4.5. Liquid–liquid Extraction Procedure

A previously validated LLE procedure for steroids was used in a comparative study [41]. Briefly, 3 mL of urine was incubated in a 10 mL glass tube at 45 °C for 30 min with E. coli in a phosphate buffer solution (pH 6.5) to achieve full deconjugation of the analytes. Then, anhydrous sodium sulfate, 1 mL of carbonate buffer solution (pH 10), and 3 mL of diethyl ether were added for steroid extraction. After 3 min of extraction on a vortex mixer, the samples were centrifuged at 3000 rpm for 10 min. For phase separation, glass tubes were put into a cryostat thermostated at −35 °C to freeze the aqueous layer. The diethyl ether layer was transferred into another flask and evaporated in a dry block heater at 60 °C under a nitrogen stream. A 100 μL aliquot of the derivatization reagent was added to the dry residue, and the tube was incubated at 60 °C for 40 min. After cooling the tube to room temperature, 0.4 mL of acetonitrile was added, and the solution was analyzed by GC-MS/MS.