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03 Q NBS SPECIAL PUBLICATION 260-80

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

Standard Reference Materials:

Evaluation by an ID/MS

Method of the AACC

Reference Method for

Serum Glucose

QC

100

.167

NO. 260-80

1982
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 — Chemical Physics —

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 — Manufacturing

Engineering — Building Technology — Fire Research — Chemical Engineering2

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, M D, unless otherwise noted; mailing address Washington, DC 20234.

! Some divisions within the center are located at Boulder, CO 80303.
Standard Reference Materials:

Evaluation by an ID/MS Method of the

AACC Reference Method for Serum Glucose

R. Schaffer, J. Mandel, T. Sun,

A. Cohen and H. S. Hertz

National Measurement Laboratory

National Bureau of Standards

Washington, DC 20234

J. W. Neese

Centers for Disease Control, Atlanta, GA 30333

In conjunction with:

R. J. L. Bondar, C. Fasce, N. Gochman,

G. Kessler, R. B. McComb, W. T. Ryan,

and R. E. Vanderlinde

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued October 1982
Library of Congress Catalog Card Number: 82-600618

National Bureau of Standards Special Publication 260-80

Natl. Bur. Stand. (U.S.), Spec. Publ. 260-80, 55 pages (Oct. 1982)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1982

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

Price $4.75

(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

therefore of more than passing interest to know the details

of the measurements made at NBS in arriving at the certified

values of the SRM's produced. An NBS series of papers, of

which this publication 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.

Inquiries concerning the technical content of this paper

should be directed to the authors. Other questions concerned

with the availability, delivery, price, and so forth 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

iii
OTHER NBS PUBLICATIONS IN THIS SERIES

Catalog of NBS Standard Reference Materials

(1981-83 edition), R. W. Seward, ed., NBS

Spec. Publ. 260 (November 1981).

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

Reference Materials: Preparation of White Cast

Iron Spectrochemical Standards. NBS Misc.

Publ. 260-1 (June 1964). COM 74- 1 1 06 1 **

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-

1 1063**

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). COM74-1 1068**

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-

1 1067**

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-I 1065**

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). COM74-1 1064**

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

IV
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-U359**

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* PB 81176620

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). COM71-50282

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). COM72-5037 1 ** 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**
McAdie, H. G., Garn, P.D., and Menis, O., Standard Reference Materials: Selection of Thermal

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

Spec. Publ. 260-40 (August 1972.) COM72-

50776**

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

Reference Materials: Use of Standard LightSensitive Paper for Calibrating Carbon Arcs Used in Testing Textiles for Colorfastness to

Light, NBS Spec. Publ. 260-41 (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. 260-47 (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. 260-48

(May 1974). 60 cents* SN003-003-0 1278-5

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

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

260-49 (November 1974). COM74-51185

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* SN003-003-

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* SN003-003-

01836-8

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

Standard Reference Materials: Standard

Thermocouple Materials, Pt.67: SRM 1967, 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* PB 277173

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 (June 1979) $2.00* SN003-

003-02077

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* SN003-

003-01975-5

vi
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* SN003-003-02 1 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, J.R., 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 (July)

1980). $4.25* SN003-003-022I4-4

Marinenko, R. B., Biancaniello, F., Boyer, P. A., Ruff, A. W., DeRobertis, L., Standard Reference

Materials: Preparation and Characterization of an Iron-Chromium-Nickel Alloy for Microanalysis, NBS Spec. Publ. 260-70 (May 1981).

$2.50* SN003-003-02328-I

Seward, R. W., and Mavrodineanu, R., Standard

Reference Materials: Summary of the Clinical Laboratory Standards Issued by the National

Bureau of Standards, NBS Spec. Publ. 260-71

(November 1981). $6.50* SN003-003-0238 1-7 Reeder, D.J., Coxon, B., Enagonio, D., Christensen,

R. G., Schaffer, R., Howell, B. F., Paule, R. C,

Mandel, J., Standard Reference Materials: SRM

900, Antiepilepsy Drug Level Assay Standard,

NBS Spec. Publ. 260-72 (June 1981). $4.25*

SN003-003-02329-9

Interrante, C. G., and Hicho, G. E., Standard Refer- ence Materials: A Standard Reference Material

Containing Nominally Fifteen Percent Austenite

(SRM 486), NBS Spec. Publ. 260-73 (January

1982). $2.75* SN003-003-02386-8

Marinenko, R. B., Standard Reference Materials:

Preparation and Characterization of K.-411 and

K.-414 Mineral Glasses for Microanalysis: SRM

470. NBS Spec. Publ. 260-74 (April 1982).

$3.50 SN003-003-023-95-7

Weidner, V. R., Hsia, J. J., Standard Reference

Materials: Preparation and Calibration of First Surface Aluminum Mirror Specular Reflectance

Standards (SRM 2003a), NBS Spec. Publ. 260-75

(May 1982). $3.75 SN003-003-023-99-0

Hicho, G. E. and Eaton, E. E., Standard Reference

Materials: A Standard Reference Material

Containing Nominally Five Percent Austenite

(SRM 485a), NBS Spec. Publ. 260-76 (in press).

Furukawa, G. T., Riddle, J. L., Bigge, W. G., and

Pfieffer, E. R., Standard Reference Materials:

Application of Some Metal SRM's as Thermometric Fixed Points, NBS Spec. Publ. 260-77 (in press).

vii
Hicho, G. E. and Eaton, E. E., Standard Reference

Materials: Standard Reference Material Containing Nominally Thirty Percent Austenite (SRM

487), NBS Spec. Publ. 260-78 (in press).

Richmond, J. C, Hsia, J. J., Weidner, V. R., and

Wilmering, D. B., Standard Reference Materials:

Second Surface Mirror Standards of Specular

Spectral Reflectance (SRM's 2023, 2024, 2025),

NBS Spec. Publ. 260-79 (in press).

Schaffer, R., Mandel, J., Sun, T., Cohen, A., and

Hertz, H. S., Standard Reference Materials:

Evaluation by an ID/ MS Method of the AACC

Reference Method for Serum Glucose, NBS

Spec. Publ. 260-80 (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

1. Introduction

2. Experimental 6

2.1 Materials and Methods for the ID/MS Method

2.1.1 Serum Samples 6

2.1.2 g-Glucose 6

2.1.3 D-Glucose-U-l3c

2.1.4 Calibration Mixtures

2.1.5 Sample Preparation

2.1.6 DAG Synthesis 8

2.1.7 MS Instrumentation

2.1.8 MS Measurements 10

2.1.9 Measurement Procedure 10

2.1.10 Calculations 11

2.1.11 ID/MS Analyses on Five Serum Pools 11

2.2 Hexokinose/Glucose - 6 -Phosphate Dehydrogenase Method

Using a Protein-Free Filtrate for the Determination

of Glucose. The Candidate Reference Method 11

3. Mult ilaboratory Studv of the Candidate Reference Method. . . 12

3.1 Round-Robin (RRI) 12

3.2 Round-Robin II (RR II) 12

3.3 Round-Robin III (RR III) 13

4. Results and Statistical Analysis 13

4.1 ID/MS 13

4.2 Candidate Reference Method 15

4.2.1 Calibration 24

4.2.2 Round Robin I 4.2.3 Round Robin II

4.2.4 Round Robin III

4.3 Comparison of Results of Round Robin III with ID/MS

Values 34

5. Discussion 37

5.1 ID/MS Method 37

5.2 Candidate Reference Method 59

5.3 Comparison of Results from the Candidate Reference and

ID/MS Method 39

6. References 42

ix
List of Tables

Table No. Page

1. Relative weights of unlabeled and labeled glucose in

standards and the relative measured intensities of

(M-15) + ions at m/z 245 and 250 from DAG 7

2. Isotope dilution mass spectrometry results (in mg/L)

from CIMS measurements 14

3. ID/MS precision parameters (mg/L) 14

4. ID/MS results from EIMS measurements 15

5. Round Robin I - individual measurements: concentration

in mg/L 16

6. Round Robin II - manual: individual measurements,

concentration in mg/L 17

7. Round Robin II - semi - automated : individual measurements,

concentration in mg/L 18

8. Round Robin III - manual: individual measurements,

concentration in mg/L 19

9. Round Robin III - semi - automated : individual measurements,

concentration in mg/L 21

10. Round Robin I - pre- and post -analys is calibration results

by least squares method 25

11. Round Robin I - comparison of two methods for calculation

of calibration parameters 25

12. Round Robin I - precision parameters and sample concentrations in mg/L, calculated by two methods 26

13. Round Robin I - percent deviations of individual

laboratory averages from all-labs average 27

14. Round Robin II - calibration results 28

15. Round Robin II - precision parameters for calculated

sample concentrations (mg/L) 29

16. Round Robin II - manual: percent deviations of individual

laboratory averages from all-labs average 29

17. Round Robin II - semi -automated : percent deviations of

individual laboratory averages from all-labs average. ... 30

18. Round Robin III - manual: calibration results. ...... 31

19. Round Robin III - semi-automated: calibration results. . . 32

x
List of Tables continued

Table No. Page

20. Round Robin III - precision parameters for calculated

sample concentrations (mg/L) 33

21. Round Robin III - manual: percent deviations of individual

laboratory averages from all-labs average 33

22. Round Robin III - semi - automated : percent deviations of

individual laboratory averages from all-labs average. ... 34

23. Comparison of Round Robin III results with ID/MS target

values: averages and standard errors (mg/L) 34

24. Comparison of Round Robin III results with ID/MS target

values: differences and standard errors in mg/L 35

25. Round Robin III - manual: percent deviations from

target value 35

26. Round Robin III - semi -automated : percent deviations

from target value 35

27. Summary of precision data - manual: coefficient of

variation (%) ..... 36

28. Summary of precision data - semi - automated : coefficient

of variation (%) 37

29. Comparison of six human serum pools by the revised DAG

ID/MS and candidate reference methods 40

30. Expected standard error of average for the candidate

reference method on performance of four or eight

replicate determinations on one or two days (mg/L) 41

xi

EVALUATION BY AN ID/MS METHOD OF THE

AACC REFERENCE METHOD FOR SERUM GLUCOSE

R. Schaffer, J. Mandel, T. Sun*, A. Cohen, and H. S. Hertz

National Bureau of Standards

Washington, DC 20234

J. W. Neese

Centers for Disease Control, Atlanta, GA 30333

In conjunction with:

R. J. L. Bondar 1

, C. Fasce 2

, N. Gochman 3

, G. Kessler 1* , R. B. McComb 5

, W. T. Ryan^ , and R. E. Vanderlinde 7

In conjunction with a study group of the Committee on

Standards of the American Association for Clinical Chemistry

working to establish a reference method for glucose in serum,

the authors from NBS developed an isotope dilution/mass

spectrometric method (ID/MS) for providing essentially biasfree, precise serum glucose analyses. This method, which is

too elaborate for clinical laboratory use as a reference

1

method, involves addition of a known amount of g-glucose-U- C

to a serum sample, conversion of the labeled and unlabeled

glucose in the sample into 1 , 2 : 5 ,6-di-O-isopropylidene-Dglucose (DAG) , and measurement of the ratio of labeled

to unlabeled DAG as the corresponding (M+l)

+ ions, by

^Present address: U.S. Department of Agriculture, Athens, GA

diagnostic Systems, Inc., Freehold, NJ

2 City of Kingston Laboratory, Kingston, NY

3 San Diego Veterans Adminstration Hospital, San Diego, CA

^Jewish Hospital, St. Louis, MO

5 Hartford Hospital, Hartford, CT

6 Beckman Instruments, Fullerton, CA

7 New York State Department of Health, Albany, NY

(present address, Hahnemann Medical College and Hospital,

Philadelphia, PA)

1
I isobutane-chemical ionization mass spectrometry. Five serum

pools having glucose concentrations ranging from 0.4 to

3.0 g/L were analyzed. The relative standard deviation among

single measurements made on different samples of the same

pool was found to range from 0.34 to 0.46 percent for four

of the pools, and was 0.79 percent for the pool with the

highest glucose concentration. Pool concentrations were also

determined from the same DAG samples using electron impact

mass spectrometry and monitoring the ratios of corresponding

(M-15)

+ ions, and the results were similar. There was no

evidence of bias.

These serum pools were used by the study group for a statistically controlled inter laboratory test to evaluate a hexokinase/glucose - 6 -phosphate dehydrogenase method using a protein-free filtrate as the reference method for glucose.

