Validation of ultra-high pressure liquid chromatography method for epoetin N-glycan profile
Objective:To analyze N-glycan of epoetin by ultra-high pressure liquid chromatography and validate the method,thus providing reference for the establishment and validation of corresponding quality control method.Methods:The epoetin sample was enzymatically digested with PNGase F,and the released N-glycan was labelled with 2-AB derivatization,followed by purification with magnetic beads.Liquid chromatography analysis with gradient elution for 70.0 min was performed,with 100 mmol·L-1 ammonium formate solution(pH 4.4)as mobile phase A and 70%acetonitrile as mobile phase B.Fluorescence detection was applied with excitation wavelength of 260 nm and emission wavelength of 430 nm.Comprehensive method validation was performed.Results:The injection and preparation repeatability were investigated,with RSDs of no more than 4.08%and 2.89%for relative peak area and retention time of peak groups 1~5 respectively.The intermediate precision analysis showed that the RSDs were no more than 11.85%and 8.05%for relative peak area and retention time of peak groups 1~5 respectively.The accuracy test demonstrated that the relative peak area recoveries were 98.28%~113.01%,98.68%~102.96%and 98.51%~100.75%for peak groups 2~4 respectively.When the protein concentration was 2.5~7.5 mg-mL-1,which corresponded to the protein content analyzed as 50~150 pg,the determination coefficients of linear regression for peak groups 1~5 were all greater than 0.99.The quantitative limit of the method was set as protein content of 50 µg.The prepared sample was stable after being placed at 4 ℃for48 h.Conclusion:The method has good specificity,precision,accuracy and linearity,providing a reference for the establishment and validation of the quality control method for epoetin.
N-glycanepoetinmethod validation2-AB labelingquality control