液相色谱-串联质谱法测定Beagle犬血浆中黄体酮浓度及其药动学

目的: 建立液相色谱-串联质谱法(LC-MS/MS)测定Beagle犬血浆中黄体酮含量。 方法: 取Beagle犬血浆样品,加入适量内标(炔诺酮),用液相萃取技术,经超高效液相色谱分离。色谱柱:Agilent ZORBAX-SB C₁₈分析柱(150 mm×2.1 mm,5 μm);保护柱:Agilent C₁₈分析柱(10 mm×2.1 mm,5 μm);流动相:甲醇-0.1%甲酸水溶液(90∶ 10);流速0.2 mL·min^-1。采用APCI离子化-串联质谱多离子反应监测(MRM)模式进行检测,放电电流(NC) =3.00,雾化气(GS1)=30.00,辅助气(GS2)=50.00,气帘气(CUR)=30.00,温度(TEM)=350.00,碰撞活化裂解(CAD) = Medium(以上单位均为仪器单位),离子选择通道分别为m/z 315.0/109.2(黄体酮)和m/z 299.0/109.1(内标)。 结果: 黄体酮血浆浓度在0.5~25 ng·mL^-1范围内线性关系良好(r=0.9991),日内和日间精密度试验的RSD均小于7.5 %,方法回收率为91.8%~100.7%,提取回收率为84.2%~91.5%,最低定量限为0.5 ng·mL^-1,最低检测限为0.2 ng·mL^-1。 结论: 该方法专属性强,灵敏度高,准确性好,适用于Beagle犬血浆中黄体酮含量的测定,可应用于黄体酮制剂生物等效性研究。

Objective: To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative of progesterone in Beagle dog’s plasma. Methods: The Beagle plasma sample, and additional internal standard (norethisterone), were treated with liquid-liquid extraction and separated by UPLC. Column:Agilent ZORBAX-SB C₁₈ (150 mm×2.1 mm,5 μm);guard column:Agilent C₁₈ (10 mm×2.1 mm,5 μm);mobile phase:methanol-0.1 % formic acid solution(90∶ 10);flow rate:0.2 mL·min^-1. Sample was analyzed by atmospheric pressure chemical ionization (APCI) applying MRM scan type. NC=3.00, GS1= 30.00, GS2=50.00, CUR=30.00, TEM=350.00, CAD=Medium. The selected ions for mass monitoring were m/z 315.0/109.2 (progesterone) and m/z 299.0/109.1 (norethisterone,internal standard). Results: The linear range of progesterone was 0.5-25 ng·mL^-1.Within day RSD and day to day RSD were both less than 7.5%. The method recovery and extraction recovery of the assay were 91.8%-100.7% and 84.2%-91.5% respectively. The detection limit was 0.2 ng·mL^-1. Conclusion: The established method of LC-MS/MS determining the concentration of progesterone in plasma has the characteristic of strong specificity, high sensitivity and good accuracy, suitable for quantitation of progesterone in Beagle plasma and can be applied to bioequivalence studies of progesterone preparation.