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.

Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is a functional food that contains several antioxidant compounds, including ferulic acid (Ogawa et al. 2017) and ergothioneine (Horie et al. 2020).In animal and cell experiments, FBRA or its aqueous extract has shown a variety of other biological functions; for example, improvement of fecal microbiota (Kataoka et al. 2007), inhibition of acute colitis (Kataoka et al. 2008), inhibition of acute hepatitis (Shibata et al. 2006), prevention of inflammation-related carcinogenesis (Onuma et al. 2015), inhibition of ACE (Nishibori et al. 2013), prevention of colon carcinogenesis (Katyama et al. 2002), and induction of apoptosis of acute lymphoblastic leukemia cells (Horie et al. 2016).Various research has suggested that some of these biological activities are derived from small peptides, such as dipeptides, generated during fermentation.X-Prolyl dipeptidyl-aminopeptidase was found from Aspergillus oryzae (Tachi et al. 1992).The anti-hypertensive peptides Leu-Arg-Ala and Tyr-Tyr were found in rice bran digested by thermolysin (Ogawa et al. 2019;Shobako et al. 2018).However, dipeptides in FBRA have been screened in only a few studies.To our knowledge, only one study has used LC/electrospray ionization (ESI)-mass spectrometry (MS), but critical identification by tandem mass spectrometry (MS/MS) and quantitation were not performed (Tanaka et al. 2017).Therefore, unique bioactive peptides in FBRA could have been overlooked.
In this study, we have screened dipeptides in the watersoluble fraction of FBRA.Five dipeptides (Ile-Arg, Ala-Phe, Ala-Tyr, Val-Phe, and Ile-Tyr) were identified by using LC/ESI-MS/MS and were confirmed by comparison with synthetic standards.A validated LC/ESI-selected reaction monitoring (SRM)/MS method using a porous graphitic carbon (PGC) Hypercarb™ column was developed for the five dipeptides without derivatization.The five dipeptides were then quantified by stable isotope dilution LC/ESI-SRM/ MS not only in FBRA, but also in related materials (malted brown rice, rice bran, brown rice, and white rice).

FBRA and Related Rice Materials
FBRA, malted brown rice, rice bran, brown rice, and white rice were obtained from Koken Co. Ltd (Hokkaido, Japan).All rice materials were prepared from local rice (Oryza sativa subsp.japonica, cultivar; Kirara 397) harvested in Hokkaido (Japan).White rice was obtained by polishing the brown rice.The rice bran containing germ and an aleurone layer was also obtained during polishing.FBRA was prepared by fermentation of steamed brown rice and steamed rice bran with Aspergillus oryzae at 38 °C for 32 h.The fermented product was then dried (Shibata et al. 2006).The brown rice for FBRA was slightly (1% of the total weight) polished to facilitate the fermentation (Horie et al. 2020).Malted brown rice was prepared by fermentation of steamed brown rice (slightly polished) with Aspergillus oryzae.All the samples were dried and finely powdered.

LC Conditions
An Agilent 1100 LC system (Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with a 1100 G1379A degasser, a 1100 G1312A binary pump, an 1100 G1367A autosampler, a 1100 G1314A UV-Vis detector, a 1100 G1315B photodiode array, and a 1100 G1316A column heater, was used for peptide identification together with an ion trap mass spectrometer (see following MS conditions).

MS Conditions for Quantification
A TSQ-Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an ESI source was used with the following conditions: Ionization mode, positive; heated capillary temperature, 350 °C; ion spray voltage, 3.5 kV; vaporizer temperature, 200 °C; sheath and auxiliary gas (N 2 ) pressures, 45 and 30 arb. units, respectively.The other conditions for SRM, such as CE, S-lens RF amplitude, and transition, were optimized by product ion scanning of each synthetic standard (Table 1) with infusion mode under a CID gas pressure of 1.5 mTorr.SRM was performed using scan width 1.0 Da and scan time 0.2 s.Data were processed using Xcalibur™ (version 2.2, Thermo Fisher Scientific Inc.).

