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Article

Characterization of Chemical Constituents of Oxytropis microphylla (Pall.) DC. by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Time-of Flight Tandem Mass Spectrometry

by
Mengqi Xia
1,2,†,
Min Yao
1,3,†,
Junmao Li
1,
Jianjian Zhang
1,2,
Yayun Yu
1,2,
Shilin Yang
1,
Guoyue Zhong
1,4,
Na Pei
1,5,
Hui Ouyang
1,6,* and
Yulin Feng
1,6,*
1
School of pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
2
Jiangxi Bencao Tiangong Technology Co., Ltd., Nanchang 330006, China
3
Jiangxi Institute for Drug Control, Nanchang 330029, China
4
Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
5
School of Pharmacy, Yichun University, Yichun 336000, China
6
State Key Laboratory of Innovative Drug and Efficient Energy-Saving Pharmaceutical Equipment, Nanchang 330006, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Separations 2022, 9(10), 297; https://doi.org/10.3390/separations9100297
Submission received: 26 August 2022 / Revised: 8 September 2022 / Accepted: 30 September 2022 / Published: 9 October 2022
(This article belongs to the Section Analysis of Natural Products and Pharmaceuticals)
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Versions Notes

Abstract

:
Oxytropis microphylla (Pall.) DC. is a traditional Tibetan medicine used as an external preparation for clearing heat and detoxification, healing sore muscles, astringent vein hemostasis, defecation, and treating plague, constipation, anthrax, and swollen and painful furuncles. It remains a challenge to comprehensively analyze and identify the chemical constituents of Oxytropis microphylla (Pall.) DC. In this study, a new analytical method using a combination of ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) and effective data mining techniques was established to identify the chemical constituents of Oxytropis microphylla. A total of 127 compounds were identified in O. microphylla extract, including 92 flavonoids, 15 indole alkaloids, and 20 others. After the oral administration of the extract to rats, 22 metabolites were identified in the plasma. The primary in vivo metabolic reactions that occurred after the administration of O. microphylla extract were glucuronidation and sulfation. Therefore, we successfully devised a high-efficiency method to distinguish compounds and used it as a source of post-study to identify the active biological components of O. microphylla extract.

1. Introduction

More than 140 species and 3 varieties of Oxytropis (Family: Leguminosae) are found in China. Oxytropis plants were used extensively in the west owing to their outstanding efficiency [1,2,3]. Oxytropis microphylla (Pall.) DC. (OMDC) is a perennial herb, locally identified as the “king of herbs” on account of its analgesic and outstanding anti-inflammatory effects. It is found in the valleys, hillsides, and meadows of the Qinghai–Tibet plateau at an altitude of 2700–4300 m [4,5,6]. In ancient books, OMDC has been recorded for its efficiency as an analgesic and for anti-inflammatory, detoxification, promotion of blood circulation, and heat-clearing effects [7,8]. Several bioactive compounds have been separated from Oxytropis using phytochemical methods. Flavones are the major active ingredients of Oxytropis that are known to exert anti-inflammatory, analgesic, ultraviolet damage–protective, and antitumor effects, and to enhance immune cofunction [9,10]. Currently, only a few reports exist in the literature on the systematic characterization of the chemical components of this plant, which do not effectively explain its active components [11]. Therefore, there is an urgent requirement to establish a scientific method to identify the chemical constituents of OMDC.
To better understand the characteristics of OMDC, it is necessary to establish a robust identification method to analyze the chemical constituents of the plant [12,13]. The current technology used to analyze the potential active components of traditional Chinese medicine is UHPLC-QTOF-MS, which can predict the elemental composition of the tested compound and accurately determine its quality [13,14,15,16]. Its high resolution, efficiency, and structure recognition ability can detect hundreds or thousands of chemical components in complex samples and rapidly characterize the extracts used in traditional Chinese medicine [17,18,19,20]. Therefore, UHPLC-QTOF-MS is widely used in the qualitative analysis of medicinal materials.
To the best of our knowledge, only a few reports have used UHPLC-QTOF-MS to identify compounds in the crude extract of OMDC, and there is no comprehensive or systematic explanation to analyze their chemical components. Therefore, there is an urgent requirement to establish a reliable method to identify the chemical components of OMDC at micro-concentration levels and to summarize MS fragmentation behaviors. To address this problem, we established a UHPLC-QTOF-MS method for the identification, classification, and systematic investigation of the chemical components of OMDC.

2. Materials and Methods

2.1. Chemicals and Reagents

Acetonitrile and formic acid used for sample processing were sourced from Fisher Scientific (Fair Lawn, NJ, USA) and Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA), respectively. Pure distilled water was obtained using a Millipore Alpha-Q water purification system. All 6 reference standards having a purity >98% (2′,4′-dihydroxychalcone, 7-hydroxyflavanone, pinocembrin, quercetin, apigenin, luteolin) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China).

2.2. Animal Experiments

Three male Sprague-Dawley rats (weighing 180 ± 20 g) were obtained from the Hunan Laike Jingda Experimental Animal Co. Ltd., Changsha, China. All rats were housed in a room (a temperature of 20 °C and humidity of 50%) and were provided access to food and water ad libitum. OMDC extract was suspended in 0.5% CMC-Na and administered orally to rats at a dose of 150 mg/kg body weight. After drug administration, blood was collected from the inner canthus vein at fixed intervals (1, 2, 4, 6, 12 h). Blood samples were centrifuged at 5000 rpm for 10 min to obtain the serum. The animal experiments were approved by the Laboratory Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (approval No. SYXK2017-0004).

2.3. Sample Preparation

OMDC was obtained from Tibet Province in 2020. The sample was identified by Professor Guo-yue Zhong and Ming Yao (Jiangxi University of Traditional Chinese Medicine). The crude extract of OMDC was prepared using the medicinal powder. Briefly, 1 g of OMDC powder was mixed with 50 mL methanol and extracted for 30 min using ultrasonication in a water bath maintained at room temperature (20–30 °C). Extracts were filtered through a 0.22-μM microporous membrane filter and 3.0 μL of the filtered sample was used for UHPLC-QTOF-MS.
All reference materials were dissolved in methanol to yield the respective stock solutions, which were stored at 4 °C. After mixing all the reference stock solutions, they were diluted with methanol to a concentration of 10.0 μg/mL, and 3.0 μL of the solution was used for UHPLC-QTOF-MS.
The plasma of the 3 rats from each time point was mixed. Plasma samples were added to 3 times their volume of methanol, vortexed for 2 min, and centrifuged at 12,000× g rpm for 10 min. This supernatant was dried under a nitrogen stream at 35 °C. The residue was redissolved in 100 μL of 50% methanol and vortexed for 2 min. The solution was centrifuged at 12,000× g rpm for 10 min, and 5 μL of the supernatant was injected for UHPLC-QTOF-MS.

