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Availability of and needs for reliable analytical methods for the assay of foods

D. A. T. Southgate
Nutrition and Food Quality Division, Agriculture Research Council, Food Research Institute, Norwich, England

The usefulness of any data base rests on the quality of the data used to construct it, and in any discussion of the design of an international or national nutritional data base there is a natural inclination to focus attention on the analytical procedures used in the analysis of nutrients in foods. While these are undoubtedly important, there are other issues that also need to be considered. Many of these are discussed in other contributions, but it is essential to consider the twin issues of variation in the composition of foods and sampling and sample preparation alongside a review of analytical procedures, because these issues are highly relevant to the choice of analytical methods and the quality of the data that they produce.

The designer of a nutrient data base must also have a clear view of how the data base will be used, as this also influences sampling and impinges on the choice of analytical procedure.

THE USES OF A NUTRIENT DATA BASE

A data base of this kind is used in two ways; first for the translation of data on food, i.e., amounts supplied or amounts eaten, into nutrients in the food supply, nutrients ingested, and second the translation of values for nutrients as amounts required or amounts desired in a controlled diet, into foods.

Nutrient data bases have also been used as standards for food composition for statutory or regulatory purposes; however, I believe that this latter type of use is undesirable, and a data base constructed with this role in mind may well be incompatible with the requirements of nutritionists using the data base (1).

The two types of use are operated at several different levels and the capacity to accomodate these different levels is an essential attribute of a comprehensive data base. Table 1 summarizes the different levels of operation and their requirements in terms of data on foods.

To meet the requirements of these different levels, a comprehensive data base must include foods at the wholesale commodity level, or foods as purchased retail, and foods at the actual level of consumption. This requirement implies that a nutrient data base for foods will be very much more extensive than a comparable one for animal feeds.

Identity of items. The very large number of food items that might be included in such a data base will make the proper identification of an entry particularly important, and the descriptors used to define entries will need to be constructed so that unambiguous identification can be made. This will, in turn, impose very precise constraints on the identity of foods analyzed and the precise identity of foods where the compositional data are derived from the literature or other sources.

Variation in composition. Foods are biological materials and there is natural variation in composition. Some constituents show considerable variation, depending on the actual conditions of husbandry, storage, and preparation. Many organic nutrients in plants are profoundly influenced by the level of solar illumination, and many inorganic constituents are affected by the soil type and fertilizer treatment used in plant production. Water content is particularly variable, and this, and the lean-to-fat proportion in meats are probably the major variables affecting the composition of foods.

Superimposed on this "natural" variation are those introduced by processing and storage and the additions of substances during the production of foods. Even for proprietary products where close quality control is exercised, variations in composition are also seen that may be of the same order as those that arise naturally. The ingredients used in a product change from time to time and these affect the nutrient composition of the final food product, e.g., the oils used in margarine production.

Ideally, the data base should provide a measure of this variation, and if this is possible it will then be practicable to assign confidence limits to the calculations of nutrients supplied by a mixed diet or by the food supply. At the present time such measures of variation are too limited to perform these calculations, but calculations from Gambian data (T. J. Cole, unpublished) show that the predictive accuracy of such calculations is not very high. For nutrients where natural variation is high, such as folates, ascorbic acid, and many trace elements, the predictive accuracy of food compositional data is too low to be of quantitative value. It would be unfortunate if the existence of a nutritional data base led users to disregard the intrinsic uncertainty of calculations made from it. For some nutrients, food compositional data can only be a semi-quantitative guide (2).

TABLE 1. Levels of Operation of Nutrient Data Base

* The requirements of the different levels are not exclusive and often merge.

The issue of variations in composition have important implications for the construction of data bases. For example, if the data base gives compositional data for representative samples, how can variation be expressed in the data base? Ideally, the data base needs associated information bases giving the causes of variation.

A further cause of variation in the reported composition of foods lies in the analytical procedures used, and very frequently in the modes of expression used for the values reported. It is thus vital for the compilers of data bases to ensure that comparable analytical data are used and that the modes of expression for a particular nutrient are identical. In many instances one convention has been followed by one group of authors and another by other authors, for example, in the calculation of energy content. Often there are equally valid scientific reasons for choosing either convention, and, provided the choice made for the data base is clearly defined, few problems should arise in using it.

