Urine testing found synthetic cannabinoid markers in 76% of clinic samples

The continuous emergence of structurally diverse indazole-based synthetic cannabinoids (SCs) challenges routine analytical detection because parent compounds are rapidly metabolized and often absent in urine. Therefore, sensitive metabolite-targeted analytical methods are required for reliable exposure assessment. In this study, we adapted and fully validated a targeted high-resolution LC-HRMS method for the quantitative determination of eight contemporary indazole-based SC markers in human urine and applied it to 50 authentic urine specimens from treatment-seeking individuals attending an addiction treatment clinic. The method allowed the identification of robust analytical performance, with LOD and LOQ values of 0.01-0.46 ng/mL and 0.04-1.53 ng/mL, respectively, along with acceptable recovery (81.54-111.72%), bias (-4.47-13.17%), precision (RSD 5.12-15.03%), and matrix effects (75.92-100.03%). All target analytes were stable for one week at room temperature, 4 °C, and -20 °C, and no significant carryover was observed. In this treatment-seeking cohort, 76% of samples were positive for at least one SC marker, with eight markers identified across MDMB-PINACA, MDMB-BINACA, and ADB-BUTINACA analogs. MDMB-4en-PINACA butanoic acid was the dominant biomarker (62%), followed by MDMB-INACA 3,3-dimethylbutanoic acid (42%), for which this study provides the first validated quantitative urine data. Other markers were detected at lower frequencies, and frequent co-detection indicates widespread polydrug SC use. The validated high-resolution LC-HRMS method enables reliable metabolite-based assessment of exposure to emerging SCs in authentic urine samples. The predominance of MDMB-type metabolites demonstrates the applicability of the method for monitoring current trends in SC exposure, while the quantitative characterization of MDMB-INACA 3,3-dimethylbutanoic acid expands the available analytical data for newly emerging SCs. This analytical platform provides a robust tool for forensic and clinical toxicology laboratories to monitor SC exposure in rapidly evolving drug markets.