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. 2023 Mar 16;13(13):8847–8862. doi: 10.1039/d2ra07591a

An integrated strategy for quality control of the multi-origins herb medicine of Gentianae Macrophyllae Radix based on UPLC-Orbitrap-MS/MS and HPLC-DAD†

Jiangyi Luo a,‡, Hanwen Yuan a,‡, Ling Liang a, Qinling Xie a, Sai Jiang a, Yangfen Fu a, Shenghuang Chen a,✉, Wei Wang a,✉
PMCID: PMC10018649  PMID: 36936846

Abstract

Gentianae Macrophyllae Radix, the dried root of Gentiana macrophylla Pall., Gentiana crassicaulis Duthie ex Burk., Gentiana straminea Maxim., or Gentiana dahurica Fisch., is a traditional Chinese medicine with multi-origins and some adulterants. Liquid chromatography coupled to electrostatic orbitrap high-resolution mass spectrometry (LC-Orbitrap-MS) was used to search the different components of Gentianae Macrophyllae Radix of the four species. High-performance liquid chromatography (HPLC) combined with fingerprint analysis, principal components analysis (PCA), and partial least-squares discrimination analysis (PLS-DA) was also utilized to distinguish them and their adulterants based on the critical components identified by LC-MS. A single standard to determine the multi-components (SSDMC) method was established for the determination of the critical markers. A total of 93 compounds were identified from Gentianae Macrophyllae Radix, including 58 common ones. Their HPLC fingerprints show a significant difference with the adulterants. In addition, PCA and PLS-DA could make a distinction among the four species. Loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside were identified as the critical markers and then quantified by the SSDMC method. The developed strategy is powerful for the quality control and authentication of Gentianae Macrophyllae Radix.

Gentianae Macrophyllae Radix, the dried root of Gentiana macrophylla Pall., Gentiana crassicaulis Duthie ex Burk., Gentiana straminea Maxim., or Gentiana dahurica Fisch., is a traditional Chinese medicine with multi-origins and some adulterants.graphic file with name d2ra07591a-ga.jpg

1. Introduction

Gentianae Macrophyllae Radix, the dried root of Gentiana macrophylla Pall., Gentiana crassicaulis Duthie ex Burk., Gentiana straminea Maxim., or Gentiana dahurica Fisch., has been used as a medicine since Han Dynasty (202 BC to 220 AD) to dispel wind-damp, clear damp-heat, ease pain, and eliminate deficiency-heat.1–4 They are cultivated in different geographic regions in China and are generally known as Qinjiao (QJ), CuJing Qinjiao (CJQJ), MaHua Qinjiao (MHQJ), and Xiao Qinjiao (XQJ) in Chinese due to their different appearances, respectively (Fig. 1).1,5–7 Numerous studies have proved that this herbal medicine is abundant in iridoids and secoiridoids, such as loganic acid and gentiopicroside, which have been recorded as quality markers in the Chinese Pharmacopoeia (2020 edition).3,8 Loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside are reported to have various excellent activities.9–13 Studies have shown that the content of active components of the herb medicine will affect their pharmacological activities, which was the reason for the differences in activity among the Gentianae Macrophyllae Radix of the four species.14–17

Fig. 1. Gentianae Macrophyllae Radix and the adulterants (A: Gentiana macrophylla Pall.; B: Gentiana crassicaulis Duthie ex Burk.; C: Gentiana straminea Maxim.; D: Gentiana dahurica Fisch.).

Fig. 1

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In addition, there are some adulterants used as Gentianae Macrophyllae Radix in the market (Table 1 and Fig. 1), such as Long dan (the rhizome of Gentiana scabra Bge.), Hong qin jiao (the root of Salvia Przewalskii Maxim.), and Ma bu qi (the root of Aconitum sinomontanum Nakai.). Therefore, the identification of different species and authentication are of great importance for the safety and effectiveness of Gentianae Macrophyllae Radix in clinical practice.

The sample information collected in this study.

Sample name Medicine Origin Sample name Medicine Origin Sample name Medicine/chinese name Origin/plant
QJ-1 QJ Yunnan QJ-15 CJQJ Sichuan QJ-29 XQJ Inner Mongoria
QJ-2 QJ Yunnan QJ-16 CJQJ Sichuan QJ-30 XQJ Qinghai
QJ-3 QJ Yunnan QJ-17 MHQJ Xinjiang QJ-31 XQJ Inner Mongoria
QJ-4 QJ Yunnan QJ-18 MHQJ Sichuan QJ-32 XQJ Inner Mongoria
QJ-5 QJ Yunnan QJ-19 MHQJ Qinghai QJ-33 XQJ Xinjiang
QJ-6 QJ Yunnan QJ-20 MHQJ Sichuan A-1 Long dan Gentiana scabra Bge.
QJ-7 QJ Yunnan QJ-21 MHQJ Qinghai A-2 Hong qin jiao Salvia Przewalskii Maxim.
QJ-8 CJQJ Yunnan QJ-22 MHQJ Sichuan A-3 Hong qin jiao Salvia Przewalskii Maxim.
QJ-9 CJQJ Yunnan QJ-23 MHQJ Sichuan A-4 Hong qin jiao Salvia Przewalskii Maxim.
QJ-10 CJQJ Sichuan QJ-24 MHQJ Sichuan A-5 Hong qin jiao Salvia Przewalskii Maxim.
QJ-11 CJQJ Sichuan QJ-25 MHQJ Sichuan A-6 Ma bu qi Aconitum sinomontanum Nakai.
QJ-12 CJQJ Sichuan QJ-26 MHQJ Tibet A-7 Du yi wei Lamiophlomis rotata (Benth.) Kudo
QJ-13 CJQJ Qinghai QJ-27 MHQJ Sichuan A-8 Bai tou wen Pulsatilla chienensis (Bge.) Regel
QJ-14 CJQJ Sichuan QJ-28 MHQJ Qinghai A-9 Bai wei Cynanchum atratum Bunge.
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Liquid chromatography coupled to electrostatic orbitrap high-resolution mass spectrometry (LC-Orbitrap-MS) has the advantages of high resolution, quality accuracy,18 and qualitative analysis of constituents by the in-house and online database. Due to its stability and controllability, high-performance liquid chromatography (HPLC) is still the classic technology for quality control of herbal medicines in pharmacopeia worldwide.

