Multi-target Screening of Toxicological Compounds in Blood on A Fully-automated Platform Consisting of Sample Preparation Module CLAM and LC-MS/MS

Significance of the Topic

Rapid and reliable multi-target screening of toxicological compounds in whole blood is a cornerstone of forensic and clinical investigations. Simultaneous detection of illicit drugs, pharmaceuticals and their metabolites enables timely decision making in emergency toxicology, workplace drug testing and postmortem analyses. Automation of sample preparation and streamlined LC-MS/MS workflows reduce turnaround time, minimize operator variability and enhance data quality.

Study Objectives and Overview

The primary goals of this study were to:

• Implement a fully automated platform combining the CLAM-2000 sample preparation module with a Shimadzu LCMS-8060 triple quadrupole mass spectrometer.
• Deploy the Rapid Toxicology Screening method package to screen 61 toxicological targets in whole blood without extensive manual method development.
• Assess analytical performance in terms of linearity, recovery, precision and data reliability across a wide concentration range.

Instrumentation Used

• CLAM-2000 automated sample preparation system (Shimadzu).
• LCMS-8060 triple quadrupole mass spectrometer with heated electrospray ionization (ESI).
• Kinetex XB-C18 analytical column (2.1 mm × 100 mm, 2.6 µm).
• LabSolutions Insight software for batch processing and Flag ID data quality alerts.

Methodology

Automated sample preparation steps on CLAM-2000:

• Dispense 50 µL whole blood and 250 µL acetonitrile into a filtering vial.
• Stir at 2000 rpm for 60 s and filter for 90 s.
• Transfer filtrate to autosampler and co-inject with 20 µL water to improve chromatographic peak shape.
• Perform direct injection (5 µL) into LCMS-8060.

Chromatographic and mass spectrometric conditions:

• Gradient elution (5 % to 95 % methanol in 0.1 % formic acid/ammonium formate) over 26 min at 0.3 mL/min.
• Positive MRM mode with two to three transitions per compound; retention time alignment using standards.
• Three calibration levels (4, 20, 100 ppb) with deuterated internal standards at 4 ppb.

Main Results and Discussion

• Linear calibration achieved for all 61 targets with correlation coefficients ≥ 0.99.
• Recoveries for 53 compounds and 23 deuterated standards fell within 70–130 %; eight analytes exhibited lower or higher recoveries, notably 6-Acetyl Morphine (58 %), Carbamazepine (67 %), Midazolam (58 %) and Morphine-D3 (152 %).
• Precision (n = 5) was < 14.4 % RSD for targets and < 17.8 % RSD for internal standards.
• Flag ID functionality successfully flagged large retention time shifts and ion ratio deviations, enhancing reliability of screening results.
• Automated workflow processed each sample in approximately 3–5 min, including standards and quality controls.

Benefits and Practical Applications

• Elimination of time-consuming manual method development through use of a ready-to-use method package.
• High throughput capability ideal for forensic casework, clinical toxicology and emergency screening.
• Improved data integrity via automated quality checks and software alerts.
• Reduced solvent consumption and consistent sample handling through automation.

Future Trends and Potential Applications

Advancements in automated sample preparation are expected to integrate with high-resolution mass spectrometry and artificial intelligence–driven data analysis for broader screening libraries. Expansion of target panels to include emerging designer drugs and metabolites will further enhance forensic and clinical toxicology capabilities. Cloud-based data sharing and real-time monitoring tools may streamline collaborative investigations across laboratories.

Conclusion

The combination of CLAM-2000 automated sample preparation and LCMS-8060 MRM screening offers an efficient and reliable solution for multi-target toxicology in whole blood. The approach reduces manual intervention, maintains robust analytical performance and supports high-throughput workflows essential to modern forensic and clinical laboratories.

References

1. Tiphaine Robin, Alan Barnes, Sylvain Dulaurent, Neil Loftus, Sigrid Baumgarten, Stéphane Moreau, Pierre Marquet, Souleiman El Balkhi and Franck Saint-Marcoux. Analytical and Bioanalytical Chemistry (2018) 410:5071–5083.
2. Shimadzu Method Package – Rapid Toxicology Screening (Version 2), Shimadzu Corporation.