Evaluation of Ultra High Resolution Mass Spectrometer in Targeted Forensic Screening Method for Urine Analysis in Comparison to Immunoassay and GC-MS Techniques

Significance of the Topic

A fast and comprehensive screening method for drugs and their metabolites in urine is crucial in forensic toxicology. Ultra high resolution mass spectrometry offers high specificity, the ability to detect multiple analytes simultaneously, and retrospective data analysis capabilities that address limitations of traditional immunoassays and GC-MS techniques.

Objectives and Study Overview

This study assesses the performance of a Thermo Exactive ultra high resolution mass spectrometer in targeted forensic screening of diluted urine samples. Results were compared to conventional immunoassay, GC-MS, and a proprietary Remedy screening approach across forty urine specimens.

Methodology and Instrumentation

The sample protocol involved diluting urine twentyfold with deionized water, spiking with deuterated internal standards, and injecting directly onto an HPLC. Chromatographic separation was achieved using a Thermo Accela 600 pump with Hypersil GOLD PFP column under a 15-minute gradient and a divert valve workflow.
The Exactive instrument operated in alternating positive and negative full-scan (50 000 resolution) and all-ion fragmentation mode (25 000 resolution) using a HESI probe. ExactFinder software reconstructed chromatograms at 5 ppm mass accuracy, applying isotopic pattern matching and fragment ion confirmation.
Comparative techniques included GC-MS analysis with SPE cleanup and BSTFA derivatization on a Thermo CSQ II with a TR-5 column, and EMIT immunoassays performed on a Siemens DB RxL platform.

Main Results and Discussion

All compounds detected by immunoassay, Remedy, and GC-MS were also identified by LC-MS, which further revealed additional metabolites and low-abundance analytes. GC-MS and LC-MS methods failed to detect low-level THC and its metabolites in one sample, whereas immunoassay demonstrated higher sensitivity for these targets. Updating the ExactFinder database to include missing compounds enhanced detection performance. The LC-MS workflow enables retrospective data mining for newly targeted analytes without re-running samples.

Benefits and Practical Applications

A simple dilution sample preparation preserves glucuronide conjugates and reduces processing time by eliminating hydrolysis and extraction steps. High resolution and accurate mass measurements ensure confident identification in complex matrices. The method supports broad-spectrum screening, rapid turnaround, and retrospective analysis, streamlining forensic laboratory operations.

Future Trends and Potential Applications

Expansion of compound databases and integration of machine learning algorithms can further enhance detection breadth and confidence. Advances in sample preparation or instrument sensitivity may lower detection limits for polar glucuronides and trace-level substances. This approach may also be applied to environmental monitoring, clinical toxicology, and anti-doping controls, leveraging retrospective analysis to address emerging threats.

Conclusion

The Exactive ultra high resolution LC-MS method surpasses traditional immunoassay and GC-MS workflows in overall compound coverage, with the exception of low-level THC detection. Its streamlined sample preparation and retrospective data analysis capabilities establish it as a powerful forensic urine screening tool.

References

1 Marta Kozak, Bénédicte Durez, Maggie Lee, Ernest Wang, Cristina Stefan. Evaluation of Ultra High Resolution Mass Spectrometer in Targeted Forensic Screening Method for Urine Analysis in Comparison to Immunoassay and GC-MS Techniques. Thermo Fisher Scientific & Center of Addiction and Mental Health.