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. 2023 Feb 9;8(7):6253–6267. doi: 10.1021/acsomega.2c05514

Qualitative and Quantitative Analyses of the Chemical Components of Peels from Different Pomelo Cultivars (Citrus grandis [L.] Osbeck) Based on Gas Chromatography–Mass Spectrometry, Ultraperformance Liquid Chromatography-Q-Exactive Orbitrap-MS, and High-Performance Liquid Chromatography-Photodiode Array Detection

Boqing Su 1, Jingyuan Tian 1, Kanghui Wang 1, Wanling Yang 1, Jinrong Ning 1, Yiyao Liang 1, Yujie Liu 1, Yongmei Li 1,*, Guodong Zheng 1,*
PMCID: PMC9948162  PMID: 36844509

Abstract

graphic file with name ao2c05514_0008.jpg

The volatile and nonvolatile phytochemicals in peels of 5 major pomelo cultivars (including Citrus grandis cv. Yuhuanyou, C. grandis cv. Liangpingyou, C. grandis cv. Guanximiyou, C. grandis cv. Duweiwendanyou, and C. grandis cv. Shatianyou) from 11 places in China were characterized. First, 194 volatile compounds in pomelo peels were identified by gas chromatography–mass spectrometry (GC–MS). Of these, 20 major volatile compounds were subjected to cluster analysis. The heatmap indicated that the volatile compounds in peels of C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou were different from those in other varieties, while there was no difference among C. grandis cv. Guanximiyou, C. grandis cv. Yuhuanyou, and C. grandis cv. Duweiwendanyou from different origins. Second, 53 nonvolatile compounds were identified in pomelo peels by ultraperformance liquid chromatography-Q-exactive orbitrap tandem MS (UPLC-Q-exactive orbitrap-MS), of which 11 components were detected for the first time. Third, six major nonvolatile compounds were quantitatively analyzed with high-performance LC-photodiode array detection (HPLC-PDA). Combining the results of HPLC-PDA and the heatmap, 6 nonvolatile compounds in 12 batches of pomelo peel were well separated among varieties. Comprehensive analysis and identification of chemical components in pomelo peels are of great significance for their further development and utilization.

1. Introduction

Citrus grandis (L.) Osbeck, also known as pomelo, is distributed worldwide, including Asia and parts of Africa and Australia. China has abundant sources of pomelo,1 and there are some famous varieties, including Citrus grandis cv. Yuhuanyou, Citrus grandis cv. Liangpingyou, Citrus grandis cv. Guanximiyou, Citrus grandis cv. Duweiwendanyou, and Citrus grandis cv. Shatianyou (Figure 1). Pomelo comprises two parts, i.e., peel and pulp, which can be easily separated from each other. Pomelo pulps are highly consumed as fresh products, while pomelo peels are often discarded as waste. However, studies have reported the anticancer,2 anti-inflammatory,3 anti-oxidant,4 and hypoglycemic activities of pomelo peels,5 which were related to essential oils, coumarins (such as auraptene,2 bergamottin,6 and d-limonene7), and flavonoids (such as naringin8 and rhoifolin3). Therefore, pomelo peel can be used as a source of functional and nutritional compounds, which is not only economic but also has health-promoting effects.9

Figure 1.

Figure 1

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Appearances of pomelo fruits and pomelo peels. (A) Citrus grandis cv. Shatianyou, (B) Citrus grandis cv. Guanximiyou, (C) Citrus grandis cv. Yuhuanyou, (D) Citrus grandis cv. Duweiwendanyou, and (E) Citrus grandis cv. Liangpingyou.

The qualitative and quantitative analyses of the compounds of citrus fruits, such as Citrus limon L.10 and Citrus reticulata L.,11 have received increasing attention. Until now, studies have reported variations in the types and contents of bioactive compounds among different citrus varieties.12 Di Rauso Simeone et al. showed that the contents of major compounds in four C. limon cultivars varied, depending on varieties and sampling time.13 Moreover, there were also studies on the phytochemicals in the peels of oranges and mandarins.14 As a common citrus variety, pomelo has been applied for its beneficiary use in the modern medical era. Thus, it is significant to identify the compounds in pomelo peels with potential therapeutic activity. This information will advance the utilization of pomelo peels in functional and pharmaceutical industries.

Gas chromatography–mass spectrometry (GC–MS) is considered a common and reliable analytical platform for volatile compounds due to its superior selectivity, separation capability, and reproducibility.15 Therefore, the chemical profile of the volatile constituents of pomelo peel samples from five cultivars was determined with GC–MS. Subsequently, the nonvolatile components of pomelo peel samples were identified with the sensitive and reliable ultraperformance liquid chromatography-Q-exactive orbitrap tandem MS (UPLC-Q-exactive orbitrap-MS) method. Furthermore, some important nonvolatile compounds in the peels of five pomelo cultivars were quantified with a novel, rapid, and sensitive high-performance LC-photodiode array detection (HPLC-PDA) method, including naringin, rhoifolin, meranzin hydrate, isomeranzin, auraptene, and bergamottin. The variations of volatile and nonvolatile compounds in the peels of different pomelo cultivars have been analyzed. This study may provide a reference for further research on pomelo peel from different varieties.

2. Results and Discussion

2.1. GC–MS of Volatile Compounds in the Peels of Five Pomelo Cultivars

The qualitative and quantitative analyses of the volatile components in pomelo peels were performed with GC–MS. The representative total ion chromatogram of the volatile compounds in pomelo peel was displayed (Figure 2). As exhibited in Table 1, the extraction rate of volatile oil was calculated with the weight of the extracted volatile oil and the weight of samples. As shown in Table 1, the extraction rate of various volatile compounds was different in the peels of five pomelo varieties. The extraction rate of volatile oils was highest (3.27%) in C. grandis cv. Liangpingyou and lowest (0.27–0.35%) in C. grandis cv. Guanximiyou.

Figure 2.

Figure 2

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Representative total ion chromatograms of volatile compounds of pomelo peels for (A) Citrus grandis cv. Shatianyou, (B) Citrus grandis cv. Guanximiyou, (C) Citrus grandis cv. Yuhuanyou, (D) Citrus grandis cv. Duweiwendanyou, and (E) Citrus grandis cv. Liangpingyou.

