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. 2020 Nov 26;33:106584. doi: 10.1016/j.dib.2020.106584

HPLC-DAD-ESI-QTOF-MS/MS qualitative analysis data and HPLC-DAD quantification data of phenolic compounds of grains from five Australian sorghum genotypes

Yun Xiong a, Pangzhen Zhang a, Robyn Dorothy Warner a, Shuibao Shen b,c, Stuart Johnson d, Zhongxiang Fang Dr a,⁎
PMCID: PMC7724171  PMID: 33318974

Abstract

Sorghum (Sorghum bicolor) grain is a rich source of bioactive phenolic compounds and understanding the phenolic profile of different sorghum genotypes is an important step towards the selection of the most appropriate genotype for industrial applications. The free and bound phenolic compounds of sorghum bran and kernel fractions from five Australian-grown sorghum genotypes (1 white, 2 red, 1 brown and 1 black coloured grain) were identified/tentatively identified by HPLC-DAD-ESI-QTOF-MS/MS and quantified/semi-quantified by HPLC-DAD. Firstly, MS chromatograms of sorghum samples and standards and the MS/MS spectra of individual detected compounds and standards are presented. Then quantification data of these compounds is provided. This dataset is supplementary to the research paper “Comprehensive profiling of phenolic compounds by HPLC-DAD-ESI-QTOF-MS/MS to reveal their location and form of presence in different sorghum grain genotypes” [1].

Keywords: Sorghum, Phenolic compounds, HPLC-DAD-ESI-QTOF-MS/MS, Comprehensive profile, Quantification

Specifications Table

Subject Agricultural and Biological Sciences (General)
Specific subject area Mass spectrometry, phytochemistry
Type of data Table, Fig.
How data were acquired The mass spectrometry data was obtained from Agilent 6520I Accurate-Mass Q-TOF LC/MS coupled to an Agilent 1200 series HPLC system (Agilent Technologies, USA). The quantification data was obtained from Agilent 1260 series HPLC-DAD (Agilent Technologies, USA). A Synergi Hydro-RP 80A LC column (4 µm, 250 × 4.6 mm) protected by an AQ C18 guard column (4.0 × 3.0 mm) (Phenomenex, Australia) was used.
Data format Raw, analysed
Parameters for data collection MS: negative mode via a dual electrospray ionisation source (ESI), drying gas N2, temperature 325 °C, gas flow 9 L/min, nebuliser 45 psi; capillary voltage 3500 V, fragmentor 175 V, MS scan range 90-1000 m/z. MS/MS: auto mode, scan range 90-850 m/z, collision energy 15-30 eV.
Description of data collection The MS chromatograms of sorghum samples and standards, and the MS/MS spectra of phenolic compounds and standards were obtained by MassHunter Qualitative software (Agilent Technologies, USA). The quantification data was analysed by Agilent OpenLAB workstation (Agilent Technologies, USA).
Data source location Liberty, Mr-Buster, Nuseed Cracka sorghum grains were obtained from Nuseed Australia (Toowoomba, QLD, Australia) in 2019. IS131C and Shawaya Short Black 1 sorghum grains were obtained from the experiment filed of Bentley campus of Curtin University, grown January to April 2019 (Bentley, WA, Australia).
Data accessibility With the article
Related research article Xiong Y, Zhang P, Warner RD, Shen S, Johnson S, Fang Z. Comprehensive profiling of phenolic compounds by HPLC-DAD-ESI-QTOF-MS/MS to reveal their location and form of presence in different sorghum grain genotypes. Food Research International. 127 (2020) 109671. DOI: 10.1016/j.foodres.2020.109671
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Value of the Data

  • •

    The MS chromatogram and MS/MS spectra data can be used as a reference, and serve as a benchmark, for the identification of phenolic compounds in sorghum grains; the quantification data provide useful information for the evaluation and estimation of individual or group of phenolic contents in sorghum grain materials.

  • •

    The qualitative and quantitative data provide valuable information/reference to researchers from various sectors (agricultural, food and pharmaceutical) for the analysis and comparison of phenolic compounds in sorghum as well as in other cereal grains or plant materials.

  • •

    The data provide a comprehensive understanding of the sorghum phenolic profile, which provides useful insights into sorghum material selection and processing design to help tailor specific industrial food or drug applications of sorghum.

1. Data Description

This present dataset provides supplementary information to our work submitted to Reference [1]. The MS chromatograms of 20 sorghum samples (i.e. free and bound phenolic extracts of bran and kernel fractions from 5 sorghum grain genotypes) and a standard sample of 27 mixed phenolic standards are provided in Fig. 1. Data in Table 1 presents the 27 phenolic standards used for identification and their retention time, precursor ion and presence in the tested sorghum samples. The MS/MS spectrum and structure of 27 standards used are provided in Fig. 2. The MS/MS spectrum and structure of 114 identified or tentatively identified compounds in sorghum samples are provided in Fig. 3. Data in Table 2 were the calibration and method validation parameters for the quantification of phenolic compounds. Data in Table 3 presents the concentration of phenolic compounds and the standards used for their quantification or semi-quantification.

