CN112485345B - Comprehensive analysis method for chemical components of compound antidotal agent of antelope horn - Google Patents

Comprehensive analysis method for chemical components of compound antidotal agent of antelope horn Download PDF

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CN112485345B
CN112485345B CN202011221582.8A CN202011221582A CN112485345B CN 112485345 B CN112485345 B CN 112485345B CN 202011221582 A CN202011221582 A CN 202011221582A CN 112485345 B CN112485345 B CN 112485345B
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acid
compound
compounds
triterpen
platycodin
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曹桂云
耿绍轩
孟兆青
宁波
田硕
庄雪松
马桂芳
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Shandong Hongjitang Pharmaceutical Group Co ltd
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Abstract

The invention aims to realize comprehensive and rapid separation and identification of chemical components of the compound sustained release capsule or tablet by taking the chemical components of phenolic acids, flavonoids and triterpenoids in the compound sustained release capsule or tablet as research objects based on a UPLC-Q-TOF-MS analyzer and an analysis strategy of target precursor ions. The research firstly integrates the information of phenolic acid, flavone and triterpenes in the compound Xiling detoxification capsule or tablet according to the literature, and summarizes the structural characteristics and the structural change rule. And (3) carrying out mass spectrum analysis on each type of representative compounds with different structural characteristics, and summarizing a mass spectrum cracking rule and a neutral loss rule. And then, constructing a target precursor ion list by taking the representative compound as a core according to the structural change rule of each type of compound. And finally, analyzing the cracking mode and neutral loss rule of the target precursor ions by adopting a secondary mass spectrum, verifying the correctness of the target precursor ions, and comprehensively analyzing information such as mass spectrum, retention time and the like to comprehensively and quickly analyze chemical components in the compound Xiling detoxification capsule or tablet.

Description

Comprehensive analysis method for chemical components of compound antidotal agent of antelope horn
Technical Field
The invention relates to a method for identifying chemical components in a traditional Chinese medicine preparation, namely, the comprehensive and rapid separation and identification of the chemical components in a compound antidotal Xiling capsule or tablet are realized based on UPLC-Q-TOF-MS technology.
Background
The compound antidotal capsule or tablet is prepared from 13 Chinese medicinal materials including honeysuckle flower, capsule of weeping forsythia, root of ballon flower, schizonepeta spike, burdock fruit (fried), licorice root, lophatherum gracile, menthol crystal, peppermint oil, fermented soybean, antelope horn, buffalo horn concentrated powder and borneol, has the functions of dispelling wind and relieving exterior syndrome and clearing away heat and toxic materials, and is clinically used for treating wind-heat type common cold, fever, headache, cough, hoarseness and sore throat. According to the literature reports, the main components of the compound are phenolic acid, flavone and triterpenoids.
The traditional Chinese medicine is a complex multi-component system, the chemical component information contained in the traditional Chinese medicine is determined, and the traditional Chinese medicine is a precondition and basis for clarifying the pharmacodynamic material basis of the traditional Chinese medicine and evaluating the quality and safety of the traditional Chinese medicine. In recent years, ultra-high performance liquid chromatography and time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) are powerful tools for analyzing complex components of traditional Chinese medicines. However, thousands of precursor ions and multi-level fragments can be detected using UHPLC-Q-TOF-MS techniques, and thus still present significant challenges in raw data mining. Today, several data post-processing techniques are available, such as mass-deficiency filtering, multi-product ion filtering, neutral-loss filtering, fragment ion diagnostic strategies are increasingly used for mass spectrometry data analysis. However, these data post-processing techniques are developed based on single chemical components, have limited ability to identify minor components and co-effluent components, and are unable to fully identify and identify complex traditional Chinese medicine components of various structural types. There are at least 3 problems: 1) Due to the co-efflux phenomenon of compounds and the complexity of mass spectrum data in the separation process of the traditional Chinese medicine, mass spectrum signals of trace components are easy to ignore in the analysis process; 2) The existing analysis strategy is generally to directly summarize the existing compound information in the literature, carry out verification analysis and cannot analyze to obtain a new compound; 3) Due to the lack of reasonable data processing strategies, chemical components in the traditional Chinese medicine cannot be comprehensively analyzed, and only a few dozens of compounds can be generally obtained by analysis. Since mass spectrum information of a large amount of trace components is lost during data processing, most studies can identify only several tens of chemical components from chinese medicines, and it is difficult to find new compounds.
Disclosure of Invention
Aiming at the defects, the invention provides a rapid separation and comprehensive identification research of chemical components of a compound Xiling detoxification capsule or tablet based on a UPLC-Q-TOF-MS technology and a target precursor ion data mining strategy. The method not only greatly simplifies the related analysis work, but also can effectively prevent the trace components from losing in the analysis process, is beneficial to the discovery of new compounds, and realizes the comprehensive analysis of the chemical components of the traditional Chinese medicine.
