Publ. 260-5 (March 1965). PB168068**

Shultz, J. I., Standard Reference Materials:

Methods for the Chemical Analysis of White

Cast Iron Standards, NBS Misc. Publ. 260-6

(July 1975). COM74-11068**

Bell, R. K., Standard Reference Materials:

Methods for the Chemical Analysis of NBS

Copper-Base Spectrochemical Standards, NBS

Misc. Publ. 260-7 (October 1965). COM74-

11067**

Richmond, M. S., Standard Reference Materials:

Analysis of Uranium Concentrates at the

National Bureau of Standards, NBS Misc. Publ.

260-8 (December 1965). COM74-1 1066**

Anspach, S. C, Cavallo, L. M., Garfinkel, S. B., Hutchinson, J. M. R., and Smith, C. N., Standard Reference Materials: Half Lives of Materials

Used in the Preparation of Standard Reference

Materials of Nineteen Radioactive Nuclides

Issued by the National Bureau of Standards,

NBS Misc. Publ. 260-9 (November 1965).

COM74-11065**

Yakowitz, H., Vieth, D. L., Heinrich, K. F. J., and

Michaelis, R. E., Standard Reference Materials:

Homogeneity Characterization on NBS Spectrometric Standards II: Cartridge Brass and LowAlloy Steel, NBS Misc. Publ. 260-10 (December

1965). COM74-11064**

Napolitano, A., and Hawkins, E. G., Standard

Reference Materials: Viscosity of Standard

Lead-Silica Glass, NBS Misc. Publ. 260-11

(November 1966). NBS Misc. Publ. 260-11**

Yakowitz, H., Vieth, D. L., and Michaelis, R. E., Standard Reference Materials: Homogeneity

Characterization of NBS Spectrometric Standards III: White Cast Iron and Stainless Steel Powder Compact, NBS Misc. Publ. 260-12

(September 1966). NBS Misc. Publ. 260-12**

Spijkerman, J. L., Snediker, D. K., Ruegg, F. C,

and DeVoe, J. R., Standard Reference Materials: Mossbauer Spectroscopy Standard for the

Chemical Shift of Iron Compounds, NBS Misc.

Publ. 260-13 (July 1967). NBS Misc. Publ.

260-13**

Menis, O., and Sterling, J. T., Standard Reference

Materials: Determination of Oxygen in Ferrous

Materials - SRM 1090, 1091, and 1092, NBS

Misc. Publ. 260-14 (September 1966). NBS Misc.

Publ. 260-14**

Passaglia, E., and Shouse, P. J. Standard Refer- ence Materials: Recommended Method of Use

of Standard Light-Sensitive Paper for Calibrating Carbon Arcs Used in Testing Textiles for

Colorfastness to Light, NBS Misc. Publ. 260-15

(June 1967). (Replaced by NBS Spec. Publ.

260-41.)

Yakowitz, H., Michaelis, R. E., and Vieth, D. L., Standard Reference Materials: Homogeneity

Characterization of NBS Spectrometric Standards IV: Preparation and Microprobe Characterization of W-20% MO Alloy Fabricated by

Powder Metallurgical Methods, NBS Spec.

Publ. 260-16 (January 1969). COM74-1 1062**

Catanzaro, E. J., Champion, C. E., Garner, E. L., Marinenko, G., Sappenfield, K. M., and Shields,

W. R. Standard Reference Materials: Boric

Acid; Isotopic and Assay Standard Reference

Materials, NBS Spec. Publ. 260-17 (February

1970). Out of Print

vii
Geller, S. B., Mantek, P.A., and Cleveland, N.

G., Standard Reference Materials: Calibration of NBS Secondary Standard Magnetic Tape

(Computer Amplitude Reference) Using the

Reference Tape Amplitude Measurement

"Process A, "NBS Spec. Publ. 260-18 (November

1969). (See NBS Spec. Publ. 260-29.)

Paule, R. C, and Mandel, J., Standard Reference

Materials: Analysis of Interlaboratory Measurements on the Vapor Pressure of Gold (Certifica- tion of Standard Reference Material 745). NBS

Spec. Publ. 260-19 (January 1970). PB190071**

Paule, R. C, and Mandel, J., Standard Reference

Materials: Analysis of Interlaboratory Measurements on the Vapor Pressures of Cadmium and

Silver, NBS Spec. Publ. 260-21 (January 1971).

COM74-11359**

Yakowitz, H., Fiori, C. E., and Michaelis, R. E., Standard Reference Materials: Homogeneity

Characterization of Fe-3 Si Alloy, NBS Spec.

Publ. 260-22 (February 1971). COM74-1 1357**

Napolitano, A., and Hawkins, E. G., Standard

Reference Materials: Viscosity of a Standard

Borosilicate Glass, NBS Spec. Publ. 260-23

(December 1970). COM71-00157**

Sappenfield, K. M., Marineko, G., and Hague, J. L., Standard Reference Materials: Comparison

of Redox Standards, NBS Spec. Publ. 260-24

(January 1972). COM72-50058**

Hicho, G. E., Yakowitz, H., Rasberry, S. D., and

Michaelis, R. E., Standard Reference Materials:

A Standard Reference Material Containing

Nominally Four Percent Austenite, NBS Spec.

Publ. 260-25 (February 1971). COM74-1 1356**

Martin, J. F., Standard Reference Materials:

National Bureau of Standards-US Steel Corportion Joint Program for Determining Oxygen and

Nitrogen in Steel, NBS Spec. Publ. 260-26

(February 1971). 85 cents* SNO03-O03-O0786-2

Garner, E. L., Machlan, L. A., and Shields, W. R., Standard Reference Materials: Uranium

Isotopic Standard Reference materials, NBS

Spec. Publ. 260-27 (April 1971). COM74-

11358**

Heinrich, K. F. J., Myklebust, R. L., Rasberry, S. D., and Michaelis, R. E., Standard Reference

Materials: Preparation and Evaluation of

SRM's 481 and 482 Gold-Silver and GoldCopper Alloys for Microanalysis, NBS Spec.

Publ. 260-28 (August 1971). COM7 1-50365**

Geller, S. B., Standard Reference Materials: Cali- bration of NBS Secondary Standard Magnetic

Tape (Computer Amplitude Reference) Using

the Reference Tape Amplitude Measurement

"Process A-Model 2," NBS Spec. Publ. 260-29

(June 1971). COM7 1-50282

Gorozhanina, R. S., Freedman, A. Y., and

Shaievitch, A. B. (translated by M. C. Selby),

Standard Reference Materials: Standard

Samples Issued in the USSR (A Translation

from the Russian). NBS Spec. Publ. 260-30 (June

1971). COM71-50283**

Hust, J. G., and Sparks, L. L., Standard Reference

Materials: Thermal Conductivity of Electrolytic Iron SRM 734 from 4 to 300 K, NBS Spec. Publ.

260-31 (November 1971). COM71-50563**

Mavrodineanu, R., and Lazar, J. W., Standard

Reference Materials: Standard Quartz Cuvettes,

for High Accuracy Spectrophotometry, NBS

Spec. Publ. 260-32 (December 1973). 55 cents* SN003-003-01213-1

Wagner, H. L., Standard Reference Materials:

Comparison of Original and Supplemental

SRM 705, Narrow Molecular Weight Distri- bution Polystyrene, NBS Spec. Publ. 260-33

(May 1972). COM72-50526**

Sparks, L. L., and Hust, J. G., Standard Reference

Materials: Thermoelectric Voltage, NBS Spec.

Publ. 260-34, (April 1972). COM72-50371**

Sparks, L. L., and Hust, J. G., Standard Refer- ence Materials: Thermal Conductivity of

Austenitic Stainless Steel, SRM 735 from 5 to 280 K, NBS Spec. Publ. 260-35 (April 1972.)

35 cents* COM72-50368**

Cali, J. P., Mandel, J., Moore, L. J., and Young, D.

S., Standard Reference Materials: A Referee

Method for the Determination of Calcium in Serum, NBS SRM 915, NBS Spec. Publ. 260-36

(May 1972). COM72-50527**

Shultz, J. I. Bell., R. K. Rains, T. C, and Menis,

O., Standard Reference Materials: Methods of

Analysis of NBS Clay Standards, NBS Spec.

Publ. 260-37 (June 1972). COM72-50692**

Richmond, J. C, and Hsia, J. J., Standard Refer- ence Materials: Preparation and Calibration of

Standards of Spectral Specular Reflectance,

NBS Spec. Publ. 260-38 (May 1972). COM72-

50528**

Clark, A. F., Denson, V.A., Hust, J. G., and

Powell, R. L., Standard Reference Materials

The Eddy Current Decay Method for Resistivity

Characterization of High-Purity Metals, NBS

Spec. Publ. 260-39 (May 1972). COM72-50529**

viii
McAdie, H. G., Garn, P. D.,and Menis,0., Standard Reference Materials: Selection of Thermal

Analysis Temperature Standards Through a Cooperative Study (SRM 758, 759, 760), NBS

Spec. Publ. 260-40 (August 1972.) COM-72

50776**

Wood, L. A., and Shouse, P. J., Standard

Reference Materials: Use of Standard LightSinsitive Paper for Calibrating Carbon Arcs

Used in Testing Textiles for Colorfastness to Light, NBS Spec. Publ. 260^1 (August 1972)

COM72-50775**

Wagner, H. L., and Verdier, P. H., eds., Standard

Reference Materials: The Characterization of

Linear Polyethylene, SRM 1475, NBS Spec.

Publ. 260-42 (September 1972). COM72-

50944**

Yakowitz, H., Ruff, A. W., and Michaelis, R. E., Standard Reference Materials: Preparation and

Homogeneity Characterization of an Austenitic

Iron-Chromium-Nickel Alloy, NBS Spec. Publ.

260-43 (November 1972). COM73-50760**

Schooley, J. F., Soulen, R. J., Jr., and Evans, G. A.,

Jr., Standard Reference Materials: Preparation

and Use of Superconductive Fixed Point

Devices, SRM 767, NBS Spec. Publ. 260-44

(December 1972). COM73-50037**

Greifer, B., Maienthal, E. J., Rains, T. C, and

Rasberry, S. D., Standard Reference Materials:

Powdered Lead-Based Paint, SRM 1579, NBS

Spec. Publ. 260-45 (March 1973). COM73-

50226**

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and

Electrical Resistivity Standard Reference Materials: Austenitic Stainless Steel, SRM's 735 and

798, from 4 to 1200 K, NBS Spec. Publ. 260-46

(March 1975). SN003-003-0 1278-5

Hust, J. G., Standard Reference Materials: Electrical Resistivity of Electrolytic Iron, SRM 797,

and Austenitic Stainless Steel, SRM 798, from 5 to 280 K, NBS Spec. Publ. 260^7 (February

1974). COM74-50176**

Mangum, B. W., and Wise, J. A., Standard Refer- ence Materials: Description and Use of Precision

Thermometers for the Clinical Laboratory,

SRM 933 and SRM 934, NBS Spec. Publ. 260^8

(May 1974). 60 cents* SN003-O03-0 1278-5

Carpenter, B. S., and Reimer, G. M., Standard

Reference Materials: Calibrated Glass Standards for Fission Track Use, NBS Spec. Publ.

260^9 (November 1974). SN003-003-0 1344-7

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and Electrical Resistivity Standard Reference Materials:

Electrolytic Iron, SRM's 734 and 797 from 4 to 1000 K, NBS Spec. Publ. 260-50 (June 1975). 1.00* SN003-003-0 1425-7

Mavrodineanu, R., and Baldwin, J. R., Standard

Reference Materials: Glass Filters As a Standard

Reference Material for Spectrophotometry;

Selection; Preparation; Certification; Use -SRM

930, NBS Spec. Publ. 260-51 (November 1975).

$1.90* SN003-003-01481-8

Hust, J. G., and Giarratano, P. J., Standard Refer- ence Materials: Thermal Conductivity and Electrical Resistivity Standard Reference Materials

730 and 799, from 4 to 3000 K, NBS Spec. Publ.

260-52 (September 1975). $1.05* SN003-003-

01464-8

Durst, R. A., Standard Reference Materials:

Standardization of pH Measurements, NBS

Spec. Publ. 260-53 (December 1975, Revised).

$1.05* SN003-003-01551-2

Burke, R. W., and Mavrodineanu, R., Standard

Reference Materials: Certification and Use of

Acidic Potassium Dichromate Solutions as an

Ultraviolet Absorbance Standard, NBS Spec.

Publ. 260-54 (August 1977). $3.00* SN003-O03-

01828-7

Ditmars, D. A., Cezairliyan, A., Ishihara, S., and

Douglas, T. B., Standard Reference Materials:

Enthalpy and Heat Capacity; Molybdenum

SRM 781, From 273 to 2800 K, NBS Spec. Publ.

260-55 (September 1977). $2.20* SN003003-

01836-8

Powell, R. L., Sparks, L. L., and Hust, J. G.,

Standard Reference Materials: Standard

Thermocouple Material, Pt.-67:SRM1967, NBS

Spec. Publ. 260-56 (February 1978). $2.20*

SN003-003-0 18864

Cali, J. P. and Plebanski, T., Guide to United

States Reference Materials, NBS Spec. Publ.

260-57 (February 1978). $2.20* SN003-O03-

01883-0

Barnes, J. D. and Martin, G. M., Standard Refer- ence Materials: Polyester Film for Oxygen Gas

Transmission Measurements. SRM 1470, NBS

Spec. Publ. 260-58 (in press).

Chang, T. and Kahn, A. H., Standard Reference

Materials: Electron Paramagnetic Resonance

Intensity Standard: SRM 2601, NBS Spec.

Publ. 260-59 (August 1978). $2.30* SN-003-

003-01975-5

ix
Velapoldi, R. A., Paule, R. C, Schaffer, R.,

Mandel, J., and Moody, J. R., Standard Refer- ence Materials: A Reference Method for the Determination of Sodium in Serum, NBS Spec.

Publ. 260-60 (August 1978). $3.00* SN003-003

01978-0

Verdier, P. H., and Wagner, H. L., Standard Refer- ence Materials: The Characterization of Linear

Polyethylene (SRM 1482, 1483, 1484), NBS

Spec. Publ. 260-61 (December 1978). $1.70*

SN003-003-02006-1

Soulen, R. J., and Dove, R. B., Standard Reference

Materials: Temperature Reference Standard

for Use Below 0.5 K (SRM 768), NBS Spec.

Publ. 260-62 (April 1979). $2.30* SN003-003-

02047-8

Velapoldi, R. A., Paule, R. C, Schaffer, R.,

Mandel, J., Machlan, L. A., and Gramlich, J. W.,

Standard Reference Materials: A Reference

Method for the Determination of Potassium in Serum, NBS Spec. Publ. 260-63 (May 1979).

$3.75* SN003-003-02068

Velapoldi, R. A., and Mielenz, K. D., Standard

Reference Materials: A Fluorescence Standard

Reference Material Quinine Sulfate Dihydrate

(SRM 936), NBS Spec. Publ. 260-64 (January

1980). $4.25* SN003-003-02 148-2 Marinenko, R. B., Heinrich, K. F. J., and Ruegg,

F. C, Standard Reference Materials: MicroHomogeneity Studies of NBS Standard Reference

Materials, NBS Research Materials, and Other

Related Samples, NBS Spec. Publ. 260-65

(September 1979). $3.50* SN003-003-021 14-1 Venable, W. H., Jr. and Eckerle, K. L., Standard

Reference Materials: Didymium Glass Filters for Calibrating the Wavelength Scale of Spectrophotometers (SRM 2009, 2010, 2013), NBS Spec.

Publ. 260-66 (October 1979). $3.50* SN003-003-

02127-0

Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel,

J., Murphy, T. J., and Gramlich, J. W., Standard

Reference Materials: A Reference Method for the Determination of Chloride in Serum, NBS Spec.

Publ. 260-67 (November 1979). $3.75* SN003-

003-02136-9

Mavrodineanu, R. and Baldwin, Jr., Standard

Reference Materials: Metal-On-Quartz Filters as a Standard Reference Material for Spectrophotometry-SRM 2031, NBS Spec. Publ. 260-68

(April 1980). $4.25* SN003-003-02 167-9

Velapoldi, R. A., Paule, R. C, Schaffer, R.,

Mandel, J., Machlan, L. A., Garner, E. L., and

Rains, T. C, Standard Reference Materials: A

Reference Method for the Determination of

Lithium in Serum, NBS Spec. Publ. 260-69 (in

press).

