certified reference materials for validating spectroscopic methods and experimental data

Certified reference materials for validating spectroscopic methods and experimental data Robert Alvarez National Bureau of Standards, Gaithersburg, MD 20899, USA

Zertifizierte Referenzmaterialien fill" die Bewertung spektroskopischer Methoden und experimenteller Daten

Zusammenfassung. Chemische Laboratorien erhalten h/iufig signifikant verschiedene Ergebnisse, wenn sie das gleiche homogene Material untersuchen. Die unterschiedlichen Er- gebnisse k6nnen verursacht sein durch ungeeignete Metho- den, falsche Ger/iteeichung, fehlerhafte experimentelle Tech- niken, unreine Reagentien oder eine Kombination dieser Faktoren. Ffir die Spurenbestimmung sind Gr613e und Auswertung des Blindwertes der Methode oft die Hauptbe- schr/inkungen bei der Erzielung genauer Ergebnisse.

Ein Schritt in Richtung auf Verbesserung der Genauig- keit von analytischen Bestimmungen ist die Benutzung zertifizierter Referenzmaterialien (CRMs), die von Organi- sationen/iberall in der Welt ausgegeben werden. Ein CRM ist ein homogenes, stabiles Material mit zertifizierten chemischen und/oder physikalischen Eigenschaften, das benutzt wird, um Ger/ite zu eichen, experimentelle Daten zu bewerten, zuverl/issige Methoden zu entwickeln und Daten verschiedener Laboratorien auf eine gemeinsame Basis zu beziehen. In den Vereinigten Staaten ist das ,,National Bu- reau of Standards" (NBS) gesetzlich autorisiert, CRMs auszugeben, die aus historischen Griinden Standardrefe- renzmaterialien (SRMs) genannt werden. In dem heute g/iltigen NBS-Katalog sind mehr als 900 SRMs aufgelistet, wovon die meisten in bezug auf ihre chemische Zusammen- setzung zertifiziert sind. Sie umfassen eine Vielzahl von Matrices mit Bestandteilen anorganischer und organischer Art und sind von grol3em Interesse fiir Spektrochemiker. Diese SRMs finden Anwendung auf derart verschiedenen Gebieten wie klinische analytische Chemie, Umweltanalytik, Nahrungsmittelchemie, landwirtschaftliche und industrielle analytische Chemie. Auch SRMs zur Beurteilung der Leistungsf/ihigkeit von Ger/iten, wie die, die entwickelt wurden, um die Leistungsf/ihigkeit von Gas-Chromato- graph-Massenspektrometern zu bewerten, sind f/Jr Spektro- chemiker von Interesse.

Summary. Chemical laboratories frequently obtain significantly different results when analyzing the same homogeneous material. The different results may be caused by poor methodology, improper instrument calibration, faulty experimental techniques, impure reagents, or a combination of these factors. For trace constituent determinations, the magnitude and evaluation of the method blank are often the main limitations toward obtaining accurate results.

One approach towards improving the accuracy of analytical determinations is by the use of Certified Reference

Materials (CRMs) issued by organizations throughout the world. A CRM is a homogeneous, stable material with certified chemical and/or physical properties used in calibrating instruments, validating experimental data, developing methods of known reliability, and referring data from different laboratories to a common base. In the United States, the National Bureau of Standards (NBS) has legal authority to issue CRMs, which for historical reasons are called Stan- dard Reference Materials (SRMs). There are more than 900 SRMs listed in the current NBS SRM catalog, the greatest number of these SRMs are certified for chemical composition. Chemical composition SRMs, which comprise a variety of matrices and constituents, inorganic and organic, are of greatest interest to spectrochemists. These SRMs have applications in such diverse areas as clinical, environmental, nutritional, agricultural, and industrial analytical chemistry. Instrument-performance SRMs, such as the one developed for validating the performance of gas chromatograph-mass spectrometers, will also be of interest to spectrochemists.

Introduction

The performance of a material is significantly affected by the concentrations of its constituents. For example, the pres- ence of hafnium in reactor-grade zirconium alloys adversely affects the performance of a reactor fuel and therefore hafnium is specified as an upper limit at gg/g levels.

On the other hand, some materials are analyzed because of their diagnostic significance. The concentrations of monitored constituents indicate conditions that may require corrective action. In the field of clinical chemistry, body fluids are analyzed for electrolytes, organic constituents, and more recently, for trace elements. However, because of the difficulty in determining trace elements reliably, the reported diagnostic significance of some elements has been controver- sial [23, 24]. Similarly, decisions regarding the toxic effects of pollutants and effectiveness of alternate environmental clean-up procedures may be invalid because of inaccurate measurements of pollutants in environmental matrices such as sediments, water, and particulate matter.