Investigators at the Centers for Disease Control (Atlanta)

had found that method the most appropriate of the several

glucose methods that were studied as possible reference

methods. [J. W. Neese et al . HEW Publication No. (CDC)

77-8330.]. Statistical analysis of the mult ilaboratory

results showed that the relative standard deviations among

single measurements made in different laboratories decreased

as glucose concentrations increased. With manual pipetting

used for performing the candidate reference method, the

relative standard deviation ranged from 4.4 to 1.2 percent;

with semi - automat ed pipetting, the range was 2.8 to 0.8

percent. Compared to the ID/MS results, the mean values

found by the candidate reference method were about 1 percent

higher at the 0.4 g/L level and changed linearly to about

2 percent lower at the 3.0 g/L level. We conclude that the

candidate reference method fulfilled our prechosen criterion

for acceptance as a reference method for serum glucose.

Key words: clinical analysis; glucose in serum; glucose

reference method; isotope dilution/mass spectrometry;

reference method; statistical analysis.

2
1. Introduction

A study group* of the Committee on Standards of the American

Association for Clinical Chemistry was organized in 1972 for the

purpose of establishing a reference method for serum glucose determinations. This required the study group to a) consider the limits for

bias and imprecision it judged would be acceptable in a glucose reference method for clinical chemistry, b) identify the potential (i.e.,

candidate) reference method, from such evidence as low susceptibility

to possible sources of interference and amenability to precise performance, c) obtain several serum pools whose glucose levels are determined

by an essentially bias-free, highly precise (i.e., definitive) method,

and d) use the serum pools in a statistically designed, mult ilaboratory

study to evaluate the candidate method as the reference method. Cali

et al . [1] employed that approach in attempting to establish the

reference method for total calcium, and it was subsequently used for

the serum sodium [2], potassium [3], chloride [4], and lithium [5]

reference methods.

Studies performed at the Centers for Disease Control (CDC) led to

the development of a modified form of Slein's method [6] as a choice

candidate reference method for serum glucose. The modified method

involves the use of a protein-free filtrate and reactions catalyzed by

hexokinase (EC 2.7.1.1 ATP :g-hexose-6-phosphotransferase) and D--glucose6-phosphate dehydrogenase (EC 1.1.1.49 D-glucose-6 -phosphate :NAD

oxidoreductase) . CDC's experimental work and description of the

candidate method have been published [7]. The study group undertook to

evaluate it as the reference method, following a multilaboratory testing

plan that was largely organized at the CDC [7]. As the criterion for

acceptability as a glucose reference method, the study group considered

a limit of bias of ±3 percent or ±30 mg/L from the definitive glucose

method, whichever was larger.

The development of a definitive method for serum glucose, carried

out at the National Bureau of Standards (NBS) , involved isotope dilution

mass spectrometry (ID/MS) although, according to the literature current

when the work was begun, "definitive quality" analyses using ID/MS had

*R. Schaffer, Chairman. Participants in addition to the authors:

B. Tejeda, Food and Drug Admins t rat ion , Washington, DC; C. Burtis,

Oak Ridge National Laboratory, TN; P. D. Schroff, Warner-Lambert Co.,

Morris Plains, NJ

3
been attained only for certain elements [8] , but not for complex

compounds like glucose [9] . The use of ID/MS offered promising

advantages: Specific ion monitoring would provide the needed ratios of

the unlabeled and labeled forms of the analyte as highly specific

measurements. With only very small quantities of the labeled and

unlabeled analyte mixture required for individual ratio measurements,

replicate ratio measurements could be performed on samples for demonstrating precision. Furthermore, small losses of analyte that might

occur prior to making ratio measurements would not affect analytical

accuracy as long as the initial ratio of labeled and unlabeled analyte

remained unaltered; hence, analyte recovery in isolation steps need not

be quantitative and the purification procedure could be extensive if

necessary to remove interfering substances. Our preliminary experiments

1

using the ID/MS method have been described [10]: B -glucose -U- C or,

alternatively, D- glucose - 6 , 6

&2 was added to the serum sample; time was

allowed for complete mixing and equilibration with the serum glucose;

and the mixture of glucose forms was converted into 1 , 2 : 5 , 6 -di -0-

isopropylidene-g-glucose (DAG) . Samples of the purified DAG were

introduced into the mass spectrometer via the spectrometer's direct

insertion probe, and subjected either to electron- impact mass spectrometry (EIMS) or chemical ionization mass spectrometry (CIMS) for

measuring the ratio of unlabeled- to- labeled glucose derivative. The

two differently labeled glucoses were used as alternatives in an attempt

to reveal evidence of interferences in the measurements. With ions of

different mass being measured, interferences should affect the ratios

and the results would be different. The initial data indicated

(incorrectly, as found later when our measurement techniques improved)

that a systematic error associated with use of the glucose-6 ,6-d ? had

13 occurred, and only glucose-U- C was used thereafter. The evidence

showing the absence of interferences in the method was then obtained,

as described in the present publication, by measuring the same DAG

samples by CIMS and alternatively by EIMS.

By the end of 1975, both the ID/MS method described in the present

publication and the mult ilaboratory analyses with the candidate reference

method had been applied to the same five bovine serum pools. However,

the results and their statistical analysis which was performed at NBS

were not published then because some additional analytical results not

in agreement with earlier data were being obtained both at NBS and at

CDC and needed to be investigated. They are discussed, in turn.

4
At NBS, some remaining samples of the serum pools which were being

stored at —20 °C were analyzed for evidence of the long-term reproducibility of ID/MS method results. However, the new results were lower

than those obtained earlier. To establish whether this was due to

changes in the stored serum samples or to defects in the method, three

separate actions were undertaken. The first was to modify the ID/MS

method in order to improve its precision [11]: For that packed-column

gas chromatography (GC) was used in place of the direct insertion probe

for introduction of samples into the mass spectrometer and measurements

were carried out with rigid adherence to an individual sample bracketing protocol like the one described with the definitive ID/MS method

for cholesterol [12] . The second action was to use the modified ID/MS

method over a 6-month period to analyze samples of six human serum pools

that were also being stored at —20 °C. (The supply of bovine serum

samples was exhausted.) These results showed a gradual decrease in

glucose values, about 1.2 percent in 6 months [11]. The third action

was to develop an alternative ID/MS method that would permit independent

confirmation of the DAG method results: For that a different glucose

derivative was used, namely, glucose 1 , 2 : 3 , 5-bis (butylboronate) -6-acetate

(glucose BBA) which is synthesized under different reaction conditions

than those required for DAG synthesis. This compound had been used in

a previously reported glucose ID/MS method [13] , but the two methods

differ. In the NBS version [11] the glucose BBA is purified by capillary

column GC and the ratios are measured by following an individual sample

bracketing protocol [12] . Details of the modified DAG and the NBS

glucose BBA ID/MS methods and evidence showing the comparability of

their results were recently published [11] . The correspondence of

results from the original and modified DAG ID/MS methods is described

in the present publication.

At the CDC, additional analyses of stored samples of the original

bovine serum pools also revealed that the glucose levels were

decreasing. This rate of decrease, about 2 percent per year, was later

observed also with the six human serum pools stored at -20 °C. In

studies conducted with these aged human serum pools, it was found that

50 percent to 75 percent of this loss could be recovered by preincubating the specimens at 25 °C for 1 to 2 days before analysis.

Normally, the samples were thawed and preincubated at 25 °C for about

one hour before analysis. CDC's study showed that these phenomena were

5
related to storage conditions. By lowering the storage temperature of

freshly prepared pools to -50 °C, glucose deterioration was prevented

[14].

The present publication covers a) the original DAG ID/MS method

and evidence showing its accuracy, and b) the statistical analysis of

the multilaboratory candidate reference method and ID/MS method data.

The data evaluated were obtained by both the ID/MS and candidate

reference methods during May and June 1975; the small glucose level

changes in the samples in that period are considered to have only a minor effect on our evaluation.

2. Experimental

2.1 Materials and Methods for the ID/MS Method

2.1.1 Serum Samples

Vials containing samples of bovine and human serum pools were

provided by the CDC where they had been prepared and stored at —20 °C.

At NBS, most pools were stored at —20 °C; others, as indicated, were

kept at -50 °C.

2.1.2 g-Glucose

NBS Standard Reference Material (SRM) 917, which is certified as

99.9 ± 0.1% pure g-glucose, was used as the primary standard material.

2.1.3 g-Glucose-U- 13

C

1

3

Crystalline D-glucose-U- C was supplied by Drs . D. Ott and

T. W. Whalley of the Los Alamos Scientific Laboratory (Los Alamos, NM

87544) . The sugar showed no evidence of impurities by thin layer

chromatography (TLC) . EIMS of samples of DAG prepared from the labeled

sugar (details given below) showed that the principal ions at high mass

correspond to the DAG-fragment ion (M-15)

+

, the molecular ion minus a methyl radical, and occurred in a cluster from m/z_ 245 to m/z_ 253 ; the

most prevalent (0.3 of the total cluster) was at m/z_ 250. The (M-15)

+

ion from unlabeled DAG was at m/z_ 245 . By CIMS with isobutane, the

(M+l)

+ ions were most intense, and the most prevalent from the labeled

and unlabeled DAG were at m/z 266 and m/z_ 261, respectively.

6
2.1.4 Calibration Mixtures

1

Known quantities of SRM glucose and glucose-U- C were combined in

a series of mixtures that ranged in proportion from about 0.85 to 1.15.

The ratios of (M-15)

+ ions at m/z^ 245 to 250 observed for these mixtures

by EIMS are shown in Table 1. The mixtures were prepared from standard

solutions of the two sugars (each about 0.4 g/L of water). Weighings

were made to 1 part in 10,000. The aliquots required for the mixtures

were delivered from burets that had been "conditioned" by filling with

one of the standard solutions, draining the solution going back into its

original container, and repeating the filling and draining again. As

each aliquot of a standard solution was transferred into a tared 300-mL

flask, the flask and contents were weighed, so that proportions in the

calibration mixtures could be calculated from the weighed quantities.

After the aliquots from both solutions were added, water was added to

the flask to wash remnants of the aliquots from the walls into the

mixture and the contents were swirled to ensure mixing. The combinations

of aliquots and washings, with total volumes about 55-75 mL each, were

freeze-dried in the flasks. The dried glucose residues were converted

into DAG, as described below under DAG synthesis.

Table 1. Relative weights of unlabeled and labeled glucose

in standards and the relative measured intensities

of (M-15)

+ ions at m/z 245 and 250 from DAG.

Standard

No.

Relative

Weights

Relative

Ion- Int ens ities

1 0 . 26672 0. 8647

2 0 . 30388 0.9566

3 0 . 31878 1.0032

4 0.32727 1. 0288

5 0 . 34378 1 . 0727

6 0. 35158 1. 0940

7 0. 36888 1. 1431

8 0. 37467 1.1554

2.1.5 Sample Preparation

(The ID/MS method requires the ratio of labeled to unlabeled sugar

in samples to be within the range of the calibration mixtures; hence,

approximate glucose concentrations in samples must be known beforehand,

for example, by use of a routine glucose method.) A serum aliquot

7
containing between 1.3 and 1.7 mg of glucose, weighed to 1 part per

1,000 or better, was combined with an exactly known amount (of the order

1

3

of 4.5 mg) of glucose-U- C, as follows: A weighed aliquot of the

1

3

standard solution of glucose-U- C from the buret was added to a 300-mL

flask. The serum aliquot added was taken from a vial that had been

allowed to warm to room temperature and gently inverted several times

for homogenizing the serum. A plastic syringe that was twice alternately

filled and emptied of serum from the vial, was used to add the aliquot

to the flask. The amount of serum transferred to the flask was obtained

by weighing the syringe when refilled with sample and then after delivering the aliquot. About 60 mL of water from a wash bottle was used to

wash any droplets of labeled glucose solution or serum from the upper

walls of the flask into the mixture. The contents were swirled for

mixing and left at room temperature for 3 h to allow the distribution of

labeled and unlabeled glucose to reach equilibrium. Then the solution

was f reeze-dried. The glucose in the dried residue was converted into

DAG, as described next.

2.1.6 DAG Synthesis

The conversion of the freeze-dried glucose (calibration mixtures or

samples) into DAG was carried out by adding 1 g of anhydrous CuSO^ , 1 g

of anhydrous CaSO^ , and 25 mL of acetone to the flask containing the

glucose; then, while swirling the slurry, adding about 0.1 mL of concentrated I^SO^ dropwise; and finally shaking the securely stoppered flask

vigorously for 2 days on a shaking machine. The reaction mixture was

neutralized (test with pH paper) by adding 1-2 g of Na2CG"2 and stirring;

then it was filtered, and the filtrate was concentrated by evaporation

at reduced pressure. The concentrate was transferred, with CHCl^ used

for rinsing, into a sublimation apparatus, where the solution was

evaporated with gentle warming under a gentle stream of N2 gas.

