DIRECT ENRICHMENT OF TRYPTOPHAN AND ITS METABOLITES IN PLASMA ONTO A PRE-COLUMN FOLLOWED BY REVERSE PHASE HPLC ANALYSIS

Abstract

A reverse phase method combined with direct pre-column enrichment and HPLC is proposed for the analysis of tryptophan (Trp) and its metabolites in plasma. A pre-column of protein-coated ODS did not adsorb plasma proteins, but quantitatively adsorbed Trp and its metabolites with an indole ring from acidified plasma in the presence of trichloroacetate anion. After elution of plasma proteins with phosphate buffer, the enriched metabolites were separated by an ODS analytical column by stepwise elution method. The HPLC analysis with native fluorescent detection showed several peaks corresponding to Trp, 5-hydroxy tryptophol, serotonin (5-HT), 5-hydroxy indole-3-acetic acid (5-HIAA), indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA). The peaks were identified by fluorescent and electrochemical characterization. The peak heights were proportional to the injected volume of plasma samples (50-500μl), and the recovery of the spiked metabolites (2 nmol each/ml plasma) was almost quantitative (99-102%) with good reproducibility (within-run c.v., less than 3%). The present method is simple and reproducible enough for routine analysis of total Trp and its metabolites in plasma.