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Or NBS SPECIAL PUBLICATION 260-

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

Standard Reference Materials:

A REFERENCE METHOD FOR THE

ION OF POTASSIUM IN SERUM
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards 1 was established by an act of Congress 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 performed by the National Measurement Laboratory, the National Engineering Laboratory,

and the Institute for Computer Sciences and Technology.

THE NATIONAL MEASUREMENT LABORATORY provides the national system of

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

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

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

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

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

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

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

calibration services. The Laboratory consists of the following centers:

Absolute Physical Quantities2 — Radiation Research — Thermodynamics and

Molecular Science — Analytical Chemistry — Materials Science.

THE NATIONAL ENGINEERING LABORATORY provides technology and technical

services to users in the public and private sectors to address national needs and to solve

national problems in the public interest; conducts research in engineering and applied science

in support of objectives in 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 utlimate user. The Laboratory consists of the following centers:

Applied Mathematics — Electronics and Electrical Engineering2 — Mechanical

Engineering and Process Technology2 — Building Technology — Fire Research —

Consumer Product Technology — Field Methods.

THE INSTITUTE FOR COMPUTER SCIENCES AND TECHNOLOGY conducts

research and provides scientific and technical services to aid Federal Agencies in the selection,

acquisition, application, and use of computer technology to improve effectiveness and

economy in Government operations in accordance with Public Law 89-306 (40 U.S.C. 759),

relevant Executive Orders, and other directives; carries out this mission by managing the

Federal Information Processing Standards Program, developing Federal ADP standards

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

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

The Institute consists of the following divisions:

Systems and Software — Computer Systems Engineering — Information Technology.

'Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D.C. 20234.

2 Some divisions within the center are located at Boulder, Colorado, 80303.

The National Bureau of Standards was reorganized, effective April 9, 1978.
NaHcnaF Boreas of Standsrtfs JUN 19

Standard Reference Materials:

A REFERENCE METHOD FOR THE DETERMINATION OF

POTASSIUM IN SERUM

Ranee A. Velapoldi, Robert C. Paule,

Robert Schaffer, John Mandel,

Lawrence A. Machlan, and

John W. Gramlich

National Measurement Laboratory

National Bureau of Standards

Washington, D.C. 20234

With the cooperation of a Committee of Experts:

George N. Bowers, Jr.,

1 Bradley E. Copeland, 2

Denis O. Rodgerson, 3 and James M. White4

'Hartford Hospital, Hartford, Connecticut

2 New England Deaconess Hospital, Boston, Massachusetts

'Department of Pathology, University of California,

Los Angeles, California

"Center for Disease Control, Atlanta, Georgia

U.S. DEPARTMENT OF COMMERCE, Juanita M. Kreps, Secretary

Jordan J. Baruch, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued May 1979
Library of Congress Catalog Card Number: 79-600022

National Bureau of Standards Special Publication 260-63

Nat. Bur. Stand. (U.S.), Spec. Publ. 260-63, 104 pages (May 1979)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1979

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

Stock No. OO3-0O3-02O68-1 Price $3.75

(Add 25 percent additional 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.

iii
Inquiries concerning the technical content of this

paper should be directed to the author(s). 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, Acting Chief

Office of Standard Reference Materials

iv
FOREWORD

A fundamental requirement for assuring adequate patient

care is the need for the accurate analysis of constituents in

body fluids. Two major functions of the National Bureau of

Standards (NBS) are to provide certified Standard Reference

Materials for the calibration of measurement systems and to

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

of known accuracy for the determination of potassium in serum.

The evaluation of a reference method by comparison to a

definitive method, used for the first time at NBS in the

development of reference methods for calcium and sodium in

serum, also was applied to this work. This hierarchy of

analytical procedures has been accepted as a valid format

for developing reference methods by the clinical community

at a recent Conference on an Understanding for a National

Reference System in Clinical Chemistry.

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

Control, the Food and Drug Administration, and a wide

spectrum of participating analytical laboratories that

included Federal, state, hospital, industrial, and academic

laboratories was essential to establish a widely accepted

reference method. It is hoped that this work will provide

an additional basis for the development of future clinical

reference methods through continued collaboration and the

concerted efforts of the individual participants.

Philip D. LaFleur, Director

Center for Analytical Chemistry
NOTE

Because of concern for the usability of the NBS sodium

and potassium methods, CDC personnel have proposed a procedure

for the concurrent determination of potassium and sodium by

FAES that differs from the separate potassium and sodium

reference methods, described here and, for sodium, in NBS

Special Publication 260-60. The CDC method includes semiautomated sample dilution, a different sample analysis format,

and different standards preparation. CDC management believes

its method will serve better as a "combined" reference method,

and as a consequence, has declined to endorse the method

described in this report and is proposing the CDC method as

the reference method. While NBS supports the evolution of

analytical methods and has agreed to participate in interlaboratory exercises that are aimed toward establishing the

transferability of the proposed CDC procedures, NBS management

believes it important that the principles of analytical

practice delineated in this present report be circulated in

a timely manner. Since the method outlined in this report

has been shown to satisfy the generally accepted criteria of

a reference method, it should function as such until the

efficacy of a subsequent method has been demonstrated. In

addition, NBS will maintain its primary role in supplying

SRM's and definitive methods.

vi
OTHER NBS PUBLICATIONS IN THIS SERIES

Catalog of NBS Standard Reference Materials

(1975-76 edition), R. W. Seward, ed., NBS

Spec. Publ. 260 (June 1975). $1.50* SN003-003-

01445-1

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

Reference Materials: Preparation of White Cast

Iron Spectrochemical Standards, NBS Misc.

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

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

Standard Reference Materials: Preparation of

NBS Copper-Base Spectrochemical Standards,

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

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). COM74-11068**

Bell, R. K., Standard Reference Materials:

Methods for the Chemical Analysis of NBS

Copper-Base Spectrochemical Standards, NBS

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

11067**

Richmond, M. S., Standard Reference Materials:

Analysis of Uranium Concentrates at the

National Bureau of Standards, NBS Misc. Publ.

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

Anspach, S. C, Cavallo, L. M., Garfinkel, S. B.,

Hutchinson, J. M. R., and Smith, C. N., Standard Reference Materials: Half Lives of Materials

Used in the Preparation of Standard Reference

Materials of Nineteen Radioactive Nuclides

Issued by the National Bureau of Standards,

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

COM74-11065**

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

Michaelis, R. E., Standard Reference Materials:

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

1965). COM74-11064**

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

Reference Materials: Viscosity of Standard

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

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

Yakowitz, H., Vieth, D. L., and Michaelis, R. E.,

Standard Reference Materials: Homogeneity

Characterization of NBS Spectrometric Standards III: White Cast Iron and Stainless Steel

Powder Compact, NBS Misc. Publ. 260-12

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

Spijkerman, J. I., 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

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

G., Standard Reference Materials: Calibra

tion 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 (Certification of Standard Reference Material 745), NBS

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

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

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

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

COM74-11359**

Yakowitz, H., Fiori, C. E., and Michaelis, R. E.,

Standard Reference Materials: Homogeneity

Characterization of Fe-3 Si Alloy, NBS Spec.

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

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

Reference Materials: Viscosity of a Standard

Borosilicate Glass, NBS Spec. Publ. 260-23

(December 1970). COM71-00157**

Sappenfield, K. M., Marineko, G., and Hague, J.

L., Standard Reference Materials: Comparison

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

(January 1972). COM 72-50058**

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). COM 74-1 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* SN003-O03-O0786-2

Garner, E. L., Machlan, L. A., and Shields, W. R.,

Standard Reference Materials: Uranium

Isotopic Standard Reference Materials, NBS

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

11358**

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

Materials: Preparation and Evaluation of

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

Publ. 260-28 (August 1971). COM71-50365**

viii

Geller, S. B., Standard Reference Materials: Calibration 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). COM7 1-50563**

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 Distribution Polystyrene, NBS Spec. Publ. 260-33

(May 1972). COM72-50526**

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

Materials: Thermoelectric Voltage, NBS Spec.

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

Sparks, L. L., and Hust, J. G., Standard Reference 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 1

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.) COM-72

50776**

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

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

Used in Testing Textiles for Colorfastness to Light, NBS Spec. Publ. 260-41 (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). SN003-003-0 1344-7

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

Electrolytic Iron, SRM's 734 and 797 from 4 to 1000 K, NBS Spec. Publ. 260-50 (June 1975).

1.00* SN003-003-0 1425-7

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

Reference Materials: Glass Filters As a Standard

Reference Material for Spectrophotometry;

Selection; Preparation; Certification; Use -SRM

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

$1.90* SN003-003-01481-8

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

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

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

01464-8

Durst, R. A., Standard Reference Materials:

Standardization of pH Measurements, NBS

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

$1.05* SN003-003-01551-2

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

Reference Materials: Certification and Use of

Acidic Potassium Dichromate Solutions as an

Ultraviolet Absorbance Standard, NBS Spec.

Publ. 260-54 (August 1977). $3.00* SN003-003-

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* SN003003-

01836-8

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

Standard Reference Materials: Standard

Thermocouple Material, Pt.-67:SRM1967, NBS

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

SN003-003-0 18864

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

States Reference Materials, NBS Spec. Publ.

260-57 (February 1978). $2.20* SN003-003-

01883-0

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

Transmission Measurements. SRM 1470, NBS

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

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

Materials: Electron Paramagnetic Resonance

Intensity Standard: SRM 2601, NBS Spec.

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

003-01975-5

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

Mandel, J., and Moody, J. R., Standard Refer- ence Materials: A Reference Method for the

Determination of Sodium in Serum, NBS Spec.

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

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

SN-003-003-02006-1

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 (in press).

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

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

Standard Reference Materials: A Reference

Method for the Determination of Potassium in

Serum, NBS Spec. Publ. 260-63 (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.

x
TABLE OF CONTENTS

PAGE

I. INTRODUCTION 2

II. DEVELOPMENT OF THE SERUM POTASSIUM REFERENCE

METHOD 4

A. Organization 4

B. Participating Laboratories, Standards, Serum

Samples, and Definitive Method 6

C. Functions of the Various Groups 9

D. Plan for Testing the Candidate Reference

Method 11

III. REFERENCE METHOD PROTOCOL FOR THE DETERMINATION

OF SERUM POTASSIUM 12

A. General 12

B. Protocol Synopsis 12

C. Detailed Protocol 14

IV. RESULTS AND STATISTICAL ANALYSIS 31

A. Round Robin Results 32

V. DISCUSSION 61

A. Candidate Protocol 61

VI. CONCLUSIONS 65

VII. REFERENCES 6 7

APPENDIX A. . 69

APPENDIX B 70

APPENDIX C 72

APPENDIX D 81

xi
LIST OF TABLES (continued)

TABLE NO . PAGE

13. Ratios of standard deviations to average standard

deviations for potassium in serum for Round

Robin II, semiautomated pipetting protocol. . . 51

14. Components of standard deviation in mmol/L for

all Round Robin II potassium levels

(1.3-7.3 mmol/L) 53

15. Summary of imprecision and bias results in mmol/L

for potassium in serum, Round Robin II 55

16. Summary of potassium in serum values ±2 standard

errors 57

17. Calibration curve data for potassium in serum,

Round Robin II using manual pipetting 58

18. Calibration curve data for potassium in serum,

Round Robin II using semiautomated pipetting. . 59

19. Calibration curve results for potassium in serum

as standard deviation of fit ( s £^ t

) i n mmol/L . 60

20. Instruments and operating conditions used by the

participating laboratories in RRII 63

xii
LIST OF TABLES

TABLE NO . PAGE

1. Preliminary results from NBS and CDC for the

determination of serum potassium . 5

2. Potassium concentrations for, the seven serum

pools as determined by IDMS, the definitive

method 9

3. Volumes of potassium standard stock solution

diluted to 250-mL that give the potassium

diluted standard solutions. . , . . 18

4. Example of intensity values for sets of readings

using a direct read-out instrument 29

5. Serum potassium concentrations reported by the

participating laboratories for the Preliminary

Round Robin, manual pipetting protocol 34

6. Concentrations of serum potassium reported by the

participating laboratories for Round Robin I,

manual pipetting protocol 37

7. Concentrations of serum potassium reported by the

participating laboratories for Round Robin I,

semiautomated pipetting protocol 38

8. Concentrations of serum potassium reported by the 43

participating laboratories for Round Robin II, §

manual pipetting protocol ...... 44

9. Concentrations of serum potassium reported by the

participating laboratories for Round Robin II,

semiautomated pipetting protocol 45

10. Percent deviations from averages for potassium in

serum from Round Robin II, manual pipetting

protocol 48

11. Percent deviations from averages for potassium in

serum from Round Robin II, semiautomated

pipetting protocol 49

12. Ratios of standard deviations to average standard

deviations for potassium in serum from Round

Robin II, manual pipetting protocol ...... 50

xiii
LIST OF FIGURES

FIGURE NO .

1. Interrelationships and functions of the various

groups in the development of a clinical

reference method for the determination of

serum potassium

2. Typical calibration curve for the determination

of serum potassium by flame atomic emission

spectroscopy

3. Percent deviations of individual results from the

collective average of the measurements obtained

in the Preliminary Round Robin test

4. Percent deviations of the Round Robin I measurements using manual pipetting from the

definitive method values

5. Percent deviations of the Round Robin I measurements using semiautomated pipetting from the

definitive method values

6. Percent deviations of the Round Robin II measurements using manual pipetting from the

definitive method values

7. Percent deviations of the Round Robin II measurements using semiautomated pipetting from the

definitive method values

xiv
ABSTRACT

Guided by a committee of experts in clinical chemistry,

a reference method was established for the determination of

serum potassium based on flame atomic emission spectroscopy

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

obtained by use of the method in 12 laboratories against the

results obtained by a definitive analytical method based on

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

with potassium concentrations in the range 1.319 to 7.326

mmol/L were analyzed. Manual and semiautomated pipetting

alternatives were tested using sample sizes of 5.0 and 0.25

mL, respectively.

The laboratories used several different FAES instruments.

The results showed that the standard error for a single

laboratory's performance of the procedure ranged from 0.049

to 0.063 mmol/L with a maximum bias of 0.065 mmol/L over the

range of concentrations studied. These values were within

the accuracy and precision goals that had been set by the

committee. The results from the two pipetting techniques

were similar. The calibration curve data showed excellent

linearity over the total concentration range, with 20 of 22

curves having standard deviations of fit of 0.075 mmol/L or

less . With appropriate experimental design, the reference

method may be used to establish the accuracy of field methods

as well as to determine reference potassium values for pooled

sera

.

Key Words: Clinical analysis; clinical chemistry; definitive

method; electrolytes; flame atomic emission spectroscopy;

reference method; semiautomated pipetting; serum potassium

analysis

.

1
I. INTRODUCTION

Serum potassium can be determined by a wide variety of

analytical methods; these include (1) separation by precipitation with measurement by photometry, gravimetry, or titrimetry, (2) separation by ion-exchange with measurement by

photometry, and (3) direct analysis by use of ion- selective

electrodes, gasometry, photometry, neutron activation, or

flame atomic emission spectroscopy (FAES) 5 [l] 6

. The use of

flame atomic emission spectroscopy has been described as a

standard method [2]. Whether the latter method or some other

should be considered by clinical laboratories as the clinical

reference method for serum potassium has not been proven; the

accuracy of none of these methods is known.

Two approaches may be used for establishing the accuracy

of analytical methods. In the first, the results obtained

from the methods in use for that analyte are compared using

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

used to express the interrelationships of the methods. A

technique is then considered to be accurate to the degree

established by knowledge of the sources of error and the

agreement of results. In the second, a single candidate

method is selected (possibly the 'best' of the methods

recognized by the first approach) and studied in detail.

