氯硝西泮与酒精联用在小鼠体内的协同代谢及行为学研究

Synergistic Metabolism and Behavioral Study of Clonazepam Combined with Alcohol in Mice

摘要: 建立血液中氯硝西泮的液相色谱–串联质谱(LC-MS/MS)检测方法，并研究氯硝西泮与酒精在小鼠体内的协同作用。优化色谱、质谱条件及血液样本前处理方法，以乙腈沉淀蛋白(血液:乙腈= 1:3，V/V)提取目标物，采用ZORBAX XDB-C18 (50 mm × 2.1 mm × 5 μm)色谱柱，以5 mmol/L甲酸铵-0.1%甲酸水溶液为流动相A，含0.1%甲酸的乙腈溶液为流动相B进行梯度洗脱；质谱采用电喷雾离子源(ESI)，正离子多反应监测(MRM)模式检测。该方法下氯硝西泮检出限为0.01 ng/mL，在1~500 ng/mL范围内线性关系良好(r > 0.9986)，相对标准偏差(RSD)为4.45%~7.56%。选取体重约30 g的SPF级昆明小鼠42只，随机分为7组(n = 6)，设空白对照组，低、中、高浓度氯硝西泮单药组，以及白酒、果酒、啤酒配制的高浓度氯硝西泮联用组。经灌胃给药后，于24 h内11个时间点眼眶静脉取血，经前处理后进行LC-MS/MS检测。结果表明，小鼠血药浓度于给药后30 min~8 h达峰，药效持续6~12 h，24 h后仅微量残留；酒精可显著升高血药浓度，且浓度随酒精度数升高而增加；氯硝西泮与酒精联用可使小鼠快速镇静昏睡、反应能力丧失。本研究建立的方法灵敏、准确、专属，可为氯硝西泮相关法医毒物鉴定及药物–酒精协同作用研究提供实验依据。

Abstract: An LC-MS/MS method for the determination of clonazepam in blood was developed, and the synergistic effects of clonazepam combined with alcohol in mice were investigated. Chromatographic and mass spectrometric conditions, as well as blood sample pretreatment methods, were optimized. The sample treatment method of acetonitrile precipitated protein (blood:acetonitrile = 1:3, V/V) was adopted to extract the target substance in blood. The chromatographic column of ZORBAX XDB-C18 (50 mm × 2.1 mm × 5 μm) was selected. The gradient elution procedure used 5 mmol/L ammonium formate-0.1% as mobile phase A and acetonitrile solution containing 0.1% formic acid as mobile phase B. The mass spectrometer uses ESI, positive ion mode scanning, and mμLtiple reaction monitoring (MRM) mode for analysis. After protein precipitation, the detection limit of clonazepam in blood was 0.01 ng/mL, and the linear relationship between 1~500 ng/mL of blood samples was good (r > 0.9986), RSD was 4.45%~7.56%. Forty-two SPF-grade Kunming mice weighing approximately 30 g were randomly divided into seven groups (n = 6): a blank control group, low-, medium-, and high-dose clonazepam monotherapy groups, and high-dose clonazepam combined with baijiu (Chinese liquor), fruit wine, and beer groups, respectively. After intragastric administration, blood samples were collected from the orbital vein at 11 time points within 24 h and analyzed by LC-MS/MS following pretreatment. The results showed that the concentration of clonazepam in the blood of mice peaked in 30 min to 8 hours, and the effect lasted for 6 to 12 hours, and only a small amount of residue existed in the blood after 24 h. Alcohol significantly elevated blood drug concentrations, with the extent of elevation increasing with alcohol content. Co-administration of clonazepam and alcohol induced rapid sedation, lethargy, and loss of responsiveness in mice. The method established in this study is sensitive, accurate, and specific, providing an experimental basis for forensic toxicological identification of clonazepam and research on drug-alcohol synergistic interactions.