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

Importance of the topic

High-throughput and reliable screening of biological samples is critical in forensic toxicology to identify a broad range of drugs and metabolites quickly and with high confidence. Ultra high resolution mass spectrometry (UHMR) offers unparalleled mass accuracy and resolution, enabling retrospective data review and expanded compound discovery without re-running samples.

Study objectives and overview

This study evaluated the performance of the Exactive™ bench-top UHMR mass spectrometer in a targeted forensic screening workflow for urine analysis. Results from LC-MS were compared against conventional immunoassay (EMIT), a laboratory Remedy panel, and EI-GC-MS across 40 authentic urine samples to assess compound coverage, sensitivity, and practicality of sample preparation.

Methodology and instrumentation

• Sample preparation: 50 µL urine mixed with 50 µL internal standard solution (Hydromorphone-D6, Methamphetamine-D5, Phenobarbital-D5) and 900 µL DI water; vortexed; 20 µL injected.
• LC system: Thermo Scientific Accela 600 pump with open autosampler; Hypersil GOLD PFP column (100 × 2.1 mm, 5 µm); mobile phases A (10 mM ammonium formate in water), B (methanol) and C (acetonitrile:1-propanol:acetone 45:45:10); 15-minute gradient; divert valve off waste 0–1.8 min, on-line thereafter.
• Mass spectrometry: Exactive Plus Orbitrap bench-top instrument with HESI source; alternating positive/negative full scan at 50 000 resolution (m/z 135–2000 and 100–2000), all-ion fragmentation at 25 000, AGC target 1e6, max IT 100 ms; NCE 35 for AIF scans.
• Comparative methods: EMIT immunoassay on Siemens DB RxL; GC-MS using Thermo CSQ II after SPE and BSTFA derivatization on TR-5 MS column.
• Data processing: ExactFinder™ software with parameter-free peak detection, background subtraction, isotopic pattern comparison and fragment matching; 5 ppm mass window; custom database of 220 compounds.

Main results and discussion

• LC-MS detected all analytes found by immunoassay, Remedy and GC-MS and additionally numerous metabolites and conjugates.
• Immunoassay remained more sensitive for low-level THC and metabolites, which were not captured by the 20×-diluted LC-MS method.
• Detection of intact glucuronides eliminated the need for hydrolysis, reducing sample preparation time and preserving labile conjugates such as nicotine and morphine glucuronides.
• GC-MS and immunoassay failed to identify several targeted compounds initially missing from the ExactFinder database; database updates and reprocessing recovered these analytes.

Practical benefits and applications

• Comprehensive single-run profiling accelerates forensic screening and reduces reliance on multiple orthogonal assays.
• Simplified dilution-only preparation minimizes sample handling, lowers risk of analyte loss, and shortens turnaround time.
• High-resolution data allow retrospective interrogation for newly added targets, supporting flexible laboratory workflows.

Future trends and applications

Advances will focus on expanding HRMS databases, integrating machine-learning-driven data mining, and automating sample preparation. Coupling UHMR screening with quantitative methods and high-throughput robotics promises seamless forensic and clinical toxicology pipelines. Improved sensitivity for low-abundance analytes such as cannabinoids may be achieved through online preconcentration and enhanced ionization strategies.

Conclusion

The Exactive UHMR platform demonstrated superior compound coverage, speed, and retrospective capabilities compared to immunoassay and GC-MS methods for targeted urine screening. While THC detection at low levels favors immunoassay, UHMR-based LC-MS offers a streamlined, high-resolution solution that can consolidate multiple workflows into a single, robust assay.

Used instrumentation

• Thermo Scientific Accela 600 LC system with open autosampler
• Thermo Hypersil GOLD PFP column (100×2.1 mm, 5 µm)
• Exactive Plus bench-top Orbitrap mass spectrometer with HESI source
• Siemens DB RxL for EMIT immunoassays
• Thermo CSQ II EI-GC-MS with TR-5 MS capillary column