高效液相色谱-串联质谱法测定人血清中替加环素的浓度

Determination of tigecycline in human serum by liquid chromatography-tandem mass spectrometry

目的:建立人血清中替加环素浓度的高效液相色谱-串联质谱法(HPLC-MS/MS)测定方法。方法:以[叔丁基-d9]替加环素为内标,将待测血清样品经氢氧化钠碱化后,加乙酸乙酯-二氯甲烷(4∶1,V/V)提取,上清液经真空干燥,得到的残渣经流动相复溶后进样。进样液经Agilent Eclipse plus C8(4.6 mm×100 mm,5μm)色谱柱分离,甲醇-水(30∶70,V/V,含0.005mol·L^(-1)甲酸铵和0.25%甲酸)为流动相,流速0.5 mL·min^(-1),进行洗脱,采用电喷雾电离源(ESI),以多反应监测(MRM)方式进行正离子检测。用于定量分析的离子分别为替加环素m/z586.4→513.3和[叔丁基-d9]替加环素m/z595.4→514.3。结果:血清中替加环素的浓度与峰面积比在10~2 000 ng·mL^(-1)范围内线性关系良好,定量下限为10 ng·mL-1,提取回收率大于70.04%,日内日间精密度小于4.54%。结论:该法简单、灵敏、快速,分离效果良好,可用于人体血药浓度测定和PK/PD研究。

OBJECTIVE To develop a HPLC-MS/MS method for determination of tigecycline in human serum.METHODSSerum samples were alkalified with sodium hydroxide,extracted with ethyl acetate-dichloromethane（4∶1,v/v）,and then separated on a Eclipse plus C_8（4.6 mm×100 mm,5μm）column with tramadol as internal standard.The mobile phase was composed of methanol and ammonium formate-formic acid buffer containing 0.005 mol·L^-1 ammonium formate and 0.25%formic acid（30∶70,v/v）,at a flow rate of 0.5 mL·min^-1.Multiple reaction monitoring（MRM）transition of m/z586.4→513.3 and m/z595.4→514.3 was performed in positive mode.RESULTS Calibration curves of tigecycline were linear in range of 10-2000 ng mL^-1.The lower limit of quantification was 10 ng mL^-1.The recovery was more than 70.04%.The intra and inter-day precisions were less than 4.54%.CONCLUSION With high selectivity,acceptable accuracy,precision and sensitivity,this validated HPLC/MS/MS method is successfully applied to pre-clinical pharmacokinetic study of tigecycline in human serum.