The technical solution of the invention is as follows: based on the UPLC-Q-TOF-MS technology and a target precursor ion data mining strategy, the chemical components of the compound Xiling detoxification capsule or tablet are comprehensively and quickly analyzed by comprehensively integrating the information such as the structural characteristics, the structural change rule, the mass spectrum cracking rule, the neutral loss rule and the like of the compound. The method comprises the following specific steps: (1) Information integration is carried out on phenolic acid, flavone and triterpenes in the compound Xiling detoxification capsule or tablet, and the structural characteristics and the structural change rule are summarized. And (3) carrying out UPLC-Q-TOF-MS analysis on each type of representative compounds with different structural characteristics, and summarizing a mass spectrum cracking rule and a neutral loss rule. (2) Taking a representative compound as a core, constructing theoretical precursor ions according to the structural change rule of each type of compound, verifying the correctness of the theoretical precursor ions by adopting a primary mass spectrum, and constructing a target precursor ion list. (3) And (3) analyzing the cracking mode and neutral loss rule of the target precursor ions by adopting a secondary mass spectrum, verifying the correctness of the target precursor ions, and comprehensively analyzing information such as mass spectrum, retention time and the like to comprehensively and quickly analyze chemical components in the compound Xiling detoxification capsule or tablet.
The separation and identification of chemical components in the compound antidotal Xiling capsule or tablet are realized based on the UPLC-Q-TOF-MS technology, and the method comprises the following conditions:
preparing a test solution of the compound Xiling detoxification capsule: weighing 1.0 g sample powder of FUFANGXILINGJIEDU Capsule, adding 25 mL methanol, and ultrasonic extracting for 30 min. It was then filtered through a 0.22 μm nylon membrane filter and then analyzed.
Preparing a test solution of the compound antidotal Xiling tablet: the compound antidotal tablet is prepared by pulverizing, weighing 1.0 g sample powder, adding 25 mL methanol, and ultrasonic extracting for 30 min. It was then filtered through a 0.22 μm nylon membrane filter and then analyzed.
And (3) UPLC: the column was an Agilent Advance Bio Peptide (2.1X 250 mm,2.7 μm); the column temperature was 25 ℃; mobile phase: the water phase (A) is 0.1% formic acid water solution, and the organic phase (B) is acetonitrile solution; the chromatographic separation is carried out by adopting a gradient elution mode, and the gradient elution program is as follows: 0-13 min,5-13% B;13-14 min,13-19% B;14-24 min,19-24% B;24-31 min,24-67% B;31-40 min,67-95% B; the flow rate was 0.4 mL/min.
Q-TOF-MS: ultra-high performance liquid chromatography coupled to Q-TOF-MS equipped with electrospray ionization source (ESI) scanning analysis using positive and negative ion modes.
The MS parameters were as follows: the capillary voltage of the positive and negative ion modes is 3500V, the capillary outlet voltage is 175V, the atomizer pressure is 35 psi, the drying gas temperature is 300 ℃, and the flow rate is 8.0L/min. Real-time mass correction uses a solution of reference ions, setting positive source reference ions 121.050873, 922.009798, and negative source reference ions 112.985587, 1033.3988109. Data were acquired at a rate of 1.0 spectrum/sec in a scan mode of m/z 50 to 3000. The collision voltages are respectively set to 10-20V, 20-30V and 30-40V.
145 compounds were screened and confirmed from the compound Xiling Jiedu capsules or tablets, including 27 phenolic acid compounds, which were protocatechuic acid (1), neochlorogenic acid (2), salidroside (3), chlorogenic acid (4), vanillic acid (5), p-hydroxybenzaldehyde (6), cryptochlorogenic acid (7), caffeic acid (8), syringic acid (9), p-hydroxyphenylacetic acid (10), p-coumaric acid (11), ferulic acid (12), isochlorogenic acid B (22), isochlorogenic acid A (23), isochlorogenic acid C (25), eugenol (40), gallic acid (52), p-hydroxybenzoic acid (54), 1-caffeoyl-beta-D-glucose (57), 2-methoxybenzoic acid (58), 4-acetylbenzoic acid (59), 3,4-dihydroxyphenylpropionic acid (60), 3-O-feruloylquinic acid (61), 5-hydroxyferulic acid (67), 4-methoxy-benzoic acid (70), methyl cinnamate (75), caffeic acid methyl ester (79), 46 compounds, genistin (13), isoquercitrin (14), isoquercitrin (17), hesperidin (24), apiose isoliquiritin (26), isoliquiritin (27), daidzein (28), liquiritigenin (30), glycitein (31), calycosin (32), luteolin (34), naringenin (36), isoliquiritigenin (38), formononetin (41), daphnetin (42), glabridin (43), glucolisoliucin apioside (63), genistein-7-O-geniposide (64), genistein-7,4' -di-O-beta-D-glucopyranoside (65), lonicerin (73), sweet pea phenol-7-O-beta-D-glucoside (77), 6' ' -O-acetylisoliquiritin apioside (88), 7-Hydroxy-3 ' -acetoxy-4 ' -methoxyisoflavone (89), butein-4 ' -O-beta-D-glucopyranoside (90), 4' -Hydroxy-7-acetoxyflavone (91), 6' ' -O-acetylisoliquiritin (92), chalconogenin-4-O-beta-D-glucopyranoside (93), 4', 3262 zft 3262-trihydroxy-2 ' -methoxychalcone (94), 4', 7-dihydroxyflavone (95), licochalcone B (96), 2(s) -3',5', 7-trihydroxyflavanone (115), 4' -O-Acetyl-naringenin (120), eugenol donkey (124), glycyrroid B (130), glycyrrone alcohol (133), licochalcone A (137), glabridin glycosides (139), glycyrroid A (140), 3,4-didehydroglaridin (141), glycyrroid B (142), isoangustine A (143), 4' -methylglabridin (144), 39 triterpenoids, platycodin L (29), descheriscoside D (33), platycodin D (35), glycyrrhizic acid (37), glycyrrhetinic acid (45), betulinic acid (46), oleanolic acid (47), isoursolic acid (48), platycodon G2 (82), deschersonine platycodine E (83), tripentene N1 (84), beta-triptycenyl (85), tripropenylic acid (86 "), tripentoyl D (99), triphylloside D (103), tripentene C-2 (99), triphylloside D (103), tripentagenin D (99), tripentene D (103), tripentene D (100), platycodin V (111), platycodi C acid D (112), glycyrrhizin A3 (113), triterpen N6 (114), glycyrrhizin G2 (116), yun Gandai K2 (117), yun Gandai G2 (119), glycyrrhizin E2 (121), glycyrrhizin K2 (125), 22-acetyl Gan Caoquan (126), 3-O- (alpha-L-arabinopyranosyl (1-2) -beta-D-glucopyranosyloxy) glycyrrhetinic acid (127), uraloside C (128), glatiramer W (129), glycyrrhizin J2 (132), 3' ' -O-beta-D-glucopyranosyl sapogenin (135), 33 other classes of compounds, respectively forsythoside A (16), verboside (20), taurin (39), diethoxylin (44), uridine lactose (49), uracil (50), uracil (51), forsythoside B-1 ' -O-D-glucopyranoside A (51), forsythoside A (66), forsythoside B (66), forsythoside A (71), matairesinol glycoside (76), 7,2',4' -trihydroxy-5-methoxy-3-aroumatin (78), forsythiside A (80), suspoiside D (81), suspoiside B (87), forsythiaside (106), arctigenin E (107), arctigenin H (108), matairesinol (118), arctigenin A (122), arctigenin F (123), glycyrrhizin I (131), glycycoumarin (134), glycyrone (136), glycyrol (138), stigmasterol (145).
The invention has the advantages that: 1. the chemical components of the traditional Chinese medicine are complex, the traditional HPLC-UV detector is adopted for analysis, the defects of low sensitivity, poor specificity, less effective information, incapability of obtaining exact structural information and the like are overcome, and a great amount of compound information can be obtained by adopting the UPLC-Q-TOF-MS technology. 2. Using a TOF analyzer, accurate molecular weights can be obtained and molecular formulas of compounds accurately inferred. 3. Chromatographic conditions are optimized, and effective separation of more than 100 compounds is realized within 40 min. 4. Integrating the structural characteristics, the structural change rule, the mass spectrum cracking rule, the neutral loss rule and other information of a compound in the compound Xiling detoxification capsule or tablet, and establishing a target precursor ion data mining strategy, 1) being beneficial to the analysis of trace components, trace components and co-effluence components; 2) Is beneficial to the discovery of new compounds; 3) Chemical components in a complex system can be rapidly and comprehensively analyzed. Therefore, the invention also provides a concept for screening target compounds in other complex samples.
The strategy is applied to the chemical component analysis of the compound antidotal Xiling capsule or tablet, and 145 chemical components are obtained by analysis, including 26 phenolic acid compounds, 46 flavonoid compounds, 39 triterpenoid compounds and 34 other compounds. Wherein 6 compounds are new compounds, 144 compounds are obtained by separating from compound antidotal capsule or tablet of cornu Saigae Tataricae for the first time, 4 compounds are obtained by separating from Glycyrrhrizae radix for the first time, and 1 compound is obtained by separating from fructus Arctii for the first time. The invention solves the problems of complex traditional Chinese medicine components and difficult identification, and can provide basis for other similar researches.
