Part II Analysis of Vitamins © 2006 by Taylor & Francis Group, LLC 16 Analytical Considerations 16.1 Bioassays Vitamins are physiologically active substances and a true assessment of the nutritional value of a food commodity or diet with respect to a particular vitamin can only be obtained by determining the vitamin’s biological activity The only direct means of determining biological activity is a bioassay based on a biological function Such assays were the means by which the vitamins were originally discovered and they are still in use today to measure the biopotency of novel vitamin analogs that may have potential therapeutic benefit A discussion of bioassay methodology is beyond the scope of this book, since such assays are in the province of specialized laboratories The result from a bioassay accounts for the combined in vivo response to all vitamin-active compounds and vitamin precursors present in the test sample The response may be either the overall growth response or a specific response, such as calcification of bone induced by vitamin D Bioassays based on biological function depend on a series of sequential physiological events: intestinal absorption, plasma transport, tissue uptake, metabolism, and biological function at the target cell Bioassays may be either curative or prophylactic In a typical bioassay of the curative type, rats or chicks are fed a formulated basal diet, which provides all their nutritional requirements except for the vitamin under assay When a decline in body weight or the appearance of a specific vitamin deficiency disorder is observed, the animals are divided into treatment groups and fed with either the test food sample or a standard preparation of the vitamin incorporated into the basal diet The extent to which the growth retardation or deficiency disorder has been cured is then estimated, and from this estimate the vitamin content of the test sample can be calculated In prophylactic assays, the animals receive the test sample and standard vitamin preparation at the beginning of the assay, and their ability to prevent the deficiency is compared In both types of assay, control animals receive only the basal diet throughout © 2006 by Taylor & Francis Group, LLC 311 312 Analytical Considerations Bioassays based on biological function can be of long duration and expensive Consequently, alternative bioassays have been developed, which are simpler to perform, more rapid, and less expensive Among the latter, assays based on elevated plasma vitamin levels take into account the efficiency of intestinal absorption and plasma transport Liver-storage assays go one step further because they depend on tissue uptake of the vitamin However, neither of these bioassays accounts for biological function at the cellular level and, therefore, they may not reflect the true physiological vitamin activity of the test sample 16.2 In Vitro Analytical Techniques Nowadays, vitamin determinations in foods are routinely performed using microbiological assays or physicochemical methods of analysis These, along with biospecific assays, can be described collectively as in vitro techniques Microbiological assays, like animal bioassays, measure the combined response of the active substances and take into account utilization at the cellular level Physicochemical assays permit the quantification of the principal substances that are responsible for the biological activity, and can achieve a high degree of precision The great drawback of in vitro analytical techniques is that they not account for the complexities of mammalian digestion and absorption and thus not provide a reliable estimate of vitamin bioavailability This is particularly the case for vitamins which are chemically bound with other constituents of the food matrix Since the mid-1970s up to the present, the method of choice for determining the fat-soluble vitamins in foods has been high-performance liquid chromatography (HPLC) This is due to the technique’s ability to separate the vitamins without the need for chemical derivatization, the nondestructive operation, and the detection selectivity HPLC can be used in the preparative mode to purify sample extracts, as well as in the quantitative mode Microbiological assays, developed in the early 1940s, remain the official (AOAC) methods for determining vitamin B6, pantothenic acid, folate, and vitamin B12 16.3 Analytical Approach The selection of a suitable analytical method depends on what needs to be measured to provide the required information to fulfil the objective For example, the chemist in the food manufacturing industry must © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 313 conform with the nutritional labeling regulations and achieve a consistent product without necessarily being concerned with bioavailability The main concern is how much of the vitamin that was added in fortification is actually present in the finished product In practice, this requirement is comparatively simple, as