Stability-Indicating Photochemical Method for the Assay of Thiamine by Spectrophotometry

A stability-indicating photochemical method has been developed for the assay of thiamine (TH) salts in aqueous solution and in fresh and aged vitamin preparations. It is based on the photooxidation of TH by UV irradiation to form thiochrome (TC) in alkaline solution./e TC : TH ratio under controlled conditions of light intensity, temperature, pH, exposure time, and irradiation distance is constant and can be used to determine the concentration of UV irradiated TH solutions. TC, on extraction with isobutanol from the photodegraded solution of TH, has been determined by the UV spectrophotometric method at 370 nm. It exhibits a high intensity of absorption in the UV region that can be used for the assay of even low concentrations of TH. Under optimum conditions, Beer’s law is obeyed in the concentration range of 0.20–2.00mg/100ml (R � 09998). /e limit of detection (LOD) and limit of quantification (LOQ) are 0.0076 and 0.0231mg/100ml, respectively. /e method has been validated and applied to aqueous solutions and vitamin preparations. /e results have statistically been compared with the United States Pharmacopeia liquid chromatography method. It has been found that there is no significant difference between the two methods at 95% confidence level.

e photochemical dehydrogenation of TH (1) to TC (3) probably proceeds through an intermediate (2) [52] as shown in Figure 1. e formation of TC is pH-dependent, and its yield is maximum at pH 10 [52].TC exhibits strong absorption in aqueous solution in the UV region [61].
e objective of this study is to develop and validate a simple, economical, and stability-indicating photochemical method for the assay of TH on the basis of the formation of TC, its spectrophotometric determination, and to establish conditions for its quantitative relationship with TH.
e method would be applied to the assay of TH in pure solutions and in vitamin preparations.

Materials.
iamine hydrochloride (TH) and thiocrome (TC) were purchased from Sigma-Aldrich (USA).All reagents and solvents were of the purest form available from Merck (Germany).e bu er systems used were Na 2 B 4 O 7 -NaOH (pH 9-10) and Na 2 HPO 4 -NaoH (pH 11); the ionic strength was maintained at 0.002 M.

Precautions.
e experimental work was carried out in a dark chamber under subdued light.e TH solutions were protected from light to avoid the e ect of any photochemical change before UV irradiation.

Photooxidation of TH.
Aqueous solutions containing 4 mg/100 ml of TH were prepared at pH 9-11 using appropriate bu ers with the addition of 2 ml of a 30% H 2 O 2 solution.A 50 ml quantity of the solution spread in a Petri dish was placed in a thermostat bath at 25 ± 1 °C and irradiated using a Philips 36 W TUV tube (100% emission at 254 nm) for 30 min.
e tube was xed horizontally at a distance of 25 cm from the centre of the Petri dish in a radiation chamber.Samples were withdrawn at appropriate intervals and subjected to chromatographic examination and spectrophotometric assay.
e compounds were detected on comparison of their R f values and uorescence colour under 365 nm (TC, blue) and uorescence quenching under 254 nm excitation (TH, dark), with those of the reference standards.

pH Measurements.
e pH of TH solutions was measured on an Elmetron LCD display pH meter (Model CP 501, sensitivity ± 0.01 pH units, Poland) using a combination pH electrode.e calibration of the electrode was carried out using commercially available bu er tablets of pH 7 and 9 (Merck).

Spectral Measurements.
e spectral measurements on TH and TC solutions were performed on a ermo Fisher Scienti c UV-Vis spectrophotometer (Evolution 201, USA) using quartz cells of 10 mm path length.

Light Intensity Measurement.
e intensity of the Philips 36 W TUV tube was determined by potassium ferrioxalate actinometry [63], and a value of 5.50 ± 0.11 × 10 18 quanta s −1 was obtained.

Spectrophotometric Assay of TH
2.8.1.Extraction of TC.A 5 ml aliquot of the photooxidized solution of TH was placed in a 100 ml separating funnel, 2 ml of dehydrated ethanol was added, and the solution extracted with 10 ml of isobutanol to isolate TC. e isobutanol layer was dried over Na 2 SO 4 and used for the determination of the concentration of TC in the solution at 370 nm using 20,400 M −1 •cm −1 as the value of molar absorptivity [61] or 654 as the value of A (1% 1 cm) at that wavelength.e same procedure was used for the determination of the concentration of TC in diluted solutions of photooxidized vitamin preparations.

Determination of TC : TH Ratios.
e TC : TH ratios are determined by the formation of a certain amount of TC on the photooxidation of a xed amount of TH under controlled experimental conditions (i.e., concentration of TH, concentration of oxidizing agent, pH of the solution, irradiation source, and irradiation time).ese values are determined from the concentrations of TC and TH and have been used for the assay of TH in di erent samples.

Assay of TH.
e spectrophotometric assay of TH in pure solutions and in vitamin preparations has been carried out on the basis of the TC : TH ratios determined under controlled conditions as described in the above section.For example, if the TC : TH ratio obtained on the irradiation of a vitamin preparation containing 30 mg TH per 100 ml is 0.405 : 1, the corresponding TH concentration can be calculated by the following formula: TH amount of TC formed TC : TH ratio × dilution factor of vitamin preparation, 1.600 0.405 × 7.5, 29.7 mg 100 ml . (1)

Identi cation of Photoproduct of TH.
e photooxidation of TH in alkaline solution leads to the formation of TC.
e identi cation of TC in UV irradiated solutions of TH at pH 9-11 has been carried out by TLC using the solvent systems (a) and (b) (Section 2.4).TC was detected by comparison of its characteristic uorescence (blue) and R f values with those of the authentic compounds (Table 1).TC was only detected in TH solutions irradiated at pH 9-11 since it is known to be formed by photooxidation in alkaline solution [52].Judging from the intensity of the uorescence of TC in the pH range studied, the maximum yield was found for the reactions carried out at pH 10.

