PUBLICATIONS

NBS SPECIAL PUBLICATION 260

U.S. DEPA^JMENT OF COMMERCE / National Bureau of Standards

Standard Referem

SRM 900, ANTIEPILEPSY

DRUG LEVEL ASSAY STANDARD
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards' was established by an act of Congress on iMarch 3, 1901

.

The Bureau's overall goal is to strengthen and advance the Nation's science and technology and facilitate their effective application for public benefit. To this end, the Bureau conducts

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

the Institute for Computer Sciences and Technology. THE NATIONAL MEASUREMENT LABORATORY provides the national system of

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

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

physical and chemical measurement throughout the Nation's scientific community, industry, and commerce; conducts materials research leading to improved methods of measurement,

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

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

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

calibration services. The Laboratory consists of the following centers: Absolute Physical Quantities2 — Radiation Research — Thermodynamics and

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

engineering standards and code changes; develops and proposes new engineering practices; and develops and improves mechanisms to transfer results of its research to the ultimate user. The Laboratory consists of the following centers: Applied Mathematics — Electronics and Electrical Engineering 2 — 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

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'Headquarters and Laboratories at Gaithersburg, MD, unless otherwise noted; mailing address Washington, DC 20234.

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

SRM 900, ANTIEPILEPSY

DRUG LEVEL ASSAY STANDARD

Dennis J. Reeder, Bruce Coxon, Delmo Enagonio, Richard G. Christensen, Barbara F. Howell, Robert C. Paule, John Mandel, and Robert Schaffer

National Measurement Laboratory

National Bureau of Standards Washington, DC 20234

Sponsored by the National Institutes of Health

National Institute of Neurological and Communicative

Disorders and Stroke

Federal Building Bethesda, MD 20205

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued June 1981
Library of Congress Catalog Card Number: 81-600062

National Bureau of Standards Special Publication 260-72

Nat. Bur. Stand. (U.S.), Spec. Publ. 260-72, 79 pages (June 1981) CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE WASHINGTON: 1981

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

Price $4.25 (Add 25 percent for other than U.S. mailing)
PREFACE

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

National Bureau of Standards are wel 1 -characteri zed 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

is carried out through' the mechanism and use of SRM's. For many of the Nation's scientists and technologists it is therefore of more than passing interest to know the details

of the measurements made at NBS in arriving at the certified

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

which this publication is a member, called the NBS Special

Publication - 260 Series , is reserved for this purpose.

This 260 Series is dedicated to the dissemination of

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

more detail will be found in these papers than is generally

allowed, or desirable, in scientific journal articles. This

enables the user to assess the validity and accuracy of the

measurement processes employed, to judge the statistical

analysis, and to learn details of techniques and methods

utilized for work entailing the greatest care and accuracy.

These papers also should provide sufficient additional information not found on the certificate so that new applications

in diverse fields not foreseen at the time the SRM was originally issued will be sought and found.

Inquiries concerning the technical content of this paper

should be directed to the authors. Other questions concerned

with the availability, delivery, price, and so forth will

receive prompt attention from:

Office of Standard Reference Materials

National Bureau of Standards

Washington, D.C. 20234

George A. Uriano, Chief

Office of Standard Reference Materials
OTHER NBS PUBLICATIONS IN THIS SERIES

Catalog of NBS Standard Reference Materials (1979-80 edition), R. W. Seward, ed., NBS

Spec. Publ. 260 (April 1979) $3.00* SN003-O03-

02048-6

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

Reference Materials: Preparation of White Cast Iron Spectrochemical Standards, NBS Misc.

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

Michaelis, R. E., Wyman, L. L., and Flitsch, R., Standard Reference Materials: Preparation of NBS Copper-Base Spectrochemical Standards, NBS Misc. Publ. 260-2 (October 1964). COM74-

11063**

Michaelis, R. E., Yakowitz, H., and Moore, G. A., Standard Reference Materials: Metallographic

Characterization of an NBS Spectrometric LowAlloy Steel Standard, NBS Misc. Publ. 260-3 (October 1964). COM74-1 1060** Hague, J. L., Mears, T. W., and Michaelis, R. E., Standard Reference Materials: Sources of

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

1965). COM74-11059

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

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

Misc. Publ. 260-5 (March 1965). PB 168068**

Shultz, J. L, 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). COM74-1 1065**

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

Michaelis, R. E., Standard Reference Materials: Homogeneity Characterization on NBS Spectrometric Standards II: Cartridge Brass and LowAlloy Steel, NBS Misc. Publ. 260-10 (December

1965). COM74-11064**

Napolitano, A., and Hawkins, E. G., Standard Reference Materials: Viscosity of Standard

Lead-Silica Glass, NBS Misc. Publ. 260-11 (November 1966). NBS Misc. Publ. 260-11** Yakowitz, H., Vieth, D. L., and Michaelis, R. E., Standard Reference Materials: Homogeneity

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

(September 1966). NBS Misc. Publ. 260-12** Spijkerman, J. L., Snediker, D. K., Ruegg, F. C,

and DeVoe, J. R., Standard Reference Materials: Mossbauer Spectroscopy Standard for the Chemical Shift of Iron Compounds, NBS Misc. Publ. 260-13 (July 1967). NBS Misc. Publ.

260-13**

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

Materials: Determination of Oxygen in Ferrous

Materials - SRM 1090, 1091, and 1092, NBS

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

Publ. 260-14**

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

of Standard Light-Sensitive Paper for Calibrat- ing Carbon Arcs Used in Testing Textiles for Colorfastness to Light, NBS Misc. Publ. 260-15 (June 1967). (Replaced by NBS Spec. Publ. 260^1.)

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

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

Powder Metallurgical Methods, NBS Spec.

Publ. 260-16 (January 1969). COM74-1 1062** Catanzaro, E. J., Champion, C. E., Garner, E. L., Marinenko, G., Sappenfield, K. M., and Shields, W. R. Standard Reference Materials: Boric

Acid; Isotopic and Assay Standard Reference

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

1970). Out of Print

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

G., Standard Reference Materials: Calibration of NBS Secondary Standard Magnetic Tape (Computer Amplitude Reference) Using the Reference Tape Amplitude Measurement

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

1969). (See NBS Spec. Publ. 260-29.) Paule, R. C, and Mandel, J., Standard Reference

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

Spec. Publ. 260-19 (January 1970). PB 190071**

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). COM7 1-00 157**

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

of Redox Standards, NBS Spec. Publ. 260-24 (January 1972). COM72-50058**

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

Michaelis, R. E., Standard Reference Materials: A Standard Reference Material Containing

Nominally Four Percent Austenite, NBS Spec.

Publ. 260-25 (February 1971). COM74-1 1356** Martin, J. F., Standard Reference Materials:

National Bureau of Standards-US Steel Corportion Joint Program for Determining Oxygen and Nitrogen in Steel, NBS Spec. Publ. 260-26

(February 1971). 85 cents* SN003-003-00786-2

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

Isotopic Standard Reference materials, NBS

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

11358**

Heinrich, K. F. J., Myklebust, R. L., Rasberry, S. D., and Michaelis, R. E., Standard Reference Materials: Preparation and Evaluation of SRM's 481 and 482 Gold-Silver and GoldCopper Alloys for Microanalysis, NBS Spec.

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

Geller, S. B., Standard Reference Materials: Cali- bration of NBS Secondary Standard Magnetic Tape (Computer Amplitude Reference) Using

the Reference Tape Amplitude Measurement

"Process A-Model 2," NBS Spec. Publ. 260-29 (June 1971). COM7 1-50282 Gorozhanina, R. S., Freedman, A. Y., and

Shaievitch, A. B. (translated by M. C. Selby), Standard Reference Materials: Standard

Samples Issued in the USSR (A Translation from the Russian). NBS Spec. Publ. 260-30 (June

1971). COM71-50283**

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

Materials: Thermal Conductivity of Electrolytic Iron SRM 734 from 4 to 300 K, NBS Spec. Publ. 260-31 (November 1971). 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-O03-O1213-1 Wagner, H. L., Standard Reference Materials: Comparison of Original and Supplemental

SRM 705, Narrow Molecular Weight Distri- bution Polystyrene, NBS Spec. Publ. 260-33 (May 1972). COM72-50526**

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

Materials: Thermoelectric Voltage, NBS Spec. Publ. 260-34, (April 1972). COM72-50371**

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

Austenitic Stainless Steel, SRM 735 from 5

to 280 K, NBS Spec. Publ. 260-35 (April 1972.) 35 cents* COM72-50368**

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

S., Standard Reference Materials: A Referee Method for the Determination of Calcium in Serum, NBS SRM 915, NBS Spec. Publ. 260-36 (May 1972). COM72-50527**

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

O., Standard Reference Materials: Methods of Analysis of NBS Clay Standards, NBS Spec.

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

Richmond, J. C, and Hsia, J. J., Standard Refer- ence Materials: Preparation and Calibration of Standards of Spectral Specular Reflectance, NBS Spec. Publ. 260-38 (May 1972). COM72-

50528**

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

Powell, R. L., Standard Reference Materials The Eddy Current Decay Method for Resistivity Characterization of High-Purity Metals, NBS

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

V
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. 260^1 (August 1972) COM72-50775**

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

Reference Materials: The Characterization of Linear Polyethylene, SRM 1475, NBS Spec. Publ. 260-42 (September 1972). COM72-

50944**

Yakowitz, H., Ruff, A. W., and Michaelis, R. E., Standard Reference Materials: Preparation and Homogeneity Characterization of an Austenitic Iron-Chromium-Nickel Alloy, NBS Spec. Publ. 260^3 (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). SNO03-OO3-0 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-O03-0 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* SN0O3-O03-0 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

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

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

01978-0

Verdier, P. H., and Wagner. H. L., Standard Refer- ence Materials: The Characterization of Linear Polyethylene (SRM 1482, 1483, 1484), NBS

Spec. Publ. 260-61 (December 1978). $1.70* SN003-003-02006-1

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

Materials: Temperature Reference Standard

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

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

02047-8 Velapolid, R. A., Paule, R. C, Schaffer, R., Mandel, J., Machlan, L. A., and Gramlich, J. W., Standard Reference Materials: A Reference Method for the Determination of Potassium in Serum, NBS Spec. Publ. 260-63 (May 1979). $3.75 SN003-003-02068

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

Reference Materials: A Fluorescence Standard

Reference Material Quinine Sulfate Dihydrate (SRM 936). NBS Spec. Publ. 260-64 (January

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

F. C, Standard Reference Materials: Micro- Homogeneity Studies of NBS Standard Reference

Materials, NBS Research Materials, and Other Related Samples, NBS Spec. Publ. 260-65

(September 1979). $3.50* SN003-003-02 1 14-1 Venable, W. H., Jr. and Eckerle, K. L., Standard

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

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

02127-0

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

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

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

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

003-02136-9 Mavrodineanu, R. and Baldwin, Jr., Standard

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

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

Velapoldi, R. A., Paule, R. C, Schaffer, R., Mandel, J., Machlan, L. A., Garner, E. L., and

Rains, T. C, Standard Reference Materials: A

Reference Method for the Determination of Lithium in Serum, NBS Spec. Publ. 260-69 (July)

1980). $4.25* SN003-003-02214-4 Marinenko, R. B., Biancaniello, F., Boyer, P. A., Ruff, A. W., DeRobertis, L., Standard Reference

Materials: Preparation and Characterization of an Iron-Chromium-Nickel Alloy for Microanalysis, NBS Spec. Publ. 260-70 (in press). Reeder, D. J., Coxon, B.,Enagonio, D.,Christensen,

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

Mandel, J., Standard Reference Materials: SRM

900, Antiepilepsy Drug Level Assay Standard, NBS Spec. Publ. 260-72 (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.

V i i
TABLE OF CONTENTS

PAGE

1 . Introduction 2 2. Development of the SRM 2 2.1 Studies of the Drugs Used in the SRM 2 A. Chemical Structure, Name, Molecular Weight, Source 2 B. Initial Stability Testing and Solubility Studies 4

C. Thin-Layer Chromatography 4

D. Pulse-Fourier Transform Proton Nuclear Magnetic Resonance Spectroscopy

at 90 MHz of Fractions Obtained by Liquid Chromatography 4 2.2 Serum Base Developments 11 A. Choice of Matrix 11 B. Procedure for Preparation of a Human Serum Base 16

C. Addition of Drugs to the Processed Serum Base 16

D. Dispensing the Drug-Spiked Serum 17

E. Freeze-Drying the Spiked Serum 17

3. Protocol for Certification of the SRM 18

3.1 Homogeneity of the Reconstituted Samples 18 3.2 Quantitative Liquid Chromatography 18

A. Qualification of the Method 18

B. High Performance Liquid Chromatography Assay 24 3.3 Gas Chromatographic Analysis and Quantitation 25

A. Derivatized Drug Methods 25

B. Underivatized Drug Determinations 26 3.4 Analysis of Drugs in Serum by the Enzyme Multiplied Immunoassay Technique . . 30

A. Experimental Procedures 30

B. Results 30

C. Interferences 35

4. Statistical Analysis for Certification of SRM 900 36

5. Summary and Conclusions 36

6. SRM Use 36

A. Water for Reconstitution 36

B. Storage 38

7. References 38 Acknowledgements 38 Appendix I - National Bureau of Standards Certificate, SRM 900 39 Appendix II - Construction of an Improved Differential Refractometer 41 Appendix III - Reagent Water Specifications - College of American

Pathologists 47

viii
LIST OF TABLES TABLE NO. PAGE

1. Proton chemical shifts of antiepilepsy drugs 10

2. Quantitative analysis of drug mixtures in Sephadex-fractionated human serum by reversed phase liquid chromatography 11

3. Target values for drugs in serum (in yg/mL) 17

4. Homogeneity of reconstituted samples 18

5. Scintillation counting of fractions from liquid chromatography of phenytoin-4- 1Lf C in ethanol 19

6. Scintillation counting of fractions from liquid chromatography of

bovine serum solutions of phenytoin-4- ll*C 20

7. Scintillation counting of fractions from liquid chromatography of

an aqueous solution of ethosuximide-l- 14 C 21 8. Scintillation counting of fractions from liquid chromatography of bovine serum solutions of ethosuximide-1 - 1U C 22

9. Scintillation counting of fractions from liquid chromatography of phenobarbital-2- 1It C solutions 23 10. Scintillation counting of fractions from liquid chromatography of primidone-4,6- 1[*C2 solutions .' 24

11. Drugs and internal standards used in GC analysis 26

12. EMIT assays of phenobarbital in bovine serum 34

13. Comparison of EMIT determinations of drugs in SRM 900 35

14. Compilation of data from certification runs - 1978 37

ix
LIST OF FIGURES

FIGURE NO. PAGE

1 . Proton NMR spectrum of phenytoin 6

2. Proton NMR spectrum of ethosuximide fraction 2 from liquid chromatography .... 7

3. Proton NMR spectrum of ethosuximide 8

4. Proton NMR spectrum of phenobarbital 9

5. Proton NMR spectrum of primidone 10

6. Comparison of bovine serum with human serum using high performance

liquid chromatography 12

7. High performance liquid chromatogram of human serum after fractionation

on Sephadex 13

8. Reversed-phase liquid chromatogram of antiepilepsy drugs in Sephadex-treated

human serum matrix (detection at 254 nm) 14

9. Preparation of serum base 15

10. Procedure for preparation of tubes containing drugs for quantitative gas chromatography employing a bracketing technique 27

11. Procedure for extraction of drugs for quantitative gas chromatography 28

12. Gas chromatogram of anitepilepsy drugs 29

13. EMIT calibration curve for phenytoin 31 14. EMIT calibration curve for phenobarbital 32

15. EMIT calibration curve for primidone 33

x
Abstract

Recognition of the efficacy of monitoring the concentrations of therapeutic drugs in the blood of patients has revealed many needs for standardization of the laboratory tests

used for such monitoring. The National Bureau of Standards was asked to provide a Standard

Reference Material (SRM) consisting of three serum samples, each to contain four anti- epilepsy drugs at different concentrations. The four drugs are phenobarbital , phenytoin,

primidone, and ethosuximide. The SRM would fill a basic role for the achievement of accurate analysis to help ensure the reliability of analyses for these drugs. The needs that had to be fulfilled to produce the SRM included: (1) analytical criteria for purity of the drugs; (2) serum to be used as a matrix for the drugs; (3) techniques

for achieving homogeneity and stability of the SRM; and (4) two independent, highly

accurate analytical methods for the certification.

This document describes development of methods and procedures used to produce and certify the SRM.

Key Words: Anticonvulsants; antiepilepsy drugs? blood analysis; chromatography, gas; chromatography, liquid; drug standards; enzyme immunoassay; epilepsy; ethosuximide; mass spectrometry; nuclear magnetic resonance spectroscopy; phenobarbital; phenytoin; primidone; reference material; serum base modification; serum matrix.

Disclaimer

Identification of commercial products does not imply recommendation or endorsement by the National Bureau of Standards nor that the product is necessarily the best available

for the purpose.

