Multiplex Enzyme Assay for Galactosemia Using Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

BACKGROUND:Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Liquid Chromatography–Tandem Mass Spectrometry Enzyme Assay for UDP-Galactose 4′-Epimerase: Use of Fragment Intensity Ratio in Differentiation of Structural Isomers

“…This contrasts with the range found for control subjects of 17.1-40.1 mol ⅐ g Ϫ1 Hb ⅐ h Ϫ1 with a HPLC method (18 ). A similar but not identical discrepancy was seen with a multiplex GALE ultraperformance liquid chromatography-MS/MS-based enzyme assay compared to a radiometric assay (15,16 ). Our method revealed a K m for UDPGal of 0.05 mmol/L.…”

Laboratory diagnosis of galactosemia: a technical standard and guideline of the American College of Medical Genetics and Genomics (ACMG)

“…43,44 These methods can be multiplexed and allow the simultaneous determination of enzyme activity for all three galactosemia types with minimal sample volume. This represents a significant improvement for diagnostic testing.…”

“…Recently, LC-MS/MS methods have been developed to measure GALK and GALE activity in RBCs and/or DBS using 13 C-labeled substrate. [43][44][45] Metabolite assays…”

Galactosemia Diagnosis Gets an Upgrade

“…In this issue of Clinical Chemistry, 2 reports push the boundaries of analytical sensitivity and versatility for galactosemia diagnosis with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (12,13 ). Both reports describe methods for measurement of GALT activity based on the use of a stable isotope-labeled substrate ([ 13 C]galactose 1-phosphate).…”

“…Both reports describe methods for measurement of GALT activity based on the use of a stable isotope-labeled substrate ([ 13 C]galactose 1-phosphate). The enzymatic assay is performed offline, and the product, 13 C-labeled UDP-galactose, is separated by a short UPLC run, ionized by negative ion electrospray ionization, and monitored by multiple reaction monitoring with appropriate isotope-labeled internal standards. Both methods produce typical values for GALT that are consistent with those obtained with the traditional enzymatic assays.…”