Analytical Method Development and Validation for the Simultaneous Quantification of Water-Soluble Vitamins (B Complex) at Single Wavelength by HPLC in Food Supplement

Background Water-soluble B-complex vitamins are essential for energy metabolism and overall health.Objectives To develop a simple, sensitive, accurate, validated and cost-effective reverse phase liquid chromatographic method for the simultaneous estimation of water-soluble vitamins (B1, B2, B3, B5, B6, B7, B9 and B12) in food supplement in a single run at single wavelength.Materials and Methods The HPLC examination was done using a C18 Phenomenex Luna column (250x4.6 mm), 5 µ particle size. The mobile phase is a mixture of 0.1% orthophosphoric acid in HPLC grade water, 80% acetonitrile with 0.1% orthophosphoric acid, filtered and degassed. A Gradient solvent system at 0.8 mL/min flow rate with detection at 210 nm wavelength was used.Results This method was validated per ICH guidelines, showing elution of vitamins in the order: B1, B3, B6, B5, B9, B12, B2 and B7. Linearity was established over a concentration range of 0.0005-0.6 mg/mL (5.0-600 ppm), which covered all the range of above vitamins in the sample with results r2>0.99. LOD ranged from 0.101 to 1.25 ppm and LOQ from 0.305 to 3.79 ppm. Developed Formulation and market samples were also analysed by this method and results were found within the range, which shows the applicability of method.Conclusion The developed in-house analytical method is simple, sensitive, accurate, fast and ICH-validated. It allows simultaneous analysis of vitamins B1, B2, B3, B5, B6, B7, B9 and B12 in a single run at single wavelength, enhancing throughput and reducing analysis time. This unique method also overcomes complications of existing approaches, sample preparation issues, complexity of mobile phase preparation (buffer system), matrix variability concerns and multiple runs for all vitamins, as it can be used for wide varieties of food matrix. It can be used for routine quality checks in various food products.