Table 1. Information of Pomelo Peel Samples from Five Cultivars.

no. cultivars sample source collecting time extraction rate (%) rate (%)
S1 C. grandis cv. Shatianyou Bingcun, Meixian District, Meizhou City, Guangdong Province 9/9/2021 1.92
S2 C. grandis cv. Shatianyou Rongxian District, Yulin City, Guangxi Province 7/9/2021 2.35
S3 C. grandis cv. Shatianyou Songkou, Meixian District, Meizhou City, Guangdong Province 6/9/2021 1.34
MH1 C. grandis cv. Guanximiyou Meizhou City, Guangdong Province 6/9/2021 0.27
MH2 C. grandis cv. Guanximiyou Da Xi, Pinghe District, Zhangzhou City, Fujian Province 7/9/2021 0.34
MB1 C. grandis cv. Guanximiyou Bingcun, Meixian District, Meizhou City, Guangdong Province 6/9/2021 0.35
MB2 C. grandis cv. Guanximiyou Daxi, Pinghe District, Zhangzhou City, Fujian Province 9/9/2021 0.28
Y1 C. grandis cv. Yuhuanyou Qinggang District, Yuhuan City, Zhejiang Province 8/9/2021 0.40
Y2 C. grandis cv. Yuhuanyou Taishan Village, Qinggang District, Yuhuan City, Zhejiang Province 9/9/2021 0.42
D1 C. grandis cv. Duweiwendanyou Licheng District, Putian City, Fujian Province 13/9/2021 1.69
D2 C. grandis cv. Duweiwendanyou Xianyou District, Putian City, Fujian Province 10/9/2021 1.72
L1 C. grandis cv. Liangpingyou Liangping District, Chongqing City 9/9/2021 3.27
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In this study, a total of 194 volatile compounds were observed in the peels of 5 pomelo cultivars, including 1 alkane, 100 alkenes, 10 ketones, 5 phenols, 52 alcohols, 7 aldehydes, 6 carboxylic acids, and 13 other compounds. Among them, 20 volatile compounds with high levels were considered the major species and analyzed using GC–MS (Table 2). Results indicated that the volatile oils in pomelo peels contained abundant d-limonene (relative content: 58.79–96.54%), which exhibited anti-inflammatory and anti-oxidative activities.16

Table 2. Summary of 20 Major Volatile Compounds in 5 Cultivars Identified by GC–MS.

no. Compound RT (min) S1 S2 S3 MH1 MH2 MB1 MB2 Y1 Y2 D1 D2 L1
Alkene
3 (1R)-α-pinene 7.22 0.39 0.44 0.34 0.21 0.26 0.25 0.33 0.31 0.27 0.45 0.41 0.45
7 β-phellandrene 8.99     0.09       0.24 0.46     0.53  
8 Sabenene 9.01 0.05 0.09   0.12 0.16 0.17     0.22 0.52   0.19
9 (−)-β-pinene 9.24 1.6 0.12 2.02 20.7 22.46 23.95 25.12 27.39 22.41 32.73 30.97 1.1
11 Myrcene 9.9   1.79     0.01 0.01 0.11     0.01 0.01 1.63
22 d-limonene 12.5 96.46 96.39 96.54 66.38 71.35 69.43 58.79 61.21 69.48 61.14 62.36 95.25
27 (Z)-ocimene 13.67 0.02 0.18 0.24 0.32 0.67 0.63 2.11 1.63 0.61 0.62 0.81 0.39
94 2-butyloctanol 39.27 0.02 0.04 0.02 0.06 0.19 0.14 0.14 0.11 0.19 0.1 0.13 0.01
108 caryophyllene 42.48 0.09 0.12 0.06 0.26 0.12 0.19 0.19 0.08 0.14 0.07 0.11  
115 1H-cubebene 44.52 0.02 0.04 0.02 1.46 0.89 1.05 0.87 0.42 1.34 0.41 0.47 0.22
153 cembrene 54.53       0.62 0.12 0.21 0.52 0.21 0.23 0.12 0.48  
Alcohol
40 linalool 17.98 0.02 0.13 0.11 0.74 0.39       0.56 0.24 0.21 0.1
62 1,5,7-octatrien-3-ol,2,6-dimethyl- 23.44       0.07 0.08 0.29 0.72 0.58 0.13   0.01  
65 4-terpinenol 26.02 0.03 0.03 0.02 0.08 0.06 0.07 0.23 0.13 0.07   0.1 0.04
69 (±)-α-terpineol 28.18 0.06 0.11 0.08 0.3 0.16 0.13 0.37 0.25 0.01 0.15 0.12 0.09
80 (Z)-nerol 32.53     0.02 0.59 0.09 0.07 0.65 0.37 0.47 0.09 0.04  
Ester
38 linalyl anthranilate 17.88 0.07 0.02 0.02     0.52 1.68       0.02 0.02
n-Hexadecanoic Acid
157 n-hexadecanoic acid 55.95       0.62   0.1 0.22 0.05 0.1      
Other Compounds
30 (Z)-linalool oxide 15.33 0.04 0.03 0.04 0.09 1 0.05 0.02 0.1 1.52 0.02 0.05 0.09
33 (E)-linalool oxide 16.61   0.01         1.13     0.72 0.66  
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The 20 major volatile compounds were subjected to multivariate statistics to determine and classify the peels from different pomelo cultivars. A heatmap was used to visualize the levels of the 20 major volatile components in the peels of 12 batches of pomelo (Figure 3). C. grandis cv. Shatianyou (S1, S2, and S3) and C. grandis cv. Liangpingyou (L1) were naturally classified as one group, whereas C. grandis cv. Guanximiyou (MH2 and MB1), C. grandis cv. Yuhuanyou (Y1 and Y2), and C. grandis cv. Duweiwendanyou (D1 and D2) were classified as one group. Moreover, C. grandis cv. Guanximiyou (MH1 and MB2) was classified as another group.

Figure 3.

Figure 3

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Heatmap and dendrogram of 20 main volatile compounds in pomelo peels from 5 cultivars.

Combining the results of the aforementioned analyses, it was seen that the profiles of 20 major volatile compounds of C. grandis cv. Shatianyou (S1, S2, and S3) and C. grandis cv. Liangpingyou (L1) were different from those of other varieties, which may be related to their high content of d-limonene (95.25–96.54%). Similarities were observed in the volatile compounds of C. grandis cv. Guanximiyou (MH2 and MB1), C. grandis cv. Yuhuanyou (Y1 and Y2), and C. grandis cv. Duweiwendanyou (D1 and D2). Moreover, C. grandis cv. Guanximiyou samples from different origins (MH1, MH2, MB1, and MB2) were not completely clustered together based on the heatmap result (Figure 3). MB2 exhibited clear separation from MH1, MH2, and MB1. This finding might be attributed to the high contents of linalyl anthranilate (1.68%) and 4-terpinenol (0.23%) in MB2, whereas MH1, MH2, and MB1 presented relatively low contents of linalyl anthranilate (0.00–0.52%) and 4-terpinenol (0.06–0.07%). Additionally, the content of n-hexadecanoic acid in MH1 was 0.62%, which was different from that in MH2 and MB1 (0.00–0.22%). The content variations of volatile components among different samples might result from several factors, including the genetic source, geographical conditions, growth environment, and instability of extracted essential oils.

