Quantification of 15 tricyclic antidepressants in human plasma by liquid chromatography-tandem mass spectrometry for clinical research

Significance of the Topic

Tricyclic antidepressants (TCAs) remain important tools in managing depression, anxiety, and chronic pain. Accurate measurement of plasma levels supports therapeutic drug monitoring, safety assessment, and pharmacokinetic studies in clinical research.

Objectives and Study Overview

This work describes development and validation of a rapid eight-minute liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantify 15 TCAs in human plasma. The approach employs simple offline protein precipitation and targeted SRM analysis, with performance evaluated using standardized calibrators, controls, and isotopically labeled internal standards.

Applied Instrumentation

• Vanquish Flex Binary UHPLC system
• Thermo Scientific Hypersil GOLD Phenyl column (100 × 2.1 mm, 1.9 µm)
• TSQ Quantis triple quadrupole mass spectrometer with heated electrospray ionization (HESI) source
• Thermo Scientific TraceFinder 5.1 software for data acquisition and processing

Methodology

• Sample Preparation: 50 µL plasma precipitated with 100 µL acetonitrile containing internal standards; vortexed, centrifuged, supernatant transferred.
• Chromatography: 2 µL injection; gradient from 10% to 100% methanol (0.1% formic acid, 5 mM ammonium formate) over 8 min; flow rate 0.5 mL/min; column temperature 40 °C.
• Mass Spectrometry: Positive‐mode SRM with quantifier and qualifier transitions; HESI parameters—spray voltage 3.5 kV, sheath gas 50 arb, auxiliary gas 10 arb; cycle time 0.3 s; Q1/Q3 resolutions 0.7/1.2 FWHM.

Results and Discussion

• Linearity: 1/x‐weighted calibration with bias within ±10% across all analytes.
• Lower Limits of Quantification (LLOQs): Ranged from 1.37 to 18.2 µg/L, enabling sensitive detection.
• Accuracy: Control sample bias between –8.6% and +9.3% at two concentration levels.
• Precision: Intra‐ and inter‐assay CVs ≤6.2% for all compounds.
• No detectable carryover was observed following the highest calibrator injection.

Benefits and Practical Applications

• High throughput: eight-minute run time for 15 analytes in a single assay.
• Robustness: protein precipitation simplifies sample handling and reduces potential errors.
• Sensitivity and reliability suitable for clinical research, therapeutic monitoring, and pharmacokinetic profiling.

Future Trends and Potential Applications

• Expansion to broader panels including metabolites and newer psychoactive drugs.
• Integration with automated sample preparation and high-throughput workflows.
• Adaptation to microflow and nano-LC platforms to reduce solvent consumption and further enhance sensitivity.
• Use in personalized medicine trials and real‐time dosing optimization.

Conclusion

A validated LC-MS/MS protocol using Vanquish Flex UHPLC and TSQ Quantis detection delivers rapid, accurate, and precise quantification of 15 TCAs in human plasma. The method meets clinical research needs for sensitivity, linearity, and reproducibility while maintaining streamlined sample preparation and high throughput.