Rapid ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and selected reaction monitoring strategy for the identification and quantification of minor spinacetin derivatives in spinach.

RATIONALE: Spinach is green leafy vegetable which is a rich source of flavonoids, phenolic acids, carotenoids, and vitamins A, C and E. It contains unique flavonoids which have significant anticarcinogenic, antiinflammatory and free radical scavenging activities. The present study reports the systematic identification and quantification of novel flavonoids by ultra-high-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UHPLC/HR-QTOFMS).
METHODS: An ultrasonication technique was used for the extraction of flavonoids from spinach. A rapid and reliable analytical method was established for the identification of flavonoids from methanolic extract. Flavonoids were characterized by their ultraviolet (UV) spectra, high-resolution accurate masses and MS/MS fragmentation pathways obtained using electrospray ionization (ESI). Furthermore, precursor ions from the intact molecule, and the resulting product ions, were monitored by selected reaction monitoring (SRM) with different collision energies in positive and negative ion mode.
RESULTS: For the first time, five minor spinacetin derivatives were identified under optimized SRM and broadband collision-induced dissociation (+bbCID) conditions. Fragmentation pathways were proposed for spectra obtained in ESI positive ion mode. The use of HR-QTOFMS and SRM allowed us to differentiate between molecules with the same nominal mass. The identified spinacetin derivatives were found to be acylated with ferulic and coumaric acids.
CONCLUSIONS: UHPLC interfaced with HR-QTOFMS in combination with SRM provides a rapid, reliable and highly sensitive method for the identification of flavonoids, and potentially other bioactive compounds, in a complex matrix.