Abstract
l-ascorbic acid 2-phosphate magnesium (APMg) salt is a vitamin C derivative frequently used in cell culture media for research purposes. It is also used as a raw material in the GMP-manufacturing of gene-, cell- and tissue advanced therapy medicinal products (ATMPs). However, quality methods are currently lacking. Therefore, a LC method was developed, based on hydrophilic interaction (HILIC)-ion exchange (IE) mixed-mode liquid chromatography. The final method consisted of an isocratic system with 15 mM KH2PO4 buffer (pH 2.5 with HCl) acetonitrile (30:70, v/v) mobile phase on a zwitterionic HILIC column, containing an hydrophilic ligand embedded cation-exchange functionality and a surface anion-exchange group. A flow rate of 0.4 mL/min and UV detection at 240 nm was applied. The assay method of APMg was validated, obtaining adequate linearity (R2 = 0.999), precision (RSD of 0.49%) and accuracy (overall recovery of 100.4%). The developed method was successfully applied on five currently marketed products from different suppliers, showing different related substance impurity profiles. Using atomic absorption spectroscopy (AAS), magnesium was found to be bound on the stationary phase, requiring a strong mobile phase to rinse the column. Finally, related impurities were identified using MS/MS and high resolution MS, and found to be ascorbic acid as well as ethyl derivatives, which was further confirmed by NMR.
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