LC-MS/MS法测定Beagle犬全血中环孢素A浓度及环孢素微乳制剂的生物等效性研究

目的:建立液相色谱串联质谱法测定Beagle犬全血中环孢素A的浓度,并考察3种环孢素微乳制剂的生物等效性。方法:全血样品中加入适量内标和氢氧化钠,以乙醚萃取后用API 3000LC-MS/MS仪进行分析。采用CAPCELLPAK(C₁₈)(2.0 mm×50 mm,5μm)色谱柱,以水(5 mmol·L^-1甲酸铵)为流动相A,甲醇-乙腈(1:1,含5 mmol·L^-1甲酸铵)为流动相B,以0.4 mL·min^-1的流速进行梯度洗脱(0~0.8 min,60%B;0.8~1.3 min,60%B→100%B;1.3~2.5 min,100%B;2.5~3.7 min,60%B),柱温65℃;质谱采用多反应离子监测(MRM)的扫描模式,以电喷雾离子源(ESI)在正离子电离模式下进行测定,选择性监测质荷比(m/z)为1220.2/1202.9(环孢素A)、1234.3/1216.9(环孢素D,内标)。结果:本方法的线性范围为20.16~2016 ng·mL^-1;最低定量浓度为:20.16 ng·mL^-1。日内、日间精密度均小于7.1%,准确度在92.9%~108.2%之间,提取回收率为63.8%~68.3%,稳定性考察结果良好。以AUC_0-t计算,受试制剂的相对生物利用度为95.42%~100.10%;以AUC_0-∞计算,受试制剂的相对生物利用度为95.82%~99.14%。剂量矫正后C_max和AUC_0-∞的90%置信区间(CI)均在80%~125%范围内,2种口服液和1种软胶囊制剂生物等效。结论:本法经方法学验证,适用于测定Beagle犬全血中环孢素A浓度及其生物等效性研究。

Objective:To develop an LC-MS/MS method for the determination of cyclosporine A(CsA)in Beagle blood and to investigate the bioequivalence of 3 kinds of cyclosporine A microemulsion.Methods:The blood samples were extracted with diethyl ethyl after addition of internal standard cyclosporine D(CsD)and sodium hydroxide solution,and then analyzed in API 3000 LC-MS/MS system.The analytical column was CAPCELLPAK(C₁₈)(2.0 mm×50 mm,5 μm).The mobile phase consisted of mobile phase A(water with 5 mmol·L^-1 ammonium formate)-mobile phase B[methanol-acetonitrile(1:1,with 5 mmol·L^-1 ammonium formate)] with the gradient elution(0-0.8 min,60%B;0.8-1.3 min,60%B→100%B;1.3-2.5 min,100%B;2.5-3.7 min,60%B)at a flow rate of 0.4 mL·min^-1,and the column temperature was controlled at 65℃.Detection was performed with multiple reactions monitoring(MRM) using positive electrospray ionization(ESI),and the selected ion mass(m/z)were 1 220.2/1 202.9(CsA)and 1 234.3/1 216.9(CsD,internal standard).Results:The calibration curves were linear over the concentration range of 20.16-2 016 ng·mL^-1 for cyclosporine A.The lower-limit-of-quantification was 20.16 ng·mL^-1.Inter-and intra-day precisions were all below 7.1% and the accuracy was ranged within 92.9%-108.2%.Extraction recoveries were 63.8%-68.3%,and the analyses were proved to be stable.The relative bioavailabilities of the test formulation was 95.42%-100.10% as calculated by AUC_0-t,and the relative bioavailabilities of the test formulation was 95.82%-99.14% as calculated by AUC_0-∞.90% confidence interval(CI)of the after dose correction C_max and AUC_0-∞ were between 80%-125%,and these three cyclosporine A microemulsion were bioequivalent.Conclusion:The established method is rapid,sensitive,and specific,which is applicable for the concentration determination of cyclosporine A and its bioequivalence study.