Table 3 (continued)

Concentration of quantified compounds (µg/g)
Class Name of Compound Peak No Standard used for quantification W-B-F 1 W-B-B 2 W-K-F 3 W-K-B 4 RM-B-F 5 RM-B-B 6 RM-K-F 7 RM-K-B 8 RC-B-F 9 RC-B-B 10 RC-K-F 11 RC-K-B 12 BR-B-F 13 BR-B-B 14 BR-K-F 15 BR-K-B 16 BL-B-F 17 BL-B-B 18 BL-K-F 19 BL-K-B 20
Flavanone (FN) Unknown flavonoid glycoside I (eriodictyol 7-O-neohesperidoside or eriodictyol type) 1 Naringenin 4.88±0.57 0 0 0 0.96±0.08 0 0 0 1.42±0.28 0 1.85±0.23 0 2.06±0.39 0 0 0 2.87±0.36 0 0 0
Eriodictyol 7-O-glucoside I 22 Naringenin 0 0 0 0 0 0 0 0 0 0 0 0 78.53±2.29 0 BDT 0 0 0 0 0
Eriodictyol 7-O-glucoside II 30 Naringenin 10.65±1.07 0 0 0 BDT 0 0 0 BDT 0 0 0 0 0 0 0 BDT 0 0 0
Eriodictyol 7-O-glucoside III 33 Naringenin 0 0 0 0 44.95±2.42 0 0 0 4.39±0.23 0 0 0 27.93±1.67 0 0 0 61.76±3.03 0 0 0
Naringenin 7-O-glucoside (prunin) I 47 Naringenin 0 0 0 0 1.15±1.81 0 0 0 BDT 0 0 0 25.11±1.08 0 0 0 64.77±3.58 0 0 0
Eriodictyol 7-O-glucoside IV 48 Naringenin 0 0 0 0 BDT 0 0 0 0 0 0 0 BDT 0 1.85±0.26 0 0 0 0 0
Naringenin 7-O-glucoside (prunin) II 50 Naringenin 0 0 0 0 163.70±2.95 0 0 0 45.58±1.06 0 0 0 269.53±4.96 0 0.17±0.38 0 17.73±3.55 0 0 0
Dihydrokaempferol 78 Naringenin 0 0 0 0 0 0 0 0 0 0 0 0 58.62±2.35 BDT 6.37±0.71 0 0 0 0 0
7-Hydroxyflavanone 7-O-beta-D-glucoside 81 Naringenin 0 6.54±0.47 0 BDT 0 BDT 0 0 0 3.84±0.52 0 0 0 9.23±0.24 0 0 0 8.50±0.87 0 0
3′,5,7-Trihydroxy-4′-methoxyflavanone (hesperetin) 83 Naringenin 0 0 0 0 BDT 0 0 0 13.91±1.23 0 BDT 0 21.33±3.25 0 0 0 27.10±0.38 0 0 0
Naringenin-7-O-glucoside (prunin) III 86 Naringenin 0 0 0 0 BDT 0 0 0 BDT 0 0 0 BDT 0 0 0 8.51±1.12 0 0 0
Eriodictyol 90 Naringenin 0 BDT 0 0 BDT 95.62±3.07 0 0 BDT 54.93±3.55 0 0 BDT 42.52±0.24 BDT 0 BDT 171.83±5.37 0 0
Unknown flavonoid (eriodictyol or flavanone type) 95 Naringenin 0 0 0 0 9.80±0.92 0 BDT 0 12.68±1.46 0 1.87±0.49 0 BDT BDT 0 0 BDT 0 0 0
Naringenin 107 Naringenin 0 0 0 0 16.64±0.93 75.39±2.71 BDT 0 BDT 119.49±6.71 BDT 0 19.99±1.84 41.49±2.96 BDT 0 BDT 106.46±2.03 0 0
5,7-Dimethoxyflavanone 108 Naringenin 0 0 0 0 0 0 0 0 0 0 0 0 0 1.89±0.22 0 0 0 BDT 0 0
Flavone (FO) Apigenin 7-O-apiosyl-glucoside I 40 Apigenin BDT 0 0 0 BDT 0 0 0 13.19±1.21 0 0 0 23.33±1.83 0 0 0 1.67±1.99 0 0 0
Apigenin 7-O-apiosyl-glucoside II 46 Apigenin 0 0 0 0 BDT 0 0 0 9.90±0.65 0 0 0 BDT 0 0 0 BDT 0 0 0
Luteolin 6-C-glucoside 8-C-arabinoside 54 Apigenin 7.07±1.61 0 0 0 0 0 0 0 BDT 0 0 0 BDT 0 0 0 BDT 0 0 0
5,4′,5′-Trihydroxy-3,6,7,8,2′-pentamethoxyflavone 60 Apigenin 6.79±0.61 0 0 0 0 0 0 0 BDT 0 0 0 0 0 0 0 0 0 0 0
Luteolin 4′-O-glucoside 61 Apigenin BDT 0 0 0 36.86±0.46 0 0 0 6.89±1.02 0 0 0 17.43±1.04 0 0 0 10.86±1.26 0 0 0
Luteolin 7-[6′'-(2-methylbutyryl)glucoside] I 94 Apigenin 0 10.15±0.49 0 0 0 20.63±2.07 0 0 0 21.23±1.81 0 0 0 BDT 0 0 0 11.98±0.37 0 0
Unknown flavonoid glycoside III (apimaysin or flavone type) 96 Apigenin 0 0 0 0 0 0 0 0 BDT 0 0 0 0 0 0 0 6.73±1.45 0 0 0
Luteolin 7-[6′'-(2-methylbutyryl)glucoside] II 97 Apigenin 0 15.87±1.33 0 0 0 46.26±2.92 0 0 0 47.45±0.88 0 0 0 7.88±1.02 0 0 0 20.06±0.89 0 0
Unknown flavonoid glycoside VI (apimaysin or flavone type) 105 Apigenin 0 0 0 0 0 0 0 0 BDT 0 0 0 0 0 0 0 7.02±0.53 118.90±3.58 0 0
Gardenin B 109 Apigenin 0 49.35±2.13 0 0 0 49.72±8.77 0 0 0 89.57±8.28 0 0 0 35.66±4.34 0 0 0 0.29±1.34 0 0
Apigenin 110 Apigenin 0 0 0 0 16.11±0.67 BDT 3.09±0.22 0 0.98±0.66 BDT 0 0 6.30±0.91 9.29±1.46 0 0 BDT BDT 0 0
Thymusin 6-isobutyrate 113 Apigenin 0 14.57±0.61 0 0 0 BDT 0 0 0 BDT 0 0 BDT 0 0 0 0 0 0 0
(continued on next page)
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Table 3(continued)