1
1. Introduction

The value of monitoring antiepilepsy drug levels in serum has been demonstrated

repeatedly over the past 10 years. This has resulted in an overwhelming number of publications describing methods for analysis. Many of these methods have been adopted by laboratories that provide clinical analyses. As demands for drug level tests have increased

by physicians who recognize their usefulness, many clinical laboratories responded by

establishing routine testing procedures for samples submitted by physicians. In many

instances, however, these laboratories did not have extensive experience in drug monitoring

techniques.

As a result of a blind survey taken in 1974 by Pippenger et al., [I],

1 it became obvious that not all laboratories were reporting accurate serum drug levels. In fact, the majority of the laboratories surveyed reported unacceptable results. Such results were

not unanticipated by researchers at the National Institutes of Health (NIH). In 1972,

Dr. J. Kiffen Penry of the National Institute of Neurological Diseases and Stroke (now the National Institute of Neurological and Communicative Disorders and Stroke) initiated efforts

to assure the development of adequate standards for clinical laboratories performing patient

serum monitoring for anticonvulsant drug levels. A research proposal was invited from the National Bureau of Standards (NBS) for work that would ultimately lead to the issuance of Standard Reference Materials (SRM's) for anticonvulsant drugs in serum. With funding from

NIH, work began in 1974 to develop methods and competences that would result in state-of- the-art analytical measurements for the drugs in a serum matrix. We present in this report information regarding the preparation, measurement, and certification of the Antiepilepsy Drug Level Assay Standard, which NBS issues as SRM 900.

2. Development of the SRM

2.1 Studies of the Drugs Used in the SRM

A. Chemical Structure, Name, Molecular Weight, 2 Source. The following four drugs were used in the SRM, after extensive characterization for

purity:

1. Phenobarbital (5-ethyl-5-phenylbarbituric acid); mol wt 232.23

C2H5JL J

c6£yH

Lot No. 3-TT-1038

Source: Gane Chemical Works, Inc. Carlstadt, NJ

1 Figures in brackets indicate the literature references at the end of this publication.

2 Data for molecular weights taken from the Merck Index, 8th ed. , published by Merck & Co. Inc. , Rahway, NJ.

2
2. Phenytoin (5,5-diphenyl-2,4-imidazolidinedione); mol wt 252.26

Lot No. 415781

Source: Parke Davis & Co. Detroit, MI

3. Ethosuximide (2-ethyl-2-methylsuccinimide) ; mol wt 141.17

Lot No. 409470

Source: Parke Davis & Co. Detroit, MI

4. Primidone (5-ethyl-5-phenylhexahydropyrimidine-4,6-dione) ; mol wt 218.25

Lot No. 81-6275-2

Source: Ayerst Laboratories

New York, NY

3
B. Initial Stability Testing and Solubility Studies

Initial testing of the stability of drugs in alkaline solution was performed by spec- trophotometry at pH 11.6 (in buffer) or at pH 12 (unbuffered). Monitoring was performed at the following wavelengths: for phenobarbital at 238 nm, phenytoin at 259 and 264 nm, ethosuximide at 243.5 nm, and primidone at 251.5, 257.5, and 264 nm. The drugs were relatively stable in these solutions for the first two to three hours (decrease in absorbance less than 1 percent), but by 24 h there was clear evidence of instability

(absorbance decrease of at least 5 percent). Some working measures of the solubility of the drugs in methanol (M), acetone (A), and chloroform (C) at about 25 °C were obtained. In mg/mL these are: for phenobarbital,

approximately 230 (M) , 275 (A), and 15 (C); Phenytoin, approximately 25 (M), 32 (A), and

1.3 (C); Primidone, approximately 6.4 (M), 2.4 (A), and 0.35 (C); Ethosuximide, greater than

1000 in (M), (A), and (C).

C. Thin- Layer Chromatography

A single, thin- layer chromatographic system separated the four drugs on the same

plate. Solvent: 14:8:1 (v/v) chloroform-l-butanol-concentrated ammonium hydroxide. Adsorbent: silica Gel GF. The drugs showed the following mobilities (Rp): phenobarbital

0.49; primidone 0.62; phenytoin 0.83; and ethosuximide 0.93. The detection level for each

neat drug was in the range of 2 to 5 ug with use of fluorescence quenching. With this system and using 0.25 mm thick layers, between 200 to 300 ug of a drug could be chromato- graphed as a discrete spot; except for primidone, whose insolubility limits the test. In the latter case, maximum feasible application is 40 ug. At maximum loading of the plates, no impurities were detected. However, concentrated

effluents from preparative columns could be spotted on the plates and the presence of impurities could be observed.

D. Pulse-Fourier Transform Proton Nuclear Magnetic Resonance Spectroscopy at 90 MHz of Fractions Obtained by Liquid Chromatography

An in-depth study of the purity of the crystalline drugs was accomplished by utilizing

the separating capabilities of liquid chromatography (LC) together with the proton nuclear magnetic resonance (proton NMR) spectra of the drugs.

1. Liquid Chromatography

A survey was made of possible liquid chromatographic conditions for characterzing the

four drugs. Four adsorbents, each with a different functional group, were identified as offering the diverse range of features needed for separating impurities: a polyvinyl pyri- dine crosslinked by di vinyl benzene (PVP), a polyvinylmethacrylate modified to have chemically

available carboxyl groups, sulfonated polystyrene as the ammonium salt, and sulfonated poly- styrene as the silver salt. Chromatography of approximately 1-gram samples (with analysis by mass balance recovery)

was undertaken on the polyvinylpyridine-divinylbenzene. Testing of chromatography fractions could then be performed by utilizing any of the other adsorbents in an analytical scale chromatographic mode, and by other techniques. Phenobarbital was run on PVP using distilled ethanol as the mobile phase. Recoveries were slightly above 100 percent. Impurities may total less than 0.3 percent. Ethosuximide was run on PVP using distilled methanol as the mobile phase. Recoveries were slightly above 100 percent. Impurities may total 0.3 percent. Phenytoin was run on PVP using distilled methanol as the mobile phase. Recovery was slightly below 100 percent. Impurities may total 0.6 percent.

4
Primidone chromatography was run using distilled ethanol. (Primidone is much more

soluble in DMSO than in most other solvents. Solutions of higher concentrations could be introduced by using this solvent, but the effect of the solvent on the adsorptive properties

of the column packing and the difficulty of recovering dry fractions from DMSO made use of

it as a solvent unattractive.) The sample for chromatography was prepared by ultrasonic mixing of 1.0 g of primidone with 50 mL of ethanol for 4-5 h at about 40 °C, and then allowing the saturated solution to cool to about 22 °C before introducing the supernatant

into the chromatographic column. Because of the limited solubility of this compound, merely dissolving all of a small, weighed quantity and then undertaking chromatography would

provide little information for measuring purity; instead the possibility of detecting

impurities was examined by attempting to extract them from an excess amount of primidone. While this approach to measurement of the purity of primidone was indirect, the evidence

indicated that the sample was of high purity.

2. Proton Nuclear Magnetic Resonance Spectroscopy of the Drugs

a) Phenytoin fractions. In general, fractions (in methanolic solution3

) were prepared

for proton NMR analysis by filtration, followed by evaporation under a stream of nitrogen. The residues that resulted were not further dried to avoid the possible loss of volatile

impurities. Each residue was extracted with acetone-d6 (3 x 0.03 mL, 100% D) and the soluble material was transferred into a cylindrical NMR~ microcel 1 (capacity 0.06 mL). Up

to 35,000 scans were taken of each fraction. The spectra of fractions 1, 2, and 4 show methyl and methylene signals at high field that indicate the presence of an ethosuximide-type of impurity in these fractions, although

the equivalence of the methylene proton signals of the ethyl group that was observed for

the fractions suggests that the impurity does not contain an asymmetric carbon atom adjacent to the ethyl group, as does ethosuximide itself. In order to confirm that ethosuximide was not a genuine impurity in phenytoin, liquid chromatography of the latter

drug was repeated with a column that .was thoroughly prewashed with acetic acid to ensure

removal of possible residues from previous analyses.

The repeated LC invariably gave a fraction 2 that contained a strongly UV absorbing

impurity, present at very low levels. Because of its strong UV absorption, this impurity

could not have been ethosuximide. However, the spectra of fraction 2 generally showed

strong proton multiplets whose chemical shift indicated the presence of aromatic molecules

other than phenytoin. The structure of the multiplets was similar to that of the proton

signals of phthalate, a common impurity in commercial organic materials. It appears probable that this material was responsible for the unusually strong ultraviolet absorption

of fraction 2 at 254 nm that was observed. The mass balance experiments indicate that the impurities present in fraction 2 comprise not more than 0.1 percent of the crystalline

phenytoin.

The acetone-soluble materials of all of the fractions also contained significant

quantities 4 of hydrocarbon impurities that originate principally from the purified methanol solvent used in the liquid chromatography. This interference problem was studied further by NMR examination of the residues obtained by (a) evaporation of purified methanol (500 mL) under a stream of nitrogen as used for the fractions, (b) evaporation of methanol (500 mL)

by means of a rotary evaporator, so as to avoid possible contamination from the nitrogen

supply, and (c) evaporation of methanol (500 mL) that had been passed through the column

of polyvinyl pyridine resin.

3The methanol used was purified at NBS by sub-boiling distillation [2].

4 In this context "significant quantities" means that the hydrocarbon impurities give proton

NMR signals that are of equal or greater intensity than those of the impurities derived

from the drug.

5
All three of these residues showed significant quantities of the same hydrocarbon con- taminants that are characterized by a methylene : methyl group ratio of 3.0-3.5:1. Examples

of structural possibilities for hydrocarbon impurities that would fit this intensity ratio

are 2-methyldodecane, 2,15-dimethylhexadecane, 2 , 17-dimethyloctadecane , and 2-methyltetradecane. A mass spectrometric total ion chromatogram obtained from a direct probe sample suggested the presence of three components including a phthalate compound that

gave a stable aromatic ion, and a molecular ion of low intensity that corresponded to a methyl tetradecane. Reports in the literature suggest that pristane (2,6,10,14-tetramethylpentadecane) and phytane (2,6,10,14-tetramethylhexadecane) occur in such commercial solvents

as methanol and pentane that have been derived from fossil hydrocarbons. However, the spectroscopic evidence indicated that the hydrocarbon impurities encountered in this work

were less highly branched than pristane and phytane. The same type of hydrocarbon

impurities (as characterized by the chemical shift of the methylene groups) had been encountered in previous work in which cholesterol was chromatographed on polyvinyl pyridine

in chloroform solution and Cortisol in ethanol solution. The proton NMR spectrum of the column residue preparation described in (c) above showed (in addition to the methanol hydrocarbon) only low levels of aromatic and methylene

proton signals that are probably due to a slow bleed of di vinylbenzene oligomers from the column material used. The intensities of the NMR signals indicate that this source of con- tamination (column bleed) is much less significant than that originating from the methanol

.

The proton NMR spectra (fig. 1) of solutions of phenytoin in acetone-d6 display a single complex multiplet for the chemically indentical phenyl groups, but separate singlets

of widely differing line width for the inequivalent NH protons. The broadness of the NH signals disappears when methyl sulfoxide-d6 is used as solvent (Table 1) suggesting that the rate of chemical exchange of the NH protons is diminished by their hydrogen bonding with

this solvent.

2 Ph

NH NH

Figure 1. Proton NMR spectrum of phenytoin.

b) Proton NMR spectroscopy of LC fraction 1 of ethosuximide indicated only the presence

of hydrocarbon artifacts derived from the redistilled solvent used for the LC. However, the proton NMR spectrum of fraction 2 (see fig. 2) showed the presence of ethosuximide, hydro- carbon artifacts, and an unknown impurity that displayed a five line, probably two-proton multiplet in the olefinic proton region, at 5.7 p. p.m. Comparison of the integrated

intensity of this multiplet with that of one of the methylene multiplets of the ethosuximide

6

ACETONE-D5 HoO TMS
indicated that the olefinic compound is present at less than one-seventh the level of the ethosuximide. Since fraction 2 represented only 0.07 percent of the mass of ethosuximide,

the olefin was present in the crystalline drug at a level of less than 0.01 percent. The proton NMR spectra of fractions 4 and 5 of ethosuximide showed only the resonances associated with the solvents used for liquid chromatography and proton NMR analysis, but

the spectrum of fraction 6 indicated that it was composed principally of ethosuximide, sug- gesting the occurrence of some long-term bleeding of the main component from the LC column packing material. As mentioned before, this problem was obviated, with respect to subse- quent usage of the same column, by washing the column packing with acetic acid prior to each

analysis. CH3

CH3 (ET)

i 5.69 P.P.M.

Figure 2. Proton NMR spectrum of ethosuximide fraction 2 from liquid chromatography.

Solutions of this drug in acetone-d6 yielded a more characteristic proton NMR spectrum

(fig. 3) in which the ring methyl substituent and methyl part of the ethyl group resonate as

a singlet and triplet, respectively, at high field. Owing to the fact that the quaternary

carbon atom of ethosuximide is asymmetric, each methylene group contains a pair of chemi- cally inequivalent protons, and the ring methylene group resonates as an AB quartet, whereas

7
the methylene part of the ethyl group gives a complex multiplet instead of the simple quartet which would be expected if the methylene protons of the ethyl group were equivalent. These observations are relevant to the characterization of fraction 2 of phenytoin

since the proton NMR spectrum of this fraction is similar to that of ethosuximide except

for the absence of inequivalent methylene proton signals at higher field. CH3

RING CH2 CH3

(Et)

NH

x8 CH2

(Et) jiiuATMS

ILL

Figure 3. Proton NMR spectrum of ethosuximide.

The NH signal of ethosuximide occurs as an extremely broad singlet at low field that

is best detected by multiple scanning of the spectrum.

c) Phenobarbital . The block averaging NMR technique was used for some of the LC fractions of phenobarbital for which spectra with a high signal: noise ratio were desired. Implementation of this technique involved signal averaging in both the time and frequency

domains. The advantage of also averaging in the frequency domain is that the dynamic range of the spectrum is not limited by the computer word-length, since, in the frequency domain, strong signals may be allowed to overflow memory. A further advantage of performing some averaging in the frequency domain is that arithmetic uncertainties in the computation of

the Fourier transforms tend to average out. The dynamic range of the transform is known to be limited by these uncertainties and by other factors. The block averaging technique has been implemented in the Nicolet data system which is being used for this work by two methods. Signal averaging is performed first in the time domain until the data almost

fills the memory. This data block is then Fourier transformed, phase corrected and either

(a) added to a second block of memory, or (b) stored on a magnetic disk and co-added with

a running data file on that disk. Automation of the whole procedure is achieved by means

of a series of linked commands. Proton NMR spectra of LC fraction 1 of phenobarbital indicated that it contained

phenobarbital as a major component, solvent hydrocarbons, and an additional aromatic compound that displayed a multiplet at 7.7 p. p.m., slightly downfield from the phenyl multiplet of phenobarbital. Comparison of the intensities of these multiplets indicates that the aromatic impurity is present at a level of one- tenth that of the phenobarbital in fraction 1. Since this fraction comprises only 0.01 percent of the mass of phenobarbital

chromatographed, the aromatic impurity is present in the phenobarbital at a level of less than 0.001 percent.

8
The proton NMR spectra of fractions 2-6 indicated only the presence of phenobarbital

and solvent impurities in each fraction. The amide form of phenobarbital contains a plane of symmetry and so only a signal at

low field is seen for the NH protons, and the usual triplet and quartet at high field for

the methyl and methylene protons, respectively, of the ethyl group (fig. 4). The very broad signal of phenobarbital at low field is removed by addition of deuterium oxide to the solution thus confirming the assignment of this signal as NH. Ph

Figure 4. Proton NMR spectrum of phenobarbital.

d) Primidone. The proton NMR spectrum of LC fraction 1 of primidone showed only the presence of solvent impurities. Spectra of fractions 2, 3, and 5 indicated the presence of primidone in each fraction in addition to solvent impurities. However, the spectrum of fraction 4 displayed an additional multiplet in the aromatic region at 7.72 p. p.m., the intensity of which indicated a level of one-fifth that of the primidone present in the

fraction. Since fraction 4 comprises 0.21 percent of the total mass of primidone chromatographed, the aromatic impurity must be present in the primidone at a level no greater than

0.04 percent. The proton NMR spectrum (fig. 5) of primidone in dimethyl sulfoxide-d6 solution displays

the NH signals (two, according to integration of the spectrum) as a doublet which suggests

spin coupling of the NH and ring methylene protons. The effect of this coupling is seen

in only one half of the ring methylene multiplet which indicates that only one of these methylene protons is favorably oriented for coupling with NH protons that are three bonds

distant. Chemical exchange of the NH protons must be a particularly slow process in dimethyl sulfoxide-d6 for coupling of the NH protons to be observed. The NH signal of primidone in acetone-d6 solution could not be detected. Moreover the

ring methylene group now resonated as a simple AB quartet. Evidently, the weaker hydrogen bonding of the NH protons in acetone-d6 permits a more rapid chemical exchange which removes

the effects of the NH-methylene coupling, and causes the NH signal to become so broad that

it is lost in the baseline noise. The phenyl group of primidone resonates as the usual complex multiplet and the ethyl group as a triplet and simple quartet at high field

(fig. 5). In summary, the proton NMR spectra of concentrated solutions of phenytoin, ethosuximide, phenobarbital, and primidone were studied and the spectra indicated that the preparations selected were of high purity. The assignments of the NH proton signals of the drugs were generally confirmed by deuterium exchange with added deuterium oxide. The proton

chemical shifts of the drugs and their spectral multiplicities and assignments are summarized in Table 1. 9
Ph

2 NH I J

Figure 5. Proton NMR spectrum of primidone.

