Chromatographic analysis of metabolites was conducted on an Agilent 1290 infinity UPLC system (Agilent Technologies, Santa Clara, CA) equipped with an XDB-C18 column (2.1 × 100mm, 1.8μM, Agilent) following a previous report with minor modification (Zhao et al., 2017b (link)). The column temperature was maintained at 45 °C. The flow rate of mobile phase was 0.3mL/min with a gradient elution ranging from 2% to 98% acetonitrile containing 0.01% formic acid in 16 min run. The Q-TOFMS (Agilent 6500, Santa Clara, CA) was operated positive mode with electrospray ionization. Nitrogen was applied as cone gas and drying gas which was set at 350 °C. The nebulizer pressure was set at 35 psi, and capillary voltage was set at 3.5 kV. The MS/MS of pazopanib metabolites was performed in the targeted mode by collision energy of 20 eV. Relative quantitation of pazopanib metabolites was determined according to the abundance of peak area normalized by the internal standard. The quantitation of metabolites in urine, serum and feces was not precise without the standards which are not available.
Protocol detail
Metabolite Profiling via UPLC-MS/MS
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Acetonitrile
Capillary
Chromatographic analysis
Cone
Feces
Formic acid
Ms ms
Nebulizer
Nitrogen
Pazopanib
Pressure
Serum
Urine
Corresponding Organization : Chinese Academy of Sciences
Other organizations : University of Chinese Academy of Sciences, National Cancer Institute, National Institutes of Health, Center for Cancer Research
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Variable analysis
independent variables
- Chromatographic analysis of metabolites
- Gradient elution ranging from 2% to 98% acetonitrile containing 0.01% formic acid in 16 min run
- Positive mode with electrospray ionization
- Collision energy of 20 eV for MS/MS of pazopanib metabolites
- Relative quantitation of pazopanib metabolites determined according to the abundance of peak area normalized by the internal standard
- Quantitation of metabolites in urine, serum and feces
- Column temperature maintained at 45 °C
- Flow rate of mobile phase at 0.3mL/min
- Nitrogen applied as cone gas and drying gas set at 350 °C
- Nebulizer pressure set at 35 psi
- Capillary voltage set at 3.5 kV
- Internal standard used for relative quantitation of pazopanib metabolites
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