Method development and validation for simulataneous estimation of biotin and folic acid in bulk and tablet dosage form by RP-HPLC

A simple, effective, economic, less time consuming and reliable chromatographic method has been developed for the simultaneous estimation of Biotin and Folic acid in bulk and tablet dosage form by RP-HPLC and validated as per guidelines. This method involves an isocratic elution carried on C₁₈ Sunfire (250 × 4.5mm I.D, 5μm particle size) column by using mobile phase of 0.05M potassium dihydrogen orthophosphate buffer: methanol (70:30v/v).The flow rate was maintained at 0.6ml/min with the UV detection of 212nm at ambient temperature. Retention time of Biotin and Folic acid was found to be 6.4 min and 3.8 min respectively. Linearity was found to be in the concentration range of 10-50μg/ml and the correlation coefficient (r²) was greater than 0.996 for both the drugs. Limit of detection and Limit of quantification were found to be 0.07,0.2 and 0.03.0.9 for Biotin and Folic acid respectively. The method was accurate with 99-100.5% recovery. The method was precise with % RSD value of Biotin and Folic acid for intraday 0.8 and 0.9, for interday 0.81 and 0.83, for repeatability 1.43 and 1.2 respectively. Validation parameters met all specifications as per ICH Guidelines.