Drawings
FIG. 1 is a total ion flow diagram of a compound Xiling detoxification capsule in positive and negative ion mode
FIG. 2 shows the cracking law of phenolic acid compounds
FIG. 3 shows the structure of the compound separated from the antidotal capsule or tablet of compound Xiling
FIG. 4 shows the cleavage pathway of the novel compound in FUFANGXILINGJIEDU Capsule or tablet
FIG. 5 shows compound information obtained by separating compound from FUFANGXILINGJIEDU Capsule or FUFANGXILINGJIEDU tablet
The specific implementation mode is as follows:
based on UPLC-Q-TOF-MS technology and target precursor ion data analysis strategy, the rapid separation and identification of chemical components in the compound antidotal Xiling capsule or tablet are realized:
1. test materials and methods
1.1 Instruments and reagents
An Agilent 1290 UPLC-Q-TOF-MS instrument, an electrospray ion source and a MassHunter workstation are configured; BSA224S-CW electronic balance (Saedodes scientific instruments, inc.); SQP electronic balance (sidoris scientific instruments ltd);
LC-MS grade acetonitrile was purchased from merck group (damstatt, germany); chromatographic grade formic acid was purchased from Tianjin Kemiou chemical reagents, inc. (Tianjin, china); purified water was purchased from drochen group ltd (guangzhou, china); the standard product is purchased from national food and drug administration, jiangsu Yongjian pharmaceutical technology, inc. and Chengdu Philippine biotechnology, inc.; compound xilingjiedu capsule (batch number: 1811001) and compound xilingjiedu tablet (2001002) were provided by Shandong Hongji Tang pharmaceutical group, inc. (Shandong Jinan).
1.2 Preparation of samples
Preparation of a standard solution: the standards were dissolved in LC-MS grade methanol to obtain respective standard stock solutions. Filtration through a 0.22 μm nylon membrane filter was performed prior to use.
Preparing a test solution of the compound Xiling detoxification capsule: weighing 1.0 g sample powder of FUFANGXILINGJIEDU Capsule, adding 25 mL methanol, and ultrasonic extracting for 30 min. It was then filtered through a 0.22 μm nylon membrane filter and then analyzed.
Preparing a test solution of the compound Xiling detoxification tablet: the compound antidotal tablet is prepared by pulverizing, weighing 1.0 g sample powder, adding 25 mL methanol, and ultrasonic extracting for 30 min. It was then filtered through a 0.22 μm nylon membrane filter and then analyzed.
1.3 Chromatographic and mass spectral conditions
In the experiment, phenolic acid, flavone, triterpene and other compounds in the compound Xiling detoxification capsule or tablet are mainly separated and analyzed, the physical and chemical properties of the compounds are comprehensively analyzed, and the chemical components are separated by using a reverse phase high performance liquid phase. The chromatographic column is one of the most important factors influencing the separation of chemical components, and the chromatographic column is repeatedly examined and finally subjected to chromatographic separation by using Agilent advanced Bio Peptide (2.1X 250 mm,2.7 mu m). The length of a chromatographic column conventionally used by UPLC-MS is 50-150 mm, and the length of the chromatographic column is 250 mm, so that a better separation effect can be achieved. Furthermore, in order to completely separate each component and shorten the peak time, gradient elution is adopted, an elution solvent and a gradient are repeatedly optimized, and finally a mobile phase is determined: the water phase (A) is 0.1% formic acid aqueous solution, and the organic phase (B) is acetonitrile solution; the gradient elution procedure was as follows: 0-13 min,5-13% B;13-14 min,13-19% B;14-24 min,19-24% of B;24-31 min,24-67% B;31-40 min,67-95% B. The flow velocity of the mobile phase has certain influence on the separation degree and the peak-off time of the compound to a certain degree, the components cannot be well separated even if the flow velocity is high or low, and finally the flow velocity of the mobile phase is determined to be 0.4 mL/min through a series of researches. In addition, the sample amount is considered, if the sample amount is too small, the detection of trace compounds is influenced, if the sample amount is too large, the column can be overloaded, and the sample amount is finally determined to be 0.5 muL after the examination. The chromatographic conditions were thus finally determined as: the column was an Agilent advanced Bio Peptide (2.1X 250 mm,2.7 μm); mobile phase: the water phase (A) is 0.1% formic acid water solution, and the organic phase (B) is acetonitrile solution; the chromatographic separation is carried out by adopting a gradient elution mode, and the gradient elution program is as follows: 0-13 min,5-13% B;13-14 min,13-19% of B;14-24 min,19-24% B;24-31 min,24-67% B;31-40 min,67-95% B. The flow rate of the mobile phase was 0.4 mL/min. The column temperature was 25 ℃.
MS conditions: mass spectrometry was performed in positive and negative ion mode, respectively. Real-time mass correction uses a solution of reference ions, setting positive source reference ions 121.050873, 922.009798, and negative source reference ions 112.985587, 1033.3988109. The capillary voltage, the capillary outlet voltage, the atomizer pressure, the drying gas temperature and the acquisition speed are optimized according to the response of the parent ions, the obtained optimized mass spectrum conditions are 3500V of the capillary voltage, 175V of the capillary outlet voltage, 35 psi of the atomizer pressure, 300 ℃ of the drying gas temperature and 8.0L/min of the flow speed, and data are acquired at the speed of 1.0 spectrum/second in a scanning mode of m/z 50-3000. Second, we optimize the parameters of the collision energy according to the intensities of the parent and fragment ions. If the collision energy is too low, the parent ions cannot be cracked into fragment ions, so that the response of the fragment ions is too low, if the collision energy is too high, the fragment ions are cracked into other ions, and proper collision energy is particularly important for obtaining the fragment ions, so that the collision energy is graded, and the collision voltage is set to be 10-20V, 20-30V, and 30-40V. The total ion flow chart of the positive and negative ions is shown in figure 1.
