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Fresenius Z Anal Chem (1988) 332:568-572

9 Springer-Verlag1988

Criteria for the development of biological reference materials Milan Ihnat Land Resource Research Centre, Agriculture Canada, Ottawa, Ontario, K1A 0C6, Canada

Kriterien fiir die Entwicklung von biologischen Referenzmaterialien Summary. A review is presented of factors to be considered in the development of biological reference materials. Some guidelines are offered regarding approaches to the generation of the varied materials required for analytical quality control. Major considerations in such an endeavour are the goal of the undertaking and role of the final product, selection of candidate materials, preparation, characterization and certification. Selection of materials should be from those important to commerce and consumers, and related to various regulatory, clinical, environmental, and research activities. They should adequately represent the different choices and types of foodstuffs, clinical materials and environmental materials, such as soils, sewage sludges, plant and animal tissues, of interest in different regions of the world. Acquisition can be from commercial sources or the result of in-house preparation, with attention to stability enhancement if required and maintenance of native analyte levels by minimization of contamination. The approach to chemical and physical characterization relies on the measurement philosophy, selection of analytes, their speciation, and selection of analytical methods and analysts for establishment of homogeneity and quantitative levels. Throughout the overall task of RM development there is a requirement for a critical approach by critical analytical and measurement scientists and the involvement of national RM agencies in order to produce top quality control materials.

a range of constituents in a wide variety of commodities of agricultural, clinical, and environmental origin with often discouraging outcomes. Accuracy is urgently needed and indeed required if rational conclusions are to be reached. The incorporation of appropriate reference materials (RM) into the scheme of analysis utilizing good methods and other aspects of a quality control program is the most convenient, cost-effective mechanism by which to assess and maintain analytical data quality. The role of a reference material within an accuracy-based measurement system is to serve as the vehicle for transfer of accuracy of a definitive method to the measurement process and the numerical data generated therefrom. These concepts have been treated in detail by Cali et al. [1], Uriano and Gravatt [21] and Huntoon [6]. In respect of the development (defined as the composite of all activities involving selection, preparation, characterization and certification) of reference materials, a number of important requirements and criteria must be taken into consideration. These considerations which also reflect the sequence of steps to be taken in RM development are: (a) reference material development philosophy, (b) end use requirements, (c) selection of materials, (d) preparation, (e) physical and chemical characterization, (f) certification, (g) documentation and (h) distribution. This report deals with a summarized discussion of these factors as applied to the development of natural matrix biological reference materials (BRM's) relevant generally to biological, environmental and clinical materials for inorganic and organic constituents.

2 Reference material development philosophy 1 Introduction Analytical chemistry plays a significant role in disciplines of agricultural, food, nutritional, clinical, medical and environmental sciences in generating the required concentration information of constituents in biological materials. Reliable measurements are mandatory for legal compliance with regulations, and essential for long term monitoring and baseline studies, standardization of laboratories over an extended period of time and on an international basis, and for research. Quite frequently contemporary analyses dealing with ultratrace, trace and even major concentrations of analytes are still plagued by uncertainty in the reported results. A great deal of concerted effort is being expended to quantify Contribution No. 88-50 from Land Resource Research Centre

Proper development of RM's requires, on the part of all of the responsible personnel, a keen, in-depth understanding of and appreciation for all the concepts behind good analytical measurement, quality control and the role of RM's. S o m e of the ideas can be summarized as the comprehension of: measurement system and RM concept and philosophy including the requirement for information of appropriate reliability; the role of RM's within an accuracy-based measurement system and data traceability with respect to definitive, reference and field methods of analysis; RM applications, uses and limitations; the need for the direct involvement of critical analytical scientists and technicians in all facets of the development; the need for involvement of a national agency in development and distribution; and the overwhelming importance of the concept of unassailable credibility in the final product.

