Abstract
Atractylodis macrocephalae rhizome (AMR) belongs to medicine food homology. Its' clinical application of invigorating the spleen-stomach of AMR was applied to various diseases. In this research, a UPLC-QTOF-MS method was developed for qualitative and quantitative analysis of AMR, simultaneously. A Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm particle size) was used for separation of AMR multi-components. The column was eluted with a mobile phase of 0.1% formic acid-water and 0.1% formic acid-acetonitrile. Electron spray ionization with positive-ion mode and external standard method was utilized for quantifying the nine analytes in AMR. Constituents of AMR were scanned by UPLC-QTOF-MS and then identified by mass fragments and chromatographic information compared with the published literature and reference standards. Under positive mode, a total of 61 chemical compositions including 16 terpenoids, 8 polyacetylenes, 6 aromatics, 5 flavonoids, 5 coumarins, 5 organic acids, 4 amino acids, 3 fatty acids, 3 aliphatics, 2 steroids, and 2 alkenes, a nucleoside and an aldehyde were identified. Simultaneously, the contents of three amino acids (L-tyrosine, L-phenylalanine, and L-tryptophan), three sesquiterpenoids (atractylenolide Ⅲ, atractylenolide Ⅱ, and atractylenolide Ⅰ), a flavonoid (rutin), an organic acid (ferulic acid), and a pentacyclic triterpenoid (oleanolic acid) were determined in seventeen AMR batches. Amino acids and triterpenoid were quantified for the first time in AMR. The UPLC-QTOF-MS method developed in this article was reliable, practical, and useful for qualitative and quantitative evaluation of AMR multi-components.
Introduction
There is a kind of substance with innocuity, nutrients, therapeutic for the sick, and pleasure for the hunger, simultaneously. The substance is defined as “medicine and food homology”, which was put forward in the ancient Huang Di Nei Jing Su Wen [1]. They are developed as health-care products broadly circulated in the market, such as ginseng, Polygonatum sibiricum, and Angelica sinensis. Atractylodis macrocephalae rhizome (AMR), as a traditional Chinese medicine (TCM), belongs to the genus Atractylodes (family Asteraceae), and is known as medicine food homology species. AMR is an indispensable herb appeared in more than 122 kinds of health-care products, 912 kinds of Chinese medicine preparation, and 4,333 kinds of herbal prescriptions for treating chronic diseases in line with the database [2]. Zhejiang and Anhui provinces of China were authentic producing areas of AMR [3]. AMR have a diversity of pharmacological effects of anti-tumor activity, enhancing immunostimulatory, improving gastrointestinal function, anti-inflammatory activity, anti-Alzheimer's disease, anti-aging, anti-oxidative and neuroprotective activities [4, 5]. Phytochemical investigation has shown that AMR contains polysaccharides, sesquiterpenoids, alkynes, amino acids, pyrazines, phenolic acids, and acyl sugar compounds [6]. Among them, the sesquiterpene-type lactones were acknowledged as principal bioactive compounds. Former literatures intensively focused on the quantitative determination and biological activities of atractylenolide I, atractylenolide II, and atractylenolide [7–9]. However, the therapeutic effects of AMR should be comprehensively revealed based on its multiple constituents.
AMR is rich in amino acids supporting human nutrition to maintain good health and prevent diseases. Tyrosine, phenylalanine, aspartic acid, tryptophan, glutamic acid, and alanine contribute to health benefits. Amino acids play important roles in the fundamental building blocks supporting life [10], preventing intestinal dysfunction [11], supporting immune function [12], and so on. Tryptophan is a precursor of serotonin that was synthesized by brain neurons [13]. Phenylalanine is a necessary amino acid for human absolutely. Yet, phenylalanine can be transformed into tyrosine, which is the precursor of epinephrine, norepinephrine, thyroxine, and neurotransmitters dopamine. From the perspective of the elementary theory of TCM, AMR possess the function of strengthening the spleen, dispel dampness for diuresis, and miscarriage prevention. The research suggested the requirement for phenylalanine during early and late gestation in healthy pregnant women [14]. Tyrosine was a versatile amino acid and proved participating in structural conformation transitions of proteins [15]. Even, it was shown that phenylalanine and tyrosine were linked with a raised risk of diabetes [16]. Chronic dietary phenylalanine, tryptophan, and tyrosine depletion brought about consequences that behavioral alterations in mice were present [14]. However, there are very rare attention on the amino acids of AMR. In addition, flavonoids and triterpenoids in AMR were also suffering a lack of attention. It is worth noting that measurement of amino acid, flavonoid, and triterpenoid in AMR is important precondition that illustrates health protection of AMR.
Previously, it has reported that gas chromatograph-mass spectrometer (GC-MS) analysis for volatile oil [17], liquid chromatography coupled with mass spectrometry (LC-MS) for multi-component characterization [18], and high performance liquid chromatography-diode array detection (HPLC-DAD) combined with chemometrics [19] have been established for composition assessment of AMR. Synchronous full-scan MS1 and MS2 capabilities of quadrupole time-of-flight mass spectrometry (QTOF-MS/MS) make both of qualitative and quantitative analyses accomplished, simultaneously [20]. To make a supplement for multi-ingredient excavation and quality control of AMR, with the intense separation ability, excellent resolution, sensitivity, and structural characterization capabilities, the strategy of UPLC-QTOF-MS was established to capture and profile the chemical components of AMR as much as possible, while we determined the contents of the representative amino acid, sesquiterpene, triterpene, and flavonoid.
