Simultaneous Analysisof Seven AmphetamineClass Drugs in Urine forForensic Toxicology

Importance of the Topic

Amphetamine-class stimulants are among the most widely abused psychoactive compounds worldwide. Their potent effects on dopamine and norepinephrine pathways pose serious health risks, including toxicity, dependence and fatal overdose. Robust analytical protocols are critical in forensic and clinical settings to detect trace levels of these substances in urine, support legal investigations and guide public health responses.

Objectives and Study Overview

This work aimed to evaluate the impact of integrating an online solid-phase extraction (SPE) system with LC/MS for the simultaneous quantification of seven amphetamine-type drugs in urine. Specifically, the study compared a simple dilute-and-shoot approach against sample cleanup using the ACQUITY Online SPE Manager, focusing on matrix effect removal, background suppression and improvement in analytical sensitivity.

Methodology

The study used human urine samples spiked at known concentrations of amphetamine, methamphetamine, MDMA, MDA, MDEA, phentermine and propylamphetamine. Two workflows were compared:

• Dilute-and-shoot: 1:10 dilution in 10 mM ammonium formate and direct injection.
• Online SPE cleanup: C18 cartridge conditioning with MeOH, ammonium hydroxide and water, sample loading at 5% NH4OH, sequential washes, then elution onto the UPLC column.

Chromatographic separation employed an ACQUITY UPLC HSS T3 column (2.1 mm×50 mm, 1.8 µm) with a water/5% NH4OH (A) and acetonitrile (B) gradient. MS detection utilized a Xevo TQD in positive-ion ESI mode, monitoring multiple reaction monitoring (MRM) transitions specific to each analyte.

Used Instrumentation

• UPLC system: Waters ACQUITY UPLC
• SPE module: ACQUITY Online SPE Manager with disposable C18 cartridges
• Mass spectrometer: Waters Xevo TQD in ESI+ mode

Main Results and Discussion

Online SPE dramatically reduced urine matrix background and ion suppression compared to direct injection. Total ion chromatograms showed near-complete removal of interfering peaks, while sensitivity for all seven analytes improved by several-fold. The method achieved detection at or below 10 ppb levels with high reproducibility across multiple SPE cartridges (RSD <5%).

Benefits and Practical Applications

• Enhanced sensitivity and lower limits of detection for forensic urine screening.
• Automated, reproducible sample cleanup reducing instrument maintenance.
• Efficient removal of matrix interferences to prolong column life and improve data quality.

Future Trends and Applications

Advances in online SPE-LC/MS integration may extend to multi-class drug panels, high-throughput clinical testing and environmental monitoring. Further miniaturization of SPE cartridges and AI-driven data processing could streamline workflows and improve automation in forensic laboratories.

Conclusion

The incorporation of an online SPE system into LC/MS analysis of amphetamine-class drugs in urine offers a significant enhancement in analytical performance. By effectively removing matrix interferences and increasing sensitivity, this approach provides a robust tool for forensic toxicology and clinical drug testing.