Capillary electrophoresis with UV detection and mass spectrometry in method development for profiling metabolites of steroid hormone metabolism

The aim of this study was to develop a method for comprehensive profiling of metabolites involved in mammalian steroid metabolism. The study was performed using the partial filling micellar electrokinetic chromatography (PF-MEKC) technique for determination of endogenous low-hydrophilic steroids. The detection techniques in capillary electrophoresis were UV absorption and electrospray mass spectrometry (ESI-MS). Thirteen steroids were included in the method development, and the selected were metabolites involved in major pathways of steroid biosynthesis. Although only eight of them could be separated and detected with UV, they could be identified by ESI-MS using selected ion monitoring (SIM) technique. Tandem MS spectra were also collected. UV detection was more sensitive than MS due to better separation of compounds and the selective signal sensitivity. The lowest limits of detection were 10–100 ng/mL for cortisone, corticosterone, hydrocortisone and testosterone. The other steroids could be detected at 500–1000 ng/mL. The identification of cortisone, corticosterone, hydrocortisone, estrogen and testosterone were made in patient urine samples and their concentrations were 1–40 μg/L.