A Fast Determination of Globotriaosylsphingosine in Plasma for Screening Fabry Disease Using UPLC-ESI-MS/MS

Abstract

: Globotriaosylsphingosine (lyso-Gb3) is considered as one of the biological marker for Fabry disease. To date, a reliable biomarker that reflects disease severity and progression has not been discovered to guide the management of Fabry disease. A new method included a simple protein precipitation with acetonitrile in 100 µL of plasma following analyte separation on an Phenomenex Kintex-C18 column using a gradient elution (0.1% formic acid in 5-90% acetonitrile). Total run time was within 12 min including sample preparation and MS/MS analysis. The limit of detection and limit of quantitation were 1 ng/mL and 2 ng/mL, respectively. The calibration curve was linear over the concentration range of 2.0-200.0 ng/mL (r 2 = 0.9999). Inter-day accuracy and precision at 7 level were 93.4-100.7% with RSD of 0.55-5.97%. Absolute recovery was 97.6-98.6%. The method was applied to human and mice plasma, proved the suitability for quantification of lyso-Gb3 for screening, diagnosis and therapeutic monitoring of Fabry disease patients.

A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3)

“…One patient from the female FD group was receiving pharmacological chaperone therapy (migalastat). Overall, lyso-Gb3 concentrations in FD samples ranged from 0.50 to 73.13 ng/mL; lyso-Gb3 concentrations in the male FD group were found to be higher than the female group ( p < 0.001), agreeing with previous published studies [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. No overlap was observed between the whole (combined male and female) FD group and the healthy controls group, or the non-pathogenic polymorphisms group ( p < 0.001).…”

“…Several quantification methods for plasma lyso-Gb3 have been published throughout the past decade, often consisting of time-consuming liquid–liquid extractions (LLE) [ 14 , 15 , 18 , 27 , 28 ], solid-phase extraction (SPE) steps [ 24 , 29 , 30 ], poorly sensitive protein precipitation-based methods (PPT) [ 31 , 32 ], or extraction protocols involving harmful solvents [ 33 , 34 ]. Here, we present a simple and sensitive method for the analysis of plasma lyso-Gb3 by liquid chromatography–tandem mass spectrometry (LC-MS/MS), with a rapid sample preparation consisting of assisted protein precipitation with Phree cartridges, and successfully validation using lyso-Gb3-D7 as an internal standard.…”