* Send order with remittance to Superintendent

of Documents, US Government Printing

Office, Washington, DC 20402. Remittance

from foreign countries should include an

additional one-fourth of the purchase price for postage. ** May be ordered from: National Technical

Information Services (NTIS), Springfield,

Virginia 22151.
TABLE OF CONTENTS

PAGE

I. INTRODUCTION 2

II. DEVELOPMENT OF THE SERUM LITHIUM REFERENCE

METHOD 4

A. Organization 4

B. Participating Laboratories, Standards, Serum

Samples, and Definitive Method 6

C. Functions of the Various Groups 10

D. Plan for Testing the Candidate Reference

Method 10

III. REFERENCE METHOD PROTOCOL FOR THE DETERMINATION

OF SERUM LITHIUM 13

A. General 13

B. Protocol Synopsis 13

C. Detailed Protocol 14

IV. RESULTS AND STATISTICAL ANALYSIS 30

A. Inter laboratory Exercise Results 30

V. DISCUSSION 67

A. Candidate Protocol 67

VI. CONCLUSIONS 73

VII. REFERENCES 76

APPENDIX A 76

APPENDIX B 77

APPENDIX C 79

APPENDIX D 91

xi
LIST OF TABLES

TABLE NO . PAGE

1. Preliminary results from NBS and CDC for the

determination of serum lithium 5

2. Lithium concentrations for the serum pools

determined by IDMS, the definitive method ... 9

3. Volumes of lithium standard stock solution

diluted to 100 mL to give the diluted

lithium standard solutions 18

4. Example of relative absorbance values for sets

of readings using a direct read-out instrument. 28

5. Serum lithium concentrations reported by the

participating laboratories for the Preliminary

Interlaboratory Exercise 32

6. Concentrations of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise I, manual pipetting protocol 36

7. The effect of sample and standard dilution on

measured serum lithium concentrations 39

8. Concentration of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise II, manual pipetting protocol 41

9. Summary of FAAS instruments and selected experimental parameters used for Interlaboratory

Exercise II 43

10. Effect of aspiration tube diameter on measured

serum lithium concentrations at several

dilutions 44

11. Concentration of serum lithium reported by the 47

participating laboratories for Interlaboratory §

Exercise III, manual method 48

12. Concentration of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise III, semiautomated pipetting protocol. 49

13. Percent deviations from averages for lithium in

serum from Interlaboratory Exercise III,

manual pipetting protocol 52

xii
LIST OF TABLES (continued)

TABLE NO. PAGE

14. Percent deviations from averages for lithium in

serum from Inter laboratory Exercise III,

semiautomated pipetting protocol 53

15. Ratios of standard deviations to average

standard deviation for lithium in serum from

Inter laboratory Exercise III, manual

pipetting protocol 54

16. Ratios of standard deviations to average

standard deviation for lithium in serum from

Interlaboratory Exercise III, semiautomated

pipetting protocol 55

17. Components of standard deviation in mmol/L for

lithium in serum, Interlaboratory Exercise III. 58

18. Summary of imprecision and bias results in

mmol/L for lithium in serum, Interlaboratory

Exercise III 60

19. Summary of lithium in serum values 62

20. Calibration curve data for lithium in serum, 63

Interlaboratory Exercise III, manual §

pipetting protocol 64

21. Calibration curve data for lithium in serum,

Interlaboratory Exercise III, semiautomated

pipetting protocol 65

22. Calibration curve results for lithium in serum

as standard deviation of fit ( s £^ t

) i- n

mmol/L 66

23. Instruments and operating conditions used by

the participating laboratories in Interlaboratory Exercises II and III 69

xiii
LIST OF FIGURES

FIGURE NO. PAGE

1. Interrelationships and functions of the various

groups in the development of a clinical

reference method for the determination of

serum lithium 11

2. Typical calibration curve for the determination

of serum lithium by flame atomic absorption

spectroscopy 26

3. Percent deviations of the two day average results

from the collective average of the measurements

obtained in the Preliminary Interlaboratory

Exercise 33

4. Percent deviations of the Interlaboratory

Exercise I measurements using manual

pipetting from the definitive method values . . 37

5. Percent deviations of the Interlaboratory

Exercise II measurements using manual

pipetting from the definitive method values . . 42

6. Percent deviations of the Interlaboratory

Exercise III measurements using manual

pipetting from the definitive method values . . 50

7. Percent deviations of the Interlaboratory

Exercise III measurements using semiautomated

pipetting from the definitive method values . . 51

xiv
ABSTRACT

A reference method was established for the determination

of serum lithium based on flame atomic absorption spectroscopy

(FAAS) . Its accuracy was evaluated by comparing the values

obtained by use of the method in 14 laboratories against the

results obtained by a definitive analytical method based on

isotope dilution-mass spectrometry (IDMS) . Ten serum pools

with lithium concentrations in the range 0.534 to 2.954

mmol/L were analyzed. Manual and semiautomated pipetting

alternatives were tested using sample sizes of 4.00 and

2.00 mL , respectively.

The laboratories used several different FAAS instruments.

The results showed that the standard error for a single laboratory's performance using either pipetting procedure was

about 1.5 percent with a negative bias of about 2.0 percent

over the range of serum lithium concentrations studied.

These values are within the accuracy and precision goals that

had been set. The calibration curve data showed excellent

linearity over the total concentration range, with 24 of 25

curves having standard deviations of fit of 0.025 mmol/L or

less . With appropriate experimental design, the reference

method may be used to establish the accuracy of field methods

as well as to determine reference lithium values for pooled

sera

.

Key Words: Accuracy; clinical analysis; clinical chemistry;

definitive method; electrolytes; flame atomic

absorption spectroscopy; inter laboratory testing;

precision; reference method; semiautomated

pipetting; serum lithium analysis; statistical

analys is

.

1
I. INTRODUCTION

Lithium was first used by Cade [l] 5 to treat and prevent

the recurrence of manic-depressive psychosis and is widely

used today. However, the margin between therapeutic dosage

and toxicity is not large [2]. Toxic symptoms have been

observed in patients with serum lithium levels of 1.6 mmol/L,

a value quite close to the 1.0-1.5 mmol/L serum level generally attained for immediate treatment [3]. It is thus

important to have an accurate analysis for serum lithium in

addition to observing the clinical symptoms of the patient

during treatment.

The quantitative analysis of serum lithium by early

analytical techniques was difficult. Attempts included separation by precipitation [4], paper chromatography [5], and

a semiquantitative colorimetric procedure [6] . The advent

of flame photometric techniques, including flame atomic

absorption spectroscopy (FAAS) e and flame atomic emission

spectroscopy (FAES) 5 has led to simple, quantitative techniques for determining serum lithium concentrations [7],

The use of flame atomic absorption spectroscopy has been

described as a standard method [8]. Whether this FAAS method

or some other should be considered by clinical laboratories

as the reference method for serum lithium has not been proven;

the accuracy of none of these methods is known.

Two approaches may be used for establishing the accuracy

of analytical methods. In the first, the results obtained

from the methods in use for that analyte are compared using

typical samples and selected samples containing known interferences for the analyses. Statistical correlations are used

5 The bracketed numerals refer to the references listed at

the end of this paper.

6 0fficial name, International Union of Pure and Applied

Chemistry, Information Bulletin Number 27, Nov. 1972.

2
to express the interrelationships of the methods. A technique

is then considered to be accurate to the degree established

by knowledge of the sources of error and the agreement of

results. In the second, a single candidate method is selected

(possibly the 'best' of the methods recognized by the first

approach) and studied in detail. Each step of the candidate

method is optimized and examined so that the systematic and

the random errors can be quantitatively expressed.

Studies have been organized using a combination of these

approaches to establish the accuracies of reference methods

for total calcium, sodium, potassium, and chloride in serum

[9-12]. For calcium, the analytical procedure was based on

the FAAS method of Pybus, Feldman, and Bowers [13], for

sodium and potassium the methods were based on FAES, and for

chloride, the method was based on coulometric titration - amperometric end-point determination. The accuracies of

these methods were assessed by comparing the results obtained

in selected clinical laboratories in statistically controlled

studies against those obtained for the same serum pools by

use of isotope dilution-mass spectrometry (IDMS) methods for

calcium, potassium, and chloride and an ion-exchange - gravimetry method for sodium. The latter analyses were

performed at the National Bureau of Standards (NBS) where

the high accuracy of those methods 7 was established by

determining their systematic and random errors [14].

Those studies, carried out with the guidance of clinical

laboratory experts, used (a) Standard Reference Materials as

pure, primary reference materials to prepare standard solutions of calcium, sodium, potassium, and chloride; (b) serum

pools prepared at the Hartford Hospital (Hartford) and the

Center for Disease Control (CDC, Atlanta); (c) definitive

7 Such methods are referred to as definitive methods because

of their high accuracy and utility for evaluating the

accuracy of a candidate reference method.

3
method analyses performed at NBS; (d) statistical analysis

of the data at NBS; and (e) accuracy and precision goals as

performance standards that the methods would have to meet to

be recommended as reference methods [15]. This same approach

was adopted to develop a clinical reference method for serum

lithium.

This work was begun with the cooperation of individuals

from the Standards Committees of the American Association for

Clinical Chemistry (AACC) and the College of American Pathologists (CAP), the CDC and the NBS. The Food and Drug Administration (FDA) provided major suppport for the NBS work.

Progress with this program was reported periodically to the

AACC Standards Committee. We present in this report the

development of a clinical reference method for serum lithium.

II, DEVELOPMENT OF THE SERUM LITHIUM REFERENCE METHOD

A. Organization

A panel of experts in clinical chemistry was invited to

meet at NBS in March 1974 to consider the development of reference methods for five serum electrolytes, namely, potassium,

sodium, chloride, lithium, and magnesium. The overall program

for the development of these reference methods was organized

by Dr. Robert Schaffer (NBS) and Dr. Ranee A. Velapoldi (NBS).

The invited experts were Dr. George N. Bowers, Jr. (Hartford

Hospital), Dr. Bradley E. Copeland (New England Deaconess

Hospital), Dr. Denis 0. Rodgerson (Center for Health Sciences,

University of California in Los Angeles), and Dr. James M.

White 8 (CDC).

8 Dr. James White died after this program was well underway.

He was recommended for membership on this Experts Committee

on electrolytes by Dr. Joseph H. Boutwell (CDC). Dr. White

made significant contributions to the protocol for the reference method. His knowledge, advice, and cooperation in

all phases of this work contributed greatly to the success

of the program.

4
Prior to the meeting, several bovine serum pools prepared

at the CDC had been analyzed for lithium by FAES, FAAS, and

IDMS. The results, summarized in Table 1, were presented at

the meeting as follows:

FAAS as obtained at the CDC, by Dr. J. White,

FAES as obtained at the NBS by Dr. R. Mavrodineanu, and

IDMS as obtained at the NBS, by Dr. L. Moore.

On consideration of these quite similar analytical results,

of the relative advances in FAAS instrumentation over those

for FAES instrumentation, and of alternative clinical laboratory procedures in use for the determination of serum lithium,

it was concluded that FAAS was the appropriate candidate

methodology to evaluate as the reference method and that its

evaluation should be made using IDMS as the definitive method.

Table 1. Preliminary results from NBS and CDC for

the determination of serum lithium.

- - - - Li, mmol/L -----

IDMS FAES FAAS

NBS CDC

1.004 0.99 1.00

1.969 1.96 1.96

2.954 2.96 2.90

Pool

I III

V

The panel of experts agreed to serve as the Committee

to oversee the development of the reference method for lithium

(as well as for the other electrolytes under consideration at

the meeting). The Committee chose Dr. Bowers as chairman.

Dr. Bowers agreed to serve as the Committee's representative

to work with those at NBS who would be involved in writing

the protocol for the lithium reference method. The Committee

agreed that the FAAS method should be based on the method of

Pybus and Bowers (using a 10-fold sample dilution) [8,16],

and should use a concentration bracketing technique rather

5
than calibration curves for determining lithium concentrations.

However, calibration curve data should be obtained as a general

check on the measurement system and to determine which of the

primary standard solutions to use for bracketing the lithium

levels in the samples being analyzed.

As goals for the candidate reference method, the maximum

bias of the method and the one- standard deviation imprecision

limit were set by the Committee at 0.2 and 0.1 mmol/L, respectively, for serum lithium at the 2.0 mmol/L level. These

goals were to be achieved by controlled, interlaboratory tests

involving a selected group of clinical chemistry laboratories

which would perform the analyses by the FAAS method according

to the written protocol. NBS would provide lithium values for

the pools by the definitive method.

B. Participating Laboratories, Standards, Serum Samples,

and Definitive Method

The laboratories that were asked to participate in the

interlaboratory study were chosen to represent a wide spectrum

of clinical chemistry interests and included government

(federal and state) and hospital laboratories, and laboratories

associated with suppliers of instruments and of test and

control materials. Two hospitals were located outside the

United States. The principal investigators at these laboratories are named in the following list. Other scientists in

each of the laboratories who contributed to this study are

acknowledged by name in Appendix A. The list includes two

laboratories that participated only in the concluding interlaboratory work. They were added to maintain a minimum

number of laboratories when some of the original laboratories

were unable to continue their participation. In alphabetical

order of the principal investigator, the laboratories that

participated in the interlaboratory studies are:

6
Dr. Huey V. Auger

Warner- Lambert

Morris Plains, NJ 07950

Dr. Eleanor Berman

Cook County Hospital

Chicago, IL 60612

Dr. George N. Bowers, Jr.

Hartford Hospital

Hartford, CT 06115

Dr. Bradley E. Copeland

Veterans Administration Hospital

Cincinnati, OH 45220

Dr. Gordon Edwards

Mr. Gary A. King

Dade Division

American Hospital Supply Co.

Miami, FL 33152

Mr. Frank J. Fernandez

The Perkin-Elmer Corp.

Norwalk, CT 06856

Dr. Nathan Gochman

Veterans Administration Hospital

San Diego, CA 92161

Mr. David Hassemer

Dr. Ronald H. Laessig

State Laboratory of Hygiene, University of Wisconsin

Madison, WI 53706

7
Dr. Frederick Mitchell

Dr. Stanley S. Brown

Clinical Research Center

Watford Road

Harrow, Middlesex, HA2 34J, England

Dr. John Pybus

Auckland Hospital

Park Road, Auckland 3, New Zealand

Mr. Theodore C. Rains

Dr. Michael Epstein

National Bureau of Standards

Washington, D. C. 20234

Dr. Denis 0. Rodgerson

Center for Health Sciences, University of California

Los Angeles, CA 90025

Dr. Barbara Tejeda

Food and Drug Administration

Washington, D. C. 20250

Dr. James M. White

Dr. Richard Carter

Center for Disease Control

Atlanta, GA 30333

Dr. Charles E. Willis

College of American Pathologists, Cleveland Clinic

Cleveland, OH 44106

NBS Standard Re

924, see Appendix B)

reference material f

ference Material Li

was to be used as

or all analyses [17

thium Carbonate (SRM

the pure, primary

] . Ten pools of

8
homogeneous, sterile, bovine serum, having different concentrations of lithium, were prepared at the CDC by Dr. David Bayse

and Ms. Sue Lewis. Samples of each pool were supplied in

approximately 7-mL volumes in sealed vials that were labeled

with computer-generated random numbers. The samples, packed

in dry ice, were shipped to NBS by air and within 24 h of

packing were placed in freezers kept at -50 °C [18]. The

pools were numbered in the codes la, 5a, and 7a for the first

series of serum pools and from 1 to 7 for the second series

of serum pools according to increasing lithium concentration.

A definitive method based on IDMS was developed at NBS

and is given in Appendix C. The lithium concentrations for

the ten serum pools were determined by this procedure and

the results obtained are summarized in Table 2.

Table 2. Lithium concentrations of the serum pools

determined by IDMS, the definitive method.

Pool [Li

+

] > mmol/L

1 0. 534 + 0 .003 a

la 1. 004 + 0.005 a

2 1.031 + 0.005 a

3 1. 290 + 0 .006 a

4 1. 546 + 0 . 008 a

5 1.809 + 0.009 a

5a 1.969 + 0 .oio a

6 2. 042 + 0 . 010 a

7 2.572 + 0 .013 a

7a 2. 954 + 0.015 a

Estimated maximum total error of 0.5 percent of the value.

This estimated error is the sum of errors due to measurement

imprecisions of ±0.3 percent (±2 sigma interval for the

ramdom error of the mean) and an estimated upper bound of

0.2 percent for possible systematic errors.
C. Functions of the Various Groups

The interrelationships and functions of the various

groups involved in developing FAAS as a reference method for

serum lithium are represented in figure 1. The Committee,

CDC, and NBS provided guidance and technical support for the

program and also served as participating laboratories. The

Experts Committee selected the candidate reference method,

set maximum bias* and imprecision goals for an acceptable

reference method, assisted NBS in selecting other participating laboratories, and reviewed all analytical results. The

CDC provided the serum pools. The participating laboratories

provided the inter laboratory test data and critiques of the

candidate reference method protocol.

At NBS, Dr. R. Schaffer served as the Reference Method

Program Manager and Dr. R. A. Velapoldi served as the coordinator. The format of the interlaboratory exercises [IE) 9 was established within the constraints imposed by protocol

requirements and sample availability by Drs. John Mandel,

Robert Paule, and Ranee Velapoldi. Dr. Velapoldi wrote the

protocol for the candidate reference method from the outline

provided by Dr. G. Bowers, Jr. Drs. Mandel and Paule performed the statistical evaluation of the results from the

interlaboratory tests. The definitive method was performed

by Mr. Lawrence A. Machlan and Mr. Ernest L. Garner.

D. Plan for Testing the Candidate Reference Method

The general plan was to evaluate the candidate reference

method by performing a series of interlaboratory exercises,

which would consist of a preliminary test (IE-P) followed by

successive interlaboratory exercises until the goals for the

reference method were reached. A main objective of the IE-P

9 In previous reports, the Interlaboratory Exercises were

called Round Robin Tests.

10
EXPERTS

COMMITTEE

GUIDE AND REVIEW

WORK

• SELECT CANDIDATE

METHOD • SET ANALYTICAL

GOALS

CDC

PROVIDE SERUM

POOLS • OUTLINED

PROTOCOL

TECHNICAL

SUPPORT

srm

NBS

> DEFINITIVE METHOD AND DETERMINE

SERUM POOL VALUES

STATISTICAL ANALYSIS • DETAILED PROTOCOL

COORDINATION

OF WORK

PARTICIPATING LABORATORIES

• ANALYZE SERUM POOLS • PROTOCOL EVALUATION

Figure 1. Interrelationships and functions of the various

groups in the development of a clinical reference

method for the determination of serum lithium.

11
was to allow participating laboratories to become familiar

with and comment on the protocol. An evaluation of the bias

was not sought in the IE-P testing phase since no definitive

method lithium values were available at the time the IE-P

was run. Thus, only interlaboratory imprecision was to be

measured. If the imprecision of the results in the IE-P was

found to be small, interlaboratory testing would begin on

samples having definitive method lithium values.

In an IE, each participating laboratory would perform

the same analyses on two separate days: i.e., analyze pairs

of samples from each serum pool on each of two days where a minimum of one day or a maximum of seven days were to elapse

between the two series of analyses. The bias and imprecision

values obtained by statistical analysis would then be compared

to the goals set by the Committee for the reference method.

If the goals were not met, additional IE's using samples

from other pools would be conducted by following the protocol

or a modified version until the bias and imprecision goals

were reached. Revisions and modifications to the protocol

could be made after an interlaboratory exercise had been

completed but would not be made after the final IE.

