Abstract

VALIDATION AND PHARMACEUTICAL APPLICATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF VITAMIN-B COMPLEX COMPONENTS AS PER ICH Q2 (R2)

Janki Joshi, Dr. Heena Patel*

Abstract

A previously optimized reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous estimation of Thiamine Hydrochloride (Vitamin B1), Riboflavin (Vitamin B2), Nicotinamide (Vitamin B3), Pyridoxine Hydrochloride (Vitamin B6), Folic Acid (Vitamin B9), and Cyanocobalamin (Vitamin B12) was subjected to comprehensive validation in accordance with International Council for Harmonisation (ICH) Q2(R2) and United States Pharmacopeia / requirements. Validation parameters assessed included system suitability, specificity, linearity and range, accuracy (recovery), precision (repeatability and intermediate precision), limit of detection (LOD), limit of quantitation (LOQ), robustness, and solution stability. Stability-indicating capability was established through forced degradation studies conducted under acidic, alkaline, oxidative, thermal, and photolytic stress conditions with degradation products successfully resolved from the intact analyte peaks. All validation parameters met the acceptance criteria prescribed by ICH Q2(R2): calibration curves for all six analytes were linear across their respective working ranges with correlation coefficients (R²) greater than 0.999; percentage recoveries fell within 98–102%; and intra-day and inter-day precision, expressed as %RSD, remained below 2%. The validated method was successfully applied to the quantitative estimation of all six vitamins in a marketed combined pharmaceutical formulation, yielding assay results within the accepted label-claim range (98.9–101.2%) with low variability. These findings confirm that the developed RP-HPLC method is accurate, precise, specific, robust, and stability-indicating, and is therefore suitable for routine quality-control analysis, stability testing, and regulatory submission for Vitamin-B complex pharmaceutical products.

Keywords: vitamins in a marketed combined pharmaceutical formulation, yielding assay results within the accepted label-claim range (98.9–101.2%) with low variability. These findings confirm that the developed RP-HPLC method is accurate, precise, specific, robust, a