Activity-Based Detection of Cannabinoids in Serum and Plasma Samples Annelies Cannaert, 1,2† Jolien Storme, 1† Cornelius Hess, 3 Volker Auwärter, 4 Sarah M.R. Wille, 2 and Christophe P. Stove 1* BACKGROUND: Synthetic cannabinoids are the largest group of new psychoactive substances monitored by the European Monitoring Centre of Drugs and Drug Addic- tion. The rapid proliferation of novel analogs makes the detection of these new derivatives challenging and has initiated considerable interest in the development of so- called “untargeted” screening strategies to detect these compounds. METHODS: We developed new, stable bioassays in which cannabinoid receptor activation by cannabinoids led to recruitment of truncated -arrestin 2 (arr2) to the can- nabinoid receptors, resulting in functional complemen- tation of a split luciferase, allowing readout via biolumi- nescence. Aliquots (500 L) of authentic serum (n = 45) and plasma (n = 73) samples were used for simple liquid– liquid extraction with hexane:ethyl acetate (99:1 v/v). Fol- lowing evaporation and reconstitution in 100 L of Opti- MEM ® I/methanol (50/50 v/v), 10 L of these extracts was analyzed in the bioassays. RESULTS: Truncation of arr2 significantly (for both can- nabinoid receptors; P = 0.0034 and 0.0427) improved the analytical sensitivity over the previously published bioassays applied on urine samples. The new bioassays detected can- nabinoid receptor activation by authentic serum or plasma extracts, in which synthetic cannabinoids were present at low- or sub-nanogram per milliliter concentration or in which  9 -tetrahydrocannabinol was present at concentra- tions 12 ng/mL. For synthetic cannabinoid detection, an- alytical sensitivity was 82%, with an analytical specificity of 100%. CONCLUSIONS: The bioassays have the potential to serve as a first-line screening tool for (synthetic) cannabinoid activity in serum or plasma and may complement conventional analytical assays and/or precede analytical (mass spectrometry based) confirmation. © 2018 American Association for Clinical Chemistry According to the latest European Drug Report, 620 new psychoactive substances (NPSs) 5 were reported to the Early Warning System of the European Monitoring Centre of Drugs and Drug Addiction, with the number of new analogs reported in the past 5 years comprising approximately 70% of the total number. The NPS mar- ket has been traditionally dominated by synthetic canna- binoids (SCs), with 169 analogs detected since 2008 (1). The SCs are designed to exert similar pharmacological effects as the traditional recreational drug cannabis but are intended to circumvent legislation (2, 3 ). The rapid proliferation of novel analogs makes the detection of these new derivatives challenging in different contexts, such as forensic, clinical, and analytical chemistry (3, 4 ). Also, the activity-based analog laws of the US (5) and UK (6) are challenged, as the pharmacology of these new derivatives is often unknown. This could be efficiently countered by applying these new substances in activity- based biological assays to establish their cannabinoid ac- tivity and therefore their illegality. The recent proliferation of NPSs has initiated con- siderable interest in the development of so-called “untar- geted” screening strategies to detect and identify novel compounds without the use of certified reference mate- rials or mass spectral libraries. High-resolution mass spec- trometry (HRMS) has been the method of choice for broad screening of NPSs in a wide range of contexts because of its ability to measure accurate masses with both data-dependent and data-independent acquisition (4). However, due to the time-consuming and expensive character of this technique, this method is not routinely 1 Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium; 2 Laboratory of Toxicology, National Institute of Crimi- nalistics and Criminology, Brussels, Belgium; 3 Institute of Forensic Medicine, University Bonn, Forensic Toxicology, Bonn, Germany; 4 Institute of Forensic Medicine, Forensic Toxicology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany. * Address correspondence to this author at: Laboratory of Toxicology, Department of Bioanaly- sis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium. Fax +32-9-264-81-83; e-mail Christophe.Stove@UGent.be. † Annelies Cannaert and Jolien Storme contributed equally to this work. Received December 1, 2017; accepted February 23, 2018. Previously published online at DOI: 10.1373/clinchem.2017.285361 © 2018 American Association for Clinical Chemistry 5 Nonstandard abbreviations: NPS, new psychoactive substance; SCs, synthetic cannabi- noids; HRMS, high-resolution mass spectrometry; CB, cannabinoid; arr2, -arrestin 2; GPCR, G-protein– coupled receptor; LgBiT, large BiT; SmBiT, small BiT; EGFP, enhanced green fluorescent protein; dNGFR, truncated nerve growth factor receptor; THC, Δ 9 - tetrahydrocannabinol; RLU, relative light units. Clinical Chemistry 64:6 918–926 (2018) Drug Monitoring and Toxicology 918 Downloaded from https://academic.oup.com/clinchem/article-abstract/64/6/918/5608707 by guest on 23 May 2020