Table 3.
Hepatic Metabolic Stability Parameters
| reaction composition | |
|---|---|
| liver microsomes | 0.5 mg/mL |
| NADPH (cofactor) | 1 mM |
| potassium phosphate, pH 7.4 | 100 mM |
| magnesium chloride | 5 mM |
| analogue 14 | 1 μM |
| testosterone control | |||
|---|---|---|---|
| control compd | half-life (min) | CLint (mL/min/mg protein) | acceptable range (t1/2, min) |
| testosterone | 3.7 | 0.372 | ≤15 |
| analytical method | ||||
|---|---|---|---|---|
| liquid chromatography | ||||
| column | Thermo BDS Hypersil C18 30 × 2.1 mm, 3 μm, with guard column | |||
| M.P. buffer | 25 mM ammonium formate buffer, pH 3.5 | |||
| aqueous reservoir (A) | 90% water, 10% buffer | |||
| organic reservoir (B) | 90% acetonitrile, 10% buffer | |||
| flow rate | 350 μL/min | |||
| gradient program | time (min) | %A | %B | |
| 0.0 | 100 | 0 | ||
| 1.0 | 0 | 100 | ||
| 1.5 | 0 | 100 | ||
| 1.6 | 100 | 0 | ||
| 2.5 | 100 | 0 | ||
| total run time | 2.5 min | |||
| autosampler | 10 μL injection volume | |||
| autosampler wash | water/methanol/2-propanol: 1/1/1; with 0.2% formic acid | |||
| mass spectrometer | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| instrument | PE SCIEX API 4000 | ||||||||
| interface | turbo ionspray | ||||||||
| mode | multiple reaction monitoring | ||||||||
| method | 2.5 min duration | ||||||||
| settings | test article | ± | Q1 | Q3 | DP | EP | CE | CXP | IS |
| analogue 14 | + | 334.3 | 198.1 | 83 | 10 | 30 | 11 | 5500 | |
| TEM: 500 | CAD: 7 | CUR: 20 | GS1: 20 | GS2: 3 | |||||