title: Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer.
abstract: 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 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R2=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.
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Xu
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Gevaert
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bracke
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
authors.forename: Han
authors.initials: H
authors.lastname: Yao
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Wynendaele
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address: Bart.DeSpiegeleer@UGent.be.
authors.forename: Bart
authors.initials: B
authors.lastname: De Spiegeleer
mesh_terms: D002021:Buffers; D002626:Chemistry, Pharmaceutical / Q000379:methods* / Q000592:standards*; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D006531:HEPES / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D015203:Reproducibility of Results
chemical_list: D002021:Buffers; D006531:HEPES
keywords: EPPS; HEPES; HILIC; N-oxide degradant; Zwitterionic