Spectrochemical methods are frequently used to analyze materials because of their speed, sensitivity and specificity of the determinations. However, in most of these methods, the instruments are calibrated with reference materials pre- pared in the laboratory or other available reference materials and the instrumental responses generated by the analytes in

Fresenius Z Anal Chem (1986) 324:376-383 6 �9 Springer-Verlag 1986

the unknown samples are compared with those generated by "known" concentrations of the same analytes in the laboratory reference materials. Consequently, a primary factor affecting the accuracy of the results is the reliability of the standards. If the analytes and matrix components of the laboratory reference materials are of questionable purity, the analytical results, especially of trace constituents, will also be questionable. That laboratory standards can be a problem is cited by Horwitz et al. [6]. The authors concluded from their interlaboratory quality control studies that "all proven causes for deviations greater than two standard deviations could be attributed to standard solutions, gas chromatographic (GC) problems, or calculation errors, with standard solution problems being the culprit about ha~ of the time". The authors further observed that if an appropriate Standard Reference Material (SRM) was available, analyses of SRMs concurrently with the samples and using the same procedure provided one of the few means for monitoring the accuracy of the analytical technique.

NBS has developed SRMs since 1906 when four cast irons certified for chemical composition were issued. One of these cast irons, which is still used in industrial quality control, has been renewed 12 times. At present, approximately 900 different SRMs are available of which those certified for chemical composition are the majority and are of most use to spectrochemists.

In this paper, the development and certification of the Certified Reference Materials (CRMs) issued by the National Bureau of Standards as Standard Reference Mate- rials (SRMs) is discussed. A reference to other sources of CRMs is also provided.

Development of an SRM

Requests for new SRMs are received from many sources - industry, government agencies, standards bodies, pro- fessional societies, trade associations, and individuals.

An SRM is produced in response to a demonstrated measurement need. The justification for its development considers such questions as: What is the measurement problem? Who is affected? And, how will the SRM assist in resolving these problems?

Data from interlaboratory studies, in which homogeneous samples are analyzed by different laboratories, often disagree greatly and confirm the need for a certified reference material as a primary control. In one of these studies, Dybczynski et al. [4] reviewed the interlaboratory analyses of a milk powder using different methods including spectrochemical. The ranges of reported laboratory means for important trace elements were: As, 5 to 544 ng/g; Cd, 1 to 1,660 rig/g; Co, 0.004 to 51 ~tg/g; Cr, 0.02 to 52 ~g/g; Cu 0.08 to 72 gg/g; Fe, 0.70 to 20 gg/g; Hg, 1 to 666 ng/g; Mn, 0.12 to 55 pg/g; Mo, 0.15 to 3.6 gg/g; Pb, 0.04 to 246 pg/g; Se, 25 to 313 rig/g; V, 0.002 to 20 pg/g; and Zn, 26 to 4,194 gg/g. For the following important macronutrients, the range of reported means, in percent by weight, were: Ca, 0.012 to 2.6; C1, 0.1] to 3.4; K, 1.27 to 2.84; and Na, 0.32 to 0.72. Parr [15] also has discussed the reliability of trace element determination in biological materials.

May et al. [11, 12] have reported the interlaboratory analyses of a shale oil for the polycyclic aromatic hydrocarbons (PAHs): fluoranthene, pyrene, benzo[a]pyrene, and

benzo[e]pyrene. PAHs are important because they are wide- spread environmental pollutants and many PAHs are reported to be either mutagenic, carcinogenic, or both. In their report, the results for fluoranthene from ten laboratories ranged from 61 (NBS value) to 380 ~tg/g. A GC/MS concentration reported by another laboratory was 247 pg/g. For pyrene, the concentrations ranged from 102 (NBS value) to 620 pg/g; in this case, 161/~g/g was the GC/MS concentration reported by another laboratory. For benzo[a]pyrene, only four laboratories reported results ranging from 3.3 to 22 pg/g (NBS value) and for benzo[a]pyrene, only two laboratories reported results, 1.3 and 21 (NBS value). For each of the four compounds, the NBS value is the mean of the results by gas chromatography (GC), high performance liquid chromatography (HPLC), and gas chromatography- mass spectrometry (GC/MS).

Once the decision has been made to develop an SRM, a study is undertaken to determine the feasibility of the pro- ject. Some of the questions considered are: Will the proposed SRM and its certified constituents be stable for its anti- cipated shelf life? Are reliable methods available to provide certified values? How will the candidate material be tested for homogeneity?

