Clinical research method for the quantitative determination of free plasma serotonin and 5-hydroxyindoleacetic acid (5-HIAA) in human plasma by UHPLC-MS/MS

Significance of the Topic

The balance of serotonin and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in plasma is critical for monitoring antidepressant treatment, assessing serotonin syndrome risk, and advancing neuropsychiatric research. A reliable, high-throughput method supports both clinical studies and therapeutic drug monitoring while minimizing sample preparation challenges and matrix interference.

Objectives and Study Overview

This study aimed to develop and validate a quantitative UHPLC-MS/MS method for simultaneous determination of free plasma serotonin and 5-HIAA in human plasma. Key goals included achieving low limits of quantification, robust performance in a 96-well format, and compatibility with clinical research workflows.

Methodology and Instrumentation

• Sample Cleanup: Solid phase extraction using Thermo Scientific SOLA SAX 10 mg/2 mL 96-well plate. Workflow involved conditioning with methanol and sodium hydroxide, sample loading, washing, elution with acetonitrile/formic acid, evaporation, and reconstitution.
• UHPLC Separation: Thermo Scientific Vanquish Horizon system equipped with Acclaim Vanquish PA2 column (150 x 2.1 mm, 2.2 µm). Mobile phases were water/formic acid (100:0.1 v/v) and methanol/formic acid (100:0.1 v/v), flow rate 0.5 mL/min, column temperature 60 °C, run time 8.5 minutes, injection volume 20 µL.
• Mass Spectrometry: Thermo Scientific TSQ Quantiva triple quadrupole with HESI-II probe. Serotonin analyzed in positive mode (m/z 177.1 → 160.0), 5-HIAA in negative mode (m/z 190.1 → 146.2). Source settings included spray voltages +2500 V/–4500 V, vaporizer temperature 320 °C, ion transfer tube temperature 330 °C, sheath and auxiliary gas pressures optimized.

Main Results and Discussion

• Calibration and Linearity: Serotonin linear over 1–100 ng/mL (1/x² weighting, R²>0.999); 5-HIAA linear/quadratic over 5–500 ng/mL (1/x², R²>0.997).
• Sensitivity: LLOQ of 1 ng/mL for serotonin and 5 ng/mL for 5-HIAA, with clear Gaussian peaks and minimal baseline noise.
• Precision and Accuracy: Serotonin bias within ±5.3% and CV 1.5–2.1%; 5-HIAA bias within ±19.5% and CV 2.2–10.5% across QC levels.
• Recovery and Matrix Effects: Absolute recoveries of 67.0% for serotonin and 58.1% for 5-HIAA. Internal standard-normalized matrix factors averaged 1.01, demonstrating effective compensation of ion suppression/enhancement.
• Specificity and Carryover: No significant interferences in six individual plasma sources; carryover below 20% of LLOQ peak area after double blanks.

Benefits and Practical Applications

• Robust SPE cleanup dramatically reduces plasma matrix effects while preserving analyte yield.
• 96-well plate format enables automation and high throughput in clinical research settings.
• Simultaneous analysis of serotonin and 5-HIAA in a single sub-9-minute run streamlines workflow.
• Superior peak shape and resolution support accurate quantitation at low concentrations.

Future Trends and Opportunities

With growing demand for precision neurochemical profiling, this approach may expand to additional neurotransmitters and metabolites. Further integration with laboratory automation, microflow and nanoscale LC formats, and real-time data processing will enhance throughput and support personalized medicine applications.

Conclusion

The validated UHPLC-MS/MS method combining SOLA SAX SPE, Vanquish PA2 chromatography, and TSQ Quantiva detection offers rapid, sensitive, and reproducible quantitation of free plasma serotonin and 5-HIAA. Its high throughput and robust performance make it well suited for clinical research and therapeutic monitoring.