In this paper, the major constituents of Gentianae Macrophyllae Radix were analyzed by LC-Orbitrap-MS. Subsequently, variable influence on projection (VIP) score, K-means calculation, and self-organizing map (SOM) were used to obtain differentials for the compounds, which were then quantified by HPLC and a single standard for determination of multiple components (SSDMC) method.19–21 Finally, the fingerprint analysis, principal components analysis (PCA), and partial least-squares discrimination analysis (PLS-DA) were performed to distinguish QJ, CJQJ, MHQJ, and XQJ, as well as the adulterants.

2. Materials and methods

2.1. Reagents and materials

The reference standards, including loganic acid, swertiamarine, gentiopicroside, and sweroside, were purchased from NatureStandard (Shanghai, China), while 6′-O-β-d-glucosylgentiopicroside was supplied by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). HPLC-grade methanol, acetonitrile, and formic acid were purchased from Sigma-Aldrich (Sigma-Aldrich, Co., Louis, USA). All aqueous solution was prepared with purified water from C'estbon (Shenzhen, China).

Thirty-three batches of Gentianae Macrophyllae Radix (including 7 batches of QJ, 9 batches of CJQJ, 12 batches of MHQJ, and 5 batches of XQJ) were collected from Xinjiang, Yunnan, Tibet, Sichuan, Inner Mongolia, and Qinghai. In addition, nine batches of its adulterants were also collected from different provinces. The details are summarized in Table 1. All the samples were authenticated by Professor Wei Wang (School of Pharmacy, Hunan University of Chinese Medicine), according to the plant morphology. Voucher specimens (QJ 1 ∼ 33, Long dan, Hong qin jiao-1 ∼ 4, Ma bu qi, Du yi wei, Bai tou wen, and Bai wei) were deposited at the TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Material Medical Research Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, China.

2.2. Sample preparation

The air-dried roots were pulverized and passed through a 50-mesh sieve. Then, 0.2 g of the powder was accurately weighed and ultrasonic-extracted with 20 mL methanol for 30 min. After 10 min centrifugation at 13 000 rpm and filtration with a 0.22 μm of filter membrane, the sample solution was collected. The quality control (QC) sample was prepared by mixing an equal volume of each sample solution.

2.3. LC-MS conditions

The LC analysis was run on a Hypersil GOLDTM Aq-C18 column (20 × 2.1 mm, 1.9 μm) (Thermo Sencitific, MA, USA) with a Vanquish™ Flex UPLC system at 30 °C, using 0.01% formic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.3 mL min−1. The gradient elution conditions were as follows: 5–30% B (0–1 min), 30–40% B (1–5 min), 40–90% B (5–6 min), 90–95% B (6–13 min), 95% B (13–21 min), and 5% B (21–24 min). The injection volume was 4 μL. MS analysis (qualitative analysis) was performed on a Orbitrap Exploris 120 in negative ion mode with a full scan MS spectrum over the m/z range 150–1000, using ion spray voltage of 2.5 kV, sheath gas of 50 Arb, aux gas of 10 Arb, sweep gas of 1 Arb, ion transfer tube temp of 325 °C, and vaporizer temp 350 °C. The orbitrap resolution of full scan MS was 60 000 and MS2 was 15 000, and HCD Collision Energies (%) was kept at 30%.

2.4. HPLC analysis

2.4.1. HPLC conditions

The HPLC analysis was conducted on an Agilent 1260 Infinity II HPLC system equipped with a binary pump, an autosampler, a thermostated column compartment, and a diode array detector (Agilent Technologies, Santa Clara, CA, USA). The compounds were separated on a Waters Atlantis® T3-C18 column (4.6 × 50 mm, 5 μm, Waters™, MA, USA) at 28 °C. The mobile phase consisted of 0.04% aqueous formic acid (A) and methanol (B) using a gradient program of 20–25% (B) in 0–13 min, 25–35% (B) in 13–20 min, and 35% (B) in 20–25 min. The flow rate was 1.0 mL min−1. The detection wavelength was 240 nm.

2.4.2. HPLC method validation

The precision of the HPLC analysis method was obtained by injecting six replicates. Six collateral sample solutions were applied to evaluate the repeatability of the approach. The durability was evaluated by analyzing the same sample solution and mix standards using three different columns, including the Waters Atlantis® T3-C18 column (4.6 × 250 mm, 5 μm), Agilent 5 TC-C18 (Agilent Technologies, Santa Clara, CA, USA) and YMC-Pack ODS-A (5 μm, 4.6 × 150 mm, YMC CO., Ltd., Kyoto 600-8106, Japan) in two HPLC systems (Agilent HPLC 1260 II and Shimadzu LC-40 D), respectively. The precision, repeatability, stability, and durability were measured by Relative Standard Deviation (RSD) values of Relative Peak Area (RPA) and Relative Retention Time (RRT). Signal-to-noise ratios (S/N) of 3 and 10 as the standard for Limits of Detection (LOD) and Limits of Quantification (LOQ), respectively. In order to evaluate the recovery, the standard with known concentration was added to the accurately weighed sample in terms of the three concentrations of the high, medium, and low, and prepared in parallel with triplicates according to the sample preparation method. Recovery was then calculated as follows:2.4.2.

In which, m1, m2, and m3 were the amount obtained, the half original amount in the sample, and the amount spiked into the sample, respectively.

2.4.3. SSDMC method development

Five reference standards at known concentrations were prepared as a mixed standard stock solution, which was then diluted to six different concentrations to obtain calibration curves for the quantitative analysis of the sample. SSDMC method was conducted by injecting one reference standard (gentiopicroside), and calculating the content of the other four components in sample solutions according to the response factor (F).2.4.3.where Ai and AS are the peak area of the corresponding compounds obtained from the sample solution and the peak area of gentiopicroside obtained from the standard solution. Ci and Cs are the concentration of components (mg mL−1) to be measured in the test sample and the concentration of gentiopicroside in the reference solution.

