SPECTROPHOTOMETRIC AND SPECTROFLUORIMETRIC METHODS FOR THE DETERMINATION OF THIAMINE IN PHARMACEUTICAL FORMULATIONS

Abstract

The objective of this research is to develop and validate simple and cheap method for determination of Thiamine (vitamin B1) in pharmaceutical formulation by means of Spectrophotometry and Spectrofluorimety. This goal has been accomplished through four methods. The first method (A1) is a direct Spectrophotometric method based on the reaction of Thiamine with 1,2-Naphthoquinone -4-sulphonic acid sodium salt (NQS) reagent in alkaline medium (pH = 11), to produce a brown-colored product exhibiting maximum absorption peak (λmax) at 487 nm. Under the optimized reaction conditions, Beer’s law was obeyed in the range of 10 – 40μg/mL. Linear regression equation of the calibration curve is: A= 0.022x + 0.171 with a linear correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.71 μg/mL and 5.18 μg/mL respectively. The second method (A2) is a direct Spectrophotometric method based on the reaction between Thiamine and 7-Chloro-4-nitrobenzoxadiazole (NBD-Cl) reagent. In alkaline medium of pH 10.5, a yellowish-brown adduct exhibiting maximum absorption peak (λmax) at 434 nm was produced. Under the optimized reaction conditions, Beer’s law was obeyed in the range of 5 – 35μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.667 μg/ml and 2.020 μg/ml respectively. The third method (B1) is Spectrofluorimetric method based on the measurement of the fluorescence activity of the product formed between Thiamine and NBD-Cl in alkaline medium. Excitation was carried out at 472 nm, with emission at 562 nm. All variables affecting the reaction were studied and optimized. Beer's law was obeyed in the concentration range of 0.2 – 1.0 μg/mL. II The fourth method (B2) is also Spectrofluorimetric method based on measuring the relative fluorescence intensity of the product formed when Thiamine reacts with NQS in alkaline conditions. The measurement was carried out with excitation at 390 nm, and emission at 460 nm. All variables affecting the reaction were studied and optimized. Beer's law was obeyed in the concentration range of 0.1 – 1.0 μg/mL. The four methods were validated with respect to accuracy, precision, linearity, sensitivity, limit of detection and limit of quantification according to the International Conference of Harmonization (ICH) guidelines for validation of analytical procedures. They successfully applied to the determination of the drug in its pharmaceutical dosage form with a high accuracy and precision, without interferences from the common pharmaceutical additives. The proposed methods are simple, sensitive, and they are practical and valuable for the routine application in quality control laboratories for the analysis of the