Thiamine determination

D Blanco, MB Llaneza, MD Gutierrez. A paired ion liquid chromatographic method for thiamine determination in selected foods. J Liq Chromatogr Rel Technol 19 2155-2164, 1996. 

Vidal-Valverde, C., and Diaz-Pollan, C., Comparison of capillary electrophoreticandhigh performance liquid chromatographic thiamin determination in milk, Milchwissenschaft, 55, 307, 2000. 

Vitamins are the foodstuff components most often quantified using fluorimetric means. There are several official fluorimetric methods for the determination of three water-soluble vitamins vitamin Bi (thiamine) (AOAC 942.23 and 957.17), B2 (rib-oflavine) (AOAC 970.65 and 981.15), and C (ascorbic acid) (AOAC 984.26). Thiamine is determined by oxidation to fluorescent thiochrome with alkaline hexacyanoferrate(III) or an alternative oxidant (Figure 1). The method is quite simple, reproducible, and selective and provides good recoveries. Many LC methods for thiamine determination in foods have... 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

The species most commonly employed for thiamine determination are some Lactobacilli such as L.fermentum and L. viridescens (ATCC 12706). The latter is the microorganism most widely used to measure thiamine concentrations. It requires the intact thiamine molecule for growth. Other organisms such as Phycomyces blakeslekanus, Kloeckera brevis (ATCC 9774), Ochromonas danica and Neurospora crassa are available, but they are less useful. 

The recently elaborated multivitamin methods for B vitamins assay using LC with MS/MS detection, micellar LC, LC-DAD/MS, LC/ESI-MS or LC-IDMS systems offer better sensitivity and selectivity, and can be potentially used for thiamine determination in fortified foods and infant formulas. These methods have potential to improve performance in the future. 

The voltammetric methods of analysis have attracted attention due to the high sensitivity, simplicity and relatively short analysis time, and the possibility of using inexpensive equipment. A refreshable mercury film silver based electrode [Hg(Ag)FE] has been suggested to be applicable for thiamine determination together with vitamin B2 and vitamin C. 

G Bertelsen, PM Finglas, J Longhridge, JM Faulks, MRA Morgan. Investigation into the effects of conventional cooking on levels of thiamin (determined by HPLC) and pantothenic add (determined by ELISA) in chicken. Food Sci Nutr 42F 83-96, 1988. 

Liquid chromatographic methods for thiamin determination in foodstuffs and other matrices are reviewed by Lynch and Yoimg [11]. 

As in thiamin determination, proposed methods for riboflavin are based on fluorimetry and HPLC. 

Abdel-Kader, Z., Comparison of AOAC and high-performance Uquid chromatographic methods for thiamin determination in foods. Food Chem., 43, 393-397, 1992. 

Determination of structural features. The ultraviolet spectrum has been of value in the determination of the structure of several vitamins. Thus the presence of an a-naphthoquinone system in vitamin K was first detected by this means. Also the 4-methylthiazole and the 2 5-dimethyl-6-aminopyridine system was first identified in vitamin Bj (thiamine), a- and /3-Ionones can be distinguished since the former contains two conjugated chromophores and the latter three conjugated chromophores. 

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

The yellow form (11) on acidification is converted to the more stable thiol form (12). On oxidation, typically with alkaline ferhcyanide, yellow form (11) is irreversibly converted to thiochrome [299-35-4] (14), a yellow crystalline compound found naturally in yeast but with no thiamine activity. In solution, thiochrome exhibits an intense blue fluorescence, a property used for the quantitative determination of thiamine. 

Important organic applications are to the determination of quinine and the vitamins riboflavin (vitamin B2) and thiamine (vitamin Bj). Riboflavin fluoresces in aqueous solution thiamine must first be oxidised with alkaline hexacyanoferrate(III) solution to thiochrome, which gives a blue fluorescence in butanol solution. Under standard conditions, the net fluorescence of the thiochrome produced by oxidation of the vitamin Bj is directly proportional to its concentration over a given range. The fluorescence can be measured either by reference to a standard quinine solution in a null-point instrument or directly in a spectrofluorimeter.27... 

