Quantification of 15 tricyclic antidepressants in human plasma by LC-HRAM-MS using the Orbitrap Exploris 120 mass spectrometer for clinical research

Significance of the topic

Tricyclic antidepressants are widely used in treating depression, anxiety and pain disorders. Reliable quantification of these compounds in biological matrices is essential for therapeutic drug monitoring and pharmacokinetic studies. High-resolution mass spectrometry coupled with rapid sample throughput can enhance data confidence and support clinical research decisions.

Goals and study overview

This work aimed to develop and validate a fast, simple and sensitive LC–HRAM–MS method to simultaneously quantify 15 tricyclic antidepressants in human plasma. The method was evaluated for linearity, lower limit of quantification, accuracy and precision to support clinical research applications.

Methodology and instrumentation

The analytical workflow included:

• Sample preparation by protein precipitation: 50 μL plasma mixed with 100 μL acetonitrile containing deuterated internal standards.
• Centrifugation and direct injection of 1 μL supernatant.
• Chromatographic separation on a phenyl UHPLC column (2.1 × 100 mm, 1.9 μm) with gradient elution using 5 mM ammonium formate/0.1% formic acid in water and methanol at 0.5 mL/min, total runtime below 8 min.
• Detection by an Orbitrap Exploris 120 mass spectrometer in positive electrospray ionization mode (resolution 60 000 at m/z 200, scan range m/z 100–500).

Used instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC system
• Hypersil GOLD Phenyl column (2.1 × 100 mm, 1.9 µm)
• Orbitrap Exploris 120 mass spectrometer with HESI source
• TraceFinder 5.1 software for data acquisition and processing

Main results and discussion

The method showed excellent performance across all 15 analytes:

• Linearity with R2 values above 0.9989 across calibration ranges.
• Accuracy between 98.0% and 102.8% and precision below 3.0% for calibrators.
• Intra- and inter-assay accuracy within 95–113% and precision (CV) below 7.8% and 6.1%, respectively, at two quality control levels.
• LLOQs extended down to 1.39–6.04 µg/L depending on the compound while retaining acceptable accuracy and precision.

Benefits and practical applications of the method

• Rapid assay turnaround (<8 min per sample) enables high throughput.
• Minimal sample volume and simple preparation reduce labor and cost.
• Simultaneous quantification of multiple tricyclic antidepressants from a single injection.
• High-resolution mass spectrometry ensures specificity and confidence in complex matrices.

Future trends and potential applications

As clinical research evolves, this platform may be integrated with automated sample handling and expanded libraries for additional psychoactive drugs. Potential developments include real-time monitoring workflows, personalized dosing strategies and AI-driven data analysis to further improve patient care.

Conclusion

A robust UHPLC–HRAM–MS method was established for the rapid, accurate and precise quantification of 15 tricyclic antidepressants in human plasma. The approach meets clinical research requirements for sensitivity, linearity and reproducibility, supporting therapeutic drug monitoring and pharmacokinetic investigations.