Methods and kits for determining protein-bound biomarkers

The invention relates to the field of diagnostic methods, in particular to detection of biological molecules using mass spectroscopy (MS).Provided is an automated high-throughput method for quantitating a low molecular weight protein-bound biomarker directly in an isolated biological sample, comprising the steps of: (a) automated prepurification of the sample using an effective amount of an acid protease under conditions that allow for digestion of one or more binding proteins; (b) applying said protease-digested sample onto an on-line SPE column to capture at least part of said biomarker, followed by sequential washing of the solid phase; (c) eluting a fraction comprising said biomarker directly onto a liquid chromatography (LC) column comprising an apolar stationary phase and subjecting it to LC-MS or LC-MS-MS measurements to determine the amount of at least one biomarker; and (d) quantitating the biomarker(s).Also provided are kits for use in such method, for instance in the determination of vitamin D derivatives, testosterone and/or or melatonin.