569 Table 1. Comparison of concentration ranges of some major and trace essential and toxic elemental constituents in foodstuffs and available biological reference materials" Element

A1 As B Ca Cd Co Cr Cu Fe Hg K Mg Mn Mo Na Ni Pb Se Sn V Zn

Range of concentrations (mg/kg or mg/1) in foodstuffs b

in reference materials ~

RM Deficiency at low (L) or High (H) end

0.02-1300 0.000 - 60 0.002-125 0-60000 0.001 -- 5 0.0001 - 5 0.002-10 0.002-140 0.09-- 1200 0.0006 - 2 0 - 47 000 0.3-- 6900 0.005- 900 0.00002-6 0-- 81000 0.004 - 8 0.001 - 1.7 0.0006-12 0.2-130 0.00005-6 0.02-1600

0.013 -775 0.047 -24.6 49.0 42-212000 0.0005-- 189 0.0012-23 0.00030- 6.5 0.13--720 1.78 --2400 0.0003 - 4.4 3 4 - 44 600 20.7-- 5940 0.0026- 3 771 0.016--3.5 6.0-- 45 500 02018 - 8.7 0.019 -64.4 0.004-6.88 0.139 0.0027-2.3 1.1-852

H L, H L, H L L H L L L L L L, H L L, H H L, H L, H

a Data taken from [7-9, 14, 19] b Estimated typical concentration ranges on fresh or dry weight basis in foods representing 12 different food classes [7] From Table 4 3 End use requirements Major uses within the measurement process of RM's with reliable certified or recommended values are quality control, analytical calibration, analytical method development and evaluation, and production and evaluation of other reference materials. End use requirements refer to the physical and chemical characteristics the RM should possess making it suitable for the above generalized end uses. End use requirements dictate an assessment of specific details such as the nature of the problem to be addressed by the product, market requirements, analytical methods to be served, analytes, forms and concentrations to be certified and the level of certification required. These considerations will have a bearing on the form of the final product, particle size and homogeneity, amount per marketable unit and quantities of starting and final product needed and will lead to an overall definition of the undertaking. 4 Selection of materials for the candidate reference material Selection of materials is made in respect of: (1) consideration of the nature and physical form of the material in relation to real world products to be analyzed; (2) representativeness of the material of the population of products in production, commerce and undergoing analysis, in respect of matrix and endogenous and adventitious levels and speciation of analytes; (3) the need for uncontaminated, endogenous-level natural materials; (4) the need for "blank" control materials

and (5) the availability of similar reference or certified reference materials. The result, after selection and pilot or large scale preparation, is a product denoted a "candidate reference material" which, depending on the outcome of final physical and chemical characterization, can become a RM. A survey of RM availability is an obvious first step prior to embarking on the development of a specific product. Listings of RMs available from government agencies are found in catalogues and information booklets [3, 12, 13, 15, 16] as well as in other compilations [2, 9, 14]. Lack of correspondence between available RMs and commodities analyzed either on the basis of matrix [8, 10] or analyte composition [8, 24] suggests the need for the development of additional, appropriate control materials. A compilation as depicted in Table 1, which presents a comparison of concentration ranges of specific elemental analytes in foodstuffs and in available BRM's, serves to illustrate the shortfall of appropriate RM's in respect of analyte composition. The last column in the Table, indicating lack of coverage by existing RM's of the low and high ends of the elemental concentration ranges of laboratory test materials, shows a deficiency of available RM's in almost every instance at the low end but also frequently at the high end of the concentration range. Representativeness with respect to matrix and analytes, of the RM with respect to the products in production and commerce is an important criteria for the development of a useful material. In this regard, consideration of production data such as those exemplified in Tables 2 and 3 is relevant. An extensive form of Table 2, dealing with the worldwide, as well as breakdown by country or continent, production of crops and livestock products was considered in choosing important candidate materials representative of the different classes of food and agricultural commodity classes [10]. Similarly, information on the available wood types in Canada (Table 3) was instructive in selecting spruce and birch as important representatives of softwoods and hardwoods, respectively, as potential candidate RM's [10]. Other such tabulations derived from national and international statistical compilations of the production of various commodities can be relevant guides for the selection of candidate RMs. Materials collected and archived under environmental specimen banking activities are good guides to the choice of required control materials; in fact surpluses of actual materials carefully collected and processed for environmental banking can serve as candidate RMs. In addition to RM's representing total (native plus adventitious) levels of analyte in the commercial material, there is a requirement for uncontaminated, native-level natural materials. This consideration can give rise to products containing low levels of analytes of natural (versus contaminated) speciation suitable for monitoring method performance in respect of these characteristics as well as serving as "blank reference materials" for blank control. Such "clean" natural-level materials may also conceivably serve the task of monitoring some sampling and presampling steps of the analytical procedure in addition to the usual laboratory measurement steps. 5 Preparation Preparation refers to all of the physical steps necessary to bring the starting material to candidate material status. It includes acquisition of commercially available, externally or

