Qualitative and Quantitative Analyses of Active Constituents in Trollius ledebourii

Qualitative and Quantitative Analyses of Active Constituents in Trollius ledebourii Abstract Trollius ledebourii has been more involved in Mongolian medicine and is often used as a type of tea for heat-clearing and detoxifying in the populus. In this study, a rapid and sensitive method was established for the qualitative and quantitative analyses of the major constituents in T. ledebourii. Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was developed for the identification of the multi-constituents in T. ledebourii. A total of 37 chemical constituents in T. ledebourii extract were unambiguously or tentatively identified, including 17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene. Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were first detected in T. ledebourii and the buttercup family. High-performance liquid chromatography–quadrupole ion trap tandem mass spectrometry was applied for the simultaneous determination of 11 compounds, which were either with high contents or strong bioactivities. Satisfactory linearity was achieved with a wide linear range and fine determination coefficient (r > 0.9987). The overall recoveries ranged from 98.07 to 101.2%, and the precision in terms of RSD was <0.74%. The results might provide the basis for quality control analysis of T. ledebourii. Introduction Trollius ledebourii, also known as Tropaeolum majus and Ludilian, is the dried flowers of Trollius chinensis Bunge and T. ledebourii Reichb in the buttercup family and was widely distributed in Inner Mongolia and the Hebei province in northern China. Trollius ledebourii is a type of traditional Chinese medicine (TCM) that is commonly found in tea with multiple functions such as the treatment of tonsillitis, periostitis and conjunctivitis (1–3). As a Mongolian medicine, it has a long history for treatments of aphtha, throat swelling, toothache, eye illness and damp-heat, which were initially recorded in Gang Mu Shi Yi. Four different ephedrine formulations of T. ledebourii prescription preparations were included in the Chinese Pharmacopoeia (2015), and orientin was set as the index component for determination (4). Modern pharmacological studies have shown that T. ledebourii possesses bioactive activities, including anti-inflammation (5), antioxidant, antimicrobial, antivirus (6) and anticancer properties (7, 8). The chemical constituents of T. ledebourii are flavonoids, phenolic acids and alkaloids (9). The flavonoids are the biologically active fractions (8–10). Previous studies have proved that orientin, vitexin and 2″-O-β-l-galactopyranosylorientin were the major constituents with high contents and strong bioactivities in T. ledebourii (11, 12). Recent studies in the qualitative analysis of T. ledebourii have not been thorough. Only Xiaolei Ren et al. (13) have identified 14 compounds including 12 flavonoids and 2 phenolic acids in the extract of the flowers of T. ledebourii by Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS). Other analyses on chemical constituents of T. ledebourii have mostly been investigated using HPLC–UV (14, 15) and HPLC–MS (16) in recent years. Almost all simultaneous determinations of the active constituents in T. ledebourii was carried on HPLC–UV (17–19). In recent years, UHPLC–Q-TOF-MS technology has been extensively applied to the component analysis of TCM (20) and compound preparations (21) with the benefits of high resolution and sensitivity. It is essential to identify complex samples with accurate molecular weights and MS2 fragment ions by TOF-MS. Quadrupole ion trap tandem mass spectrometry (QTRAP–MS-MS) is often used for the quantitative analysis of multiple components storing ions in a trap and selecting expectant mass charge ratio ions. The major advantage of it is strong specificity and sensitivity. In our approach, a total of 37 compounds in T. ledebourii were identified or tentatively characterized by UHPLC–Q-TOF-MS. Their structures were elucidated based on accurate masses, chromatographic behaviors and MS2 fragment ions. Then, a rapid and specific HPLC–QTRAP-MS-MS method was validated for the simultaneous determination of 11 major flavonoids and phenolic acids compounds in T. ledebourii. This method was validated in terms of good linear correlation, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) that could be used for the quality control analysis of T. ledebourii from different habitats. Material and Methods Standards, reagents and samples Orientin (15102309), vitexin (15120611) and diosmetin (15011627) were purchased from Shanghai Shifeng Biological Technology Co., Ltd. Apigenin (101129) and acacetin (100929) were purchased from Shanghai Winherb Medical Technology Co., Ltd. Protocatechuic acid (20151228) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Quercetin (100081-200907), ferulic acid (110773-201313), luteolin (111520-200504), pectolinaringenin (111815-201001), pectolinarin (111728-201001), isorhamnetin (110860-201410), campherenol (110861-201310), caffeic acid (110885-200102) and vanillic acid (110776-200402) were purchased from the National Institute for Food and Drug Control. The purity was higher than 98.0% by the normalization of peak areas detected by the HPLC analysis of these standards. 2″-O-β-l-galactopyranosylorientin (purity > 99.0% by HPLC) was isolated from T. ledebourii in our laboratory. Hyperoside and naringenin were laboratory-made in the medicinal chemistry of natural products laboratory by isolation from Fructus Evodia and Smilax glabra Roxb, respectively. The methods of isolation were reflux extraction, extraction and column chromatographic separation, referring to the previous research by Langsheng Pan et al. (22) and Lei Li (23). The purities were more than 98.0%, as detected by nuclear magnetic resonance (NMR). Acetonitrile was purchased from JT-BAKER Company (USA), formic acid (HPLC grade) was purchased from DIKMA Company (USA). Purified water was obtained from Wahaha (Gangzhou Wahaha Group Co., Ltd., China). The other chemicals were of analytical grade. Five batches of the dried flowers of T. ledebouri Reichb were collected from Chengde of Hebei province (HB-1), Ximeng of Inner Mongolia (NM), Taiyuan of Shanxi province (SX), Kunming of Yunnan province (YN) and Zhangjiakou of Hebei province (HB-2) in China and identified by Ding Zhao, Professor of pharmacognosy of School of Pharmacy in Hebei Medical University. The verified specimens were dried and readied in the Laboratory of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University. The detailed plant source information including collection time, specific location, gathering person and appraiser are listed in Table I. Table I. List of Trollius ledebourii Samples No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  Table I. List of Trollius ledebourii Samples No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  Instrumentation The qualitative analysis was conducted on a UHPLC system (Agilent 1290, USA), which was coupled with a triple TOF™ 5600+ MS/MS system (AB SCIEX, USA); the system was used especially for its Duo-Spray™ source. The UHPLC instrument included a binary pump with an online degasser, an auto plate-sampler and a thermostatically controlled column compartment. Data acquisition was processed on Analyst 1.6.1 software (AB SCIEX, CA, USA). A total of 37 compounds were identified using Masterview 1.1 software and Peakview 2.2 software (AB SCIEX, CA, USA). Quantitative analysis was performed on an LC system (Agilent 1200, USA) equipped with a quaternary solvent delivery system, an autosampler, an automatic degasser, and a column compartment. Mass spectrometric detection was composed of a 3200 QTRAP™ system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion equipped with a Turbo V source, and a TurboIonSpray interface. Data acquisition and procession was performed with Analyst 1.5.2 software (AB SCIEX, Ontario, Canada). Standard solutions and sample preparations Solid portions of quantitative standards (2″-O-β-l-galactopyranosylorientin, orientin, vanillic acid, vitexin, hyperoside, ferulic acid, luteolin, quercetin, apigenin, naringenin and acacetin) were weighed and dissolved in 80% methanol to prepare stock solutions directly. An accurate volume of each standard solution was transferred to the combined solution and diluted step-by-step to prepare a sequence of working mix solutions regularly. The max concentration of each standard solution was 489.4, 350.0, 20.64, 34.22, 33.33, 5.100, 3.267, 1.410, 0.6075, 0.0235 and 1.697 μg/mL. For the qualitative analysis, the other standards were weighed appropriately and dissolved in 80% methanol to determine the limit of detection. Five batches of T. ledebourii samples were comminuted into a fine powder with a size that should be <60 meshes. The powder (1.0 g) was accurately weighted and extracted with 25 mL of 70% methanol in an ultrasonic bath (25 kHz, 100 w) for 30 min, yielding the highest extraction efficiency and lowest noise level. Then, the samples were removed, and any evaporated solvent was compensated with 70% methanol. The mixed solution was vortex blended, allowed to sit and then filtered through a 0.22 μm nylon membrane. The primary filtrate was discarded, and the continuous filtrate was collected for qualitative analysis. The samples for quantitative analysis were similar except for the half-weighing samples. All the samples were stored at 4°C before analysis. UHPLC–Q-TOF-MS analysis The chromatography separation was performed with a Poroshell 120 EC-C18 column (100 mm × 2.1 mm, 2.7 μm) coupled with a C18 pre-column (Security Guard®) with a column temperature of 40°C. It was found that the peak shape and response of analytes were enhanced with a mobile phase of a mixture 0.1% formic acid–water (A) and acetonitrile (B) with an optimized linear gradient elution as follows: 0–14 min, 10–20% B; 14–15 min, 20–25% B; 15–20 min, 25–40% B; 20–26 min, 40–60% B; 26–27 min, 