2.5. Data analysis

The MS/MS data was analyzed by Freestyle 1.8 and Compounds discover 3.3 software. The compounds were identified by mzVault, mzCloud, ChemSpider, and Mass List Search. The VIP scores, K-means Clustering, SOM, and Pearson correlations were analyzed by Metaboanalyst 5.0. The fingerprint was generated by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2012A) software. PCA, PLS-DA, and HCA were obtained by SIMCA 14.1 software.

3. Results and discussion

3.1. LC-MS analysis

The response of Gentianae Macrophyllae Radix under the negative ion mode was better than the positive model in LC-MS analysis (Fig. S1†). The total ion chromatograms of QJ, CJQJ, MHQJ, XQJ, QC sample, and the adulterants are shown in Fig. 2 and S2.† The total ion chromatograms between Gentianae Macrophyllae Radix and its adulterants exhibit great difference in triterpenoid with retention time from 10 to 20 min (Fig. 2E). On the basis of reference standards, literature data,8,22,23 and the inhouse and online database (inluding mzVault, mzCloud, ChemSpider, and Mass List Search with scores of more than 90), a total of 93 compounds were identified from Gentianae Macrophyllae Radix, including 9 iridoids, 10 secoiridoids, 12 flavonoids, 6 lignans, 38 terpenes, and 18 other types of compounds (Table 2). There were 58 common compounds in QJ, CJQJ, MHQJ, and XQJ (Fig. 3A). Their peak areas data was uploaded to Metaboanalyst 5.0 for statistical analysis (one factor) to screen out the differential components through VIP scores (Fig. 3B). Compounds 18 (gentiopicroside), 12 (6′-O-β-d-glucosylgentiopicroside), and 13 (swertiamarine) were the critical markers due to their high VIP scores. In addition, K-means calculation and SOM specified that compounds 6 (loganic acid), 18 (gentiopicroside), 12 (6′-O-β-d-glucosylgentiopicroside), 13 (swertiamarine), and 83 (soyasapogenol B) were the most critical components (Fig. 3C and D). Therefore, the HPLC analysis focused on these compounds.

Fig. 2. TIC chromatogram of QJ (A), CJQJ (B), MHQJ (C), XQJ (D), and quality control sample (E).

Fig. 2

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The compounds identified by LC-MS.