Sublimation was performed at reduced pressure (7 Pa; about 0.05 mm Hg)

and 88 ± 2 °C. The DAG in the sublimate was separated from acetone selfcondensation products and the diacetone derivatives of other hexoses

possibly present in serum samples by performing three sequential TLC

steps: the first on a 20 x 20-cm, 500-ym SiG^ GF plate, with benzenemethanol (90:7.5 by vol.) used as developer; the second on a 20 x 20-cm,

500 urn A1

2 0

3

G plate, with CHCl^ as developer; and the third TLC on a 20 x 20-cm, 500 ym SiC^ GF plate, by double-development using benzenemethanol (4:1 by vol.). The DAG was extracted from the Si0

2 with CHC1

3

8
and methanol and the solution was filtered and then blown with ^ gas

down to a concentration of about 1 mg of DAG per mL of CHCl^ for mass

spectrometry. The DAG in the calibration mixtures required only the

first TLC step for its purification, because no other hexoses were present

The location of the DAG on SiG^-coated plates was detected indirectly

by gently pressing a strip of adhes ive - coated , transparent tape to the

SiC^ coating, to remove a thin covering of SiG^ from the plate, and then

charring the tape after spraying the adhering Si02 with H^SO^. On an

A^O^-coated plate, the DAG was detected indirectly by pressing a SiC^-

coated plate to the wet A^O^-coated plate as soon as the development

was finished, so that the SiG^-coated plate became wet with developer.

When dry, the Si02"Coated plate was charred with F^SO^.

2.1.7 MS Instrumentation*

We used a Model CH 7A mass spectrometer (Varian MAT; now, Finnigan

MAT) equipped with a combined chemical ionization/electron impact ionsource and a multi-ion selection device, with modifications and

additions, as noted. The standard ion- detect ion system for the CH 7A

was used, including the electron multiplier, preamplifier, and amplifier.

The output of this amplifier is connected to three devices in parallel:

a mass-peak display, the multi-ion selection device (the output of

which was used only for qualitative data), and a mult i - channel scaler

for quantitative data.

The mass-peak display device permitted the selection and observation

of a region of the ion beam about one atomic mass unit wide. From a lowfrequency function generator, we applied a voltage of triangular wave

form (55 Hz, with ±20 V maximum amplitude) to the beam-deflection plates

located in front of the exit slit, so that an ion- intensity signal was

obtained by sweeping a small mass range. The signal was displayed on an

oscilloscope, providing continuous visual monitoring of peak shapes and

positions. The advantages for quantitative application of combining

continuous display with selected ion monitoring have been previously

noted for systems having other methods of display [15,16].

^Certain commercial equipment, instruments, or materials are identified

in this report to specify adequately the experimental procedure. Such

identification does not imply recommendation or endorsement by the

National Bureau of Standards, nor does it imply that the materials or

equipment identified are necessarily the best available for the

purpose

.

9
The multi-ion selection device controlled the switching of the

magnetic field, for monitoring two masses, and the timing, for acquisition of intensity data. The device also provided for acquisition of

intensity data for each mass selected, and for the data to be displayed

in the form of gas - chromatographic peak profiles.

Data for quantification were collected in parallel by two channels

of an eight-channel sealer-timer (built at NBS) , which converts voltage

to frequency and then counts, giving 100 counts per millivolt -second

Two masses were monitored and the separately accumulated counts were

transferred to a Model 9830A Calculator (Hewlett-Packard) for data

reduction

2.1.8 MS Measurements

The direct insertion probe of the mass spectrometer was used for

introduction of DAG samples. About 3 pg of purified DAG in CHCl^ solution was pipetted into a short gold capillary tube, sealed at one end.

When the CHCl^ had evaporated, the tube was inserted in the probe, and

the latter was introduced into the ion-source. For isobutane - chemical

ionization measurements, the ion-source temperature was 200 °C, the

_ 5 source manifold pressure was about 5 x 10 mm Hg , the electron energy

was 400 eV, and the probe heating current was varied to volatilize the

DAG over a 0.5 to 1.0 min interval. For measurements made in the

electron- impact mode, the ion-source was at 200 °C and the probe-heating

current was raised to volatilize the DAG at a fairly constant source

- 6

pressure (about 3 x 10 mm Hg) to allow the volatilization to proceed

between 0.5 to 1.0 min. The multi-ion selection device was set to monitor

the two selected ions alternately by switching the magnetic field every

second. The peak-display device was operated simultaneously, with the

width of the display set so that, for each mass peak, all of that peak

and some of the adjacent baseline on each side were displayed. Data were

collected from the onset of sample ionization until the ion-intensities

were very low (about 1 percent of maximum) . Measurements using electronimpact ionization were carried out using the same measurement procedure

and calculation method as for isobutane-chemlcal ionization.

2.1.9 Measurement Procedure

As a preliminary for each sample, the intensity ratio of the

selected labeled to unlabeled DAG ions was measured once. The pair of

calibration standards whose previously measured intensity ratios of the

10
same selected ions most closely bracketed the ratio observed for each

sample were identified. The duplicate ion- intens ity ratios of the two

standards and the sample were measured, usually not in sequential order,

and measurements were accepted only if duplicates agreed within 1 percent.

2.1.10 Calculations

RW , the weight-ratio of unlabeled to labeled glucose for a calibration standard or sample can be equated to a constant b plus the product

of a constant m by the quantity RI_, the unlabeled to labeled ion intensity

ratio for that standard or sample; thus, RW = b + mRI . The values of m

and b are obtained from RW and R_I values for the calibration standards,

and are then used for calculating RW for the sample. The product of RW

1

and the amount (mass) of glucose-U- C added to that sample gives the

concentration of glucose in the sample, in terms of mass of glucose per

gram of serum. The specific gravity of the serum is used to obtain the

glucose concentration in terms of mass per unit volume of serum.

2.1.11 ID/MS Analyses on Five Serum Pools

Single vials of bovine serum pools labeled A, CGSP, 2774, 2974, and

3074 respectively were warmed to room temperature (from —20 °C) , weighed

aliquots were taken from each, and these were spiked with labeled glucose

on days 1, 8, 15, 140, and 290. The spiked samples were allowed to

equilibrate, then freeze-dried and treated to convert the glucose into

DAG. Four replicate CIMS measurements of each DAG sample were obtained

within a two-day period. Later, two to four replicate EIMS measurements

were made on the same DAG samples. Only the CIMS data from days 1, 8,

and 15 were used for calculating the target values for the serum pools,

because the glucose concentrations in the stored samples were slowly

falling. The EIMS values were used only for confirming the accuracy

of the CIMS-derived results.

2.2 Hexokinose/Glucose- 6-Phosphate Dehydrogenase Method Using a Protein-Free Filtrate for the Determination of Glucose. The

Candidate Reference Method

The method with all details has been published [7]; hence, the

description here is a synopsis. Included with the published method are:

a) recommendations and precautions for sampling, handling, and storage

of glucose standard solutions and biologic specimens; b) specifications

for pipets and semi -automated pipetting and diluting devices, spectrophotometry instruments and cuvetts, other glassware, and reagent chemicals;

11
c) procedures for preparing stock and working solutions of D-glucose

(SRM 917a is used) , standard solutions of protein precipitating reagents

and buffers; and d) pre-assay test procedures for the components of the

enzyme reagents and for the enzyme reagent.

The method is performed either with glass pipets (pipetting

manually) or with a semi-automated pipetor/dilutor . With the former,

the sample volume is 1,000 yL and is only 500 yL with the latter — but

then the reagent volumes are proportionally less.

Vials of frozen serum are thawed and kept in a 25 °C water bath for

about 1 h before aliquots are removed. The aliquot of sample is mixed

with a tenfold larger volume of standardized barium hydroxide solution

and immediately thereafter also with a like volume of standardized zinc

sulfate. The mixture is centrifuged and placed in the 25 °C water bath.

A 1-mL aliquot of the deproteinized supernatant solution is incubated

with 5 mL of enzyme reagent, and after 30 min (when the absorbance is no

longer changing) the photometric measurement at 340 nm is made. Calibra

tion data are collected by running the series of working solutions of

standard glucose before and again after the samples. Tests for the

validity of the calibration data are applied before the sets of calibration data are considered acceptable.

3. Multilaboratory Study of the Candidate Reference Method

3.1 Round-Robin I (RR I)

Participating laboratories were supplied with six standard glucose

solutions, prepared enzyme and protein-precipitating reagents, and

samples of five unknown specimens. The materials had been prepared at

the CDC according to the directions given in the procedure for the

candidate reference method and were distributed by the CDC. The

laboratories analyzed the standard solutions in duplicate and the

unknowns in quadruplicate in a single run. Only manual pipetting was

used with the method.

3.2 Round-Robin II (RR II)

The participating laboratories were supplied by the CDC with six

standard glucose solutions, prepared enzyme reagent, samples of 5 unknowns and 2 controls, and with crystalline Ba(OH)2 and ZnSO^ . The

laboratories prepared their own Ba(OH)2 and ZnSO^ solutions as directed

in the procedure for the method. They analyzed the standard solutions

12
in duplicate and the unknowns in quadruplicate in a single run. Manual

pipetting and, alternatively, semi - automated pipetting were used.

3.3 Round-Robin III (RR III)

The participating laboratories were supplied by the CDC with

samples of 5 unknowns. Each laboratory prepared its own standard solutions and reagents, following the procedures in the candidate method.

The laboratories analyzed the standard solutions in duplicate and the

unknowns in quadruplicate on five separate days. Manual and,

alternatively, semi-automated pipetting were used.

4. Results and Statistical Analysis

4.1 ID/MS

The target values used for evaluating the candidate reference

method were obtained by the ID/MS procedure described in the present

report. The complete data obtained with the use of CIMS are shown in

Table 2. However, only the data obtained on days 1, 8, and 15 were used

for the target values, because of the instability of the glucose in the

stored serum over longer periods. A statistical analysis was made of

these data for the purpose of calculating the components of variance due

to within-day measurement - replication error and between-day (samplepreparation and measurement) error. Table 3 gives four standard

deviations for each of the five levels: s is the standard deviation

e among replicate measurements on the same DAG sample; s^ is the square

root of the component of variance due to sample preparation and measurement effects (different DAG preparations from a pool) ; s^, is the total

standard deviation (for single measurements made on different samples of

V2

2 s + s ; and, s is the standard error of the

e p av

average value over all replicate measurements and samples. Since the

average value at each level is the average of 12 individual measurements

s s rf~~

+ The EIMS measurements of the same DAG samples are summarized in

Table 4. Only averages and not the components of variance of these

measurements are given, because the number of replicate measurements

differed (from 2 to 4) from sample to sample. The precision of the

EIMS measurements appears to be similar to the precision of the CIMS

measurements

.

13
Table 2. Isotope dilution mass spectrometry results

(in mg/L) from CIMS measurements.

Pools

Day Pool A CGSP 2774 2974 3074

1 408 . 5 789.9 1366 1980 2945

407 .5 786 .6 1341 1981 2983

406.9 784. 5 1372 1986 2989

406.9 794. 7 1358 1975 2987

8 410. 8 786 . 7 1340 1979 2940

413.0 788 .6 1349 1982 2963

412.5 793. 7 1349 1978 3001

411.3 793. 0 1335 1978 2997

15 413.0 789. 1 1344 1988 3016

412.8 790. 4 1357 1987 3014

412.9 787 .1 1352 1982 3014

413.5 791. 0 1351 1982 3011

140 a 403. 7 775.6 1338 1957 2969

403.5 774. 8 1337 1951 2953

402 .8 775 .0 1338 1948 2969

403.8 775.6 1338 1953 2953

290 a 400. 5 778 . 5 1325 1940 2936

405.4 776. 7 1322 1923 2918

406. 7 781.6 1334 1945 2930

406. 8 785.1 1322 1938 Li? QJ J3 id?

cl Data obtained on days 140 and 290 were not used for the

target value calculations.

Table 3. ID/MS precision parameters 3

" (m£l/D • Pool Average s s - s e P av

Pool A 410.8 1.4 1.2 1.9 0. 8

CGSP 789. 6 2.1 1 . 7 2 . 7 1. 2 2774 1351.2 3.7 2 . 9 4.7 2 . 1 2974 1981 . 5 7.0 5.2 8.8 3. 8

3074 2988.