Concentration of quantified compounds (µg/g)
Class Name of Compound Peak No Standard used for quantification W-B-F 1 W-B-B 2 W-K-F 3 W-K-B 4 RM-B-F 5 RM-B-B 6 RM-K-F 7 RM-K-B 8 RC-B-F 9 RC-B-B 10 RC-K-F 11 RC-K-B 12 BR-B-F 13 BR-B-B 14 BR-K-F 15 BR-K-B 16 BL-B-F 17 BL-B-B 18 BL-K-F 19 BL-K-B 20
Flavonol (FOL) Quercetin 3,4′-O-di-beta-glucoside I 3 Quercetin 3-O-glucoside 61.07±0.97 0 0 0 23.24±1.54 0 0 0 37.81±1.52 0 6.72±1.28 0 27.57±1.89 0 5.04±0.30 0 42.26±1.97 0 6.25±0.33 0
Quercetin 3,4′-O-di-beta-glucoside II 4 Quercetin 3-O-glucoside 5.58±0.64 0 0 0 0 0 0 0 4.12±0.26 0 0 0 BDT 0 0 0 0 0 0 0
Taxifolin 3-glucopyranoside 11 Quercetin 3-O-glucoside 53.21±2.65 0 0 0 31.43±2.21 0 0 0 20.75±1.61 0 0 0 1993.21±14.55 0 160.42±6.88 0 78.71±2.98 0 0 0
Taxifolin I 51 Quercetin 3-O-glucoside 0 0 0 0 0 0 0 0 0 0 0 0 1479.26±16.05 74.63±12.29 219.99±8.64 16.78±3.08 BDT 0 0 0
3-Methylquercetin (isorhamnetin) I 52 Quercetin 3-O-glucoside 0 0 0 0 0 0 0 0 0 0 0 0 0 BDT 0 5.38±0.59 0 BDT 0 0
Quercetin 3‐O‐glucoside (isoquercitrin) 62 Quercetin 3-O-glucoside 0 0 0 0 0 0 0 0 0 0 0 0 48.09±3.15 0 BDT 0 0 0 0 0
Taxifolin II 70 Quercetin 3-O-glucoside BDT 0 0 0 BDT 0 0 0 BDT 0 5.59±0.83 0 BDT 0 0 0 BDT 0 0 0
Unknown flavonoid glycoside II (patuletin 7-galactoside or flavonol type) 84 Quercetin 3-O-glucoside 0 0 0 0 0 0 0 0 0 0 0 0 0 95.59±6.31 0 0 0 34.87±2.43 0 0
Quercetin 99 Quercetin BDT 0 0 0 0 0 0 0 0 0 0 0 BDT 84.63±10.36 0 0 BDT BDT 0 0
Kaempferol 100 Kaempferol 14.30±1.12 BDT 4.79±0.35 0 34.64±3.81 112.54±5.22 4.49±0.68 0 35.86±3.45 32.40±2.73 5.18±0.72 0 53.63±2.60 16.68±5.10 3.58±0.83 0 101.36±6.96 43.36±3.82 0 0
3-Methylquercetin (isorhamnetin) II 102 Quercetin 3-O-glucoside 0 0 0 0 0 0 0 0 BDT 0 0 0 BDT 0 0 0 31.53±3.39 BDT 0 0
Proanthocyanidin (P) Procyanidin B1 8 Procyanidin B1 0 0 0 0 BDT 0 0 0 0 0 0 0 1283.49±14.19 0 63.22±3.23 0 549.49±7.45 0 0 0
Procyanidin I 36 Procyanidin B1 0 0 0 0 0 0 0 0 0 0 0 0 162.78±11.25 0 BDT 0 BDT 0 0 0
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0 = not detected.

BDT = below the set UV-Vis detection threshold but confirmed by the mass spectrum.

UQ = unable to quantify due to large background noise/interference.

Peak number and 20 sorghum sample acronyms referring to Fig. 1.

W-B-F 1 = white colour Liberty sorghum, bran fraction, free form extract.

W-B-B 2 = white colour Liberty sorghum, bran fraction, bound form extract.

W-K-F 3 = white colour Liberty sorghum, kernel fraction, free form extract.

W-K-B 4 = white colour Liberty sorghum, kernel fraction, bound form extract.

RM-B-F 5 = red colour Mr-Buster sorghum, bran fraction, free form extract.

RM-B-B 6 = red colour Mr-Buster sorghum, bran fraction, bound form extract.

