Amino Acid Analysis in Rat Wound Exudate by High-Performance Liquid Chromatography-Fluorescence Detection

Abstract

Wound exudate is produced during the healing process and plays a crucial role in natural defense and repair mechanisms. Proper wound management is necessary by monitoring exudate levels. Amino acid concentrations in wound exudates are altered under wound conditions during the wound-healing process. Thus, analysis of amino acids in wound exudates is useful for determining wound conditions. In this study, we developed a validated analytical method for amino acids in rat wound exudates by high-performance liquid chromatography (HPLC)-fluorescence detection using the fluorescence derivatization reagent, 2,3-naphthalenedicarboxaldehyde (NDA). Chromatographic separation of 18 NDA-amino acids and 5-aminovaleric acid (internal standard) was achieved within 100 min on an octadecylsilyl column under gradient elution using an eluent consisting of water, acetonitrile, and trifluoroacetic acid. The calibration curves were linear (0.25–25 pmol/injection) and exhibited high correlation coefficients (r2 > 0.998) for each amino acid. The developed method was applied to a wound exudate sample and 18 amino acids were quantified. The amino acid concentrations in the wound exudate samples were 24.2–1694 µM. This method was successfully applied to amino acids quantification in wound exudates, showing its potential for clinical application.