19.6 13.3 23.6 10 . 1

a Based on data shown in Table 2 for days 1, 8, and 15.

14
Table 4. ID/MS results from EIMS measurements.

Average concentrations (mg/L)

.

Pools

Day Pool A CGSP 2774 2974 3074

1 409.3 784. 5 1346 1982 2962

8 410.8 786.4 1338 1966 2993

15 413.3 787 .3 1360 1967 3013

140 405.4 775.6 1330 1947 2956

. EIMS a 411 .

786 .

1348. 0 1971

.

7 2989. 7

,

. CIMS a 410. 8 789.6 1351. 2 1981

.

5 2988. 3

cl Only data from days 1, 8, and 15.

Other results giving evidence of the accuracy of the ID/MS method,

are the following: a) Four, weighed, 1.5-mL aliquots of a dialyzed serum

were analyzed after adding about 1.5 mg (exactly weighed) of SRM glucose

to each aliquot and also adding to two of the aliquots about 0.03 g each

of D-fructose, Q- galactose , and D-mannose. The recoveries of glucose

were 100.4, 100.5, 100.2, and 100.3 percent for the four samples,

respectively; b) Two 2.2-mL aliquots (exactly weighed) of Pool GCSP were

analyzed after 1.5 mg (exactly weighed) of SRM glucose was added to each.

These analyses were begun on day 150 in the time sequence indicated in

Table 2. By assuming 100 percent recovery of the added glucose, the

glucose levels in the serum at day 150 was 774 and 776 mg/L. The average

CGSP values at 140 days from Table 2 is 775.2 mg/L and from Table 4 is

775.6 mg/L.

4.2 Candidate Reference Method

Tables 5-9 exhibit all the mult ilaboratory sample values obtained

in the three round robins of this study. These values were derived from

the calibration experiments (see below) , using a least squares linear

fit for the derivation of the blank and the conversion factor. The data

are presented as a series of two-way tables in which the rows represent

laboratories (and, where applicable, days within laboratories) and the

columns represent samples or pools. The laboratories are designated by

code numbers from 1 to 11. The code is used consistently for the three

round robins. The replicates obtained on a particular day, for a particular sample, in a particular laboratory constitute a "cell" of

the two-way table.

15
Table 5. Round Robin I - individual measurements:

concentration in mg/L.

Pools

1-1 1-2 1-3 1-4 1-5

Laboratory

1 429. 2 820 . 7 1101. 9 2143. 4 5856. 8

429

2 820

7 1101

9 2147 . 2 58568 429

2 813

1 1098 . 1 2147 . 2 5879 . 6

429

2 824

5 1094 . 3 2154 . 8 5853 . 0

2 464. 4 849. 4 1118. 2 2040. 9 5702 . 3

468 . 0 8313 1103 . 7 2055. 4 5651 . 5 457 . 1 831

3 1114. 6 2062 . 6 5684. 1 453

5 820 . 4 11073 20445 56660

3 415. 8 781. 1 1068. 0 2063. 2 5574. 2 419

5 7811 10531 2078 . 1 5555

6

419. 5 788 . 5 1060

6 20595 5555

6

427

0 773 . 6 1068

0 20744 5533 . 2

4 421. 7 809. 2 1111. 4 2111. 0 5753. 0

425

6 820

8 10998 20916 5799

5

3520 754 . 9 10106 2037 . 4 5660

0

359. 7 754. 9 1026. 1 20451 5784

0

5 407. 3 799 . 6 1071. 4 2074. 9 5705. 6

388 . 1 789

7 1062 . 1 20838 5695

0

4045 792

5 1062 . 1 20749 56950

406

2 790 . 1 1066

4 20554 5673 . 7

6 421. 5 799. 9 1080. 9 2100. 0 5614. 4 406. 5 799 . 9 1084. 6 2107. 5 5839 . 2

417. 7 781. 1 1077. 1 2081. 3 5558. 2 414. 0 769. 9 1092. 1 2096. 2 5876. 7

7 412. 2 778. 0 1063. 9 2069. 9 5667. 3

416. 0 778. 0 1079. 1 2081. 3 5644. 4

412. 2 778. 0 1067 . 7 2077 . 5 5655. 9

412. 2 781. 9 1063. 9 2085. 1 5499. 6

16
Table 6. Round Robin II - manual: individual measurements,

concentration in mg/L.

Pools

II -1 1 1

- 2 1 1 - 3 II -4 1 1 - 5 Laboratory

1 410

.

8 755

.

4 1321

.

3 2057

.

6 4513.3

4T 4 At 748 /to. 7 1 Jit q Z.7 0U 4t D^ . iX 4 c: 1 7 7 t D X D . D 41Ut rO 7/to. 4 R ? i1^74 jt t qy 7L 0^4 U D t . n at q i ft q D X o . y 4H J.1 RO . 1X

7/1 O /to. L7 1 ^ ^ 7 7L

? n ft /i Z, U D 4 . Qy

/I C 1 7 7 4 D X D . D

2 408

.

7 760

.

4 1377

.

8 2063 . 4 4540 .

j nu 7 ft ^ / O D . cD

1 7 7/1 X D / t . 7L z, U o 4 . Q /l Q 1 A Q 4 D X 4 . y 4 7 ^ u

7 7 4 / / t . 00 X D o D . u? n /i q n

U

/i tr 7 a. c 4 D D 0 . D HUD. a n r iX 7/ 7/ Ro • t

1IJOJ, u L X u z.

.

qyA Q 7 7 q t D D z. . y

3 397 . 3 739

.

3 1339

.

7 2038 . 3 4501 .

6

X Q 7 7D 7/ jy , 7D J.1 D^ D^ L7 . t Z, U Z. D . 7/ t D U X . D

d. n n q 7^0 / D U . 7 1^17 _L D X / . qy L7 0^4 u D t . 7/ 4^10 t d x y . Ro Q ^ Au 7^0 Z.7 1J1 ^ 7L J5 X 7Zi U0 41t X . qy 41 41 Ro 7 1

/ . X 4 440

.

7 791

.

5 1357 . 1 2119 . 6 4442.8

444 t 4- t • 7 66 4 7/ 7Z. 0U 1X D^ . 7i 4t 4^7 t D 1 / . X 4^7 1X Au X1 D^ D^ RO . nu 7Zi 0U X1^D . 7/ 1 1 D / . X 4t L? Qz> . Qy 7/ 6?uz. qy 1 ^ ^ q 7L zuyu ? n o o . y

a s n x ft t D U D . D

5 408 . 4 760

.

9 1351

.

9 2077 . 8 4498 . 0

4 n q cD 7/ 6u 7/ . AU 1X D\ D^ 7Z, . AD 7 0 ft ^ 7D at

c; n i ft D u X . D

4 1 n L 7^6 / D U • qy X1^44 D 1 1 . OO 7 0 ft 1

Z. U O X . 0o 4^1? t d x z, q . y 4 0 7 A 7/ u66u . qy ij1 ^ oy^q i 1X Z.7 0u 7/ 7/ . 0O

zi n 7 7 t D X Z . D

6 372 . 9 724 . 7 1339

.

2 2100

.

6 4547.2

D ftU QV . i 7?0 Ro 1ij"\ jy\ q i L7 Z,7 0U RO D . 7z

/lt D

c o

o D

c

. qy JUJ. 9

Z 7/ ?Z RO . CD X1 D Z,7 7/ . f.U 7 0 7*. L U / D . Au t D D o . o ODD. L 7/ 7£ 4t . rD

i : X D D D . 7D 7 n R q u/I cor q t D o D . y

* a

6 388

.

9 753 . 8 1318

.

0 2089 . 1 4425.0

Ann 4UU . 7L 7/DO. X R oo X D L D . cD iyy j . 1 /1 :7q c t D / D . D Q ? 7

/ 7/Do. ^ R 0o X D Z, X . 0o 7z 0u ftu z.7 . Q0 4t D^ DX 1/ . qz> D O X . A4

7/Do. ^ R Qo1

"5 7 Q D 700? z, u y z,

.

qy

/| T7 7 Q t d d / . y

7 413. 1 749. 6 1344. 8 2051. 0 4376.

402. 1 760. 7 1348. 5 2002 . 9 4365.3

405. 8 764. 4 1315. 2 2069. 4 4383.8

420. 5 764. 4 1333. 7 1999. 2 4413.4

8 471. 1 810. 6 1401

.

9 2073. 4 4549 . 3

425. 3 795. 4 1340. 9 2088. 6 4564.

5

455. 8 806. 8 1409. 6 2096. 3 4610.

3

429. 1 864 . 0 1352 . 3 2035. 2 4511 .

cL Different spectrophotometers.

17
Table 7. Round Robin II - semi - automated : individual

measurements, concentration in mg/L.

Pools

Laborat o ry

T I -

J. J. x 1 1 - 2 1 1 - 3 1 1 -4 TXXT - «J5

A 1 O 412

1

H £L 7 763 . 21 7 7 H 1337 . 6o r\ *7 o

2 0 7 2. 8 4498 . 1

A f\ O 4 08. 4n r c\ 7 59. 6 1366 . o8o n £ r 2 065. 5 4516

4 412 " X u • 1 7 59 6 1337 . 6 2065 . 5 4516

4T 412. 1 759 . 6 1337. 6 2065! 5 4516. 4

4 04. y / 5o . y loUo . o8 Z U Z 1 . 0 4510 . o8

4 U 1 . o / 0 u

r5 1 J Z 1 . A4 Z U o 0 . 7o 4 5 Z 5 . rb 401

3 749

5 1317

7 2029

o 4514 . 5 404. 9 756. 9 1325 . 1 2047. 3 4532 . 8

A A 1 4 4 Z . 6 / 6 / . 1 l j y oio Z U / 5 . r5 44 /0 . / 413

7 7 53. 73

~\ 1 1 A 1334 . y 2 0 50 . 2

4 r c a 4564 . 6

4137

"j r 7 56. 9 1342 . 1

o r\ 7 o 203Z . 2A r 7 c\ 4 539. 3

410. 1 760. 5 1338. 5 2071 . 9 4546. 6

420. 5 754. 7 1323. 9 2003 . 3 4416. 1

420. 5 762 . 0 1320 . 2 2036. 4 4441. 8

413. 1 754. 7 1323. 9 1984. 9 4460. 2 416. 8 758. 3 1327 . 6 2008 . 2 4452. 8

18
Table 8. Round Robin III - manual:

concentration in mg/L.

individual measurements,

Pools

III- 1 III - 2 III- 3 III- 4 III- 5 atory Day

1 1 412. 1 780 . 9 1336. 5 1948. 0 2940. 0

408

.

5 789 . 8 1340 . 1 1948

.

0 2940 . 9

1 2 416. 3 786. 6 1336. 5 1940 . 4 2928 . 9

412 . 7

f-j r\ f 786

.

6 1336

.

5 1940 . 4 2928

.

9

1 3 410

.

8 7 83. 5 1328

.

3 1937 . 7 2923

.

3 410. 8 780. 0 1324. 8 1934. 1 2919. 7

X AH- X1 O^ • ? 7 84 q 1i J

^

JUn . cj 1 QAD 7

/ ?£ qy £? 7/ • ft0 415. 2 784. 9 1334. 1 1933 . 5 2927 . 8

1X

cD A 1 ^ 71 Q

0 i j ji

.

Q i

j. q a n y h- u . tX ? q l i l y x x . i > 413. 7 782 . 8 1331

.

0 1932 . 9 2921

.

8

2 1 402. 5 800. 1 1363 . 5 1959. 9 3005. 6

420 . 0 807 . 5 1374

.

5 2048 . 3 3005

.

6

2 2 406. 0 800. 9 1364. 0 1930. 7 2950. 8 417 . 0

T A A

7 89 . 9 1364 . 0 1949 . 0 29 50. 8

2 3 399

.

1 7 81

.

4 1321. 9 19 7 2. 6 2935 . 8 391. 7 7 74. 1 1336. 6 1910. 1 2928. 5

L

A J v o .

1 7 Q ? U ijji < cD l q n 7 un ?L q 1 ft ZJ X O . 7

385. 7 767

.

0 1320. 5 1910. 7 2918. 7

L

cD a ? n u 7/ Qy Qy . i j ji i 0o 1 Q A 1 ?La ? qn q Qo 420. 0 795. 7 1348 . 1 1944. 8 2935 . 6

3 1 424. 3 817. 0 1348. 2 1925. 7 2927 . 5 428 . 4 799 . 2 1358

.

