Drug metabolite identification using ultrahigh-performance liquid chromatography-ultraviolet spectroscopy and parallelized scans on a tribrid Orbitrap mass spectrometer.

Abstract

To capture all metabolites in metabolite identification studies, MS/MS information is required in both positive and negative ionization mode, usually involving several sample injections to gain all information about samples. A high-resolution and high mass accuracy quadrupole/linear trap/Orbitrap tribrid instrument was used to gain this information in a novel single injection 'capture-all' approach to metabolite identification. Diclofenac, a model compound, was incubated in human and rat hepatocytes. These incubated samples were run using an ultrahigh-performance liquid chromatography/ultraviolet (UHPLC-UV) system coupled to a Thermo Fusion tribrid mass spectrometer. Five parallel scans were used: positive and negative ion full scan, data-dependent MS/MS, both high energy dissociation and collision-induced dissociation, and data-independent all ion fragmentation (AIF) spectra were collected in positive and negative ion mode. Nine metabolites were identified; a metabolite observed in the UV trace, but not positive ion full scan MS, was detected in the same sample injection by negative ion full scan MS. This was identified as a sulphate metabolite, and the corresponding negative ion AIF allowed for some structural elucidation. The use of a photo-diode array (PDA) detector allowed for spectral assessment in case of changes in absorbance spectra, and the subsequent semi-quantification of metabolites. This method provided good-quality MS/MS data across the m/z range in both positive and negative ion mode. The addition of both negative ion full scan MS and negative ion MS/MS allowed for the detection and structural elucidation of metabolites not observed in positive ion mode. The use of the PDA detector allowed for the semi-quantification of metabolites.