Experimental
Materials and reagents
Twelve standards of L-tyrosine (2), L-phenylalanine (6), L-tryptophan (9), rutin (14), ferulic acid (18), luteolin (21), baicalein (26), wogonin (27), atractylenolide Ⅲ (34), atractylenolideⅡ (37), atractylenolide Ⅰ (43), and oleanolic acid (53) with 98% purity were obtained from Weikeqi Biological Technology Co., Ltd. (Sichuan, China). HPLC-grade acetonitrile and methanol were procured from Merck (Darmstadt, Germany). Formic acid for LC-MS analysis was supplied by Fluka (Steinheim, Germany). Distilled water was fetched from an Aquapro water purification system (Aquapro, Chongqing, China).
Sample preparation
Stock standard solution of compounds 2, 6, 9, 14, 18, 34, 37, 43, and 53 were prepared in pure methanol at 3.28, 21.27, 16.2, 3.8, 6.8, 201.3, 25.55, 25.7, and 13.15 ug/mL, respectively. Proper concentration levels (n = 6) for determining calibration curves were obtained by diluting mixed stock solutions. The commercial AMRs from Anhui (S1–S4), Gansu (S5–S6), Henan (S7–S8), Sichuan (S9–S11), Shanxi (S12–S13), Yunnan (S14), and Zhejiang (S15–S17) were collected from different medicinal stores (Table S1). 0.5 g herb powder of seventeen AMR batches ground into 80 mesh were weighted, and added into 20 mL pure methanol, shaken, stood still for 20 min, and weighted again. After vortexed for 30 min, cooled down, weighed, and made up for weight loss with methanol, the supernatant was passed through a 0.22 μm microporous membrane before UPLC-QTOF-MS/MS analysis.
UPLC-QTOF-MS/MS conditions
The UPLC separation was conducted using a Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation) on the Shimadzu LC-30AD system (Shimadzu, Japan). The column was eluted with mobile phase of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B), which was conducted as follows: 90% A (0–2 min), 79%–75% A (2–4 min), 75%–55% A (4–6 min), 55%–45% A (6–14 min), 45%–43% A (14–17 min), 43%–42% A (17–19 min), 42%–30% A (19–20 min), 30–20% A (20–30 min), 20%–5% A (30–31 min), and 5% A (31–33 min). The flow rate, column temperature, and injection volume were set at 0.3 mL min−1, 40 °C, and 5 μL, respectively.
QTOF-MS/MS analysis was performed using a Triple TOFTM 5600+ mass spectrometer (AB Sciex, Foster City, CA). The mass acquisition conditions were as follows: Ion source, DuoSpray ion source; polarity, positive mode; ion source gas 1, 55 psi; ion source gas 2, 55 psi; curtain gas, 35 psi; temperature, 550 °C; Ion Spray Voltage Floating, 4500 V; declustering potential, 90 V; collision energy, 5 V. For the information dependent acquisition criteria, the eight most intense fragmentation ions of each target were chosen to conduct a product ion scan when they exceeded 100 cps counts. MS and MS/MS scan range were 100–1550 and 50–1000 with a 250 ms accumulation time, respectively. Dynamic background subtraction was turned on during the full scans. Calibration delivery system was applied for precursor and product ion calibration at every 4 h. Data acquisition and processing were executed by Analyst®TF 1.7 and PeakView® 2.0 software, respectively.
Results and discussion
Optimization of UPLC-QTOF-MS method
The pivotal UPLC-QTOF-MS parameters such as mobile phase composition (0.1% formic acid-water/0.1% formic acid-methanol, 0.1%-formic acid water/0.1%-formic acid acetonitrile), analytical columns Waters Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm) and Waters Cortecs UPLC C18 (2.1 mm × 100 mm, 1.6), MS acquisition modes (negative or positive), CE values (25V/35V/45V) were inspected for obtaining an advantageous acquisition method. Taking separation performance, chromatographic peak shape, response intensity, and fragment ions into consideration, a Waters Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm) column at 0.1%-formic acid-water/0.1%-formic acid-acetonitrile with the optimized gradient elution was applied for the UPLC analysis. The CE value of 35V was recommended for qualitative analysis. Both negative and positive modes were adopted for acquiring chromatograms and MS data facilitating mutual verification. However, extract ion chromatograms (EICs) of active compounds 37 and 43 in AMR were unable to be detectable in ESI‒ mode, while the EICs of the two compounds were extracted perfectly in ESI+ mode (Fig. S1). Therefore, a quantitative analysis of nine compounds was detected in positive acquisition mode.
Qualitative analysis of AMR
Before characterization of chemical ingredients in AMR, a lot of beforehand work is necessary. A database of AMR components covering names, formulas, original literatures, CAS number, and compound structures was established for identification. For QTOF-MS/MS was acclaimed as a high-resolution technique, in the first round, molecular weights with the error (ppm >5) were excluded based on the full scan mass spectra. In the second round, the final identified component corresponding to single or multiple EIC was found out according to characteristic fragments and retention time (Rt) existing in published literature or standards. Additionally, the cracking law conforming to chemical structure was employed for inferring target compound, which particularly suits the circumstance of no contrast reference. As a result, the total ion chromatogram of AMR in positive mode was displayed in Fig. 1A. And the chromatograms of the quantified nine analytes were seen in Fig. 1B. A total of 61 chemical compositions (16 terpenoids, 8 polyacetylenes, 6 aromatics, 5 flavonoids, 5 coumarins, 5 organic acids, 4 amino acids, 3 fatty acids, 3 aliphatics, 2 steroids, and 2 alkenes, a nucleoside and an aldehyde) were tentatively identified, and their Rt, formula, ppm, fragmentation, mass value, as well as references were described in Table 1. The chemical structures of 61 identified compositions are shown in Fig. S2. Fragmentation pathways and MS2 spectrum of atractylenolide VI, (8R,9R)-8,9-dihydroxylatractylodinol-9-O-β-D-glucopyranoside, scoparone, and proline were displayed in Fig. S3.
