UPLC-Q-Exactive Orbitrap MS Analysis for Identification of Lipophilic Components in Citri Sarcodactylis Fructus from Different Origins in China Using Supercritical CO2 Fluid Extraction Method.

To thoroughly evaluate the quality of Citri Sarcodactylis Fructus (CSF) and acquire knowledge of the lipophilic components of CSF from different origins, a simple and efficient approach based on supercritical fluid extraction (SFE) combined with ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) detection for the discrimination of components from CSF was set up for the first time in this work. Eight batches of CSF samples from five main producing areas were extracted by SFE under optimized conditions, and then SFE extracts were dissected via UPLC-Q-Exactive Orbitrap/MS. The results indicated that 39 lipophilic compounds were successfully separated and unambiguously or tentatively identified, where 4 coumarins, 6 polymethoxyflavones, 3 phthalides, 6 terpenes, and 4 phenolics were not reported formerly. It was illustrated that CSF may be abundant in polymethoxyflavones, as in coumarins. Moreover, there were significant differences in the components of CSF from different origins. Especially, coumarin, dehydrocostus lactone, atractylenolide II, and atractylenolide I were exclusively found in CSF from the Guangdong province; isopsoralen was almost exclusively found in CSF from the Guangxi province; and ferulic acid was exclusively found in CSF from the Zhejiang province. These observations indicated that SFE joint with UPLC-Q-Exactive Orbitrap/MS owing to the potential of characterizing the lipophilic components could be used to promote quality assessment and chemotaxonomic investigation in phytology sciences of CSF.

Introduction

Citri Sarcodactylis Fructus (CSF), the dried fruit of Citrus medica L. var. sarcodactylis Swingle, which belongs to Citrus botany in Rutaceae,[1] is widely cultivated in Guangdong, Guangxi, Zhejiang, Sichuan, and Yunnan provinces of China.[2] It can be utilized as a traditional medicine for the cure of stomach ache, headache, edema, rheumatism, arthritis, and infectious hepatitis[3] and also as a tonic material to make crispy cookies.[4] As it is considered to be one of the homologies in medicine and food, CSF is full of potential for vast development and application prospects.[5] However, the chemical composition of CSF from different origins has not been fully clarified. Therefore, a rapid and efficient method should be built for the systematic analysis and identification of phytochemicals in CSF.

As reported previously, many compounds such as coumarins and limonoids were separated and distinguished in the CSF extracts by use of methanol,[6−8] although it is worth considering that some lipophilic components like nonpolar and low-polarity substances may not be easily obtained by the use of common organic solvents. Up to now, there have been a few studies wherein supercritical fluid CO2 extraction (SFE) was used to extract the chemical compounds of CSF prior to ingredient analysis. SFE is a low-temperature extraction technology and widely implemented to extract chemical components in plants, due to the potential to extract nonpolar and low-polarity substances.[9,10] In addition, although high-performance liquid chromatography mass spectrometry (HPLC-MS) and gas chromatography mass spectrometry (GC-MS) are usually used for the analysis of plant chemicals, recently, ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) has proved to be a more rapid, efficient, and sensitive implement than HPLC-MS and GC-MS in the field of the analysis of extracts and bioactive constituents from medicine and food plants.[11,12]

Herein, the chemical components of CSF were extracted by SFE and discriminated via UPLC-Q-Exactive Orbitrap/MS technology in eight batches of CSF samples from several main producing areas in China. Notably, 39 lipophilic components were isolated and identified, some of which were first reported, for instance, 4 coumarins, 6 polymethoxyflavones (PMFs), and 3 phthalides. This study could lay the foundation for the quality evaluation and chemotaxonomic exploration of CSF in phytology sciences.

Results

Optimization of SFE and UPLC-Q-Exactive Orbitrap/MS Conditions

The SFE extracts of 8 batches of CSF (seen from Table ) were analyzed via UPLC-Q-Exactive Orbitrap/MS. To acquire more precise information about compounds of CSF, analytical conditions were determined by optimizing a sequence of parameters like the elution gradient and flow rate of the mobile phase, whereby the components of SFE extracts in CSF were able to be isolated in the positive mode where the detection signal was better than that in the negative-ion mode within 30 min. The application method was efficient, and the mean SFE extraction rate of CSF was 1.42% (1.19–1.73%). Herein, the total ion chromatogram (TIC) of samples and mixed standard substances are displayed in Figure . Totally, 39 compounds were separated and identified and labeled 1–39 in accordance with the sequence of the peak time (seen in Table ). In general, these extraction constituents were divided into several groups consisting of 14 coumarins, 7 PMFs, 3 phthalides, 3 limonoids, 7 terpenes, and 5 phenolics. Among these, 7 compounds were discerned via retention times and fragments by comparison with standard substances and 32 compounds were distinguished according to MS2 positive-ion fragments and the related information from these studies was reported formerly. All of these chemical structures are displayed in Figure .

