3.2.4. Analysis of **2**, **2m**, **18, 19, 20, 21, 22** and **23** in Serum

Methods for the preparation of **2**, **2m**, **18**, **19**, **20** and **21** serum sample are delineated as follows. 50 μL of acetate buffer (pH 5.0), 50 μL of ascorbic acid (200 mg/mL), and 50 μL of 0.1 N HCl solution was added into 100 μL of serum which was collected at different time points). The resulting mixture was partitioned with 250 μL of ethyl acetate (containing 5.0 μg/mL of butyl paraben as the internal standard). After centrifuging at 10,000× *g* for 15 min, the upper organic layer was separated and dried under nitrogen gas to provide the crude sample, which was diluted with an appropriate volume of acetonitrile for LC-MS analysis.

The amount of **22** and **23** were analyzed by the serum samples before and after treatment with enzyme solution. The procedure for enzyme treatment is shown as follows. 100 μL of serum was treated with enzyme solution (50 μL; containing 1000 units/mL of sulfatase and 39861 units/mL of β-glucuronidase in pH 5.0 acetate buffer), 50 μL of 0.1 N HCl solution and 50 μL of ascorbic acid (200 mg/mL) in the light protected tubes at 37 ◦C. For optimal hydrolysis efficiency, the incubation time was decided as 2 h based on a previous study [35]. The mixture was then partitioned with 250 μL of ethyl acetate (containing 5.0 μg/mL of butyl paraben as the internal standard). After centrifuging at 10,000× *g* for 15 min, the upper organic layer was separated and dried under nitrogen gas to provide the crude sample, which was diluted with an appropriate volume of acetonitrile for LC analysis.
