以氘代利伐沙班为内标物的UHPLC-MS/MS法测定Beagle犬血浆中利伐沙班及其药动学研究

Determination of Rivaroxaban in Beagle Dog Plasma by UHPLC-MS/MS with Deuterium Substituted Rivaroxaban as Internal Standard and Its Application in Pharmacokinetics Studies

关键词:  UHPLC-MS/MS  利伐沙班  血药浓度  药动学  Beagle犬

Key words:  UHPLC-MS/MS  rivaroxaban  drug concentration  pharmacokinetics  Beagle dog

摘要:

目的 建立测定Beagle犬血浆中利伐沙班浓度的UHPLC-MS/MS法。方法 以甲基叔丁基醚-二氯甲烷（2：1）沉淀蛋白处理血浆样品，氘代利伐沙班（利伐沙班-d₄）为内标，采用Thermo BDS Hypersil C₁₈色谱柱（100 mm×4.6 mm，2.4 μm）；流动相：A相为甲醇溶液（含0.015 mol·L^-1甲酸铵和0.1%甲酸）；B相为水-甲醇（90：10）溶液（含0.015 mol·L^-1甲酸铵和0.1%甲酸），梯度洗脱。质谱条件为电喷雾离子源，正离子模式检测，扫描方式为多反应离子监测。用于定量检测的离子反应分别为[M+H]⁺ m/z 436@30 eV→m/z 144.900（利伐沙班）和[M+H]⁺ m/z 440@24 eV→m/z 144.900（利伐沙班-d₄）。结果 血浆利伐沙班浓度在0.5~400 ng·mL^-1内线性关系良好（r=0.996 1），定量下限为0.5 ng·mL^-1，批内、批间精密度均<13%，提取回收率89.4%~96.5%，基质效应94.7%~97.2%。血浆样品冻存（-80℃）33 d稳定性良好，反复冻融3次及提取后室温放置12 h条件下，样品浓度均无显著变化。将本法应用于Beagle犬口服给予利伐沙班片后的药动学考察，主要药动学参数如下：T_max（2.11±1.133） h，C_max（179.3±50.65） ng·mL^-1，t_1/2（10.05±4.34） h，AUC_0-t（1 161.6±339.74） h·ng·mL^-1。结论 试验结果表明本法操作简单，灵敏度高，重复性良好，结果准确可靠，可用于测定Beagle犬血浆中利伐沙班浓度和其体内的药动学研究。

Abstract:

OBJECTIVE To establish an UPLC-MS/MS method for the determination of rivaroxaban in Beagle dog plasma. METHODS The plasma samples were treated with protein precipitated by methyl tert-butyl ether-dichloromethane (2:1), the deuterium substituted rivaroxaban(rivaroxaban-d₄) was selected as internal standard. The chromatographic separation was performed with a Thermo BDS Hypersil C₁₈ column (100 mm×4.6 mm, 2.4 μm) and gradient mobile phase (mobile phase A was methanol solution containing 0.015 mol·L^-1 ammonium formate and 0.1% formic acid; mobile phase B was water-methanol (90:10) solution containing 0.015 mol·L^-1 ammonium formate and 0.1% formic acid.). Mass spectrometry conditions were ESI⁺ with multiple reaction monitoring(MRM) mode. The ion reactions used for quantitative detection were[M+H]⁺ m/z 436@30 eV→ m/z 144.900 (rivaroxaban) and[M+H]⁺ m/z 440@24eV→m/z 144.900 (rivaroxaban-d₄). RESULTS Good linearity was obtained for rivaroxaban in the range of 0.5-400 ng·mL^-1(r=0.996 1), the lower quantification of current method was 0.5 ng·mL^-1 for rivaroxaban. The intra- and inter-batch precision were <13%. The absolute recoveries ranged from 89.4% to 96.5% and the matrix effects were 94.7%-97.2%. The plasma samples were stored frozen (-80℃) for 33 d with good stability. After three freeze-thaw cycles of the samples and at room temperature for 12 h after extraction, the change in samples concentration were not obvious. It was applied in dog pharmacokinetic study after oral administration of rivaroxaban tablets. The main pharmacokinetic parameters were as follows:T_max (2.11±1.133)h, C_max (179.3±50.65)ng·mL^-1, t_1/2(10.05±4.34)h, AUC_0-t (1161.6±339.74)h·ng·mL^-1. CONCLUSION The trial results show that the method is simple in operation, high in sensitivity, good in reproducibility, accurate and reliable, and can be used to determine the concentration of rivaroxaban in Beagle dog plasma and in vivo pharmacokinetic studies in Beagle dog.