The ion chromatograms of ARM (A) and reference standards (B) by UPLC-QTOF-MS/MS analysis. (peak 2: L-tyrosine; 6: L-phenylalanine; 9: L-tryptophan; 14: rutin; 18: ferulic acid; 34: atractylenolide Ⅲ; 37: atractylenolide Ⅱ; 43: atractylenolide Ⅰ; 53: oleanolic acid)
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01151
Characterisation of the chemical constituents of AMR by UPLC-QTOF-MS method
| No. | Compounds | Rt (min) | Theoretical mass | Detected mass | ppm | Detected mode | Formula | Fragments | Type | Reference |
| 1 | Proline | 0.88 | 115.0633 | 116.0711 | 4.5 | [M+H]+ | C5H9NO2 | 70.0735,53.0483 | amino acids | [33] |
| 2 | L-tyrosine | 0.90 | 181.0739 | 182.0805 | −3.6 | [M+H]+ | C9H11NO3 | 147.0444,136.0759,123.0448,119.0501, 107.0508,103.0562,95.0508,77.0412 | amino acids | * |
| 3 | citric acid | 1.13 | 192.0270 | 193.0345 | 1.0 | [M+H]+ | C6H8O7 | 193.0952,175.0864,147.0943,139.0018 129.0172,111.0090,93.0331,83.0494,68.9989 | organic acids | [37] |
| 4 | aconitic acid | 1.16 | 174.0164 | 175.0235 | −1.0 | [M+H]+ | C6H6O6 | 175.1191,130.0994,116.0747, 104.0508 | organic acids | [37] |
| 5 | Uridine | 1.18 | 244.0695 | 245.0774 | 2.3 | [M+H]+ | C9H12N2O6 | 245.1161,217.0954,142.0891,113.0371 | nucleoside | [38] |
| 6 | L-phenylalanine | 1.41 | 165.0790 | 166.0856 | −4.0 | [M+H]+ | C9H11NO2 | 120.0821,119.0749,118.0673,103.0564 102.0492 | amino acids | * |
| 7 | 5-hydroxymethyl furaldehyde | 1.56 | 126.0317 | 127.0390 | 0.4 | [M+H]+ | C6H6O3 | 109.0324 | aldehyde | [39] |
| 8 | atractyloside A | 1.87 | 448.2309 | 449.2379 | −0.3 | [M+H]+ | C21H36O10 | 287.0572,269.1752,251.1682,233.1590 223.1689 215.1457,187.1565,147.1209 | terpenoids | – |
| 9 | L-tryptophan | 2.05 | 204.0899 | 205.0969 | −3.7 | [M+H]+ | C11H12N2O2 | 143.0738,142.0662,132.0825,130.0662 128.0514, | amino acids | * |
| 10 | scopoletin-D-xylopyranosyl -(1→6)-D-glucopyranoside | 2.27 | 486.1373 | 487.1448 | 0.4 | [M+H]+ | C21H26O13 | 355.0976,193.0539,163.0440,133.0678 87.0647 | coumarins | – |
| 11 | (8R,9R)-8,9-dihydroxylatractylodinol-9-O-β-D-glucopyranoside | 2.30 | 394.1264 | 395.1320 | −4.2 | [M+H]+ | C19H22O9 | 232.0632,215.0721,198.1827,164.0443 114.1107 | polyacetylenes | – |
| 12 | chlorogenic acid | 2.31 | 354.0951 | 355.1013 | −4.5 | [M+H]+ | C16H18O9 | 163.0392,145.0296,135.0460,117.0358 107.0523 | organic acids | [40] |
| 13 | 5-O-feruloylquinic acid | 3.23 | 368.1107 | 369.1176 | −1.1 | [M+H]+ | C17H20O9 | 177.0546,149.0628,145.0313,117.0387 | organic acids | [33] |
| 14 | Rutin | 3.51 | 610.1534 | 611.1602 | −0.4 | [M+H]+ | C27H30O16 | 465.1037,303.0537,129.0578,71.0537 | flavonoids | * |
| 15 | 1-(2-Furyl)-(1E,7E)-nonadiene-3,5-diyne-9-yl 4-methylbenzoate or isomers | 3.61 | 314.1307 | 315.1368 | −3.5 | [M+H]+ | C22H18O2 | 283.1007,247.0852,235.0827,222.0770 211.0851 206.0838,193.0768,167.0604 | polyacetylenes | – |
| 16 | Puerarin | 3.61 | 432.1057 | 433.1127 | −0.5 | [M+H]+ | C21H20O10 | 415.0701,397.0648,379.0570,361.0456, 337.0506,323.0739,309.0626,283.0486, 255.0580,165.0197,149.0250,121.0339 | flavonoids | [41] |
| 17 | scopoletin | 3.88 | 192.0423 | 193.0493 | −1.1 | [M+H]+ | C10H8O4 | 193.0507,178.0266,165.0574,161.0246, 150.0326,137.0609,133.0298,122.0381, 107.0507,105.0360 | coumarins | [42] |
| 18 | ferulic acid | 3.93 | 194.0579 | 195.0652 | 0.2 | [M+H]+ | C10H10O4 | 152.0364,149.0598,145.330,134.0391, 117.0352,106.0433 | organic acid | * |
| 19 | scoparone | 5.46 | 206.0579 | 207.0553 | −4.9 | [M+H]+ | C11H10O4 | 191.0322,163.0394,151.0760,146.0394, 135.0483,133.0324,117.0365,107.0551 105.0400 | coumarins | [32] |
| 20 | 4-methylumbelliferone | 5.83 | 176.0473 | 177.0543 | −2 | [M+H]+ | C10H8O3 | 149.0636,145.0293,134.0372,117.0361, 115.0533,106.0435,105.0357 | coumarins | [43] |
