Determination of cannabinoids in 50 μL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases

IF 2.6 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS Drug Testing and Analysis Pub Date : 2023-06-21 DOI:10.1002/dta.3532

Flavio Zancanaro, Gianpaola Tedeschi, Luca Zamengo, Samuela Frasson, Giampietro Frison

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Abstract

The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH) in 50 μL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow—Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10 min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

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利用湍流色谱和 LC-HRAM-Orbitrap-MS 在线萃取法测定 50 μL 全血样本中的大麻素：在毒品影响下驾车案例中的应用。

分析全血中的大麻素通常采用传统的质谱（MS）技术，然后进行离线净化或衍生化步骤，这些步骤耗时长、费力且昂贵。我们提出了一种简单、快速、高特异性和高灵敏度的方法，用于测定 50 μL 全血样本中的Δ9 -四氢大麻酚（THC）、大麻二酚（CBD）、大麻酚（CBN）、11-羟基-Δ9 -四氢大麻酚（11-OH-THC）和 11-去甲-9-羧基-Δ9 -四氢大麻酚（THC-COOH）。在加入氚代内标（IS）和简单的蛋白质沉淀步骤后，使用湍流色谱法（TurboFlow-Thermo Scientific）对样品上清液进行在线提取。分析物在 C18 分析柱上分离，并使用 Thermo Scientific Q Exactive Focus MS 系统进行 LC-HRAM-Orbitrap-MS 检测。质谱检测采用极性切换和选择离子监测（SIM）模式，使用五个特定的采集窗口，分辨率为 70,000 (FWHM)。包括分析前步骤在内的总运行时间约为 10 分钟。通过确定所有分析物的检测限 (LOD)、定量下限 (LLOQ)、线性范围、分析准确度、测定内和测定间精密度、迁移量、基质效应、萃取回收率和选择性，对方法进行了验证。此外，还对测量的不确定性进行了评估，并为法医目的设定了一个有把握的判定规则。该方法所需的基质体积小、样品制备过程简单且成本效益高、分析运行时间短，因此可能适用于临床和法医毒理学应用。对该方法的性能进行了长期监测，并对 7620 份毒品影响下驾驶（DUID）样本进行了测试，其中包括 641 份阳性样本。

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来源期刊

Drug Testing and Analysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

5.90

自引率

24.10%

发文量

191

审稿时长

2.3 months

期刊介绍： As the incidence of drugs escalates in 21st century living, their detection and analysis have become increasingly important. Sport, the workplace, crime investigation, homeland security, the pharmaceutical industry and the environment are just some of the high profile arenas in which analytical testing has provided an important investigative tool for uncovering the presence of extraneous substances. In addition to the usual publishing fare of primary research articles, case reports and letters, Drug Testing and Analysis offers a unique combination of; ‘How to’ material such as ‘Tutorials’ and ‘Reviews’, Speculative pieces (‘Commentaries’ and ‘Perspectives'', providing a broader scientific and social context to the aspects of analytical testing), ‘Annual banned substance reviews’ (delivering a critical evaluation of the methods used in the characterization of established and newly outlawed compounds). Rather than focus on the application of a single technique, Drug Testing and Analysis employs a unique multidisciplinary approach to the field of controversial compound determination. Papers discussing chromatography, mass spectrometry, immunological approaches, 1D/2D gel electrophoresis, to name just a few select methods, are welcomed where their application is related to any of the six key topics listed below.