CH»

RING H2° CH2

(Et) CH,

TMS

A_AJL

Table 1. Proton chemical shifts of anti epilepsy drugs.

CH3 CH2 CH3 CH2 Drug Solvent (Et) (Et) n ring) (in ring) Ph Phenytoin (CD3 ) 2 C0

(CD3 ) 2 S0

Ethosuximide (CD3 ) 2 C0 0.89t 1.64m

Phenobarbital (CD3 ) 2 C0 0.97t 2.44q

Primidone (CD3 ) 2 C0 0.93t 2.07q

(CD3 ) 2 S0 0.86t 1.98q

1.27s 2.56q

7.42mb 8.23s,

C 9.93sd 7.38m 9.32s, 11.11s

7.40m

9.92s'

10.23s

l

4.25q 7.36m 4.03sx 7.32m 8.68d (x2)

Parts per million from internal tetramethylsilane.

'Multiplicities of NMR signals are indicated by s (singlet), d (doublet), t (triplet),

q (quartet), sx (sextet), and m (complex multiplet).

'Broad singlet. Very broad singlet.

10
2.2 Serum Base Developments

A. Choice of Matrix. Since the intended SRM was to have a serum matrix, procedures were established to assure that the drugs would be soluble in serum, that they would disperse homogeneously,

that they would not be irreversibly bound to protein, and that they would not precipitate

or segregate when the matrix was freeze-dried and subsequently reconstituted with water. Bovine serum at first appeared to be an adequate serum base. It is easy to obtain in bulk quantities and is generally free from the myriad of drugs found in pooled human

serum. One concern was that the bovine serum would not have the same handling properties

and characteristics as those of clinical (human) samples. High performance liquid chroma- tography using an octadecyl si 1 ane (C 18 ) column in a reversed-phase system revealed the relative complexities of the candidate sera (figure 6). The liquid chromatogram of the bovine serum demonstrated that the serum is quite free of interfering peaks in the region where the drugs are eluted. In contrast, chromatograms of human serum show a variety of peaks in this region. They were found to cause substantial interference in quantitative

determinations, especially of phenobarbital , which has a similar retention volume to that

of the strongly absorbing serum component labeled A in the human serum chromatogram. Although the human serum is more complex than bovine serum and contains many more inter- ferences, we nevertheless decided to pursue the preparation of human serum processed to remove those interferences. A critical factor in this decision was the finding that preparations of a bovine-based drug preparation did not give accurate quantitation with a currently-used, enzyme-based, immunoassay method, due to cross-reacting antibodies to bovine components. We first attempted to prepare interference-free human serum by passing it through a 1.5 x 30 cm column of Sephadex G-50 (Pharmacia, Piscataway, NJ), equilibrated with 0.15 mol/L NaCl. The liquid chromatogram of the serum protein peak is shown in figure 7. It may be seen by comparison with figure 6 that the serum components that elute in the region

of the drugs were removed by passage of the serum through the Sephadex. The treated serum

was then spiked with known amounts of all four drugs and subjected to quantitation by liquid chromatography. A typical chromatogram of this spiked, treated matrix is shown in figure 8 and the quantitative results are shown in Table 2. A relative precision of 2-3 percent was achieved during the first quantification efforts for phenytoin, ethosuximide, and phenobarbital, but quantification of primidone was more difficult owing to its low concentration at therapeutic levels.

Table 2. Quantitative analysis of drug mixtures in Sephadex-fractionated human serum

by reversed phase liquid chromatography.

Drug Concentration of djrug injected (ug/mL) Drug found

(ug/mL) ± a

RSD%

Phenytoin 16.6 16.6 0.3 1.9 Ethosuximide 78 75.0 2.5 3.3 Phenobarbital 22 23.6 0.7 3.0 Primidone 4

Volume injected: 0.2 mL.

b Relative standard deviation.

CThis concentration of primidone gave a peak that was too small to be reliably recognized

by the integrator in use. Measurement of a higher concentration of primidone (9 ug/mL) yielded a found value of 9.05 ug/mL. At this level, the error was estimated to be 5-10 percent and presumably could be diminished in proportion to the injection of larger volumes of serum.

11

ot=l

START OF

GRADIENT

Figure 7. High performance liquid chromatogram of human serum after fractionation on Sephadex.

13
I I I I 1 0 10 20 30 40 TIME, MINUTES

Figure 8. Reversed-phase liquid chromatogram of antiepilepsy drugs in Sephadex-treated human serum matrix (detection at 254 nm).

The process of fractionation of human serum on Sephadex suffered from the disadvantage

that it appeared to be applicable only to rather small volumes of serum. The processing of batches of serum with total volume requirements approximating fifty liters would require

exceedingly large columns or many individual separations on smaller columns. A procedure

for serum treatment that was applicable to large batch-mode processing was developed by making modifications to portions of a human serum pool and then following the disappearance

of serum components that elute in the same region as the drugs. Some treatments were more effective than others in removing specific components from the serum. A sequence of opera- tions was finally devised that consistently produced serum suitable for f reeze-drying and substantially free from interferences that elute in the same region as the drugs. The procedure is outlined in figure 9 and described in the following section.

14
POOLED PLASMA (certified hepatitis-free)

4- Clot with CaCl 2 (1% w/v)

OR POOLED SERUM (certified hepatitis free)

Centrifligation 4000 x g, 30 min

Dialysis

followed by Dialysis vs 0.15 mol/L NaCl

Add Aerosil 380 (Si02 ) (10 g/L)

4- Heat (56 °C, 30 min) (stir occasionally)

4- Centrifugation

4000 x g, 30 min

Diaf i ltration in a commercially available

hollow fiber unit (Retention of Proteins with

MW >50,000)

Circulate through

Charcoal Columns

or Commercial "Detoxifying"

Columns

Add Charcoal

1% w/v

4- Stir 30 min Filter Paper Separation

High Speed Centrifugation

(15,000 x g, 1 hr or more)

4- Filter through Previously Washed 0.22 urn Filter

Figure 9. Preparation of serum base.

15
B. Procedure for Preparation of a Human Serum Base Certified hepatitis-free plasma from several donors is combined in a single con- tainer. Calcium chloride is added to a final concentration of about one percent (w/v), or

until clotting is observed. The addition of glass microscope slides or glass beads often enhances the clotting. After the clot is retracted, the supernatant liquid is centrifuged

at 4000 x g to remove debris and provide a clarified serum. Aerosil 380, (Degussa, Inc., Teterboro, NJ, 07608) a Si02 product, is added to a level of about 10 g/L and the mixture is heated to 56 °C and stirred for 30 minutes. This step removes substantial portions of lipoproteins and deactivates some enzymes. The removal of lipoproteins is necessary to obtain a clear solution upon reconstitution from the freeze- dried state. Alternative delipidation procedures consisting of treatment with dextran or

freon were judged to be less effective. They also require extra steps of processing. The mixture is centrifuged at 4000 x g for 30 min to pellet the Aerosil. The super- natant liquid is transferred into previously boiled and washed dialysis bags, for dialysis

against deionized or distilled water at 4 °C. This procedure is continued with periodic

changes of water until the conductivity of the dialysate is constant for two hours or more

and is near to the conductivity of the water used for the dialysis. The serum, while still in the dialysis bags, is equilibrated with 0.15 mol/L NaCl at

pH 6.5 to 7.0 until the conductivity of the dialysate is constant (±1 percent) for two hours or more. The contents of each dialysis bag are pooled and passed through charcoal filters to reduce the serum content of non-protein substances. For this procedure coconut

charcoal (6-14 mesh) may be packed in a long glass column and the serum pumped through the charcoal packing. Commercial columns such as the Hemodetoxif ier unit (B-D Life Support

Systems, Sharon, MA 02067) were found useful. Alternatively, finely-divided charcoal such

as Norit may be added (1 percent w/v) and then removed by centrifuging at high speed (15000 x g) for one hour or more. Finally, the material is filtered through 0.22-um pore-size membrane filters into sterile, tared glass containers.

C. Addition of Drugs to the Processed Serum Base

The direct addition of crystalline drugs to a serum base imposes the possibility of obtaining a nonhomogenous mixture due to poorly solubilized drugs. Conversely, the addition

of drugs dissolved in an organic solvent risks protein denaturation and precipitation.

Further, solvent impurities might contribute to the problem of non-specific interference in analytical procedures.

We found that ethyl alcohol (95%) was a suitable solvent for all four drugs, and that

it was consistently free from interfering peaks in both liquid and gas chromatography.

Ethyl alcohol was found to precipitate proteins from the serum bases but only when added at

a rapid rate or if its proportion exceeded about five percent (v/v). Lower alcohol concen- trations, e.g., 1 percent in serum, obtained by slow, dropwise addition with constant

stirring, resulted in no change in the optical clarity of the serum base. Some SRM target values for the concentrations of the four drugs in serum, expressed

in ug/mL are listed in Table 3.

16
Table 3. Target values for drugs in serum (in ug/mL). Subtherapeutic Therapeutic Toxic Phenytoin 4 17 62 Ethosuximide 11 78 177 Phenobarbital 5 22 104 Primidone 3 8 19

In order to achieve these concentrations of drugs in the serum matrix, appropriate

quantities of the drugs are dissolved in alcohol at 100 times the target level; an excess volume of the solution is prepared. The drugs are dissolved with the aid of ultrasonic oscillation to effect their complete dissolution. The volume of alcohol-drug solution added is one percent of the serum base volume. For example, 60 mL of the alcohol -drug solution is added dropwise to 6000 mL of processed serum.

D. Dispensing the Drug-Spiked Serum

The homogeneity of the standard is dependent upon uniform quantities of the drug-spiked

serum being dispensed into vials prior to freeze-drying. Experience with manual dispensors

such as hand-held pipettes has shown that precision of dispensing is limited to about 1.0 percent relative standard deviation (RSD) at 5.0 mL volumes. However, use of automated pipettor-dilutors has resulted in RSD's in the range of 0.01 - 0.05 percent with similar

volumes. These automated instruments must be pre-cal ibrated to assure that delivery of the serum is accurate. The instrument must be operated in a uniform manner in order to obtain consistent dispensing. For example, there needs to be 1) a routine sequence for handling

the vials and dispensing the serum so that the delivery stream does not splash; 2) a routine

of touching the end of the delivery tip to the vial to remove the nascent drop; and 3) a fixed, limited time interval between dispensing so that evaporation from the tip is minimized.

E. Freeze-Drying the Spiked Serum

Since there are known to be differences in residual moisture in separate lots of protein-based products that are freeze dried in the same freeze-dryer, it is essential that

the protocol for freeze-drying be designed to minimize lot-to-lot variations. For example,

residual moisture can be affected by the length of time the dryer is in operation, the degree of vacuum employed, the final shelf and product temperature, the size of the "plug"

of dried serum, the capacity of the freeze-dryer, and the final conditions inside the chamber at the time the vials are sealed. As an additional concern, if the residual moisture is too high, shelf life of the product could be decreased; whereas if the moisture

is too low, the dried protein may not dissolve readily in the reconstituting water. Stoppering the vials under a high vacuum may be a source of problems if there is subse- quent leakage of moisture- laden air into the vial. However, if the freeze-dry chamber is repressurized with dry nitrogen to only slightly below atmospheric pressure (approxmately

0.9 atmosphere) before the vials are stoppered, there is less likelihood of significant moisture entering the vials. The design of the freeze-dryer is an important factor in the achievement of a dry, nitrogen atmosphere. Most commercial freeze-dryers have the condenser

situated in close proximity to the product shelves, and when nitrogen is admitted, moisture

may be picked up by the gas so that moisture may re-enter the product. This is obviated if the design of the freeze-dryer provides for the physical separation of the product chamber

from the condenser chamber. At the completion of the freeze-dry cycle, the condenser

chamber is partitioned from the product chamber with a valve, the dry nitrogen can be admitted directly to the product chamber, bypassing ice in the condenser chamber.

17
3. Protocol for Certification of the SRM

3.1 Homogeneity of the Reconstituted Samples

It is essential that each level of drug be homogeneous in the entire lot of serum and that the serum be dispensed uniformly with respect to fill volume into each vial. While

local inhomogeneities of drug in serum may initially exist when the drugs are added, stir- ring before and during the filling operation overcomes that. Inhomogeneity due to filling

can be detected by weighing. After f reeze-drying is completed, homogeneity among vials can

be determined after reconstituting by testing with a sensitive differential ref ractometer

(see Appendix I for a description of the instrument built at NBS). Random sampling of individual production lots is required to ensure a meaningful estimation of vial-to-vial variations in concentration. A sampling of at least 25 vials from each 1000 vials is required. Sample vials are selected from the lot of vials as identified from a list generated from a table of random numbers. The contents of the vials

are then reconstituted with 5 mL of water, which is dispensed from a constant volume pump

or pipette. The vials are weighed before and after the water is added to correct for variance in the addition of the water. The standard deviation we found in the amount dispensed was about 0.5 microliter. The refractive indices of individual, reconstituted

sera obtained in a differential ref ractometer are then compared with that of a reference

solution which contains about 100 g of KC1 per liter of water. The refractive index of this solution differs from water by about 0.012 units. The measured differences are then corrected for weight and instrument drift, if any, and the values are then analyzed for evidence of systematic filling errors or drift in the dispensed volumes. The sensitivity of this method is sufficient to determine vial-to-vial differences in concentration as small as 0.03 percent. The reproducibility of the differential ref ractometer we used was about

3 x 10 6 refractive index units. Since the lot of unspiked (blank) serum was dispensed in the same manner as the drug-spiked serum, and the inhomogeneity in the latter was less than

0.5 percent in all cases, we did not derive homogeneity figures for the blank. The findings

are tabulated in Table 4.

Table 4. Homogeneity of reconstituted samples.

Percent Standard Deviation

Sample by Differential Refractometry

Blank (not run) Subtherapeutic 0.086 Therapeutic 0.080 Toxic 0.305

3.2 Quantitative Liquid Chromatography

A. Qualification of the Method

Preliminary studies were performed to determine whether each of the four drugs could

be quantitatively separated from serum proteins by reversed phase liquid chromatography. Bovine serum was used as a base in these initial experiments. The drugs labeled with 14 C or

3 H were chromatographed concurrently and the fractions were counted. The data provided

evidence for the purity of these labeled drugs.

18
1. Phenytoin-4- 14 C A solution of 0.56 uCi of phenytoin-4- 14 C in ethanol was chromatographed on uBondapak

C-18 using a water-methanol gradient of 0-50 percent methanol, and a flow rate of 2 mL/min. An aliquot (1 mL) from each of 35 fractions was mixed with counting cocktail

(10 mL) before scintillation counting was carried out. The results for phenytoin-4- 14 C are shown in Table 5 in which it may be seen that the only significant activity other than that present in the major component (fractions 20-27) occurs in fractions 2 and 3. The latter fraction contained only 0.05 percent of the total activity injected. Table 5. Scintillation counting of fractions from liquid

chromatography of phenytoin-4- 14 C in ethanol.

FifArt inn uCi x 10

i 3.2

2 127.3

3 149.

1

4 18.

6

5 15. 5

6 12.6

7 11.8

8 7.7

9 10.9

10 11.6

11 17.8

12 18.0

13 18.

1

14 16.5

15 19.4

16 18.3

17 19.9

18 24.9

19 41.2

20 123.2

21 341.3

22 519.3

23 122.8

24 2,470.3

25 84,355.1

26 292,634.6

27 216,773.4

28 759.9

29 562.4

30 381.8

31 187.6

32 226.8

33 234.5

34 61.0

35 57.0

Total 600,373.9 (0.600 uCi) Activity injected 0.56 uCi

19
Results from the liquid chromatography of a solution of phenytoin-4- 14 C in bovine serum

are shown in Table 6. Chromatographic conditions are the same as those used to separate

the drug dissolved in ethanol (Table 5). It may be seen that practically all (99.5%) of the radioisotope appears in one peak (fractions 25-26) and only 0.1 percent of the activity

is associated with the protein peak (fractions 2-3). Table 6. Scintillation counting of fractions from liquid chromatography

of bovine solutions of phenytoin-4- 14 C.

Fraction Counts (total uC -

1 ) 2 483.0

3 142.5

5 46. 6 6 15.6

7 42.8

8 37.7

9 22.0

1 u lb. /

11 37.9

12 24.4

13 30.3

14 18.7

1 3 \C. 0 16 12.5

17 19.2

18 26.1

19 45.1 20

21 69.5

22 987.3

23 80.1 24 139.6

25 12,175.6

26 604,663.4

27 2,878.9

28 327.7

29 255.0

Total 622,209.8 (0.622 yCi) Activity injected 0.62 uCi

20
2. Ethosuximide-l- 14 C Results from scintillation counting of fractions from liquid chromatography of 0.013

uCi of ethosuximide-l- 14 C in aqueous solution are shown in Table 7 and, for chromatography

of similar solutions of this drug in bovine serum, in Table 8. The activities found in fractions 2 and 3, 5, and 8 in Table 7 indicate the presence

of impurities in the ethosuximide-l- 14 C corresponding to levels of less than 0.25, 0.1, and

0.1 percent, respectively.