Peak no. RT (min) Reference ion m/z Diff. (ppm) Formula Fragment ions (m/z) Identification QJ CJQJ MHQJ XQJ
1 1.886 [M − H]− 421.1351 −0.11 C17H26O12 375.12955, 310.12399, 255.08681, 229.04193, 213.07692, 169.05067, 125.0245 Lamiide √ √ √
2 2.176 [M − H]− 315.0724 0.78 C13H16O9 272.01117, 259.91162, 229.02588, 180.87544, 165.01967, 153.01947, 109.02966 Gentisic acid-5-O-β-glucoside √ √
3a 2.377 [M + FA-H]− 391.12454 −0.35 C15H22O9 246.82138, 229.03535, 211.06192, 183.06647, 167.07111, 137.06094, 121.06605 Aucubin √ √ √ √
4 2.439 [M − H]− 375.12949 −0.4 C16H24O10 229.03514, 213.07697, 169.08714, 151.0766, 125.06094 Mussaenosidic acid √ √ √
5 2.499 [M − H]− 188.03529 −0.12 C10H7NO3 181.76221, 159.87849, 144.04565, 116.05076, 89.20306, 81.13036 Kynurenic acid √
6a 2.544 [M − H]− 375.12921 −0.63 C16H24O10 310.93152, 273.17215, 229.03088, 213.077, 169.08717, 151.07664, 125.06109 Loganic acid √ √ √ √
7a 2.584 [M + FA-H]− 725.21478 0.25 C28H40O19 415.73318, 383.1196, 323.09967, 229.04402, 221.06702, 179.0564, 149.06079, 131.03517 Scabrans G3 √ √ √ √
8a 2.601 [M − H]− 373.11379 −0.32 C16H22O10 364.96799, 229.02231, 211.06148, 193.05083, 167.07156, 149.06096, 123.04534 Geniposidic acid √ √ √ √
9a 2.607 [M − H]− 593.15125 0.09 C27H30O15 503.12003, 473.10944, 375.12659, 311.05649, 282.05389, 229.02704, 213.0777 Vicenin-2 √ √ √ √
10a 2.65 [M − H]− 405.13995 −0.6 C17H26O11 281.06702, 243.09027, 229.04758, 221.0452, 197.08226, 179.03546, 155.03568, 141.05588 Shanzhiside methyl ester √ √ √ √
11 2.682 [M + FA-H]− 729.26111 0.17 C32H44O16 383.11844, 359.1492, 329.13965, 310.11627, 139.07669, 101.02464 (+)-Lariciresinol-4,4′-O-β-d-diglucopyranoside √ √
12a 2.707 [M + FA-H]− 563.16179 0.09 C22H30O14 374.97791, 304.93506, 229.04768, 221.06778, 193.04979, 179.05632, 149.06085 6′-O-β-d-glucosylgentiopicroside √ √ √ √
13a 2.754 [M + FA-H]− 419.11919 −0.65 C16H22O10 361.99875, 229.04565, 169.6095, 149.06107, 141.01965 Swertiamarin √ √ √ √
14 2.791 [M − H]− 403.12551 1.13 C17H24O11 273.46982, 249.06258, 229.02711, 195.06598, 179.05597, 161.04498, 153.01945, 149.06076 Gardenoside √
15a 2.798 [M − H]− 681.23997 1.3 C32H42O16 519.13965, 501.16895, 381.1348, 357.13501, 339.12378, 323.07669, 309.11362, 229.02699, 203.07179, 179.05647 Pinoresinol diglucoside √ √ √ √
16a 2.832 [M + FA-H]− 639.15704 0.64 C27H30O15 519.11658, 477.10452, 459.09302, 433.11359, 323.07831, 315.0726, 283.26459, 229.05029, 153.01952 Saponarin √ √ √ √
17 2.867 [M − H]− 447.09268 −1.34 C21H20O11 429.08356, 357.06143, 327.05136, 297.04062, 285.0405, 269.10281, 229.03467, 161.04578 Isoorientin √ √ √
18a 2.881 [M + FA-H]− 401.10856 −1.04 C16H20O9 295.08347, 235.06143, 229.02054, 193.05118, 175.04045, 149.06096, 121.06655 Gentiopicroside √ √ √ √
19a 2.984 [M − H]− 357.11896 0.66 C16H22O9 269.11877, 259.0976, 229.03552, 195.0313, 177.05588, 153.01932, 133.06612 Sweroside √ √ √ √
20 3.026 [M + FA-H]− 581.1883 1.58 C26H32O12 373.12875, 355.11829, 343.1185, 313.1084, 229.02733, 209.08191, 193.0506, 163.04053, 151.04045, 137.02448 8-Hydroxypinoresinol-4′-O-β-d-glucopyranoside √ √
21a 3.062 [M − H]− 431.09847 0.24 C21H20O10 413.0878, 387.07269, 341.06653, 327.05112, 311.0564, 283.05881, 255.06628, 229.04552, 205.01439, 178.99817 Isovitexin √ √ √ √
22 3.083 [M − H]− 359.13467 −0.15 C16H24O9 271.37622, 232.08966, 229.03514, 197.08215, 153.09233, 135.08171, 109.06606, 119.03494 7-Deoxyloganic acid √ √ √
23a 3.093 [M − H]− 521.20322 0.75 C26H34O11 477.13144, 329.13983, 325.05759, 229.02888, 192.07962, 178.06396, 175.07663 Lariciresinol-4-O-glucoside √ √ √ √
24a 3.278 [M − H]− 417.15476 −1.74 C22H26O8 402.13223, 387.10843, 236.06927, 229.04073, 190.0636, 181.05084, 166.02734, 152.04797 (+)-Syringaresinol √ √ √ √
25a 3.28 [M − H + HAc]− 579.20863 0.69 C26H32O11 357.13419, 342.11081, 311.12927, 229.04831, 151.04018, 135.04543 (−)-Pinoresinol glucoside √ √ √ √
26a 3.333 [M − H]− 191.03512 0.72 C10H8O4 176.04822, 163.95222, 147.04532, 144.86652, 111.00893, 87.00882 6,7-Dihydroxy-4-methylcoumarin √ √ √ √
27a 3.404 [M − H]− 609.18261 0.21 C28H34O15 503.62744, 488.21753, 367.08380, 343.082, 325.07181, 301.07217, 286.04901, 257.08224, 229.04509, 179.78441, 125.02504 Hesperidin √ √ √ √
28 3.452 [M + FA-H]− 569.15155 0.16 C24H28O13 476.10995, 474.09473, 388.09415, 374.32324, 289.98453, 229.04309, 137.02455, 93.03461 (+)-Seguinoside D √ √
29a 3.473 [M − H]− 397.11412 0.34 C18H22O10 356.28165, 328.25037, 326.10419, 235.0983, 229.04501, 153.01874, 149.06119 6′-O-Acetyl-gentiopicroside √ √ √ √
30a 3.483 [M + FA-H]− 417.21305 0.1 C19H32O7 349.17096, 252.32874, 229.04927, 161.04509, 141.23784, 123.11726 Blumel C glucoside √ √ √ √