Homogeneity

Evaluating the homogeneity of the candidate SRM material is a prerequisite to its further characterization. The anti- cipated minimum mass of the sample that is to be specified in the certificate, or less, is used for homogeneity testing. Preliminary testing of randomly selected samples for a number of elements is done most efficiently by rapid multielement methods capable of high precision. A number of analytical techniques are used depending on the material and concentration levels of the analytes. For metal ingots, optical emission spectroscopy (OES) using point-to-plane techniques is still used at NBS, as it has been for about 40 years, to analyze samples from different locations of an ingot. X-ray fluorescence (XRF) has been used recently to test the homogeneity of complex alloys, fertilizers, cements, glasses, and silicon carbide. Spark source mass spectroscopy (SSMS) is also used for homogeneity testing of metal by direct sparking of the metal in the form of pins. SSMS methods are particularly valuable for high-purity metals because of their sensitivity. Instrumental neutron activation analysis (INAA) and atomic absorption spectroscopy (AAS) are used in the analysis of a variety of matrices, including biologicals, because of their applicability to many elements and high sensitivity.

Analysis of the candidate SRM

Whenever possible, SRMs are certified on the basis of accuracy, rather than method-dependent analyses. Exceptions are those constituents that cannot be determined accurately because of the analytical state-of-the-art. The determination of enzymes in human serum (SRM 909) is an example.

Certified concentrations are based either on the con- cordant analytical results of two or more independent methods, or on the results of a definitive method. A definitive method has a valid theoretical foundation, negligible systematic errors, and high precision.

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For certification by the use of two or more methods, the methods should be completely different in theory and experimental technique - for example, neutron activation analysis (NAA) and atomic emission spectroscopy (AES). The advantage of NAA methods for trace element determinations is freedom of these methods from reagent contamination when the sample is irradiated initially without pretreatment. For certification of organic constituents, GC, HPLC, GC/MS, and HPLC/mass spectrometry have been used [2, 5, 8, 11, 12].

For elemental determinations, a variety of analytical methods are used. For example, for the certification of elemental concentration in SRM 1549, Non-Fat Milk- Powder, the methods used, grouped under six main headings were: 1. atomic absorption spectrometry (AAS), 2. atomic emission spectrometry (AES), 3. ion chromatography, 4. isotope dilution mass spectrometry (ID-MS), 5. neutron activation (NA), and 6. photon activation. Variants of the AAS and AES methods were used depending on sample treatment and excitation. For the ID-MS methods, different methods of ionization were used.

ID-MS is considered a definitive method for the determination of certain elements and organic constituents. For elemental determinations, a known amount of an isotopically enriched element known as a "spike", is added to the sample to be analyzed. After chemically processing the sample to equilibrate the spike with the naturally occurring element in the sample, the ratio of the "spike" isotope to another isotope is measured. From this altered isotopic ratio, mass of the sample, mass of the added spike and additional data, the concentration of the element originally present in the sample can be calculated. For organic constituents, isotopically labelled compounds are used for equilibration with the compound to be determined [20, 25].

In comparison with other trace analytical methods, ID- MS has several advantages. First, analyte losses do not affect the reliability of the final analytical results providing that isotopic equilibration has occurred. This follows because the altered isotopic ratios are unaffected by losses of the isotopically equilibrated elements. Secondly, quantitative separation of the trace element analytes or a determination of their recoveries is not required. And, finally ID-MS methods do not require calibration with laboratory reference materials containing known concentrations of the analytes.

Equilibration of the enriched isotope spike with the naturally occurring element is essential to obtain valid results by an isotope dilution method. Confidence that this vital step has occurred successfully is increased when the spike is added at the beginning and end of separate experiments and the analytical results agree.

Loss of an analyte before equilibration with the enriched isotope of the analyte is of particular concern when determining a volatile element, such as mercury. For the determination of the submicrogram-per-gram concentration of mercury in a plant tissue SRM, wet-oxidation was done under reflux and with a trap [1]. Experiments in which the spikes were added at the beginning and at the end of the wet oxidation procedure yielded compatible results.

The accurate determination of trace elements by ID-MS is limited by the analytical or method blank. Contamination is minimized during chemical treatment of the sample by selecting laboratory vessels of inert material, filtering the air in the laboratory environment with class 100 filters, and by limiting and carefully purifying the number of reagents used.

At NBS, high-purity acids are produced by sub-boiling dis- tillation and analyzed by ID-MS [10]. Veillon and Alvarez [22] have reviewed the use of stable isotope techniques for the determination of trace metals in biological materials. These techniques are discussed in connection with spectroscopic methods, neutron activation analysis, and mass spectrometry.

For the problem of accurately determining Hf in zirconium metal and zircaloy, Powell and Paulsen [17] developed a method based on ID-SSMS. These results and those obtained by NAA were used to provide certified concentrations of Hf ranging from 31 _+ 3 lag/g in SRM 1237 to 198.6 ~tg/g in SRM 1236. The use of these SRMs by both buyer and seller in international trade, has substantially reduced disputes regarding the Hf content of zirconium and zircaloy.

In addition to analyses made at NBS by NBS scientists and guest workers, analyses are made by outside cooperators. The cooperators are requested or volunteer to perform particular analytical determinations because of their expertise. If the same constituent has also been determined at NBS by an independent method and the results agree, the concentration can be certified. If the constituent has been determined only by the cooperator, the concentration will be listed as a non-certified value.

