LC/MS-MS procedures for the identification and quantitation of metabolites after incubation of the substrates with various cDNA-expressed dog P450 proteins
Substrate | HPLC Column | Flow Rate | LC Gradienta | Metaboliteb Q1 → Q3 | Internal Standard Q1 → Q3 | |
|---|---|---|---|---|---|---|
| ml/min | m/z | |||||
| DZ, TMZ, NDZ | Extend-C18 3.5 μm, 4.6 × 50 mm | 1.5 | 30% B to 70% B 7.0 min linear | TMZ 270.9 → 140.0 NDZ 300.9 → 255.2 OX 292.0 → 246.2 | Lorazepam 320.9 → 275.2 | |
| Diclofenac | BDS Hypersil C8 5 μm, 2 × 50 mm | 1.5 | 10% B to 70% B 1.9 min linear | 4′-OH Diclofenac 312 → 231.1 | Flufenamic acid 282.1 → 264.1 | |
| (S)-Mephenytoin | Zorbax SB-Aq 5 μm, 4.6 × 50 mm | 1.5 | 10% B to 80% B 1.9 min linear | 4′-OH-Mephenytoin 235 → 150.1 | Phenytoin 253 → 182.2 | |
| Bufurarol | BDS Hypersil C8 5 μm, 2 × 50 mm | 1.5 | 0% B to 50% B 1.9 min linear | 1′-OH-Bufurarol 278.1 → 186.1 | DL-Propanolol 260.2 → 155.1 | |
| Dextromethorphan | XDB C8 5 μm, 2.1 × 50 mm Agilent | 1.5 | 0% B to 60% B 2.1 min linear | Dextrorphan 258.2 → 157.0 3-Methoxymorphinan 258.2 → 171.1 | Levallorphan 284.1 → 199.2 | |
|
Testosterone
|
SB-C8 5 μm, 4.6 × 75 mm
|
0.75
|
25% B to 60% B 7.0 min linear
|
2α-, 2β-, 6β-, 16α-, 7α-, or
16β-OH-Testosterone 305.5 → 269.0
|
Cortisone 361.0 → 163.2
| |
↵ a Solvents used for chromatography were A (10% methanol/90% water/ 0.05% formic acid, v/v) and B (90% acetonitrile/10% water/0.05% formic acid)
↵ b A PerkinElmer HPLC system, composed of a series 200 LC pump and autosampler and an API Triple Quadrupole LC/MS-MS Mass Spectrometer (Sciex API 2000) with atmospheric pressure chemical ionization ion source was used to perform the qualitative and quantitative analyses