17 Extraction Techniques for the Water-Soluble Vitamins In vitro analytical techniques require prior extraction of the vitamins from the food matrix in order to facilitate their measurement The appropriate method of extraction depends upon the following criteria: the analytical information required, the nature of the food matrix, the form in which the vitamin occurs naturally or is added (different bound forms of vitamins are often found in meat, plant, and dairy products), the nature and relative amounts of potentially interfering substances, the stability of the vitamin towards heat and extremes of pH, and the selectivity and specificity of the analytical method to be used Extraction procedures for water-soluble vitamins include hydrolysis of the sample with a mineral acid [hydrochloric acid (HCl) or sulfuric acid (H2SO4)], alkaline hydrolysis with calcium hydroxide, deproteinization with trichloroacetic acid or similarly acting agent, and digestion with an appropriate enzyme 17.1 Vitamin B1 The extraction procedure generally used for the determination of total vitamin B1 by fluorometry, GC, HPLC, and microbiological assay involves hot mineral acid digestion to release the thiamin and thiamin phosphate esters from their association with proteins, followed by enzymatic hydrolysis of the phosphate esters to complete the liberation of thiamin Food samples of animal origin can be autoclaved at 1218C for 30 with 0.1 N HCl, as the phosphorylated forms of thiamin present in such samples are not degraded under these conditions For the majority of cereals and cereal products, which contain mostly nonphosphorylated thiamin, it is necessary to lower the autoclaving temperature to 1088C in order to avoid vitamin loss A commercial diastatic enzyme preparation of fungal origin (e.g., Takadiastase, Claradiastase, or Mylase) is suitable for the hydrolysis step, as such preparations contain phosphatase activity in addition to © 2006 by Taylor & Francis Group, LLC 321 322 Extraction Techniques for the Water-Soluble Vitamins a-amylase and other enzymes [1] The enzyme treatment can be omitted for the analysis of those grain products that not contain phosphorylated thiamin For proteinaceous samples such as meat, the proteolytic enzyme papain is sometimes added to the diastase in order to dissolve the proteins that have been denatured during the previous acid digestion Instead of using an enzyme hydrolysis procedure for thiamin extraction prior to HPLC, rice flour samples can be refluxed with a mixture of hydrochloric acid and methanol (0.1 N HCl –40% aqueous methanol) for 30 at 608C [2] For the analysis of milk, the extraction procedure simply entails precipitation of the protein by acidification at room temperature, and filtration This nonhydrolytic extraction procedure has the advantage of leaving the biologically inactive thiamin monophosphate intact, so this compound can be excluded from the measurement 17.2 Vitamin B2 When carrying out physicochemical or microbiological assays for vitamin B2, it is necessary to release the flavins from their intimate association with proteins and to completely convert the FAD to FMN Both of these requirements are readily accomplished (for noncovalently bound flavins) by autoclaving food samples at 1218C for 30 with dilute mineral acid (usually 0.1 N HCl) at a pH of ,3 During acid digestion some of the FMN is hydrolyzed to riboflavin, and a small fraction of the FMN is converted to the isomeric 20 -, 30 -, and 40 -phosphates [3] The complete conversion of FMN to riboflavin can only be achieved by subsequent enzymatic hydrolysis, for which a standardized diastatic enzyme preparation such as Takadiastase or Claradiastase is used Watada and Tran [4] reported that Mylase was as effective as Takadiastase, the latter being unobtainable at that time These are relatively inexpensive and crude preparations that contain varying degrees of phosphatase activity In practice, the complete enzymatic conversion of FMN to riboflavin may not always be achieved, the degree of hydrolysis depending on the source and batch-to-batch phosphatase activity of the enzyme and on the incubation conditions For the analysis of milk, eggs, and dairy products, it is common practice to determine the riboflavin specifically, on the assumption that free or loosely bound riboflavin is the predominant naturally occurring flavin present In this case, the extraction procedure simply entails precipitation of the protein by acidification and filtration, omitting the acid and enzyme digestion steps Rashid and Potts [5] removed the protein from milk and milk products by filtration after treatment with acidified lead acetate solution © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 323 Acid and enzymatic hydrolysis carried out successively are incapable of liberating the covalently bound FAD of certain enzymes, and hence this source of FAD will not be measured This is perhaps fortuitous when the nutritional value of the food sample is under assessment, as there is evidence that