Offline and online sample extraction for the quantification of tricyclic antidepressants in human plasma or serum for clinical research

Importance of the topic

Tricyclic antidepressants (TCAs) remain important in clinical research for therapeutic drug monitoring, personalized dosing, and pharmacokinetic investigations. Precise quantification of TCAs and their metabolites in human plasma or serum is essential to ensure patient safety, optimize therapeutic regimens, and support investigational studies of drug behavior in biological systems.

Objectives and Study Overview

This study aimed to develop a single robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method capable of quantifying twelve TCAs in human plasma or serum. The targeted compounds include amitriptyline, clomipramine, clozapine, desipramine, doxepin, imipramine, maprotiline, norclomipramine, norclozapine, nordoxepin, nortriptyline, and trimipramine. Two sample preparation strategies were compared: fully automated direct injection and offline protein precipitation.

Methodology

• Two sample workflows:

• Direct injection: Automated addition of deuterated internal standards followed by online solid-phase extraction and LC–MS/MS analysis without pre-extraction.
• Offline protein precipitation: Manual addition of internal standards, precipitation with an organic solvent, centrifugation, and transfer of supernatant for LC–MS/MS.

• Chromatography: A 5.0-minute runtime for direct injection and 2.9 minutes for offline prep using a dedicated SPE cartridge (direct mode) and an analytical column driven by tailored mobile phases.

Used Instrumentation

• Thermo Scientific Transcend II TLX-1 system for online SPE and autosampler functions.
• Thermo Scientific TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization, operated in positive ion mode.
• SRM acquisition with two transitions per analyte and matching deuterated internal standards.

Main Results and Discussion

• Linearity: Both approaches exhibited linear responses across calibration ranges—direct injection from ~2–1 200 ng/mL, offline precipitation down to sub-ng/mL levels for enhanced sensitivity.
• Sensitivity: Offline precipitation achieved lower limits of quantification (below 1 ng/mL for most analytes) compared to direct injection.
• Accuracy: Bias values ranged from –7.6 % to +9.2 % (offline) and –7.2 % to +17.1 % (direct), meeting typical bioanalytical acceptance criteria.
• Precision: Intra-assay relative standard deviations were under 7.2 %, and inter-assay precision remained below 9.4 % for all compounds.

Benefits and Practical Applications

• Dual workflow offers flexibility: high throughput direct injection for large sample loads and enhanced sensitivity offline preparation when detecting low-concentration analytes.
• Comprehensive panel of twelve TCAs in a single method streamlines laboratory operations.
• Automated direct injection reduces hands-on time and potential for human error, supporting routine clinical research.

Future Trends and Opportunities

• Integration of microflow or nanoflow LC could further lower solvent consumption and enhance ionization efficiency.
• Expansion to include additional psychotropic or co-medicated compounds may broaden clinical utility.
• Combining high-resolution mass spectrometry for untargeted metabolite screening alongside targeted TCA quantification.

Conclusion

A unified LC–MS/MS protocol was established for quantitative analysis of twelve tricyclic antidepressants using both direct injection and offline sample preparation. The method delivers high throughput, excellent sensitivity, and robust accuracy and precision, making it well suited for clinical research and therapeutic drug monitoring of TCAs.