Ultrafast Analysis of a Tricyclic Antidepressant Drug Panel in Human Serum by the Agilent RapidFire High-Throughput Triple Quadrupole Mass Spectrometry System

Significance of the Topic

The rapid and accurate quantitation of tricyclic antidepressants (TCAs) in human serum is critical in forensic toxicology for cases such as driving under the influence, violent crimes, sexual assault, and unexplained deaths. Traditional methods based on HPLC or immunoassay suffer from long run times, high solvent use, potential cross-reactivity, and false positives. An ultrafast SPE/MS/MS approach addresses these limitations by combining minimal sample preparation with high-throughput analysis, improving laboratory turnaround and reducing operational costs.

Objectives and Study Overview

This study aimed to develop and validate a simple, robust, and sensitive method for simultaneous quantitation of eight TCAs—amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, and norclomipramine—in human serum. Key goals included achieving a broad linear range (10–500 ng/mL), excellent accuracy and precision, minimal carryover, and a sample cycle time below 15 seconds to enable >260 samples per hour throughput.

Methodology and Used Instrumentation

A protein precipitation protocol was employed: 150 µL serum mixed with 150 µL 0.2 M zinc sulfate, followed by addition of methanol containing deuterated internal standards and centrifugation. A 10-fold dilution into water preceded direct injection onto the SPE/MS/MS system.

• Sample cleanup: RapidFire SPE cartridge C18 (Agilent G9205A)
• Instrumentation: Agilent RapidFire 360 coupled to Agilent 6460 Triple Quadrupole MS
• Mobile phases: 0.1% formic acid in water and methanol
• Flow rates: 1.5 mL/min (aqueous), 1.25–0.8 mL/min (organic)
• Injection volume: 10 µL
• Ionization: Electrospray in positive mode; gas temperature 300 °C; gas flow 10 L/min; capillary voltage 3,500 V

Main Results and Discussion

Calibration curves over 10–500 ng/mL exhibited outstanding linearity (R2 > 0.995) for all eight analytes. Accuracy across low, mid, and high QC levels remained within ±10%, and precision (CV) was below 6%. Carryover was negligible (<1% of the highest calibrator). A reproducibility study of >2,000 sequential injections demonstrated stable signal responses with CVs between 2.2% and 4.2% (example: norclomipramine CV = 3.1%). The ultrafast cycle time of 13 seconds per sample represented a >10-fold increase in throughput compared to conventional LC/MS/MS.

Benefits and Practical Applications

The described SPE/MS/MS workflow combines minimal preparation with high-throughput MS analysis, delivering rapid, reliable TCA quantitation with significant savings in time and solvent consumption. This approach is well suited for forensic and clinical laboratories requiring large-scale screening and confirmation of antidepressant levels in serum.

Future Trends and Opportunities

Advances may include automation of the protein precipitation step, integration with robotic liquid handlers, and expansion of the panel to additional drug classes (e.g., benzodiazepines, opioids). Coupling ultrafast SPE/MS/MS with high-resolution MS or multiplexed ion sources could further enhance specificity and throughput.

Conclusion

An ultrafast SPE/MS/MS method using the Agilent RapidFire system enables simultaneous, precise measurement of eight TCAs in human serum with a sample cycle time of 13 seconds. The method achieves broad linearity, low carryover, and high reproducibility, representing a significant improvement over traditional HPLC or LC/MS/MS workflows for forensic toxicology.

References

1. K. Titier et al. Quantification of Tricyclic Antidepressants and Monoamine Oxidase Inhibitors by High-Performance Liquid Chromatography-Tandem Mass Spectrometry in Whole Blood. Journal of Analytical Toxicology. 31:200–207 (2007).