60–90% B; and 27–30 min, 90% B. The flow rate was set to 0.30 mL/min. The injection volume was 2 μL. The mass spectrometer was operated in positive mode because of the instrument status and response values, and the parameters of the MS/MS detector were set as follows: electrospray ionization (ESI) source with a turbo spray temperature of 550°C; ion spray voltage (IS): 5,500 V, nebulizing gas (Gas1): 55 psi, TIS gas (Gas2): 55 psi, curtain gas: 35 psi, and declustering potential (DP): 60 V. The IDA (information dependent acquisition) criteria were provided for the ions that match the mass defect window to obtain the MS/MS spectra. The experiments were run with scans of 100–1,000 and 50–1,000 amu for the full MS and MS/MS experiments, respectively. Additionally, the MS/MS experiments were run with 200 and 70 ms of accumulation time for the full MS and MS/MS experiments. The collision energy spread (CES) was set at 35 ± 15 eV, which had been optimized for observing better MS2 spectra. Simultaneously, the calibration delivery system was acquired for calibrating mass numbers online. HPLC–QTRAP-MS-MS analysis The chromatography separation was processed on a Diamonsil C18 column (150 mm × 4.6 mm, 5μm), and the column was maintained at room temperature. The formulation of the mobile phase was 0.1% formic acid–water (A) and acetonitrile (B). The gradient elution mode was as follows: 0–6 min, 20–40% B; 6–12.5 min, 40–75% B; 12.5–13 min, 75–90% B; and 13–15 min, 90% B. The injected sample volume was 10 μL, and the flow rate was 0.8 mL/min. The mass spectrometer was operated in negative mode, and the parameters of the MS/MS detector were set as follows: source temperature: 650°C, ESI source voltage: −4,500 V, nebulizer gas (Gas1): 60 psi, turbo gas (Gas2): 65 psi, and curtain gas (CUR): 30 psi. Nitrogen gas was used for the entire analysis, and the heating of the electrospray interface was continuous. The collision activated dissociation (CAD) gas level was set at medium. Multi-reaction monitoring (MRM) technology for the triple-quadrupole tandem mass spectrometer was applied for quantitative analysis. Q1 and Q3 were operated at unit mass resolution. The dwell time was 50 ms, with a 5 ms pause between scans. Monitoring ion-pairs, the value of DP and the value of CE of each desired component in multicomponent are listed in Table II. The structures of the 11 compounds, MRM chromatograms of reference substances and T. ledebourii samples, second mass spectra of the reference substances and chromatograms of the standard mixture under the optimal separation conditions are shown in Figure 1. Table II. HPLC–ESI-MS-MS Data of 11 Constituents From Trollius ledebourii Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  aMonitored MRM transitions. Table II. HPLC–ESI-MS-MS Data of 11 Constituents From Trollius ledebourii Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  aMonitored MRM transitions. Figure 1. View largeDownload slide Multiple-reaction monitoring chromatograms of reference substances (A) and Trollius ledebourii samples (B) and second mass spectra of reference substances (C). Figure 1. View largeDownload slide Multiple-reaction monitoring chromatograms of reference substances (A) and Trollius ledebourii samples (B) and second mass spectra of reference substances (C). Establishment of T. ledebourii chemistry database The related chemical composition knowledge about of different growth parts for T. ledebourii and similar plants in the buttercup family were gathered by analysing the massive literature. Each compound with its name and molecular formula were inputted into the Masterview 1.1 software, which can produce the exact mass number automatically. According to the information, a comprehensive database including molecular formula, English name, exact mass number and ion mode was developed. Results Analysis of chemical constituents in T. ledebourii extract Through the above analysis strategies, a total of 37 chemical constituents in T. ledebourii extract were identified by UHPLC–Q-TOF-MS based on PeakView™ 2.2 (AB Sciex) data processing and 18 of them (compounds 2, 6, 7, 9, 10, 15, 16, 17, 20–22, 26, 28–31, 35 and 36) were unambiguously confirmed by comparing their retention times, as well as MS and MS2 fragment ions, with those of the reference standards; 19 compounds were tentatively assigned based on their MS2 fragment ions, after referring to previous studies. Compared with the database of compounds from the plants of T. ledebourii and other plants in the buttercup family, 5 flavones (compounds 20, 28 and 35–37), 2 flavonols (compounds 21 and 29), 16 flavonoid glycosides (compounds 7–9, 11–16, 19, 23–25, 27, 32 and 34), 4 phenolic acids (compounds 3, 5, 10 and 18), 1 amide (compound 1) and 1 triterpene (compound 33) have already been reported (7, 12, 13, 23–36). Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were detected in T. ledebourii and the buttercup family for the first time. The mass error for practical molecular ions of all identified compounds was within ±5 ppm. The retention times, mass number, mass error and MS2 fragments of the identified compounds in T. ledebourii are summarized in Table III. The UHPLC–Q-TOF-MS total ion chromatograms of T. ledebourii samples in positive ion mode are displayed in Figure 2. The chemical structures and MS2 spectra of the 37 compounds are shown in Figures 3 and 4. Table III. Qualitative Analysis of Chemical Constituents in Trollius ledebourii No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  *Compared with reference standards. Table III. Qualitative Analysis of Chemical Constituents in Trollius ledebourii No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  *Compared with reference standards. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of Trollius ledebourii samples in positive ion mode. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of Trollius ledebourii samples in positive ion mode. Figure 3. View largeDownload slide View largeDownload slide Chemical structures of the 37 compounds in Trollius ledebourii. Figure 3. View largeDownload slide View largeDownload slide Chemical structures of the 37 compounds in Trollius ledebourii. Figure 4. View largeDownload slide View largeDownload slide View largeDownload slide MS2 spectra of 37 compounds in Trollius ledebourii. Figure 4. View largeDownload slide View largeDownload slide View largeDownload slide MS2 spectra of 37 compounds in Trollius ledebourii. Mass spectra for fragmentation behavior of 37 compounds Flavonoid glycosides Compound 7 (tR = 7.25 min) and compound 8 (tR = 7.59 min) yielded a [M + H]+ at m/z 611.1632 with the molecular formula C27H30O16 and a [M + H]+ at m/z 581.1513 with the molecular formula C26H28O15, which gave rise to fragment ions at m/z 449, m/z 431, m/z 413 and m/z 329, corresponding to the characteristics of orientin. Comparing with the retention time and MS2 fragment ions of the reference substance or the data reported in the literature, compound 7 was unequivocally identified as 2″-O-β-l-galactopyranosylorientin, and compound 8 was tentatively identified as 2″-O-β-d-xylopyranosylorientin (26). Compound 9 (tR=7.86 min) produced [M + H]+ at m/z 449.1102 with the molecular formula C21H20O11. Furthermore, the fragmentation ions at m/z 431, m/z 413, m/z 353 and m/z 329 corresponded to orientin. This compound has previously been isolated from T. ledebourii (27). Compound 11 (tR=8.30 min) and compound 12 (tR=8.31 min) were comprised of the same aglycone, quercetin and exhibited an identical [M + H]+ at m/z 597.1402 with the molecular formula C26H28O16 and [M + H]+ at m/z 465.1202 with the molecular formula C21H20O12, respectively. The same characteristic ion observed at m/z 303, corresponded to the complete loss of glycosides in the structure. Thus, compounds 11 and 12 were assigned as 3-O-sambubiosylquercetin (28) and 3-O-glucopyranosylquercetin (29, 30), according to previous literature. Compound 13 (tR=8.50 min) and compound 14 (tR=9.23 min) displayed a [M + H]+ at m/z 595.1690 with the molecular formula C27H30O15 and a [M + H]+ at m/z 565.1530 with the molecular formula C26H28O14, both of which showed a common MS2 fragment at m/z 433 ascribed to the loss of a glucose moiety [M + H-C6H10O5]+ and a xyloside unit [M + H-C5H8O4]+, respectively. The fragment ions of compounds 13 and 14 at m/z 415 and m/z 397 were both the characteristic ions of vitexin. Therefore, compounds 13 and 14 were tentatively identified as 2″-O-β-l-galactopyranosylvitexin (28) and 2″-O-β-d-xylopyranosylvitexin (24). Compound 15 (tR=9.61 min) produced [M + H]+ at m/z 433.1178 with the molecular formula C21H20O10. Furthermore, the fragmentation ions at m/z 415, m/z 397, m/z 367 and m/z 283 corresponded to vitexin. Then, by comparing with the retention times and MS2 fragment ions of the reference substances, compound 15 was confirmed as vitexin. This compound also has previously been isolated from T. ledebourii (13, 25, 27). Compound 16 (tR=10.65 min) gave a [M + H]+ at m/z 465.1021 with the molecular formula C21H20O12, and showed a fragment ion at m/z 303.0530 [M + H-C6H10O5]+, the special fragment ion of the aglycone quercetin. Further comparing with the reference substances, it was definitively identified as hyperoside (13). Compound 19 (tR=17.51 min) and 27 (tR=19.42 min) presented the same aglycone orientin and gave a [M + H]+ at m/z 533.1652 with the molecular formula C26H28O12 and a [M + H]+ at m/z 547.1816 with the molecular formula C27H30O12, respectively. Furthermore, comparing the MS2 spectra with the literature (27, 30), compounds 19 and 27 were tentatively identified as 2″-O-(2‴-methylbutanoyl) orientin and 7-methoxy-2″-O-(2‴-methylbutanoyl) orientin. Compound 23 with the molecular formula C28H32O14 generated the characteristic ions of m/z 593.1858 [M + H]+ and m/z 285.0762 that were the neutral loss of a neohesperidoside. Compound 23 was identified as 7-O-neohespeidosylacacetin by comparison with that of the literature (13). Compounds 24 (tR=18.13 min) and 25 (tR=18.45 min) possessing the same aglycone vitexin presented a [M + H]+ at m/z 597.1594 with the molecular formula C30H28O13 and a [M + H]+ at m/z 517.1707 with the molecular formula C26H28O11, respectively. Compound 24 had the fragment ions at m/z 579.1439 [M + H-H2O]+ and m/z 477.1162 [M + H-C4H8O4]+, and compound 25 had fragment ions at m/z 499.1587 [M + H-H2O]+ and m/z 433.1602 [M + H-C5H8O]+. By comparing with the data reported, compounds 24 and 25 were tentatively identified as 2″-O-(3″, 4‴-dimethoxybenzoyl) vitexin and 2″-O-(2‴-methylbutanoyl) vitexin respectively (28). The fragment ions at m/z 477.1375 [M + H-Rha]+ and m/z 315.0864 [M + H-Rha-Glu]+ were the characteristic ions of compound 22 producing a [M + H]+ at m/z 623.1964 with the molecular formula C29H34O15. After a comprehensive analysis of the mass data with the references, compound 22 was identified as pectolinarin, and pectolinarin has not been reported to be present in T. ledebourii previously. Compounds 32 (tR=20.18 min) generated a [M + H]+ at m/z 531.1879 with the molecular formula C27H30O11. The characteristic fragment ions were m/z 447.1691 [M + H-C5H8O]+ and m/z 429.1191 [M + H-C5H8O-H2O]+. According to the knowledge in the literature (31, 32), compounds 32 was tentatively identified as 2″-O-(2‴-methylbutanoyl) isoswertisin or 3″-O-(2‴-methylbutanoyl) isoswertisin. Compound 34 (tR=20.81 min) showed a [M + H]+ at m/z 447.0680 with the molecular formula C22H22O10. It yielded the diagnostic fragment ions at m/z 429.1151, which lost a H2O from the parent ion. Compound 34 were tentatively identified as isoswertisin (31, 33). Flavones Compound 20 at 17.86 min presented a molecular ion at m/z 287.0542 [M + H]+ with the molecular formula C15H10O6. Comparing with the retention time and MS2 fragment ions of the reference substance, compound 20 was unequivocally identified as luteolin (23). Compound 26 (tR=19.29 min) displayed a [M + H]+ at m/z 271.0597 with the molecular formula C15H10O5, which yielded a fragment ion at m/z 243.0666 corresponding to the loss of a CO. By comparing our data with its standard, compound 26 was identified as apigenin. Compound 30 (tR=19.78 min) showed a [M + H]+ at m/z 301.0709 with the molecular formula C16H12O6, which presented fragment ion at m/z 286.0476 and 258.0521 corresponding to the loss of a methyl group and a CO, respectively. After comparing with the reference substances, the species was confirmed as diosmetin, which has not been previously identified in T. ledebourii. Compound 35 (tR=23.11 min) was the aglycone of compound 23 (tR=17.98 min) and gave a [M + H]+ at m/z 285.0758 with the molecular formula C16H12O5, exhibiting ions at m/z 270.0514 [M + H-CH3]+ and m/z 242.0560[M + H-CH3-H2O]+. It was identified as acacetin upon comparison with the standards. Compound 36 (tR=23.47 min) was the aglycone of compound 22 (tR=17.97 min) which displayed a [M + H]+ at m/z 315.0864 with the molecular formula C17H14O6, observed at m/z 300.0628 [M + H-CH3]+ and m/z 282.0804 [M + H-CH3-H2O]+. It was tentatively identified as pectolinaringenin (13). The final compound 37 (tR=25.39 min) was also the flavonoid compound producing a [M + H]+ at m/z 329.1038 with the molecular formula C18H16O6, which observed fragment ions at m/z 314.0799 [M + H-CH3]+, m/z 296.0694 [M + H-CH3-H2O]+ and m/z 268.0743 [M + H-CH3-CO]+. The characteristic ions were ascribed to salvigenin that has been reported in T. ledebourii in a previous study (13). Flavonols Compound 21 (tR=17.90 min) yielded a [M + H]+ at m/z 303.0483 with the molecular formula C15H10O7, which was similar to the fragment pattern of luteolin. It gave fragment ions at m/z 285.0434 and m/z 257.0431, corresponding to the neutral loss of an H2O and a CO. Furthermore, by comparing with the standards, compound 21 was identified as quercetin (13). Compound 29 was eluted at 19.43 min yielding a molecular ion at m/z 287.0539 [M + H]+ with the molecular formula C15H10O6. By comparing with the retention time and MS2 fragment ions of the reference substances, compound 29 was unequivocally identified as campherenol (34, 35). Compound 31 (tR=20.06 min) yielded a [M + H]+ at m/z 317.0651 with the molecular formula C16H12O7, which gave the MS2 ion at m/z 302.0468 corresponding to the loss of a methyl group. By comparing with standards, compound 31 was identified as isorhamnetin. Dihydroflavones Compound 28 (tR=19.43 min), the derivative of dihydro-flavonoids, gave a precursor ion [M + H]+ at 273.0771 with the molecular formula C15H12O5. The characteristic ion at m/z 255.0757 was inferred as a loss of an H2O. The compound was identified as naringenin according to the reference substances. This is the first time this compound is identified in T. ledebourii. Phenolic acids Compound 2 (tR=2.02 min) generated a [M + H]+ at m/z 155.1029 with the molecular formula C7H6O4. Then, the [M + H]+ ion fragmented into two characteristic ions at m/z 137.0232 and m/z 109.0287, which corresponded to [M + H-H2O]+ and [M + H-H2O-CO]+. This was confirmed by preparing the compound with a reference standard. Accordingly, the structure was identified as protocatechuic acid. Compound 3 (tR=3.14 min) showed a [M + H]+ at m/z 139.0392 with the molecular formula C7H6O3. The ion at m/z 121.0284 could be formed by the loss of a molecule of H2O. Compound 3 was identified as hydroxybenzoic acid (12) based on comparisons with the reference substances. Compound 4 (tR=3.47 min) gave a [M + H]+ at m/z 167.0681 with the molecular formula C9H10O3. Its MS2 spectrum gave a characteristic ion at m/z 149.0591, which was identified as the loss of a molecule of H2O. The chromatogram retention time and MS2 fragments correspond to those of the reference solution. Therefore, it was validated as paeonol, and it is the first time it is identified in T. ledebourii. Compound 5 (tR=4.10 min) displayed a [M + H]+ at m/z 183.0661, and its molecular formula was C9H10O4. The fragment ions observed at m/z165.0546 and m/z 139.0758 were the loss of an H2O and a carboxyl group, respectively. Therefore, compound 6 was tentatively identified as veratric acid (25) by comparing the MS/MS fragmentation pattern with those found in the literature. Compound 6 (tR=4.23 min) displayed a molecular ion at m/z 181.0493 [M + H]+ with the molecular formula C9H8O4 in positive ionization mode. Further MS2 scans showed that it produced similar fragment ions at m/z 163.0384 [M + H-H2O]+ and m/z 145.0281 [M + H-2H2O]+, indicating the loss of an H2O group. Thus, compound 6 was identified as caffeic acid, upon comparison with the standards. However, this is the first study identifying caffeic acid in T. ledebourii. Compound 10 (tR=3.88 min) presented a [M + H]+ at m/z 169.1023 with the molecular formula C8H8O4, producing fragments at m/z 151.0392 and m/z 125.0607 that were ascribed to [M + H-H2O]+ and [M + H-COO]+, respectively. By comparing with the standards, it was confirmed as vanillic acid. Compound 17 (tR=8.09 min) presented a [M + H]+ at m/z 195.0634 with the molecular formula C10H10O4, producing fragments at m/z 180.0540 and m/z 151.0613 that were ascribed to [M + H-CH3]+ and [M + H-COO]+, respectively. The retention time and MS2 fragment ions were consistent with a reference substance. Therefore, compound 17 was identified as ferulic acid. To our knowledge, this compound has only been reported in the genus but not in T. ledebourii. Compound 18 (tR=13.43 min) showed a precursor ion [M + H]+ at m/z 237.1118 with the molecular formula C13H16O4, and yielded a MS2 fragment at m/z 219.0985, corresponding to the loss of an H2O, suggesting that this compound was proglobeflowery acid per the literature (26). Amides Compound 1 (tR=1.95 min) yielded a [M + H]+ at m/z 182.0211 with the molecular formula C9H11NO3, which gave characteristic MS2 fragments at m/z 166.9899, m/z 164.0356, m/z 137.0595 and m/z 119.0483 corresponding to the loss of one methyl and one H2O. Therefore, compound 1 was tentatively proposed to be veratrum amide (7, 24) according to the literature. Triterpenes Compound 33 (tR=20.43 min) was a phenolic acid compound that generated [M + H]+ at 457.2314 and a fragment ion at m/z 439.2022 [M + H-H2O]+. Based on the above data, compound 33 was identified as ursolic acid (36). Fragmentation patterns of the representative biologically active components in T. ledebourii The fragmentation patterns of flavone C-glycosides and flavones are shown in Figures 5 and 6, and 2″-O-β-l-galactopyranosylorientin and luteolin were used as examples for detailed explanations, respectively. Figure 5. View largeDownload slide Hypothesized fragmentation pattern of protonated 2″-O-β-l-galactopyranosylorientin generated by UHPLC–Q-TOF-MS. Figure 5. View largeDownload slide Hypothesized fragmentation pattern of protonated 2″-O-β-l-galactopyranosylorientin generated by UHPLC–Q-TOF-MS. Figure 6. View largeDownload slide Hypothesized fragmentation pattern of protonated Luteolin generated by UHPLC–Q-TOF-MS. Figure 6. View largeDownload slide Hypothesized fragmentation pattern of protonated Luteolin generated by UHPLC–Q-TOF-MS. Methodological validation of the quantitative analysis 2″-O-β-l-Galactopyranosylorientin, orientin, vanillic acid, vitexin, hyperoside, ferulic acid, luteolin, quercetin, apigenin, naringenin and acacetin were quantified by HPLC–QTRAP-MS-MS, which showed high sensitivity in the qualitative and quantitative determination of the analytes (Figure 7). The total values of these 11 compounds are listed in Tables IV and V. The 11 compounds exhibited good linear regression (r > 0.9987) with in the detected ranges. Additionally, the LODs of the 11 constituents were estimated to be 0.0128–8.600 ng/mL. The LOQs were 0.1809–76.00 ng/mL. The relative standard deviation (RSD) values of all these 11 compounds observed from intra-day and inter-day precision studies were <1.76%. The recovery values were 98.07–101.2% with an RSD less than 2.00%, suggesting that the method was accurate for determining the analytes. The RSD of the storage stability was