In summary, the types of main volatile components were similar in pomelo peels, but their contents were different in peels from different pomelo cultivars. C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou were different from other cultivars. Furthermore, no difference was found in the samples of C. grandis cv. Guanximiyou, C. grandis cv. Yuhuanyou, and C. grandis cv. Duweiwendanyou from different origins.

2.2. Analysis of the Nonvolatile Compounds in the Peels of Five Pomelo Cultivars

The representative total ion chromatogram of nonvolatile compounds in the pomelo peel in the positive mode is shown (Figure 4). According to the retention time and fragment ion information of constituents provided by UPLC-Q-exactive orbitrap-MS analysis, 53 components were determined from the methanol extracts from peels of 5 pomelo cultivars, including 21 flavonoids, 21 coumarins, 2 organic acids, and 9 other compounds. All the compounds were identified in comparison with the Orbitrap Traditional Chinese Medicine Library (OTCML), standard reference, and literature information. The molecular ions, related product ions, and retention time of 53 compounds observed in UPLC-Q-exactive orbitrap-MS are shown in Table 3, and their chemical structures are illustrated in Figure 5. A total of 11 identified compounds were first reported in pomelo peels, including 7 flavonoids, 2 coumarins, and 2 other compounds. These compounds were determined as vitexin, apigenin 7-glucoside, isorhamnetin, pinocembrin, tectorigenin, iristetrigenin B, 7-methoxy-4-methylcoumarin, columbianetin acetate, N,N-dimethyl-l-proline, and curdione.

Figure 4.

Figure 4

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Representative total ion chromatograms of nonvolatile compounds of pomelo peels for (A) Citrus grandis cv. Shatianyou, (B) Citrus grandis cv. Guanximiyou, (C) Citrus grandis cv. Yuhuanyou, (D) Citrus grandis cv. Duweiwendanyou, and (E) Citrus grandis cv. Liangpingyou.

Table 3. Non-Volatile Compounds Identified in Five Cultivars by UPLC-Q-Exactive Orbitrap-MS.