Three kinds of information were to be supplied by each

participating laboratory after finishing an interlaboratory

exercise

:

1. General Data — a list of the instrumental parameters

used and comments on the protocol including problems

encountered during the analysis;

2. Calibration Curve Data - a list of the FAAS relative

absorbance values versus the lithium concentrations of

the standards calculated from the Li 2 C0 3 used; and

3. Valid Measurement Data - a list of the sets of data

that constituted the five 'valid measurements' (see

section IIIC-5e for discussion).

Examples of the data sheets on which the information was

collected are shown in Appendix D, Note 8.

12
III. REFERENCE METHOD PROTOCOL FOR THE DETERMINATION

OF SERUM LITHIUM

A. General

This protocol for the analysis of serum lithium by flame

atomic absorption spectroscopy provides for the optional use

of either manual or semiautomated pipetting.

B. Protocol Synopsis

The protocol must be followed exactly. The reference

method is used to analyze four samples of a serum or pool:

two on one day and the other two on a subsequent day.

Approximately 10 mL of serum is needed to carry out the semiautomated pipetting procedure and 25-30 mL of serum is needed

to carry out the manual pipetting procedure.

1. Use an analytical balance to weigh the SRM Li2C0 3 in

appropriate quantities to prepare a stock standard

lithium solution;

2. To dilute to the lithium concentrations that are used as

working solutions for FAAS, use either a single pipet or

a pipettor-dilutor to obtain a) aliquots of the serum

to be analyzed, b) aliquots of the stock standard lithium

solutions, and c) the solution used as a blank;

3. Obtain calibration curve data on the working blank and

standards for use as a check on standards preparation

and instrument stability and linearity;

4. Measure the FAAS signals of the working solutions of the

serum sample; select the pair of working standards whose

signals most closely bracket the signal for each sample;

5. For each sample to be analyzed, obtain five valid measurement sets from repeated sequential measurements of the

13
working solutions of the low bracketing standard, the

sample, and the high bracketing standard;

6. Calculate the lithium concentration of the sample for

each set by mathematical interpolation;

7. Average the five calculated values to obtain a 'single

measurement' of that sample; (in the statistical analysis,

each such average is designated a 'single measurement');

8. Perform steps (4) through (7) for each sample to be

analyzed on the first day;

9. Repeat steps CI) through (8) on the subsequent day to

obtain the second pair of measurements needed for each

sample

10. Average the four values obtained by the replicate

determinations to obtain the lithium concentration

for each serum pool.

C. Detailed Protocol

The selection of the specific alternatives of the protocol

to be used dictates the glassware and diluent volumes needed.

These needs are summarized in the protocol or in Appendix D

notes. Stock solutions and working solutions are to be

prepared at and maintained at a room temperature that is

constant within ±2 °C [see Appendix D, Note 1).

1 . Reagent Specifications

a. Water : At the time of preparation, the distilled

and/or deionized water used should exhibit a specific

resistance of at least 10 k£>m at 23 ± 2 °C. At the

time of use, this water should show a FAAS signal

that is less than 0.1 percent of the expanded

absorbance scale (Section IIIC-7c- (2) ) . A large

quantity of this water (more than 50 L) must be

available for use as diluent and for the final
rinsings of all glassware and other apparatus that

come in contact with the solutions involved. Unless

specified otherwise, the water referred to in this

protocol is this tested water.

Lithium Standard Solutions : Use Standard Reference

Material Lithium Carbonate (originally issued as SRM 924, Certificate reproduced in Appendix B) [17]

certified by the National Bureau of Standards

.

(Note: see Appendix D, Note 5.) Dry the SRM Li 2 C0 3 at 200 °C for four hours in a loosely capped container, allow to cool, and then store it tightly

capped in a desiccator containing CaSCs or an

equivalent desiccant.

Potassium and sodium chlorides, hydrochloric and

nitric acids, chloroform, methanol and 95-percent

ethanol (v:v) meeting ACS [20] or equivalent

specifications are to be used.

Dilute nitric acid (0.77 mol/L) is prepared by

making a twenty-fold dilution of concentrated

HN0 3 CIS. 4 mol/L) with water.

Dilute hydrochloric acid (2.3 mol/L is prepared by

making a five-fold dilution of concentrated HC1

(11.6 mol/L) with diluent solution (see IIIC-3a).

Prepare approximately 25 mL

.

Glassware Specifications

Volumetric glassware (Appendix D, Note 2) should be

of borosilicate material and meet NBS Class A [21]

or equivalent specifications. All glass or plastic

surfaces that come into contact with reagents,

water, diluent, or sample must be clean (Appendix

D, Note 3).

15
b. Pipettor-dilutor Device : The volumetric delivery

of the pipettor-dilutor device must have a tested

maximum inaccuracy of 2 percent and a maximum

imprecision of ±0.2 percent relative standard

deviation at the pump setting used. (The test

procedures are in Appendix D, Note 4.)

3 . Preparation of Reagents

a. Sodium Chloride-Potassium Chloride Diluent Solution

(NaCl - 140 mmol/L and KC1 - 5.0 mmol/L Diluent : Add 16.36 g of NaCl and 0.746 g of KC1 to a 2-liter

volumetric flask, dissolve in water, and dilute to

the calibrated volume with water. Stopper and

invert the flask and shake 10 times; repeat the

inversion and shaking 10 times. (NOTE: Approximately 6 liters of this solution are needed.)

b . Lithium Standard Stock Solutions (10 mmol/L) : Prepare separately two stock solutions each containing approximately 10.0 mmol/L LiCl. Weigh

accurately (to 0.1 mg) approximately 0.74 g of

lithium carbonate (molecular weight = 73.94855,

Appendix D, Note 5b) and transfer quantitatively

into a 2-liter volumetric flask. Dissolve the

Li2CC>3 by just covering the bottom of the flask

with diluent solution (IIIC-3a) and slowly adding

10 mL of dilute HC1 (IHC-le). Swirl until dissolved. Fill to the calibrated mark using diluent

solution and mix thoroughly. Repeat these steps

to prepare the second lithium standard stock

solution. Label the solutions I and II. From the

weighed quantities of Li 2 C0 3 taken, calculate

their lithium concentrations in mmol/L to four

decimal places.

16
(1) Intercomparison of Standard Stock Solutions:

Transfer by pipet using the "to deliver mode",

5.00-mL of stock solution I into a one-liter volumetric flask. Dilute to the calibrated volume

with water, stopper, invert, shake, and mix as

described above to give a working solution with a lithium concentration of 0.05 mmol/L. Repeat

these dilution steps for stock solution II.

Immediately aspirate each of the 0.05 mmol/L lithium

solutions and measure their expanded absorbance

values (see Section IIIC-7d) under the instrumental

settings used for this analysis. If the expanded

absorbance values corrected for concentration

differences agree to within 0.5 percent for both

solutions, lithium stock standard I may be used

for the analyses on the first day and stock

standard II may be used for the analyses on the

subsequent day subject to temperature restrictions.

If the values do not agree within 0.5 percent,

discard both stock standard solutions and repeat

their preparation and the intercomparison test

until the requirement of 0.5 percent agreement is

obtained

.

Diluted Lithium Standard Solution : Prepare the

various diluted lithium standard solutions by

transferring the volumes of the lithium stock

standard solution listed in Table 3 into 100-mL

volumetric flasks and diluting each to the calibrated

volume with the NaCl-KCl diluent. Mix thoroughly.

CNOTE: These dilutions are made using volumetric

pipets in the "to deliver" mode (Section IIIC-4) - not the "to contain" mode, Section IIIC-5.

17
Table 3. Volumes of lithium standard stock solution diluted

to 100 mL to give the diluted lithium standard

solutions

.

Stock Solution to be Concentration of Diluted Standards

Transferred, mL LiCl, mmol/L

5.00 0.50

10.00 1.00

15.00 1.50

20.00 2.00

25.00 2.50

30.00 a 3.00

a See Appendix D, Note 6.

Pipetting and Diluting Procedures to Prepare Diluted

Lithium Standard Solutions (To Deliver Mode) : General: Transfer of the lithium stock standard

solution to prepare the diluted lithium standard

solutions is performed by using the appropriate

Class A pipet in the to deliver mode (see Table 3)

.

Procedure: Add approximately 15 mL of the NaCl-KCl

diluent solution to each 100-mL volumetric flask.

Condition the pipet as in Section I IIC- 5b- (3) (b)

with approximately 3 mL of the lithium standard

stock solution to be transferred and discard this

solution. Repeat twice more. Fill the pipet to

approximately 1.0 cm above its calibration mark.

Withdraw the pipet from the container, and wipe

the delivery end of the pipet with a clean, absorbent

paper. Contact the tip of the pipet with. the side

of the container or a clean waste container and

deliver excess solution until the meniscus is at

the calibrated mark on the pipet. Remove the pipet

18
from contact with the container and direct the

delivery end of the pipet into the receiver.

Deliver the solution into the volumetric flask by

allowing the solution to run down the side of the

flask. When the solution level has reached the

low end of the pipet (just below the bulb), contact

the pipet tip to the side of the flask. (NOTE: As

a minimum, allow the pipet to drain for the delivery

time inscribed on the pipet.) After drainage is

complete, remove the tip from contact with the

flask wall, dilute to volume using the diluent

solution, and mix. Repeat these steps until the

six diluted lithium standard solutions are prepared

and labeled appropriately.

5 . Pipetting and Diluting Procedures to Prepare Working

Solutions ('to contain' mode) :

a. General : A 25-fold dilution is to be used.

b. Manual Pipetting Alternative : The blank, the standard, and the sample solutions are diluted by

employing only one 4-mL pipet with a wash-out

technique and 100-mL volumetric flasks. Working

solutions are prepared with the one pipet and the

wash-out technique to eliminate errors that may be

caused by differences in drainage between aqueous

and serum solutions.

(1) Twenty- five- fold Dilutions: Transfer approximately 25 mL of NaCl-KCl diluent into a 100-mL

volumetric flask and then add a 4 mL aliquot of the

sample or diluted lithium standard solution by the

procedure described in step (2) below.

(2) Pipetting Procedure: Fill the 4-mL pipet to

approximately 1.0 cm above its calibration mark,

withdraw the pipet from the container, and wipe the

19
delivery tip with a clean, absorbent paper. Contact

the tip to the side of a clean waste container and

allow excess solution to drain until the meniscus

is at the calibrated mark on the pipet. Remove

the pipet from contact with the container and

direct the delivery tip of the pipet into the

receiver. Deliver the sample by contact of the

pipet tip with the wall inside the volumetric flask

and allow the solution to drain fully. After

drainage stops, gently expel the residual liquid.

Wash off the outside of the pipet tip into the

receiver with about 4 mL of NaCl-KCl diluent delivered, for example, from a wash bottle or a disposable

Pasteur or similar pipet. (Caution: New, disposable

pipets need to be cleaned.) Rinse the 4-mL volumetric pipet three times by filling with fresh

NaCl-KCl diluent-from a separate beaker, each time „ delivering the contents into the receiver by drainage against the inner wall of the flask above the

liquid level. Dilute to the calibrated volume with

the NaCl-KCl diluent and mix thoroughly.

(3) Preparation of Working Solutions:

(a) Working Blank Solution and Working Standard

Solutions: Prepare the working solutions of the

blank and of the 0. 50-, 1. 00-, 1.50-, 2 . 00-, 2 . 50-,

and 3.00-mmol/L lithium standards by making dilutions in appropriately labeled volumetric flasks in

the order cited. Condition the 4-mL pipet by

filling it with the solution to be diluted.

Discard this pipetful and repeat filling and

discarding twice more. Then refill the pipet with

the solution, adjust to the calibrated volume, and

deliver into the volumetric flask to be used for

the dilution. Rinse the pipet by filling it three

20
times with the NaCl-KCl diluent, each time delivering

the rinse solution into the volumetric flask. Fill

the flask to the calibrated volume with the NaCl-KCl

diluent. Wash out the pipet three times with water

(see Appendix D, Note 7) and expel the residual

liquid.

(b) Working Sample Solutions: Condition the 4-mL

pipet with some of the sample to be diluted in the

following way: 1) Draw ^1 mL of the sample into

the pipet, 2) withdraw the pipet from the container,

3) wipe off the tip with a clean, absorbent paper,

4) tilt the pipet to a horizontal position, 5) allow

a small volume of air to leak in and rotate the

pipet so that the conditioning liquid wets all the

internal surface to approximately 0.5 cm above the

calibration mark, 6) discard this conditioning

solution, and 7) repeat steps (1-6). Then prepare

the working solutions as described in sections

IIIC-5b-(l) and (2), i.e., fill the 4-mL pipet with

the sample, adjust volume to the mark, deliver,

rinse three times into the volumetric flask with

NaCl-KCl diluent, dilute to the calibrated volume,

and mix, Finally, wash out the pipet three times

witji water (Appendix D, Note 7). For each of the

next sample solutions to be diluted, repeat step

IIIC-5b- (3) (b)

.

Semiautomated Pipetting Alternative : To prepare

working solutions, the blank, standard, and sample

solutions are diluted by using a pipettor-dilutor

device to deliver 2.000 mL into appropriately

labeled 50-mL volumetric flasks. A single delivery

tube on the pipettor-dilutor and the wash-out

technique are used throughout.

21
(1) Twenty- five-fold Dilutions: Transfer approximately 15 mL of NaCl-KCl diluent into a 50-mL

volumetric flask and then add 2.000 mL of the

appropriate solution by the procedure described in

step (2) below.

(2) Procedure: The pipettor-dilutor is set to

sample 2.000 mL and to dilute with 5 mL of NaCl-KCl

diluent. After conditioning the pipettor-dilutor

as in Appendix D, Note 3b, dip the delivery tip of

the pipettor-dilutor into the solution to be transferred. Draw up the desired volume of solution

(2.000 mL) . Care must be taken to avoid air

bubbles in the tubing before or during this

operation. Withdraw the tip of the delivery tube

from the solution, touch the tip to the container

side, and remove the container. With care not to

touch the open end of the tip of the tube, wipe

the outside of the delivery tube, direct the tip

of the tube into the 50-mL volumetric flask, and

deliver the aliquot and diluent solution into the

flask. Rinse the delivery tube twice more by

delivering two additional 5-mL portions of diluent

through the tube into the 50-mL volumetric flask.

(NOTE : To minimize foaming and spattering, deliver

the stream of solution and diluent on the wall

inside the neck of the flask.) After delivery is

complete, touch the tip of the tube to the inside

wall of the flask to transfer any solution remaining outside the tube. Remove the volumetric

flask, dilute to the calibrated volume with the

NaCl-KCl diluent, and mix.

22
(3) Preparation of Working Solutions:

(a) Prepare the working blank, standard, and

sample solutions using the semiautomated pipetting

procedure described above.

Total Solutions : At the conclusion of the dilution procedure, appropriately labeled flasks with the following working

solutions should be ready for analysis:

a. For the Manual and/or Semiautomated

Pipetting Alternatives:

(1) One working blank;

(2) Six working standards; and

(3) A working solution for each serum

sample to be analyzed.

Flame Atomic Absorption Spectroscopy Measurement

Procedures

It is not possible to provide detailed instructions

for each type of instrument to assure necessary

instrument stability, linearity, flame conditions,

etc. The operator must be familiar with the instru

ment used. The instrument should meet all the

manufacturer's specifications. In general, the

accuracy of the method cannot be attained unless

the instrument is in optimum operating condition.

Air and acetylene are used as oxidant and fuel,

respectively

Instrument and Electrical Adjustment . Prepare the

atomic absorption spectrometer for operation

according to instructions provided in the operator'

manual. Select the optimum current for lamp, and

allow ample "warm-up" time for the lamp to become

stable. Adjust the monochromator slit and set the

23
wavelength selector to the lithium resonance line

o at 670.8 nm (6708 A). Adjust the photomultiplier

dynode voltage to give optimum current output with

minimum dark current.

b. Flame Condition . Adjust the secondary regulators

for the air and acetylene and adjust the airacetylene flow rates to those recommended for the

instrument. Stabilize the temperature of the

burner head by aspirating water into the flame for

10 min before proceeding to the next step. (NOTE:

A fuel- rich air-acetylene flame gives optimum

sensitivity for the measurement of lithium; however,

it may be difficult to obtain the precision specified

in this method with a fuel-rich flame. Therefore,

it is suggested that a stoichiometric or only

slightly fuel-rich (slight yellow streaking) flame

be used to obtain the highest precision for lithium

in serum.) Check the flame appearance and aspiration

rate to assure that the nebulizer burner system is

free of foreign materials.

c. Instrument Stability . Determine the stability and

repeatability of the instrument as follows:

(1) Adjust the instrument to a zero absorbance

reading while nebulizing the NaCl-KCl diluent.

(NOTE: Always nebulize NaCl-KCl diluent when

measurements of the working blank, standard, or

sample solutions are not being made. Adjust the

instrument so that the NaCl-KCl diluent reads

'zero' at all times.)

(2) Nebulize the working lithium standard solution

obtained from the 3.000 mmol/L standard solution,

maximize the signal by adjusting the appropriate

parameters, and adjust direct read-out instruments

24
so that a relative absorbance of at least 1.500

is observed.

(3) Check the instrument zero with NaCl-KCl

diluent and readjust as necessary.

(4) Repeat steps (c)(1) -(3) until stable conditions are achieved. Readings for the same solution

should agree within 0.5 percent of full scale.

Determination of the Calibration Curve .

(1) Nebulize the working solutions of the blank and

the lithium standards and record their relative

absorbance values. (A typical data sheet is given

in Appendix D.)

(2) Subtract the value for the blank (if any) from

the values obained with the standard solutions, and

plot these corrected relative absorbance values

versus the calculated lithium concentrations on

rectilinear graph paper. A typical calibration

curve is shown in figure 2. The calibration curve,

using a least squares linear fit, should show a standard deviation of fit of 0.03 mmol/L or less.

(3) The standard deviation of fit can be calculated

from the deviations, d^, of the N points from the

least squares fitted calibration line:

N

S fit ^(di

2 )/(N-2). (1)

i = l

If on visual inspection, one point of the plot

exhibits a large residual from a straight line

drawn through the remaining points, remeasure that

standard solution. If the remeasured value for

25
LITHIUM CONCENTRATION, mmol/L

Figure 2. Typical calibration curve for the determination

of serum lithium by flame atomic absorbance

spectroscopy; wavelength = 670.8 nm; oxidantfuel = air-acetylene.

