液相色谱-串联质谱法检测大鼠血浆盐酸妥布特罗含量及其贴剂药代动力学的研究

Simultaneous determination of tulobuterol in rat plasma by LC-MS/MS and its application in pharmacokinetic study of tulobuterol patch

目的建立测定大鼠血浆中盐酸妥布特罗浓度的方法,并对盐酸妥布特罗贴剂药代动力学进行研究。方法采用液相色谱-串联质谱法进行检测,以克仑特罗为内标,安捷伦氨基C18分析柱为色谱柱,A(0.1%甲酸+100%水)及B(甲醇)为流动相进行色谱分离。色谱条件为0~2.5 min,70%A→40%A;2.5~2.6 min,40%A→70%A;2.6~5 min,70%A。采用电喷雾电离源和多反应监测的方式进行正电荷检测,用作定量分析的盐酸妥布特罗和克仑特罗的离子反应对m/z分别为228.2→154.0和277.1→203.0。结果盐酸妥布特罗在0.5~100 ng/m L范围内线性关系良好;最低检测限分别为0.5 ng/m L;盐酸妥布特罗样本的日内及日间精密度和准确度均符合相关生物样品分析的要求,且RSD<10%。盐酸妥布特罗大鼠血浆样品在室温放置7 h、经历3次冷冻-解冻循环及在-80℃中放置120 d等条件下均可保持稳定;该方法成功运用于盐酸妥布特罗贴剂的药代动力学研究。结论该方法具有检测快速且灵敏性好等特点,可对其人体临床相关药动学研究展开探讨。

Objective To establish a method for simultaneous determination of tulobuterol in rat plasma and study the pharmacokinetics of tulobuterol patch. Methods After plasma sample was treated with protein precipitation combined with extraction,the determination was performed on Angilent Venusil MP C18 column with clenbuterol as the internal standard and 0. 1% formic acid ＋ 100% water（ A） and methanol（ B） as the mobile phase（ 0 ~ 2. 5 min,70%A→40% A; 2. 5 ~ 2. 6 min,40% A →70% A; 2. 6 ~ 5 min,70% A）. Tulobuterol（ 228. 2 → 154. 0） and clenbuterol（ 277. 1→203. 0） were detected by electrospray ionization（ ESI） with positive ion in MRMmode quantitatively. Results The linear range was 0. 5 ~ 100 ng/m L for tulobuterol. The lower limits of quantitation was 0. 5 ng/m L. The intraday and inter-day precision and accuracy were all in line with the requirements of biological sample analysis. The plasma samples were stable after three freeze-thawcycles and being stored for 7 h at room temperature and 120 days at-80℃. Conclusion The method has the advantages of fast detection and good sensitivity,which can be used to investigate the pharmacokinetics of the human body in clinic.