Preparation of Standard Stock Solutions and QC Samples
Synthetic peptides are normally supplied as lyophilized products of the TFA salts and are not pure enough to prepare standard stock solutions.Therefore, the following calibrations were made to obtain accurate concentrations without the effects of moisture and TFA.For Ala-Tyr and Ile-Tyr, the peak areas were compared with an authentic Tyr standard using LC-UV at 275 nm.For Ile-Arg, Ala-Phe, and Val-Phe, the peak areas were compared with an authentic dansyl-Phe standard using LC-UV at 325 nm after derivatization with dansyl chloride.The roughly prepared stock solutions (ca. 100 µg/mL in water) had the following concentrations: Ile-Arg, 51.12 µg/mL; Ala-Phe, 54.78 µg/mL; Ala-Tyr, 66.30 µg/mL; Val-Phe, 58.39 µg/ mL; and Ile-Tyr, 50.90 µg/mL.The five stock solutions (each 50 µL) were mixed together to give the standard mixture solution (× 1) followed by serial dilutions (× 2.7, 4, 10, 20, 40, 100, and 200) to give seven calibration points.The HQC, MQC, and LQC samples were independently prepared from the five stock solutions followed by mixing together and serial dilutions of × 2.7, × 10, and × 40, respectively.Each internal standard (IS) solution was prepared in water using the corresponding stable isotopelabeled dipeptide as follows: Ile-[ 13 C 6 , 15 N 4 ]Arg, 11.90 µg/ mL; Ala-[ 13 C 9 , 15 N 1 ]Phe, 9.85 µg/mL; Ala-[ 13 C 9 , 15 N 1 ]Tyr, 10.49 µg/mL; Val-[ 13 C 9 , 15 N 1 ]Phe, 10.97 µg/mL; and Ile-[ 13 C 9 , 15 N 1 ]Tyr, 12.18 µg/mL.The five stock solutions (each 25 µL) were mixed together followed by dilution with water (500 µL) to give the IS mixture solution.

Preparation of Calibration and QC Samples
Each standard mixture solution for calibration or QC (20 µL) and IS mixture solution (20 µL) were mixed, and an aliquot of the solution (10 μL) was injected into the LC/ESI-SRM/MS system.

Sample Preparation and Quantification of the Water-soluble Fraction from FBRA
The water-soluble fractions from FBRA were obtained by using the extraction conditions reported by Horie et al. (2016) with some modification.Each sample (dried fine powder, 100 was accurately weighed, and dispersed in water (1 mL).Therefore, the relationship between the concentration of each peptide in water-soluble fraction (µg/  Day 1 y = 0.0124x-0.0029(R 2 = 0.9976) Day 2 y = 0.0122x + 0.0006 (R 2 = 0.9992) Day 3 y = 0.0121x + 0.0002 (R 2 = 0.9994) mL) and the amount of each peptide in powder (µg/g) is as follows: x µg/mL (extract from 100 mg powder) = 10 × µg/g (powder).The solution was then heated at 40 °C for 30 min and ultrasonicated at room temperature for 30 min, and this cycle was repeated three times.The solution was centrifuged at 15,000 × g at 4 °C for 15 min, the supernatant was transferred to another tube and recentrifuged at 15,000 × g at 4 °C for 15 min.The recentrifuged supernatant was filtered through a 0.45 µm PTFE syringe filter and stored at − 85 °C until the analysis.Water-soluble fractions of malted brown rice, rice bran, brown rice, and white rice were also prepared in the same manner as those of FBRA, except the second centrifugation and filtration were omitted for malted brown rice, brown rice, and white rice.The filtrate was diluted five times with water.The diluted filtrate (20 µL) and IS mixture solution (20 μL) were mixed, and an aliquot of the solution (10 μL) was injected into the LC/ESI-SRM/ MS system.

