2. Academic Validation
  3. Zwitterionic-hydrophilic interaction liquid chromatography for l-ascorbic acid 2-phosphate magnesium, a raw material in cell therapy

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

  • J Pharm Biomed Anal. 2019 Feb 20:165:338-345. doi: 10.1016/j.jpba.2018.12.010.
Xiaolong Xu 1,  Jonas Urlaub 2,  Magdalena Woźniczka 3,  Evelien Wynendaele 1,  Karen Herman 1,  Curd Schollmayer 2,  Bernd Diehl 4,  Serge Van Calenbergh 5,  Ulrike Holzgrabe 2,  Bart De Spiegeleer 6
Affiliations

Affiliations

  • 1 Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
  • 2 Institute of Pharmacy & Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.
  • 3 Department of Physical and Biocoordination Chemistry, Faculty of Pharmacy, Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland.
  • 4 Spectral Service AG, Emil-Hoffmann-Straße 33, D-50996 Cologne, Germany.
  • 5 Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
  • 6 Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address: [email protected].
PMID: 30579234 DOI: 10.1016/j.jpba.2018.12.010
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.

Keywords

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

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