Table 1

Representative Samples of CSF Investigated in This Studya

no.	collecting area (name)	sample source	collection time (yy/mm/dd)
S1	Guangdong province	Lecheng town, Zhaoqing city, Guangdong province	2019/09/25
S2	Guangxi province	Yongfu county, Guilin city, Guangxi province	2019/10/29
S3	Zhejiang province	Luodian town, Jinhua city, Zhejiang province	2019/11/07
S4	Zhejiang province	Chisong town, Jinhua city, Zhejiang province	2019/11/15
S5	Sichuan province	Huidong county, Liangshan prefecture, Sichuan province	2019/10/21
S6	Sichuan province	Peng’an county, Nanchong city, Sichuan province	2019/11/17
S7	Sichuan provinceb	Baiyang town, Wanzhou county, Chongqing city	2019/10/28
S8	Yunnan province	Huaning county, Yuxi city, Yunnan province	2019/10/28

Guangdong province, Guangxi province, Zhejiang province, Sichuan province, and Yunnan province are the main producing areas.

Collecting area of Chongqing city was classified into main producing area of the Sichuan province by considering the geography.

Figure 1

Representative total ion chromatogram of CSF in positive ionization mode. Compound 6: scopoletin; compound 9: scoparone; compound 14: 5,7-dimethoxycoumarin; compound 16: limonin; compound 20: nomilin; compound 29: nobiletin; compound 33: tangeretin.

