Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry

Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann–Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC–MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann–Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC–MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann–Pick and successfully distinguished different subtypes reflecting the disease severity.