HPLC-MS/MS测定大鼠血浆及组织中盐酸戊乙奎醚浓度

Detecting Penehyclidine Hydrochloride in Mouse Plasma and Tissues with HPLC-MS/MS

目的建立用高效液相色谱质谱联用(HPLC-MS/MS)技术测定大鼠血浆、脑组织及肺组织中戊乙奎醚浓度的方法。方法大鼠血浆、脑组织及肺组织样品中戊乙奎醚及内标苯海拉明经石油醚∶乙醚(70∶30)提取后,采用HPLC-MS/MS技术进行测定。以Allure C18(50×2.1 mm,5μm)为色谱柱,以甲醇∶5 mmol/L醋酸胺∶三乙胺=90∶10∶0.05(甲酸调pH5.8)为流动相进行分离,检测离子为质荷比(m/z)316.2/128.3(戊乙奎醚),256.3/167.1(内标)。结果标准曲线线性范围为血浆0.391～200 ng/mL,脑组织及肺组织0.391～100 ng/mL。预处理回收率为血浆67.32%～70.80%,肺组织84.99%～89.27%,脑组织76.86%～81.98%;方法回收率为血浆97.66%～99.97%,肺组织96.55%～101.65%,脑组织95.18%～100.21%;日内精密度RSD分别为:血浆1.94%～2.93%,肺组织1.73%～3.70%,脑组织2.35%～2.79%;日间精密度RSD分别为:血浆3.00%～3.85%,肺组织2.86%～3.94%,脑组织2.77%～5.00%;血浆和组织样品保存于-30℃低温冰箱7、21 d,分别测定浓度以考察稳定性,RSD分别为:血浆1.52%～5.26%,肺组织1.88%～2.93%,脑组织2.22%～3.76%;反复冻融1次和2次测定样品浓度,血浆RSD为0.38%～3.55%,肺组织为2.79%～9.60%,脑组织为1.35%～2.29%。结论成功建立HPLC-MS/MS法测定大鼠血浆及组织中戊乙奎醚浓度的方法,该法简便、准确、重现性好,可用于盐酸戊乙奎醚药动学和生物利用度研究。

Objective To develop a method for detecting penehyelidine hydrochloride （PH） in mouse plasma and tissues using HPLC-MS/MS. Methods The plasma and tissue samples were extracted with petroleum ether： ethyl ether （70 ： 30）. The isolation of PH was achieved on an Allure Cls （50 ×2.1mm,5μm） column, with methanol/5 mmol/L ammonium acetate/triethylamine= 90/10/0. 05 （adjusted pH5. 8 by formic acid as mobile phase. Benzhydramine was used as the internal standard. Results The calibration curves were linear over the range of 0.39-200 ng/mL for plasma and 0. 391-100 ng/mL for tissues. The extraction recovered 67.32%-70.80% of PH in plasma, 84. 99%-89. 27% of PH in lung tissues, and 76.86%-81.98% of PH in brain tissues. The HPLC detected 97.66%-99.97% of PH in extracted samples of plasma, 96.55%-101.65% extracted samples of lung and 95.18%-100.21% of extracted samples of brain. The within-day RSD were 1.94%-2.93%, 1.73%-3.70% and 2.35%-2.79% for plasma, lung, and brain, respectively, The betweewday RSD were 3. 00%-3. 85% 2. 86%- 3.94% and 2.77%-5.00% for plasma, lung, and brain, respectively. Both the long-term and freeze-thaw stabilities were acceptable. The long-term RSD were 1.52%-5.26%, 1.88%-2.93%, and 2.22%-3.76% for plasma, lung, and brain, respectively. The freeze-thaw RSD were 0. 38%-3. 55%, 2. 79%-9. 60%, and 1. 35%-2. 29% for plasma, lung, and brain, respectively. Conclusion The assay is simple and accurate, with good reproducibility, and can satisfy to the needs of pharmacokinetics and relative bioavailability studies on penchyclidine hydrochloride.