Table 7. Scintillation counting of fractions from liquid chromatography

of an aqueous solution of ethosuximide-l- 14 C.

Fraction uCi x 10

1 3.57

2 23.53

3 17.37

4 7.27

b 21 . 31 6 11.33

7 6.65

8 15.40

9 6.28

10 4.68

11 2.83

12 0.99

13 0 14 0.99

15 1.36

16 2.09

17 1.97

18 105.09

19 15,029.29

20 773.45

21 147.96

22 51.13

23 11.70

24 4.44

25 3.82

26 2.59

Total 16,257.10 (0.016 pCi) Activity injected 0.013 pCi

21
Table 8. Scintillation counting of fractions from liquid chromatography of bovine

serum solutions of ethosuximide-l- 14 C.

Fraction Run 1 Run 2 uCi x 10~^

U 1 X

1 0. 74

2 23. 65 34. 74

3 20.94 19! 59

4 3.33 5.05

5 9. 73 6. 90

5 7. 76 30. 31 7 29. 94 7. 02

8 8! 38 17^99

9 14.91 5.17

10 2. 09 0 . / u

1

1

3. 70 0. 99

12 0. 74 0. 99

13 o" 1 !97

14 0 2.34

15 o 0 16 1 . 23 2. 96

17 3. 45 4. 19 18 45. 46 9 374.53

19 14,720.06 e'045. 79

20 '555.88 '210.92

21 97.82 65.91

22 35.73 22.05

23 9.61 7.89

24 5.05 4.56

25 2.71 3.94

26 3.45 3.82

Total 15,606.4 (0.016 uCi) 15,883.3 (0.016 uCi) Activity injected 0.013 uCi 0.013 uCi

The fact that the percentage of radioactivity in fractions 2 and 3 (which also contain

the protein) is only 0.3 percent of the total activity (Table 8) and is not significantly

greater than the radioactivity found in fractions 2 and 3 when the labeled drug is chromatographed in the absence of protein, indicates that this drug is separated virtually

quantitatively from the protein and that the drug-protein binding is negligible under the conditions of the chromatography.

3. Phenobarbital-2- 14 C The results from scintillation counting of fractions from separations of phenobarbital-2- 14 C from ethanolic and bovine serum solutions are shown in Table 9. Again the activity at the protein location (fractions 2 and 3) is comparable to the activities in fractions 2 and 3 in the absence of protein (~0.5 percent). Other minor quantities of impurities were detected at fraction 23 or 24 (<0.3 percent) and at fraction 27 or 28 (<0.2 percent).

22
Table 9. Scintillation counting of fractions from liquid chromatography of phenobarbital-2- 14 C solutions.

Fraction Run 1 (in serum) Run 2 (in serum) Run 3 (in EtOH)

uCi x

10' 6 uCi x

10~ 6 uCi x lO-6

1 24.0 49.4 52.

5

2 439.3 501.4 462! 8

3 142.7 189.8 142.1 4 53.0 39.9 29.8

5 57.9 41.0 27.0

6 35.8 35.5 34.8

7 32.5 89.7 39.7

8 73.7 54.4 48.6

9 73.4 44.8 71.4

10 48.7 28.5 51.2

11 50.3 84.2 63.5

12 113.6 91.2 92.2

13 109.8 204.4 91.7

14 181.1 96.5 144.3

15 158.3 123.0 516.7

16 105.2 50.9 3,086.6

17 71.6 83,113.4 11,410.8

18 113,545.3 45,122.4 32,601.9

19 13,506.6 158.0 60,456.8

20 152.4 86.7 20,974.6

21 104.0 46.4 104.

22 109.2 43.0 69.3

23 176.8 396.7 121.9

i yb. 5 25 82.5 34.6 295.8

26 106.9 163.0

27 200.2 208.6

28 246.6 121.3

29 130.6 106.2

30 60.3 23.7

Total 130,637.6 (0.13 uCi) 131,544.7 (0.13 uCi) 131,286.1 (0.13 uCi) Activity injected 0.13 uCi 0.13 uCi 0.13 uCi

4. Primidone-4,6- 14 C2 Chromatography of 0.025 uCi aliquots of primidone-4,6- 14 C 2 afforded similar activities

(•vO.2 percent) in fractions 2 and 3, whether the drug was dissolved in bovine serum (left- most columns of Table 10) or aqueous methanol (rightmost column of Table 10), and again indicated virtually complete separation of the drug from proteins, with negligible

binding. The activity in other fractions, e.g., 11-13 with 0.4-0.6 percent of total activity, indicates the presence of small proportions of impurities in the labeled drug.

23
In summary, the data from scintillation counting of the fractions obtained from liquid

chromatography of separate solutions of the four labeled drugs in bovine sera demonstrated

that the complete separation of drugs from protein can be achieved. The drugs in serum behaved like drugs in aqueous or alcoholic solutions under the conditions of the reversed

phase liquid chromatography. The degree of binding between the drugs and serum proteins is insignificant.

Table 10. Scintillation counting of fractions from liquid chromatography of primidone-4,6- 14 C

? solutions.

Fraction Run 1 (in serum) Run 2 (in serum) Run 3 (in H?0-MeOH)

uCi x 10

0

_ c uCi x 10

0 uCi x

10'

1 6.0 7.3 1.3

2 31.8 34.0 34.0

3 17.1 15.5 11 7 4 7A K0 2^3

5 1.2 2.1 3.5

6 2.8 1.6 3.0

7 6.8 5.3 7.4

g 18.3 17.

1

9 9!l 8^6 7.5

10 5.8 4.9 2.8

11 29.6 72.7 60.9

12 56.0 43.7 61.7

i j 6.911 . AH 1 D. D 9C\ ft 14 11.1 7.7 13.5

15 11.9 9.2 10.6

16 37.0 97.7 69.8

17 20,925.7 23,264.5 23,139.4

18 2,934.2 335.6 1,052.5

19 73.1 24.5 27.3

20 11.8 10.4 10.9

21 11.8 15.7 11.8

22 6.7 7.1 5.3

23 3.0 4.7 5.4

24 3.2 10.3 3.0

25 1.8 5.0 3.4

26 2.8 2.8 2.2

27 5.8 8.4 9.5

28 5.0 3.5 4.0

29 3.4 4.2 3.5

30 2.3 3.5 1.2 Total 24,770.6 (0.0248 uCi) 24,045.1 (0.0240 uCi) 24,591.9 (0.024 uCi) Activity injected 0.0211 uCi 0.0211 uCi 0.018 uCi

B. High Performance Liquid Chromatography Assay

The liquid chromatographic (LC) assays of the drug concentrations in the three human serum samples containing drugs were made directly on the reconstituted serum, without an intermediate extraction step. Reversed-phase LC was employed with a 0.01 mol/L phosphate

buffer at pH 6.5 and a water- to-methanol gradient. The flow-rate was 2.0 mL/min and the gradient was advanced linearly from 0 to 60 percent methanol in 30 minutes. The column was then flushed with 100 percent methanol for 1-2 minutes, a reverse gradient was run from

24
TOO to 0 percent methanol in 5 minutes, and another 5 minutes was allowed for equilibration

to ready the column for another run. A Waters Associates (Milford, MA 01757) u-Bondapak

C- 18 column was used throughout. The volumes of serum injected were 200 uL for the sub- therapeutic, 100 uL for the therapeutic, and 50 uL for the toxic levels. Detection was by absorbance at 254 and 210 nm. A digital integrator was used with the 254-nm absorbance

detector to calculate peak areas. Peak heights were also measured at 254 nm. Peak heights alone were measured at 210 nm. Three solutions were used as calibrating standards with each of the serum samples. They were made up to contain slightly less, about the same, and slightly more of each drug

than the target value for the sample under analysis. The standards were made up by weighing

the drugs in aluminum-foil boats, on a gram electrobalance which had been calibrated with a carefully checked weight. The boats containing drugs were then placed into 500-mL volu- metric flasks. Since the mass of each boat was about 10 mg, no significant volume error

was introduced. Flasks were then made up to volume with distilled water containing 20 percent (v/v) of methanol. This was found necessary in order to keep the phenobarbital and phenytoin in solution. Tests showed that the injection of methanol with the drugs did not measurably alter the peak heights unless a methanol concentration above 30 percent was reached, i.e. , with the injection of 200 uL of solution. On each day when measurements were made, a vial of the serum was reconstituted and analyzed alternately with the calibrating standard solutions for as many runs as could be made in one day. On succeeding days other vials were similarly prepared and analyzed until six or more vials had been measured. On a typical day, about five calibrating standards and about four serum samples were analyzed. A calibration factor for each drug was calculated

daily. In those cases where drift was evident, the calibration factors were calculated as

a function of time. It is assumed that drift up to 1 percent per hour arose because of a decreasing flow rate as the pumping system warmed up.

3.3 Gas Chromatographic Analysis and Quantitation

A. Deri vati zed Drug Methods

Initially, the method used to analyze these samples by gas chromatography was based on

a procedure reported by Kupferberg [3]. In that procedure, 1 mL of serum is pipetted into 37-mL screw-capped centrifuge

tubes. To the serum aliquots are added internal standards (at levels close to the nominal levels of the drugs in the serum), buffer solution (pH 7.2), and chloroform (10 mL). The samples are extracted for 15 minutes by mechanical shaking. The aqueous phase is aspirated off and discarded. The CHC1 3 layer is transferred to smaller centrifuge tubes and

the CHC1 3 evaporated by means of a rotary evaporator. Dilute HC1 and n-heptane are added to the residue and the tubes are shaken. Serum-derived components, particularly lipids, are extracted into the n-heptane. The heptane is aspirated off and the aqueous layer is extracted with 1 ,2-dichloroethane. The aqueous layer is aspirated off and the organic layer

is taken to dryness on the Buchler apparatus. Methylating reagent is added to the residue

and the mixture is vortex-mixed. The reagent for methylation is trimethylphenylammonium

hydroxide (TMPAH), prepared by reacting trimethylphenylammonium iodide with silver oxide in methanol over a period of several hours. The solids are filtered off and the reagent is stored in a brown screw-capped bottle at 4 °C. A "control" or "reference" sample is taken through the procedure at the same time, and

in the same way. This reference sample consists of unspi ked serum and measured amounts of both the drugs and the internal standards. Injected 1.0-uL volumes of the solution are chromatographed and the peak areas of the drugs and internal standards are measured. The chromatography is performed using an all glass column packed with 3 percent 0V-17

on Chromsorb WHP 100/120 mesh (Supelco, Inc., Bellefonte, PA 16823). The oven temperature

is programmed from 200 to 290 °C at 8°/min with an initial hold period of 2 minutes and a final hold of 4 minutes at 290 °C. 25
The amounts of the drugs in the unknown sample are calculated from the peak area

ratios (drug to internal standard) measured in the control and unknown samples. The procedure was modified for ethosuximide because of its high volatility and the volatility of its internal standard. Before evaporation of the dichloroethane in the final recovery of the drugs, four drops of iso-amyl acetate is added. The dichloroethane is evaporated, leaving an iso-amyl acetate solution of the drugs. Methyl ating reagent is added and the mixture is chromatographed.

The procedure gives reasonably well-shaped peaks with some tailing, particularly those

of the internal standards. The peaks for phenobarbital and phenytoin always have additional

small peaks associated (fused) with them. The early attempts at quantification showed considerable imprecision, although by making several injections in the chromatography it was usually possible to arrive at average values that were within a few percent of the "target" value.

B. Underi vatized Drug Determinations

1. Conditions

Our best analytical results were obtained using a special column packing, GP 2 percent

SP-2510 DA on 100/120 Supelcoport, (Supelco, Inc., Bellefonte, PA 16823) which had been developed to separate anticonvulsant drugs and their metabolites in underi vatized forms.

A gas chromatograph equipped with flame ionization detectors (FID) was used under the following conditions: initial temperature 150 °C; temperature program rate 16 °C/min; final temperature 265 °C; final temperature-hold 4 minutes; injector temperature 300 °C; FID temperature 265 °C; and helium carrier gas at 50 mL/min. The signal output is interfaced with a computing integrator.

2. Bracketing Technique

a) Tube preparation. Analytical accuracy is improved by using a bracketing method performed as follows: Amounts of internal standard for each drug, calculated to be equivalent to the target values are placed into a 100 mL volumetric flask. The internal standards

used for each of the four drugs in the SRM are listed in Table 11. The flask is filled to the mark with ethyl alcohol and the contents are mixed thoroughly. Ethyl alcohol was used

for solubilizing drugs because it was found to have fewer contaminating peaks. An automatic pipettor is used to dispense 100-uL volumes of the solutions into clean screw-capped

centrifuge tubes (29 mm x 140 mm).

Table 11. Drugs and internal standards used in GC analysis.

Drug Internal Standard

Phenytoi n 5- (p-Methyl phenyl )-5-phenyl hydantoi n Ethosuximide a-Methyl-orpropylsuccinimide

Phenobarbital 5-Ethyl-5-p-toly-barbituric acid Primidone 4-Methylprimidone

Standard solutions of the drugs are prepared similarly, one at a level about two percent lower than the target value and another about two percent higher than the target. The automatic pipettor is used to dispense 100-uL volumes of these drug solutions into the separate tubes containing internal standards (see the outline in figure 10). For example,

26
80 tubes are loaded with the internal standards in ethanol. Forty of these are reserved as Tube A, 20 are loaded with the mixture having the higher concentration of drugs (Tube B), and 20 others are loaded with the mixture having the lower level of drugs (Tube C). The

alcohol is allowed to evaporate to dryness, and the tubes are then capped. The internal standards and drugs may be kept for several weeks in a dried state.

Figure 10. Procedure for preparation of tubes containing drugs for quantitative

gas chromatography employing a bracketing technique.

27
b) Extraction and use. For use, 1.0 mL of reconstituted SRM is added to a tube that contains only the internal standard (Tube A, fig. 10). At the same time, 1.0 mL of a serum blank is pipetted into the tubes containing both the internal standards and the drugs at high and low bracketing concentrations (Tubes B and C). Usually, four tubes with SRM and

two each of the high and low bracketing tubes are processed as a group. The samples are acidified with three drops of 3 mol/L HC1 to pH 1-2 and then extracted with high-purity chloroform (10 mL). After shaking and centrif ugation of the tubes, the aqueous upper layer is aspirated and discarded and the chloroform is decanted into a 15 mL

conical centrifuge tube. The chloroform is evaporated at 20 °C under vacuum on a roto- evaporator. The concentrate is redissolved in 50 uL of chloroform and vortex-mixed. One microliter of this residue is then injected into the gas chromatograph. This procedure is summarized in figure 11.

TUBE B

IB I IB

I

I

TUBE A TUBE C

ADD:

\_

SERUM

BLANK

UNKNOWN SERUM j SERUM

CONTAINING DRUG i BLANK

• ACIDIFY TO pH 1-2 • CHLOROFORM EXTRACTION

• EVAPORATION OF CHLOROFORM

• RECONSTITUTE WITH 50|jLCHLOROFORM

• INJECT 1-2 |iL INTO G.C.

Figure 11. Procedure for extraction of drugs for quantitative gas chromatography.

c) Results and calculations. A typical separation of drugs and their internal stan- dards is illustrated in figure 12. Peak areas are obtained and ratios of drugs to internal standard are calculated. Concentration of drugs in the serum is then calculated using the

formula:

R high- R low

X ( WC low>

+ C low

28
where

"high " til of ?nternafstandard Peak «<* M &

R !ow " ATM of ^nteVnafStandard Peak < f° r '» sta "dard >

D _ Area of Drug Peak „ Rx " Area of Internal Standard Peak (for sample)

^high

= ^ oncentratlon °f ^ ru9 in MQ/nL (for high standard)

Sow

= ^ oncentratlon °f Drug in M9/m L (for low standard)

Cx = Concentration of Drug in ug/mL (for sample)

Alternatively, peak height ratios may be used instead of peak area ratios.

Ethosuximide

Internal Standard (i.s.)

Phenobarbital

Primidone

i.s. Phenytoin

i i i i i i i i i i 1 2 3 4 5 6 7 8

Time, Minutes

Figure 12. Gas chromatogram of anti epilepsy drugs. Conditions described in Section 3.3,B,1.

29
3.4 Analysis of Drugs in Serum by the Enzyme Multiplied Immunoassay Technique4 Analysis of three of the drugs in bovine serum by the enzyme multiplied immunoassay

technique (EMIT) was first studied to determine whether this serum is a suitable matrix for preparation of standard drug solutions that could be used to calibrate all of the methods

for analysis of anti-epilepsy drugs that are used in the field, including the recently introduced EMIT procedure. Assays by the EMIT method could only be performed for phenytoin,

phenobarbital , and primidone due to the non-availability (at that time) of commercial EMIT reagents for the assay of ethosuximide.

