Thiochromes fluorescence

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,... 

Visual measurement of the thiochrome fluorescence is possible using narrow non-fluorescent tubes, the contents of which are matched in a dark room with an ultra-violet lamp. The test solution and a strong aneurine standard are oxidised and extracted under exactly similar conditions together with a blank from which the ferricyanide has been omitted a measured volume of the test solution is matched against addition of the standard to the blank. The tubes are inclined at an angle of 60°, but they must not be exposed longer than necessary, since the fluorescence is unstable in ultra-violet light. The final comparison is made when the volumes have been equalised. 

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. 

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... 

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. 

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... 

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... 

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. 

Thiamine triphosphate, diphosphate, and monophosphate were separated as the thiochrome derivatives on Hamilton PRP-1 column. The mobile phase was 15 mill phosphate buffer (pH 8.5) containing 1% tetrahydrofuran. The solvent flow rate was 0.5 mL/min, and a 20 fiL sample loop was used. Detection was by fluorescence using excitation and emission wavelengths of 365 and 433 nm, respectively. 

The reaction mixture was composed of 50 p.L of membrane preparation, 10 mM Hepes-Tris buffer (pH 6.8), 1.5 mM MgQ2, 1.5 mM EGTA, and 0.1 mM thiamine triphosphate in a total volume of 100 fiL. After 15 minutes of incubation at 25°C, the reaction was stopped by addition of 500 fiL of 6% trichloroacetic acid. The supemate obtained by centrifugation was extracted with 4 volumes of diethyl ether. The thiamine derivatives were transformed into fluorescent thiochromes by the addition of 50 fiL of oxidant [4.3 mJW K3Fe(CN)6 in 15% NaOH] to 80 fiL of sample. Thiamine diphosphatase activity was minimized by using magnesium as the metal and EGTA to chelate calcium. 

In experimental animals and in depletion studies, measurement of the concentration of thiamin in plasma or whole blood provides an indication of the progression of deficiency. The normal method is by the formation of thiochrome, which is fluorescent ordy free thiamin, and not the phosphates, undergoes... 

The thiochrome method involves the addition of alkaline ferricyanide to the biological sample containing thiamin. This treatment results in the ostidahve conversion of thiamin to thiochrome, which can be measured by fluorescence. 

Vitamin Bi, vitamin B2, and nicotinic acid, all of which frequently occur together in foods, were separated by TLC and fluorimetrically determined by using a commercially available fiber optic-based instrument. A fluorescent tracer (fluoresceinamine, isomer II) was used to label the nicotinic acid. Vitamin B1 was converted to fluorescent thiochrome by oxidizing with potassium ferricyanide solution in aqueous sodium hydroxide. These vitamins were separated by HPTLC on silica gel using methanol-water (70 30 vol/vol) as mobile phase. Under these conditions, the Rf values of the vitamin Bi, vitamin B2, and nicotinic acid derivatives were 0.73, 0.86, and 0.91, respectively. 

Fluorescence Kinetic-Based Measurements. Our studies of the reaction rate determination of thiamine (vitamin Bl) will be used to demonstrate the unique capabilities of rapid acquisition of spectra in kinetic measurements. The kinetic method is based on the oxidation of thiamine by Hg + in basic solutions to highly fluorescent thiochrome (16) The initial rate, taken as the change in fluorescence signal at 444 nm that occurs in a fixed time after mixing the sample and reagents, is directly proportional to the thiamine concentration. 

Figure 2. Fluorescence emission spectra taken during the course of the thiochrome reaction. The sample contained thiamine and riboflavin. (Reproduced with permission from Ref. 13. Copyright 1981, Pergamon Press.)...

The diagnostic capabilities of IDA detection were further revealed when applying the thiochrome rate method to other matrices such as urine and cereal (13). Fluorescence spectra of samples can be rapidly obtained at different pH s or excitation wavelengths. Shifts in the maxima of emission spectra under these different conditions reveal the presence of additional fluorescent species. Spectra of potential interferents can be compared to the spectrum of the sample to confirm the presence of certain species. 

In the case of urine samples, a fluorescent compound was formed at a rate comparable to the thiochrome reaction when the pH was increased. The appearance of this fluorescence peak was readily observed when the difference in spectra taken at different times was calculated and displayed. This information could not be obtained with a conventional scanning spectrofluorometer. 

Chemical ionization mass spectrometric detection has been explored for the detection of methyl hydroperoxides However, fluorometry has dominated the current detection schemes for the organic peroxides. Typically, a nonfluorescent substrate is oxidized by the peroxide to generate a fluorescent product. These methods are sufficiently sensitive for accurate measurement of the peroxides in the low ppt by volume. For example, the peroxidase-catalyzed dimerization of p-hydroxyphenylacetic acid (POPHA) occurs in the presence of a peroxy group at elevated pH. The formation of the fluorescent dimer, detected by excitation at 310 nm and emission at 405 nm, is proportional to the concentration of the peroxide. The most common peroxidase catalyst used for this reaction is horseradish peroxidase (HRP). Cost and stability issues with the use of HRP led to the use of other catalysts, such as metalloporphyrins or phthalocyanine complexes. Another fluorescent reaction scheme involves the oxidation of the nonfluorescent thiamine (vitamin Bi) to the fluorescent thiochrome by the peroxide group. This reaction is catalyzed by bovine hematin. This reaction is 25-fold more sensitive for H2O2 than for the organic peroxides. 

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... 

Thiamin (vitamin Bi) Thiamin in the body is chiefly found in the phosphorylated form thiamin pyrophosphate (TPP) which is a coenzyme. The majority (80%) of thiamin in the blood is found in the erythrocytes and assay of blood thiamin is a more reliable indicator of deficiency than assay of erythrocyte transketolase. The phosphorylated vitamers are enzymically converted to thiamin in samples using diastase following deproteinization. To reach the low picomolar concentrations the thiamin compounds are oxidized by ferricyanide to form thiochromes, which are highly fluorescent. The thiochromes are then separated by reversed-phase HPLC and detected by their emission at 425-450 nm. 

Thiamin in aqueous solution is most stable between pH 2 and 4, and labile at alkaline pH. It is also heat labile. TPP is stable in aqueous solution at pH 2-6 and at 0°C. In alkaline oxidation these form fluorescent thiochromes. 

Figure 1 Reversed-phase LC separation of thiamin and riboflavin from food samples. Column Waters rBondapak Cis (3.9 x300 mm). Eluent 28% methanol in 5 mmol P ammonium acetate, pH 5.0. Flovr rate 1.5 ml min Detector Waters 470 fluorescence detector. (A) Flour 370 nm excitation, 430 nm emission for thiochrome 370 nm excitation, 520 nm emission for riboflavin. 1, thiamin (measured as thiochrome) 0.704 mg per 100 g. 2, riboflavin 0.442 mg per 100g. (B) Bread 1, thiamin (measured as thiochrome) 0.499mg per lOOg. 2, riboflavin 0.288mg per 100g. (C) Cornflakes 1, thiamin (measured as thiochrome) 3.05 mg per lOOg. 2, riboflavin 1.93 mg per lOOg. (Reproduced with permission from Millipore Corporation, Milford, MA.)...

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