However experience with the use of data from McCance and Widdowson's The Composition of Foods (2) has shown that problems arise when users are not familiar with the conventions, and in some cases are unaware that they are using a convention at all.

SAMPLING

The major requirements for sampling of foods for food composition tables (1) are applicable to sampling for a data base. The primary aim is to draw representative samples for analysis and to describe the sample adequately. The essential specifications of samples are given in table 2.

The data base will require a range of sampling protocols in order to encompass foods at the "wholesale commodity" level, the "retail purchase" level, and the "as consumed" level. For the first two of these, the AOAC lays down the principles that should be followed.

CHOICE OF ANALYTICAL PROCEDURES

The primary objectives of the analyses are nutritional, and the most appropriate analytical procedure for the measurement of a nutrient is one that reflects the nutritional value of the food for man most closely. In many instances, nutritional analyses call for a more biochemically oriented approach than has been the rule in food analysis for legislative or quality control.

Laboratories that provide data for a data base have the responsibility to ensure that:

(a) appropriate analytical quality control procedures are used routinely, i.e., analysis of standards and reference materials and recovery tests;

(b) where different analysts are involved, the methods used give reproducible and consistent values within laboratories and between laboratories in collaborative analyses.

In the compilation of the data base it will be essential for the compilers to be fully conversant with the analytical procedures used, and full descriptions of the procedures used to obtain data will be necessary.

TABLE 2. Specification of Samples: Details of the Description of Samples. Ideally Required by Compilers of Food Composition Tables

* Whenever results are expressed on a basis other than fresh weight, details should be given so that the result' can be calculated back to this basis.

Choice of procedures. For many nutrients a number of procedures are available that give comparable results. Most recently developed procedures require the use of extensive instrumentation and therefore of capital investment; older procedures often give similar results but have been discontinued because of their time-consuming nature. It would be unwise, therefore, to restrict the approved procedures to those requiring expensive instrumentation, because this would exclude contributions from laboratories without this instrumentation and would discount the value of a large body of analytical information obtained by these equally valid, but simpler, methods.

For briefly reviewing available procedures, the nutrients have been grouped conventionally. The first section deals with established procedures and the second attempts to identify areas where the present methods have important limitations and where methodological studies are required. The assumption has been made that the data base will provide comprehensive coverage of nutrients-in practice, other constituents, contaminants, pesticide residues could be included-but these are not part of a nutritional data base (sensu stricto) and are outside my competence.

ESTABLISHED METHODS FOR MAJOR COMPONENTS

Wherever possible, alternative procedures are suggested with comments on their limitations. The procedures for the major components are summarized in table 3.

Water. Although not strictly a nutrient, because water is a major variable in foods, it is useful to give a value for this in any data base. Expression on a dry matter basis is not appropriate for a data base where foodstuffs will be measured in their normal states.

For most nutritional purposes the accuracy of the Karl Fischer types of measurements is not usually necessary, and conventional drying procedures are adequate. Near infrared reflectance procedures (NIR) provide significant advantages in speed of analysis where large numbers of samples of the same type are being analysed, but this has to be set against a high capital cost.

Total nitrogen. All methods are based on the Kjeldhal procedure and automation at as many levels of complexity as possible. Interference from inorganic nitrogen can occur with some procedures, but this is rarely of real quantitative nutritional significance.

Protein. Conventionally, protein is calculated from total N values using an appropriate factor (3), and where the nonprotein nitrogen is amino acid in origin, this approach is nutritionally sound. However, the calculation of protein in this way is a convention and does not measure protein in the biochemical sense Calculation from the nonprotein nitrogen (NPN) is intuitively preferable, but all procedures for separating NPN from protein N are empirical. The factor used in the calculation should ideally be derived from the amino acid composition, but this is frequently incomplete; for example, amide nitrogen is rarely quoted, and tryptophan values are frequently not available. The conventional factors are quite close approximations to values derived from amino acid values and, provided the nature of the convention is accepted, little real error is produced. Colorimetric and dye-binding procedures can be used for specific foods to give protein values, but colour yields and binding values are specific for particular proteins and proper calibration is essential.

TABLE 3. Procedures for Major Components of Foods

NIR gives good agreement and rapid analysts where large numbers of similar samples are being measured.