Screening and Identification of Dipeptides in FBRA
Five dipeptides were identified from the water-soluble fraction by our preliminary experiment using data-dependent LC/ESI-MS/MS with ion trap MS.The dipeptides were screened by manual monitoring with 19 y 1 -ions derived from the second amino acids.All the candidate dipeptides were confirmed by comparing the retention time and MS/MS results with synthetic standards (Fig. S1).Isomeric pairs of dipeptides (Ile-Arg vs Leu-Arg and Ile-Tyr vs Leu-Tyr) were identified further by co-injection of the authentic standard and the water-soluble fraction of FBRA (Fig. S2).For Ile-Tyr and Leu-Tyr (Fig. S2B), dansylation followed by LC separation with an ODS column was used to improve the LC resolution.

Optimization of SRM Conditions
Because protonated molecules ([M + H] + ) appeared as the most intense peaks from the five peptides and the corresponding stable-isotope-labeled dipeptides (ISs), product ion scans were performed using [M + H] + as the precursor ions (Q1).Consequently, the y 1 -ions (derived from the second amino acids) were observed clearly from all the dipeptides as relatively intense peaks (Fig. 1).Therefore, the y 1 -ions were selected as the Q3 settings for sensitive SRM.Each corresponding IS showed a 10 Da shift for both the Q1 and Q3 settings because the second amino acid (y 1 -ion source) was labeled with stable isotopes of 13 C and 15 N. To increase the intensity of the y 1 -ions, the CE and S-lens were optimized further for SRM (Table 1).

Optimization of LC Conditions
Dipeptides are generally too polar to be retained on the versatile reversed-phase ODS column.Therefore, we examined hydrophilic interaction chromatography (HILIC), multimode (ODS + strong anion and strong cation exchange), and PGC LC columns without derivatization.Among of these, the PGC column was chosen because of the separation between isomeric peptides (Ile-Arg vs Leu-Arg and Ile-Tyr vs Leu-Tyr) and the lower ion suppression from co-eluting substances.PGC columns have been used for highly polar compounds, such as dipeptides (Nèmeth-Kiss et al. 1997).
In addition, Piovesana et al. reported that using THF as an
Quantification of Five Dipeptides in FBRA and Related Materials (Malted Brown Rice, Brown Rice, Rice Bran, and White Rice) The five dipeptides were quantified in water-soluble fractions of FBRA, malted brown rice, brown rice, rice bran, and white rice (Table 3).Figure 4 shows a typical LC/ESI-SRM/ MS chromatogram of the water-soluble fraction of FBRA.No interfering peaks were observed.The dipeptides were detected in both FBRA and malted brown rice in higher amounts (> 10 µg/g powder), and the amounts were 1.8-5.1 times higher in FBRA.In the water-soluble fractions of brown rice, rice bran, and white rice, the dipeptides were not detected or were under LQC.Therefore, these dipeptides were generated during the fermentation by Aspergillus oryzae.The biological activities of the five dipeptides in FBRA were found in the BIOPEP-UWM database (Iwaniak et al. 2016;Minkiewicz et al. 2019) and related articles (Cheung et al. 1980;Matsumura et al. 1993   and Ile-Tyr, 3.7 µmol/L.Peptides with lower IC 50 values were found in higher concentrations in FBRA (Val-Phe, 46.3 µg/g and Ile-Tyr, 49.9 µg/g), suggesting that these peptides in the water-soluble fraction of FBRA could inhibit ACE (Nishibori et al. 2013).In addition, copper complex of Ala-Phe was reported as a candidate drug against breast cancer (Facchin et al. 2016) and Ala-Tyr was reported as an antioxidant (Tkaczewska et al. 2019).

Conclusion
A validated LC/ESI-SRM/MS method was developed for five dipeptides found in the water-soluble fraction of FBRA.A PGC Hypercarb™ column made it possible to analyze polar dipeptides without derivatization.The mobile phases of 0.1% FA/0.1% TFA/1% THF in H 2 O and MeCN further improved peak shape and reproducibility.This method was simple and robust and would be applicable for other polar peptides overlooked in fermented foods.

Table 1
SRM conditions for five dipeptides and the corresponding stable isotope-labeled ISs

Table 3
Amounts of dipeptides in water-soluble fraction of FBRA, malted brown rice, rice bran, brawn rice, and white rice *The experiments (n = 3) were performed using 100 mg powder samples, and calculated to shown as μg/g powder