DETERMINATION OF TRYPTOPHAN IN HUMAN SERUM BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PRECOLUMN FLUORESCENCE DERIVATIZATION USING PHENYLGLYOXAL

A high-performance liquid chromatographic method based on pre-column fluorescence derivatization with phenylglyoxal is described for the sensitive and selective quantification of total (free plus albumin-bound) and free tryptophan in serum. Serum was deproteinized with perchloric acid for total tryptophan and by ultrafiltration for free tryptophan, and then subjected to fluorescence derivatization. The reaction mixture was separated on a reversed-phase column by isocratic elution and the tryptophan derivative was then detected by fluorimetry. The method was at least ten-fold more sensitive than other conventional chromatographic methods with no derivatization. The lower limit of determination (signal-to-noise ratio = 3) for endogenous tryptophan in human serum was 72 nmol/l, which corresponds to 200 fmol on-column.