Zwitterionic-hydrophilic interaction liquid chromatography for L-ascorbic acid 2-phosphate magnesium, a raw material in cell therapy

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作   者：

Xu, Xiaolong;  Urlaub, Jonas;  Wozniczka, Magdalena;  Wynendaele, Evelien;  Herman, Karen;  Schollmayer, Curd;  Diehl, Bernd;  Van Calenbergh, Serge;  Holzgrabe, Ulrike;  De Spiegeleer, Bart; 

作者机构：

Dept Phys & Biocoordinat Chem;  Emil Hoffmann Str 33;  D-97074 Wurzburg;  Fac Pharmaceut Sci;  PL-90151 Lodz;  Ottergemsesteenweg 460;  Drug Qual & Registrat DruQuaR Grp;  Med Univ Lodz;  Inst Pharm & Food Chem;  Lab Med Chem;  D-50996 Cologne;  B-9000;  Fac Pharm;  Germany;  Univ Ghent;  Spectral Serv AG;  B-9000 Ghent;  Muszynskiego 1;  Univ Wurzburg;  Poland;  Belgium; 

关键词：

Hydrophilic interaction liquid chromatography;  Advanced therapy medicinal products;  Impurities;  Quality control;  L-ascorbic acid 2-phosphate magnesium;  Mass spectrometry; 

期刊名称：

Journal of Pharmaceutical and Biomedical Analysis: An International Journal on All Drug-Related Topics in Pharmaceutical, Biomedical and Clinical Analysis

i s s n：

0731-7085

年卷期：

2019 年 165 卷

页   码：

338-345

页   码：

摘   要：

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 KH2 PO4 buffer (pH 2.5 with HCI) 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 (R-2 = 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. (C) 2018 Elsevier B.V. All rights reserved.