Standard Reference Materials for Clinical Measurements - Analytical

Standard Reference Materials for Clinical Measurements W. WAYNE MEINKE Analytical Chemistry Division National Bureau of Standards Washington, D. C. 20234

TK 1967 THE National Bureau of -*- Standards issued its first bio medical standard produced specif ically for use in clinical laborato ries. This standard, cholesterol (SRM 911), has been widely ac cepted as a primary standard by the clinical laboratories, and was followed in 1968 by urea (SRM 912), uric acid (SRM 913), and cre atinine (SRM 914), and in 1969 by calcium carbonate (SRM 915). NBS traditionally has been ori ented toward basic physical and chemical standards for science, in dustry, and technology, but entered the biomedical standards field at the urging of the College of American Pathologists and the American As sociation of Clinical Chemists. One may ask, "What is the func tion of the National Bureau of Stan dards in the area of clinical chemis try?" While we certainly are not clinical chemists, we are experts in measurement and have a very broad set of competences represented throughout the Analytical Chemis try Division. Furthermore, we have found that many of the prob lems of accurate measurement en countered by clinical chemists are amenable to the same competences we have applied in other areas such as metals analysis, oceanography, lunar samples, or air pollution. Thus, in addition to the five above-mentioned standard reference materials (SRMs), a glucose (SRM 917) and a potassium chloride (SRM 918) have been made avail 28 A .

able. Recently the Bureau com pleted a detailed study of bilirubin and made samples of this material available as SRM 916. At the same time, an extensive study of spectro photometry measurements has led to the issuance of a set of neutral density filters for the calibration of spectrophotometers. NBS also has available many other SRMs which, though not aimed specifically for use in the clinical laboratory, are prerequisites for accurate quality control in any laboratory performing chemical analyses. Basic standardization of titrimetric procedures should be es tablished via NBS primary stan dards. pH meters should be calibrated against NBS pH stan dards. Of the more than 700 SRMs available from NBS, many have particular applicability in estab lishing and maintaining the highest quality control in clinical or bio medical research laboratories. NBS Standard Reference Materi als provide unequivocal "thirdparty" certification of composition, purity, or some physical property. A number of other government agencies tie their regulations to NBS-SRMs. Sixty-five years of ex perience ha\re built a tradition of "accuracy awareness" into the SRM program which is unique through out the world. Experience has shown that this vector toward ac curate measurement can be applied with important consequences to many fields. History

The Analytical Chemistry Divi sion of the Bureau first became in terested in the problems of measure ment in clinical chemistry about five years ago when Bradley E. Copeland, M.D., Chairman of the

ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971

Standards Committee of the College of American Pathologists, pointed out the major discrepancies found in cholesterol measurements per formed in different laboratories throughout the country. Our at tention was also called to the diffi culties various clinical laboratories were having in standardizing spec trophotometers. Indeed, it was through Dr. Copeland's energetic persuasions that we became con vinced that we could make an im portant contribution by providing an SRM of cholesterol. Results of the cholesterol study, as we learned from Dr. Copeland, were indeed startling. In 1962 the Standards Committee of the Col lege of American Pathologists sur veyed a large number of clinical laboratories in this country with re gard to the measurement of choles terol. Over 6000 clinical labora tories were invited to participate in an intcrcomparison, and 1088 use ful replies were received. Two 5ml serum samples with different concentrations of cholesterol were distributed for measurement, one containing 152 mg/100 ml, and the other 259 mg/100 ml. The labora tories were requested to measure each sample routinely and to re turn the results to the College of American Pathologists. The results of the 1088 laboratories are plotted in Figure 1, in which the reported value on Sample A is plotted against that for Sample B. Wide scatter ing around the "true value" is ap parent. Indeed, some results were as much as 35-50% in error in the intercomparison. Dr. Copeland also emphasized the results of another intercompari son made by his Standards Com mittee in which samples οξ potas sium nitrate and pyrene were sent

REPORT FOR ANALYTICAL CHEMISTS Pressures on the modern clinical laboratory to provide rapid and accurate analyses have produced a great demand for standard reference materials. New SRMs being developed at the National Bureau of Standards will have particular applicability in establishing and maintaining the highest quality control in clinical or biomedical research laboratories

to 94 different laboratories for measurement on recording spectrophotometers. Figure 2 shows t h e r e sults of this intercomparison in a plot of potassium nitrate vs. pyrene measurements. T h e 94 results fall onto 11 points on t h e plot, each point corresponding to a particular make of recording spectrophotometer. I n other words, comparisons on this simple measurement system were easily intercomparable between different laboratories, when made with t h e same make a n d model of instrument, b u t comparisons between different types of instruments introduced significant errors. Soon after our initial contacts with D r . Copeland, we also began a series of very fruitful discussions with George N . Bowers, Jr., M . D . , Chairman of the Standards Committee of the American Association of Clinical Chemists. This active group heartily endorsed t h e u r gency of efforts on cholesterol a n d spectrophotometry standards. T h e y went further and provided a priority list of m a n y other clinical measurements which they felt could be markedly improved by S R M s . T h e list included clinical standards which they felt were needed prior to 1970, another list needed prior t o 1975, a n d a third list which they felt would be important to have prior t o 1980. This long-range a d vice and perspective in clinical chemistry have been particularly helpful t o us. I n clinical measurement there is an increased use of automation and computer control without commensurate attention to meaningful standardization. With t h e number of clinical analyses being performed in this country each year approaching one billion, it is obvious t h a t in-

VALUE FOUND - S T D . A Figure 1. National Cholesterol Survey—1962, by Standards Committee of College of American Pathologists Courtesy, Dr. B. E. Copeland

PYRENE Figure 2 . Spectrophotometer Survey-^-1965, by Standards Committee of College of American Pathologists Courtesy, Dr. B. E. Copeland ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971

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Report for Analytical Chemists

creased automation and computerization is essential. However, it is also essential that automated instruments be subjected to accurate standardization, or the increasing flow of data may be meaningless. Indeed, in a field such as clinical chemistry erroneous data may be more than meaningless; it may be actually harmful to a patient. Within the last decade NBS has become increasingly aware of its responsibilities and opportunities for assistance in improving measurement capabilities in national problem areas such as biomaterials, air and water pollution, and safety. [Indeed it is this awareness that has led to our cosponsorship this summer of the Analytical Summer Symposium with the topic, "Analytical Chemistry—Key to Progress in National Problem Areas" (1).] Thus as an outgrowth of this trend within NBS the Analytical