2.4. UHPLC-QTOF-MS/MS

A Shimadzu system (Kyoto, Japan) was used for separation. The other systems included a CTO-30AC column oven, a DGU-20A3 degasser, an LC-3AD solvent delivery system, a CBM-20A controller, and a SIL-30ACXR auto-sampler. A Welch UHPLC AQ-C18 (100 mm × 2.1 mm, 1.8 μm) was used at a temperature of 40 °C. The mobile phase was composed of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) and the flow rate was 0.3 mL/min. The elution conditions were as follows: 0.1–2.0 min, 5–10% B; 2.0–10.0 min, 10–14% B; 10.0–13.0 min, 14–20% B; 13.0–30.0 min, 20–40% B; 30.0–37.0 min, 40–50% B; 37.0–45.0 min, 50–65% B; 45.0–50.0 min, 65–95% B; 50.0–53.0 min, 95% B; 53.0–58.0 min, 5% B.
UHPLC-QTOF-MS/MS analyses were performed in the negative electrospray ionization mode using a Triple TOF™ 5600+ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA). The mass spectrometry conditions were as follows: ion spray voltage, −4500 V; ion source temperature, 550 °C; curtaingas, 25 psi; nebulizer gas (GS1), 55 psi; heater gas (GS2), 55 psi; and decluster potential (DP), −100 V. The mass ranges of TOF-MS and TOF MS/MS experiments were all set at 50–1250 Da.

3. Results and Discussion

3.1. Screening and Identification of Chalcone

In the mass spectrometry of protonated chalcones, phCO+ and ph’CH=CHCO+ were observed as the major fragments. Compound 82 was obtained as a quasi-molecular ion at m/z 239.0713, which was in accordance with the chemical formula of C15H12O3 (Figure 1, Figure 2 and Figure 3; Table 1). The characteristic ions at m/z 135.0090 [M-H-C8H8]−, 197.0600 [M-H-C2H2O]−, 148.0169 [M-H-C7H7]−, and 109.0308 [M-H-C9H6O]− were yielded by C-C bond. Thus, the compound was identified as 2′,4′-dihydroxychalcone by retention time of the standard. Compound 58 was inferred by sifting using the DPI acquired at m/z 239.0712 and the NL at 162 Da (C6H10O5). The chemical formula was computed as C21H22O8 based on the exact mass precursor ion. This compound was determined to be 2′,4′-dihydroxychalcone-glucoside. Compound 43 has two more OH radicals than 2′,4′-dihydroxychalcone. The molecular fragment had m/z 253.0507 [M-H-H2O]−, 151.0038 [M-H-C8H8O]−, 125.0244 [M-H-C9H6O2]−, 119.0508 [M-H-C7H4O4]−, 93.0354 [M-H-C9H6O4]− and it was structurally similar naringenin chalcone that is reported in the literature [21]. Compound 80 had a mass of 2 Da (2H) more than 2′,4′-dihydroxychalcone and was identified as 2′,4′-dihydroxydihydrochalcone [22]. The deprotonation molecular ion of compound 59 (C15H12O4) was discovered at m/z 256.0735. The fragment ions (m/z 237.056 [M-H-H2O]−, 135.0093 [M-H-C8H8O]−, 119.0512 [M-H-C7H4O3]−, and 91.0204 [M-H-H2O-C9H7O2]−) were consistent with isoliquiritigenin reported in the literature [23]. Compounds 81 had a mass of 28 Da (C2H2), more than isoliquiritigenin. This generated fragment ions at m/z 225.0916[M-H-CO2]−, 148.0151[M-H-CH3-C7H6O]−, 119.0507 [M-H-C8H6O3]−, which was substantially identical with those reported previously, and was tentatively identified as 2′,4-dihydroxy-4′-methoxy chalcone (Figure 2).

3.2. Screening and Identification of Flavone

The fragmentation of A1,3− and B1,4− was formed by Retro-Diels-Alder (RDA) reaction on the C ring of flavone (Figure 4) and flavonol aglycones. Furthermore, isoflavone aglycones appeared specific fragmentations of B0,3− and A1,3−, which derived from the C ring’s breakage (Figure 4). The reason may be that the various conjugated systems belong to flavone and isoflavone aglycones influenced the fragmentation behavior of C ring. They also tend to lose some small neutral molecules, for example CO, CHO, C3O2, CO2, C2H2O, etc. The ion fragments of some compounds are listed in this work.
Compound 53 had a deprotonated molecular ion at m/z 238.0629 and its molecular formula was determined to be C15H10O3. This compound generated fragment ions at m/z 208.0531, 193.0658, 180.0573, 165.0721, and 91.0209, and was tentatively identified as 7-hydroxyflavone (Figure 3) [22]. The molecular formulae for 69, 45, and 17 were C15H10O4, C15H10O5, and C15H10O6, respectively, and the compounds were identified as chrysin, apigenin, and luteolin, respectively, by DPIs (chrysin: m/z 225.0561 [M-H-CO]−, 209.0610 [M-H-CO2]−, 151.0034 [M-H-C8H6]−, 143.0504 [M-H-C6H6O2]−, 107.0147 [M-H-C9H6O2]−; apigenin: m/z 225.0554 [M-H-CO2]−, 151.003 [M-H-C8H6O]−, 117.0347 [M-H-C7H4O4]−, 107.0154 [M-H-C9H6O3]−; luteolin: m/z 257.0048 [M-H-H2O]−, 241.0504 [M-H-CO2]−, 217.0504 [M-H-CO2-C2H2]−, 149.0239 [M-H-C7H4O3]−, 133.0293 [M-H-C7H4O4]−) and NL [44 Da (CO2) and 68 Da (C4H4O)], the results were consistent with those reported in the literature [24,25]. (Compound 61) The quasi-molecular ions acquired at m/z 267.0670 were 14 Da (CH2) more than chrysin and the compound was determined to be formononetin via the DPIs (252.0424 [M-H-CH3]−, 223.0391 [M-H-CO2]−, 195.0442 [M-H-CO2-CO]−, 132.0214 [M-H-C7H3O3]−, 91.0181 [M-H-CH3-C9H5O3]−) [26]. Compound 24 was screened in OMDC extracts based on the precursor ions at m/z 283.0609 and the chemical formula was computed as C16H12O5. The fragment ions (m/z 268.0375 [M-H-CH3]−, 239.0344 [M-H-CO2]−, 184.0526 [M-H-C4H4O3]−, 148.0526 [M-H-C7H3O3]−, 135.0089 [M-H-C9H8O2]−) of these compounds were the same as those reported in the literature [4,27]. Compound 24 was, therefore, tentatively deduced to be 5,7-dihydroxy-4′-methoxy isoflavones (Figure 3 and Figure 5). The molecular formula for compound 51 was determined to be C16H12O6 based on the accurate mass of the quasi-molecular ions at m/z 299.0558. This organic compound was confirmed to be 5,7,2′-trihydroxy-4′-methoxy flavone (Figure 3) by the DPI m/z 284.0323, 255.0261, 227.0338, 211.0398, 148.0180 [2]. Compound 57 was determined to be pseudobaptigenin based on the parent ions at m/z 281.0455, and its molecular formula was deduced as C16H10O5. It generated fragment ions at m/z 253.0504 [M-H-CO2]−, 225.0557[M-H-CO2-CO]−, 135.0088[M-H-C9H6O2]−, and 91.0201[M-H-C10H6O4]−, and its structure was consistent with that reported in the literature [28,29].
Compounds 71 and 18 yielded parent ions at m/z 255.0656, corresponding to the formula C15H12O4. Compound 71 was determined to be pinocembrin based on its chromatographic data and comparison with the fragment ions with standard pinocembrin. Compounds 26, 28, and 38 were determined to be pinocembrin derivatives based on their DPIs acquired at m/z 211.0790, 171.0418, and 151.0031. These compounds were tentatively classified as pinocembrin-glucoside due to the DPIs and NL 162 Da (C6H10O5). Compound 18 and isoliquiritigenin were structural isomers. It found to be liquiritigenin by the DPIs acquired at m/z 135.0093[M-H-C8H8O]−, 119.0512[M-H-C7H4O3]−, and 91.0204[M-H-C9H8O3]−. These compounds were similar, as reported in the literature [30]. Two compounds (31 and 84) had a deprotonated molecular ion at m/z 269.0815 in the MS data, which matched with the chemical formula C16H14O4. Based on the DPIs (m/z 163.0397[M-H-C6H5O]−, 148.0151 [M-H-C6H3O2]−, 135.0085 [M-H-C8H8O]−, 119.0507 [M-H-C7H4O3]−, and 91.0188 [M-H-C9H8O3]−) of the compound 31 was identified as 7-hydroxy-4′-methoxy dihydroflavone (Figure 6) [31], and compound 84 was identified as 5-hydroxy-7-methoxy dihydroflavone by DPIs (m/z 165.0196, 149.9946, 122.0012, and 65.0046) [32]. Compound 89 had a mass of 68 Da (C5H8), more than pinocembrin and its chemical formula was calculated to be C20H20O4. This compound was provisionally designated as isobavachin based on DPIs (m/z 255.0667 [M-H-C5H8]−, 203.0706 [M-H-C6H3O2]−, 159.0805 [M-H-C9H8O3]−, 119.0506 [M-H-C5H8-C7H3O3]−, and 93.0361 [M-H-C14H14O3]−) [33].

