目的 建立尿液中扎来普隆和5-氧-扎来普隆的液相色谱-串联质谱检验法。方法 尿液用乙腈直接沉淀蛋白并通过96孔板去磷酯后,选用ZORBAX Eclipse Plus C18色谱柱,以0.1%甲酸水(A相)和乙腈(B相)作为流动相,进行梯度洗脱分离。采用液相色谱-串联质谱仪的电喷雾电离,正离子模式扫描、多反应监测(MRM)模式检测扎来普隆及5-氧-扎来普隆,并用外标法定量。结果 该方法可有效分离尿液中的扎来普隆及5-氧-扎来普隆,保留时间分别为2.48min和1.96min,样品检验时间仅需4min。尿液中扎来普隆及5-氧-扎来普隆分别在0.1~50ng/mL和0.25~50ng/mL范围内线性关系良好,回归方程分别为y=70393x+33700 和y= 34491x+16854,检出限分别为0.05ng/mL和0.1ng/mL。扎来普隆及5-氧-扎来普隆的回收率均在90%以上,日内与日间精密度均小于10%。结论 本文所建方法简便、快速、分离度好,适用于尿液中的扎来普隆和5-氧-扎来普隆检测。

Objective Zaleplon, a pyrazolopyrimidine drug, is one of non-benzodiazepine sedative hypnotic drugs. As a new kind of hypnotic, it is an improvement of traditional benzodiazepines in treatment of insomnia. In 2007, the International Association of Forensic Toxicologists (TIAFT) listed zaleplon as one of the new type of abuse drugs since it was often used in drug-facilitated cases. Zaleplon acted rapidly, and victims were less able to recall the circumstances under which the offence occurred due to its amnesic properties. The hypnotic acts within 0.5h and has short half-life (0.9~1.1h). Because its ultra-short half-life, low frequency of use, and short window of detection, zaleplon has been detected in few clinical and forensic cases. Urine samples are most likely to be useful in cases of drug-facilitated crimes as its detection window is longer than that of blood or plasma samples. 5-O-zaleplon is a main metabolite of zaleplon in biological body. In order to improve detectable rate of zaleplon poisoning cases, we established an ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) assay for simultaneous determination of zaleplon and its main metabolite 5-O-zaleplon in urine samples.Methods Aliquots of 0.2mL urine samples were used for the analysis. Zaleplon and its main metabolite 5-O-zaleplon were extracted with two-step method using acetonitrile to precipitate protein and 96-well plates to deplete phospholipid. The separation was performed on a ZORBAX Eclipse Plus C18 (2.1mm×100mm, 1.8µm) analytical column by Shimadzu 30A-LC ultra-performance liquid chromatography. The MS/MS analysis was performed on API 5500 QTRAP tandem mass spectrometry. The column temperature was maintained at 40℃, while the sample plate was maintained at 4℃. By comparing different chromatographic column, different mobile phase, different gradient, and optimization conditions of mass spectrometry, we established the liquid chromatography-tandem mass spectrometry method. The mobile phases consisted of acetonitrile (mobile phase B) and water containing 0.1% formic acid (mobile phase A), and the flow rate was 0.4mL/min. MS-MS in the multiple reaction monitoring (MRM) mode via positive electrospray ionization mode (ESI+) was used. The retention times and the two parent/daughter ion pairs were used for qualitative analysis, and the first parent/daughter ion pair was used for quantitative analysis.Results Zaleplon and 5-O-zaleplon in urine samples were separated well. There were no interferences of endogenous impurity. The chromatographic separation time was 4 min. Calibration curve of zaleplon was linear within the range of 0.1~50ng/mL and calibration curve of 5-O-zaleplon was linear within the range of 0.25~50ng/mL. Regression equations were y=70393x+33700 and y=34491x+16854, respectively. The limits of detection for zaleplon and 5-O-zaleplon were 0.05ng/mL and 0.1ng/mL. The recoveries of zaleplon and 5-O-zaleplon were more than 90%. The inter-day and intra-day precisions were less than 10%.Conclusions This method is rapid, sensitive and effective. It is suitable for determination of zaleplon and its main metabolite 5-O-zaleplon in urine sample. It can be a solution to prolong the window of detection and applied to forensic toxicological analysis.