Chemistry Division in 1966 made the decision to enter the field of clinical standardization with high purity SRMs. In 1969 the National Institute for General Medical Sciences of NIH initiated a substantial cooperative program with the Bureau to provide increased impetus in this SRM area. Organic SRMs for Clinical Chemistry

Robert Schaffer, Chief of the Organic Chemistry Section, has provided the dedicated leadership for the certification of organic SRMs for clinical chemistry. Thomas W. Mears, Organic Standards Coordinator of the NBS Office of Standard Reference Materials, has worked with suppliers to provide large quantities of high-quality material. Dr. Schaffer and associates in organic chemistry have then made appropriate measurements to guaran-

tee homogeneity throughout the supply and then, along with Division experts in a number of other competences, have exhaustively studied the material for purity (and impurities). It is this Divison and NBS capability to make measurements on a material by many different independent competences which is unique and which permits us to pursue the concepts of accuracy in depth. Application of our broad Division experience in accurate measurement to clinical SRMs has proved very fruitful. Cholesterol. The determination of cholesterol in blood is very important in the clinical laboratory. Often cholesterol determinations are made in routine screening of adult populations. Above-normal cholesterol levels in blood may be symptomatic of atherosclerosis or other clinical conditions; belownormal values may occur with he-

Standard Reference Materials for Clinical Measurements· SRM No.

Name

Property certified

Purity, %

Amount, g

Date issued

40h

Sodium oxalate

99.95

Reductometric standard

60

April 24, 1969

83c

Arsenic trioxide

99.99

Reductometric standard

75

Feb. 6, 1962

84h

Acid potassium phthalate

99.993

Acidimétrie standard

60

July 9, 1969

136c

Potassium dichromate

99.98

Oxidation standard

60

March 24, 1970

1861c

Potassium dihydrogen phosphate

99.9

30

July 29, 1966

18611c

Disodium hydrogen phosphate

99.9

pH pH

30

Sept. 1, 1970

350

Benzoic acid

99.98

Acidimétrie standard

30

April 15, 1958

911

Cholesterol

99.4

Identity and purity

912

Urea

99.7

Identity and purity

25

Sept. 24, 1968

913

Uric acid

99.7

Identity and purity

10

Sept. 24, 1968

914

Creatinine

99.8

Identity and purity

10

Sept. 24, 1968

915

Calcium carbonate

99.9

Identity and purity

20

March 4, 1969

916

Bilirubin

99

Identity and purity

917

D-Glucose

99.9

Identity and purity

25

Nov. 18, 1970

918

Potassium chloride

99.9

Identity and purity

20

Jan. 22, 1971

922

tris(Hydroxymethyl)aminomethane

99.9

pH

25

May 1, 1971

923

tris(Hydroxymethyl)aminomethane hydrochloride

99.7

PH

35

May 1, 1971

930

Glass filters for spectrophotometry

Absorbance

3 filters

Feb. 24, 1971

1571

Orchard leaves

Major and trace constituents

75

Jan. 28, 1971

2201

NaCI

99.9

pNa pCI

120

April 15, 1971

2202

KCI

99.9

pK pCI

160

May 1, 1971

0.5

0.1

Oct. 20, 1967

March 10, 1971

a Orders and requests for information about these SRM1's should be directed to the Office of Standard Refe rence Materials, National Bureau of Standards, Washington, D. C. 20234.


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patic disease and hyperthyroidism. Cholesterol is estimated by a number of procedures. Cholesterol and esters of cholesterol are extracted from the serum, and then either separated from each other and estimated independently or the combined total is estimated together by the color produced on reaction with acetic anhydride and sulfuric acid (Liebermann-Burchard reaction) or by the color produced on reaction with FeCl 3 , acetic acid, and sulfuric acid. Although the sensitivity is greater with the latter reagent mixture, the method of Abell et al. (2), which employs the Liebermann-Burchard reagent, is usually recognized as the preferred method. Because their modification is not easily adapted to use with large numbers of determinations, quicker methods are utilized for routine analyses. The survey carried out in 1962 by the College of American Pathologists on the proficiency of laboratories testing for cholesterol led to the conclusion that an essential step needed for improving cholesterol determinations would be the provision of a cholesterol preparation of assured high purity to be used as a standard. The cholesterol of high purity which NBS has certified provides a uniform base line to which clinical laboratories can standardize their procedures. The purity of the cholesterol was originally certified as 99.4 ± 0.3%, but is now known to be 99.7 ± 0.2% (95% confidence level). The cholesterol had been purified according to the method of L. F. Fieser (3)—i.e., preparing cholesterol dibromide, regenerating the cholesterol by reacting the dibromide with zinc dust, and crystallizing. Reliance on the use of this procedure was incorporated into a set of criteria (4) proposed for obtaining and characterizing a highly pure, crystalline preparation of cholesterol to be used in standardizing the clinical measurement. The SRM cholesterol was analyzed for conformance to the expected elemental composition and tested for melting behavior and optical rotation. It was also examined by spectrophotometry (ultraviolet and infrared), by spectrometry (mass and nmr), and by

gas-liquid and thin-layer chromatography (GLC and TLC). For revealing impurities in the very pure cholesterol, ultraviolet spectra and TLC proved most useful, but it required use of differential scanning calorimetry and phase solubility analysis to secure estimates of the purity. These techniques showed 99.8 mol % and 99.8 wt % purities, respectively. We regard these methods to be accurate to within ±0.2% for the cholesterol. The standard material shows a 0.013 ± 0.005% bromine content, a trace indication of the purification procedure employed. The cholesterol (SRM 911) is packaged in an amber bottle under nitrogen. It had been suggested that the material should not be issued after one-year storage. We have now followed the shelf life of the SRM cholesterol for over three years and have found no decomposition (less than 0.2% when stored below 0°C). Indeed, at this high purity, cholesterol is reasonably stable. Apparently, impurities in cholesterol catalyze its decomposition. Since November 1967, we have distributed about 600 samples of cholesterol to over 300 different clinical laboratories within the United States. Within the next six months we expect to issue a renewal of the cholesterol SRM packaged in individual-use vials. A detailed paper, "Cholesterol: Standard Reference Material 911" by R. Schaffer et al. has been submitted to the American Journal of Clinical Pathology (5). Urea. The clinical test for urea is one of the most frequently performed in the clinical laboratory. Urea is formed in the liver from the amino groups of amino acids as the end product of nitrogen metabolism and is subsequently excreted by the kidney. Impaired kidney function leads to high urea in the serum. In clinical procedures urea is determined either by reaction with diacetyl monoxime followed by measurement of the color, or by use of the enzyme urease and measurement of the liberated ammonia by the Berthelot reaction or by Nesslerization. The urea SRM was certified to be