3.3. Screening and Identification of Flavonol

The molecular formula of compound 7 was calculated as C15H10O7, and the ion fragments that were obtained were mainly m/z 283.0248[M-H-H2O]−, 255.0286[M-H-H2O-CO]−, and 151.0003 [M-H-C8H6O3]−. Based on the results that were consistent with those reported in the literature, the compound was identified as quercetin [34]. Compound 79 had a deprotonated molecular ion at m/z 299.0565 and its molecular formula was calculated as C16H12O6. These fragments (m/z 284.0320 [M-H-CH3]−, 271.0610 [M-H-CO]−, 165.0190 [M-H-C8H6O2]−, 151.0033 [M-H-CH3-C8H6O2]−, and 93.0355 [M-H-C10H6O5]−) were determined to be those of rhamnocitrin (Figure 3 and Figure 7), which was consistent with that reported in the literature [35]. Compound 9 was identified as dihydrokaempferol (Figure 8) by DPIs (m/z 259.0605 [M-H-CO]−, 269.00435[M-H-H2O]−, 177.0558[M-H-H2O-C6H4O]−, and 151.0031[M-H-H2O-C8H8O]−) and its molecular formula was C15H12O6.

3.4. Screening and Identification of Isoflavane

The molecular ion peak of compound 67 at m/z 301.1048 was determined based on quantitative data and its molecular formula was estimated to be C17H18O5. The compound was confirmed as 5,7-dimethoxy-2′,4′-dihydroxy isoflavane (Figure 3 and Figure 9) by the DPIs at 286.0853[M-H-CH3]−, 271.0602[M-H-CH3-CH3]−, 135.0451[M-H-C9H10O3]−, and 109.0293[M-H-C11H12O3]− (Figure 2), and further confirmed by comparison with fracture modes reported in the literature [4,36]. The parent ion of Compound 39 was identified as m/z 287.0912 (C16H16O5) and it had a mass of 14 Da (CH2) less than that of compound 67. The compound was provisionally identified as 7,2′,3′-trihydroxy-4′-methoxy isoflavane (Figure 3) by DPIs (m/z 257.0820 [M-H-CH3O]−, 239.0712 [M-H-CH3O-H2O]−, 136.0170[M-H-C8H7O3]−, and 109.0298[M-H-C10H10O3]−) [36].

3.5. Screening and Identification of Indole Alkaloids

Compound 112 was an isomer with a quasi-molecular ion m/z 238.0867, which was consistent with the chemical formula of C15H11NO2. It was determined to be 3-hydroxy-N-benzoyl indole based on the resulting DPIs (m/z 165.0705, 105.0342, 77.0397) [36]. The deprotonated molecular ions at m/z 254.0821 (C15H11NO3) for two compounds (93 and 98) were 16 Da (O) more than that of 3-hydroxy-N-benzoyl indole. The structure of Compound 93 was deduced from the DPIs (165.0695, 121.0288, 107.0498) and speculated as 3-hydroxy-N-(3-hydroxybenzoyl) indole. Based on the DPIs at m/z 135.0449, Compound 98 was tentatively identified as 3-hydroxy-N-(p-hydroxybenzoyl) indole [36]. Compounds 108 had a mass of 30 Da (CH2O) more than that of 3-hydroxy-N-benzoyl indole and was identified as 3-methoxy-N-(p-hydroxybenzoyl) indole by the fragmentation ions (m/z 253.0742, 121.0291, 93.0347) [36]. The precursor ion of Compound 118 was identified as m/z 322.1456, and had a mass of 84 Da (C5H8O) more than that of 3-hydroxy-N-benzoyl indole. It was provisionally identified as 3-hydroxy-N-(3-isopentenyl-4-hydroxybenzoyl) (Figure 10 and Table 2) indole on the basis of DPIs (m/z 266.0825 [M+H-C4H7]+, 238.0865 [M+H-C4H8-CO]+, 189.0913, 133.0289).

3.6. Identification of 22 Metabolites (M1-M22) in Mice

Since drug metabolism largely determines the pharmacokinetic characteristics and bioavailability of most drugs, in order to better understand the metabolic pathway of OMDC, in vivo metabolite analysis was carried out. To analyze the in vivo metabolites, a megadose of OMDC solution (150 mg/kg body weight) was administered orally to rats. Plasma samples from three animals at different sampling sites (1, 2, 4, 6, and 12 h) were mixed and gathered for LC/MS. In total, 22 metabolites from plasma were tentatively identified (Table 3) based on fragment ions. These compounds were metabolized by glucuronidation and sulfation (Figure 11 and Figure 12).
Eight glucuronidated, 9 sulfated, 3 both glucuronidated and glucuronidated, and 2 both glucuronidated and sulfated metabolites were identified. M10 showed [M-H]− at m/z 431.0984 and its molecular formula was predicted as C21H20O10. The fragment ion was formed by the neutral loss of 176.0326 Da (C6H8O6) in the MS2 spectra; thus, it was determined to be pinocembrin glucuronide. M20 showed [M-H]− at m/z 335.0231 and its molecular formula was predicted to be C15H12O7S. Based on the fragment ion at m/z 255.0650, 171.0463, and 151.0000, the molecule was determined to be pinocembrin sulfate. M1 showed [M-H]− at m/z 607.1304 and its chemical formula was C27H28O16. The fragment ion at m/z 255.0650, forecasted as C15H12O4, was generated by the loss of two 176.0326 Da (C6H8O6). Subsequently, the molecule was provisionally determined to be pinocembrin diaglucuronide. M9 showed [M-H]− at m/z 511.0552 and its chemical formula was C21H20O13S. It was generated by a loss of 79.95 Da (SO3) and 176.0326 Da (C6H8O6). Hence, the metabolite was provisionally confirmed as pinocembrin glucuronide sulfate. Four corresponding metabolites (M4, M7, M8, and M18) of 2′,4′-dihydroxychalcone were detected (Figure 11). Other metabolites are also derived from flavonoids. As the major chemical components and major in vivo metabolites of OMDC, flavonoids may be crucial for the pharmacological effects of OMDC.