less than 1.96% within 48 h. Figure 7. View largeDownload slide Chromatograms of standard mixture under the optimal separation conditions by HPLC–QTRAP-MS-MS. 7: 2″-O-β-L-galactopyranosyl-orientin, 9: orientin, 10: vanillic acid, 15: vitexin, 16: hyperoside, 17: ferulic acid, 20: luteolin, 21: quercetin, 26: apigenin, 28: naringenin, 35: acacetin. Figure 7. View largeDownload slide Chromatograms of standard mixture under the optimal separation conditions by HPLC–QTRAP-MS-MS. 7: 2″-O-β-L-galactopyranosyl-orientin, 9: orientin, 10: vanillic acid, 15: vitexin, 16: hyperoside, 17: ferulic acid, 20: luteolin, 21: quercetin, 26: apigenin, 28: naringenin, 35: acacetin. Table IV. Regression Equations, Linear Ranges, Correlation Coefficients, LODs and LOQs of 11 Compounds Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  aY, peak area and X, concentration (μg/mL). bLOD (S/N = 3). cLOQ (S/N = 10). Table IV. Regression Equations, Linear Ranges, Correlation Coefficients, LODs and LOQs of 11 Compounds Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  aY, peak area and X, concentration (μg/mL). bLOD (S/N = 3). cLOQ (S/N = 10). Table V. Precision, Accuracy and Atability of the 11 Components From Trollius ledebourii Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    aRecovery (%) = (detected amount-original amount)/spiked amount × 100. bRSD (%) = (SD/mean) × 100. Table V. Precision, Accuracy and Atability of the 11 Components From Trollius ledebourii Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    aRecovery (%) = (detected amount-original amount)/spiked amount × 100. bRSD (%) = (SD/mean) × 100. Sample analysis In this study, the HPLC–QTRAP-MS-MS analytical method was first applied to simultaneously determine 11 compounds including flavones, flavonoid glycosides and phenolic acids in T. ledebourii samples collected from various locations. The contents of the 11 investigated compounds in 5 batches of T. ledebourii samples (n = 3) are exhibited in Table VI. However, there was a difference between the chemical constituents of the five samples collected from different geographical locations, especially for the contents of flavonoid constituents. The total contents of each batch of the 11 investigated constituents ranged from 17.02 to 28.60 mg/g, and the content of the flavonoid constituents in the five batches ranged from 16.72 to 28.29 mg/g. The content of nine flavonoid compounds was considerably more than those of two phenolic acid compounds. Orientin, vitexin and 2″-O-β-l-galactopyranosylorientin are the characteristic components of T. ledebourii and presented the highest contents among the 11 components. The contents of the flavonoid compound orientin were all higher than 10.00 mg/g, which could be considered as the quality control component. T. ledebourii, which was collected from Taiyuan city of Shanxi province (SX), was tentatively considered to be the best quality with the content of 15.97 mg/g orientin. From the above data, the difference among the contents of each component in different batches was closely related to the geographical location and collection time of the medicinal herb T. ledebourii. Table VI. Contents of the 11 Active Components in Trollius ledebourii Samples Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Table VI. Contents of the 11 Active Components in Trollius ledebourii Samples Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Discussion According to our knowledge, there are too many compounds in herbal medicine. Which compound should be quantified and how to avoid its cross-talk become significant issues. A large number of papers have determined that flavones, flavonoid glycosides and a part of phenolic acids were biologically active constituents of T. ledebourii (9–12). In our assay, a quantification of nine flavonoids and two phenolic acids that are considered as active and with high levels in T. ledebourii extract were developed that can be useful for quality control. However, two different techniques were applied for qualitative and quantitative analyses depending on their specific benefits. Though UHPLC–Q-TOF-MS has the high resolution to identify compounds that hold similar MS ions, it is easily disrupted by temperature so that it has poor reproducibility with quantified analytes. HPLC–QTRAP-MS-MS possesses high sensitivity and reproducibility contributing to its MRM pattern using ion-pairs. To achieve low detection limits required for all analytes, methods of qualitative and quantitative analyses of analytes in positive and negative ionization modes were developed. In consideration of their stability and high abundance, positive mode was chosen for the qualitative analysis by UHPLC–Q-TOF-MS, and negative mode was chosen for the quantitative analysis by HPLC–QTRAP-MS-MS. To achieve a better peak shape and a high response for the analysis of most compounds, optimization of the mobile phase was conducted by comparing types and additions. It was found that the best composition consisted of acetonitrile and 0.1% formic acid aqueous solution. Conclusions In this study, an UHPLC–Q-TOF-MS method was developed to investigate the chemical constituents in T. ledebourii. Based on exact masses, retention times, chromatographic behaviors and characteristic fragment ions, a total of 37 compounds (17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene) were identified or tentatively identified in 25 min, and their representative fragmentations were summarized. The fragment patterns of the major compounds might contribute to its metabolites identification in a later study. To the best of our knowledge, protocatechuic acid, paeonol, caffeic acid, ferulic acid, pectolinarin, naringenin, isorhamnetin and diosmetin have not been reported in T. ledebourii and the buttercup family. HPLC–QTRAP-MS-MS was established for the first time, which was successfully applied to simultaneously determine 11 compounds in T. ledebourii. This full scale qualitative and quantitative study sheds some new light on the quality control of T. ledebourii. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Qualitative and Quantitative Analyses of Active Constituents in Trollius ledebourii

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmy035
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Abstract

Abstract Trollius ledebourii has been more involved in Mongolian medicine and is often used as a type of tea for heat-clearing and detoxifying in the populus. In this study, a rapid and sensitive method was established for the qualitative and quantitative analyses of the major constituents in T. ledebourii. Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was developed for the identification of the multi-constituents in T. ledebourii. A total of 37 chemical constituents in T. ledebourii extract were unambiguously or tentatively identified, including 17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene. Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were first detected in T. ledebourii and the buttercup family. High-performance liquid chromatography–quadrupole ion trap tandem mass spectrometry was applied for the simultaneous determination of 11 compounds, which were either with high contents or strong bioactivities. Satisfactory linearity was achieved with a wide linear range and fine determination coefficient (r > 0.9987). The overall recoveries ranged from 98.07 to 101.2%, and the precision in terms of RSD was <0.74%. The results might provide the basis for quality control analysis of T. ledebourii. Introduction Trollius ledebourii, also known as Tropaeolum majus and Ludilian, is the dried flowers of Trollius chinensis Bunge and T. ledebourii Reichb in the buttercup family and was widely distributed in Inner Mongolia and the Hebei province in northern China. Trollius ledebourii is a type of traditional Chinese medicine (TCM) that is commonly found in tea with multiple functions such as the treatment of tonsillitis, periostitis and conjunctivitis (1–3). As a Mongolian medicine, it has a long history for treatments of aphtha, throat swelling, toothache, eye illness and damp-heat, which were initially recorded in Gang Mu Shi Yi. Four different ephedrine formulations of T. ledebourii prescription preparations were included in the Chinese Pharmacopoeia (2015), and orientin was set as the index component for determination (4). Modern pharmacological studies have shown that T. ledebourii possesses bioactive activities, including anti-inflammation (5), antioxidant, antimicrobial, antivirus (6) and anticancer properties (7, 8). The chemical constituents of T. ledebourii are flavonoids, phenolic acids and alkaloids (9). The flavonoids are the biologically active fractions (8–10). Previous studies have proved that orientin, vitexin and 2″-O-β-l-galactopyranosylorientin were the major constituents with high contents and strong bioactivities in T. ledebourii (11, 12). Recent studies in the qualitative analysis of T. ledebourii have not been thorough. Only Xiaolei Ren et al. (13) have identified 14 compounds including 12 flavonoids and 2 phenolic acids in the extract of the flowers of T. ledebourii by Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS). Other analyses on chemical constituents of T. ledebourii have mostly been investigated using HPLC–UV (14, 15) and HPLC–MS (16) in recent years. Almost all simultaneous determinations of the active constituents in T. ledebourii was carried on HPLC–UV (17–19). In recent years, UHPLC–Q-TOF-MS technology has been extensively applied to the component analysis of TCM (20) and compound preparations (21) with the benefits of high resolution and sensitivity. It is essential to identify complex samples with accurate molecular weights and MS2 fragment ions by TOF-MS. Quadrupole ion trap tandem mass spectrometry (QTRAP–MS-MS) is often used for the quantitative analysis of multiple components storing ions in a trap and selecting expectant mass charge ratio ions. The major advantage of it is strong specificity and sensitivity. In our approach, a total of 37 compounds in T. ledebourii were identified or tentatively characterized by UHPLC–Q-TOF-MS. Their structures were elucidated based on accurate masses, chromatographic behaviors and MS2 fragment ions. Then, a rapid and specific HPLC–QTRAP-MS-MS method was validated for the simultaneous determination of 11 major flavonoids and phenolic acids compounds in T. ledebourii. This method was validated in terms of good linear correlation, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) that could be used for the quality control analysis of T. ledebourii from different habitats. Material and Methods Standards, reagents and samples Orientin (15102309), vitexin (15120611) and diosmetin (15011627) were purchased from Shanghai Shifeng Biological Technology Co., Ltd. Apigenin (101129) and acacetin (100929) were purchased from Shanghai Winherb Medical Technology Co., Ltd. Protocatechuic acid (20151228) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Quercetin (100081-200907), ferulic acid (110773-201313), luteolin (111520-200504), pectolinaringenin (111815-201001), pectolinarin (111728-201001), isorhamnetin (110860-201410), campherenol (110861-201310), caffeic acid (110885-200102) and vanillic acid (110776-200402) were purchased from the National Institute for Food and Drug Control. The purity was higher than 98.0% by the normalization of peak areas detected by the HPLC analysis of these standards. 