Targeted MRM Screening for Forensic Toxicology in Negative Electrospray Ionization Mode Using the Xevo TQD or Xevo TQ-S micro

Importance of the Topic

Forensic toxicology demands sensitive and reliable methods to identify negatively ionizing compounds—such as diuretics, barbiturates, and NSAIDs—in complex biological samples. Enhanced detection of these substances supports accurate casework, compliance with regulatory standards, and improved public safety outcomes.

Objectives and Study Overview

The study aimed to adapt and evaluate a previously published targeted MRM screening protocol for negative electrospray ionization on two tandem mass spectrometers (Xevo TQD and Xevo TQ-S micro). Fifty-three analytes were spiked into urine at WADA’s minimum required performance level (200 ng/mL) to compare detection capabilities and explore method robustness in forensic toxicology settings.

Methodology and Instrumentation

Sample Preparation:

• Urine (200 µL) diluted with 200 µL 4 % phosphoric acid and centrifuged.
• Solid-phase extraction using Oasis PRiME HLB µElution plates.
• Wash with 5 % methanol; elution with acetonitrile/methanol (90:10).
• Evaporation under nitrogen at 50 °C; reconstitution in 0.001 % formic acid with 10 % acetonitrile.

LC Conditions:

• System: ACQUITY UPLC I-Class with flow-through-needle.
• Column: ACQUITY UPLC HSS C18, 1.8 µm, 2.1×150 mm at 50 °C.
• Mobile phases: 0.001 % formic acid in water (A) and acetonitrile (B); flow rate 0.4 mL/min.
• Injection volume: 10 µL; sample temperature 10 °C.

MS Conditions:

• Instruments: Xevo TQD or Xevo TQ-S micro in negative ESI mode.
• Capillary voltage 2.5 kV; source temp. 150 °C; desolvation temp. 400 °C.
• MRM transitions: quantifier and qualifier with preconfigured cone voltages and collision energies.

Key Results and Discussion

Of the 53 target analytes spiked at 200 ng/mL, 51 were detected on both platforms using the supplied method. Amiloride and canrenoic acid required additional dilution for reliable detection. In negative control urine, common endogenous metabolites (salicylic acid, gentisic acid, naproxen) were identified. Certified positive controls confirmed detection of barbiturates and carboxy-THC at concentrations meeting EWDTS screening and confirmation cut-offs.

Benefits and Practical Applications

• Broad analyte coverage in negative ion mode enhances screening scope.
• High sensitivity and reproducibility across two MS platforms.
• Efficient sample preparation compatible with routine forensic workflows.
• Compliance with WADA and EWDTS guidelines for urine analysis.

Future Trends and Applications

• Extension of the workflow to serum, plasma, and other matrices.
• Integration with high-resolution MS for non-targeted screening.
• Automation of sample preparation to increase throughput.
• Development of expanded libraries for emerging toxicants.

Conclusion

The validated UPLC-MS/MS method in negative ESI mode provides a robust, sensitive, and high-throughput solution for targeted forensic toxicology screening of negatively ionizing compounds, meeting regulatory requirements and supporting complex case analyses.

Reference

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4. Lee R, Freeto S, Wakefield M, Wood M. Improved Sensitivity for Negatively Ionising Substances. Waters Application Note. 720005479EN. 2015.
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6. European Workplace Drug Testing Society Guidelines. 2018.