4. Conclusions

A rapid method using UHPLC-QTOF-MS was established for the isolation and authentication of the chemical constituents of OMDC. The compounds in OMDC extract were identified by NL and DPI sifting schema. Our results indicated that a total of 127 compounds, including 92 flavonoids, 15 indole alkaloids, and 20 others, could be identified in the OMDC extract. After the oral administration of OMDC extract to rats, 22 different compounds were found in the plasma, which appeared to be flavonoid metabolites. The primary in vivo metabolic reactions undergone by OMDC were glucuronidation and sulfation. This UHPLC-MS method is the first of its kind to determine the chemical constituents of OMDC in the positive and negative ion modes. Our findings revealed that the combination of UHPLC-MS and effective data mining is a logical, practical, and systematic method for the characterization of the chemical constituents and metabolites of OMDC.

Author Contributions

Conceptualization: Y.F. and H.O.; methodology: J.L.; validation: J.Z., N.P. and Y.Y.; formal analysis: M.X. and M.Y.; investigation: M.X. and M.Y.; writing—original draft preparation: M.X. and M.Y.; writing—review and editing: M.X., M.Y., H.O. and J.L.; supervision: S.Y. and G.Z.; project administration: Y.F. and H.O.; funding acquisition: Y.F. and H.O. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the National Key R and D Program of China (2019YFC1712302; Yulin Feng); Jiangxi University of Traditional Chinese Medicine 1050 youth talent project and Jiangxi University of Chinese Medicine Science and Technology Innovation Team Development Program (Hui Ouyang).