31 3.485 [M − H]− 697.19905 0.69 C31H38O18 655.1897, 571.16766, 535.14624, 475.12473, 409.11395, 367.10291, 349.09293, 315.07251, 229.03539, 153.01949 Gentistraminoside A √ √
32a 3.571 [M − H]− 447.0936 0.82 C21H20O11 357.06213, 327.05191, 285.04074, 229.03545, 177.01965, 116.92889 Kaempferol-7-O-glucoside √ √ √ √
33 3.658 [M − H]− 521.16652 0.31 C25H30O12 359.1142, 357.11865, 315.12424, 297.11343, 229.0264, 213.07632, 195.06635, 163.04022, 151.07657 2′-O-(4′′-Hydroxycinnamoyl)mussaenosidic acid √ √
34 3.993 [M − H]− 755.2045 0.64 C33H40O20 713.19714, 613.17859, 593.15192, 571.16803, 533.12988, 451.12473, 409.11401, 391.10428, 367.10297, 349.09274, 315.07257, 229.0226, 153.01955 Gentistraminoside B √ √
35 4.3 [M − H]− 479.15596 0.44 C23H28O11 357.11929, 273.11514, 234.43077, 229.03462, 195.0663, 151.07655, 121.02967 Albiflorin √
36a 4.306 [M + FA-H]− 493.22922 0.43 C21H36O10 315.18182, 285.11374, 229.05032, 191.05629, 161.04556, 131.03499, 113.02454, 101.02451 Atractyloside A √ √ √ √
37a 4.692 [M − H]− 301.03555 0.57 C15H10O7 286.04874, 257.04626, 242.0584, 233.0457, 193.01447, 164.01154, 151.00389, 125.02461 Quercetin √ √ √ √
38 5.234 [M − H]− 301.14468 0.48 C18H22O4 283.13483, 257.15469, 229.03175, 213.16476, 193.01418, 177.09232, 149.81148, 106.89589 Terbucromil √ √ √
39a 5.272 [M − H]− 797.21476 0.21 C35H42O21 755.20563, 655.18994, 635.16437, 613.17889, 593.15143, 493.13611, 451.12488, 409.11401, 315.07251, 153.01958 Rindoside √ √ √ √
40a 5.385 [M − H]− 269.04572 0.64 C15H10O5 225.0562, 200.88235, 181.91168, 159.04614, 151.00385, 117.03601 Aloe-emodin √ √ √ √
41 5.55 [M − H]− 299.05627 0.54 C16H12O6 284.0329, 256.03848, 229.02341, 190.84854, 169.60602, 134.90634 Hispidulin √ √ √
42a 5.615 [M − H]− 285.04067 0.74 C15H10O6 257.04556, 241.05099, 199.04027, 193.01436, 177.0195, 151.00386, 133.02988 Luteolin √ √ √ √
43 5.847 [M − H]− 781.21987 0.26 C35H42O20 697.20068, 655.1911, 619.16736, 577.15662, 493.13516, 451.12427, 315.07239, 153.0195 Trifloroside √ √
44 6.087 [M − H]− 955.49086 0.08 C48H76O19 835.44794, 793.4389, 731.43817, 613.37518, 569.38464, 523.37915, 455.35229, 229.04059 Gensenoside Ro √
45a 6.782 [M − H]− 519.33282 0.11 C30H48O7 501.32266, 453.3027, 451.28586, 435.28998, 389.28601, 365.28641, 229.04555, 152.99568 Cucurbitacin P √ √ √ √
46 6.833 [M − H]− 793.43851 0.67 C42H66O14 733.41687, 673.39227, 631.38538, 613.37506, 569.38501, 455.3537, 356.71674, 317.46048, 229.02242, 175.02512, 157.01413 Fatsiaside C √
47 6.898 [M − H]− 821.39696 0.54 C42H62O16 759.39392, 645.37128, 627.3584, 351.05676, 333.04721, 289.0556, 229.04875, 193.03555 Glycyrrhizic acid √
48 6.989 [M − H]− 319.1188 0.27 C17H20O6 287.09247, 275.12903, 243.10214, 207.06651, 205.05106, 191.03516, 179.03526, 148.05307 Mycophenolic acid √ √
49a 7.2 [M − H]− 325.20226 0.62 C18H30O5 307.19183, 289.18204, 263.20181, 229.03473, 195.10229, 171.1026, 151.11298, 137.09737, 125.09727, 111.08197 2,3-Dinor-11-β-prostaglandin F2α √ √ √ √
50a 7.265 [M − H]− 485.32726 0.13 C30H46O5 407.29648, 373.28903, 273.11261, 231.10339, 193.05061, 179.03552, 155.53018, 111.57957 (3β,4)-3,23-Dihydroxy-1-oxoolean-12-en-28-oic acid √ √ √ √
51a 7.311 [M + FA-H]− 549.34344 0.11 C30H48O6 470.01324, 441.30182, 393.09708, 349.1084, 285.0416, 229.04987, 193.01363, 111.00887 Arjungenin √ √ √ √
52a 7.329 [M − H]− 299.05623 0.4 C16H12O6 284.03311, 271.06165, 240.04305, 207.03024, 191.03517, 176.01167, 165.01964, 139.0403, 133.02956 Kaempferide √ √ √ √
53 7.4 [M − H]− 373.16547 −0.51 C21H26O6 355.15488, 329.17667, 285.18646, 246.0903, 229.03528, 191.03514, 178.02711 Ustosolate E √
54a 7.407 [M − H]− 487.34297 0.17 C30H48O5 373.74661, 251.06982, 229.02534, 86.7402 Asiatic acid √ √ √ √
55a 7.412 [2 M − H]− 499.30671 0.47 C15H22O3 229.03546, 205.16005, 189.12825, 163.00533, 141.0988, 121.06618, 116.97608 2-[(1S,2S,4aR,8aS)-1-Hydroxy-4a-methyl-8-methylidene-decahydronaphthalen-2-yl]prop-2-enoic acid √ √ √ √
56 7.536 [M − H]− 503.33804 0.85 C30H48O6 490.36255, 301.03766, 247.0618, 229.04099, 193.01413, 152.99648, 116.92847 Sericic acid √
57a 7.656 [M − H]− 269.04576 0.8 C15H10O5 251.20149, 229.0273, 211.13416, 197.15456, 185.11882, 150.9537, 130.08713, 119.43418 Apigenin √ √ √ √
58a 7.693 [M − H]− 499.30665 0.38 C30H44O6 455.31589, 423.28726, 409.31454, 247.75471, 229.04976, 139.07742, 100.93333 11-Deoxocucurbitacin I √ √ √ √
59 7.731 [M − H]− 403.1185 −0.53 C24H20O6 388.09537, 357.11511, 319.18762, 298.02972, 229.04922, 217.0874, 201.05643, 151.96779 Tribenzoin √ √
60 7.777 [M − H]− 343.22738 −1.42 C22H32O3 315.25467, 297.24362, 287.22321, 269.21329, 229.02066, 201.11327, 187.0975, 139.11281 Medroxyprogesterone √ √
61 7.81 [M − H]− 265.12357 0.62 C18H18O2 247.1111, 229.04784, 117.73475, 111.88721 Magnolol √