1.4 Data processing
The data were processed using Agilent MassHunter Qualitative Analysis (B.08.00) software. The target precursor ion data resolution strategy mainly comprises three steps. Firstly, the components of the antidotal compound capsules or tablets of cornu saigae tataricae are divided into several basic structural types according to the literature. And (3) carrying out mass spectrum analysis on the representative compounds of each type of structure, and summarizing a cracking rule and a neutral loss rule. Secondly, the target compounds are oriented through the structural characteristics and structural correlation of each class of compounds, and a target precursor ion list is constructed. Third, the structure of the target precursor ion is verified by secondary mass spectrometry.
2. Test results
145 compounds are separated from the compound Xiling detoxification capsule or tablet by adopting a compound analysis strategy based on target precursor ions, wherein the compounds comprise 26 phenolic acid compounds, 46 flavonoid compounds, 39 triterpenoid compounds and 34 other compounds.
2.1 Basic structure and cracking rule of chemical components in compound Xiling detoxification capsule or tablet
The compound antidotal capsule or tablet comprises 13 traditional Chinese medicines of honeysuckle, weeping forsythia, balloonflower root, schizonepeta spike, great burdock achene (fried), liquorice, lophatherum gracile, menthol, peppermint oil, fermented soybean, antelope horn, buffalo horn concentrated powder and borneol, and the main components of the single traditional Chinese medicines are phenolic acid, flavone and triterpenoid according to literature reports. The chemical components in the Chinese patent medicine are derived from a single Chinese medicine, so that the main components of the compound Xiling detoxification capsule or tablet are phenolic acid, flavone and triterpene. Through literature search, the three classes of compounds are respectively divided into several basic structural frameworks. Standard compounds (Compounds 1-48) were purchased for mass spectrometry analysis to summarize the cleavage pattern. The standard compound meets the following two requirements: 1) Is the main component of the antidotal capsule or tablet of compound antelope; 2) Has a basic structure skeleton.
2.1.1 Phenolic acid compound
Protocatechuic acid (1), neochlorogenic acid (2), chlorogenic acid (4), vanillic acid (5), p-hydroxybenzaldehyde (6), cryptochlorogenic acid (7), caffeic acid (8), syringic acid (9), p-hydroxyphenylacetic acid (10), p-coumaric acid (11), ferulic acid (12), isochlorogenic acid B (22), isochlorogenic acid A (23), isochlorogenic acid C (25) and eugenol (40) are main phenolic acid compounds in the compound Xiling detoxification capsule or tablet. The compounds are classified into class 2 according to whether caffeoyl group is contained, and the main structural changes comprise addition and subtraction of hydroxyl, methoxy, ethoxy, aldehyde group, carboxyl and the like. The analysis of the 15 compounds in the compound Xiling Jiedu capsule or tablet is confirmed by the cracking rule, reference substance and reference substance. For example, neochlorogenic acid (2), chlorogenic acid (4), and cryptochlorogenic acid (7) are 3 phenolic acid isomers having a caffeoyl group in the structure. All 3 of these compounds produced [ M-H at M/z 353] - Peak at M/z 191 [ M-H-C 9 H 6 O 3 ] - ，179 [M-H-C 7 H 10 O 5 ] - ，173 [M-H-C 9 H 6 O 3 -H 2 O] - ，161 [M-H-C 7 H 10 O 5 -H 2 O] - Generating debris peaks. The results are summarized and the cleavage pattern of phenolic acids is shown in FIG. 2, which mainly includes 2 cleavage pathways. Fragment ion m/z 191 [ quinic acid-H [ ]] - 173 [ quinic acid-H ] 2 O] - Indicating that a quinic acid structural fragment exists in the structure of the compound. The type of substituents on the phenyl ring in the structure of the compound can be determined by the fragment ion generated by cleavage pathway B. The cracking rule of phenolic acid compounds without caffeoyl in the structure is generally easy to lose CO 2 、H 2 O、CH 3 、C 2 H 2 A series of neutral groups are mixed to produce [ M-H-44 (CO) 2 )] - ，[M-H-18(H 2 O)] - ，[M-H-15(CH 3 )] - ，[M-H-28(C 2 H 2 )] - Fragment ions.
