LC-ESI-MS测定新的抗胆碱能化合物苯环喹溴铵在大鼠胆汁、尿液和粪便中的含量

目的：首次建立了LC-ESI-MS法测定大鼠胆汁、尿液和粪便中苯环喹溴铵的浓度,对该药在大鼠体内的排泄规律进行研究,为该药在大鼠体内的代谢转化途径的研究和临床试验提供依据。方法：生物样品经过C18固相小柱提取后,采用LC-ESI-MS法进行测定。以1-乙基-苯环喹溴铵为内标,在Hanbon Lichrospher5-C18色谱柱上,甲醇-（含40mmol/L醋酸铵和1%甲酸）水溶液（70：30,v/v）为流动相,流速为0.8mL/min分析胆汁样品;甲醇-（含40mmol/L醋酸铵和1%甲酸）水溶液（75：25,v/v）为流动相,流速为1.0mL/min分析尿液、粪便样品;以气动辅助电喷雾离子化（ESI）为接口技术,选择性正离子监测方式,苯环喹溴铵的分子离子（M]^＋m/z330.2）和内标1-乙基-苯环喹溴铵的分子离子（M]＋m/z344.2）作为测定离子。结果：胆汁、尿样、粪便中苯环喹溴铵的回收率均大于77%,日间和日内的变异系数均小于15%。胆汁中苯环喹溴铵在30.15～1507.50ng/mL浓度范围内线性关系良好（r=0.9995）,最低定量限为30.15ng/mL。尿液中苯环喹溴铵在3.02～1507.50ng/mL浓度范围内线性关系良好（r=0.9997）,最低定量限为3.02ng/mL。粪便中苯环喹溴铵在3.02～1507.50ng/mL浓度范围内线性关系良好（r=0.9995）,最低定量限为3.02ng/mL。结论：该方法专属性强,准确可靠,灵敏度高,可满足大鼠鼻腔给药排泄研究中药物浓度测定的要求。

Determination of bencycloquidium bromide, a novel anticholinergic compound, in rat's bile, urine and feces by LC-ESI-MS

AIM:This study was undertaken to study the excretion rule of bencycloquidium bromide(BCQB)in rats,and a LC-ESI-MS method was developed for the determination of BCQB in bile,urine and feces sample of rats.METHODS:A liquid chromatography-electrospray ionization-mass spectrometry(LC-ESI-MS)assay for the determination of BCQB in rats bile,urine and feces was firstly developed and validated.After addition of 1-ethyl-bencycloquidium bromide as an internal standard(I.S.),the biological samples were prepared by using solid phase extraction on a C18-cartridge column.Chromatographic separation was analyzed on a Hanbon Lichrospher 5-C18 column.The mobile phase consisted of methanol-40 mmol/L ammonium acetate buffer-formic acid(70:30:0.3,v/v/v)was pumped at 0.8 mL/min for bile.The mobile phase consisted of methanol-40 mmol/L ammonium acetate buffer-formic acid(75:25:0.25,v/v/v)was pumped at 1.0 mL/min for urine and feces.LC-ESI-MS was carried out on a single quadrupole mass spectrometer using electrospray ionization(ESI)and positive selected-ion monitoring(SIM).Target ions were monitored at M]+ m/z 330.2 for BCQB and M]+m/z 344.2 for I.S..RESULTS:The extraction recoveries of BCQB in bile,urine and feces were upper 77%.The RSD of intra-run and inter-run precisions were all below 15%.The calibration curves were linear over the concentration ranges of 30.15-1507.50 ng/mL(r=0.9995)for the BCQB in bile,and the low limit of quantitative(LLOQ)was 30.15 ng/mL.The calibration curves were linear over the concentration ranges of 3.02-1507.50 ng/mL(r=0.9997)for the BCQB in urine,and the LLOQ was 3.02 ng/mL.The calibration curves were linear over the concentration ranges of 3.02-1507.50 ng/mL(r=0.9997)for the BCQB in feces,and the LLOQ was 3.02 ng/mL.CONCLUSION:The method is specific,accurate,reliable and sensitive.The established method has successfully been applied to study the excretion of BCQB in rats after intranasal administration.