99.7 ± 0.1% pure. A satisfactory method for drying the urea was not found. Either incomplete drying, or excessive (and progressive) weight loss occurred, depending on the degree and intensity of heating and evacuation. However, there was little tendency to gain or lose weight (±0.003%) when the SRM was exposed to laboratory air for a two-week period. Hence, analyses should be performed on the SRM urea without attempting to lower its moisture content. The reported moisture content of 0.18% was determined by the Karl Fischer titration method. The apparent purity of the urea was determined by two methods. Differential scanning calorimetry indicated an apparent purity of 99.82 mol %. Phase solubility analysis of the urea in isopropyl alcohol indicated an apparent purity of 99.82 wt %. These values are designated as "apparent purity" because neither method accounted for the moisture content of the urea. Biuret in the urea was estimated spectrophotometrically to be 0.07%. Neither paper chromatography nor TLC indicated any evidence of biuret or cyanuric acid. Neither infrared (ir) absorption nor nmr spectroscopy revealed any unexpected peaks, and hence, they gave no evidence of impurity. For trace metallic constituents, analysis by emission spectrometry and activation analysis showed the following to be present : aluminum, silicon, iron, sodium, nickel, calcium, chlorine, magnesium, manganese, copper, and zinc. Uric Acid. Measurements of uric acid in serum are made only 10-20% as often as measurements of urea. This can still amount to several thousand tests a year in a large hospital. Uric acid is the metabolic end product of purines and is found in the blood in increased concentration in patients with gout, or undergoing rapid release of nucleic acids owning to drug treatment—i.e., for leukemia —and in cases of renal failure. The clinical procedure using the enzyme uricase follows the decrease in absorbance at about 290 nm due to uric acid as it is converted into other substances. Alternatively, its reaction with alkaline phospho-


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tungstate produces tungsten blue, which is measured. T h e uric acid S R M was certified 99.7 ± 0 . 1 % pure. T h e uric acid used in this standard reference material was found to be homogeneous by paper chromatography and T L C with several solvent systems. This homogeneity was further verified by GLC of the trimethylsilylated material. T h e 0.14% volatile material was determined by heating the sample overnight at 110°C. T h e ash, 0.057% of the total weight, was composed principally of sodium and potassium salts. Aluminum, calcium, iron, phosphorus, and silicon comprise a few percent of the ash; cobalt, copper, manganese, nickel, and zinc are present as traces. A 100-gram sample of uric acid was extracted successively with 500 ml each of water, absolute ethanol, benzene, and water. T h e ultraviolet absorption spectrum of each of these extracts showed no absorption bands other t h a n those of uric acid. Only uric acid was detected by T L C of these same extracts. Creatinine. Between 6000 and 10,000 creatinine measurements m a y be made each year by a large hospital. Creatinine is studied to aid in the diagnosis of kidney disfunction and uremia. I n the clinical procedures, the creatinine is treated with alkaline picrate (the Joffé reaction) and forms a measurable red pigment. M a n y modifications are used. T h e purity of the creatinine S R M was certified at 99.8 ± 0 . 1 % . The homogeneity of the creatinine was determined bv paper chromatography, T L C , and GLC. Volatile m a t t e r (0.03%) was determined by measurement of loss of weight of the creatinine after heating for 24 hr atll0°C. Phase solubility analysis of an oven-dried sample with absolute methanol as the solvent indicated the purity to be 99.82 wt %. Phase solubility analysis of the undried creatinine with 9 5 % methanol and 9 5 % ethanol indicated purities of 99.81 and 99.76 wt %, respectively. Potentiometric titration required 99.82% of the theoretical amount of hydrochloric acid. T h e certified value for chloride (0.07%) was obtained by titration 34 A ·

with mercuric nitrate of a solution of the products resulting from an oxygen-flask combustion. T h e ultraviolet absorption spect r u m of a solution of the S R M in water showed a molar absorptivity (cmax) of 7140 ± 30 a t 234 nm. Infrared absorption and nmr spect r a provided no evidence of impurities. Analysis for trace metallic constituents by emission spectroscopy and activation analysis showed the following to be present: silicon, chlorine, aluminum, sodium, titanium, copper, iron, manganese, and magnesium. D-Glucose. I n most general hospitals, the glucose measurement is the single most prevalent clinical determination made. Approximately 50,000 measurements m a y be made in one year in a 1000-bed hospital. For about 60 years, the National Bureau of Standards has made available a standard reference material dextrose for the purpose of standardization of chemical methods of analysis, primarily for the sugar industry. I n recent years, developments in the standardization of clinical analysis techniques have resulted in a national requirement for a more highly characterized D-glucose, suitable for use in both chemical and biochemical methods of analysis, and particularly in those in which enzymes are employed. T h u s , clinical chemists require a D-glucose standard reference in connection with analyses of blood and urine in the detection and t r e a t ment of diabetes mellitus. T h e clinical test for glucose is performed by using it as a reducing agent with iron or copper containing oxidants or by using the enzyme glucose oxidase for its conversion to gluconic acid. Each method is subject to interferences. T h e D-glucose S R M is certified at 99.9 ± 0 . 1 % purity. The only impurities detected in the glucose were moisture and traces of inorganic compounds. Paper, thin-layer, and high-pressure ion-exchange chromatographic techniques revealed no organic impurities. T o establish its purity, it was necessary to know the proportion of the a-D-anomcr in the glucose. This was estimated


