UPLC-MS/MS法同时检测人血浆中卡马西平和苯巴比妥浓度

Simultaneous Determination of Carbamazepine and Phenobarbital in Human Plasma by UPLC-MS/MS

目的:建立同时检测人血浆中卡马西平和苯巴比妥浓度的UPLC-MS/MS方法,应用于临床卡马西平和苯巴比妥血药浓度监测。方法:以地西泮为内标,血浆样品经乙腈沉淀蛋白,运用Waters XEVO TQD三重四级杆液质联用仪检测,色谱柱为ACQUITY UPLC~■ BEH C_(18)柱(50 mm×2.1 mm,1.7μm);流动相为乙腈(10 mmol·L^(^(-1))甲酸铵)-水(10 mmol·L^(^(-1))甲酸铵,含0.1%甲酸),梯度洗脱,流速为0.4 ml·min^(-1),柱温40℃;采用电喷雾离子化源(ESI),正离子多反应监测模式扫描分析;加入200μl乙腈沉淀蛋白,12 000×g离心10 min转移至1.5 ml EP管中取2μl上样。结果:卡马西平的保留时间为1.23 min,线性范围为0.25~25μg·ml^(-1)(r=0.999 7),最低定量限为0.01μg·ml^(^(-1));苯巴比妥的保留时间为1.11 min,线性范围为0.5~50μg·ml^(-1)(r=0.999 6),最低定量限为0.05μg·ml^(-1)。卡马西平的高、中、低浓度的回收率为(82.1±6.83)%,(82.91±4.3)%和(84.35±3.09)%;苯巴比妥的高、中、低浓度的回收率为(84.27±6.91)%,(84.32±7.74)%和(89.07±6.24)%。卡马西平和苯巴比妥的日内、日间RSD均<10%,血浆样品体系中的其他内源性物质不干扰测定。结论:该方法准确可靠,操作简便,重复性好,适于检测人血浆中卡马西平和苯巴比妥的血药浓度测定。

Objective： To establish an ultra performance liquid chromatography-tandem quadrupole mass spectrometry （UPLC- MS/MS） method to determine the concentration of carbamazepine and phenobarbital in human plasma, and apply in the clinical moni- toring. Methods： Diazepam was used as the internal standard, and the samples were precipitated by acetonitrile. An ACQUITY UPL- C BEH C18（50 mm ×2.1 mm, 1.7 μm） column was used as the stationary phase at 40℃ with a Waters XEVO TQD. The mobile phase consisted of acetonitrile （containing 10 mmol · L-1 ammonium formate） and water （containing 10 mmol · L-1 ammonium for- mate and 0.1% formic acid） with gradient elution pumped at a flow rate of 0.4 ml · min-1. ESI was applied and the samples were scanning analyzed by positive ion multi-reaction monitoring mode. The plasma was precipitated by 200 μl acetonitrile and centrifugated at 12 000 × g for 10 min and tranfer it into an Ep tube. The sample size was 20 μl. Results： The retention time of carbamazepine was 1.23 min. Excellent linear calibration curves of carbamazepine were obtained within the concentration range of 0.25-25 μg · ml - 1 （ r = 0. 999 7 ）. The lower limit of quantification of carbamazepine was 0.01 μg·ml-1. The retention time of phenobarbital was 1. 11 min. Excellent linear calibration curves of carbamazepine were obtained within the concentration range of 0.5-50μg· ml - 1 （ r = 0.999 6）. The lower limit of quantification of carbamazepine was 0.05 μg ·ml-1. The recovery of three concentrations of carbamazepine was （ 82.1 ± 6.83 ） %, （ 82.91 ± 4.3 ） % and （ 84.35 ± 3.09 ） %, and the recovery of three concentrations of phenobarbital was （ 84.27 ± 6.91 ）%, （84.32 ± 7.74）% and （89.07 ± 6.24）% , respectively. The intra- and inter-day RSDs were all less than 10%. There were no endogenous substances existing in the incubation system, therefore, there was no interference with the determination. Conclu- sion： The simple, accurate and rapid method is suitable for the determination of carbamazepine and phenobarbital in human plasma, which can contribute greatly to the therapeutic drug monitoring service for patients.