Abstract
This study establishes the first reversed-phase HPLC-UV method for simultaneous baseline separation of 12 structurally diverse water-soluble vitamins (WSVs) within 25 min. The eco-friendly approach utilizes a methanol-potassium dihydrogen phosphate (pH 4.85) gradient on a ChromCore C18 column (250 × 4.6 mm, 5 μm) with dual-wavelength detection (210/266 nm), eliminating environmentally detrimental ion-pairing reagents. Rigorous validation per ICH Q2(R1) guidelines confirmed excellent linearity (R² > 0.999), precision (RSD < 2.1%), accuracy (spike recoveries: 93.9-106.8%), and sensitivity (LODs: 0.01-0.78 µg/mL). The method resolved key analytical challenges including polarity disparities, matrix interference, and vitamin instability. Successful quantification of endogenous vitamins in DMEM medium showed ≤ 5.1% deviation from certified values for 11/12 analytes. Applied to five commercial MDCK cell culture media, the method revealed significant inter-product variability in vitamin composition - notably universal under-supplementation of riboflavin (0.576-0.741 µg/mL, 10× lower than other vitamins) and inconsistent biotin/adenine supplementation. This robust, cost-effective platform provides biomanufacturers with a powerful quality control tool for optimizing vitamin supplementation in vaccine production systems, addressing a critical gap in cell culture analytics.