peak no. RT (min) experimental [M + H]+ (m/z) major secondary fragment ions (m/z) molecular formula identification reference
Flavonoids
3 0.39 595.1663 577.1547, 541.1345, 511.1235, 481.1129, 457.1133, 427.1026, 409.0917, 391.0814, 379.0813, 349.0707, 337.0708, 325.0707, 295.0603, 283.0601, 271.0603, 257.0441, 229.0498, 219.0291, 203.0343, 195.0288, 145.0285, 121.0287, 103.0390, 85.0291, 57.0339 C27H30O15 vicenin (24)
6 0.5 433.1132 415.1024, 397.0919, 379.0812, 367.0813, 349.0705, 337.0707, 323.0913, 313.0707, 297.0757, 283.0602, 271.0602, 271.0602, 256.0728, 243.0284, 213.0548, 183.0291, 165.0184, 145.0285, 121.0286, 109.0284, 79.0185 C21H20O10 vitexin (25)
9 0.64 579.1710 433.1128, 344.1442, 313.0717, 283.0602, 271.0601, 243.0653, 203.0698, 171.0288, 153.0183, 119.0494, 85.0290 C27H30O14 rhoifolin a
10 0.65 271.0599 253.0490, 243.0650, 225.0547, 203.0699, 187.0394, 171.0288, 163.0387, 153.0184, 145.0286, 129.0181, 119.0494, 109.0284, 91.0548, 67.0186 C15H10O5 apigenin (26)
11 0.65 433.1132 332.4244, 313.0716, 283.0602, 271.0603, 261.1127, 243.0661, 225.0536, 189.0553, 171.0291, 153.0184, 119.0494, 91.0547, 67.0187 C21H20O10 apigenin 7-glucoside (27)
12 0.66 273.0756 255.0648, 231.0651, 207.0654, 189.0545, 179.0337, 171.0287, 157.0650, 153.0182, 147.0440, 129.0182, 123.0441, 119.0493, 107.0494, 91.0547, 83.0134, 67.0186, 55.0186 C15H12O5 naringenin a
13 0.68 581.1873 527.1444, 443.1302, 417.1174, 401.1236, 383.1126, 365.1016, 339.0863, 315.0862, 285.0755, 273.0757, 263.0549, 219.0289, 195.0289, 171.0289, 153.0183, 129.0547, 119.0493, 91.0547, 85.0290, 71.0499 C27H32O14 naringin a
14 0.71 609.1819 463.1243, 361.0900, 301.0708, 286.0473, 258.0524, 229.0494, 201.0560, 153.0184, 129.0546, 85.0290 C28H32O15 diosmin (28)
15 0.72 303.0865 285.0755, 261.0773, 244.1052, 219.0647, 201.0548, 189.0548, 177.0549, 171.0290, 163.0392, 153.0185, 149.0600, 137.0598, 123.0444, 117.0339, 111.0080, 89.0392, 83.0134, 67.0187, 55.0186 C16H14O6 hesperitin (29)
16 0.72 611.1978 449.1441, 413.1235, 395.1128, 369.0969, 345.0969, 315.0864, 303.0865, 281.0660, 263.0551, 219.0292, 195.0291, 177.0548, 153.0184, 129.0548, 111.0444, 85.0291, 71.0499 C28H34O15 hesperidin a
22 1.44 317.0660 302.0424, 285.0400, 273.0391, 257.0449, 229.0499, 217.0500, 203.0341, 189.0548, 165.0184, 153.0185, 139.0390, 121.0285, 105.0340, 92.0263, 68.9980 C14H12O7 isorhamnetin (30)
25 1.94 257.0812 239.0706, 215.0707, 191.0703, 179.0348, 173.0291, 171.0291, 153.0186, 145.0656, 131.0495, 123.0444, 107.0496, 103.0548, 97.0290, 91.0549, 79.0550, 67.0187, 57.1876 C15H12O4 pinocembrin (31)
27 3.48 301.0709 286.0473, 258.0523, 240.0417, 229.0500, 213.0548, 195.0444, 184.0514, 168.0051, 147.0440, 121.0285, 91.0552, 68.9974 C16H12O6 tectorigenin (32)
31 5.16 331.0815 316.0580, 301.0346, 288.0636, 273.0396, 245.0444, 231.0498, 203.0342, 189.0550, 173.0600, 159.0442, 147.0443, 139.0028, 121.0287, 107.0492, 91.0548, 68.9977 C17H14O7 iristetrigenin B (33)
35 7.63 343.1180 328.0945, 313.0709, 299.0899, 285.0759, 270.0529, 257.0811, 243.0659, 211.0764, 199.0240, 181.0132, 171.0288, 153.0184, 144.1716, 133.0651, 125.0236, 108.8341, 90.3219, 69.0341 C19H18O6 5,7,8,4′-tetramethoxyflavone b
38 8.21 361.0921 346.0677, 331.0439, 317.0650, 303.0493, 287.0538, 275.0546, 257.0442, 229.0489, 197.0443, 181.0130, 164.0466, 137.0596, 121.0283, 93.0337, 65.0392 C18H16O8 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone b
39 8.55 345.0972 330.0736, 315.0502, 301.0704, 287.0553, 272.0319, 259.0601, 231.0652, 213.0549, 189.0548, 181.0136, 153.0184, 131.0494, 121.0287, 107.0496, 85.0291, 65.0393 C18H16O7 lysionotin (34)
40 8.6 403.1391 388.1156, 373.0921, 358.0683, 345.0971, 327.0863, 313.0710, 301.0709, 287.0539, 274.0840, 258.0524, 229.0342, 211.0239, 193.0136, 183.0291, 165.0549, 148.0517, 127.0393, 99.0443, 69.0344 C21H22O8 nobiletin a
41 8.77 375.1077 360.0841, 345.0607, 330.0370, 302.0418, 274.0474, 257.0449, 229.0502, 201.0545, 181.0136, 151.0392, 137.0234, 109.0288, 74.4930 C19H18O8 chrysosplenetin B (35)
43 9.54 373.1284 358.1048, 343.0814, 328.0579, 315.0857, 297.0759, 283.0603, 271.0602, 244.0733, 229.0321, 211.0240, 193.0139, 183.0290, 168.0052, 145.0647, 135.0442, 127.0393, 99.0441, 69.0341 C20H20O7 tangeretin a
45 10.55 389.1236 373.0987, 374.0998, 343.0455, 331.0815, 301.0354, 275.0919, 257.0815, 242.0583, 217.0495, 181.0133, 165.0549, 153.0185, 137.0236, 109.0287, 79.0546 C20H20O8 artemetin (36)
Coumarins
5 0.48 191.0704 173.1327, 163.0755, 158.1095, 149.0599, 145.1010, 133.1013, 123.0813, 119.0495, 115.0547, 105.0703, 93.0704, 91.0548, 86.0242, 79.0549, 69.0342, 55.0551 C11H10O3 7-methoxy-4-methylcoumarin b
8 0.61 247.0965 229.0860, 211.0756, 201.0547, 187.0753, 175.0391, 163.0390, 159.0441, 147.0441, 142.0776, 131.0493, 119.0494, 103.0546, 91.0547, 85.0651, 65.0393 CH14H14O4 marmesin (37)
17 0.78 193.0498 178.0262, 161.0598, 150.0314, 154.0782, 143.0598, 137.0598, 133.0286, 122.0365, 115.0546, 105.0703, 95.0496, 89.0391, 79.0548, 66.0472, 55.0187 C10H8O4 isoscopoletin a
19 0.91 409.1492 391.1861, 261.1112, 247.0965, 229.0859, 211.0751, 201.0547, 187.0390, 159.0441, 131.0492, 97.0289, 85.0290, 69.0343 C20H24O9 nodakenin (38)
