Evaluation of the ACQUITY UPLC H-Class System with ACQUITY QDa Mass Detector for the Determination of Amphetamine, Methamphetamine, Ketamine, and Norketamine in Human Urine for Forensic Toxicology

Significance of the Topic

Amphetamine and methamphetamine are among the most frequently abused illicit substances worldwide, with ketamine increasingly encountered in forensic toxicology. Conventional immunoassays are rapid but suffer from selectivity issues, leading to false positives and inefficiencies due to confirmatory testing. The application of a compact UPLC system coupled with a single quadrupole mass detector addresses these limitations by offering enhanced specificity and streamlined workflows.

Objectives and Study Overview

The study aimed to evaluate the Waters ACQUITY UPLC H-Class system combined with the ACQUITY QDa mass detector as an alternative to immunoassay methods for the qualitative and quantitative analysis of amphetamine, methamphetamine, ketamine, and norketamine in human urine. Performance criteria included selectivity, linearity, sensitivity, and agreement with established high-resolution methods.

Methodology and Instrumentation

A series of calibrators (40–2000 ng/mL) and quality control (QC) samples (75–1600 ng/mL) were prepared by spiking pooled drug-free human urine. Samples, including a commercial QC at 150 ng/mL, were diluted 5-fold in aqueous solution containing deuterated internal standards (500 ng/mL). Chromatographic separation was achieved on a BEH C18 UPLC column (2.1×50 mm, 1.7 µm) with a 3-minute gradient. Data acquisition used the ACQUITY QDa mass detector in positive ionization mode under selected ion recording (SIR) with in-source collision-induced dissociation. MassLynx and TargetLynx software facilitated data processing and identification based on retention time, precursor/product ions, and ion ratios.

• UPLC system: ACQUITY UPLC H-Class
• Mass detector: ACQUITY QDa in positive SIR mode
• Column: BEH C18, 2.1×50 mm, 1.7 µm
• Software: MassLynx and TargetLynx

Main Results and Discussion

All four analytes exhibited excellent linearity (R² > 0.99) across 40–2000 ng/mL, with retention times spanning 0.85 to 1.60 minutes. Analysis of a commercial QC yielded measured concentrations within 12% of expected values. Spiking blank urine with 21 potential interferents at 10 µg/mL produced no false positives, demonstrating robust selectivity. Comparison with high-resolution LC–MS screening (Xevo G2-XS QTof) on authentic urine specimens showed strong concordance, confirming the method’s accuracy.

Benefits and Practical Applications

• Improved selectivity over immunoassays, reducing false positives
• Rapid analysis with a total run time of 3 minutes per sample
• Simplified sample preparation with direct dilution and internal standardization
• Cost-effective alternative to tandem mass spectrometry
• Suitable for routine forensic and clinical toxicology workflows

Future Trends and Potential Applications

With ongoing advancements in compact mass detection, single quadrupole-based UPLC–MS methods are poised for broader adoption in high-throughput toxicology labs. Future directions include expanding analyte panels, integrating automated sample handling, and coupling with informatics platforms for real-time data review and reporting.

Conclusion

The ACQUITY UPLC H-Class system paired with the QDa mass detector offers a reliable, selective, and efficient approach for illicit drug screening and quantification in urine. This workflow presents a viable alternative to immunoassay, improving laboratory throughput and data confidence in forensic toxicology settings.

References

• United Nations Office on Drugs and Crime. World Drug Report 2018. Global Overview of Drug Demand and Supply.
• European Monitoring Centre for Drugs and Drug Addiction. European Drug Report 2018, Trends and Developments.
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• Marin SJ et al. One Hundred False-Positive Amphetamine Specimens Characterized by LC–TOF MS. J. Anal. Toxicol. 2016;40(1):37–42.
• Lee C-H et al. False Positive Ketamine Urine Immunoassay Screen Induced by Quetiapine: A Case Report. J. Formosan Med. Assoc. 2017;116(9):1–3.
• Goshawk J, Lee R, Wood M. Evaluation of the Potential of the ACQUITY QDa Mass Detector for Use in Forensic Chemistry and Drug Control Laboratories. Waters Technology Brief 720006004EN, 2017.
• Lee R, Wood M. Systematic Toxicological Screening Using the ACQUITY UPLC I-Class/Xevo TQ-S micro. Waters Technology Brief 720005661EN, 2016.
• Waters Corporation. Forensic Toxicology Screening Application Solution with UNIFI [Brochure]. 2014.