26
the solution continues to exhibit the large

deviation, prepare that standard solution again,

remeasure it, and compare the values obtained,

as in steps d- (1) and (2). (See Results and

Statistical Analysis Section IVA-4f.)

Sample Measurements .

CI) Nebulize a working sample solution and select

the two working standard solutions whose relative

absorbance values most closely bracket that of the

s amp 1 e

(2) Nebulize the lower working standard, the

working sample, and the higher working standard in

that order and record each reading in the set.

C3) Repeat step e-(2) until five valid sets are

obtained, as illustrated in section f, below.

(4) Repeat steps e- (1) , (2), and (3) for all of

the samples.

Valid Sets of Readings .

Sets of readings are considered valid if the

following condition is met:

The relative absorbance values for the sample and

the two standards in a set may not differ by more

than 2 percent from any of the corresponding values

in the previous valid set. (NOTE: Initially, the

first set measured is considered to be valid, but

may be discarded if subsequent sets prove it to be

non-valid. Non-valid sets are discarded.)

Five valid sets must be obtained to complete a measurement. For example: In Table 4, set 2 is

valid since each difference between the relative

absorbances for the Low Standard (Set

2

_ Set

1

-0.001), the Sample (Set^Se^ = +0 . 005) and the

27
2 percent. Note, however that set 4 is not valid

because two differences, i.e., between the Low

I Standard values (Set^-Set^ = +0.015), and Sample

values (Set.-Set 7 = +0.018), are outside the 2 percent limit. Just one such difference would

have disqualified set 4. Thus, sets 1, 2, 3, 5,

and 6 comprise the group of 5 valid sets.

i Table 4. Example of relative absorbance values for sets of

readings using a direct read-out instrument.

Set

Low Standard

1.001 mmol/L S amp 1

e

High Standard

1.5 01 nmol/L

1 0. 523 0 .639 0.753

2 0 . 522 0.644 0. 758

3 0. 525 0.641 0.750

4 0. 539 0.659 0. 753

5 0. 530 0. 643 0.752

6 0. 539 0.645 0. 748

f . Data Recording and Calculations :

(1) On the data sheet, record the concentrations

of the standard solutions in mmol/L of lithium to

four significant figures and the measured relative

absorbance values to as many figures as given by

the instrument.

i 28
(2) Calculate the concentration C of lithium

present in the sample in mmol/L by mathematical

interpolation as follows:

(C

2 -C )(Y-X ) C = C, + — — (2)

(x

2

- Xl )

where

A,C is the sample concentration of lithium in

mmol/L

,

is the low standard concentration of lithium

in mmol/L,

C

2 is the high standard concentration of lithium

in mmol/L,

Y is the relative absorbance value of the sample

minus that of the blank (the NaCl-KCl diluent

reading that was initially set at '0')

X^ is the relative absorbance value of the low

standard minus that of the blank (the NaCl-KCl

diluent blank) , and

X

2 is the relative absorbance value of the high

standard minus the blank.

(3) Record the C values calculated to four

significant figures in the column provided on the

data sheet.

(4) Average the results for the four samples of

the serum pool analyzed to obtain the 'final concentration 1 .

29
IV. RESULTS AND STATISTICAL ANALYSIS

The main objective of the statistical analyses of the

interlaboratory exercise data is to derive measures of precision and accuracy for the manual and semiautomated versions

of the reference method. Precision is characterized by the

variability of the protocol measurements within a single

includes the variability of 'between laboratory' measurements. Accuracy relates to the comparison between reference

method and definitive method values and is related to the

magnitude of the bias.

Each reported data point (test result) is the end product

of five valid flame atomic absorbance spectrometer reading

sets, the number of valid readings specified by the protocol.

For simplicity of discussion, each reported data point is

referred to as a single measurement , meaning that each is the

product of a single run-through of the protocol. When "replication" is mentioned, replication of the entire protocol

process is meant, and "replication error" thus refers to the

variability among the end results of repeated run-throughs

of the protocol. Each interlaboratory exercise is discussed

separately; the final, detailed statistical analysis is

reported for the results from IE-III.

A. Interlaboratory Exercise Results

1. Preliminary Interlaboratory Exercise (Dates Run:

October- January 1975)

.

a. Obj ect ives : To allow the participating laboratories to become familiar with and comment on the

protocol and to determine interlaboratory precision.

and by the total variability of a labora-

, cr. . -, . This latter uncertainty

30
Samples : Six vials, two of three different pools

each containing a sample from the same serum pool.

Each participating laboratory was to analyze a single vial of each concentration on each of two

days, with seven days between the two analyses.

Procedure : The manual pipetting protocol was used.

Comments and Protocol Deviations : The following

laboratory comments or protocol deviations were

received

(1) Lab 5 : Called attention to error in manuscript,

corrected same.

(2) Lab 6 : Prefer use of a single stock standard

solution with volumetric dilutions.

(Original protocol called for weighings

and dissolution to prepare each standard solution. Subsequently changed

as presented in this protocol.)

(3) Lab 8 : Performed extra work. Showed that

using a 10-fold dilution, a difference

between sample and standard with same

lithium concentrations may be observed

due to protein effect.

(4) Lab 9 : Instrumental problems. Dilutions for

two samples, 2nd day analyses stored

overnight in refrigerator.

(5) Lab 11 : One sample had solid suspended in it,

and showed 'many long gram positive

rods '

.

Data : The six data points reported by the individual

laboratories are summarized in Table 5. The data

are presented graphically in figure 3 as the percent

differences from the collective average of the

31
reported values. All reported values except one

from laboratory 11 for pool B are within three

percent of the collective average with a standard

deviation of ±0.03 mmol/L.

Table 5. Serum lithium concentrations reported by the

participating laboratories for the Preliminary

Interlaboratory Exercise. -------- [Li] , mmol/La --------

Laboratory Pool A Pool B Pool C

Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

1 1. 021 0. 996 1. 524 1.477 1.985 1.960

2 0. 99 1. 00 1.49 1.46 1.95 1.91

3 0. 99 0.96 1.46 1.46 1.95 1.91

4 0. 999 0. 990 1. 502 1.468 1.941 1.942

6 1. 026 1.00 1. 505 1.493 1.991 1.968

7 1.00 0.95 1.49 1.46 1.98 2.02

8 1. 043 1.01 1. 504 1. 50 1.997 1.998

9 1. 019 1. 013 1. 511 1.492 1.990 1.962

10 1. 033 1. 008 1. 502 1.488 1.971 1.960

11 1. 03 1.03 1. 59 1. 53 2. 04 2.02

13 0. 995 0. 995 1. 481 1. 500 1.956 1.960

X 1. 004 1. 495 1. 971

S

X

0. 022 0. 029 0. 033

Each value represents a single measurement on one sample.

32
LITHIUM, IE-P

MANUAL PIPETTING PROTOCOL

I I I I I I I I I I I

1 2 3 4 6 7 8 9 10 11 13

LABORATORY NUMBER

3. Percent deviations of the two day average results

from the collective average of the measurements

obtained in the Preliminary Interlaboratory

Exercise. The letters A, B ., and C next to the

results from Lab 1 refer to three different

sample pools.

33
£. Based on the good interlaboratory agreement for

IE-P and discussions with the statisticians and

Experts Committee, it was concluded that an

interlaboratory exercise should be undertaken using

samples with lithium concentration values determined

by the definitive method. (NOTE : IDMS values for

the serum pools were not available at this time, so

a preliminary estimation of the bias could not be

made. It was expected that few problems would be

encountered in the lithium analyses.)

2. Interlaboratory Exercise I (IE-I. Dates Run:

January 19 75 - May 1975.)

a. Obj ectives . To test the manual pipetting protocol

on serum samples for which definitive lithium

values had been obtained and determine the imprecision and bias of the test results.

b. Samples : IE-I was a test series run on 12 samples — four vials (samples) of each of three different

concentrations (Pools 1, 4, and 5). Each laboratory

was to analyze two vials of each pool on one day

and the remaining pairs of samples on a subsequent

day with the requirement that a minimum of one day

and a maximum of seven days should elapse between

analyses

.

c. Protocol : A revised manual pipetting protocol was

used. A 10-fold dilution for samples and standards

was used (same as IE-P) . Five valid measurement

sets rather than ten were required.

d. Comments and Protocol Deviations : The following

laboratory comments or protocol deviations were

received

:

34
(1) Lab 1 : Many readings were taken to obtain

'valid measurement sets' using 0.5

percent agreement criteria. (NOTE:

Requirement changed to 1.0 percent

for IE-II and to 2.0 percent for

IE-III .

)

(2) Lab 7 : Instrument noisy if scale expansion

is large.

(3) Lab 10 : Fifty mL of working standard insufficient volume; although only five valid

measurement sets required, agreement

of 0.5 percent too stringent; use of

single slot burner head resulted in

more stable operation compared to

stability with use of triple slot

head.

e. Data : The single-measurement data reported by the

laboratories for this procedure are summarized in

Table 6. The data are presented graphically in

figure 4 as percent deviations of each one-day

'single measurement' average from the definitive

method value.

35
Table 6. Concentrations of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise I, manual pipetting protocol.

- - - - - - - - [Li], mmol/La -------

Laboratory Pool 1 Pool 4 Pool 5 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

1 u

.

49 6 1

.

AC! 4 51 l

.

/ oU

0. 516 _

2 0

.

50 0 o

.

49 8 1

.

A O 7 4 83 1

.

a n 1 49 3 1

.

7 3 8 1 . 719

0. 502 0. 500 1. 461 1. 485 i. 738 1. 720

3 0

.

500 0

.

F *7 O 538 1 . a r o 458 1

.

500 1

.

1 . H O C 7 86

0. 500 0. 538 1. 458 1. 500 1. 726 1. 771

4 0

.

518 0

.

r o 520 1

,

497 1

.

p r\ o 502 1

.

T F O 75 2 1 .

*7 A C\ 749

0. 518 0. 519 1. 496 1. 490 1. 751 1. 747

6 0

.

F 1 1 511 0 . FIT 51

1

1

.

505 1

.

493 1

.

T "7 7

7 73 1 . h n a 766

0. 521 0. 510 1. 505 1. 511 1. 788 1. 758

7 0

.

49 5 0

.

524 1 . 437 1 . 540 1 . 76 2 1 . n A 7 783

0. 499 0. 511 1. 445 1. 595 1. 725 1. 823

9 0. 524 0. 524 1, 514 1. 508 1. 768 1. 758

0. 525 0. 521 1, 506 1. 507 1. 755 1. 761

10 0. 514 0. 505 1. 476 1. 474 1. 737 1. 739

0. 511 0. 508 1. 456 1. 492 1. 760 1. 777

13 0. 512 0. 524 1. 488 1. 488 1. 731 1. 744

0. 512 0. 524 1. 488 1. 488 1. 731 1. 750

14 0. 513 0. 517 1. 500 1. 510 1. 762 1. 759

0. 516 0. 515 1, 505 1. 487 1. 760 1. 758

Definitive

Method Values 0.534 1.546 1.809

Each value represents a single measurement on one sample.

36
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Discussion : Except for a few scattered values,

all the results from the laboratories showed an

average, negative bias of approximately five

percent. This was surprising in view of the

results reported earlier [8]

.

Although estimation of the bias and imprecision

values for the results from IE- I show the values

to be within the original goals set by the Experts

Committee, the negative bias is somewhat disconcerting. Upon inquiry, it was found that the

results presented in Table 1 for FAAS and FAES

were obtained using a 25-fold dilution of sample

and standard rather than the expected 10-fold

dilution as performed in references 8 and 16.

Based on this observation, and the observation of

Laboratory 8 while performing extra work during

IE-P and IE- I which showed a possible protein

effect, a ministudy to determine the effect of

dilution was performed by Laboratory 9. The

results are summarized in Table 7. As can be

seen, increasing the dilution ratios decreases the

negative bias. Similar studies performed at two

other laboratories support this observation. A

25-fold dilution was chosen to be tested rather

than a 50-fold dilution since the latter required

a large electronic scale expansion resulting in

greatly increased instrument noise.

38
Table 7. The effect of sample and standard dilution on

measured serum lithium concentrations.

Dilution

50:

1

25:1

12. 5: 1

25:1

25:1

25:1

10:la

25:1

10:l a

25:1

10:la

[Li] , mmol/L

FAAS

1. 999

1.992

1.963

1.010 ± 0.007

1.999 ± 0.004

1.025 ± 0.007

1.016 ± 0.005

1.535 ± 0.010

1.500 ± 0.009

2.092 ± 0.006

1.970 ± 0.007

IDMS

2. 000

2. 000

2. 000

1.004

1. 969

1. 032

1. 032

1. 542

1. 542

2.041

2.041

% Difference

-0.05

-0.40

-1.85

+ 0.55

+ 1.50

-0.67

-1.55

-0.45

-2.72

+ 2.45

-3.48

From IE-P

g. Direction : Based on the results of the dilution

minitests by Lab 9 and supporting data from Labs

and 8, the statisticians and Experts Committee

decided to incorporate a 25-fold dilution step

into the protocol and to run IE- II using the

manual pipetting protocol.

3. Interlaboratory Exercise II : (IE-II. Dates run:

May - July 1975.)

a. Ob j ective : To test the revised manual pipetting

protocol on samples with lithium concentrations

determined by the definitive method.

39
b. Samples : IE-II was a test series run on a total

of 12 samples — four vials (samples) of each of

three different lithium concentrations (Pools 3, 4,

and 7) . Each laboratory was to analyze two vials

of each concentration on the first day and the

remaining pairs of samples after the elapse of a minimum of one day and a maximum of seven days.

c. Protocol : The revised (25-fold dilution) manual

pipetting protocol was used.

d. Comments and Protocol Deviations : The following

laboratory comments or protocol deviations were

received

:

(1) Lab 3 : All samples were run on one day.

(2) Lab 4 : Suggested semiautomated procedure be

added to protocol. (NOTE: See

general comments for IE _ III.)

e. Data : Results from IE-II are given in Table 8 and

illustrated in figure 5 in the usual format.

f. Discussion : The majority of laboratories returned

results that showed a negative bias of ^3 percent.

Laboratories 9 and 10 showed excellent agreement

with the IDMS values; this was expected in Lab 9

since it performed the mini-test to determine the

effect of dilution. No explanation could be found

to explain the results obtained by Lab 8. All

other laboratories verified that they performed

the test using the revised protocol with the

25-fold dilution. The average negative bias did

decrease from about -5 to about -2.3 percent, a decrease in the right direction but certainly not

as large as expected.

40
Table 8. Concentration of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise II, manual pipetting protocol.

- - - - - - - - [Li], mmol/L --------

Laboratory Pool 3 Pool 4 Pool 7 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

1X -L « 7L* O5 3-J 1 Li774I T- 1. 240 1

.

241

? 1 2Lt *JSO\J i

J- • L2 J5 0U 1. 223 1. 261

7. 1

J- • Li2 *JSO\J 2L, _>50\J 1. 250 1. 214

4 1 . 273 1 265

1. 272 1. 263

6 1 . 271 1 . 274

1. 267 1. 292

7 1 . 2 73 1

.

265

1. 266 1. 264

8 1. 350 1

.

34 5 1. 350 1. 336

9 1. 292 1. 294

1. 291 1. 292

10 1. 296 1. 308

1. 302 1. 307

11 1. 251 1 . 253

1. 247 1. 261

13 1. 259 1. 284

1. 239 1. 288

14 1. 259 1. 264

1. 256 1. 261

Definitive

Method Values 1 . 291

1

.

490 1

.

487 2 428 2 461

1. 492 1. 497 2. 440 2 .483

1

.

453 1

.

400 2

.

441 2 .523

1. 473 1. 420 2 .403

1

.

429 1

.

464 2 . 500 2 .465

1. 464 1. 464 2. 465 2 . 500

1

.

511 1

.

491 2

.

494 2 . 490

1. 513 1. 505 2. 502 2 . 484

1

.

510 1

.

521 2 . 497 2 .525

1. 504 1. 519 2. 509 2 . 537

1

.

514 1

.

465 2

.

523 2 .419

1. 504 1. 459 2 .419

1

.

580 1

.

584 2

.

626 2 . 629

1. 580 1. 580 2. 625 2 .626

1. 539 1

.

538 2 . 552 2 .539

1. 540 1. 530 2 . 550 2 . 541

1. 525 1. 538 2. 547 2 . 569

1. 543 1. 553 2. 533 2 . 567

1. 475 1. 495 2. 480 2 . 499

1. 488 1. 493 2. 495 2 .480

1. 468 1. 506 2. 469 2 . 518

1. 474 1. 506 2. 473 2 . 516

1. 492 1. 496 2. 491 2 .488

1. 491 1. 498 2. 494 2 .491

1. 546 2. 572

41
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Variations in instruments, burner heads, hollow

cathode sources, and bandpasses do not appear to be

the cause of the slight negative bias found during

IE^-II as shown in Table 9. All laboratories used

acetylene as the fuel with variable air-acetylene

pressures and flow rates.

Table 9. Summary of FAAS instruments and selected

experimental parameters used for IE- II.

Lab

Average

% Bias Instrument Burner HL BP,

b

nm

1 -3. 71 IL 453 Hi Solid Varian 0 . 24

2 -5.02 PE 303 3 slot Sing. Ele. 4 . 0

3 -4. 39 IL 153 3 slot Pyrex-neon 0 .43

4 -2. 50 PE 403 1 slot PE 1 .4

6 -1. 80 IL 353 Boling

Laminar Flow IL 0 .43

7 -3. 46 IL 402 3 slot Low. Int.

8 + 2. 50 TECH AA5 1 slot TECH

9 -0. 49 PE 403 3 slot J, A. 1 .4

10 -0. 05 PE 403 1 slot WE 1 .4

11 -3. 32 PE 503 1 slot PE 1 .4

13 -2.85 PE 403 1 slot PE 1 . 4

14 -2.94 PE 403 3 slot PE 1 .4

To describe instruments, it was necessary to identify commercial products by manufacturer's name. In no instances does

such identification imply endorsement by the National Bureau

of Standards, nor does it imply that the particular product or

equipment is necessarily the best available for that purpose.