9 1940 . 0 2920

.

4

3 2 411. 9 795. 4 1345. 3 1964 . 0 2962 . 5 408

.

3 788 . 1 1370 . 6 1953 . ] 2948

.

]

3 3 424

.

3 802 . 7 1352 . 5 1955 . 9 2930 . 5 431. 4 777. 7 1341. 8 1941

.

6 2916. 2

7J

/i 4 u ftout0 A • H i ^ a n f

; u. x1 Qy 1x Qj • qy ? q ? 7

c.J 408 . 6 790. 0 1333. 4 1934 . 3 2949. 1

o

c ad? 7.J 7/ ft6 o u . O0 Q ix qy nv m-A . AU ?L ftO qZ) 71 . QO

405. 8 772 . 5 1324

.

5 1901

.

0 2905. 0

4 1 397. 4 823. 5 1305. 1 1990. 5 2909 . 3 390

.

0 849

.

4 1305 . 1 1975 . 6 2994 . 4

4 2 416. 6 815. 6 1395. 9 1940 . 0 2970 . 1

434

.

7 822 . 8 1374

.

2 1987 . 2 2966

.

5

4 3 430. 3 813. 1 1352 . 7 1961. 5 3018. 7 415. 7 805 . 8 1334. 4 1961

.

5 2960. 4

4 4 435. 4 802. 6 1358. 7 1958 . 5 2943. 6

417. 2 806. 2 1355. 1 1958 . 5 2958. 1

4 5 414. 7 782. 4 1330. 3 1982. 8 2981. 4

432 . 7 789. 6 1337. 5 1975 . 6 2927 . 3

19
Continuation of Table 8.

Pools

III- 1 III- 2 III- 3 III- 4 1 1 1 - 5 at ory Day

5 1 414. 8 787. 2 1339. 5 1993 . 4 2943 .

422. 3 804. 5 1338. 4 1947 . 3 2958.

5 2 410. 8 7812 1328 . 0 1915 . 1 2917 .

413. 0 782. 0 1347. 1 1936 . 7 2960.9

5 5 411. 9 778. 2 1336. 5 1930. 1 2911.4

408. 0 773. 9 1338. 7 1898 . 8 2938 .

5 4 415. 5 782 . 6 1306. 3 1960. 2 2930. 1 410

5 784

0 13316 1938 . 5 2955.5

5 5 414. 6 788. 2 1341. 4 1960 . 3 2929. 5 413

9 786

4 13414 1942 . 2 2953.0

6 1 424. 6 828 . 5 1380 . 5 1980 . 8 2951.

420. 9 810. 0 1369. 4 1984 . 5 3003.4

6 2 427 . 9 795. 1 1355. 1 1947 . 9 2929 . 7 431. 5 791. 5 1362 . 4 1958. 8 2977 .

6 3 440. 1 826. 7 1360. 6 1968. 1 2888.6

425. 4 808. 3 1342 . 2 1979. 1 2918.0

6 4 399. 4 796. 0 1379. 6 2023. 1 2958.4

410

7 777

3 1364 . 7 1978

2 2950.9

6 5 416. 7 794. 5 1366. 8 1975. 8 2958 .9

420

4 790

9 13595 1986 . 8 2977.2

7 1 405. 4 792 . 3 1347 . 4 1942. 0 2930.

416. 2 803. 0 1351. 0 1934 . 8 2944.8

7 2 409. 1 805. 6 1359 . 1 1935. 9 3012 .

416. 3 823 . 4 1348. 4 1957. 4 2939.

7 3 399. 8 794. 2 1326. 6 1936. 8 2886.6

408. 7 778. 5 1303. 6 1928 . 0 2900 . 7

7 4 383. 1 772 . 6 1317. 7 19217 2911.

397 . 4 7 74 . 4 1314

1 1918 . 1 2900.9

7 5 336. 0 741. 0 1302 . 5 1876. 7 2873.8

3306 712 . 2 1316

9 1865

9 28 72.0

8 1 4398 8041 1336 . 3 1964. 8 2982.

427. 3 800 . 6 1336. 3 1939. 8 2939.8

8 2 426 . 5 802 . 6 13 57. 8 1959 . 5 2940.

426. 5 813. 4 1366. 7 1991. 7 2955.

0o x 7 ft 1 A o 1 O . Q 1 z x ? Q I J .) L . 7 ^ n o ? ^ 426. 4 791. 8 1343. 6 2063. 8 2966. 7

8 4 426. 1 785 . 4 1354. 6 1966. 9 2939. 5 451. 0 796. 1 1354. 0 1959. 4 2966.

8 5 419. 0 792 . 7 1337. 2 1967. 1 2974. 2 415. s 803 . 4 1365 . 6 1952 . 8 2949.3

20
Table 9. Round Robin III - semi -automated : individual

measurements, concentration in mg/L.

Pools

III -1 III-2 III-3 III-4 III-5

Laboratory Day

1 1 419. 0 794. 1 1329. 7 1938. 6 2929 1

4 2 3. 6 1 94 . 1

T 7 7 7 133/ ut n 7 o 19 38 0 2936 4

1 2 412. 6 788. 9 1340 6 1932 5 2915 3 412. 6

•7 O O

7 8 8. 9

1 7 /I A 1340 . 6 1936 2 O A A f) 2908 0

1 3

J 1 T 412. L 7 0 0

/ 8 o . 0

T 7 O "7 13 2 7 9 1926 3 2914 9

419. 5 791. 6 1331. 6 1926. 3 2914 9

1 4 410 •"t _L W 4 4 78 5 4 _L1 .J339mj +7 0 1918 -L Zf X O

nU ? Q 0 8

414. 9 781. 8 1324 4 1918 0 2919 5

X •J 415 QJ 778 7 J.1328 *J Li \J nu X19J L21J. nu 791 L, Zf X 7/ 7

418. 6 778. 7 1328 0 1921 0 2903 1

3 1 427. 8 795. 0 1325. 7 1932. 7 2932 4 4 0 9. 6 7ni / 91

.

3 1340 . 2 10 7/ 19 36 4

O A 7 *<1 2936 0

3 2 405. 2 791. 2 1344. 7 1941. 9 2932 5

j no 4 0 8. o0

7 O 7

1 8 3 . 9

1 7 7 T 133 7. 4 1931

.

0 2921

,

5

7 3 4 U 4 . A4 Z1 7. 7 A 1326 . 4 1921 . / o n o 7 292 / 1 404. 4 771. 0 1326. 4 1921

.

7 2923 5

4 426 "T L* \J « 7 771 / / X \ Q 1X *J320Li \J i 1909 7. 291 ? 2

412. 2 771. 9 1320. 6 1916. 5 2926 7

•J •J 40 2 8 7 7 8 Q-7 1315 R X192Zf Li %J5 8o 297 L* Zt d 2L, 424. 7 778. 9 1330. 4 1940. 4 2933 8

6 1 420. 0 798. 1 1355. 9 1966 2 2939 6

< no 4 0 o . / 7 O /I / y 4

.

4 1 b b b . 9

i o a n 1969 . n

y

o n 7 n 2939 6

6 2 410. 4 787 8 1359 6 1957. 5 2932 8

/I 1 /I 414 . 1 ion 787 o8i 7 r n 1359 6

1 n / i 1961

.

2 Tata 2929 1

6 3 416

.

4 795 . 0 17// 1366 6 1964 2 i o c\ r* 289 5 9

401. 5 791. 3 1366. 6 1930 8 3007 5 AU

At 4 ? ^ Q 8o 08\j o i 7 1 "^74 X1 Q70 Zs 1 u

t:J 7QQ2 7 423. 9 812. 4 1363 2 1970 5 2981 4 Au D 40Q T- \J Z> * 791 7 113J J59J

7. X19Z* 791 z* « 2967 L\ Z/ \J / 417. 1 799 3 1359 3 1960 s 2940 8

7 1 405. 4 797 2 1306 2 1958 0 2902 7

379

.

3 764 3 1320 8 1910 4

i a r 7 2953 9

7 2 426. 7 802 6 1387 7 1958 6 2955 0

430 2

T O O 7 88 4 1359 3 1923 1 2912 r5

7 3 445 3 820 9 1342 , 5 1914 1 2957 0

427 8 799 4 1324 6 1932 0 2900 2

7 4 411 4 777 3 1358 .4 1914 .3 2900 7

418 .6 780 . 9 1329 . 7 1925 1 2925 S

7 5 396 . 1 784 . 4 1323 .8 1917 1 2931 1 424 . 8 780 . 8 1341 . 8 1913 . 5 2895 2

21
Continuation of Table 9.

Pools

T111" T T ±

f T T

j. 1 1

- 9ZT111- T T 7JT111- T T A4T111- T T 5 Laboratory Day

Qoii 40 / . 9L oUZ q n ? . 11 777 1 J J J . 9Z1 Q77 iy j /

z

o o o a z y z D . z 435

1 798 . 6 1351

0 1939 . 0 2933 . 2

Qo L A ? X 1 1 v 4 , c0

1 X/l Q ± j 4 y . QO

1 O 9 Q i y z o . 0

1(1/1 7 zy4o

oO

426 . 6 783

7 1360 . 7 1955

7 2954 . 6

8 3 384. 7 813. 9 1303 . 0 1940 . 5 2931. 2

395 . 4 742. 4 1296. 7 1901. 2 2916. 9

8 4 432. 8 798. 0 1342. 2 1958 . 1 2967 . 7

432. 8 801. 6 1342 . 2 1961. 6 2946 . 2

8 5 425. 1 782 . 6 1333. 2 1919 . 5 2934 . 9

460. 8 793. 3 1343. 9 1951. 7 2938 . 5

9 1 418. 5 816. 1 1356. 9 1947. 9 2932 .

447 . 2 787 . 4 1339 . 8 1947 . 9 2950.7

9 2 408. 7 785. 3 1341. 3 1947 . 4 2923.0

408. 7 785. 3 1341. 3 1947. 4 2923.0

9 3 419. 3 796. 4 1342 . 4 1949. 5 2930.1

422. 9 796. 4 1346. 0 1953 . 1 2937.

9 4 416. 6 782. 2 1361. 5 1951

7 2951.0

420. 2 778. 6 1405. 0 1958 . 9 2925.6

9 5 402 . 5 780 . 2 1337. 7 1913. 3 2920.5

420. 5 780. 2 1348 . 5 19313 2920. 5

10 1 426. 4 789. 4 1346. 8 1940 . 8 2930.9

415. 4 793. 1 1346. 8 1940 . 8 2930.9

10 2 421. 0 792 . 5 1340. 5 1925. 3 2907 . 2 428. 4 785. 1 1344. 2 1925 . 3 2918 .3

10 3 414. 4 790. 3 1328. 8 1925 . 1 2890.0

414. 4 786. 7 1343. 2 1925 . 1 2890.0

10 4 415. 7 778. 5 1324. 5 1932 . 8 2918. 5 426. 7 789. 5 1324. 5 1932. 8 2911.2

10 5 428. 5 800. 4 1349 0 1949. 1 2932 . 2 446. 9 800. 4 1349. 0 1949. 1 2932 . 2

11 1 411. 3 789. 3 1343. 7 1952 . 1 2945.6

407. 7 803. 7 1343. 7 1944. 9 2949. 2 11 2 421. 2 790. 9 1350. 8 1950 . 2 2933.6

414. 0 787. 3 1340. 0 1935. 8 2926.4

11 3 409. 4 798. 7 1339 . 3 1941. 3 29 5 0.4

409. 4 805. 9 1339. 3 1926. 8 2932 .4

11 4 407. 5 793. 1 1335. 7 1939. 1 2931.0

421. 8 782. 4 1332 . 2 1932 . 0 2931.0

11 5 411. 3 786. 5 1343. 8 1911

9 2930.

414. 8 797. 2 1336. 7 1929 . 7 2908.6

22
The method of analysis applied to each two-way table is the

"weighted linear model" analysis [17,18], where, however, the method for

the calculation of weights was a modified version of that given in [19].

The rationale underlying this method of analysis is simple. Essentially,

a plot is made of the results obtained by a particular laboratory on a particular day versus the corresponding target values or, in a study of

precision, versus the average values obtained for each pool by all

laboratories. Ideally, the points of such a plot would be expected to

fall on a straight line going through the origin and of unit slope. In

practice, the line may deviate from this ideal line both in location and

in slope. Thus, each day- laboratory combination is characterized by an

intercept and a slope. The variability between the lines representing

the various laboratory-day combinations is the systematic betweenlaboratory (and/or day) variability. In addition to the systematic

component of variability, one must consider another component resulting

from the scatter of the observed points about the lines fitted to them.