Table 2

Compounds Identified in CSF by UPLC-Q-Exactive Orbitrap/MSaFour

no.	tR (min)	theoretical [M + H]+ (m/z)	experimental [M + H]+ (m/z)	major secondary fragment ions (MS/MS)	compound formula	proposed compound	ref
Coumarins
3	1.51	193.0495	193.0498	178.0263, 165.0548, 161.0539, 150.0313, 147.1172, 137.0599, 133.0286, 122.0365, 117.0337, 109.0651, 105.0339, 94.0420, 77.0393, 66.0472, 56.9658	C10H8O4	isoscopoletin	(14)
6	1.70	193.0495	193.0498	178.0263, 165.0549, 161.0235, 150.0313, 137.0598, 133.0286, 122.0365, 117.0336, 105.0341, 89.0391, 77.0393, 66.0471, 53.0395	C10H8O4	scopoletin	c
8	3.17	147.0440	147.0443	132.9590, 119.0858, 114.9491, 105.0703, 103.0546, 95.0494, 91.0548, 84.9603, 79.0553, 73.9930, 65.0393, 60.9770, 53.0395	C9H6O2	coumarin	(15)
9	3.60	207.0651	207.0654	192.0417, 191.0340, 181.4489, 179.0704, 163.0390, 151.0755, 148.0520, 136.0520, 121.0651, 107.0496, 91.0549, 79.0549	C11H10O4	scoparoneb	c
10	3.96	223.0601	223.0604	208.0370, 190.0263, 179.0343, 162.0314, 153.0105, 149.0964, 134.0364, 125.0154, 106.0418, 91.0547, 78.0472,58.0658	C11H10O5	fraxinolb	(16)
11	5.50	177.0546	177.0547	162.0311, 149.0598, 133.0648, 121.0650, 118.0416, 103.0546, 91.0548, 79.0549, 66.0472, 53.0394	C10H8O3	7-methoxycoumarin	(17)
12	6.86	187.0389	187.0392	173.1181, 159.0441, 143.0493, 131.0494, 115.0546, 109.1017, 103.0548, 95.0495, 88.0224, 67.0550, 55.0550	C11H6O3	isopsoralenb	(18)
13	8.71	305.1019	305.1020	287.1166, 203.0340, 192.0297, 175.0392, 159.0441, 147.0441, 131.0493, 119.0494, 91.0548, 59.0500	C16H16O6	oxypeucedan hydrate	(19)
14	9.18	207.0651	207.0654	192.0418, 179.0703, 164.0469, 151.0755, 148.0520, 133.0650, 121.0651, 118.0471, 103.0547, 91.0548, 79.0549, 65.0393	C11H10O4	5,7-dimethoxycoumarinb	c
15	9.57	217.0495	217.0495	202.0261, 174.0312, 173.0598, 167.9620, 161.0596, 146.0361, 131.0491, 115.0545, 105.0704, 91.0548, 79.0549, 53.4664	C12H8O4	bergapten	(7)
17	12.98	317.1019	317.1020	299.0896, 273.075, 245.0441, 233.0446, 218.0211, 203.0339, 188.0106, 175.0392, 162.0310, 147.0441, 119.0496, 91.0548, 85.0655, 67.0550, 57.0706	C17H16O6	byakangelicol	(20)
19	12.99	287.0914	287.0913	203.0338, 175.0390, 174, 159.0441, 147.0440, 131.0492, 119.0491, 91.0546, 85.0653, 67.0550, 59.0500	C16H14O2	oxypeucedanin	(20)
34	23.40	193.0495	193.0497	178.0261, 165.0547, 149.0598, 137.0598, 134.0364, 121.0650, 109.0652, 106.0418, 91.0549, 79.0549, 67.0186, 53.0394	C10H8O4	5,7-dihydroxy-4-methylcoumarinb	(21)
38	28.77	163.0389	163.0391	149.0450, 139.9821, 135.0441, 119.0493, 107.0494, 95.0495, 91.0547, 84.9604, 79.0548, 68.9979, 61.6360, 53.0395	C9H6O3	7-hydroxycoumarin	(22)
Methoxyflavonoids
23	18.02	361.0917	361.0920	346.0685, 331.0449, 315.0510, 303.0501, 285.0396, 257.0445, 229.0495, 201.0546, 181.0133, 169.0133, 121.0288, 105.0336, 68.9977	C18H16O8	5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone	(8)
25	18.72	343.1176	343.1178	328.0944, 313.0707, 285.0758, 270.0524, 257.0806, 242.0570, 211.0751, 199.0237, 181.0132, 153.0183, 135.0442, 125.0235, 107.0126, 85.0287, 69.0339	C19H18O6	6-demethoxytangeretinb	(24)
28	19.93	331.0812	331.0812	316.0578, 301.0347, 285.0393, 273.0393, 257.0445, 242.0571, 214.0622, 186.063, 169.0133, 135.0443, 121.0650, 108.0572, 91.0549, 68.9977	C17H14O7	iristectorigeninab	(25, 26)
29	20.43	403.1387	403.1388	388.1152, 373.0919, 358.0682, 355.0811, 327.0860, 301.0706, 284.0680, 258.0524, 229.0340, 211.0237, 193.0129, 183.0290, 165.0546, 127.0392, 99.0441, 69.0344	C21H22O8	nobiletinb	c
30	20.92	331.0812	331.0813	316.0579, 301.0344, 288.0629, 273.0395, 257.0448, 245.0444, 229.0493, 199.0389, 169.0133, 148.0520, 135.0442, 121.0651, 108.0574, 86.9453	C17H14O7	jaceosidinb	(27)
32	22.31	389.1230	389.1234	374.0998, 359.0763, 341.0659, 331.0810, 316.0582, 285.0755, 260.0676, 227.0552, 215.0189, 197.0083, 163.0755, 148.0521, 113.0238, 85.0285	C20H20O8	5-demethylnobiletinb	(28)
33	22.51	373.1281	373.1286	358.1050, 343.0816, 328.0579, 297.0762, 285.0401, 271.0603, 254.0570, 229.0322, 211.0240, 183.0292, 135.0443, 127.0392, 99.0441, 69.0339	C20H20O7	tangeretinb	c
Phthalides
18	12.66	191.1066	191.1068	173.0963, 163.1328, 158.0728, 149.0598, 145.1012, 130.0778, 117.0702, 111.0443, 105.0702, 99.0444, 91.0547, 79.0549, 71.0489, 55.0551	C12H14O2	3-n-butylphthalideb	(30, 31)
21	13.61	193.1223	193.0498	175.1120, 157.1016, 147.1170, 137.0599, 133.0286, 123.0444, 119.0859, 109.0652, 105.0703, 97.0652, 93.0705, 91.0548, 81.0705, 69.0706, 67.0550, 53.0395	C12H16O2	senkyunolide Ab	(31)
24	18.42	191.1066	191.1068	173.0963, 163.1118, 149.0599, 145.1013, 135.0442, 130.0778, 121.0650, 117.0702, 105.0703, 91.0548, 79.0549, 67.0550, 55.0549	C12H14O2	ligustilideb	(30, 31)
Limonoids
16	9.69	471.2013	471.2013	453.1893, 435.1798, 425.1957, 409.2002, 391.1882, 367.1909, 339.1952, 213.0910, 205.0498, 187.0754, 169.1006, 161.0598, 133.0649, 119.0858, 105.0702, 95.0132, 79.0549, 69.0706	C26H30O8	limoninb	c
20	13.08	515.2272	515.2272	497.2172, 469.2216, 455.2065, 437.1960, 419.1853, 411.2165, 393.2059, 369.2074, 341.2097, 279.1383, 243.1000, 231.0645, 187.0754, 161.0598, 133.0649, 105.0703, 95.0132, 79.0548, 69.0705	C28H34O9	nomilinb	c
22	17.73	455.2064	455.2068	437.1958, 419.1862, 409.2012, 391.1910, 377.1368, 359.1272, 349.1434, 331.1335, 303.1391, 273.1281, 235.1120, 209.0965, 187.0757, 161.0599, 133.0650, 119.0860, 105.0703, 95.0133, 81.0341, 67.0551	C26H30O7	obacunone	(33)
Terpenoids
5	1.66	153.1273	153.1274	135.1169, 125.0599, 120.9808, 111.0444, 109.1015, 107.0859, 95.0860, 93.0704, 88.9530, 84.9603, 81.0705, 79.0549, 65.0394, 59.0500, 55.0551	C10H16O	camphor	d
27	19.59	231.1379	231.1381	213.1277, 203.1432, 195.1168, 189.0915, 185.1328, 175.0752, 163.0391, 157.1013, 145.1015, 143.0857, 131.0858, 119.0859, 105.0702, 91.0548, 79.0549, 69.0707, 55.0550	C15H18O2	dehydrocostus lactoneb	(42)
31	22.13	233.1536	233.1539	215.1434, 197.1322, 187.1486, 177.0912, 173.0965, 159.0810, 151.0756, 145.1015, 131.0858, 105.0703, 95.0861, 91.0549, 81.0706, 67.0549, 56.9656	C15H20O2	atractylenolide IIb	(34)
35	24.70	231.1379	231.1380	213.1274, 203.1068, 189.0910, 185.1325, 175.0756, 161.1327, 157.1013, 147.0800, 143.0856, 129.0700, 105.0702, 91.0548, 81.0705, 67.0549, 55.0549	C15H18O2	atractylenolide Ib	(35)
36	27.43	219.1743	219.1745	203.1433, 187.0761, 173.0234, 163.0390, 145.0284, 131.0857, 119.0858, 107.0859, 95.0860, 91.0547, 81.0705, 79.0549, 69.0706, 67.0550, 55.0551	C15H22O	(+)-nootkatoneb	(43)
37	28.37	219.1743	219.1745	203.1430, 191.1794, 187.0763, 173.1328, 161.1326, 145.1013, 135.1169, 119.0858, 107.0859, 95.0860, 93.0704, 91.0548, 81.0705, 67.0550, 55.0551	C15H22O	α-cyperoneb	(44)
39	28.85	219.1743	219.1746	203.1433, 201.1436, 191.1792, 187.0761, 175.1479, 161.1328, 159.1017, 151.1120, 145.1013, 133.1017, 121.1016, 107.0860, 95.0861, 91.0548, 81.0705, 67.0550, 55.0551	C15H22O	germacroneb	(36)
Phenolics
1	0.48	123.0553	123.0555	106.0292, 96.0448, 95.0498, 90.9482, 81.0706, 80.0501, 79.0549, 72.9379, 67.0550, 60.9389, 56.9430, 53.0395	C6H6N2O	nicotinamideb	d
2	1.49	153.0546	153.0547	135.1170, 125.0599, 111.0444, 107.0859, 93.0340, 84.9602, 81.0706, 79.0548, 71.0500, 65.0394, 59.0500	C8H8O3	vanillinb	(45)
4	1.59	183.0651	183.0653	165.0911, 155.0704, 140.0468, 137.0959, 125.0235, 123.0443, 111.0443, 105.0451, 97.0284, 95.0496, 90.9480, 81.0704, 71.0501, 67.0550, 53.0394	C9H10O4	syringaldehydeb	(45)
7	1.71	195.0651	195.0544	187.4464, 177.0549, 163.0392, 153.0553, 149.0599, 145.0286, 135.0443, 125.0599, 117.0339, 107.095, 95.0494, 89.0392, 84.9606, 79.0549, 63.0237	C10H10O4	ferulic acid	(46)
26	18.85	179.0702	179.0710	161.0599, 147.0442, 136.0521, 133.0651, 119.0495, 115.0548, 105.0703, 95.0499, 91.0549, 84.9603, 79.0550, 65.0394, 55.0187	C10H10O3	ferulaldehydeb	(47)

