Further sample preparation was performed on an automated system, as previously described (30). In short, linkage-specific sialic acid stabilization (ethyl esterification) was performed by adding 60 µL 250 mM EDC 250 mM HOBt in ethanol to 3 µL released glycan sample. This mixture was incubated for 75 min at room temperature, after which 120 µL ACN was added. To purify the samples, a GHP membrane (GHP plate, Pall AcroPrep Advance 96 Filter plate, Pall Corporation, Ann Arbor, MI, USA) was prewetted with 70% ethanol, activated with MQ and equilibrated with 100% ACN. The complete sample was transferred to the GHP plate and incubated for 5 min. Subsequently the GHP plate was washed three times with 100 µL 96% ACN. Elution was performed by adding 30 µL of MQ to the GHP plate, incubating at room temperature for 5 min, followed by centrifugation into a PCR plate.