Spectral Characteristics of TH and TC.
TH exhibits absorption maximum at pH 7 at 246 nm with a molar absorptivity value of 15,200 M −1 •cm −1 and an A (1% 1 cm) value of 450 [64].TC shows an absorption maximum at 370 nm with a molar absorptivity of 20,400 M −1 •cm −1 [61] and an A (1% 1 cm) value of 654 (Figure 2).In the present study, a molar absorptivity value of 19,800 M −1 •cm −1 and an A (1%, 1 cm) value of 635 for TC at 370 nm have been obtained (Table 2).In view of the very high value of the molar absorptivity of TC at 370 nm, it can be conveniently used for the assay of TH in vitamin preparations on its photooxidation to TC under controlled conditions as described in Section 3.5.e absorption maximum of TC at 370 nm provides maximum speci city and sensitivity for absorbance measurements in the method.
e validation data for the test method is reported in Table 2.

Yield of TC.
e photooxidation reaction of TH has been carried out at pH 9-11.It has been observed that the yield of TC on UV irradiation for a xed period of time is maximum at pH 10 compared to that of pH 9 and 11 (Figure 3).erefore, pH 10 was considered suitable for the photooxidation reaction of TH under the conditions used in this    study (Table 3).It has earlier been reported that the formation of TC depends on the pH of the medium, and its yield is maximum at pH 10 [52].Above pH 10, TH is ionized to the negatively charged thiol form (pK a 11.6) which undergoes hydrolytic degradation to thiazole and pyrimidine moieties [53].erefore, there is a decrease in the formation of TC with an increase in pH above 10.

Application of the Assay Method.
e photochemical method has been applied to the assay of TH in fresh and aged pharmaceutical vitamin preparations, and the results are compared with the United States Pharmacopeia liquid chromatography method (Table 4).At 95% confidence level, it has been found that the calculated t value for each concentration is less than the tabulated t value, indicating no significant difference between the two methods.e accuracy and precision of the method has been determined, and its sensitivity has been calculated (Table 2).e method is stability-indicating for the assay of TH in vitamin preparations on the basis of the amount of TC photochemically formed under controlled conditions and its quantitative relationship with TH, that is, TC : TH ratio.A similar photochemical method for the assay of riboflavin (RF) on the basis of the formation of its photoproduct, lumichrome (LC) and LC : RF ratio, has been reported [65].

Radiation Source.
A 36 W TUV tube (100% emission at 254 nm) has been found to be suitable for the photooxidation of TH.
e absorption maximum of TH (246 nm) [64] overlaps the emission band of the TUV tube that provides maximum radiation energy for the photochemical reaction and the formation of TC.

Radiation Source-Vessel Distance.
e distance between the radiation source and the reaction vessel must be constant for a fixed number of quanta to be absorbed to perform a uniform photochemical reaction.A distance of 25 cm between the radiation source and the reaction vessel has been found to be appropriate for this work.

Temperature.
e control of temperature is essential for photochemical studies to avoid any thermal effect on the rate of reaction.is has been achieved by irradiation of the solutions in a thermostat bath maintained at a constant temperature, that is, 25 ± 1 °C during the reaction.

Irradiation Time.
e irradiation of the solutions for a fixed period of time is necessary to achieve uniform results e concentration of TC in photooxidized solution of TH has been calculated from the increase in absorbance at 370 nm of the extracted solution at 30 min irradiation using 654 as the value of A (1% 1 cm) [61].e labeled amount of thiamin is the amount (mg/100 ml) stated on the label of the vitamin preparations.b n � 3. c Percent recovery of TH in vitamin preparation � ((amount found in mg/100 ml)/(labeled amount in mg/100 ml)) × 100. 4 Journal of Spectroscopy in the formation of TC.In the present study, a time period of 30 min has been found to be sufficient to avoid any photodegradation of TC [52].

Oxidizing Agent.
e oxidation of TH in alkaline solutions occurs in the presence of an oxidizing agent, that is, potassium ferricyanide [66] or hydrogen peroxide [67].In the present study, the photooxidation of TH has been carried out in the presence of hydrogen peroxide to avoid any interference of the oxidizing agent in the absorbance of TC.

Conclusion
A simple photochemical method has been developed and validated for the assay of TH in aqueous solution and pharmaceutical vitamin preparations.It is based on the photooxidation of TH to TC, determination of their quantitative relationship (TC : TH ratio) under controlled conditions and on this basis the assay of TH in different pharmaceutical samples.TC has a high value of A (1%, 1 cm) and can be conveniently determined by spectrophotometry at 370 nm.
e method is comparable to the United States Pharmacopeia method.

Figure 3 :
Figure 3: Yield of TC as a function of reaction pH.

Table 2 :
Analytical parameters for the assay of thiochrome (TC).Accuracy, n 6 at 100% concentration.c Precision, n 6 at 100% concentration.d Limit of detection (LOD) 3.3 × σ/S, where σ is the standard deviation and S is the slope of the standard curve.e Limit of quantitation (LOQ) 10 × σ/S, where σ is the standard deviation and S is the slope of the standard curve.
a Linearity, n 5. b