Fat. Values for total lipid are of questionable nutritional significance, but nevertheless are widely used. The choice of method is very important; some conventional continuous extraction procedures give low extraction. Mixed-solvent procedures are to be preferred, but some re-extraction of the initial extract may be necessary to eliminate non-lipid material.

Several automated systems are available, and these offer considerable advantages in speed of analysis with only moderate capital investment. NIR techniques are applicable to some foods.

Carbohydrates. Determination of carbohydrate by difference is nutritionally irrelevant, and these values have no place in a nutritional data base.

A variety of procedures for free sugars are available. Of these, specific enzymatic procedures and HPCL seem satisfactory from the point of view of specificity. Good resolution of sugar derivatives is also possible with gas chromatographic methods, but the derivatization process is time-consuming and the best procedures are based on the alditols and do not distinguish between glucose and fructose.

In the measurement of starch, acid hydrolysis is acceptable where small amounts of cell wall polysaccharides or other non-starch polysaccharides are present, especially if a glucose-specific assay is used. Enzymatic procedures offer specificity in the presence of non-starch glucans, but methodological studies are not quite complete (see below).

In the case of the cell wall polysaccharides and polysaccharide food additives falling within the definition of dietary fibre (4), methodological problems exist and further work is required. This will be discussed later.

COMPOSITION OF MAJOR COMPONENTS

Amino acid composition. This is most commonly measured by chromatographic procedures, liquid chromatography being the most established procedure, but both gas chromatographic and HPLC techniques are available. All these procedures require acid hydrolysis and the conditions of hydrolysis are critical-serial hydrolysis is necessary to establish release patterns and to estimate hydrolytic losses of labile amino acids. Sulphur-containing acids are best measured after oxidation and hydrolysis. Tryptophan can be measured calorimetrically or after alkaline hydrolysis. At the present time, collaborative trials have not yet established a preferred method.

Fatty acid composition. This is most conveniently measured by gas chromatography, usually of the methyl esters. This provides a convenient route for estimating triglyceride content.

The other lipid components, sterols and phospholipids, are of nutritional interest and the data base should be designed to include these components.

Carbohydrates. Estimation of the component sugars and polysaccharides is the best procedure for total carbohydrate, and it is envisaged that the data base should include a detailed breakdown of the various carbohydrate species present.

INORGANIC CONSTITUENTS

Most procedures for inorganic constituents require the preliminary destruction or removal of organic matter. This is most frequently performed by incineration (usually in a muffle furnace) to produce an ash residue that is then dissolved in acid prior to the measurement of the constituents. The sample is incinerated in a crucible, often of silica, but porcelain or platinum can be used. The temperature of incineration is critical, as losses of alkali metals occur at quite low temperatures (ca. 525 to 550"C) and at/or below those used in many procedures for measuring ash. Losses of trace constituents because of interactions with the crucible can occur, and analysed reference samples within a matrix are essential as quality control procedures.

The inorganic constituents are most conveniently measured instrumentally by atomic absorption spectrometry or emission techniques. Many trace inorganic constituents require electro-thermal (i.e., flameless) techniques. A variety of calorimetric procedures give comparable values, but these have generally been abandoned in favour of the instrumental procedures that require medium to high capital investment. Anions of nutritional interest ret quire individual procedures (table 5).

TABLE 4. Methods for Composition of Major Components

TABLE 5. Methods for Anions of Nutritional Interest

Oxidation of the organic matter with concentrated acids provides an alternative to dry ashing and may be more convenient where one or two constituents are to be measured.

Plasma emission techniques that require major capital investment in instrumentation are available that permit a large range of constituents to be measured simultaneously. The results obtained are profoundly influenced by the experimental conditions, and many workers have experienced working-up problems with these instruments. Values derived by this technique can be different from those obtained using conventional procedures, and care is needed in interpretation of the results by this procedure. It is, however, reasonably certain that, in laboratories with sufficient capital allocations, these techniques will be widely adopted once these initial problems are resolved.

VITAMINS

The range of procedures in use for the different vitamins is summarized in table 6. The more recently developed chromatographic procedures give greater specificity and are the methods of choice where there is more than one form of the vitamin. The older chemical procedures give comparable results but are distinctly more time-consuming. Microbiological assay is still the only practicable procedure for vitamin B₁₂ and, although HPLC separations of folates can be achieved, the sensitivity and specificity of the detectors is too low at present for these procedures to be applied generally to foods. Microbiological assay gives acceptable values for most of the B vitamins and is the better alternative where capital restraints preclude instrumental procedures. Radio-immune assay techniques may well provide the better approach in the future.

