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 in situ diazotization to 2-chloro-3-phenylpropionic acid (CPP). A single solvent extraction is then used to isolate the isotopomers of CPP along with the trans-cinnamic 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 microM.
Sarkissian
Christineh N
CN
Departments of Biology, Human Genetics and Pediatrics, McGill University and Debelle Laboratory, McGill University-Montreal Children's Hospital Research Institute, Montreal, QC, H3H 1P3, Canada.
Scriver
Charles R
CR
Mamer
Orval A
OA
D000818:Animals; D019817:Benzoic Acid / Q000032:analysis*; D002934:Cinnamates / Q000032:analysis*; D003903:Deuterium; D004359:Drug Therapy, Combination; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006626:Hippurates / Q000032:analysis*; D051379:Mice; D008817:Mice, Mutant Strains; D010649:Phenylalanine / Q000032:analysis* / Q000493:pharmacokinetics; D010650:Phenylalanine Ammonia-Lyase / Q000493:pharmacokinetics
D002934:Cinnamates; D006626:Hippurates; C029010:cinnamic acid; D010649:Phenylalanine; D019817:Benzoic Acid; D003903:Deuterium; D010650:Phenylalanine Ammonia-Lyase; C030514:hippuric acid