UPLC-Q-TOF-MS法测定绵羊血浆中阿苯达唑及其3种代谢产物的浓度及药代动力学研究

Simultaneous determination of albendazole and its three metabolites in sheep plasma by UPLC-Q-TOF-MS and pharmacokinetic study

目的建立同时测定绵羊血浆中阿苯达唑(ABZ)、阿苯达唑亚砜(ABZSO)、阿苯达唑砜(ABZSO_2)、阿苯达唑氨基砜(ABZSO_2-NH_2)浓度的超高效液相色谱-飞行时间质谱联用(UPLC-Q-TOF-MS)分析方法,并研究阿苯达唑脂质体混悬液在健康绵羊体内的药代动力学特征。方法 6只绵羊单剂量灌胃给予阿苯达唑脂质体混悬液(10mg/kg)后,不同时间点颈静脉采血,血浆样品经乙腈沉淀蛋白法处理后,采用UPLC-Q-TOF-MS法测定血药浓度,内标为甲苯咪唑(MBZ),色谱柱为ACQUITY UPLC~? BEH C_(18)(2.1mm×50mm,1.7μm),流动相为甲醇-0.1%甲酸,梯度洗脱,流速为0.4mL/min,进样量为2μL,柱温为40℃,采用电喷雾离子化(ESI)源,正离子扫描模式,选择监测离子检测(SIM),ABZ m/z 266.096,ABZSO m/z 282.091,ABZSO_2m/z 298.086,ABZSO_2-NH2m/z 240.081,内标MBZ m/z 296.104,应用Kinetica程序计算药代动力学参数。结果阿苯达唑浓度为6.2～798.0ng/mL,阿苯达唑亚砜浓度为5.2～5 370.0ng/mL,阿苯达唑砜浓度为3.3～3 168.0ng/mL,阿苯达唑氨基砜浓度为8.6～1 100.0ng/mL,线性关系良好(r=0.999 5、0.998 1、0.999 4、0.998 8),定量下限分别为6.2、5.2、3.3、8.6ng/mL,提取回收率均>95.3%,日间、日内精密度RSD均<15.0%,在本实验涉及的条件下样品稳定。绵羊灌胃给予阿苯达唑脂质体混悬液后,血浆中检测不到ABZ母体药物;给药后36、48h可检测到较低浓度ABZSO_2-NH_2。用非房室模型测得主要药代动力学参数,ABZSO和ABZSO_2的T_(1/2)、C_(max)、AUC_(0-∞)分别为(5.13±2.00)h、(3.78±0.78)μg/mL、(59.10±6.24)μg×h/mL和(3.29±0.09)h、(0.53±0.070)μg/mL、(13.66±1.54)μg×h/mL。结论本方法可靠、专属性强,适用于ABZ及其代谢物的血药浓度测定和药代动力学研究,为综合评价阿苯达唑脂质体混悬液提供参考。

Objective To develop a UPLC-Q-TOF-MS method for the determination of albendazole(ABZ),ricobendazole(ABZSO),albendazole sulfone(ABZSO2)and albendazole sulfone amine(ABZSO2-NH2)in sheep plasma and study pharmacokinetic characteristics of liposome-entrapped albendazole in healthy sheep.Methods A single oral dose of10mg/kg liposome-entrapped albendazole was administered to6healthy sheep.The blood for test was collected at the jugular vein.The plasma samples were pretreated by acetonitrile for protein precipitation and the plasma concentration was determined by UPLC-Q-TOF-MS.Mebendazole was used as an internal standard(IS).The ACQUITY UPLC^■BEH C(18)column(2.1mm×50mm,1.7μm)was used with the mobile phase of methanol and water containing0.1%formic acid at the flow rate of0.4mL/min.The injection volume was2μL and the column temperature was40℃.The detection was operated using target ions of[M+H]+at m/z266.096for ABZ,m/z282.091for ABZSO,m/z298.086for ABZSO2,m/z240.081for ABZSO2-NH2and m/z296.104for IS in selective ion mode,respectively.The main parameters of pharmacokinetics were calculated by kinetica5.1software.Results The linear calibration curves were obtained.The lowest limit of quantification of ABZ,ABZSO,ABZSO2and ABZSO2-NH2were6.2ng/mL,5.2ng/mL,3.3ng/mL and8.6ng/mL,respectively.The intra-day and inter-day precision(RSDs)were lower than15.0%and the extraction recovery were higher than95.3%.The stability of this method was satisfactory too.T(1/2),C(max)and AUC(0-∞)of ABZSO and ABZSO2was(5.13±2.00)h,(3.78±0.78)μg/mL,(59.10±6.24)μg×h/mL and(3.29±0.09)h,(0.53±0.070)μg/mL,(13.66±1.54)μg×h/mL respectively.ABZ was not detected in plasma at any time post-treatment.ABZSO2-NH2was at low levels at36h,48hafter the administration or was not visible in the chromatograms.The main pharmacokinetic parameters were calculated by kinetica5.1software using non-compartmental analysis.The T(1/2),C(max)and AUC(0-∞)of ABZSO were(5.13±2.00)h,(3.78±0.78)μg/mL and(59.10±6.24)μg×h/mL,respectively.The T(1/2),C(max)and AUC(0-∞)of ABZSO2were(3.29±0.09)h,(0.53±0.070)μg/mL and(13.66±1.54)μg×h/mL,respectively.Conclusion The method is reliable and selective for plasma concentration determination and pharmacokinetic research of ABZ,ABZSO,ABZSO2and ABZSO2-NH2,providing reference for comprehensive evaluation of liposome-entrapped albendazole.