covalently bound FAD is largely unavailable to the host 17.3 Niacin In order to assess the nutritional value of a foodstuff with respect to its niacin content, it is necessary to determine the niacin that is biologically available As discussed in Section 9.5.1, the majority of the niacin in mature cereal grains exists in chemically bound forms of nicotinic acid that are not biologically available Therefore, measurement of total niacin (i.e., free plus bound) provides a gross overestimate of the biologically available niacin of several staple cereal-based foods The terms “total” and “free” (bioavailable) niacin are defined by the extraction methods employed in the analysis Total niacin generally refers to the niacin that is extractable by autoclaving the sample with alkali or N mineral acid; free niacin is frequently defined as the niacin extractable by heating or autoclaving with 0.1 N mineral acid In the AOAC colorimetric method for determining total niacin [6], noncereal foods are extracted by autoclaving for 30 at 1218C in the presence of N (0.5 M) H2SO4 This same procedure is used in the AOAC microbiological method for determining niacin in milk-based infant formulas [7] The acid treatment liberates nicotinamide from its coenzyme forms and simultaneously hydrolyzes it to nicotinic acid; it does not, however, completely liberate the bound nicotinic acid from cereal products A procedure that has been used for extracting total niacin from cereal products is autoclaving at 1218C for h in the presence of 0.22 M calcium hydroxide [8,9] This alkali treatment readily liberates the nicotinic acid from its chemically bound forms; it also converts nicotinamide to nicotinic acid, but with a yield lower than 80% Sodium hydroxide, although more effective at hydrolyzing nicotinamide, is not used because it induces gelation of the cereal sample If the microbiological assay with Lactobacillus plantarum or the AOAC colorimetric assay are to be used, complete conversion of nicotinamide to nicotinic acid is not necessary, as these procedures account for both vitamers Autoclaving meat samples with N HCl in the presence of urea resulted in a significant increase in the niacin content when compared with extraction using N acid alone [10] This suggests the release of © 2006 by Taylor & Francis Group, LLC 324 Extraction Techniques for the Water-Soluble Vitamins niacin from nonester conjugates by the acid – urea combination, possibly from amide-linked forms Windahl et al [11] found that autoclaving food samples at 1218C for 30 in the presence of N H2SO4 did not completely hydrolyze nicotinamide to nicotinic acid These authors ensured complete hydrolysis by autoclaving samples in the presence of 1.6 N (0.8 M) H2SO4 for h at 1218C They also performed alkaline extraction by autoclaving samples in the presence of saturated calcium hydroxide for h at 1218C Acid and alkali extractions gave similar levels of niacin in foods as determined by capillary electrophoresis and HPLC In meat samples, acid extraction resulted in slightly higher niacin values compared with alkali extraction Conversely, in cereal samples, alkali extraction yielded slightly higher values compared with acid extraction Among the many published HPLC methods for determining niacin in foods, several have used extraction procedures designed to yield a value for bioavailable niacin Lahe´ly et al [12] added 0.1 N HCl to ground food samples and heated the suspensions in a water-bath at 1008C for h A portion of the diluted and filtered digest was then autoclaved at 1208C in a medium of 0.8 N NaOH for h to ensure complete conversion of nicotinamide to nicotinic acid Thus, ultimately, only nicotinic acid needed to be measured chromatographically The application of this method to beef liver and yeast gave comparable niacin values to those obtained when simulating gastric digestion conditions (0.1 N HCl hydrolysis at 378C for h, followed by an alkaline treatment) However, when the method was applied to cereal products, the alkaline treatment induced the formation of impurities, which interfered with the chromatography Rose-Sallin et al [13] found that a one-step acid hydrolysis (0.1 N HCl, h, 1008C water-bath) yielded similar concentrations of niacin to those following two-step acid-alkaline or acid-enzymatic hydrolysis in a range of fortified foods, including cereal products The one-step procedure also yielded slightly better recoveries for niacin compared to the two-step methods Rose-Sallin et al [13] adopted the one-step extraction and calculated bioavailable niacin from the nicotinic acid and nicotinamide peaks in the chromatogram Vidal-Valverde and Reche [14] found that treatment of acid hydrolysates with Takadiastase was absolutely necessary in the case of legume samples, because the high starch content made the hydrolysate extremely viscous Ndaw et al [15] replaced the usual 0.1 N acid extraction by enzymatic hydrolysis, using a NADase that hydrolyses only the bound forms of niacin clearly bioavailable (i.e., NAD and NADP) This