Biological SRMs certified for nutrients and potentially toxic constituents

Table 1 lists the chemical compositions of biological SRMs that are available at present. An element is listed when at least one SRM has a certified concentration. An oyster tissue SRM is being renewed as SRM 1566a.

The SRMs in Table I may be divided into two general groups according to application. One group, consisting of the powdered plant tissues, citrus leaves, tomato leaves, and pine needles, is of interest to agronomists, horticulturists, phytopathologists and other plant scientists.

Reports that diet may influence the onset of cancer has led to an increased interest in the chemical composition of foods. Selenium, in particular, has been the subject of much research because of its possible anticancer effects. The metabolism of other trace element nutrients is also of interest to establish estimated safe and adequate daily dietary in- takes. The food SRMs in Table I provide a range of certified concentrations for Se and other essential trace elements, such as Cu, and Fe. The plant tissue SRMs may also be useful in analyzing plant tissues used as foods. However, the plant tissue SRMs were developed primarily to assist scientists in obtaining reliable foliar analyses which can indicate nutrient needs or other plant stress factors. Because of the certified concentrations of such elements as Cd, Ba, and Pb, the plant tissue SRMs are also used in environmental studies. A range of certified concentrations in plant tissue SRMs is desirable to validate measurements at different concentration levels. For these three plant tissue SRMs, ranges are provided for a number of important elements. For example, Ca ranges from 0.41 to 3.15%; K, from 0.37 to 4.46%; Sr, from 4.8 to 100 lag/g; and Mn from 23 to 675 gg/g.

Reference Material 8412 Corn (Zea Mays) Stalk and Reference Material 8413 Corn (Zea Mays) Kernel are being distributed by NBS. As defined by the International Organ- ization for Standardization (ISO), a Reference Material

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Table 1. Composit ion of Biological SRMs (Concentration in I~g/g or where noted, in percent by weight)

Element Non-fat milk Bovine liver Wheat flour Rice flour Brewers Citrus Tomato Pine needles powder SRM 1577a SRM 1567 SRM 1568 yeast leaves leaves SRM 1575 SRM 1549 SRM 1569 SRM 1572 SRM 1573

Aluminum (2) (2) - - - 92 (0.12%) 545 Arsenic (0.0019) 0.047 (0.006) 0.41 - 3.1 0.27 0.21 Barium . . . . . 21 -- - Cadmium 0.0005 0.44 0.032 0.029 - 0.03 (3) ( < 0.5) Calcium 1.30% 120 0.019% 0.014% - 3.15% 3.00% 0.41% Chlorine 1.09% 0.28% - - - (414) - - Chromium 0.0026 - - - 2.t2 0.8 4.5 2.6 Cobalt (0.0041) 0.21 - 0.02 - 0.2 0.6 0.1 Copper 0.7 158 2.0 2.2 - 16.5 11 3.0 Iodine 3.38 . . . . 1.84 - - Iron 1.78 194 18.3 8.7 - 90 690 200 Lead 0.019 0.135 0.020 0.045 - 13.3 6.3 10.8 Magnesium 0.120% 600 - - - 0.58% (0.7%) - Manganese 0.26 9.9 8.5 20.t - 23 238 675 Mercury 0.0003 0.004 0.001 0.0060 - 0.08 (0.1) 0.15 Molybdenum (0.34) 3.5 (0.4) (1.6) - 0.17 - - Nickel - - (0.18) (0.16) - 0.6 - (3.5) Phosphorus 1.06% 1.11% - - - 0.13% 0.34% 0.12% Potassium 1.69% 0.996% 0.136% 0.112% - 1.82% 4.46% 0.37% Rubidium 11 12.5 (1) (7) - 4.84 t 6.5 11.7 Selenium 0.11 0.71 1.1 0.4 - 0.038 - - Silver ( < 0.0003) 0.04 . . . . . . Sodium 0.497% 0.243% 8.0 6.0 - 160 - - Strontium - 0.138 - - - 100 44.9 4.8 Sulfur 0.351% 0.78% - - - 0.407% (0.590) (0.117) Thorium . . . . . 0.015 0.17 0.37 Uran ium - 0.00071 - - - (_< 0.15) 0.061 0.020 Vanadium - 0.0978 - - - 0.245 - - Zinc 46.1 123 10.6 19.4 - 29 62 -

Certificates of Analysis provide estimated uncertainties for the certified concentrations. Values in parentheses are not certified

