A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry

•A fully automated mass spectrometry method for determining steroids in serum was developed and validated.•The method includes protein precipitation, filtration and online 2D chromatography.•A biphenyl stationary phase demonstrated acceptable analytical performance for steroid separation.•Method comparison with immunoassay highlights improved performance of mass spectrometry.