enzymatic hydrolysis (incubation at 378C for 18 h) did not induce any subsequent conversion of nicotinamide into nicotinic acid The one-step enzymatic treatment was always sufficient, even when the foodstuff contained large quantities of starch (rice, wheat flour) or proteins (wheat germ, © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 325 TABLE 17.1 Influence of the Extraction Protocol on the Niacin Concentration in Various Foodstuffs as Determined by HPLC with Fluorometric Detection Food Peas Spinach French beans Sweet corn Rice Wheat flour Wheat germ Peanuts Yeast Beef fillet Pork escalope Extraction Protocola Nicotinic Acid (mg/g) Nicotinamide (mg/g) Niacin (mg/g of Nicotinic Acid Equivalents) NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl NADase 0.1 N HCl 0.29 (0.01) 1.22 (0.08) 0 0.19 (0.01) 0.37 (0.06) 3.6 (0.2) 4.3 (0.4) 10.3 (0.2) 10.0 (0.5) 3.4 (0.1) 5.7 (0.4) 10.8 (0.2) 13.8 (0.5) 26.5 (0.9) 93.4 (0.7) 17 (1) 22.0 (0.3) 3.8 (0.2) 3.6 (0.7) 0.2 (0.1) 11.0 (0.1) 10.2 (0.4) 0.72 (0.06) 0.69 (0.04) 2.8 (0.2) 2.6 (0.2) 13.8 (1.0) 12.7 (0.9) 0 1.7 (0.1) 1.9 (0.1) 0 3.7 (0.3) 1.9 (0.3) 182 (5) 174 (5) 53 (1) 50 (2) 64 (2) 57 (1) 11.3 (0.1) 11.4 (0.4) 0.72 (0.06) 0.69 (0.04) 3.0 (0.2) 3.0 (0.2) 17.4 (1.0) 17.0 (1.0) 10.3 (0.2) 10.0 (0.5) 5.2 (0.1) 7.6 (0.4) 10.8 (0.2) 13.8 (0.5) 30.2 (1.0) 95.8 (0.8) 199 (5) 196 (5) 57 (1) 54 (2) 64 (2) 58 (1) Note: Concentrations are averages of three determinations (standard deviations in parentheses) a (1) NADase (pH 4.5, 18 h, 378C); (2) 0.1 N HCl (water-bath at 1008C during h) Source: From Ndaw, S et al Food Chem, 78, 129–134, 2002 With permission from Elsevier peanuts, beef fillet) Table 17.1 compares the niacin contents of various foods extracted either by NADase treatment or acid hydrolysis (0.1 N HCl, h, 1008C water-bath) Acid hydrolysis led to significantly higher niacin contents in the analysis of wheat flour, wheat germ, and peanuts, attributable to the release of nicotinic acid from bound forms that are probably nonbioavailable On analysis of peas, French beans, and yeast (foods in which nicotinamide is by far the major vitamer), nicotinic acid contents were slightly higher after acid hydrolysis than they were after enzymatic hydrolysis This increase most probably resulted from a partial conversion of the nicotinamide to nicotinic acid When the acid hydrolysis was applied to standard solutions of NAD (1.35 mM) and NADP (1.17 mM), about 10% of the nicotinamide liberated was converted to nicotinic acid © 2006 by Taylor & Francis Group, LLC 326 Extraction Techniques for the Water-Soluble Vitamins For the determination of added nicotinic acid as a color fixative in fresh meat (illegal in Japan), meat samples have been extracted by boiling with 96% ethanol [16] and blending with water [17 –19], acetonitrile [20], methanol [21], or methanol after addition of a small amount of phosphoric acid [22] 17.4 Vitamin B6 Because animal and plant tissues differ greatly with respect to the forms of vitamin B6 contained in them, there is no single set of conditions that can quantitatively extract vitamin B6 from both plant and animal products In the AOAC microbiological method [23] for determining total vitamin B6 in food extracts, animal-derived foods are autoclaved with 0.055 N HCl for h at 1218C This treatment hydrolyzes phosphorylated forms of vitamin B6, whilst also liberating PL from its Schiff base and substituted aldamine bound forms Plant-derived foods are autoclaved with 0.44 N HCl for h at 1218C, the stronger acid environment being necessary to liberate PN from its glycosylated form Autoclaving whole-wheat samples with 0.055 N HCl, instead of 0.44 N HCl, yielded a similar PL value, but lower values of PN and PM [24] Conversely, autoclaving meat products with 0.44 N HCl, instead of 0.055 N HCl, gave approximately the same PN and PL values, but only about half of the PM [25] The superiority of the lower concentration of acid used for animal products does not result from destruction of vitamin B6 by the stronger acid; rather, it is due to the incomplete liberation of the vitamin by the more concentrated acid [26] The optimum release occurs between pH 1.5 and 2.0, with a maximum at pH 1.7 –1.8 [27] To satisfy these strict pH criteria, one must always ensure that the acid is added in amounts that exceed the buffering capacity of the sample Another factor to consider is that PMP is more resistant to acid hydrolysis than is PLP Autoclaving for h at 1258C in 0.055 N HCl was required for complete hydrolysis of PMP, while PLP was completely hydrolyzed in 30 under the same conditions [28] The possibility of interaction of PL or PLP with amino acids during the AOAC extraction procedure for animal foods has been investigated [29] No loss of activity for Saccharomyces cerevisiae was observed when PL or PLP was autoclaved in the presence of a relatively high concentration of glutamic acid, which indicated that transamination does not occur under these conditions PN-glucoside exhibits around 60% bioavailability relative