Abstract: The present invention relates to an on-line detection method comprising the steps of: contacting an effluent of a fractionation step with a controlled amount of an enzyme; allowing the enzyme to interact with analytes suspected to be present in the effluent; addition of a controlled amount of a substrate for said enzyme; allowing a reaction of the enzyme with the substrate providing one or more modified substrate products; and detection of unreacted substrate, or a modified substrate product using a mass spectrometer.