LC-MS/MS法同时测定人血浆中的咖啡因和氯苯那敏

Simultaneous determination of caffeine and chlorpheniramine in human plasma by LC-MS/MS

目的采用LC-MS/MS法同时测定人血浆中咖啡因和氯苯那敏的浓度。方法人血浆样本经液液萃取后,选用InertsilODS-SP色谱柱(75 mm×2.1 mm,3μm),流动相为甲醇-5 mmol.L-1乙酸铵(含0.5%甲酸)(55∶45),流速0.3 mL.min-1,选用API3200型三重四极杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式,选择监测离子反应分别为m/z 195.3→110.2(咖啡因)、m/z 275.2→230.2(氯苯那敏)和m/z 256.2→167.3(苯海拉明内标)。结果血浆中咖啡因和氯苯那敏的线性范围分别为4.0～1.2×103、0.05～15.0μg.L-1(r>0.999);批内、批间RSD均小于13%;相对误差(RE)均在±5%的范围内;平均提取回收率分别为67.6%±1.3%、69.1%±3.4%。结论所用方法快速、灵敏、专属性强、重复性好,适用于人血浆中咖啡因和氯苯那敏的测定,并可用于小儿氨酚烷胺泡腾片的人体生物等效性研究。

OBJECTIVE To develop a method for simultaneous determination of caffeine and chlorpheniramine in human plasma by LC-MS/MS.METHODS After liquid-liquid extraction,caffeine,chlorpheniramine and diphenhydramine（IS） were separated on an Inertsil ODS-SP analytical column using the mobile phase of methanol-5 mmol·L-1 ammonium acetate（containing 0.5% formic acid）（55：45） at a flow rate of 0.3 mL·min-1.Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring（MRM） mode.The MRM transitions of m/z 195.3→110.2,m/z 275.2→230.2,m/z 256.2→167.3 were used to quantify caffeine,chlorpheniramine and IS,respectively.RESULTS The method was linear（r0.999） in the concentration ranges of 4.0-1.2×103,0.05-15.0 μg·L-1 for caffeine and chlorpheniramine,respectively.Intra-batch and inter-batch RSD were both less than 13%,and the RE were within ±5%.The mean extract recoveries were 67.6%±1.3%,69.1%±3.4% for caffeine and chlorpheniramine,respectively.CONCLUSION The method is a rapid,sensitive,selective and reliable method for the determination of caffeine and chlorpheniramine in human plasma.It was successfully applied to a bioequivalence study of the two analyses in healthy Chinese volunteers after administration of pediatric paracetamol and amantadine hydrochloride effervescent tablets.