All the procedures for vitamins involve careful sample preparation and sometimes complex extraction procedures. In most cases these procedures represent the limiting stage in the through-put of an analytical laboratory; the question of sample preparation is raised below.

ORGANIC ACIDS

Few compilations of nutritional data include values for organic acids; however, in many plant foods and proprietary products such as soft drinks and yogurt, organic acids may make a major contribution to the energy content. Specific enzymatic methods are available and these are possibly the method of choice, although chromatographic procedures are also available.

ALCOHOL

Ethanol may be measured in alcoholic beverages by the traditional distillation procedures; however, sensitive gas chromatographic and enzymatic assays are available.

REQUIREMENTS IN RELATION TO ANALYTICAL PROCEDURES

In this section L would like to discuss the developments that appear to be necessary in relation to the provision of sound nutritional data. In the main, these relate to reducing the time spent in analysis because at the present time the resources required to generate nutritional data are limited (and are likely to remain so), and there are insufficient data on natural variation, which are vital to the question of accuracy in use of a data base.

Sampling and sample Preparation. This usually is the limiting factor in determining the rate at which analyses can be completed. Correct sampling procedures at all stages are absolutely essential, and it is difficult to see how the stage from food, as received, to material suitable for analysis can be greatly shortened. Some rationalization of extraction procedures is, however, possible, and if a few procedures could replace the many required at present, considerable savings in time could be achieved. Much sample preparation is carried out manually at present, and some attention to automating this is desirable.

Automation of procedures. This will need to be applied more extensively and this will create a considerable need for improved quality control. There is a need for analyzed reference materials that are stable and readily available for all the major types of food product for all laboratories who wish to use them. Standard materials are available for some trace elements, but the range is rather limited.

Dietary fibre (non-starch polysaccharides and lignin). A range of procedures has been suggested, but the ones that give estimates of the components (cellulosic, noncellulosic polysaccharides, and lignin) are preferable on nutritional grounds. These procedures also give the composition of the non-cellulosic fraction in terms of monosaccharide and uronic acids, which is useful in relation to studies of the physiological function of dietary fibre. Collaborative trials are in progress and it is anticipated that the bases for reference procedures will be established and tested soon.

Folates. Microbiological assay alones has the sensitivity for assaying many foods, and development of HPLC detector systems or on-line reactions is essential if detailed information on the different forms of folates in foods is to be obtained. Competitive protein-binding assays may also be possible for the routine assay of folates in foods.

CONCLUSIONS

Acceptable procedures are available for measuring most nutrients, but methodological studies need to be completed in relation to dietary fibre and folates. The major requirements relate to improved procedures for sample preparation, the wider use of automated procedures, and the associated need for reference foodstuffs.

The wider issues of the presentation of data on bioavailability of nutrients and other biological measurements of nutritional value have relevance in a comprehensive data base. For most nutrients, insufficient experimental data are available to provide numerical values suitable for use in a data base; furthermore, the complex interactions in diets and between diet and man still need to be resolved conceptually before such experimental work can be properly evaluated.

TABLE 6. Procedures for Vitamins

ACKNOWLEDGEMENTS

I would like to acknowledge the contribution of my colleagues at the Food Research Institute for their helpful suggestions and comments. Miss J. M. Penson also contributed to the task of assembling the paper.

REFERENCES

1. D.A.T. Southgate, Guidelines for the Preparation of National Tables of food Composition (Karger, Basle 1974).

2. A.A. Paul and D. A. T. Southgate, McCance and Widdowson's The Composition of Foods 4th ed. (Her Majesty's Stationery Office, London, U. K., 1978).

3. Energy and Protein Requirements, Report of a Joint FAO/ WHO Ad Hoc Expert Committee, Wld Hlth. Org. Techn. Rep. Ser. No. 522 (WHO, Geneva, 1973).

4. H. C. Trowell, D. A. T. Southgate, T. M. S. Wolever, A. R. Leeds, J. A. Gassull, and D. A. Jenkins, "Dietary Fibre Redefined," Lancet i: 967 (1976).