Each step of the candidate method is optimized and examined

so that the systematic and the random errors can be quantitatively expressed.

Studies have been organized using a combination of these

approaches to establish the accuracy of a clinical chemistry

5 0fficial name, International Union of Pure and Applied

Chemistry, Information Bulletin Number 27, Nov. 1972.

6 The bracketed numerals refer to the references listed at

the end of this paper.

2
method for total calcium and sodium in serum [3,4]. For

calcium, the analytical procedure was based on the flame

atomic absorption spectrometr ic (FAAS) method of Pybus,

Feldman, and Bowers [5] , while for sodium it was based on

FAES. The accuracies of these methods were assessed by

comparing the results obtained using them on several human

serum pools for calcium and bovine serum pools for sodium in

selected clinical laboratories against those obtained for the

same pools by an isotope dilution-mass spectrometry (IDMS)

method for calcium and an ion-exchange gravimetry method for

sodium. These analyses were performed at the National Bureau

of Standards (NBS) where the high accuracy of those methods 7

were established by the second approach of determining their

systematic and random errors [4,6],

These studies, carried out with the guidance of clinical

laboratory experts, used (a) Standard Reference Materials as

the pure primary reference material to prepare standard solutions of calcium and sodium for all the analyses; (b) serum

pools prepared at the Hartford Hospital (Hartford) and the

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

method analysis for calcium or sodium on these pools at NBS;

(d) statistical analysis of the data at NBS; and (e) accuracy

and precision goals as performance standards that the methods

would have to meet to be recommended as the clinical reference

method for total calcium [7] or sodium [8] in serum.

This same approach was adopted to develop clinical

reference methods for a number of other serum electrolytes

including rodium, chloride, lithium, and magnesium. This

work was begun with the cooperation of individuals from the

Standards Committees of the American Association for Clinical

7 Such methods are referred to as definitive methods because

of their high accuracy and utility for evaluating the

accuracy of a candidate reference method.

3
Chemistry (AACC) and the College of American Pathologists

(CAP), the CDC and the NBS. The Food and Drug Administration

(FDA) provided major suppport for the NBS work. We present

in this report the development of a clinical reference

method for serum potassium.

II. DEVELOPMENT OF THE SERUM POTASSIUM REFERENCE METHOD

A. Organization

A panel of experts in clinical chemistry was invited

to meet at NBS in March 1974 to consider the development of

reference methods for five serum electrolytes, namely,

potassium, sodium, chloride, lithium, and magnesium. The

development of these reference methods was organized by

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

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

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

Deaconess Hospital), Dr. Denis 0. Rodgerson (Center for

Health Sciences, University of California in Los Angeles),

and Dr. James M. White 8 (CDC).

Prior to the meeting, several bovine serum pools prepared

at the CDC had been analyzed for potassium by FAES and IDMS.

The results, summarized in Table 1, were presented at the

meeting as follows:

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

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

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

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

He was recommended for membership on this Experts Committee

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

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

all phases of this work contributed greatly to the success

of the program.

4
On consideration of these quite similar analytical results

and of alternative clinical laboratory procedures in use for

the determination of serum potassium, -it was concluded that

FAES was the appropriate candidate methodology to evaluate

as the reference method and that its evaluation should be

made using IDMS as the definitive method.

Table 1. Preliminary results from NBS and CDC for the

determination of serum potassium.

- - - K in Serum, mmol/L - - - Pool IDMS a FAES

NBSb CDC C

I 2. 794 2 . 73 2.8

III 4. 765 4. 76 4. 76

V 6. 867 6.72 6.8

Data from L. J. Moore (NBS).

Data from R. Mavrodineanu (NBS)

.

c Data from J. White (CDC).

The experts agreed to serve as the Committee to oversee

the development of the reference method for potassium (as

well as for the other electrolytes discussed at the meeting)

.

The Committee chose Dr. Bowers as chairman. Dr. White agreed

to serve as the Committee's representative to work with those

at NBS who would be involved in writing the protocol for the

potassium reference method. The Committee agreed that the FAES

method should use a concentration bracketing technique rather

than calibration curves for determining potassium concentrations. However, calibration curve data would be obtained as

a general check on the measurement system and to determine

which of the primary standard solutions would be used to

bracket the potassium levels in the samples being analyzed.

5
As goals for the candidate reference method, the maximum

bias of the method and its one-standard deviation imprecision

limit were set by the Committee at 0.2 and 0.1 mmol/L, respectively, for serum potassium at the 2.5 and 6.5 mmol/L levels.

These goals were to be achieved by controlled, inter laboratory

tests involving a selected group of clinical chemistry laboratories which would perform the analyses by the FAES method

according to the written protocol while NBS would provide

potassium values by the definitive method.

B. Participating Laboratories, Standards, Serum Samples,

and Definitive Method

The laboratories that were asked to participate in the

interlaboratory study were chosen to represent a wide spectrum

of clinical chemistry interests and included government

(federal and state) and hospital laboratories, and laboratories

associated with suppliers of instruments and suppliers of

test and control materials. One hospital was located outside

the United States. The principal investigator at each

laboratory is named in the list below. Other scientists in

each of the laboratories who contributed to this study are

acknowledged by name in Appendix A. The list includes three

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

number of laboratories when some of the original laboratories

were unable to continue their participation. In alphabetical

order of the principal investigator, the laboratories that

participated in the interlaboratory studies are:

Dr. George N. Bowers, Jr.

Hartford Hospital

Hartford, CT 06115

6
Dr. Bradley E. Copeland

New England Deaconess Hospital

Boston, MA 02215

Professor Lorentz Eldjarn

Rikshospitalet , University of Oslo

Oslo, Norway

Mr. David Hassemer

Dr. Ronald H. Laessig

State Laboratory of Hygiene, University of Wisconsin

Madison, WI 53706

Mr. Theodore C. Rains

Dr. Michael Epstein

National Bureau of Standards

Gaithersburg, MD 20760

Dr. Denis 0. Rodgerson

Center for Health Sciences, University of California

Los Angeles, CA 90025

Mr. William Ryan

Beckman Instruments

Fullerton, CA 92634

Mr. Leonard Sideman

Pennsylvania Department of Health

Philadelphia, PA 19130

Dr. Barbara Tejeda

Food and Drug Administration

Washington, D. C. 20250

7
Dr. James M. White

Dr. Richard Carter

Center for Disease Control

Atlanta, GA 30333

Ms. Peg T. Whittemore

Instrumentation Laboratories

Lexington, MA 02173

Dr. Charles E. Willis

College of American Pathologists, Cleveland Clinic

Cleveland, OH 44106

NBS Standard Reference Material, Potassium Chloride

(SRM 918, see Appendix B) was to be used as the pure primary

reference material for all analyses [9]. Seven pools of

homogeneous, sterile, bovine serum, having different concentrations of potassium, were prepared at the CDC by

Dr. David Bayse and Miss Sue Lewis. Samples of each pool

were supplied in approximately 7-mL volumes in stoppered

vials that were labeled with computer generated random

numbers. The samples, packed in dry ice, were shipped to

NBS by air and within 24 h of packing were placed in freezers

kept at -50 °C [10]. The pools were numbered in code from

1 to 7 according to increasing potassium concentration.

A definitive method based on IDMS was developed at NBS.

The definitive method is given in Appendix C. The potassium

concentrations for the seven serum pools were determined by

this procedure and the results obtained are summarized in

Table 2.

8
Table 2. Potassium concentrations for the seven

serum pools as determined by IDMS, the

definitive method.

Pool [K

+ ], mmol/L

1 1.319 ± 0.003 a

2 2.540 ± 0.006

3 3.448 ± 0.009

4 4.323 ± 0.011

5 5.501 ± 0.014

6 6.092 ± 0.015

7 7.326 ± 0.018

Estimated at ±0.25 percent for all concentrations at the 95 percent confidence limit.

C. Functions of the Various Groups

The interrelationships and functions of the various

groups involved in developing FAES as a reference method for

serum potassium are represented in figure 1. The Committee,

CDC, and NBS provided guidance and technical support for the

program and also served as participating laboratories. The

Experts Committee selected the candidate reference method,

set maximum bias and imprecision goals for an acceptable

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

The CDC provided the serum pools. The participating laboratories provided the interlaboratory test data and critiques

of the candidate reference method protocol.

9
Figure 1. Interrelationships and functions of the various

groups in the development of a clinical reference

method for the determination of serum potassium.

10
At NBS, Dr. R. Schaffer served as the Reference Method

Program Manager and Dr. R. A. Velapoldi served as the coordinator. The format of the round robin tests was established

within the constraints imposed by protocol requirements and

sample availability by Drs. John Mandel, Robert Paule, and

R. A. Velapoldi. Dr. Velapoldi wrote the protocol for the

candidate reference method from the outline provided by

Dr. J. White. Drs. Mandel and Paule performed the statistical

evaluation of the results from the interlaboratory tests. The

definitive method was performed by Mr. Larry Machlan and

Dr. John W. Gramlich.

D. Plan for Testing the Candidate Reference Method

The general plan was to evaluate the candidate reference

method by performing a series of interlaboratory test exercises, which would consist of a preliminary round robin test

(PRR) followed by successive round robin tests (RR) until the

goals for the reference method were reached. A main objective

of the PRR test was to allow participating laboratories to

become familiar with and comment on the protocol. Since an

evaluation of the bias was not sought in the PRR testing phase,

normal bovine serum samples [11] not having definitive method

analyses were to be used. However, interlaboratory imprecision

was to be measured. If the imprecision of the results in the

PRR was found to be small, round robin (RR) testing would

begin on samples having definitive method potassium values.

In a RR, each participating laboratory would perform the

same analyses on two separate days: i.e., analyze a pair of

aliquots from each serum pool on each of two days where a

minimum of one day or a maximum of seven days were to elapse

between the two series of analyses. The bias and imprecision

values obtained by statistical analysis would then be compared

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

If the goals were not met, additional RR tests using samples

11
from other pools would be conducted by following the protocol

or a modified form of it until the bias and imprecision goals

were reached. Revisions and modifications to the protocol

could be made after a round robin test had been completed

but would not be made after the final RR.

Three kinds of information were to be supplied by each

participating laboratory after finishing a round robin:

1. General Data — a list of the instrumental parameters

used and comments on the protocol including problems

encountered during the analysis;

2. Calibration Curve Data — a list of the FAES relative

intensity values versus the potassium concentrations of

the standards; and

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

that constituted the five 'valid measurements' (see

section IIIC-5e for discussion).

Examples of the data sheets on which the information was

collected are shown in Appendix D, Note 8.

III. REFERENCE METHOD PROTOCOL FOR THE DETERMINATION

OF SERUM POTASSIUM

A. General

This protocol for the analysis of serum potassium by

flame atomic emission spectroscopy provides for the optional

use of either manual or semiautomated pipetting and also for

one hundred-fold or two hundred-fold dilutions of samples to

prepare working solutions. The pipetting alternatives are

discussed separately in detail whereas the dilution alternatives are not discussed since they are prescribed by the

instrument used.

B. Protocol Synopsis

The protocol must be followed exactly.

12
The reference method is used to analyze four aliquots

of a serum: two on one day and the other two on a subsequent

day

.

1. Use an analytical balance to weigh the SRM KC1 in

appropriate quantities and to prepare a stock standard

potassium solution;

2. Use either a single pipet or a pipettor-dilutor to

dilute to the potassium concentrations that are used as

working solutions for FAES a) aliquots of the serum

to be analyzed, b) aliquots of the stock standard

potassium solutions, and c) the solution used as a

blank;

3. Obtain calibration curve data for the working blank and

standards

;

4. Measure the emission signals of the working solutions

of the serum; select the pair of working standards

whose emission signals most closely bracket the signal

of each aliquot;

5. For each aliquot to be analyzed, obtain five valid

measurement sets by measuring the emission signals

obtained from repeated sequential measurements of the

working solutions of the low bracketing standard, the

sample, and the high bracketing standard;

6. Calculate the potassium concentration of the aliquot

for each set in the valid measurement set by

mathematical interpolation;

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

measurement' for that aliquot; (in the statistical

analysis, each such average is designated a 'single

measurement ' )

;

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

analyzed on the first day;

13
9. Repeat steps (1) through (8) on the subsequent day to

obtain the second pair of measurements needed for each

aliquot

;

10. Average the four values obtained by the replicate

determinations to obtain the potassium concentration

for each serum.

C. Detailed Protocol

The selection of the specific alternatives of the

protocol to be used (i.e., the pipetting and the dilution)

dictates the glassware and diluent volumes needed. These

needs are summarized in the protocol or in Appendix D notes.

Stock solutions and working solutions are to be prepared at

and maintained at a room temperature that is constant within

±2 °C (see Appendix D, Note 1). I

1 . Reagent Specifications

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

and/or deionized water used should exhibit a

specific resistance of at least 0.01 MQ*m at

23 ± 5 °C. At the time of use, this water should

show a flame emission signal that is less than 0.1

percent of full scale at the instrumental settings

used for the analysis. A large quantity of this

water (more than 50 L) must be available for use

as diluent and for the final rinsings of all

glassware and other apparatus that come in contact

with the solutions involved. Unless specified

otherwise, the water referred to in this protocol

is this tested water.

.

b. Potassium Standard Solutions : Use Standard Reference

Material, Potassium Chloride (originally issued as

SRM 918, Certificate reproduced in Appendix B) [9]

certified by the National Bureau of Standards. Dry

14
the SRM KC1 at 110 °C for four hours in a loosely

capped container and then store it in a desiccator

containing CaSOi* or an equivalent desiccant.

c. Lithium carbonate, sodium chloride, hydrochloric

acid, nitric acid, chloroform, methanol and 95-

percent ethanol meeting ACS [12] (or equivalent)

specifications are to be used.

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

making a twenty-fold dilution of concentrated

HN0 3 (15.4 mol/L) with water.

2 . Glassware Specifications

a. All volumetric glassware (Appendix D, Note 2)

should be of borosilicate material and meet NBS

Class A [13] (or equivalent) specifications. All

glass or plastic surfaces that come into contact

with reagents, water, diluent, or sample must be

clean (Appendix D, Note 3)

.

b. Pipettor-dilutor Device : The volumetric delivery

of the pipettor-dilutor device must have a tested

maximum inaccuracy of 2 percent and a maximum

imprecision of ±0.2 percent relative standard

deviation at the pump setting used. (The test

procedures are in Appendix D, Note 4.)

3 . Preparation of Reagents

If the instrument employed in the analyses does

not use lithium as an internal standard, water

is substituted for the aqueous lithium chloride

diluent solution in this protocol.

a . Lithium Chloride Diluent Solution (LiCl Diluent , 15 mmol/L ) : The homogeneity of this solution is

critical if an internal standard instrument is to

15
be used. The required volume may be prepared in

eleven 2-liter batches and then mixed thoroughly.

For each 2-liter volume, weigh 1.1082 g of dried

Li 2 C0 3 (mw = 73.8912, Appendix D, Note 5b); however,

if NBS SRM 924 is used, weigh 1.1092 g (see Appendix

D, Note 5) . Transfer the weighed Li 2 C0 3 quantitatively into a 2-liter volumetric flask. Add water

to just cover the bottom of the flask and, with

swirling, carefully add 4 mL of concentrated HC1 to

dissolve the LiaCCh. Dilute to the calibration

mark with water, stopper, and mix thoroughly by

inverting the flask and shaking ten times. Repeat

the inverting and shaking steps nine more times.

1) Manual pipetting alternative: Since all standards and samples are to be diluted with this

reagent, approximately 22 liters will be needed on

day 1 and 18 liters will be needed on day 2.