to PN in humans [30] Since the AOAC extraction procedure for plant foods hydrolyzes glycosylated forms of PN, analyses based on this procedure © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 327 would overestimate the biologically available vitamin B6 in foods that contain significant quantities of b-glucoside conjugates The AOAC acid hydrolysis procedures have no effect upon the peptidebound 1-pyridoxyllysine and its 50 -phosphate derivative, which are formed during the heat-sterilization of evaporated milk and other animal-derived canned foods (Section 10.3.2) These conjugates, which possess anti-vitamin B6 activity under certain conditions, exhibit 75 –80% stability when subjected to N HCl at 1058C for 48 h [31] Bogna˚r and Ollilainen [32] investigated the use of hydrochloric acid and trichloroacetic acid alone, and in combination with several commercial enzyme preparations, as extractants for the determination of total vitamin B6 in food by HPLC Three reference materials were tested: CRM 121 (wholemeal flour), CRM 485 (lyophilized mixed vegetables) and CRM 487 (lyophilized pig liver) Also included in the investigation were broccoli, Brussels sprouts, kidney beans, spelt (a kind of wheat), potatoes, sunflower seeds, pork meat, cod, and milk The highest values of total vitamin B6 were achieved by autoclaving samples at 1208C for 30 in 0.1 N HCl, followed by incubation with acid phosphatase and b-glucosidase at 378C for 18 h after adjustment to pH 4.8 Enzymatic hydrolysis of food by Takadiastase, degraded PL distinctly and also produced a compound that interfered with the PN peak during gradient elution The content of glycosylated PN could be determined by analyzing the acid hydrolysate before and after the double enzyme treatment The difference in PN content before and after enzyme treatment gives an estimate of glycosylated PN The simultaneous separation of all six B6 vitamers, plus pyridoxic acid, can be achieved using HPLC Treatment of samples with deproteinizing agents such as metaphosphoric, perchloric, trichloroacetic, or sulfosalicylic acid at ambient temperature readily hydrolyzes Schiff bases, whilst preserving the phosphorylated vitamers These acids also preserve PN-glucoside, and hence their use provides better estimates of available vitamin B6 than the use of mineral acids The high efficiency of extraction using these acidic reagents is partly due to the conversion of the pyridine bases to quaternary ammonium salts, thereby increasing their solubility in water Their use as extracting agents also prevents enzymatic interconversion of B6 vitamers during homogenization of samples In such procedures it is usually necessary to remove excess reagent, which might otherwise interfere with the analytical chromatography Trichloroacetic acid can be removed by extraction with diethyl ether; perchloric acid by reaction with M potassium hydroxide and precipitation as insoluble potassium perchlorate; and sulfosalicylic acid by chromatography on an anion exchange column [33] An extraction procedure using 5% sulfosalicylic acid has been successfully applied to such complex foods as pork, dry milk, and cereals [34] Recoveries of B6 vitamers added to © 2006 by Taylor & Francis Group, LLC 328 Extraction Techniques for the Water-Soluble Vitamins samples were 95– 105% for all vitamers except for PNP, where the recovery was 85% Other workers [35,36] have found perchloric acid to be a better extracting agent of the B6 vitamers for animal tissues than sulfosalicylic acid 17.5 Pantothenic Acid Before pantothenic acid can be determined by methods other than an animal bioassay, it is necessary to liberate the vitamin from its bound forms, chiefly coenzyme A Neither acid nor alkaline hydrolysis can be used, as the pantothenic acid is degraded by such treatments The only practicable alternative is enzymatic hydrolysis, and this was successfully accomplished through the simultaneous action of intestinal phosphatase and an avian liver enzyme [37] This double enzyme combination liberates practically all of the pantothenic acid from coenzyme A, but it does not release the vitamin from acyl carrier protein [38] The phosphatase splits the coenzyme A molecule between the phosphate-containing moiety and pantethiene, while the liver enzyme breaks the link in pantethiene between the pantothenic acid and b-mercaptoethylamine moieties The double enzyme combination is used in the AOAC microbiological method for determining pantothenic acid in milk-based infant formula [39] 17.6 Biotin Bound forms of biotin, including biocytin, cannot be utilized by L plantarum, the organism usually employed in microbiological biotin assays, and strong mineral acid hydrolysis at elevated temperature is required to liberate biotin completely from natural materials [40] Animal tissues require more stringent hydrolysis conditions than plant tissues, because the latter contain a higher proportion of free water-extractable biotin [41] Experimental studies with meat and meat products [42] and feedstuffs