RM-K-F 7 = red colour Mr-Buster sorghum, kernel fraction, free form extract.

RM-K-B 8 = red colour Mr-Buster sorghum, kernel fraction, bound form extract.

RC-B-F 9 = red colour Nuseed Cracka sorghum, bran fraction, free form extract.

RC-B-B 10 = red colour Nuseed Cracka sorghum, bran fraction, bound form extract.

RC-K-F 11 = red colour Nuseed Cracka sorghum, kernel fraction, free form extract.

RC-K-B 12 = red colour Nuseed Cracka sorghum, kernel fraction, bound form extract.

BR-B-F 13 = brown colour IS131C sorghum, bran fraction, free form extract.

BR-B-B 14 = brown colour IS131C sorghum, bran fraction, bound form extract.

BR-K-F 15 = brown colour IS131C sorghum, kernel fraction, free form extract.

BR-K-B 16 = brown colour IS131C sorghum, kernel fraction, bound form extract.

BL-B-F 17 = black colour Shawaya Short Black 1 sorghum, bran fraction, free form extract.

BL-B-B 18 = black colour Shawaya Short Black 1 sorghum, bran fraction, bound form extract.

BL-K-F 19 = black colour Shawaya Short Black 1 sorghum, kernel fraction, free form extract.

BL-K-B 20 = black colour Shawaya Short Black 1 sorghum, kernel fraction, bound form extract.

Fig. 1.

Fig 1

Fig 1

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MS chromatograms of 20 sorghum samples (a-t) and mixed standards (u). Peak numbers 1-114 referring to Table 1 in Reference [1] and Fig. 3; peak numbers S1-27 referring to Fig. 2.

Table 1.

Authentic standards used for identification.

Peak number Standards Retention time (min) [M-H]- (m/z) Detected in sorghum samples
S1 Gallic acid 9.456 169.0121 ND
S2 Protocatechuic acid 14.669 153.0203 Yes
S3 Procyanidin B1 18.480 577.1430 Yes
S4 4-hydroxybenzoic acid 21.147 137.0248 Yes
S5 Catechin 21.680 289.0748 Yes
S6 Procyanidin B2 24.632 577.1456 ND
S7 Caffeic acid 25.987 179.0342 Yes
S8 Syringic acid 26.725 197.0441 ND
S9 Epicatechin 17.574 289.0716 ND
S10 p-coumaric acid 34.927 163.0385 Yes
S11 Epicatechin gallate 37.935 441.0833 ND
S12 trans-Ferulic acid 38.300 193.0485 Yes
S13 trans-Sinapic acid 39.072 223.0597 ND
S14 Luteolinidin 40.247 269.0443 Yes
S15 Quercetin 3-O-galactoside 41.326 463.0871 ND
S16 Quercetin 3-O-glucoronide 41.868 477.0672 ND
S17 Quercetin 3-O-glucoside 42.046 463.0890 Yes
S18 Apigeninidin 44.727 253.0497 Yes
S19 Quercetin 3-O-rhaminoside 45.994 447.0931 ND
S20 Kaempferol 3-O-glucoside 45.994 447.0931 ND
S21 7-Methoxyapigeninidin 49.340 267.0669 Yes
S22 Resveratrol 51.444 227.0720 ND
S23 Quercetin 56.240 301.0376 Yes
S24 Kaempferol 56.587 285.0421 Yes
S25 Naringenin 60.160 271.0628 Yes
S26 Apigenin 61.847 269.0477 Yes
S27 Luteolin 62.239 285.0429 ND
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Standard peak numbers S1-27 are shown in Fig. 1 (u).

ND = not detected

Fig. 2.

Fig 2

Fig 2

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The MS/MS spectrum and structure of 27 standards. Standard peak numbers S1-27 are shown in Fig. 1.

Fig. 3.

Fig 3

Fig 3

Fig 3

Fig 3

Fig 3

Fig 3

Fig 3

Fig 3

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The MS/MS spectrum and structure of 114 identified or tentatively identified compounds in sorghum samples. Compound peak numbers 1-114 are shown in Fig. 1.

Table 2.

HPLC-DAD method validation parameters for the quantification of phenolic compounds.

