SOFT: Toxicology Screening of Human Blood using Quadrupole-Time of Flight (QTOF) Mass Spectrometry

Importance of the Topic

Initial toxicology screening plays a critical role in forensic and clinical laboratories by rapidly detecting a wide range of abused substances, prescription medications, and emerging novel psychoactive compounds in human blood. Traditional immunoassay methods are limited by cross-reactivity and restricted analyte coverage, leading to false positives and undetected substances. Implementation of high-resolution mass spectrometry workflows addresses these limitations by providing sensitive, accurate and untargeted screening capabilities. The enhanced identification confidence and expanded compound libraries empower analysts to meet the growing demands for comprehensive toxicological assessments.

Study Objectives and Overview

This study aimed to develop and validate a robust toxicology screening workflow using quadrupole-time of flight (QTOF) mass spectrometry combined with data-independent acquisition (DIA) and a high-resolution MS/MS library. Objectives included:

• Establishing a method for simultaneous targeted quantitation and non-targeted screening of over 900 toxicologically relevant compounds.
• Optimizing chromatographic separation to handle diverse chemical classes in blood matrices.
• Demonstrating confident compound identification with stringent mass accuracy, retention time, and spectral matching criteria.

Methodology

Solid phase extraction was performed on spiked human blood samples covering concentrations from 5 to 5000 ng/mL for a panel of nine common drugs of abuse, including benzoylecgonine, amphetamine, cocaine, and morphine. Chromatographic separation utilized a polar-embedded Shim-pack Velox column (2.1 x 100 mm, 2.7 µm) with a binary gradient of water and methanol containing 2 mM ammonium formate and 0.002% formic acid. Mass spectrometric data acquisition employed:

• MS TOF full scan (m/z 40–900).
• DIA-MS/MS with variable precursor isolation windows and collision energy spread of 5–55 V in positive mode.

Instrumentation Used

The workflow was implemented on a Shimadzu LCMS-9030 QTOF mass spectrometer interfaced with a high-performance liquid chromatography system. Data review and spectral library matching were performed using LabSolutions Insight software, leveraging a comprehensive library containing accurate mass MS/MS spectra, retention times, and fragmentation energies for over 900 compounds.

Main Results and Discussion

The method successfully detected and positively identified all target analytes across the tested concentration range with library match scores exceeding 80%. Key findings included:

• High sensitivity and selectivity in detecting low ng/mL levels of analytes such as benzoylecgonine and methamphetamine.
• Retention time deviations within ±0.2 minutes and mass errors below 5 ppm, ensuring accurate compound confirmation.
• Effective DIA-MS/MS spectral quality for both targeted quantitation and non-targeted screening workflows.

Chromatograms and MS/MS spectra demonstrated clear signal separation and minimal interference, supporting the method’s reliability for complex blood matrices.

Benefits and Practical Applications

This QTOF-based screening approach offers multiple advantages:

• Comprehensive coverage of a broad chemical space, including novel psychoactive substances.
• Reduced risk of false positives compared to immunoassays through high-resolution accurate mass measurements and robust library verification.
• Streamlined workflow enabling simultaneous targeted analysis and untargeted screening in a single run.

The method is suitable for forensic toxicology, clinical toxicology, workplace drug testing, and anti-doping applications.

Future Trends and Opportunities

Ongoing developments in high-resolution mass spectrometry and spectral libraries will further enhance toxicology screening capabilities. Potential future directions include:

• Expansion of libraries to include emerging designer drugs and metabolic biomarkers.
• Integration of machine learning algorithms for automated spectral interpretation and suspect screening.
• Coupling with high-throughput sample preparation systems for increased laboratory efficiency.

Conclusion

A QTOF-DIA mass spectrometry workflow was established for comprehensive toxicology screening of human blood, demonstrating high confidence in compound identification, sensitivity across a wide dynamic range, and suitability for both targeted quantitation and untargeted library-based verification. This method addresses the limitations of traditional immunoassays and meets the growing demands for rapid, accurate, and comprehensive toxicological analysis.