UPLC-QToF/MS Determination of 14 Endogenous Steroids in Human Urine

The sample (5.000 0 g) was mixed with 2.0 mL of phosphate buffer solution (pH 6.8) and 150 μL of β-glucuronidase, and the mixture was cultured for 2 h at 55℃. After cooling to the room temperature, the solution was extracted ultrasonically twice, with 5 mL of ether for 5 min. The combined extract was dried by N₂-blowing, and the residue was dissolved with 5 mL of methanol (3+7) solution. The solution was purified on Oasis HLB solid phase extraction (SPE) column and the analytes were eluted from the column with 5 mL of a mixture of methanol-acetonitrile (1+1) solution. The purified solution was dried by N₂-blowing, and the residue was dissolved and diluted to 1 mL with a mixture of acetonitrile (95+5) solution, and the diluted solution was then separated by UPLC using HSS T3 column as stationary phase and a mixture of acetonitrile and 0.1% (φ) formic acid-5 mmol·L^-1 ammonium acetate solution in various ratios as mobile phase in gradient elution. ESI and MS^E were adopted in MS. Linear relationships between values of peak area and mass fraction of 14 endogenous steroids were found in definite ranges, with detection limits (3S/N) in the range of 0.5-5.0 μg·kg^-1. Test for recovery was made by standard addition at 3 concentration levels, giving results in the range of 80.6%-99.2%, with RSDs (n=6) ranged from 1.1% to 7.9%.