2) Semiautomated pipetting alternative: Prepare

approximately six liters of the LiCl diluent.

b . Sodium Chloride Diluent Solution (NaCl Diluent

140 mmol/L ) : Weigh 8.182 g of NaCl (mw = 58.4428

Appendix D, Note 5b) and trans-fer it quantitatively

to a one-liter volumetric flask. Dilute to the

calibration mark with water, stopper, invert, and

mix as described above. Four liters will be needed

on day 1 and 2 liters will be needed on day 2.

c. Potassium Standard Stock Solutions : Weigh accurately

(to 0.1 mg) approximately 5.96 g of dried potassium

chloride (mw = 74.5513, Appendix D, Note 5b) and

transfer it quantitatively into a 1-liter volumetric

flask. Dissolve and dilute to the mark with the

140 mmol/L sodium chloride diluent solution. Mix

thoroughly as described above. Repeat these steps

16
to prepare a second potassium standard stock solution. Label the solutions I and II. This weight

will give a solution of approximately 80 mmol of

potassium per liter. From the weighed quantities

of KC1 taken, calculate the exact potassium concentrations in mmol/L to three decimal places.

(1) Intercomparison of Standard Stock Solutions:

Transfer by pipet, 25.00-mL of stock solution I into a one-liter volumetric flask. Dilute to the

calibrated volume with LiCl diluent, stopper,

invert, shake, and mix as described above. Transfer

by pipet, 25-mL of the diluted solution to a oneliter volumetric flask. Dilute to the calibrated

volume with LiCl diluent, stopper, invert, shake,

and mix to give a working solution with a potassium

concentration of 0.05 mmol/L. Repeat these dilution

steps for stock solution II. [NOTE: Care must be

exercised in the mixing step so that the analysts

hands do not touch the rim of the flask since the

solution, when poured out, will become contaminated

with potassium.]

Immediately aspirate each of the 0.05 mmol/L

potassium solutions and measure their relative

intensity values under the instrumental settings

used for this analysis. If the relative intensity

values corrected for concentration differences for

both solutions agree to within 0.5 percent, potassium stock standard I may be used for the analyses

on day 1 and stock standard II may be used for the

analyses on day 2 subject to temperature restrictions. If the relative intensity values do not

agree within 0.5 percent, discard both stock

standard solutions and repeat their preparation

17
and the intercompar ison test until the requirement

of 0.5 percent agreement is obtained.

d. Diluted Potassium Standard Solution : Prepare the

various diluted potassium standard solutions by

transferring the appropriate volumes of the potassium

stock standard solution listed in Table 3 to 250-mL

volumetric flasks and dilute to the calibrated volume

with the 140 mmol/L NaCl diluent. Mix thoroughly.

[NOTE: These dilutions are made using volumetric

pipets in the "to deliver" mode, rather than the

"to contain" mode discussed in 4b- (3) below.]

Table 3. Volumes of potassium standard stock solution

diluted to 250 mL that give the potassium

diluted standard solutions.

Stock Solution to be

Transferred, mL

3. 00

5.00

10 . 00

15.00

20. 00

25.00

Concentration of Diluted Standards

KC1, mmol/L

0.96

1.60

3.20

4.80

6.40

8.00

4 . Dilution and Pipetting Procedures

a. General : A one hundred-fold or two-hundred fold

dilution is to be used as required by the instrument employed.

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

one hundred- fold "or two hundred-fold by employing

only one 5-mL pipet with a wash-out technique and

either 500-mL or 1-liter volumetric flasks. (The

18
working solutions are prepared with the one pipet

and wash-out technique to eliminate errors that may

be caused by differences in drainage between aqueous

and serum solutions.) Two blanks are necessary with

instruments using lithium as an internal standard:

1) the LiCl diluent (IIIC-3a) used as the blank for

samples and standards, and 2) the NaCl diluent

(IIIC-3b) diluted with the LiCl diluent used as a

blank for the potassium standards (see Appendix D,

Note 6)

.

(1) One Hundred-fold Dilutions: Transfer approximately 400 mL of LiCl diluent into a 500-mL

volumetric flask and then add 5 mL of the sample

or stock standard solution by the procedure

described in step (3) below.

(2) Two Hundred-fold Dilutions: Transfer approximately 900 mL of LiCl diluent into a 1-liter

volumetric flask and then add 5 mL of the sample

or stock standard solution by the procedure

described in step (3) below.

(3) Pipetting Procedure: Fill the 5-mL pipet to

approximately 1.0 cm above its calibration mark,

withdraw the pipet from the container, and wipe

the delivery tip with a clean, absorbent paper.

Contact the tip to the side of a clean waste

container and allow excess solution to drain until

the meniscus is at the calibrated mark on the pipet.

Remove the pipet from contact with the container

and direct the delivery tip of the pipet into the

receiver. Deliver the sample by contact of the

pipet tip with the wall inside the volumetric flask

and allow the solution to drain fully. After

drainage stops, gently expel the residual liquid.

Wash off the outside of the pipet tip into the

19
receiver with about 4 mL of LiCl diluent delivered,

for example, from a wash bottle or a disposable

Pasteur or similar pipet. (Caution: New, disposable pipets need to be cleaned.) Rinse the 5-mL

volumetric pipet three times by filling with fresh

LiCl diluent from a separate beaker, each time

delivering the contents into the receiver by

drainage against the inner wall of the flask above

the liquid level. Dilute to the calibrated volume

with the LiCl diluent and mix thoroughly.

(4) Preparation of Working Solutions:

(a) Working Blank Solution and Working Standard

Solutions: Prepare the working solutions of the

blank solution and the working 0.96-, 1.60-, 3. 20-,

4. 80-, 6. 40-, and 8.00-mmol/L potassium standard

solutions by making dilutions in appropriately

labeled volumetric flasks in the order cited.

Condition the 5-mL pipet by filling it with the

solution to be diluted. Discard this pipetful and

repeat filling and discarding twice more. Then

refill the pipet with the solution, adjust to the

calibrated volume, and deliver into the volumetric

flask to be used for the dilution. Rinse the pipet

by filling it three times with the LiCl diluent,

each time delivering the rinse solution into the

volumetric flask. Fill the flask to the calibrated

volume with the LiCl diluent. Wash out the pipet

three times with water (see Appendix D, Note 7)

and expel the residual liquid.

(b) Working Sample Solutions: Condition the 5-mL

pipet with some of the sample to be diluted in the

following way: (1) Draw ^2 mL of the sample into

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

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

20
(4) tilt the pipet to a horizontal position,

(5) allow a small volume of air to leak in and

rotate the pipet so that the conditioning liquid

wets all the internal surface to approximately

0.5 cm above the calibration mark, (6) discard this

conditioning solution, and (7) repeat steps (1-6) . Then prepare the working solutions as described in

sections IIIC-4b-(l) or (2) and (3), i.e., fill the

5-mL pipet with the sample, adjust volume to the

mark, deliver, rinse three times into the volumetric

flask with LiCl diluent, dilute to the calibrated

volume, and mix. Finally, wash out the pipet three

times with water (Appendix D, Note 7) . For each

of the next sample solutions to be diluted, repeat

step (4) -(b).

Semiautomated Pipetting Alternative : To prepare

working solutions, the blank, standard and sample

solutions are diluted either one hundred-fold or

two hundred-fold by using a pipettor-dilutor

device to deliver either 0.250 or 0.500 mL into

appropriately labeled 50-mL volumetric flasks. A

single delivery tube on the pipettor-dilutor and

the wash-out technique are used throughout. Two

blanks are prepared for instruments using lithium

as an internal standard: i.e., the LiCl diluent

(Section IIIC-3a) is used as the blank for samples

and standards and the NaCl diluent (IIIC-3b),

diluted with the LiCl diluent, is used as a blank

for the potassium standards (see Appendix D,

Note 6)

.

(1) One Hundred-Fold Dilutions: Transfer approximately 20 mL of LiCl diluent (or water) into a

50-mL volumetric flask and then add 0.500 mL of

21
the appropriate solution by the procedure described

in step (3) below.

(2) Two Hundred-Fold Dilutions: Transfer approximately 20 mL of LiCl diluent (or water) into a

50-mL volumetric flask and then add 0.250 mL of

the appropriate solution by the procedure described

in step C3) below.

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

sample either 0.250 or 0.500 mL and to dilute with

5 mL of diluent. After conditioning the pipettordilutor as in Appendix D, Note 3b, dip the delivery

tip of the pipettor-dilutor into the solution to

be transferred. Draw up the desired volume of

solution (0.250 or 0.500 mL) . Care must be taken

to avoid air bubbles in the tubing before or

during this operation. Withdraw the tip of the

delivery tube from the solution, touch the tip to

the container side, and remove the container.

With care not to touch the open end of the tip of

the tube, wipe the outside of the delivery tube,

direct the tip of the tube into the 50-mL volumetric flask, and deliver the aliquot and diluent

solution into the flask. Rinse the delivery tube

twice more by delivering two additional 5-mL portions of diluent through the tube into the 50-mL

volumetric flask. [NOTE : To minimize foaming and

spattering, deliver the stream of solution and

diluent on the wall inside the neck of the flask.]

After delivery is complete, touch the tip of the

tube to the inside wall of the flask to transfer

any solution remaining outside the tube. Remove

the volumetric flask, dilute to the calibrated

volume with the appropriate diluent, and mix.

22
(4) Solution Preparation:

(a) Prepare the working blank, standard, and

sample solutions by the procedures described in

Sections c(l), (2), and (3).

(b) At the conclusion of the dilution procedure,

appropriately labeled flasks with the following

working solutions should be ready for analysis:

(1) For the Manual Pipetting Alternative:

(a) One (or two) working blank(s)

;

(b) Six working standards;

(c) A working solution for each serum

sample to be analyzed.

(2) For the Semiautomated Pipetting Alternative:

(a) One (or two) working blank(s)

;

(b) Six working standards;

(c) A working solution for each serum

sample to be analyzed.

5. Flame Atomic Emission Spectroscopy Measurement

Procedures

It is not possible to provide detailed instructions

for each type of instrument to assure necessary

instrument stability, linearity, flame conditions,

etc. The operator must be familiar with the

instrument used. The instrument should meet all

the manufacturer's specifications. In general, the

accuracy of the method cannot be attained unless the

instrument is in optimum operating condition. Air

and propane are used as oxidant and fuel, respectively. The instruments that are currently in use

for FAES measurements may be classified into two

groups: internal standard and non- internal standard instruments. Each group is considered briefly.
For the internal standard instruments, the concentration of the internal-standard LiCl must be kept

uniform throughout the analysis since the potassium

emission signal is measured relative to the lithium

emission signal.

a . Internal Standard Instruments :

(1) Instrument Adjustment:

The most commonly used internal standard

instruments employ filter ' monochromators '

,

automatic gas-flow control systems and automatic ignition devices. Choose the correct

series of filters for the analyses. After

starting the instrument, turn on the air

supply (adjust to manufacturer's recommended

pressure) , open the valve on the propane fuel

tank, and allow the instrument to warm-up for

at least 15 minutes while aspirating the LiCl

diluent. Check the flame appearance and

aspiration rate to assure that the nebulizer

burner system is free of foreign materials.

(2) Instrument Stability:

Determine the stability and repeatability of

the instrument as follows:

(a) Adjust the instrument to a zero reading

while nebulizing the LiCl diluent. [NOTE:

Always nebulize LiCl diluent when measurements

of the working blank, standard or sample

solutions are not being made. Adjust the

instrument so that the LiCl diluent reads

•zero' at all times.]

(b) Nebulize the working potassium standard

solution obtained from the 8.000 mmol/L

24
standard solution and adjust direct read-out

instruments so that a reading of 8.000 units

is observed.

(c) Check the instrument zero with LiCl

diluent and readjust as necessary.

(d) Repeat steps (2) (a) -(c) until stable

conditions are achieved. Readings for the

same solution should agree within 0.5 percent

of full scale.

Non-Internal Standard Instruments :

Instrumental Adjustments:

(a) After turning on the instrument and

adjusting the wavelength to 766.5 nm, adjust

the slit as recommended by the manufacturer.

(b) Open the propane and air supply valves

and adjust the secondary regulators as recommended by the manufacturer.

(c) Ignite the gas and adjust the flow rates

for the fuel and oxidant as recommended for

the instrument. Check the flame appearance

and nebulization rate to assure that the

nebulizer burner system is free of foreign

materials

.

(d) Nebulize water into the flame for at

least 10 min; then make a fine adjustment of

wavelength by nebulizing one of the working

standards and adjusting the wavelength

selector until a maximum signal is obtained.

25
(2) Instrument Stability:

Determine the stability and repeatability of

the instrument as follows:

(a) Adjust the instrument to zero while

nebulizing water. [NOTE: Always nebulize

water when measurements of working standard,

blank, or sample solutions are not being

made. Water should give a reading of 'zero'

at all times .

]

(b) Nebulize the working standard obtained

from dilution of the 8.00 mmol/L standard and

adjust the instrumental gain so that for

digital read-out instruments a reading of at

least 2.000 units is observed.

(c) Check the instrument zero with water and

readjust as necessary.

(d) Repeat steps (2) (a) -(c) until stable

conditions are achieved. Readings should

reproduce within 0.5 percent of full scale.

c . Determination of the Calibration Curve :

(1) Nebulize the working solutions of the blank and

the potassium standards and record their relative

intensity values. (A typical data sheet is given

in Appendix D .

)

(2) Subtract the value for the blank from the

values obtained with the standard solutions, and

plot these corrected relative intensity values

versus the calculated potassium concentrations on

rectilinear graph paper. A typical calibration

curve is shown in figure 2. The calibration curve,

using a least squares linear fit, should show a

standard deviation of fit of 1 percent or less.

26
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

POTASSIUM CONCENTRATION, mmol/L

Figure 2. Typical calibration curve for the determination

of serum potassium by flame atomic emission

spectroscopy

.

27
The standard deviation of fit can be calculated

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

least squares fitted calibration line:

S fit "J 2J Cdi

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

If on visual inspection, one point of the plot

exhibits a large residual from a smooth curve

drawn through the remaining points , remeasure that

standard solution. If the value for the solution

continues to exhibit the large deviation, prepare

that standard solution again, remeasure it, and

compare the values obtained, as in steps c(l) and

(2). (See Statistical Analysis Section V-A-3d.)

d . Sample Measurements :

(1) Nebulize a working sample solution and select

the two working standard solutions whose emission

intensities most closely bracket that of the

s amp 1 e

.

(2) Nebulize the lower working standard, the

working sample, and the higher working standard in

that order and record each reading in the set.

(3) Repeat step d(2) until 5 valid sets are

obtained, as illustrated in section e, below.

(4) Repeat steps d(l), (2), and (3) for all of

the samples.

28
e . Valid Sets of Readings :

Sets of readings are considered valid if the

following condition is met:

The emission intensities for the sample and the

two standards in a set may not differ by more than

2 percent from any of the corresponding values in

the previous valid set. [NOTE: The first set

measured is considered to be valid. Non-valid

sets are discarded.

]

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

since each difference between the intensities for

the Low Standard (Set2"Set^ = +0.01), the Sample

(Set

2

-Set

1 = +0.01) and the High Standard (Set^Se^

= -0.01) is less than 2 percent. Note, however that

set 4 is not valid because two differences, i.e.,

between the Low Standard values (Set^-Set^ = +0.05),

and Sample values (Set^-Set^ = +0.08), are outside

the 2 percent limit. Just one such difference would

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

6 comprise the group of 5 valid sets.

Table 4. Example of intensity values for sets of readings

using a direct read-out instrument.

