LC-MS/MS法同时测定兔血浆中甲硝唑和盐酸克林霉素的浓度

Simultaneous determination of metronidazole and clindamycin hydrochloride in rabbit plasma by LC-MS/MS

目的建立同时测定兔体内血浆中甲硝唑和克林霉素浓度的LC-MS/MS法,并进行方法学验证,然后应用于兔体内药动学研究。方法本试验采用LC-MS/MS法同时测定阴道给予兔复方甲硝唑原位凝胶后血浆中甲硝唑和克林霉素的浓度。采用液-液萃取法对血浆样品进行前处理,色谱柱为Thermo C_(18)column(10 mm×4.6 mm,5μm),流动相:0.05%甲酸-乙腈(V∶V=30∶70),等度洗脱,流速为0.2 mL·min^(-1)。质谱条件为ESI源,扫描方式为选择反应监测,正离子模式,定量分析的离子反应对分别为甲硝唑:m/z 172→128(15 eV),克林霉素:m/z 425→126(21 eV),替硝唑(内标):m/z 248→121.1(21 eV)。结果甲硝唑和克林霉素的线性范围分别在25~1500μg·L^(-1)(r=0.9932)和50~3000μg·L^(-1)(r=0.9920)内线性关系良好,两者定量下限分别为25μg·L^(-1)和50μg·L^(-1),准确度(RE)均在-5.7%~3.7%之间,日内、日间精密度(RSD)均小于6.9%,提取回收率均在86.0%~101.1%之间。结论该方法专属性强、准确性好、灵敏度高,可用来同时测定甲硝唑和克林霉素的血药浓度,为探讨复方甲硝唑原位凝胶在兔体内的药动学行为提供依据。

Objective To establish and validate a method for the simultaneous determination of metronidazole and clindamycin in rabbit plasma by LC-MS/MS method,and apply it to the in vivo pharmacokinetics of rabbits.Methods The concentrations of metronidazole and clindamycin were measured simultaneously after administration the compound metronidazole in situ gel to rabbits via vagina by LC-MS/MS method.Plasma samples were pretreated by liquid-liquid extraction.Metronidazole and clindamycin in rabbit plasma were separated on Thermo C_(18) column(10 mm×4.6 mm,5 μm) with the mobile phase consisting of 0.05% formic acid-acetonitrile(V∶V=30∶70),iso-elution,and the flow rate was 0.2 mL·min^(-1).With an electrospray source interface in positive ionization mode,detection and quantification were carried out in selected reaction-monitoring mode at m/z 172→128(15 eV) for metronidazole,m/z 425→126(21 eV) for clindamycin,m/z 248→121.1(21 eV)for tinidazole(internal standard),respectively.Results The linear ranges of metronidazole and clindamycin were within the range of 25 to 1 500 μg·L^(-1)(r=0.993 2)and 50 to 3 000 μg·L^(-1)(r=0.993 2) respectively,and the lower limits of quantification were 25 μg·L^(-1)and 50 μg·L^(-1),respectively.The accuracy(RE)was-5.7% to 3.7%,the intra-day and inter-day precision(RSD) were less than 6.9%,and the extraction recovery was 86.0% to 101.1%.Conclusion This method has strong specificity,high sensitivity and good accuracy.It is suitable for simultaneous determination of metronidazole and clindamycin in rabbits plasmas,and can be investigated the pharmacokinetics of compound metronidazole in situ gel in rabbits.