| 21 | luteolin | 6.20 | 286.0477 | 287.0547 | −1.0 | [M+H]+ | C15H10O6 | 287.0457,269.0372,241.0458,153.0196, 139.0584,135.0468, | flavonoids | [42]* |
| 22 | 2-[(2′E)-3′,7′-dimethyl-2′,6′-octadienyl] -4-methoxy6-methylphenol | 6.35 | 272.2140 | 273.2208 | −1.7 | [M+H]+ | C19H28O | 220.1760,182.0983,165.0724,160.1258, | aromatics | – |
| 23 | eudesm-4(15),7-diene-9α,1 1-diol or isomers | 6.47 | 236.1776 | 237.1846 | −1.3 | [M+H]+ | C15H24O2 | 201.1597,173.1307,161.1351,145.1041 119.0902,107.0936 | terpenoids | – |
| 24 | umbelliferone | 6.99 | 162.0317 | 163.0387 | −1.6 | [M+H]+ | C9H6O3 | 135.0465,133.0284,105.0349,103.0554 | coumarin | [40] |
| 25 | (4E,6E,12E)-tetradeca-4,6, 12-trien-8,10-diyne-1,3,14-triol | 7.01 | 232.1099 | 233.1166 | −2.6 | [M+H]+ | C14H16O3 | 215.1072,187.1096,175.1111,169.1042 159.1181,153.0705,141.0718,131.0877 129.0725,119.0885,115.0578,105.0747, | polyacetylenes | [37] |
| 26 | baicalein | 7.32 | 270.0528 | 271.0589 | −4.3 | [M+H]+ | C15H10O5 | 271.0560, 253.0458,225.0520, 179.0477 169.0115, 151.0026, 123.0077, 95.0141 | flavonoids | * |
| 27 | wogonin | 8.05 | 284.0685 | 285.0752 | −2.0 | [M+H]+ | C16H12O5 | 270.0443,253.0438,242.0519,168.0051 140.0119 | flavonoids | * |
| 28 | 2-methoxy-4-methyl-1-(1-methylethyl)benzene | 8.29 | 164.1201 | 165.1275 | 0.5 | [M+H]+ | C11H16O | 165.0689,163.0529,119.0826,109.0649, 107.0872,105.0707 | terpenoids | – |
| 29 | ethyl 3-(4-Hydroxyphenyl) acrylate or isomers | 8.36 | 192.0786 | 193.0857 | −1.1 | [M+H]+ | C11H12O3 | 161.0597,135.0497,133.0670,131.0500, 118.0442,115.0571,105.0731,103.0578 | aromatics | – |
| 30 | β-elemene or isomers | 9.16 | 204.1878 | 205.1950 | −0.3 | [M+H]+ | C15H24 | 149.1343,121.1009,119.0887,107.0865, | terpenoids | – |
| 31 | 4α,7α-epoxyguaiane-10α,11-diol or isomers | 9.31 | 254.1882 | 255.1955 | 0.3 | [M+H]+ | C15H26O3 | 158.0252,141.0051,133.1038,128.0633, 117.0704,107.0893 | terpenoids | – |
| 32 | (4E,6E,12E)-1-acetoxy-3-(2-methylbutyryloxy) −4,6,12-trien-8,10-diyn-14-ol or isomers | 9.53 | 358.1780 | 359.1856 | 0.8 | [M+H]+ | C21H26O5 | 359.1732,341.1554,331.1816,313.1741, 311.1558,295.1587,271.1628,243.1348, 225.1588,217.1617, 211.1128,105.0704 | polyacetylenes | – |
| 33 | Safrole | 9.74 | 162.0681 | 163.0754 | 0.4 | [M+H]+ | C10H10O2 | 163.0764,135.0829,117.0731,115.0559, 107.0509,103.0564 | aromatics | [44] |
| 34 | atractylenolide Ⅲ | 9.80 | 248.1412 | 249.1481 | −1.6 | [M+H]+ | C15H20O3 | 231.1394,213.1286,203.1434,198.1056, 189.0929,175.0764,163.0761,155.0870, 142.0788,129.0709,117.0710,105.0708 | terpenoids | * |
| 35 | (6E,12E)-1-acetoxytetradeca −6,12-dien-8,10-diyn-3-ol or isomers | 10.02 | 260.1412 | 261.1487 | 0.7 | [M+H]+ | C16H20O3 | 261.1412,243.1405,219.1771,201.1634, 187.0770,173.1337,159.1172,145.1035, 131.0875,115.0575 | polyacetylenes | – |
| 36 | 5-Isopropyl-2-methyl-2,4-cyclohexadien-1-one | 11.10 | 150.1045 | 151.1116 | −0.7 | [M+H]+ | C10H14O | 117.0711,115.0555,109.0644,105.0705, 103.0531 | terpenoids | – |
| 37 | atractylenolide Ⅱ | 12.66 | 232 .1463 | 233.1533 | −1.5 | [M+H]+ | C15H20O2 | 233.1493,215.1396,197.1312,187.1462, 167.0861,159.0830,145.1020,141.0722, 117.0734,115.0579,105.0741 | terpenoids | * |
| 38 | azulene | 13.06 | 128.0626 | 129.0698 | −0.6 | [M+H]+ | C10H8 | 128.0632,117.0701,115.0577,113.0462, 103.0537, 102.0487 | terpenoids | [37] |
| 39 | (4E,6E,12E)-tetradecatrien e−8,10-diyne-1,3-diyl diacetate | 13.44 | 300.1362 | 301.1426 | −2.7 | [M+H]+ | C18H20O4 | 199.1113,178.0808,165.0705,152.0629, 141.0694, 128.0633,115.0557,105.0706 | polyacetylenes | [37] |
| 40 | furanodiene | 13.78 | 216.1514 | 217.1582 | −2.3 | [M+H]+ | C15H20O | 157.1003,143.0871,119.0879,105.0727 | terpenoids | [45] |
