LC-MS/MS toxicology workflow on the new Orbitrap Exploris 120 mass spectrometer for screening, quantitation, and confirmation of drugs

Significance of the Topic

The identification and quantitation of drugs of abuse and toxins in biological matrices are critical for clinical, forensic, and anti-doping laboratories. The complexity of emerging designer and illicit substances demands robust, high-throughput workflows that combine comprehensive screening, accurate confirmation, and retrospective analysis while maintaining cost efficiency and fast turnaround times.

Aims and Study Overview

This work demonstrates a targeted screening and quantitation method for over 1,800 drugs of abuse and related toxins in urine using the Thermo Scientific Tox Explorer Collection. The workflow integrates high-resolution, accurate-mass data acquisition on the Orbitrap Exploris 120 mass spectrometer with spectral library matching and a compound database in TraceFinder software for rapid identification, confirmation, and quantitation.

Methodology

• Sample Preparation
  Urine samples were spiked with over 100 analytes covering diverse drug classes and eight isotopically labeled internal standards. After dilution (1:20 in water), calibration curves from 0.1 to 1,000 ng/mL were prepared and analyzed in triplicate.


• Liquid Chromatography
  A Vanquish Flex UHPLC system with an Accucore Phenyl-Hexyl column (100 × 2.1 mm, 2.6 μm) employed a 15.5-minute gradient (mobile phase A: 2 mM ammonium formate/0.1% formic acid in water; B: 1:1 acetonitrile/methanol) for efficient separation of isomers and a broad polarity range.


• Mass Spectrometry
  The Orbitrap Exploris 120 operated in full scan (60,000 FWHM at m/z 200) and targeted data-dependent MS/MS (15,000 FWHM) modes with an inclusion list specifying exact masses, polarities, and retention times. Polarity switching enabled acquisition of positive and negative ions in a single run. Stepped collision energies (18.75, 37.5, 56.25 eV) produced rich fragment ion spectra.


• Software and Library Generation
  TraceFinder 5.1 provided integrated data acquisition, processing, and reporting. An mzVault spectral library with over 1,800 HRAM MS/MS spectra (including 200+ fentanyl analogs) and a compound database containing molecular formulae, retention times, and fragment lists facilitated targeted screening and confirmation. New compounds were added without extensive method revalidation.

Instrumentation Used

• Thermo Scientific Vanquish Flex UHPLC System


• Accucore Phenyl-Hexyl Column (2.6 μm, 100 × 2.1 mm)


• Thermo Scientific Orbitrap Exploris 120 Mass Spectrometer


• TraceFinder Software 5.1 and mzVault Library

Main Results and Discussion

• Screening Performance
  Compounds were identified within ±5 ppm mass accuracy and ±30 s retention time windows. Chromatographic resolution enabled separation of isomeric drugs, such as morphine isomers, with reliable library matching (spectral score >70%) and isotope/fragment verification.


• Quantitation
  Limits of detection and quantitation (LOD, LOQ) were achieved down to 0.1 ng/mL for multiple analytes with intra-assay precision (CV <20%) and inter-day accuracy within standard criteria. Calibration curves (0.1–1,000 ng/mL) exhibited linearity with back-calculated concentrations within ±20% (±30% for analytes lacking labeled IS).


• Robustness
  Continuous operation over 48 hours showed mass accuracy stability (<1.3 ppm, RSD <0.1%) and retention time repeatability (±0.01 min). Internal standard peak areas remained consistent (CV <13%).


• Retrospective Analysis
  Non-targeted data mining enabled the discovery of unexpected compounds, such as dibutyl phthalate, with high mass accuracy (<2 ppm) without additional experiments.

Benefits and Practical Applications

• Comprehensive all-in-one workflow from screening to reporting on a single platform.


• High throughput (15.5-minute run) with minimal sample prep (dilute-and-shoot).


• Expanded spectral coverage (>1,800 compounds including new fentanyl analogs).


• Simultaneous positive/negative ion analysis and retrospective data mining.


• Space-efficient instrumentation suitable for routine toxicology and anti-doping labs.

Future Trends and Applications

The integration of alternative sample preparation strategies (e.g., solid-phase extraction) and further enrichment of spectral libraries will enhance sensitivity and expand analyte coverage. Advances in machine learning for spectral deconvolution and automated database updates promise more rapid adaptation to emerging substances. Coupling HRAM platforms with ambient ionization techniques may further streamline workflows for point-of-care toxicology.

Conclusion

The Thermo Scientific Tox Explorer Collection combined with the Orbitrap Exploris 120 and TraceFinder software delivers a robust, high-resolution LC-MS/MS method for targeted screening, quantitation, and confirmation of a broad drugs-of-abuse panel in urine. The workflow’s sensitivity, speed, and adaptability address the evolving demands of toxicology, forensics, and anti-doping laboratories.

References

• SAMHSA, Guidelines for Workplace Drug Testing, U.S. Department of Health and Human Services, 2021.