Analysis of organic acid markers relevant to inherited metabolic diseases by ultra-performance liquid chromatography/tandem mass spectrometry as benzofurazan derivatives

We describe a new approach applicable to the determination of organic acids that serve as diagnostic markers for several inherited metabolic disorders. We utilized liquid chromatography/tandem mass spectrometry for analysis of organic acid derivatives of a recently described benzofurazan reagent. The derivatization step was necessary to obtain organic acid derivatives suitable for analysis by reversed-phase liquid chromatography with high ionization efficiency for mass spectrometry in the positive-ion mode. In this work, a group of related dicarboxylic acid markers containing five or six carbon atoms were analyzed and validation was performed for glutaric and 3-hydroxyglutaric acids, the specific markers for glutaric acidemia type 1. Derivatization was achieved by reacting untreated urine with the derivatization reagent under mild conditions. The reaction mixture was analyzed on a C18 ultra-performance liquid chromatography (UPLC) column (50 x 2.1 mm, 1.7 mu m) and detected in the multiple reaction monitoring mode in 5 min. Calibration curves were linear up to at least 1000 mu M with detection limits for glutaric and 3-hydroxyglutaric acids of 0.025 and 0.02 mu M, respectively (signal-to-noise ratio of 3). Intra-day (n =11) and inter-day (n = 6) coefficients of variation were better than 11.2%. The assay was successfully applied to control (n = 134) and glutaric acidemia type 1 (n = 55) urine samples. Copyright (C) 2007 John Wiley & Sons, Ltd.