Low Standard

Set 1.601 mmol/L

1 1.61

2 1.62

3 1.61

4 1.66

5 1.61

6 1 .60

Sample

High Standard

3.201 mmol/L

1.98 3.22

1. 99 3.22

2.00 3.20

2. 08 3. 21

1.99 3.21

2.00 3.22

29
£ . Data Recording and Calculations:

(1) On the data sheet, record the concentrations

of the standard solutions in mmol/L of potassium

to four significant figures and the measured

relative intensity values to as many figures as

given by the instrument.

(2) Calculate the concentration C of potassium

present in the sample in mmol/L by mathematical

interpolation as follows:

(C

2 -C,).(Y-X,)

C = C, + — — (2)

(x

2

-x l}

where

C is the sample concentration of potassium in

mmol/L

,

C^ is the low standard concentration of potassium

in mmol/L,

is the high standard concentration of

potassium in mmol/L,

Y is the relative emission intensity of the

sample minus that of the blank (the LiCl

diluent or water reading that was initially

set at '0')

X^ is the relative emission intensity of the low

standard minus both blanks (the diluted

sodium chloride solution blank and the LiCl

diluent blank) , and

%2 is the relative emission intensity of the

high standard minus both blanks.

30
(3) Record the calculated C values to four

significant figures in the column provided on the

data sheet.

(4) Average the results for the four aliquots of

the serum analyzed to obtain the 'final concentration' .

IV. RESULTS AND STATISTICAL ANALYSIS

The main objective of the statistical analyses of the

round robin data is to derive measures of precision and

accuracy for the manual and semiautomated versions of the

reference method. Precision is characterized by the variability of the protocol measurements within a single laboratory, Within* and by the total variability of a laboratory's

protocol measurements, °^ 0 ^ a i'This latter uncertainty

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

method and definitive method values and is related to the

magnitude of the bias.

Each reported data point (test result) is the end

product of five valid flame atomic emission spectrometer

reading sets, the number of valid readings specified by the

protocol. For simplicity of discussion, each reported data

point is referred to as a single measurement , meaning that

each is the product of a single run-through of the protocol.

When "replication" is mentioned, replication of the entire

protocol process is meant, and "replication error" thus

refers to the variability among the end results of repeated

run-throughs of the protocol. Each round robin is discussed

separately; the final, detailed statistical analysis is

reported for the results from RRII.

31
A. Round Robin Results

1. Preliminary Round Robin (Dates Run: June-August

1975) .

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

protocol and to determine inter laboratory precision.

b. Samples : Three vials, each containing a sample

from the same serum pool. Each participating

laboratory was to analyze a single portion of each

sample within one day.

c. Procedure : The manual pipetting protocol was used.

d . Data : The three data points reported by the individual laboratories are summarized in Table 5. The

data are presented graphically in figure 3 as the

percent differences from the collective average of

the reported values. All reported values except

those from laboratory 10 are within 4 percent of

the collective average with a standard deviation

of ±0.05 mmol/L. No explanation could be determined

as to why the results from laboratory 10 were so

different from the average. These results were

considered to be outliers and were not included in

the calculation of the standard deviation. (NOTE:

see comments on protocol deviation after RRI . ) No

major problems were encountered in the performance

of the protocol.

e. Direction : On examining these results with the

statisticians and the Experts Committee, it was

concluded that a round robin should be undertaken

using samples with potassium concentration values

determined by the definitive method. A semiautomated pipetting alternative was written into

the protocol.

32
POTASSIUM, PRELIMINARY ROUND ROBIN

E -1

-2

-3

W

/ \

• CO CO CO N N N

'

I

'

J J I 3457891011 12 13

LABORATORY NUMBER

Figure 3. Percent deviations of individual results from the

collective average of the measurements obtained

in the Preliminary Round Robin test.

33
Table 5. Serum potassium concentrations reported by the

participating laboratories for the Preliminary

Round Robin, manual pipetting protocol.

[K

+

] , mmol/L a

Laboratory Vial 1 Vial 2 Vial 3 Laboratory Av

3 4.62 4.62 4. 59 4.61

4 4.77 4.70 4.81 4. 76

5 4. 70 4.64 4.66 4.67

7 4.68 4.65 4.75 4.69

8 4. 64 4.62 4.62 4.63

9 4. 73 4. 74 4.74 4.73

10 3. 35 3. 36 3.35 3.35

11 4.66 4.67 4.66 4.66

13 4.60 4.80 4.60 4.67

Collective Average 4.68b

cl Each value represents a single measurement on a sample.

b Does not include value from laboratory 10.

2. Round Robin I (RRI . Dates Run: November 1975 -

January 1976.)

a - General : The addition of the semiautomated pipetting alternative to RRI was considered advantageous

because the manual and semiautomated pipetting

versions could be evaluated simultaneously on the

same serum samples. The semiautomated version would

be used in suitably equipped laboratories with

consequent economies in reagents and labor; whereas

the manual version would be used in laboratories

having equipment basic to the method but lacking the

appropriate seimautomated sampling device.

A review and test of the capabilities of positive

displacement pipettor-dilutors demonstrated that

34
the precision and accuracy requirements listed in

the protocol could be met. Consequently, a method

for testing the pipettor-dilutor was included in

the protocol.

b. Ob j ect ives : To test the manual and semiautomated

pipetting alternatives on serum samples having a

wider range of potassium values and determine the

imprecision and bias of the test results.

c. Samples : RRI was a test series run on 12 samples — four vials of each of three different concentrations (Pools 1, 4, and 5). Each laboratory was to

analyze two vials of each pool on one day and the

remaining pairs of samples on a subsequent day

with the requirement that a minimum of one day and

a maximum of seven days should elapse between

analyses

.

d. Protocol : The manual and semiautomated pipetting

protocols were used.

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

laboratories for both pipetting alternatives are

summarized in Tables 6 and 7. The data are presented

graphically in figures 4 and 5 as percent deviations

of each one-day 'single measurement' average from

the definitive method value. In general, the data

reported by most laboratories were within 5 percent

of the definitive method values, although several

results for the manual pipetting alternative (lab 7 — pool 1 — day 1; lab 8 — pool 4 — day 1; lab 15 —

pools 1, 4, 6 — day 2) were outside this limit.

Excluding the excessively high values, statistical

analysis of the remaining results showed that the

imprecision and bias goals set by the Experts Committee had been reached in RRI testing. The

35
statistical analysis of the data for RRI yields

imprecision and bias values that are similar to

those obtained for RRI I. [NOTE: The RRII data are

discussed in Section IV-A-3.] It is interesting

to note that the results that differed extensively

from the definitive method values were all high —

in fact, for each pool in RRI, the average difference

for all the laboratories results from the definitive

method values (i.e., ^ bs""^^) wer e positive by an

average of approximately 1.3 percent. One laboratory

showed that contact of the analysts hand with the

rim of the volumetric flasks used for the preparation of the working solutions can result in potassium

contamination, which in turn, leads to potassium

values that can be 10 percent high. It is suggested

that care be exercised in sample and glassware

handling

.

A complete statistical analysis for the final round

robin, RRII, is presented later; therefore no

detailed tables summarizing these results are presented here. Considering the caveat that statistical

sampling in terms of the number of participating

laboratories and samples is considered to be

'limited', the following observations could be made

for RRI: a) at the highest potassium concentration,

the imprecision value approached the goal of 0.1

mmol/L; b) the imprecisions increase with increasing

potassium concentration; and c) the bias becomes

more positive as the potassium concentration

increases

.

Laboratory 15 repeated the analysis (data labelled

15R) since on the initial analysis instrumental

and blank problems were encountered.

36
Table 6. Concentrations of serum potassium reported by the

participating laboratories for Round Robin I,

manual pipetting protocol.

Laboratory Pool 1

[K], mmol/L'

Pool 4 Pool 6

Day 1 Day 2 Day 1 Day 3 Day 1 Day 2

4 1. 325

1.311

1. 306

1.314

4. 323

4.345

4.332

4. 390

6. 130

6. 092

6.127

6.142

4XC1. 306

1.311

1. 299

1. 310

4. 341

4. 362

4. 311

4. 305

6. 191

6. 134

6. 114

6.118

5 1. 292

1. 272

1.312

1. 370

4. 386

4.404

4. 370

4. 374

6. 034

6.102

6. 204

6. 192

7 1. 796

1.656

1. 356

1. 316

4. 504

4.334

4. 404

4. 338

6. 106

6. 172

6. 044

6.098

8 1 .398

1.328

1.374

1 .370

4.568

4 .736

4 .468

4.390

6 .388

6 .406

6 .198

6 .114

11 1. 304,

b

1. 301

1.291

4. 293

4. 399

4.352

4. 391

6.109

6. 060

6.182

6. 144

13 1. 335

1. 350

1 . 326

1. 326

4. 335

4. 344

4. 360

4. 320

6. 166

6.159

6.135

6. 120

15RC

1. 352

1.377

1.652

1.429

4.420

4. 390

4.932

4.200

6.098

6.087

6. 855

6. 395

Definitive

Method Values 1

.

319 4. 323 6. 092

a Each value is the single measurement average of five valid

FAES readings made on a single sample dilution.

Value not reported.

c Repeated analysis, instrumental problems.

37
Table 7. Concentrations of serum potassium reported by the

participating laboratories for Round Robin I,

semiautomated pipetting protocol.

- " [K], mmol/L a

- Laboratory Pool 1 Pool 4 Pool 6

Day 1 Day 2 Day 1 Day 3 Day 1 Day 2

4 1. 349

1. 343

1.321

1. 319

4. 312

4. 291

4. 397

4. 412

6. 155

6.095

6. 206

6. 246

4X C1. 311

1. 353

1. 298

1. 306

4. 373

4. 389

4. 353

4.450

6. 030

6. 149

6. 183

6.168

10 1. 300

1. 300

1. 380

1.304

4. 202

4. 316

4. 346

4.358

6. 154

6. 214

6. 134

6.136

11 1.264ub

1. 374

1.409

4. 387

4. 387

4. 576

4. 576

6.418

6.251

6.228

6. 284

15RC 1.297

1. 384

1. 371

1. 364

4.372

4. 374

4 . 337

4.402

6. 167

6. 151

6. 150

6. 144

15Ad 1.298

1. 312

1. 386

1.299

4. 329

4. 337

4.377

4.380

6. 139

6. 137

6.138

6. 125

Definitive

Method Values 1. 319 4. 323 6. 092

Each value is the single measurement average of five valid

FAES readings made on a single sample dilution.

Value not reported.

Repeated analysis, instrument problems.

^ Dilution was 1 in 201 and not 1 in 200.

38
10

uj 8

M 6

z

°

I— "J

< s

>UJ 2

UJ 2* O p £5 0

4J Tt O UJ

1 Q -2

4

6- j_L

1 2

4

POTASSIUM, ROUND ROBIN I MANUAL PIPETTING PROCEDURE

JUUL

in

CO

1

r

1 J-L-'-

1 2

4X

1 2

5

1 2

7

1 2

8

1 2

11

1 2

13

p=, SEiricei

\ /

1 2

15X

LABORATORY NUMBER AND DAY RUN

Figure 4. Percent deviations of the Round Robin I measurements using manual pipetting from the definitive

method values. The analyzed pools are identified

by the numbers 1, 4, and 6 next to the data from

laboratory 4. The designations are similar for

the remaining results. The numbers 1 and 2,

placed directly above the laboratory number,

designate the first day and subsequent day test

results, respectively. The letter 'X' after the

laboratory number designates a repeat analysis.

39
10

8

POTASSIUM, ROUND ROBIN I SEIVi (AUTOMATED PIPETTING PROCEDURE

2 P

< S

> UJ

O uj

as a

6-

4

2 -

0

-2

-4 -

1

6

n

- 4

JUIn

i Ml j

-6 -

JL 1 ±1 JL j-Ll

12 12 12 12 12 12

4 4X 10 11 15X 15A

LABORATORY NUMBER AND DAY RUN

Figure 5. Percent deviations of the Round Robin I measurements using semiautomated pipetting from the

definitive method values. The analyzed pools are

identified by the numbers 1, 4, and 6 next to the

data from laboratory 4. The designations are

similar for the remaining results. The numbers

1 and 2, placed directly above the laboratory

number, designate the first day and subsequent

day test results, respectively. The letter 'X'

after the laboratory number designates a repeat

analysis and 'A' designates a 201-fold dilution.

40
Comments and Protocol Deviations : The following

laboratory comments germane to changing the

protocol or signifying deviations from the protocol

were received:

(1) Lab 4 : Encountered instrument problems;

consequently, working samples and standards

for day 1 manual procedure, had been prepared

8 days before being measured. The diluted

samples had been stored at 4 °C. A similar

problem was encountered during performance of

the semiautomated pipetting alternative. RRI

was repeated with new set of samples.

(2) Lab 7 : Noted working standard solutions do not

aspirate the same as working serum solutions.

(3) Lab 10 : Dilutions of the stock standard solutions were made to 200 mL rather than 250 mL

.

Direction : A second round robin test (RRII) was to

be run using both the semiautomated and manual

pipetting alternatives. Test samples would cover

the full range of potassium concentrations.

Round Robin II : (RRII. Dates Run: July -

November 1976.)

Ob j ect ive : To test both the manual and semiautomated pipetting alternatives of the protocol on

samples with potassium concentrations over the

nominal range of 1.32 to 7.33 mmol/L.

Samples : RRII was a test series run on a total of

20 samples — four vials of each of five different

potassium concentrations (Pools 1, 2, 4, 5, and

7) . Each laboratory was to analyze two vials of

each concentration on the first day and the

41
remaining pairs of samples after the elapse of a

minimum of one day and a maximum of seven days.

c. Protocol : The manual and semiautomated pipetting

versions of the protocol were used.

d. Data and Statistical Analysis : Results from RRII

are given in Tables 8-9 and illustrated in Figures

6-7. The data are presented as two-way tables in

which the rows represent the different participating laboratories and the columns represent the

different sample pools. The sample pool concentrations ranged from approximately 1.3 to 7.3 millimoles

of potassium per liter of serum. The results for

the manual procedure and for the semiautomated

procedure are listed separately, and all single

measurements reported are included in the tables.

The definitive method values for the potassium

concentrations in the sample pools are listed at

the bottom of Tables 8-9.

A detailed statistical analysis was made. First

the data were inspected by calculating the percent

deviation of each day's results for each pool from

an average for that sample pool. These percent

deviation values for all laboratories and the two

pipetting procedures are listed in Tables 10-11.

42
Table 8. Concentrations of serum potassium reported by the

participating laboratories for Round Robin II,

manual pipetting protocol.

