HPLC and LC/MS/MS methods
Parameter | HPLC Method 1 | HPLC Method 2 | LC/MS/MS | |
|---|---|---|---|---|
| Apparatus | Agilent 1100 HPLC system with binary pump, auto sampler, column thermostat, and diode array detector. | Waters Alliance HT 2790 coupled to a Micromass Quattro Ultima II triple quadrupole mass spectrometer. | ||
| Solvent A | 0.02% HFBA in water (v/v) | 0.1% TFA in water (v/v) | 0.02% HFBA in 5:95 MeOH/water (v/v/v) | |
| Solvent B | 0.02% HFBA in 10:90 water/MeOH (v/v/v) | 0.1% TFA in 10:90 water/MeOH (v/v/v). | 0.02% HFBA in 10:90 water/MeOH (v/v/v) | |
| Column | Luna C18(2), 150×2 mm, 3 μm (30°C) (Phenomenex, Torrance, CA) | Vydac 218MS52 C18, 250×2.1 mm, 5 μm (30°C) (Vydac, Hesperia, CA) | XterraMS C18, 50×3 mm, 3.5 μm (50°C) (Waters, Milford, MA) | |
| Flow rate | 0.200 ml/min | 0.200 ml/min | 0.200 ml/min | |
| Gradient | Initial: 5% B | Initial: 0% B | Initial: 0% B | |
| Linear gradient: 0-5 min, 5 to 30% B | Linear gradient: 2-14 min, 0 to 25% B | Linear gradient: 0-4 min, 0 to 100% B | ||
| Isocratic: 5-15 min, 30% B | Wash: 15-16 min, 100% B | Wash: 4-4.9 min, 100% B | ||
| Wash: 16-17 min, 95% B | Equilibration: 17-30 min, 0% B | Equilibration: 5-10 min, 0% B | ||
| Equilibration: 18-35 min, 5% B | ||||
| Detection | UV at 214.5 nm | UV at 214.5 nm | Parent > daughter m/z, (collision energy): | |
| ZP123: 618.10 > 413.15 (15 eV) | ||||
| AAP10: 576.30 > 391.30 (20 eV) | ||||
| Capillary and Cone Voltage: 3,000 and 45 V | ||||
| Cone Gas (N2): 100 l/hr (100°C) | ||||
| Desolvation Gas (N2): 600 l/h (350°C) | ||||
| I.S.: 624.1 > 419.15 (15 eV) | ||||
|
|
|
|
Collision gas: Ar (1 × 10−3 mbar)
| |