Fully automated platform for determination of Tricyclic Antidepressant in serum

Importance of the Topic

Monitoring tricyclic antidepressants (TCAs) in serum is critical due to their narrow therapeutic window and potential for serious adverse effects at overdose levels. Accurate, high-throughput quantification of TCAs supports therapeutic drug monitoring in clinical and research settings, ensuring patient safety and treatment efficacy.

Aims and Study Overview

This study presents a fully automated workflow for the quantitation of over 13 common TCAs in human serum. The primary objectives were to integrate sample preparation with LC-MS/MS analysis, eliminate manual handling steps, and achieve robust analytical performance suitable for routine applications.

Used Instrumentation

• CLAM-2000 automated sample preparation unit (Shimadzu) equipped with refrigerated sample carousel at 8 °C
• Nexera X2 UHPLC system coupled to LCMS-8060 triple-quadrupole mass spectrometer (Shimadzu)
• ClinMass® TDM Kit System (Recipe Chemicals + Instruments) including extraction reagents, calibrators, and internal standards
• Analytical column: Recipe MS9030 maintained at 40 °C

Methodology

Serum samples, calibrators, and internal standard mixtures were loaded into the CLAM-2000 unit. Automated operations included protein precipitation, internal standard spiking, stirring, incubation, and filtration. Prepared extracts were transferred directly to the Nexera X2 LC for separation by a binary gradient over 3.8 minutes at 0.6 mL/min. Detection employed positive-ion electrospray and multiple reaction monitoring (MRM) transitions optimized for each TCA.

Main Results and Discussion

Linearity and accuracy were verified using three reference serum calibrators, with correlation coefficients (R2) exceeding 0.999 and accuracy between 85 % and 115 % for all analytes. Intra- and inter-day precision (CV%) remained below 7 % in most cases, confirming reproducible quantitation across low and high concentration levels. Representative chromatograms at the lower limit of quantification demonstrated clear separation and reliable signal intensity for each compound.

Benefits and Practical Applications

• Complete elimination of manual sample handling steps reduces operator variability and contamination risk.
• Throughput of one fully processed and analyzed sample every 5 minutes supports high-volume laboratories.
• Reduced reagent consumption lowers per-sample costs.
• Automated traceability enhances data integrity for clinical research and QA/QC environments.

Future Trends and Potential Applications

Further integration of automated sample preparation with data analysis software and laboratory information systems can streamline workflows. Expanding the platform to include additional drug classes and metabolites will broaden its clinical utility. Advances in miniaturized and multiplexed LC-MS technologies may further increase throughput and reduce solvent consumption.

Conclusion

The fully automated CLAM-2000/Nexera X2-LCMS-8060 platform offers a reliable, high-throughput solution for quantifying multiple TCAs in serum without manual preparation steps. The method delivers excellent linearity, accuracy, and precision, making it suitable for routine therapeutic drug monitoring and research applications.

References

The summary is based on a research presentation titled “Fully automated platform for determination of Tricyclic Antidepressant in serum,” ASMS 2017, by Vecchietti et al.