Determination of unbound L‐tryptophan in human plasma using high‐performance liquid chromatography with fluorescence detection

Abstract

An assay for the measurement of unbound L‐tryptophan concentrations in plasma was developed using reverse‐phase HPLC with fluorescence detection. Unbound L‐tryptophan from plasma was obtained using the Amicon MPS‐1 ultrafiltration device. L‐tryptophan binding to the membrane in the ultrafiltration device was not significant. A linear relationship between the peak‐area ratios (peak‐area of L‐tryptophan to internal standard) and concentrations of L‐tryptophan was obtained in the range of 0.1 μg/mL to 2.5 μg/L. The intra‐assay precision was less than 10% for each control (0.18 μg/mL, 0.75 μg/mL and 2.0 μg/mL). The inter‐assay precision was less than 15% for each control; the accuracy for each control, expressed as percent difference from nominal (%DFN), was less than 12%. This method was used to determine unbound L‐tryptophan concentrations in plasma from nine male subjects who participated in a clinical study.