62 7.86 [M − H]− 285.04067 0.72 C15H10O6 267.19684, 257.0451, 241.21785, 229.04376, 223.20557, 174.75847, 112.1844 Kaempferol √ √
63 7.89 [M − H]− 235.09763 0.54 C13H16O4 229.02434, 199.85121, 189.85136, 176.08412, 163.64058, 134.89507 4-(3-Hydroxy-1-buten-1-yl)-3-methoxy-5-methylbenzoic acid √ √
64 7.956 [M − H]− 299.20142 −0.77 C20H28O2 281.21219, 255.23141, 237.22255, 229.03529, 197.14368, 173.10722, 157.88564 Tretinoin √
65a 7.991 [M − H]− 471.34794 −0.05 C30H48O4 318.6073, 229.02156, 195.29953, 152.99611, 144.25693 Colosolic acid √ √ √ √
66 8.071 [M − H]− 503.33821 0.78 C30H48O6 473.32901, 459.35007, 441.33878, 425.30679, 341.26569, 279.23495, 237.15018, 152.18785 2,3,19,23-Tetrahydroxyolean-12-en-28-oic acid √ √ √
67 8.241 [M − H]− 315.19681 0.77 C20H28O3 296.23151, 271.20706, 243.1758, 229.05154, 120.11904 15d-PGA2 √
68a 8.249 [M − H]− 469.33264 0.66 C30H46O4 451.32596, 425.34454, 383.35666, 280.06595, 229.03511 Enoxolone √ √ √ √
69a 8.341 [M − H]− 455.35293 0 C30H48O3 393.27533, 375.2677, 327.8494, 279.23322, 257.23996, 229.04012, 175.06078, 114.02003 β-Boswellic acid √ √ √ √
70 8.471 [M − H]− 301.18104 0.4 C19H26O3 272.23172, 254.2231, 229.03221, 218.09511, 204.11674, 189.09203 2-Methoxyestradiol √
71a 8.702 [M − H]− 487.3429 0.08 C30H48O5 469.33502, 443.34897, 425.34457, 369.31638, 353.28592, 229.0455 Arjunic acid √ √ √ √
72a 8.734 [M − H]− 455.35301 −0.05 C30H48O3 437.3428, 379.05026, 365.32156, 297.94916, 285.42944, 229.03523, 160.8452 Oleanolic acid √ √ √ √
73a 8.762 [M − H]− 323.25943 0.75 C20H36O3 295.26431, 265.25485, 238.83621, 229.02774, 165.12883, 125.39846 Labdanolic acid √ √ √ √
74a 8.857 [M − H]− 455.35294 −0.24 C30H48O3 437.34277, 411.33011, 229.03543, 214.91963, 144.2729, 128.21217 Ursolic acid √ √ √ √
75 9.112 [M − H]− 485.32726 0.04 C30H46O5 441.33749, 423.3273, 407.29764, 381.31573, 365.28815, 229.02361, 177.2758 Melaleucic acid (6CI) √
76a 9.457 [M − H]− 391.28268 −6.88 C24H40O4 363.28711, 355.32327, 343.26233, 229.02185, 191.03249, 172.81613, 142.60358 Deoxycholic acid √ √ √ √
77a 9.53 [M − H]− 453.33751 0.38 C30H46O3 435.32776, 391.28375, 355.77863, 298.2478, 229.0423, 171.10327 Pinicolic acid √ √ √ √
78a 9.602 [M − H]− 469.33236 0.17 C30H46O4 425.3428, 411.29059, 397.31042, 367.30142, 339.26877, 229.04851 18-β-Glycyrrhetinic acid √ √ √ √
79a 9.664 [M − H]− 453.33746 0.11 C30H46O3 435.32736, 393.31696, 336.5705, 247.89471, 229.03549, 165.65364, 157.5334 Glycyrrhetaldehyde √ √ √ √
80a 10.104 [M − H]− 471.34791 −0.03 C30H48O4 441.33719, 427.35895, 413.30646, 397.35464, 341.28409, 251.1653, 229.02982, 191.1433, 152.99596 Bourjotinolone A (7CI) √ √ √ √
81a 10.432 [M − H]− 783.4906 0.74 C42H72O13 737.48523, 600.46472, 575.43262, 484.41187, 323.10037, 221.06688, 179.05632, 161.04568 Ginsenoside F2 √ √ √ √
82a 12.115 [M − H]− 455.353 0.02 C30H48O3 437.34271, 408.33716, 383.33078, 312.17545, 229.02316, 175.14978 3-Hydroxyurs-12-en-23-oic acid √ √ √ √
83a 13.162 [M − H]− 457.36858 −0.21 C30H50O3 439.36069, 399.32846, 333.66791, 293.06851, 229.04471, 153.84746, 120.77197 Soyasapogenol B √ √ √ √
84a 13.44 [M − H]− 439.35802 −0.25 C30H48O2 313.57648, 263.74875, 229.02229, 194.77271, 163.58023, 137.89886, 120.79447 Roburic acid √ √ √ √
85 13.618 [M − H]− 437.34251 0.02 C30H46O2 419.33331, 365.32208, 361.2926, 345.65881, 229.03558, 152.99625, 127.24102 2,2′-Ethylidene-bis(4,6-di-tert-butylphenol) √
86a 13.873 [M − H]− 415.32155 −0.51 C27H44O3 380.88861, 326.64908, 229.02951, 216.85457, 163.04048, 145.02974, 118.04257 Calcitriol √ √ √ √
87a 13.951 [M − H]− 441.33739 −0.07 C29H46O3 402.7934, 383.35178, 355.32266, 260.00064, 243.20294, 229.04424, 193.1041, 163.04025, 145.02969 4-α-Methylzymosterol-4-carboxylate √ √ √ √
88a 14.016 [M − H]− 425.34254 0.16 C29H46O2 407.33124, 379.39474, 363.36276, 349.29794, 238.83777, 229.03508, 152.10498, 134.22285 4-β-Methylzymosterol-4-carbaldehyde √ √ √ √
89a 14.923 [M − H]− 427.35806 −0.2 C29H48O2 367.33734, 288.5162, 229.05119, 174.3268, 116.92864 (3β,24R,24′R)-fucosterol epoxide √ √ √ √
90a 16.017 [M − H]− 443.35303 −0.06 C29H48O3 305.3432, 300.46295, 252.77142, 229.05147, 201.34343, 163.04047, 145.03, 139.7027, 118.04246 3-β-Hydroxy-4β-methyl-5α-cholest-7-en-4α-oic acid √ √ √ √
91a 17.338 [M − H]− 433.36878 0.19 C28H50O3 397.36902, 389.37885, 322.08398, 258.96381, 229.03616, 180.1319, 152.99579, 146.96379 6-Deoxoteasterone √ √ √ √
92a 18.95 [M − H]− 471.38424 −0.25 C31H52O3 417.68497, 300.66922, 229.03661, 163.04039, 145.02951, 118.04212 (22S,24R)-24-Methyllanosta-8-en-22,28-epoxy-3β,28α-diol √ √ √ √
93 20.902 [M − H]− 485.39998 −0.07 C32H54O3 440.36252, 397.52771, 344.73618, 302.97443, 229.05005, 145.02914, 116.92834 6-Deoxy-16-β-O-acetyl-leucotylin √ √
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a