20 1.31 261.1123 261.1123, 243.1017, 231.1023, 217.0860, 201.0548, 189.0548, 177.0547, 159.0442, 145.0648, 131.0493, 128.0625, 115.0544, 103.0547, 95.0491, 85.0655, 67.0550 C15H16O4 isomeranzin a
21 1.32 279.1228 261.1121, 243.1064, 231.1020, 217.0861, 201.0548, 189.0547, 177.0547, 159.0441, 145.0647, 131.0492, 117.0702, 103.0546, 95.0497, 77.0388, 67.0550 C15H18O5 meranzin hydrate a
24 1.92 305.1024 203.0342, 189.0550, 175.0393, 159.0443, 147.0443, 131.0494, 119.0493, 91.0549, 67.0549, 59.0500 C16H16O6 oxypeucedanin hydrate (39)
26 2.18 289.1073 261.1127, 248.1003, 243.1019, 228.0784, 213.0549, 189.0549, 185.0603, 173.0600, 159.0443, 155.0856, 145.0650, 131.0494, 127.0397, 117.0702, 103.0547, 91.0547, 81.0705, 69.0707 C16H16O5 columbianetin acetate (40)
28 3.57 179.0342 169.9784, 161.0600, 151.0388, 138.0549, 135.0443, 128.9511, 123.0441, 119.0494, 111.0443, 107.0495, 95.0495, 91.0548, 83.0496, 68.9979, 55.9353 C9H6O4 esculetin (41)
29 3.71 209.0448 194.0214, 191.0711, 181.0499, 166.0265, 163.0390, 153.0550, 149.0236, 138.0313, 131.0493, 121.0288, 110.0367, 92.0259, 82.0419, 68.9980, 55.0188 C10H8O5 fraxetin a
30 5.09 217.0499 217.0499, 202.0264, 189.0543, 178.0263, 174.0314, 161.0600, 146.0364, 131.0494, 118.0419, 115.0546, 105.0702, 91.0547, 74.0971, 55.0185 C12H8O4 bergapten a
32 5.41 247.0603 235.0233, 232.0368, 229.0856, 217.0134, 203.0692, 189.0185, 175.0392, 164.5641, 161.0235, 147.0442, 133.0288, 119.0860, 104.1075, 95.0132, 60.0816 C13H10O5 isopimpinellin (42)
34 7.06 231.1014 223.9490, 189.0543, 175.0393, 147.0443, 131.0491, 119.0491, 112.0470, 103.0547, 91.0548, 65.0392 C14H14O3 7-demethylsuberosin b
37 7.91 287.0918 203.0343, 175.0392, 159.0444, 147.0443, 131.0494, 119.0497, 91.0548, 85.0655, 67.0549, 59.0501 C16H14O5 oxypeucedanin (39)
42 9.48 187.0393 163.9645, 159.0443, 143.0494, 135.9704, 131.0494, 118.9676, 115.0547, 103.0547, 95.0497, 81.0705, 55.9351 C11H6O3 isopsoralen (43)
44 9.72 193.0498 178.0258, 175.0384, 165.0545, 161.0229, 150.0310, 137.0595, 133.0283, 122.0362, 117.0335, 105.0335, 94.0416, 77.03912, 66.04706 C10H8O4 scopoletin a
47 10.85 245.1176 226.2699, 203.0706, 189.0549, 183.6220, 174.0308, 159.0443, 147.0438, 131.0494, 121.9895, 115.0547, 103.0547, 95.0498, 77.0392, 53.0394 C15H16O3 osthole (44)
49 11.31 271.0970 241.9205, 209.7384, 203.0342, 175.0392, 159.0443, 147.0443, 131.0494, 119.0495, 103.0548, 91.0548, 84.4042, 69.0707, 65.0393 C16H14O4 isoimperatorin (39)
51 16.93 163.0391 135.0442, 121.2155, 119.0495, 114.8570, 107.0496, 105.0451, 95.0498, 91.0548, 79.0548, 72.2852, 65.0394, 53.0394 C14H14O3 7-hydroxycoumarin a
52 16.95 299.1644 256.9647, 231.1012, 203.0700, 189.0551, 175.0392, 163.0392, 159.0449, 137.1326, 119.0495, 107.0496, 95.0861, 91.0548, 81.0706, 69.0707 C19H22O3 auraptene a
53 17.6 339.1594 203.0342, 175.0392, 159.0444, 147.0443, 131.0494, 119.0495, 91.0548, 81.0706, 69.0707 C21H22O4 bergamottin a
Organic acids
7 0.6 225.0758 207.0651, 201.7923, 192.0414, 189.0544, 181.0854, 175.0389, 164.0465, 151.1117, 147.0440, 132.0207, 123.0441, 119.0493, 113.9640, 105.0701, 95.0496, 91.0547, 81.0703, 69.0340, 65.0391, 61.0403 C11H12O5 sinapic acid (45)
18 0.78 195.0655 186.0556, 180.0312, 177.0545, 163.0386, 154.0585, 149.0598, 145.0283, 135.0441, 125.0598, 117.0337, 111.0443, 107.0494, 95.0499, 91.0545, 89.0390, 79.0548, 65.0392, 57.0706 C10H10O4 ferulic acid a
Other Compounds
1 0.31 144.1019 128.0712, 116.1075, 102.0552, 84.0813, 81.0343, 70.0658, 58.0659, 55.0550 C7H13NO2 N,N-dimethyl-l-proline a
2 0.33 127.0393 122.4651, 109.0286, 99.0807, 97.0289, 88.7007, 85.0652, 81.0340, 79.0548, 71.0497, 67.0549, 57.0342, 53.0393 C6H6O3 5-hydroxymethylfurfural (46)
4 0.42 237.1851 222.1535, 219.1746, 212.4210, 201.1639, 191.1796, 177.0538, 173.1326, 151.1119, 145.1013, 137.0964, 133.1014, 123.1170, 119.0859, 109.1016, 105.0704, 95.0861, 93.0705, 83.0496, 81.0706, 71.0499, 55.0551 C15H24O2 curdione (47)
23 1.7 134.0602 132.2048, 116.0498, 106.0654, 99.0060, 95.0495, 89.0391, 79.0548, 76.5491, 71.5103, 64.7083 C8H7NO 6-hydroxyindole b
33 6.06 153.1275 141.7384, 135.1169, 115.9385, 111.0806, 107.0859, 105.0705, 97.0652, 95.0860, 93.0704, 91.0546, 83.0860, 81.0705, 79.0549, 73.0656, 71.0499, 69.0706, 67.0549, 59.0500, 55.0551 C10H16O camphor b
36 7.67 471.2021 453.1918, 435.1810, 425.1965, 409.2016, 391.1884, 367.1910, 349.1818, 339.1961, 321.1861, 279.1387, 251.1058, 227.1071, 213.0914, 205.0499, 175.0756, 161.0600, 145.0651, 133.0651, 119.0860, 105.0704, 95.0134, 79.0549, 69.0707 C26H30O8 limonin a
46 10.62 175.0390 147.0443, 134.0603, 131.0496, 121.0291, 119.0495, 105.0335, 103.0549, 95.0499, 91.0549, 79.0549, 70.0659, 65.00394, 58.3657 C10H6O3 lawsone (48)
48 11 151.1121 133.1011, 131.0858, 123.1168, 121.1012, 113.9638, 109.0649, 107.0857, 105.0700, 98.5123, 97.0650, 95.0494, 93.0702, 91.0546, 87.0045, 83.0495, 81.0704, 79.0547, 71.0497, 69.0341, 67.0548, 65.0392, 57.0342, 55.0550 C10H14O perillene b
50 11.62 219.1745 201.1635, 191.1790, 177.127, 163.1115, 159.1168, 149.0959, 145.0959, 135.0803, 131.0856, 123.1168, 119.0855, 111.0805, 107.0856, 97.0651, 95.0858, 93.0702, 91.0546, 81.0704, 19.0547, 69.0705, 67.0549, 57.0704, 55.0550 C5H22O α-cyperone (49)
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a