( R M ) is a m a t e r i a l one or m o r e p r o p e r t i e s o f w h i c h are suff ic ient ly well e s t ab l i s hed to be used fo r the c a l i b r a t i o n o f a n a p p a r a t u s , t he a s s e s s m e n t o f a m e a s u r e m e n t m e t h o d , or the a s s i g n m e n t o f va lues to ma te r i a l s . R M s 8412 a n d 8413 were p r e p a r e d a t A g r i c u l t u r e C a n a d a u n d e r the d i r e c t i o n o f M. I h n a t , w h o w i t h W. R. Wolf , U S D e p a r t m e n t o f Agr i cu l t u r e , c o o r d i n a t e d the i n t e r l a b o r a t o r y ana lyses to c h a r a c t e r i z e the p o w d e r e d ma te r i a l s . Six l a b o r a t o r i e s p a r t i c i p a t e d in the ana ly t i ca l p r o g r a m . T h e R e p o r t s o f In- v e s t i g a t i o n fo r R M s 8412 a n d 8413 b o t h p r o v i d e re- c o m m e n d e d c o n c e n t r a t i o n s , w h i c h a re n o t cer t i f ied by N B S , for these e l emen t s : K, Mg, Ca , M n , Fe, Cu, Z n a n d Se. In a d d i t i o n , R M 8412, p r o v i d e s r e c o m m e n d e d c o n c e n t r a t i o n s for t h r ee a d d i t i o n a l e l emen t s ; N a , Sr, a n d C1. T h e re- c o m m e n d e d c o n c e n t r a t i o n s are b a s e d o n resul t s o b t a i n e d by two or m o r e i n d e p e n d e n t , re l iab le ana ly t i ca l m e t h o d s . B o t h R M s also list i n f o r m a t i o n va lues for N a n d F ; a n d in a d d i t i o n R M 8413 lists i n f o r m a t i o n va lues for A1 a n d C1. I n f o r m a t i o n va lues a re b a s e d e i t he r o n resu l t s o f on ly one m e t h o d or l imi ted d a t a f r o m two m e t h o d s .

Clinical laboratory SRMs

Clin ica l l a b o r a t o r y S R M s for s p e c t r o c h e m i c a l a p p l i c a t i o n s m a y be classif ied i n to two gene ra l types :

M a t r i x m a t e r i a l cons i s t i ng o f h u m a n se rum, ur ine , or a n i m a l b l o o d w i t h a c c u r a t e l y cer t i f ied cons t i t uen t s .

H i g h - p u r i t y o r g a n i c a n d i n o r g a n i c c o m p o u n d s for pre- p a r i n g so lu t i on c a l i b r a t o r s or sp ik ing m a t r i x so lu t ions .

T h r e e lyoph i l i zed h u m a n s e r u m S R M s are ava i lab le . O f these, S R M 909 is o f g rea t e s t in t e res t to spec t roscop is t s . Th i s S R M was d e v e l o p e d o r ig ina l ly to p r o v i d e cer t i f ied con- c e n t r a t i o n s o f selected e lec t ro ly tes a n d o rgan i c c o n s t i t u e n t s p r e s e n t in the s e r u m a t n o r m a l levels. H i g h h o m o g e n e i t y o f the s e r u m m a t e r i a l was ach i eved b y p roces s ing it i n to spheru les , a p p r o x i m a t e l y 1 m m in d iamete r , a n d m i x i n g the en t i r e lo t be fo re bo t t l ing . T h e la tes t rev ised Cer t i f i ca te o f Ana lys i s lists cer t i f ied c o n c e n t r a t i o n s a n d unce r t a in t i e s fo r ca lc ium, ch lor ide , cho les te ro l , c rea t in ine , glucose, l i th ium, m a g n e s i u m , p o t a s s i u m , sod ium, urea , ur ic acid, a n d six t r ace e lements . T h e n o m i n a l c o n c e n t r a t i o n s o f the t race e l emen t s in the r e c o n s t i t u t e d se rum, w h i c h s h o u l d n o t be c o n s i d e r e d " n o r m a l va lues" in h u m a n se rum, are : c o p p e r (1.1 ~tg/ml), i r o n (2.0 gg/ml) , c a d m i u m (1.2 ng /ml ) , c h r o m i u m (91.3 rig/ ml), l ead (20 ng /ml ) , a n d v a n a d i u m (2.7 ng /ml ) . E x c e p t fo r sod ium, a m o n o n u c l i d i c e l e m e n t n o t d e t e r m i n a b l e b y iso- t o p e d i l u t i o n m a s s spec t rome t ry , all c o n s t i t u e n t s i n c l u d i n g the t r aee e l emen t s were d e t e r m i n e d b y these def in i t ive m e t h o d s . S o d i u m was d e t e r m i n e d by a m e t h o d in w h i c h the s o d i u m was s e p a r a t e d by ion exchange , c o n v e r t e d to s o d i u m sulfate, a n d d e t e r m i n e d grav imet r ica l ly . T h e s o d i u m con- c e n t r a t i o n was c o n f i r m e d by a t o m i c a b s o r p t i o n spec t ros - copy.