the vitamin in its parent form will be added as a stabilized preparation, which can be readily extracted from an analytical sample in a measurable quantity The fortified product should be reasonably consistent for every batch, and therefore, no unforeseen problems associated with a changing food matrix should be encountered once a suitable analytical method has been established The quality control manager will need a quick and reliable result on a regular basis, with minimum cost per analysis; therefore, the method should be simple and robust, automated as far as possible, and provide good repeatability (i.e., within-laboratory precision) The investigator who needs to determine the nutritional value of a food commodity or diet is faced with a more demanding analytical challenge Most of the vitamins occur naturally in trace amounts and in a variety of forms (vitamers), and some are chemically bound to proteins or carbohydrates in the food matrix Moreover, the matrix will vary enormously in composition if different diets are being studied For nutritional purposes, the accuracy of the method (i.e., nearness to truth) is more important than the precision 16.4 Preparation of Sample Extracts for Analysis Before a food sample can be analyzed, consideration must be given to obtaining a representative sample [1,2] In fortified foods, there is a tendency for the beadlets containing the vitamin to separate during mixing and handling, and then to agglomerate if exposed to moisture The amount of sample material to be taken for analysis depends on the inherent homogeneity of the material or the homogeneity achieved after comminution Most of the vitamins are photosensitive and, therefore, vitamin solutions must be protected from light throughout the analysis This can be achieved by artificial lighting provided by gold fluorescent tubes that exclude radiation wavelengths of less than 500 nm, and by the use of low-actinic amber glassware Several of the water-soluble vitamins are rapidly destroyed by alkali, and glassware should be acid-washed before rinsing to prevent inactivation of vitamins by surface films of alkaline detergents The general analytical procedure for microbiological and physicochemical assays can be broken down into five main stages: sampling, extraction, © 2006 by Taylor & Francis Group, LLC Analytical Considerations 314 cleanup, measurement, and calculation of results Other factors to be considered are maintenance of sample integrity, storage of the sample pending analysis, and preparation of the sample for analysis In analysis for carotenoids, some plants must be blanched to inactivate lipoxidase enzymes that otherwise would accelerate destruction of carotenes during storage and mixing 16.4.1 Extraction Often, the extraction step in a method is designed to liberate vitamin that is chemically bound, thus providing an assay value that represents the total amount of vitamin present For some vitamins, especially niacin, this approach can lead to a gross overestimation of the amount of biologically available vitamin in the food Some methods employ more selective extraction procedures, which simulate (as far as possible) mammalian digestion, in attempts to estimate the biologically available vitamin content 16.4.2 Cleanup The extracts prepared by treatment of the test material may require some form of cleanup before the vitamins are measured The requirement for cleanup depends upon the ratio of analyte to interfering substances, and also upon the sensitivity and selectivity of the analytical technique employed Colorimetric, titrimetric, and spectrophotometric techniques have rather poor specificity and require extensive sample cleanup Direct fluorometric techniques are rather more specific, while chromatographic techniques, particularly HPLC, have relatively high specificity Microbiological assays are subject to interference from many substances, although these can usually be diluted out owing to the high sensitivity of such assays Nonchromatographic cleanup techniques include deproteinization, sterol precipitation, solvent extraction, and dialysis Chromatographic techniques include open-column chromatography, solid-phase extraction, and high-pressure gel permeation chromatography 16.5 Method Evaluation 16.5.1 Measurement Value and Uncertainty All scientific measurements have an uncertainty associated with the measured value This uncertainty represents the total error of the analytical method, comprising systematic errors and random errors Systematic © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 315 errors are nonrandom and are due to bias Causes are exemplified by defects in the methodology, instrument malfunction, improper calibration, and the presence of interfering compounds These errors are usually constant for a given method and it is sometimes possible to reduce or eliminate them Random errors are unpredictable and cannot be eliminated Typical causes are sample inhomogeneity, incomplete