Institutional Review Board Statement

The animal experiments were approved by the Laboratory Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (approval No. SYXK2017-0004).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data related to the manuscript are available in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Separations 09 00297 g001 550
Figure 1. Representative chromatograms from the analysis of OMDC extracts. (A,B): extracted ion chromatogram (EIC) of reference standards; (C): positive ion mode; (D): negative ion mode) total ion chromatogram (TIC) of OMDC extract.
Figure 1. Representative chromatograms from the analysis of OMDC extracts. (A,B): extracted ion chromatogram (EIC) of reference standards; (C): positive ion mode; (D): negative ion mode) total ion chromatogram (TIC) of OMDC extract.
Separations 09 00297 g001
Separations 09 00297 g002 550
Figure 2. Structures of compounds identified from OMDC.
Figure 2. Structures of compounds identified from OMDC.
Separations 09 00297 g002
Separations 09 00297 g003 550
Figure 3. Proposed fragmentation pathways of 2′,4′-dihydroxychalcone.
Figure 3. Proposed fragmentation pathways of 2′,4′-dihydroxychalcone.
Separations 09 00297 g003
Separations 09 00297 g004 550
Figure 4. RDA collision of flavone and isoflavone aglycones at C ring (A, B, and C stand for different six-membered rings).
Figure 4. RDA collision of flavone and isoflavone aglycones at C ring (A, B, and C stand for different six-membered rings).
Separations 09 00297 g004
Separations 09 00297 g005 550
Figure 5. Proposed fragmentation pathways of 5,7-dihydroxy-4′-methoxy isoflavones.
Figure 5. Proposed fragmentation pathways of 5,7-dihydroxy-4′-methoxy isoflavones.
Separations 09 00297 g005
Separations 09 00297 g006 550
Figure 6. Proposed fragmentation pathways of 7-hydroxy-4′-methoxy dihydroflavone.
Figure 6. Proposed fragmentation pathways of 7-hydroxy-4′-methoxy dihydroflavone.
Separations 09 00297 g006
Separations 09 00297 g007 550
Figure 7. Proposed fragmentation pathways of rhamnocitrin.
Figure 7. Proposed fragmentation pathways of rhamnocitrin.
Separations 09 00297 g007
Separations 09 00297 g008 550
Figure 8. Proposed fragmentation pathways of dihydrokaempferol.
Figure 8. Proposed fragmentation pathways of dihydrokaempferol.
Separations 09 00297 g008
Separations 09 00297 g009 550
Figure 9. Proposed fragmentation pathways of 5,7-dimethoxy-2′,4′-dihydroxy isoflavane.
Figure 9. Proposed fragmentation pathways of 5,7-dimethoxy-2′,4′-dihydroxy isoflavane.
Separations 09 00297 g009
Separations 09 00297 g010 550
Figure 10. Proposed fragmentation pathways of 3-hydroxy-N-(3-isopentenyl-4-hydroxybenzoyl) indoles.
Figure 10. Proposed fragmentation pathways of 3-hydroxy-N-(3-isopentenyl-4-hydroxybenzoyl) indoles.
Separations 09 00297 g010
Separations 09 00297 g011 550
Figure 11. Structures of identified metabolites of drugs.
Figure 11. Structures of identified metabolites of drugs.
Separations 09 00297 g011
Separations 09 00297 g012 550
Figure 12. The EICs of 22 metabolites in rat plasma after administration of OMDC.
Figure 12. The EICs of 22 metabolites in rat plasma after administration of OMDC.
Separations 09 00297 g012
Table
Table 1. Chromatographic and mass spectrometric data (negative ion) of compounds identified from OMDC using UHPLC-QTOF-MS/MS.
Table 1. Chromatographic and mass spectrometric data (negative ion) of compounds identified from OMDC using UHPLC-QTOF-MS/MS.
No.Formula[M-H]-Error (ppm)tR (min)MS/MS (Characteristic Product Ions)IdentityIntensity
1C15H12O5271.0612−0.98.17243.0656, 227.0728, 185.0579, 164.0089, 136.0160, 109.0292, 91.0215, 65.04172′,3,4,4′-Tetrahydroxychalcone7488
2C15H12O6287.0561−0.712.52269.0428, 259.0615, 243.0683, 203.0330, 173.0608, 151.0031, 125.0255(−)-dihydrokaempferol3122
3C15H14O5273.0769−412.87255.0650, 243.0622, 109.0287,2,4,2’,5’ -Tetrahydrodihydrochalcone 1984
4C15H12O5271.0612−0.712.94253.0493, 243.0659, 227.0566, 13.0819, 164.0107, 136.0168, 109.0303, 91.0195, 67.01902′,3,5,4′-Tetrahydroxychalcone21,430
5C15H10O5269.0456−0.913.75241.0515, 225.0545, 197.0611, 181.0653, 135.0096, 91.02052’,5,7-Trihydroxyflavone5705
6C21H22O10433.114023.213.84271.0590, 165.0192Naringenin-glucose 1139
7C15H10O7301.0354−0.614.08283.0248, 255.0286, 215.0340, 151.0003, 145.9294Quercetin; 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one3244
8C15H14O5273.0769−1.414.12227.0768, 167.0349, 149.0244, 137.0246, 123.0453, 109.03022’,4’,6’,4-Tetrahydroxydihydrochalcone3608
9C15H12O6287.0561−0.514.79269.0435, 259.0605, 243.0667, 201.0534, 177.0558, 151.0031, 125.0242, 83.0133, 63.0250Dihydrokaempferol; 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one6907
10C17H16O6315.08741−1.214.83300.0646, 257.0497, 178.9552, 149.02713’,5-Dihydroxy-4’,7-Dimethoxyflavanone1362
11C16H14O6301.07176−0.715.1286.0473, 283.0595, 271.0616, 255.0702, 191.0359, 179.0344, 176.0093, 164.9257, 151.0394, 135.0081, 121.02953’,5,7-Trihydroxy-4’-Methoxyflavanone4211
12C15H10O5269.0456−0.715.26251.0317, 241.0492, 225.0556, 181.0643, 149.0225, 135.0082, 91.02327,8,3’-Trihydroxyflavone8460
13C21H22O10433.114020.715.42271.0601, 165.0179Naringenin-glucose 1470
14C15H14O5273.0769−0.915.62255.0670, 227.0683, 167.0347, 148.0172, 137.0131, 123.0455, 109.02862,4,2′,5′-Tetrahydrodihydrochalcone 5233
15C17H16O6315.08741−0.816.23300.0618, 257.0438, 178.9567, 149.0228, 121.03094’,6-Dihydroxy-5,7-Dimethoxyflavanone1794
16C16H14O6301.07176−0.916.39283.0612, 271.0613, 225.0554, 179.0352, 151.0418, 136.0136, 121.0305, 93.03537-methoxy-3’,4’,5-trihydroxyflavanone4732
17C15H10O6285.0405−0.816.9257.0449, 217.0506, 199.0390, 175.0396, 149.0229, 133.0292, 83.0140, 65.0201Luteolin; 3’,4’,5,7-Tetrahydroxyflavone42,838
18C15H12O4255.0663−1.417.65135.0088, 119.0504, 91.0198Liquiritigenin; 4’, 7-dihydroxyflavanone114,322