2.1.2 Flavonoid compounds
Rutin (13), isovitexin (14), liquiritin (15), hyperoside (17), isoquercitrin (18), luteolin (19), genistin (21), hesperidin (24), apioside isoliquiritin (26), isoliquiritin (27), daidzein (28), liquiritigenin (30), glycitein (31), calycosin (32), luteolin (34), naringenin (36), isoliquiritigenin (38), formononetin (41), genkwanin (42) and glabridin (43) are main flavonoids compounds of the antidotal capsule or tablet of compound Saigo. Is structurally characterized by having C 6 -C 3 -C 6 Structural skeleton, substituent change includes hydroxylation, hydroxymethylation, methoxylation, carboxylation, etc. Three examples are: 1. compound 34 has a retention time of 27.812 min, producing an excimer ion peak ([ M-H ] 3242 at M/z 285.0407] - ). At M/z 241.0491 [ M-H-CO ] 2 ] - ，257.0453 [M-H-CO] - ，199.0400 [M-H-C 3 H 2 O 3 ] - ，151.0030 [M-H-C 8 H 6 O 2 ] - A fragment peak was observed, and compound 34 was thus determined to be luteolin. 2. The retention time of compound 24 was 22.036 min, producing an excimer ion peak ([ M-H ] 3242 at M/z 609.1826] - ) At M/z 301.0724 [ M-H-C 6 H 10 O 4 -C 6 H 10 O 5 ] - ，151.0031 [M-H-C 6 H 10 O 4 -C 6 H 10 O 5 -C 9 H 10 O 2 ] - ，107.0135 [M-H-C 6 H 10 O 4 -C 6 H 10 O 5 -C 10 H 10 O 4 ] - A debris peak was observed, thus identifying compound 24 as hesperidin. 3. The retention time of compound 27 was 25.629 min, producing an excimer ion peak ([ M-H ] 3242 at M/z 417.1198] - ) A debris peak 255.0668 [ M-H-C ] was observed at M/z 6 H 10 O 5 ] - ，135.0087 [M-H-C 6 H 10 O 5 -C 8 H 9 O] - ，119.0505 [M-H-C 6 H 10 O 5 -C 7 H 4 O 3 ] - Determining that compound 27 is isoliquiritin. Thus, the major cleavage pathways for flavonoids are RDA cleavage and-CH 3 And CO, the main fragment ion of flavonoid glycoside is generated by sugar chain cleavage.
2.1.3 Triterpenoid
Platycodin D (35), glycyrrhizic acid (37), glycyrrhetinic acid (45), betulinic acid (46), oleanolic acid (47) and ursolic acid (48) are main triterpenoid components in the compound antidotal capsules or tablets. Their structures were determined by mass spectrometry cleavage route, standard controls, literature controls. The structure is characterized in that the structure has a framework formed by 5 six-membered rings, and the structural changes comprise hydroxylation, methylation, acetylation and the like. For example, compound 35 has a retention time of 27.813 min, producing an excimer ion peak ([ M-H ] at M/z 1225.5854] - ) At M/z 1093.5430 [ M + H-C 5 H 8 O 4 ] + ，1093.5365 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 ] + ，961.4945 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 ] + , 815.4418 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 ] + ，683.3996 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 -C 5 H 8 O 4 ] + ，521.3484 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 -C 5 H 8 O 4 -C 6 H 10 O 5 ] + ，503.3381 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 -C 5 H 8 O 4 -C 6 H 10 O 5 -H 2 O] + The fragment peak was generated, and compound 35 was identified as platycodin D. Cracking crushing of triterpenoidFlakes are mainly produced by the cleavage of sugar residues.
2.2 Construction of a list of target precursor ions
On the basis of the representative compounds (compounds 1-48) in 2.1, theoretical precursor ions are constructed according to the change characteristics (such as hydroxylation, dehydroxylation, acetylation, hydrogenation and the like) of each class of compounds, the rationality of the theoretical precursor ions is verified by adopting a primary mass spectrum, the theoretical precursor ions with unmatched molecular weights are excluded, and a target precursor ion list is constructed.