This scatter is only partly explained by the replication error, i.e.,

the variability among replicate measurements (same day, same laboratory).

The portion of the scatter that is not explained by the replication

error is referred to as lambda variability [19] and constitutes the

random part of between- laboratory (and/or day) variability. When

combined with the systematic component of between- laboratory (and/or day)

variability, it yields the total between- laboratory (and/or day) variability. The combined component is then partitioned into two parts:

between-days within- laboratories and between- laboratories . To summarize:

at each level, the imprecision is made up of three components: 1) the

replication error (within-days within-laboratories) , 2) the between-days

within- laboratories component, and 3) the between- laboratories component.

One important aspect of this analysis must be mentioned. In each

of the individual round robins, five samples are used as representatives

of five different levels, covering a wide range of concentrations of

glucose, and the statistical analysis used a smoothing procedure in

order to express each component of variability as a function of level

only. In other words, the idiosyncrasies of the individual samples are

considered unimportant, since each sample merely represents a particular

concentration level and is not an object of specific interest.

23
4.2.1 Calibration

The calibration curve for the conversion of optical absorbance

into glucose concentration is based on absorbance measurements made

after performing the analysis on aqueous glucose solutions of known

concentration. The protocol for RR I prescribed a specific calculation

procedure for the determination of the blank and conversion factor.

However, the least squares method can be used for fitting a straight

line to the absorbance-concentration points, and the least squares

method was subsequently adopted in the final version of the protocol.

In the present report, we include for RR I a comparison between the

concentration data for the five unknown pools derived from both methods

of calibration calculations. A similar comparison for RR II and the

corresponding data and calculations are available, but are not provided

in this report. A detailed comparison of all calibration curves

obtained in RR III in which each laboratory performed calibrations on

each of five days, is included in this report.

4.2.2 Round Robin I Each calibration line is based on the linear relation: A = B + k*C, where C is concentration in mg/L, A is absorbance, B represents

the blank and is also the intercept of the calibration line, and k is

the slope of the calibration line. For the derivation of concentration

A- B

C from absorbance measurement A, we write: C = —^— = F(A-B), where

F = . F is the conversion factor of absorbance (corrected for blank)

to concentration. Values for B and F, for the two calibrations in each

laboratory, obtained by the least squares fitting procedure, are shown

in Table 10. It is apparent that with the exception of laboratory 7,

the two calibrations by each laboratory are in excellent agreement with

each other. We discarded the first calibration curve for laboratory 7,

and the concentration values given in Table 5 for this laboratory were

based on the data from the second calibration curve exclusively. For

all other laboratories, the conversion to concentration was based on

the averages of the blanks and conversion factors of the two curves.

Table 11 exhibits the averages obtained by both methods of calculation.

It is seen that the two methods of calculation give similar, though

not identical, results.

24
Table 10. Round Robin I - pre- and post - analys is calibration

results by least squares method.

Lab

Calibration Intercept (B)

Time abs a Conversion Factor

mg/L/abs

1 Before

After

.056

.058

3770

3830

2 Before

After

.052

.052

3610

3610

3 Before

After

.064

. 065

3730

3730

4 Before

After

. 061

.057

3890

3860

5 Before

After

. 096

. 097

3570

3530

6 Before

After

. 061

.062

3750

3750

7 Before

After

.074

. 077

3980

3810

abs == absorbance units

Table 11. Round Robin I - comparison

calculation of calibration

of two methods for

parameters

.

Least Squares Method RR I -Protocol Method

Lab

B

abs a

F

mg/L/abs

B

abs

F

mg/L/abs

1 . 057 3800 .055 3780

2 .052 3630 . 051 3640

3 . 064 3730 .062 3710

4 .059 3870 . 055 3820

5 . 097 3550 .095 3530

6 . 062 3750 . 064 3770

7 b

. 074 3980 .076 4010

7 C .077 3810 .078 3810

a abs = = absorbance unit

.

Before sample measurements.

After sample measurements.

25
Two tables can be constructed from the data in Table 5: One containing the 35 cell -averages and the other the 35 standard deviations

within cells. The latter forms the basis for the calculation of the

replication error at each of the five levels of concentration. The

table of cell-averages forms the basis for the between-laboratory

comparisons. The calculations were carried out with the data in Table

5 and with the analogous data obtained by using the RR I protocol method

for the calculation of the calibration parameters. These results are

shown in Table 12, where parameter s q

is the standard deviation of

replication error (i.e., the standard deviation among replicate analyses

made at the same time, in the same location, on the same pool); parameter

s^ is the square root of the component of variance due to laboratory - to

Taboratory variability; and parameter Srp is the square root of the sum

V~2

2

s g

+ s^ . The parameter

Srp thus represents the standard deviation among single measurements

oFtained on the same pool in different laboratories. For all practical

purposes, the two methods of calculation of calibration factors are

seen to give identical results, and henceforth only the results based

on the least squares fit of calibration data are discussed. It is

important to note that all sets of standard deviations are functions of

the concentration level of the sample, and to recall that the values

shown are "smoothed" values, in which all effects of the samples due to

factors other than their concentration levels have been eliminated.

Later in this report, a comparison of all three round robins is made in

terms of coefficients of variation as well as standard deviations.

Table 12. Round Robin I - precision parameters and sample

concentrations in mg/L, calculated by two methods.

Least Squares Method Protocol

Pools Average s e fx

Avera'g e s e fL

1-1 420.5 10 22 24 421. 2 10 21 23

1-2 800.1 14 20 25 798 . 7 14 21 25

1-3 1082.9 18 20 27 1079 . 9 17 22 28

1-4 2092.

29 29 41 20818 29 30 42

1-5 5714.3 69 87 111 5680. 1 69 83 108

26
Table 13 was prepared to provide a more detailed picture of the

laboratory -to- laboratory differences in RR I. It shows the extent by

which each laboratory average differs from the overall average of all

laboratories at every concentration level. The deviations are

expressed as percentages of the overall average at each level.

Table 13. Round Robin I - percent deviations of individual

1j. d^hoTP L/ u i ci fu uOTVi y CIV -L CL Li »_) f rnm cx 1 1 J_ 1 L7111 d _L _L

r KJ VJ _L C5

Laboratory

1-1 1-2 1-3 1-4 I -5

i

_L 2 5 5 2 86 1 fi 7 J us L q n

2 i o . o y 4.54 2.97 -1.58 - . 35

3 .46 -1.99 -1.53 - .72 -2 .48

4 -6. 88 -1 . 50 -1.57 - .60 .93

5 -4.06 - .50 -1.24 - .55 .07

6 - .86 -1.16 . 44 .60 .46

7 -1. 29 -2.25 - .95 - .25 -1 . 39

Average

all labs

mg/L 420.5 800. 1 1082.9 2092 . 0 5714 .3

4.2.3 Round Robin II

A statistical analysis was made separately for the data from the

so-called manual and semi -automated methods. Table 14 lists the parameters B, F and standard deviation of fit s^ (generally referred to in the

statistical literature as standard error bT estimate) for each calibration

curve. The calibration data consists of two sets of single measurements

for each of 7 solutions. The least squares fit is therefore a linear

regression of absorbance on concentration made on 14 experimental points.

It is seen that, with the exception of laboratory 6, all calibrations

were made with comparable precision (average standard deviation approximately 0.0030 A, or approximately 10 mg of glucose per liter per point).

The calibration precision for laboratory 6 (which made measurements with

two different spectrophotometers) was poorer, with a standard deviation

of the order of 50 mg/L per point. The' results reveal little difference

in the precision obtained in calibration between the manual and the

semi -automated methods.

27
Table 14

Lab

lMb

2M

3M

4M

5M

6MC

6MC

7M

8M

Round Robin II

B

abs a

- calibration results.

. 052

.053

.059

. 057

. 097

. 084

. 084

. 051

. 060

mg/L/abs

3630

3590

3640

3580

3570

3860

3760

3700

3810

abs

.0018

.0043

.0026

. 0047

.0030

. 0136

. 0162

.0043

.0032

ISA

3SA

7SA

8SA

. 053

.054

. 057

. 056

abs = absorbance unit.

} M = manual.

'Different spectrophotometers

*SA = semi -automated

3660

3660

3610

3670

. 0032

.0018

.0014

.0034

Table 15 lists the precision parameters s , s^, and derived from

the data of Tables 6 and 7. Examination showsthat both for within- and

between- laboratory comparisons, the precision of the serum glucose values

determined with the semi-automated method is somewhat better than with

the manual method. Tables 16 and 17 exhibit, at each concentration

level, the percent deviations of individual laboratory averages from

the al 1 - laboratories average for the manual and semi -automated method,

respectively

28
Table 15. Round Robin II - precision parameters for

calculated sample concentrations (mg/L)

Manua

Ave! d g t: fL fl

1 1 -

409. 7 12 25 28

1 1 - 2 762 . 0 14 22 26

1 1 - 3 1343.9 17 20 26

T11-4 T A 20 24 31

T T - ^ 4 50 5 7 32 59 67

Semi -Automated

II-l 413.0 8 6 10

1 1 - 2 758.4 9 9 13

1 1 - 3 1334.9 12 14 18

1 1 - 4 2041.6 15 21 26

1 1 - 5 4501.3 25 42 49

Round Robin

laboratory

II - manual:

averages from

percent

all - labs

deviations

average

of

Pools

II- 1 1 1 - 2 1 1 - 3 1 1 - 4 II

Laboratory

1 .94 -1.57 -1.34 - .28 . 19

2 1.28 1.67 2.72 .69 . 56

3 -3.03 -1.61 -1.12 -1.27 - .07

4 6. 90 1.52 - .44 - .01 - .90

5 - .20 .14 .61 . 48 .01

6 a -10.15 -4. 78 - .64 1 .28 1.42

6 a

- 4.61 -2. 56 -1. 58 - .04 . 30

7 . 17 - .30 - .62 -1.46 -2.68

8 8. 70 7. 50 2.40 .61 1. 18

Average all

labs (mg/L) 409. 7 762.0 1343.

2060.8 4505.7

Different spectrophotometers.

29
Table 17. Round Robin II - semi - automated : percent deviations

of individual laboratory averages from all-labs

average

Pools

Laboratory 1 1 -

1 1 - 2 1 1 - 3 1 1 - 4 1 1 - 5

1 - .44 . 28 .75 1.25 . 23

3 -2.40 - . 32 -1. 28 - . 39 .48

7 1 . 70 . 16 1.35 . 78 . 64

8 1.14 - .12 - .82 -1. 63 -1.30

Average all

labs (mg/L) 413.0 758 . 4 1334.

2041.4501.3

4.2.4 Round Robin III

In this round robin the manual and semi -automated methods were

performed in seven laboratories. Each laboratory analyzed the five

samples in duplicate on each of five days, resulting in 35 analytical

runs for each method. New calibration curves were made each day. Each

calibration consisted of measurements made on seven standard solutions,

run before and again after the serum samples. The manual and the semiautomated methods were calibrated in the same way. All the data were

used for the statistical analysis although the calibration curves for

eight runs by the manual method and three runs by the semi-automated

method did not meet all the criteria for acceptability.

Tables 18 and 19 are summaries of the calibration results for the

manual and semi - automated methods, respectively. For each laboratory,

the calibration parameters B, F and s^ were calculated for each of the

five days. As in the previous roundrobins , the precision of calibration by the manual method, from the standard deviations of fit, is quite

uniform over all labs, with the exception of laboratory 6, for which the

values of s^ are somewhat higher than for the other laboratories. It is

also apparent from Tables 18 and 19 that systematic differences exist

between the calibration lines of the various laboratories, with much

smaller variations existing between calibrations made in the same

laboratory on different days.

30
Table 18. Round Robin III - manual: calibration results.

Lab Day 1 Day 2 Day 3 Day 4 Day 5

ix Odd 044 044 • UH J 0 4 4

Pr 56 o n j5^90 j .7 u 5 q n J JoU

S fI .0013 .0012 .0014 . 0017 . 0009

7L D 04R 049 046 • 0UJO58 . 0U JU6

P 36R0 3660 56R0 JU56 7/ u0 56R0

J.