A. Experimental Procedures Analysis of the drug solutions by the EMIT method was performed at 30 °C using the equipment combination for this method recommended by the SYVA Corporation (Palo Alto, CA

94304), and also with a Gilford Model 250 spectrophotometer (Gilford Instrument Lab., Inc., Oberlin, OH 44074) in combination with a Micromedic pipettor-di lutor. The spectrophotom- meter was equipped with a chart recorder instead of the calculator and digital print-out of the SYVA EMIT system. The EMIT method was adapted to the Gilford instrument as follows.

It was found that the Micromedic pipettor-di lutor could be used for accurate dispensing of reagents if the samples were shaken gently by inversion when aliquots were to be removed

for a subsequent assay step. In order to obtain reproducible sampling from the conical shaped plastic beakers, it was necessary to make a diagonal cut at the tip on the intake tube of the rapid sampler of the Gilford, and use a vacuum setting of 55 kPa. The volume

of diluent was increased to 300 uL instead of 250 uL as specified in the EMIT system instructions for addition of reagents. The larger sample size (1.1 mL) was necessary because the Gilford 250 system has a greater volume between the sample and cell measurement

region than does the SYVA rapid sampling device, which employs the Gilford Model 200-N. The sample size is controlled by adjustment of sample timing. A 1-mL dilutor pump, 50-uL sampling pump and 0.03-cm tip were used with the Micromedic equipment for dispensing

the volumes of reagents. Absorbance values were measured on the chart recorder 15 and 90 seconds after mixing of the reagents. If the values for replicate samples did not agree within six mil 1 iabsorbance units, additional samples were analyzed until this precision

was attained.

By using these procedural modifications, it was possible to make equally accurate measurements with either the Gilford 250 spectrophotometer in combination with the Micro- medic pipettor-dilutor, or the EMIT equipment marketed by SYVA.

B. Results Examples of EMIT calibration curves for phenytoin, phenobarbital, and primidone are shown in figures 13, 14, and 15, respectively. The ordinate represents the difference (AA-AA

Q

) between the change in absorbance (AA) at 340 nm for various standard solutions containing a drug in a range of known concentrations and the change in absorbance (AA

Q

) of a blank solution containing no drug. The abscissa represents the drug concentration in ug/mL. The differential change in absorbance at 340 nm which is measured indicates the quantity of NADH produced by reduction

of NAD on release of the drug-labeled enzyme from a drug-labeled enzyme-antibody complex.

EMIT is a patented procedure marketed by Syva Corp. , Palo Alto, CA.

30

°vv- vv

32
°vv- vv

33
The EMIT method was applied to solutions of phenytoin, phenobarbital , and primidone in bovine serum and to freeze-dried preparations of these materials. Typical results for phenobarbital are shown in Table 12 in which may be compared the drug concentrations determined by the EMIT method and those weighed into the solutions. The limited agreement

between these concentrations is thought to be due to cross reactions caused by use of bovine albumin as a base for the drug standards. Bovine serum had been used as a carrier

for the production of antibodies in the commercial reagents for EMIT assays.

Table 12. EMIT assays of phenobarbital in bovine serum.

Drug Level from Weight

Matrix Sample (ug/mL) (ug/mL) Percent Difference

Serum A 7.49 2.65 64.6

B 48.68 28.2 42.1

C 12.33 13.5 9.5

D 113.31 85 25.0

E 41.37 41 0.9

F 80.11 80 0.1 G 8.05 10.5 30.4

Freeze-dried E 41.37 56 35.4

E 41.37 50 20.9

F 80.11 84 4.9

F 80.11 Off-scale

G 8.05 13.5 67.7

G 8.05 13.3 65.2

Average of duplicate measurements.

In order to provide a check on the accuracy of the commercial calibrating solutions

for EMIT analyses of the drugs, we prepared calibrator solutions for use with the EMIT

reagents. Liquid chromatographic analysis showed reasonably good linearity for these solutions within the relevant range of drug concentrations. The drug concentrations in the human serum based SRM were determined at NBS and in •four other laboratories by use of the EMIT system. Results are shown in Table 13. The averaged values obtained were close to the values obtained by liquid chromatography and gas chromatography but did not have the precision necessary for including them in the certification. They are useful, however, as information values and indicators of the kind of results that might be seen in routine use of the EMIT technique in a clinical laboratory.

34
Table 13. Comparison of EMIT determinations of drugs in SRM 900. Weighed- In Target

Drug Value NBS Lab A Lab B Lab C Lab D (ug/mL) (ug/mL)

Phenytoin 4.2 4.3(2.4)

a 4.4(4.8) 4 (-4.8) 5 (19) 4.2(0)

16.4 17.4(6.1) 17.0(3.7) 17.8(8.5) 18.0(9.8) 16 (-2.4) 59.8 64.0(7.0) 62.0(3.7) 65.3(9.2) 60.5(1.2) 62 (3.7)

Ethosuximide 10.9 11.1(1.8) 11.0(0.9) 14.0(28.4) 14.0(28.4)

74.4 79.2(6.5) 78.0(4.8) 80.5(8.2) 72.5(-2.6)

170.5 181 (6.2) 181 (6.2) 180.5(5.9) 140 (-17.9)

Phenobarbital 5.0 5.4(8.0) 5.3(6.0) 4.2(-16) 5.5(10) 5.2(4) 21.2 20.2(-4.7) 20.0(-5.7) 19.4(-8.5) 21.0(-0.9) 19 (-10.4) 100.2 102 (1.8) 94.0(-6.2) 101.8(1.6) 104 (3.8) 110 (9.8)

Primidone 3.4

7.9

18.5

3.3(-2.9)

7.7(-2.5)

19.2(3.8)

3.2(-5.9)

9.0(13.9)

20.0(8.1)

3.7(8.8) 9.6(21.5)

20.0(8. 1)

3.5(2.9) 9.0(13.9) 20.0(8.1)3.6(5.9) 7.4(-6.3)

20.0(8.1)

Figures in brackets indicate the percent deviation from target.

C. Interferences

During the early production phase of the SRM, samples were sent to selected labora- tories for testing in a variety of analytical systems as well as for comments on the ease

of reconstitution, handling characteristics, and clarity of the reconstituted serum. Those

laboratories, using off-column methyl ati on procedures, reported that they experienced

problems with phenobarbital quantitation. Investigation revealed a problem due to the presence of an extraneous peak that co-el uted with phenobarbital under most gas chromatographic column conditions. Further identification of this interfering compound was made by use of gas chroma- tography/mass spectrometry. The spectrum from the isolated peak was indicative of phthalic

anhydride (MW 148), probably derived from a common plasticizer during our serum work-up

procedure. The source of the plasticizer may have been the original plastic storage bags for the

serum, since all materials coming in contact with the serum subsequent to pooling had been thoroughly conditioned and washed with large amounts of distilled water. Although charcoal was used in the processing of the serum, the quantity may have been insufficient to eliminate phthalate esters from the product. Subsequent production lots of serum were more thoroughly treated with the charcoal and were monitored more stringently for interferences. Experience with the later material with a variety of laboratory analytical methods has not revealed any further serious interferences.

35
4. Statistical Analysis for Certification of SRM 900

The determination of satisfactory sample homogeneity is the first step in the certifi- cation process. This determination was described in Section 3.1 where the refractive index

of a random sampling of each of the three concentration levels of SRM 900 was tested. The standard deviations of these measurements were found to be between 0.1 and 0.3 percent of the value. This was considered to represent satisfactory sample homogeneity. The concentrations of the four drugs at the three concentration levels used in SRM 900 were measured at NBS by liquid chromatography using as many as three separate peaks, and by

gas chromatography using as many as three separate runs. For each drug and level, the standard error of concentration at each peak or run was calculated. These values, which were calculated from the detailed laboratory records, represent the internal variability

for each peak or run. The measured peak or run values and their standard errors are reported in Table 14 in the "NBS LC" and "NBS GC" columns. Only these data were used for

the SRM 900 Certificate values and uncertainties. For comparison purposes, however, values

from other measurement sources as well as the "NBS EMIT" values, are also listed in Table

14. In general, the agreement is quite satisfactory. The certified concentrations for each drug and level were obtained as weighted

averages of the measured values. For each average the relative weights were calculated, using both the internal variances and the between peak and or run component of variance. The standard errors of the certified values were also calculated using combinations of both

of these variances. These standard errors were then smoothed by a linear least squares

fit of the standard errors for each drug verses the concentrations. The standard errors

listed on the SRM certificate are from this fitting process and are believed to be a better representation of the uncertainty of the overall NBS measurement and certification

process.

5. Summary and Conclusions

SRM 900 represents the first SRM issued by NBS that is certified for trace amounts of organic compounds (in this case, drugs) in a complex matrix. Careful characterization of the drugs and their purity was carried out before analytical methods were developed.

Total separations by liquid chromatography of the drugs from serum were experimentally

proven with radioactive drugs. Methods for "stripping" the serum matrix to provide an interference-free base were developed. The correspondence of results from independent

analytical methods assured that the certified values are accurate. The SRM is intended for use (1) in the calibration and standardization of procedures

employed in clinical laboratories for the determination of drugs in serum, and (2) for the critical evaluation of working or secondary reference solutions prepared either in-house

or supplied commercially.

6. SRM Use

A. Water for Reconstitution

It is recommended that high-purity water be used for reconstitution of the freeze-dried

materials. A low-quality water may contribute substances that may cause false or spurious

peaks to appear in the gas or liquid chromatograms. Water that is equivalent to Type II reagent grade water as specified by the College of American Pathologists (CAP) (see Appendix IV) is sufficiently pure. Permission to include the CAP specifications for water

in this publication was kindly granted by the College of American Pathologists. The

National Committee for Clinical Laboratory Standards (Villanova, PA 19085) has also published guidelines for water as NCCLS Tentative Standard, TSC-3, "Specifications for Reagent Water Used in the Clinical Laboratory".

36
Drug Phenytoin

Blank Sub-Therapeutic

Therapeutic

Toxic

Ethosuximide

Blank Sub-Therapeutic

Therapeutic

Toxic

Phenobarbital Blank Sub-Therapeutic

Therapeutic

Toxic

Primidone Blank Sub-Therapeutic

Therapeutic

Toxic

Table 14. Compilation of data from certifiation runs - 1978. Weighed- in Target Value . ^ (g/mL) NBS LC

a NBS GC

D Other GCC Final

. Certified

NBS EMIT Value6

04.2

16.4

59.8

010.9 74.4

170.5

05.0

21.2

100.2

03.4

7.9

18.5

4.10±0.06

16.45±0.11

62.1810.80

60.94±0.81

11.74±0.13 75.7410.84 72.8510.68

175.41+0.52 175.5311.13

174.0910.67

5.4410.10 5.6210.08

5.2910.13 21.4410.15

103.7710.26 103.3510.54

3.6210.07 7.9810.14

19.2010.13

4.2110.04

17. 3810. 37 16.6410.21 16.6910.05 59.3810.45

11.94+0.28 76.4610.78 78.2810.16

75.81+0.31

4.3 +0.05

16.2 +0.50

60.8 10.61

13.6 10.25

74.5511.35

174.0010.58 178.4313.58

4.9210.08

21.8210.17

102.7710.66

3.3410.38 8.0810.06

17.8810.38

5.60+0.05

22.1310.13

106.1510.85

3.2810.03 6.6810.13

16.2810.28

04.3

64

0

11.1 79.2

181

03.3

7.7

19.2

1 One standard error.

0 4.2 10.1 17.4 16.7 10.3

60.7 10.9

0 11.8 10.4

75.8810.5

174.6710.6

0 0 5.4 5.3 +0.2

20.2 21.6 10.2

102 103.6 10.3

0 3.6 10.

1

8.1 +0.2

18.6 10.7

Underivatized value 1 one standard error.

cDerivatized value 1 one standard error.

dFrom duplicate measurements.

eThe certified value is based on the NBS LC and NBS GC values. The i value is one standard

error and includes all known sources of imprecision.

37
B. Storage

Once reconstituted, the SRM should be used within one day. Beyond that time, several variables may be encountered: 1) evaporation leading to concentration of the drugs;

2) deterioration of the drugs; 3) degradation of the serum base. Should a laboratory decide to store the reconstituted SRM for additional studies with

the material, the reconstituted serum should be distributed among small, screw-top vials, which are tightly sealed and stored at -20 °C. Avoid repeated freezing and thawing. Material thus stored should not be used in critical calibration procedures or regarded as long-term stable controls.

7. References

[1] Pippenger, C. E. ; Penry, J. K. ; White, B. G. ; Daly, D. D. ; Buddington, R. Inter- laboratory Variability in Determination of Plasma Antiepi leptic Drug Concentrations.

Arch. Neurol. 33: 351-355; 1976. [2] Kuehner, E. C. ; Alvarez, R. ; Paulsen, P. J.; Murphy, T. J. Production and Analysis

of Special High-Purity Acids Purified by Sub-Boiling Distillation. Anal. Chem. 44: 2050-2056; 1972. [3] Kupferberg, H. J. Quantitative Estimation of Diphenylhydantoin, Primidone, and Pheno- barbital in Plasma by Gas-Liquid Chromatography. Clin. Chem. Acta 29: 283-288;

1970.

We would like to thank the following scientists for their help in evaluating early prototype reference materials and providing comparison analyses after production of the

SRM: Dr. Kenneth Dudley and associates, particularly Mr. Daniel L. Bius at the University

of North Carolina School of Medicine, Chapel Hill, NC; Dr. Harvey J. Kupferberg, National Institutes of Health, Bethesda, MD; Dr. Marjorie Horning, Baylor College of Medicine,

Houston, TX; Dr. Charles E. Pippenger, Columbia-Presbyterian Medical Center New York, NY;

Dr. Henn Kutt, Cornell University Medical College, New York, NY; Dr. Dorothy D. Schottelius, University of Iowa Hospital, Iowa City, IA; Ms Elizabeth B. Solow, Indiana University School of Medicine, Indianapolis, IN; Drs. Richard Bastiani and John G. Miller, The SYVA Co., Palo Alto, CA; Dr. Donald J. Fishman, The Neurology Center, Bethesda, MD; Dr. Steven J. Soldin, The Hospital for Sick Children, Toronto, Canada; Dr. Robert W. Burnett, Hartford

Hospital, Hartford, CT; Dr. Reginald J. Adams, the Perkin-Elmer Corp., Norwalk, CT;

Dr. H. Bryan Bente, Hewlett-Packard Analytical Instrument Group, Avondale, PA; Dr. Erich L. Gibbs, Gibbs Laboratories, Wilmette, IL. The work at NBS was performed in the Center for Analytical Chemistry. Special thanks

go to Ms. Rosalie Angeles, Dr. Lorna Sniegoski, Dr. Alex Fatiadi, and Mr. Alex Cohen for their work in characterizing a variety of pure drug samples sent for evaluation. We thank Drs. Philip D. LaFleur and Curt W. Reimann from the Center for Analytical

Chemistry for their support and encouragement throughout this program. Support from the Office of Standard Reference Materials was continually present from Mr. J. Paul Cali,

Mr. George Uriano and from Mr. R. Keith Kirby. Special thanks are extended to Mrs. Joy Shoemaker and Ms. Teresa Sperow of the Text

Editing Facility for their efforts in manuscript production which included typing, editing,

and formatting.

Funding for the project was initiated by the National Institute of Neurological and Communicative Disorders and Stroke, the National Institutes of Health.

38
APPENDIX I U.S. Department of Commerc

Juanita M. Kreps

Secretary

j

" «• I National Bureau of Standards

Ernest Ambler. Director Rational ^mtm of j§>tantlards

Certificate

Standard Reference Material 900

Antiepilepsy Drug Level Assay Standard

This Standard Reference Material (SRM) is certified for concentrations of four antiepilepsy drugs-phenytoin,

ethosuximide, phenobarbital, and primidone-in a processed human serum base. It is intended for use ( 1 ) in the

calibration and standardization of procedures employed in clinical laboratories for the determination of these drugs in serum, and (2) for the critical evaluation of working or secondary reference solutions prepared either in- house or supplied commercially. The certified concentrations apply to the four materials supplied, after each serum is reconstituted, following the procedures described in "Instructions for Use." Concentration Level, ng/mL

Drug Toxic Therapeutic Subtherapeutic Blank

Phenytoin 60.7 ± 0.9 16.7 ± 0.3 4.210.1 0 Ethosuximide 174.7±0.6 75.9 ± 0.5 11.8 10.4 0

Phenobarbital 103.6 ± 0.3 21.610.2 5.3 1 0.2 0

Primidone 18.6 ±0.7 8.1 10.2 3.610.1 0

The uncertainties represent one standard error for the above certified values. The imprecisions observed both

within and between the liquid and gas chromatographic analyses used for this certificate are included in the standard errors. The statistical analyses were made by R. C. Paule and J. Mandel. The modified human serum base was processed, vialed and packaged by Microbiological Associates, Walkersville, Maryland. Analyses leading to certification were performed in the NBS Center for Analytical Chemistry by R. Angeles, R. G. Christensen, A. Cohen, B. Coxon, D. Enagonio, D. J. Reeder, and L. T. Sniegoski. Valuable correlating analyses were also performed by: E. Berman, Cook County Hospital, Chicago, Illinois; K. Dudley, The University of North Carolina at Chapel Hill; and H. J. Kupferberg, National Institute of Neurological and Communicative Disorders and Stroke, NIH. The overall direction and technical measurements leading to the certification were under the chairmenship of D. J. Reeder and R. Schaffer. 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 R. Keith Kirby. Washington, D.C. 20234 J. Paul Cali, Chief

April 5, 1 979 Office of Standard Reference Materials

(Editorial Revision of Certificate dated 11-17-78) (over)

39
The analytical techniques used in the certification of this Standard Reference Material were liquid chromatography, calibrated with external standards, and gas chromatography, calibrated by bracketing and internal standards. The drug concentrations have also been determined with commercially available reagents that are based on immunoenzymatic assay principles. The mean values obtained by this methodology were close to the

certified values; however, the precision was not sufficient to be included in the certification data. The Standard Reference Material is intended for "in vitro" diagnostic use only. WARNING: HANDLE AS IF CAPABLE OF TRANSMITTING HEPATITIS. Source material from

which this serum base was derived was found non-reactive for Hepatitis B antigen when tested with licensed third-generation reagents. No known test method can provide complete assurance that products derived from human blood will not transmit hepatitis.