coumarins (compounds 9, 10, 12, and 34), 6 polymethoxyflavones (compounds 25, 28, 29, 30, 32, and 33), 3 phthalides (compounds 18, 21, and 24), 6 terpenes (compounds 27, 31, 35, 36, 37, and 39), and 4 phenolics (compounds 1, 2, 4, and 26) were not reported previously.

Compounds were not reported in CSF extracts previously.

Further confirms the comparison with standard compounds.

Refers to the database.

Figure 2

Chemical structure of 39 compounds that were unequivocally and tentatively identified.

Identification of Coumarins

Coumarins are a kind of natural compound with benzo-α-pyranone parent nucleus, which are the primary constituents in the SFE extracts of CSF. Fourteen coumarins were identified in CSF extracts. In the positive mode, the fragmentation of coumarins from mass spectrometry was characterized by the loss of neutral molecules, such as CO, CO2, and CH3, because of high-energy collisions.[13]

Compound 3 produced a protonation ion at m/z 193.0498 and fragment ions at m/z 178.0263 [M + H – CH3]+, 165.0548 [M + H – CO]+, 150.0313 [M + H – CH3 – CO]+, and 147.1172 [M + H – CO2]+. Referring to mass spectrum cracking information from a previous study,[14] compound 3 was identified as isoscopoletin. The precursor ion of compound 8 was m/z 147.0443, which gave daughter ions such as m/z 132.9590, m/z 119.0858, m/z 103.0546, and m/z 91.0548 successively. According to the structure cracking rule of coumarins, compound 8 was detailed to be coumarin as done previously.[15] Meanwhile, compound 10 produced a strong molecular ion at m/z 223.0604 and formed fragments at m/z 208.0370 [M + H – CH3]+ and 179.0343 [M + H – CO2]+. Thus, compound 10 was tentatively identified as fraxinol from a previous study.[16] Moreover, compound 11 produced a molecular ion at m/z 177.0547 and formed fragments at m/z 162.0311 [M + H – CH3]+, 133.0647 [M + H – CO2]+, and 121.0650 [M + H – 2CO]+. Therefore, compound 11 was identified as 7-methoxycoumarin as before.[17]

Compound 6, 9, and 14 were unambiguously identified as scopoletin, scoparone, and 5,7-dimethoxycoumarin, respectively, by referring to the document and the standard substance with the same fragmentation traits.