Although reliable methods (3) for the determination of ascorbic acid, thiamine,... 

Evaluation of kinetic data. Rate constants were determined for 2-H exchange from 3-R-4-methylthiazolium ions, catalyzed by D2O (pseudo first order) and DO- (second order).154 The observed rate constants for the pD-independent exchange reaction were corrected for the solvent isotope effect ( h2o/ d2o = 2.6), and the reverse protonation of the carbene by H30+ was assumed to be diffusion-controlled (k = 2 x 1010 M-1 s-1). A similar analysis was performed for the exchange catalysed by DO-. The results agreed nicely, giving pAfa = 18.9 for 213 and p/sfa = 18.0 for thiamine.154 The thiazolium ion 213 seems to be less acidic in water154 than in DMSO152 (Ap/fa = 2.4). Aside from the... 

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

We have determined thiamine levels of mother and infant at parturition (Bll). Thiamine in the fetal circulation is 5 times higher than in the mother s. A similar situation exists in the guinea pig (B35). 

Polarography has been used to study the structure of thiamine phosphates190 and for the determination of phosphatase activity of milk.191... 

The crystal structure of MPT synthase and the simultaneously determined NMR structure of the MoaD-related ThiS protein involved in thiamine biosynthesis [37] unambiguously demonstrated the evolutionary relationship between a subset of enzymes involved in the biosynthesis of S-containing cofactors (e.g. Moco, thiamine and certain EeS-clusters) and the process of ubiquitin activation. MoaD displays significant structural homology to human ubiquitin (Figure 3.3B and C), resulting in a superposition with a root mean square (rms) deviation of 3.6 A for 68 equivalent Ca atoms out of 76 residues in ubiquitin. The key secondary structure... 

Typical FI A manifolds are shown in Fig. 13.10 with two general alternatives depending on whether injection takes place before or after the continuous extractor device. The most common situation is when prior injection of the sample take place. Figure 13.10(a) depicts a manifold for the determination of vitamin Bi in pharmaceuticals [178], based on the oxidation of thiamine to thiochrome in a carrier of potassium ferricyanide in a basic medium (NaOH). The thiochrome is continuously extracted into a chloroform stream and the fluorescence of the organic phase is measured continuously. 

Acid dye method for the analysis of thiamin, 18A, 73 electrophoretic separation and fluorometric determination of thiamin and its phosphate esters, 18A, 91 catalytic polarography in the study of the reactions of thiamin and thiamin derivatives, 18A, 93 preparation of thiamin derivatives and analogs, 18A, 141 preparation of the mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamin biosynthesis, 18A, 162 formation of the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine by enzymes from brewers yeast in thiamin biosynthesis, 18A, 203 resolution, reconstitution, and other methods for the study of binding of thiamin pyrophos-... 

The chromatography conditions applied are usually the same used for thiamine analysis. A RP chromatography is used for analytical determination, such as ODS stationary phase, C18 or amide phases. The mobile phases are methanol, water or acetonitrile with addition of buffer. 

Thiamine, riboflavin, nicotinamide, pyridoxine, and folic acid can be determined together by using DAD, but pantothenic acid and biotin do not have adequate sensitivity for UV detection in complex matrices. 

More recently [635], a unique extraction step in supplemented foods, by using hot water and a precipitation solution, following by HPLC-ELD/UV analysis has been performed for the simultaneous determination of pyridoxine, thiamine, riboflavin, niacin, pantothenic acid, folic acid, cyanoco-balamin, and ascorbic acid. The mobile phase consisting of phosphate buffer and methanol has been modified in order to perform ion-liquid chromatography by adding l-octanesulfonic acid sodium salt. Furthermore, triethylamine has been also added to improve peak symmetry. 

Thiamine deficiencies are determined In the clinical laboratory by measuring the activity of transketolase In the RBC... 

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