Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity
Küçük Resim Yok
Tarih
2023
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Academic Press Inc.
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
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 ?mol/gHb/hour. The linear range was 0.4–50 ?M (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. © 2023 Elsevier Inc.
Açıklama
Anahtar Kelimeler
Dried blood spot, GALT, LC-MS/MS, Newborn screening, Blood, Liquid chromatography, Mass spectrometry, Carbohydrate metabolism, Dried blood spot, Enzymes activity, GALT, LC-MS-MS, Lc.ms/ms, Liquid chromatography - mass spectrometries, Newborn screening, Optimisations, Spot samples, Enzyme activity, acetonitrile, galactose 1 phosphate uridylyltransferase, galactose 1 phosphate uridylyltransferase, adolescent, adult, aged, Article, child, controlled study, dried blood spot testing, enzyme activity, female, fluorometry, human, infant, intermethod comparison, liquid chromatography-mass spectrometry, male, matrix effect, measurement accuracy, measurement repeatability, newborn, normal human, quantitative analysis, storage, validation process, dried blood spot testing, galactosemia, liquid chromatography, procedures, reproducibility, tandem mass spectrometry, Chromatography, Liquid, Dried Blood Spot Testing, Galactosemias, Humans, Reproducibility of Results, Tandem Mass Spectrometry, UTP-Hexose-1-Phosphate Uridylyltransferase
Kaynak
Analytical Biochemistry
WoS Q Değeri
Scopus Q Değeri
Q3
Cilt
678