Bandpass

.

Additional testing performed by Lab 9 showed that

the diameter of the aspiration tubing and the

aspiration rate of the solution affects the lithium

analysis and could decrease the bias by approximately one percent, Table 10.

43
Table 10. Effect of aspiration tube diameter on measured

serum lithium concentrations at several

dilutions

.

Tube Diameter Dilution [Li] , mmol/L % Difference

cm

0.038

0.058

0.086

FAAS IDMS

50 : 1 1. + 0 . 005 a

1. 546 -1. 75

25: 1 1. r c\ c 0 . 009 1. 546 -2. 59

12.5: 1 1.

/in/ 496 + 0

.

006 1. 546 -3. 23

6.25: 1 1. 482 + 0. 008 1 . 546 -4. 14

50 : 1 1. 525 + 0. 011 1. 546 -1. 36

25: 1 1. 516 + 0. 005 1. 546 -1. 94

12.5: 1 1. 502 + 0. 006 1. 546 -2. 85

6.25: 1 1. 474 + 0. 013 1. 546 -4. 66

50: 1 1. 530 + 0. 005 1. 546 -1. 03

25: 1 1. 529 + 0. 004 1. 546 -1. 10

12.5: 1 1. 537 + 0. 003 1. 546 -0. 58

6.25: 1 1. 498 + 0. 018 1. 546 -3. 10

cL

* Standard deviation of a single measurement.

g. Direction : Based on the results of IE-II, the

studies by Lab 9, and the laboratory comments, the

statisticians and Experts Committee decided to run

one additional inter laboratory exercise, IE-III

which would include a semiautomated pipetting

procedure as well as a manual pipetting procedure.

An aspiration tube with ID of ^0.076 cm was supplied

with the samples with instructions for its use as instructions to maximize the solution aspiration

rate to ^8-10 mL/min. It was emphasized again that

the flame should be stoichiometric or slightly

fuel-rich since Lab 9 demonstrated that a fuel-lean

flame caused an error of -1.3 percent in lithium

concentration compared to the results from a slightly fuel-rich flame.

44
4. Interlaboratory Exercise III (IE-III. Dates Run:

December 1975 -March 1976.)

a. General : The addition of the semiautomated pipetting alternative to IE-III was considered advantageous because the manual and semiautomated pipetting

versions could be evaluated simultaneously on the

same serum samples. The semiautomated version would

be used in suitably equipped laboratories with

consequent economies in reagents and labor; whereas

the manual version would be used in laboratories

having equipment basic to the method but lacking

the appropriate seimautomated sampling device.

A review and test of the capabilities of positive

displacement pipettor-dilutors demonstrated that

the precision and accuracy requirements listed in

the protocol could be met. Consequently, a method

for testing the pipettor-dilutor was included in

the protocol.

b. Obj ective : To test the revised manual and semiauto^

mated pipetting protocols on serum samples having

a wider range of lithium values and to determine

the imprecision and bias of the test results.

c. Samples : IE-III was a test series run on the total

of 16 samples — four vials (samples) of each of

four different lithium concentrations (Pools 1, la,

5, and 7a) . Each laboratory was to analyze two

vials of each concentration on the first day and

the remaining pairs of samples after the elapse of

a minimum of one day and a maximum of seven days.

d. Protocol : The manual and semiautomated pipetting

versions of the protocol were used.

45
Data : Results from IE- III are given in Tables

11-12 and illustrated in figures 6-7. The data

are presented as two-way tables in which the rows

represent the different participating laboratories

and the columns represent the different sample

pools. The sample pool concentrations ranged from

approximately 0.5 to 3.0 millimoles of lithium per

liter of serum. The results for the manual procedure and for the semiautomated procedure are

listed separately, and all single measurements

reported are included in the tables. The definitive

method values for the lithium concentrations in

the sample pools are listed at the bottom of

Tables 11-12.

A detailed statistical analysis was made. First

the data were inspected by calculating the percent

deviation of each day's results for each pool from

an average for that sample pool. These percent

deviation values for all laboratories and the two

pipetting procedures are listed in Tables 13-14.

46
Table 11. Concentrator! of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise III, manual method.

[Li], mmol/L - -

• Laboratorya Pool 1 Pool la Pool 5 Pool 7a

1-1 . 566 . 987 1. 772 3. 078

. 520 . 991 1. 744 2 . 904

1-2 . 525 1 . 000 1. 760 2 . 918

. 520 . 989 1. 750 2 . 898

2-1 . 500 . 987 1. 771 2 . 973

. 510 . 995 1. 790 3. 012

2-2 . 500 1. 013 1. 772 2. 962

. 506 . 984 1. 762 2. 993

4-1 . 517 . 983 1.750 2 .873

. 517 .976 1. 743 2.866

4-2 . 521 . 988 1. 769 2 . 914

. 520 .990 1. 768 2. 918

5-1 . 533 . 981 1. 820 2 .896

. 504 1.035 1. 749 2.996

5-2 . 500 1.022 1.827 2 . 954

.523 1.008 1.785 2. 896

8-1 . 533 1.035 1.850 3 . 007

.539 1.028 1.837 3.032

8-2 . 537 1.029 1. 826 3. 046

. 535 1. 030 1 . 826 3.057

9-1 . 521 . 998 1 .778 2 . 922

. 524 . 990 1. 784 2 . 930

9-2 . 521 . 997 1.776 2. 934

. 525 . 995 1. 769 2. 918

10-1 . 523 1.008 1. 786 2 .952

.529 1. 004 1. 893 2. 947

10-2 .528 1. 000 1.770 2 . 948

. 525 . 998 1. 768 2 . 959

47

continued
Continuation of Table 11.

[Li], mmol/L - Laboratory Pool 1 Pool la Pool 5 Pool 7a

"1*7 1 13-1 r 7 n .5 20 o 7 7 1 . / 76 2.916

. 520 . 982 1. 767 2.913

13-2 r o o .52 2

r\ o i .98 31 7 7 f\ 1.770 2.902

.519 . 985 1. 770 2.898

14-1 . 521 n o c .986 T 7 C 7 1.767 2.898

.520 . 982 1. 759 2.899

14-2 . 524 . 985 1. 764 2.900

. 522 .986 1. 763 2.904

15-1 . 522 .981 1. 765 2. 899

. 518 .977 1. 760 2. 887

15-2 . 521 . 988 1. 761 2. 895

,521 . 992 1. 760 2.890

Definitive

Method Values . 534 1. 004 1. 809 2.954

The laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or the

second day's results.

i

48
Table 12. Concentration of serum lithium reported by the

participating laboratories for Interlaboratory

Exercise III, semiautomated pipetting protocol.

[Li] , mmol/L

Laboratory Pool 1 Pool la Pool 5 Pool 7a

4-1 . 513 . 986 1. 759 2.901

. 517 . 981 1. 759 2.906

4-2 . 517 . 981 1. 744 2 . 888

. 516 . 976 1. 741 2 .874

6-1 . 542 1. 015 1.805 2.965

.536 1. 014 1.805 2.964

6-2 . 525 .994 1.802 2.927

. 520 .996 1.801 2. 937

9-1 . 523 . 991 1. 786 2 . 931

. 524 . 996 1. 773 2.921

9-2 . 525 . 997 1. 775 2.924

. 521 . 998 1. 775 2 . 924

10-1 , 535 1.011 1. 792 2, 942

.520 . 998 1. 786 2.942

10-2 . 525 . 995 1. 779 2.950

.525 . 988 1.777 2.985

11-1 X JL _L c 9 n • D L U Q 7 7 . v 1 1 1 7 A A 1. / DO 2 .873

.01/ 077 . y / s

t n r o 1.752 2.865

11-2 . 520 .995 1. 762 2. 891

. 516 . 991 1. 773 2.911

15-1 . 521 .986 1. 759 2. 914

. 521 . 988 1. 757 2.910

15-2 . 520 . 985 1. 759 2.972

. 517 . 986 1.753 2.898

Definitive

Method Values . 534 1. 004 1. 809 2.954

The laboratory designation consists of two parts; the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or the

second day's results.

49
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Table 13. Percent deviations from averages for lithium in

serum from Interlaboratory Exercise III,

manual pipetting protocol.

Laboratory^ Pool 1 Pool la Pool 5 Pool 7a

1-1 4 . 06 - .12 -1.19 1.82

1-2 . 13 - . 16 -1.36 -1.01

2-1 -3.22 - . 51 . 07 1.87

2-2 -3.60 - . 68 - . 68 1 . 36

4- 1 - . 92 -1.67 -1.84 -2.32

4- 2 - . 25 - . 72 - . 60 - . 74

5-1 - . 63 1 . 19 . 30 . 29

5-2 -1.97 1 . 89 1 . 51 - . 43

8- 1 2.72 3. 55 3. 62 2 . 79

8-2 2.72 3. 35 2 . 63 3.88

9- 1 . 13 - . 21 . 10 - . 39

9- 2 . 23 * . 01 - . 38 - . 39

10-1 . 80 .99 3 . 39 .41

10-2 . 90 . 29 - . 57 . 54

13- 1 - . 34 -1.67 - . 71 - . 79

13-2 -.25 -1.22 - .52 -1.28

14-1 - .25 -1.22 - .91 -1.33

14-2 . 23 -1.07 - . 88 -1.21

15-1 - . 34 -1.72 - . 94 -1. 52

15-2 -.15 — .61 -1.05 -1. 54

Average

used in . 522 . 996 1. 779 2.938

calculations

,

mmo 1 /

L

The laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

52
Table 14. Percent deviations from averages for lithium in

serum from Interlaboratory Exercise III,

semiautomated pipetting protocol.

Laboratory^ Pool 1 Pool la Pool 5 Pool 7a

4-1 -1.40 - .82 -.76 -.61

4-2 -1.12 -1.32 -1.69 -1. 38

6-1 3.19 2. 31 1.83 1.47

6-2 . 03 .34 1. 64 .36

9-1 . 22 .19 . 39 .16

9-2 .13 .60 .14 .09

10-1 . 99 1. 30 .93 .70

10-2 , 51 - . 01 . ol 1 CO 1 . DO

11-1 -.73 -1.67 -.76 -1.80

11-2 - . 83 .14 -.28 - . 70

15-1 -.26 -.46 -.82 *.32

15-2 -.73 -.61 -.93 .46

Average

used in , 522 ,992 1. 773 2.921

calculations

,

mmol/L

aThe laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day' s results

.

A comparison was next made of the ability of each

laboratory to replicate its values relative to that

of the average replication ability of all laboratories. This was done by comparing the standard

deviation for each day's measurements for each pool

against the laboratory averaged standard deviation

for that pool (see Tables 15-16). If all of the

participating laboratories were of the same population in regard to replication error, the standard

deviation ratios reported in Tables 15-16 would be

5 3
Table 15. Ratios of standard deviations to average standard

deviation for lithium in serum from Interlaboratory Exercise III, manual pipetting protocol.

Laboratory Pool 1 Pool la Pool 5 Pool 7a

1-1 5.97 A S~ . 46 1.65 6.47

1-2 . 65 1.27 . 59 . 74

2-1 1 . 30 . 92 1.12 1 .45

2-2 . 78 3. 35 . 59 1. 15

4-1 . 00 . 81 . 41 . 26

4-2 . 13 . 23 . 06 . 15

5-1 3.77 6 . 24 4 . 19 3. 72

5-2 2 . 99 1.62 2.48 2 . 16

8-1 .78 . 81 . 77 . 93

8-2 . 26 . 12 . 00 . 41

9-1 . 39 . 92 . 35 . 30

9-2 . 52 . 23 . 41 . 59

10-1 . 78 . 46 6 . 31 . 19

10-2 . 39 . 23 . 12 . 41

13-1 . 00 r o

. 58 . 06 . 11

13-2 . 39 .23 .00 .15

14-1 .13 .46 .47 .04

14-2 . 26 .12 . 06 .15

15-1 .52 .46 .29 .45

15-2 . 00 .46 . 06 .19

Average

Standard . 00545 . 00612 . 01199 . 01902

Deviation

,

mmol/L

aThe laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

54
Table 16. Ratios of standard deviations to average standard

deviation for lithium in serum from Interlaboratory Exercise III, semiautomated pipetting

protocol

.

Laboratory3 Pool 1 Pool la Pool 5 Pool 7a

4-1 1. 04 1.20 .00 . 33

4-2 . 26 1.20 .62 . 93

6-1 1. 57 . 24 . 00 .07

6-2 1. 30 .48 .21 . 66

9-1 . 26 1. 20 2.69 .66

9-2 1. 04 . 24 . 00 .00

10-1 3. 91 3. 12 1 . 24 . 00

10-2 . 00 1.68 .41 2. 32

11-1 . 78 . 96 2. 90 . 53

11-2 1. 04 . 96 2. 28 1.33

15-1 .00 .48 .41 .27

15-2 . 78 .24 1. 24 4. 91

Average

Standard . 00271 .00295 . 00342 .01067

Deviation

,

mmol/L

aThe laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

larger than 2.44 and 2.36, respectively, only about

one percent of the time. In practice, it is not

too uncommon to encounter a few standard deviation

ratios that are somewhat larger as this is a reflection of some heterogeneity of the laboratory population in regard to replication error. (As long as

the standard deviation ratios are not too large,

this is normally not used as a reason for rejection

of a laboratory. It is advised, however, that

55
laboratories with large standard deviation ratios

should reexamine their procedures for possible

sources of excessive replication error.)

The calculations on IE- III data were made on the

data in the two-way tables using a weighted least

squares fit to the following model [22]:

Y

ijk - V± * BjCXj - X) X i} e.- k

^

where

th

Y.., = the sample concentration reported by the i

1 3 K th laboratory, for the j sample, and for the

t h

k replicate measurement,

u. = a constant factor associated with the

average bias for laboratory .i,

3^ = a slope factor for laboratory .i, expressing

the relation of bias to concentration,

X. = the observed average concentration for

sample pool j (this average is taken over

all laboratories)

X = the weighted average concentration for all

samples (this average is taken over all

laboratories and over all sample pools)

,

= a random sample interference factor (matrix

effect) for laboratory i and sample pool j

,

and

e^jk = a random replication error.

The above model is quite general and extensive

experience has shown that it is well suited to

describe a number of measurement factors in

interlaboratory tests [23],

56
Weighted analyses of variance were made on the data

in the two-way tables using the fits to the above

model. (A modified version of the weighting procedure reported in reference 24 was used.) From the

analyses it is possible to derive the following

estimates for three components of variability, each

characterized by its standard deviation:

°e ~ G e(Repl)

= t ^ie uncerta i nty observed for

replicate measurements in a given

laboratory on a given day,

°D

= °Day

= t ^ie additional uncertainty that is

observed when measurements are

made on different days within the

same laboratory, and

°L

= °Lab= t ^ie ac^ (ii't i ona l uncertainty that is

observed when measurements are

made by different laboratories.

All three of these components of standard deviation

were observed to systematically increase with

increasing lithium concentration. The smoothed

values of these components of standard deviation

are given in Table 17.

5 7
Table 17. Components of standard deviation in mmol/L for

lithium in serum, Inter laboratory Exercise- II I

.

Manual Pipetting Protocol

(Pooled Results from 10 Laboratories)

Lithium Level, mmol/L

. 5

1.0

1.8

2.9

£ (Repl.

)

. 008

. 013

. 022

. 034

u Day

0

0

0

. 003

u Lab

. 007

. 013

.026

.044

Semiautomated Pipetting Protocol

(Pooled Results from 6 Laboratories)

Level, mmol/L CT e(Repl.

)

Day °Lab

. 5 .002 . 004 .005

1.0 . 004 . 006 .009

1.8 . 009 . 009 .016

2.9 . 016 . 012 .027

Because of the relatively small size of the lithium

interlaboratory exercises, the individual components

of standard deviation are considered to be only

advisory in nature. The final, practical statements

of uncertainty are made through the recombination

of these components. One such final statement is

awithin' exP ec

'tecl uncertainty within a single

laboratory from running the complete protocol (2

replicates/day for 2 days) . The o^-thin results

are reported in columns three and seven in the top

section of Table 18, and are calculated as follows:

58
2

(4)

These are the expected uncertainties that a single

average laboratory could see by repeating the complete protocol a number of times and observing the

variability of its results. This awi t j1 ^ n

i- s not tne

total uncertainty since there is also a "between

laboratory" component, a

L at>' ^ne standard deviation

of the total uncertainty expected as a result of a single laboratory running the complete protocol is

calculated as follows:

Columns four and six in the top section of Table 18

list such standard deviations for the manual and

semiautomated data from IE-III. The precision

goal for the reference method is listed in column

five. Comparison of the tabulated standard deviations and the goal shows that the precision goals

have been met.

a Total

59
Table 18. Summary of imprecision and bias results in mmol/L

for lithium in serum, Interlaboratory Exercise-III

.

Manual

Pipetting Protocol

Semiautomated

Pipetting Protocol

Li

Level a comp

awithin a total Goal a total

-A. a within a comp

0.5 0. 002 0. 004 0.008 0.1 0.005 0. 003 0.003

1.0 . 005 . 006 . 014 0,1 . 010 .004 .004

1.8 .009 .011 . 028 0.1 .017 .007 .007

2.9 . 015 .017 . 047 0.1 .029 .011 .012

Li

Level

0.5

1.0

1.8

2.9

Manual

Pipetting Protocol

Accuracy

Semiautomated

Pipetting Protocol

Interlaboraotry Exercise

Composite Bias

(X obs" X

DM)

. 012

.008

.030

.016

Interlaboratory Exercise

Composite Bias

Goal

±.2

±.2

±.2

±.2

(X obs^ XDM)

-.012

- .012

- .036

- .033

60
The standard errors of the IE- III composite values

are given in columns two and eight of the top

section of Table 18. These standard errors are

calculated from the components of standard deviation as follows:

where N represents the 10 or 6 laboratories participating in the manual or semiautomated procedures,

respectively. The bottom section of Table 18 lists

the observed biases between the reference method

interlaboratory exercise composite values and the

definitive method values. A consistent, small

negative bias is observed. The observed biases,

however, are easily within the goals for the

reference method.