extraction, and the inherent variability of the measurement system Random errors follow the normal distribution curve and hence can be expressed in units of standard deviation Precision is an estimate of the variability of a method and deals only with random errors Accuracy is the nearness of an individual measured value, or the arithmetic mean of a set of values, to the true, expected, or accepted value Statistically, accuracy represents the total error of the analytical method A measurement is accurate when the determined value is both precise and free of systematic error Precision is a statistically defined term, stated as a standard deviation of the mean, and can be ascertained by multiple analysis of an homogenous sample The relative standard deviation (RSD) and coefficient of variation (CV) are synonymous terms for standard deviation  100 divided by the mean The RSD allows the analyst to tell quickly if the measured value falls within the acceptable precision of the method, independent of the size of the value The precision depends on the concentration range of the analyte Under optimal conditions the standard deviation should increase linearly with the concentration, while the RSD should remain more or less constant When the RSD begins to increase rapidly with lower concentration or amount, it determines the limit of reliable measurement With regard to accuracy, it is impossible to determine with absolute certainty the true value of a nutrient in a natural food product Accuracy can be assessed by comparing the results with those obtained using a method that is different in principle to the method normally employed If two independent methods give numerically similar results, it is more probable that the measured values are near to the true value than that both methods have the same systematic error by chance If a certified standard reference material is available, whose compositional properties simulate those of the samples of interest, its “correct” measurement implies correct measurement of the samples The sample itself can serve as the matrix for recovery studies to ensure that at least the added analyte is recovered in a satisfactory amount Ideally, recovery experiments should be carried out using a zero reference material (i.e., a manufactured product matrix lacking the vitamin analyte), since such materials can be spiked with analyte at levels below those of the finished product Recovery data provide useful information regarding the completeness and precision of the extraction process, but the recovery © 2006 by Taylor & Francis Group, LLC Analytical Considerations 316 of exogenously added vitamin does not necessarily reflect the extraction efficiency of the indigenous vitamin The method of standard additions is an important technique for evaluating systematic error caused by proportional matrix bias [3] To perform this method, three or four identical aliquots of the sample extract are spiked with increasing known amounts of the pure analyte (aliquots from a standard solution), and each solution is analyzed The analytical response (e.g., HPLC peak height) is plotted as a function of the added amount of analyte The graph obtained represents the standard curve of the analyte in the presence of matrix, offset by the amount of endogenous analyte in the sample The linearity and slope of this curve, when compared to an equivalent standard curve in matrix-free solution, gives information about the presence or absence of matrix effects The method of standard additions also allows for correction for proportional error if such error is consistent over the entire calibration range (i.e., the curve is linear) 16.5.2 Quality Assurance Quality assurance is the broad management concept of maintaining the ability of a laboratory to furnish reliable information A quality assurance program encompasses the entire laboratory operation from sampling to application of the analytical result The analyst is usually confronted with the problem of having too few data with which to apply statistical analysis to a set of results, and hence other techniques for verifying how a method behaves in actual practice must be used A laboratory that routinely performs a particular determination should periodically analyze a working standard (house standard) along with routine samples, and monitor such analyses using a statistical control chart The working standard consists of a large amount of a homogeneous product such as nonfat dry milk or flour, which is dispensed into small sealed bottles and stored under conditions that inhibit deterioration In the absence of a suitable working standard, previously analyzed samples, on which several analyses have been performed, can be used instead 16.5.3 Food Reference Materials Food reference materials with certified contents of vitamins are needed to validate the analytical methods that are used to ensure compliance with nutrition labeling laws In 1989, a project funded by the European Commission’s