. 0042 .0042 . 0052 . 0031 . 0029

J D • nU Jrro 0 50 UJO o R . 060 u u u • 06U U 7L P j5^70 j / u 36 7 0 5 5 7 0 7Ann JJUU

I .0036 .0019 . 0022 . 0016 .0019

A4 . Uo J50U I V J J . U J L 06 4 4 U JO

pr 0^ 7/ 1J. nu \(\ ^0 7 n OD'tU jOUU

S fI .0044 .0061 . 0052 . 0078 .0036

CJ D 044 046 • 04U H J5 . 04U *T RO 04 7 P J3660 U U U 3660 3 590 36 7 0 »_J 3670 W i- U

S fI

. 0020 . 0060 .0035 .0094 . 0045

0 D i U j J 0 ^ 4 , 0^7 U JL . 0U 61O X • 0UJO^ R pr 77i n

j / 1 u "^640 ^6 r o ^740 J56u 7/ u0

r

. 0066 . 0061 .0101 .0067 .0030

7

/ 0 5 7 0 59 . 06u u 0u . 0U J59J • 076 u / u pr J JOU J3 J5 70/ U J5 JtU 540 J5 J5 70/ u J56u 0u 0u

cs £

. 0022 . 0069 .0105 . 0062 .0192

8 B .043 .041 .042 .046 .041

F 3570 3580 3580 3560 3560

s , . 0042 . 0028 .0065 .0022 . 0036

31
Table 19. Round Robin III - semi- automated

cal ibrat ion results

T ah Do. y ± liny l UcLy 3 jjay 4 Day b

1 B . 044 . 045 .045 . 045 .045

F . 3670 3650 3650 3640 3640

s £

nm n o m n n n i f\ n n i 7 . U U 1 L . U U 1 4

5 B . 063 .058 .052 . 063 . 062

!-' 3640 3640 3630 3630 3650

S

f

. UUIO n m i

. U U 1 3 . U U o u

6 B .050 .055 .055 . 055 .061

F 3740 3740 3710 3770 3780

cs f

0 ft ^ A , uutu o n 4 n niUlii 7/ n n j 7 . UU4 i . u U b 4

7 B . 080 .059 .056 .059 .060

F 3660 3550 3570 3590 3600

S f, uuyy n n a n n a ^ . UU4 j . U U D 1 . U U b4

8 B . 049 . 048 .042 . 039 .041

F 3560 3610 3580 3580 3580

cs £

. uut j , UU J J n n ? a n n 7 7 n n 7 ^

9 B .035 . 041 .044 .044 . 048

F 3580 3590 3590 3610 3600

s £

n n a f\ n n ? 7 n n 7 . U U L O . nn?Q uuzy . u U 4 0

10 B .053 .050 .055 .056 .054

F 3670 3680 3610 3660 3680

cS

f

. nuutu DAD n n ^ ^ n n 7 A . UUZ4 . U U L 1

11 B .055 .056 .053 .056 .055

F 3660 3590 3660 3570 3570

S

f

.0036 . 0039 .0030 . 0025 .0026

Table 20 lists the precision parameters for the calculated concentration values for both the manual and semi - automated methods, at each

concentration level. The parameters are: s e , s q/l> the square root of

the component of variance due to day-to-day variability within

V2

2 2 s e

+ S D/L+ S

L

' Thus, s^,

represents the standard deviation among single measurements made on

samples of the same pool in different laboratories. Percent deviations

from the all - laboratories averages were also calculated for each

laboratory at each level. These results are given in Tables 21 and 22

for the manual and the semi-automated methods, respectively.

32
Table 20. Round Robin III - precision parameters for

calculated sample concentrations (mg/L)

Manual

Pools AvPTfl Q P cj e

S D/L S

L

cO rp Fool A 415.4 7 14 9 18

CGSP 793.4 9 15 9 20

27 74 1343.3 13 16 10 7 3

2974 1950.5 1

17 13 71

3074 2941 . 8 23 19 18 34

Semi -Automat ed

Pool A 417. 9 9 9 0 12

CGSP 789.9 10 9 0 13

2774 1341.

12 8 6 15

2974 1937.5 13 8 11 19

3074 2930.

16 6 18 25

Table 21. Round Robin III - manual: percent deviations

of individual laboratory averages from all-labs

average

Pools

Laboratory

Pool A CGSP 2774 2974 3074

1 - .13 -1. 04 - . 77 - .56 - .50

2 -1. 90 - .33 . 33 - .16 .14

3 . 40 0 .08 - .84 - .45

4 1.22 2.22 .12 . 96 . 72

5 . 04 -1. 09 - .62 - .42 - .07

6 2.01 1. 06 1. 55 1. 43 - .32

7 -5. 61 -1.73 -1. 08 -1.47 - .83

8 3.98 .91 .39 1.07 .68

Average all

labs (mg/L) 413. 4 793. 4 1343.

1950 . 5 2941 .

33
Table 22. Round Robin III - semi -automated : percent deviations of

individual laboratory averages from all-labs average.

Pools

ratory

Poo 1 A Vj VJ U I 2 7 74 ?Q 74 J^0U 74/ *t

1 - .49 - .37 - .65 - .51 - .45

3 • -1.25 - .97 - .94 - .51 - .11

6 - .80 . 85 1.54 1.32 .77

7 - .30 - .04 - .14 - .56 - .22

8 2. 50 .14 - .42 .09 .32

9 .15 - .14 .79 .38 .05

10 1.41 .08 - .12 - .15 - .47

11 -1.20 .45 - .06 - .06 .13

Average all

labs (mg/L) 417. 9 789. 9 1341.4 1937.5 2930.1

4.3 Comparison of Results of Round Robin III with ID/MS Values

Table 23 presents the averages obtained at each concentration

level by ID/MS and the RR III manual and semi-automated candidate

reference methods. The standard error of each average is also listed

Table 24 lists the differences between the RR III. results and the

target values, together with their standard errors.

Table 23. Comparison of Round Robin III results with ID/MS

target values: averages and standard errors (mg/L).

Round Robin III

ID /MS Manual Semi - automated

Average Std. Error Average Std. Error Average Std. Error

410. 8 0.8 413.4 3.9 417.9 1.7

789 .6 1.2 793.4 4.1 789 .9 1.8

1351.2 2 .1343.3 4.7 1341 .4 2.8

1981. 5 3.8 1950.5 5.6 1937.

4.2

2988.

10.1 2941.8 7.4 2930.1 6.8

34
Table 24. Comparison of Round Robin III results with ID/MS target

values: differences and standard errors in mg/L.

(ID/MS) - (Manual) (ID/MS) - (Semi -Automated)

Pool Difference

Std. Error

of Diff. Difference

Std. Error

of Diff.

Pool A -2.6 4 . 0 - 7 1 CGSP -3.8 4.3 -0.3 2.2

2774 7.7 5.1 9.6 3.5

2974 31 . 5 6.8 44. 5 5.7

3074 46. 2 12.5 57.9 12.2

For a laboratory-by-laboratory comparison with the target values,

the individual percent deviations of each laboratory average from the

corresponding target value are listed in Tables 25 and 26.

Table 25. Round Robin III - manual: percent deviations from

target value.

_. Target Value (mg/L)

Laboratory 410 ' 8 789 ' 6 1351 ' 2 1981 ' 5 2988 ' 3

1 0.51 -0.56 -1.35 -2.12 -2.05

2 -1.27 0.16 -0.26 -1.72 -1.42

3 1.04 0.48 -0.51 -2.40 -2.00

4 1.87 2.72 -0.47 -0.62 -0.85

5 0.68 -0.61 -1.21 -1.98 -1.62

6 2.67 1.56 0.95 -0.16 -1.24

7 -5.00 -1.25 -1.66 -3.02 -2.38

8 4.64 1.41 -0.20 -0.51 -0.89

Table 26. Round Robin III - semi - automated : percent deviations

from target value.

Target Value (mg/L)

410.8 789.6 1351.2 1981.5 2988.3

Laboratory

1 1 . 23 -0. 33 -1. 37 -2.72 -2.39

3 0.45 -0.92 -1. 66 -2. 71 -2.06

6 0.92 0. 89 0. 80 -0.92 -1. 19

7 1. 43 0.00 -0. 86 -2.77 -2 . 17

8 4.27 0.19 -1.15 -2. 13 -1.64

9 1. 88 -0. 10 0. 56 -1.85 -1.90

10 3. 16 0. 13 -0.85 -2.37 -2.41

11 0. 50 0.49 -0. 79 -2.28 -1.82

35
Table 27 is a summary of the precision results by the manual method

for all three round robins. The table lists the glucose contents of all

samples in increasing order of magnitude and exhibits the corresponding

coefficients of variation, both for the replication error and for the

total error (single determinations made in different laboratories)

Table 28 shows the same information for the semi - automat ed results for

RR II and RR III.

Table 27

Glucose

Qg/L)

409. 7 413. 4

420. 5 762.0

793 .4

800.1

1082.

1343.

1343.

1950.5

2060.

2092.

2941.8

4505.

5714.

Summary of precision data - manual: coefficient of

variation (%)

2.4

1.8

1.7

1.4

1.2

Replication'

RR I RR II RR III

2.9

1.

1.3

1.0

0.7

1.7

1 .

1.0

0.9

0.8

5.7

3.1

2.5

2.0

1.9

Total

RR I RR II RR III

6.8

3.4

1.9

1.5

1 . 5

4.4

2.5

1.7

1.4

1.2

Corresponds to s q

. 'Corresponds to s^.

36
Table 28. Summary of precision data - semi -automated

coefficient of variation ($).

Glucose

a h Replication Total

(mg/L) RR II RR III RR II RR III

413.0 1.9 2.4

417.9 2.2 2.9

758.4 1.2 1.7

789.9 1.3 1.6

1334.9 0.9 1.4

1341.4 0.9 1.1

1937.5 0.7 1.0

2041.6 -0.7 1.3

2930.1 0.6 0.9

4501.3 0.6 1.1

Corresponds to s . b

^* Corresponds to s^.

5. Discussion

5.1 ID/MS Method

Some of the practices employed for optimizing ID/MS analyses for

elemental composition [8] were adapted to the glucose method, e.g.,

a) a magnetic sector mass spectrometer was used; b) each analysis

involved weighed aliquots of serum and labeled glucose solution; c) the

proportions of labeled and unlabeled glucose in mixtures were controlled

so that the signal intensities of the two monitored ions, corresponding

to the labeled and unlabeled DAG, were approximately alike (within ±10

percent) ; and d) the measurements were made on purified DAG samples

inserted directly into the mass spectrometer. In addition, the DAG

samples were measured together with standards having ion- intensity ratios

bracketing those of the samples, so that interpolation of the known

weight-ratios of a pair of standards bracketing a sample and the measured

ion- intensity ratios for those standards and that sample, would give the

weight-ratio of unlabeled to labeled glucose in the sample. The analyst

monitored the replicate ion- intensity ratios as acquired, for acceptability according to a simple protocol.

The precision of the ID/MS analyses is evident from the entries in

Tables 2 and 3 for days 1, 8, and 15. The standard deviations for

single determinations were .less than 1 percent of the average glucose

37
content of the pool; and the standard errors were less than 0.5 percent

of the pool averages.

The small variability in these results lies mainly between the

values for the successively analyzed samples of the pools. There was no

directly observable evidence of interferences in the CIMS measurements;

nor were interference effects evident by comparison with results obtained

using EIMS (Table 4) . The latter results agreed closely with the CIMS

values; even the pattern of between- sample variability was similar.

Although some of the variability must be due to random effects that

occurred during sample preparation, the same sample preparation method

was subsequently used for the 6 human serum pools that were analyzed by

the revised ID/MS method, and there was much less variation in those

results [11] . We believe the somewhat better precision obtained with

these human serum samples was due in part to the use of the gas chromatography coupled to the mass spectrometer and in part to the use of a measurement protocol requiring, the immediate bracketing of each sample

with standards.

The accuracy of the ID/MS method was assessed from the following

results: 1) The near identity of the CIMS and EIMS results for the serum

pools; 2) The 100 . 2—100 . 5

• percent glucose recoveries of the quantities

added to four samples of dialyzed serum, two of which also contained

three other hexoses as possible interfering substances. In calculating

these results, it was assumed that no glucose remained in the serum after

dialysis; 3) The finding in two samples of Pool CGSP to which glucose

had been added on day 150 (as numbered in Table 2) , of 774 and 776 mg of

glucose per liter, essentially the value that had been directly ' determined by ID/MS for that pool at day 140. For this calculation it

was assumed that 100.0 percent of the added glucose was recovered.

The original and revised DAG ID/MS methods have not been compared

directly. However, the results from each have been verified independently by EIMS to CIMS comparisons as described here for the original

method and in reference [11] for the revised method. The revised DAG

method has been used also for determining the glucose in several pools

whose glucose content had been measured at the CDC by the candidate

reference method; but that data will be discussed after we consider

the mult ilaboratory , candidate reference method results, and compare

them with the DAG method results.

