Development, optimization and validation of LC-MS/MS method for the determination of erythrocyte GALT enzyme activity
Project no. 20812 · Status: Kapanmış · 2019
Summary
The diagnosis of, galactosemia which mostly develops because of galactose-1-phosphate uridyltransferase(GALT) deficiency, is possible by determining erythrocyte GALT-enzyme activity. In this disease, where early diagnosis and treatment is critical, determination of the enzyme activity with high accuracy and reliability is crucial for an effective screening program and the differential diagnosis of the disease. In this study, it was aimed to develop a liquid chromatography-mass spectrometry(LC-MS/MS) based method for the determination of GALT activity in hemolysate and dry blood spot(DBS) samples; to validate the developed method and compare it with the fluorometric method. Within the scope of method validation parameters, measurement limits, measurement limits, recovery, repeatability, matrix effect, selectivity, carryover and reference interval studies were performed and the performance data of the developed method were found to be very satisfactory. When enzyme activities of hemolysate and dried blood spot(DBS) samples obtained from tubes with different anticoagulants were measured and interpreted, whole blood sample containing K2EDTA was determined to be the most appropriate tube to collect the samples. The developed LC-MS/MS method was found analytically superior to the fluorometric method compared. It is predicted that the use of LC-MS/MS method in determining GALT enzyme activity will provide great convenience in diagnosis and differential diagnosis of the disease, as well as increasing the effectiveness of newborn scans. Keywords: GALT; LC-MS/MS; newborn screening; dried blood spot