Community Bureau of Reference (BCR) (in 1993 renamed as the Standard, Measurement and Testing programme) was initiated The main objective was to improve the reliability and accuracy of methods for the determination © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 317 of vitamins in food The project included research into methodology (extraction and cleanup, end-method of determination, and calibration), inter-laboratory comparison of the methods, and the preparation of homogeneous food reference materials having good stability properties Six reference materials were selected to cover the range of food matrices in which vitamins are commonly determined, namely, Brussels sprouts, mixed vegetables, wholemeal flour, milk powder, pig’s liver, and margarine With the exception of margarine, which is a canned product, these materials can be prepared in the form of dry powders The vitamin contents of the materials have been certified on the basis of acceptable statistical agreement of results produced by collaborating laboratories using technically valid analytical procedures Certified reference materials (CRMs) available in Europe are listed in Table 16.1 The preparation and certification of the six listed CRMs are described in two reports [4,5] The National Institute of Standards and Technology (NIST) in the United States is in the process of introducing standard reference materials (SRMs) for use in determining the nutritional contents of foods, and developing or adapting reliable methods with which to analyze these TABLE 16.1 European Certified Reference Materials (CRMs) with Certified Vitamin and Provitamin Constituents Material Certified Constituents a Wholemeal flour Margarine Milk powderb Lyophilized Brussels sprouts powder Lyophilized mixed vegetablesc Lyophilized pig’s liver a Designation Vitamin B1, vitamin B6, and folate Vitamin D3 and a-tocopherol Vitamin D3, a-tocopherol, vitamin B1, B2, B6, B12, niacin, folate, and vitamin C Niacin and vitamin C CRM 121 CRM 122 CRM 421 Vitamin B1, B6, folate, trans-a-carotene, total a-carotene, trans-b-carotene, and total b-carotene Vitamin B1, B2, B6, B12, and folate CRM 485 CRM 431 CRM 487 Commercially obtained wheat flour Spray-dried and vitamin enriched powder produced from cow’s milk c Sweet corn, carrot, and canned tomatoes Source: From Finglas, P.M., van den Berg, H., and de Froidmont-Go¨rtz, I., The certification of the mass fractions of vitamins in three reference materials: margarine (CRM 122), milk powder (CRM 421), and lyophilized Brussels sprouts powder (CRM 431), Report EUR 18039 EN, Office for Official Publications of the European Communities, Luxembourg, 1997 and Finglas, P.M., Scott, K.J., Wittho¨ft, C.M., van den Berg, H., and de Froidmont-Go¨rtz, I., The certification of the mass fractions of vitamins in four reference materials: wholemeal flour (CRM 121), milk powder (CRM 421), lyophilized mixed vegetables (CRM 485), and lyophilized pigs liver (CRM 487), Report EUR 18320 EN, Office for Official Publications of the European Communities, Luxembourg, 1998 With permission b © 2006 by Taylor & Francis Group, LLC Analytical Considerations 318 TABLE 16.2 Standard Reference Materials (SRMs) with Certified Vitamin and Provitamin Constituents Certified Constituents Material Coconut oil (fortified) Infant formula Baby food composite Slurried spinach DL -a-Tocopheryl acetate a-Tocopherol, vitamin C, vitamin B2, vitamin B6, and niacin all-trans-Retinol, a-tocopherol, g-tocopherol, d-tocopherol, total a-carotene, and total b-carotene Total b-carotene Certificate Issue Date Designation 28 February 2003 SRM 1563 14 January 2004 SRM 1846 26 July 2002 SRM 2383 December 2003 SRM 2385 materials Certified values for a given SRM are the equally weighted means of the measurements made by the NIST laboratory and the grand-mean of measurements made by collaborating laboratories The expanded uncertainties are expressed at the 95% level of confidence and include within- and between-laboratory uncertainties SRMs that have certified values for vitamins and provitamins are listed in Table 16.2 Reference values are noncertified values that are the best estimate of the true values based on available data 16.5.4 Method Validation Method validation is the process used to confirm that the analytical procedure is acceptable for its intended purpose A valid method is necessary, but not sufficient, for the production of reliable data; for instance, a degree of skill is required on the part of the analyst Data obtained by a valid method used in a well-defined quality assurance program should allow the assignment of limits of uncertainty that can be used to judge the reliability of the data First, the scope of the method and its validation criteria should be defined These include analytes, matrices, type of equipment, quantitation limits, precision, and accuracy The parameters for method validation are: Specificity: A specific method is one in which an analyte measurement is not