Screening and Semi-Quantitation by LC/MS/MS in Whole Blood Using Rapid Tox Screening

Significance of the Topic

Forensic toxicology requires rapid and sensitive detection of toxic compounds in limited-volume whole blood samples. Advances in LC/MS/MS technology have enabled comprehensive screening of multiple analytes, addressing the need for timely and accurate toxicological information in death investigations and clinical cases.

Objectives and Overview of the Study

This study presents the development and application of an LC/MS/MS Rapid Toxicology Screening System (version 3.01) for the qualitative screening and semi-quantitative analysis of 231 forensic compounds in whole blood using a minimal sample volume. The approach eliminates the need for standard references during screening and streamlines forensic workflows.

Methodology and Instrumentation

• Sample Preparation: A Micro Volume QuEChERS extraction kit was used to process 100 µL of whole blood with acetonitrile, water, and internal standards (Diazepam-d5 and Phenobarbital-d5), followed by phase separation, evaporation, reconstitution in methanol, and centrifugation.
• Liquid Chromatography: Shim-pack Velox SP-C18 column (2.1 × 100 mm, 2.7 µm) with gradient elution (10 mM ammonium formate/0.1% formic acid) at 0.3 mL/min and 5 µL injection volume on a Nexera X2 system.
• Mass Spectrometry: LCMS-8045 operated in ESI positive/negative switching. The Synchronized Survey Scan (SSS) method was used for library-based compound identification, and Multiple Reaction Monitoring (MRM) for semi-quantitative analysis.
• Data Processing: LabSolutions Insight Library Screening software for spectral matching and rapid review of screening results.

Main Results and Discussion

• Screening Performance: The SSS method detected 7-Aminoflunitrazepam, Biperiden, and Haloperidol in an autopsy blood sample, with library match scores up to 95%.
• Semi‐Quantitation: MRM analysis yielded concentrations of 0.0051 ng/µL for 7-Aminoflunitrazepam (14% error), 0.0007 ng/µL for Biperiden (65% error due to proximity to the LLOQ), and 0.0034 ng/µL for Haloperidol (3% error), closely matching fully validated quantitation.
• Analytical Throughput: The workflow processes small sample volumes rapidly, combining extraction and analysis in under 30 minutes per sample.

Benefits and Practical Applications

• High Sensitivity: Detects compounds at trace levels using only 100 µL of blood.
• Efficiency: Eliminates individual standard preparation by employing stored calibration curves and spectral libraries.
• Versatility: Capable of screening a wide range of forensic analytes in clinical and forensic laboratories.

Future Trends and Possibilities of Use

• Expansion of Screening Panels: Inclusion of emerging psychoactive substances and metabolites.
• Automation and High-Throughput Integration: Coupling with robotic sample preparation for large case loads.
• Advanced Data Analysis: Implementation of machine learning for improved spectral interpretation and lower false positives.
• Cross-Matrix Applications: Adapting the workflow to other biological samples such as urine or tissues.

Conclusion

The LC/MS/MS Rapid Toxicology Screening System provides a streamlined and robust workflow for the rapid screening and semi-quantitative analysis of forensic compounds in whole blood. It demonstrated high sensitivity, accuracy, and operational efficiency, supporting its adoption in forensic toxicology laboratories.

Used Instrumentation

• Nexera X2 UHPLC System
• LCMS-8045 Triple Quadrupole Mass Spectrometer
• Shim-pack Velox SP-C18 Column
• Micro Volume QuEChERS Kit

Reference

1) Waters B et al., Tissue distribution of suvorexant in three forensic autopsy cases, Journal of Analytical Toxicology 42 (2018) 276-283.