| 41 | amylcinnamyl alcohol or isomers | 14.74 | 204.1514 | 205.1588 | 0.6 | [M+H]+ | C14H20O | 187.1524,161.1366,149.0260,141.9626, 128.9546,119.0892,100.9363,97.9725, 81.0740,55.9393 | alkenes | – |
| 42 | curcumene or isomers | 15.62 | 202.1722 | 203.1791 | −1.6 | [M+H]+ | C15H22 | 147.1176,133.1014,119.0875,105.0729 | terpenoids | [46] |
| 43 | atractylenolide Ⅰ | 15.62 | 230.1307 | 231.1376 | −1.7 | [M+H]+ | C15H18O2 | 231.1393,215.1089,201.0933,188.0854, 185.1326,175.0779,165.0710,155.0857, 142.0780,129.0700,115.0546,105.0709 | terpenoids | * |
| 44 | selina-4(14),7(11)-dien-8-one | 17.32 | 218.1671 | 219.1740 | −1.7 | [M+H]+ | C15H22O | 177.1255,141.0732,131.0856,119.0868, 107.0863, 105.0708 | terpenoids | [22] |
| 45 | (E,E,E)-2,4,6-octatriene | 17.36 | 108.0939 | 109.1011 | −0.3 | [M+H]+ | C8H12 | 100.9589 | alkenes | – |
| 46 | (1S,4S)-Bicyclo[2.2.1]hept-5-en-2-one | 18.12 | 108.0575 | 109.0652 | 4 | [M+H]+ | C7H8O | 109.0839,100.9582 | alkenes | – |
| 47 | diisobutyl phthalate or isomers | 18.26 | 278.1518 | 279.1591 | 0.2 | [M+H]+ | C16H22O4 | 201.0448,149.0245,121.0306, | aromatics | [47] |
| 48 | diethyl phthalate | 18.31 | 222.0892 | 223.0965 | −0.1 | [M+H]+ | C12H14O4 | 223.1675,207.0263, 191.0000, 149.0245,121.0316 | aromatics | [48] |
| 49 | atractylodin | 19.85 | 182.0732 | 183.0806 | 0.6 | [M+H]+ | C13H10O | 152.06017,141.0767,139.0542,128.0632 115.0564 | polyacetylenes | [37] |
| 50 | (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol diacetate | 20.38 | 302.1518 | 303.1587 | −1.2 | [M+H]+ | C18H22O4 | 243.1329,172.8635,135.0464 | polyacetylenes | [28] |
| 51 | methyl linolenate | 22.25 | 292.2402 | 293.2470 | −1.7 | [M+H]+ | C19H32O2 | 293.2021,145.0908,121.1054,109.1036, 107.0918,105.0757 | aliphatics | [49] |
| 52 | Sitosterol | 22.31 | 414.3862 | 415.3934 | 4.2 | [M+H]+ | C29H50O | 397.2353,369.2470,341.2659,313.3311, | steroids | [50] |
| 53 | oleanolic acid | 23.63 | 456.3604 | 457.3667 | −2 | [M+H]+ | C30H48O3 | 411.3532,333.1758,315.2569,297.2513, 269.2217, 31.2073,217.1558,203.1818, 189.1646,163.1486,149.1339,135.1182, 119.0892,107.0880 | terpenoids | * |
| 54 | atractyline | 23.94 | 216.1514 | 217.1584 | −1.5 | [M+H]+ | C15H20O | 199.1529,161.1019,147.1244,143.0913, 133.1072,105.0777,95.0565,77.0465, 67.0629 | terpenoids | [51]* |
| 55 | stigmasterol | 25.22 | 412.3705 | 413.3798 | 3.6 | [M+H]+ | C29H48O | 395.3149,383.2175,365.1762,294.1277, 273.1184,243.1100,229.0963,215.0814, 202.0743,165.0706,141.0720,115.0577 | steroids | [52] |
| 56 | monoolein | 26.125 | 356.2927 | 357.3000 | 0.1 | [M+H]+ | C21H40O4 | 357.2905,310.1592,247.2414,149.1308 135.1218,107.0918 | aliphatics | [37] |
| 57 | linoleic acid | 26.27 | 280.2402 | 281.2475 | −0.1 | [M+H]+ | C18H32O2 | 263.2292,245.2210,219.2089,203.1790, 189.1640,175.1473,165.1255,149.1339, 147.1179,133.1031,119.0881,105.0733 | fatty acids | [53] |
| 58 | palmitic acid | 29.04 | 256.2402 | 257.2471 | −1.4 | [M+H]+ | C16H32O2 | 257.1879,178.0783,165.0697,128.0630, 115.0553 | fatty acids | [53] |
| 59 | oleic acid | 29.72 | 282.2559 | 283.2623 | −3.1 | [M+H]+ | C18H34O2 | 263.2288,245.2234,219.2126,203.1795 189.1614,175.1468,161.1329,149.1342 147.1178,135.1181,133.1032, 119.0874, 105.0728 | fatty acids | [53] |
| 60 | atractylenolide VI | 31.04 | 202.1722 | 203.1791 | −1.6 | [M+H]+ | C15H22 | 173.0990,147.1182,133.1031,119.0880 105.0729 | terpenoids | [21] |
| 61 | methyl octadeca-9,12-dienoate | 32.31 | 294.2559 | 295.2628 | −1.4 | [M+H]+ | C19H34O2 | 295.1587,280.1339,237.1231,226.0852, 199.0727,187.0738,159.0804,142.0815, 141.0720,128.0639,115.0562,119.0867 | aliphatics | [54] |
*Compounds 2, 6, 9, 14, 18, 21, 26, 27, 34, 37, 43 and 53 were identified by comparison with reference standards.