Confirmation in comparison with standard substances.

b

Refers to the database.

Figure 5.

Figure 5

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Chemical structures of the 53 components that were tentatively identified.

In addition, as shown in Table 4, some compounds were detected in all 12 batches of samples, such as rhoifolin, naringenin, naringin, nobiletin, tangeretin, chrysosplenetin B, isomeranzin, meranzin hydrate, bergapten, osthole, 7-hydroxycoumarin, auraptene, and N,N-dimethyl-l-proline. These compounds may be closely associated with the biological activity of pomelo peels, such as anti-inflammatory and anti-oxidant properties.17,18

Table 4. Non-Volatile Compound Differences among Pomelo Peels from Five Cultivars.

peak no. identification RT (min) molecular formula S1 S2 S3 MH1 MH2 MB1 MB2 Y1 Y2 D1 D2 L1
Flavonoids
3 vicenin 0.39 C27H30O15       √ √ √ √ √ √ √ √ √
6 vitexin 0.50 C21H20O10       √ √ √ √ √ √ √ √  
9 rhoifolin 0.64 C27H30O14 √ √ √ √ √ √ √ √ √ √ √ √
10 apigenin 0.65 C15H10O5       √ √ √ √ √ √ √ √  
11 apigenin 7-glucoside 0.65 C21H20O10         √              
12 naringenin 0.66 C15H12O5 √ √ √ √ √ √ √ √ √ √ √ √
13 naringin 0.68 C27H32O14 √ √ √ √ √ √ √ √ √ √ √ √
14 diosmin 0.71 C28H32O15             √ √ √   √  
15 hesperitin 0.72 C16H14O6       √                
16 hesperidin 0.72 C28H34O15       √                
22 isorhamnetin 1.44 C14H12O7       √ √ √ √ √ √ √ √ √
25 pinocembrin 1.94 C15H12O4 √ √ √ √ √ √   √ √ √   √
27 tectorigenin 3.48 C16H12O6                       √
31 iristetrigenin B 5.16 C17H14O7 √ √ √                 √
35 5,7,8,4′-tetramethoxyflavone 7.63 C19H18O6     √                  
38 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone 8.21 C18H16O8 √ √ √             √ √ √
39 lysionotin 8.55 C18H16O7 √ √ √         √ √      
40 nobiletin 8.6 C21H22O8 √ √ √ √ √ √ √ √ √ √ √ √
41 chrysosplenetin B 8.77 C19H18O8 √ √ √ √ √ √ √ √ √ √ √ √
43 tangeretin 9.54 C20H20O7 √ √ √ √ √ √ √ √ √ √ √ √
45 artemetin 10.55 C20H20O8 √ √ √                  
Coumarins
5 7-methoxy-4-methylcoumarin 0.48 C11H10O3 √ √   √   √   √        
8 marmesin 0.61 CH14H14O4 √ √ √       √ √ √ √ √ √
17 isoscopoletin 0.78 C10H8O4 √     √     √     √ √ √
19 nodakenin 0.91 C20H24O9   √                    
20 isomeranzin 1.31 C15H16O4 √ √ √ √ √ √ √ √ √ √ √ √
21 meranzin hydrate 1.32 C15H18O5 √ √ √ √ √ √ √ √ √ √ √ √
24 oxypeucedanin hydrate 1.92 C16H16O6 √ √ √                 √
26 columbianetin acetate 2.18 C16H16O5 √   √                  
28 esculetin 3.57 C9H6O4 √     √ √ √ √ √ √ √ √ √
29 fraxetin 3.71 C10H8O5 √ √ √         √ √ √ √ √
30 bergapten 5.09 C12H8O4 √ √ √ √ √ √ √ √ √ √ √ √
32 isopimpinellin 5.41 C13H10O5       √ √              
34 7-demethylsuberosin 7.06 C14H14O3 √ √ √ √ √ √   √ √ √ √  
37 oxypeucedanin 7.91 C16H14O5   √ √                 √
42 isopsoralen 9.48 C11H6O3     √             √    
44 scopoletin 9.72 C10H8O4 √     √     √     √ √ √
47 osthole 10.85 C15H16O3 √ √ √ √ √ √ √ √ √ √ √ √
49 isoimperatorin 11.31 C16H14O4 √ √ √ √ √ √   √   √ √ √
51 7-hydroxycoumarin 16.93 C14H14O3 √ √ √ √ √ √ √ √ √ √ √ √
52 auraptene 16.95 C19H22O3 √ √ √ √ √ √ √ √ √ √ √ √
53 bergamottin 17.60 C11H6O3       √ √ √ √ √ √ √ √ √
Organic Acids
7 sinapic acid 0.60 C11H12O5       √   √ √ √ √ √ √  
18 ferulic acid 0.78 C10H10O4     √ √   √ √ √       √
Other Compounds
1 N,N-dimethyl-l-proline 0.31 C7H13NO2 √ √ √ √ √ √ √ √ √ √ √ √
2 5-hydroxymethylfurfural 0.33 C6H6O3 √ √ √ √ √   √ √ √ √ √ √
4 curdione 0.42 C15H24O2 √       √             √
  curcumol           √                
23 6-hydroxyindole 1.70 C8H7NO     √ √                
33 camphor 6.06 C10H16O √ √   √ √       √   √  
36 Limonin 7.67 C26H30O8 √ √ √     √ √ √ √ √ √ √
46 Lawsone 10.62 C10H6O3             √     √ √  
48 Perillene 11.00 C10H14O     √         √   √ √  
50 α-cyperone 11.62 C5H22O √       √   √   √ √    
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Furthermore, the content difference of different components was observed among five different varieties. For instance, C. grandis cv. Shatianyou contained the greatest number of coumarins (18), whereas C. grandis cv. Shatianyou contained the least number of flavonoids (12). Some compounds, including vicenin and isorhamnetin, were not found in C. grandis cv. Shatianyou. Except for C. grandis cv. Shatianyou, all varieties contained no artemetin, which might be one of the distinctive compounds to differentiate C. grandis cv. Shatianyou from other cultivars. Moreover, 14 flavonoids were observed in C. grandis cv. Yuhuanyou and C. grandis cv. Duweiwendanyou, which were the most abundant. Additionally, C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou had the most and least types of nonvolatile compounds, respectively.

2.3. Simultaneous Quantification of Six Bioactive Compounds by HPLC-PDA

In this study, six major nonvolatile compounds in pomelo peels were determined with HPLC-PDA using external standard methods based on corresponding calibration curves. As exhibited in Table 5, the calibration equations and correlation coefficients (R2) of naringin, rhoifolin, meranzin hydrate, isomeranzin, auraptene, and bergamottin were provided. There was good linearity across the tested concentration ranges, and all R2 values were above 0.999. The limits of detection (LOD) and limits of quantification (LOQ) were designated signal-to-noise (S/N) ratios of 3 and 10, respectively. Moreover, the method showed good reliability and feasibility. The mean extraction recovery was within the range of 90.13–105.87%. In addition, the relative standard deviations of repeatability (0.72–2.84%), precision (0.12–0.60%), stability (0.37–2.13%), and recovery (1.07–2.81%) were below 3.00% for all standards.

Table 5. Linear Correlation, Repeatability, Precision, Stability, and Recovery Investigation of Seven Chemical Compounds.

compounds calibration curves correlation coefficients (R2) linear range (μg/mL) LOD (μg/mL) LOQ (μg/mL) repeatability RSD (%), n = 6 precision RSD (%), n = 6 stability RSD (%), n = 6 extraction recovery (%), n = 6 recovery RSD (%),n = 6
naringin y = 20053x + 4000000 0.999 500.00–3133.08 0.007 0.024 1.91 0.45 2.13 91.07 1.30
rhoifolin y = 31529x + 24430 1.000 22.19–412.00 0.007 0.024 2.76 0.34 0.37 92.38 1.65
meranzin hydrate y = 51450x + 64334 1.000 0.78–386.00 0.003 0.009 1.54 0.60 1.62 90.13 1.07
isomeranzin y = 53693x – 11226 1.000 4.00–112.00 0.005 0.018 2.84 0.60 1.20 105.87 2.78
auraptene y = 49794x + 43015 0.999 6.25–246.00 0.003 0.009 2.50 0.28 0.68 94.72 2.67
bergamottin y = 26185x – 4122 0.999 2.54–42.20 0.013 0.043 0.72 0.58 1.37 96.71 2.81
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The developed analytical method was subsequently applied to analyze the peels of five pomelo cultivars. The quantitative analysis results of six major nonvolatile compounds are displayed in Table 6. As displayed in Table 6, the content of naringin was the highest in all 12 batches of samples, varying from 84.44 to 288.40 mg·g–1. It was followed by meranzin hydrate (0.01–37.97 mg·g–1), auraptene (0.52–23.05 mg·g–1), rhoifolin (1.42–19.67 mg·g–1), isomeranzin (0.49–10.89 mg·g–1), and bergamottin (0.35–3.88 mg·g–1). Studies revealed that these Citrus flavonoids and coumarins exhibited diverse biological activities.19 Other researchers have reported that naringin exhibited bone regeneration,20 anti-inflammatory,21 and anticancer effects.22 Moreover, some pharmacological research demonstrated that meranzin hydrate possessed anti-anxiety and anti-depressant effects.23

Table 6. Results of Determination of Non-Volatile Constituents in Five Cultivars by HPLC-PDAa.

