Thiochrome

Thiochrome (377), the well-known derivative of the 2-imino-4-thiazoline ring, is synthesized according to the set of reactions shown in Scheme 216 (704, 732). 

Reduction of A-4-thiazoline-2-one by zinc dust gives low yields of the corresponding thiazoles (36, 231). The formation of thiochrome (95) results from an intramolecular nucleophilic attack (Scheme 47) (232). 

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. 

The thiol form (12) is susceptible to oxidation (see Fig. 2). Iodine treatment regenerates thiamine in good yield. Heating an aqueous solution at pH 8 in air gives rise to thiamine disulfide [67-16-3] (21), thiochrome (14), and other products (22). The disulfide is readily reduced to thiamine in vivo and is as biologically active. Other mixed disulfides, of interest as fat-soluble forms, are formed from thiamine, possibly via oxidative coupling to the thiol form (12). 

Thiochrome (2,7-dimethyl-5//-thiachromine-8-ethanol 3,8-dimethyl-2-hydroxyethyl-5//-thiazolo[2,3 l, 2 ]pyrimido[4, 5 -d]pyrimidine [92-35-3] M 262.3, m 227-228 , pK st 5.8 (thiazol-N protonation). Crystd from chloroform. 

Potassium hexacyanoferrate(III) forms, for example, fluorescent thiochrome with vitamin Bi ... 

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... 

Tetrahydrocannabinol (= THC)-11-carboxylic acid 290 Tetrahydrocortiso 221 Tetrahydrocortisone 221 Tetrahydrosteroids 222 Tetrazolium salts, reduction 61 Thalidomide 45 -, hydrolysis products 45 Thiabendazole 307, 308 Thiamine 235, 236, 397 Thickening agents 179 Thin-layer chromatography, advantage 5 -, numbers of publications per year 6 Thiobarbiturates 45,66 Thiocarbamide derivatives 322 Thiocarbamides, N -ary I-N -benzenesulfo-nyl- 248,249 Thiochrome 395... 

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. 

Extraction with HCl and sonication addition of K4Fe(CN)g with NaOH and MeOH to oxidase thiamine to thiochrome filtration... 

Extraction is commonly carried out by hydrolysis in boiling acid such as chloridric acid or sulfuric acid. To release thiamine bonded to phosphate enzyme, hydrolysis with phosphatase, alone or together with claradiastase or takadiastase, is carried out. After the enzymatic digestion, an acid treatment is applied in order to precipitate the protein and denaturate the enzymes. Ndaw et al. [603] proved that for extraction of vitamins Bj, B, and Bg, acid hydrolysis is always superfluous if the activity of the enzymes chosen is sufficiently high. SPE or column chromatography may be used in further purification, mainly to remove excess of derivatization reagents used to convert thiamine to a highly flnorescent thiochrome derivatives. lEC may be used in purification step, as well. 

When the amount of thiamine is relatively high, U V detection at 254nm can be applied. Fluorometric detection, however, enables to reach higher sensitivity, when coupled with precolumn or postcolumn derivatization. The derivatization is carried out by using potassium ferricyanide under alkaline conditions to convert thiamine to highly fluorescent thiochrome derivatives. 

A fluorometric method was developed for determination of atmospheric H2O2 simultaneously with other species present at ppbv or lower levels, avoiding chromatographic separation. H2O2 is selectively collected by diffusion through a Nafion membrane, and is carried by a water stream into a reactor where it oxidizes thiamine hydrochloride (117) to a fluorescent ionic form of thiochrome (118), catalyzed by bovine hematin (75b) in alkaline solution, as shown in equation 40. The end solution containing 118 is passed through... 

Thiochrom-4-ones have also been prepared by the amine-promoted elimination of hydrogen halide from 3-haIothiochroman-4-ones,188 by the Puinmerer reaction on thiochroman-4-one S-oxides [Eq. (20)],98 from a ring expansion of activated benz[6]thiophenes [Eq. (21)],189 by the reaction of o-mercaptoaryl alkyl ketones with ethyl esters of... 

Thiamin is unstable at high pH90 91 and is destroyed by the cooking of foods under mildly basic conditions. The thiol form undergoes hydrolysis and oxidation by air to a disulfide. The tricyclic form (Eq. 7-19) is oxidized to thiochrome, a fluorescent compound... 

Bioassay methods include yeast fermentation polyneuritic rate of cure in rat bacterial metabolism. Physicochemical methods include thiochrome fluorescence polarography chromatography absorption in neutral and acid solutions,... 

Thiamine shows a pH-dependent UV absorbance range of 230-270 nm. However, its UV absorbance is prone to interference by other endogenous UV absorbers in foods, such as nucleic acids (67,68). In a recent interlaboratory comparison of thiamine methods (42), the results obtained from an HPLC method using UV absorbance detection were rejected due to the presence of peaks that interfered with thiamine. In the interests of increased sensitivity and selectivity, the thiamine vitamers are generally converted to their thiochrome derivatives by alkaline oxidation and determined fluorimetrically (42,70). The thiochrome derivatives of thiamine and its phosphate esters all fluoresce at nearly identical excitation (365-375 nm) and emission (425-435 nm) maxima at pH over 8. The thiochrome derivatives are all relatively stable in alkaline solution at pH greater than 9 and room temperature. 

Recently developed HPLC methods determine thiamine either alone or concomitantly with other vitamins. Tables 6-10 review HPLC methods, published from 1992 to 1997, for the determination of total thiamine. All but one method (79) uses fluorescence detection of the thiochrome derivative. Those methods that determine thiamine simultaneously with other B vitamins are reviewed in Sec. XI of this chapter. 

Total thiamine Milk Enzymatic hydrolysis of protein with trypsin and thiamine phosphates to thiamine with claradiastase oxidation of thiamine to thiochrome using ferricyanide (derivatization stopped with sodium sulphite) thiochrome extracted with 1-butanol Analytical Nucleosil Phenyl (150 mm, 5 fi Macherey-Nagel). Isocratic methanol + acetonitrile + isobutanol + water (80 +10+10+5 v/v/v/v). 0.7 ml/min. Fluorescence 375/430 nm (ex/em). External standardization. 76 Recoveries 95% thiamine as thiochrome from milk. 

Thiochrome Variety of spots under long-wave UV 137... 

Thiochrome A 0.3 M aqueous potassium ferricyanide solution that is 15% in sodium hydroxide is sprayed on the plate, yielding a variety of spots under long-wave UV. 

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