Compounds 12, 13, 15, 17, and 19 belong to the linear furanocoumarins that are rich in Citrus genus. Compound 12 produced a molecular ion at m/z 187.0392 and formed characteristic fragmentations at m/z 159.0441 [M + H – CO]+, 143.0493 [M + H – CO2]+, 131.0494 [M + H – 2CO]+, and 115.0546 [M + H – CO – CO2]+. Thus, compound 12 was identified as isopsoralen from a previous study.[18] Because of an [M + H]+ ion at m/z 305.1020, the fragment ion at m/z 203.0340 indicating loss of the neutral substituent [M + H – C5H10O2]+, and sequential cleavage losing the CO radical and generating the typical [203-2CO]+ fragment at m/z 147.0441, compound 13 was tentatively identified as oxypeucedanin hydrate, which was in accordance with a previous report.[19] Similarly, compound 15 generated a precursor ion m/z 217.0495, while m/z 202.0261, m/z 174.0312, m/z 173.0598, and m/z 146.0361 were characteristic fragment ions that matched with the MS2 information as reported earlier.[7] Therefore, compound 15 was identified as bergapten (fragmentation pattern is shown in Figure A). Compound 17 exhibited a precursor ion at m/z 317.1020 and fragment ions at m/z 299.0896, m/z 273.0750, and m/z 233.0446, while compound 19 exhibited a precursor ion at m/z 287.0913 and fragment ions at m/z 203.0338 and m/z 175.0390. According to the MS2 information supported by the previous investigation,[20] compounds 17 and 19 were confirmed to be byakangelicol and oxypeucedanin, respectively.

Figure 3

Proposed fragmentation pathways of the main fragment ions in the positive-ion mode. (A) Bergapten; (B) nobiletin; (C) 5-demethylnobiletin; (D) ligustilide; (E) senkyunolide A; (F) limonin; (G) vanillin; and (H) atractylenolide II.

In addition, compound 34 exerted a protonated ion at m/z 193.0497 and fragment ions at m/z 178.0261, m/z 165.0547, m/z 149.0598, and m/z 137.0598. Thus, compound 34 was confirmed as 5,7-dihydroxy-4-methylcoumarin, as reported previously.[21] Also, compound 38 produced a precursor ion at m/z 163.0391 and fragment ions at m/z 149.0450, m/z 135.0441, m/z 119.0493, and m/z 107.0494. Following the characteristic fragmentation profiles and a previous study,[22] compound 38 was assigned as 7-hydroxycoumarin.

It was manifested that compounds 9, 10, 12, and 34 were not reported previously. These four coumarins have various functions; for example, compound 12 as isopsoralen has antibacterial, anti-inflammatory, antitumor, and antiosteoporotic properties.[23]

Identification of PMFs

PMFs are discovered nearly exclusively in Citrus species and possess the basic aglycone structure and are different from each other in the location and amount of substituted −OCH3 and/or −OH on the flavonoid mother nucleus. It was believed that characteristic fragment ions were formed by removal of −CH3, CO, and H2O. After scanning, seven PMFs were identified or tentatively discriminated in CSF.

Because of standard reference, compounds 29 and 33 were determined as nobiletin and tangeretin, respectively. The comprehensive fragmentation pattern of compound 29 is shown in Figure B, which included daughter ions at m/z 388.1152, m/z 373.0919, m/z 355.0811, and m/z 327.0860.

Compound 23 was assigned to be 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone with reference to the literature,[8] which gave a protonated molecular ion at m/z 361.0920 and fragment ions at m/z 346.0685, m/z 331.0449, and m/z 303.0501. Besides, compound 25 generated a protonated ion at m/z 343.1178 and fragment ions at m/z 328.0944, m/z 313.0707, and m/z 285.0758. Thus, compound 25 was indicated as 6-demethoxytangeretin from reference literature.[24] Compound 28 was assigned as isoflavone for the protonated ion at m/z 331.0812 and fragment ions like m/z 316.0578, m/z 301.0347, m/z 285.0393, and m/z 273.0393. As a result, compound 28 was assigned as iristectorigenina from the literature reported.[25,26] Compound 30 was plausibly regarded as jaceosidin from reference literature,[27] which exhibited the [M + H]+ at m/z 331.0813 and MS2 ions at m/z 316.0597 [M + H – CH3]+, 301.0344 [M + H – 2CH3]+, and 288.0629 [M + H – CH3 – CO]+. The precursor ion of compound 32 was at m/z 389.1234, as well as fragment ions, at m/z 374.0998, m/z 359.0763, m/z 341.0659, and m/z 331.0818. By comparison with the previous report,[28] compound 32 could be unambiguously identified as 5-demethylnobiletin (fragmentation pattern is shown in Figure C).

