超高效液相色谱-串联质谱法测定氯氮平中基因毒性杂质

Determination of genotoxic impurities in clozapine by UPLC-MS/MS

目的建立超高效液相色谱-串联质谱法(UPLC-MS/MS),测定氯氮平中基因毒性杂质。方法采用Inert Sil-ODS-3(75 mm×4.6 mm×3 mm)色谱柱;以0.1%甲酸水溶液和乙腈为流动相,流速为0.7 mL·min^(-1);质谱采用ESI负离子模式,进行多反应监测。结果通过外标法计算,邻氨基苯甲酸、环合物缩合物和环合物酸析物分别在20~100、0.16~3.2、0.48~3.2 ng·mL^(-1)范围内线性良好;低(80%)、中(100%)、高(120%)3个浓度的加样回收率为90.8%~117.2%,相对标准偏差(RSD)为3.9%~12.8%;检测限范围为0.08~10 ng·mL^(-1),邻氨基苯甲酸、环合物缩合物和环合物酸析物的定量限分别是20、0.16、0.48 ng·mL^(-1)。不同批次样品检测结果均低于限度值。结论本方法操作简便,结果可靠,适用于氯氮平中基因毒性杂质检测。

Objective To establish an analytical method based on UPLC-MS/MS for the determination of genotoxic impurities in clozapine.Methods The impurities were separated using an Inert Sil-ODS-3(75 mm×4.6 mm×3 mm)with a mobile phase composed of 0.1%formic acid aqueous solution(mobile phase A)and acetonitrile(mobile phase B)by gradient elution at a flow rate of 0.7 mL·min^(-1).Multiple reaction monitoring(MRM)was performed on a mass spectrometer equipped with an ESI source in the negative mode.Results The linearity of anthranilic acid,cyclized and condensed compounds,and cyclized acid precipitates was desirable within ranges of 20 to 100 ng·mL^(-1),0.16 to 3.2 ng·mL^(-1) and 0.48 to 3.2 ng·mL^(-1).The recoveries of spiked samples at low(80%),medium(100%)and high(120%)concentrations ranged from 90.8%to 117.2%,and RSD was between 3.9%and 12.8%.The limit of detection ranged from 0.08 to 10 ng·mL^(-1) and the limits of quantification of anthranilic acid,cyclized and condensed compounds,and cyclized acid precipitates were 20 ng·mL^(-1),0.16 ng·mL^(-1) and 0.48 ng·mL^(-1),separately.The detected concentration was below the limit.Conclusion The established method is simple and reliable,which is applicable to quantifications of three genotoxic impurities in clozapine.