Repeatability
Peak number Standards Quantification λ (nm) Calibration curve R2 Linearity range (µg/mL) LOD (µg/mL) LOQ (µg/mL) Conc. (µg/mL) RSD% (n=3)
S2 Protocatechuic acid 280 y = 2.7382x + 0.8849 0.9997 0.4-100 0.28 0.84 50 3.84
25 4.03
12.5 1.78
S3 Procyanidin B1 280 y = 1.0122x - 0.0470 0.9986 0.7-40 0.37 1.12 40 3.45
20 3.21
10 1.49
S4 4-hydroxybenzoic acid 280 y = 2.6027x + 0.2256 0.9996 0.4-100 0.31 0.93 50 3.78
25 4.11
12.5 5.91
S5 Catechin 280 y = 1.1088x + 1.0751 0.9999 0.8-200 0.23 0.71 200 0.24
100 1.72
50 2.72
S7 Caffeic acid 320 y = 5.0965x - 0.3624 0.9998 0.4-100 0.31 0.93 50 4.53
25 3.45
12.5 2.88
S10 p-coumaric acid 320 y = 7.7790x + 0.2580 0.9999 0.4-100 0.19 0.57 50 4.04
25 3.57
12.5 2.19
S12 trans-Ferulic acid 320 y = 5.2207x + 2.5300 0.9999 0.8-200 0.27 0.82 200 0.06
100 2.75
50 1.79
S14 Luteolinidin 485 y = 2.9103x - 2.2840 0.9999 0.8-200 0.36 1.09 200 0.11
100 3.28
50 1.66
S17 Quercetin 3-O-glucoside 370 y = 1.5905x - 0.1083 0.9983 0.4-100 0.67 2.02 50 4.60
25 7.30
12.5 4.82
S18 Apigeninidin 485 y = 5.0106x +1.0940 0.9999 0.8-200 0.20 0.60 200 0.07
100 3.00
50 1.93
S21 7-Methoxyapigeninidin 485 y = 2.2525x - 0.2609 0.9999 0.4-100 0.12 0.35 50 4.58
25 3.52
12.5 4.79
S23 Quercetin 370 y = 0.6596x - 0.4096 0.9980 0.4-100 0.73 2.20 50 4.07
25 8.95
12.5 1.78
S24 Kaempferol 370 y = 1.2170x - 0.0813 0.9995 0.4-100 0.35 1.06 50 2.17
25 1.29
12.5 6.29
S25 Naringenin 280 y = 4.7151x + 0.1801 0.9999 0.4-100 0.10 0.32 50 3.96
25 3.71
12.5 2.06
S26 Apigenin 340 y = 4.8070x - 1.6658 0.9998 0.4-100 0.24 0.73 50 4.09
25 2.74
12.5 3.10
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Standard peak numbers S1-27 are shown in Fig. 1.

LOD = limits of detection; LOQ = limits of quantification; RSD = relative standard deviation.

Table 3.

Quantification of sorghum phenolic compounds by HPLC-DAD.

