Tandem mass spectrometric assay of succinylacetone in urine for the diagnosis of hepatorenal tyrosinemia

Abstract

We describe an isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of succinylacetone (SA) in urine for the diagnosis of hepatorenal tyrosinemia (HT1). The method used 15N-labeled 5(3)-methyl-3(5)-isoxazole propionic acid as internal standard. Urine samples were oximated with hydroxylamine hydrochloride at 80 °C, extracted by solvent–solvent extraction, and followed by derivatization of the butyl ester. The butylated isoxazole derivatives of SA and its internal standard were detected and quantified using positive ion electrospray LC–MS/MS with selected reaction monitoring. The turnaround time between injections was 10 min. Calibration curves were linear over the range of 0.0633–63.3 μmol/L. The intra- and interday assay variations were less than 7%. Mean recoveries of SA at three different concentrations ranged from 96 to 109%. During the course of this study, we identified 12 new patients with HT1 and applied this method to follow up the treatment of 4 of these patients as well as previously diagnosed HT1 patients.