液相色谱-串联质谱法测定大鼠血浆中黄体酮的浓度

Determination of progesterone in rat plasma by LC-MS/MS

目的建立测定大鼠血浆中黄体酮的液相色谱-串联质谱（LC-MS/MS）法。方法血浆样品采用液-液萃取法,经正己烷-二氯甲烷-异丙醇（100∶50∶5,V/V/V）提取后,以甲醇-水（75∶25,V/V）为流动相,以Hypersil ODS柱（50 mm×2.1 mm,5μm）为分析柱,采用ESI源以多反应监测（MRM）方式进行正离子检测。用于定量分析的离子反应为m/z 315.4→m/z（97.0＋109.2）（黄体酮）和m/z 385.4→m/z 267.4（醋酸甲地孕酮,内标）。结果黄体酮血浆浓度测定方法的线性范围为0.5～200 ng/ml,日内、内间精密度（RSD）均〈6.7%,准确度（RE）在±6.2%之间。结论该方法分析时间短、操作简便、灵敏度高、专属性强,适用于黄体酮的药动学研究。

Objective To develop and validate an LC-M S/MS method for determination of progesterone in rat plasma.Methods Plasma samples were prepared by liquid-liquid extraction with a mixture of n-hexane/dichloromethane/isopropanol（100∶50∶5,V/V/V）. Isocratic chromatographic separation was performed on a Hypersil ODS column（50 mm × 2.1 mm,5 μm,Thermo）.The mobile phase was methanol-water（75∶25,V/V）. Detection of progesterone and the internal standard（IS） megestrol acetate was achieved by ESI MS/MS in the positive ion mode using m/z 315.4 → m/z（97.0 ＋ 109.2） and m/z 385.4 → m/z 267.4 transitions,respectively. Results The method was linear from 0.5 to 200 ng/ml when 100 μl plasma was analyzed. The lower limit of quantification was 0.5 ng/ml. Both the inter- and intra-day precision values were below 6.7%,and the accuracy（relative error）was within ±6.2% in all quality control samples. Conclusion The method provides a specific,sensitive and rapid means for the determination of progesterone in rat plasma,and completely meets the requirements for pharmacokinetic study.