QUANTIFICATION OF THE D-(-ENANTIOMER OF PHENYLALANINE IN PHYSIOLOGICAL FLUIDS USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH COLUMN-SWITCHING())

A procedure is described whereby the chiral resolution of racemic phen ylalanine is achieved at trace levels in physiological samples, such a s urine, following precolumn derivatisation using o-phthalaldehyde (OP A) and 2-mercaptoethanol to produce highly fluorescent thio-substitute d isoindole derivatives, A pa-controlled ionisation suppression techni que allowed the retention of OPA-derivatised rac-phenylalanine on a Sp herisorb 30DS2 guard column, while the urine matrix components were fl ushed to waste. Zone transfer of the retained band of the amino acid f rom the guard column was made onto an analytical beta-Cyclobond column , using a stronger eluent and a column switching procedure, This allow ed 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 pretreatm ent. The fluorescence responses of both the D-(+)- and L-(-)-phenylala nine derivatives are linear up to 15 mu g ml(-1).