LC-APCI-MS/MS法研究维氨酯在大鼠体内的排泄

Study on the excretion of viaminate in rats by LC-APCI-MS/MS

目的:建立LC-APCI-MS/MS法测定大鼠胆汁、尿液和粪便中维氨酯的浓度,对该药在大鼠体内的排泄规律进行研究,为临床试验提供依据。方法:生物样本经处理后,采用AgilentTCC18色谱柱,甲醇-水-甲酸(9370.1)为流动相,辛伐他汀为内标,在三重四极杆串联质谱仪上,采用大气压化学离子化(APCI)、正离子方式选择性离子监测模式,维氨酯和内标的检测离子分别为m/z448/283和m/z419/285,分别测定尿液、胆汁和粪便中原型物的含量。结果:胆汁、尿样、粪便中维氨酯的回收率均大于89%,日间和日内的变异系数均小于10%。胆汁和尿液中维氨酯在0.0808～40.4ng/mL浓度范围内线性关系良好(r=0.9972和0.9949),粪便中维氨酯在0.2525～101ng/mL浓度范围内线性关系良好(r=0.9990),维氨酯的定量下限为0.2pg,方法符合生物样品分析要求。维氨酯在粪便中72h内、在尿液中96h内、在胆汁中24h内原型药的平均累积排泄量分别为12.9%、0.003%、0.0001%。结论:该方法灵敏度高,专属性强,样品前处理简便,分析速度快,可满足大鼠排泄研究中药物浓度测定的要求,大鼠灌胃给药(5mg/kg)后维氨酯从尿液和胆汁中排泄的原型药物很少。

AIM： To establish a highly sensitive, rapid and selective liquid chromatography-tandem mass spectrometry （LC-MS/MS） method to determinate the viaminate in bile, urine and feces sample of rats. And to study the excretion of viaminate in rats in order to provide a guidance for the clinical trial. METHODS： The concentration of viaminate was determinated on an Aglilent TC C17 column and the mobile phase consisted of methanol-water and formic acid （93 ： 7 ： 0.1, V/ V/V） using simvastatin as internal standard. The detection was performed on a trip-quadrupole tandem mass spectrometer by selected ion monitoring （SRM） scan mode via atmospheric pressure chemical ionization （APCI）. The detected ions were m/z 448/283 （viaminate） and m/z 419/285（internal standard） respectively. The accumulative excretion amount of viaminate was calculated in the urine, feces and bile, respectively. RESULTS： The extraction recoveries of viaminate in bile,urine and feces were more than 89%. The RSD of intra-run and inter-run precisions were all less than 10%. The calibration curves were linear over the concentration ranges of 0.0808-40.4 ng/mL（ r = 0.9972 and 0. 9949） for the viaminate in bile and urine. The calibration curve was linear over the concentration ranges of 0.505 - 50.5 ng/mL （ r = 0.9990） for the viaminate in feces, The limit of quantitation of method for viaminate was 0.2 pg. 12.9% viaminate was found from feces in 72 hours, 0.003 % was found from urine in 96 hours and 0.0001% was detected from bile in 24 hours. CONCLUSION： The method is proved to be accurate, sensitive, selective and convenient. It has successfully been applied to study the excretion of viaminate in rats. The amount of viaminate recovered from bile and urine after orally administrated to rat was very low.