38
5.2 Candidate Reference Method

The method and organization of the int erlaborat ory testing were

discussed at length in the CDC publication [7]; here we consider the

data from the three round robins, for evaluating precision and bias.

The first two round robins were run primarily for familiarizing laboratory participants with the reagent and instrumental requirements and

with the performance of the method. The data from those early rounds

showed on statistical analysis that the method run using manual pipetting

was fairly . precise , and that did not change from RR I to RR II. Also,

although based on data obtained in only 4 of the 8 laboratories, the

statistical analysis showed that somewhat more precise results were

obtained in RR II when the method was run using semi -automated pipetting. (The number of laboratories performing the method with each

alternative pipetting version was equalized for RR III. Before participating, the additional laboratories familiarized themselves with the

method.

)

In RR III, probably because the laboratories ran the method for

5 days and developed more skill with the procedure, the precision was

better than observed in the preliminary rounds, variances declining more

for the method run with manual pipetting. Although the differences are

not spectacular in RR III, the semi - automated version provided somewhat

better precision.

5.3 Comparison of Results from the Candidate Reference and ID/MS Method

The differences between the round robin III results and the ID/MS

values, and the corresponding standard errors, which are listed in

Table 24, reveal that at low concentration levels, the differences are

generally of the order of their standard errors. At h.igh concentrations,

they are appreciably larger. It would be erroneous to conclude that the

candidate reference method is biased only at high concentration. When

the average results obtained in round robin III are plotted against the

ID/MS values, straight lines result, both for the manual and for the

semi - automated methods, but the. lines have non-zero, positive intercepts

and slopes that are significantly less than unity. The least squares

fits yield the following equations, where x is the ID/MS value and y

the average obtained in round robin III:

Manual: y = 1.6 + 0.979 x Semi-automated: y.= 2.0 + 0.973 x

39
The pattern of increasing differences with increasing glucose level

was found also with the six human serum pools that were being stored at

—20 °C and were analyzed at the same time by the candidate reference

method and the revised DAG ID/MS method, ID/MS giving the higher results

(Table 29). In another comparison [11] involving three human serum

pools which were related by spiking with added glucose and where the

thawed samples were left at room temperature for 20 hours before analysis

by the reference method and revised DAG ID/MS method, the revised DAG

ID/MS to reference method differences were —0.2 mg/L at 700 mg/L, +8.3

mg/L at 1840 mg/L and +7.8 mg/L at 2980 mg/L. Also, with a bovine serum

pool (i.e., the WHO reference serum) having glucose at 980 mg/L, the

ID/MS value was higher by 9.3 mg/L [11]. Ill general the relationship

between the revised ID/MS , results and CDC results with the candidate

reference method is in comformity. with the differences found between the

original ID/MS method and the mult i laboratory study. The bias in the

candidate reference method is less than 1 percent with serum glucose

concentrations below 1500 mg/L and below 2 percent at higher concentrations. From the precision, given in Table 20, and the bias just recounted

we conclude that the candidate reference method meets our prechosen

criterion for an acceptable glucose reference method.

Table 29. Comparison of six human serum pools by the revised DAG ID/MS

and candidate reference methods (data from [12 and 15]).

Method Difference

Pool ID/MS Reference ID/MS—Reference

No

mg/L mg/L mg/L

3077 667.7 665.6 2.1

3177 1180.7 1171.9 8.8

3277 1682.9 1672.1 10.8

3377 2194.3 2171.7 22.6

3477 2872.2 2848.0 24.2

Laboratories that intend to perform the reference method, must

carry out the method in all detail, as described [7]. As initial

evidence of appropriate performance of the method, calibration results

should be examined in terms of closeness to straight line fits, in the

light of the data given in Tables 18 and 19. When a laboratory performs

the reference method for 5 days on different serum pools and also on

the Human Serum Reference Material (SRM 909) for which NBS provides a

40
definitive glucose value and has results that are comparable in precision

and accuracy to those in this report, they may wish to reduce the amount

of testing per sample by using Table 30 as a guide. Table 30 exhibits

the standard error that may be expected for the average in a laboratory

that is able to perform the reference method as well as those that

participated in this study, when running four or eight replicate analyses

on only one or two days. The standard errors were calculated from the

following equation ft ^

^

+ in which the values for s , L e

m

S D/L' anc^ S

L

are ^ rom Table 20, n is the total number of replicate

analyses, and m is the number of days.

Table 30. Expected standard error of average for the candidate

reference method on performance of four or eight

replicate determinations on one or two days (mg/L)

With Manual Pipetting

Sample Average 4 in 1 day 4 in 2 days 8 in 2 days

1 1 1 -413.4 17 14 14

III - 2 793.4 18 15 14

III -3 1343.

20 16 16

1 1

1 -4 1950.

23 20 19

1 1 1 - 5 2941 .

29 25 24

With Semi -Automated Pipetting

1 1 1 -

• 419.9 10 8 7

1 1 1 - 2 789. 9 11 8 7

1 1 1 - 3 1341.4 12 10 9

III-4 1937.

15 14 13

1 1 1 - 5 2930. 1 21 20 19

The Bureau of Medical Devices and Diagnostic Products of the Food

and Drug Administration provided support for the work performed by the

National Bureau of Standards,

41
6. References

[1] Cali, J. P.; Mandel, J.; Moore, L. J.; Young, D. S. A referee

method for the determination of calcium in serum. Nat. Bur.

Stand. (U.S.) Spec. Publ. 260-36; 1972.

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

Moody, J. R. A reference method for the determination of sodium

in serum. Nat. Bur. Stand. (U.S.) Spec. Publ. 260-60; 1978.

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

Machlan, L. A.; Gramlich, J. W. A reference method for the

determination of potassium in serum. Nat. Bur. Stand. (U.S.)

Spec. Publ. 260-63; 1979.

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

Murphy, T. J.; Gramlich, J. W. A reference method for the

determination of chloride in serum. Nat. Bur. Stand. (U.S.) Spec.

Publ. 260-67; 1979.

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

Machlan, L. A.; Garner, E. L.; Rains, T. C. A reference method

for the determination of lithium in serum. Nat. Bur. Stand.

(U.S.) Spec. Publ. 260-69; 1980.

[6] Slein, M. W. D-Glucose determination with hexokinase and glucose6-phosphate dehydrogenase, chapter in Methods of Enzymatic

Analysis, 2nd ed. H. U. Bergmeyer, ed. New York: Academic Press;

1965. 117-122.

[7] Neese, J. W. ; Duncan, P.; Bayse, D. ; Robinson, M. ; Cooper, T;

Stewart, C. Development and evaluation of a hexokinase/glucose-6

phosphate dehydrogenase procedure for use as a national glucose

reference method. U. S. Dept. Health, Education, and Welfare

Publ. No. (CDC) 77-8330; 1976.

[8] Moore, L. J.; Machlan, L. A. High accuracy determinations of

calcium in blood serum by isotope dilution mass spectrometry.

Anal. Chem. 44:2291-2296; 1972.

[9] Bjorkhem, I.; Blomstrand, R. ; Lantto, 0.; Svensson, L.;

Ohman, G. Toward absolute methods in clinical chemistry: application

of mass f ragmentography to high-accuracy analysis. Clin. Chem.

22:1789-1801; 1976.

42
[10] Schaffer, R. The development of a clinical reference method for

glucose in serum. Pure Appl. Chem. 45:75-79; 1976.

[11] White V, E. W. ; Welch, M. J.; Sun, T.; Sniegoski, L. T.;

Schaffer, R. ; Hertz, H. S.; Cohen, A. The accurate determination

of serum glucose by isotope dilution mass spectrometry — two

methods. Biomed. Mass Spectrora. (in press).

[12] Cohen, A.; Hertz, H. S.; Mandel , J.; Paule, R. C; Schaffer, R. ; Sniegoski, L. T. ; Sun, T. ; Welch, M. J.; White, V, E. Total

serum cholesterol by isotope dilution/mass spectrometry:

A candidate definitive method. Clin. Chem. 26:854-860; 1980.

[13] Bier, D. M. ; Leake, R. D. ; Gruenke , L. D. ; Sperling, M. A. The

in vivo measurement of alanine and glucose turnover with deuterium

labeled metabolites. Klein, P. D. ; Peterson, S. V., eds. Proceedings of the first international conference on stable isotopes in

chemistry, biology and medicine; 1973.

[14] Neese, J. W. ; Roark, B. R. ; Turner, W. E. Use of the glucose

reference method to characterize biological reference materials.

Clin. Chem. 28:1655; 1982.

[15] Klein, P. D.; Haumann, J. R. ; Eisler, W. J. Gas chromatographmass spectrometer-accelerating voltage alternator system for the

measurement of stable isotope ratios in organic molecules. Anal.

Chem. 44: 490-493; 1972.

[16] Gruenke, L. D.; Craig, J. C; Bier, S. M. Multiple ion detection

by acceleration voltage alternations in conjunction with voltage

sweeping. Biomed. Mass Spectrom. 1:418-422; 1974.

[17] Mandel, J. Models, transformations of scale, and weighting. J.

Quality Technol. 8(2):86-97; 1976.

[18] Mandel, J. The interlaboratory evaluation of testing methods.

ASTM Bulletin 239:53-61 and 133; 1959.

[19] Mandel, J. Estimation of weighting factors in linear regression

and analysis of variance. Technometrics 6:1-25; 1964.

ft U.S. GOVERNMENT PRINTING OFFICE : 1982 O—360-997 (2280) 43
NBS-114A (REV. 2-8C) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See instructions)

1. PUBLICATION OR

REPORT NO.

NBS SP 260-80

2. Performing Organ. Report No. 3. Publication Date

October 1982

4. TITLE AND SUBTITLE

Standard Reference Materials:

Evaluation by an ID/MS Method of the AACC Reference Method for Serum Glucose

5. AUTHOR(S)

R. Schaffer, J. Mandel, T. Sun, A. Cohen, and H. S. Hertz

6. PERFORMING ORGANIZATION (If joint or other than NBS. see instructions) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE 8. Type of Report & Period Covered

WASHINGTON, D.C 20234

Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City, State, ZIP)

Same as 6.

10. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number: 82-600618

~J Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here) In conjunction with a study group of the Committee on Standards of the American Association for

Clinical Chemistry working to establish a reference method for glucose in serum, the authors from MBS

developed an isotope dilution/mass spectrometry c method (ID/MS) for providing essentially bias-free,

precise serum glucose analyses. This method, which is too elaborate for clinical laboratory use as a reference method, involves addition of a known amount of D-gl ucose-U 1

C to a serum sample, conversion

of the labeled and unlabeled glucose in the sample into 1 ,2:5,6-di-0-isoprcpylidine-D-g!ucose (DAG),

and measurement of the ratio of labeled to unlabeled DAG as the corresponding (M+l) ions, by isobutane-chemical ionization mass spectrometry. Five serum pools having glucose concentrations ranging

from 0.4 to 3.G g/L were analyzed. The relative standard deviation among single measurements made on different samples of the same pool was found to range from 0.34 to 0.45 percent for four of the pools,

and was 0.79 percent for the pool with the highest glucose concentration. Pool concentrations were

also determined from the same DAG samples using electron impact mass spectrometry and monitoring the

ratios of corresponding (M-15)

+ ions, and the results were similar. There was no evidence of bias.

These serum pools were used by the study group for a statistically controlled inter! aboratory test

to evaluate a hexokinase/glucose-5-phosphate dehydrogenase method as the reference method for glucose.

Investigators at the Centers for Disease Control (Atlanta) had- found that method the most appropriate

of the several clinical glucose methods that were studied as possible reference methods. [J. W. Neese

et al . HEW Publication No. (CDC) 77-8330. J. Statistical analysis of the mul ti laboratory results showed

that the relative standard deviations among single measurements made in different laboratories

creased as glucose concentrations increased. With manual pipetting used for performing the candidate

reference method, the relative standard deviation ranged from 4.4 to 1.2 percent; with semi -automated

pipetting, the range was 2.8 to 0.8 percent. Compared to the ID/MS results, the mean values found by

the candidate reference method were about 1 percent higher at the 0.4 g/L level and changed linearly to about 2 percent lower at the 3.0 g/L level. We conclude that the candidate reference method fulfilled

our prechosen criterion for acceptance as a reference method for serum glucose.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

"clinical analysis; glucose in serum; glucose reference method; isotope dilution/mass

spectrometry; reference method; statistical analysis.

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