Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer

作者：Xu Xiaolong; Gevaert Bert; Bracke Nathalie; Yao Han; Wynendaele Evelien; De Spiegeleer Bart

来源：Journal of Pharmaceutical and Biomedical Analysis, 2017, 135: 227-233.

DOI：10.1016/j.jpba.2016.11.054

• HILIC
• HEPES
• EPPS
• N-oxide degradant
• Zwitterionic

HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5 mL/min and UV detection at 195 nm. The assay method of HEPES was validated, obtaining adequate linearity (R-2 =0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.

• 出版日期2017-2-20