of animal origin [43] showed that maximum liberation of biotin in animal-derived products is obtained by autoclaving with N H2SO4 for h at 1218C This procedure promotes losses of biotin in plant materials, which are extracted more efficiently by autoclaving with N H2SO4 for h at 1218C [41] or with N H2SO4 for h at 1218C [43] Because of the differences in extractability between animal and plant tissues, a single acid extraction procedure to cover all food commodities must be a compromise, and no such procedure has © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 329 been universally adopted Representative methods for extracting foods of any type entail autoclaving with N H2SO4 at 1218C for h [41,44] or N H2SO4 at 1218C for h [45] or 30 [46] Hydrolysis with N H2SO4 destroys the synthetic sodium salt of biotin added to feed premixes A suggested procedure for extracting feed premixes with biotin potencies up to g/lb entailed the addition of 50 ml of 0.1 N NaOH and 250 ml of water to g of sample, shaking vigorously, and then standing for 30 at room temperature with occasional swirling [47] Sulfuric acid, rather than hydrochloric acid, is invariably used for sample hydrolysis, as the biotin content of dilute (30 ng/ml) solutions is almost completely destroyed by autoclaving with N HCl [48] Evidence from differential microbiological assay points to the oxidation of biotin to a mixture of its sulfoxide and sulfone derivatives, possibly caused by trace impurities (e.g., chlorine) in the acid This loss of vitamin activity does not necessarily occur when autoclaving actual food samples, as many natural products are capable of preventing this oxidation [49] Finglas et al [50] reported no loss of biotin from liver using N HCl It is evident from the foregoing that sulfuric acid hydrolysis is an unreliable way of extracting biotin from food The results depend on both the concentration of acid and the duration of autoclaving This makes the microbiological assay of biotin problematic, since acid hydrolysis is used to convert biocytin to biotin A proposed HPLC method [51] solves the problems associated with acids by eliminating acid extraction Instead, food samples are digested with papain for 18 h, a treatment that releases biotin from its association with proteins, but leaves biocytin intact There is no degradation of biotin during the digestion at 378C Biotin and biocytin are measured separately after postcolumn conversion to fluorescent derivatives The addition of Takadiastase is necessary for starchy foods such as cereals and yeast 17.7 Folate The AOAC microbiological method for determining folic acid in infant formula [52] employs a single-enzyme digestion with folate conjugase (pteroylpoly-g-glutamyl hydrolase; EC 3.4.22.12) The chicken pancreas conjugase specified in the method converts folylpolyglutamates to diglutamates, which can be utilized by the assay organism, L rhamnosus HPLC methods for determining folate require deconjugation of folylpolyglutamates to monoglutamates, and therefore chicken pancreas conjugase is unsuitable Conjugases from hog kidney and human or rat plasma yield folylmonoglutamates and can be used in HPLC and other © 2006 by Taylor & Francis Group, LLC 330 Extraction Techniques for the Water-Soluble Vitamins nonmicrobiological methods Chicken pancreas conjugase is most active at neutral pH, in contrast to hog kidney conjugase and plasma (human or rat) conjugase whose pH optimum is 4.5 [53] The various conjugases are not commercially available in purified form and enzyme solutions have to be prepared in the laboratory from their crude sources, such as lyophilized human or rat plasma and hog kidney acetone powder In 1990, DeSouza and Eitenmiller [54] reported that increased folate levels could be obtained in microbiological and radioassays by including protease (EC 3.4.24.31) and a-amylase (3.2.1.1) with the conjugase treatment Martin et al [55] then published a tri-enzyme digestion procedure using chicken pancreas conjugase, a-amylase, and protease in the microbiological determination of total folate in foods This was followed by reports from other laboratories advocating tri-enzyme treatment as a means of extracting the maximum possible amount of folate from foods as diverse as cereal-grain products [56], American fast foods [57], dairy products [58], foods commonly consumed in Korea [59], and complete food composites [60] Folate values in of 16 fortified bakery products, and of 13 fortified products in the rice, macaroni, and noodle category were significantly higher following the additional protease and a-amylase treatments [61] In order to achieve maximum extraction of bound folate from the food matrix, food samples suspended in buffered aqueous medium are first autoclaved to break up particles, gelatinize starch, and denature folatebinding proteins and enzymes that may catalyze folate degradation or interconversion The inclusion of an antioxidant is essential in preventing the destruction of labile folates during heat treatment The