高效液相色谱-质谱联用法测定Beagle犬血浆中黄体酮的浓度

Determination of Progesterone in Beagle Dog Plasma by High Performance Liquid Chromatography-Tandem Mass Spectrometry

目的 建立灵敏的高效液相色谱-质谱联用法(HPLC-MS/MS),测定Beagle犬血浆中的黄体酮浓度。方法 选用Thermo Accucore C_(18)色谱柱(100 mm×2.1 mm,2.6μm),以0.1%甲酸水(A)-乙腈(B)为流动相,采用梯度洗脱进行分离,流速为0.4 ml/min,进样量10μl。样品采用乙腈进行蛋白沉淀,通过电喷雾电离源,以多重反应监测(MRM)方式进行正离子检测,用于定量分析的离子对为m/z 315.1→109.2(黄体酮)和m/z 271.4→171.9(内标,甲苯磺丁脲)。结果 黄体酮的线性范围为0.5~100 ng/ml,定量下限(LLOQ)可达0.5 ng/ml,提取回收率(n=6)分别为95.97%,105.20%,108.09%,日内、日间精密度试验RSD均小于15%。结论 该方法操作简单,灵敏度高,准确,重现性好,适用于Beagle犬血浆样品中黄体酮的测定和药动学研究。

Objective To develop a sensitive high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method for determination of progesterone in Beagle dog plasma.Methods The separation was accomplished in a Thermo Accucore C18 column(100 mm×2.1 mm,2.6 μm),using 0.1 % formic acid-water(A)-acetonitrile(B) as mobile phase of gradient elution.The flow rate was 0.4 ml/min,and the injection volume was 10 μl.The samples were treated with acetonitrile to precipitate the proteins,then the positive ion detection was carried out by the electrospray ion source in the multiple reaction monitoring(MRM) mode.MRM transitions of m/z 315.1→109.2(progesterone) and m/z 271.4→171.9(tolbutamide) were used for quantitative analysis.Results The calibration curve for progesterone was linear over the range of 0.5-100 ng/ml and the lower limit of quantification(LLOQ) was 0.5 ng/ml.The recoveries were 95.97 %,105.20 %,108.09 % respectively and the RSDs of intra-day and inter-day were less than 15 %.Conclusion The method is simple,sensitive,accurate and reproducible.It is applicable for determination and pharmacokinetic study of progesterone in Beagle dogs.