Quantitative toxicology screening for over 1000 compounds in whole blood samples; is there a better way of reporting results?

Importance of the topic

Clinical toxicology laboratories must rapidly and accurately screen biological samples for a wide range of toxicants. Traditional methods relying on two MRM transitions per analyte can suffer from false positives or negatives, leading to misdiagnosis and inappropriate treatment. Developing a comprehensive spectral library combined with multi-transition monitoring enhances confidence in both identification and quantitation of over 1,000 compounds in whole blood.

Study objectives and overview

This work aimed to build a mass spectrometry database of 1,222 clinical and forensic toxins with full-scan and MRM spectrum data, optimize a single 17-minute LC-MS/MS method, and evaluate whether monitoring more than five precursor-fragment ion transitions per compound improves reporting accuracy without compromising quantitative performance.

Methodology

• Sample preparation: Whole blood spiked with target compounds and extracted using a QuEChERS protocol, including deuterated internal standards where available.
• Chromatography: Single 17-minute gradient on a Restek Raptor Biphenyl column (2.7 µm, 100 × 2.1 mm).
• Mass spectrometry: Shimadzu LCMS-8060 triple quadrupole system operating in MRM Spectrum mode, acquiring more than five optimized transitions per analyte in both positive and negative ionization.
• Data processing: Library matching using retention time, accurate precursor and fragment mass, and optimized collision energies to generate high-confidence product ion spectra.

Instrumentation used

• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• Restek Raptor Biphenyl 2.7 µm, 100 × 2.1 mm LC column

Main results and discussion

• A total of 3,010 MRM transitions targeted 616 compounds, with retention time windows of ±0.5 min and sub-millisecond dwell times, yielding a loop time of ~1.14 s.
• Quantitative performance for benzoylecgonine and other analytes showed R2 values >0.99, matching conventional two‐transition methods.
• Comparative analysis of patient samples (e.g., morphine, methadone, ecgonine methyl ester) demonstrated close agreement between the MRM Spectrum and validated 2-transition assays.
• PATIENT STUDY: Psychiatric patient whole blood profiling successfully identified and quantified opioids and benzodiazepines (buprenorphine, norbuprenorphine, oxazepam, temazepam, nordiazepam) with similarity scores ≥95.
• High-density multi‐transition spectra improve specificity and reduce false reporting without thresholds for signal triggering.

Benefits and practical applications

• Enhanced confidence in compound identification via library‐searched MRM spectra.
• Maintained quantitative accuracy while expanding screening scope to over 1,000 analytes.
• Single LC method simplifies workflow in routine clinical and forensic laboratories.
• Reduction of false positives and negatives minimizes unnecessary follow‐up testing and inappropriate patient care.

Future trends and potential uses

• Expansion of spectral libraries to include metabolites and novel psychoactive substances.
• Integration with high-resolution mass spectrometry and advanced data analytics for real-time screening.
• Use of machine learning algorithms to refine collision energies and retention time predictions.
• Development of fully automated workflows for point-of-care toxicology screening.

Conclusion

Combining a large MRM Spectrum library with optimized multi‐transition monitoring on a single LC-MS/MS platform delivers reliable, high‐confidence toxicology screening. This approach maintains quantitative performance comparable to conventional assays while significantly reducing the risk of false reporting, making it well suited for routine clinical and forensic toxicology laboratories.