The proliferation of new psychoactive substances (NPS) in recent years has resulted in the development of numerous analytical methods for the detection and identification of known and unknown NPS derivatives. High-resolution mass spectrometry (HRMS) has been identified as the method of choice for broad screening of NPS in a wide range of analytical contexts due to its ability to measure accurate masses using data-independent acquisition (DIA) techniques. Additionally, it has shown promise for non-targeted screening strategies that have been developed in order to detect and identify novel analogues without the need for certified reference materials (CRMs) or comprehensive mass spectral libraries. This paper reviews the applications of HRMS for the analysis of NPS in forensic drug chemistry, clinical and forensic toxicology. It provides an overview of the sample preparation procedures in addition to data acquisition, instrumental analysis and data processing techniques.Furthermore, it will give an overview of the current state of non-targeted screening strategies with discussion on future directions and perspectives of this technique.3

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Hallucinogenic phenethylamines such as 2,5-dimethoxyphenethylamines (2C-X) and their N-(2-methoxybenzyl) derivatives (25X-NBOMe) have seen an increase in novel analogues in recent years. These rapidly changing analogues make it difficult for laboratories to rely on traditional targeted screening methods to detect unknown new psychoactive substances (NPS). In this study, twelve 2C-X, six 2,5-dimethoxyamphetamines (DOX), and fourteen 25X-NBOMe derivatives, including two deuterated derivatives (2C-B-d and 25I-NBOMe-d ), were analyzed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Collision-induced dissociation (CID) experiments were performed using collision energies set at 10, 20, and 40 eV. For 2C-X and DOX derivatives, common losses were observed including neutral and radical losses such as NH (17.0265 Da), •CH N (32.0500 Da), C H N (45.0578 Da) and C H N (47.0735 Da). 2C-X derivatives displayed common product ions at m/z 164.0837 ([C H O ] ), 149.0603 ([C H O ] ), and 134.0732 ([C H O] ) while DOX derivatives had common product ions at m/z 178.0994 ([C H O ] ), 163.0754 ([C H O ] ), 147.0804 ([C H O] ), and 135.0810 ([C H O] ). 25X-NBOMe had characteristic product ions at m/z 121.0654 ([C H O] ) and 91.0548 ([C H ] ) with minor common losses corresponding to 2-methylanisole (C H O, 122.0732 Da), 2-methoxybenzylamine (C H NO, 137.0847 Da), and •C H NO (152.1074 Da). Novel analogues of the selected classes can be detected by applying neutral loss filters (NLFs) and extracting the common product ions. Copyright © 2017 John Wiley & Sons, Ltd.

Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked samples inserted amongst authentic plasma samples, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a sample, as well as the confidence in detections that are made.

An analytical method was developed and validated for the purpose of detecting and quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines and piperazines. Using only 100 µL whole blood, a salting-out-assisted liquid-liquid extraction with acetonitrile was performed to isolate target compounds followed by chromatographic separation using a Waters ACQUITY ultra performance liquid chromatograph coupled to a Waters XEVO quadrupole time-of-flight mass spectrometer. Mephedrone-d3 was used as an internal standard. A gradient elution was used in combination with a Waters ACQUITY HSS C18 column (2.1 × 150 mm, 1.8 µm). Samples were analyzed using the detector in positive electrospray ionization mode with MS(E) acquisition. All compounds of interest were resolved in a 15 min run time and positively identified based on accurate mass of the molecular ion, two product ions and retention time. All analyte calibration curves were linear over the range of 0.05-2 mg/L with most correlation coefficient (r(2)) values >0.98. The limits of detection were within the range of 0.007-0.07 mg/L and limits of quantification within 0.05-0.1 mg/L. All analytes were stable 48 h after extraction and most were stable in blood after 1 week stored in a refrigerator and 3 freeze-thaw cycles. No carryover was observed up to 10 mg/L and no interferences from common therapeutic drugs or endogenous compounds. Recoveries ranged from 71 to 100% and matrix effects were assessed for blank, post-mortem and decomposed blood. All bias and % coefficient of variation values were within the acceptable values of ±15 and ≤15%, respectively (±20 and ≤20% at lower limit of quantification). The method was applied to several forensic cases where the subject exhibited behavior characteristic of designer drug intoxication and where routine screening for a panel of drugs was negative.

An analytical method using ultra performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) was developed and validated for the targeted toxicological screening and quantification of commonly used pharmaceuticals and drugs of abuse in postmortem blood using 100 µL sample. It screens for more than 185 drugs and metabolites and quantifies more than 90 drugs. The selected compounds include classes of pharmaceuticals and drugs of abuse such as: antidepressants, antipsychotics, analgesics (including narcotic analgesics), anti-inflammatory drugs, benzodiazepines, beta-blockers, amphetamines, new psychoactive substances (NPS), cocaine and metabolites. Compounds were extracted into acetonitrile using a salting-out assisted liquid-liquid extraction (SALLE) procedure. The extracts were analyzed using a Waters ACQUITY UPLC coupled with a XEVO QTOF mass spectrometer. Separation of the analytes was achieved by gradient elution using Waters ACQUITY HSS C18 column (2.1 mm x 150 mm, 1.8 μm). The mass spectrometer was operated in both positive and negative electrospray ionization modes. The high-resolution mass spectrometry (HRMS) data was acquired using a patented Waters MSE acquisition mode which collected low and high energy spectra alternatively during the same acquisition. Positive identification of target analytes was based on accurate mass measurements of the molecular ion, product ion, peak area ratio and retention times. Calibration curves were linear over the concentration range 0.05-2 mg/L for basic and neutral analytes and 0.1-6 mg/L for acidic analytes with the correlation coefficients (r2) > 0.96 for most analytes. The limits of detection (LOD) were between 0.001-0.05 mg/L for all analytes. Good recoveries were achieved ranging from 80% to 100% for most analytes using the SALLE method. The method was validated for sensitivity, selectivity, accuracy, precision, stability, carryover and matrix effects. The developed method was tested on a number of authentic forensic samples producing consistent results that correlated with results obtained from other validated methods.

Synthetic cannabinoids (SCs) remain the largest class of new psychoactive substances (NPS), and while the number of NPS that are reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) for the first time each year declines, the number of newly reported SCs still exceeds other NPS classes. This decline can be seen as a result of legislative changes by different jurisdictions which have sometimes transitioned to a more generalized approach when controlling substances by defining common structural scaffolds rather than explicit structures. While the consequences of such legislative changes have been expected over the years, the introduction of so‐called “class‐wide” bans puts further pressure on clandestine laboratories to synthesize compounds which are out of the scope of the legislation, and thus, these compounds are initially harder to detect and/or identify in the absence of analytical data. Recently, a SC with an indole‐3‐acetamide core‐linker scaffold, AD‐18 (i.e., ADB‐FUBIATA or ADB‐FUBIACA), was reported for the first time in China in 2021. Here, an additional cannabinoid with the indole‐3‐acetamide scaffold, N‐cyclohexyl‐2‐(1‐pentyl‐1H‐indol‐3‐yl)acetamide (CH‐PIACA), is reported which was identified for the first time in a seized material in Denmark. Structural characterization was performed using gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐high‐resolution mass spectrometry (LC‐HRMS), and nuclear magnetic resonance (NMR) spectroscopy.

The use of LC-HRAM spectrometry to identify ‘unknown’ compounds by non-targeted screening provides a potential advantage for forensic toxicology.