The common compounds identified from Gentianae Macrophyllae Radix.

Fig. 3. The analysis of LC-MS data (A: the common peaks in Gentianae Macrophyllae Radix; B: the VIP score of 58 common peaks; C: K-means clustering; D: SOM).

Fig. 3

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3.2. HPLC analysis

After optimization, methanol-0.04% formic acid water was finally selected as the elution system (Fig. S3†) for HPLC analysis. Five of the peaks were identified to belong to loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside by comparison with the reference standards (Fig. 4A and B). The precision, stability, and repeatability results are shown in Tables 3 and S1.† The durability result is shown in Table S2.† All the RSD values of the five compounds were less than 3.0%, which indicated this developed method was sensitive, precise, and robust.

Fig. 4. The HPLC chromatograms of Gentianae Macrophyllae Radix (A) and mixed standards (B), the fingerprint of Gentianae Macrophyllae Radix (C), and comparison of Gentianae Macrophyllae Radix and its adulterants (D).

Fig. 4

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The precision, repeatability, and stability of the analysts.

Analyst Precision (n = 6) Repeatability (n = 6) Stability (n = 8)
RRT RRA RRT RRA RRT RRA
RSD (%) RSD (%) RSD (%) RSD (%) RSD (%) RSD (%)
Loganic acid 0.10 0.09 0.28 1.82 0.75 1.63
6′-O-β-d-Glucosylgentiopicroside 0.10 0.31 0.30 2.23 0.66 1.68
Swertiamarine 0.07 0.30 0.26 2.12 0.54 1.59
Gentiopicroside 0.04 0.11 0.18 2.12 0.31 1.73
Sweroside 0.03 0.30 0.14 2.19 0.23 1.71
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All the collected samples were analyzed according to the HPLC method. Thereafter, the data was used to establish the fingerprints. As a result, 16 common peaks were observed in QJ, CJQJ, MHQJ, and XQJ (Fig. 4C). Loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside were relatively abundant in Gentianae Macrophyllae Radix compared with it adulterates, which are the key ingredients for the authentication (Fig. 4D). The content of active compounds is the linchpin for distinguishing Gentianae Macrophyllae Radix of the four species. Gentiopicroside had a significant relationship with swertiamarine (p < 0.01), and sweroside had a significant negative relationship with 6′-O-β-d-glucosylgentiopicroside (p < 0.05) (Table S3† and Fig. 5B). The average content of each component in XQJ is far lower than QJ, CJQJ, and MHQJ, and the content of sweroside in MHQJ is the highest (Fig. 5C). In addition, the five active components of Gentianae Macrophyllae Radix in Yunnan, Sichuan, and Qinghai all show high content, and the content of sweroside in Gentianae Macrophyllae Radix of Qinghai is the highest (Fig. 5D). In order to further confirm the findings, the peak area data of 16 common compounds was used for PCA and PLS-DA, which could also distinguish QJ, CJQJ, MHQJ, and XQJ (Fig. 6).

Fig. 5. The contents of five iridoids in Gentianae Macrophyllae Radix (A), the co-relationships of five active compounds (B), comparison of the contents of five active components among the Gentianae Macrophyllae Radix of the four species (C) and different regions (D).

Fig. 5

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Fig. 6. PCA (A) and PLS-DA (B) analyses.

Fig. 6

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SSDMC method based on the optimized HPLC was developed for their simultaneous detection of the compounds. The calibration curves, linear ranges, LOD, and LOQ of the analytes are shown in Table 4. The average relative response factors (F) for loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, and sweroside were 0.78, 1.97, 0.67, and 0.81, with an RSD of 1.31%, 0.89%, 0.08%, and 1.76%, respectively (Table S4†). Additionally, the recovery of loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside was 102.96–104.52%, 100.74–103.44%, 100.26–105.93%, 98.07–101.45%, and 99.62–102.83%, respectively (Table 5). Combined with the results from method validation in the HPLC fingerprint study, the described SSDMC approach proved to be robust, sensitive, precise, and accurate. As shown in Table 6, the results calculated by the SSDMC method showed no significant difference from the calibration curve method.

The calibration curves of the analysts.

Analyst RT (min) Calibration curve R 2 Linear range (mg mL−1) LOD (mg mL−1) LOQ (mg mL−1)
Loganic acid 14.91 y = 7472.7x + 1.9765 0.9996 0.0065625–0.21 1.09 × 10−5 3.62 × 10−5
6′-O-β-d-Glucosylgentiopicroside 16.51 y = 3007.1x − 0.5279 0.9995 0.004688–0.15 2.81 × 10−5 9.36 × 10−5
Swertiamarine 17.56 y = 8048.6x + 5.1576 0.9994 0.001894–0.0606 1.00 × 10−5 3.34 × 10−5
Gentiopicroside 20.34 y = 5858.9x + 3.1785 0.9996 0.025313–0.81 1.14 × 10−5 3.79 × 10−5
Sweroside 22.06 y = 7385.1x − 1.0209 0.9994 0.001656–0.053 8.35 × 10−6 2.78 × 10−5
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The recovery of the analysts.

Analytes Level Original (mg) Spiked (mg) Found (mg) Average (%) RSD (%)
Loganic acid High 2.8498 2.1 3.684 104.52 1.157
Medium 1.575 3.104 103.47 1.846
Low 1.05 2.548 102.96 1.211
6′-O-β-d-Glucosylgentiopicroside High 0.4512 0.46 0.695 101.44 1.368
Medium 0.23 0.471 103.44 1.926
Low 0.15 0.378 100.74 1.395
Swertiamarine High 0.4905 0.505 0.752 100.26 1.199
Medium 0.2525 0.508 102.09 1.464
Low 0.101 0.367 105.93 1.564
Gentiopicroside High 7.6598 4.536 8.487 101.45 1.169
Medium 3.8475 7.754 101.00 1.822
Low 1.9278 5.647 98.07 1.280
Sweroside High 0.2691 0.212 0.356 102.83 1.191
Medium 0.1378 0.271 99.62 1.853
Low 0.0636 0.203 102.63 1.616
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Comparsion of the contents determined by calibration curve and SSDMC methods.