It was indicated that six PMFs including compounds 25, 28, 29, 30, 32, and 33 were not reported previously.

Identification of Phthalides

Phthalides are also known as γ-hydroxymethyl benzoate lactones, which are characterized by the double ring fusion of the inner ester and benzene. The fragment ions of H2O, CO, and alkyl chain are easily formed under high-energy collision.[29] Herein, three phthalides were identified.

The protonated ions of compounds 18 and 24 were both at m/z 191.1068, with similar second-order fragment ions at m/z 173 [M + H – H2O]+, 149 [M + H – C3H6]+, 145 [M + H – H2O – CO]+, and 130 [M + H – H2O – C3H6]+; they were found to be isomers. Further, the retention times of compound 18 and compound 24 was 12.66 and 18.42 min, respectively; according to the polarity of the compound combined with the above information, compound 18 was finally determined as 3-n-butylphthalide and compound 24 (fragmentation pattern shown in Figure D), as ligustilide.[30,31] Compound 21 had a precursor ion at m/z 193.0498 and secondary fragment ions at m/z 175.1120, m/z 147.1170, m/z 137.0599, and m/z 105.0703. Based on previous research,[31] compound 21 was tentatively characterized as senkyunolide A (fragmentation pattern is shown in Figure E).

It was clarified that three phthalides including compounds 18, 21, and 24 were not reported previously in CSF. Phthalides, especially ligustilide, were effective in improving microcirculation, protecting against cerebral ischemia-reperfusion injury, and treating tumor.[32]

Identification of Limonoids

Three limonoids, including limonin, nomilin, and obacunone, were identified from the SFE extracts of CSF. Via standard reference, compounds 16 and 20 were identified as limonin and nomilin, respectively. The detailed cracking process of compound 16 is shown in Figure F, which included son ions at m/z 425.1957, m/z 409.2002, m/z 367.1909, m/z 339.1952, and m/z 161.0598. In addition, compound 22 had an [M + H]+ ion at m/z 455.2068 and a series of MS2 ions at m/z 437.1958, m/z 419.1862, and m/z 409.2012; thus, compound 22 was determined to be obacunone, which is also a known ingredient in Citrus fruits as reported previously.[33]

Identification of Terpenoids

In this study, seven terpenoids were identified from CSF where compounds 5, 27, 31, 35, 36, 37, and 39 were unequivocally identified as camphor, dehydrocostus lactone, atractylenolide II, atractylenolide I, (+)-nootkatone, α-cyperone, and germacrone, respectively.

Compound 31 (fragmentation pathway is shown in Figure H) exhibited the [M + H]+ ion at m/z 233.1539 in the MS spectrum, and the main fragment ion peaks were at m/z 187.1486, m/z 177.0712, m/z 151.0756, m/z131.0858, m/z 105.0703, and m/z 91.0549. Therefore, compound 31 was confirmed as atractylenolide II from a previous report.[34] Compound 35 produced a molecular ion peak at m/z 231.1380 for [M + H]+ and daughter ions like m/z 213.1274, m/z 185.1325, and m/z 147.0800. On the basis of characteristic fragment profiles, compound 35 was determined to be atractylenolide I, as reported earlier.[35]

In addition, the deprotonated ion of compound 39 appeared at m/z 219.1746, further producing MS2 fragmentation at m/z 201.1436 and m/z 159.1017. According to the literature,[36] compound 39 was determined to be germacrone.

Note that compounds 27, 31, 35, 36, 37, and 39 were not found in previous studies for identification of components in CSF extracts.

Identification of Phenolics

In addition, five constituents including compounds 1, 2, 4, 7, and 26 were unequivocally assigned as nicotinamide, vanillin, syringaldehyde, ferulic acid, and ferulaldehyde, respectively, integrated with the literature and the corresponding formula supplied by MS.

Taking compound 2 as an example, a precursor ion sign was observed at m/z 153.0547, while in the MS2 spectrum, the primary fragment ions were at m/z 125.0599 [M + H – CO]+, m/z 111.0444 [M + H – CO – CH2]+, and m/z 93.0340 [M + H – C2H4O2]+. Compound 2 was identified as vanillin by comparing with a previous study,[37,38] which included a protonated ion at m/z 153.0547 and son ions at m/z 125.0599, m/z 111.0444, and m/z 93.0340. The fragmentation pathways of vanillin are shown in Figure G.

Of note, compounds 1, 2, 4, and 26 were not reported previously in CSF extracts.

