Development and Validation of Simultaneous Determination of Vitamins B1, B2, B3, B6 and Folic Acid in Multivitamin Tablet by RP-HPLC

A. Sankara Avudaiappan¹, A. Chenthilnathan^1* and S. Vikraman^2 
1Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University, Tirunelveli – 627 012, Tamil Nadu, India.
²Apex Laboratories Private Limited, Alathur, Kancheepuram District-603110 Tamil Nadu, India.
Received: 29 April 2014, Accepted: 15 June 2014, Published Online: 27 July 2014

Abstract
A simple, efficient and reproducible RP-HPLC method for simultaneous determination of vitamins B₁ (Thiamine mononitrate), B₂ (Riboflavin), B₃ (Nicotinamide), B₆ (Pyridoxine hydrochloride) and Folic acid in a combined multivitamin tablet dosage form has been developed and validated. The Chromatographic Separation was carried out on Water C18 (250 × 4.6mm; 5µm) column using the mobile phase consists of water, methanol and acetic acid in the ratio 820:170:10. The mobile phase was flowed at the rate of 0.6 ml/min and effluent was detected at 280 nm. The retention times of Thiamine mononitrate, Riboflavin, Nicotinamide, Pyridoxine hydrochloride and Folic acid were 13.056 min, 6.128 min, 3.154 min, 4.306 min and 4.860 min respectively. The method was validated according to ICH guidelines and the acceptance criteria for system suitability, specificity, linearity, precision, robustness and ruggedness were met in all cases. The method was linear in the range of 80-120 µg/ml for Thiamine mononitrate (r² = 0.9991), Riboflavin (r² = 0.9998), Nicotinamide (r² = 0.9996) Pyridoxine hydrochloride (r² = 0.9995) and Folic acid (r² = 0.9997). The percentage relative standard deviation for Intermediate precision was found to be less than 2.0%. Hence, the method could be successfully applied for routine analysis of Thiamine mononitrate, Riboflavin, Nicotinamide, Pyridoxine hydrochloride and Folic acid from multivitamin tablets.
Keywords: Vitamins B_1, B_2, B_3, B₆ and Folic acid, Multivitamin tablets, RP-HPLC, Validation