most effective reducing conditions are provided by the presence of both ascorbic acid and mercaptoethanol, with the air displaced by nitrogen The autoclaved samples are digested with protease to liberate the folate bound to proteins, then heated to inactivate the protease Digestion with a-amylase then follows to liberate the folate bound to starch Prolonged digestion with conjugase completes the tri-enzyme treatment A variety of foods cause detectable inhibition of conjugase activity [62], but this problem can be partly overcome by extracting at near neutral pH using a large excess of conjugase [63] In 2000, the American Association of Cereal Chemists (AACC) [64] published a microbiological assay using tri-enzyme extraction for the determination of total folate in cereal products The extraction procedure is as follows: Weigh an amount of ground sample equal to 0.25 – 1.0 g dry solids and containing about mg folic acid into a 125-ml conical flask Add 20 ml 0.1 M phosphate buffer (pH 7.8) containing 1% ascorbic acid, mix thoroughly, then add enough water to bring the total volume to 50 ml Add 0.1– 1.0 ml octanol (antifoaming agent), cover flasks with 50-ml © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 331 beakers, and autoclave for 15 at 121 – 1238C Cool and add a further 10 ml of the pH 7.8 buffer Add ml protease solution, cover the flask, and incubate for h at 378C Autoclave for at 1008C, then cool Add ml a-amylase solution and incubate for h at 378C Add ml chicken pancreas conjugase solution and incubate for 16 h (or overnight) at 378C Inactivate the enzymes by autoclaving for min, then cool Adjust to pH 4.5, dilute to 100 ml with water, and filter approximately 20 ml through 2V filter paper Dilute an aliquot of the clear filtrate with 0.1 M phosphate buffer (pH 6.7 + 0.1) to a final volume such that the folate concentration is about 0.2 ng/ml, and assay microbiologically using L rhamnosus Rader et al [61] tested the efficiency of the tri-enzyme extraction using chicken pancreas conjugase at four different pHs (pH 4.3, 6.0, 6.8, and 7.8) for the microbiological assay of four cereal-grain products and found no significant differences among folate values A pH 7.8 buffer is used in the AACC method, but this pH is not optimal for the tri-enzyme extraction of all food types Tamura et al [60], for example, found that complex food composites were extracted more efficiently at pH 4.1 than at pH 6.3 or 7.85 The pH optima and incubation times for protease and a-amylase can vary, depending on the substrates present in the foods [65], and this creates a dilemma in deciding which conditions should be used for the tri-enzyme treatment 17.8 Vitamin B12 Procedures for extracting vitamin B12 generally have the dual purpose of liberating protein-bound cobalamins and converting the labile naturally occurring forms to a single, stable form — cyanocobalamin or sulfitocobalamin Conversion to the sulfitocobalamin by reaction with metabisulfite avoids the use of lethally toxic cyanide solutions required to form cyanocobalamin The extraction procedure employed in the AOAC microbiological method for determining vitamin B12 activity in vitamin preparations [66] is also applicable to foods, having been found satisfactory by interlaboratory collaborative analysis of a crude liver paste, condensed fish solubles, and a crude vitamin B12 fermentation product [67] The procedure entails homogenizing the sample with 0.1 M phosphate – citrate buffer at pH 4.5 containing freshly prepared sodium metabisulfite (Na2S2O5), and then autoclaving the mixture for 10 at 1218C The heat treatment denatures the proteins, inactivates the enzymes, and accelerates the conversion of liberated cobalamins to sulfitocobalamin © 2006 by Taylor & Francis Group, LLC 332 Extraction Techniques for the Water-Soluble Vitamins In the AOAC method for determining vitamin B12 in milk-based infant formula [68], protein is removed by filtration after adjustment of the autoclaved extract to the point of maximum precipitation (ca pH 4.5) Methods, in which the sample is heated on a boiling water-bath, rather than autoclaved, may not completely extract all of the bound vitamin [69] 17.9 Vitamin C An effective means of extracting vitamin C from foods is homogenization with a solution of 3% (w/v) metaphosphoric acid dissolved in 8% glacial acetic acid [70] This extracting solution denatures and precipitates proteins (thereby inactivating all enzymes) and provides a medium below pH 4, which favors the stability of ascorbic acid and dehydroacorbic acid Furthermore, metaphosphoric acid prevents catalysis of the oxidation of ascorbic acid by copper(II) or iron(III) ions [71] Addition of ethanol or acetone to the metaphosphoric extract precipitates solubilized starch [72] Dilute (5 or 6%) metaphosphoric acid forms precipitates when mixed with certain ion-pairing reagents [73], which warrants caution in its use for ion-pair chromatography It is recommended to deoxygenate extracting solutions by bubbling an inert gas (e.g., oxygenfree