2.3 Comprehensively resolving chemical components in compound Xiling detoxicating capsule or tablet
The collision energy was optimized with 2.2 constructs of target precursor ions set to 10-20V, 20-30V, 30-40V, respectively. The 97 compounds (excluding compounds 1-48 in 2.1) including 11 phenolic acid compounds, namely gallic acid (52), p-hydroxybenzoic acid (54), 1-caffeoyl-beta-D-glucose (57), 2-methoxybenzoic acid (58), 4-acetylbenzoic acid (59), 3,4-dihydroxyphenylpropionic acid (60), 3-O-feruloylquinic acid (61), 5-hydroxyferulic acid (67), 4-methoxycinnamic acid (70), methyl cinnamate (75) are obtained by co-analysis according to the information of secondary mass spectrum, retention time, ultraviolet absorption and the like of target precursor ions, caffeic acid methyl ester (79), 26 flavonoids, glucoisoliquidin apioside (63), genistein-7-O-gentioside (64), genistein-7,4' -di-O-beta-D-glucopyranoside (65), lonicerin (73), cyanopsis-7-O-beta-D-glucoside (77), 6' ' -O-acetylisoidin apioside (88), 7-Hydroxy-3 ' -acetoxy-4 ' -methoxyisoflavone (89), butein-4 ' -O-beta-D-glucopyranoside (90), 4' -Hydroxy-7-acetoxyflavone (91), 6' ' -O-acetylisoxanthin (92), chalconoargingenin-4-O- β -D-glucopyranoside (93), 4',6,7-trihydroxy-2 ' -methoxychalcone (94), 4', 7-dihydroxyflavone (95), licochalcone B (96), 2(s) -3',5', 7-trihydroxyflavanone (115), 4' -O-Acetyl-naringenin (120), equol (124), licoisoflavone B (130), licoflavonol (133), licochalcone a (137), glaridin glycosides (139), licoisoflavone a (140), 3,4-didehydroglobin (141), licoflavone B (142), isoangustine a (143), 4' -methylglabridin (144), 31 triterpenoids, platycodin G2 (82), apigenin E (83), triterpen N1 (84), beta-genistein plantylcodin (85), platycodin E (86), triterpen N2 (97), apigenin D3 (98), platycode D2 (99), platycodin H (100), triterpen N3 (101), triterpen N4 (102), deapi-2' ' -O-Acetyl plantylcodin D2 (103), triterpen N5 (104), 3' ' -O-Acetyl-GAGalacin D2 (105), platycode C (109), platycodin C (110), platycodin V (111), platycodonic acid D (112), glycyrrhizin A3 (113), triterpen N6 (114), glycyrrhizin G2 (116), yun Gandai K2 (117), yun Gandai G2 (119), glycyrrhizin E2 (121), glycyrrhizin K2 (125), 22-acetyl Gan Caoquan (126), 3-O- (alpha-L-arabinopyranosyl (1-2) -beta-D-glucopyranosuronyl) glycyrrhetinic acid (127), uralen glycyrrhizin C (128), uralen glycyrrhizin W (129), glycyrrhizin J2 (132), 3' ' -O-beta-D-glucopyranosyl platycodigenin (135), 29 other compounds which are respectively lactose (49), uridine (50), uracil (51), forsythic acid-1 ' -O-B-D-glucoside (53), loganin acid (55), forsythoside E (56), forsythiade (62), forsythoside C (66), secologanin acid (68), forsythenide A (69), akebia saponin B (71), forsythoside G (72), akebia saponin A (74), matairesinol (76), 7,2',4' -trihydroxy-5-methoxy-3-aromicin (78), forsythoside A (80), susponoidide D (81), susponoidide B (87), forsythiaside (106), arctigenin E (107), arctigenin H (108), matairesinol (118), arctigenin A (122), arctigenin F (123), liquiritin I (131), glycycoumarin (134), glycyrone (136), glycyrol (138), stigmasterol (145). Wherein, the compounds 84, 97, 101, 102, 104 and 114 are six new compounds, and the structures cannot be inquired in the Scifinder database. The compounds 77, 89, 91 and 120 are firstly separated from liquorice, and the compound 139 is firstly separated from burdock. The compound analysis strategy will be described below by analyzing a new compound.
2.3.1 Structure analysis of Triterpen N1 (84) and Triterpen N2 (97)
Target precursor ions of the compound 84 and the compound 97 are respectively added with CH on the basis of platycodin D (35) and the platycodin D (33) without apiose 2 And (4) O construction. The retention times for these two compounds were 24.5 min and 27.0 min, respectively. The excimer ion peaks ([ M + H ] s) are given at M/z 1255.5959, 1123.5537, respectively] + ) And the peak value is 30 Da larger than the quasi-isolated ion peaks of two known compounds, and the correctness of the target precursor ions is preliminarily verified. Compound 84 is at M/z 1123.5536 [ M + H-C 5 H 8 O 4 ] + 、961.5008 [M+H-C 5 H 8 O 4 -C 6 H 10 O 5 ] + 、683.4006 [M+H-C 5 H 8 O 4 -C 6 H 10 O 5 -C 6 H 10 O 4 -C 5 H 8 O 4 ] + Fragment ions are observed, which are generated by the fragmentation of sugar residues in the structure. In the secondary mass spectrum of platycodin D, M/z 1093.543 [ M + H-C ] can be observed 5 H 8 O 4 ] - （M+H-Api），961.5008 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 ] - （M+H-Api-xyl），815.4429 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 ] - （M+H-Api-xyl-Rha），683.4006 [M+H-C 5 H 8 O 4 -C 5 H 8 O 4 -C 6 H 10 O 4 -C 5 H 8 O 4 ] - Fragment ion of (M + H-Api-xyl-Ara), indicating hydroxymethyl group (CH) in Compound 84 2 OH) to a xylose residue on the sugar chain. Similarly, by comparing Compound 97 with the Desapiose Platycodon Saponin D SecondaryThe spectrum can confirm that the hydroxymethyl group in the structure is also linked to the xylose residue. The structures of compounds 84 and 97 are shown in FIG. 3, and the mass spectrometry cleavage pathway is shown in FIG. 4. These 2 compounds are new and named Triterpen N1 (84) and Triterpen N2 (97).
