METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF NICOTINIC ACID, FOLIC ACID AND CYANOCOBALAMIN USING RP-HPLC

Abstract

A simple Re verse phase liquid chromatographic method has been developed and subsequently validated for simultaneous determination of nic otinic acid, folic acid and cyanocobalamin in combination. The separation was carried out using a mobile phase consisting of sodium acetate buffer of pH 2.5 and Acetonitrile in the ratio of 90 : 10. The column used was phenomenex Luna 5 µ c18 (2) 250x4.6 mm, 100 A with flow rat e of 1.5 ml/min, and the detection was done at 278 nm. The retention times of nicotinic acid, folic acid and cyanocobalamin were found to be 3.79, 6.01 and 14.54 min respectively. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification and ru ggedness. The limit of detection for nicotinic acid, folic acid and cyanoc obalamin was found to be 100 ng/ml, 1 µg/ml and 10 µg/ml and the limit of Quantification was found to be 1 µg/ml, 10 µg/ml and 100 µg/ml respectively. Linearity for nicotinic acid, folic acid and cyanocobalamin has bee n observed in the range of 1 - 5 µg/ml, 10 - 50 µg/ml and 100 - 500 µg/ml and the respective R 2 values are observed at 0.996, 0.997 and 0.99 respectively. The relative standard deviation for intra and interday precision were <5%. Hence the proposed RP - HPLC method wa s sensitive, rapid, cost - effective and accurate for the simultaneous determination of nicotinic acid, folic acid and cyanocobalamin in both bulk materials and pharmaceutical dosage forms.