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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 establish a liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the determination of ciclosporin A concentration in whole blood of Beagle dogs and investigate the bioequivalence of the three cyclosporine microemulsion formulations. Methods: Whole blood samples were spiked with appropriate amount of internal standard and sodium hydroxide. After extraction with ether, analysis by API 3000 LC-MS / MS. The mobile phase A was consisted of CAPCELLPAK (C18) (2.0 mm × 50 mm, 5 μm), mobile phase A (5 mmol·L -1 formic acid), methanol-acetonitrile Ammonium) was used as mobile phase B and gradient elution was carried out at a flow rate of 0.4 m L · min -1 (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 65 ℃. The mass spectrometry was performed with multi-reactive ion monitoring (MRM) scan mode and electrospray ionization (ESI) The mass-to-charge ratio (m / z) was 1 220.2 / 1 202.9 (cyclosporine A) and 1 234.3 / 1 216.9 (cyclosporin D, internal standard). Results: The linear range of this method was 20.16 ~ 2 016 ng · m L-1; the lowest concentration was 20.16 ng · m L-1. The intra-day and inter-day precision were less than 7.1% and the accuracy was between 92.9% and 108.2%. The extraction recoveries ranged from 63.8% to 68.3%. The stability was good. The relative bioavailability of the tested formulations was 95.42% -100.10% by AUC0-t. The relative bioavailability of the tested formulations was 95.82% -99.14% based on AUC0-∞. The 90% Confidence Intervals (CI) for Cmax and AUC0-∞ after dose correction ranged from 80% to 125%, and two oral solutions and one soft capsule formulation were bioequivalent. Conclusion: This method is validated by methodology and is suitable for the determination of ciclosporin A concentration and its bioequivalence in whole blood of Beagle dogs.