*2.6. Characterization of Carboxylesterases Involved in the Formation of 4-Hydroxybenzoic Acid from Catalposide*

To identify the CES enzymes involved in hydrolysis of catalposide to 4-hydroxybenzoic acid, 100 µL reaction mixtures containing human liver S9 fractions; human intestinal microsomes; or human CES1b, CES1c, or CES2 enzymes (50 µg protein), and catalposide (200 or 400 µM) in 50 mM phosphate buffer (pH 7.4) were incubated at 37 ◦C for 30 min. Reactions were stopped by addition of 100 µL 4-methylumbelliferone (internal standard, 10 ng/mL) in methanol. After vortex-mixing and centrifugation, 50 µL of each supernatant was diluted with 50 µL of deionized water. Each mixture was transferred to an injection vial, and a 5 µL aliquot was injected into the LC-HRMS system.
