LC-MS/MS法测定人血浆中苯海拉明血药浓度及其制剂的生物等效性研究

Determination of Diphenhydramine in Human Plasma by LC-MS/MS and Bioequivalence Study of Its Preparation

目的:建立测定人血浆中苯海拉明血药浓度的方法,并进行其生物等效性研究。方法:血样经处理后,采用高效液相色谱串联质谱电喷雾(LC-MS/MS)法进样测定。色谱柱为Intersil Hilic(150mm×3.0mm,5μm),流动相为乙腈-水(10mmol/L乙酸铵)-甲酸(40:60:1,V/V/V);电喷雾电离(ESI)离子源,正离子模式,多级反应监测(MRM)方式检测,离子对分别为m/z256→167(苯海拉明)、m/z344→215(氯马斯汀,内标)。结果:苯海拉明血药浓度在0.5～125ng/ml范围内线性关系良好(r=0.9965);日内、日间RSD均<8%,方法回收率为99.4%～103.4%,提取回收率为71.9%～75.3%。受试者口服氨麻苯美片受试制剂与参比制剂后苯海拉明的主要药动学参数分别为:t1/2(9.892±1.615)、(10.745±3.227)h,tmax(2.00±0.81)、(1.78±0.55)h,cmax(90.4±20.6)、(103.0±30.1)h,AUC0-36h(806.293±211.453)、(827.856±223.996)h。结论:本方法灵敏、准确,适用于苯海拉明血药浓度测定及其生物利用度研究;2种制剂生物等效。

OBJECTIVE： To develop a method for the determination of diphenhydramine in human plasma, and to study bioequivalence of its preparation in healthy volunteers. METHODS： After blood sample processing, LC-MS/MS method was used. The determination was performed on Inertsil Hilic column （150 min×3.0 mm, 5 μm） with mobile phase consisted of acetonitrile-water （10 mmol/L ammonium acetate）-formic acid （40：60：1, V/V/V）. ESI was applied and operated in positive ion mode： m/z 256→167 for diphenhydramine and m/z 344→215 for clemastine （IS） under multiple reaction monitoring （MRM） mode. RESULTS： The linear ranges of diphenhydramine were 0.5-125 ng/ml （r=0.996 5）; RSDs of intra-day and inter-day were less than 8%, and method recoveries were 99.4 % - 103.4 % and extraction recoveries were 71.9 % -75.3 %. Main pharmacokinetic parameters of test preparation and reference preparation after oral administration were as follows： t1/2 were（9.892 ± 1.615） h and （10.745 ± 3.227）h; t_max were （2.00 ± 0.81） h and （1.78 ± 0.55）h; C_max were （90.4±20.6）h and （103.0±30.1） h; AUC0-36 h were （806.293 ± 211.453） h and （827.856 ± 223.996）h. CONCLUSIONS： The method is sensitive and accurate, which is suitable for the determination of plasma concentration and bioequiavailability study of diohenhvdramine. Two preparations are bioeauivalent.