nitrogen) through the solution before use Krall and Andersen [74] extracted fruits and vegetables with an aqueous solution of 1% (w/v) metaphosphoric acid and 0.5% (w/v) oxalic acid adjusted to pH Homogenization of high-starch food samples with aqueous 2% metaphosphoric acid and 1% oxalic acid mixed : with ethanol resulted in precipitation of starch Use of these extractants was compatible with the reversed-phase ion-pair HPLC system Bogna´r and Daood [75] compared two solvent systems, A and B, for their effect on the stability of vitamin C derivatives in standard solutions and spiked extracts of fruits and vegetables Solvent A was the classic solution of 3% metaphosphoric acid in 8% acetic acid; solvent B was solvent A mixed with acetonitrile (1 : 2) In solvent A, dehydroascorbic acid was unstable in standard solution: only 64 and 40% of the initial concentration was retained after and 24 h of ambient storage, respectively In contrast, the corresponding retentions in solvent B were 100 and 98% In fruit and vegetable extracts prepared in solvent A, 36 –54% of the spiked quantity of dehydroascorbic acid was lost after 12 h of storage time These decreases in dehydroascorbic acid were accompanied by remarkable increases in ascorbic acid concentration (29 – 53%), most probably due to the presence of reducing agents in the extracts of fruits and vegetables There was little or no loss of dehydroascorbic acid added to © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 333 food extracts prepared in solvent B After a 24-h storage, ascorbic acid was highly stable in all of the standard solutions and food extracts tested Taking these findings into account, Bogna´r and Daood [75] added acetonitrile to the standard solutions or to food extracts immediately after preparation or extraction with 3% metaphosphoric acid in 8% acetic acid This modification of the extraction procedure resulted in complete recoveries of dehydroascorbic acid and total vitamin C In an interlaboratory study [76], fruit juices and processed foods were extracted by diluting or blending with water, then immediately adding dithiothreitol to reduce dehydroascorbic acid to ascorbic acid, thereby stabilizing the vitamin C Proteinaceous samples were treated with 5% trichloroacetic acid to precipitate the proteins References Defibaugh, P.W., Evaluation of selected enzymes for thiamine determination, J Assoc Off Anal Chem., 70, 514, 1987 Ohta, H., Maeda, M., Nogata, Y., Yoza, K.-I., Takeda, Y., and Osajima, Y., A simple determination of thiamine in rice (Oryza sativa) by high-performance liquid chromatography with post-column derivatization, J Liq Chromatogr., 16, 2617, 1993 Nielsen, P., Rauschenbach, P., and Bacher, A., Preparation, properties, and separation by high-performance liquid chromatography of riboflavin phosphates, Meth Enzymol., 122G, 209, 1986 Watada, A.E and Tran, T.T., A sensitive high-performance liquid chromatography method for analyzing riboflavin in fresh fruits and vegetables, J Liq Chromatogr., 8, 1651, 1985 Rashid, I and Potts, D., Riboflavin determination in milk, J Food Sci., 45, 744, 1980 AOAC official method 961.14 Niacin and niacinamide in drugs, foods, and feeds Colorimetric method Final action 1962, in Official Methods of Analysis of AOAC International, Indyk, H and Konings, E., Eds., 17th ed., AOAC International, Gaithersburg, MD, 2000, p 45-12 AOAC official method 985.34 Niacin and niacinamide (nicotinic acid and nicotinamide) in ready-to-feed milk-based infant formula Microbiological-turbidimetric method Final action 1988, in Official Methods of Analysis of AOAC International, Phifer, E., Ed., 17th ed., AOAC International, Gaithersburg, MD, 2000, p 50-21 Tyler, T.A and Shrago, R.R., Determination of niacin in cereal samples by HPLC, J Liq Chromatogr., 3, 269, 1980 van Niekerk, P.J., Smit, S.C.C., Strydom, E.S.P., and Armbruster, G., Comparison of a high-performance liquid chromatographic and microbiological method for the determination of niacin in foods, J Agric Food Chem., 32, 304, 1984 © 2006 by Taylor & Francis Group, LLC 334 Extraction Techniques for the Water-Soluble Vitamins 10 Roy, R.B and Merten, J.J., Evaluation of urea-acid system as medium of extraction for the B-group vitamins Part II Simplified semi-automated chemical analysis for niacin and niacinamide in cereal products, J Assoc Off Anal Chem., 66, 291, 1983 11 Windahl, K.L., Trenerry, V.C., and Ward, C.M., The determination of niacin in selected foods by capillary electrophoresis and high performance liquid chromatography: acid extraction, Food Chem., 65, 263, 1998 12 Lahe´ly, S., Bergaentzle´, M., and Hasselmann, C., Fluorimetric determination of niacin in foods by high-performance liquid chromatography with postcolumn derivatization, Food Chem., 65, 129, 1999 13 Rose-Sallin, C., Blake, C.L., Genoud, D., and Tagliaferri, E.G., Comparison of microbiological and HPLC — fluorescence detection methods for determination of niacin in fortified food products, Food Chem., 73, 473, 2001 14 Vidal-Valverde, C and