Identification of terpenoids
Sixteen compounds (8, 23, 28, 30, 31, 34, 36, 37, 38, 40, 42, 43, 44, 53, 54, and 60) were identified as terpenoids, which have no irregular parent nucleus structure. These compounds were assigned as sesquiterpenoids (23, 30, 31, 34, 37, 40, 42, 43, 44, 54, and 60), diterpenes (8, 28, 36, and 38), and pentacyclic triterpene (53). Compound 60 exhibited a m/z 203.1791[M+H]+ at a Rt of 31.04 min. By comparing Rt and the characteristic ions at m/z 173.0990 [M+H–2CH3]+, 147.1182[M+H–C3H5–CH3]+ with literature data [21]. Compound 60 was tentatively identified as atractylenolide VI. Compound 44 was inferred as selina-4(14), 7(11)-dien-8-one by consulting literature in the same way [22]. Compound 23 (ppm −1.3) with an adduct ion at m/z 237.1846 [M+H]+ produced ions at m/z 201.1597[M+H–2H2O]+, 173.1307[M+H–2H2O–2CH2]+, and 161.1351[M+H–H2O–C3H6O]+. Compound 23 with the core skeleton similar to compounds 60 and 44 was identified as eudesm-4(15),7-diene-9α,11-diol or its isomers. Compounds 34, 37, 43, and 53 were confirmed as atractylenolide Ⅲ, atractylenolideⅡ, atractylenolide Ⅰ, and oleanolic acid by comparison to standards. Atractylenolide Ⅲ, atractylenolide Ⅱ, and atractylenolide Ⅰ possess multiple activities. Atractylenolide Ⅰ and atractylenolide Ⅱ have noticeable anti-tumor activities [9, 23]. Atractylenolide Ⅰ and atractylenolide Ⅲ have excellent anti-inflammatory and neuroprotective activities [24]. Oleanolic acid exerted beneficial bio-active effects including anti-viral, antibacterial, anticarcinogenic, anti-atherosclerotic, anti-diabetes, etc [25]. Mass spectral and chromatographic information, and reference literatures for identification of compounds 30, 31, 36 38, 40, 42, and 54 were listed in Table 1.
Identification of polyacetylenes
Polyacetylenes are a class of vigorous compounds consisting of carbon-carbon triple bond that are abundant in natural medicine. Diverse biological functions including immune regulation, tumor suppression, anti-depressant, and neuroprotection have attracted extensive attention [26]. Compounds 11, 15, 25, 32, 35, 39, 49, and 50 were judged as polyacetylenes. Compound 11 (Rt 2.3 min) displayed an [M+H]+ precursor ion peak at m/z 395.1320 (error ppm −4.2). The formula was calculated as C19H22O9. For the characteristic ion at m/z 232.0632 corresponds to the loss of glucopyranoside, compound 11 was conducted as (8R,9R)-8,9-dihydroxylatractylodinol-9-O-β-D-glucopyranoside. Compound 50 present a precursor ion [M+H]+ at m/z 303.1587 (error ppm −1.2, C18H22O4). The mass spectrum showed the primary fragment ion was m/z 243.1329 by losing of terminal methyl ester. By comparing Rt of literature [27, 28], the compound 50 was considered as (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol diacetate. Other identified polyacetylenes were introduced in Table 1, correspondingly.
Identification of flavonoids and coumarins
Flavonoids have a wide variety of bioactivities including antioxidation, cardio-protective, anti-inflammatory, anticancer, and other properties [29, 30]. Flavonoids possessed a characteristic “C6–C3–C6” skeleton, easy to break glycosidic bonds and Diels-Alder (RDA) reaction generating major fragment ions [29]. Compounds 14, 16, 21, 26, and 27 belong to flavonoids, divided into flavone aglycones (14 and 16) and glycosyl flavonoids (21, 26, and 27). Compound 14 was ascribed to rutin compared with reference standard, which yielded an [M+H]+ ion at m/z 611.1602 and produced fragments ions [M+H–C6H10O4]+ at m/z 465.1037, [M+H–C6H10O4–C6H10O5]+ at m/z 303.0537. As a result, compounds 16, 21, 26, and 27 were identified as puerarin, luteolin, baicalein, and wogonin based on the differentiated data in Table 1.