Instructions for Use

This Standard Reference Material is supplied as a set of four different freeze-dried preparations. They should

be stored at refrigerator temperature (~ 4 °C) and should not be exposed to sunlight or ultraviolet radiation. Under such storage, the SRM is expected to be stable for at least 2 years. Samples of this SRM will be

monitored. Should statistical evidence indicate a degradation of the certified properties purchasers will be

notified by NBS. It is recommended that the material not be used after 2 years from the date of purchase. For use, it is necessary to reconstitute the freeze-dried materials with high-purity water.* When a vial is opened, remove the rubber stopper carefully so as not to dislodge any serum particles that may adhere to the stopper. Add 5.0 mL of water to the vial from a calibrated volumetric pipet or other dispenser of known accuracy. Replace the stopper and allow the contents to stand at room temperature for 20-30 min. Finally, mix the contents by gentle swirling. DO NOT shake vigorously because denaturation and frothing may result. After reconstitution, the contents should be used within one day; otherwise, the certified values cannot be

assured. Storage of the reconstituted material beyond 1 2-24 hours may result in deterioration of the drugs or in degradation of the serum base. 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 is November 17, 1978. Equivalent to Type II reagent grade water as specified by the College of American Pathologists (CAP).

SRM 900

40
APPENDIX II CONSTRUCTION OF AN IMPROVED DIFFERENTIAL REFRACTOMETER

A. Introduction

Several years ago, it was decided to construct a differential refractometer which

would be an improvement over the modified Brice-Phoenix [1.2] 1 refractometer constructed

earlier by Wagner [3]. The improvements desired were to provide easier sample introduction

and an objective means of readout. It was felt that the former could best be provided by means of flow- through sample cells; and that the latter would require some sort of photocell

to detect the position of the image, such as that described by Penther and Noller [4]. Since the design criteria included the specification that the instrument should be operable

up to 150 °C, the use of an absolute interferometric refractometer was ruled out. The larger cells required would be too difficult to thermostat and to provide with flow-through

sample introduction.

A commercial instrument embodying flow-through cells and photocell readout of image position, the Waters Associates Model R-4, was available, but it lacked sufficient range and readout precision for use as a general purpose differential refractometer. It was designed

for use as a liquid chromatography detector. Also, because of the compact arrangement of

its components, it did not lend itself to direct modification for high temperature use.

B. Details of Modifications to the Refractometer

It was decided to incorporate the sample cells and photocell readout of the R-4 instrument into a new instrument with a linear rather than a folded optical path. The folded optical path was eliminated by removing the focusi ng-coll imati ng lens and the mirror

from the original cell chamber and drilling a hole in it to permit light to pass straight

through the cell. The components are all bolted to a steel bar 2.5 x 12.5 x 60 cm which

serves as an optical bench (figure 1). The original rotating null-glass block was removed

and another substituted which was about four times as thick, thereby increasing the range. The original screw-driven lever which rotated the block was replaced by. a slightly longer

one and the screw was replaced by a 0-2 inch micrometer head. The face of the micrometer

bears against a small ball imbedded in the lever. Thus the micrometer reading, L, is a linear function of sin 0 (see glossary of symbols). Temperature stability was achieved as follows: The original cell chamber was removed

from the heat exchanger block of the R-4 and installed in a copper box of external dimen- sions 12 x 12 x 10 cm, having walls 1.25 cm thick. The cell chamber is mounted in the box

by attaching it to a brass plate, which is in turn affixed to the bottom of the copper box

by push-pull alignment screws. The stainless steel inlet tubings to the cell are soldered

to the lid of the box for about 30 cm to provide heat exchange for the entering liquids. The inlet tubings pass through the cell chamber to the bottom of the cell holder where they

are connected to the cell compartments with a teflon gasket. The outlet tubings are con- nected to the top of the cell compartments with another teflon gasket. The copper box is attached to the optical bench with legs of bakelite and is further insulated with about

4 cm of balsa wood. Glass windows are fitted in holes in the insulation along the optical

path. Thermal coupling between the box and the cell chamber was adjusted by packing in wads of copper turnings to give a time constant of about 10 minutes. This was found by experiment to be the optimum. Longer time constants gave rise to drift of the cell temperature and shorter time constants permitted ripples in the temperature of the cell chamber

due to temperature oscillations of the outer box. Heating of the copper box is accomplished by cartridge heaters inserted in bores, one

in each side. The two main heaters are variable up to 50 W each, and the two control heaters are 15 W each. The control thermistor is placed in a well about 2 mm from one- of

the control heaters. Tests with a thermistor bridge sensitive to about 0.0002 degrees

showed no periodic variation in the temperature of the cell chamber.

x Figures in brackets indicate the literature references at the end of this appendix.

41

The light source is the same as that used in the R-4, a tungsten filament bulb. Since

high intensity is not required, an interference filter can be placed in the optical path to give the desired wavelength. A slit is placed near the bulb and a simple lens of 50-mm

focal length is used as a collimator. Between the collimator and the cell box is mounted

a holder for interference filters. The cell, also from the Waters R-4 instrument, is their "90 degree" cell. It is a rectangular glass box divided into two compartments by a glass partition at such an angle

that the angle of incidence upon it of light from the source is 63 degrees. Each com- partment has a hole at the top and at the bottom where thf> outlet and inlet tubings,

respectively, are connected. The volume of each compartment is about 70 microliters. When the collimated beam exits from the copper box, it is focused by a 150-mm focal length lens through a glass block which can be rotated to nullify any displacement of the

image. At the focal plane of this lens is a beam splitter in the form of the edge of a front surface mirror which throws light from a part of the image onto a photoresistive

element. The remainder of the light passes the beam splitting mirror and falls on another photoresistive element. These are two arms of a Wheatstone bridge. The output of this bridge is amplified and displayed on a stripchart recorder. It was found necessary to modify the original photocell block as shown in figure 2 in order to correct its sensitivity

to changes in the state of polarization of the incident light.

In addition to the micrometer which rotates the glass block to return the image to a null position, there is a zero adjustment screw which bears directly on the photocell

block. It is used to position the photocell block so that when the refractive index dif- ference is zero and the setting of the micrometer is zero, the bridge is balanced. The system was aligned by use of a laser beam to define the optical axis. The noise level of the instrument corresponds to about 2 x 10"7 in refractive index. If the sensitivity is taken as 2 times the noise level, the sensitivity is about 4 x 10~7

, corresponding to about

0.0001 inch on the micrometer. The range of the instrument is about 0.004 in refractive

index, corresponding to about 1 inch on the micrometer.

C. Operation of the Differential Refractometer

In use, the device is turned on and allowed to warm up. This usually takes several

hours. The end of the warm-up period is determined by cessation of baseline drift. With

solvent in both sides of the cell and with the micrometer set at zero, the zero adjusting

screw is used to balance the bridge. Then the recorder trace is set to near the center of the paper. The solution to be measured is introduced into one compartment of the cell. Since the combined volumes of the inlet tubing and cell are only about 350 microliters,

three rinses of 1 mL each suffice to flush the cell adequately. The trace is then brought

back to near the center of the paper, the micrometer being set to the nearest whole thousandth of an inch, the ten thousandths being interpolated by the position of the chart

trace. After several solutions have been measured, solvent is re-introduced and a baseline

is drawn. Interpolations of the micrometer readings are made with reference to this baseline, thus eliminating the effect of any drift in the instrument.

D. Calibration of the Refractometer

Probably the most satisfactory liquids to use in calibrating differential refrac- tometers are the salt solutions which have been studied by Kruis [5] who has measured An

of solutions of a number of salts at various wavelengths. His instrument was an absolute interferometric differential refractometer of great sensitivity and considerable range. Since the optical relationships of the various components of the refractometer are readily derived, it was at first felt that it would be desirable to fit the calibration

data to an equation, the functional form of which was known a priori . The image displace- ment given by a rotation of the glass block is given by: (see glossary of symbols)

43

d = t sin (

1 - ^n' 2 - sin:

fl sin2 *

n

2

<t> (1)

a relation readily derived from application of Snell's Law. The angle of deviation of the

light beam induced by the refractive index difference is not precisely proportional to the refractive index, but is given by:

which is also straightforwardly arrived at from the laws of refraction. Since for small angles:

it is possible to obtain a relation between the reading of the micrometer and An. A complication arises when e is not zero, because the angle of incidence on the glass block

becomes 0 + 4>. This is easily taken into account, but proves to be negligible. The calibration constant thus obtained is a value of f, the image distance of the focusing

lens. Slight errors in other measured quantities can be lumped into it without causing

serious difficulty, since the function is nearly linear. A possible refinement is to calculate values of 6 from equation 2 and fit them to a function of L. This will allow the dependence of 6 on n as well as An to be taken into account. Since it is so nearly

negligible, the fact that the angle of deviation is dependent on n as well as An seems to have been generally disregarded in discussions of prism-cell refractometers. In this instrument, errors in An as large as 0.1 percent can arise when calibrating with water

solutions and measuring benzene solutions. Attempts to put the foregoing into practice were not successful. Standard deviations

of An about the calculated line were about 4 x 10~6. Astigmatism in the image produced by

the rotation of the glass block is likely to be the cause. Since the image is perhaps 0.2

mm wide and the photo cells are attempting to determine the position of the center to about

0.1 micrometer, any change of focus is critical.

In lieu of the above procedure, An vs. L were fitted by least squares to a third order

polynomial. Using a wavelength of 546 nm, seven potassium chloride solutions were measured

against water and against each of the other solutions. Fitting these 28 data points to a polynomial in L yielded this calibration equation:

with an estimated standard deviation in An about the calculated line of 1.6 x 10~6 in refractive index.

(2)

e

d

f (3)

An x 106 = 0.2 + 4443.9 L - 172.5 L

2 + 311 L

3

45
References

[1] Brice, B. A. and Halwer, M. , J. Opt. Soc. of America, 4J, 1033 (1951).

[2] Certain commercial instruments are identified by manufacturer's name or label. In no instance does such identification imply endorsement by the National Bureau of Standards

nor does it imply that the particular products or equipment are necessarily the best available for that purpose.

[3] Wagner, H. L. , J. Res. Nat. Bur. Stand. (U.S.), 76A, No. 2, 163 (1972).

[4] Penther, C. J. and Noller, G. W. , Rev. Sci. Instr. , 29, 43 (1958).

[5] Geffken, W. and Kruis, A., Z. Physik. Chem., B23_, 175 (1933).

Glossary of Symbols

A Angle of incidence at the septum of the prism-cell, here about 63°

d Lateral displacement of the image

L Micrometer reading * Angle of rotation of the glass block

9 Angle of deflection by the prism-cell

t Thickness of the glass block

f Image distance of the focusing lens

n' Refractive index of glass block

n Refractive index of solvent An Refractive index difference between solvent and solution

46
APPENDIX III COLLEGE OF AMERICAN PATHOLOGISTS

REAGENT WATER

SPECIFICATIONS

COMMISSION ON LABORATORY INSPECTION AND ACCREDITATION

47
OFFICERS TYRA T. HUTCHENS, M.D.

LAWRENCE J. McCORMACK, M.D. JAMES D. BARGER, M.D.

PRESIDENT—1978-79 University of Oregon

Health Sciences Center

Portland, Oregon 97201

VICE PRESIDENT Cleveland Clinic Cleveland, Ohio 44106

SECRETARY-TREASURER

Sunrise Hospital Las Vegas, Nevada 89114 HEADQUARTERS OFFICE HOWARD E. CARTWRIGHT

FRANCES RYAN

RITA MUELLER

Executive Director Manager, Inspection and

Accreditation Applications Coordinator,

Inspection and Accreditation

Reprinted with permission of the College of American Pathologists,

7400 Skokie Blvd., Skokie, IL 60077.

Copyright 1978 College of American Pathologists. All rights re- served. None of the contents of this publication may be repro- duced in a retrieval system or transmitted in any form or by any means (electronic, mechanical, photocopying, recording or other- wise) without the prior written permission of the publisher.

48
COMMISSION ON

LABORATORY INSPECTION AND ACCREDITATION

MAJ. GEN. JOE M. BLUMBERG, M.D. JOHN K. DUCKWORTH, M.D. LESTER KIEFER, M.D. NORTHEAST REGION

ISRAEL DIAMOND, M.D. RICHARD W. BARR, M.D. MIDDLE EAST REGION ROBERT R. RICKERT, M.D. JOHN J. MORAN, M.D. LESTER KIEFER, M.D. ROBERT J. HARTSOCK, M.D. MANUEL A. BERGNES, M.D. ALFRED SCHWARZ, M.D. DONALD B. POLAN, M.D. STEPHEN HANSON, M.D. SOUTHEAST REGION VERNON E. MARTENS, M.D. DEZSO K. MERENYI, M.D. DENNIS S. O'CONNOR, M.D. GEORGE P. VENNART, M.D.

E. ARTHUR DRESKIN, M.D. GULF REGION HANS J. PETERS, M.D. ROBERT DELASHMUTT, M.D. DONALD M. TAFT, M.D. ANTHONY R. CLERCH, M.D.

G. LESTER FORBES, JR., M.D. JOSEPH C. ERWIN, M.D. THOMAS F. PUCKETT, M.D.

Chairman

Vice-Chairman and

Interstate Commissioner

Chairman, 1 Checklist Sub-Committee

Commissioner

State Commissioner

(Massachusetts)

Commissioner

Deputy Commissioner

Deputy Commissioner

State Commissioner

(W. Pennsylvania)

State Commissioner

(Philadelphia) State Commissioner (New York City and E. New York)

State Commissioner

(W. New York)

State Commissioner

(Delaware)

Commissioner

State Commissioner

(Maryland)

State Commissioner

(West Virginia) State Commissioner

(Virginia) State Commissioner

(South Carolina)

Commissioner

Deputy Commissioner

State Commissioner

(Alabama)

State Commissioner

(Florida) State Commissioner

(Georgia)

State Commissioner

(Louisiana) State Commissioner

(Mississippi)

49
SOUTH CENTRAL REGION FRANK M. TOWNSEND, M.D. MICHAEL H. SULAK, M.D. RICHARD J. TAYLOR, M.D. RONALD E. BUCHANAN, M.D. CLAUDE K. LEEPER, M.D. ALVIN SMITH, M.D. GLEN F. BAKER, M.D. WEST CENTRAL REGION

C. W. REIQUAM, M.D. NORMAN E. POND, M.D. ROBERT FENNELL, M.D. FRED MIYA, M.D. GREGORY BRONDOS, M.D. NORTH CENTRAL REGION

MILTON SIMONS, M.D. CRAIG W. FREEMAN, M.D. JOHN GRUHN, M.D.

D. B. CLAUDON, M.D. GREAT LAKES REGION

WILLIAM A. HAWK, M.D. GEORGE C. HOFFMAN, M.D. ROBERT L. COSTIN, M.D. ROBERT S. HOWELL, M.D. AARON LUPOVITCH, M.D. NORTHWEST REGION

WILLIAM B. HAMLIN, M.D. ARCHIE Y. HAMILTON, M.D. DELBERT SCOTT, M.D. SOUTHWEST REGION ERNEST E. SIMARD, M.D. GERALD F. SIMON, M.D. MARK G. KARTUB, M.D. THOMAS KIDDIE, M.D.

Commissioner

Deputy Commissioner

State Commissioner

(Kansas and Oklahoma)

Asst. State Commissioner

(Texas)

State Commissioner

(Arkansas and Missouri)

Asst. State Commissioner

(Kansas and Oklahoma)

Asst. State Commissioner

(Arkansas and Missouri)

Commissioner

State Commissioner (New Mexico) Deputy Commissioner

State Commissioner

(Utah) State Commissioner

(Wyoming)

Commissioner

Deputy Commissioner

State Commissioner

(Illinois) State Commissioner

(Wisconsin)

Commissioner

Deputy Commissioner

State Commissioner

(Indiana) State Commissioner

(Kentucky)

State Commissioner

(Michigan)

Commissioner

Deputy Commissioner

Deputy Commissioner

Commissioner

State Commissioner

(N. California) State Commissioner

(Nevada and Arizona)

State Commissioner

(S. California)

50
ACKNOWLEDGEMENTS

This manual was originally written by Alton Stier, M.D., Larry Miller, M.T.(ASCP) and Robert Smith, M.S. Their efforts were instrumental

in filling a significant void in the information available to laboratories. This revision is intended to update that information and is in no way

intended to detract from their initial, valuable efforts. Likewise, Martha Winstead, M.T.(ASCP), who has been instru- mental in causing people to be concerned about the water they use, cannot be praised enough for her efforts over many years in the face

of overwhelming "disinterest." Her contributions indeed are the very

basis for much of the material contained herein.

