Quantification of the D-(+)-enantiomer of phenylalanine in physiological fluids using high-performance liquid chromatography with column switching

Abstract

A procedure is described whereby the chiral resolution of racemic phenylalanine is achieved at trace levels in physiological samples, such as urine, following precolumn derivatisation using o-phthalaldehyde (OPA) and 2-mercaptoethanol to produce highly fluorescent thio-substituted isoindole derivatives. A pH-controlled ionisation suppression technique allowed the retention of OPA-derivatised rac-phenylalanine on a Spherisorb 3ODS2 guard column, while the urine matrix components were flushed to waste. Zone transfer of the retained band of the amino acid from the guard column was made onto an analytical β-Cyclobond column, using a stronger eluent and a column switching procedure. This allowed the chiral resolution and quantification of the enantiomers of the amino acid using reversed-phase high-performance liquid chromatography and fluorescence detection. The limit of detection achieved for this method was estimated as 24 ng ml^–1 in urine without sample pretreatment. The fluorescence responses of both the D-(+)- and L-(–)-phenylalanine derivatives are linear up to 15 µg ml^–1.