Table 2.
Optimized parameters of the established LC–MS/MS methodology
| LC parameters | MS/MS parameters | |||||
|---|---|---|---|---|---|---|
| HPLC | Agilent 1200 (Agilent Technologies, CA, USA) | Mass spectrometer | Agilent 6410 QqQ (Agilent Technologies, CA, USA) | |||
| Mobile phase (gradient) | Aqueous phase: 10 mM Ammonium formate in H2O (pH: 4.1 using Formic acid) | Ionization source | Positive electrospray ionization (ESI) | |||
| Organic phase: ACN (0.1% Formic acid) |
Drying gas: N2 gas Pressure (55 psi) Flow rate (12 L/min) |
|||||
| Flow rate: 0.2 mL/min | ||||||
| Elution time: 90 min | ||||||
| Agilent eclipse plus C18 Column | In vitro | In vivo | ESI temperature: 350 °C | |||
| Length (mm) | 50 | 150 | Capillary voltage: 4000 V | |||
| Internal diameter (mm) | 2.1 | 2.1 | Collision gas | N2 (high purity) | ||
| Particle size (μm) | 1.8 | 3.5 | Modes | Product ion (PI) and full mass scan and | ||
| Temperature: | 22 ± 2 °C | Software | Mass Hunter software | |||
| Elution system | Time (min) | %B (ACN) | Analyte | Vandetanib, in vivo, in vitro and reactive metabolites | ||
| 0 | 5 | |||||
| 60 | 25 | Mass conditions | Fragmentor voltage (V) | 140 | ||
| 80 | 70 | |||||
| 90 | 5 | |||||
| Post time 15 | 5 | Collision energy (eV) | 15 | |||