570 Table 2. Annual worldwide production of crops and livestock products, 1980 (1000 metric tons) a Commodity Cereals Sugar cane Roots and tubers Wheat Cow milk, whole, fresh Paddy rice Maize Vegetables and melons Sugar beets Potatoes Barley Horsemeat Cassava Sweet potatoes Sugar, centrifugal, raw Soybeans Grapes Sorghum Pigmeat Tomatoes Pulses Beef and veal Oats Seed cotton Bananas

Production

Commodity

1 570 673 730723 487113 444 534

Oranges Apples Coconuts Cabbages Wine

39798 35660 35422 35139 33 921

Millet Hen eggs Rye

28 918 27456 27 368

Buffalo milk Cottonseed Watermelons Plantain Dry onions Groundnuts in shell Dry beans

27 209 27155 25071 21265 19410 18901 14 664

Cotton lint Mangoes Sunflower seed Sugar, noncentrifugal Cheese (all kinds) Dry peas Rapeseed Olives Cucumbers and Gherkins Carrots

14391 14342 13174 12362 11376 11085 10 574 10 544

427 887 399 779 392249 347 859 268 722 225 718 162402 142166 122134 107254 85431 83481 65255 58435 54999 50153 47 408 45 350 42 647 42111 39254

Table 3. Inventoried, productive and economically accessible wood volume in Canada - all maturity classes on Provincial and Federal Crown production and private forest land a

Production

Species

Softwoods Spruce Fir Jack and lodgepole pine Hemlock Eastern white and western red cedar Douglar fir White pine Yellow cedar Larch Ponderosa and red pine Other softwoods Total Hardwoods Poplar White birch Aspen Maple Yellow birch Beech Black cottonwood Ash Other hardwoods Total Total all species

10 524 10087 a

a Adapted from [5]. Only commodities with annual production in excess of 10000 (• 1000 metric tons) are listed here; complete tabulation down to less than 100 ( x 1000 metric tons) was, however, used in this work

in-house custom prepared starting material and steps dealing with collection, cleaning, component separation, comminution, drying, sieving, blending, packaging; selection, testing and assessment of preparation pilot runs and selection of final large scale preparative procedures; control and monitoring of contamination; assessment of stability expected during long-term storage and steps for stability enhancement such as drying, defatting, use of antioxidants, preservatives, sterilization by gamma ray irradiation, hermetic packaging under inert atmosphere and low temperature storage. Attention to detail in all of these steps is mandatory. Several recent preparatory undertakings have addressed the question of identity of the reference material to the native material from the point of view of uncontaminated native analyte levels [18, 23] and physical form [20]. Second generation R M ' s are defined by Versieck et al. [23] and Stoeppler et al. [18] as those with trace element and c o m p o u n d concentrations close to those in real samples, that is absence o f noticeable contamination, and of utmost homogeneity with respect to analyte concentration and particle composition. Preparation and certification of such a freeze-dried human serum has been described by Versieck et al. [22, 23] while Stoeppler et al. [18] discuss prerequisites for second generation environmental RM's. Consideration of the need for close matching of R M and analytical sample matrix

Volume, millions of m 3 4759 2 580 2413 1806 1010 505 179 170 45 43 4 13514 1354 964 567 358 290 44 31 22 86 3 715 17 229

Adapted from [17]

composition and physical form has lead Sturgeon and Berman to prepare a non-defatted lobster hepatopancreas tissue with physical and chemical characteristics similar to those of the original material [20].

