Abstract:

To evade legal controls, new psychoactive substances (NPS), which have been designed as substitutes for traditional and synthetic drugs, are gradually dominating the drug market. Synthetic cannabinoids (SCs), which account for the majority of NPS, are rapidly being derivatized; consequently, controlling increasing abuse by merely listing individual compounds is difficult. Therefore, China has included the entire SC category of SCs listed as legal controlled substances since July 1, 2021. However, new SCs obtained through structural modification are still appearing and pose significant analytical challenges for forensic laboratories. Therefore, an efficient, green, and automated detection method is urgently required to provide technical support for the accurate screening actual samples. Meanwhile, the number of indazole-type SCs has increased sharply since 2013, which is ascribable to their stronger psychoactive effects. Indeed, forensic laboratories mainly analyze this key SC subclass. Therefore, in this study, we developed a new method for analyzing 51 indazole-type SCs in human urine and blood, which involves online solid-phase extraction (online SPE) as the preprocessing technology, with analysis performed using liquid chromatography-linear ion trap mass spectrometry. Deproteinization was achieved by adding acetonitrile, with dilution performed using 10 mmol/L ammonium acetate solution (pH 4.8) containing 0.1% formic acid. Samples were then analyzed directly using acetonitrile-10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) as the mobile phase. The mass-to-charge ratios of protonated molecular ions ([M+H]⁺) in the mass spectra acquired in full-scan mode, and the retention times in the chromatograms of the analytes were selected with the aim of monitoring the MS² ions of the various compounds. Characteristic fragment ions of the various SC structures were summarized, with five groups of isomers, each containing ten compounds, successfully distinguished using multistage mass spectrometry and their retention times. Multistage MS was used to qualitatively screen 51 indazole-type SCs, which were then quantitatively analyzed using MS² ion pairs (as quantitative ion pairs). The analytes exhibited limits of detection (LODs) of 0.02-1 ng/mL, with limits of quantification (LOQs) of 0.04-4 and 0.1-4 ng/mL in urine and blood, respectively. Linear fitting (weighting factor 1/x) revealed good linearity for each analyte within its respective linear range, with correlation coefficients (R²) greater than 0.99 in both urine and blood. The validity of the analytical method was tested by determining precision and spiked recovery values (n=6). Recoveries of 83.47%-116.95% were obtained at LOQ levels, with precisions of 2.29%-13.40%. In addition, recoveries of 86.63%-113.38% and precisions of 0.58%-13.79% were obtained at low, medium, and high levels. The method described herein is not only easy to operate but also can be automated. Indeed, high-throughput sample analysis was achieved when sample extraction, enrichment, and analysis were performed in dynamic mode through valve switching. Meanwhile, the method exhibited good sensitivity and is applicable to a wider range of compounds than those previously reported; it also provides a scientific basis and technical support for the rapid screening and quantitative analysis of SCs in actual relevant cases.

Key words: synthetic cannabinoids (SCs), indazole-type, urine, blood, online solid-phase extraction (online SPE), liquid chromatography-linear ion trap-mass spectrometry (LC-LIT-MS)