Quantification of glutaric and 3-hydroxyglutaric acids in urine of glutaric acidemia type I patients by HPLC with intramolecular excimer-forming fluorescence derivatization

Abstract

Glutaric aciduria type I (GA1) is an autosomal recessive disorder that usually causes neurological damage. Early diagnosis of the disease prior to the appearance of clinical symptoms can lead to better outcomes. We describe a simple and selective HPLC method with intramolecular excimer-forming fluorescence derivatization to diagnose GA1. Glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in urine and an internal standard were derivatized with 1-pyrenebutyric hydrazide (PBH). The derivatives were separated on a C18 column and fluorometrically detected at 475 nm (excitation of 345 nm) with a run time of 18 min. Excellent linearity over a wide range, reproducibility (coefficient of variation < or =14.5%), and sensitivity (limit of detection 0.4 micromol/l 3HGA and 0.2 micromol/l GA) were obtained. A retrospective study on previously diagnosed GA1 patients' urine from our laboratory archives between 1999 and 2004 was performed by analysts blinded to the study. The method enabled us to differentiate GA1 cases (n=36) from controls (n=99), regardless of the years of urine storage. The method is valuable for both retrospective and prospective diagnoses of GA1.