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

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.

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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.).

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.

Table 1 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:

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).
where A_i and A_S are the peak area of the corresponding compounds obtained from the sample solution and the peak area of gentiopicroside obtained from the standard solution. C_i and C_s 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).

Table 2 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
a The common compounds identified from Gentianae Macrophyllae Radix.
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	√	√
3^a	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	√
6^a	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	√	√	√	√
7^a	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	√	√	√	√
8^a	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	√	√	√	√
9^a	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	√	√	√	√
10^a	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			√	√
12^a	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	√	√	√	√
13^a	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			√
15^a	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	√	√	√	√
16^a	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		√	√	√
18^a	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	√	√	√	√
19^a	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	√	√
21^a	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	√	√	√
23^a	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	√	√	√	√
24^a	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	√	√	√	√
25^a	3.28	[M − H + HAc]^−	579.20863	0.69	C26H32O11	357.13419, 342.11081, 311.12927, 229.04831, 151.04018, 135.04543	(−)-Pinoresinol glucoside	√	√	√	√
26^a	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	√	√	√	√
27^a	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			√	√
29^a	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	√	√	√	√
30^a	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			√	√
32^a	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			√
36^a	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	√	√	√	√
37^a	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	√		√	√
39^a	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	√	√	√	√
40^a	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	√	√	√
42^a	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		√
45^a	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		√		√
49^a	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	√	√	√	√
50^a	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	√	√	√	√
51^a	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	√	√	√	√
52^a	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		√
54^a	7.407	[M − H]^−	487.34297	0.17	C30H48O5	373.74661, 251.06982, 229.02534, 86.7402	Asiatic acid	√	√	√	√
55^a	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	√
57^a	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	√	√	√	√
58^a	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			√
65^a	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				√
68^a	8.249	[M − H]^−	469.33264	0.66	C30H46O4	451.32596, 425.34454, 383.35666, 280.06595, 229.03511	Enoxolone	√	√	√	√
69^a	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				√
71^a	8.702	[M − H]^−	487.3429	0.08	C30H48O5	469.33502, 443.34897, 425.34457, 369.31638, 353.28592, 229.0455	Arjunic acid	√	√	√	√
72^a	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	√	√	√	√
73^a	8.762	[M − H]^−	323.25943	0.75	C20H36O3	295.26431, 265.25485, 238.83621, 229.02774, 165.12883, 125.39846	Labdanolic acid	√	√	√	√
74^a	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)				√
76^a	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	√	√	√	√
77^a	9.53	[M − H]^−	453.33751	0.38	C30H46O3	435.32776, 391.28375, 355.77863, 298.2478, 229.0423, 171.10327	Pinicolic acid	√	√	√	√
78^a	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	√	√	√	√
79^a	9.664	[M − H]^−	453.33746	0.11	C30H46O3	435.32736, 393.31696, 336.5705, 247.89471, 229.03549, 165.65364, 157.5334	Glycyrrhetaldehyde	√	√	√	√
80^a	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)	√	√	√	√
81^a	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	√	√	√	√
82^a	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	√	√	√	√
83^a	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	√	√	√	√
84^a	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)				√
86^a	13.873	[M − H]^−	415.32155	−0.51	C27H44O3	380.88861, 326.64908, 229.02951, 216.85457, 163.04048, 145.02974, 118.04257	Calcitriol	√	√	√	√
87^a	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	√	√	√	√
88^a	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	√	√	√	√
89^a	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	√	√	√	√
90^a	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	√	√	√	√
91^a	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	√	√	√	√
92^a	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	√	√

---

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).

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).

Table 3 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

---

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

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.

Table 4 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

---

Table 5 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

---

Table 6 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

---

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.

Acknowledgements

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.

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Footnotes

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

‡ Two authors contributed equally to this work.

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