METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF NICOTINIC ACID, FOLIC ACID AND CYANOCOBALAMIN USING RP-HPLC
International Journal of Research in Ayurveda and Pharmacy
A simple Reverse phase liquid chromatographic method has been developed and subsequently validated for simultaneous determination of nicotinic 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 Å with flow rate of 1.5 ml/min, and the detection was done at 278 nm. The retention times of
... on 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 ruggedness. The limit of detection for nicotinic acid, folic acid and cyanocobalamin 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 been 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 was 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.