Development and validation of a novel method for serotonin and 5-hydroxyindole-acetic acid determination in plasma using liquid chromatography tandem mass spectrometry

Abstract

5-Hydroxytryptamine (5-HT) also known as serotonin is a biomarker in gastrointestinal disorder and several other pathological diseases where 5-HT and its metabolite 5-hydroxyindole-acetic acid are implicated. A sensitive and precise method has been developed and validated for the determination of serotonin and 5-hydroxyindole-acetic acid in human plasma. The method involves a simple protein precipitation step requiring no further downstream sample preparation. The method was developed using a Zorbax Eclipse-C8 RP (150mm × 4.6mm, 5µm) column (Agilent Technologies, Palo Alto, CA, USA). The column temperature was kept at 20°C and the solvent in gradient mode consisted of water with 0.1% formic acid (v/v) (B), Acetonitrile with 0.1% formic acid (v/v) (C) and ethanol (D) and a UV detector at 254nm and the flow rate was maintained at 0.2mL/min. Linearity of the method was studied over the range of 0.5-50µg/mL. The regression coefficients was r2 = 0.9823 for serotonin and r2 = 0.9892 for 5-hydroxyindole-acetic acid which indicates strong correlation between the studied concentration of the analytes and the signal. The precision of the method for 5-HT and 5-HIAA were achieved based on repeatability with RSD of 3.07- 7.73% and 3.93-9.99% respectively. The percentage recoveries were 83-119% and 84-116% for 5-HT and 5-HIAA respectively, which shows good accuracy of the developed method. The limit of detection and limit of quantification were 0.5µg/mL and 1µg/mL respectively suggesting good sensitivity of the method. The developed method was applied in the analysis of human plasma samples from a project related to the determination of serotonin and its metabolite in plasma from pathological patients subjected to a diet rich in vegetables.