  HPLC-PDA (mg·g–1)
sample naringin rhoifolin meranzin hydrate isomeranzin auraptene bergamottin
S1 161.95 ± 0.93a 1.42 ± 0.04e 20.39 ± 0.07c 4.27 ± 0.01A 1.75 ± 0.03a 0.00 ± 0.00a
S2 163.16 ± 1.04a 2.94 ± 0.03A 18.33 ± 0.06d 4.50 ± 0.04B 1.62 ± 0.07b 0.00 ± 0.00a
S3 84.44 ± 1.26c 4.47 ± 0.04a 17.22 ± 0.05e 4.06 ± 0.01C 1.90 ± 0.01c 0.00 ± 0.00a
MH1 242.17 ± 0.77b 17.45 ± 0.22b 3.94 ± 0.02A 0.73 ± 0.02a 2.91 ± 0.01d 0.87 ± 0.00c
MH2 218.07 ± 0.21d 9.20 ± 0.03B 3.25 ± 0.00B 0.65 ± 0.02a,b 1.83 ± 0.00e 0.35 ± 0.01b
MB1 261.16 ± 0.26e 15.60 ± 0.16C 2.76 ± 0.03C 0.49 ± 0.01b 1.73 ± 0.00a 0.38 ± 0.00b
MB2 244.95 ± 1.03A 17.34 ± 0.19b 0.10 ± 0.00a 0.82 ± 0.00a 7.76 ± 0.01f 1.51 ± 0.00d
Y1 256.16 ± 1.55B 17.83 ± 0.23c 0.16 ± 0.00a 1.67 ± 0.10c 6.40 ± 0.02A 1.45 ± 0.03A
Y2 240.21 ± 0.75b 17.81 ± 0.16c,d 0.14 ± 0.00a 1.79 ± 0.05c 8.85 ± 0.03B 1.86 ± 0.00B
D1 288.40 ± 0.70C 19.67 ± 0.04D 0.01 ± 0.00b 3.65 ± 0.30d 16.32 ± 0.04C 2.75 ± 0.03C
D2 253.71 ± 0.77D 17.52 ± 0.23b,d 0.01 ± 0.00b 3.60 ± 0.01d 23.05 ± 0.04D 3.88 ± 0.04D
L1 263.41 ± 1.42E 4.24 ± 0.02a 11.38 ± 0.01D 2.73 ± 0.03D 0.52 ± 0.01E 0.00 ± 0.00a
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a

Different letters indicate significant differences (P < 0.05). Data are presented as mean ± standard deviation (n = 3).

The contents of the above-mentioned 6 nonvolatile compounds were different in 12 batches of pomelo peels, suggesting differences among cultivars and origins. The quantification results were compared and displayed with a heatmap (Figure 6). Samples were divided into two main clusters (clusters A and B). Cluster A included C. grandis cv. Shatianyou (S1, S2, and S3) and C. grandis cv. Liangpingyou (L1), and this classification might be related to the low content of rhoifolin and the high content of meranzin hydrate. Cluster B comprised C. grandis cv. Guanximiyou (MH1, MH2, MB1, and MB2), C. grandis cv. Yuhuanyou (Y1 and Y2), and C. grandis cv. Duweiwendanyou (D1 and D2). This classification might be because these cultivars contained a high content of naringin and a low content of meranzin hydrate. In short, C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou contained similar levels of six major nonvolatile compounds, whereas C. grandis cv. Guanximiyou, C. grandis cv. Yuhuanyou, and C. grandis cv. Duweiwendanyou had relatively consistent levels of the six chemicals.

Figure 6.

Figure 6

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Heatmap and dendrogram of six major nonvolatile compounds in pomelo peels from five cultivars.

3. Conclusions

The volatile and nonvolatile compounds were determined and analyzed in 12 batches of pomelo peel samples from 5 different cultivars obtained from 11 major origins around China. A total of 194 volatile compounds were observed with GC–MS. The main volatile compounds in all pomelo peel samples were similar, but their contents were different. The contents of volatile compounds in C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou were different from those in other cultivars. No difference was found in samples of C. grandis cv. Guanximiyou, C. grandis cv. Yuhuanyou, and C. grandis cv. Duweiwendanyou from different origins. For nonvolatile compounds, 53 chemicals were identified with UPLC-Q-exactive orbitrap-MS. Additionally, the quantification of six major nonvolatile compounds based on HPLC-PDA and the heatmap revealed good separation between different cultivars and batches of pomelo. Notably, C. grandis cv. Shatianyou and C. grandis cv. Liangpingyou exhibited similarity in both volatile and nonvolatile compounds based on the results of GC–MS and HPLC-PDA. This study may provide some information for the development and application of peels from different pomelo cultivars in functional and medicinal industries, thus facilitating their recycling.

4. Materials and Methods

4.1. Plant Materials

A total of 12 batches of pomelo peel samples from 5 different cultivars were obtained from 11 major origins around China in September 2021 (Table 1). All these samples were selected as experimental materials for subsequent analysis. Samples were identified and authenticated by Prof. Guodong Zheng of the Laboratory of Pharmacognosy, Guangzhou Medical University in Guangdong Province, China.

4.2. Chemical Materials

Eighteen (18) commercial standards (≥98% purity) were obtained from Weikeqi (Sichuan, China). These standards were naringin, naringenin, rhoifolin, hesperidin, nobiletin, tangeretin, fraxetin, meranzin hydrate, isomeranzin, bergapten, isoscopoletin, auraptene, bergamottin, scopoletin, 7-hydroxycoumarin, ferulic acid, N,N-dimethyl-l-proline, and limonin. Methanol and acetonitrile of HPLC grade were obtained from Merck (Germany). Hexane and formic acid were provided by Honeywell (USA) and Thermo Fisher Scientific (China), respectively. Water was purified using the Milli-Q system from Millipore (Milford, MA, USA).

