HPLC-MS/MS法定量研究氧化白藜芦醇对沙奎那韦在大鼠体内药动学的影响

目的:应用高效液相色谱-串联质谱(HPLC-MS/MS)技术建立大鼠血浆中沙奎那韦的定量测定方法,并用于氧化白藜芦醇对沙奎那韦在大鼠体内药动学研究。方法:液相色谱分离采用C₁₈反相色谱柱(150mm×2.1mm,5.0μm),流动相为乙腈/水(含0.2mmol·L^-1甲酸铵),流速为300μL·min^-1;质谱检测采用电喷雾正离子(ESI^+)模式,多反应监测。血浆样品采用液-液萃取方式提取,检测反应为m/z671.4→570.4(沙奎那韦),m/z721.4→296.2(利托那韦,内标)。将10只SD大鼠分为2组,对照组与实验组分别灌胃给予沙奎那韦(30mg·kg^-1)、沙奎那韦(30mg·kg^-1)+氧化白藜芦醇(40mg·kg^-1),于不同时间取血,测定血药浓度。结果:沙奎那韦在1.0~100.0ng·mL^-1范围内呈良好的线性关系(r=0.998 5),日内和日间精密度均小于12.16%,偏差为0.89%~6.24%,回收率为67.85%~82.38%,血浆样品稳定性好。同时服用氧化白藜芦醇时,大鼠体内沙奎那韦达峰浓度C_max、C_max1显著降低,而C_max、T_max、t_1/2、CL/F均无显著差异。结论:HPLC-MS/MS测定方法准确度高、专属性强、灵敏度高、快速高效。沙奎那韦的药时曲线存在双峰现象;氧化白藜芦醇可以显著降低沙奎那韦的达峰浓度,但对口服生物利用度等其他药动学参数没有显著性影响。

HPLC-MS/MS analysis and evaluation of the effects of oxy-resveratrol on pharmacokinetics of saquinavir by quantification of saquinavir in rat plasma

OBJECTIVE To develop a sensitive and rapid liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of saquinavir in rat plasma samples, and investigate the pharmacokinetics characteristics of saquinavir in rats after co-administrating with oxy-resveratrol. METHODS The analyte saquinavir and the internal standard (IS, ritonavir) were separated on a reversed phase C₁₈ column (150 mm×2.1 mm,5.0 μm) with mobile phase of acetonitrile/water containing 0.2% mmol·L^-1 ammonium formate at a flow rate of 300 μL·min^-1. Electrospray ionization source was applied and operated in the positive ion mode using multiple reactions monitoring (MRM). The plasma samples were extracted by liquid-liquid extraction method and the reactions monitored were m/z 671.4→570.4 for saquinavir and m/z 721.4→296.2 for ritonavir(IS). Ten rats were randomized into 2 groups equally, and administered orally 30 mg·kg^-1 SQV with or without 40 mg·kg^-1 oxy-resveratrol. RESULTS The method exhibited a good linearity over the concentration range of 1.0-100.0 ng·mL^-1. The values on both the occasions (intra-and inter-day) were all within 12.16%, and the accuracy was 0.89%-6.24%. The extraction recoveries were 67.85%-82.38% and the plasma samples were stable. When co-administrating with oxy-resveratrol, C_max and C_max1 of saquinavir in rats were significantly reduced while no significant difference was observed in C_max, T_max, t_1/2, or CL/F value. CONCLUSION The HPLC-MS/MS method is specific, accurate, reliable, rapid and sensitive for the pharmacokinetic study of saquinavir in rat plasma. Double peak phenomenon is observed in the plasma SQV profiles and the results demonstrate that oxy-resveratrol can significantly reduce the peak concentration but cannot significantly affect the SQV oral bioavailability and other pharmacokinetic characteristics.