[K] , mmol/L

Laboratory Pool 1 Pool 2 Pool 4 Pool 5 Pool 7

1-1 1.319

1.318

2. 572

2.553

4. 375

4. 349

5. 565

5. 546

7. 495

7.394

1-2 1. 279

1.353

2. 653

2 .697

4.411

4.416

5.202

5.631

7. 133

7.306

2-1 1.318

1.381

2.628

2. 589

4.368

4. 366

5. 514

5.522

7 .386

7. 360

2-2 1. 473

1.421

2 . 566

2.601

4.498

4. 340

5.640

5.660

7 .396

7 .409

5-1 1.278

1.283

2. 520

2 . 527

4.299

4.272

5.532

5. 508

7. 347

7 .396

5-2 1.378

1.359

2 . 582

2 . 592

4.401

4.355

5.712

5 . 507

7. 507

7. 460

7-1 1.340

1. 344

2. 573

2 . 572

4.334

4. 342

5. 539

5. 557

7. 386

7 .418

7-2 1.372

1. 363

2. 600

2 .600

4. 393

4.394

5 .604

5.595

7.392

7 .386

8-1 1.324

1.336

2 . 552

2 . 548

4.338

4.328

5. 508

5. 520

7.362

7 .344

8-2 1.298

1.290

2. 538

2 . 532

4. 314

4. 304

5. 480

5.488

7 .340

7.316

9-1 1.292

i ? ft n 1 . L O U

2. 509

? A7 A

4.257

A ? ft 1 5. 543

J t J JO

7 .467

7 466

9-2 1.293

1.274

2. 502

2. 509

4 .251

4.202

5.423

5.423

7 .458

7. 420

10-1 1. 306

1.312

2. 560

2.540

4.440

4.412

5. 586

5.602

7.340

7 .314

10-2 1.328

1. 334

2 . 542

2 . 536

4.334

4.336

5.478

5.480

7.384

7 .380

continued

43
Continuation of Table 8.

[K] , mmol/L

Laboratorya Pool 1 Pool 2 Pool 4 Pool 5 Pool 7

11-1 1.323

1. 309

2.597

2. 574

4 . 443

4.425

5.613

5.644

7 . 393

7. 399

11-2 1.302

1.297

2.526

2 . 563

4.283

4. 258

5 . 536

5. 515

7 . 364

7. 360

13-1 1. 344

1.280

2. 580

2 . 527

4. 338

4. 400

5.611

5. 589

7. 480

7.493

13-2 1.404

1.417

2.622

2 . 588

4. 331

4. 387

5.467

5.465

7. 453

7.383

Definitive

Method Values 1.319 2 . 540 4.323 5 . 501 7 . 326

a The laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

44
Table 9. Concentrations of serum potassium reported by the

participating laboratories for Round Robin II,

semiautomated pipetting protocol.

[K] , mmol/L

Laboratory Pool 1 Pool 2 Pool 4 Pool 5 Pool 7

1-1 1.349

1. 359

2.629

2.626

4.428

4.420

5. 574

5. 588

7.350

7. 395

1-2 1.261

1. 288

2 . 456

2.475

4.285

4.205

5. 346

5. 349

7. 082

7.112

2-1 1.375

1.372

2. 582

2 . 528

4.388

4.355

5.573

5. 548

7.423

7. 390

2-2 1.398

1. 501

2. 586

2 . 578

4.378

4. 549

5.573

5. 569

7.425

7.409

9-1 1.269

1.235

2 .474

2. 467

4. 356

4.289

5. 517

5. 505

7.461

7.474

9-2 1.206

1.205

2 .441

2 . 388

4. 211

4. 204

5. 553

5. 544

7. 465

7.463

10-1 1. 294

1. 292

2. 514

2.484

4.224

4. 168

5.460

5. 506

7. 434

7 . 382

10-2 1.240

1.276

2 . 496

2 . 394

4.312

4. 264

5. 518

5. 406

7 . 294

7.262

11-1 1. 269

1 292

2 . 543

2.588

4.178

4.280

5.463

5 .424

7.308

7 . 415

11-2 1.274

1.250

2 .462

2 .463

4. 295

4.277

5.451

5. 494

7 . 359

7.366

15-1 1.377

1.430

2 . 503

2 .482

4.271

4.241

5.467

5.513

7.424

7.390

15-2 1.291

1.344

2.577

2 . 582

4.320

4. 309

5.497

5. 487

7.350

7 . 403

Definitive

Method Values 1 . 319 2 . 540 4 .323 5.501 7 .326

a The laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

45
anivA aoHiaiAi aAiiiNuaa ihi

IAIOUJ NOI1VIA3Q IN33U3d

nd a3 P

bfl 0 +-> P X ' P

•H +-> a3 CD +J P 3 m >^ bond cr (D,DH 0) CD P, tfl u ifl HU CD aj X)

Ph CD Td rH

p

•H fl, «

i—I <-H 0

03 -H rCj Csl rrj P p

03 CD £ • o3

X

,|—' i—| o3 bO HTj P CD •h fn u in +j w tij o ^ in 3 +J CD h

i/i nj Xl -H

(fl

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CD Ph aj

X

g rH CD +-> CD Td X fn CD g H CD P N O +-> in o3 03 rH <-H • Pi

CD 03 i/i

M

g

p (/)P -H

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CO ,p •H

g

CD +J

P

P

Tj 3 -Hp. P rH fn 03 >^ P 03 03 g >,rH O > CD CD fn CD C£ P fH O >

TJ

P -H

<D

O IN (D Oj fJ

X rP X) fiO

PP id

P

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CD

P

X Ph

o

g aj fn o3 t/)

COPo

•HP

03

O rH CD CD *<4H fn > LO CD •H fn XI - P •» 03 P CO rH P P -H CD rH •H - g > p

•H 4H N-H

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CD f-i >-,+->

P

X to aj rH t/i

p

p fn -P CD CD CD to O P U g X P CD

fH

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CD fn

p .H -H 03 Cm m P +-> T3 Td

CD

fnP

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46
<

a

o

10

8

6

4

2

0

-2

-4

-6

-8

POTASSIUM, ROUND ROBIN II SEMIAUTOMATED PIPETTING PROCEDURE

oo

I

CO

o

QC

o

<

o

QC

1 2*7

F

i i

1 1

1

CO

CO

I

J I L I ' j_L J-L.L 12 12 12 12 12 12

1 2 9 10 11 15

LABORATORY NUMBER AND DAY RUN

Figure 7. Percent deviations of the Round Robin II measurements using semiautomated pipetting from the

definitive method values. The analyzed pools are

identified by the numbers 1, 2, 4, 5, and 7 near

the results from laboratory 1. The designations

are the same for the remaining results. The

numbers 1 and 2, placed directly above the laboratory number, designate the first day and

subsequent day test results, respectively.

47
Table 10. Percent deviations from averages for potassium in

serum from Round Robin II, manual pipetting

protocol

.

Laboratory3 Pool 1 Pool 2 Pool 4 Pool 5 Pool 7

1-1 - .95 - . 10 . 33 . 72

1-2 -1.14 4.28 1.48 -2. 18 -2. 32

2-1 1.38 1.69 .41 - .35 - .25

2-2 8. 70 .72 1 .60 2 .04 .15

5-1 -3.81 -1.62 -1.47 - .31 - .27

5-2 2 . 80 .85 .66 1.30 1.25

7-1 .81 . 29 - ,26 . 19 .14

7-2 2 . 73 1.36 1.02 1 .12 - .03

8-1 - .09 - .59 - .37 -.42 - .52

8-2 -2 . 79 -1.17 - .93 - .96 - .86

9-1 -3. 39 -2.87 -1.85 .06 1.02

9-2 -3.58 -2 . 32 -2.82 -2.06 .65

10-1 -1.67 - .59 1 . 76 1.02 -.87

10-2 - .01 -1.02 - . 33 -1,05 - .13

11-1 - 1 14 79 1 95 1 65 06

11-2 -2 . 38 - .80 -1.81 - .21 - .40

13-1 -1.44 - .45 .45 1.13 1.29

13-2 5.96 1. 56 .22 -1.29 .36

Averages

used in 1 . 332 2 . 565 4 . 349 5 .537 7. 391

calculations

The laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

48
Table 11. Percent deviations from averages for potassium in

serum from Round Robin II, semiautomated pipetting

protocol

.

Laboratory8 Pool 1 Pool 2 Pool 4 Pool 5 Pool 7

1-1 3.34 4.55 2.88 1.53 .06

1-2 -2.73 -1 .90 -1.29 -2.72 -3.68

2-1 4.82 1.66 1 .66 1.16 .52

2-2 10 .62 2 . 74 3.80 1.35 .66

9-1 -4.45 -1 . 70 .52 .26 1 .35

9-2 -8.00 -3.93 -2 .16 .94 1 . 30

10-1 -1.32 - .57 -2.43 - .25 . 54

10-2 -3.99 -2.72 -.29 - .63 -1.22

11-1 -2.27 2 . 08 -1.66 - 97 - 09

11-2 -3 .69 -2.02 - .33 - .44 - .08

15-1 7.11 - .83 -1.03 - .13 . 53

15-2 .55 2 .64 .33 - .09 .11

Averages

used in 1 .310 2.513 4.300 5.497 7.368

calculations

The laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory number

and the last digit represents either the first or second

day 1 s results

.

A comparison was next made of the ability of each

laboratory to replicate its values relative to that

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

deviation for each day's measurements for each

pool against the laboratory averaged standard

deviation for that pool (see Tables 12-13), If all

of the participating laboratories were of the same

population in regard to replication error, the

standard deviation ratios reported in Tables 12-13

49
Table 12. Ratios of standard deviations to average standard

deviations for potassium in serum from Round Robin

II, manual pipetting protocol.

Laboratory Pool 1 Pool 2

1-1 .05 .90

1-2 3.45 2 .08

2-1 2.94 1.85

2-2 2.42 1.66

5-1 . 23 .33

5-2 .89 .47

7-1 .19 .05

7-2 .42 .00

8-1 . 56 .19

8-2 .37 .28

9-1 . 56 1.66

9-2 .89 .33

10-1 . 28 .95

10-2 .28 .28

11-1 .65 1 .09

11-2 .23 1 .75

13-1 2 .98 2 .51

13-2 .61 1.61

Average

Standard

Deviation,

mmo 1 /

L

.0152 .0149

Pool 4 Pool 5 Pool 7

.84 .40 2.79

.16 9.07 4 78

. 06 • 17J- / 72

5.11 42 36

.87 * 51*J -X. 1 35

1 .49 4 . 34 1 .30

.26 . 38 88

.03 19 • 17J- / .32 . 2 5 50

.32 .17 .66

. 78 11 .03

1 CO

. UU 1.05

.90 .34 .72

.06 .04 .11

.58 .66 .17

.81 .44 .11

2.00 .47 .36

1 .81 .04 1.94

.0219 .0334 .0256

The laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

50
Table 13. Ratios of standard deviations to average standard

deviations for potassium in serum for Round Robin

II, semiautomated pipetting protocol.

Laboratory^ Pool 1 Pool 2 Pool 3 Pool 5 Pool 7

1-1 .33 .10 .15 .46 1.27

1-2 .88 .66 1 . 52 .10 .85

2-1 .10 1.86 .63 .83 .93

2-2 3.35 .28 3.25 .13 .45

9-1 1.11 .24 1 .27 .40 .37

9-2 .03 1.83 .13 .30 .06

10-1 .07 1.03 1 .06 1 .52 1.47

10-2 1.17 3.52 .91 3.70 .91

11-1 .75 1.55 1 .94 1.29 3.03

11-? 7 R 0 X X A 1 A 1 1 . 4 L . 2 0

15-1 1 .72 .72 .57 1.52 .96

15-2 1 .72 .17 .21 .33 1.50

Average

Standard

Deviation

,

.0217 .0205 .0372 .0214 . 0250

mmol/L

The laboratory designation consists of two parts: the

initial digit (s) represents the assigned laboratory number

and the last digit represents either the first or second

day's results.

would be larger than 2.43 and 2.36, respectively,

only about one percent of the time. In practice, it

is not too uncommon to encounter a few standard

deviation ratios that are somewhat larger as this is

a reflection of some heterogeneity of the laboratory

population in regard to replication error. (As long

as the standard deviation ratios are not too large,

this is normally not used as a reason for rejection

of a laboratory. It is advised, however, that

51
laboratories with large standard deviation ratios

should reexamine their procedures for possible

sources of excessive replication error.)

The calculations on round robin II data were made

on the data in the two-way tables using a weighted

least squares fit to the following model [14]

:

Y. ., = y . + 3- (X. - X) + A. . + e. (3) ijk K

i i

v j

J ij ilk *

where

:

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

laboratory, for the j sample, and for the

k

1^

replicate measurement,

y^ = a constant factor associated with the

average bias for laboratory i,

(3^ = a slope factor for laboratory i_ f expressing

the relation of bias to concentration,

X. = the observed average concentration for

sample pool j (this average is taken over

all laboratories)

,

X = the weighted average concentration for all

samples (this average is taken over all

laboratories and over all sample pools)

,

= a random sample interference factor (matrix

effect) for laboratory i and sample pool j

,

and

e^jk = a random replication error.

The above model is quite general and extensive experience has shown that it is well suited to describe a

number of measurement factors in interlaboratory tests

[15].

52
Weighted analyses of variance were made on the data

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

model. (A modified version of the weighting procedure reported in reference 16 was used.) From the

analyses it is possible to derive the following

estimates for three components of variability, each

characterized by its standard deviation:

°e

= °e(Repl)

= t ^ie uncer 'ta i n

't: y observed for

replicate measurements in a given

laboratory on a given day,

a

D

= a Day= t ^ie a dditional uncertainty that is

observed when measurements are

made on different days within the

same laboratory, and

a T = ^

= the additional uncertainty that is

observed when measurements are

made by different laboratories.

These components of standard deviation are given

in Table 14.

Table 14. Components of standard deviation in mmol/L for all

Round Robin II potassium levels (1.3-7.3 mmol/L).

a e(Repl) °Day °Lab

Manual Pipetting Protocol .039 .048 .030

(Pooled results from 9 labs)

Semiautomated Pipetting Protocol .034 .065 .039

(Pooled results from 6 labs)

53
From the analyses, it was observed that the ranges

of values for the a

£

, a^, and c? L components of

standard deviation were small, and that the values

did not depend significantly on the potassium concentration. Because of this, only average a , a n , and

values are reported.

Because of the relatively small size of the potassium

round robin tests, the individual components of

standard deviation are considered to be only advisory

in nature. Nevertheless, they do seem to indicate

that the three components (o , a^, and a^) are all of

about the same order of magnitude. The final,

practical statements of uncertainty are made through

the recombination of these components. One such

a final statement is a

w ^-t j1 ^ n

> the expected uncertainty

within a single laboratory from running the complete

protocol (2 replicates/day for 2 days). The a

w^-(- n ^ n

results are reported in columns three and seven

in the top section of Table 15, and are calculated

as follows:

"2 " :

°within = ^

+ — *

These are the expected uncertainties that a single

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

A

variability of its results. This a

w^ t }1 jLn

i s not tne

total uncertainty since there is also a "between

laboratory" component, ^ Lab - The standard deviation

of the total uncertainty expected as a result of a

single laboratory running the complete protocol is

calculated as follows:

54
a Total

a

D

+ a. (5)

Columns four and six in the top section of Table 15

list such standard deviations for the manual and

semiautomated data from round robin II. The precision goal for the reference method is listed in

column five. Comparison of the tabulated standard

deviations and the goal shows that the precision

goals have been met.

Table 15. Summary of imprecision and bias results in mmol/L for

potassium in serum, Round Robin II.

K Level

mmol/L

1.3-7.3

Manual

Pipetting Protocol

la Precision

Q

comp °within

.016 .039

total

.049

Semiautomated

Pipetting Protocol

Goal

.100

a

total

.063

gwithin q

comp

.049 .026

K Level

mmol/L

1.3

2.5

4.3

5.5

7.3

Manual

Pipetting Protocol

Round Robin

Composite Bias

(Xobs"

X

DM}

.013

.025

.026

.036

.065

Accuracy

Goal

+ .200

±.200

±.200

±.200

±.200

Semiautomated

Pipetting Protocol

Round Robin

Composite Bias

^obs'^W

-.009

-.027

-.023

-.004

.042

55
The standard errors of the round robin composite

values are given in columns two and eight of the

top section of Table 15. These standard errors

are calculated from the components of standard

deviation as follows:

where N represents the 9 or 6 laboratories participating in the manual or semiautomated procedures,

respectively. The bottom section of Table 15 lists

the observed biases between the reference method

round robin composite values and the definitive

method values. The observed biases are within the

goals for the reference method.

Table 16 lists the composite round robin II sample

averages ± twice the standard error for the manual

and for the semiautomated versions, and for the

corresponding definitive method values.

The accuracy of the round robin results is within

the recommended goal of the reference method. The

biases tend to become more positive at the higher

potassium concentrations. The biases, however,

are very close to zero and are in general not

significant

.

