Fully Automated LC-MS/MS Analysis of Anticoagulants Using a Stable Isotope Labelled Internal Standards

Significance of the topic

Novel oral anticoagulants and traditional vitamin K antagonists require accurate, rapid quantification in clinical pathology to guide patient management, especially in emergency bleeding or preoperative settings. Conventional LC-MS/MS workflows are labor-intensive, vulnerable to manual errors, and demand highly trained personnel. Automating sample preparation and analysis can improve throughput, reproducibility, and safety.

Objectives and Overview

This study presents a fully automated LC-MS/MS method for simultaneous quantification of nine anticoagulant drugs in plasma using stable isotope labeled internal standards. The workflow combines a dedicated DOSINACO™ kit, the CLAM-2000 automated preparation system, a UHPLC C18 column, and a triple-quadrupole mass spectrometer to deliver a walk-away solution for routine therapeutic drug monitoring.

Methodology and Instrumentation

• Sample Preparation: CLAM-2000 performs protein precipitation and filtration by mixing 20 µL plasma with extraction buffer and stable isotope labeled standards in an automated sample cup, then transfers the filtrate directly to the LC system without manual intervention.
• Chromatography: Separation occurs on a DOSINACO™ C18 column (2.1 × 50 mm, 5 µm) at 50 °C using a 7-min gradient from 2% to 98% organic phase at 0.5 mL/min.
• Mass Spectrometry: Shimadzu LCMS-8045 with positive electrospray ionization (2.5 kV) acquires MRM transitions for each unlabeled analyte and its corresponding labeled internal standard.

Main Results and Discussion

Calibration curves covering 10–500 µg/L for NOACs and 100–5000 µg/L for coumarins exhibited excellent linearity (r2 > 0.99). Automated preparation achieved precision with CVs below 8.7% and accuracy within ±5% deviation across four concentration levels. Parallel processing of sample cleanup and LC-MS/MS runs reduced total assay time and minimized human error, enabling consistent performance even by non-expert operators.

Benefits and Practical Applications

• Walk-away automation eliminates manual pipetting steps, reducing operator exposure and variability.
• High throughput supports routine and emergency clinical laboratories with rapid turnaround.
• Standardized methodology enhances reproducibility and streamlines therapeutic drug monitoring workflows.

Future Trends and Applications

Future developments may extend the automated platform to larger analyte panels, integrate with laboratory information management systems, and utilize machine-learning for method optimization. Miniaturization and point-of-care adaptations could broaden its use to bedside testing and decentralized labs.

Conclusion

The fully automated LC-MS/MS method using DOSINACO™ standards and the CLAM-2000 system offers a robust, user-friendly solution for quantitative anticoagulant analysis in clinical settings. It delivers high precision, safety, and throughput, facilitating faster clinical decisions without requiring specialized personnel.

References

• Minohata T., Uno Y., Baumgarten S., Moreau S., Dayot F., Hoeffler J.-F. Fully Automated LC-MS/MS Analysis of Anticoagulants Using Stable Isotope Labelled Internal Standards. ASMS 2019.