HPLC-MS/MS测定人体血浆中克拉霉素浓度的方法及方法学确证

Determination of clarithromycin in human plasma by LC-MS/MS

目的建立测定人体血浆中克拉霉素浓度的高效液相色谱-串联质谱联法(HPLC-MS/MS)法。方法血浆样本用甲醇沉淀蛋白后,选用色谱柱Agilent-XDB-C8柱(2.1mm×150mm,5μm),以甲醇(A相):水(含0.1%甲酸)=85:15(V:V)的比例为流动相,流速为0.2ml/min,选用岛津(SHIMADZU)高效液相色谱系统,串联API3200型三重四极杆质谱仪,采用多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源(ESI源),正离子方式,用于定量分析的离子反应对分别为质荷比m/z748.4→m/z158.2(克拉霉素)和m/z515.3→m/z276.2(替米沙坦,内标)。结果本方法血浆中克拉霉素的线性范围为10～3000ng/ml(r>0.99),定量下限为10ng/ml,批内和批间相对标准偏差均<15%,准确度(相对回收率)相对标准偏差也均<15%。稳定性试验中,血浆中克拉霉素在方法学要求的各种贮存条件下均较稳定。结论该方法快速、灵敏、专属性强、重现性好,适用于人体内克拉霉素的药代动力学研究。

Objective LC-MS/MS method for determination of clarithromycin in human plasma was developed.Methods After protein precipitation with acetonitrile,the analyte and internal standard（I.S.）,diphenhydramine,were separated on a Shim-pack VP-ODS analytical column using the mobile phase of V（methanol）：V（water（containing 0.1% formic acid） =85：15 at a flow rate of 0.2ml/min.detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring（MRM） mode.The MRM transitions of（m/z） 748.4→m/z 158.2（clarithromycin） and m/z 515.3→m/z 276.2（telmisartan） were used to puantify clindamycin and I.S.,respectively.Results The calibration curve is linear over the concentration range of 10ng/ml ～ 3,000ng/ml with the lower limit of quantitation（LLOQ） 10ng/ml.Inter-and intra-day relative standard deviations are both less than 15%,and the relative recovery is less than 15% too.In the stability studies,clarithromycin in plasma is found to be stable under various storage conditions.It is rapid,sensitive,selective and reliable method for the determination of clarithromycin in human plasma.Conclusion The method is successfully applied to applied to a pharmacokinetic study in healthy volunteers.