Additional General Bibliography for Food Tables and Food Analysis

Association of Official Analytical Chemists, Official Methods of Analysis. W. Horwitz (Ed.). (AOAC, Washington, D. C., 1980).

H. Egan, R. S. Kirk, and R. Sawyer, Pearson's Chemical Analysis of Foods (Churchill Livingstone, Edinburgh, 1981).

D. R. Osborne and P. Voogt, The Analysis of Nutrients in Food (Academic Press, New York and London, 1978).

Moisture

B. Makower and E. Nielsen, "Use of Lyophilisation in Determination of Mositure Content of Dehydrated Vegetables," Anal. Chem. 29 (9): 856 (1948).

S.B. Thung, "Comparative Moisture Determination in Dried Vegetables by Drying after Lyophilisation or by the Karl Fischer Method," J. Sci. Food Agric. 15: 236 (1964).

Total N2

D. B. Jones, "Factors for Converting Percentages of Nitrogen in Foods and Feeds into Percentages of Protein," U S Dept Agric. Cir. 183 (1941).

B. K. Watt and A. L. Merrill, Composition of Foods (U. S. Dept. Agric. Handbook No. 8, Washington, D. C., 1963).

Non-protein Nitrogen

G. I. Stekelenburg and J. Desplanque, "Deproteination by Ultra Filtration with Centrifugal Force, Techniques in Amino Acid Analysis" (Technican Instruments Co. Ltd., Chertsey, U. K. 1966).

Protein

O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, "Protein Measurement with the Follin Phenol Reagent," J. Biol. Chem. 193: 265 (1951).

J. S. Noll, D. H. Simmonds, and W. Gushuk, "A Modified Biuret Method for Determination of Protein," Cereal Chem. 51: 610 (1974)

D. C. Udy, "Improved Dye Method for Estimating Protein," J. Amer. Oil Chem. Soc. 48: 29A (1971).

Near infra-red Reflection

A. M. C. Davies, "Near Infra Red Analysis," Nutr. Food Sci. Sept./Oct. 5-6 (1982).

B. G. Osborne, "Principles and Practice of Near Infra-Red (NIR) Reflectance Analysis," J. Food Technol. 16: 13 (1981).

Total Fat

T. Atkinson, V. R. Fowler, G. A. Garton, and A, K. Lough, "A Rapid Method for the Accurate Determination of Lipid in Animal Tissues," Analyst 97: 562 (1972).

L. V. Cocks and C. van Rede, Laboratory Handbook for Oil and Fat Analysis (Academic Press, New York, 1966).

J. Folch, M. Lees, and G. H. S. Stanley, "A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues," J. Biol. Chem. 226:497 (1957).

D. A. T. Southgate and J. V. G. A. Durnin, "Calorie Conversion Factors. An Experimental Reassessment of the Factors Used in the Calculations of the Energy Value of Human Diets," Brit. J. Nutr. 24:517 (1970).

D. A. T. Southgate, "A Procedure for the Measurement of Fats in Foods," J. Sci. Food Agric. 22: 590 (1971).

Supers-HPLC

K. Aitzet Muller, "Sugar Analysis by High-Performance Liquid Chromatography Using Silica Columns," J. Chromatogr. 156: 354 (1978).

S. C. C. Bandad, M. L. Richmond, J. L. Gray, and C. M. Stine, "Separation of Mono- and Di-saccharides by High Performance Liquid Chromatography," J. Food Sci. 45: 1492 (1980) .

D. L. Dunmire and S. E. Otto, "High Pressure Liquid Chromatographic Determination of Sugars in Various Food Products," J. Assn Offic.. Anal. Chem. 62: 1 (1979).

M. L. Richmond, S. C. C. Bandad, J. I. Gray, P. Markskis, and C. M. Stine, "Analysis of Simple Sugars and Sorbitol in Fruit by High-Performence Liquid Chromatography," J. Agric food Chem. 29:4 (1981).

Starch

H. V. Bergmeyer, Methods of Enzymatic Analysis, 4 Vols. (Academic Press, New York, 1974).

K, M. Clegg, "The Application of the Anthrone Reagent to the Estimation of Starch in Cereals," J. Sci. Food Agric. 7: 40 (1956).

