Targeted Forensic Screening and Semi-Quantitation of Drugs in Urine Using an HRAM Mass Spectrometer

Importance of the Topic

Forensic toxicology laboratories face growing demands to rapidly detect and quantify a broad range of drugs of abuse in complex biological matrices such as urine. The emergence of designer drugs, high sample volumes, and the need for retrospective data mining drive the adoption of high-resolution accurate-mass (HRAM) mass spectrometry combined with ultra-high-performance liquid chromatography (UHPLC). A single-run workflow that unifies targeted screening, semi-quantitation, and the ability to re-interrogate data for unforeseen analytes can significantly enhance laboratory throughput, data confidence, and operational cost-effectiveness.

Aims and Study Overview

This study evaluates the performance transfer of an established UHPLC-HRAM MS screening method from a first-generation Q-Orbitrap platform to a next-generation orbitrap instrument. Key objectives include:

• Assessing detection, identification, and semi-quantitation of 101 drugs of abuse spiked into urine.
• Comparing HRAM MS2 spectral matches between the legacy and new orbitrap systems.
• Demonstrating method robustness over extended injection sequences without recalibration.

Methodology and Instrumentation

Sample Preparation

• Eight internal standards (e.g., cotinine-d3, amphetamine-d5) and 101 analytes representing opiates, amphetamines, benzodiazepines, cannabinoids, cathinones, barbiturates, and common medications were spiked into human urine at 0.1–2000 ng/mL.
• Samples were diluted 1:20 with water (dilute-and-shoot) and injected in triplicate.

UHPLC Conditions

• Thermo Fisher Vanquish Flex UHPLC with an Accucore phenyl hexyl, 100 × 2.1 mm, 2.6 µm column.
• Mobile phase A: 2 mM ammonium formate/0.1% formic acid in water; phase B: methanol:acetonitrile (1:1) with 2 mM ammonium formate/0.1% formic acid.
• Gradient elution optimized for broad chemical diversity; typical run time ~15 minutes.

HRAM MS Acquisition

• Thermo Scientific Orbitrap Exploris 120 operated in full-scan/data-dependent MS2 (FS-ddMS2) mode.
• Resolution: 60,000 for full scan, 15,000 for MS2; isolation window m/z 1.5; stepped collision energies 18.75, 37.5, 56.25 eV.
• Polarity switching to capture both positive and negative ions in a single analysis.

Data Processing

• Thermo Scientific TraceFinder 5.1 software with an integrated compound database and mzVault spectral library.
• Screening criteria:
  • Precursor mass deviation ≤ ±5 ppm.
  • Retention time tolerance ±30 s.
  • Isotopic pattern match >70% fit, ≤10 ppm mass deviation, ≤20% intensity deviation.
  • At least two fragment ions within ±10 ppm and library match score ≥70.
• Quantitation criteria: back-calculated concentration within ±30% of nominal, passing isotopic pattern, fragment, and library scores.

Main Results and Discussion

Targeted Screening

• All 101 analytes were successfully detected and identified in urine at concentrations as low as 0.1 ng/mL for many compounds.
• MS2 spectra collected on the Exploris 120 matched the legacy library with high confidence, confirming seamless method transfer.
• Extracted ion chromatogram overlays demonstrated clear chromatographic separation of isomeric analytes (e.g., morphine isomers).

Semi-Quantitation

• Calibration curves from 0.1 to 1000 ng/mL showed linearity and precision for diverse drug classes.
• Over 70% of compounds met the limits of detection (LOD), quantitation (LOQ), and identification (LOI) criteria.
• Example: 7-amino-flunitrazepam displayed a robust calibration with R² > 0.995 and inter-day precision %RSD < 10%.

Robustness

• Mass accuracy for internal standards remained within ±5 ppm over 48 hours and >300 injections without recalibration.
• Retention time stability was ±0.01 min, and peak area %RSD remained below 10% for most analytes.

Benefits and Practical Applications of the Method

The unified UHPLC-HRAM MS approach offers significant advantages:

• Simultaneous targeted screening and semi-quantitation in one run reduces sample turnover time.
• High resolution and mass accuracy enhance confidence in compound identification and isomer separation.
• Integrated data processing streamlines reporting and retrospective data mining for emerging substances.
• Simple dilute-and-shoot sample preparation accelerates throughput and reduces solvent usage.

Future Trends and Possibilities

Advances in HRAM instrumentation and data analytics will drive further improvements:

• Integration of machine-learning algorithms for automated spectral deconvolution and novel compound discovery.
• Application of microflow UHPLC and ion mobility separation to enhance sensitivity and selectivity.
• Expansion of spectral libraries to cover new psychoactive substances and metabolites.
• Cloud-based data sharing platforms for collaborative forensic toxicology networks.

Conclusion

The migration of a comprehensive urine drug screening and semi-quantitation method to the Orbitrap Exploris 120 demonstrated equivalent or improved performance compared to a first-generation system. The workflow delivers reliable identification of 101 drugs of abuse, robust calibration across sub-ng/mL to µg/mL levels, and exceptional method stability over extended runs. This platform supports forensic and clinical toxicology laboratories seeking high throughput, data confidence, and flexible retrospective analysis.

Used Instrumentation

• Thermo Fisher Vanquish Flex UHPLC system with Accucore phenyl hexyl column.
• Thermo Scientific Orbitrap Exploris 120 HRAM mass spectrometer.
• Thermo Scientific TraceFinder software version 5.1 with Compound Database and mzVault library.

References

1. Technical Note 73771: LC-MS/MS Toxicology Platform and Method for High-resolution, Accurate Mass Detection, Screening, and Quantitation of Drugs, Thermo Fisher Scientific.