56
Table 16. Summary of potassium in serum values ±2 standard

errors

Round Robin II - Composite Values Definitive Method

(mmol/L) _ Values (mmol/L)

Manual Semiautomated

1.332 ± .032 1.310 ± .052 1.319 ± .003 a

2.565 ± .032 2.513 ± .052 2.540 ± .006

4.349 ± .032 4.300 ± .052 4.323 ± .011

5.537 ± .032 5.497 ± .052 5.501 ± .014

7.391 ± .032 7.368 ± .052 7.326 ± .018

a Estimated maximum error of 0.25 percent as reported by NBS

Analytical Mass Spectrometry Section. This estimated

maximum error includes both imprecision and an estimated

upper bound for possible systematic errors. The estimated

maximum error is believed to be equal to or greater than

the true error for the 95 percent confidence limits.

Auxiliary Statistical Analysis

The protocol requires a check on the flame emission

spectrometer by running a calibration curve each

day using freshly prepared standard solutions. The

necessity of these curves also provides a check on

the correct preparation of the standard solutions.

The data reported here on the calibration curve

check are advisory in nature since in the actual

analytical procedure only the pair of calibrating

solutions nearest to the unknown concentration is

used. The calibration curve data for the manual

and semiautomated sodium procedures were reported

and are given in Tables 17-18. Straight line least

square fits were made to these data and the resultant

standard deviations of fit are given in Table 19.

These standard deviations of fit are expressed in

units of potassium concentration (mmol/L) . Our

57
Table 17. Calibration curve data for potassium in serum, Round Robin

II using manual pipetting.

Laboratory3

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

1-1

h

.96 1.60 3.20 4.80 6.40 8.00

Y

c

94 1 54 J3 • It14 4 74 7 Q7

1-2 X .96 1.60 3.20 4.80 6.40 8.00

YX 92 1 57 J»3 XU1 8 4 97 \jfS • *JU 36 o . uj

2-1 X .96 1.60 3.20 4.80 6.40 8.00

Y 1 00 1 70 3 24 4 83 6 4? 7 QQ

2-2 X .96 1.60 3.20 4.80 6.40 8.00

YJ. 96 1 62 3 24 4 82 \J6 m TX41 7 97

5-1 X .96 1.60 3.20 4.80 6.40 8.00

Y 98 1 59 3 22 4 78 6 37 7 99

7-1 X .96 1.60 3.20 4.80 6.40 8.00

Y1 1 91 3 20 6 40 9 68 1 2 83 1 5 99

8-1 X .96 1.60 3.20 4.80 6.40 8.00

Y1 97 1X • 61U X O3 • 19X z? 4 80 6 40

8-2 X .96 1.60 3.20 4.80 6.40 8.00

Y 97 1 6? 3 ?f) t4 . 8?O L fS 4? o . uu

10-1 X .96 1.60 3.20 4.80 6.40 8.00

Yi . vo -L.J/ 1 Q 4 8? 6 4Q o . UJ

10-2 X .96 1.60 3.20 4.80 6.40 8.00

Y .96 1.59 3.18 4.79 6.40 7.95

11-1 X .96 1.60 3.20 4.80 6.40 8.00

Y .93 1.60 3.21 4.73 6.41 8.01

11-2 X .96 1.60 3.20 4.80 6.40 8.00

Y .94 1.62 3.21 4.80 6.45 8.02

The laboratory designation consists of two parts: the initial

digit (s) represents the assigned laboratory number and the last

digit represents either the first or second day's results.

k X = Standard solution values in mmol/L.

r

Y = Instrument reading.

58
Table 18. Calibration curve data for potassium in serum, Round Robin

II using semiautomated pipetting.

Laboratory5

Std. 1 Std. 2

1-1 x

b

.96 1.60

vc

Y .94 1.60

1-2 X .96 1.60

Y .95 1.62

2-1 X .96 1.60

Y 1 .02 1.64

2-2 X .96 1.60

Y 1 .05 1.64

10-1 X .96 1.60

Y .95 1.58

10-2 X .96 1.60

Y .94 1.57

11-1 X .96 1.60

Y 1 .01 1.55

11-2 X .96 1.60

Y .90 1.58

15-1 X .959 1.598

Y .932 1.614

15-2 X .959 1.598

Y .978 1.664

Std. 3 Std. 4 Std. 5 Std. 6

3.20 4.80 6.40 8.00

3.16 4.74 6.44 8.05

3,20 4.80 6.40 8.00

3.23 4.74 6.42 8^06

3. 20 4.80 6.40 8 00

3.21 4.80 6.40 7.98

3.20 4.80 6 40 8 00

3.25 4.84 6.42 7.97

3.20 4 80 6 40 8 00

3.21 4.85 6.44 8.01

3. 20 4.80 6.40 8 00

3.26 4.86 6.45 7.99

3.20 4.80 6.40 8 00

3 19 4 90 6 30 7 96

3.20 4.80 6.40 8.00

3.26 4.85 6.53 8.07

3.197 4.795 6.394 7.992

3.302 4.928 6.404 7.980

3.197 4.795 6.394 7.992

3.210 4.826 6.404 7.960

The laboratory designation consists of two parts: the initial

digit (s) represents the assigned laboratory number and the last

digit represents either the first or second day's results.

X = Standard solution values in mmol/L.

r Y = Instrument reading.

59
Table 19. Calibration curve results for potassium in serum

as standard deviation of fit Cs-£- t

) in mmol/L.

Manual Semiautomated - ory Number S fit Laboratory Numbera

s fit

1-1 .025 1-1 .039

1-2 . 086 1-2 .043

2-1 . 025 2-1 .012

2-2 .023 2-2 .020

5-1 .018 10-1 .024

7-1 .019 10-2 .045

8-1 . 007 11-1 .077

8-2 .011 11-2 . 041

10-1 .024 15-1 .073

10-2 . 017 15-2 .024

11-1 . 035

11-2 .021

The laboratory designation consists of two parts: the

initial digit(s) represents the assigned laboratory

number and the last digit represents either the first

or second day's results.

analysis indicates that if in the calibration step

it is found that any calibration point deviates

from the calibration curve by more than 0.08 mmol/L,

then the standard solutions and the instrument

should be checked for sources of excessive error

before proceeding further into the analysis.

60
V. DISCUSSION

A . Candidate Protocol :

1. Preliminary Tests

Generally, in the development of a reference method

where the state of analytical knowledge leaves an uncertainty

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

analytical conditions, minimized interferences, and freedom

from other sources of bias. Such preparation helps avoid

initiating the interlaboratory testing process with inappropriate procedures. However, in the case of potassium, the

similarity of results obtained by White and Mavrodineanu

using FAES and the similarity of their results with those

obtained using the highly specific IDMS method, led the

Committee to decide to proceed directly into the round robin

testing phase with the FAES method, without further preliminary

studies

2. Specifications

In light of the prior experience [3,4,7,17], the written

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

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

grade or equivalent chemicals, 'tested' water, analytical

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

In addition, the reference method provides for the use of

analytical techniques that should reduce the combined error

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

3. Flame Atomic Emission Spectroscopy

Specific instructions are not given for the use of

flame emission instruments. In general, all the instruments

used in the laboratories that participated in this study

61
provided excellent results. The FAES instruments that were

used are listed in Table 20. Internal and non-internal standard

instruments for which eight laboratories used 200-fold dilutions

and two laboratories used 100-fold dilutions provided essentially similar results. One laboratory used air-acetylene rather

than air-propane as oxidant-fuel without a problem. Thus

specifications other than the requirement for stable instrument

operating conditions are not presented. As in sample preparation and handling, the human element in achieving accuracy and

precision is critical. It is essential that operators be

thoroughly familiar with their instruments and alert to the

onset of instrumental difficulties.

The protocol initially required a one-percent agreement

for measurement sets to be considered valid. That requirement was changed to two percent at the July 1975 meeting of

the representatives from the participating laboratories. In

the discussion that led to this protocol change, the representatives affirmed that if their instruments were operating

optimally, agreement of successive sets of readings could be

obtained to within 0.5 percent. However, the precision of the

round robin results was not significantly degraded due to this

change

.

Instrument linearity requirements were not included in

the protocol since the bracketting method for obtaining valid

measurements was used to minimize the errors attributable to

instrumental drift. Nevertheless, on examination of the data

reported for the calibration curves, excellent linearity was

found over the range of potassium concentrations from 1.3 to

7.3 mmol/L. More than 85*percent of the calibration curves

showed standard deviations of fit of about 0.04 mmol/L or

less. A standard deviation of fit larger than 0.08 mmol/L

would clearly warrant a laboratory's investigation of its

operation of the procedure and/or preparation of the standard

solutions

.

62
coCU

•S

4-> cd

CU

4-1

OfC

•H

4-1 4J

CUP-

(3O

•H

4J3

rH

•HO

0)u

tico

encu

5-i P-l TCO

0) 4JiSPd

ISo <

C/3

5-1 CUc

5-1

PQ

CO

4-1 c

CU

5-i 4-1 CO

cO

CUa

cO&.o

5-i P-t U

•H

XX

00

•rlCOa

•H

COa.

•rl0

a)

5-i

X

X!

•He

CM

•He

CU

5-i P-l

CN

X

X

X

X

X

PS55

PS

55

Pi

55 CO • o

vo vO 00

VO vO vO PS

55

•He

(U

5-1 P-i

•rl0

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63
The use of the bracketing criterion for valid sets

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

needed for the semiautomated pipetting protocol. About 25

mL of working solution is required to obtain five sets of

valid measurements, assuming a nebulization rate of 2-4

mL/min for approximately 45 s to obtain a single reading.

(That time- interval is necessary for the instrument and

flame to be stabilized and for actual integration of the

signal.) Much larger volumes of diluted sample were available

with the manual pipetting protocol because of the large

aliquot volumes taken to ensure pipetting accuracy.

4. Statistical Analysis

All of the results discussed here are based on the analysis of four replicate samples analyzed as pairs on two separate

days. Adherence to this pattern of replicate analysis helped

assure the reliable performance of the reference method.

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

respectively, were in fact reached over the total concentration range by the laboratories using either the manual or

semiautomated pipetting protocols. Additionally, there were

no significant differences in the imprecision values obtained

by the two pipetting alternatives as evident in Table 15.

The imprecision values for both pipetting procedures are

approximately twice as good as the original goal set for the

reference method by the Experts Committee. Although the

observed composite bias values listed in Table 15 for both

the manual and semiautomated pipetting procedures are quite

small and considered inconsequential, the manual pipetting

procedure appears to show biases that are somewhat more

positive. This could be due to increased human contact with

the solution or glassware (manual pipetting) which results

in potassium contamination. The bias values are about five

times better than the goals, with the largest bias being about

equal to Q^otal ' Thus the agreement between the reference

64
method values and the definitive method values are considered

to be quite acceptable. From this statistical analysis, it

is concluded that for laboratories in the population typical

of those participating in this study (i.e., clinical laboratories that have practiced the reference method and are in

good quality control), imprecisions (^ total ) within 0.063

mmol/L and biases within 0.065 mmol/L can be expected in the

performance of this reference method (refer to Table 15)

.

VI. CONCLUSIONS

A 'candidate' reference method, specified by a written

protocol for the determination of serum potassium by flame

atomic emission spectroscopy was evaluated by analyzing serum

and aqueous samples in a selected group of laboratories. The

results for samples having potassium concentrations in the

1.3 to 7.3 mmol/L range showed a total imprecision of

approximately 0.06 mmol/L or less and a maximum bias of 0.06

mmol/L as compared to definitive method values for these

samples. The observed bias values, converted to a 'percent'

bias, are in general approximately one percent or less over

the total potassium concentration range. Similar imprecisions

were found whether manual pipetting, requiring large sample

volumes, or semiautomated pipetting, requiring small sample

volumes, was used. The imprecision and bias values for both

pipetting procedures were well within the goals set by the

experts committee. An isotope dilution — mass spectrometric

procedure was used as the definitive method to determine

potassium values in the pooled sera.

Statistical analysis of the results shows that the

candidate reference method can be carried out with the accuracy and precision expected of a reference method for serum

potassium. Hence, the 'candidate' method should be considered

to be the reference method. This reference method may be

used to establish the accuracy of field methods for potassium

65
by comparative testing. It may also be used to determine

reference serum potassium values. Each of these uses would

require an appropriate experimental design to ensure achievement of the desired accuracy and precision goals.

We would like to especially thank the principal

investigators and other scientists in the participating

laboratories (listed in Appendix A), who, through their

efforts, made this work meaningful and possible. We thank

Dr. David Bayse and Ms. D. Sue Lewis, CDC, for providing

excellent, homogeneous serum pools used in the interlaboratory testing process.

The work at NBS was carried out in the Institute for

Materials Research and we acknowledge Ms. Nancy Steele for

initial reduction of the PRR and RRI data. We thank

Dr. Philip D. LaFleur, Director, Center for Analytical

Chemistry, and Mr. J. Paul Cali, Chief, Office of Standard

Reference Materials, for their support and encouragement

throughout this program. Special thanks go to

Dr. Radu Mavrodineanu , Mr. Theodore Rains, and

Dr. Michael Epstein for discussions and suggestions in the

area of flame atomic emission spectroscopy. Acknowledgement

is also made to Mr. John Matwey for help in sample handling.

Special thanks are due to Mrs. Joy Shoemaker of the Text

Editing Facility for he-r efforts in manuscript production

which included formatting, editing, and typing.

The Bureau of Medical Devices of The Food and Drug

Administration is acknowledged for their financial support

of this project.

66
VII. REFERENCES

[1] Henry, R. J., Clinical Chemistry: Principles and

Technics, 1st Edition, Harper and Row, Publishers,

New York, N.Y. 1964, p. 350; Weissman, N. and Pileggi,

V. J. in Clinical Chemistry: Principles and Technics,

Henry, R. J., Cannon, D. C, and Winkelman, J. W. , Eds., 2nd Edition, Harper and Row Publishers, New York,

N.Y. 1974, p. 641.

[2] Benotti, J. in "Standard Methods of Clinical Chemistry",

Vol. I, M. Reiner, Ed. Academic Press, New York, N.Y.

1953, p. 102; Hold, P. M. and Mason, W. B. in "Standard

Methods of Clinical Chemistry", Vol. II, D. Seligson,

Ed., Academic Press, New York, N.Y. 1958, p. 165.

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

"A Referee Method for the Determination of Calcium in

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

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

Documents, U.S. Government Printing Office, Washington,

D.C. 20402 SD Cat. No. C13 . 10 : 260 - 36 .

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

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

Special Publication, 260 Series (1978) in process.

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

Chem. , 16_, 998 (1970) .

[6] Moore, L. J., in Reference 3, page 63.

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

Chem. , 1_9 (10) , 1208 (1973) .

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

and Moody, J. R., to be published.

[9] Available from: Office of Standard Reference Materials,

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

67
[10] Serum samples have been stored frozen for several years

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

electrolyte concentration. White, J. CDC, Private

Communication

.

[11] Thawed serum samples were mailed to the participating

laboratories. In previous experiences at CDC, thawed

samples mailed to the west coast and returned after two

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

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

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

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

Washington, D.C. 20025.

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

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

No. 2 , 86-97 (1976) .

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

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

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

Regression and Analysis of Variance", Technometrics

,

6 , 1-25 (1964) .

[17] Clinical reference methods are also being developed at

NBS for lithium, chloride, and magnesium.

