Determination of Choline-Containing Compounds in Rice Bran Fermented with Aspergillus oryzae Using Liquid Chromatography/Tandem Mass Spectrometry

Abstract

Choline-containing compounds are essential nutrients for human activity, as they are involved in many biological processes, including cell membrane organization, methyl group donation, neurotransmission, signal transduction, lipid transport, and metabolism. These compounds are normally obtained from food. Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is a fermented food product derived from rice and rice ingredients. FBRA exhibits a multitude of functional properties with respect to the health sciences. This study has a particular focus on choline-containing compounds. We first developed a simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis method for seven choline-containing compounds. The method was subsequently applied to FBRA and its ingredients. Hydrophilic interaction chromatography (HILIC) and selected reaction monitoring were employed for the simultaneous analysis of seven choline-containing compounds. MS ion source conditions were optimized in positive ion mode, and the product ions derived from the choline group were obtained through MS/MS optimization. Under optimized HILIC conditions, the peaks exhibited good shape without peak tailing. Calibration curves demonstrated high linearity across a 300- to 10,000-fold concentration range. The application of the method to FBRA and other ingredients revealed significant differences between food with and without fermentation. In particular, betaine and α-glycerophosphocholine were found to be highest in FBRA and brown rice malt, respectively. The results indicated that the fermentation processing of rice ingredients results in alterations to the choline-containing compounds present in foods. The developed HILIC/MS/MS method proved to be a valuable tool for elucidating the composition of choline-containing compounds in foods.

Keywords: HILIC/MS/MS; choline-containing compounds; fermented brown rice and rice bran with Aspergillus oryzae; simultaneous analysis.

Fig. 1. Chemical structure of analytes. (A) choline, (B) acetylcholine, (C) betaine, (D) phosphocholine, (E) glycerophosphocholine, (F) lysophosphatidylcholine (sn-1, 16:0), and (G) lysosphingomyelin (d18:1).

Fig. 2. Optimized SRM chromatograms of all compounds. SRM, selected reaction monitoring.

Fig. 3. Summary of fermentation effect on the content of choline-containing compounds in rice ingredients. FBRA, fermented brown rice and rice bran with Aspergillus oryzae.