by three methods. T h e ratio of the /3-anomer to the α-anomer was found to be 0.5:100 by GLC after per (trimethylsilyl) ation of the solid standard material a t 0°C using Λ Γ -(trimethylsilyl) imidazole in an hydrous pyridine to minimize pos sible mutarotation. Use of this GLC technique on p a r t l y melted standard material showed t h a t the proportion of the /?-anomer in creases markedly during melting. Differential scanning calorimetry of the standard material showed the α-D-glucose content to be 99.4%. This value represents only the pro portion of anhydrous a-D-glucopyranose present, since the method treats /î-D-glucose, or hydrates of α-D-glucose t h a t are stable up to the melting point, as impurities. However, proton magnetic reso nance spectroscopy a t 90 M H z indi cated the ratio of β-anomer to a anomer to be 0.9:100:' This de termination wyas performed 10 min after dissolution of 100 mg of D-glu cose in 0.5 ml of methyl sulfox i d e - ^ , by integration of the doub lets due to the anomeric hydroxyl groups. T h e moisture content (0.06%) was determined by the K a r l Fischer and near-infrared methods. As only 0.01-0.02% in weight was lost on drying a t 70°C for 100 hr, the analyses reported herein were per formed on the undried standard ref erence material. T h e ash content was determined by ignition of 20-gram samples at 750°C. Turbidimetric assays of solutions of the standard showed the presence of chloride at 2 p p m and sulfate at 3 ppm. Neutron activation indicated chloride at 4 ppm. Emission spectrometric analysis of the ash showed calcium to be less t h a n 5 ppm ; magnesium and silicon each less t h a n 0.1 p p m ; aluminum, boron, and iron each less t h a n 0.05 p p m ; and copper, less t h a n 0.01 ppm. Atomic absorption spectrom etry indicated t h a t the S R M con tains less t h a n 0.5 ppm of mag nesium. F l a m e emission spectrom etry indicated the content of cal cium to be about 1 ppm, and sodium to be about 2.9 ppm. A detailed paper, "Characteriza tion and Quantitative Analysis of D-Glucose for Use in Clinical Analy-


sis" by B. Coxon and R. Schaffer has been submitted to ANALYTICAL CHEMISTRY

(ff).

Bilirubin. T h e clinical test for bilirubin m a y be performed 10,000 times a y e a r in a large hospital. Jaundice, the yellowing of certain tissues due to excessive levels of bilirubin in blood (a particularly serious condition for the newborn), results from certain hemolytic or liver conditions. Serum analyses help to diagnose the cause. T h e most commonly employed clinical test for bilirubin involves reaction with diazotized sulfanilic acid under conditions t h a t afford a measure of the uronic acid—bilirubin conjugate and then of the total bilirubin. Some use is made of a direct spectrophotometric estimation at the absorption maximum of the bilirubin. T h e S R M bilirubin (Figure 3) is provisionally certified for a purity of 9 9 % with a possible estimated inaccuracy of 2 % , due to a detected impurity which it is presently impossible to quantitate. The S R M contains 0.8% chloroform. I t was prepared from material isolated from hog bile and was crystallized as the acid. I t was purified further b y treatment in chloroform solution with sodium sulfate ac-

Figure 3.

cording to Fog (7) and recrystallization from chloroform. T h e ash, 0.01% of total weight, was determined on 100-mg samples heated overnight a t 250°C and then for 3 hr a t 650°C. Flame photometry and atomic absorption measurements on a solution of the ash showed trace amounts of lithium, sodium, potassium, calcium, and magnesium. Elemental composition found for the material was close to theoretical, assuming 0.8% chloroform. I n these analyses, nitrogen was determined by the Kjeldahl technique; chlorine was determined by Carius digestion followed by gravimetric determination as silver chloride. During gradual heating of the material in a quadrupole mass spectrometer, only ion fragments characteristic of chloroform were observed until the sample was heated above 300°C, whereupon water and carbon dioxide were detected. At a somewhat higher temperature, the mass spectrum typical of bilirubin was obtained. Gas chromatography and nmr spectroscopy with the material dissolved in methyl sulfoxide provided further evidence in confirmation of the presence of chloroform. Because the chloroform is so firmly held and

Bilirubin—SRM 916—recently issued by NBS

homogeneously dispersed, it is recommended t h a t the material be used as supplied. Thermogravimetric analysis with sample heated in a dry nitrogen atmosphere at 5 ° C / m i n showed the initiation of the loss of a large proportion of sample weight a t temperatures between 319° and 323°C ; run in air, however, comparable weight loss began between 288° and 292°C. T h e molar absorptivity of the S R M in chloroform was found to be 61,100. T h e measurements of a b sorbances were recorded by use of a C a r y 16 spectrophotometer on solutions in a capped 1.00 ± 0.001-cm path-length cuvet in a cell holder thermostated a t 25.0 ± 0.1 °C. Solution preparation and handling were performed under low intensity, incandescent light; a t other times, solutions were totally protected from light. T o preclude any change in concentration of chloroform solutions owing to evaporation of the solvent during solution transfer, nitrogen gas was used to force solution from the flask through Teflon tubing into the cuvet and, to effect rinsing of the cuvet, from the latter to an overflow. Bicarbonate extraction provided no evidence of impurities more acidic t h a n bilirubin. Carbonate extraction showed a negligible proportion of nonacidic impurities. An impurity in the standard m a terial m a y be detected by T L C with polyamide as the absorbent and 3:1 ( v / v ) methanol—aqueous ammonium hydroxide (3.3%) as the developer (8). T h u s , 0.05 mg of the material (spotted from a chloroform solution) provides, on development, an elongated, yelloworange streak ahead of which (and usually separate) is a faintly visible yellow spot. Under 366-nm irradiation, a pink fluorescence develops very quickly a t t h e location of this spot. T h e material responsible for the pink fluorescence and its precursor have not been characterized. A means for obtaining a reliable estimate of this contribution to overall impurity has not been ascertained, but is under study. Repurification of the standard material does not affect this behavior. I t is recommended t h a t bilirubin


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and its solutions be handled only under low-intensity incandescent light. T h e bilirubin should be stored under conditions t h a t totally exclude light. Once it is removed from the ampul, the vial should be replaced for storage in an air-tight container and stored a t low temperature. T h e vial must be allowed to return to room t e m p e r a t u r e before opening. A s u m m a r y of our work on bilirubin is contained in the report, "A Crystalline Bilirubin S t a n d a r d for Clinical Analysis, S R M 916," by R. Schafïer et al. submitted to Science (9). Another paper "Bilirubin Stability—Spectral Shifts of Transition and R a r e E a r t h Element Complexes" by R. A. Velapoldi and 0 . Menis has been submitted to Clinical Chemistry {10). A third paper, "Periodic Acid as a N e w Oxidant for the D e g r a d a t i o n of Bile Pigments. Isolation of a Biliverdine T y p e of Reaction I n t e r m e d i a t e on Oxidation of Bilirubin with Periodic Acid," by A. J. F a t i a d i and R. Schaffer has been submitted to Experientia (11). Inorganic SRMs for Clinical Chemistry Calcium C a r b o n a t e . T h e rapid and accurate analysis of calcium in serum is an i m p o r t a n t clinical test. Hypercalcemia when supported by other evidence is often indicative of hyperparathyroidism, and medical decisions involving immediate surgery are often made on the basis of the serum-calcium value. In clinical procedures, calcium in serum is often precipitated as the oxalate and titrated with perm a n g a n a t e , complexed with E D T A in the presence of metal indicator dyes, or complexed with dyes to produce fluorescence or color. F l a m e emission and atomic a b sorption methods are also used. T h e N B S calcium carbonate serves as the p r i m a r y standard for the calibration of the instruments and methods used. While the N B S calcium carbonate (SRM-915) is certified at only 99.9% purity at this time, work under way a t N B S indicates t h a t the purity is, in fact, in excess of 9 9 . 9 9 % . T h e material was found to meet or exceed the m i n i m u m requirements for ACS Reagent Grade

