LC-MS/MS parameters for quantitation of the known microsomal P450 probe metabolites
Microsomal P450 activity was assessed by the conversion of a known specific probe substrate to metabolite: phenacetin → acetaminophen for CYP1A2, bupropion → OH-bupropion for CYP2B6, rosiglitazone → 5-OH-rosiglitazone for CYP2C8, diclofenac → 4′-OH-diclofenac for CYP2C9, S-mephenytoin → S-4-hydroxy mephenytoin for CYP2C19, dextromethorphan → dextrorphan for CYP2D6, midazolam → 1′-OH-midazolam for CYP3A4/5.
| Acetaminophen (CYP1A2) | OH-bupropion (CYP2B6) | 5-OH-rosiglitazone (CYP2C8) | 4′-OH-diclofenac (CYP2C9) | S-4-OH-mephenytoin (CYP2C19) | Dextrorphan (CYP2D6) | 1′-OH-midazolam (CYP3A4/5) | |
|---|---|---|---|---|---|---|---|
| Internal standard | Acetaminophen-d4 | OH-bupropion-d6 | 5-OH-rosiglitazone-d4 | N/Aa | N/Aa | N/Aa | 1’-OH-midazolam-d4 |
| MS mode | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
| Analyte m/z transition | 152.04 > 109.93 | 256.22 > 238.23 | 374.19 > 151.07 | 312.05 > 231.15 | 235.24 > 150.09 | 258.24 > 157.08 | 341.93 > 324.14 |
| Internal standard m/z transition | 156.1 > 114.0 | 262.25 > 244.27 | 378.24 > 155.06 | N/Aa | N/Aa | N/Aa | 346.17 > 328.13 |
| Capillary voltage (Kv) | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| Cone voltage (V) | 25 | 16 | 38 | 22 | 30 | 30 | 34 |
| Collision energy (Ev) | 15 | 12 | 24 | 18 | 18 | 35 | 20 |
| Desolvation temperature (°C) | 350 | 350 | 350 | 350 | 350 | 350 | 350 |
| Source temperature (°C) | 120 | 120 | 120 | 120 | 120 | 120 | 120 |
| Standard curve range (nM) | 20 ∼2000 | 5 ∼2000 | 2∼2000 | 100 ∼2000 | 50 ∼2000 | 2 ∼2000 | 5 ∼2000 |
| Retention time (min) | 2.9 | 8.1 | 6.8 | 4.2 | 3.7 | 6.9 | 9.3 |
| Mobile phase gradient: %B (min)b | 55 (0) → 100 (10) → 55 (10.1) → 100 (16) | ||||||