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Lecture8

Certified concentrations of constituents are listed in the certificate in two ways corresponding to whether reconstitu- tion is done with or without weighing the freeze-dried serum contents of a bottle. Concentration values having smaller uncertainties are obtained by weighing the freeze-dried serum and multiplying this mass by the certified concentrations of the analytes per unit mass of dried serum. The mass of freeze-dried serum is determined by difference. The procedure consists essentially of weighing the bottle of freeze-dried serum, reconstituting the serum with the water supplied, analyzing the reconstituted serum, and finally washing, drying, and weighing the bottle.

The certificate for SRM 909 also provides a method- dependent concentration for total protein and uncertified catalytic concentrations of seven enzymes. Enzymes, for which definitive methods are not available, were determined cooperatively by teams of experts using "best available" methodology.

The other two lyophilized human serum SRMs are for use as primary controls for drugs. Antiepilepsy Drug Level Assay standard, SRM 900, is certified for the toxic, ther- apeutic, and subtherapeutic levels of phenytoin, ethos- uximide, phenobarbital, and primidone. Anticonvulsant Drug Level Assay, SRM 1599, is certified for low, medium, and high levels ofvalproic acid and carbamazepine. Valproic acid has also been determined in SRM 1599 at NBS by a reversed-phase liquid chromatography mass spectrometric method [3].

Lead in Blood, SRM 955, consists of four levels of lead in porcine blood. For the production of this SRM, hogs were fed diets containing various quantities of lead acetate for three weeks. Three days after the final feeding, blood fi'om the hogs was collected directly into clean Teflon con- tainers and treated with heparin, an anticoagulant. The blood was then subjected to repeated freeze-thawing, followed by flow-through sonication to assure complete rupture of the red cells. The blood was then stored frozen for several years while stability studies were performed.

Finally, selected lots of blood from different animals were blended to provide the four levels of lead. The pooled blood was centrifuged to remove any particulate matter, clots, etc. Approximately 2 ml of blood was dispensed into polyethylene bottles and stored frozen at - 2 0 ~

Samples were analyzed by thermal ionization stable isotope dilution mass spectrometry (IDMS) and by atomic absorption spectrometry (AAS) using electrothermal atomization. Because IDMS is inherently more accurate for the determination of lead in blood than other analytical methods, the certified concentrations are the means of the IDMS results. However, because AAS procedures are more rapid, they were used to determine Pb in a sufficient number of random samples to establish the homogeneity of the entire lot.

The certified concentrations of lead at 22 ~ in gg/dl and gmol/1, respectively, and their uncertainties are: Level A, 5.7 _+ 0.5, 0.27 +_ 0.02; Level B, 30.5 • 0.3, 1.47 • 0.02; Level C, 49.4• 2.38_+0.04; and Level D, 73.2_+0.7, 3.53 _+ 0.03. The uncertainties are expressed as two standard deviations of the mean and include allowances for between- bottle variability.

SRM 2670 is a freeze-dried urine certified primarily for elements of toxicological significance. It consists of two concentration levels for trace elements, an elevated level and a normal level. The low level is normal human urine; the

elevated level is normal human urine with added amounts of elements of interest. For the low level, selenium and copper are certified; and for the elevated level, arsenic, cadmium, chromium, copper, lead, mercury, and selenium are certified. In addition to the trace elements the matrix elements, calcium, magnesium, and sodium, which are present at the same concentration in both levels, are certified. The other matrix elements, chloride, potassium and sulfate are not certified but their concentrations are listed for information only. The concentrations of the trace elements alu- minium, beryllium, nickel and platinum are listed for information only.

A Bovine Serum Reference Material (RM 8419), developed under the technical guidance of C. Veillon, US Depart- ment of Agriculture, has been issued by NBS. The Report of Analysis lists recommended concentrations, which are not certified by NBS, with estimated uncertainties for 14 elements. The elements are: sodium, potassium, calcium, magnesium, iron, copper, zinc, aluminum, cobalt, chromium, manganese, molybdenum, nickel, and selenium. The recommended concentrations are based on determinations of each element by at least three different methods. The methods used include atomic absorption spectrometry, atomic emission spectrometry and isotope dilution mass spectrometry. The pooled bovine serum, which tested satis- factorily for homogeneity, was analyzed by chemists from 14 organizations.

A comparison of the reported major (matrix) constituents of human and bovine serum with respect to total protein, albumin, fat, glucose, sodium, chloride, and inorganic phosphorous indicates human and bovine serum to be very similar. In addition the measured specific gravity of each is identical, as is the moisture content.

Environmental SRMs

The reliable determination of pollutants in a variety of environmental materials is necessary to establish accurate baseline concentrations of pollutants in these materials; to determine effectiveness of pollution control measures; to assemble reliable data on the emission, transport, and fate of pollutants; and to issue environmental regulations based on sound experimental data. Quality assurance of environmental measurements, was addressed at a conference sponsored by the American Society for Testing and Materi- als (ASTM) and the information was published in a special publication [18].