m a t e r i a l in every respect. E x a m ination by thermogravimetric analysis indicated the loss of a minute proportion of weight below 175°C (volatile m a t t e r ) , and the composition was stable above this t e m p e r a t u r e until the t e m p e r a t u r e of 625°C, above which decomposition (evolution of C 0 2 ) set in. Replicate samples t a k e n from a randomly selected region of the u n dried material were assayed by a coulometric acidimétrie procedure. T h e results from nine independent determinations, based on expression of the assay as calcium carbonate, indicate a purity of 9 9 . 9 9 + % with a s t a n d a r d deviation of 0.003%. Samples equilibrated a t a relative humidity of 9 0 % and assayed by this coulometric procedure showed a maximum moisture adsorption of 0.02% as compared to samples t h a t were dried for 6 hr at 210°C. T h e moisture content on samples equilibrated a t 7 5 % relative humidity was found to be 0 . 0 1 % . This water content was determined by the K a r l Fischer method. A semiquantitative survey for trace contaminants b y emission spectroscopy indicated the presence of less t h a n 10 ppm of copper, iron, magnesium, manganese, and silicon in the material. B y atomic absorption, magnesium was evaluated at 1.0, sodium a t 0.4, and strontium at 2.1 p p m ; potassium was less t h a n 0.4, lithium less t h a n 0.05, and barium much less t h a n 10 ppm. N e u t r o n activation analysis indicated copper 0.9, manganese 0.6, and sodium 0.5 ppm. Copper was determined spectrophotometrically at 1 ppm. Potassium Chloride. D e t e r m i n a tion of both potassium and chloride ions are among some of the most common measurements made in clinical chemistry. W i t h i n the cell, potassium is the major cation, but outside the cell in the serum its concentration is much lower. Acute renal failure, dehydration, and cellular breakdown result in increased potassium concentration in serum. Conditions which promote excessive excretion can result in decreased serum potassium, although much potassium can be lost from the intracellular fluid without decreasing the serum potassium.

I n the clinical laboratory, p o tassium in urine and serum is almost always measured by flame photometry. Chloride is often measured by t i t r a t i o n with mercuric nitrate using diphenylcarbazone as an indicator or by coulometric generation of silver ions and an amperometric end point detection. T h e S R M is certified a t the 99.9 ± 0.0% level for use in the calibration and standardization of procedures employed in the determination of potassium and chloride ions in clinical analyses. T h e sample consists of highly purified p o t a s sium chloride. Chemical assay as well as analyses for specific impurities indicate t h a t the m a t e r i a l m a y be considered to be essentially pure, except for moisture due to occlusion. T h e value for the p u r i t y of the material is based on a sample dried over magnesium perchlorate for 24 hr. Potassium chloride is hygroscopic when the relative humidity at room t e m p e r a t u r e exceeds 7 5 % , but can be dried to the original weight by desiccation for 24 hr over freshly exposed P 2 0 5 or M g (C10 4 ) 2 · T h e m a t e r i a l should be stored with such a desiccant. T h e potassium assay was determined by a combination of gravimetric and isotope dilution analyses. More t h a n 9 9 % of t h e potassium was precipitated, filtered, and weighed as potassium perchlorate. The weight of potassium perchlorate was corrected for rubidium perchlorate. T h e soluble potassium was determined by isotope-dilution mass spectrometry. T h e total potassium was the sum of the potassium from the potassium perchlorate and the potassium from the filtrate. T h e chloride assay was determined by a coulometric argentometric procedure. Based on 12 independent measurements for each ion, t h e sample was judged to be homogeneous. M a t e r i a l dried a t 500°C for 4 hr in a platinum or Vycor crucible (Pyrex is unsatisfactory) was assayed a t 99.98 ± 0 . 0 1 % . T h e loss of moist u r e by this procedure was about 0.07%. T h e S R M met or exceeded the minimum ACS Reagent Grade requirements in every respect. A semiquantitative survey for trace


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contaminants by emission spectroscopy indicated the presence of less t h a n 10 ppm of aluminum, copper, iron, and magnesium. B y atomic absorption, magnesium was evaluated a t 0.24 ppm. F l a m e emission spectrometry indicated the presence of the following elements: rubidium, 27 p p m ; sodium, 9 p p m ; lithium, 0.6 p p m ; and cesium and calcium, less t h a n 2 ppm. Spectrophotometry

Spectrophotometry is an import a n t measuring technique in m a n y branches of science, but its potentialities for high precision and high accuracy have not been fully exploited. Clinical chemists early realized the limitations on accuracy when they tried to m a k e intercomparisons between laboratories. More recently scientists seeking accurate measurements in water and air pollution studies have also encountered these limitations. At N B S , Oscar Menis, Chief of the Analytical Coordination Chemistry Section, and his associates have provided perceptive leadership in this area. R a d u Mavrodineanu has designed and assembled a highaccuracy spectrophotometer and has been responsible for the selection and calibration of glass filters. Robert B u r k e leads the work on the development and characterization of liquid S R M s for spectrophotometry. I n 1969 we made a broad evaluation of the present status of spectrophotometry and considered designs for the construction of a special high-accuracy spectrophotometer which would be used for the accurate calibration of spectrophotometric standard reference materials. There are today a number of clinical chemistry measurements which require an accuracy better t h a n 0.3%. After consultation with F . J. J. Clarke at the National Physical Laboratory in England and with scientists in the N B S Institute for Basic Standards, we have designed and constructed an instrument which is now capable of making transmittance measurements at the 0 . 1 % accuracy level; with additional modifications we expect an accuracy of 0.02%. I n this instrument, samples can be interchanged in a m a t t e r of min40 A

·

Figure 4.

