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

LC- MS/MS determination of cyclosporine A in Beagle blood and bioequivalence study of cyclosporine A microemulsion

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）（2.0mm×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 m L·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1 220.2/1 202.9（环孢素A）、1 234.3/1 216.9（环孢素D,内标）。结果：本方法的线性范围为20.16~2 016 ng·m L-1;最低定量浓度为：20.16 ng·m L-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AUC0-t计算,受试制剂的相对生物利用度为95.42%~100.10%;以AUC0-∞计算,受试制剂的相对生物利用度为95.82%~99.14%。剂量矫正后Cmax和AUC0-∞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-1ammonium formate）- mobile phase B [ methanolacetonitrile （ 1 ： 1, with 5 mmol · L - 1 ammonium formate ） ] with the gradient elution （ 0- 0. 8 min,60% B;0. 8-1.3 rain,60% B→00% 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-l. 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 AUC0-t ,and the relative bioavailabilities of the test formulation was 95.82%-99. 14% as calculated by AUC0-∞. 90% confidence interval （CI） of the after dose correction Cmax and AUC0-∞ 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.