19C15H10O6285.0405−0.817.71257.0504, 271.0504, 199.0396, 175.0391, 149.0239, 133.0293, 83.0159, 65.00472’,4’,5,7-Tetrahydroxyisoflavone56,144
20C16H14O6301.07176−1.318.06191.0353, 176.0103, 164.9285, 148.0148, 109.0297, 67.02083′,4′,7-Trihydroxy-5-methoxyflavanone2937
21C9H8O2147.04515−0.818.3103.0556, 77.0391, 61.9902Cinnamic acid8302
22C16H12O5283.0612−0.518.42268.0377, 239.0341, 215.0326, 211.0400, 195.0450, 184.0531, 147.0451, 112.9849, 61.99026,7-dihydroxy-5-methoxyflavone9831
23C15H12O4255.0663−1.318.64237.0560, 209.0608, 199.0761, 167.0861, 149.0247, 135.0090, 109.0301, 91.02066,7-dihydroxyflavanone225,821
24C16H12O5283.0612−118.73268.0376, 239.0345, 211.0394, 184.0519, 148.0168, 135.00895,7-dihydroxy-4′-Methoxy isoflavones91,281
25C16H16O5287.0925−1.418.91272.0683, 257.0439, 163.0405, 150.0321, 135.0439, 121.0306, 109.0280, 91.05807,2′,3′-Trihydroxy-4′-methoxyisoflavane 4783
26C21H22O9417.11910.618.96255.0659, 177.0147, 151.0057Pinocembrin-glucoside 4369
27C16H12O5283.0612−0.419.22268.0375, 239.0344, 211.0399, 184.0526, 148.0168, 135.00894’,7-Dihydroxy-2’-methoxyisoflavon131,630
28C21H22O9417.11910.419.47255.0669, 177.0191, 151.0029Pinocembrin-glucoside 4277
29C16H16O5287.0925−0.419.48272.0690, 257.0435, 163.0397, 150.0317, 135.0463, 121.0292, 109.0279, 91.06157,2′,3′-Trihydroxy-6-Methoxy isoflavane5798
30C15H12O4255.0663−1.119.52237.0610, 209.0610, 199.0765, 165.0709, 135.0089, 109.0302, 91.02023’, 6-dihydroxyflavanone279,373
31C16H14O4269.0819−119.75253.0522, 163.0401, 148.0178, 135.0084, 119.0499, 109.0299, 91.02247-hydroxy-4’-methoxy dihydroflavone36,522
32C16H12O6299.0561−119.96109.0297, 65.04337,3’,4’-trihydroxy-5-methoxy flavone13,371
33C16H14O4269.0819−0.620.26253.0499, 225.0541, 163.0397, 148.0158, 135.0085, 119.0508, 109.0294, 91.01882’,4’-Dihydroxy-2-methoxychalcone46,597
34C21H20O10431.098370.120.32269.0456, 226.9665, 158.9784, 140.9788Apigenin-glucoside 1697
35C21H22O9417.11910.720.33255.0658, 145.0644Pinocembrin-glucoside 9326
36C16H12O6299.0561−0.420.45109.0299, 65.04112’,4’,5-Trihydroxy-7-methoxyisoflavone14,610
37C21H20O10431.098371.520.62269.0439, 223.0971, 140.9806Apigenin-glucoside 1657
38C21H22O9417.11910.920.76255.0659, 211.0790, 171.0418, 151.0031Pinocembrin-glucoside 10,565
39C16H16O5287.0925−0.720.83257.0818, 239.0698, 224.0470, 136.0171, 109.0297, 91.02107,2′,3′-Trihydroxy-4′-methoxyisoflavane11,412
40C15H12O5271.0612−0.420.84253.0506, 225.0542, 215.0712, 197.0600, 177.0190, 161.0607, 151.0033, 119.0504, 107.0144, 93.0352Naringenin; 4’,5,7-trihydroxyflavanone196,476
41C16H12O5283.0612−0.821.16268.0372, 239.0360, 211.0406, 184.0529, 146.9650, 135.0069, 61.99163’-methoxy-5,7-dihydroxyflavone9696
42C16H16O5287.0925−0.721.26257.0820, 239.0712, 224.0484, 136.0170, 109.0298, 91.02047,2′,3′-Trihydroxy-4′-methoxyisoflavane 13,087
43C15H12O5271.0612−0.721.32253.0507, 225.0548, 215.0706, 197.0598, 185.0599, 177.0191, 161.0605, 151.0038, 119.0508, 107.0143, 93.0354, 63.0268Naringenin chalcone; 2’,4,4’,6’-tetrahydroxychalcone300,571
44C16H12O5283.0612−0.721.53268.0375, 239.0348, 211.0406, 195.0449, 184.0515, 146.9653, 135.0073, 61.98926,4’-dihydroxy-7-methoxyflavone11,665
45C15H10O5269.0456−121.83225.0554, 201.0550, 181.0660, 151.0031, 149.0242, 117.0347, 107.0154, 87.0472Apigenin; 4’,5,7-Trihydroxyflavone48,246
46C17H16O6315.08741−0.221.84297.0389, 269.0453, 109.0294, 65.04045,4’-dihydroxy-7,3’-dimethoxy-flavanone6428
47C16H12O7315.0510.222.08297.0397, 269.0450, 254.0218, 226.0249, 165.0199, 109.0293, 65.0390Rhamnetin4246
48C15H12O5271.0612−0.822.1253.0502, 227.0706, 185.0606, 151.0034, 143.0499, 107.0138, 83.0146, 65.00447,3’,5’-trihydroxyflavanone 46,118
49C17H16O6315.08741−0.622.14297.0401, 269.0448, 254.0230, 226.0184, 198.0315, 165.0202, 109.0293, 65.04333’,4’-Dihydroxy-6,7-methoxyflavanone6592
50C15H12O5271.0612−0.822.43253.0500, 227.0706, 185.0601, 151.0030, 143.0495, 107.0143, 83.0157, 65.00533,7,4’-Trihydroxyflavanone55,627
51C16H12O6299.0561−0.523.26284.0323, 255.0261, 227.0338, 211.0398, 148.0180, 91.02005,7,2′-trihydroxy-4’-methoxy flavone8908
52C15H14O4257.0819−1.123.56163.0400, 151.0405, 135.0088, 107.0511, 93.0358, 65.04142’,4’,4-Trihydroxydihydrochalcone218,604
53C15H10O3237.0557−1.223.69208.0531, 193.0658, 180.0573, 165.0721, 132.0213, 91.02097-Hydroxyflavone52,893
54C16H12O4267.0663−0.324.19252.0418, 223.0387, 196.0520, 168.0583, 135.0085, 117.0344, 91.01927-methoxy-4’-hydroxyisoflavone26,052
55C15H10O3237.0557−2.424.54208.0515, 193.0641, 180.0579, 135.0094, 117.0348, 91.02686-Hydroxyflavone10,396
56C16H14O5285.07685−124.56267.0644, 163.0393, 135.0452, 121.0306, 109.03222, 91.0673Vestitone; 2’,7-dihydroxy-4’-methoxyisoflavanone5450
57C16H10O5281.0456−0.924.73253.0504, 224.0477, 209.0602, 195.0446, 167.0493, 135.0088, 117.0337, 91.0201Pseudobaptigenin158,719
58C21H22O8401.12420.824.76239.0712, 197.0607, 135.0087, 112.98512′,4′-Dihydroxychalcone-glucose13,190
59C15H12O4255.0663−1.225.06135.0093, 119.0512, 91.0204Isoliquiritigenin; 2’,4,4’-Trihydroxychalcone518,486
60C16H12O6299.0561−0.925.39284.0315, 256.0351, 165.0191, 149.9952, 121.0293, 65.02623’-Methoxy-4’,5,7-trihydroxyflavone39,790
61C16H12O4267.0663−1.225.45252.0424, 223.0391, 208.0522, 195.0442, 167.0485, 132.0214, 91.0181Formononetin; 7-Hydroxy-4’-methoxyisoflavone101,086
62C15H12O3239.0714−2.325.85197.0603, 169.0658, 148.0164, 135.0090, 109.0302, 91.02077-Hydroxyflavanone1,785,720
63C16H14O4269.0819−0.825.9225.0899, 148.0184, 119.05104,4’-dihydroxy-2’-methoxychalcone20,639
64C15H12O4255.0663−1.126.12237.0560, 209.0605, 193.0657, 169.0661, 145.0294, 135.0088, 119.0503, 109.0302, 91.0201(2R)-Pinocembrin; (2R)-5,7-dihydroxy-2-phenyl-2,3-dihydrochromen-4-one202,511
65C16H12O6299.0561−0.726.31284.0326, 255.03040, 227.0347, 211.0384, 148.0152,3′,4′,7-Trihydroxy-5-methoxyflavone12,184
66C15H10O4253.0506−0.826.78223.0384, 208.0526, 195.0450, 180.0582, 152.0633, 132.0422, 116.9286, 92.03184’,7-Dihydroxyisoflavone26,456