Table 19 lists the composite IE-III sample averages

± twice the standard error for the manual and for

the semiautomated versions, and for the corresponding

definitive method values.

The accuracy of the IE-III results is within the

recommended goal of the reference method.

61
Table 19. Summary of lithium in serum values.

IE-III - Composite Values

(mmol/L)

Definitive Method Values

(mmol/L)

Manual Semiautomated

0.522 ± 0.004a 0.522 ± 0.006 a 0.534 ± 0.003b

0.996 ± 0.010 0.992 ± 0.008 1.004 ± 0.005

1.779 ± 0.018 1.773 ± 0.014 1.809 ± 0.009

2.938 ± 0.030 2.921 ± 0.024 2.954 ± 0.015

Twice the standard error of the composite average, i.e.,

2 o comp

Estimated maximum error of 0.5 percent of the value. This

estimated error is the sum of errors due to measurement

imprecisions of ±0.3 percent (±2 sigma interval for the

random error of the mean) and an estimated upper bound of

0.2 percent for possible systematic errors.

f. Auxiliary Statistical Analysis : The protocol

requires a check on the flame atomic absorption

spectrometer by running a calibration curve each

day using freshly prepared standard solutions. The

necessity of these curves also provides a check on

the correct preparation of the standard solutions.

The data reported here on the calibration curve

check are advisory in nature since in the actual

analytical procedure only the pair of calibrating

solutions nearest to the unknown concentration is

used. The calibration curve data for the manual

and semiautomated lithium procedures were reported

and are given in Tables 20 and 21. Straight line

least squares fits were made to these data and the

resultant standard deviations of fit are given in

Table 22. These standard deviations of fit are

expressed in units of lithium concentration (mmol/L)

.

Our analysis indicates that if in the calibration

62
Table 20. Calibration curve data for lithium in serum, Interlaboratory Exercise III, manual pipetting protocol.

Lab* No. Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6

1- 1 X

b

0. 5000 1. 0000 1. 5000 2 .0000 2. 5000 3. 0000

Y

c

0. 254 0. 505 0. 761 1 .034 1. 293 1

.

519

1- 2 X 0. 5000 1. 0000 1. 5000 2 . 0000 2. 5000 3 . 0000

Y 0. 254 0. 507 0. 765 1 .020 1. 269 1. 509

2- 1 X 0. 5001 1. 0002 1. 5003 2 .0004 2. 5005 3. 0000

Y 0. 288 0. 527 0. 788 1 .028 1. 280 1

.

516

2- 2 X 0. 5001 1. 0002 1. 5003 2 .0004 2. 5005 3. 0000

Y 0. 289 0. 530 0. 785 1 .025 1. 277 1. 510

4- 1 X 0. 5000 1. 0000 1. 5000 2 .0000 2. 5000 3. 0000

Y 0. 2595 0. 5225 0. 7775 1 .035 1. 2865 1. 5535

4- 2 X 0. 5000 1. 0000 1. 5000 2 . 0000 2. 5000 3. 0000

Y 0. 257 0. 5175 0. 779 1 .036 1. 300 1

.

5565

5- 1 X 0. 5001 1. 0002 1. 5003 2 .0004 2. 5005 3. 0006

Y 0. 0381 0. 0716 0. 1090 0 .1475 0. 1884 0. 2328

8- 1 X 0. 500 1. 000 1. 500 2 .000 2. 500 3. 000

Y 0. 100 0. 200 0. 300 0 .398 0. 500 0 . 601

8- 2 X 0. 500 1. 000 1. 500 2 .000 2. 500 3. 000

Y 0. 100 0. 200 0. 300 0 .399 0. 500 0. 602

9- 1 X 0. 500 1

.

000 1. 500 2 .000 2. 500 3. 000

Y 0. 265 0. 531 0. 796 1 .059 1. 326 1

.

585

10- 1 X 0. 5000 1. 0000 1. 5000 2 .0000 2. 5000 3. 0001

Y 0. 299 0. 591 0. 880 1 .165 1. 458 1. 744

10- 2 X 0. 5000 1

.

0000 1. 5000 2 .0000 2. 5000 3. 0001

Y 0. 284 0. 554 0. 831 1 .104 1

.

373 1. 642

J. o

i

J- YYY 0. 5000 1. 0000 1. 5000 2 . 0000 2. 5000 3. 0001

Y 0. 255 0. 510 0. 764 1 .015 1. 260 1. 505

14- 1 X 0. 5000 1

.

0000 1. 5000 2 . 0000 2. 5001 3. 0001

Y 0. 259 0. 518 0. 770 1 .022 1. 273 1. 524

14- 2 X 0. 5000 1. 0000 1. 5000 2 . 0000 2. 5001 3. 0001

Y 0. 255 0. 512 0. 762 1 .011 1. 260 1

.

512

continued

63
Continuation of Table 20.

Lab. No Std Std Std. 3 Std. 4 Std. 5 Std. 6

15-1 X 0.5001

Y 0.015

1.0001

0. 0305

1. 5002

0. 045

2.0002

0.0605

2. 5003

0. 075

3.0003

0.090

15-2 X

Y

0.4999

0. 016

0.9999

0.031

1. 4998

0. 046

1.9998

0.061

2. 4997

0. 075

2.9997

0.089

The laboratory designation consists of two parts: the initial

digit (s) represents the assigned laboratory number and the

last digit represents either the first or second day's

results

.

L X = Standard solution values in mmol/L.

CY = Instrument readings.

64
Table 21. Calibration curve data for lithium in serum,

Inter laboratory Exercise III, semiautomated

pipetting protocol.

Lab

.

No.

a Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6

4- 1 X

b

0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3.0000

Y

c

0. 259 0. 5125 0. 768 1.0165 1. 274 1. 534

4- 2 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3. 0000

Y 0. 2565 0. 5075 0. 764 1.0195 1. 269 1.5285

6- 1 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3.0000

Y 0. 231 0. 647 1. 056 1.430 1. 855 2. 238

6- 2 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3. 0000

Y 0. 489 0. 918 1. 338 1.815 2. 205 2.622

10- 1 X 0

.

5000 1

.

0000 1

.

5000 2. 0000 2

.

5000 3.0001

Y 0. 293 0. 572 0. 842 1. 104 1. 400 1.665

10- 2 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3.001

Y 0. 261 0. 5126 0. 773 1.029 1. 279 1. 507

11- 1 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3. 0000

Y 0. 252 0. 503 0. 756 0.993 1. 237 1.494

11- 2 X 0. 5000 1. 0000 1. 5000 2.0000 2. 5000 3.0000

Y 0. 255 0. 509 0. 766 1.003 1. 255 1. 510

The laboratory designation consists of two parts: the initial

digit(s) represents the assigned laboratory number and the

last digit represents either the first or second day's

results

.

X = Standard solution values in mmol/L.

CY = Instrument readings.

65
Table 22. Calibration curve results for lithium in serum as

standard deviation of fit (S-c-.,-) in mmol/L.

Manual ----- ___ Semiaut omated - - - Laboratory Number S fit

cl Laboratory Number S fit

-L1-1X 0VJ • 0248 VJ Li *+ (J 4-1 n 0064

1-2 _1_ Li 0VJ • 01VJ X 24L> *+ 4 - ? n n n 4 7

Li2-1X 0VJ a 012 VJ X La 71 6U - 1J. n m ^ q

Li Lt n oiii 6-1 U J. 4-1 0 0 0 7 6 i n -

1

n fii Aft 4-2 "T Li 0VJ o 00\J \J *J"h *J^ i n - ? U . U L _L J

5 -

1

0VJ • 0482 \J tT O L, 11-1 n mil

8-VJ 1X 0VJ • 00^4 VJ VJ *J *+ 11-2 XX L. oU • nU1UU l 06

8-2 0VJ • 0048 VJ VJ *T VJ 9-1 •J X 0VJ • 00^9 VJ VJ <J *7 10-1 VJ0 • 00^6 VJ «J VJ 10-2 0. 0052

1^-1 o n n q ^ 14-1 0.0055

14-2 0. 0051

• 15-1 0. 0094

15-2 0. 0178

The laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

step it is found that any calibration point deviates

from the calibration curve by more than 0.03 mmol/L,

then the standard solutions and the instrument

should be checked for sources of excessive error

before proceeding further into the analysis.

66
V. DISCUSSION

A. Candidate Protocol :

1. Preliminary Tests

Generally, in the development of a reference method

where the state of analytical knowledge leaves an uncertainty

in the choice of a 'candidate' reference method, it is essential that investigations be undertaken to assure optimized

analytical conditions, minimized interferences, and freedom

from other sources of bias. Such preparation helps avoid

initiating the inter laboratory testing process with inappropriate procedures. In the case of lithium, the similarity

of results obtained by White and Mavrodineanu using FAES and

FAAS, the similarity of their results with those obtained

using the highly specific IDMS method, and previous work [8]

led the Committee to decide to proceed directly to the interlaboratory exercise phase with the FAAS method, without

further preliminary studies. With this electrolyte, however,

the inter laboratory exercises revealed a negative bias unless

at least 25-fold dilutions of sample and standard are made

and slightly fuel-rich flame, high aspiration rate, and large

aspiration tube operating conditions are used.

2. Specifications

In keeping with prior experience [9-12], the written

protocol is explicit as to reagent and glassware specifications, pipetting, and directions for dilution of the standard

and sample. Thus, Class A or equivalent glassware, reagent

grade or equivalent chemicals, 'tested' water, analytical

balances with a ±0.1 mg weighing capability, and a pipettordilutor with tested accuracy and precision are specified.

In addition, the reference method provides for the use of

analytical techniques that should reduce the combined error

due to weighing, pipetting, and dilution to below one percent.

67
3. Flame Atomic Absorption Spectroscopy

In general, all the FAAS instruments used in the laboratories that participated in this study provided excellent

results. The instruments that were used are listed in

Table 23 and encompass several types. Thus, specific

instructions are not given for the use of flame atomic

absorption instruments; only a requirement for stable instrument operating conditions is presented. As in sample

preparation and handling, the human element in achieving

accuracy and precision is critical. It is essential that

operators be thoroughly familiar with their instruments and

alert to the onset of instrumental difficulties.

The protocol initially required a 10-fold sample dilution

and a one-percent agreement for measurement sets to be

considered valid. The dilution of sample and standards was

subsequently changed to 25-fold in order to decrease the

negative bias that was observed and the one percent requirement was changed to two percent at the July 1975 meeting of

the representatives from the participating laboratories to

facilitate the analysis. In the discussion that led to this

protocol change, the representatives affirmed that if their

instruments were operating optimally, agreement of successive

sets of readings could be obtained to within 0.5 percent.

It was found that the overall precision of the inter laboratory

exercise results did not significantly degrade due to this

change

.

Instrument linearity requirements were not included in

the protocol since the bracketing method for obtaining valid

measurements was used to minimize the errors attributable to

instrumental drift. Examination of the calibration data

which were requested showed excellent linearity over the

range of lithium concentrations • from 0.5 to 3.0 mmol/L.

More than 95 percent (24 of 25) of the calibration curves

showed standard deviations of fit of about 0.025 mmol/L or

68
CD CD *H 4J a

f—< o

CD| 1 •H

CD CO

>—(

•H

+-> 1—1

4_) CD PS Ph •H cd

r-H

CD

CD

drH

CD rH

CO

toCD CD >i O

Ph <

CD

4-> cd P<o •H

rH < X

XH6\-JLO

LOp

•H6 X

X

X

Xp

•H

vO o

COPn

00

to

X

H6

CNl

(VI

X

X

X

Xp

•H6

LO

00

XP

•H6

LO

LO

XP

•H6

to

p Pi PI Pi P

•H

u •H •H •H

H 6 1—1 6 s s

CO

rJ Ph 1 LO oo LO LO 00

LO vO vo <* I—1

CNl CNl rg CNl

X

PI •H6

LO

to

PI •H6o

vO P

•H6

3-P

•H6

CNl

X

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XH

co

vou

I—I

CO

PhO

to

CD

PIUpPI

?-< P

7)

PIO

00 oo 00 00 00 00 00 oo 00 CO 00 o o O o o o o o o o o o O

vO vO vO vO vO vO vO vO vO vO vO vO P P p P P P p P P P P p P O O o O O o o O O O O o O

rH i—1

I—1i—1i—1i—1I—1

I—1

i—1

i—1

i—1

rH i—1

10 CO (0 CO to to to to to to to to to to to to to to to rH to i—l i—l rH to to

LO

to to to to to o to to to to o to

LO LO LO o LO "3- o o o o vO o

-=* CNl I—1•* to 1—1LO LO

l-H

w rJuww

w

mm

W

1—Ir-1

Ph 1—1 w Ph PL, Ph Pu Ph Ph

CNl to vO 00 to LO

to

CD

Ph0

i-H

CT3 H

CD POapoom

CDCD CO

69
less. A standard deviation of fit larg er than 0.03 iriinol/L

would clearly warrant a laboratory's investigation of its

operation of the procedure and/or preparation of the standard

solutions

.

The use of the bracketing criterion for valid sets

determined that a 50-mL minimum volume of working sample was

needed for the semiautomated pipetting protocol. About 25 mL

of working solution is required to obtain five sets of valid

measurements, assuming a nebulization rate of 2-4 mL/min for

approximately 45 s to obtain a single reading. (That timeinterval is necessary for the instrument and flame to be

stabilized and for actual integration of the signal.) Larger

volumes of diluted sample were available with the manual

pipetting protocol because of the large aliquot volumes

taken to ensure pipetting accuracy.

4. Statistical Analysis

All of the results discussed here are based on the

analysis of four replicate samples analyzed as pairs on two

separate days. Adherence to this pattern of replicate analysis helped assure the reliable performance of the reference

method

.

The imprecision -and bias goals of 0.1 and 0.2 mmol/L,

respectively, were reached over the total concentration

range by the laboratories using either the manual or semiautomated pipetting protocols. As is evident from Table 18,

the imprecision values for both pipetting procedures are at

least twice as good as the original goal set for the

reference method by the Experts Committee.

The

-total.) P re cision values were constant over the

lithium concentration range of 0.5-3.0 mmol/L when expressed

as CV and were 1.6 and 1.0 percent for the manual and semiautomated pipetting protocols, respectively. A negative

bias of one to two percent of the serum lithium concentration

was observed for both pipetting protocols. This bias is

70
small when compared to the original accuracy goal of ±10

percent. The agreement between the reference method values

and the definitive method values are considered to be

acceptable

.

The above precision and accuracies can be expected for

laboratories in the population typical of those participating

in this study (i.e., clinical laboratories that have practiced

the reference method and are in good quality control)

.

VI. CONCLUSIONS

A 'candidate' reference method, specified by a written

protocol for the determination of serum lithium by flame

atomic absorption spectroscopy was evaluated by analyzing

serum and aqueous samples in a selected group of laboratories.

The results for samples having lithium concentrations in the

0.53 to 2.95 mmol/L range showed a total imprecision of about

1.5 percent and a negative bias of about 2 percent of the

serum lithium concentration as compared to definitive method

values for these samples. Slightly smaller imprecisions were

found for the semiautomated pipetting procedure compared to

the manual pipetting procedure. The imprecision and bias

values for both pipetting procedures were well within the

goals set by the experts committee. An isotope dilution —

mass spectrometric procedure was used as the definitive method

to determine lithium values in the pooled sera.

Statistical analysis of the results shows that the flame

atomic absorption candidate reference method can be carried

out with the accuracy and precision expected of a reference

method for serum lithium. Hence, the 'candidate' method

should be considered to be the reference method. This

reference method may be used to establish the accuracy of

field methods for lithium by comparative testing. It may

also be used to determine reference serum lithium values.

Each of these uses would require an appropriate experimental

71
design to ensure its achievement of accuracy and precision

equal to those demonstrated here.

We would like to especially thank the principal investigators and other scientists in the participating laboratories

(listed in Appendix A), who, through their efforts, made this

work meaningful and possible. We thank Dr. David Bayse and

Ms. Sue Lewis, CDC, for providing excellent, homogeneous

serum pools used in the inter laboratory testing process.

The work at NBS was carried out in the National Measurement Laboratory and we acknowledge Ms. Mary Nan Steel

for initial reduction of the IE-P data. We thank

Dr. Philip D. LaFleur and Mr. J. Paul Cali, who were at

the time this program was carried out, Director, Center

for Analytical Chemistry and Chief, Office of Standard

Reference Materials, respectively, for their support and

encouragement throughout this program. Special thanks go

to Dr. Radu Mavrodineanu and Dr. Michael Epstein for discussions and suggestions in the area of flame atomic absorption

spectroscopy. Acknowledgement is also made to Mr. John Matwey

for help in sample handling. Special thanks are due to

Mrs. Joy Shoemaker of the Text Editing Facility for her

efforts in manuscript production which included formatting,

editing, and typing.

The Bureau of Medical Devices of The Food and Drug

Administration is acknowledged for their financial support

of this project.

72
VII. REFERENCES

[1] Cade, J. F. J., Med. J. Aust. 2, 349 (1949).

[2] Davis, J. M. , Janowsky, D. S., and El-Yousef, M. K.,

Psychiatric Annals, 3, 78 (1973); Koch-Weser, J.

New Engl. J. Med., 287_, 227 (1972).

[3] Admisen, A., Scand. J. Clin. Lab. Invest., 2_0, 104

(1967)

.

[4] Good, C. A., Amer. J. Med. Sci., 12S_, 273 (1903).

[5] Cavello, M. and Ciampa, G., J. Chromat., 2£, 201 (1965).

[6] Plum, C. M., Clinica Chim. Acta, 2, 67 (1957).

[7] Coombs, H. I., Coombs, R. R. H., and Nee, U. G., in

"Lithium Research and Therapy", F. N. Johnson, Ed.,

Academic Press, New York, NY, 1979, p. 165 and references

therein; Pybus , J. and Bowers, G. N., Jr., Clin. Chem.

,

16, 139 (1970); Levy, A. L. and Katz, E. M., Clin.

Chem. , 15^, 787 (1969) .

