Rapid screening and semi-quantitative analysis for forensic drugs in blood using liquid chromatography triple quadrupole mass spectrometry

Importance of the Topic

The rapid identification and semi-quantitative measurement of drugs in whole blood are crucial in forensic toxicology for timely decision making in criminal and clinical investigations. Traditional sample preparation methods for complex matrices such as whole blood often involve laborious, time-consuming procedures that can limit throughput and introduce variability. The adaptation of streamlined extraction techniques coupled with high-speed triple quadrupole mass spectrometry addresses the need for a robust, high-throughput workflow enabling simultaneous analysis of multiple drug classes.

Objectives and Study Overview

This study aimed to develop and validate an efficient analytical workflow integrating a modified QuEChERS extraction method with liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) for forensic drug screening in blood samples. Key targets included optimizing sample pretreatment, establishing rapid screening parameters using MRM-triggered product ion scanning, and evaluating method recovery and inter-instrument reproducibility across multiple laboratories.

Methodology and Instrumentation

• Sample Preparation: A modified QuEChERS protocol was applied to 0.5 mL whole blood. The procedure involved aqueous dilution, acetonitrile extraction with internal standard (Diazepam-d5), salt-induced phase separation, TFA-assisted cleanup, nitrogen evaporation, and reconstitution in methanol.
• Instrumentation: Analysis was performed on a Shimadzu Nexera UHPLC coupled to an LCMS-8040 triple quadrupole MS. Chromatographic separation used a Shim-pack FC-ODS column (2.0×150 mm, 3 μm) with a 5–95% methanol gradient in 10 mM ammonium formate at 0.3 mL/min and 40°C.
• MS Conditions: Electrospray ionization in both positive and negative modes with fast polarity switching (15 ms) enabled simultaneous acquisition of MRM transitions and product ion spectra. Optimal parameters included a nebulizing gas flow of 1.5 L/min and drying gas pressure of 10 L/min.

Main Results and Discussion

The method demonstrated average recoveries of 57–98% for 24 representative drugs spanning basic, acidic, and neutral compounds. The MRM-triggered product ion scanning provided confirmatory spectral information matching a library of 106 forensic analytes. Calibration curves exhibited linearity with correlation coefficients exceeding 0.99. Inter-laboratory testing across three different LC–MS models yielded consistent semi-quantitative results, confirming the robustness of the workflow.

Benefits and Practical Applications

• High throughput screening of over 100 drugs in a single run reduces instrument time per sample.
• Modified QuEChERS extraction streamlines sample cleanup, minimizing solvent use and hands-on time.
• MRM-triggered product ion scanning enhances specificity, supporting reliable compound identification in complex matrices.
• Semi-quantitative calibration using a single internal standard simplifies quantitation for routine forensic casework.

Future Trends and Opportunities

Advancements may include integration with high-resolution mass spectrometry for improved selectivity, automation of QuEChERS sample preparation to further increase throughput, and expansion of spectral libraries to cover emerging psychoactive substances. Implementation of data processing software with machine learning algorithms could streamline screening workflows and enhance identification confidence.

Conclusion

The combination of a modified QuEChERS sample preparation and high-speed triple quadrupole LC–MS/MS provides a reliable, efficient, and high-throughput method for the rapid screening and semi-quantitative analysis of forensic drugs in whole blood. This workflow meets the demands of forensic laboratories by delivering accurate results with reduced turnaround times.

References

1. Usui K., Shima N., Katagi M., Tsuchihashi H., Suzuki K., Hirano I., Ikeda N. Modified QuEChERS extraction method for blood sample preparation. Legal Medicine. 2012;14:286-296.
2. Hayes T., Kudo K., Minohata T., Usui K., Shima N., Katagi M., Tsuchihashi H., Suzuki K., Hirano I., Ikeda N. Rapid screening and semi-quantitative analysis for forensic drugs in blood using LC–MS/MS. ASMS 2013;WP-121.