Coumarins were part of benzopyrone family. Natural coumarins have extensive pharmacological activities such as antifungal, antiviral, Alzheimer's disease inhibition, etc. [31]. The ordinary fragmentation pathway was observed losing neutral molecules carbon monoxide, carbon dioxide, methyl, H2O, and sugar, etc. [30]. For example, compound 19 gave an [M+H]+ ion at m/z 207.0553 and generated fragments ions at m/z 191.0322[M+H–CH4]+, 151.0726[M+H–C3H4O]+, and 145.0237[M+H–C2H6O2]+ (seen in Table 1). Thus, compound 19 was identified as scoparone according to previous literatures [32]. Compounds 10, 17, 20, and 24 were recognized as scopoletin-D-xylopyranosyl-(1→6)-D-glucopyranoside, scopoletin, 4-methylumbelliferone, and umbelliferone in comparison with standard or literatures, respectively.
Identification of organic acids, amino acids, fatty acids, steroids, and the others
Five compounds (3, 4, 12, 13, and 18) were identified as organic acids in the positive mode. Through the characteristic ions at m/z 175.0864 [M+H–H2O]+, 147.0943[M+H–H2CO2]+, 129.0172[M+H–H2O–H2CO2]+, and precursor ion at m/z 193.0345[M+H]+, compound 3 was predicted as citric acid. Compound 4 had deprotonated molecular at m/z 175.0235[M+H]+ at 1.16 min. The loss of carboxyl (HCO2) generated a major fragment ion at m/z 130.0994. Compound 4 was identified as aconitic acid. Fragmentary ions at m/z 145, 149 and 117 can result from compounds 12, 13 and 18, because they have a common mono-acyl chlorogenic acid. Compounds 12, 13, and 18 were recognized as chlorogenic acid, 5-O-feruloylquinic acid, and ferulic acid. Compounds 1, 2, 6, and 9 were classified as amino acids. A parent ion at m/z 116.0711[M+H]+ was observed with Rt of 0.88 min, with the predicted formula C5H9NO2. The fragmentation ion at m/z 70.0735 [M+H–H2CO2]+ was similar to that in documents [33] and human metabolome database. Compound 1 was attributed to proline. Compounds 57, 58, and 59 belong to fatty acids, while compounds 52 and 55 are steroids. By consulting the reference substances with the same MS2 spectra and Rt (seen in Table 1), compounds 2, 6, 9, 52, 55, 57, 58, and 59 were identified as L-tyrosine, L-phenylalanine, L-tryptophan, sitosterol, stigmasterol, linoleic acid, palmitic acid, and oleic acid, respectively.
There are thirteen additional chemicals including 5 (alkaloid), 7 (aldehyde), 22 (aromatics), 29 (aromatics), 33 (aromatics), 41 (alkenes), 45 (alkenes), 46 (alkenes), 47 (aromatics), 48 (aromatics), 51 (aliphatic), 56 (aliphatic), and 61 (aliphatic). On basis of the data found in literature or regular MS splitting decomposition law, compounds 5, 7, 22, 29, 33, 41, 45, 46, 47, 48, 51, 56, and 61 were assigned to uridine, 5-hydroxymethyl furaldehyde, 2-[(2′E)-3′,7′-dimethyl-2′,6′-octadienyl]-4-methoxy6-methylphenol, ethyl 3-(4-Hydroxyphenyl) acrylate or isomers, safrole, amylcinnamyl alcohol or isomers, (E,E,E)-2,4,6-octatriene, (1S,4S)-Bicyclo[2.2.1]hept-5-en-2-one, diisobutyl phthalate or isomers, diethyl phthalate, atractylodin, methyl linolenate, monoolein, and methyl octadeca-9,12-dienoate, respectively.
Quantitative analysis
Method validation
Six different concentrations of mixed standard solutions were applied for constructing standard curves by plotting the peak area (y) versus the concentration (x). Table 2 shows the linear equation with good correlation coefficient (0.9990–0.9999) over a wide linear range of the nine standards. The limit of detection (LOD) and the limit of quantification (LOQ) of analytes were calculated as signal-to-noise of 3:1 and 10:1, respectively. The LODs and LOQs of nine analytes were in the range of 1.86–31.64 ng mL−1 and 7.42–126.56 ng mL−1, respectively. The intra- and inter-day precision of the developed method were analyzed by repeated injection for six times in one day or over three consecutive days, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions of nine targets were 0.71%–2.81% and 1.01%–4.51%, respectively. The repeatability was examined by continuously injecting the same sample for six times. The same sample was analyzed at 0, 4, 8, 12, 24 and 48 h to examine stability. The RSDs of repeatability and stability performed on S7 were ranged from 1.56%–3.30% and 1.02%–3.54%, respectively. A certain quantity of the nine analytes at low, medium, and high concentrations were added into a 0.25 g powder of S7. The recovery was analyzed by comparing the detected amounts of reference compounds with the spiked amount (Tables S2). The average recoveries (n = 3) of the nine analytes were in the range of 95.61%–103.57% with the RSD <4.66. The results indicated that the analytical methods are appropriate for determination of three amino acids, three sesquiterpenoids, a flavonoid, an organic acid, and a pentacyclic triterpenoid, simultaneously.