Glass Filters for Spectrophotometry—SRM 930—recently issued by NBS

utes; more importantly, measurements m a y be performed not only on solid filters, but on liquid filters as well. As the first output of the spectrophotometry program, we have made available as of March 1, a set of three neutral glass filters. These filters, designated S R M 930 and shown in Figure 4, serve to check the photometric scale of spectrophotometers in the visible region of the spectrum from 400—700 nm. This range will be extended, in the near future, to include the ultraviolet region to 200 nm. T h e accuracy of these certified transmittance measurements is based on the N B S instrument which is calibrated by independent physical means to assure a minimum of systematic errors.

ANALYTICAL CHEMISTRY, VOL. 4 3 , NO. 6, MAY 1 9 7 1

T h e neutral N G - 4 glass for the filters was provided by Schott of Mainz, Germany, and is designated as "Jena Colored and Filter Glass." Nominal transmittance for a filter 1 mm thick is 2 0 % at 400 n m wavelength and 3 2 % at 700 nm wavelength. Between these limits the transmittance varies in a monotonous manner {12). T h e transmittance of the filters depends on the intrinsic properties of the material. Spectral band pass (12, 13), geometry of t h e optical beam, surface conditions, and positioning of the filter also affect the transmittance measurements. T h e certified data will be reproduced when transmittance measurements are made under the conditions specified. The filter is positioned perpen-


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dicularly to the optical beam and at the point where the image of the slit is formed. Under these conditions, the vertical image is about 13 m m x 2 m m and is located in the center of the opening in the metal holder. T h e filter holder and the size and shape of the filters were selected to conform to the dimensions of the sample compartment of most conventional spectrophotometers. The filters are approximately 1.0, 1.5, and 2.0 m m thick. Corresponding to these thicknesses are nominal transmittances of 30, 20, and 10%, respectively. These thicknesses were selected to provide a means for calibrating the photometric scale at three different levels. A point of philosophy should be brought up at this time in connection with the spectrophotometry standardization. T h e set of neutral glass filters has been top priority in our program because of the urgent requests of the Standards Committees. However, it appears t h a t these glasses will be used by research laboratories and by clinical analytical laboratories who need to standardize rather elaborate spectrophotometers. A glass filter will be less suitable to standardize the thousands of automated systems which rely on continuously circulating discrete volumes of solutions through plastic tubes and finally through small colorimeter cells. Thus, as we see it now, the primary effort of our spectrophotometry program will be oriented toward providing the standardization means necessary for these automated systems. This is a major problem and will be a difficult one to solve. Our present schedule calls for the first liquid spectrophotometry standards to be available by July of this year. These will be composite solutions available in disposable ampuls and containing either (a) chromium ( I I I ) , cobalt ( I I ) , and p-nitrophenol, or (b) Thomson solution (14)—a mixture of chromium ( I I I ) , chromium ( V I ) , cobalt ( I I ) , and copper ( I I ) . A number of these samples are being evaluated by clinical laboratories. T h e results of these cooperative tests, together with accurate measurements obtained on our high-accuracy instrument will be combined

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with our studies of stability and storage characteristics to provide the certification for these N B S liquid spectrophotometry S R M s . Spectrofluorometry

M u c h of the current work presented . a t scientific meetings and in publications points out the growing usefulness of fluorescence measurements in the biochemical and environmental fields. These measurements complement the present capabilities of spectrophotometry by permitting measurements a t lower concentrations, smaller sample sizes, and with greater specificity. However, conventional spectronuorometric instruments are not capable of making absolute measurements, and standards are therefore urgently needed to provide a means of calibration. Within the last year, Division scientists have begun studying some of the problems associated with fluorescence measurements. Preliminary work has led to the selection of several materials as potential q u a n t u m efficiency standards. I t is proposed t h a t cerium, lead, and thallium glass filters, and solutions, such as quinine sulfate, 3-aminophthalimide, the aluminum chelate of Pontochrome Blue Black R, and m-nitrodimethylaniline, be evaluated. T h e measurement of quant u m yields of these compounds will depend on the development and construction of a high-accuracy spectrofluorometer which will be calibrated at N B S by independent physical means in a similar manner to the high-accuracy spectrophotometer. pH and Ion-Selective Electrodes

p H . I t has long been recognized t h a t the p H of blood reflects the acid-base balance of the body. T h e p H level is controlled between relatively narrow limits by intricate mechanisms involving the production, elimination, and buffering of acid by the body. Its measurement is of great significance and m a y be related to a large number of specific pathologic states including respiratory, gastrointestinal, and renal diseases. I n the clinical laboratory, p H is one of the most commonly performed determinations using an


electrometric procedure based on the glass electrode. T o calibrate these electrodes accurately, the NBS several years ago certified a special ratio of the phosphate buf fer ( S R M 186 Ic and 186 He) for the physiologic range of p H from 7.35 to 7.45 at 37°C. I n spite of the fact t h a t this material was not compatible with blood (phosphate causes calcium precipitation), it has been widely used as a primary standard in the clinical laboratory. T o alleviate this and other prob lems, the N B S has been working for some time to evaluate and certify special S R M s of tris(hydroxymethyl) aminomethane and its hydrochloride salt. These new physiologic p H standards (SRM 922 and 923) are now available in solid form, and work is in progress to certify this material in isotonic saline solution for greater accuracy and convenience for the clinical chemist. Ionic Activity Standards. The recent development of ion-selective electrodes for such physiologically important ions as calcium, sodium, potassium, and chloride has re sulted in a need for activity stan dards comparable to the clinical p H standards. Although still not widely or routinely used in the clinical laboratory, perhaps be cause, to some extent, standards were not available, such sensors have been extremely useful to reseach physiologists and biochem ists in monitoring the activities of these ions. As has been discussed in a variety of biologic studies, it is ionic activity, rather t h a n con centration, t h a t is of utmost im portance in a wide variety of physiologic phenomena. I t has been suggested t h a t more accurate measurements and interpretations could be made from activities than from concentrations. Ion-selective electrodes offer a very simple and convenient way of determining ionic activities. Not only are the measurements rapid and precise, but these sensors easily lend themselves to automation us ing continuous flow-through analy sis systems requiring submilliliter sample volumes. Using procedures similar to those developed in the certification of the p H standards, N B S has recently

Figure 5. Ion-Selective Electrodes Standards—SRM 2 2 0 1 and 2 2 0 2 — r e cently issued by NBS

issued the first in a series of ionselective electrode standards (Fig ure 5 ) . Two standards, N a C l (SRM 2201) and KC1 (SRM 2202), have been certified for the activities of sodium, potassium, and chloride over the range from 0.001 to 2m. Although not specifically developed for clinical applications, the certified ranges encompass the normal physiologic ranges for these ions, which are the three most abundant electrolytes in the body. We cite just a few diseases accom panied by changes in the activities of these ions: diabetic coma, toxemia, alimentary tract infec tions, renal and cardiac failure, cellular breakdown, and adrenal cortical insufficiency. Of all the recently developed ion-selective electrodes, the calcium electrode may prove to be the most useful for diagnosis in the clinical laboratory. Again, the measure ment of ionic calcium, believed to be the physiologically active com ponent of the total serum calcium, is of considerable value in diagnos ing such conditions as hyperpara thyroidism, tetany, bone diseases, and cardiac dysrhythmias. W o r k is in progress to develop a calcium-ion activity standard in solution form. Our work is ulti mately directed toward the devel opment of a multicomponent clini cal standard in which the activities of several of the most important biologic electrolytes are certified and can be used for the calibration of clinical analyzer systems based on ion-selective electrodes.