68
APPENDIX A

Scientists not previously acknowledged who contributed to

this study are:

Dr. Robert Moore

Hartford Hospital

Mrs. Harriet Bailey

Dr. Daniel Grisley

New England Deaconess Hospital

Dr. Johan Kofstad

University of Oslo

Mr. Orlando Flores

Mr. James North

Beckman Instruments

Mr. Richard Schlough

University of Wisconsin

Miss Mary Dassow

Mrs. Shirley Wertlake

University of California

Mr. Frank Doherty

Pennsylvania Department of Health

Mr. Claude Walker

Food and Drug Administration

69
APPENDIX U. S. Department of Commerce

Frederick B. Dent

Secretary

National Bureau of Standards

Richard W. Roberts, Director

This standard reference material is certified as a chemical of known purity. It is intended

primarily for use in the calibration and standardization of procedures employed in the determination of potassium and chloride ions in clinical analyses. The sample consists of highly purified

potassium chloride. Chemical assay as well as analyses for specific impurities indicate that the

material may be considered essentially pure, except for moisture due to occlusion.

Purity 99.9 ± 0.0 percent

The above value for the purity of the material is based on a sample dried over magnesium

perchlorate for 24 hours. Potassium chloride is hygroscopic when the relative humidity at room

temperature exceeds 75 percent, but can be dried to the original weight by desiccation over freshly

exposed P2O5 or Mg(C104 ) 2 for 24 hours. The material should be stored with such a desiccant. The

potassium was determined by a combination of gravimetric and isotope dilution analyses. More than

99 percent of the potassium was precipitated, filtered, and weighed as potassium perchlorate. The

weight of potassium perchlorate was corrected for rubidium perchlorate. The soluble potassium was

determined by isotope dilution mass spectrometry. Total potassium was the sum of the potassium

from the potassium perchlorate and the potassium from the filtrate. The chloride was determined

by a coulometric argentimetric procedure.

Based on 12 independent measurements for each ion, the sample was considered homogeneous.

Material dried at 500 °C for 4 hours in a platinum or Vycor crucible (Pyrex is unsatisfactory) was

assayed at 99.98 ± 0.01 percent. The loss of moisture by this procedure was about 0.07 percent.

The potassium chloride used for this standard reference material was obtained from the J. T. Baker Chemical Company, of Phillipsburg, New Jersey. Analyses were performed by G. Marinenko,

T. J. Murphy, T. C. Rains, T. A. Rush, W. P. Schmidt, and V. C. Stewart.

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

The technical and support aspects concerning the preparation, certification, and issuance of this

standard reference material were coordinated through the Office of Standard Reference Materials

by J. L. Hague.

Potassium Chloride

(Clinical Standard)

Washington, D. C. 20234

January 22, 1971

Revised November 23, 1973

J. Paul Cali, Chief

Office of Standard Reference Materials

(over)

70
This material was examined for compliance with the specifications for reagent grade potassium

chloride as given in Reagent Chemicals, 4th edition, published by the American Chemical Society.

The material met or exceeded the minimum requirements in every respect.

A semi-quantitative survey for trace contaminants by emission spectroscopy indicated the

presence of less than 0.001 percent aluminum, copper, iron, and magnesium. A value of 0.24 parts

per million (ppm) of magnesium was obtained by atomic absorption spectrometry. Flame emission

spectrometry indicated the presence of the following elements: rubidium, 27 ppm; sodium, 9 ppm;

lithium, 0.6 ppm; and cesium and calcium, less than 2 ppm.

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

This material is intended for use as a standard for potassium and, to a lesser degree, for chloride

determination in clinical chemistry.

Potassium is most frequently determined by flame emission photometry. The operative details

of this methodology vary from instrument to instrument and are discussed at length in their

respective operating manuals. A standard solution of potassium chloride (10 mmol potassium per

liter) may be prepared as follows: transfer 0.746 g of SRM 918 that has been dried to constant

weight quantitatively to a 1-liter volumetric flask, dilute to volume with water, and make the

solution uniform by inverting the flask at least 30 times. The concentrations required for analysis

may be prepared by accurate dilution of this standard with deionized water.

A standard solution of chloride containing 100 mmol per liter may be prepared by transferring

quantitatively 7.46 g of SRM 918 to a 1-liter volumetric flask and adding 3 ml of concentrated

nitric acid (ACS Reagent Grade) and 100 ml of deionized water. After all the salt is dissolved, dilute to volume with deionized water. It should be noted that a chloride standard solution prepared from

sodium chloride contains the respective ions in a ratio more nearly that of normal serum than a solution prepared from potassium chloride.

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

normal laboratory conditions. It is recommended that weighing and other manipulations of the

solid SRM not be made when the relative humidity exceeds 75 percent.

The solutions of SRM 918 are stable indefinitely when stored in a well-stoppered, all-glass

container. All such solutions should be clear and display no turbidity.

References:

[1] P. M. Hald and W. B. Mason, "Sodium and potassium by flame photometry", in Standard

Methods of Clinical Chemistry, Vol. 2, David Seligson, editor-in-chief, pp. 165-185, Academic

Press, Inc., New York, (1958).

[2] N. W. Tietz, Fundamentals of Clinical Chemistry, pp. 621-625, W. B. Saunders Company,

Philadelphia, Pa. (1970).

This Standard Reference Material has been measured and certified at the laboratories of the

National Bureau of Standards, Gaithersburg, Maryland. All inquiries should be addressed to:

Office of Standard Reference Materials

Room B311, Chemistry Building

•National Bureau of Standards

Washington, D. C. 20234

The date of issuance and certification of this Standard Reference Material was January 22,

1971.

71
APPENDIX C

Isotopic Dilution Mass Spectrometry

The use of thermal - ionization mass spectrometry for

isotope analysis has a valid and well-described theoretical

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

accurately known systematic errors and high levels of

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

Isotope dilution analyses are performed by measuring the

change in the relative magnitude of two isotopes of the analyte

when a measured amount of one of these isotopes is added to

the sample. The method consists of the following steps:

(1) The addition of a known amount of a separated isotope

(spike) of the analyte to be determined to a weighed

serum sample. For high accuracy, this addition is

made as a weighed portion of a spike solution having

known isotopic composition and analyte concentration.

(2) Dissolution of the sample by appropriate means and

thorough mixing of the resulting solution to ensure

equilibration of the separated isotope with the

analyte in the sample. This may involve chemical

treatment to convert the analyte and the separated

isotope to the same oxidation state.

(3) Chemical separation of the isotopically altered

analyte from possible interfering elements and

into a form suitable for mass spectrometric analysis.

A major advantage of isotope dilution mass spectrometry (IDMS) is the fact that recoveries need not

be quantitative since only the ratios of the

isotopes are measured.

(4) Measurement of the altered isotopic ratio by

thermal ionization mass spectrometry.

72
(5) Calculation of the amount of the analyte in the

sample using equation 1:

W C[A - R B ] Concentration, yg/g = -5£

R ^

p

_ 5E- • g- (1)

s

where

W = Weight of spike solution, grams

sp

C = Concentration of spike, ymoles/gram of solution

A = Atomic fraction of isotope A in spike sp

B = Atomic fraction of isotope B in spike

A = Atomic fraction of isotope A in sample

B = Atomic fraction of isotope B in sample

R = Experimentally measured ratio

M = Atomic weight of element

W

g = Weight of sample, grams

This calculated concentration must be corrected for

the blank.

The possible sources of systematic error in isotope

dilution mass spectrometry (IDMS) are:

(1) Error in the calibration of the concentration of

the spike isotope. The spike solution is calibrated

against at least two different solutions of the pure

analyte containing 'natural' isotopic abundances

by what might be called reverse isotope dilution.

Whenever possible, NBS Standard Reference Materials

are used as the 'natural' material. The error from

this source will be the same as for the analyte

being determined and is due to the imprecision of

the ratio measurement.

(2) Chemical errors. In a well designed analysis, an

undetected chemical error should not arise if adequate precautions are taken against the following

73
potential sources of error. Errors might be

caused by:

a) Incomplete decomposition or dissolution of the

sample, a problem common to all wet analytical

methods

.

b) Loss of the analyte from the sample or spike due

to volatility or adsorption during dissolution.

These losses can usually be detected by spiking

some samples before dissolution and others after

dissolution

;

c) Incomplete mixing or equilibration of the spike

and the 'natural' analyte. This can be caused by

differences in oxidation state or the presence of

the 'natural' analyte in a complex or chelated form

This source of error can be eliminated by proper

chemical treatment, for example, by oxidation or

reduction and wet-ashing;

d) Isotope fractionation in the chemical treatment

if the separation is not quantitative. This is

seldom a problem but can occur with some techniques

Fractionation can be detected by isotopic analysis

of small amounts of 'natural' materials before and

after being subjected to the non-quantitative

separation procedure.

(3) Contamination or blank. Sources of contamination

or blank may be reagents, apparatus, or fall-out

from the laboratory atmosphere. The problem can be

minimized by carrying out the chemical operations

in a carefully controlled atmosphere and by using

special, high-purity reagents. The total blank

may be estimated by carrying a number of 'blanks'

through all the steps of the analysis. The average

blank value can be treated as a systematic error

74
and the average value obtained for the analyte in

the 'blanks' is used as a correction. The uncertainty of this correction is equal to the randomness

of its measurement, i.e., its coefficient of variation. For concentrations where the blank amounts

to a significant fraction of an analytical value,

the blank may become the largest source of error.

Interferences. Interference usually occurs between

elements with isobars; i.e., isotopes with the same

mass to charge ratio, and may be avoided by either

selecting, where possible, an isotope of the element

without isobaric interference, or by chemically

removing the interfering element. Conveniently,

most of the elements containing isobars are in different groups of the Periodic Table and separations

are not difficult. Thus, a concealed systematic

error should not arise from this source. For

example, although ^Ca and are isobaric, Ca

can be separated easily from K by cation exchange

40 chromatography. To ensure that the amount of K

is insignificant when measuring ^Ca, the mass

39 spectromist can monitor for K which is four orders

40 of magnitude more abundant than K in natural

potassium. In the present case, where potassium

39 concentrations are to be determined, isotopes K

and 41

K, which do not have isobaric interferences,

are used.

Instrumental errors. Instrumental errors may be

caused by mass discrimination or fractionation,

but usually cancel since the same percent error is

present in the ratio measurement for the spike

calibration. With some analytes, impurities in a

sample can cause a different fractionation pattern

from the pure material. These effects are usually

75
small (less than 0.11) and can be corrected by

repurifying the sample.

This review of possible sources of systematic error

shows that these errors can be eliminated or measured accurately for correction. However, random error components are

present in the isotope ratio measurements for the analyte

determination, the spike calibration, and the blank correction. If the blank correction is insignificant, then the

total error in a careful determination reduces to the combined

random errors for the spike calibration and the analyte

determination. The error of either measurement is reflected

in the precision of the isotopic measurements; for many

analytes, it is on the order of 0.05 to 0.25 percent at the

95 percent confidence level. Therefore, absolute accuracies

of 0.1 to 0.5 percent are possible even for very low concentrations in complex matrices. When the blank correction is

significant, the uncertainty from this source must be added

to the uncertainties from the ratio measurements.

Reagents, Columns, and Clean Laboratory

(1) Reagents : All acids and water were purified by a

sub-boiling distillation technique utilizing quartz

stills [1]

.

(2) Cation Exchange Column : A 0.7-cm ID ion exchange

column filled to an approximately 10-cm height with

100-200 mesh, strongly-acidic, cation- exchange resin

having eight percent crosslinkage was used for the

separations. The column of resin was cleaned by eluting

with 60 g of 5 mol/L HC1, followed by 10 g of H

2

0.

(3) Clean Laboratory : To reduce particulate contamination,

all the chemical preparations were carried out in a

Class-100, clean-air hood located in a vertical flow

clean room [ 2 ]

.

76
Procedure

The frozen serum samples were allowed to come to room

temperature and mixed by repeated O20) careful inversions

of the vials. A sample was quickly withdrawn from each vial

through a platinum needle (18 gauge) into a 10-mL plastic

syringe after the septum was opened just far enough to allow

the needle to enter the vial. Approximately 5 g samples,

weighed to 0.01 mg, were transferred to 50- or 10-mL Teflon

beakers. Weighed aliquots of separated- isotope solution

sufficient to give a

39 K/ 41

K ratio of approximately one, were

added to each sample. The samples then were decomposed by

adding 5 g of HNO^ and 5 g of HCIO^ and heating in the covered

beakers. After decomposition, the covers were removed and

the samples were evaporated to dryness. The acid on the sides

of the beakers was rinsed down with a minimum amount of f^O

and the samples were again evaporated to dryness. Each

residue was dissolved in 10 mL of L^O and the solution was

transferred to a cation exchange column. Approximately 10 mL

of was used to rinse the beaker and complete the transfer

of the sample to the column. Then 0.3 mol/L HC1 was added

as an eluting agent. The first 50 mL of the eluent was

discarded and the next 40 mL of eluent which contained the

K fraction was collected in a Teflon beaker. (Those volumes

may vary depending on the particular lot of resin; nearly

all of the Na should have been eluted and K should just be

starting to elute with this volume; this can be checked with

a flame test.) The solution was then evaporated to dryness.

To aid in the decomposition of the organic material from the

column, a few drops of HNO^ were added and the sample was

heated and evaporated to dryness. The residue was converted

to the chloride form by adding a few drops of 5 mol/L HC1

and evaporating to dryness. The residue was dissolved in

enough 0.2 mol/L HC1 to give a solution containing approximately 2.5 mg K/mL

.

77
Mass Spectrometry

Isotopic ratios were determined by solid sample, thermal

ionization mass spectrometry on 30-cm radius-of- curvature

,

90°-analyzer tube, mass spectrometers equipped with thin-lens

"Z"-£ocusing ion-sources and multielement, deep-bucket,

faraday-cage collectors. Details of the procedure for measuring the isotopic K ratios have been published [3]

.

Results

The potassium concentrations determined by isotope dilution mass spectrometry on seven lots of serum are given in

Table 1. Exclusive of Lot P3, the precisions (one standard

deviation) of the concentration determinations are equal to

or less than 0.05 percent. However, a realistic estimate of

the imprecision must also include uncertainty components for

the spike calibration, analytical blank variability and the

effects of impurities [4]. The estimated uncertainty at the

95 percent confidence level for the potassium concentrations

in Table 1 is ±0.25 percent and includes allowances for

unknown sources of systematic error.

78
Table I

Sample Sample

Lot No. Ug K/mL 23 °C Lot No. yg K/mL 23 °C

PI 1 51.583 P5 1 215.04

2 51.558 2 215.10

3 51.574 3 215. 10

4 51.578 Average 215.10

Average 51.573 a ±.03 (0.02%)

a ±.011 (0.02%)

P2 1 99.332 P6 1 238.12

2 99.364 2 238.26

3 99.272 3 238. 20

Average 99.323 Average 238.19

a ±.047 (0.05%) a ±.07 (0.03%)

P3 1 134.79 P7 1 286.34

2 134.70 2 286.49

3 154.94 3 286.49

Average 135.81 Average 286.44

a ±.12 (0.09%) a ±.09 (0.03%)

P4 1 168.97

2 168.99

3 169.09

4 169.02

Average 169.02

a ±.05 (0.03%)

79
References

[1] Kuehner, E. C, Alvarez, R. , Paulsen, P. J., and Murphy,

T. J., Anal. Chem. , 44_, 2050 (1972).

[2] Murphy, T. J., The Role of the Analytical Blank in

Accurate Trace Analysis, Nat. Bur. Stand. (U.S.) Special

Publication 422, Accuracy in Trace Analysis: Sampling,

Sample Handling, and Analysis, p. 509-539 (1900).

[3] Garner, E. L., Murphy, T. J., Gramlich, J. W., Paulsen,

P. J., and Barnes, I. L., J. Res. Nat. Bur. Stand.

(U.S.) 79A (Phys. and Chem.) 713-725 (Nov. -Dec. 1975).

