Quantification of 15 tricyclic antidepressants in human plasma or serum by LC-HRAM-MS for clinical research

Significance of the topic

Tricyclic antidepressants (TCAs) remain widely used for managing depression, anxiety disorders and neuropathic pain despite potential adverse effects. Accurate therapeutic drug monitoring (TDM) of TCAs in plasma or serum is essential to ensure patient safety, verify compliance and optimize dosing. Traditional HPLC-UV methods provide limited sensitivity and selectivity, driving demand for high-resolution mass spectrometry approaches that can deliver faster analysis, improved specificity and structural confirmation.

Objectives and Study Overview

This work describes the development and validation of a quantitative LC-HRAM-MS method for simultaneous determination of 15 TCAs in human plasma or serum. Using a Thermo Scientific Orbitrap Exploris 120 mass spectrometer and a Vanquish Flex Binary UHPLC system, the study aims to demonstrate the method’s linearity, sensitivity, precision, accuracy and absence of carryover in a clinical research context.

Methodology and Instrumentation

Sample preparation employs offline protein precipitation of 50 µL plasma or serum with a proprietary precipitating solution containing deuterated internal standards from the ClinMass® TDM Platform. Following vortexing and centrifugation, the supernatant is injected directly into the UHPLC-MS system.

• Chromatography: Vanquish Flex Binary UHPLC, RECIPE column, 4 min gradient from 15 % to 75 % organic at 0.8 mL/min.
• Mass Spectrometry: Orbitrap Exploris 120 with heated electrospray ionization (H-ESI) in positive mode. Full-scan acquisition at 60,000 resolution (m/z 200) combined with data-dependent MS2 at 15,000 or targeted MS2 at 30,000 resolution for co-eluting isobaric analytes.
• Data Analysis: TraceFinder™ 5.1 software with ClinMass calibration and quality control materials.

Main Results and Discussion

All 15 analytes exhibited linear calibration curves with 1/x weighting across therapeutic concentration ranges. Lower limits of quantification ranged from 0.68 to 24.2 µg/L, meeting the ≤ 20 % CV criterion. No carryover was detected in blank injections following high-level calibrators. Accuracy evaluations showed biases within ±12 %, while intra- and inter-assay precision remained below 7.1 % and 4.2 % CV, respectively. Representative chromatograms confirmed baseline resolution and reliable detection of target compounds and internal standards.

Benefits and Practical Applications

• High throughput: 4-minute analysis time supports large sample batches.
• Enhanced selectivity: HRAM reduces matrix interferences and enables confident compound identification.
• Robust performance: stringent validation ensures consistency for clinical research and TDM studies.
• Streamlined workflow: simple protein precipitation accelerates sample turnaround.

Future Trends and Potential Applications

Integration of high-resolution Orbitrap technology into routine clinical laboratories may expand TDM to additional psychoactive and cardiovascular drugs. Emerging microflow LC and ion mobility techniques promise even greater sensitivity and multiplexing capacity. Data-independent acquisition and retrospective screening workflows could facilitate identification of novel or off-label compounds in biological samples. Automated sample handling and cloud-based data processing will further enhance laboratory efficiency and data sharing.

Conclusion

The presented LC-HRAM-MS method on an Orbitrap Exploris 120 coupled to a Vanquish UHPLC system offers a rapid, sensitive and precise solution for quantifying 15 tricyclic antidepressants in human plasma or serum. Its validated performance confirms its suitability for clinical research applications and therapeutic drug monitoring.