Determination of plasma C16-C24 globotriaosylceramide (Gb3) isoforms by tandem mass spectrometry for diagnosis of Fabry disease

ABSTRACT

PURPOSE: A simple, rapid, and highly sensitive analytical method for Gb3 in plasma was developed without labor-extensive pre-treatment by electrospray ionization MS/MS (ESI-MS/MS). Measurement of globotriaosylceramide (Gb3, ceramide trihexoside) in plasma has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ??-galactosidase A (alpha-Gal A). The lack of alpha-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. METHODS: Only simple 50-fold dilution of plasma is necessary for the extraction and isolation of Gb3 in plasma. Gb3 in diluted plasma was dissolved in dioxane containing C170 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. RESULTS: Eight isoforms of Gb3 were completely resolved from plasma matrix. C160 Gb3 occupied 50% of total Gb3 as a major component in plasma. Linear relationship for Gb3 isoforms was found in the range of 0.001-1.0 microgram/mL. The limit of detection (S/N=3) was 0.001 microgram/mL and limit of quantification was 0.01 microgram/mL for C160 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9678 to 0.9982. CONCLUSION: This quantitative method developed could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.