*2.4. Incubation Procedure and CYP450 Activity Assay*

The effects of CTE, NGTS, CNP, and 18 representative compounds (A1–A18, Supplementary Materials) on CYP450 activities were investigated using a pool of SD RLM following the procedure in [11]. Incubations were conducted at 37 ± 1 ◦C in 200 µL of incubation mixtures containing RLM (0.2 mg/mL), phosphate buffer saline (PBS) (pH 7.4, 0.1 mM), MgCl<sup>2</sup> (5 mM), nicotinamide adenine dinucleotide phosphate (NADPH) (1 mM), and CYP450 probe substrates (90/1.07/18/0.13/0.02/3.6/90 µM of phenacetin/omeprazole/ tolbutamide/dextromethorphan/midazolam/chlorzoxazone/ bupropion, respectively). CYP450 inhibitors (triethylenethiophosphoramide/sulfaphenazole/ticlopidine/furafylline/

ketoconazole/quinidine/4-methylpyrazole for CYP2B6/2C9/2C19/1A2/3A4/2D6/2E1, respectively) were added as the positive control, and blank solvents (PBS containing methanol and/or dimethyl sulfoxide (DMSO)) were used as the negative control. The incubation mixtures also contained eight concentrations for CTE (2.5–200 µg/mL), NGTS (2.5–200 µg/mL), CNP (2.5–200 µg/mL), A1–A17 (5–200 µM), and A18 (0.25–10 µM). Reactions were initiated by adding the NADPH-generating system and terminated after 15 min by 200 µL of cold methanol containing 105 nM hydroxybupropion-[D6] (OHBUP-[D6]) and 5 nM 4 ′ -hydroxydiclofenac-[ <sup>13</sup>C6] (OHDIC-[ <sup>13</sup>C6]) as internal standards (ISs). The mixture was placed in an ice bath for 30 min, and the precipitated protein was removed by centrifugation (12,000 rpm for 10 min at 4 ◦C) three times. Then an aliquot of 100 µL supernatant was diluted with 100 µL ultrapure water, and centrifuged at 12,000 rpm for 10 min, before being subjected to LVDI-UHPLC-MS/MS analysis.
