Simultaneous quantification of 12 water-soluble vitamins in MDCK cell culture media by eco-friendly ion-pairing reagent-free HPLC-UV

Simultaneous quantification of 12 water-soluble vitamins in MDCK cell culture media by eco-friendly ion-pairing reagent-free HPLC-UV

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 colu...

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Bibliographic Details
Main Authors: Lei Zhou, Mengting Zhang, Huinan Sun, Liyuan Duan, Qingwei Meng
Format: Article
Language:English
Published: Nature Portfolio 2025-07-01
Series:Scientific Reports
Subjects:
HPLC-UV
Water-soluble vitamins
Green analytical chemistry
MDCK cell culture media
Simultaneous quantification
Online Access:https://doi.org/10.1038/s41598-025-12536-z
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Summary: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.
ISSN:2045-2322

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