Optimized enzymatic digestion
To optimize the hydrolysis process for determination of conjugated metabolites, enzymatic digestion with glucuronidase and sulfatase was performed under different incubation conditions. The hydrolysis reaction was initiated by adding 50 µL of either enzyme into a 50 µL of plasma or urine sample using different enzyme concentrations (25, 50, 100, 200, and 400 units/mL for sulfatase and 200, 500, 1000, 2000 and 4000 units/mL for The standard calibration curves of each analyte for determining conjugated glucuronide and sulfate metabolites were carried out using the same optimized method as plasma and urine incubation. Briefly, working standard solutions were added into blank plasma and urine samples and then the process of incubation was performed before extraction with protein precipitation.