Table 2 Optimized parameters of the established LC–MS/MS methodology

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