Tandem Mass Spectrometry Quantitation of Lyso-Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients

Fabry disease is an X-linked lysosomal storage disorder, caused by a deficit in α-galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso-Gb₃), globotriaosylceramide (Gb₃), and galabiosylceramide (Ga₂) in organs, tissues and biological fluids. A recent metabolomic study performed in plasma revealed lyso-Gb₃ analogs as novel Fabry disease biomarkers. These molecules correspond to lyso-Gb₃ with different chemical modifications on the sphingosine chain (−C₂H₄, −H₂, +O, +H₂O, +H₂O_2, and +H₂O₃). An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the multiplex analysis of lyso-Gb₃ and its 6 analogs in plasma. The samples are prepared by solid phase extraction using mixed-mode strong cation exchange (MCX) cartridges. An in-house synthesized N-glycinated lyso-Gb₃ derivative was used for the internal standard. The limits of detection (LODs) measured for lyso-Gb₃ and its analogs ranged from 0.06 to 0.29 nM. © 2016 by John Wiley & Sons, Inc.