subject to interference from other sample components Linearity: Linearity is achieved when results are directly or, after mathematical transformation, linearly proportional to analyte concentration within a given range © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 319 Precision: Precision is achieved when results of multiple analyses of an homogenous sample fall within the acceptance criteria for the method’s precision The relative standard deviation can be subdivided into three categories: repeatability, intermediate precision, and reproducibility Repeatability refers to the withinlaboratory precision and is determined from a set of replicate results obtained simultaneously or in quick succession by one operator using the same equipment throughout over a relatively short time span Intermediate precision is the long-term variability of the measurement process and is determined by comparing the results of a method run within one laboratory over a number of weeks A method’s intermediate precision may reflect discrepancies in results obtained by different analysts using different standards, reagents, HPLC columns, and so on Reproducibility refers to the between-laboratory precision and is determined from results obtained by a number of analysts in different laboratories The 95% confidence intervals of the repeatability and reproducibility for the expected assay concentration are useful information of the precision Accuracy: This is assessed by recovery experiments Range: The range of a method is the interval between the upper and lower analyte concentrations (inclusive) over which acceptable linearity, precision, and accuracy can be obtained Detection limit: The detection limit of a method is the lowest con- centration of analyte in a sample that can be detected (but not necessarily quantified) and is typically three times the noise level of the measurement system Quantification limit: This is the lowest concentration of analyte that can be precisely measured Robustness: The robustness of a method is its ability to remain unaffected by small changes in operational parameters, such as mobile-phase composition and flow-rate, column temperature, and injection volume in chromatographic systems The reproducibility of a candidate method is determined in an interlaboratory collaborative study using identical samples provided by the peer laboratory The ideal sample is a certified food reference material Such a study will reveal any bias in the method A method is considered to have been successfully validated when all participants in the study can obtain results comparable to those of the peer laboratory Statistical equivalence can be used to compare results; alternatively, the peer laboratory stipulates a range of values as a criterion of acceptability When the © 2006 by Taylor & Francis Group, LLC 320 Analytical Considerations candidate method has been validated and standardized by an organization such as AOAC International (AOAC) in the United States or the International Organization for Standardization (ISO), it becomes a standard test method References Lento, H.G., Sample preparation and its role in nutritional analysis, in Modern Methods of Food Analysis, Stewart, K.K and Whitaker, J.R., Eds., AVI, Westport, CT, 1984, p 71 Elkins, E.R and Dudek, J.A., Sampling for vitamin analyses, in Methods of Vitamin Assay, Augustin, J., Klein, B.P., Becker, D., and Venugopal, P.B., Eds., 4th ed., Wiley, New York, 1985, p 135 LaCroix, D.E., Wolf, W.R., and Chase, G.W., Jr., Determination of niacin in infant formula by solid-phase extraction/liquid chromatography: peer-verified method performance — interlaboratory validation, J AOAC Int., 85, 654, 2002 Finglas, P.M., van den Berg, H., and de Froidmont-Go¨rtz, I., The certification of the mass fractions of vitamins in three reference materials: margarine (CRM 122), milk powder (CRM 421), and lyophilized Brussels sprouts powder (CRM 431), Report EUR 18039 EN, Office for Official Publications of the European Communities, Luxembourg, 1997 Finglas, P.M., Scott, K.J., Wittho¨ft, C.M., van den Berg, H., and de FroidmontGo¨rtz, I., The certification of the mass fractions of vitamins in four reference materials: wholemeal flour (CRM 121), milk powder (CRM 421), lyophilized mixed vegetables (CRM 485), and lyophilized pigs liver (CRM 487), Report EUR 18320 EN, Office for Official Publications of the European Communities, Luxembourg, 1998 © 2006 by Taylor & Francis Group, LLC ...16 Analytical Considerations 16.1 Bioassays Vitamins are physiologically active substances and a true assessment... for determining vitamin B6, pantothenic acid, folate, and vitamin B12 16.3 Analytical Approach The selection of a suitable analytical method depends on what needs to be measured to provide the... general analytical procedure for microbiological and physicochemical assays can be broken down into five main stages: sampling, extraction, © 2006 by Taylor & Francis Group, LLC Analytical Considerations