2″-O-β-l-galactopyranosylorientin (purity > 99.0% by HPLC) was isolated from T. ledebourii in our laboratory. Hyperoside and naringenin were laboratory-made in the medicinal chemistry of natural products laboratory by isolation from Fructus Evodia and Smilax glabra Roxb, respectively. The methods of isolation were reflux extraction, extraction and column chromatographic separation, referring to the previous research by Langsheng Pan et al. (22) and Lei Li (23). The purities were more than 98.0%, as detected by nuclear magnetic resonance (NMR). Acetonitrile was purchased from JT-BAKER Company (USA), formic acid (HPLC grade) was purchased from DIKMA Company (USA). Purified water was obtained from Wahaha (Gangzhou Wahaha Group Co., Ltd., China). The other chemicals were of analytical grade. Five batches of the dried flowers of T. ledebouri Reichb were collected from Chengde of Hebei province (HB-1), Ximeng of Inner Mongolia (NM), Taiyuan of Shanxi province (SX), Kunming of Yunnan province (YN) and Zhangjiakou of Hebei province (HB-2) in China and identified by Ding Zhao, Professor of pharmacognosy of School of Pharmacy in Hebei Medical University. The verified specimens were dried and readied in the Laboratory of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University. The detailed plant source information including collection time, specific location, gathering person and appraiser are listed in Table I. Table I. List of Trollius ledebourii Samples No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  Table I. List of Trollius ledebourii Samples No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  No.  Code  Specific location  Collection time  Gathering person  Appraiser  1  HB-1  Chengde, Hebei Province, China  2016.05  Man Liao  Professor Lianhuai Li  2  NM  Ximeng, Inner Mongolia Province, China  2016.05  Man Liao  Professor Lianhuai Li  3  SX  Taiyuan, Shanxi Province, China  2016.06  Man Liao  Professor Lianhuai Li  4  YN  Kunming, Yunnan Province, China  2016.06  Xiaoye Cheng  Professor Lianhuai Li  5  HB-2  Zhangjiakou, Hebei Province, China  2016.06  Xia Zhang  Professor Lianhuai Li  Instrumentation The qualitative analysis was conducted on a UHPLC system (Agilent 1290, USA), which was coupled with a triple TOF™ 5600+ MS/MS system (AB SCIEX, USA); the system was used especially for its Duo-Spray™ source. The UHPLC instrument included a binary pump with an online degasser, an auto plate-sampler and a thermostatically controlled column compartment. Data acquisition was processed on Analyst 1.6.1 software (AB SCIEX, CA, USA). A total of 37 compounds were identified using Masterview 1.1 software and Peakview 2.2 software (AB SCIEX, CA, USA). Quantitative analysis was performed on an LC system (Agilent 1200, USA) equipped with a quaternary solvent delivery system, an autosampler, an automatic degasser, and a column compartment. Mass spectrometric detection was composed of a 3200 QTRAP™ system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion equipped with a Turbo V source, and a TurboIonSpray interface. Data acquisition and procession was performed with Analyst 1.5.2 software (AB SCIEX, Ontario, Canada). Standard solutions and sample preparations Solid portions of quantitative standards (2″-O-β-l-galactopyranosylorientin, orientin, vanillic acid, vitexin, hyperoside, ferulic acid, luteolin, quercetin, apigenin, naringenin and acacetin) were weighed and dissolved in 80% methanol to prepare stock solutions directly. An accurate volume of each standard solution was transferred to the combined solution and diluted step-by-step to prepare a sequence of working mix solutions regularly. The max concentration of each standard solution was 489.4, 350.0, 20.64, 34.22, 33.33, 5.100, 3.267, 1.410, 0.6075, 0.0235 and 1.697 μg/mL. For the qualitative analysis, the other standards were weighed appropriately and dissolved in 80% methanol to determine the limit of detection. Five batches of T. ledebourii samples were comminuted into a fine powder with a size that should be <60 meshes. The powder (1.0 g) was accurately weighted and extracted with 25 mL of 70% methanol in an ultrasonic bath (25 kHz, 100 w) for 30 min, yielding the highest extraction efficiency and lowest noise level. Then, the samples were removed, and any evaporated solvent was compensated with 70% methanol. The mixed solution was vortex blended, allowed to sit and then filtered through a 0.22 μm nylon membrane. The primary filtrate was discarded, and the continuous filtrate was collected for qualitative analysis. The samples for quantitative analysis were similar except for the half-weighing samples. All the samples were stored at 4°C before analysis. UHPLC–Q-TOF-MS analysis The chromatography separation was performed with a Poroshell 120 EC-C18 column (100 mm × 2.1 mm, 2.7 μm) coupled with a C18 pre-column (Security Guard®) with a column temperature of 40°C. It was found that the peak shape and response of analytes were enhanced with a mobile phase of a mixture 0.1% formic acid–water (A) and acetonitrile (B) with an optimized linear gradient elution as follows: 0–14 min, 10–20% B; 14–15 min, 20–25% B; 15–20 min, 25–40% B; 20–26 min, 40–60% B; 26–27 min, 60–90% B; and 27–30 min, 90% B. The flow rate was set to 0.30 mL/min. The injection volume was 2 μL. The mass spectrometer was operated in positive mode because of the instrument status and response values, and the parameters of the MS/MS detector were set as follows: electrospray ionization (ESI) source with a turbo spray temperature of 550°C; ion spray voltage (IS): 5,500 V, nebulizing gas (Gas1): 55 psi, TIS gas (Gas2): 55 psi, curtain gas: 35 psi, and declustering potential (DP): 60 V. The IDA (information dependent acquisition) criteria were provided for the ions that match the mass defect window to obtain the MS/MS spectra. The experiments were run with scans of 100–1,000 and 50–1,000 amu for the full MS and MS/MS experiments, respectively. Additionally, the MS/MS experiments were run with 200 and 70 ms of accumulation time for the full MS and MS/MS experiments. The collision energy spread (CES) was set at 35 ± 15 eV, which had been optimized for observing better MS2 spectra. Simultaneously, the calibration delivery system was acquired for calibrating mass numbers online. HPLC–QTRAP-MS-MS analysis The chromatography separation was processed on a Diamonsil C18 column (150 mm × 4.6 mm, 5μm), and the column was maintained at room temperature. The formulation of the mobile phase was 0.1% formic acid–water (A) and acetonitrile (B). The gradient elution mode was as follows: 0–6 min, 20–40% B; 6–12.5 min, 40–75% B; 12.5–13 min, 75–90% B; and 13–15 min, 90% B. The injected sample volume was 10 μL, and the flow rate was 0.8 mL/min. The mass spectrometer was operated in negative mode, and the parameters of the MS/MS detector were set as follows: source temperature: 650°C, ESI source voltage: −4,500 V, nebulizer gas (Gas1): 60 psi, turbo gas (Gas2): 65 psi, and curtain gas (CUR): 30 psi. Nitrogen gas was used for the entire analysis, and the heating of the electrospray interface was continuous. The collision activated dissociation (CAD) gas level was set at medium. Multi-reaction monitoring (MRM) technology for the triple-quadrupole tandem mass spectrometer was applied for quantitative analysis. Q1 and Q3 were operated at unit mass resolution. The dwell time was 50 ms, with a 5 ms pause between scans. Monitoring ion-pairs, the value of DP and the value of CE of each desired component in multicomponent are listed in Table II. The structures of the 11 compounds, MRM chromatograms of reference substances and T. ledebourii samples, second mass spectra of the reference substances and chromatograms of the standard mixture under the optimal separation conditions are shown in Figure 1. Table II. HPLC–ESI-MS-MS Data of 11 Constituents From Trollius ledebourii Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  aMonitored MRM transitions. Table II. HPLC–ESI-MS-MS Data of 11 Constituents From Trollius ledebourii Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  Compounds  tR (min)  MW  MS1 (m/z)  MS2 (m/z)  DP (V)  CE (eV)  2′′-O-β-l-galactopyranosylorientin  3.53  610.52  609.4a  327.1a  −65  −45  Orientin  4.69  448.38  447.3a  327.2a  −64  −32  Vanillic acid  5.07  168.15  167.0a  107.9a  −27  −25  Vitexin  5.37  432.38  431.0a  311.1a  −74  −32  Hyperoside  5.62  464.38  463.2a  300.1a  −64  −42  Ferulic acid  6.84  194.18  193.0a  134.0a  −32  −24  Luteolin  9.55  286.23  284.9a  132.9a  −51  −47  Quercetin  9.79  302.00  301.0a  150.9a  −75  −31  