Table 2.
Metabolic pathways, substrates, metabolites and LC/MS-MS parameters for the quantification of the drug metabolizing enzyme activities of cryopreserved human enterocytes. Tolbutamide was used as an internal standard with the MRM (Mass Transition Monitoring) at m/z 271.2 to 91.3 and m/z 269.1 to 105.9 for positive mode and negative mode, respectively. The concentrations used for each substrate is as indicated. The total incubation time was 1 hour.
| Drug Metabolizing Enzymes | Substrate and Concentrations | Marker Metabolite Analyzed |
Ion Mode
Application |
Mass Transitions
Monitoring |
|---|---|---|---|---|
| CYP2C9 | Diclofenac (25 μM) | 4-Hydroxydiclofenac | Negative | m/z 309.8 to 265.9 |
| CYP2C19 | S-Mephenytoin (250 μM) | 4-Hydroxy-S-Mephenytoin | Positive | m/z 235.2 to 150.0 |
| CYP3A4-1 | Midazolam (20 μM) | 1’-Hydroxymidazolam | Positive | m/z 342.1 to 203.1 |
| CYP3A4-2 | Testosterone (200 μM) | 6β-Hydroxytestosterone | Positive | m/z 305.2 to 269.1 |
| CYP2J2 | Astemizole (50 μM) | O-Demethylastemizole | Positive | m/z 445.0 to 204.2 |
| UGT | 7-Hydroxycoumarin (100 μM) | 7-Hydroxycoumarin Glucuronide | Negative | m/z 336.9 to 160.9 |
| SULT | 7-Hydroxycoumarin (100 μM) | 7-Hydroxycoumarin Sulfate | Negative | m/z 240.9 to 161.0 |
| CES2 | Irinotecan (50 μM) | SN38 | Positive | m/z 393.0 to 349.3 |
• SULT: SULT activity was evaluated by quantifying the formation of 7-hydroxycoumarin sulfate from 7-hydroxycoumarin. The specific activity (pmole/min/106 cells) of MMHH was 13 ± 0.69 which was 179% of that of CCHE.
• CES-2: CES-2 activity was evaluated by quantifying the formation of SN38 from irinotecan. The specific activity (pmole/min/106 cells) of MMHH was 0.38 ± 0.27 which was similar (102%) to that of CCHE.