67C17H18O5301.1082−0.927.07286.0853, 271.0602, 179.0728, 164.0482, 149.0234, 135.0451, 109.02937,3 ‘-dihydroxy-2’, 4 ‘-dimethoxy isoflavane5886
68C16H8O6295.02481−0.827.88267.0292, 266.0218, 239.0352, 211.0407, 195.0285, 158.9768, 114.9876Medicagol2425
69C15H10O4253.0506−0.929.23225.0561, 209.0610, 181.0659, 143.0504, 119.0504, 107.0147, 63.0270Chrysin; 5,7-Dihydroxyflavone872,015
70C16H14O5285.07685−0.129.5269.0472, 165.0191, 119.0505, 97.0290, 89.0041, 65.0129Sakuranetin, 4’,5-Dihydroxy-7-methoxyflavanone52,887
71C15H12O4255.0663−1.329.68227.0708, 213.0549, 211.0762, 185.0603, 171.0447, 169.0655, 151.0035, 145.0659, 107.0146, 83.0160, 65.0068Pinocembrin; 5,7-Dihydroxyflavanone2,838,626
72C15H12O5271.0612−229.72253.0504, 243.0659, 227.0706, 185.0603, 173.0606, 152.0107, 124.0162, 95.0139, 65.00507,3’,4’-Trihydroxyflavanone45,968
73C15H10O3237.0557−0.329.79208.0519, 193.0671, 180.0560, 165.0724, 135.0106, 107.0156, 91.0193, 65.01035-Hydroxyflavone13,845
74C15H12O3239.0714−1.730.26211.0796, 197.0601, 195.0805, 169.0653, 148.0164, 135.0089, 109.0305, 91.0203, 65.00572’,5’-Dihydroxychalcone74,066
75C15H10O5269.0456−0.130.44252.0426, 239.0369, 224.0454, 200.8814, 169.0643,3’,4’,5-Trihydroxyflavon7107
76C16H14O5285.07685−1.930.57267.0656, 145.0293, 139.0398, 124.0168, 96.02293′,4′-Dihydroxy-5′-methoxyflavanone 63,776
77C15H14O4257.0819−2.230.73239.0721, 213.0925, 195.0814, 173.0610, 151.0039, 122.0377, 107.0153, 81.0367, 65.00432’,4’,6’-Trihydroxydihydrochalcone127,231
78C16H8O6295.02481−0.830.79267.0293, 266.0216, 237.0185, 211.0393, 135.0100Medicagol; 7-Hydroxy-11,12-methylenedioxycoumestan23,276
79C16H12O6299.0561−0.630.99284.0320, 271.0610, 256.0367, 240.0415, 178.0270, 165.0190, 151.0033, 122.0014, 93.0355, 65.0054Rhamnocitrin; 3,4’,5-Trihydroxy-7-methoxyflavone64,943
80C15H14O3241.087−1.233.52223.0762, 197.0970, 150.0321, 135.0091, 122.0380, 109.0307, 91.0209, 65.04362′,4′-Dihydroxydihydrochalcone2,684,260
81C16H14O4269.0819−1.233.69225.0916, 148.0151, 119.0507, 93.03372′,4-dihydroxy-4′-Methoxy chalcone108,605
82C15H12O3239.0714−1.433.95211.0761, 197.0600, 195.0812, 169.0662, 148.0169, 135.0090, 109.0308, 91.0210, 65.00742′,4′-Dihydroxychalcone3,251,005
83C15H10O5269.0456−0.535.22241.0498, 225.0544, 197.0596, 181.06453’,4’,7-Trihydroxyflavone9165
84C16H14O4269.0819−1.235.86254.0574, 226.0620, 198.0652, 165.0185, 149.9948, 122.0016, 65.00805-hydroxy-7-methoxy dihydroflavone5383
85C16H16O4271.09758−0.236.11256.0734, 165.0207, 152.0110, 139.0396, 124.01664,5-Dihydroxy-2-Methoxy dihydrochalcone7714
86C16H16O4271.09758−1.436.66256.0752, 253.0872, 165.0202, 151.0116, 139.0392, 124.01584,5-Dihydroxy-2-Methoxy dihydrochalcone7147
87C16H14O4269.0819−0.938.02254.0580, 226.0628, 177.0184, 165.0196, 149.9946, 122.0012, 65.00465-hydroxy-7-methoxy dihydroflavone40,090
88C16H14O5285.07685−0.938.02270.0547, 165.0202, 145.0286, 139.0408, 93.03533’,4’-Dihydroxy-5’-methoxyflavanone3772
89C20H20O4323.1289−0.739.75255.0667, 203.0706, 159.0805, 119.0506, 93.0361isobavachin; 4’,7-dihydroxy-8-prenylflavanone25,579
90C16H14O5285.07685−0.940.66267.0642, 241.0865, 176.0101, 148.0137, 109.01913’,4’-Dihydroxy-6-methoxyflavanone3395
91C20H20O4323.1289−0.443.34305.1204, 277.1659, 255.0628, 219.0698, 186.93124,2′,4′-trihydroxy-3′-isopentenyl chalcone2040
92C16H14O6301.07176−0.852.85283.0601, 257.0847, 192.0055, 173.0594, 164.01034’,5,7-trihydroxy-3’-methoxyflavanone9203
Table
Table 2. Chromatographic and mass spectrometric data (positive ion) of compounds identified from OMDC using UHPLC-QTOF-MS/MS.
Table 2. Chromatographic and mass spectrometric data (positive ion) of compounds identified from OMDC using UHPLC-QTOF-MS/MS.
No.Formula[M+H]-Error (ppm)tR (min)MS/MS (Characteristic Product Ions)IdentityIdentity
93C15H11NO3254.08120.521.29236.0703, 208.0760, 165.0695, 121.0288, 107.0498, 93.0677, 65.03833-Hydroxy-N-(3-hydroxybenzoyl) indole6243
94C15H26O2239.2006−0.822.19221.1896, 203.1798, 147.1171, 135.1166, 133.1010, 107.0862, 95.0863, 81.07103-Methyl-5-(1,3,3-trimethyl-7-oxabicyclo [2.2.1] hept-2-yl)-pent-1-en-3-ol18,924
95C15H11NO3254.08120.422.85236.0713, 208.0754, 165.0694, 121.0289, 107.0493, 93.0342, 65.03963-Hydroxy-N-(3-hydroxybenzoyl) indole33,513
96C16H13NO4284.09170.722.91269.0690, 226.0628, 150.0299, 121.0280,3-Methoxy-4-hydroxy-n-(p-hydroxybenzoyl) indole 9202
97C16H13NO4284.09170.823.79269.0685, 150.0309, 120.04423-Methoxy-4-hydroxy-n-(p-hydroxybenzoyl) indole 165,711
98C15H11NO3254.08120.924.9236.0716, 208.0768, 181.0657, 165.0705, 135.0449, 121.0291, 107.0499, 93.0341, 65.03993-Hydroxy-N-(p-hydroxybenzoyl) indole 251,175
99C15H15NO226.12260.725.7122.0602, 105.0342, 103.0553, 77.0399N-Benzoyl-phenylethylamine1,428,756
100C15H11NO3254.08120.926.75236.0714, 209.0585, 181.0654, 165.0708, 153.0689, 135.0453, 121.0286, 107.0482, 93.0338, 65.04013-Hydroxy-N-(p-hydroxybenzoyl) indole 50,895
101C32H32O9561.2119−1.327.96509.1637, 424.1442, 385.1068, 373.1072, 259.0970, 167.0699, 123.0433, 107.0498(3R)-Propterol-B-(α,6)-(−)-isomucronulatol4620
102C15H26O2239.20060.628.48221.1925, 203.1806, 161.1315, 147.1172, 119.0849, 95.0860, 81.07223-Methyl-5-(2,2,4-trimethylcyclohexanol-3-yl)-pent-l-ene-3-ol18,924
103C16H13NO4284.0917128.57269.0685, 137.0245, 91.05303-Methoxy-4-hydroxy-n-(p-hydroxybenzoyl) indole 7924
104C17H19NO254.15390.829.84131.0490, 122.0962, 105.0699, 103.0545, 79.0548N-Hydrocinnamoyl-2-phenylethylamine113,125
105C17H17NO252.13831.130.75148.0760, 131.0492, 105.0699, 103.0543, 79.0550, 77.0395N-Cinnamoyl-2-phenylethylamine5,724,222
106C15H11NO2238.08630.830.84220.0760, 165.0700, 121.0291, 91.0548N-p-Hydroxybenzoyl indole369,512
107C15H11NO2238.0863031.83220.0758, 165.0698, 121.0289, 93.0336, 65.0405,N-p-Hydroxybenzoyl indole52,164