Lastly, mention must be made of the literally hundreds of technical people and chemists who have questioned, complained and persistently requested information. They, through these mechanisms,

have served as the stimulus that has kept me working to complete

this revision.

All of the above have contributed mightily to laboratory im- provement!

WILLIAM B. HAMLIN, M.D.

51
INTRODUCTION

Water, the most commonly used reagent in the clinical laboratory, has been

consistently neglected over the years with respect to the investigation and

definition of the required degree of purity that will insure reliable test results.

Historically, Winstead's (1) publication in 1967 was the first major effort to call attention to the effect of water on clinical laboratory test results and

suggests ways to produce and monitor adequate water. In 1972 Stier et al. (2) published a manual which expanded considerably the definition of the problem and suggested specifications and testing methods for controlling water purity in the clinical laboratory. This effort was followed shortly by the 1973 American Society of Testing Methods (ASTM) Standards (3); and fi- nally the National Committee for Clinical Laboratory Standards (NCCLS)

has produced tentative specifications for reagent water which are appended

to this manual (4). The uncomfortable fact is that almost all the interest in the water used

in clinical laboratories has developed within the last 10-15 years; and fur- ther, there is still today only scant, published, scientific information indicat- ing what effect, and at what level, various contaminants in reagent water will have on clinical laboratory test results. Many of the current specifications are speculations as to the effect that a given contaminant will potentially have on clinical laboratory testing methods or procedures. The specifications and grades defined herein represent suggestions for the minimum quality of water to be used in the Clinical Laboratory. The

criteria proposed should be satisfactory for mosf uses within the laboratory. There may be some special uses that will require additional purification. The specifications are for the end product, i.e. the water produced, re- gardless of the production method. If a production method gives water that

is always negative for some contaminant, it may not be necessary to test for that contaminant as frequently as is suggested. There is no specification for Type I water because the level of resistance precludes the presence of sufficient ions to obtain reliable pH readings. Possible contaminants such as sodium, chloride, sulfate, etc. have not been shown in the specifications. If such ions were present to a significant degree

in the Type I water the resistance specifications could not be met.

52
These are not standards but recommendations of the Commission

on Inspection and Accreditation for Water Quality Control. REAGENT WATER

1. To suggest minimum specifications for various grades of water

suitable for use in the clinical laboratory.

2. To suggest utilization limitations for the various grades of water. 3. To describe testing methods for insuring that water conforms to the minimum specifications. The minimum specifications suggested are as follows: Water Grade or Type

Specification

1. Resistivity - Specific Resistance Type 1 Type II Type III (megohms© 25°C)

a. In line 10

b. Effluent (As Used) 2.0 0.1

2. Silicate (mg/iiter S:02) 0.05 0.10 1.00

3. pH N.A. N.A. 5.0-8.0

4. Microbiological Content

(cfu/ml; max.) 10 104 N.A. NOTES:

1) Particulate Matter — Type I water should be "free" of particulate matter larger than 0.2 micron. Free of particulate matter is defined as less than 500 particles/liter which are greater than 0.2 micron.

2) Other parameters such as particle size, heavy metals and/or

trace elements, and carbon dioxide content should be considered and monitored when appropriate for special or specific uses.

3) Organics, and other contaminants, may be reduced with the use

of activated carbon. Any method of preparation of these three types of water is acceptable provided the end product meets the specifications. Deionization, distillation and reverse osmosis either alone or in various combinations are considered capable of providing water

that conforms to the specifications. Each of these processes has its own requirements for source water; and each has its particular re- sidual contaminants that must be considered.

53
REAGENT Suggestions for the utilization of the various types of water WATER specified is as follows: UTILIZATION: Type I — Tissue and/or cell culture methods; ultra-micro chemical analyses, special and critical analytical chemical analyses where accuracy at nanogram

and sub-nanogram levels is necessary; preparation of standard solutions. Type II — Most routine clinical laboratory methods in chemistry, immunology, hematology and other

areas. Type III — Most qualitative procedures; most procedures in urinalysis, parasitology and histology; glassware

washing; general laboratory tests not requiring

Type II or better water. TESTING: The methods listed are basically those suggested in the NCCLS

Tentative Standard PSC-3: Specifications for Reagent Grade

Water Used In The Clinical Laboratory; although some modifications have been included in this manual.

1. RESISTIVITY

A. Equipment and Materials

1) Resistivity meter, temperature compensated, capable of measuring resistivity higher than that specified for the designated water. The meter should be used in accord- ance with the manufacturer's instructions.

2) Thermometer with graduations to 0.5°C and calibrated to conform to National Bureau of Standards (NBS)

standards.

3) Potassium Chloride Stock Standard is prepared by dis- solving 10.0 g. of pure, dry KC1 in one liter of Type I water at 25°C. (Alternately, a stock standard solution can be prepared using NaCl. Dissolve 9.25 g. of pure, dry

NaCl in one liter of Type I water at 25°C).

B. Sampling and Measurement

1) The specification for resistivity of all the types of water

is at 25°C. If meters are used to measure resistivity which do not include an automatic temperature compensating circuit, an accurate measurement of temperature must be made simultaneously with resistivity measurements. Whenever the temperature of the water measured is outside the 25±2°C range, the resistance should be cor- rected to 25°C according to the directions provided with

the meter.

2) The resistivity of all the types of water should be measured as close as possible to the time of use of the water.

54
The measurement should be taken in accordance with

the manufacturer's directions.

3) The resistivity of all types of water can be measured by

using either an in-line meter or an off-line meter using

the following precautions:

a. For in-line meters, follow the manufacturer's instruc- tions carefully.

b. For off-line meters:

1) Rinse the cell and container at least three times with separate aliquots of the water to be tested.

2) Fill the cell and read as rapidly as possible. C. Calibration

1) The resistivity meter should be calibrated and/or checked periodically in accordance with the manufacturer's instructions.

2) For some off-line meters, a KC1 (or NaCl) solution may

conveniently be used to monitor the day-to-day operation of the meter-electrode combination. Appendix II contains the details of preparing and using the stock standard solutions for this purpose. Read the resistance of the standard solutions in the same manner outlined

for the water samples. Day-to-day consistency is more

important than a precisely accurate resistance reading. SILICATE

A. Method

1) Silicates react with molybdate ion to form a complex which can be reduced by 1-amino, 2-naphthol, 4-sulfonic acid to produce a blue color. The intensity of the blue

color is proportional to the concentration of soluble

silica. B. Reagents

Prepare and store in plastic containers.

1) 1-amino, 2-naphthol, 4-sulfonic acid solution. Dissolve 1 g. of sodium sulfite (Na2S03) in 50 ml. of Type I water and add 0.5 g. 1-amino, 2-naphthol, 4-sulfonic acid. Mix

to dissolve. Add this solution to 100 ml. of a solution containing 30 g. of sodium hydrogen sulfite (NaHSOa).

Dilute to 200 ml. with water. Store in a dark, polyethylene bottle at room temperature (stable for two

weeks).

2) Ammonium molybdate solution, 100 g./liter. Dissolve 10

g. of ammonium molybdate [(NH4) eMo7O24-4H20] in 100 ml. water. Store in a polyethylene bottle at room

temperature (stable for three months).

3) Hydrochloric acid, approximately 6 mol/liter. Mix one volume of concentrated HC1 with one volume of water.

55
Store in a polyethylene bottle at room temperature (sta- ble indefinitely).

4) Oxalic acid solution, 100 g./liter. Dissolve 10 g. of oxalic acid in 100 ml. of water. Store in polyethylene bottle at room temperature (stable for three months).

5) Silica, standard solution (1 ml.= 1 mg. SiCh). Dissolve

4.732 g. of sodium metasilicate (Na2Si02-9H20) in water and dilute to one liter. Store in polyethylene bottle at room temperature (stable for three months).

C. Equipment and Materials Spectrophotometer. Ideally a setting of 802 nm. is desirable

since that is the maximum absorbance wavelength for the compound to be measured. However, a setting of 700 nm.

can be used for silica concentrations of 0.10 mg./liter or

greater. The limit of detectability of this method using a wavelength setting of 802 nm. is .01 mg./liter. D. Sampling

Collect 500 ml. samples of the water to be tested in chemically clean polyethylene bottles at 35°C or below.

E. Standardization

1) Prepare a series of standards covering a concentration range of 0.05 to 1.0 mg./liter of Si02 by proper dilution of

the standard silica solution. Treat 50 ml. aliquots of each

dilution as described in the procedure.

2) Use a 50 ml. aliquot of silica-free water carried through

the procedure as a blank.

3) Prepare a calibration curve, plotting absorbance against

silicate concentration as Si02 in mg./liter.

F. Procedure

1) Transfer, quantitatively, 50 ml. of water to be tested to a polyethylene or other suitable plastic container.

2) Add in quick succession 1 ml. of HC1 and 2 ml. of am- monium molybdate solution. Mix well.

3) After exactly 5 minutes, add 1.5 ml. of oxalic acid solu- tion and mix.

4) After one minute, add 2 ml. of the amino-naphtholsulfonic acid solution. Mix well and allow to stand for 10

minutes.

5) Treat a 50 ml. aliquot of silica-free water exactly as described above and use to zero the spectrophotometer.

6) Measure the absorbance of the sample at 802 nm. (or 700

nm.).

7) Silica concentration in milligrams per liter, may be read

directly from the calibration curve. Record the milli- grams per liter in a permanent bench log.

56
NOTE: If desired, a visual assessment can be made by comparing

the 0.1 mgm./liter standard with the test sample. If the

test water is no darker than the standard color, the "water

is acceptable for Type II Reagent Water."

3. pH

Pure water (2 megohm +) contains so few ions that it is unnecessary to obtain pH measurements.

A. Reagents

1) Reference buffer solutions. At least two buffers should be chosen: one with a pH of 7.0 and one with a pH

lower than 7.0. Commercially available buffers may be

used if verified against NBS reference buffers.

2) Potassium chloride, saturated. B. Standardization The room temperature must be recorded and the exact pH

for, the buffers at the temperature must be used to standardize the pH meter. Standardization should be done

in accordance with the manufacturer's instructions. C. Procedure

1) Standardize the pH meter.

2) Rinse the electrodes with at least three changes of water

using a flowing stream.

3) Add one drop' of saturated KC1 to 50 ml. of water sample. Take the pH reading as quickly as possible, in ac- cordance with the manufacturer's instructions.

4) Record the pH to the nearest 0.1 unit.

4. MICROBIOLOGICAL CONTENT

A. Definition Microbiological content must include the evaluation of total colony count as determined by an accepted method after incubation at (36± 1°C) for 24 hours and at 25± 1°C for 48

hours and reported as colony forming units per milliliter (cfu/ml.) B. Sampling

1) Use a sterile closed container of adequate size to hold

the entire sample and leave ample air space to permit

mixing of the sample before examination. Sample sizes which result in 10 to 100 colonies on the membrane cul- ture or plate are optimum for enumeration of results (ap- proximately 0.1 ml. for Type II water and 5-10 ml. for Type I water).

2) Open tap fully for one minute, then restrict flow to allow

filling of bottle without splashing.

3) The sample should be processed within one hour of col- lection or stored at 2 to 8°C for no longer than six hours. C. Total Bacterial Count Methodology The current NCCLS Tentative Standards describe three (3)

57
alternative methods for ascertaining total bacterial count in terms of colony forming units per milliliter. These are:

1) Standard Plate Count (Reference Method)

2) Membrane Filtration Method

3) Calibrated Wire Loop Method

It is our opinion that any of these three methodologies would

be adequate for monitoring purposes and the specific technique used will depend on the type of laboratory, the nature or type of water being cultured (for example, a calibrated wire loop

technique should not be used to monitor Type I water because of

the very low bacterial concentration anticipated), resources avail- able etc. Whatever method is selected, the appropriate and cus- tomary records of tests performed, results, actions taken etc. must

be carefully documented. Appendix III is a compendium of these three methods cur- rently described in the NCCLS Tentative Standards.

DEFINITIONS

1. Microbiological Content - The microbiological content of via- ble organisms, as determined by total colony count after incu- bation at 35-37°C for 24 hours; reported as colony forming units per milliliter. 2. Particulate Matter - A discrete quantity of matter existing in a

solid state, dispersed in water to give a heterogeneous mixture.

3. Free of Particulate Matter - Defined as less than 500 particles/

liter which are greater than 0.2 micron.

4. pH - The negative logarithm of the hydrogen ion activity measured electrometrically.

5. Silicate - The content of soluble silica; reported in milligrams per liter, as SiCh.

6. Specific Resistivity - The electrical resistance in ohms measured between opposite faces of a 1.00 centimeter cube of an aqueous solution at a specified temperature. For these specifi- cations the specific resistivity will be corrected to 25°C and re- ported in megohms-cm.

7. Specific Conductance - The electrical conductivity, or the re- ciprocal of the resistance in ohms measured between opposite

faces of a centimeter cube of an aqueous solution at a specified temperature. The measurement may be in part due to dissolved

gases that ionize in solution. Unionized materials are not measured. The specific conductance is expressed in micromhos per

centimeter at 25°C. (3)

58
APPENDIX I

SPECIFICATIONS:

PREPARATION:

INTENDED USE:

The current NCCLS publication Specifications For Reagent

Water Used In The Clinical Laboratory (NCCLS Tentative Standard: PSC-3), lists the following specifications, usage recommendations and storage and handling requirements. A complete copy of the publication including testing mechanisms may be obtained by

writing to: National Committee For Clinical Laboratory Standards

771 East Lancaster Avenue

Villanova, Pennsylvania 19085

All specifications are stated for water as measured at the time of

production. The resistivity of Type I water must be measured inline; all other specifications relate to the samples measured "off- line."

Microbiological content, colony forming

units/milliliter (maximum) PH

Resistivity megohms-cm. room

temperature (25°C)

Silicate, mg./liter Si02 (maximum) NOTE: Type I water must be free of particulate matter larger than 0.2 micron. Any method of preparation of these three types of water is ac- ceptable provided the specifications are met. The following proc- esses are considered capable of providing water that conforms to the specifications. These processes are examples only and any

combination of them is satisfactory as long as the resulting water meets the specifications: Reverse Osmosis

Distillation Deionization Manufacturers of in vitro diagnostic products and scientists using clinical chemical laboratory testing procedures should

specify those interferences which must be avoided such as heavy

metals, NH3 and COz. Type I water should be used in test methods requiring minimal

interference and maximum precision and accuracy. Examples of

Type 1 Type II Type III

10 103 N.A. N.A. N.A. 5.0-8.0 10 2.0 0.1 0.05 0.10 1.00

59
such uses might be:

1. Atomic absorption and flame emission spectrometry.

2. Enzymatic procedures sensitive to trace metals.

3. Electrophoretic procedures.

4. High sensitivity chromatographic procedures.

5. Fluorometric procedures.

6. Buffer solutions (special buffers may also require C02-free water).

7. Preparation of standard solutions. NOTE: Even greater purity than Type I water will be required

for selected purposes, such as pesticide analysis. Type II water may be used for general laboratory testing other than the above and for glassware rinsing. Type III water may be used for glassware washing, for prelimi- nary rinsing of glassware (final rinsing should be with the water

grade suitable for intended glassware use), and for feedwater for production of higher grade water. STORAGE AND Type I water should preferably be used immediately after proc- HANDLING: essing. Storage of Type II water should be kept to a minimum in order to provide the quality consistent with the specific intended

use. Storage and distribution systems should be constructed of

materials that will protect the water from contamination.

60
APPENDIX II USING STANDARD

SOLUTIONS TO

VERIFY THE

RELIABILITY OF CONDUCTANCE

METERS:

The most practical and at the same time reliable means of ver- ifying the function of a conductance meter is to use standard solutions of known resistance. This can be done easily and cheaply by

using either KC1 or NaCl solutions. The principle involved is based on the formula: M.W.

Cppm = T

~

Where: Cppm = Concentration in parts per million M.W. = Molecular weight = Equivalent conductance Rs = Specific resistance in ohms

In the use of KC1, the ^-o for KC1 at 25°C is 149.86; while NaCl has an at 25°C of 126.45 (these values are listed in the Handbook of Chemistry and Physics.). The molecular weight of KC1 is 74.56 while NaCl has a molecular weight of 58.44. Thus, one has sufficient information to prepare a standard solu- tion of any resistance desired assuming that the water used in pre- paring the solution is so pure as to not interfere in any way with

the resistance of the prepared solution.