6 Characterization Both physical and chemical characterization are required to establish material properties essential for decisions regarding suitability, instructions on handling, and assignment of reference values. Physical characterization includes sieving, visual observation and examination by light and electron microscopy, assessment of particle size distribution, shape and density and effects of particle size on chemical composition; moisture loss/pickup and moisture determination techniques/conditions, and demixing/settling during storage and transport. Visual and light microscopical examinations have been instrumental in recent work in guiding the selection and preparation of potential R M ' s and in physical characterization [10, 11]. Chemical characterization deals with the establishment of analyte concentration values, following an appropriate protocol to establish material homogeneity and to provide data for recommendation/certification. This task, the most demanding component of the R M development undertaking, encompasses analyte selection based on nutritional, toxicological, environmental, clinical, medical, and occupational health significance as well as availability of suitable analytical methodologies and analyst competences;

571 Table 4. Ranges of certified and recommended elemental concentration values (mg/kg or rag/l) reported for biological reference materials available from government agencies ~ Ele- Range ment

No. of Ele- Range RM's ment

No. of RM's

Ag A1 As B Ba Br Ca Cd C1 Co Cr Cs Cu F Fe Hg I K La Li

5 4 14 1 5 8 32 27 13 12 17 2 37 3 32 27 4 27 1 1

27 29 8 4 25 13 14 31 14 6 2 1 20 1 14 2 4 4 30

0.027- 0.89 0.013-775 0.047-24.6 49.0 6-125 0.38-357.8 42-212000 0.0005- 189 550-55800 0.0012-23 0.00030-6.5 0.0763-0.12 0.13-720 0.55-5.87 1.78-2400 0.0003-4.4 1.29-5~35 34-44600 0.0864 11.5

Mg Mn Mo N Na Ni P Pb Rb S Sb Sc Se Sn Sr Th U V Zn

20.7- 5940 0.0026-3771 0.016-3.5 42790-163980 6.0 - 45 500 0.0018-8.7 592-10200 0.019- 64.4 0.48-75 3510--15660 0.005-0.07 0.014 0.004-6.88 0.139 0.138-113 0.037-0.17 0.00071-0.116 0.0027-2.3 1.1-852

a Adapted from data in [8, 9, 14, 19] selection of characterization and certification protocols based on in-house and cooperative use of definitive, reference and validated methodologies and selected expert analysts applying conceptually different approaches, organization of cooperative efforts; selection, development, assessment and validation of methodologies; adaptation of statistical protocols for homogeneity testing and data analysis; homogeneity assessment and evaluation of results. Elemental analyte selection can be aided by reference to information such as that in Tables I and 4 indicating coverage (and gaps) of elements and concentration ranges reflected by the current world repertoire of BRM's. From the perspective of foodstuff and RM elemental concentration ranges (Table 1) although a good general indication of comparability is evident, several elements (A1, As, B, Cr, Na, Se, Sn, V, Zn) are not covered at the high end of the concentration range whereas a larger number (As, B, Ca, Co, Cu, Fe, K, Mg, Mo, Na, Pb, Se, V, Zn) do not have coverage at low concentrations. With respect to the number of available BRM's with certified or recommended elemental concentrations, although fairly good coverage of a number of elements (39) and concentration ranges is evident, some 15 elements (AI, B, Cs, F, I, La, Li, N, Sb, Sc, Th, Sn, Sr, U, V) are reflected in only 1 - 4 reference materials. Both of these observations can guide effort towards the selection of required, but poorly-represented elements. This thrust is, however, tempered by the need for and possible shortage of highly competent analysts, the difficulties of good work at trace and ultratrace levels and methodologic deficiencies for specific elements. The three principal approaches for certification/quantitation are (1) in-house use of definitive and reference methods with supplementary input from cooperating outside expert laboratories, (2) selected "expert" cooperating laboratories working independently and using different methods, (3) volunteer cooperating laboratories (round robin). The

first of these approaches is preferred but is often impossible to put into practice due to the unavailability of expertise concentrated in one location. The second alternative is then a viable one as long as the selection process is geared to the choice of analytical chemists with the requisite expertise, using definitive, reference or validated methods of analysis. Whereas the round robin approach whereby a (random) selection of volunteer laboratories is amassed can produce usable information, it cannot be construed generally to lead to assigned values of the high quality demanded of RMs and is not advocated. Inherent in the selected approach is the need for use of proven methods of analysis for total analyte concentrations and the application of appropriate computational techniques for manipulation of the multilaboratory data. The subject of accurate estimation of analyte speciation as well as method-dependent measurements such as extractable elements in soils for reference purposes begs definition and solution. 7 Certification

