自动固相萃取/液相色谱-串联质谱法测定血液中氯硝西泮及其代谢产物7-氨基氯硝西泮

Quantitation of Clonazepam and Its Major Metabolite 7-Aminoclonazepam in Blood by Automated Solid Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

建立了血液中氯硝西泮及其代谢产物7-氨基氯硝西泮的自动固相萃取/液相色谱-串联质谱（ASPE/LC-MS/MS）分析方法.样品经C18固相萃取柱提取后,采用LC-MS/MS进行测定,外标法定量.在Waters AtlantisTM dC18反相柱上分离,梯度洗脱,流动相为甲醇和0.1％甲酸水溶液,质谱采集为电喷雾正离子多反应监测模式.2种目标物在2～1 000 μg/L范围内具有良好的线性关系,相关系数为0.995 9～0.998 2,检出限为0.2～0.5 μg/L;加标水平为50,200,1 000 μg/L时,方法的回收率为72.6％ ～ 96.3％,相对标准偏差为4.2％～10.3％.本方法可用于法庭与临床的毒物分析.

An automated solid phase extraction/liquid chromatography-tandem mass spectrometric （ASPE/LC-MS/MS） method for the quantitative determination of clonazepam and its major metabolite 7-aminoclonazepam in blood.The samples were cleaned up with a C18 solid phase extraction cartridge using methanol as elute solution,and separated on a Waters AtlantisTM dC18 column（150 mm ×3.9 mm,5.0 μm） by gradient elution using methanol and water （containing 0.1％ formic acid） as mobile phase.The analysis of target compounds were carried out by LC-MS/MS under multiple reaction monitoring（MRM） in positive ionization mode.The calibration curves showed good linearities in the concentration range of 2-1 000 μg/L with correlation coefficients more than 0.995 9.The detection limits of the method were in the range of 0.2-0.5 μg/L.The mean recoveries at three spiked levels of 50,200,1 000 μg/L were in the range of 72.6％-96.3％,with relative standard deviations of 4.2％-10.3％.The proposed method was rapid,sensitive,simple and accurate,and could be used to detect clonazepam and 7-aminoclonazepam in forensic toxicology.