[4] Garner, E. L., Machlan, L. A., Gramlich, J. W. Moore,

L. J., Murphy, T. J., and Barnes, I. L., An Accurate

Determination of Electrolyte Concentrations in Blood

Serum by Isotope Dilution Mass Spectrometry, Nat. Bur.

Stand. Special Publication 422, 951-960 (1900).

80
APPENDIX D

Note 1:

A temperature range of room ±2 °C is designated as the

operating temperature. In this temperature range the maximum

difference in aqueous solution volumes due to thermal expansion

of the liquid is 0.102 percent and the difference in volume

due to the volumetric glassware is very small since the coefficient of expansion for borosilicate glass is 0.00001 per °C.

(J. Lembeck, "Calibration of Small Volumetric Laboratory

Glassware", NBSIR Report 74-461, 1974, Institute for Basic

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

20234) . We judge these errors to be acceptable for this

reference method. Larger temperature variations may necessitate

appropriate correction.

Note 2:

Glassware Required:

a) Manual pipetting alternative:

Volumetric Flasks : (for one hundred-fold dilutions)

one 2-L; seven 1-L; six 250-mL; seven 500-mL plus one

additional 500-mL volumetric flask for each sample.

Volumetric Flasks : (for two hundred-fold dilutions):

one 2-L; six 250-mL; fourteen 1-L plus one additional

1-L volumetric flask for each sample.

Pipets : two 5-mL, five 25-mL, and one each of 3-, 10-,

15-, and 20-mL.

b) Semiautomated pipetting alternative:

Volumetric Flasks : (for one hundred-fold or two

hundred-fold dilutions): One 2-L; seven 1-L; six

250-mL; and seven 50-mL plus one 50-mL volumetric flask

for each sample.

81
Pipe ts : five 25-mL; and one each of 3-, 5-, 10-, 15-,

and 20-mL.

Note 3:

Cleaning of Glassware and the pipettor-dilutor:

a) Clean the glassware in the following manner:

(1) Soak glassware for 60 min in 0.77 mol/L HNO^.

(2) Rinse six times with a volume of water equal to at

least 10 percent of the container volume.

(3) Use immediately or air dry (inverted in a dustfree environment) for later use.

b) Clean the pipettor-dilutor device as follows:

(1) Rinse the tubing with water by delivering at least

four 5-mL water samples.

(2) Rinse the tubing with 0.77 mol/L HNO^ by drawing

into the delivery tube a volume of HNO^ equal to

the volume of sample pipetted and then delivering

four 5-mL portions of HNO^ through the system.

(3) Repeat step (2) using ^0, ethanol, and FL^O

sequentially.

(4) Repeat step (2) with the diluent to be used for

preparing the working solutions of the sample,

standards, and blank. The pipettor-dilutor is

then ready for the preparation of the working

solutions

.

Note 4:

Procedure for Testing Pipettor-Dilutor Devices : The accuracy

and precision of the device is determined by weighing fixed

volumes of water repetitively delivered by the device.

1. The water that is delivered in tared, stoppered flasks

is to be weighed on an analytical balance capable of

82
being read to the nearest one-tenth milligram. Measure

the temperature of the delivered water to the nearest

0.1 °C just before or after delivery.

2. Test the delivery of the 0.250 mL or 0.500 mL volume

(as will be used) as follows:

a. (1) Number and tare ten, clean, dry, stoppered,

glass or plastic weighing bottles of approximately

10-20 mL volume.

(2) Sample 0.250 or 0.500 mL of water and deliver

it together with 5 mL of diluent water into the

first bottle. Stopper immediately.

(3) Repeat step '2' with the remaining 9 bottles.

(4) Weigh each of the 10, filled bottles.

(5) Calculate the weight of each aliquot plus

diluent

.

b. Repeat steps 1-5 of part a, but in step 2 omit the

sampling of the 0.250 or 0.500 mL of water by

allowing air to be sampled rather than water; thus

only the 5 mL of diluent water is collected in the

tared bottles. Calculation then gives the weights

of diluent.

c. Calculate from part b the mean weight for the

diluent

.

d. Calculate the differences between the individual

weighings obtained in part a step (5) and the

mean weight of the diluent (from part c) to obtain

the weights of the water aliquots delivered at

the 0.250-mL or 0.500-mL setting that was used.

e. Calculate the mean and standard deviation for the

weights of water samples (from part d)

.

f. Use the attached table (#43) from Circular #19,

"Standard Density and Volume Tables," [National

Bureau of Standards, Washington, D.C. 20234] to

83
£oo CD i—i

CD

f-i 00

M CD •HU mo

ccj U CO

+-> CD c

4-> <u

cti

H

U

•Hrd

p) i—i

CTl (NI to

o oo r-» oo CTl rH OO to CTl i—1

to H" vO oo CTi rH to LO oo

o (NJ LO t~-- CTl (Nl CNI CNI (Nl (Nl CNI to to to to H" -5t O

r-- rH vO rH oo \o LO vo r>. cn (Nl vo rH vO

r—Ito VO CTl I—1to LO

O (NJ LO c-« oo (Nl CNI (Nl (Nl CNI (Nl to to to to H- o

vooo to vO CTl to oo

rH to vO

t

ct rH to LO cti (Nl

(Nl CNI (Nl (Nl CNI (Nl to to to to to ^f" ^ H" O

LO oo to 00 LO to CNI rH CNI rH vO rH I—1

(Nl LO CTi rH to LO CTl (N) t-- vo OO (Nl (Nl (Nl (N] (Nl to to to to to o

to t~~~ rH r-- to rH

O CTlo (Nl LO co to CTi rH (NJ LO cti rH CNI LO o- CTl rH vO

LO CNI CNI CN1 (N) CNI CNI to to to to to O

CNI LO

o LO CNI CTl OO ooo (Nl vOo vO

rH CNI LO 00

o CNl r~- CTi rH vO

•Sfr (Nl (NJ CNI (Nl (Nl (Nl to to to to to

to

(Nl I u

cd

CCj Pi H -H

CD

CL, CD CD S H H

<D 3 H +-> 00

CD

rH "=3" OO to

o vO LO 00

o 00 to

rH CN1 to LO 00

O (Nl vO CTi rH to vO

(Nl CNI (Nl (Nl CNI (NJ to to to to to

LO vO 00 CTi

O rH

(Nl (NJ

(Nl tO

CNI (Nl H"

(Nl

o to (Nl 00 LO to LO 00 rH vO rH

rH (Nl to LO vO OO

o (Nl vO 00 rH to vO 00

(Nl (Nl CNI (Nl (NJ CNI to to to to to ^t- H" o

00 rH LO

o vO <=3" (Nl rH (Nl to vO CTi to 00

o (Nl to LO vO 00

o (Nl vO 00

O to LO oo

(Nl (N| CNI (Nl (Nl CNI to to to to to H" H" H" H" o o H" CTi LO CNI o CTi o rH to vO rH vO (NJ o (Nl to vO 00 o rH vO 00 O to LO OO

(Nl (Nl (Nl (NJ (Nl (Nl to to to to to "=3"

LO vO C~- 00 CT>

(N] (NJ CNI CNI (Nl

84
convert the mean of the diluent weights (from part

c) and the mean of the sample weights (from part e)

into volumes at 20 °C, in the following manner:

1) Determine the volume of the nominally 0.250-mL

sample at 20 °C by adding to the mean value of the

delivered sample, (from part e) an amount equal

to the product of 0.0025 and the value for the

appropriate water temperature read from Table 43.

2) Determine the volume of the nominally 0.500-mL

sample at 20 °C by adding to the mean value of the

delivered sample, (from part e) an amount equal to

the product of 0.0050 and the value for the

appropriate water temperature read from Table 43.

The sums obtained are in milliliters.

3. The requirements for the bias and imprecision of the

pipettor-dilutor are listed in Table 1. The pipettordilutor may be used in the semiautomated pipetting

alternative if these requirements are fulfilled.

Table 1. Bias and imprecision requirements for the volume

of sample delivered by the pipettor-dilutor

device, Section III-A2.

Imprecision, Relative

Sample Size, mL Bias, mL Standard Deviation

0.250 ±0.005 0.21

0.500 ±0.010 0.21

Note 5

:

The use of SRM Li^CO^ is not recommended for this purpose.

However, if it is used, note the following:

a) The Li

2

c0 3 in NBS SRM 924 has been de P leted in the 6 Li

isotope. Thus the atomic weight of lithium in this SRM

85
is 6.9696 rather than the usual 6.941, and the molecular

weight of this Li

2

C0

3

is 73.9484 rather than 73.8912.

Thus, more of the SRM 924 Li

2

C0

3

is needed to obtain the

lithium diluent solution with the desired concentration.

b) The atomic weights used in this report are those reported

in: Pure and Applied Chemistry, 4J7 , 75 (1976).

Note 6:

There can be two blanks for the standards. The lithium

chloride diluent solution (or water) blank is nebulized to

set the instrument reading to zero. If the reading of this

blank is not zero, then its value and the blank for the

working solution of the sodium chloride diluent are to be

subtracted from the readings for the standards. Additionally,

if the lithium chloride blank reading is not zero, then its

value must also be subtracted from the readings obtained for

the working samples.

Note 7:

If the wash solution does not drain cleanly from the

pipet, wash with 0.77 mol/L HN0

3 , H

2 0, MeOH, 70:30 v/v

CHCl

3 :MeOH, MeOH, and H

2

0 in that order. Then repeat the

water wash and check that the pipet does drain properly.

Note 8:

The three following pages are examples of the data

sheets returned from each laboratory after each round robin

test

.

86
ELECTROLYTES IN SERUM - CLINICAL REFERENCE METHOD

I ON K

LABORATORY 2 ANALYST FD - LS

EXERCISE NO. RR II

DATE SAMPLES RECEIVED 777 DATES ANALYZED CD 8/2 ( 2 ) 8/4

INSTRUMENT MANUFACTURER IL, Inc. MODEL 143-02

WAVELENGTH NM SCAN SIT ON PEAK MAX

SLIT WIDTH yM

BURNER TYPE

OXIDANT Propane F LOW RATE 0-45 |_/MIN

FUEL Air FLOW RATE 1-42 L/MIN

INSTRUMENT TIME CONSTANT S

RECORDER TIME CONSTANT S

READOUT: RECORDER , DIGITAL , OTHER

LABORATORY TEMPERATURE 2 4 .

0

°c TO 26 .0 ° C (VARIATION DURING

ROUND ROBIN)

COMMENTS: Aspiration tube length- 9Q mm; inside diameter- 0.5 mm.

SRM 918 KC1 dried over anhydrous AI2O3. See attached sheet for

additional comments. [NOTE: Additional comments concerned

the protocol] .

87
DATA SHEET: STANDARD CURVE

Lab 2 PROTOCOL USED MANUAL SEMI-AUTOMATED X Day 1

CALCULATED I ON RELATIVE CORRECTED RELATIVE

STANDARD CONCENTRATION, MMOL/L INTENSITY VALUES INTENSITY VALUES

1

2

3

5

6

0.960

1.600

3.200

4.800

6.400

8.000

1.04

1.66

3.23

4.82

6.42

8 .00

1.02

1.64

3.21

4.80

6 .40

7.98

DILUENT BLANK

0.02

LITHIUM BLANK 0 .00

88
DATA REPORTING SHEET FOR VALID MEASUREMENTS

PROTOCOL USED: MANUAL SEMI-AUTOMATED

LAB 2 ION K ROUND ROBIN 11 DATE ANALYZED 8 / 2 OPERATOR FD - LS

SAMPLE # 236725 RELATIVE INTENSITIES

STANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

(X, )

1

C AMDI P (Y)

n 1 b 1 U

(x

2

)

C

LO 1-600 CCi 5 1 . 1.59 2 .57 3.18 2 .586

HI 3.200 (Co) 2 . 1.57 2.57 3.19 2 ,588

3. 1 .58 2 .56 3 .19 2 .574

4. 1.59 2.57 3 . 19 2 .580

5. 1 .59 2.57 3 . i y 2 .580

SAMPLE tt -2;?8737 RELATIVE INTENS ITIES

b 1 ANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

Cx

1 )

C AUDI P (Y)

(-IT n 1 CTH b 1 U

(x

2

)

C

LO 4 - 800 CC

X ) 1 . 4.80 5.57 6 .41 5 . 565

HI 6 ' 400 CCO 2 . 4.78 5 .57 6 .41 5 .575

3.

4.78 5.57 6.41 r r n r 5.575

4.

4.78 5.57 6 .43 5.566

5 . 4.78 D . D / J ,003

Q /I A Q

SAMPLE # y4Dy RELATIVE INTENSITIES

STANDARD

CONCENTRATIONS

MMOL/L (CALCULATED)

VALID

SET

LO STD

CX.)

1

SAMPLE

(Y)

HI STD

(x

2

)

A,c

LO 6.400 CCj)

HI 8.000 CC„)

1 . 6.43 7 .44 8.02 7 .416

2 . 6 .43 7.47 8.02 7 .447

3. 6 .44 7.43 8 .03 7 .396

4. 6.42 7 .46 8.05 7.421

5. 6 .43 7.46 8.02 7.436

89
NBS-114A (REV. 9-76)

U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET

1. PUBLICATION OR REPORT NO.

SP 260-63

2.Gov'L Accession No, 3, Recipient's Accession No.

—

4. title and subtitle standard Reference Materials:

A Reference Method for the Determination of Potassium

in Serum

5. Publication Date May 1979

S. Performing Organization Code

7. author(S) Ranee A. Velapoldi, Robert C. Paule, Robert

Schaffer, John Mandel, Lawrence A. Machlan, & John W. Grarolidh

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, DC 20234

10. Project/Task/Work Unit No.

11. Contract/Grant No.

12. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street, City, State, ZIP) 13. Type of Report & Period Covered

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

Library of 1 ongress Catalog Card Number: 79-600022

|

| Document describes a computer program; SF-185, FIPS Software Summary, is attached.

16. ABSTRACT (A 200-word or less factual summary of most si gni ficant information. If document includes a s i gni ficant bibliography or

literature survey, mention it here.) Guided by a committee of experts in clinical chemistry, a reference method was

established for the determination of serum potassium based on flame atomic emission

spectroscopy (FAES) . Its accuracy was evaluated by comparing the values obtained by

use of the method in 12 laboratories against the results obtained by a definitive

analytical method based on isotope dilution-mass spectrometry (IDMS) . Seven serum

pools with potassium concentrations in the range 1.319 to 7.326 mmol/L were analyzed.

Manual and semiautomated pipetting alternatives were tested using sample sizes of

5.0 and 0.25 ml, respectively.

The laboratories used several different FAES instruments. The results showed

that the standard error for a single laboratory's performance of the procedure ranged

from 0.049 to 0.063 mmol/L with a maximum bias of 0.065 mmol/L over the range of

concentrations studied. These values were within the accuracy and precision goals

that had been set by the committee. The results from the two pipetting techniques

were similar. The calibration curve data showed excellent linearity over the total

concentration range, with 20 of 22 curves having standard deviations of fit of 0.075

mmol/L or less.

With appropriate experimental design, the reference method may be used to

establish the accuracy of field methods as well as to determine reference sodium

values for pooled sera.

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name;

separa ted by semicolons) Clinical analysis; clinical chemistry; definitive method;

electrolytes; flame atomic emission spectroscopy; reference method; semiautomated

pipetting; serum potassium analysis.

18. AVAILABILITY [Xj Unlimited

I | For Official Distribution. Do Not Release to NTIS

LXl Order From Sup. of Doc, U.S. Government Printing Office, Washington, DC

20402, SD Stock No. SN003-003-02068-1

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