Certification implies the assignment of a reliable, unassailable numerical value to a property of a material. It should be generally accepted that certification should be backed by legal mandate under the auspices of a national agency with demonstrated technical competence and an established credibility; information produced outside of such legally-authorized, technically-competent agencies should be denoted "recommended". Certification (or recommendation) deals with the assessment of analytical information, selection of data and computation and assignment of certified (recommended) values as well as associated uncertainties or confidence limits. Credibility of the certified information and of the product is of overwhelming importance and has an immense bearing on the value of the RM. Characterization approaches 1 and 2 mentioned above, comprising in-house capability or external expert cooperators using definitive, reference and validated methods of analysis can lead to a strong data base for certification. The definitive method mode, based on a sound theoretical foundation and thoroughly tested, provides results of high precision and essentially free of systematic error. Measurement by the selected laboratory - different method route makes use of two or more methods independent both in theory and experimental procedure, thoroughly validated, yielding highly precise data having small inaccuracies relative to end-use certification requirements. In both cases analyst training, expertise, competence and motivation are prerequisites. The premise behind use of different analytical methods is that independently-different techniques are not likely to experience identical systematic errors, and agreement of results from the two strengthens the belief that the data are good approximations of the true value. At least two, preferably three or more different techniques as well as overall procedures should be applied by as many analysts. Although only one definitive method can be applied, rigid certification criteria utilized by some agencies such as USNBS require that analyses performed by the definitive method are performed by two or more analysts working independently. Continual reassessment, reevaluation and updating of assigned values, as occurs with data generated by round robin approaches, should not be allowed for truly bona fide RMs for which the assigned numerical values of properties must stand the test of time.

572 A relevant consideration is which analytes to determine and certify. With regard to elemental analytes, a wide range of elements reflecting nutritionally, toxicologically and environmentally pertinent analytes are in demand, covering about one half of the periodic table. Increasing interest is being expressed in elements at ultratrace levels (gg kg-1) and also in the actual forms (speciation) in which these elements occur in the native state. Based on views expressed in the literature [4] and at conferences, it would seem advisable to certify new R M ' s for as many elements as possible. Thus not only would new matrices be made available but certified values would be available for a larger number of elements in addition to the small number of core elements typical of many current RM's. Reference to Table 4 indicates a fair number (15) of elements including N, an economically important major constituent, reflected in only 1 - 4 available RM's, which should be emphasized. Availability of suitable analytical methods and competent analysts will, however, play a role in certification possibilities. 8 Documentation Adequate documentation is required in respect of the numerical values assigned to the product, instructions on its use and information on its production and characterization. The documentation is typically a certificate issued by the certifying agency or a report of investigation whose sole authority is the author. Critically important information should be included in the certificate or report. Such information will define and describe the material, describe its preparation and certification, list numerical values for properties together with the associated uncertainties (as well as their definitions), stipulate minimum weight to be taken for analysis, indicate conditions of storage and include other details necessary for the analyst to correctly and fully utilize the material. In-depth treatment of all aspects of the development procedures should be available in other publications (e.g. NBS 260 series o f special publications and B C R and I A E A documents) in order for the analyst/user to fully appreciate the R M concept and apply it to the fullest benefit. 9 Distribution The production and analytical effort will be futile should the final product not find its way to the analyst. A distribution/ marketing strategy is indicated with all the related infrastructure of storage, pricing, marketing, handling and shipping, most effectively managed by a national or international standards agency. The material must be available in sufficient overall quantity and quantity per unit, and must be accessible. 10 Conclusions This summary of criteria for the development of B R M ' s is intended for the benefit of those who wish to augment the current shortage of materials and certified analytes. Material and information credibility is an overriding requirement. To maintain the requisite credibility of new products, throughout the overall task of R M development there is a requirement for a critical approach by critical, expert analytical and measurement scientists using proven definitive, reference and validated methods of analysis and the involvement of national R M agencies of acknowledged good re-

