Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry methodology (LC–MS-MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC–MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

中文翻译：

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LC-MS/MS-MS3 用于测定和定量血液样本中的 Δ9-四氢大麻酚和代谢物

由于法医毒理学分析中大麻素的普遍存在，因此拥有一种有效的方法至关重要，该方法允许使用少量样品，并且需要最少的样品制备来测定和定量低浓度。开发了一种简单、高选择性和高通量的液相色谱串联质谱方法 (LC-MS-MS-MS3)，用于测定和定量 Δ9-四氢大麻酚 (THC)、11-羟基-Δ9-四氢大麻酚 (THC- OH) 和 11-正-9-羧基-Δ9-四氢大麻酚 (THC-COOH) 存在于血液样本中。在对 THC、THC-OH 和 THC-COOH 及其氘代内标进行色谱分析之前，先用乙腈对 0.1 mL 血液样品进行蛋白质沉淀 (PPT)。通过使用 Acquity UPLC® HHS T3（100 mm × 2.1 mm id，1.8 μm）反相色谱柱，使用 2 mM 甲酸铵水溶液、0.1% 甲酸和甲醇的流速进行梯度洗脱，实现色谱分离0.4 mL/min，运行时间为 10 分钟。对于 MS/MS-MS3 分析，通过电喷雾电离 (ESI) 使用 SCIEX QTRAP® 6500+ 三重四极杆线性离子阱质谱仪，在多反应监测 (MRM) 和线性离子阱模式 (MS3) 下运行。该方法根据国际公认的标准和指南进行了验证，并证明在 0.5 至 100 ng/mL 之间具有选择性和线性（r2 > 0.995）。定量下限 (LLOQ) 对应于校准曲线所用的最低浓度。获得的准确度和精密度的变异系数＜15％。研究浓度水平（1 ng/mL、5 ng/mL 和 50 ng/mL）的平均回收率在 88.0% 至 117.2% 之间。没有观察到显着的干扰化合物、基质效应或残留。经过验证的方法使用 LC-MS-MS-MS3 和 0.1 mL 的样品量，为血液中大麻素的定量提供了一种灵敏、高效和稳健的程序。这项工作也是使用 LC-MS3 技术测定生物样品中药物的概念证明。