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CIRCLE 126 O N READER SERVICE CARD

ANALYTICAL CHEMISTRY, CHEMI VOL. 4 3 , NO. 6, MAY 1 9 7 1

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43 A

Report for Analytical Chemists Basic Chemical Standards

There are also a number of the traditional chemical standards which are as applicable in the clinical laboratory as in any other chemical laboratory. These include the redox reagents of potassium dichromate ( S R M 136b), arsenic trioxide ( S R M 83c), and sodium oxalate (SRM 40h), as well as the acidimétrie S R M s , benzoic acid ( S R M 350), and potassium acid phthalate ( S R M 84h). These materials are certified accurate to a few parts in 10 4 and provide the basic standardization for titrimetric and gravimetric analyses. Trace SRMs—Biological Material

T h e Bureau has been working for the past seven years to develop standard reference materials certified for trace (less t h a n ppm) amounts of different elements in matrices such as metals, inorganic materials, and biological systems. Several high-purity metals have already been certified and a series of trace glasses with some 60 elements dissolved a t the 500, 50, 1, and 0.02 ppm levels are now available. W e have just recently made available our first botanical material, S R M 1571 orchard leaves, with informa-

tion reported on both major constituents and trace elements (Figure 6 ) . This S R M is certified (through measurements by two independent methods) for calcium, potassium, iron, sodium, copper, and nickel, while supporting information is given for nitrogen, magnesium, phosphorus, arsenic, bismuth, boron, chromium, cobalt, fluorine, lead, manganese, selenium, uranium, and zinc. To achieve these analyses the following techniques were used: atomic absorption spectrometry, flame emission spectrometry, gravimetry, isotope-dilution sparksource mass spectrometry, Kjeldahl-nitrogen, neutron activation, nuclear-track technique, and polarography. The evaluation of the homogeneity of the sample was based on the magnesium, nitrogen, and potassium analyses using a minimum sample size of 250 mg. T h e average content for the entire lot has been determined to be within ± 1 % (relative) or better for the major elements and to within ± 3 % relative for the minor elements (95% confidence). T h e same techniques as used on the orchard leaves will in the near future be used on freeze-dried liver and freeze-dried bovine serum.

CALIFORNIA EASTERN LABORATORIES, INC.

SRM 1571-ORCHARD LEAVES (AS OF 1 OCTOBER 1970) CONCENTRATION CERTIFIED J PRELIMINARY VALUES ELEMENTS TO BE STUDIED

At

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Figure 6. Orchard Leaves—SRM 1 5 7 1 — t r a c e biological SRM recently issued by NBS. Freeze-dried liver and freeze-dried bovine serum SRMs now being initiated by NBS will be certified in a similar manner CIRCLE 73 ON READER SERVICE CARD 44 A

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ANALYTICAL CHEMISTRY, VOL. 4 3 , NO. 6, MAY 1 9 7 1


NBS has contracted for 125 kg of freeze-dried beef liver. Homo geneity tests are now being run on representative samples and work will begin soon on the certification. Negotiations are now in progress for the freeze-dried bovine serum. Thus, within the next six months or a year, we expect to have avail able several materials certified for trace elements of interest to the clinical laboratory and to the bio medical specialist.

A Compact FID is Available From G O W - M A C

Future

The detailed priority list pro vided by the Standards Committee of the American Association of Clinical Chemists identifies many important clinical measurements for which SRMs are required. Of particular importance in the near future are the areas of protein and enzyme chemistry. As many as 25% of the tests performed daily in the clinical chemistry laboratory are protein and enzyme assays, yet standardization is very difficult. Several years ago Theodore Peters published a description of a pro posed protein standard consisting of bovine serum albumin, which would provide an initial material for standardization (IS). We plan to begin work on such a protein standard very shortly. Similarly, work will be initiated on several steroids during the next year as well as on NADH (nicotinamideadenine dinucleotide), which gives every evidence of being a very dif ficult material to certify. In spectrophotometry our plans include extension of the calibration work to the uv region of the spec trum to provide solid and liquid fil ters in the 250-nm and higher wave length region. Molar absorptivity and linearity data will be certified for well-characterized benzoic acid, potassium dichromate, acid potas sium phthalate, and other stoichio metric compounds of interest. In a related area, the evaluation and certification of selected mate rials to assess stray light in the uv region will serve to eliminate a ma jor source of error. KC1, a wellcharacterized compound, will be one of the first materials certified. Perhaps a statement by a clini cal chemist member of the NBS Analytical Chemistry Division Ad-

Heart of the GC, FID Detector 12-700 In response to demand. Called the 69-700, it has many desirable fea tures. For example, operation in excess of 3 0 0 °C. Three-step input attenuator. Ten step, binary output attenuator. Sensitivity of 1 χ 10 " g/sec hydrocarbon. Pyrometer readout, coarse and fine suppression con trols and many other features which will provide rugged, continuous performance. The 69-700, Standard FID is a companion to GOW-MAC's compact thermal conductivity instru ments. These units are all tough, inexpensive, reli able and deliberately simple in design. They don't replace research gas chromatographs but they can perform equally with them in routine analysis. This makes the research units avail able for the more sophisticated work for which they were de signed. It is not economical to do r o u t i n e a n a l y s i s on a re search instrument. Send f o r o u r l i t e r a t u r e . The technical facts are worth having.