Quality assurance depends on the use of reliable methodology and also certified reference as primary control standards to improve data accuracy. The use of ID-MS [13] for the certification of environmentally important elements in SRMs and the use of AAS and plasma emission spectrometry [19] for the same purpose have been described.

Environmental matrix SRMs, include a number certified for toxic constituents designated as "priority pollutants" by the US Environmental Protection Agency (EPA). The matrices are: atmospheric dust, water, sediments, biological materials, fuels, and gases. In addition, calibrator solution SRMs of inorganic and organic toxic constituents, such as halocarbons, pesticides, and polycyclic aromatic hydrocarbons PAHs, are available for determining instrumental response factors and adding accurate amounts of these compounds to samples. The most recent water SRM was

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Table 2. SRMs certified for organic pollutants

SRM No. SRM name Certification

1580 1581 1582 1649 1650 1644 b

1647 1583 1584 1585 1586

1639 1614 1587

Organics in shale oil Polychlorinated biphenyls in oils Petroleum Crude Oil Urban dust/organics Diesel particulate matter Polynuclear aromatic hydrocarbon generator columns

Priority pollutant polynuclear aromatic hydrocarbons in acetonitrile Chlorinated pesticides in 2,2,4-trimethylpentane Priority pollutant phenols in methanol Chlorinated biphenyls in 2,2,4-trimethylpentane Isotopically labeled and unlabeled priority pollutants in methanol

Halocarbons in methanol Dioxin (2,3,7,8-TCDD) in isooctane Nitrated polycyclic aromatic hydrocarbons in methanol

5 PAHs"; 3 phenols + 1 additional compound Aroclors 1242 and 1260 in two oils 5 PAHs plus dibenzothiophene 5 PAHs 5 PAHs plus 1-nitropyrene Anthracene, benzo(a)anthracene, and benzo(a)- pyrene 16 PAHs 5 pesticides 10 phenols 8 chlorinated biphenyls 10 isotopically labeled compounds 10 unlabeled compounds 7 halocarbons 2,3,7,8-TCDD; 2,3,7,8-TCDD-13C 6 nitrated PAHs

a Polycyclic aromatic hydrocarbons b SRM 1644 generates known concentrations of three PAHs in water

developed because of world-wide concern for the effects of acid precipitation on forests and other vegetation [14]. SRM 2694, a simulated rainwater, consists of two different solutions. At present, the Certificate of Analysis for SRM 2694 provides certified values, at two levels, for pH, specific conductance, acidity, F - , NO~-, SO42-, Na +, K +, C a 2+ and Mg 2 +. The background and research leading to the production of this stable simulated rainwater SRM has been published [9].

As reported by O'Sullivan [14] the complexity of acid rain is reflected by the many theories that have been proposed to explain its mode of action. For example, one such theory implicates not only nitrogen oxides, sulfur dioxide, and ozone as primary pollutant factors but also trace metals. Although, transition and heavy metals could not be included in SRM 2694, because of their instability at the certified pH levels, 3.59 4- 0.02 and 4.30 4- 0.02, certified concentrations of 17 trace elements are listed in the Certificate for SRM 1643 b, Trace Elements in Water.

X-ray fluorescence spectrometers can be used for the elemental analysis of particulate matter collected on filter media. Two SRMs have been developed for standardization of this instrumentation. SRMs 1832 and 1833 each consists of a silica based glass film that has been deposited onto a polycarbonate filter. The glass film is a continuous layer approximately 0.55 gm thick containing known concentrations of the oxides of selected elements. For SRM 1832, the certified elements are: Na, A1, Si, Ca, V, Mn, Co, and Cu; for SRM 1833, the certified elements are: Si, K, Ti, Fe, Zn, and Pb.

In recent years, methods have been developed to certify toxic organic constituents in SRMs. Matrix SRMs and calibrator solution SRMs, certified for toxic organic compounds, are listed in Table 2.

A number of SRMs have been certified for PAHs. The concentrations of PAHs at different levels are certified in two oils, SRMs 1580 and 1582; and, they are certified in an urban dust and in a particulate matter from a diesel engine exhaust. In addition, SRM 1647 provides certified concentration of 16 EPA Priority Pollutant PAHs in acetonitrile,

and SRM 1644 Polynuclear Aromatic Hydrocarbon Gen- erator was developed to generate known concentrations of three PAHs.

