Using Ion Mobility to separate different designer drug metabolites

Significance of the Topic

Screening for designer drugs in biological fluids demands not only detection of parent compounds but also their diverse metabolites. The metabolic transformations of synthetic cannabinoids often yield structural isomers that complicate conventional LC-MS analysis. Integrating ion mobility separation (IMS) with high-resolution time-of-flight mass spectrometry enhances confidence in identifying known and emerging metabolites, thereby strengthening forensic and clinical toxicology workflows.

Study Objectives and Overview

This work aims to demonstrate how IMS coupled with a timsTOF instrument resolves isomeric hydroxylated metabolites of the synthetic cannabinoid AKB-48. Two metabolite mixtures were prepared in urine-mimicking matrices to assess chromatographic and mobility separation. Key goals include mapping collision cross section (CCS) groupings, distinguishing positional isomers via adduct species, and verifying identification parameters for forensic screening.

Methodology

Metabolites of AKB-48, not commercially available, were synthesized in-house. Two mixtures containing multiple hydroxylated isomers at 125 ng/mL each were analyzed. Initial separation employed reversed-phase liquid chromatography. Ion mobility separation preceded time-of-flight detection to add an orthogonal separation dimension. Data processing focused on extracted ion chromatograms (EICs), extracted ion mobilograms (EIMs), accurate mass, isotopic pattern matching, and CCS evaluation.

Applied Instrumentation

• Ultra-Performance LC Ultimate 3000 Rapid Separations system (Thermo Scientific)
• Column: Waters HSS T3, 2.1×150 mm, 1.8 µm at 60 °C
• Mobile phase A: water with 10 mM ammonium formate and 0.05% formic acid
• Mobile phase B: acetonitrile with 0.05% formic acid; gradient from 57% to 93% B over 8 min; flow rate 0.5 mL/min; injection volume 1 µL
• timsTOF mass spectrometer (Bruker Daltonics) operating in ESI+ mode at 2500 V
• MS calibration with sodium formate cluster; IMS calibration with tuning mix

Main Results and Discussion

Chromatographic separation yielded baseline peaks for most hydroxylated isomers, ionizing as both [M+H]+ and [M+Na]+. Mass accuracies better than 1 ppm and tight isotopic pattern overlays confirmed high data quality. IMS resolved protonated and sodiated species into distinct CCS clusters: protonated isomers exhibited inverse mobility (1/K0) around 1.00–1.04 V·s/cm2, while sodium adducts ranged 1.04–1.08 V·s/cm2. Metabolites hydroxylated on the alkyl chain (B1–B4) formed a separate mobility group from those modified on the adamantyl moiety (B5–B7). Notably, isomer B1 displayed intermediate mobility values, diverging from both clusters. Co-eluting isomers B4 and B7, indistinguishable by LC alone, achieved baseline separation in both adduct forms via IMS. Combined metrics—accurate mass, isotope distribution, MS/MS fragmentation, and CCS—provided robust identification criteria.

Benefits and Practical Applications

• Enhanced isomer discrimination in complex urine matrices improves forensic screening reliability.
• CCS measurements serve as an additional identification parameter, reducing false positives/negatives.
• Orthogonal separation via IMS complements existing LC-MS/MS workflows without extensive method modification.
• Applicability extends to other designer drug classes and unknown metabolite discovery.

Future Trends and Applications

Emerging directions include expanding CCS libraries for a broader range of synthetic cannabinoids and their metabolites. Integration with high-throughput data processing and machine learning will accelerate unknown analyte annotation. Miniaturized or field-deployable IMS-MS platforms could enable on-site screening in forensic or clinical settings. Collaborative databases sharing CCS and fragmentation data will facilitate cross-laboratory harmonization.

Conclusion

IMS coupled to high-resolution MS demonstrably separates positional isomers of AKB-48 metabolites, overcoming limitations of LC-MS alone. The combined use of accurate mass, isotopic pattern matching, MS/MS fragmentation, and CCS values delivers a comprehensive workflow for confident metabolite identification. This approach enhances forensic toxicology capabilities to address the dynamic landscape of designer drug use.

References

1. Jakob Wallgren et al. Synthesis and identification of an important metabolite of AKB-48 with a secondary hydroxyl group on the adamantyl ring. Tetrahedron Letters. 2017;58:1456-1458.
2. Svante Vikingsson, Martin Josefsson, Henrik Green. Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry. Journal of Analytical Toxicology. 2015;39(6):426-435.