1. The document identifies the primary metabolite of clonidine as hydroxyclonidine and develops an LC-MS method to quantify both compounds using a deuterated internal standard. 2. It characterizes the kinetic parameters of clonidine hydroxylation in human liver microsomes and screens 17 cytochrome P450 isoforms, finding five that hydroxylate clonidine with varying potency. 3. Selective inhibition experiments show that CYP2D6, CYP1A2, and CYP3A4/5 contribute most to clonidine hydroxylation in human liver microsomes.

4. Kinetic parameters of clonidine hydroxylation in pooled HLM 26 Analyzed Number of points Km > 0.0 Km Constraints 2.527 Sy.x 153.3 Absolute Sum of Squares 0.9735 R² 24 Degrees of Freedom Goodness of Fit 1.577 to 2.508 Km 40.03 to 44.77 Vmax 95% Confidence Intervals 0.2255 Km 1.147 Vmax Std. Error 2.043 Km 42.40 Vmax Best-fit values

5. Screen of 17 common cytochrome P450 isoforms (activity expressed in pmol/nmol P450/min )

6. Screen of 17 common cytochrome P450 isoforms (activity expressed in pmol/nmol P450/min )

8. Kinetic parameters of clonidine hydroxylation in rCYP2D6 26 Analyzed Number of points Km > 0.0 Km Constraints 0.9244 Sy.x 20.51 Absolute Sum of Squares 0.9881 R² 24 Degrees of Freedom Goodness of Fit 8.835 to 11.85 Km 25.00 to 27.15 Vmax 95% Confidence Intervals 0.7314 Km 0.5191 Vmax Std. Error 10.34 Km 26.07 Vmax Best-fit values