Chapter 17 extraction techniques for the water soluble vitamins

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 hyd

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 vita-mins 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 trichloro-acetic 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 B1by 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 min 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

a-amylase and other enzymes [1] The enzyme treatment can be omitted for the analysis of those grain products that do not contain phosphory-lated 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 hydro-chloric acid and methanol (0.1 N HCl –40% aqueous methanol) for 30 min

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 min 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 Takadias-tase, the latter being unobtainable at that time These are relatively inexpensive and crude preparations that contain varying degrees of phos-phatase 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

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 biologi-cally 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 1 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 min at 1218C in the presence of 1 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 1 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 nico-tinamide 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 microbiologi-cal 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 1 N HCl in the presence of urea resulted in a significant increase in the niacin content when compared with extraction using 1 N acid alone [10] This suggests the release of

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 min in the presence of 1 N H2SO4did not completely hydrolyze nico-tinamide to nicotinic acid These authors ensured complete hydrolysis

by autoclaving samples in the presence of 1.6 N (0.8 M) H2SO4for 2 h at 1218C They also performed alkaline extraction by autoclaving samples

in the presence of saturated calcium hydroxide for 2 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 1 h A portion of the diluted and filtered digest was then auto-claved at 1208C in a medium of 0.8 N NaOH for 1 h to ensure complete conversion of nicotinamide to nicotinic acid Thus, ultimately, only nic-otinic 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 3 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 chromato-graphy Rose-Sallin et al [13] found that a one-step acid hydrolysis (0.1 N HCl, 1 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 extrac-tion and calculated bioavailable niacin from the nicotinic acid and nicoti-namide 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,

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, 1 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

TABLE 17.1

Influence of the Extraction Protocol on the Niacin Concentration in Various Foodstuffs as Determined by HPLC with Fluorometric Detection

Food

Extraction Protocola

Nicotinic Acid (mg/g) Nicotinamide(mg/g)

Niacin (mg/g of Nicotinic Acid Equivalents)

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 1 h).

Source: From Ndaw, S et al Food Chem, 78, 129–134, 2002 With permission from Elsevier.

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 B6contained in them, there is no single set of conditions that can quantitatively extract vitamin B6from both plant and animal pro-ducts 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 5 h at 1218C This treatment hydrolyzes phosphorylated forms of vitamin B6, whilst also liberating PL from its Schiff base and sub-stituted aldamine bound forms Plant-derived foods are autoclaved with 0.44 N HCl for 2 h at 1218C, the stronger acid environment being necess-ary 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 approxi-mately 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 pro-ducts 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 3 h at 1258C in 0.055 N HCl was required for complete hydrolysis of PMP, while PLP was completely hydrolyzed in 30 min 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 hydro-lyzes glycosylated forms of PN, analyses based on this procedure

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 peptide-bound 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 B6activity under certain conditions, exhibit

75 –80% stability when subjected to 6 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 commer-cial 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 B6were achieved by autoclaving samples at 1208C for

30 min 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 pro-duced a compound that interfered with the PN peak during gradient elution The content of glycosylated PN could be determined by analyz-ing 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 B6vitamers, plus pyridoxic acid, can be achieved using HPLC Treatment of samples with deproteinizing agents such as metaphosphoric, perchloric, trichloroacetic, or sulfosa-licylic 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 B6than 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 intercon-version 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 Trichloro-acetic acid can be removed by extraction with diethyl ether; perchloric acid

by reaction with 6 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% sulfo-salicylic acid has been successfully applied to such complex foods as pork, dry milk, and cereals [34] Recoveries of B6 vitamers added to

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 liber-ates 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 micro-biological 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 do 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 6 N H2SO4for 2 h at 1218C This procedure promotes losses of biotin in plant materials, which are extracted more efficiently

by autoclaving with 4 N H2SO4for 1 h at 1218C [41] or with 2 N H2SO4 for 2 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

been universally adopted Representative methods for extracting foods of any type entail autoclaving with 2 N H2SO4at 1218C for 2 h [41,44] or 3 N

H2SO4at 1218C for 1 h [45] or 30 min [46]

Hydrolysis with 6 N H2SO4destroys the synthetic sodium salt of biotin added to feed premixes A suggested procedure for extracting feed premixes with biotin potencies up to 1 g/lb entailed the addition of

50 ml of 0.1 N NaOH and 250 ml of water to 5 g of sample, shaking vigor-ously, and then standing for 30 min 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 2 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 3 N HCl

It is evident from the foregoing that sulfuric acid hydrolysis is an unre-liable 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 diglu-tamates, which can be utilized by the assay organism, L rhamnosus HPLC methods for determining folate require deconjugation of folylpoly-glutamates to monofolylpoly-glutamates, and therefore chicken pancreas conjugase

is unsuitable Conjugases from hog kidney and human or rat plasma

do yield folylmonoglutamates and can be used in HPLC and other

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 treat-ment Martin et al [55] then published a tri-enzyme digestion procedure using chicken pancreas conjugase, a-amylase, and protease in the micro-biological 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 8 of 16 fortified bakery products, and 4 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 folate-binding 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 pro-teins, 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 1 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