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

Importance of the Topic

Forensic toxicology is essential for determining the presence and levels of drugs and poisons in biological specimens during death investigations. Rapid, reliable screening and semi-quantitation of whole blood samples can accelerate decision-making for forensic scientists and medical examiners, especially when sample volume is limited and time is critical.

Objective and Study Overview

This study presents the development of a streamlined LC/MS/MS workflow for rapid toxicology screening and semi-quantitative analysis of 231 forensically relevant compounds in only 100 µL of whole blood. The system combines synchronized survey scan (SSS) for identification and multiple reaction monitoring (MRM) for semi-quantitation in a single 15 min run.

Methodology

Sample pretreatment employs a micro-volume QuEChERS kit. Whole blood is mixed with acetonitrile, water, internal standards, and extraction salts; vortexed and centrifuged. The supernatant is treated with 0.1% TFA in acetonitrile, centrifuged again, evaporated to dryness under nitrogen, reconstituted in methanol, and filtered. Analysis uses gradient UHPLC separation on a Shim-pack Velox SP-C18 column, with two data-acquisition modes: SSS triggers MS/MS for compounds exceeding a threshold for library-based identification, while MRM monitors predefined transitions and uses stored calibration curves for semi-quantitative results.

Instrumentation

• UHPLC System: Shimadzu Nexera X2
• Mass Spectrometer: Shimadzu LCMS-8045 triple quadrupole with ESI positive/negative switching
• Column: Shim-pack Velox SP-C18 (2.1 mm × 100 mm, 2.7 µm)
• Sample Prep: Micro-volume QuEChERS kit
• Operating Parameters: 0.3 mL/min flow, 5 µL injection, gas flows (nebulizing 3 L/min, drying 10 L/min, heating 10 L/min), temperatures (DL 250 °C, block heater 400 °C, interface 300 °C)

Main Results and Discussion

Application to an authentic autopsy blood sample resulted in SSS identification of three drugs: 7-aminoflunitrazepam, biperiden, and haloperidol. Semi-quantitative MRM analysis yielded concentrations of 0.0059 ng/mL for 7-aminoflunitrazepam, 0.0007 ng/mL for biperiden (below the method’s LLOQ, leading to a 65% error relative to reference data), and 0.0034 ng/mL for haloperidol. The MRM approach demonstrated improved accuracy at low concentration levels compared to SSS-based estimates.

Benefits and Practical Applications

Rapid sample preparation and low blood volume requirements reduce resource consumption and turnaround time. Integration of identification and semi-quantitation in one run enhances laboratory throughput. The pre-configured method package streamlines routine forensic analyses and supports timely casework decisions without the need for extensive external calibration.

Future Trends and Applications

• Expansion of compound panels to include emerging psychoactive substances
• Integration of high-resolution mass spectrometry for improved specificity
• Automation of sample prep and data processing for higher throughput
• On-site or near-patient screening platforms for rapid field analysis
• Machine-learning algorithms for enhanced library matching and decision support

Conclusion

The presented LC/MS/MS screening system effectively identifies and semi-quantitates dozens of forensic drugs from minimal blood volumes with high sensitivity and acceptable accuracy. The combined SSS and MRM workflow delivers reliable data suitable for forensic investigations, demonstrating strong agreement with fully validated reference methods.

Reference

1. Waters B et al., Tissue distribution of suvorexant in three forensic autopsy cases, J. Anal. Toxicol. 42 (2018) 276-283.