Volumetric absorptive microsampling coupled with UHPLC-MS/MS for the determination of Lyso-Gb3 as Fabry disease diagnostic biomarker

A volumetric absorptive microsampling (VAMS) combined with UHPLC-MS/MS method was validated for the analysis of lyso-Gb3 in capillary blood. Lyso-Gb3, a biomarker for Fabry disease (FD), was extracted from VAMS device with methanol/acetonitrile/water mixture, in a temperature-controlled ultrasonic bath and the solution was analysed on a Kinetex C18 in gradient elution with MRM detection. The VAMS method was fully validated according to EMA guidelines. No hematocrit effect was observed over a range of 20-70 %. The method showed linearity from 0.25 to 100.00 ng/mL, the lower limit of quantitation was 0.25 ng/mL, both precision and accuracy were <= 15 % and the average extraction recovery was 94.6 %. The VAMS UHPLC-MS/MS method was successfully compared with a reference plasma method in a series of 13 Fabry patients and 12 healthy volunteers. This is the first validated UHPLC-MRM VAMS-based method, hematocrit-independent, for capillary blood lysoGb3 quantification in FD patients.