Tetrazolium blue reagent

Fig. 4. Part of a 20 X 20 cm thin-layer chromatography plate for the assay in urine of B, THS C, allo-THE E, THE F, allo-THP G, THF. Standard compounds have been run in positions 2, 4, 6, and 8. Cortisol is shown in position D and is measurable only if present in abnormally increased amount. The compounds shown in position A are mainly Cm steroids with an a-ketol group. Duplicate extracts from normal adult urines have been run in positions 1 and 3 from a case of adrenal carcinoma in position S from a normal infant in position 7 and from an infant with adrenogenital syndrome in position 9. The plate was sprayed with blue tetrazolium reagent, then heated. From Shackleton et al. (SIO).

It was reported that superoxide anion was formed in the oxidation of ASA in an aqueous solution, and the superoxide was further involved in the oxidation of ASA (10). Our previous study agreed to this finding and it was further confirmed in our experiment during the autoxidation of ASA in MeOH by using nitro blue tetrazolium reagent (11) (data not shown). From the results of MO calculations, superoxide anion might be produced by direct release from the peroxy anion type intermediate, or more likely from the dianion form of the peroxy anion type intermediate. Further investigations, however, are by all means needed to establish the mechanism of formation of superoxide anion. 

In the presence of the C-20-ketogroup at the side chain, tetrazolium salts are reduced to the corresponding strongly colored formazans. Mader and Buck were the first to apply tri-phenyl tetrazolium chloride to assay pharmaceutical steroids [83], but it was later found that air affects the procedure leading to poor reproducibility [84,85]. More stable reagents such as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium bromide [86] and 2,5-diphenyl-3-(p-strylphenyl)tetrazolium chloride [87] were employed successfully. Good results were also obtained with blue tetrazolium, or 3,3 -(3,3 -dimethoxy-4,4 -biphenylene) bis 2,5-diphenyl-... 

Oxidation, reagents Barium permanganate. Bismuth trIoxide. Blue tetrazolium. N-Bromo-acetamide. Bromocarbamide. N-Bromoiucclnlmldc. l-Butyl chromate. r-Butyl hypochlorite. r-Butylhydroperoxide. Caro i. acid. Ceric ammonium nitrate. o-Chloranll. m-Chloropei benzoic acid. NChloroauccinlmlde. Chromic Mid In dll. HiSOi, in acetone (Jonee reagent)i... 

Owing to the presence of acidic groups in melanins (carboxyls, phenolic groups) positively charged reagents react faster than anions or neutral species, especially in basic media. Thus, cationic nitroxides react much faster than anionic ones, and the reaction is twofold faster at pH 10 than pH 5. The slow reaction with Nitro Blue Tetrazolium is dramatically accelerated in the presence of a cationic detergent (92). 

Dimethoxyhydroxyphenylflurone has been proposed as a chromogenic reagent for the spectrophotometric determination (e = 1.3610 1 mof cm ) of trace amounts of Mo in steel and pure iron [4]. Thiazolylazo compounds were used as chromogenic reagents to determine Mo in human urine [5]. Thiazolyl blue tetrazolium bromide and 3,4,5-trihydroxybenzoic acid and nitrobluetetrazolium chloride were employed to determine Mo in soil [6,7]. 

Blue tetrazolium (BT) and Phenylhydrazine H2SO4 (PH) reagents react with the intact side chain at C17 of prednisolone while isonicotinic acid hydrazide (INH) and UV-absorption depend upon conjugation in Ring A at the other end of the prednisolone molecule. Since... 

The zones or spots of separated compormds are generally visualized by detecting dehydrogenase activity with tetrazolium salt-based reagents. The metabolically active bacteria convert the tetrazolium salt into red formazan dye (2,3,5-triphenyl-2H-tetra-zolium chloride, tetrazolium red). A number of tetrezolium salts were evaluated as potential substrates for the enzymatic reaction, but p-iodonitrote-trazolium violet, tetranitro blue tetrazolium, and MTT were found to be suitable substrates. The equations below show the principle of the reaction with tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5 dip-henyltetrazolium bromide (MTT, tetrazolium dye). 

To enhance the applicability of UV spectrophotometry in pharmaceutical steroid analysis by increasing its selectivity and sensitivity, various methods have been developed based on chemical reactions leading to colored derivatives. Although their importance has decreased considerably, some of these methods are still in use in pharmacopoeias, mainly in the assay of formulations. For example, the A" -3-oxo and A " -3-oxo steroids can be determined as iso-nicotinoyl hydrazones (/Uax 380 and 410 nm 11500 and 17000, respectively). The dihydroxy-acetone side chain of corticosteroids at C17 reduces tetrazolium reagents to colored formazans (Amax 485 and 525 nm with triphenyl tetrazolium chloride and Tetrazolium Blue reagents, respectively e 16000 and 24000), thus creating the basis for a stability-indicating indirect colorimetric assay. Corticosteroids... 

Other colour tests which do not interfere in the subsequent quantitative determination, can be of only limited application. An example is with organic phosphorus compoimds, where the substance can be ignited and determined through its phosphorus content [166, 258]. Glycolipoid fractions have been localised with ammoniacal bromothymol blue indicator and then, after extraction, evaluated with anthrone/sul-phuric acid [324]. 17-Hydroxycorticosteroids have been sprayed with blue tetrazolium and then determined with phenylhydrazine/sulphuric acid [11] the steroids partly reacted with the identification reagent which led to difficulties. 

This reagent is quite specific for corticosteroids producing blue spots on a white background. The tetrazolium spray is used in a test for related foreign steroids in fluclorolone acetonide. 

Early applications of thin-layer chromatography in corticosteroid analysis were mainly based on use of phenylhydrazine in sulfuric acid as a spray reagent for derivatization of the analytes (529, 530). Later applications involved use of other spray reagents including phosphoric acid in methanol, tetrazolium blue. 

Bioindicators. Water-soluble, colorless tetrazolium salts can be reduced to water-insoluble, deeply colored formazans. The reduction of tetrazolium salts in plant tissue at pH 7.2 was first demonstrated in 1941 [77], Tetrazolium salts have since been used in biochemistry, cytochemistry, and histochemistry because of the great sensitivity of this reaction. They can be used to detect biological redox systems in blood serum, in living cells, tissues, tumors, and bacteria. Tetrazolium Blue [167429-81-7] (48) is a particularly sensitive reagent. 

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

An interesting example is die assay of clonidine hydrochloride in injections and tablets (British Pharmacopoeia, 1980). In 0.01 Mhydrochloric acid, clonidine exhibits two sharp maxima ne - 272 nm and 279 nm, which are not suitable for precise measurement. However, clonidine forms an ion pair with bromothymol blue, and this can be readily extracted into chloroform for subsequent measurement of the broad maximum near 420 nm. Because of the intiinsic variabihty of reagents used in such methods, a pharmacopoeial reference standard is employed for cahbration. A similar policy is adopted for assays involving chemical modification of the drug, as in the tetrazolium assay for corticosteroids, the assay for folic acid involving hydrolysis, diazotisation, and coupling with N-(l-naphthyl)ethylenediamine, and the reaction of penicillins with imidazole and mercuric salts. 

Systems 3-6 Kieselguhr GF 254 plates Spray reagent Tetrazolium blue Solvent systems 3 - 1,2-Dichloroethane/methanol/water 95 5 0.2 4 - 1,2-... 

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