Quantitation of phenylalanine and its trans-cinnamic, benzoic and hippuric acid metabolites in biological fluids in a single GC-MS analysis

We describe a sensitive, simple and convenient stable isotope dilution assay developed to study endogenous metabolism of administered stable isotope-labeled phenylalanine (Phe) in phenylketonuric (PKU) mice treated experimentally with phenylalanine ammonia lyase (PAL). Mouse urine and plasma containing endogenous and administered labeled Phe together with internal standard Phe bearing a different pattern of labeling are converted by insitu diazotization to 2-chloro-3-phenylpropionic acid (CPP). A single solvent extraction is then used to isolate the isotopomers of CPP along with the transcinnamic acid (TCA) produced from Phe by PAL, as well as the TCA metabolites benzoic and hippuric acids. This procedure eliminates the need for a separate ion-exchange isolation step for Phe on a second sample aliquot and separate GC-MS analysis. Extracted CPP and the Phe metabolites are then measured by conversion to the pentafluorobenzyl esters and a single analysis by electron capture negative ion GC-MS. The estimated lower limit of quantitation is 0.1 mu m. Copyright (c) 2007 John Wiley & Sons, Ltd.