Fast Screening Method for 86 Forensic Analytes in Human Urine using the QTRAP® 4500 LC-MS/MS System

Significance of the Topic

In forensic toxicology, efficient screening of biological samples is essential to rapidly identify a wide variety of compounds. LC-MS/MS techniques offer superior selectivity and multiplexing compared to immunoassays, overcoming limitations such as the need for multiple antibody kits and long development times.

Objectives and Overview

This study presents a fast screening workflow to detect 86 forensic targets in human urine within a single 2.5-minute injection. It integrates multiple reaction monitoring (MRM) with an Enhanced MS (EMS) scan, enabling comprehensive data collection for both targeted compounds and potential unknowns.

Methodology

Sample Preparation:

• Hydrolysis of urine (100 µL) with beta-glucuronidase (IMCSzyme) and buffer at 55 °C for 30–60 min.
• Addition of internal standards and dilution (0.8 mL diluent), followed by centrifugation at 21,000 g for 15 min.
• Direct injection of supernatant into the LC-MS/MS system.

Chromatography and MS Acquisition:

• 2.5 min gradient on Phenomenex Luna C18 column at 40 °C, flow 0.2–1 mL/min, mobile phases: ammonium formate/water (A) and 0.1% formic acid/methanol (B).
• Injection volume: 5 µL; autosampler rinse with MeOH/ACN/IPA (1:1:3).
• Scheduled MRM algorithm with optimized DP, CE, and CXP for 86 analytes, ensuring ≥5 data points per peak.
• EMS scan in linear ion trap mode (mass range 100–600 Da, scan rate 20,000 Da/s) for full spectral screening.

Used Instrumentation

• SCIEX ExionLC AC HPLC system
• SCIEX QTRAP 4500 hybrid triple quadrupole/linear ion trap mass spectrometer
• Phenomenex Luna C18 column

Main Results and Discussion

The method achieved consistent retention for all 86 analytes between 0.7 and 1.86 min. Single-point calibration at cutoff levels provided accurate quantification across low, medium, and high quality controls, with a representative calibration curve for alpha-PVP showing excellent linearity through zero. EMS data generated a total ion chromatogram and extracted ion chromatograms that enable the detection and tentative identification of unexpected compounds, with the potential for subsequent library matching via EPI scans.

Benefits and Practical Applications

• High throughput screening with minimal sample preparation.
• Comprehensive coverage of multiple drug classes in one injection.
• Dual data acquisition (MRM and EMS) improves target identification and retrospective analysis.
• Suitable for forensic laboratories requiring rapid turnaround and robust data quality.

Future Trends and Opportunities

• Expansion of compound panels and inclusion of emerging novel psychoactive substances.
• Integration with automated sample handling platforms for further throughput gains.
• Enhanced data processing workflows combining library matching and machine learning for unknown identification.
• Application to alternative matrices (blood, oral fluid) and use of high-resolution MS for broader screening.

Conclusion

The described LC-MS/MS approach offers a rapid, sensitive, and versatile screening tool for forensic urine analysis. By leveraging Scheduled MRM and EMS scanning on the SCIEX QTRAP 4500, laboratories can achieve fast runtimes, reliable quantitation, and the ability to detect both known and unknown analytes within a single injection.