Vitamin alkaline hydrolysis

Retinoids The challenge in fat-soluble vitamins analysis is to separate them from the lipid fraction that contains interferents. Alkaline hydrolysis, followed by LLE, is widely applied to remove triglycerides. This technique converts the vitamin A ester to all-trani-retinol. A milder process, which does not hydrolyze vitamin A ester, is alcoholysis carried out with metha-nolic KOH solution under specific conditions that favor alcoholysis rather than saponification. A more accurate explanation of this technique is reported in the book Food Analysis by FIPLC [409]. For some kind of matrices a simple liquid extraction can be sufficient with [421-423] or without [424,425] the purification... 

Methods of extracting the fat-soluble vitamin from food matrices include alkaline hydrolysis, enzymatic hydrolysis, alcoholysis, direct solvent extraction, and supercritical fluid extraction of the total lipid component. 

Vitamin B3 Vitamin B3 (niacin) has different physiologically active forms, nicotinic acid, nicotinamide, and their coenzymes, which are very stable at ambient temperature. Usually acid or alkaline hydrolysis are used to convert nicotinamide to nicotinic acid for quantitation of both vitamers as nicotinic acid the first treatment (acid) is used to quantitate biologically available niacin, while alkaline hydrolysis provides an estimate of the total niacin content. Nicotinic acid has been analyzed using ion exchange or RP chromatography with amino columns. Detection is performed with UV absorbance at 254 nm. 

In the biosphere, triesters of orthophosphoric acid are unknown, however, this does not exclude the possibility that this binding may be present in certain macromolecules. The diesters of orthophosphoric acid which exist in the biosphere are often mixed esters. Acid or alkaline hydrolysis slowly transforms them into monoesters. Most of the complex lipides are diesters and vitamin also falls into this category. The mono-phosphoric esters of alcohols form a very important biochemical group. The two free acid groups are more strongly acidic than when they were... 

Niacin refers to a group of compoimds also known as vitamin B3, presenting similar biological activity, including nicotinamide, nicotinic acid, as well as other pyridine nucleotide structures. In the body, these compounds act as cofactors in oxidation—reduction reactions. To determine the total vitamin B3 content, either an acid or alkaline hydrolysis is necessary. The separation is normally performed by RPLC with fluorescence (322 nm ex., 380 nm em.) or UV detection (254 nm). 

Hot saponification [1,3,77—79] is the most effective tool for removing the majority of fatty material, hydrolyzing ester linkages of glycerides, phospholipids, esterified sterols, and carotenols. Moreover, alkaline hydrolysis frees boimd forms of vitamirrs (for instance, esterified and protein-bound forms) and degrades chlorophylls in water-soluble products. Also gelatine of the vitamin premix, added to supplemented... 

Food sample pretreatment may consist of either (a) saponification to quantify the free forms (retinol or xanthophylls may occur free or ester-ified in foods) [95,96] or (b) direct extraction to determine the unaltered A vitamers [84,88]. Alkaline hydrolysis is also an expedient to simplify the vitamin A analysis, since retinol is the only form to be quantified nevertheless, due to its sensitivity to light and oxygen, it is important to prevent photo-oxidation by inclusion of a antioxidant (ascorbic acid, hydroquinone, or pyrogallol). A drawback of hot saponification is the generation of artifacts, such as geometric isomers of retinol and carotenoids [97]. 

The extraction of vitamin D from fatty foods necessitates alkaline hydrolysis [1,3,85]. Thermal isomerization of vitamin D to previtamin D during hot saponification entails losses of 10—20% making its quantification difficult. Overnight cold saponification (prolonged digestion at... 

The addition of potassium cyanide to the acid (77) and subsequent acid (not, as before, alkaline) hydrolysis, led to a mixture of the cis- and transisomers from which the pure trans-isomer, forming the trans-diester (80) on esterification, was isolated by crystallization. Reduction of the keto group, Dieckmann cyclization of the hydroxydiester formed, and decarboxylation led to the Z-trans-C/D ketol (79). Resolution of the racemic diacid corresponding to the diester (80) by crystallizing its brucine salts and performing the reactions described above with the optical isomers obtained enabled the tZ-enantiomer of the ketol (79) to be obtained [898], this being identical with the product formed in the oxidation of vitamin D2 [899]. This enantiomer has also been used as the CD fragment in the synthesis of vitamin D (Scheme 90). 

Pantothenate in blood and tissues is bound (R9) and released by autolysis or hydrolysis. More vitamin could be released by use of an alkaline phosphatase and an enzyme from avian liver (L6). This method liberates pantothenate from coenzyme A in a variety of foods and tissues (N3, N4). A comparison of hydrolytic methods in blood suggested autolysis to be the most advantageous method (N3) in our hands, treatment with Clarase gave more reliable results as compared with autolysis, acid hydrolysis, treatment with Mylase P, or combination of Clarase and papain, or liver enzyme and alkaline phosphatase. In urine, pantothenic acid is unbound our results show no increase with Clarase treatment. The vitamin has presumably a low threshold. Pantothenic acid shows the same concentration in blood and cerebrospinal fluid. 

