180 Chapter 5 SM3: Detailed procedure of Lab GMD (Amsterdam UMC) lysoGb3 assay LysoGb3 is measured using LC-MS/MS ((Xevo TQ MS, Waters Inc.) in multiple reaction monitoring(MRM) mode. Sample preparation: • Pipette 50 µL plasma into a 2 mL vial. • Add 25 µL Internal Standard (IS) (concentration: 0.1 µM). • Add 25 µL of MilliQ water. • Add 300 µL MeOH and 150 µL CHCl3 and vortex for 30 sec. • Centrifuge at 16060 g (14000 rpm) at 4°C for 10 min. • Transfer the supernatant to a 2 mL vial. • Add 150 µL CHCl3 and 225 µL MilliQ water and vortex for 30 sec. • Centrifuge at 16060 g (14000 rpm) at 4°C for 2 min. • Take 400 µL of the supernatant and transfer to a 1.5 mL screw cap vial. • Evaporate to dryness at 35°C under N2. • Take up the residue in 60 µL MeOH by vortexing (30 sec) and sonicating (1 min) in a sonication bath. • Centrifuge for 10 min at 16060 g at 4°C. • Transfer 50 µL to a gilson vial and cap and add 50 µL MilliQ water. • Mix by vortexing briefly. (Internal) standards and LC-column • Internal standard: Gly-lysoGb3 (Matreya, 1530) • Standard: LysoGb3 (Sigma, G9534) • LC-column: Acquity BEH C18 200x2.1 mm, 1.7µm particles (Waters, 186002350) Liquid chromatography settings
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