Apigenin  10.86  270.24  269.0a  117.0a  −55  −50  Naringenin  11.08  272.25  271.0a  150.9a  −62  −25  Acacetin  13.72  284.26  283.0a  268.0a  −64  −31  aMonitored MRM transitions. Figure 1. View largeDownload slide Multiple-reaction monitoring chromatograms of reference substances (A) and Trollius ledebourii samples (B) and second mass spectra of reference substances (C). Figure 1. View largeDownload slide Multiple-reaction monitoring chromatograms of reference substances (A) and Trollius ledebourii samples (B) and second mass spectra of reference substances (C). Establishment of T. ledebourii chemistry database The related chemical composition knowledge about of different growth parts for T. ledebourii and similar plants in the buttercup family were gathered by analysing the massive literature. Each compound with its name and molecular formula were inputted into the Masterview 1.1 software, which can produce the exact mass number automatically. According to the information, a comprehensive database including molecular formula, English name, exact mass number and ion mode was developed. Results Analysis of chemical constituents in T. ledebourii extract Through the above analysis strategies, a total of 37 chemical constituents in T. ledebourii extract were identified by UHPLC–Q-TOF-MS based on PeakView™ 2.2 (AB Sciex) data processing and 18 of them (compounds 2, 6, 7, 9, 10, 15, 16, 17, 20–22, 26, 28–31, 35 and 36) were unambiguously confirmed by comparing their retention times, as well as MS and MS2 fragment ions, with those of the reference standards; 19 compounds were tentatively assigned based on their MS2 fragment ions, after referring to previous studies. Compared with the database of compounds from the plants of T. ledebourii and other plants in the buttercup family, 5 flavones (compounds 20, 28 and 35–37), 2 flavonols (compounds 21 and 29), 16 flavonoid glycosides (compounds 7–9, 11–16, 19, 23–25, 27, 32 and 34), 4 phenolic acids (compounds 3, 5, 10 and 18), 1 amide (compound 1) and 1 triterpene (compound 33) have already been reported (7, 12, 13, 23–36). Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were detected in T. ledebourii and the buttercup family for the first time. The mass error for practical molecular ions of all identified compounds was within ±5 ppm. The retention times, mass number, mass error and MS2 fragments of the identified compounds in T. ledebourii are summarized in Table III. The UHPLC–Q-TOF-MS total ion chromatograms of T. ledebourii samples in positive ion mode are displayed in Figure 2. The chemical structures and MS2 spectra of the 37 compounds are shown in Figures 3 and 4. Table III. Qualitative Analysis of Chemical Constituents in Trollius ledebourii No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  *Compared with reference standards. Table III. Qualitative Analysis of Chemical Constituents in Trollius ledebourii No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  No.  tR (min)  Observed mass (m/z)  Theoretical mass (m/z)  Error (ppm)  Formula  MS2 (m/z)  compounds  1  1.95  182.0871  182.0812  −0.5  C9H11NO3  182,167,164  Veratrum amide  2*  2.02  155.0334  155.0339  −3.2  C7H6O4  155,137,109  Protocatechuic acid  3  3.14  139.0387  139.0390  −2.1  C7H6O3  139,121  Hydroxybenzoic acid  4  3.47  167.0708  167.0703  −0.8  C9H10O3  167,149  Paeonol  5  4.10  183.0650  183.0652  0.8  C9H10O4  183,165,139,124  Veratric acid  6*  4.23  181.0493  181.0495  −1.1  C9H8O4  181,163,145  Caffeic acid  7*  7.25  611.1601  611.1607  −0.8  C27H30O16  611,449,431,413, 353,329,311,287  2″-O-β-l-Galactopyranosylorientin  8  7.59  581.1495  581.1501  −1.1  C26H28O15  581,449,431,413, 353,329  2″-O-β-d-Xylopyranosylorientin  9*  7.86  449.1076  449.1078  −0.5  C21H20O11  449,431,413,353, 329,299  Orientin  10*  7.95  169.0494  169.0495  −1.1  C8H8O4  169,151,125,110  Vanillic acid  11  8.30  597.1445  597.1450  −0.9  C26H28O16  597,477  3-O-Sambubiosylquercetin  12  8.31  465.1022  465.1028  −1.2  C21H20O12  465,303  3-O-Glucopyranosylquercetin  13  8.50  595.1660  595.1658  0.4  C27H30O15  595,433,415,397  2″-O-β-l-Galactopyranosylvitexin  14  9.23  565.1545  565.1552  −1.2  C26H28O14  565,433,415  2″-O-β-d-Xylopyranosylvitexin  15*  9.61  433.1124  433.1129  −1.1  C21H20O10  433,415,397,367,337,283  Vitexin  16*  10.65  465.1021  465.1028  −1.4  C21H20O12  465,303  Hyperoside  17*  13.00  195.0647  195.0652  −2.7  C10H10O4  195,180,151,136  Ferulic acid  18  13.43  237.1119  237.1121  −0.9  C13H16O4  237,219  Proglobeflowery acid  19  17.51  533.1639  533.1654  −2.8  C26H28O12  533,515,431,413  2″-O- (2‴-methylbutanoyl)Orientin  20*  17.86  285.0545  285.0550  −1.8  C15H10O6  287,269,241  Luteolin  21*  17.90  303.0488  303.0499  −3.7  C15H10O7  303,285,257  Quercetin  22*  17.97  623.1946  623.1971  −3.9  C29H34O15  623,477,315  Pectolinarin  23  17.98  593.1852  593.1865  −2.2  C28H32O14  593,285  7-O-Neohespeidosylacacetin  24  18.13  597.1584  597.1603  −3.1  C30H28O13  597,579,477,415  2″-O-(3″,4‴-dimethoxybenzoyl)Vitexin  25  18.45  517.1703  517.1704  −0.4  C26H28O11  517,499,433,415,397  2″-O- (2‴-methylbutanoyl)Vitexin  26*  19.29  271.0599  271.0601  −0.8  C15H10O5  271,243  Apigenin  27  19.42  547.1811  547.1810  0.1  C27H30O12  547,445,385  7-methoxy-2″-O- (2‴-methylbutanoyl)Orientin  28*  19.43  273.0754  273.0758  −1.3  C15H12O5  273,255  Naringenin  29*  19.77  287.0547  287.0550  −1.0  C15H10O6  287,259,213,153  Campherenol  30*  19.78  301.0704  301.0707  −1.0  C16H12O6  301,286,258  Diosmetin  31*  20.06  317.0654  317.0656  −0.9  C16H12O7  317,302  Isorhamnetin  32  20.18  531.1843  531.1861  −3.4  C27H30O11  531,447,429  2″-O- (2‴-methylbutanoyl)Isoswertisin or 3″-O- (2‴-methylbutanoyl)Isoswertisin  33  20.43  457.3647  457.3676  −6.4  C30H48O3  457.439  Ursolic acid  34  20.81  447.1265  447.1286  4.6  C22H22O10  447,429  Isoswertisin  35*  23.11  285.0756  285.0758  −0.4  C16H12O5  285,270,242  Acacetin  36*  23.47  315.0863  315.0863  −0.1  C17H14O6  315,300,282,257  Pectolinaringenin  37  25.39  329.1022  329.1020  0.6  C18H16O6  329,314,296,268,240  Salvigenin  *Compared with reference standards. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of Trollius ledebourii samples in positive ion mode. Figure 2. View largeDownload slide UHPLC–Q-TOF-MS total ion chromatogram of Trollius ledebourii samples in positive ion mode. Figure 3. View largeDownload slide View largeDownload slide Chemical structures of the 37 compounds in Trollius ledebourii. Figure 3. View largeDownload slide View largeDownload slide Chemical structures of the 37 compounds in Trollius ledebourii. Figure 4. View largeDownload slide View largeDownload slide View largeDownload slide MS2 spectra of 37 compounds in Trollius ledebourii. Figure 4. View largeDownload slide View largeDownload slide View largeDownload slide MS2 spectra of 37 compounds in Trollius ledebourii. Mass spectra for fragmentation behavior of 37 compounds Flavonoid glycosides Compound 7 (tR = 7.25 min) and compound 8 (tR = 7.59 min) yielded a [M + H]+ at m/z 611.1632 with the molecular formula C27H30O16 and a [M + H]+ at m/z 581.1513 with the molecular formula C26H28O15, which gave rise to fragment ions at m/z 449, m/z 431, m/z 413 and m/z 329, corresponding to the characteristics of orientin. Comparing with the retention time and MS2 fragment ions of the reference substance or the data reported in the literature, compound 7 was unequivocally identified as 2″-O-β-l-galactopyranosylorientin, and compound 8 was tentatively identified as 2″-O-β-d-xylopyranosylorientin (26). Compound 9 (tR=7.86 min) produced [M + H]+ at m/z 449.1102 with the molecular formula C21H20O11. Furthermore, the fragmentation ions at m/z 431, m/z 413, m/z 353 and m/z 329 corresponded to orientin. This compound has previously been isolated from T. ledebourii (27). Compound 11 (tR=8.30 min) and compound 12 (tR=8.31 min) were comprised of the same aglycone, quercetin and exhibited an identical [M + H]+ at m/z 597.1402 with the molecular formula C26H28O16 and [M + H]+ at m/z 465.1202 with the molecular formula C21H20O12, respectively. The same characteristic ion observed at m/z 303, corresponded to the complete loss of glycosides in the structure. Thus, compounds 11 and 12 were assigned as 3-O-sambubiosylquercetin (28) and 3-O-glucopyranosylquercetin (29, 30), according to previous literature. Compound 13 (tR=8.50 min) and compound 14 (tR=9.23 min) displayed a [M + H]+ at m/z 595.1690 with the molecular formula C27H30O15 and a [M + H]+ at m/z 565.1530 with the molecular formula C26H28O14, both of which showed a common MS2 fragment at m/z 433 ascribed to the loss of a glucose moiety [M + H-C6H10O5]+ and a xyloside unit [M + H-C5H8O4]+, respectively. The fragment ions of compounds 13 and 14 at m/z 415 and m/z 397 were both the characteristic ions of vitexin. Therefore, compounds 13 and 14 were tentatively identified as 2″-O-β-l-galactopyranosylvitexin (28) and 2″-O-β-d-xylopyranosylvitexin (24). Compound 15 (tR=9.61 min) produced [M + H]+ at m/z 433.1178 with the molecular formula C21H20O10. Furthermore, the fragmentation ions at m/z 415, m/z 397, m/z 367 and m/z 283 corresponded to vitexin. Then, by comparing with the retention times and MS2 fragment ions of the reference substances, compound 15 was confirmed as vitexin. This compound also has previously been isolated from T. ledebourii (13, 25, 27). Compound 16 (tR=10.65 min) gave a [M + H]+ at m/z 465.1021 with the molecular formula C21H20O12, and showed a fragment ion at m/z 303.0530 [M + H-C6H10O5]+, the special fragment ion of the aglycone quercetin. Further comparing with the reference substances, it was definitively identified as hyperoside (13). Compound 19 (tR=17.51 min) and 27 (tR=19.42 min) presented the same aglycone orientin and gave a [M + H]+ at m/z 533.1652 with the molecular formula C26H28O12 and a [M + H]+ at m/z 547.1816 with the molecular formula C27H30O12, respectively. Furthermore, comparing the MS2 spectra with the literature (27, 30), compounds 