Enzymatic hydrolysis is a nondestructive alternative to saponification for removing triglycerides in vitamin K determinations. For the simultaneous determination of vitamins A, D, E, and K in milk- and soy-based infant formulas and dairy products fortified with these vitamins (81), an amount of sample containing approximately 3.5-4.0 g of fat was digested for 1 h with lipase at 37°C and at pH 7.7. This treatment effectively hydrolyzed the glycerides, but only partially converted retinyl palmitate and a-tocopheryl acetate to their alcohol forms vitamin D and phyllo-quinone were unaffected. The hydrolysate was made alkaline in order to precipitate the fatty acids as soaps and then diluted with ethanol and extracted with pentane. A final water wash yielded an organic phase containing primarily the fat-soluble vitamins and cholesterol. 

An improved route to 2a-hydroxycholesterol has been devised as part of the preparation of 2a-hydroxy-vitamin D3 (263 R1 = R4 = R5 = R6 = H, R2 = R3 = OH).123 Hydroxylation of the A bond of cholesta-l,5-dien-3/3-ol by means of 9-borabicyclo[3,3,l]nonane followed by reaction with alkaline hydrogen peroxide produced the 2-equatorial 2a,3a-diol in 70—80% yield. The conventional four-step sequence, acetylation, bromination, dehydrobromination, and hydrolysis, gave 2a -hydroxycholesta-5,7-dien-3/3-ol which was converted into 2a-hydroxy-vitamin D3. The isomeric 2/3-hydroxy-vitamin D3 has also been reported.124 Reaction of the 1/6,2/3-oxide obtained by peroxidation of the adduct (265) with lithium aluminium hydride results in a mixture of 2/3,3/3-dihydroxycholest-5,7-diene and its 1/3,3/3-dihydroxy-epimer in the ratio 8 1. Irradiation of the former 5,7-diene furnished the expected previtamin, which on equilibration gave 2/3-hydroxy-vitamin D3 (263 R1 = R4 = R5 = R6 = H, R2 = a-OH, R3 = OH). 

Extraction of Active Compounds from Food. Vitamins are the group of compounds more usually extracted from foods using SFE (83). A method for the analysis of the natural contents of vitamins A and E in milk powder based on SFE, a miniaturized alkaline saponification procedure, and HPLC was proposed by Turner and Mathiasson (84). Modifications of the sample matrix, the combination of static and dynamic extraction modes, and the effect of changes in extraction parameters such as temperature, flow rate, time, collection solvent, and collection temperature were optimized, obtaining recoveries of 99% and 96% for vitamins A and E, respectively. Another method for the determination of vitamins A and E based on the coupling of SFE-enzymic hydrolysis-HPLC has also been proposed providing recoveries between 79% and 152% (85). 

Daniel, H., Binninger, E., and Rehner, G., 1983. Hydrolysis of FMN and FAD by alkaline phosphatase if the intestinal brush-border membrane. International Journal of Vitamin and Nutrition Research. 53 109-114. 

In the case of milk samples the problem occurs that the K vitamins, together with triglycerides, are encapsulated by membranes. By breaking up these membranes, one should avoid strong alkaline media. In the chromatographic determination, following lipase hydrolysis, one has to prepurify samples of an excess of coextracted lipid material. 

Vitamin B5 occurs in three biologically active forms in foods [1] pantothenic acid, coenzyme A (CoA), and acyl carrier protein (ACP). Calcium or sodium pantothenate are the forms generally used as supplements in infant formula [4], The total quantification of vitamin B5 requires the release of pantothenic acid from CoA and ACR Since it consists of pantoic acid linked through an amide linkage to p-alanine, chemical hydrolysis cannot be used. The only alternative to free pantothenic acid from CoA is the digestion with a number of enzymes (pepsin, alkaline phosphatase, pantetheinase) nevertheless, this treatment is unable to release the vitamin from ACP [27,28]. For the extraction of free pantothenic acid from milk and calcium pantothenate from infant formula an acidic deproteination is often used, followed by centrifugation and filtration [29,30]. 

En2ymatic hydrolysis [81,82] with lipase (from Candida rugosa or from porcine pancrease) is an alternative procedure to remove glycerides in vitamin K determinations. Addition of papain (from Carica Papaya) aids the digestion of meat and foods of animal origin. The hydrolysate is first alkalinized (potassium carbonate in ethanol) to precipitate fatty acids as soaps then extracted with a water immiscible organic solvent (hexane or pentane). It has also been used in combination with supercritical fluid extraction (SEE) [83]. 

The stability of pantothenic add in aqueous solutions depends greatly on the pH value. The vitamin is most stable in weakly acidic (pH 4 5) solutions, but in acidic and alkaline media the amide Hnkage is hydrolysed and pantothenic add yields pantoic add (or its salt) and fl-alanine. The enzyme pantothenase of some baderia specifically cleaves pantothenic add into the same products. In acidic solutions, pantoic acid spontaneously dehydrates to form lactone, (R)-2-hydroxy-3,3-dimethylbutano-4-lactone, which is called pantoyllactone or pantolactone (5-73). Analogously, products of panthenol hydrolysis are pantoic add... 

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