Stable Isotope Dilution LC/ESI-SRM/MS Analysis for Highly Polar Bioactive Dipeptides Found in Fermented Brown Rice Product using a Porous Graphitic Carbon Column

In this study, a validated analytical method based on liquid chromatography/electrospray ionization-selected reaction monitoring/mass spectrometry (LC/ESI-SRM/MS) was developed for dipeptides in a functional food, fermented brown rice and rice bran with Aspergillus oryzae (FBRA). The aim of this study was to screen and quantify dipeptides in the water-soluble fraction of FBRA. There are few studies focusing on dipeptides in FBRA, even though FBRA is expected to contain bioactive peptides: FBRA or its aqueous extract has shown variety of biological activities. An anti-hypertensive dipeptide was found in similar rice bran products digested by thermolysin. Dipeptides are the smallest class of peptides, and many show a variety of biological activities. Some bioactive peptides are widely found in fermented foods. However, because dipeptides are generally too polar to be retained on versatile LC columns, such as octadecylsilyl columns, dipeptides have been often overlooked and not quantified because of the analytical difficulties. The LC was performed using a porous graphitic carbon column with the mobile phases of 0.1% formic acid/0.1% trifluoroacetic acid/1% tetrahydrofuran in water and acetonitrile. The stable isotope-labeled internal standards and SRM for dipeptides made it possible to develop a validated method. Five dipeptides were identified and quantified in FBRA in much higher concentrations than related materials (malted brown rice, rice bran, brown rice, and white rice) as follows: Ile-Arg, 82.1 µg/g; Ala-Phe, 27.8 µg/g; Ala-Tyr, 31.6 µg/g; Val-Phe, 46.3 µg/g; and Ile-Tyr, 49.9 µg/g. This method was simple and robust and would be applicable for other functional foods. Graphical Abstract