Reche, A., Determination of available niacin in legumes and meat by high-performance liquid chromatography, J Agric Food Chem., 39, 116, 1991 15 Ndaw, S., Bergaentzle´, M., 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Harris, R.S., Eds., 2nd ed., Vol II, Academic Press, New York, 1968, pp 294 50 Finglas, P.M., Faulks, R.M., and Morgan, M.R.A., The analysis of biotin in liver using a protein-binding assay, J Micronutr Anal., 2, 247, 1986 51 Lahe´ly, S., Ndaw, S., Arella, F., and Hassellmann, C., Determination of biotin in foods by high-performance liquid chromatography with post-column derivatization and fluorimetric detection, Food Chem., 65, 253, 1999 52 AOAC official method 992.05 Folic acid (pteroylglutamic acid) in infant formula Microbiological methods Final action 1995, in Official Methods of Analysis of AOAC International, Phifer, E., Ed., 17th ed., AOAC International, Gaithersburg, MD, 2000, p 50-21 53 Gregory, J.F., III, Chemical and nutritional aspects of folate research: analytical procedures, methods of folate synthesis, stability, and bioavailability of dietary folates, Adv Food Nutr Res., 33, 1, 1989 54 De Souza, S and Eitenmiller, R., Effects of different enzyme treatments on extraction of total folate from various foods prior to microbiological assay and radioassay, J Micronutr Anal., 7, 37, 1990 55 Martin, J.I., Landen, W.O., Jr., Soliman, A.-G.M., and Eitenmiller, R.R., Application of a tri-enzyme extraction for total folate determination in foods, J Assoc Off Anal Chem., 73, 805, 1990 56 Pfeiffer, C.M., Rogers, L.M., and Gregory, J.F., III, Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography, J Agric Food Chem., 45, 407, 1997 57 Johnston, K.E., Lofgren, P.A., and Tamura, T., Folate concentrations of fast foods measured by trienzyme extraction method, Food Res Int., 35, 565, 2002 58 Johnston, K.E., DiRienzo, D.B., and Tamura, T., Folate content of dairy products measured by microbiological assay with trienzyme treatment, J Food Sci., 67, 817, 2001 59 Yon, M and Hyun, T.H., Folate content of foods commonly consumed in Korea measured after trienzyme extraction, Nutr Res., 23, 735, 2003 60 Tamura, T., Mizuno, Y., Johnston, K.E., and Jacob, R.A., Food folate assay with protease, a-amylase, and folate conjugase treatments, J Agric Food Chem., 45, 135, 1997 © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 337 61 Rader, J.I., Weaver, C.M., and Angyal, G., Use of a microbiological assay with tri-enzyme extraction for measurement of pre-fortification levels of folates in enriched cereal-grain products, Food Chem., 62, 451, 1998 62 Engelhardt, R and Gregory, J.F., III, Adequacy of enzymatic deconjugation in quantification of folate in foods, J Agric Food Chem., 38, 154, 1990 63 Goli, D.M and Vanderslice, J.T., Investigation of the conjugase treatment procedure in the microbiological assay of folate, Food Chem., 43, 57, 1992 64 AACC method 86-47 Total folate in cereal products — microbiological assay using trienzyme extraction, in Approved Methods of the American Association of Cereal Chemists, 10th ed., Vol 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determination of vitamin B12 in foods, J Assoc Off Anal Chem., 65, 85, 1982 70 International Organization for Standardization Fruits, vegetables and derived products Determination of ascorbic acid Part 1: reference method International Standard ISO 6557/1, 1986 71 Ponting, J.D., Extraction of ascorbic acid from plant materials, Ind Eng Chem Anal Edu., 15, 389, 1943 72 Eitenmiller, R.R and Landen, W.O., Jr., Vitamin Analysis for the Health and Food Sciences, CRC Press, Boca Raton, FL, 1999, pp 223 73 Liau, L.S., Lee, B.L., New, A.L., and Ong, C.N., Determination of plasma ascorbic acid by high-performance liquid chromatography with ultraviolet and electrochemical detection, J Chromatogr., Biomed Appl., 612, 63, 1993 74 Kall, M.A and Andersen, C., Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid, and dehydroisoascorbic acid in food and biological samples, J Chromatogr B., 730, 101, 1999 75 Bogna´r, A and Daood, H.G., Simple in-line postcolumn oxidation and derivatization for the simultaneous analysis of ascorbic and dehydroascorbic acids in foods, J Chromatogr Sci., 38, 162, 2000 76 Brause, A.R., Woollard, D.C., and Indyk, H.E., Determination of total vitamin C in fruit juices and related products by liquid chromatography: interlaboratory study, J AOAC Int., 86, 367, 2003 © 2006 by Taylor & Francis Group, LLC ...322 Extraction Techniques for the Water-Soluble Vitamins a-amylase and other enzymes [1] The enzyme treatment can be omitted for the analysis of those grain products... Taylor & Francis Group, LLC 328 Extraction Techniques for the Water-Soluble Vitamins samples were 95– 105% for all vitamers except for PNP, where the recovery was 85% Other workers [35,36] have found... Extraction Techniques for the Water-Soluble Vitamins In the AOAC method for determining vitamin B12 in milk-based infant formula [68], protein is removed by filtration after adjustment of the autoclaved