Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity.

Autor: Topbaş M; Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Izmir, 35100, Turkey., Canbay E; Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Izmir, 35100, Turkey. Electronic address: erhan.canbay@ege.edu.tr., Sezer E; Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Izmir, 35100, Turkey., Canda E; Department of Pediatric Metabolic Disease, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey., Kalkan Uçar S; Department of Pediatric Metabolic Disease, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey., Çoker M; Department of Pediatric Metabolic Disease, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey., Yıldırım Sözmen E; Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Izmir, 35100, Turkey.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2023 Oct 01; Vol. 678, pp. 115284. Date of Electronic Publication: 2023 Aug 10.
DOI: 10.1016/j.ab.2023.115284
Abstrakt: Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 μmol/gHb/hour. The linear range was 0.4-50 μM (2.4-310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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