LC/MS/MS analytical methods
Catalytic Activity | P450-Selective Activity | IS | Sample Preparationa | Column and Flow Rate | Mobile Phases | Gradient Conditions | Ionization Mode | Ions Monitored (m/z) |
|---|---|---|---|---|---|---|---|---|
| R-95913→R-138727 | Not applicable | 2H4-R-138727 | A | Monochrome C18, 5 μm, 100 × 2.0 mm (Metachem); 0.2 ml/min | A: 5% methanol in 0.2% formic acid; B: 95% methanol in 0.2% formic acid | 6 min: escalation from 10% B to 95% B and back to 10% B | Positive electrospray | 498→348 for derivatized R-138727; 502→111 for derivatized IS |
| Phenacetin→acetaminophen | CYP1A2 | 2H4-acetaminophen | B | Monochrome C18, 5 μm, 50 × 2.0 mm (Metachem); 0.2 ml/min | A: 5% methanol in 5 mM ammonium acetate; B: 95% methanol in 5 mM ammonium acetate | 3.6 min: escalation from 10% B to 80% B and back to 10% B | Positive electrospray | 152→110 for acetaminophen; 155.95→114.05 for IS |
| S-Mephenytoin→4′-hydroxy S-mephenytoin | CYP2C19 | phenytoin | B | Monochrome C18, 5 μm, 50 × 2.0 mm (Metachem); 0.2 ml/min | A: 5% methanol in 5 mM ammonium acetate; B: 95% methanol in 5 mM ammonium acetate | 3.6 min: escalation from 10% B to 80% B and back to 10% B | Negative electrospray | 233→189.9 for 4′-hydroxy S-mephenytoin; 250.9→102.1 for IS |
| Midazolam→1′-hydroxy-midazolam | CYP3A | α-hydroxy-triazolam | C | YMC-AQ, C18, 5 μm, 100 × 2.0 mm (Waters); 0.22 ml/min | A: 5% methanol in 50 mM ammonium acetate; B: 95% methanol in 50 mM ammonium acetate | 6.0 min: escalation from 65% B to 95% B and back to 65% B | Positive electrospray | 342→324 for 1′-hydroxy-midazolam; 359→331 for IS |
↵ a Sample Preparation Methods: A, internal standard (IS) and 10 mM BMAP (derivatizing agent) added in an equal volume of acetonitrile (ACN) to stop reactions; samples centrifuged and 30-μl supernatant diluted with 170 μl of deionized water. B, IS added in an equal volume of ACN to stop reactions; samples centrifuged and 30-μl supernatant diluted with 170 μl of deionized water. C, equal volume of ACN added to stop reactions; IS (20 μl) added, samples centrifuged, and 30-μl supernatant diluted with 170 μl of deionized water.