Simultaneous estimation of Pyridoxine HCl and FMOC-Leucine using derivative and chromatographic approach in pharmaceutical dosage form

This work developed a fast and simple method for quantifying two important amino acids, Pyridoxine Hydrochloride and Leucine, to treat obesity. In the current study, the active hydrogen of a free amino group at Leucine was only replaced by non-polar group using N-(9-Fluorenylmethoxycarbonyloxy) succinimide (FMOC-OSU) as a derivatizing agent, resulting in conversion of Leucine to FMOC-Leucine derivative to make an analyte less reactive, volatile, and thus improve its chromatographic behavior. The aim of the present study is to develop accurate, precise, and selective first order UV-Visible spectroscopic and HPLC methods for the simultaneous estimation of Leucine and pyridoxine HCl and to validate these methods as per International Conference on Harmonization (ICH) Q2 (R1) guidelines. In the developed method the correlation co-efficient was found to be 0.999; 0.999 and 0.995; 0.997 for FMOC-leucine and pyridoxine HCl respectively. The %RSD for accuracy was found to be less than 2. The detection limit and quantification limit for validation in UV-Visible spectroscopy and HPLC for Pyridoxine HCl and FMOC-Leucine were found under limit. The methods were also found to be precise and robust (% RSD= < 2%) and % assay of the marketed formulation containing both the drugs was found to be 98.6%; 98.7%; 99.2%; 99.3% for Pyridoxine HCl and FMOC-Leucine respectively. Comparison between both the methods was done by one-way ANOVA using % assay values. There was not any significant difference found. Both the methods can use for simultaneous estimation of Leucine and Pyridoxine HCl.