J. C. MacRae and D. G. Armstrong, "Enzyme Method for the Determination of Alpha Linked Glucose Polymers in Biological Materials" J. Sci. Food Agric. 19: 578 (1968).

Dietary Fibre

H. Englyst, H. S. Wiggins, and J. H. Cummings, "Determination of NonStarch Polysaccharides in Plant Foods by Gas-Liquid Chromatography of Constituent Sugars as Alditol-Acetates," Analyst 107: 307 (1982).

R. R. Selvendran and M. S. DuPont, "Problems Associated with the Analysis of Dietary Fibre and Some Recent Developments," in: Developments In Food Analysis Techniques, R. D. King (Ed.). (Applied Science, London, 1983).

Amino Acids

A. Khayat, P. K. Redenz, and L. A. Gorman, "Quantitative Determination of Amino Acids in Food by High Pressure Liquid Chromatography," Food Technol. 36: 46 (1982).

Fatty Acids, Phospholipids, Sterols

International Union of Pure and Applied Chemistry (IUPAC), Standard Methods for the Analysis of Oils, Fat, and Derivatives, 6th ed. (Pergamon Press, Oxford, 1979).

A. Pryde and M. T. Gilbert, Applications of High Performance Liquid Chromatography (Chapman and Hall, London, 1979),

S. Spanner, "Separation and Analysis of Phospholipids," in: Form and Function of Phospholipids, G. B. Ansell, R. M. C. Dawnson, and J. N. Hawthorne (Eds.), (Elsevier Scientific Publishing Co., Amsterdam/North Holland, 1973).

Phosphorus

C. H. Fiske and Y. Subbarow, "The Colorimetric Determination of Phosphorus," J. Biol. Chem. 66: 375 (1925).

Halides

H. J. M. Bowen, "The Determination of Chlorine, Biomine, and iodine in Biological Materiels by Education Analysis," Biochem. 1 73: 381 (1959).

E. Cotlove, H. V. Trantham, and R. L. Bowman, "An Instrument and Method, Rapid, Accurate and Sensitive for the Titration of Chloride in Biologic Samples," J. Lab. Clin. Mod 51: 461 (1958).

R. A. Durst, ea., Ion Selective Electrodes (Proceedings of e Symposium Held at the National Bureau of Standards, Gaithersburg, Maryland, Jan. 30-31, 1969).

T. S. Ma and S. S. M. Hassan, Organic Analysis Using lon-Selective Electrodes, Vols. l and 11 (Academic Press, London, New York 1982).

S. Mergregian, "Rapid Spectrophotometric Determination of Fluoride with Zirconium-Eriochrome Cyanine R. Lake. Anal. Chem. 26:1161 (1954).

L. Singer and W. D. Armstrong, "Determination of Fluoride in Blood Serum," Anal. Chem. 31: 105 (1959),

M. J. Follett and P. W. Ratcliff, "Determination of Nitrite and Nitrate in Meet Products," J. Sci. Food Agric. 14: 138 (1963),

J. D. Henshall, M. Ongley, and H. Hall, "The Analysis of Nitrates in Plant Material," Campden Food Preservation Research Association, Technical Memorandum No, 179 (1971).

M. Nakamura and A. Murata, "Spectro photometric Determination of Nitrite Using 4.5-Dihydroxycoumarin " Analyst 104: 985 (1979),

Vitamins

E.E. Snell, Microorganisms for Assay of Vitamins, "Physiol. Rev. 28: 255 (1948).

R.C. Williams, D.R. Baker, and E.l. Schmidt, "Analysis of Water-Soluble Vitamins by High-Speed lon-Exchange Chromatography," J. Chromotogr. Sci. 11: 618 (1973).

R.C. Williams, J.A. Schmit, and R.A. Henry, "Quantitative Analysis of the Fat-Soluble Vitamins by High-Speed Liquid Chromatography," J. Chromatogr. Sci. 10: 494 (1972).

Vitamin A

E. Brunius, "Assay of Vitamin A Oils," Nature (London) 181: 395 (1958).

F. H. Carr and E.A. Price "Colour Reactions Attributed to Vitamin A," Biochem. J. 20: 497 (1926).

J. W. Erdman, Jr., S-h. F. Mov, and P.A. La Chance, "Fluorimetric Determination of Vitamin A in Foods," J. Food Sci. 38: 447 (1973).