Linear equation, correlation coefficient, linear range, LODs, LOQs, precision, repeatability, stability, and recovery of nine reference standards
| NO. | Linear equation | Correlation coefficient | Linear range (μg mL−1) | LODs (ng mL−1) | LOQs (ng mL−1) | Precision RSD (%) | Repeatability RSD (%) | Stability RSD (%) | Recovery (%) | ||
| Intra-day | Inter-day | Mean | RSD | ||||||||
| 2 | y = 2.42684e5x+19475.34990 | 0.9994 | 0.10–3.28 | 12.81 | 51.25 | 1.02 | 1.43 | 2.71 | 1.45 | 98.92 | 2.51 |
| 6 | y = 2.81161e5x+5.21415e4 | 0.9999 | 0.66–21.27 | 10.39 | 83.09 | 1.99 | 2.16 | 2.43 | 3.02 | 103.57 | 1.75 |
| 9 | y = 4.90522e5x+17522.03931 | 0.9997 | 0.51–16.20 | 31.64 | 126.56 | 1.46 | 1.31 | 3.30 | 3.54 | 102.32 | 4.66 |
| 14 | y = 7.76104e5x + −4218.35468 | 0.9992 | 0.01–0.48 | 1.86 | 7.42 | 2.81 | 4.51 | 2.75 | 2.65 | 95.61 | 0.41 |
| 18 | y = 2.01936e5x + −4679.18156 | 0.9996 | 0.11–3.40 | 13.28 | 106.25 | 0.92 | 1.20 | 2.15 | 2.00 | 96.47 | 3.87 |
| 34 | y = 6.88885e5x+1.64143e6 | 0.9996 | 1.57–50.31 | 3.07 | 12.28 | 0.79 | 2.12 | 3.10 | 1.16 | 98.89 | 1.45 |
| 37 | y = 3.01940e6x+2.53101e6 | 0.9990 | 0.40–12.78 | 3.12 | 12.48 | 1.54 | 1.71 | 3.15 | 2.86 | 99.10 | 3.36 |
| 43 | y = 2.25477e6x+1.91026e6 | 0.9994 | 0.80–25.70 | 6.27 | 25.10 | 1.25 | 1.60 | 3.01 | 2.51 | 99.63 | 2.87 |
| 53 | y = 6.20298e4x+3.72172e4 | 0.9993 | 0.21–6.58 | 25.68 | 102.73 | 0.71 | 1.01 | 1.56 | 1.02 | 97.82 | 1.13 |
Quantitative determination of nine components in AMR
As obviously displayed in Fig. 2 and Table S3, atractylenolide Ⅲ (150.30–303.20 μg g−1), atractylenolide Ⅱ (119.60–210.80 μg g−1) and atractylenolide Ⅰ (72.84–185.76 μg g−1) were the major bioactive components, which was consistent with previous reports [34]. Aractylenolide Ⅲ, atractylenolide Ⅱ, and atractylenolide Ⅰ were frequently considered as key chemicals for AMR quality control. Few reports concerning quantification of amino acids in AMRs were published. As shown in Table S3, L-tyrosine, L-phenylalanine, and L-tryptophan (14.60–40.00 μg g−1, 117.00–245.60 μg g−1, 27.00–211.60 μg g−1, respectively) were detected in seventeen AMR batches. L-phenylalanine and L-tryptophan were observed abundant in AMR samples. The level of tryptophan in the body is both tightly relevant to depression pathophysiology [35]. The exogenous amino acid could be supplied from the diet of AMR for disease prevention. In addition, the amount of oleanolic acid (2.44–144.36 μg g−1) was reported for the first time. As a note, the amounts of oleanolic acid in S2, S3, S9, S11, and S16 were unable to be calculated for their content was not up to LOQ standard. Rutin, as a familiar flavonoid, had low content (0.4–3.10 μg g−1) in seventeen AMR batches (seen in Table S3). An assessment of the amount of ferulic acid had also been observed at 1.46–3.60 μg g−1. Ferulic acid has multiple functions such as anti-oxidant and anti-inflammatory [36]. The detailed composition profiles of the nine quantified components in seventeen batches of AMR were presented in Fig. 2.
Composition profiles of the quantified nine components in AMR extracts
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01151
Conclusion
In this research, a UPLC-QTOF-MS method was developed for qualitative and quantitative analysis of AMR, simultaneously. The results highlighted nutritional composition such as amino acids, which significantly complement for the chemical profiling of AMR. A total of 61 chemical compositions including 16 terpenoids, 8 polyacetylenes, 6 aromatics, 5 flavonoids, 5 coumarins, 5 organic acids, 4 amino acids, 3 fatty acids, 3 aliphatics, 2 steroids, and 2 alkenes, a nucleoside and an aldehyde were identified. Simultaneously, the contents of three amino acids, three sesquiterpenoids, a flavonoid, an organic acid, and a pentacyclic triterpenoid were determined in seventeen AMR batches. Amino acids and triterpenoid were quantified for the first time in AMR. The established UPLC-QTOF-MS method in this article was practical, useful, and reliable for qualitative and quantitative evaluation of AMR multi-components.
Acknowledgements
This work was funded by grants from the National Science Foundation of China (No. 81973510), the Specialized Research Fund for the Doctoral Program of Wannan Medical College (No. WYRCQD2019008), and the Project of top talents in universities of Anhui Province (No. gxbjZD2022043).
Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2023.01151.
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