[8] Pybus, J. and Bowers, G. N. , Jr., in "Standard Methods

of Clinical Chemistry", Vol. 6, R. P. MacDonald, Ed.,

Academic Press, New York, NY, 1970, p. 189.

[9] Cali, J. P., Mandel, J., Moore, L. J., and Young, D. S.,

"A Referee Method for the Determination of Calcium in

Serum" Nat. Bur. Stand. (U.S.) Special Publication

260-36 (1972). For sale by: Superintendent of Documents,

U.S. Government Printing Office, Washington, D.C. 20402

SD Cat. No. C13. 10: 260-36.

[10] Velapoldi, R. A., Paule, R. C, Schaffer, R. , Mandel, J.,

and Moody, J. R. , "A Reference Method for the Determination of Sodium in Serum", Nat. Bur. Stand. (U.S.)

Special Publication 260-60 (1978). For sale by:

Superintendent of Documents, U.S. Government Printing

Office, Washington, D. C. 20402 SD Cat. No.

C13. 10: 260-60.

73
[11] Velapoldi, R. A., Paule, R. C, Schaffer, R. , Mandel, J.,

Machlan, L. A., and Gramlich, J. W. , "A Reference Method

for the Determination of Potassium in Serum", Nat. Bur.

Stand. (U.S.) Special Publication 260-63 (1979). For

sale by: Superintendent of Documents, U.S. Government

Printing Office, Washington, D. C. 20402 Stock No.

003-003-02068-1.

[12] Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel, J.,

Murphy, T. J., and Gramlich, J. W. , "A Reference Method

for the Determination of Chloride in Serum", Nat. Bur.

Stand. (U.S.) Special Publication 260-67 (1979). For

sale by: Superintendent of Documents, U.S. Government

Printing Office, Washington, D. C. 20402 Stock No.

003-003-02136-9.

[13] Pybus , J., Feldman, F. J., and Bowers, G. N. Jr., Clin.

Chem. , 16, 998 (1970)

.

[14] See sections on Definitive Methods in references 9-12

and references cited therein.

[15] Cali, J. P., Bowers, G. N., and Young, D. S., Clin.

Chem., 19 (10), 1208 (1973).

[16] Pybus, J. and Bowers, G. N. , Jr., Clin. Chem., 139

(1970) . [17] Available from: Office of Standard Reference Materials,

National Bureau of Standards, Washington, D.C. 20234.

[18] Serum samples have been stored frozen for several years

at temperatures below -20 °C with no apparent affect on

electrolyte concentration. White, J. CDC, Private

Communication.

[19] Thawed serum samples were mailed to the participating

laboratories. In previous experiences at CDC, thawed

samples mailed to the west coast and returned after two

weeks showed no change in serum electrolyte concentration. White, J., CDC, Private Communication.

74
[20] Reagent Chemicals, American Chemical Society Specifications ACS Publications, Washington, D.C. (1968).

[21] Hughes, J. C, "Testing of Glass Volumetric Apparatus,"

NBS Circular 602, 1959. For sale by: The Superintendent of Documents, U.S. Government Printing Office,

Washington, D.C. 20025.

[22] Mandel, J., "Models, Transformations of Scale, and

Weighting", Journal of Quality Technology, Vol. 8,

No. 2, 86-97 (1976).

[23] Mandel, J., "The Inter laboratory Evaluation of Testing

Methods", ASTM Bulletin 239, 53-61 and 133 (1959).

[24] Mandel, J., "Estimation of Weighting Factors in Linear

Regression and Analysis of Variance", Technometrics

,

6, 1-25 (1964).

75
APPENDIX A

Scientists not previously acknowledged who contributed to

this study are:

Dr. Mogens Herders 1 Hartford Hospital

Mrs. Harriet Bailey

New England Deaconess Hospital

Ms . Joan Schmit

z

Veterans Administration Hospital

Ms. Leyda A. Ortega

American Hospital Supply Corp.

Mr. Richard Schlough

Mr. Gary Hoffman

University of Wisconsin

Mr. J. R. Brown

Center for Disease Control

Ms . Loretta Wong

University of California

Mr. Nelson T. Lao

Food and Drug Administration

Visiting Scientist from Odense University Hospital,

Denmark

.

76
U. S. Department of Commerce ADD

TM

nT v r> Frederick B. Dent ArrtNUlA H

Secretary

National Bureati of Standards

Richard W. Robe.tu, Director Rational ££ttram xxi j^tandartte

(tterttftcate of ^naltjsts

Standard Reference Material 924

Lithium Carbonate

This Standard Reference Material is certified as a chemical of known purity. It is intended

primarily for use in calibration and standardization of procedures employed in clinical analysis and

for the routine critical evaluation of daily working standards used in these procedures.

Purity 100.05 ± 0.02 Percent

The purity shown is based on the determination of the carbonate ion by coulometric aci- dimetry. The molecular weight for lithium carbonate employed in the calculations is 73.9486. This

value is based on a mass-spectrometrically determined value of 6.9696 for the atomic weight of

lithium in this sample. The uncertainty shown represents the 95-percent confidence interval of the mean based on 16 determinations. The assay in excess of 100 percent may be due to anion

impurities of lower molecular weight than the carbonate ion, e.g., hydroxide.

This Standard Reference Material is of limited certification because no actual determination of

lithium content was made. The certification is based on the analysis of the carbonate anion and the proven absence (or presence in trace quantities) of metallic cations.

The lithium carbonate used for this Standard Reference Material was obtained from the J. T. Baker Chemical Company of Phillipsburg, New Jersey. Analyses were performed by G. Marinenko, M. Darr, E. L. Garner, T. C. Rains, and T. A. Rush.

The overall direction and coordination of technical measurements leading to certification were under the chairmanship of R. A. Durst.

The technical and support aspects concerning preparation, certification, and issuance of this Standard Reference Material were coordinated through the Office of Standard Reference Materials by T. W. Mears.

Washington, D. C. 20234 J. Paul Cali, Chief

February 23, 1972 Office of Standard Reference Materials Revised November 23, 1973

(over)

77
The lithium carbonate meets or exceeds the specifications for reagent grade lithium carbonate

as given in Reagent Chemicals, 4th edition, published by the American Chemical Society. A semi- quantitative survey for trace contaminants by emission spectroscopy showed no significant metallic

impurities. Atomic absorption and flame emission spectrometry showed neither alkali metal nor

alkaline-earth impurities in excess of 1 ppm except calcium (4 ppm).

This Standard Reference Material is intended for "in vitro" diagnostic use only.

This material is intended for use as a standard for determination of lithium in clinical chemistry. For best results using either atomic absorption spectroscopy or flame emission photometry it

is necessary that lithium be determined against a background of sodium and potassium.

A "standard" solution containing 1.00 mmol of lithium per liter may be prepared as follows. Dry SRM 924 for 4 hours at 200 °C, then cool to room temperature in a desiccator. Dissolve 73.91

mg of SRM 924 in 50 ml of deionized water and 20 ml of 0.1N HC1 (ACS Reagent Grade). Dilute to the mark with deionized water and mix well in a 2-liter class-A volumetric flask. A "blank" solution containing 140 mmol of sodium per liter and 5 mmol of potassium per liter may be prepared as follows: Dissolve 8.18 g of sodium chloride (SRM 919) and 0.373 g of

potassium chloride (SRM 918) in deionized water. Bring to the mark of a 1-liter flask with de- ionized water and mix well.

Working standards containing 0.10 or 0.1*0 mmol of lithium, 14 mmol of sodium and 0.5 mmol

of potassium per liter may be prepared as follows. To each of two 100-ml volumetric flasks add 10

ml of "blank solution". Add exactly 10.00 ml of "standard solution" to one flask and exactly

20.00 ml of "standard solution" to the second flask using class-A volumetric pipettes. Dilute each

flask to the mark with deionized water and shake well.

This Standard Reference Material should be stored in the well-closed original bottle under

normal laboratory conditions.

The solutions of SRM 924 are stable indefinitely when stored in a well-stoppered, all-glass container. All such solutions should be clear and display no turbidity.

References:

[1] J. Pybus and G. N. Bowers, Jr., Serum lithium determination by atomic absorption spec- troscopy, in Standard Methods of Clinical Chemistry, Vol. 6, R. P. McDonald, editor-in-chief,

pp 189-192, Academic Press, Inc., New York, N. Y. (1970).

[2] A. L. Levy and E. M. Katz, A comparison of serum lithium determinations using flame photometry and atomic absorption. Clin. Chem. 15, 787 (1969).

This Standard Reference Material has been measured and certified at the laboratories of the National Bureau of Standards, Gaithersburg, Maryland. All inquiries should be addressed to:

Office of Standard Reference Materials Room B311, Chemistry Building

National Bureau of Standards

Washington, D. C. 20234

The date of issuance and certification of this Standard Reference Material was February 23,

1972.

78
APPENDIX C

Isotope Dilution Mass Spectrometry

The use of thermal- ionization mass spectrometry for

isotope analysis has a valid and well-described theoretical

foundation. The methodology has been experimentally evaluated so that results from the procedure have negligible or

accurately known systematic errors and high levels of

precision. It is regarded at NBS as a definitive method.

Isotope dilution analyses are performed by measuring the

change in the relative magnitude of two isotopes of the analyte

when a measured amount of one of these isotopes is added to

the sample. The method consists of the following steps:

(1) The addition of a known amount of a separated isotope

(spike) of the analyte to be determined to a weighed

serum sample. For high accuracy, this addition is

made as a weighed portion of a spike solution having

known isotopic composition and analyte concentration.

(2) Dissolution of the sample by appropriate means and

thorough mixing of the resulting solution to ensure

equilibration of the separated isotope with the

analyte in the sample. This may involve chemical

treatment to convert the analyte and the separated

isotope to the same oxidation state.

(3) Chemical separation of the isotopically altered

analyte from possible interfering elements and

into a form suitable for mass spectrometric analysis.

A major advantage of isotope dilution mass spectrometry (IDMS) is the fact that recoveries need not

be quantitative since only the ratios of the

isotopes are measured.

(4) Measurement of the altered isotopic ratio by

thermal ionization mass spectrometry.

79
(5) Calculation of the amount of the analyte in the

sample using equation 1:

W C[A - RB ] Concentration, yg/g = -52

RR

S P 5E_ . g_ Q)

s where

W = Weight of spike solution, grams bp

C = Concentration of spike, ymoles/gram of solution

A = Atomic fraction of isotope A in spike sp

B = Atomic fraction of isotope B in spike

sp

A = Atomic fraction of isotope A in sample

B = Atomic fraction of isotope B in sample

R = Experimentally measured ratio

M = Atomic weight of element

W

s

= Weight of sample, grams

This calculated concentration must be corrected for

the blank.

The possible sources of systematic error in isotope

dilution mass spectrometry (IDMS) are:

(1) Error in the calibration of the concentration of

the spike isotope. The spike solution is calibrated against at least two different solutions of

the pure analyte containing 'natural' isotopic

abundances by what might be called reverse isotope

dilution. Whenever possible, NBS Standard Reference

Materials are used as the 'natural' material. The

error from this source will be the same as for the

analyte being determined and is due to the imprecision of the ratio measurement.

(2) Chemical errors. In a well designed analysis, an

undetected chemical error should not occur if adequate precautions are taken against the following

80
potential sources of error. Errors might be

caused by:

a) Incomplete decomposition or dissolution of the

sample, a problem common to all wet analytical

methods

.

b) Loss of the analyte from the sample or spike due

to volatility or adsorption during dissolution.

These losses can usually be detected by spiking

some samples before dissolution and others after

dissolution.

c) Incomplete mixing or equilibration of the spike

and the 'natural' analyte. This can be caused by

differences in oxidation state or the presence of

the 'natural' analyte in a complex or chelated form

This source of error can be eliminated by proper

chemical treatment, for example, by oxidation or

reduction and wet-ashing.

d) Isotope fractionation in the chemical treatment

if the separation is not quantitative. This is

seldom a problem but can occur with some techniques

Fractionation can be detected by isotopic analysis

of small amounts of 'natural' materials before and

after being subjected to the non-quantitative

separation procedure.

Contamination or blank. Sources of contamination

or blank may be reagents, apparatus, or fall-out

from the laboratory atmosphere. The problem can be

minimized by carrying out the chemical operations

in a carefully controlled atmosphere and by using

special, high-purity reagents. The total blank

may be estimated by carrying a number of 'blanks'

through all the steps of the analysis. The average

blank value can be treated as a systematic error

81
and the average value obtained for the analyte in

the 'blanks' is used as a correction. The uncertainty of this correction is equal to the randomness

of its measurement, i.e., its coefficient of variation. For concentrations where the blank amounts

to a significant fraction of an analytical value,

the blank may become the largest source of error.

(4) Interferences. Interference usually occurs between

elements with isobars; i.e., isotopes of different

elements that have the same mass to charge ratio,

and may be avoided by either selecting for the

analysis, where possible, an isotope of the element

without isobaric interference, or by chemically

removing the interfering element. Fortunately, most

of the elements containing isobars are in different

groups of the Periodic Table and separations are not

difficult. Thus, a concealed systematic error should

not arise from this source. [For example, although

lt0 Ca and lt0 K are isobaric, Ca can be separated

easily from K by cation exchange chromatography.

To ensure that the amount of 11 °K is insignificant

when measuring lf0 Ca, the mass spectromist can

monitor for 39 K which is four orders of magnitude

more abundant than k °K in natural potassium.] In

the present case, where lithium concentrations are

to be determined, there are no isobaric interferences with the two lithium isotopes, 6 Li and 7 Li.

(5) Instrumental errors. Instrumental errors may be

caused by mass discrimination or fractionation,

but usually cancel since the same percent error is

present in the ratio measurement for the spike

calibration. With some analytes, impurities in a sample can cause a different fractionation pattern

from the pure material. These effects are usually

82
small Cless than 0.11) and can be corrected by

repurifying the sample.

This review of possible sources of systematic error

shows that for IDMS these errors can be eliminated or measured accurately for correction. Thus the absolute accuracy

for the analyte concentration is determined by the random

error (imprecision) components in the measurements. The

imprecision components are present in the isotope ratio

measurements for the analyte, the spike calibration, and the

blank correction. If the blank correction is insignificant,

as is the case for lithium, the total error in a careful

determination reduces to the combined imprecisions for the

spike calibration and the analyte determination. The imprecision of the analyte determination for Li has been determined to be on the order of 0.30 percent (relative standard

deviation of a single measurement) . For an average of five

replicates (as done in these measurements) the imprecision of

the mean, due to analytical replication error, was 0.3//5 or

0.13 percent. The standard deviation for a single spike

calibration was 0.12 percent and for four replicate calibrations was thus 0.06 percent. Quadrature addition of these

two random error sources results in a combined standard

deviation of 0.14 percent. The ± 2-sigma interval for the

random error of the mean is approximately 0.3 percent. To

this is added an estimated upper bound for possible systematic

errors of 0.2 percent to give the estimated maximum total

error of 0.5 percent used in Table 1. When the blank correction is significant, the uncertainty from this source must be

added to the uncertainties from the ratio measurements.

83
Reagents, Columns, and Clean Laboratory

(1) Reagents : All acids and water were purified by a sub-boiling distillation technique utilizing quartz

stills [1J.

(2) Cation Exchange Column : A 0.7-cm ID ion exchange

column filled to an approximately 10-cm height with

100-200 mesh, strongly-acidic, cation- exchange resin

(Dowex 50x8AG) having eight percent crosslinkage was

used for the separations. The column of resin was

cleaned by eluting with 60 g of 5 mol/L HC1, followed

by 10 g of H-2 0.

(3) Clean Laboratory : To reduce particulate contamination,

all the chemical preparations were carried out in a Class-100, clean-air hood located in a vertical flow

clean room [ 2]

.

Procedure

The frozen serum samples were allowed to come to room

temperature and mixed by repeated (^20) careful inversions

of the vials. A sample was quickly withdrawn from each vial

through a platinum needle (18 gauge) into a 10-mL plastic

syringe after the septum was opened just enough to allow the

needle to enter the vial. Approximately 5 g samples, weighed

to 0.01 mg, were transferred to 50- or 100-mL Teflon beakers.

Weighed aliquots of 6 Li separated isotope solution sufficient

to give a

6 Li/ 7 Li ratio of approximately 1-1.5, were added to

each sample. The samples then were decomposed by adding 5 g

of HN0 3 (15.6 mol/L) and 5 g of HC1CK (11.7 mol/L) and heating

in the covered beakers. After decomposition, the covers were

removed and the samples were evaporated to dryness. The acid

on the sides of the beakers was rinsed down with a minimum

amount of H 2 0 and the samples were again evaporated to

dryness. Each residue was dissolved in 10 mL of H2O and

84
transferred to a cleaned cation exchange column. Approximately

10 mL of H 2 0 was used to rinse the beaker and complete the

transfer of the sample to the column. Then 0.2 mol/L HC1 was

added as an eluting agent. The first 12 mL of eluent was

discarded and the next 12 mL 1 of eluent, containing the Li

fraction, was collected in a Teflon beaker. The Li fractions

were evaporated to dryness. To aid in the decomposition of

organic material that elutes from the column, a few drops of

HNO3 (15.6 mol/L) were added and the sample was heated and

evaporated to dryness. The Li residues were converted to the

chloride form by adding a few drops of 5 mol/L HC1 and

evaporating to dryness. The residues were dissolved in

enough 0.005 mol/L HC1 to give a solution containing

approximately 10 yg Li/mL.

Mass Spectrometry

Isotopic ratios were determined by solid sample, thermal

ionization mass spectrometry on 15-cm-radius of curvature,

60°-analyzer tube, mass spectrometers equipped with thin-lens

"Z"-focusing ion-sources and multielement, deep-bucket,

faraday-cage collectors. The mass spectrometric technique

for lithium is similar to a tantalum triple- filament procedure

developed for potassium analysis [3] . The sample size per

analysis was reduced to approximately 50 ng

.

: The volumes may vary depending on the particular lot of

resin and on the sample loading; most of the Li should

have been eluted before Na starts; the start of Na elution

can be checked by a flame test.