Comparison of Citrus Samples from Different Origins

The established UPLC-Q-Exactive Orbitrap/MS method was applied to the coinstantaneous determination of the chemical markers in eight batches of CSF samples from several main producing areas, such as the Guangdong province, Guangxi province, Zhejiang province, Sichuan province, and Yunnan province. The results are shown in Table as the mean values of two repeated injections. Twenty-five compounds were found in all of these CSF samples, which showed that they were common in places. Of course, there were differences among these samples. Coumarin, dehydrocostus lactone, atractylenolide II, and atractylenolide I were exclusively found in CSF samples from the Guangdong province by comparison with others. Moreover, compared with others, isopsoralen was exclusively found in CSF samples from the Guangxi province and ferulic acid was exclusively found in CSF samples from the Zhejiang province. In parallel, it should be noted that nicotinamide was not found in CSF samples from the Yunnan province, syringaldehyde was not found in CSF samples from the Guangdong province, and both 3-n-butylphthalide and ligustilide were deficient in CSF samples from a region of the Sichuan province (Nanchong city).

Table 3

Differences in Chemical Components Among Different Originsa

peak	compound	S1	S2	S3	S4	S5	S6	S7	S8
1	nicotinamidec	*	*	*	*	*	*	*
2	vanillin	*	*	*	*	*	*	*	*
3	isoscopoletin	*	*	*	*	*	*	*	*
4	syringaldehydec		*	*	*	*	*	*	*
5	camphor	*	*	*	*	*	*	*	*
6	scopoletin	*	*	*	*	*	*	*	*
7	ferulic acidb			*	*
8	coumarinb	*
9	scoparone	*	*	*	*	*	*	*	*
10	fraxinol		*	*		*	*		*
11	7-methoxycoumarin	*	*	*	*	*	*	*	*
12	isopsoralenb		*
13	oxypeucedan hydrate	*	*	*	*	*	*	*	*
14	5,7-dimethoxycoumarin	*	*	*	*	*	*	*	*
15	bergapten	*	*	*	*	*	*	*	*
16	limonina	*	*	*	*	*	*	*	*
17	byakangelicol	*	*	*	*	*	*	*	*
18	3-n-butylphthalidec	*	*	*	*	*		*	*
19	oxypeucedanin	*	*	*	*	*	*	*	*
20	nomilin	*	*	*	*	*	*	*	*
21	senkyunolide A	*	*	*	*
22	obacunone	*	*	*	*	*	*	*	*
23	5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone	*	*	*	*	*	*	*	*
24	ligustilidec	*	*	*	*	*		*	*
25	6-demethoxytangeretin	*		*		*	*	*	*
26	ferulaldehyde	*				*	*	*	*
27	dehydrocostus lactoneb	*
28	iristectorigenina	*	*	*	*	*	*	*	*
29	nobiletinb	*	*	*	*	*	*	*	*
30	jaceosidin	*	*	*	*	*	*	*	*
31	atractylenolide IIb	*
32	5-demethylnobiletin	*	*	*	*	*	*	*	*
33	tangeretin	*	*	*	*	*	*	*	*
34	5,7-dihydroxy-4-methylcoumarin	*	*	*	*	*	*	*	*
35	atractylenolide Ib	*
36	(+)-nootkatone	*	*	*	*	*	*	*	*
37	α-cyperone	*	*	*	*	*	*	*	*
38	7-hydroxycoumarin	*	*	*	*	*	*	*	*
39	germacrone	*	*	*	*	*	*	*	*

S1, CSF samples from the Guangdong province; S2, CSF samples from the Guangxi province; S3 and S4, CSF samples from the Zhejiang province; S5, S6, and S7, CSF samples from the Sichuan province; and S8, CSF samples from the Yunnan province.

The compound is exclusively found in the corresponding origins or main producing area.

The compound is exclusively deficient in the corresponding origins or main producing area.

*The compound is found in the corresponding origins or main producing area.

Discussion

The most widespread technique for isolation of bioactive components from plant materials is the traditional organic solvent extraction. The major drawbacks of this extraction method are high time consumption and relatively poor selectivity. Noticeably, the new generation of extraction techniques are evolving to overcome these shortcomings.[39] SFC has proved to be a powerful separation technique. Because of the low viscosity and high diffusivity of supercritical fluids’ mobile phases, high flow rates and thus short analysis time as well as high efficiency could be easily achieved.[40] The SFE approach showed an excellent functioning of extraction of nonpolar and some low-polarity substances. Herein, SFE combined with UPLC-Q-Exactive Orbitrap/MS method was established for identification of components from CSF, whereby 39 lipophilic compounds in SFE extracts were isolated and identified via UPLC-Q-Exactive Orbitrap/MS, among which 23 compounds including 4 coumarins, 6 PMFs, and 3 phthalides were first identified in CSF extracts.