The two most recent SRMs listed in Table 2 are SRM 16/4, Dioxin (2,3,7,8-TCDD) in Isooctane and SRM 1587 Nitrated Polycyclic Aromatic Hydrocarbons in Methanol. SRM 1614 consists of separate solutions of unlabeled and 13C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin. The nominal certified concentrations of both solutions are 100 ng/g, which are also listed in ng/ml at 23~ The t3C-labeled compound can be used as an internal standard in GC/MS methods. SRM 1587 was developed because nitrated PAHs are reported to exhibit direct acting Ames mutagenic activ- ity. The Certificate of Analysis for SRM 1587 lists certified concentrations, in ~tg/g and gg/ml, of six nitrated PAHs in methanol. The compounds are: 2-nitrofluoranthene, 9-nitroanthracene, 3-nitro-fluoranthene, 1-nitropyrene, 7- nitrobenz[a]anthracene, and 6-nitrochrysene.

GC/MS system performance standard, SRM 1543

SRM 1543 was developed for evaluating the sensitivity of GC/MS instrumentation. The SRM consists of four solutions: two concentrations of methyl stearate in hexane, and two, of benzophenone in hexane. The nominal concentrations of each compound is 1 and 5 ng/~d.

Industrial application SRMs

This category includes metals, alloys, glasses, ores, cements, and wear metals in oil. Most of these materials are analyzed in a cooperative industry-NBS program conducted with the assistance of the American Society for Testing and Materials (ASTM). An ASTM Committee, composed of industry, ASTM, and NBS representatives establishes priorities for the development of industrial type SRMs based on justifications received from industry. Some recent SRMs in this category are described.

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High-alloy white cast irons, C 1290 "- C 1292

Three high-alloy white cast irons have been certified for chemical composition. Issued in the form of disks 32 mm in diameter, they are for use mainly with atomic emission and X-ray fluorescence spectrometry. The Certificates of Analy- sis provides a range of certified concentrations for ten elements: carbon, manganese, phosphorus, sulfur, silicon, copper, nickel, chromium, vanadium, and molybdenum.

Ductile irons, SRMs C 2424 and C 2425

Two new ductile irons have been issued in the form of disks, 32 mm in diameter, for use in atomic emission and X-ray fluorescence spectrometric methods of analysis. The disks were chill cast on a water-cooled copper plate mold to provide rapid unidirectional solidification. The SRMs provide certified concentrations for 14 elements; carbon, manganese, phosphorus, sulfur, silicon, copper, nickel, chromium, vanadium, molybdenum, magnesium, cerium, lanthanum, and titanium.

CM 2423, another member of this ductile iron series, is a low sulfur iron with a high silicon content. Because the range of sulfur concentrations reported by laboratories was large, approximately 2 to 90 gg/g, sulfur was determined by an isotope dilution, thermal ionization mass spectrometric method [7, 16]. A preliminary concentration, obtained by this method, is 6.4 ~g/g.

Aluminum alloys (Grade 3004) SRMs 853, 1240, and (Grade 5182) SRMs 854, 1241

These alloys were developed for the analysis of aluminum can stock alloys. SRMs 853 and 1240 have the chemical composition of aluminum alloy grade 3004 while SRMs 854 and 1241, the composition of grade 5182. SRMs 853 and 854 are in the form of millings; and SRMs 1240 and 1241, in the form of disks. The four SRMs are certified for Si, Fe, Cu, Mn, Mg, Cr, Ni, Zn, Ti, V, Ga, and Zr.

Glass SRMs

Two glass SRMs are being issued. SRM 1411, Soft Borosilicate Glass, is certified for twelve elements; and SRM 1412; Multicomponent Glass, for eleven elements. XRF was used to test the homogeneity of the materials.

Sources of other certified reference materials

In addition to the SRMs discussed in this paper, other CRMs are issued by organizations throughout the world. Directory of Certified Reference Materials, lists available CRMs according to category and the names and addresses of organizations issuing them. It is available from International Organization for Standardization, Case postale 56, CH- 1211, Geneva 20, Switzerland.

Conclusions

The use of spectrochemical or other analytical methods, no matter how well written and tested, is not sufficient to assure analysts of the accuracy of their data. Neither are SRMs or other CRMs by themselves sufficient to ensure accurate data

if the method is biased or not under statistical control. However, a comparison of the results obtained with the certified concentrations for a matrix SRM or other CRM analyzed at the same time as similar materials, can reveal a measurement problem. But the cause may not be known. Based on the conclusions of Horwitz et al. [6], it may be an inaccurate standard solution, which causes an inaccurate instrument calibration. This possible source of error can be checked with calibrator solution SRMs or other CRMs where the concentrations of the analytes of interest in solution are certified.

The proper use of reference methods of proven and demonstrated accuracy together with primary reference materials (SRMs or other CRMs) yield an accuracy-based measurement system. The matrix reference materials serve as primary controls to indicate whether the entire measurement system is "in control". Reference methods can then be used to develop field methods of proven but lesser accuracy. The field methods and CRM-traceable, secondary reference materials are used to establish a quality control system for field use. Uriano and Gravatt [21] have discussed the relationship and hierarchy of methods and reference materials to establish accuracy-based measurement systems.

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Received October 28, 1985

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certified reference materials for validating spectroscopic methods and experimental data

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