Quantification of monosialogangliosides in human plasma through chemical derivatization for signal enhancement in LC–ESI-MS

Abstract

Gangliosides are found in abundance in the central nervous system of vertebrates. Their metabolic disruption and dysfunction are associated with various neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In order to improve our understanding of the etiology of these diseases, analytical ganglioside assays with sufficient specificity and sensitivity in relevant biological matrices are required. In the present work we have developed and validated a reverse-phase ultra-performance liquid chromatography (UPLC)/tandem mass spectrometry (MS) method for determining monosialogangliosides GM1, GM2, and GM3 present in human plasma. Compared with our previous method, this method enhanced, by 15 fold, MS responses of the analytes by employing 2-(2-Pyridilamino)-ethylamine (PAEA) & 4-(4, 6-Dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)-based derivatization. The analytes and internal standards were derivatized with PAEA&DMTMM after extraction from plasma using a protein precipitation procedure. They were then purified using liquid-liquid partitioning. When the samples were then analyzed by UPLC-MS/MS with a multiple reaction monitoring (MRM) mode, we achieved superior sensitivity and specificity. This method was evaluated for extraction recovery, calibration linearity, precision, accuracy, and lower limit of quantification (LLOQ). The validated method was successfully applied to monitor monosialoganglioside levels in the plasma from patients with GM3 synthase deficiency. With significantly increased sensitivity, we have, for the first time, detected a significant amount of GM3 in the affected patients.

Enhanced Susceptibility to Chemoconvulsant-Induced Seizures in Ganglioside GM3 Synthase Knockout Mice

“…In patients with ST3GAL5 deficiency, GM3 and all downstream biosynthetic derivatives in the circulation are hardly detectable even with some recently improved analytic assays ( Huang et al., 2014 ). The lack of a-, b-, and c-series gangliosides are replaced by overexpression of asialo- and α-series gangliosides ( Seyfried et al., 1994 ; Huang et al., 2016 ). The discovery of ST3GAL5 deficiency, a recessive genetic disorder, provided the first evidence for a congenital disorder of glycosylation that affects the biosynthesis of complex glycosphingolipids ( Bowser et al., 2019 ).…”

Quantitative LCMS for ceramides/dihydroceramides: pregnancy baseline biomarkers and potential metabolic messengers

“…According to previous studies [24][25][26][27], a prior chromatographic separation step is essential to alleviate the charging competition that occurs between co-eluting analytes and matrix components and minimize the extent of ionization suppression introduced onto the targeted analytes, thus improving the sensitivity, specificity, and robustness of the assay.…”

“…Among multiple chromatographic platforms, reverse phase chromatography is known for its capability to retain and separate lipophilic molecules in order of increasing hydrophobicity, and its application has been documented in multiple studies to be fundamental for the chromatographic separation of glycosphingolipids [24][25], lipophilic vitamins [26], and steroids [27] to take place in a highly reproducible manner. As a result, in view of the hydrophobic nature of ceramides and dihydroceramides as complex sphingolipids, an ACE Excel SuperC18 UPLC column (1.7 µm, 100 mm´2.1 mm) was selected for chromatographic separation to provide reliable matrix cleanup and analyte enrichment prior to the ionization.…”

“…13 Moreover, in the existing methodologies, gradient elusion programs were preferentially employed for the analysis of ceramides to improve chromatographic resolution and detection sensitivity. Based on published data, utilization of gradient elusion programs allows the targeted ceramides to be retained, separated, and eluted reproducibly with excellent run-to-run consistency [24][25][26][27]. However, as the tradeoff of improved chromatographic resolution and detection sensitivity, gradient elusion is also technically subjected to a number of limitations.…”

Chemical Derivatization-Based LC–MS for Metabolomics: Advantages and Challenges

“…Large quantity of emerging derivatization reagents for metabolites have been synthesized over the last decade [3], they were designed for respective metabolites with different functional groups such as amines, phenols, carboxylic acids, steroids, thiols and alcohols [4]. The enhancement of detectability is the main purpose of derivatization-based [4][5][6]. Interestingly, a new strategy that integrates several subsets of metabolites with different functional groups together by derivatization-based LC-MS for untargeted metabolomics has been proposed [7].…”