Determination of endogenous steroids in urine by liquid chromatography-tandem mass spectrometry

A method was developed for the determination of endogenous steroids in urine using liquid chromatography-tandem mass spectrometry LC-MS/MS with methyltestosterone as internal standard. After enzymatic hydrolysis by ss-glucuronidase and liquid-liquid extraction the urine sample was chromatographed on a Cosmosil C-18 column with a mixture of methanol and ammonium acetate-formic acid 68: 32 v/v as mobile phase then detected using MS/MS system with electrospray ionization ESI in multi-reaction monitoring MRM mode. The detection limits ranged from 0. 01 ng/mL to 10 ng/mL. The recoveries ranged from 96. 7% to 106.5% and the intra-and inter-day precisions measured as relative standard deviations were less than 7% and 11% respectively. With simple and fast sample preparation the method was sensitive and specific for simultaneous determination of these 5 kinds of endogenous steroids in urine. The method has been successfully applied in pharmacokinetic study and is thus a potential alternative for gas chromatography-mass spectrometry GC-MS based procedures in routine analysis of endogenous steroids such as DHEA in human urine.