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BUCHLER UNIVERSAL DENSITY GRADIENT MIXER FORMS GRADIENTS FROM 5 to 50 ml WITHIN THE SAME MIXER

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visory Panel regarding t h e N B S standardization of bilirubin (16) will p u t the N B S contribution into perspective: " T h e program in clinical standards illustrates dramatically t h e unique role and capability of the Bureau in providing materials standards. T h e Analytical Division has been involved for just over a year in clinical chemical s t a n d a r d s ; several new S R M s are now offered, with another soon to be offered— bilirubin. Bilirubin standardization has been an extremely recalcit r a n t problem which has been a t tacked over the last decade by cooperative groups of scientists, both here and abroad, with incomplete success. " I n one year the Bureau has achieved a bilirubin S R M (solid) plus a tested new method of preparing a stable concentrated standard solution which can be added to serum or plasma without denaturing protein. T h e Division has thus unmistakably demonstrated t h a t (1) the Bureau's competence is qualitatively superior to t h a t in the field, (2) the Bureau acts as a stimulus to industry—there are now three bilirubin suppliers, (3) the Bureau can provide an unbiased unifying authority in the clinical field (which is very sensitive to authority) , (4) the Bureau can provide not only an S R M , but can also provide a method of use t h a t makes practical both t h e S R M and industrial secondary standards." One thing is certain, with the pressures on t h e clinical laboratory to be able to prove its capability, there will be a great demand for standard reference materials. Traceability to the National Bureau of Standards will ensure consistent quality control between individual laboratories. Ultimately this will lead to upgrading of t h e measurements made in the clinical laboratory. Acknowledgment

BUCHLER INSTRUMENTS BUCHLER INSTRUMENTS DIVISION NUCLEAR-CHICAGO CORPORATION ASUBSIDIARY OF G.D.

SEARLE

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T h e clinical S R M program is D i vision-wide in scope. Names of several major contributors have been mentioned in the text but over half of the 100 scientists in our N B S Analytical Chemistry Division have been involved in this program at one time or another during the past

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five years. This report and t h e series of S R M s in clinical chemistry are indeed a tribute to the harmonious interaction of these diverse scientific efforts within our Division. References (1) "Analytical Chemistry: Key to Progress in National Problem Areas," announcement of 24th Annual Summer Symposium on Analytical Chemistry to be held June 16-18, 1971, a t the National Bureau of Standards, Gaithersburg, Md. (2) L. L. Abell, B. B. Levy, B. B. Brodie, and F . E. Kendall, J. Biol. Chem., 195,357 (1952). (3) L. F . Fieser, / . Amer. Chem. Soc, 75,5421 (1953). (4) "Selection of Criteria for a Pure Cholesterol Preparation to Be Used for Standardizing Serum Cholesterol Measurements for Medical Diagnosis and Therapy," Amer. J. Clin. Pathol., 47, 654 (1967). (5) R. Schaffer, A. Cohen, R. F . Brady, Jr., "Cholesterol: Standard Reference Material 911," ibid., submitted for publication. (6) B. Coxon, R. Schaffer, "Characterization and Quantitative Analysis of DGlucose for Use in Clinical Analysis," ANAL. CHEM., submitted for publica-

tion. (7) J. Fog, Scand. J. Clin. Lab. Invest., 16,49 (1964). (8) Z. J. Petryka, C. J. Watson, J. Chromatog., 37, 76 (1968). (9) R. Schaffer, R. F. Brady, Jr., A. J. Fatiadi, B. A. Johnson, B. F. West, "A Crystalline Bilirubin Standard for Clinical Analysis, SRM 916," Science, submitted for publication. (10) R. A. Velapoldi, O. Menis, "Bilirubin Stability—Spectral Shifts of Transition and Rare Earth Element Complexes," Clin. Chem., submitted for publication. (11) A. J. Fatiadi, R. Schaffer, "Periodic Acid as a New Oxidant for the Degradation of Bile Pigments. Isolation of a Biliverdine Type of Reaction Intermediate on Oxidation of Bilirubin with Periodic Acid," Experientia, submitted for publication. (12) R. Mavrodineanu, "Solid Materials to Check the Photometric Scale of Spectrophotometers," NBS Technical Note 544, Menis, O., Shultz, J. I., Ed., pp. 6-17, U. S. Government Printing Office, Washington, D. C. 20402 (September 1970). (13) K. S. Gibson, "Spectrophotometry," NBS Circ. 484 (September 1949). (14) L. C. Thomson, Trans. Faraday Soc, 42, 663 (1946). (15) T. Peters, Jr., Clin. Chem., 14, 1147 (1968). (16) NBS Analytical Chemistry Division Advisory Panel Report, January 1970. Certain commercial materials and instruments are identified in this paper in order to specify the experimental procedure adequately. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.


W. WAYNE MEINKE is presently Chief of the Analytical Chemistry Division in the Institute for Materials Research of the National Bureau of Standards, Washington, D. C, where he leads a program encompassing work in some 60 different areas of materials characterization with particular emphasis on trace analysis. In 1964—69 he also held the position of Chief of the

NBS Office of Standard Reference Materials. In this program he had responsibility for the preparation, analysis, and distribution of all types of well-characterized materials that can be used to calibrate measurement systems or to produce reference scientific data. Dr. Meinke received his A.B. degree in chemistry at Oberlin College in 1947 and his Ph.D. in nuclear chemistry under Professor Glenn T. Seaborg at the University of California at Berkeley in 1950. He served on the faculty of the Department of Chemistry at the University of Michigan, 1950—63, progressing from Instructor to Full Professor. In 1963 he joined the National Bureau of Standards. Throughout his career, Dr. Meinke's professional interests have been in the fields of radio chemistry and analysis. He has authored singly or in collaboration over 125 scientific papers. He has edited with B. F. Scribner a book entitled "Trace Characterization—Chemical and Physical." He serves on the Editorial Advisory Board of ANA-

LYTICAL C H E M I S T R Y , and

is

a

Re-

gional Advisory Editor of the Analyst. He is active in the affairs of the American Chemical Society and the International Union of Pure and Applied Chemistry. At present he is Chairman of the Steering Panel on the Characterization of Pure Materials Program within the Organization for Economic Cooperation and Development (OECD). Dr. Meinke has been honored by the American Nuclear Society through its 1968 Special Award for Distinguished Service in the Advancement of Nuclear Science—specifically in Industrial Applications of Radiation Technology through Activation Analysis. In 1968 he received the first George Hevesy Medal for Radio analytical Chemistry. Dr. Meinke was also honored with the 1968 Edward Bennett Rosa Award of the National Bureau of Standards for outstanding leadership of the Standard Reference Materials Program. He is an elected, honorary member of the Society for Analytical Chemistry, London.

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Standard Reference Materials for Clinical Measurements - Analytical

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