Concentration of quantified compounds (µg/g)
Class Name of Compound Peak No Standard used for quantification W-B-F 1 W-B-B 2 W-K-F 3 W-K-B 4 RM-B-F 5 RM-B-B 6 RM-K-F 7 RM-K-B 8 RC-B-F 9 RC-B-B 10 RC-K-F 11 RC-K-B 12 BR-B-F 13 BR-B-B 14 BR-K-F 15 BR-K-B 16 BL-B-F 17 BL-B-B 18 BL-K-F 19 BL-K-B 20
Phenolic acid
Hydroxybenzoic acid (B) Salicylic acid 5 4-Hydroxybenzoic acid 41.93±2.80 0 0 0 11.07±1.69 0 0 0 27.61±0.71 0 0 0 40.29±0.89 0 0 0 109.98±3.11 0 0 0
Protocatechuic Acid 6 Protocatechuic Acid 0 0 0 0 0 0 0 0 0 0 0 0 0 22.83±0.60 0 0 0 26.10±3.45 0 0
4-Hydroxybenzoic acid 14 4-Hydroxybenzoic acid 121.01±2.32 91.69±1.74 0 0 59.32±1.03 36.44±1.78 0 0 74.57±1.54 48.85±2.20 0 0 BDT 18.20±3.07 0 0 BDT 36.67±2.40 0 0
Benzoic acid 26 4-Hydroxybenzoic acid BDT UQ 0 0 BDT UQ 0 0 BDT UQ 0 0 BDT UQ 0 0 BDT 64.97±6.00 0 0
2,5-Dihydroxybenzoic acid 32 4-Hydroxybenzoic acid 0 0 0 0 0 BDT 0 0 0 BDT 0 0 0 14.03±3.02 0 BDT 0 24.12±3.30 0 0
Hydroxycinnamic acid (C) Caffeic acid 3-glucoside 16 Caffeic Acid BDT 0 0 0 3.82±0.19 0 0 0 4.31±0.95 0 0 0 0 0 0 0 0 0 0 0
2-O-Caffeoylglycerol 19 Caffeic Acid BDT 0 4.63±0.55 0 BDT 0 7.84±0.16 0 BDT 0 6.05±0.55 0 BDT 0 5.14±0.70 0 BDT 0 0 0
1-O-Caffeoylquinic acid 20 Caffeic Acid 0 0 0 0 BDT 0 0 0 0 0 0 0 22.99±0.91 0 0 0 9.84±2.98 0 0 0
1-O-Caffeoylglycerol I 23 Caffeic Acid BDT 0 10.21±2.29 0 BDT 0 BDT 0 0 0 BDT 0 0 0 BDT 0 0 0 0 0
Caffeic Acid 24 Caffeic Acid 10.68±1.06 UQ BDT 0 19.2±1.07 UQ 2.17±1.80 UQ 19.53±0.73 UQ 32.75±1.74 UQ 23.55±3.28 UQ 7.93±0.71 UQ BDT 78.12±1.87 13.24±0.23 0
1-O-Caffeoylglycerol II 25 Caffeic Acid 55.80±1.56 0 21.82±1.48 0 45.94±1.77 BDT 42.9±1.31 0 40.73±0.69 0 35.18±0.82 0 30.03±2.07 0 21.81±0.46 0 68.51±3.74 BDT 19.24±0.88 0
1-O-Coumaroyl-2-O-glucosylglycerol 28 p-Coumaric Acid 6.12±0.17 0 0 0 0 0 0 0 BDT 0 0 0 0 0 0 0 0 0 0 0
Dihydroferulic acid 4-O-glucuronide 29 trans-Ferulic Acid 1.68±0.46 0 0 0 4.64±0.29 0 0 0 BDT 0 0 0 17.41±0.94 0 0.17±0.23 0 15.56±0.51 0 0 0
N1,N4-Dicaffeoyl spermidine 31 Caffeic Acid BDT 0 0 0 BDT 0 BDT 0 BDT 0 0 0 BDT 0 4.85±0.24 0 0 0 0 0
2-O-Coumaroylglycerol 37 p-Coumaric Acid BDT 0 2.34±0.73 0 0 0 1.48±0.27 0 0 0 1.27±0.05 0 0 0 0 0 0 0 0 0
1-O-(4-Coumaroyl)-beta-D-glucose 38 p-Coumaric Acid 0 11.05±0.92 0 BDT 0 24.47±2.96 0 2.35±0.77 0 11.85±0.67 0 0 0 9.05±2.11 0 8.28±3.14 0 7.46±0.70 0 BDT
N1,N8-Dicaffeoyl spermidine 39 Caffeic Acid 49.14±0.88 0 BDT 0 73.37±1.71 0 20.62±1.07 0 46.61±1.31 0 20.09±1.53 0 45.89±4.66 0 18.91±0.70 0 36.64±2.78 0 8.40±1.24 0
1-O-Coumaroylglycerol 41 p-Coumaric Acid BDT BDT 6.41±0.71 0 BDT BDT BDT 0 BDT BDT BDT 0 BDT 0 BDT 0 BDT BDT 4.04±0.29 0
p-Coumaric Acid 43 p-Coumaric Acid 0 91.67±2.37 0 0 8.98±0.94 66.21±4.01 0 0 0 52.66±2.42 BDT 0 0 BDT 2.88±0.16 0 0 BDT 0 0
trans-Ferulic Acid 49 trans-Ferulic Acid 0 273.82±2.42 0 17.68±1.41 0 391.41±9.60 0 30.03±2.17 0 256.20±3.13 0 27.14±0.79 0 80.71±6.04 0 27.74±0.98 0 234.19±5.61 0 22.65±1.72
Caffeoylferuloylspermidine 53 Caffeic Acid BDT 0 0 0 22.89±0.87 0 0 0 BDT 0 0 0 0 0 0 0 0 0 0 0
Cinnamic acid 58 trans-Ferulic Acid 0 0 0 0 0 5.90±1.02 0 0 0 8.42±0.96 0 0 0 0 0 0 0 BDT 0 0
3-(6-hydroxy-7-methoxy-2H-1,3-benzodioxol-5-yl)prop-2-enoic acid 63 trans-Ferulic Acid 0 3.12±0.83 0 0 0 BDT 0 0 0 2.34±0.29 0 0 0 BDT 0 0 0 0 0 0
Linocinnamarin 68 trans-Ferulic Acid 0 20.02±1.11 0 0 0 15.06±4.80 0 0 0 9.17±1.08 0 0 0 BDT 0 0 0 BDT 0 0
N1,N10-Diferuloylspermidine 72 trans-Ferulic Acid 1.57±0.37 0 0 BDT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Isoferulic acid 79 trans-Ferulic Acid 0 13.96±1.44 0 BDT 0 28.17±3.10 0 0 0 20.35±1.30 0 0.28±0.17 0 6.47±1.11 0 BDT 0 7.95±1.34 0 BDT
8-4′-Dehydrodiferulic acid 98 trans-Ferulic Acid 0 43.62±1.25 0 0 0 BDT 0 0 0 25.55±0.58 0 BDT 0 20.27±0.34 0 0 0 61.82±1.32 0 0
4-Methoxycinnamic acid 101 trans-Ferulic Acid 0 19.32±0.53 0 0 0 7.90±1.29 0 0 0 BDT 0 0 0 BDT 0 0 0 BDT 0 0
Coumaroyl-caffeoylglycerol 104 p-Coumaric Acid 12.63±1.00 BDT 3.39±0.26 0 0.85±2.04 0 6.61±0.22 0 14.22±0.77 BDT 9.71±0.58 0 12.96±0.70 0 6.81±0.11 0 4.74±0.71 BDT 1.64±0.19 0
1,3-O-Dicoumaroylglycerol 111 p-Coumaric Acid 6.49±0.34 0 0 0 13.35±0.92 0 0 34.25±0.18 0 0 0 6.06±0.97 0 0 0 44.51±2.40 0 0 0
1,3-O-Coumaroyl-feruloylglycerol 112 p-Coumaric Acid 8.63±0.61 0 0 0 18.45±0.48 0 BDT 0 54.06±0.48 0 BDT 0 16.84±0.77 0 1.59±0.18 0 90.37±1.74 0 BDT 0
1,3-O-Diferuloylglycerol 114 trans-Ferulic Acid 1.42±0.23 0 0 0 3.80±0.93 0 0 0 14.93±0.40 0 BDT 0 4.93±0.66 0 0 0 31.09±0.89 0 0 0
Flavonoids
3-Deoxyanthocyanidin (3DA) Luteolinidin 56 Luteolinidin 18.18±0.20 0 0 0 104.37±2.83 54.29±2.19 8.26±0.44 0 61.68±2.35 36.92±1.86 9.45±0.61 0 37.57±1.62 13.62±0.97 9.89±0.52 0 171.59±4.43 76.35±1.27 7.99±0.31 0
Apigeninidin 73 Apigeninidin 0 0 0 0 242.08±7.39 30.35±2.24 3.84±0.45 0 244.77±2.45 52.85±2.73 2.05±0.41 0 372.51±5.41 BDT 0 0 38.88±4.38 22.37±1.29 0 0
5-Methoxy-luteolinidin 75 Luteolinidin BDT 0 0 0 25.22±4.06 0 6.67±0.70 0 18.61±1.58 BDT 8.15±0.66 0 48.92±1.90 0 0 0 28.35±3.21 0 5.23±0.18 0
7-Methoxy-apigeninidin 85 7-Methoxy-apigeninidin 0 0 0 0 53.02±1.52 BDT 8.25±0.87 0 37.57±1.34 0 3.87±0.74 0 0 0 0 0 BDT 0 0 0
Anthocyanidin (A) Cyanidin 59 Luteolinidin 0 BDT 0 0 0 0 0 0 0 0 0 0 0 192.71±24.06 0 56.01±4.12 0 236.57±6.86 0 0
Flavan-3-ol (F3OL) 3′-O-Methyl-(-)-epicatechin 7-O-glucuronide 7 Catechin 1.15±0.29 0 0 0 0 0 0 0 BDT 0 0 0 13.40±1.19 0 0 0 11.45±1.93 0 0 0
Catechin 15 Catechin 0 0 0 0 0 0 0 0 0 0 0 0 1210.70±24.39 0 106.37±4.24 0 116.11±2.73 0 0 0
Catechin I 45 Catechin 0 0 0 0 BDT 0 10.24±0.80 0 BDT 0 13.23±1.44 0 BDT 0 0 0 BDT 0 0 0
(-)-Epiafzelechin 65 Catechin 0 0 0 0 31.47±0.99 0 7.23±0.53 0 BDT 0 0 0 0 0 0 0 0 0 0 0
3′-O-Methyl-(-)-epicatechin 80 Catechin BDT 0 0 0 217.54±4.19 0 7.87±0.76 0 80.15±2.86 0 5.53±0.70 0 69.68±6.95 0 BDT 0 95.79±5.01 0 BDT 0
Flavonoid glycoside (catechin or catechin type) 103 Catechin 0 0 0 0 0 936.08±27.12 0 0 0 783.84±20.40 0 0 0 403.78±34.04 0 0 0 BDT 0 0
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2. Experimental Design, Materials and Methods