If one wishes to have a standard KC1 solution with a resistance of 1.0 megohm at 25°C, substitution in the formula can provide the necessary information as follows: Cppm = — = 0.4974ppm

(149.86) (1.0) Therefore a solution containing 0.4974 mgm/liter of KC1 will have a specific resistance of 1 megohm. By preparing an adequate

stock standard solution and with appropriate dilutions, one can

thus prepare the desired standard solution. In this case, the stock standard solution mentioned in the text contains 10,000 milligrams per liter. 1 ml. of the stock (10 mgm) diluted to 100 ml. would

result in a solution containing 100 mgm/liter or 0.1 mgm/ml. Thus,

5.0 ml. of the diluted stock solution would contain 0.5 mgm. and

that amount diluted to one liter produces the desired 1 megohm

standard (0.5 mgm/liter -KC1).

Since the resistance of the water used to make the standard solu- tions will have an impact oh the expected reading, the effect should be taken into consideration. This can be done by using the

61
formula based upon the fact that the resistance of two substances

(the standard solution as calculated and the water used) is equal to the resistance of two resistances in parallel which is equal to the product over the sum: Rs = (Rm)(Rt) Rm + Rt Where: Rs = The specific resistance Rm

= The resistance of the make-up water (one solution) Rt = The resistance of the test solution (KCI solution) If the water used (after equilibration with air from CO2) has a resistance of 2.0 megohms and the calculated resistance of the KCI

solution as described above is 1.0 megohm that the actual resist- ance is: (2.0) (1.0) = 2, = , 666 ohm

2.0 + 1.0 3 Or if the make-up water has a resistance of 5 megohms, then: Rs= (5-0) (1-0) . 5, = , 8333megohm

5.0 + 1 .0 6

62
APPENDIX III TOTAL BACTERIAL COUNT METHODS: A. Standard Plate Count (Reference Method)

1. Equipment

a. Petri plates, sterile and disposable, 15x 100 mm.

b. Laboratory incubator capable of maintaining temperature

at 35 to 37 C and moisture content at 90% humidity.

Periodic calibration is recommended.

c. Magnification. 10x to 15x lens or dissecting microscope.

2. Reagents

a. Nutrient medium

1) Tryptone glucose extract agar (TGE)*, or

2) Tryptone glucose yeast agar (TGY)*.

b. Buffered dilution water

1) Stock phosphate buffer solution*. Dissolve 34.0 g. of potassium dihydrogen phosphate (KH2PO4) in 500 ml. of Type I water. Adjust to pH 7.2 with 1.0 N NaOH.

Dilute to 1 liter with water.

2) Working buffer solution. Add 1.25 ml. of stock phosphate buffer solution and 5.0 ml. magnesium sulfate (50 g. MgSO4-7Ha0 per liter of water), to 1 liter of

water. Dispense 100 ml. amounts in capped bottles. Autoclave at 121 C for 15 minutes. Store at room temperature, tightly capped.

3. Procedure

a. Shake sample vigorously (approximately 25 times) to en- sure proper distribution of bacteria within the sample.

b. Into 15x100 mm Petri dishes, pipet, in duplicate, ap- propriate aliquots of:

1) Type I water: 1 ml. of undiluted sample.

2) Type II water: 1 ml. of 1:100 dilution of sample in working buffer solution.

c. Overlay unknown sample with 15 ml. of TGE of TGY

agar, tempered to 45±1 C.

d. Mix sample and agar. Solidify.

e. Incubate at 36±1 C. for 24 hours, and follow at 25±1 C

for 48 hours.

B. Membrane Filtration

1. Equipment

a. Petri plates, sterile and disposable, 15x60 mm.

b. Filtration unit. Seamless funnel which fastens to porous

plate for support of filter membrane.

*Available from BBL, Cockeysville, MD; or equivalent. 63
c. Filtration receptacle. One-liter flask with side arm or other suitable device which will exert a pressure dif- ferential on the membrane filter. d. Vacuum source.

e. Sterile filter membranes, nontoxic, with 0.45 micron

pore size.

f. Sterile absorbent pads. Nontoxic, 48 mm in diameter, and capable of absorption of 1.8 to 2.2 ml. of nutrient.

g. Instead of (a) through (f) above, a bacteriological

monitoring kit** or equivalent, including sterile monitors, ampoules of total count medium, and presterilized disposable polypropylene syringes.

h. Forceps, stainless steel, with smooth tips.

i. Laboratory incubator capable of maintaining temperature at 35 to 37 C and moisture content at 90% humidity.

Periodic calibration is recommended.

j. Magnification. 10 x to 15 x lens or dissecting microscope.

2. Reagents

a. Nutrient medium

1) M-Tryptone glucose yeast broth (M-TGY)*.

Prepare according to manufacturer's instructions.

After sterilization, store tightly capped under refriger- ation (stable for 30 days), or

2) M-TGE medium supplies in 1.8 ml. glass ampoules**

(stable for 1 year).

b. Buffered dilution water

1) Stock phosphate buffer solution*. Dissolve 34.0 g. of potassium dihydrogen phosphate (KH2PO4) in 500 ml. of Type I water. Adjust to pH 7.2 with 1.0 N NaOH.

Dilute to 1 liter with water.

2) Working buffer solution. Add 1.25 ml. of stock phosphate buffer solution and 5.0 ml. magnesium sulfate (50 g MgS04 -7H20/l water), to 1 liter of water. Dis- pense 100 ml. amounts in capped bottles, autoclave at 121 C for 15 minutes. Store at room temperature,

tightly capped.

c. Stain (supplementary material to aid in colony counting). Aqueous solution Malachite Green, 0.1 mg/liter, or stain supplied with the kit**. Store at room temperature in capped bottle.

3. Procedure

a. Shake sample vigorously (approximately 25 times) to en- sure proper distribution of bacteria within the sample.

•Available from BBL, Cockeysville, Maryland; or equivalent. **Available from Millipore Corporation, Bedford, Massachusetts; or equivalent.

64
b. Preparation of nutrient pad. Place sterile absorbent pad

in 15x60 mm Petri dish. Add 1.8 to 2.2 ml. M-TGE

broth; remove surplus liquid.

c. Filtration of sample.

1) Filtration units should be sterile at the beginning of each filtration series. A filtration series is considered

to be interrupted when an interval of 30 minutes or longer elapses between sample filtrations. After such

interruption, any further sample filtration is treated as a new filtration series that requires resterilization of

all membrane filter holders in use.

2) Place porous plate on filtration receptacle.

3) Using sterile forceps (dip in 95% ethyl or absolute methyl alcohol and then ignite fluid), place sterile membrane filter over porous plate.

4) Place funnel unit over plate and lock in place.

5) Filtration is then accomplished by passing sample (10

to 100 ml.) through filter under partial vacuum.

6) Between samples, rinse funnel and membrane three times with at least 30 ml. of buffered dilution water.

7) Unlock and remove funnel. Immediately remove membrane with sterile forceps, and place on sterile nutrient pad with rolling motion to avoid entrapment

of air. 8) Invert and incubate at 36 C± 1C with atmosphere of at least 90% humidity for 24 hours, followed by 25±1C

for 48 hours.

C. Calibrated Wire Loop Method

1. Equipment

a. Sterile Petri plates, disposable, 15x100 mm.

b. Calibrated loops — APHA platinum wire with 5%

rhodium and checked monthly for proper calibration —

to deliver; 0.01 ml. 19 gauge wire; inside loop, 4 mm.

c. Laboratory incubator capable of maintaining temperature

at 35 to 37 C and moisture content at 90% humidity.

Periodic calibration is recommended.

d. Magnification. 10x to 15x lens or dissecting microscope.

2. Reagents

a. Nutrient medium

1) Tryptone glucose extract agar (TGE)*, or

2) Tryptone glucose yeast agar (TGY)*.

Available from BBL, Cockeysville, Maryland; or equivalent.

65
b. Loop calibration dye

1) Prepare stock dilution of Evans blue dye by adding

0.75 g. of Evans blue dye to 100 ml. distilled water.

2) Filter through No. 40 Whatman filter paper.

3) Store in stoppered bottle at room temperature. Good

for 1 year.

3. Procedure

a. Calibration of loop should be done monthly.

1) Prepare accurate 1:500, 1:1000, 1:2000, and 1:4000 di- lutions of stock dye solution and determine optical densities in a spectrophotometer at 600 nm. Plot OD

on a linear scale against the concentration of dye.

2) Delivery of loop (0.01) can then be checked by trans- ferring 10 loopsful of stock solution to 100 ml. distilled water, flamed, and cooled between each loopful. OD of this solution is determined in the spec- trophotometer and should correspond to that of the 1:1000 dilution on the standard curve within ±20%. If the error is greater, the loop should be replaced or an

appropriate correction be made in colony counts

using that loop.

b. Calibrated loop culture procedure

1) Shake sample vigorously (approximately 25 times) to ensure proper distribution of bacteria within the sample.

2) Label two agar plates for each sample.

3) Flame and cool 0.01 loop, hold vertically and im- merse just below surface of the sample. The loop is moved straight up and down.

4) Place this loopful of water sample on the center of the surface of the plate. Streak entire surface with a

sterile bent glass rod.

5) Incubate at 36±1C for 24 hours, followed by 25±1C

for 48 hours.

4. Counting Procedure

a. Using 10 x to 15 x magnification, or dissecting scope, or

counter, count all colonies.

b. Colony counting on membrane filters may be aided by

adding 2-4 ml. stain. After 8-10 seconds, pour off stain. Colonies appear white or yellow with a green

background.

c. Results are calculated to represent cfu/ml.

66
REFERENCES

1. Winstead, M.; "Reagent Grade Water: How, When and Why?"; American Society of Medical Technologists, Bellaire, Texas 1967. Published by Steck Company, Austin, Texas.

2. Stier, A. R.; Miller, L. K.; and Smith, R. T.; "Water Specifications" College of Ameri- can Pathologists, Commission on Laboratory Inspection and Accreditation, Chicago,

Illinois, 1972.

3. 1973 Annual Book of ASTM Standards, Part 23, Water: Atmospheric Analysis. American Society for Testing and Materials, Philadelphia, Pennsylvania, 1973.

4. NCCLS-PSC-3: Specifications for Reagent Water Used in the Clinical Laboratory.

5. Otten, Gerard; "Measuring Water Purity by Specific Resistance"; American Laboratory, p.41, July 1972.

NOTES

67
COLLEGE OF AMERICAN PATHOLOGISTS

7400 N. SKOKIE BOULEVARD/ SKOKIE. ILLINOIS 60077

68
NBS-114A (rev

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET (See instructions)

PUBLICATION OR REPORT NO. NBS SP 260-722. Performing Organ. Report No 3. Publication Date Junes 1981

4. TITLE AND SUBTITLE Standard Reference Materials: SRM 900, Anti epilepsy Drug Level Assay Standard

5. author(S) Dennis J. Reeder, Bruce Coxon, Delmo Enagonio, Richard G. Christensen,

Barbara F. Howell, Robert C. Paule, John Mandel , and Robert Schaffer

6. PERFORMING ORGANIZATION (If joint or other than NBS. see instructions) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS DEPARTMENT OF COMMERCE 8.' Type of Report & Period Covered WASHINGTON, D.C. 20234 Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City. State, ZIP) National Institutes of Health

National Institute of Neurological and Communicative Disorders

and Stroke, Federal Building

Bethesda, MP 20205

10. SUPPLEMENTARY NOTES Library of Congress Catalog Card Number: 81-600062

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

11. ABSTRACT (A 200-word or less factual summary of'most significant information. If document includes a significant bibliography or literature survey, mention it here) Recognition of the efficacy of monitoring the concentrations of therapeutic drugs

in the blood of patients has revealed many needs for standardization of the laboratory

tests used for such monitoring. The National Bureau of Standards was asked to provide a Standard Reference Material (SRM) consisting of three serum samples, each to contain four antiepilepsy drugs at different concentrations. The four drugs are phenobarbi tal , phenytoin, primidone, and ethosuximide. The SRM would fill a basic

role for the achievement of accurate analysis to help ensure the reliability of

analyses for these drugs. The needs that had to be fulfilled to produce the SRM included: (1) analytical

criteria for purity of the drugs; (2) serum to be used as a matrix for the drugs;

(3) techniques for achieving homogeneity and stability of the SRM; and (4) two independent, highly accurate analytical methods for the certification.

This document describes development of methods and procedures used to produce

and certify the SRM.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons) Anticonvulsants; antiepilepsy drugs; blood analysis; chromatography, gas; chromatography, liquid; drug standards; enzyme immunoassy; epilepsy; ethosuximide; mass spectrometry; nuclear magnetic resonance spectroscopy; phenobarbi tal ; phenytoin; primidone;

13. availability reference material; serum base modification; serum matrix! 14. no. of

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NBS TECHNICAL PUBLICATIONS

PERIODICALS

JOURNAL OF RESEARCH—The Journal of Research of the National Bureau of Standards reports NBS research and develop- ment in those disciplines of the physical and engineering sciences in which the Bureau is active. These include physics, chemistry,

engineering, mathematics, and computer sciences. Papers cover a broad range of subjects, with major emphasis on measurement

methodology and the basic technology underlying standardization. Also included from time to time are survey articles on topics

closely related to the Bureau's technical and scientific programs. As a special service to subscribers each issue contains complete

citations to all recent Bureau publications in both NBS and non- NBS media. Issued six times a year. Annual subscription: domestic

$13; foreign $16.25. Single copy, $3 domestic; $3.75 foreign. NOTE: The Journal was formerly published in two sections: Section A "Physics and Chemistry" and Section B "Mathematical

Sciences." DIMENSIONS/NBS—This monthly magazine is published to in- form scientists, engineers, business and industry leaders, teachers,

students, and consumers of the latest advances in science and

technology, with primary emphasis on work at NBS. The magazine

highlights and reviews such issues as energy research, fife protection, building technology, metric conversion, pollution abatement,

health and safety, and consumer product performance. In addition, it reports the results of Bureau programs in measurement

standards and techniques, properties of matter and materials, engineering standards and services, instrumentation, and

automatic data processing. Annual subscription: domestic $11;

foreign $13.75. NONPERIODICALS

Monographs—Major contributions to the technical literature on

various subjects related to the Bureau's scientific and technical activities. Handbooks—Recommended codes of engineering and industrial

practice (including safety codes) developed in cooperation with in- terested industries, professional organizations, and regulatory

bodies.

Special Publications—Include proceedings of conferences sponsored by NBS, NBS annual reports, and other special publications appropriate to this grouping such as wall charts, pocket cards, and

bibliographies. Applied Mathematics Series—Mathematical tables, manuals, and

studies of special interest to physicists, engineers, chemists,

biologists, mathematicians, computer programmers, and others engaged in scientific and technical work.

National Standard Reference Data Series—Provides quantitative data on the physical and chemical properties of materials, compiled from the world's literature and critically evaluated. Developed under a worldwide program coordinated by NBS under

the authority of the National Standard Data Act (Public Law

90-396).

NOTE: The principal publication outlet for the foregoing data is the Journal of Physical and Chemical Reference Data (JPCRD)

published quarterly for NBS by the American Chemical Society (ACS) and the American Institute of Physics (AIP). Subscriptions,

reprints, and supplements available from ACS, 1 155 Sixteenth St., NW, Washington, DC 20056.

Building Science Series—Disseminates technical information developed at the Bureau on building materials, components,

systems, and whole structures. The series presents research results,

test methods, and performance criteria related to the structural and

environmental functions and the durability and safety characteristics of building elements and systems. Technical Notes—Studies or reports which are complete in themselves but restrictive in their treatment of a subject. Analogous to monographs but not so comprehensive in scope or definitive in treatment of the subject area. Often serve as a vehicle for final reports of work performed at NBS under the sponsorship of other government agencies. Voluntary Product Standards—Developed under procedures

published by the Department of Commerce in Part 10, Title 15, of

the Code of Federal Regulations. The standards establish nationally recognized requirements for products, and provide all concerned interests with a basis for common understanding of the

characteristics of the products. NBS administers this program as a supplement to the activities of the private sector standardizing

organizations. Consumer Information Series—Practical information, based on NBS research and experience, covering areas of interest to the con- sumer. Easily understandable language and illustrations provide

useful background knowledge for shopping in today's technological marketplace. Order the above NBS publications from: Superintendent of Documents, Government Printing Office, Washington, DC 20402. Order the following NBS publications—Fl PS and NBSIR's—from

the National Technical Information Services, Springfield, VA 22161

.

Federal Information Processing Standards Publications (FIPS PUB)—Publications in this series collectively constitute the Federal Information Processing Standards Register. The Register

serves as the official source of information in the Federal Govern- ment regarding standards issued by NBS pursuant to the Federal Property and Administrative Services Act of 1949 as amended.

Public Law 89-306 (79 Stat. 1127), and as implemented by Executive Order 11717(38 FR 12315, dated May 1 1, 1973) and Part 6 of Title 15 CFR (Code of Federal Regulations). NBS Interagency Reports (NBSIR)—A special series of interim or

final reports on work performed by NBS for outside sponsors

(both government and non-government). In general, initial dis- tribution is handled by the sponsor; public distribution is by the National Technical Information Services, Springfield, VA 22161,

in paper copy or microfiche form.
U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Washington, D.C. 20234

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