19 and 27 were tentatively identified as 2″-O-(2‴-methylbutanoyl) orientin and 7-methoxy-2″-O-(2‴-methylbutanoyl) orientin. Compound 23 with the molecular formula C28H32O14 generated the characteristic ions of m/z 593.1858 [M + H]+ and m/z 285.0762 that were the neutral loss of a neohesperidoside. Compound 23 was identified as 7-O-neohespeidosylacacetin by comparison with that of the literature (13). Compounds 24 (tR=18.13 min) and 25 (tR=18.45 min) possessing the same aglycone vitexin presented a [M + H]+ at m/z 597.1594 with the molecular formula C30H28O13 and a [M + H]+ at m/z 517.1707 with the molecular formula C26H28O11, respectively. Compound 24 had the fragment ions at m/z 579.1439 [M + H-H2O]+ and m/z 477.1162 [M + H-C4H8O4]+, and compound 25 had fragment ions at m/z 499.1587 [M + H-H2O]+ and m/z 433.1602 [M + H-C5H8O]+. By comparing with the data reported, compounds 24 and 25 were tentatively identified as 2″-O-(3″, 4‴-dimethoxybenzoyl) vitexin and 2″-O-(2‴-methylbutanoyl) vitexin respectively (28). The fragment ions at m/z 477.1375 [M + H-Rha]+ and m/z 315.0864 [M + H-Rha-Glu]+ were the characteristic ions of compound 22 producing a [M + H]+ at m/z 623.1964 with the molecular formula C29H34O15. After a comprehensive analysis of the mass data with the references, compound 22 was identified as pectolinarin, and pectolinarin has not been reported to be present in T. ledebourii previously. Compounds 32 (tR=20.18 min) generated a [M + H]+ at m/z 531.1879 with the molecular formula C27H30O11. The characteristic fragment ions were m/z 447.1691 [M + H-C5H8O]+ and m/z 429.1191 [M + H-C5H8O-H2O]+. According to the knowledge in the literature (31, 32), compounds 32 was tentatively identified as 2″-O-(2‴-methylbutanoyl) isoswertisin or 3″-O-(2‴-methylbutanoyl) isoswertisin. Compound 34 (tR=20.81 min) showed a [M + H]+ at m/z 447.0680 with the molecular formula C22H22O10. It yielded the diagnostic fragment ions at m/z 429.1151, which lost a H2O from the parent ion. Compound 34 were tentatively identified as isoswertisin (31, 33). Flavones Compound 20 at 17.86 min presented a molecular ion at m/z 287.0542 [M + H]+ with the molecular formula C15H10O6. Comparing with the retention time and MS2 fragment ions of the reference substance, compound 20 was unequivocally identified as luteolin (23). Compound 26 (tR=19.29 min) displayed a [M + H]+ at m/z 271.0597 with the molecular formula C15H10O5, which yielded a fragment ion at m/z 243.0666 corresponding to the loss of a CO. By comparing our data with its standard, compound 26 was identified as apigenin. Compound 30 (tR=19.78 min) showed a [M + H]+ at m/z 301.0709 with the molecular formula C16H12O6, which presented fragment ion at m/z 286.0476 and 258.0521 corresponding to the loss of a methyl group and a CO, respectively. After comparing with the reference substances, the species was confirmed as diosmetin, which has not been previously identified in T. ledebourii. Compound 35 (tR=23.11 min) was the aglycone of compound 23 (tR=17.98 min) and gave a [M + H]+ at m/z 285.0758 with the molecular formula C16H12O5, exhibiting ions at m/z 270.0514 [M + H-CH3]+ and m/z 242.0560[M + H-CH3-H2O]+. It was identified as acacetin upon comparison with the standards. Compound 36 (tR=23.47 min) was the aglycone of compound 22 (tR=17.97 min) which displayed a [M + H]+ at m/z 315.0864 with the molecular formula C17H14O6, observed at m/z 300.0628 [M + H-CH3]+ and m/z 282.0804 [M + H-CH3-H2O]+. It was tentatively identified as pectolinaringenin (13). The final compound 37 (tR=25.39 min) was also the flavonoid compound producing a [M + H]+ at m/z 329.1038 with the molecular formula C18H16O6, which observed fragment ions at m/z 314.0799 [M + H-CH3]+, m/z 296.0694 [M + H-CH3-H2O]+ and m/z 268.0743 [M + H-CH3-CO]+. The characteristic ions were ascribed to salvigenin that has been reported in T. ledebourii in a previous study (13). Flavonols Compound 21 (tR=17.90 min) yielded a [M + H]+ at m/z 303.0483 with the molecular formula C15H10O7, which was similar to the fragment pattern of luteolin. It gave fragment ions at m/z 285.0434 and m/z 257.0431, corresponding to the neutral loss of an H2O and a CO. Furthermore, by comparing with the standards, compound 21 was identified as quercetin (13). Compound 29 was eluted at 19.43 min yielding a molecular ion at m/z 287.0539 [M + H]+ with the molecular formula C15H10O6. By comparing with the retention time and MS2 fragment ions of the reference substances, compound 29 was unequivocally identified as campherenol (34, 35). Compound 31 (tR=20.06 min) yielded a [M + H]+ at m/z 317.0651 with the molecular formula C16H12O7, which gave the MS2 ion at m/z 302.0468 corresponding to the loss of a methyl group. By comparing with standards, compound 31 was identified as isorhamnetin. Dihydroflavones Compound 28 (tR=19.43 min), the derivative of dihydro-flavonoids, gave a precursor ion [M + H]+ at 273.0771 with the molecular formula C15H12O5. The characteristic ion at m/z 255.0757 was inferred as a loss of an H2O. The compound was identified as naringenin according to the reference substances. This is the first time this compound is identified in T. ledebourii. Phenolic acids Compound 2 (tR=2.02 min) generated a [M + H]+ at m/z 155.1029 with the molecular formula C7H6O4. Then, the [M + H]+ ion fragmented into two characteristic ions at m/z 137.0232 and m/z 109.0287, which corresponded to [M + H-H2O]+ and [M + H-H2O-CO]+. This was confirmed by preparing the compound with a reference standard. Accordingly, the structure was identified as protocatechuic acid. Compound 3 (tR=3.14 min) showed a [M + H]+ at m/z 139.0392 with the molecular formula C7H6O3. The ion at m/z 121.0284 could be formed by the loss of a molecule of H2O. Compound 3 was identified as hydroxybenzoic acid (12) based on comparisons with the reference substances. Compound 4 (tR=3.47 min) gave a [M + H]+ at m/z 167.0681 with the molecular formula C9H10O3. Its MS2 spectrum gave a characteristic ion at m/z 149.0591, which was identified as the loss of a molecule of H2O. The chromatogram retention time and MS2 fragments correspond to those of the reference solution. Therefore, it was validated as paeonol, and it is the first time it is identified in T. ledebourii. Compound 5 (tR=4.10 min) displayed a [M + H]+ at m/z 183.0661, and its molecular formula was C9H10O4. The fragment ions observed at m/z165.0546 and m/z 139.0758 were the loss of an H2O and a carboxyl group, respectively. Therefore, compound 6 was tentatively identified as veratric acid (25) by comparing the MS/MS fragmentation pattern with those found in the literature. Compound 6 (tR=4.23 min) displayed a molecular ion at m/z 181.0493 [M + H]+ with the molecular formula C9H8O4 in positive ionization mode. Further MS2 scans showed that it produced similar fragment ions at m/z 163.0384 [M + H-H2O]+ and m/z 145.0281 [M + H-2H2O]+, indicating the loss of an H2O group. Thus, compound 6 was identified as caffeic acid, upon comparison with the standards. However, this is the first study identifying caffeic acid in T. ledebourii. Compound 10 (tR=3.88 min) presented a [M + H]+ at m/z 169.1023 with the molecular formula C8H8O4, producing fragments at m/z 151.0392 and m/z 125.0607 that were ascribed to [M + H-H2O]+ and [M + H-COO]+, respectively. By comparing with the standards, it was confirmed as vanillic acid. Compound 17 (tR=8.09 min) presented a [M + H]+ at m/z 195.0634 with the molecular formula C10H10O4, producing fragments at m/z 180.0540 and m/z 151.0613 that were ascribed to [M + H-CH3]+ and [M + H-COO]+, respectively. The retention time and MS2 fragment ions were consistent with a reference substance. Therefore, compound 17 was identified as ferulic acid. To our knowledge, this compound has only been reported in the genus but not in T. ledebourii. Compound 18 (tR=13.43 min) showed a precursor ion [M + H]+ at m/z 237.1118 with the molecular formula C13H16O4, and yielded a MS2 fragment at m/z 219.0985, corresponding to the loss of an H2O, suggesting that this compound was proglobeflowery acid per the literature (26). Amides Compound 1 (tR=1.95 min) yielded a [M + H]+ at m/z 182.0211 with the molecular formula C9H11NO3, which gave characteristic MS2 fragments at m/z 166.9899, m/z 164.0356, m/z 137.0595 and m/z 119.0483 corresponding to the loss of one methyl and one H2O. Therefore, compound 1 was tentatively proposed to be veratrum amide (7, 24) according to the literature. Triterpenes Compound 33 (tR=20.43 min) was a phenolic acid compound that generated [M + H]+ at 457.2314 and a fragment ion at m/z 439.2022 [M + H-H2O]+. Based on the above data, compound 33 was identified as ursolic acid (36). Fragmentation patterns of the representative biologically active components in T. ledebourii The fragmentation patterns of flavone C-glycosides and flavones are shown in Figures 5 and 6, and 2″-O-β-l-galactopyranosylorientin and luteolin were used as examples for detailed explanations, respectively. Figure 5. View largeDownload slide Hypothesized fragmentation pattern of protonated 2″-O-β-l-galactopyranosylorientin generated by UHPLC–Q-TOF-MS. Figure 5. View largeDownload slide Hypothesized fragmentation pattern of protonated 2″-O-β-l-galactopyranosylorientin generated by UHPLC–Q-TOF-MS. Figure 6. View largeDownload slide Hypothesized fragmentation pattern of protonated Luteolin generated by UHPLC–Q-TOF-MS. Figure 6. View largeDownload slide Hypothesized fragmentation pattern of protonated Luteolin generated by UHPLC–Q-TOF-MS. Methodological validation of the quantitative analysis 2″-O-β-l-Galactopyranosylorientin, orientin, vanillic acid, vitexin, hyperoside, ferulic acid, luteolin, quercetin, apigenin, naringenin and acacetin were quantified by HPLC–QTRAP-MS-MS, which showed high sensitivity in the qualitative and quantitative determination of the analytes (Figure 7). The total values of these 11 compounds are listed in Tables IV and V. The 11 compounds exhibited good linear regression (r > 0.9987) with in the detected