No. Loganic acid (mg mL−1) 6′-O-β-d-Glucosylgentiopicroside (mg mL−1) Swertiamarine (mg mL−1) Gentiopicroside (mg mL−1) Sweroside (mg mL−1)
Calibration curve SSDMC Calibration curve SSDMC Calibration curve SSDMC Calibration curve Calibration curve SSDMC
QJ-1 0.1893 0.1872 0.0355 0.0354 0.035 0.032 0.6393 0.0045 0.0044
QJ-2 0.1985 0.1963 0.0721 0.0722 0.034 0.032 0.6421 0.0068 0.0067
QJ-3 0.1421 0.1406 0.1208 0.1210 0.023 0.022 0.4400 0.0030 0.0029
QJ-4 0.1846 0.1826 0.1196 0.1198 0.032 0.030 0.5406 0.0061 0.0060
QJ-5 0.1901 0.1880 0.0589 0.0590 0.031 0.029 0.5897 0.0064 0.0063
QJ-6 0.1459 0.1444 0.0565 0.0565 0.027 0.025 0.4982 0.0052 0.0051
QJ-7 0.1385 0.1370 0.0759 0.0760 0.035 0.032 0.6899 0.0073 0.0073
QJ-8 0.1891 0.1870 0.0476 0.0476 0.024 0.023 0.4250 0.0063 0.0062
QJ-9 0.1259 0.1246 0.0578 0.0579 0.023 0.022 0.4030 0.0040 0.0039
QJ-10 0.1814 0.1794 0.0718 0.0719 0.028 0.026 0.4835 0.0065 0.0065
QJ-11 0.0870 0.0861 0.0800 0.0801 0.030 0.028 0.5489 0.0038 0.0037
QJ-12 0.1284 0.1271 0.0711 0.0712 0.023 0.022 0.4566 0.0046 0.0045
QJ-13 0.1840 0.1819 0.0928 0.0930 0.024 0.023 0.4654 0.0056 0.0055
QJ-14 0.1810 0.1790 0.0638 0.0638 0.025 0.024 0.5344 0.0035 0.0034
QJ-15 0.0849 0.0841 0.0558 0.0559 0.025 0.024 0.4274 0.0042 0.0041
QJ-16 0.0911 0.0902 0.0330 0.0329 0.038 0.035 0.6545 0.0058 0.0057
QJ-17 0.1425 0.1410 0.0226 0.0225 0.025 0.023 0.3830 0.0135 0.0135
QJ-18 0.1297 0.1284 0.0332 0.0331 0.021 0.020 0.3502 0.0179 0.0180
QJ-19 0.1531 0.1514 0.0275 0.0275 0.024 0.022 0.3795 0.0267 0.0269
QJ-20 0.0835 0.0827 0.0242 0.0241 0.020 0.019 0.3851 0.0063 0.0063
QJ-21 0.1192 0.1180 0.0396 0.0396 0.033 0.031 0.5625 0.0231 0.0233
QJ-22 0.1325 0.1312 0.0441 0.0441 0.029 0.027 0.5574 0.0111 0.0111
QJ-23 0.1026 0.1016 0.0397 0.0397 0.024 0.022 0.3759 0.0287 0.0290
QJ-24 0.0933 0.0924 0.0430 0.0430 0.023 0.021 0.4458 0.0071 0.0071
QJ-25 0.1103 0.1091 0.0694 0.0695 0.034 0.032 0.6359 0.0104 0.0104
QJ-26 0.1383 0.1368 0.0999 0.1001 0.028 0.026 0.5438 0.0054 0.0053
QJ-27 0.1717 0.1698 0.0278 0.0277 0.024 0.022 0.4260 0.0200 0.0201
QJ-28 0.0875 0.0867 0.0484 0.0484 0.020 0.019 0.3991 0.0295 0.0298
QJ-29 0.0230 0.0230 0.0480 0.0480 0.011 0.011 0.2360 0.0073 0.0073
QJ-30 0.0783 0.0775 0.0240 0.0239 0.020 0.019 0.3645 0.0081 0.0080
QJ-31 0.0282 0.0281 0.0320 0.0320 0.001 0.002 0.0810 0.0035 0.0034
QJ-32 0.0288 0.0287 0.0463 0.0463 0.008 0.008 0.1744 0.0061 0.0060
QJ-33 0.0427 0.0424 0.0232 0.0232 0.007 0.007 0.0926 0.0142 0.0143
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4. Conclusion

In this paper, a LC-Orbitrap-MS method was established to analyze the common or characteristic components of Gentianae Macrophyllae Radix originated from four species, which led to the identification of 93 components, including 58 common ones in the four species. It also proved that Gentianae Macrophyllae Radix mainly contains terpenes (iridoids and triterpenes), flavonoids, alkaloids, lignans, and sterols. The terpenes (with retention time between 10 to 20 min) were the characteristic compounds to identify Gentianae Macrophyllae Radix and its adulterants. The established HPLC fingerprint could also distinguish this medicine and its adulterants depended on the five critical compounds of loganic acid, 6′-O-β-d-glucosylgentiopicroside, swertiamarine, gentiopicroside, and sweroside. Another compound (peak 8) is also one of the specific components of Gentianae Macrophyllae Radix, but it has not been identified (Fig. 4D). In addition, HPLC combined with PCA and PLS-DA could identify QJ, CJQJ, MHQJ, and XQJ based on the content of 16 common peaks. The SSDMC method is also powerful for the determination of five main compounds. It is very important to select the authentic and high-quality medicinal materials because the level of the compounds is directly related to the clinical efficacy.6,7,14,15,24,25

In conclusion, the developed LC-Orbitrap-MS and HPLC strategy is of great importance for quality control and authentication of Gentianae Macrophyllae Radix. The further study is needed for the comparison of pharmacological effects of Gentianae Macrophyllae Radix of the four species and the impact of geographical and ecological environment on its chemicals.

Conflicts of interest

All the authors have declared no conflict of interest.

Supplementary Material

RA-013-D2RA07591A-s001

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2018YFC1707903), Provincial Natural Science Foundation of Hunan (2022JJ40318), Scientific Research Fund of Hunan University of Chinese Medicine (2021XJJJ006), Changsha Municipal Science and Technology Bureau (kq1901095), and Innovation and Entrepreneurship Training Plan for College Students of Hunan University of Chinese Medicine.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2ra07591a

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