Rethinking library identication in quantitative clinical toxicology – transitioning towards MRM Spectrum mode

Importance of the topic

Clinical toxicology relies on accurate, sensitive, and specific assays to guide patient management and forensic investigations. Traditional triple quadrupole MRM methods deliver robust quantitation but may lack comprehensive spectral information for unambiguous compound confirmation. By integrating multiple precursor–fragment transitions into a single workflow, MRM Spectrum mode enhances identification confidence without sacrificing quantitation performance.

Objectives and study overview

This work aims to evaluate the potential of MRM Spectrum mode for simultaneous quantitation and library-based identification of drugs in whole blood, comparing it to conventional 2-MRM and MRM-triggered product ion acquisition strategies. The study assesses linearity, precision, accuracy, and spectral matching performance for a panel of clinical and forensic markers.

Methodology and Instrumentation

Whole blood samples were spiked with standards of cocaine, amphetamines, opiates, antipsychotics, and stable isotope internal standards. Extraction employed a QuEChERS protocol. Chromatographic separation was performed on a Nexera UHPLC system with a Restek Raptor Biphenyl column (2.7 μm, 100 × 2.1 mm) at 50 °C using a water/methanol gradient containing ammonium formate and formic acid. Mass analysis used a Shimadzu LCMS-8060 triple quadrupole in heated ESI mode. Three acquisition schemes were compared:

• Conventional 2-MRM quantifier/qualifier pairs
• MRM-triggered product ion scans at multiple collision energies
• MRM Spectrum mode with 6–10 optimized transitions per compound

Main Results and Discussion

Calibration curves for benzoylecgonine (5–500 µg/L) showed equivalent slopes, intercepts, and R² (>0.997) across all modes. Accuracy (85–115%) and precision (<10% RSD) criteria were met. Both MRM Spectrum and triggered scans yielded library-searchable spectra with high similarity scores (>99 for Spectrum mode vs. 78 for triggered scans at low levels), demonstrating superior specificity. Quantitation remained consistent regardless of acquisition approach, while MRM Spectrum offered rich fragment ion patterns for confident identification even at trace concentrations.

Benefits and Practical Applications

• Enhanced compound confirmation through extensive fragment spectra
• Unchanged quantitation figures of merit compared to conventional methods
• Streamlined workflow enabling simultaneous data acquisition for quantitation and identification
• Improved reporting confidence and reduced false positives

Future Trends and Possibilities

Expansion of MRM Spectrum libraries to cover broader analyte panels or alternative matrices (urine, oral fluid) could further improve clinical and forensic workflows. Advances in data processing, structural analytics, and automation will accelerate method deployment. Integration with high-throughput platforms and hybrid instruments may extend applicability across therapeutic drug monitoring and doping control.

Conclusion

MRM Spectrum mode offers a unified solution for quantitative clinical toxicology, retaining the sensitivity and precision of traditional MRM while delivering comprehensive fragment ion spectra for robust library-based identification. This approach enhances analytical confidence and supports rapid, reliable reporting in patient care and forensic investigations.

References

Loftus N., Barnes A., Robin T., Marquet P., Dulaurent S., Saint-Marcoux F. Rethinking library identification in quantitative clinical toxicology – transitioning towards MRM Spectrum mode. MSACL 2017.