2.1. Chemicals and reagents

Standards of apigeninidin chloride, 7-methoxy-apigeninidin chloride and luteolinidin chloride were obtained from ChromaDex (Los Angeles, CA, USA). All other standards and chemicals were obtained from Sigma- Aldrich (Castle Hill, NSW, Australia). All chemicals used for the HPLC-DAD-ESI-QTOF-MS/MS and HPLC-DAD analyses were LC-MS grade.

2.2. Samples and preparation and phenolic extraction

Five different coloured sorghum grains were used. Liberty (White, W), Mr-Buster (Red, RM), Nuseed Cracka (Red, RC) sorghum grains were obtained from Nuseed Australia (Toowoomba, QLD, Australia) in 2019. IS131C (Brown, BR) and Shawaya Short Black 1 (Black, BL) sorghum grains were obtained from the experiment filed of Bentley campus of Curtin University, grown January to April 2019 (Bentley, WA, Australia). A TM05C SATAKE Testing Mill equipped with an #36 abrasive roller (SATAKE Corporation, Hiroshima, Japan) was used for grain decortication. Sorghum grains (200 g) were decorticated for 60 s to collect the bran fraction. The remaining grains were collected and further decorticated for 45 s to remove uncleared bran residues to give the kernel samples. Both bran and kernel fractions were ground by an EM0405 Multigrinder II grinder (Sunbeam, FL, USA), sieved 100% through a 500 µm brass sieve, and stored at –20 °C in vacuum bags in the dark before extraction.

The free and bound phenolic compounds were extracted according to previously published work [2]. For the extraction of free phenolic compounds, the ground sorghum sample (4 g) was mixed with 30 mL of 80% methanol solution under nitrogen gas, and the mixture was shaken at 25 °C and 150 rpm in the dark for 2 h. The mixture was centrifuged at 3500 g and 4 °C for 10 min to collect the supernatant, and the residue was re-extracted with 35 mL 80% methanol two more times. All supernatants were combined and evaporated to dryness by a rotary evaporator at 39–40 °C and 100 rpm for 10–15 min, and the resulting solid was re-dissolved in 20 mL of 100% methanol and stored under nitrogen gas at −20 °C in the dark for 1–3 day until analysis. For the extraction of free phenolic compounds, the residue remaining after the free phenolic extraction was mixed with 30 mL of 2 M HCl under nitrogen gas and heated at 100 °C for 60 min for hydrolysis. Then, 40 mL ethyl acetate was added and mixed thoroughly and wait for about 5 min for partition. After partitioning, the ethyl acetate fraction was collected, and the hydrolysate was re-extracted with 50 mL ethyl acetate five more times. All ethyl acetate fractions were pooled and evaporated to dryness by a rotary evaporator at 39–40 °C and 100 rpm for 10–15 min, and the resulting solid was re-dissolved in 20 mL of 100% methanol and stored under nitrogen gas at −20 °C in the dark for 1–3 day until analysis.

2.3. HPLC-DAD-ESI-QTOF-MS/MS qualitative analysis

The identification of phenolic compounds was performed by an Agilent 1200 series HPLC system, equipped with a vacuum degasser, auto-sampler, binary pump and diode-array detection (DAD), and coupled with an Agilent 6520I Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved on a reverse phase Synergi Hydro-RP 80A LC column (4 µm, 250 × 4.6 mm) protected by an AQ C18 guard column (4.0 × 3.0 mm) (Phenomenex, Lane Cove, NSW, Australia).

