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Abstract Introduction: Immunoassay-based (IA) detection for drugs of abuse is commonly used as an initial screening step in urine-based drugs testing due to rapid generation of results and ease of automation. However, IAs suffer from significant issues with cross-reactivity leading to false positives, thus requiring costly and time-consuming chromatography-based confirmatory testing. As a cost-effective alternative, DART-MS provides quantitative and highly selective results greatly reducing or eliminating false positives compared to conventional IA-based drug screening. In this work, we report on the development of a rapid, chromatography-free screening approach for eighteen synthetic cannabinoids in urine: (4-cyano-CUMYL-BUTINACA (1), 4-fluoro ABUTINACA N-(4-hydroxybutyl) metabolite (2), 4-fluoro MDMB-BUTICA (3), 4-fluoro BUTICA butanoic acid metabolite (4), 4-fluoro MDMB-BUTINACA (5), 4-fluoro MDMB-BUTINACA N-butanoic acid metabolite (6), 5-fluro ADB metabolite (7), 5-fluoro MDMB-PICA (8), 5-fluoro MDMB-PICA metabolite (9), ADB-4en-PINACA (10), ADB-BINACA (11), ADB-BUTINACA (12), ADB-BUTINACA N-(4-hydroxylbutyl) METABOLITE (13), ADB-HEXINACA (14), AMP-4en-PINACA (15), JWH 018 N-pentanoic acid METABOLITE (16), MDMB 4en-PINACA-butanoic acid metabolite (17), MDMB-CHMICA METABOLITE (18)). This DART-MS screening method successfully measures the targeted synthetic cannabinoids in 96 samples at a rapid throughput of 23 seconds per sample.
Methods: For method development, triplicate calibration series were prepared by spiking certified drug-free urine with standards 1-18 (0.1-2500 ng/mL) using deuterated AB-PINACA as an internal standard. Hydrolysis was performed by adding 50 µL Kura enzyme to 500 µL pre-spiked certified aliquots of drug-free urine and incubated at room temperature for 20 minutes. After hydrolysis, 500 µL 0.1 M Borax buffer (pH=10.4) and 2.5 mL 30:70 (ethyl acetate:n-chlorobutane) were added to each sample followed by a 30 second agitation. Samples were centrifuged at 4000 RPM for 10 minutes and the organic layer was transferred to glass vials and evaporated to dryness under N2 at 40°C followed by reconstitution in 100 µL MeOH. Reconstituted samples were vortexed for 30 seconds, and 2 µL aliquots of each sample were transferred onto a Bruker DART QuickStrip HTS-96 screen and allowed to dry under N2 gas at 40°C for 15 minutes. For analysis, the prepared QuickStrip-HTS 96 screen was loaded onto the automated XY transmission stage of a TQ-Plus (Bruker Daltonics) triple quadrupole mass spectrometer for DART-MS-MS analysis. Accuracy was determined in triplicate using certified drug-free urine without detectable levels of 1-18 at 2 levels for each analyte within the linear range of each calibration series. Results were validated against LC-MS that was performed using 20 urine samples confirmed as positive for one or more analytes.
Results and Discussion: DART and TQ-plus parameters were optimized for high sensitivity, precision, and fast analysis time. With DART gas temperature and grid voltage optimized at 350°C and 100 V, respectively, unique MS/MS transitions, collision energies, and MS scan times were successfully identified for 1-18. DART-MS analysis of the synthetic cannabinoid panel resulted in a good linear correlation of R2 > 0.95 and an accuracy between 87 and 107% for all 18 analytes across the defined calibration ranges. The lower level of quantitation (LLOQ) of between 0.1 to 5 ng/mL and cross-validation of the samples showed good correlation with LC-MS data, indicating that this rapid chromatography-free workflow is sufficient in detecting all 18 analytes at or below the common cutoff values without the high rate of false positives associated with IA based screening approaches.
The results presented herein demonstrate the suitability of the DART-MS workflow as a rapid, quantitative, and selective alternative to conventional IA-based urine screening by offering a quantitative method with the benefits of minimizing false positives typically associated with IA based screening, avoiding costly and unnecessary chromatography-based confirmatory testing.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
