Fully automated LC-MS/MS analysis of anticoagulants using a novel reagent kit

Importance of the Topic

Novel oral anticoagulants (NOACs) have become an essential alternative to vitamin K antagonists in preventing and treating thromboembolic events. Precise measurement of these drugs in plasma is critical for confirming anticoagulant effects, guiding dosing decisions, and ensuring patient safety. Integrating a fully automated sample preparation with LC–MS/MS analysis can streamline workflows, reduce human errors, and accelerate turnaround times in clinical and research laboratories.

Goals and Overview of the Study

This study describes the development and validation of a fully automated, multi-analyte LC–MS/MS workflow for quantifying nine anticoagulants using the DOSINACO™ reagent kit and the Shimadzu CLAM-2000 autosampler coupled to an LCMS-8045 system. The objectives were to demonstrate linearity, accuracy, precision, and throughput of the integrated platform, and to compare it with conventional manual preparation.

Methodology and Instrumentation

The automated protocol combines protein precipitation, filtration, and direct injection into an LC–MS/MS system:

• Reagent kit: DOSINACO™ including nine target anticoagulants (acenocoumarol, apixaban, argatroban, betrixaban, dabigatran, edoxaban, fluindione, rivaroxaban, warfarin) and corresponding stable isotope–labeled internal standards.
• Sample processing: Shimadzu CLAM-2000 performing automated precipitation and filtration; sample arm transfers filtrate to the NEXERA X2 LC system.
• Chromatography: C18 column (2.1×50 mm, 5 µm) at 50 °C, gradient from 5 % to 100 % methanol with 5 mM ammonium formate/0.1 % formic acid, total runtime 5 min.
• Mass spectrometry: LCMS-8045 in positive ESI MRM mode, optimized source and gas parameters; two transitions per compound for quantifier and qualifier MRM.

Main Results and Discussion

Calibration and quality control data demonstrated robust analytical performance:

• Linearity: Correlation coefficients (R²) ranged from 0.998 to 0.999 across analytes and concentration ranges (10–5 000 µg/L).
• Accuracy: Mean recoveries between 90.6 % and 108.6 % for all compounds.
• Precision: Intra- and inter-day CVs below 8.5 % for all quality control levels.
• Throughput: One complete analysis every five minutes, enabling high-capacity sample processing without manual intervention.

These results confirm that the automated workflow matches or exceeds conventional manual methods in speed, reproducibility, and traceability.

Benefits and Practical Applications of the Method

By eliminating manual pipetting and centrifugation steps, the integrated system offers:

• Reduced operator bias and improved traceability of each sample.
• Enhanced laboratory safety by minimizing exposure to biofluids and reagents.
• Accessibility for non-specialist users, lowering training requirements for LC–MS/MS operation.
• Scalability to other analyte panels without hardware modifications.

Future Trends and Potential Applications

Advances expected in clinical and research settings include:

• Expansion of automated workflows to cover broader therapeutic drug panels and biomarkers.
• Seamless integration with laboratory information management systems (LIMS) for real-time data reporting.
• Use of machine learning to optimize sample preparation steps and predict maintenance requirements.
• Miniaturized or multiplexed platforms for point-of-care anticoagulant monitoring.

Conclusion

The described fully automated LC–MS/MS method using the DOSINACO™ kit and Shimadzu CLAM-2000 provides accurate, precise, and high-throughput quantitation of nine anticoagulants. This streamlined workflow enhances laboratory efficiency, minimizes manual errors, and supports rapid clinical decision-making while maintaining robust analytical performance.