4.3. Preparation of Standard Solutions

Appropriate amounts of 18 reference substances were separately dissolved in methanol to obtain solutions with a concentration of 100 μg·mL–1. The resulting solutions were used as standard stock solutions for UPLC-Q-exactive orbitrap-MS analysis. The contents of six main compounds (including naringin, rhoifolin, meranzin hydrate, isomeranzin, auraptene, and bergamottin) in pomelo peel were calculated using external standard methods based on corresponding calibration curves. The standard substances of these six main compounds were separately prepared in methanol and diluted to appropriate concentrations for HPLC-PDA.

4.4. Sample Preparation

Samples of pomelo were cleaned using distilled water and peeled. Pomelo peels were collected, sliced, and air-dried to constant weight for about 2 weeks for further analysis. Then, dry pomelo peels were comminuted and passed through a 40-mesh sieve to obtain a fine powder for the following UPLC-Q-exactive orbitrap-MS and HPLC-PDA detection.

The volatile compounds in pomelo peels were extracted with water and then determined with GC–MS. 100 g of the powdered sample was added in approximately 1.5 L of water and transferred into a 2 L round-bottomed distillation flask. The mixture was heated to reflux for 1 h and filtered to obtain the extract. Then, 50 μL of the extract was transferred into a test tube containing 950 μL of hexane. The mixture was filtered with a 0.22 μm PTFE membrane before performing GC–MS.

The nonvolatile compounds in pomelo peels were extracted with methanol and then analyzed with UPLC-Q-exactive orbitrap-MS. 0.5 g of the dried and powdered sample was mixed with 50 mL of methanol in a conical flask. The flask with samples was immersed into an ultrasonic bath (KQ-800KDE instrument, Kunshan Ultrasonic Instruments Co. Ltd., China) for 30 min at 320 W (40 kHz) for the extraction of nonvolatile compounds. Afterward, the sample was filtered and analyzed with the UPLC-Q-exactive orbitrap-MS system.

The main nonvolatile compounds were quantified with HPLC-PDA. 0.5 g of the powdered sample was mixed with 10 mL of methanol. The mixture was treated in an ultrasonic bath for 30 min. Afterward, the supernatant was collected in a conical flask and passed through a 0.22 μm PTFE membrane to obtain the methanol extract for further experiments.

4.5. Qualitative and Quantitative GC–MS System for the Analysis of Volatile Compounds in Pomelo Peels

The GC system consisted of a TRACE DSQ GC instrument (Thermo Finnigan, USA), and a TG-5SILMS GC capillary column (0.25 mm × 30 m, 0.25 μm) was used to analyze the volatile compounds of pomelo peels. GC–MS operating conditions were as follows: the initial temperature was set at 60 °C, increased to 80 °C at a rate of 1 °C·min–1, further increased to 250 °C at a rate of 5 °C·min–1, and finally increased to 260 °C at a rate of 20 °C·min–1 and held for 1 min. Temperatures at the injection port and detector were maintained at 270 °C. The solvent delay was fixed at 4 min. High-purity helium (1 mL·min–1) was used as carrier gas at a split ratio of 30:1. Moreover, the injection volume for samples was 1 μL. Detection was performed in the full-scan mode ranging from m/z 35 to m/z 300, with a scan speed of 0.2 amu·s–1, and the electron impact (EI+) mode (70 eV) was used. All volatile compounds were identified by comparing their recorded mass spectra with the NIST08.L database.

4.6. Qualitative UPLC-Q-Exactive Orbitrap-MS System for the Analysis of Methanol Extracts of Pomelo Peels

Samples were first separated on a ZORBAX Eclipse Plus C18 column with a stable flow rate of 0.4 mL·min–1 and a column temperature of 40 °C. The mobile phase included 0.1% formic acid aqueous solution (A) and acetonitrile (B). The nonvolatile compounds of pomelo peels were eluted under the following conditions: 25% B at 0–4 min, 25–50% B at 4–10 min, 50% B at 10–14 min, 50–85% B at 14–18 min, and 85–100% B at 18–25 min. The injection volume was 2 μL for both the sample and standard solutions.

The full-scan mode was applied for data acquisition in positive ionization modes ranging from m/z 70 to m/z 1000 with a resolution of 70,000. The spray voltage was 35 kV, the capillary temperature was 320 °C, and the auxiliary gas heating temperature was 300 °C. The flow rate of the sheath, sweep, and auxiliary gases was 10.0, 1.7, and 3.3 L·min–1, respectively.

4.7. Quantitative HPLC-PDA System for the Analysis of Methanol Extracts of Six Main Nonvolatile Compounds

Chromatographic separation was carried out at 30 °C on a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase included 0.1% (v/v) phosphoric acid water solution (pH 3.70, A) and 30% methanol +70% acetonitrile (B). The nonvolatile compounds of pomelo peels were eluted by using a gradient program: 0–20% B at 0–5 min, 20–40% B at 5–15 min, 40% B at 15–30 min, 40–60% B at 30–40 min, 60–70% B at 40–45 min, 70–80% B at 45–50 min, 80% B at 50–55 min, and 80–100% B at 55–60 min. The flow rate was 1 mL·min–1. A 15 μL aliquot was used for injection and analysis. Furthermore, naringin (283 nm), rhoifolin (325 nm), meranzin hydrate (325 nm), isomeranzin (325 nm), auraptene (325 nm), and bergamottin (325 nm) were monitored using a PDA detector.

4.8. Statistical Analysis

Data were statistically analyzed using Statistical Product and Service Solutions version 25 (SPSS Inc, Chicago, USA). Analysis of variance was performed to determine any significant difference in measurement, and P < 0.05 indicated statistical significance. Clustering analysis allowed the grouping of different cultivars based on their similarities.

Acknowledgments

This work was primarily supported by the Undergraduate Teaching Quality and Teaching Reform Project of Guangzhou Medical University in 2021 (no. GYDF[2021]160) and Special Fund for Pharmaceutical Research of Guangdong Hospital Association (2021YMS04).

Glossary

Abbreviations

GC–MS

gas chromatography–mass spectrometry

HPLC-PDA

high-performance liquid chromatography-photodiode array detection

OCTML

Orbitrap Chinese Traditional Medicine Library

TIC

total ion chromatogram

UPLC-Q-Exactive Orbitrap/MS

ultraperformance liquid chromatography-Q-Exactive Orbitrap tandem mass spectrometry

Author Contributions

† B.S. and J.T. contributed equally to this work.

The authors declare no competing financial interest.

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