Quantification of succinylacetone in urine of hepatorenal tyrosinemia patients by HPLC with fluorescence detection.

BACKGROUND: Hepatorenal tyrosinemia (HT1) is considered a treatable inherited metabolic disease, particularly when detected early in life. Succinylacetone (SA), a unique metabolic marker for HT1, is normally circulating or excreted at very low physiological concentrations and is significantly increased in HT1 patients. METHODS: We developed and validated a new method for the determination of SA in urine using high-pressure liquid chromatography with fluorescence detection. SA and its homologue 5,7-dioxooctanoic acid used as internal standard (IS) were extracted from urine, derivatized with pyrenebutyric hydrazide and separated on a C18 column within 11 min. Calibration curves were linear between 0.025 to 100 micromol/l. Within- and between-day variations were <5% and results obtained by the current method compared favorably with a reference liquid chromatography tandem mass spectrometric method. The method was applied retrospectively to the analysis of urine samples from HT1 patients. CONCLUSIONS: The method requires a minimal sample volume (0.1 ml) with simple instrumentation. The method enabled us to differentiate HT1 cases (n=14) from controls (n=104), regardless of the years of urine storage.

Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry.

Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a (13)C(4)-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 microM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 microM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.

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

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 degrees 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 micromol/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.