ranges. Additionally, the LODs of the 11 constituents were estimated to be 0.0128–8.600 ng/mL. The LOQs were 0.1809–76.00 ng/mL. The relative standard deviation (RSD) values of all these 11 compounds observed from intra-day and inter-day precision studies were <1.76%. The recovery values were 98.07–101.2% with an RSD less than 2.00%, suggesting that the method was accurate for determining the analytes. The RSD of the storage stability was less than 1.96% within 48 h. Figure 7. View largeDownload slide Chromatograms of standard mixture under the optimal separation conditions by HPLC–QTRAP-MS-MS. 7: 2″-O-β-L-galactopyranosyl-orientin, 9: orientin, 10: vanillic acid, 15: vitexin, 16: hyperoside, 17: ferulic acid, 20: luteolin, 21: quercetin, 26: apigenin, 28: naringenin, 35: acacetin. Figure 7. View largeDownload slide Chromatograms of standard mixture under the optimal separation conditions by HPLC–QTRAP-MS-MS. 7: 2″-O-β-L-galactopyranosyl-orientin, 9: orientin, 10: vanillic acid, 15: vitexin, 16: hyperoside, 17: ferulic acid, 20: luteolin, 21: quercetin, 26: apigenin, 28: naringenin, 35: acacetin. Table IV. Regression Equations, Linear Ranges, Correlation Coefficients, LODs and LOQs of 11 Compounds Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  aY, peak area and X, concentration (μg/mL). bLOD (S/N = 3). cLOQ (S/N = 10). Table IV. Regression Equations, Linear Ranges, Correlation Coefficients, LODs and LOQs of 11 Compounds Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  Analytes  Regression equationa  Linear range (μg/mL)  r2  LODb (ng/mL)  LOQc (ng/mL)  2′′-O-β-l-galactopyranosylorientin  Y = 9.980 × 103X + 1.421 × 106  61.17–489.4  0.9995  5.098  76.00  Orientin  Y = 2.822 × 104X + 1.490 × 106  13.12–350.0  0.9987  0.8006  22.00  Vanillic acid  Y = 2.410 × 104X + 1.222 × 103  0.2580–20.64  0.9991  8.600  36.90  Vitexin  Y = 1.786 × 105X + 3.938 × 104  0.0535–34.22  0.9993  0.2673  2.139  Hyperoside  Y = 1.432 × 105X + 9.679 × 104  2.083–33.33  0.9989  1.042  5.208  Ferulic acid  Y = 1.046 × 105X − 6.893 × 103  0.0797–5.100  0.9990  3.984  9.375  Luteolin  Y = 1.165 × 106X + 3.644 × 104  0.0102–3.267  0.9992  0.0128  0.3403  Quercetin  Y = 5.731 × 105X − 1.246 × 106  0.0220–1.410  0.9998  0.0881  2.203  Apigenin  Y = 3.128 × 106X + 2.783 × 103  0.0038–0.6075  0.9998  0.0632  0.1898  Naringenin  Y = 3.985 × 106X − 2.266 × 102  0.0006–0.0235  0.9989  0.0392  0.1809  Acacetin  Y = 2.285 × 106X + 5.612 × 103  0.0053–1.697  0.9997  0.0353  0.2651  aY, peak area and X, concentration (μg/mL). bLOD (S/N = 3). cLOQ (S/N = 10). Table V. Precision, Accuracy and Atability of the 11 Components From Trollius ledebourii Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    aRecovery (%) = (detected amount-original amount)/spiked amount × 100. bRSD (%) = (SD/mean) × 100. Table V. Precision, Accuracy and Atability of the 11 Components From Trollius ledebourii Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    Analytes  Precision (n = 6)  Accuracy (n = 3)  Stability 24 h, n = 3    Intra-day RSD%  Inter-day RSD%  Original mean (mg)  Spiked mean (mg)  Detected mean (mg)  Recoverya (%)  RSDb (%)  RSDb (%)  2′′-O-β-l-  0.60  0.71  7.465  5.970  13.45  100.3  1.96  1.36  Galactopyranosylorientin        7.462  14.86  99.10  1.06            8.955  16.35  99.21  0.99    Orientin  1.48  1.23  6.875  5.500  12.27  98.09  0.43  1.45          6.875  13.63  98.25  0.07            8.250  15.22  101.2  0.51    Vanillic acid  1.92  0.80  0.1502  0.1204  0.2710  100.3  0.84  1.96          0.1505  0.3000  99.53  0.34            0.1806  0.3277  98.28  0.20    Vitexin  1.09  0.59  0.3108  0.2494  0.5578  99.04  2.00  1.85          0.3118  0.6210  99.49  1.47            0.3741  0.6880  100.8  1.73    Hyperoside  1.46  1.66  0.2136  0.1717  0.3836  99.01  1.27  1.42          0.2146  0.4267  99.30  1.32            0.2576  0.4696  99.38  1.19    Ferulic acid  1.34  0.28  0.0132  0.0105  0.0237  100.0  1.05  1.21          0.0132  0.0262  98.48  0.33            0.0158  0.0290  100.0  0.64    Luteolin  1.88  1.00  0.0131  0.0106  0.0237  100.0  1.14  1.45          0.0132  0.0262  99.24  0.81            0.0158  0.0286  98.10  1.13    Quercetin  1.95  0.42  0.0043  0.0034  0.0077  100.0  1.11  1.73          0.0043  0.0086  100.0  1.31            0.0052  0.0094  98.07  0.59    Apigenin  1.80  1.69  0.0010  0.0008  0.0018  100.0  1.81  1.56          0.0010  0.0020  100.0  0.25            0.0012  0.0022  100.0  0.59    Naringenin  1.85  1.75  0.00008  0.00006  0.00014  100.0  0.39  1.72          0.00008  0.00016  100.0  0.82            0.00009  0.00017  100.0  0.81    Acacetin  1.57  0.44  0.0056  0.0045  0.0101  100.0  0.65  1.63          0.0057  0.0113  100.0  0.57            0.0068  0.0124  100.0  1.63    aRecovery (%) = (detected amount-original amount)/spiked amount × 100. bRSD (%) = (SD/mean) × 100. Sample analysis In this study, the HPLC–QTRAP-MS-MS analytical method was first applied to simultaneously determine 11 compounds including flavones, flavonoid glycosides and phenolic acids in T. ledebourii samples collected from various locations. The contents of the 11 investigated compounds in 5 batches of T. ledebourii samples (n = 3) are exhibited in Table VI. However, there was a difference between the chemical constituents of the five samples collected from different geographical locations, especially for the contents of flavonoid constituents. The total contents of each batch of the 11 investigated constituents ranged from 17.02 to 28.60 mg/g, and the content of the flavonoid constituents in the five batches ranged from 16.72 to 28.29 mg/g. The content of nine flavonoid compounds was considerably more than those of two phenolic acid compounds. Orientin, vitexin and 2″-O-β-l-galactopyranosylorientin are the characteristic components of T. ledebourii and presented the highest contents among the 11 components. The contents of the flavonoid compound orientin were all higher than 10.00 mg/g, which could be considered as the quality control component. T. ledebourii, which was collected from Taiyuan city of Shanxi province (SX), was tentatively considered to be the best quality with the content of 15.97 mg/g orientin. From the above data, the difference among the contents of each component in different batches was closely related to the geographical location and collection time of the medicinal herb T. ledebourii. Table VI. Contents of the 11 Active Components in Trollius ledebourii Samples Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Table VI. Contents of the 11 Active Components in Trollius ledebourii Samples Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Sample No.  Content(mg/g, n = 3)  2′′-O-β-l-Galactopyranosylorientin  Orientin  Vanillic acid  Vitexin  Hyperoside  Ferulic acid  Luteolin  Quercetin  Apigenin  Naringenin  Acacetin  1  14.99  11.81  0.2975  0.6189  0.4157  0.0262  0.0249  0.0086  0.0021  0.00015  0.0114  2  12.50  14.08  0.2948  0.9021  0.1693  0.0168  0.0251  0.0078  0.0021  0.00012  0.0134  3  8.854  15.97  0.3693  1.116  0.4610  0.0287  0.0280  0.0194  0.0019  0.00008  0.0096  4  12.93  14.20  0.2931  0.8961  0.2199  0.0161  0.0250  0.0087  0.0021  0.00012  0.0122  5  5.653  10.22  0.2696  0.4211  0.3977  0.0299  0.0131  0.0029  0.0010  0.00008  0.0101  Discussion According to our knowledge, there are too many compounds in herbal medicine. Which compound should be quantified and how to avoid its cross-talk become significant issues. A large number of papers have determined that flavones, flavonoid glycosides and a part of phenolic acids were biologically active constituents of T. ledebourii (9–12). In our assay, a quantification of nine flavonoids and two phenolic acids that are considered as active and with high levels in T. ledebourii extract were developed that can be useful for quality control. However, two different techniques were applied for qualitative and quantitative analyses depending on their specific benefits. Though UHPLC–Q-TOF-MS has the high resolution to identify compounds that hold similar MS ions, it is easily disrupted by temperature so that it has poor reproducibility with quantified analytes. HPLC–QTRAP-MS-MS possesses high sensitivity and reproducibility contributing to its MRM pattern using ion-pairs. To achieve low detection limits required for all analytes, methods of qualitative and quantitative analyses of analytes in positive and negative ionization modes were developed. In consideration of their stability and high abundance, positive mode was chosen for the qualitative analysis by UHPLC–Q-TOF-MS, and negative mode was chosen for the quantitative analysis by HPLC–QTRAP-MS-MS. To achieve a better peak shape and a high response for the analysis of most compounds, optimization of the mobile phase was conducted by comparing types and additions. It was found that the best composition consisted of acetonitrile and 0.1% formic acid aqueous solution. Conclusions In this study, an UHPLC–Q-TOF-MS method was developed to investigate the chemical constituents in T. ledebourii. Based on exact masses, retention times, chromatographic behaviors and characteristic fragment ions, a total of 37 compounds (17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene) were identified or tentatively identified in 25 min, and their representative fragmentations were summarized. The fragment patterns of the major compounds might contribute to its metabolites identification in a later study. To the best of our knowledge, protocatechuic acid, paeonol, caffeic acid, ferulic acid, pectolinarin, naringenin, isorhamnetin and diosmetin have not been reported in T. ledebourii and the buttercup family. HPLC–QTRAP-MS-MS was established for the first time, which was successfully applied to simultaneously determine 11 compounds in T. ledebourii. This full scale qualitative and quantitative study sheds some new light on the quality control of T. ledebourii. 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Published: Apr 13, 2018

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