Determination of Bioactive Betaines in <i>Aspergillus oryzae</i>-Fermented Rice Bran Foodstuff by HILIC/ESI-MS/MS

Various betaines (amino acid derivatives possessing a quaternary ammonium group) are present in various foods and have significant roles in health maintenance and enhancement for humans. Although rice bran (RB) fermented with Aspergillus oryzae (A. oryzae) is expected to be a foodstuff rich in the bioactive betaines, its betaine contents have not been elucidated. To address this concern, a hydrophilic interaction liquid chromatography (HILIC)/electrospray ionization-tandem mass spectrometry method was developed and validated for the determination of the five bioactive betaines, i.e., γ-butyrobetaine, L-carnitine, L-stachydrine, L-ergothioneine and L-hercynine, in the A. oryzae-fermented RB products. These betaines were satisfactorily retained and separated on an InertSustain Amide column with an acetonitrile-rich mobile phase. A solvent extraction-based pretreatment, HILIC separation and internal standard calibrations provided the precise (relative standard deviations: ≤ 5.6 %) and accurate (analytical recovery rates: 97.1–100.6 %) measured values. No significant matrix effect was observed. By using this method, we demonstrated that the A. oryzae-fermentation significantly increased the contents of all five betaines in the RB. The A. oryzae-fermented RB products had compatible or greater betaine contents compared to the plant-based foods that have been reported to be rich in betaines.