SIMULTANEOUS DETERMINATION OF 5-HYDROXYTRYPTAMINE, 5-HYDROXY-TRYPTOPHAN, 5-HYDROXYINDOLEACETIC ACID, DOPAMINE, AND HOMOVANILLIC ACID IN WHOLE BLOOD, USING ISOCRATIC HPLC WITH ELECTROCHEMICAL DETECTION

Abstract

A sensitive and a rapid procedure is described using high performance liquid chromatography combined with electro-chemical detection (HPLC-ECD), for quantification of whole blood biogenic amines and their metabolites. Serotonin (5-hydroxytryptamine, 5-HT), its precursor 5-hydroxytryptophan (5-HTRP), and metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA) and its metabolite homovanillic acid (HVA), were extracted from an aliquot of whole blood. After precipitation of proteins with 20% zinc sulfate and centrifugation the clear supernatant was treated with 10% sodium hydroxide and centrifuged. The supernatant was filtered and injected into the HPLC system. Separation of the analytes of interest was carried out with a mobile phase containing sodium acetate, citric acid, sodium octyl sulfate, disodium-EDTA, dibutylamine, and 15% methanol, at a flow rate of 0.5mL/min and at a potential of +0.60 V. Peaks of each compound were identified by their respective retention times established separately with each standard, as well as with a mixture of all standards. Calibration was carried out by injecting the extract prepared by spiking 0.5 mL of 5% bovine serum albumin (BSA) solution with known concentrations of standards (8.0 ng/mL for each, except that of HVA with a higher concentration of 20 ng/mL) and a fixed concentration of internal standard (8.0 ng/mL). Concentration in the samples were calculated by the data module using the ratio of the detector response (area under the peak) of each compound relative to that of the internal standard. The lower limits of sensitivity for 5-HTRP, DA, 5-HIAA and 5-HT was 0.5 ng/mL. For HVA, the lower limit of sensitivity was 2.0 ng/mL. Recovery of all compounds ranged between 80.0–96.0% and was tested by adding the known concentration of standards to either a solution of BSA or to an aliquot of whole blood. All components from the extract were eluted within 25 minutes. This procedure has been found to be rapid, simple and reliable for investigating simultaneously the status of biogenic amines, 5-HT and DA and their metabolites, in whole blood.