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Quantitation of phenylalanine and its trans ‐cinnamic, benzoic and hippuric acid metabolites in biological fluids in a single GC‐MS analysis
Author(s) -
Sarkissian Christineh N.,
Scriver Charles R.,
Mamer Orval A.
Publication year - 2007
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1218
Subject(s) - chemistry , hippuric acid , phenylalanine , chromatography , benzoic acid , cinnamic acid , isotopomers , detection limit , isotope dilution , phenylalanine ammonia lyase , urine , mass spectrometry , organic chemistry , biochemistry , amino acid , molecule
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 µ M . Copyright © 2007 John Wiley & Sons, Ltd.