HPLC determination of serotonin and its metabolites from human platelet-rich plasma; shift to 5-hydroxytryptophol formation following alcohol consumption

Abstract

A sensitive, simple, and reliable high-performance liquid chromatographic method with electrochemical detection is developed for the measurement of four natural products, the serotonin-related indols from human platelet-rich plasma (PRP) using N-methylserotonin as internal standard. Separation of serotonin (5HT), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL), and 5-hydroxyindole-acetic acid (5HIAA) is carried out on Supelcosil LC-18DB stationary phase. A mixture of 48mM citric acid, 28mM sodium phosphate dibasic, 0.027mM Na2EDTA, and 3% methanol (pH 3.18) serves as the mobile phase. Measurements are carried out at 25°C at Eox = 0.65 V. The calibration curves are linear through the range of 10-200 pg/mL. Method validation is performed according to internationally accepted criteria. Blood is collected from healthy controls and schizophrenic subjects. Significantly higher PRP serotonin is measured in schizophrenics; patients with recent alcohol consumption could be characterized with significantly elevated 5HTOL/5HIAA ratio.