Acid hydrolysis riboflavin

The alcoholysis of the cyclic phosphate of catechol by alditols can lead, after acid hydrolysis of intermediate, cyclic phosphates, to the selective formation of phosphoric esters of the primary hydroxyl groups in the alditols. Thus, erythritol and D-mannitol afford, after chromatographic purification of the reaction products, their 1-phosphates in yields of 31 and 38%, respectively.217 The method was used to convert riboflavine into riboflavine 5 -phosphate.218 1-Deoxy-1-fluoro-L-glycerol has been converted into the 3-(dibenzyl phosphate) in 54% yield by selective reaction with dibenzyl phosphorochloridate. 219... 

Total riboflavin Bread Acid hydrolysis with 0.1 N Analytical Bon-... 

Forrest and Todd developed a procedure for treatment of riboflavin with phosphoryl chloride in pyridine containing a small amount of water to form the cyclic riboflavin-4, 5 -phosphate (2). which on acid hydrolysis yields ribaflavln-.5-phos-phate (3), identical with the natural coenzyme. 

The most common forms of vitamin B2 are riboflavin 5 -phosphate (FMN) and flavin adenine dinucleotide (FAD), which are best known for their participation as co-factors (ligands) to some of the enzymes involved in electron transfer chains. The ligand is usually coupled to enzymes through the phosphate moiety and therefore isolation from tissue can be achieved by either mild acid hydrolysis or by enzymatic digestion with an acid phosphatase. 

Milk samples were prepared by acid hydrolysis but without an enzymatic hydrolysis step in order not to convert FMN into free riboflavin. Relatively low amounts of flavocoenzymes compared to free riboflavin were found in dairy products. 

In the last few years a further type of modified flavocoenzyme has beeen discovered which is structurally related to the coenzymes of succinate dehydrogenase and monoamine oxidase, but differs considerably in its chemical properties. Early literature reports indicated the presence of a flavin in cytochrome C552 from Chromatium which could not be extracted with trichloroacetic acid or acidic ammonium sulfate (7), but could be released, for example by trypsin digestion or by incubation with saturated urea solutions (2). Absorption, fluorescence and ESR behaviour were closely similar to those of 8a-cysteinyl-ribo-flavin (12) and indicated the presence of a covalent link to the protein, through position 8a (185). Strong acid hydrolysis of these peptides liberated the flavin as mixture of riboflavin derivatives oxidation with per-formic acid and acid dephosphorylation yielded a homogeneous riboflavin derivative, which was identical with 8-nor-8-carboxy-riboflavin (185). 

Hence the difference between 8a-cysteinyl-riboflavin (12) and the flavin from eyt C552 must be in the oxidation state of the 8a-carbon it should be higher than 8a-CH2 —SR and lower than 8a-COOH, namely 8-CH = O. A flavin mixture at the 8a-oxo- level was first obtained in the lumiflavin series upon acid hydrolysis of 8a-dibromo-lumiflavins (67, 154). This strongly electron deficient aldehyde exists in aqueous... 

The fact however, that this group cannot be cleaved by nucleophiles such as RS or NH2OH remains very puzzling. Edmondson (24) has shown that 8a-oxoriboflavin, in analogy to 8a-oxo-lumiflavin mentioned above, is in slow equilibrium with its 5 -hemiacetal. This in turn is identical with the mixture of riboflavin derivatives obtained from acid hydrolysis of cyt C552 flavin. 

The most thoroughly studied of the phenoxy acids is 2,4-D. Its photochemical decomposition by hydrolysis and oxidation leads, through various intermediate products, to chlorine-free polyquinoidal humic adds. The cleavage of the phenyl— carbon bond, leading to the 2,4-dichlorbphenol intermediate, is sensitised by riboflavin. 1,2,4-Trihydroxybenzene formed by hydroxy substitution is oxidised by air to 2-hydroxybenzoquinone, which is then polymerised (Crosby and Tutass,... 

Minerals and vitamins are usually found in BSG [27]. The mineral elements include aluminum, barium, calcium, chromium, cobalt, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, silicon, sodium, strontium, sulfur, and zinc, typically all in concentrations lower than 0.5%, except for silicon that is the major mineral present. The vitamins include biotin, choline, folic acid, niacin, pantothenic acid, riboflavin, thiamine, and pyridoxine. Although, many of the vitamins can be destroyed during the hydrolysis... 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Two studies have looked for the influence of alternative extraction procedures in some detail. Three different approaches—acid digestion, hot water extraction, and pepsin digestion—were compared for the efficient extraction of low concentrations of riboflavin from casein (55). The pepsin extraction gave about 17% higher riboflavin values than did acid or hot water extraction. Experiments to optimize the extraction procedure clearly showed the considerable influence of enzyme concentration and the incubation time both for the pepsin-catalyzed peptide hydrolysis and the takadiastase dephosphorylation. According to the authors of this study, the new approach seems to be superior to the traditional acid extraction. 

A second remark concerns the rapid isomerization of riboflavin phosphates in acid solution (pH < 2), especially at elevated temperature. The thermodynamic equilibrium is characterized by the presence of about 65% 5 -FMN, 11 % 4 -FMN, 8% 3 -FMN, and 15% 2 -FMN. The rate constants for the various isomerization reactions under a variety of experimental conditions have been determined by reversed-phase HPLC (83). According to the official method of the American Association of Analytical Chemists, protein-bound flavins are extracted from biological samples by treatment with 0.1 M HCl at 121°C for 15 to 30 min. It is known that this treatment leads to hydrolysis of FAD. As shown in Figure 9, it should also be noted that a substantial fraction of 5 -FMN is converted to other isomeric phosphates under these conditions. 

Pantothenic acid and pantothenates may also be analyzed following derivatization to extend the chromophore and hence allow UV detection at higher wavelengths or fluorometric detection. Hudson et al. (63) have attempted to analyze the vitamin as a P-alanine-fluorescamine complex. The derivatization procedure was lengthy and required extensive sample cleanup before the hydrolysis step due to the interference of riboflavin, niacinamide, and some minerals such as zinc, copper, manganese, and molybdenum. Although these interferences were eliminated, the method did not yield reproducible results. 

Flavin coenzymes are very susceptible to enzymatic or chemical hydrolysis. FAD is hydrolysed in acidic solutions to FMN. In acidic solutions, the phosphate group of FMN migrates from the C-5 position to C -4, C -3 and C -2 positions and subsequent hydrolysis of phosphates yields free riboflavin. 

Oxiranes are highly toxic alkylating agents and the hydrolysis product of ethylene oxide ethylene glycol and reaction products of oxiranes with chloride ions is likewise toxic. The latter reaction yields 2-chloroethanol, which arises from oxirane, and isomeric vicinal chloropropanols (chlorohydrins) resulting from methyloxirane (Figure 11.3). Oxiranes react with a number of other food components, such as vitamins (riboflavin, pyridoxine, niacin and folic acid) or amino acids (methionine and histidine) to form biologically inactive products. 

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