The HPLC-DAD-ESI-QTOF-MS/MS (and also the HPLC-DAD in Section 2.4) analysis was based on previously published work [3,4], with modifications and optimisation. The LC and MS conditions, mobile phases, and elution program were optimised for maximum peak separation and signal intensity and quality. The LC conditions: column temperature 30 °C, injection volume 10 μL. DAD settings: scan range 190–720 nm at 2.0 nm step, and monitoring wavelength at 280 nm for hydroxybenzoic acid, flavan-3-ol and flavanone, 320 nm for hydroxycinnamic acid, 340 nm for flavone, 370 nm for flavonol and 485 nm for 3-deoxyanthocyanidin. The mobile phase A was 1.0% formic acid in milli-Q water and mobile phase B was LC-MS grade acetonitrile. The flow rate was 0.650 mL/min, with an 80 min elution program was set as follows: 5% B (0 min), 5–8% B (5 min), 8–21% B (30 min), 21–35% B (19 min), 35–60% B (9 min), 60–100% B (4 min), 100% B (5 min), 100–5% B (0.1 min), 5% B (7.9 min). For MS analysis, negative mode via a dual electrospray ionisation source (ESI) was employed. The MS acquisition parameters are as follows: drying gas N2, temperature 325 °C, gas flow 9 L/min, nebuliser 45 psi; capillary voltage 3500 V, fragmentor 175 V; MS scan range 90–1000 m/z. The MS/MS was performed in auto mode with MS/MS scan range 90–850 m/z and collision energy 15–30 eV.

The data was analysed by MassHunter Qualitative software (Agilent Technologies, Santa Clara, CA, USA). The integration thresholds were set as peak area > 30000 counts for UV–Vis chromatogram and > 1 counts for MS chromatogram, and only the MS and UV-Vis matched peaks, i.e. peaks that are present in both MS and UV-Vis chromatograms with the peak area above the thresholds, were selected for further analysis. Compound identification and characterisation were based on comparing the retention time, UV-Vis, MS and MS/MS spectra with authentic standards, database, and published literature as follows:

  • (1)

    Standards: a total of 27 standards were used for identification, of which 15 matching compounds were identified in the tested sorghum samples, as shown in Table 1.

  • (2)

    Published literature: some compounds were identified by comparing their data profile with that reported in published literature (of sorghum studies), and these compounds were double checked by database for verification in the following step.

  • (3)

    Database: MS-DIAL 4.0 coupled with MS-FINDER 3.24 software using MSMS-Public-Neg-VS14 database was the main tool used for identification [5,6]. The settings were MS-DIAL score > 80 and MS-FINDER score > 7.5, and compounds/peaks below these scores were not selected for identification. Besides, the UV-Vis spectrum of each compound was used to assign it to a subclass according to its specific UV-Vis absorption/peak pattern [7], and compounds without matched subclass UV-Vis absorption/peak pattern were not selected for identification. Also, online UV–Vis (SpectraBase) and Mass (ChemSpider, Phenol-Explorer and MassBank) database were used for double verification when available.

  • (4)

    Mass error: only compounds with mass error ≤ ±10 ppm, and compounds with mass error > ±11 ppm but identified by standards or having a high MS-DIAL score > 90, were selected for identification and verification.

2.4. HPLC-DAD quantitative analysis

The quantification of phenolic compounds was performed by an Agilent 1260 series HPLC system equipped with a DAD (Agilent Technologies, Santa Clara, CA, USA), and the same column, mobile phase and conditions were applied as described above in Section 2.3. The data was intergraded by Agilent OpenLAB Workstation software (Agilent Technologies, Santa Clara, CA, USA), and the integration threshold was set as peak area > 1. Compounds with standards were directly quantified by the standards, and compounds without available standards were semi-quantified by selecting structurally similar standards or the standards of the same subclass based on their functional group and chemical structure (i.e. core structure and functional group), as shown in Table 3. Compounds without structurally matched standards were not quantified. The calibration curves of standards were created at their specific monitoring wavelengths as described above in Section 2.3, and compounds were quantified/semi-quantified at their selected monitoring wavelengths. The semi-quantification was performed on the basis of that phenolic compounds of the same subclass with similar core structure and functional group have similar UV-Vis absorption pattern/peaks at 200–600 nm [5], and this method has been used in many studies [8], [9], [10].

The quantification method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ) and precision (repeatability). Calibration curves were obtained at eight levels of concentration of standards, except for procyanidin (seven levels of concentration). Method linearity was tested on the basis of calibration curves, which were processed using linear regression. LOD and LOQ were calculated based on the standard deviation of the regression line (SD) and the slope (S) according to the formulae: LOD = 3.3(SD/S) and LOQ = 10(SD/S). Precision (repeatability) was evaluated by analysing three replicates (consecutive injections) of three different concentrations of standards according to Table 2, and the relative standard deviation (RSD) at each concentration of standard was calculated. All the calibration and method validation parameters for the quantification of phenolic compounds were presented in Table 2. The experiment was carried out in triplicate and data were expressed as mean ± standard deviation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Acknowledgments

This work was financially supported by Taiyuan Brand Will Firm Biotechnology Development Co., Ltd, China (project No. GL 022055 - TA 39201).

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