Rositano Joanna, Harpas Peter, Kostakis Chris, Scott Timothy
Forensic Science SA, Adelaide, South Australia, Australia.
Forensic Science SA, Adelaide, South Australia, Australia; Flinders University of South Australia, Bedford Park, South Australia, Australia.
Forensic Sci Int. 2016 Aug;265:125-30. doi: 10.1016/j.forsciint.2016.01.017. Epub 2016 Jan 28.
Since 2006, the South Australian Government has been conducting roadside oral fluid testing of drivers for the illicit drugs methylamphetamine (MA), methylenedioxymethylamphetamine (MDMA) and Δ(9)-tetrahydrocannabinol (THC) using the Securetec Drugwipe II Twin and Alere DDS 805 AP saliva collection kit. Forensic Science South Australia carries out the confirmatory analysis by LC/MS for the positive test results of oral fluid roadside testing along with the pre-screened ELISA positive road traffic accident blood samples. The number of blood and oral fluid samples received in the laboratory has been steadily increasing during this time, and over 10,000 samples were received in 2014. The proportion of positive results from these samples has also been increasing over the decade of driver drug testing, and this data is presented. A simple and efficient method has been developed for the analysis of the three drugs using Biotage Isolute(®) SLE+ 96-well plates. Sample preparation included 1:1 dilution with a dilute ammonia solution for buffered oral fluids (1:3 dilution for blood samples), and addition of deuterated internal standards. Samples were loaded onto the phase, left to absorb for 5min then eluted with methyl t-butyl ether (MTBE). The samples were evaporated and reconstituted in methanol. LC/MS analysis was performed on an AB Sciex 5500 Q-Trap in positive ion mode, monitoring 3 transitions for each analyte. Separation was achieved on a Restek Ultrabiphenyl 50×2.1mm column with a gradient system of acetonitrile/0.1% formic acid over 5min. Method validation and recoveries were carried out on drug free ante mortem blood and DDS buffer solution provided by Alere, Australia. Recoveries above 80% were achieved for MA and MDMA at a concentration of 25ng/mL, whilst recoveries of greater than 65% were achieved for THC at 4.5ng/mL. Accuracy and precision were acceptable down to the LLOQ for all three analytes (5, 5 and 1ng/mL for MA, MDMA and THC, respectively). Mean matrix effects were 1.0, 0.97 and 0.78 in DDS buffer and 0.96, 0.96 and 0.62 in blood for MA, MDMA and THC, respectively. Linearity was achieved up to 1250ng/mL for MA and MDMA, and 112ng/mL for THC (r(2)>0.999 for all analytes). The method is designed for easy transfer to an automated liquid handling platform.
自2006年以来，南澳大利亚州政府一直在使用Securetec Drugwipe II Twin和Alere DDS 805 AP唾液采集试剂盒对驾驶员进行路边口腔液非法药物检测，检测的药物包括甲基苯丙胺（MA）、亚甲基二氧甲基苯丙胺（MDMA）和Δ(9)-四氢大麻酚（THC）。南澳大利亚法医科学部对路边口腔液检测的阳性结果以及预先筛选的酶联免疫吸附测定（ELISA）阳性的道路交通事故血液样本进行液相色谱/质谱（LC/MS）确证分析。在此期间，实验室收到的血液和口腔液样本数量一直在稳步增加，2014年收到了超过10000个样本。在驾驶员药物检测的十年中，这些样本的阳性结果比例也一直在上升，并展示了该数据。已开发出一种使用Biotage Isolute(®) SLE+ 96孔板分析这三种药物的简单高效方法。样品制备包括用稀氨溶液对缓冲口腔液进行1:1稀释（血液样本为1:3稀释），并添加氘代内标。将样品加载到固定相上，吸附5分钟，然后用甲基叔丁基醚（MTBE）洗脱。样品蒸发后用甲醇复溶。在AB Sciex 5500 Q-Trap上以正离子模式进行LC/MS分析，监测每种分析物的3个跃迁。在Restek Ultrabiphenyl 50×2.1mm柱上，通过乙腈/0.1%甲酸在5分钟内的梯度系统实现分离。使用澳大利亚Alere公司提供的无药物生前血液和DDS缓冲溶液进行方法验证和回收率测定。MA和MDMA在浓度为25ng/mL时回收率高于80%，而THC在4.5ng/mL时回收率大于65%。所有三种分析物在定量下限（LLOQ）时的准确度和精密度均可接受（MA、MDMA和THC分别为5、5和1ng/mL）。MA、MDMA和THC在DDS缓冲液中的平均基质效应分别为1.0、0.97和0.78，在血液中的平均基质效应分别为0.96、0.96和0.62。MA和MDMA线性范围高达1250ng/mL，THC为112ng/mL（所有分析物的r(2)>0.999）。该方法设计用于易于转移到自动化液体处理平台。
