A UPLC/MS Approach for the Diagnosis of Inborn Errors of Metabolism

IN T RO DU C T IO N LC-ESI-MS is currently the most prevalent separation technique employed in neonatal screening, used to diagnose over 30 inborn errors of amino acid, organic acid, and fatty acid metabolism. Recent analytical improvements in this approach have reduced the false-positive result rate, as compared to other conventional techniques. 2 However, UPLC/MS has not yet been used for analyzing inborn errors of metabolism. UPLC uses columns packed with sub-2 μm particles, resulting in better chromatographic resolution, excellent separation, and increased sensitivity than conventional high performance liquid chromatography (HPLC). The combination of quadrupole time-of-flight (Q-Tof) mass spectrometers with UPLC systems provides the power of accurate mass measurements with fragmentation information, and has proved successful for the identification and structural elucidation of metabolites in complex biofluids. This application note illustrates the value of UPLC/MS when applied to a blind study of urine samples from 25 patients with five unknown inborn errors of metabolism (IEM). A 12 min UPLC/QTof/MS assay was developed for the targeted profiling of organic acids using an ACQUITY UPLC C18 BEH, 2.1 x 100 mm, 1.7 μm Column, but was designed to also provide information on other classes of urinary metabolites. All five IEMs were identified correctly, and could be differentiated through principal component analysis (PCA) from data generated in MarkerLynx XS Application Manager. All organic acids were identified through a combination of MS and MS/MS and compared with authentic standards. This work illustrates the capability of UPLC/MS for the rapid and accurate diagnosis of inborn errors of metabolism.