It is noteworthy that it is well known that coumarins are the main components of CSF and that PMFs may not be abundant; however, this study raised the novel interpretation that SFE extracts of CSF may contain a series of PMFs as well as coumarins. Interestingly, seven PMFs composed of 6-demethoxytangeretin, iristectorigenina, nobiletin, jaceosidin, 5-demethylnobiletin, and tangeretin were identified in SFE extracts, illustrating that CSF may contain a series of PMFs. The result was different from that of the study performed by Li et al.,[6] whose investigation demonstrated that PMFs were hardly detected in certain Citrus herbs including CSF based on LC-QTOF-MS/MS. The present study was of great significance for a strong supplement to the previous conclusion. In addition, these PMFs have beneficial pharmacodynamics, such as tangeretin exhibiting anti-inflammation, anti-oxidation, and neuroprotection properties.[41]

Moreover, a correlation between their composition and the collection areas was demonstrated. To a great extent, coumarin, dehydrocostus lactone, atractylenolide II, or atractylenolide I could be the marker compounds used to distinguish CSF of the Guangdong province from that of the other origins, while isopsoralen and ferulic acid may be the marker compounds that are exclusively found in CSF from the Guangxi province and Zhejiang province, respectively.

Conclusions

SFE coupled with UPLC-Q-Exactive Orbitrap MS analysis helped to explore more novel information on lipophilic components in CSF extracts: 23 compounds were not reported in CSF extracts earlier. Meanwhile, it was indicative that PMFs should be abundant in CSF. Moreover, some compounds may be recognized as a marker to distinguish CSF from different origins in China. In brief, the present study can boost the quality of assessment as well as future pharmacodynamics investigation of CSF and provide chemotaxonomic botanical knowledge for this functional food. Further, developing quality control methods is indispensable to ensure the identification of the product from different origins.

Materials and Methods

Reagents and Chemicals

Formic acid and methanol of MS grade were purchased from Merck KGaA Company of Germany, Ltd., and ThermoFisher Scientific (China) Co., respectively. Standard compounds 5,7-dimethoxycoumarin (compound 14), nomilin (compound 20), scopoletin (compound 6), and scoparone (compound 9) were provided by Sichuan Vicky Biotechnology Co. Ltd., and limonin (compound 16), nobiletin (compound 29), and tangeretin (compound 33) were provided by Chengdu Mansite Biotechnology Co. Ltd. The purity of these standard substances was over 98%. There were eight batches of CSF samples (shown in Table ), which were identified from the dried fruits of Citrus medica L. var. sarcodactoxylis Swingle in Rutaceae by Professor Wu Bo of the School of Pharmacy, Guangzhou Medical University. The samples were kept in the Pharmacognosy Laboratory, School of Pharmacy, Guangzhou Medical University.

Instrumentation

SFE was operated with an ASI Supercritical Fluid Extraction system (SPE-ed SFE-2) (Applied Separations, Inc., Allentown, PA) equipped with a high-pressure CO2 ASF-100 pump. Hk-04b swing crusher was produced from Guangzhou Xuyang Machinery Equipment Co. Ltd., and the ME 104 electronic analytical balance was provided by Mettler Toledo Instrument Co. Ltd. The UPLC system mainly consisted of an Ultimate 3000 series ultra-high-performance liquid chromatograph (ThermoFisher Scientific) equipped with an online degasser, a quaternary pump, an autosampler, and a column temperature compartment; it was connected to a Q-Exactive Orbitrap tandem mass spectrometer (ThermoFisher Scientific) via an electrospray ionization interface. Besides, the ZORBAX C18 column (2.1 mm × 50 mm, 1.8 μm) was offered by Agilent Technologies.

SFE Conditions

All SFE extractions were carried out at 50 °C in a dynamic mode with a total CO2 flow rate of 3 L/min and extractive pressure of 33 MPa. The total process was within 120 min for each extraction.

Sample Preparation

These reference substances were weighed appropriately and then dissolved in dichloromethane. The CSF samples collected from different origins were cut into smaller pieces and smashed into powder by a swing crusher and sifted through a 40-mesh sieve. The accurately weighed powder (50 g) was directly put into the SFE ax, without any type of support materials or entrainers. SFE extracts were harvested, put into vials, and then stored at 4 °C. Prior to the detection test; the stock solution was diluted with dichloromethane to a suitable concentration. The diluent solution was filtered by a 0.22 μm microporous membrane and then shifted into an autosampler vial for MS analysis.

Analytical System

The separation was performed on a ZORBAX Rclipse Plus C18 column at 35 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and methanol (phase B), using a gradient elution of 30% B at 0–3 min; 30–45% B at 3–8 min; 45% B at 8–12 min; 45–50% B at 12–16 min; 50–70% B at 16–25 min; and 70–90% B at 25–30 min. The flow rate was maintained at 0.3 mL/min, and the injected sample volume was 1 μL.

For Q-exactive Orbit-MS analysis, the source settings were as follows: The spray voltage was 3.5 kV. The capillary temperature was 320 °C, and the auxiliary gas heating temperature was 300 °C. Sheath gas, auxiliary gas, and purge gas were set at 30 units, 10 units, and 5 units, respectively. Full scan data collection and dependent scan event data collection were executed from m/z 100 to m/z 1000 in the dd-MS2 mode with a resolution of 70 000. The system was regulated by Xcalibur software and collected data was processed by Metworks software (ThermoFisher Scientific).