LC-MS/MS法测定犬体内托拉塞米浓度及相对生物利用度研究

目的:建立LC-MS/MS法测定Beagle犬体内托拉塞米的浓度,并研究托拉塞米缓释片的相对生物利用度。方法:色谱柱为Waters Sym-metry-C18(100mm×4.6mm,5μm);流动相为甲醇∶20mmol/L甲酸铵(65∶35,V∶V);流速0.4mL/min;柱温30℃。采用两制剂双周期随机交叉试验设计,分别给予6只Beagle犬受试制剂或参比制剂5mg,采用LC-MS/MS法测定给药后不同时间的血药浓度。结果:托拉塞米线性范围为0.02～5μg/mL,最低定量限为0.02μg/mL,分析方法灵敏、稳定、特异性高。受试制剂和参比制剂的主要药动学参数:峰浓度(Cmax)、达峰时间(tmax)和药-时曲线下面积(AUC0-48h)分别为(2.6±0.5)g/mL和(3.0±0.6)g/mL、(3.3±1.4)h和(1.2±0.8)h、(36.1±11.0)g.h.mL-1和(32.1±13.1)g.h.mL-1。以AUC0-48h计算,受试制剂的相对生物利用度F为(118.0±28.3)%。结论:该方法操作简单、准确、重复性好,并成功地运用于犬体内托拉塞米相对生物利用度的研究。

LC-MS/MS determination of torasemide in Beagle dog plasma and bioavailability evaluation

AIM: To establish an LC-MS/MS method for the determination of torasemide in Beagle dog plasma,and study the relative bioavailability of the torasemide sustained release tablets.METHODS: A reversed phase HPLC column of Waters Symmetry-C18(100 mm×4.6 mm,5 μm) was used in the experiment.Acetonitrile-20 mmol/L ammonium formate(65∶35,V/V) was used as mobile phase at the flow rate of 0.4 mL/min,the column temperature was set at 30 ℃.A single oral dose of 5 mg torasemide release tablets or reference tablets were given to 6 Beagle dogs in an open randomized two way crossover design.The concentrations of plasma samples collected at the different time were determined by LC-MS/MS method.RESULTS:With the linear range of 0.02-5 μg/mL and the lower limit quantification of 0.02 μg/mL,the LC-MS/MS method had high sensitivity,stability and specificity.The main pharmacokinetic parameters of test sustained release tablets and reference tablets were as follows:the Cmax were(2.6±0.5) μg/mL and(3.0±0.6) g/mL,the tmax were(3.3±1.4) h and(1.2±0.8) h,the AUC0-48 h were(36.1±11.0) μg·h·mL-1 and(32.1±13.1) μg·h·mL-1.Compared to regular tablets,the bioavailability of sustained release tablets was(118.0±28.3)%.CONCLUSION: The method is simple,accurate and robust for the determination of torasemide in Beagle dog plasma,and successfully applied it to the bioavailability evaluation of torasemide sustained release tablets given to Beagle dogs.