β-Glucuronidase (G0251) and sulfatase (S9626 and S9754) activity analysis. A, Deconjugation of phenolphthalein β-d-glucuronide by β-glucuronidase. The deconjugation system included 1 mm phenolphthalein β-d-glucuronide, 40 U/ml β-glucuronidase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 30 min with shaking and terminated by adding 1 ml 1 m NaOH and tested by spectrophotometry at 540 nm. B, Deconjugation of p-nitrocatechol sulfate by sulfatase. The deconjugation system included 1 mm p-nitrocatechol sulfate, 40 U/ml sulfatase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 30 min with shaking and terminated by adding 1 ml 1 m NaOH and tested by spectrophotometry at 515 nm. C, Deconjugation of SaMT by sulfatase. The deconjugation system included standard SaMT, 40 U/ml sulfatase or β-glucuronidase, with or without 1 mm SAL, 200 mm sodium acetate buffer, and the total volume was 1 ml. The incubation proceeded at 37 C for 6 h with shaking and terminated by adding 2 ml ethyl acetate/tert-butyl methyl ether. The deconjugation mixture was extracted and 6-HMEL monitored by LC-MS/MS.