LC-MS/MS法测定微透析样品中酮康唑的浓度

目的: 建立微透析样品中酮康唑的LC-MS/MS检测方法,并将其应用于酮康唑经皮肤给药后大鼠体内药动学研究。方法: 选用Agilent ZORBAX-C₁₈(4.6 mm×50 mm,1.8 μm)色谱柱,流动相为乙腈-0.1%甲酸水(70:30),流速0.3 mL·min^-1,柱温30℃,进样量10 μL;质谱条件采用ESI源正离子扫描,多级反应检测模式(MRM)。载气为氮气,载气压力为206.85 kPa;脱溶剂气体(载气)温度350℃,脱溶剂气流速11 L·min^-1;酮康唑裂解电压205 V,碰撞能量46 eV;地西泮裂解电压140 V,碰撞能量30 eV;酮康唑和内标地西泮的扫描离子片段分别为m/z 531.2→82.1、m/z 284.5→193.2。结果: 该方法专属性良好,空白基质对测定无干扰,酮康唑在1~5 000 ng·mL^-1范围内线性良好(r=0.997 8),最低定量限为1 ng·mL^-1,日内和日间精密度试验的RSD均<3.62%,基质效应范围在90%~110%之间。测得大鼠颈静脉微透析样品中酮康唑浓度范围为18.61~211.88 ng·mL^-1,大鼠腹部皮下微透析样品中酮康唑浓度范围为438.57~3 004.27 ng·mL^-1,所建立的方法能满足样品检测要求。结论: 本文所建立的方法准确快速,灵敏度高,专属性强,可用于微透析实验样品中酮康唑浓度的测定及酮康唑经透皮吸收药动学研究。

Objective: To develop an LC-MS/MS method for the determination of ketoconazole in microdialysis sample after transdermal application, and to apply it to the pharmacokinetics study of ketoconazole transdermal experiment. Methods: The separation was achieved on an Agilent ZORBAX-C₁₈(4.6 mm×50 mm, 1.8 μm) column with the mobile phase consisting of acetonitrile-water with 0.1% formic acid (70:30) at a flow rate of 0.3 mL·min^-1.The column temperature was 30℃, and the injection volume was 10 μL.An electrospray ionization(ESI)source was applied and operated in the positive multiple reaction monitoring (MRM) mode.Nitrogen was used as nebulizer gas and nebulizer pressure was set at 206.85 kPa.Cesolvation gas (nitrogen) was heated to 350℃ and delivered at a flow rate of 11 L·min^-1. Fragmentor and collision energy was 205 V and 46 eV, respectively; Fragmentor and collision energy of diazepam(internal standard)was 140 V and 30 eV, respectively.The fragment ion for ketoconazole and diazepam was m/z 531.2→82.1 and m/z 284.5→193.2, respectively. Results: Chromatograms showed no endogenous interfering peaks with blank samples.The linear calibration curve was obtained within the concentration range of 1-5 000 ng·mL^-1(r=0.997 8), and the limit of quantification was 1 ng·mL^-1.Both of the intra-day and inter-day precisions (RSDs) were less than 3.62%.The average matrix effects ranged from 90% to 110%.The concentration range of ketoconazole in the jugular vein samples and abdomen subcutaneous samples were 18.61-211.88 and 438.57-3 004.27 ng·mL^-1, respectively. Conclusion: The developed and validated method is rapid, sensitive, selective and reliable for the determination of ketoconazole in microdialysis sample after transdermal application, which can be applied to the pharmacokinetic study of ketoconazole transdermal experiment.