Determination of Ketosteroids in Human Urine Using Dispersive Liquid-Liquid Microextraction and Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry

A method is proposed for the determination of some ketosteroids in human urine, including enzymatic hydrolysis using β-glucuronidase from E. coli followed by dispersive liquid-liquid microextraction, derivatization of analytes with hydroxylamine, and detection by reversed-phase ultra-HPLC–quadrupole time-of-flight mass spectrometry. Optimization of extraction and derivatization conditions of the studied compounds made it possible to find that the highest recoveries were achieved using an acetone–chloroform mixture as a dispersant and an extractant, and the completeness of the derivatization reaction was achieved by thermostating the sample at 70°C for 90 min. The proposed method has high sensitivity (limits of detection in the range of 0.1–0.25 ng/mL) and a wide linearity range.