Development and validation of a RP-HPLC method for simultaneous estimation of pyridoxine hydrochloride (Vitamin B6) and its degraded products formed under effect of different solvents

The aim of the present study was to develop a relatively simple, sensitive, validated, and reliable HPLC method for the determination of pyridoxine hydrochloride substance and drug product. Very simple, rapid, sensitive, accurate, and less expensive HPLC method was develop for the simultaneous estimation of pyridoxine hydrochloride (vitamin B6). Pyridoxine hydrochloride has absorbance at wavelength 254 nm in a mixture of potassium dihydrogen phosphate (pH 3±0.2) and methanol with a ratio 70:30 respectively, mobile phase was delivered at the flow rate of 1.0 ml/min. Substance was resolved on a C18 column (thermo 250∗4.6 mm id, 5 μm). The linearity was obtained over the concentration ranges 10-50μg/ml with linear regression (r²) = 0.9996. Separation completed within 3.5 min ± 0.02, no interference for any excipients observed. Accuracy (recovery) calculated as percentage found to be between 98.8 100.86 %. Method found to be reproducible with relative standard deviation (RSD) for intra and interday precision < 2 over the concentration range. The results obtained from the analysis of pyridoxine by developed validated method were successfully to the determination of pyridoxine substance, drug product and it can be useful for their combination.