International Union of Pure and Applied Chemistry (IUPAC), The Assay of Vitamin A In Oils (Butterworth, London, 1959),

A.E. Sobel and H. Webbin, "Activated Glycerol Dichlorohydrin, A New Calorimetric Reagent for Vitamin A," Industr. Engin. Chem. (Analytical Edition) 18: 570 (1946).

G. Vastagh, "Die Berslimmung von Vitamin A, besonders in Arzneipräpareten 1 Teil," Arch. Pharm. (Weinheim) 279: 51 (1976).

Carotene

V. H. Booth, Carotene: Its Determination in Biological Materials (Hefter and Sons, Cambridge, U.K., 1957).

V. H. Booth, "The Extraction of Pigments from Plant Materials," Analyst 84: 464 (1959).

J. P. Sweeney and A.C. Marsh, "Separation of Carotene Stereoisomers in Vegetables," J. Assn Anal Chem. 53: 937 (1970).

Vitamin C

Association of Vitamin Chemists. Methods of Vitamin Assay, 3rd ed. (Interscience, Inc., New York, 1966).

M. J. Deutsch and L.E. Weeks, "Microfluorimetric Assay of Vitamin C," J. Assn Offic. Agric. Chem. 48: 1248 (1965).

I. H. Ror and C.A. Kuetner, "The Determination of Ascorbic Acid in Whole Blood and Urine through the 2,4-Dinitro-phenylhydrezine Derivative of Dehydro-ascorbic Acid," J. Biol. Chem. 147: 399 (1943).

E. Stahl, Thin Layer Chromatography. A Laboratory Handbook (Springer, Berlin, Academic Press, New York, 1965).

J. Vuilleumier, "Analytische Probleme bei der Bestimunung von vitaminen in Zusanimenhangmit Ernahrungserkebungen," Z Vitamin. Hormon, Fermentforsch, 37: 504 (1967).

Vitamin D

T. K. Murray, K.C. Day, and E. Kodicek, "The Differentiation and Assay of Vitamins D2 and D3 by Gas-Liquid Chromatography," Biochem. J. 98: 293 (1966).

R. A. Wiggins, "Separation of Vitamin D₂ and Vitamin D₃ by High-Pressure Liquid Chromatography," Chem. Industr. 20: 840 (1977),

P. W. Wilson, D.E.M. Lawson, and E. Kodicek, "Gas-Liquid Chromatography of Ergocalciferol and Chole Calaferolin Nanogram Quantities," J. Chromatogr. 39: 75 (1969).

B Vitamins

C. Y. Ang and F.A. Moseley, "Determination of Thiamin and Riboflavin in Meat and Meat Products by High-Pressure Liquid Chromatography," J. Agric. Food Chem. 28: 483 (1980).

Vitamin B₆

J. T. Vanderslice, K.K. Steward, and M.M. Varmas, "Liquid Chromatographic Separation and Quantification of B6 Vitamins and Their Metabolite, Pyridoxic Acid," J. Chromatogr. 176: 280 (1979).

F. F. Wong, "Analyses of Vitamin B6 of Food Materials by High Performance Liquid Chromatography," J. Agric. Food Chem. 26: 1444 (1978).

Vitamin B₁₂

M. J. Thompson, L.S. Dietrich, and C.A. Elvehjem, "The Use of Lactobacillus leichmaninii in the Estimation of Vitamin B12 Activity," J. Biol. Chem. 184: 175 (1950).

P. J. Casey, K.R . Speckmen, F.J. Ebert, and W. E. Hobbs, "Radioisotope Dilution Technique for Determination of Vitamin B12 in Foods," J. Assn Offic. Anal. Chem. 65: 85 (1982).

Tocopherol (Vitamin E)

C. H. McMurray and W.J. Blanchflower, "Determination of Alpha" Tocopherol in Animal Feedstuffs Using High-Performance Liquid Chromatography with Spectro-fluoresence Detection," J. Chromatogr. 176: 488 (1979).

C. H. McMurray, W.J. Blanchflower, and D.A. Rice, "Influences of Extraction Techniques on Determination of Alpha-Tocopherol in Animal Feedstuff," J. Assn Offic. Anal. Chem. 63: 1258 (1980).

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