Sample preparation
Steroid extraction strategy should be developed, taking into
consideration nature of the matrix, cleaning and pre-concentration step,
and detection of conjugated steroid1,15,16. 240 µL
urine were first mixed with the internal standard mixture solution (2.4
uL) (see EXPERIMENTAL SECTION in the “Supplemental
information ”), mixed with 240 µL of freshly prepared 0.15 M sodium
acetate buffer (pH 4.6) containing 1.5 mg L -ascorbic acid and 4
µL of β-glucuronidase-arylsulfatase solution. After the reaction, the
solution was allowed to stand at 37 °C for 16 h, 960 µL of ACN was mixed
in a 2 mL Eppendorf tube (Eppendorf AG, Hamburg, Germany), vortexed for
30 s and allowed to stand at 4 °C for 30 min. The mixture was
centrifuged at 21,000 x g for 15 min at 4°C and the protein precipitate
was spun down. The supernatant was transferred to a 15 mL Eppendorf tube
and 8.16 mL of H2O was added to give a final
concentration of 10% ACN (v/v). The mixture was centrifuged at 19,000 x
g for 15 min at 4°C and the supernatant was loaded on a Bond Elute C18
column (50 mg) that had been washed with 1 mL of aqueous 80% ACN twice
and equilibrated with 1 mL of aqueous 10% ACN twice. Samples were
washed three times with 1 mL of 10% ACN and collected with 1 mL of 80%
ACN in 1.5 mL Eppendorf tubes. The eluate was evaporated to dryness
using a speed-vac and the resulting residue was redissolved in 24 µL of
40% MeOH. The mixture was centrifuged at 20,000 x g for 15 min at room
temperature and the supernatant was transferred to a 250 µL inactivated
glass insert. The insert was placed in a vial and subjected to LC-MS/MS.