Analysis of “Bath Salt” Compounds from Urine for Forensic Toxicology Using μElution Mixed-mode SPE Combined with UPLC/MS/MS Detection

Importance of the Topic

With the rapid emergence of synthetic cathinones marketed as bath salts, forensic toxicology faces new challenges in detecting these stimulants in biological fluids. The structural diversity and low dosage levels demand sensitive, selective, and high-throughput analytical methods for reliable identification and quantitation in urine samples.

Objectives and Study Overview

This study aimed to develop a single, streamlined workflow for the extraction and UPLC/MS/MS analysis of a panel of ten forensic-relevant synthetic cathinones and a key metabolite in human urine. The goal was to achieve rapid separation, high recovery, minimal matrix effects, and quantitation down to 1 ng/mL.

Methodology

Mixed-mode strong cation exchange µElution SPE was used to isolate analytes from 100 µL of acidified urine without evaporation or reconstitution. Extracts were directly injected onto a UPLC system equipped with a 1.7 µm BEH C18 column and analyzed by positive ESI MRM on a Xevo TQD mass spectrometer. Method parameters, including mobile phases, gradient, cone voltages, transitions, and collision energies, were optimized for all targets.

Instrumentation Used

• Oasis MCX µElution 96-well plates
• ACQUITY UPLC BEH C18 column, 1.7 µm, 2.1 x 100 mm
• ACQUITY UPLC system
• Xevo TQD tandem quadrupole mass spectrometer

Main Results and Discussion

All ten cathinones and one metabolite were separated within two minutes, with a total cycle time of four minutes. Baseline resolution was achieved for isobaric pairs such as ethylone and butylone. Use of 40% IPA in the SPE elution solvent improved recoveries to 87.4–100.5% and reduced matrix effects to between –3.6% and 12.4%. Calibration curves were linear across 1–500 ng/mL (R2 ≥ 0.990), with accuracy within 15% of nominal values and limits of quantitation at 1 ng/mL.

Benefits and Practical Applications

• High throughput with no evaporation or reconstitution steps
• Robust recoveries and minimal ion suppression
• Rapid chromatographic run time enhances sample throughput
• Broad applicability to emerging cathinone analogs with minimal method adjustments

Future Trends and Applications

Further developments may include integration with high-resolution mass spectrometry for non-targeted screening, automation of the µElution SPE workflow, extension to other biological matrices, and adaptation for point-of-care or field-deployable platforms to support real-time forensic investigations.

Conclusion

The combined µElution SPE and UPLC/MS/MS approach delivers a rapid, sensitive, and reliable assay for the comprehensive analysis of bath salt compounds in urine, supporting forensic laboratories in meeting growing demands for synthetic cathinone monitoring.

References

1. Coppola M, Mondola R. Synthetic cathinones chemistry, pharmacology and toxicology of a new class of designer drugs of abuse marketed as bath salts or plant food. Toxicology Letters. 2012;211(2):144–149.