putation in order to produce top quality control materials. Such materials will continue to find acceptance by the community of analysts and lead to continual improvements in analytical information. References 1. Cali JP, Mears TW, Michaelis RE, Reed WP, Seward RW, Stanley CL, Yolken HT, Ku HH (1975) The role of standard reference materials in measurement systems, National Bureau of Standards Monograph 148, NBS, Washington, DC 2. Cantillo AY (1986) Standard and reference materials for use in marine science, National Oceanic and Atmospheric Administration, Rockville, MD, in press 3. Community Bureau of Reference (BCR) (Undated) BCR reference materials, Brussels 4. Dams R (1983) Pure Appl Chem 55:1957-1968 5. Food and Agriculture Organization of the United Nations (1981) FAO Production Yearbook Vol. 34, Rome 6. Huntoon RD (1975) In: Seward RW (ed) Standard reference materials and meaningful measurements, National Bureau of Standards Spec Publ 408, NBS, Washington, DC, pp 4 - 56 7. Ihnat M (1982) In: Cantle JE (ed) Atomic absorption spectrometry, Elsevier, Amsterdam, pp 139- 210 8. Ihnat M (1988) Sci Total Environ 71:85-103 9. Ihnat M (1988) In: McKenzie HA, Smythe LE (eds) Quantitative analysis of biological materials. Elsevier, Amsterdam, in press 10. Ihnat M (1988) Fresenius Z Anal Chem 332: 539-545 11. Ihnat M, Cloutier R, Wood D (1987) Fresenius Z Anal Chem 326: 627- 633 12. International Atomic Energy Agency (1988) Analytical Quality Control Services, Intercomparison Runs, Reference Materials 1988 : IAEA, Vienna 13. Mavrodineanu R, Gills TE (1985) Standard reference materials: summary of the coal, ore, mineral, rock and refactory standards issued by the National Bureau of Standards, National Bureau of Standards Spec Publ 260-97, NBS, Gaithersburg, MD 14. Muramatsu Y, Parr RM (1985) Survey of currently available reference materials for use in connection with the determination of trace elements in biological and environmental materials, IAEA/RL/128 :IAEA Vienna 15. Seward RW (ed) (1988) NBS standard reference materials catalogue 1988- 89, National Bureau of Standards Spec Publ 260, NBS, Gaithersburg, MD 16. Seward RW, Mavrodineanu R (1981) Standard reference materials: summary of the clinical laboratory standards issued by the National Bureau of Standards, National Bureau of Standards Spec Publ 260-71, NBS, Washington, DC 17. Statistics Canada (1981) Canadian Forestry Statistics 1979, Ottawa 18. Stoeppler M, Backhaus F, Mohl C, Schladot J-D (1988) 3rd International Symposium on Biological Reference Materials, May 4 - 7 , 1988, Bayreuth, FRG 19. Sturgeon RE, McLaren JW, Willie SN, Beauchemin D, Berman SS (1987) Can J Chem 65:961-964 20. Berman SS, Sturgeon RE (1988) Fresenius Z Anal Chem 332: 546- 548 21. Uriano GA, Gravatt CC (1977) CRC Crit Rev Anal Chem 6:361-411 22. Versieck J, Hoste L, De Kesel A, Van Renterghem D (1987) J Radioanal Nucl Chem 113 : 299 23. Versieck J, Vanballenberghe L, De Kesel A, Hoste J, Wallaeys B, Vandenhaute J, Baeck N, Steyaert H, Byrne AR, Sunderman FW Jr (1988) Anal Chim Acta 204:63-75 24. Wolf WR, Ihnat M (1985) In: Wolf WR (ed) Biological reference materials: Availability, uses and need for validation of nutrient measurement. John Wiley and Sons, New York, pp 89-105 Received July 29, 1988