High-Throughput Quantitative Analysis of Tricyclic Antidepressants and Selective Serotonin Re-uptake Inhibitors in Human Serum Using Ultrafast SPE/MS/MS

Importance of the Topic

Accurate and rapid quantification of tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs) in human serum is critical for forensic toxicology, clinical pharmacokinetics, and therapeutic drug monitoring. Traditional LC/MS/MS methods, while sensitive and selective, often suffer from long analysis times and limited throughput. The adoption of ultrafast solid-phase extraction coupled to tandem mass spectrometry addresses these challenges by combining high analytical performance with sample cycle times under 13 seconds.

Objectives and Study Overview

This study aimed to develop and validate a high-throughput analytical workflow for simultaneous measurement of eight TCAs (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine) and six SSRIs (citalopram, N-desmethyl citalopram, fluoxetine, norfluoxetine, sertraline, paroxetine) in human serum. The performance of the RapidFire SPE/MS/MS system was benchmarked against conventional HPLC-MS/MS in terms of linearity, sensitivity, precision, and throughput.

Methodology and Instrumentation

Sample preparation involved protein precipitation with zinc sulfate and organic solvent, followed by online SPE on a RapidFire system. Key methodological points included:

• Sample volume: 100–150 µL serum spiked with deuterated internal standards
• Precipitation: addition of ZnSO₄ and methanol or acetonitrile
• Online SPE cartridge: C18 sorbent
• Elution and analysis: RapidFire RF states with aqueous/formic acid and organic/formic acid solvents
• Instrument setup: Agilent RapidFire High-Throughput SPE coupled to Agilent 6460 or 6490 triple quadrupole MS using ESI+ mode
• Data acquisition: multiple reaction monitoring transitions optimized for each analyte

Key Results and Discussion

• Linearity: All TCAs and SSRIs showed excellent linear response over 10–500 ng/mL (R²>0.995).
• Limit of quantification: 5 ng/mL for all compounds.
• Precision and accuracy: Intra- and inter-day accuracy within ±10%, coefficient of variation below 10% across concentration ranges.
• Throughput: Analysis time below 13 seconds per sample, enabling >240 injections per hour.
• Carryover and reproducibility: Negligible carryover observed; repeatability maintained over >2000 sequential injections.

Benefits and Practical Applications

The ultrafast SPE/MS/MS workflow provides significant advantages:

• High sample throughput for large-scale toxicology or clinical studies.
• Robust quantitation with minimal sample volume.
• Reduced solvent consumption and waste compared to traditional LC/MS/MS.
• Applicability to forensic casework, therapeutic monitoring, and pharmacokinetic profiling.

Future Trends and Potential Applications

Ongoing developments could further enhance this platform:

• Integration with automated sample handling for 24/7 operation.
• Expansion to other drug classes and metabolites.
• Miniaturized or multiplexed SPE cartridges for tailored applications.
• Coupling with high-resolution MS for comprehensive screening workflows.

Conclusion

This study demonstrates that ultrafast SPE combined with tandem mass spectrometry on Agilent RapidFire platforms delivers comparable sensitivity and precision to traditional LC/MS/MS methods, while achieving >10-fold faster analysis speeds. The method supports high throughput, robust quantitation of TCAs and SSRIs in human serum, meeting the demands of forensic and clinical laboratories.

References

• Youssef MG, Parikh NR, Miller VP, LaMarr WA. High-Throughput Quantitative Analysis of Tricyclic Antidepressants and Selective Serotonin Re-uptake Inhibitors in Human Serum Using Ultrafast SPE/MS/MS. MSACL 2014 Poster #27.a.