About 620 new psychoactive drugs (NPS) have appeared on the worldwide drug market the last decade. Several European countries have recently published a generic legislation, making a broad range of NPS illegal. The major challenge is the continuous chemical development of NPS and the rapid turnover of the drug market, making it difficult to obtain up-to-date monitoring techniques. The aim of this presentation is to discuss the challenges of the NPS detection workflows for toxicological analysis of biological matrices.

A literature overview of the current methodology for NPS detection will be achieved via a Pubmed search. In addition, the experience of the researchers’ group concerning the detection of NPS will be discussed using either (a) high-resolution mass spectrometry via the (LC-HRMS) Xevo-G2-QTOF-XS (Waters, Manchester, UK) with UNIFI software (Wille et al., https://doi.org/10.1002/dta.2232) or (b) a newly developed cell-based G-protein coupled receptor activation assay using the NanoLuc^® Binary technology (Promega), detecting in vitro cannabinoid (CB1 and 2) or μ-opioid receptor (MOR) activation, via the recruitment of β-arrestin 2 and resulting in a luminescent signal (Cannaert et al., https://doi.org/10.1373/clinchem.2017.285361).

Amongst the major challenges for NPS detection continuous adaptation of the methodology to ensure up-to-date screening of correct target compounds and their metabolites with adequate sensitivity, limited research concerning stability, the lack of reference materials or their access, and the development of up-to-date libraries. Immunoassays focusing on classical drugs have insufficient NPS cross-reactivity, while immunoassays focusing on specific NPS can be quickly outdated as they target specific chemical structures. The bioassay developed by Cannaert et al. detects cannabinoid or opioid receptor activity. The detection of synthetic cannabinoids resulted in an overall sensitivity of 75% and a specificity of 91% in urine and a sensitivity of 82% and a specificity of 100% in serum. Several semi-synthetic opioids (hydromorphone and desomorphine) and synthetic opioids (fentanyl and derivatives) and other unrelated opioids (U-49900 and U-47700) demonstrated different levels of MOR activation in the opioid bioassay. A sensitivity and specificity of 100% was obtained for authentic blood samples from morphine users and non-users. In the NPS detection workflow of a toxicological laboratory, this technique may be of interest as an initial screening technique. However, chromatographic detection will still be necessary to obtain identification and quantification of NPS. Advanced techniques involving high-resolution mass spectrometry combine the detection using developed standard libraries with possible elucidation of unknown chemical structures via accurate mass. In addition, NPS metabolites can be in vitro produced, identified and subsequently added in mass libraries to improve NPS detection. One of the challenges of accurate mass instruments is the development of an adequate procedure to generate a time-efficient interpretation of the obtained analytical data. A practical and time-efficient workflow for detection of NPS in biological matrices using time-of-flight is described and applied to blood (n=558) as well as oral fluid (n=199) samples obtained during roadside testing by Wille et al. An NPS positivity rate of 7% in blood and 11% in oral fluid was observed for the cohort that was examined.

A feasible and straightforward workflow for the detection of NPS in biological matrices for toxicological screening consisting of first-line screening tools using receptor activity in combination with a practical workflow for accurate mass detection is demonstrated.