108C16H13NO3268.09680.732.1253.0742, 225.0792, 210.0675, 149.0602, 134.0366, 121.0291, 107.0496, 93.0347, 65.03973-Methoxy-N-(p-hydroxybenzoyl) indole520,562
109C16H13NO3268.09681.132.63253.0745, 150.0318, 121.0298, 105.0342, 77.03983-Methoxy-hydroxy-n-benzoyl indole503,247
110C30H48O3457.3676−133.09439.3568, 421.3458, 376.1913, 245.1907, 233.1914, 185.1312, 163.1505, 147.1145, 109.0989, 81.0724Soyasapogenol E13,542
111C16H13NO3268.09680.533.3253.0737, 255.0783, 150.0320, 134.0367, 121.0285, 105.0349, 93.0333, 77.0394, 65.03903-Methoxy-hydroxy-n-benzoyl indole 29,002
112C15H11NO2238.0863133.57221.0604, 220.0762, 193.0655, 165.0705, 135.0442, 105.0342, 77.03973-Hydroxy-N-benzoyl indole1,270,624
113C16H14O3255.10161.634.48240.0793, 209.0972, 194.0735, 177.0560, 165.0707, 151.0395, 131.0500, 103.0553, 95.0504, 77.04002′-Hydroxy-4′-methoxychalcone2,708,762
114C30H46O4471.3469−135.03453.3366, 435.3233, 395.2965, 199.1518, 173.1328, 159.1160, 145.0998, 97.06543,22,24-Trihydroxy-γ-lactone-olean-12-en-29-oic acid15,709
115C30H48O3457.3676−0.735.18439.3575, 421.3461, 409.3465, 381.3164, 309.2588, 259.2056, 245.1891, 233.1904, 205.1586, 145.1007, 135.1165, 119.0863, 81.0704Melilotigenin C64,956
116C16H13NO3268.09680.835.67253.0736, 165.0544, 150.0313, 137.121.0291, 105.0341, 77.03933-Methoxy-hydroxy-n-benzoyl indole 59,907
117C20H19NO3322.1438135.79266.0819, 238.0874, 211.0753, 133.0322, 121.002863-Hydroxy-N-(3-isopentenyl-4- hydroxybenzoyl) indole 11,031
118C20H19NO3322.14380.737.49266.0825, 254.0814, 211.0757, 189.0913, 165.0703, 133.0289, 105.0336, 77.03893-Hydroxy-N-(3-isopentenyl-4-hydroxybenzoyl) indole261,939
119C16H14O3255.10160.941.5240.0783, 209.0972, 194.0725, 177.0551, 165.0699, 151.0392, 131.0492, 103.0545, 95.0498, 77.03852′-Hydroxy-4′-methoxychalcone129,251
120C30H46O4471.3469−0.642.6453.3360, 435.3254, 423.3261, 395.2930, 287.2016, 259.1681, 247.1705, 201.1631, 189.1624, 147.1173, 109.1043, 95.0854Unknown17,953
121C30H48O3457.3676−0.945.42421.3458, 399.2707, 297.2570, 215.1794, 173.1332, 135.1171, 109.1006Unknown54,664
122C30H46O4471.3469−5.745.93387.2866, 325.1404, 233.1514, 148.085224-Hydroxy-3-oxoolean-12-en-29-oic acid52,681
123C30H50O3459.3833−0.546.13441.3717, 423.3615, 355.1895, 335.2005, 247.2064, 203.1779, 163.0758, 131.0486, 81.0691Soyasapogenol B8837
124N19H21NO4366.2127−1.746.43244.1758, 145.0256, 121.0996, 85.06513-Methoxyaegeline64,028
125C30H50O4475.3782−0.546.52457.2366, 321.1122, 267.0679, 219.1767, 179.0354Wistariasapogenol B12,829
126C30H50O3459.3833−0.247.04441.3741, 423.3649, 323.1291, 311.1293, 219.0657, 203.1810, 161.1330, 135, 1163, 123.1181, 95.0873Unknown25,684
127C30H50O3459.3833−0.448.77441.3746, 323.1950, 311.1284, 293.1184, 201.1654, 179.0698, 109.1012, 107.0856Unknown18,190
Table
Table 3. In vivo metabolites after the administration of OMDC to rats.
Table 3. In vivo metabolites after the administration of OMDC to rats.
No.Formula[M-H]−Error (ppm)tR (min)MS/MS (Characteristic
Product Ions)		Identity
M1C27H28O16607.130463.315.94431.0981, 255.0655Dia-glucuronidation of 71
M2C22H20O11459.093290.416.3283.0594, 268.0382Glucuronidation of 24
M3C21H20O11447.09329−4816.42271.0603, 151.0036, 119.0506Glucuronidation of 43
M4C27H28O15591.135542.716.67415.1037, 239.0711Dia-glucuronidation of 82
M5C27H30O15593.151194.817.05417.1143, 241.0861Dia-glucuronidation of 80
M6C21H18O10429.082723.517.36253.0505, 195.0454Glucuronidation of 69
M7C21H20O9415.103461.419.78239.0709, 197.0611, 135.0085Glucuronidation of 82
M8C21H20O12S495.060275.221.37319.0283, 239.0682Sulfation and glucuronidation of 82
M9C21H20O13S511.055191.122.12431.0999, 255.0656Sulfation and glucuronidation of 71
M10C21H20O10431.098371.923.03255.0667, 213.0552, 211.0769, 171.0448Glucuronidation of 71
M11C22H20O12475.0882−61.923.27299.0558, 284.0336, 255.0281, 227.0366Glucuronidation of 51
M12C15H12O8S351.018010.423.58271.0618, 177.0213, 151.0031, 119.0517, 107.0156Sulfation of 43
M13C22H22O10445.114020.925.99269.0817, 254.0572, 226.0632, 165.0196Glucuronidation of 84
M14C21H22O9417.119110.227.33241.0865, 197.0986, 150.0330Glucuronidation of 80
M15C16H12O8S363.01801127.59283.0616, 268.0378Sulfation of 24
M16C16H10O8S361.00236−128.34281.0453, 253.0508, Sulfation of 57
M17C16H12O9S379.01293029.92299.0560, 284.0319, 165.0181Sulfation of 51
M18C15H12O6S319.02818030.36239.0707, 197.0598, 148.0168, 135.0084Sulfation of 82
M19C16H14O7S349.03875−0.732.49269.0813, 165.0194, 149.9945Sulfation of 84
M20C15H12O7S335.0231−0.535.08255.0653, 213.0538, 171.0463, 151.0000, 145.0642, 107.0139Sulfation of 71
M21C15H14O7S337.038750.340.05257.0818, 239.0714, 151.0031Sulfation of 52
M22C15H14O6S321.04383−0.841.09241.0864, 197.0967, 150.0318, 135.0087Sulfation of 80
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Xia, M.; Yao, M.; Li, J.; Zhang, J.; Yu, Y.; Yang, S.; Zhong, G.; Pei, N.; Ouyang, H.; Feng, Y. Characterization of Chemical Constituents of Oxytropis microphylla (Pall.) DC. by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Time-of Flight Tandem Mass Spectrometry. Separations 2022, 9, 297. https://doi.org/10.3390/separations9100297

AMA Style

Xia M, Yao M, Li J, Zhang J, Yu Y, Yang S, Zhong G, Pei N, Ouyang H, Feng Y. Characterization of Chemical Constituents of Oxytropis microphylla (Pall.) DC. by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Time-of Flight Tandem Mass Spectrometry. Separations. 2022; 9(10):297. https://doi.org/10.3390/separations9100297

Chicago/Turabian Style

Xia, Mengqi, Min Yao, Junmao Li, Jianjian Zhang, Yayun Yu, Shilin Yang, Guoyue Zhong, Na Pei, Hui Ouyang, and Yulin Feng. 2022. "Characterization of Chemical Constituents of Oxytropis microphylla (Pall.) DC. by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Time-of Flight Tandem Mass Spectrometry" Separations 9, no. 10: 297. https://doi.org/10.3390/separations9100297

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