Rapid and Sensitive Analysis of a 93-Compound Forensic Panel in Urine

Importance of the Topic

Analysis of complex forensic drug panels in urine is critical for toxicology, clinical studies and legal investigations. A rapid, sensitive and high-throughput LC-MS/MS method allows laboratories to confirm diverse analytes from opioids to synthetic cannabinoids in a single run, reducing turnaround time and minimizing manual effort.

Objectives and Study Overview

This work aimed to develop and validate a robust assay for simultaneous quantitation of 93 forensic compounds in human urine. Key goals included:

• Achieving full coverage of a broad drug panel within a short chromatographic runtime
• Maintaining adequate sensitivity, specificity and quantitative performance
• Implementing an efficient sample preparation protocol

Methodology and Instrumentation

Sample preparation involved hydrolysis of blank urine at 55 °C, followed by dilution with methanol and water, centrifugation and direct injection. Chromatography was performed on an ExionLC AC HPLC system using a Kinetex Phenyl-hexyl column at 1 mL/min with ammonium formate in water (A) and formic acid in methanol (B). The MS platform was a SCIEX QTRAP®/Triple Quad™ 4500 operated under Scheduled MRM™ with fast polarity switching. A total of 212 transitions (including internal standards) were monitored in a 6.5-minute method.

Main Results and Discussion

• Baseline separation of isobaric pairs was achieved despite the brief 6.5 min gradient, with potential reduction to 5.5 min for smaller panels.
• Scheduled MRM™ ensured at least 10–15 data points per peak even with over 200 transitions, supporting precise quantitation.
• Limit of quantitation reached as low as 1 ng/mL for acetyl fentanyl, and strong signal at the lowest calibrator suggested further sensitivity gains.
• Excellent linearity and reproducibility were demonstrated across the dynamic range for all 93 compounds.

Benefits and Practical Applications

This method offers forensic and clinical laboratories a fast, dilute-and-shoot workflow that minimizes sample handling. High throughput supports large screening batches, while sensitivity and specificity meet regulatory and quality assurance requirements in toxicology, workplace testing, doping control and emergency medicine.

Future Trends and Opportunities

Advancements may include expanding panels via Scheduled MRM™ optimization, lowering limits of detection through enhanced ion optics, integrating high-resolution mass spectrometry for non-targeted screening and applying machine learning for automated data review. Automation of sample prep and miniaturized LC platforms could further increase throughput.

Conclusion

A fast, sensitive and high-throughput LC-MS/MS assay was established for quantifying 93 forensic analytes in urine. The combination of Scheduled MRM™, fast polarity switching and dilute-and-shoot prep delivers reliable results in under seven minutes, making it highly suitable for modern forensic and clinical laboratories.