Guan F, Seno H, Ishii A, Watanabe K, Kumazawa T, Hattori H, Suzuki O
Department of Legal Medicine, Hamamatsu University School of Medicine, Japan.
J Anal Toxicol. 1999 Jan-Feb;23(1):54-61. doi: 10.1093/jat/23.1.54.
Benzodiazepines are common drugs that cause intoxication. Benzodiazepines and their metabolites can be converted by hydrolysis in acid to the corresponding benzophenones, which are easier to be separated from matrices because of their hydrophobic properties. In this study, a new separation technique called solid-phase microextraction (SPME), which can integrate extraction, concentration, sampling and sample introduction into one single procedure, has been employed to extract the products of benzodiazepines from urine after acid hydrolysis. The extracts were determined by gas chromatography with electron-capture detection (GC-ECD). The hydrolysis conditions were optimized by a statistic orthogonal design. Factors influencing direct-immersion (DI)-SPME process were also checked and chosen experimentally. The method was evaluated with spiked human urine samples. The recoveries of nine benzodiazepines ranged from 1 to 25%, with the highest for oxazolam and the lowest for bromazepam. The calibration curves were linear from 10 to 500 ng/mL for oxazolam, haloxazolam, flunitrazepam, nimetazepam, and clonazepam and from 20 to 1000 ng/mL for the others except bromazepam. The detection limits were 2-20 ng/mL for most drugs tested. The intraday and interday coefficients of variation of the developed method were within 10 and 17%, respectively. In addition, the utility of the method was confirmed by determining two ingested benzodiazepines (flunitrazepam and oxazolam) in a volunteer's urine; urine flunitrazepam was still detectable 32 h after a therapeutic dose (1.2 mg) of the drug. Finally, the DI-SPME was compared with the conventional liquid-liquid extraction with regard to detection limits and extraction efficiency of the analytes. By DI-SPME, more amounts of analytes could be introduced into GC column than by conventional liquid-liquid extraction, and thus lower detection limits of the analytes were reached, although benzophenone recoveries by DI-SPME were rather low.
苯二氮䓬类药物是常见的能导致中毒的药物。苯二氮䓬类药物及其代谢物在酸性条件下可通过水解转化为相应的二苯甲酮，由于其疏水性，二苯甲酮更容易从基质中分离出来。在本研究中，一种名为固相微萃取（SPME）的新型分离技术被用于在酸性水解后从尿液中提取苯二氮䓬类药物的产物，该技术可将萃取、浓缩、取样和进样整合为一个单一过程。提取物通过带有电子捕获检测的气相色谱法（GC - ECD）进行测定。水解条件通过统计正交设计进行了优化。还通过实验检查并选择了影响直接浸入（DI）-SPME过程的因素。该方法用加标的人尿样进行了评估。九种苯二氮䓬类药物的回收率在1%至25%之间，奥沙唑仑的回收率最高，溴西泮的回收率最低。奥沙唑仑、卤沙唑仑、氟硝西泮、硝甲西泮和氯硝西泮的校准曲线在10至500 ng/mL范围内呈线性，除溴西泮外的其他药物校准曲线在20至1000 ng/mL范围内呈线性。大多数测试药物的检测限为2 - 20 ng/mL。所建立方法的日内和日间变异系数分别在10%和17%以内。此外，通过测定一名志愿者尿液中的两种摄入的苯二氮䓬类药物（氟硝西泮和奥沙唑仑）证实了该方法的实用性；在服用治疗剂量（1.2 mg）的该药物32小时后，尿液中的氟硝西泮仍可检测到。最后，在分析物的检测限和萃取效率方面，将DI - SPME与传统的液 - 液萃取进行了比较。通过DI - SPME比通过传统液 - 液萃取能将更多量的分析物引入GC柱，因此达到了更低的分析物检测限，尽管DI - SPME对二苯甲酮的回收率相当低。