85
Results

The lithium concentrations determined by isotope dilution mass spectrometry on seven lots of serum are given in

Table 1. The blank correction based on three blank

determinations was less than 0.1 ng or less than 0.001

percent of the amount of lithium present in any sample.

Sources of error in the lithium concentration determination

are calibration of the 6 Li spike, the ratio measurement of

the spiked sample, and the effects of the impurities [4].

The 5 Li spike solution was calibrated with two natural

solutions prepared from SRM 924, Lithium Carbonate. The 95

percent confidence limits for a single analysis of the spike

calibration is 0.3 percent. While this is a relatively

large uncertainty in the calibration of a spike solution, it

is consistent with the precision of the s Li/ 7 Li ratio of the

standard. The chief source of imprecision is the inability

to adequately control the isotopic fractionation between

analyses. The estimated maximum error for the measured

lithium concentrations in Table 1 is ±0.5 percent which

includes allowances for measurement imprecision and

systematic errors.

86
Table 1. Lithium concentrations in CDC bovine serum.

Pool Sample No. yg L-i/g mmol/L

1 1 3.592

2 3.628

3 3.608

4 3.615

= 3.611 0.534 ± 0.003a

la 1 6.817

2 6.783

3 6.810

4 6.809

5 6.804

6 6.788

6.802 1.004 ± 0.005 a

1 6.994

2 6.987

3 6.963

4 6.960

6.976 1.031 ± 0.005 a

1 8.720

2 8.741

3 8.710

4 8.732

x = 8.726 1.290 ± 0.006 a

continued

87
Continuation of Table 1.

Pool Sample No. yg Li/g mmol/L

4 la 10.419

lb 10.440

2a 10.493

2b 10.453

3a 10.433

3b 10.445

4a 10.494

4b 10.474

x =

x = 10.456 1.546 ± 0.008 a

1 12.249

2 12.186

3 12.219

4 12.326

x = 12.245 1.809 ± 0.009 a

5a 1 13.331

2 13.364

3 13.366

4 13.357

5 13.363

6 13.378

13.360 1.969 ± 0.010 a

1 13.764

2 13.765

3 13.888

4 13.823

x = 13.812 2.042 ± 0.010 a

88

continued
Continuation of Table 1.

Pool Sample No. yg Li/g mmol/L

1 17.287

2 17.426

3 17.425

4 17.465

= 17.401 2.572 ± 0.013 a

7a 1 20.058

2 20.005

3 20.072

4 20.002

5 20.020

6 20.020

x = 20.030 2.954 ± 0.015 a

Estimated maximum error of 0.5 percent of the value. This

estimated error is the sum of errors due to measurement

imprecisions of ±0.3 percent (± 2-sigma interval for the

random error of the mean) and an estimated upper bound of

0.2 percent for possible systematic errors.

89
The isotopic composition of lithium was determined for

each lot of serum and found to be experimentally identical

to the reference standard, 7.562 atom percent s Li and

92.438 atom percent 7 Li. A significant amount of the high

purity lithium that is commercially available is depleted in

6 Li; e.g., as much as 50 percent depletion has been found on

a limited number of samples. Thus, the only methods for

obtaining reliable knowledge of the isotopic composition in

samples of this element are by isotopic analysis or use of a standard of known composition.

References

[1] Kuehner, E. C, Alvarez, R. , Paulsen, P. J., and Murphy,

T. J., Anal. Chem., 44, 2050 (1972).

[2] Murphy, T. J., The Role of the Analytical Blank in

Accurate Trace Analysis, Nat. Bur. Stand. (U.S.) Special

Publication 422, Accuracy in Trace Analysis: Sampling,

Sample Handling, and Analysis, p. 509-539 (1976).

[3] Garner, E. L., Murphy, T. J., Gramlich, J. W. , Paulsen,

P. J., and Barnes, I. L., J. Res. Nat. Bur. Stand.

(U.S.) 79A (Phys. and Chem.) 713-725 (Nov. -Dec. 1975).

[4] Garner, E. L., Machlan, L. A., Gramlich, J. W. Moore,

L. J., Murphy, T. J., and Barnes, I. L., An Accurate

Determination of Electrolyte Concentrations in Blood

Serum by Isotope Dilution Mass Spectrometry, Nat. Bur.

Stand. Special Publication 422, 951-960 (1976).

90
APPENDIX D

Note 1 : A temperature range of room ±2 °C is designated as the

operating temperature. In this temperature range the maximum difference in aqueous solution volumes due to thermal

expansion of the liquid is 0.102 percent and the difference

in volume due to the volumetric glassware is very small

since the coefficient of expansion for borosilicate glass is

0.00001 per °C. (J- Lembeck, "Calibration of Small Volumetric

Laboratory Glassware", NBSIR Report 74-461, 1974, Institute

for Basic Standards, National Bureau of Standards,

Washington, D.C. 20234). We judge these errors to be

acceptable for this reference method. Larger temperature

variations may necessitate appropriate correction.

Note 2 : Glassware Required:

a) Manual pipetting alternative:

Volumetric Flasks : three 2-L; two 1-L; seven 100- plus

one additional 100-mL volumetric flask for each sample.

Pipets : two 5-mL, and one each of 4-, 10-, 15-, 20-,

25-, and 3Q-mL.

b) Semiautomated pipetting alternative:

Volumetric Flasks : three 2-L; two 1-L; and seven 50-mL

plus one 50-mL volumetric flask for each sample.

Pipets : two 5-mL; and one each of 10-, 15-, 20-, 25-,

and 30-mL.

91
Note 3 : Cleaning of Glassware and the pipettor-dilutor

a) Clean the glassware in the following manner:

(1) Soak glassware for 60 min in 0.77 mol/L HN0 3 . (2) Rinse six times with a volume of water equal to at

least 10 percent of the container volume.

(3) Use immediately or air dry (inverted in a dustfree environment) for later use.

b) Clean the pipettor-dilutor device as follows:

(1) Rinse the tubing with water by delivering at least

four 5-mL water samples.

(2) Rinse the tubing with 0.77 mol/L HN0 3 by drawing

into the delivery tube a volume of HN0 3 equal to

the volume of sample pipetted and then delivering

four 5-mL portions of HN0 3 through the system.

(3) Repeat step (2) using H 2 0, ethanol, and H2O

sequentially

.

(4) Repeat step (2) with the diluent to be used for

preparing the working solutions of the sample,

standards, and blank. The pipettor-dilutor is

then ready for the preparation of the working

solutions

.

Note 4 : Procedure for Testing Pipettor-Dilutor Devices : The accuracy

and precision of the device is determined by weighing fixed

volumes of water repetitively delivered by the device.

1. The water that is delivered in tared, stoppered flasks

is to be weighed on an analytical balance capable of

being read to the nearest one-tenth milligram. Measure

the temperature of the delivered water to the nearest

0.1 °C just before or after delivery.

92
Test the delivery of the 2.00 mL volume (as will be used)

as follows:

a. (1) Number and tare ten, clean, dry, stoppered,

glass or plastic weighing bottles of approximately

10-20 mL volume.

(2) Sample 2.00 mL of water and deliver it together

with 5 mL of diluent water into the first bottle.

Stopper immediately.

(3) Repeat step '2' with the remaining 9 bottles.

(4) Weigh each of the 10, filled bottles.

(5) Calculate the weight of each aliquot plus

di luent

.

b. Repeat steps 1-5 of part a, but in step 2 omit the

sampling of the 2.00 mL of water by allowing air

to be sampled rather than water; thus only the 5.00

mL of diluent water is collected in the tared bottles. Calculation then gives the weights of diluent.

c. Calculate from part b the mean weight for the

diluent

.

d. Calculate the differences between the individual

weighings obtained in part a step (5) and the mean

weight of the diluent (from part c) to obtain the

weights of the water aliquots delivered at the

2.00-mL setting that was used.

e. Calculate the mean and standard deviation for the

weights of water samples (from part d)

.

f. Use the attached table (#43) from Circular #19,

"Standard Density and Volume Tables," [National

Bureau of Standards, Washington, D.C. 20234] to

convert the mean of the diluent weights (from part

c) and the mean of the sample weights (from part e)

into volumes at 20 °C, in the following manner:

93
Jso to o CD i—i

CD

bfi

4-> CD •H n3 U

cti 4H

P_

O

crj u (/}

4-> <D PI P CD

crj

H

U

•H

i—(

CTi

00

no o o IX «3- to NO to 00

I—1

to NO lx o> I—1

to LO rx. CTi (XI IX (XI (XI CM (X] (XI to to to to to -3- -3-

NO

to i u U -h in

CD CD On CD CD E Sh h

CD 3 M H 4-> CD t3

(X] to

O oo o- oo cn rH oo to CT>

r—

to NO 00 rH to LO 00 o (XI LO

(XI (XI (XI (XI (XI (XI to to to to "3- o

rH NO I—1

CO NO LO NO tx CT> (XI NO rH nO

i—i

to NO i—1

to tx- o LO tx-

(X] (XI (XI (XI (X] (XI to to to to -3-

LO 00 to 00 LO to Cxi rH (XI IX. rH NO rH 1 !—t

(XI rt LO [X- O") rH to LO IX Cn (X] (X] (XI (X] (XJ CX1 CX] to to to to to "3- O

to IX rH IX- to rH o CT> o (X) LO 00 to Cn |1 rH (XI LO tx Cn rH CX) LO rx cn I—1

NO

(XI (XI CX] (XI (X] ex] to to to to to "3- ^t- O

(XI LO

O LO CX] at 00 IX- 00 o CXI NO o NO |

rH (XI LO rx- 00 o CX1 rx- Cn rH ^> NO ; (X) (X] CXJ CXI (X] (X] to to to to to *=* "3- O

rH oo to

O tx NO LO NO CO o «tf oo to

rH (XI to LO IX- 00 o CX] NO Cn rH to NO

(XI (X] (X] (XJ CX] (XI to to to to to "St -3- OO to (XJ oo LO to "3- LO 00 rH NO rH tx

rH (XI to LO NO 00 o CX) NO 00 rH to NO 00

(XI (X] Cxi CXI CXI CX) to to to to to ri- "3- ^ O

OO rH LO o NO «* CX) rH (X) to NO oi to 00 -3- o (XI to LO NO 00 O (X) NO oo o to LO oo

(XI (X] (XI (XI (XJ (X] to to to to to o

IX o «=* Cn LO (X) o o rH to NO rH NO (X) o (X] to NO oo o rH rf NO 00 o to LO oo

(X] (X] (X] CXI (XI CX) to to to to to "5t

LO NO OO Cn o

CX] rH CX) tO

(X) CX] CX] (X) lo no ix- oo cn

CXI (X) CX] (X) (X)

94
(1) Determine the volume of the nominally 2.000-mL

sample at 20 °C by adding to the mean value of the

delivered sample, (from part e) an amount equal to

the product of 0.020 and the value for the appropriate water temperature read from Table 43. The

sums obtained are in milliliters.

3. The requirements for the bias and imprecision of the

pipettor-dilutor are listed in Table 1. The pipettordilutor may be used in the semiautomated pipetting

alternative if these requirements are fulfilled.

Table 1. Bias and imprecision requirements for the volume

of sample delivered by the pipettor-dilutor

device, Section IIIC-2b.

Imprecision, Relative

Sample Size, mL Bias, mL Standard Deviation

2.00 0.04 0.2%

Note 5 :

a) The Li 2 C0 3 in NBS SRM 924 has been depleted in the 6 Li

isotope. Thus the atomic weight of lithium in this SRM

is 6.9696 rather than the usual 6.941, and the molecular

weight of this Li 2 C0 3 is 73.9484 rather than 73.8912.

b) The atomic weights used in this report are those reported

in: Pure and Applied Chemistry, 4_7, 75 [1976) .

Note 6 :

In some cases, two pipets will be needed to transfer

the desired volume; e.g., a 30-mL aliquot may be transferred

by using a combination of a 20-mL pipet and a 10-mL pipet.

The statistical limit of error for this dual pipetting is

0.032 mL or 0.107 percent of the total volume transferred

95
and is not very different from the limit of error in using

one pipet, 0.03 mL or 0.10 percent for a 30-mL pipet. No

estimate of operator error is included in this calculation

Note 7 :

If the wash solution does not drain cleanly from the

pipet, wash with 0.77 mol/L HN0 3 , H 2 0, MeOH, 70:30 v/v

CHCl 3 :MeOH, MeOH, and H 2 0 in that order. Then. repeat the

water wash and check that the pipet does drain properly.

Note 8 : The three following pages are examples of the data

sheets returned from each laboratory after each interlaboratory exercise.

96
ELECTROLYTES IN SERUM - CLINICAL REFERENCE METHOD

I ON Ll

LABORATORY 10 ANALYST XY

EXERCISE NO. IE ~ 11

DATE SAMPLES RECEIVED^ 2/9/75 DATES ANALYZED (1)1 2/16/7 5(2)1 2/18/7 5

INSTRUMENT MANUFACTURER Perkin-Elmer MODEL 403

WAVELENGTH 670.8 NM SLIT WIDTH 1.4 M Bandpass yM

TYPE HOLLOW CATHODE LAMP Westinghouse

CURRENT 12 MA

BURNER TYPE Single Slot

OXIDANT Air FLOW RATE 27 . 5 L/MIN

FUEL Acetylene FLOW RATE 5 . 5 L/MIN

INSTRUMENT TIME CONSTANT NA S SCALE EXPANSION

RECORDER TIME CONSTANT NA s READOUT: RECORDER , DIGITAL X , OTHER

LABORATORY TEMPERATURE 23 °c TO 26 °C (VARIATION DURING IE)

BACKGROUND CORRECTION? No HOW?

COMMENTS: Use plastic aspiration tube supplied. Aspiration

rate =8.0 mL/min.

97
DATA SHEET: STANDARD CURVE

PROTOCOL USED: MANUAL SEM I AUTOMATED

CALCULATED ION EXPANDED CORRECTED EXPANDED

STANDARD CONCENTRATION, MMOL/L ABSORBANCE VALUES ABSORBANCE VALUES

1.

2.

3.

k.

5.

6.

DILUENT BLANK

0.5000 0.293 0.293

0.000

1.0000 0.572 0.572

1.5000 0.842 0.842

2.0000 1.104 1.104

2.5000 1.400 1.400

3.0001 1.665 1.665

98
DATA REPORTING SHEET FOR VALID MEASUREMENTS

PROTOCOL USED: MANUAL __X SEM I AUTOMATED

LAB 10 ION Li IE III DATE ANALYZED 5/12/75 OPERATOR XY

SAMPLE # 802876 EXPANDED ABSORBANCES

STANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

(xp

SAMPLE

(Y)

HI STD

(x

2

)

C

LO 0.5000 CCj) 1

.

n 0 *7 H

L) . Z / / U . ZUU U . 5 0 / U . r o i o 5232

hi 1.0000 eco 2. 0. 281 0. 292 0. 556 0. 5200

3. 0.279 0. 296 D. 564 0. 5298

tf. 0.277 0. 297 0. 559 0. 5354

5. 0.278 0. 295 0. 550 0. 5312

SAMPLE # 76331 EXPANDED ABSORBANCES

STANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

(x

x ) SAMPLE

(Y)

HI STD

(x

2

)

c

LO 1. 5000 (C

1 ) 1

.

0.870 1 .020 1.159 1. 7595

HI 2.0000 (C„) 2. 0. 876 1.022 1.154 1. 7692

3. 0. 874 1.018 1.132 1. 7790

k. 0. 859 1.005 1.126 1. 7734

5. 0. 854 1.003 1.129 1. 7709

SAMPLE tt 35 EXPANDED ABSORBANCES

STANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

(x

x )

SAMPLE

(Y)

HI STD

(x

2

)

C

LO ^ • 5000

1. 1.412 1.654 1. 700 2. 9202

HI 3.0001 Cc

^

2.

1.406 1. 643 1. 689 2. 9506

3.

1.399 1 .654 1.671 2. 9688

4.

1.400 1.662 1. 674 2. 9781

5.

1. 397 1.658 1,672 2. 9746

99
NBS-114A (Rev. 9-7ei

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PUBLICATION OR REPORT NO.

NBS SP 260-69

3 ft*cip

4. TITLE AND SUBTITLE

Standard Reference Materials:

A Reference Method for the Determination of

Lithium in Serum

5. Publication Date

7. AUTHOR(S)

R.A. Velapoldi, R.C. Paule, R. Schaffer, J. Mandel

L.A. Machlan, E.L. Garner and T.C. Rains

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. Project/Task/Work Unit (to.

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, DC 20234

11. Contract/Grant No.

12. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street, City, state. ZIP)

Same as above

13. Type of Report & Period Covered

Final

14. Sponsoring

1 1 )1 111 1 1

15. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number: 80-600091

I | Document describes a computer program; SF-185, Fl PS Software Summary, is attached.

16. ABSTRACT (A 200-word or less factual summary oi most significant information. If document includes a significant bibliography or

literature sur^y. mention it here.) A reference method was established for the determination

of serum lithium based on flame atomic absorption spectroscopy (FAAS). Its accuracy

was evaluated by comparing the values obtained by use of the method in 14

laboratories against the results obtained by a definitive analytical method based

on isotope dilution-mass spectrometry (IDMS). Ten serum pools with lithium

concentrations in the range 0.534 to 2.954 mrnol/L were analyzed. Manual and semiautomated pipetting alternatives were tested using sample sizes of 4.00 and 2.00 mL,

respectively.

The laboratories used several different FAAS instruments. The results

showed that the standard error for a single laboratory's performance using either

pipetting procedure was about 1.5 percent with a negative bias of about 2.0 percent

over the range of serum lithium concentrations studied. These values are within the

accuracy and precision goals that had been set. The calibration curve data showed

excellent linearity over the total concentration range, with 24 of 25 curves

having standard deviations of fit of 0.025 mmol/L or less.

With appropriate experimental design, the reference method may be used to establish the accuracy of field methods as well as to determine reference lithium

values for pooled sera.

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name;

separated by semicolons) Accuracy; clinical analysis; clinical chemistry; definitive method; electrolytes;

flame atomic absorption spectroscopy; interlaboratory testing; precision; reference

method; semi automated pipetting; serum lithium analysis; statistical analysis.

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