2.3.2 Structure analysis of Triterpen N1 (101) and Triterpen N2 (114)
The target precursor ions of compound 101 and compound 114 were obtained by hydrogenation and water based on the structure of glycyrrhizic acid. The glycyrrhizic acid has a retention time of 29.8 min, and produces an excimer ion peak ([ M-H ] at M/z 821.3960] - ). Major fragment ion 645.3639 [ M-H-GluA] - ，469.3320 [M-H-GluA-GluA] - ，351.0564 [2GluA-H] - Is generated by breaking sugar chains in the structure. Compounds 101 and 114 had retention times of 27.8 min and 28.4 min, producing excimer peaks at m/z 823.4117 (2 Da greater than the excimer peak for glycyrrhizic acid) and m/z 839.4066 (18 Da greater than the excimer peak for glycyrrhizic acid), respectively. The above information preliminarily verifies the rationality of its target precursor ions. In the secondary mass spectrum of the compound 101, M/z 647.3796 [ M-H-GluA ] can be observed] - ，471.3475 [M-H-GluA-GluA] - The fragment peak of (1) was observed in the secondary mass spectrum of Compound 114, and M/z 663.3745 [ M-H-GluA ] was observed] - ，487.3424 [M-H-GluA-GluA] - Debris peak of (2), description of H 2 And H 2 O is added on the aglycone of glycyrrhizic acid. The 2 compounds are new compounds named Triterpen N3 and Triterpen N6, and the structures and cleavage routes of the compounds are shown in FIGS. 3 and 4.
2.3.3 Structure analysis of Compounds Triterpen N4 (102) and Triterpen N5 (104)
The target precursor ions of compounds 102 and 104 were subtracted by CH from the compounds des-apiose platycodin D (33) and Platycoside L (29), respectively 2 And (4) constructing. The peak of the excimer ion of the compound 102 is m/z 1063.5325 (Rt = 27.85 min), the excimer ion peak of compound 104 is m/z 815.4429 (m/z 5363: (m/z)Rt = 27.87 min), all 30 Da less than 2 known compounds, confirming the rationality of their target molecular weights. By comparing the 2 piecesMass spectra fragmentation of compound and 2 known compounds, determined that 2 compounds all lost hydroxymethyl groups on xylose residues compared to the known compound. The structures of their compounds are shown in FIG. 3, and the cleavage pattern is shown in FIG. 4. These 2 compounds are new and named Triterpen N4 and Triterpen N5.

Claims (5)

1. A rapid separation and identification method of traditional Chinese medicine chemical components based on an UPLC-Q-TOF-MS analytical instrument and an analysis strategy of target precursor ions is characterized by comprising the following steps: (1) Firstly, information integration is carried out on the traditional Chinese medicine components, and the structural characteristics and the structural change rule of the traditional Chinese medicine components are summarized; performing mass spectrometry on each type of representative compounds with different structural characteristics by using a UPLC-Q-TOF-MS (ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry) analyzer, and summarizing a mass spectrometry cracking rule and a neutral loss rule; (2) Constructing theoretical precursor ions according to the structural change rule of each class of compounds by taking a representative compound as a core, verifying the correctness of the theoretical precursor ions by adopting a primary mass spectrum, and constructing a target precursor ion list; (3) Analyzing the cracking mode and neutral loss rule of the target precursor ions by adopting a secondary mass spectrum, verifying the correctness of the target precursor ions, and comprehensively analyzing the mass spectrum and retaining time information to comprehensively and quickly analyze chemical components of the traditional Chinese medicine;
the traditional Chinese medicine is compound Xiling detoxification capsule or compound Xiling detoxification tablet;
the method comprises the following conditions: and (3) UPLC: the chromatographic column is Agilent advanced Bio Peptide 2.1X 250 mm,2.7 μm; mobile phase: the water phase A is 0.1% formic acid water solution, and the organic phase B is acetonitrile solution; the chromatographic separation is carried out by adopting a gradient elution mode, and the gradient elution program is as follows: 0-13 min,5-13% B;13-14 min,13-19% B;14-24 min,19-24% B;24-31 min,24-67% B;31-40 min,67-95% B.
2. The separation and identification method according to claim 1, wherein the sample and standard solutions are ultrasonically extracted with methanol.
3. A separation and identification method as claimed in claim 1 wherein the mass spectrometry ionization source is an ESI source; the MS parameters were as follows: capillary voltage 3500V, atomizer pressure 35 psi, drying gas temperature 300 ℃, flow rate 8.0L/min.
4. The isolation and identification method according to any one of claims 1 to 3, wherein 145 compounds including 27 phenolic compounds, each of which is protocatechuic acid, neochlorogenic acid, salidroside, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, cryptochlorogenic acid, caffeic acid, syringic acid, p-hydroxyphenylacetic acid, p-coumaric acid, ferulic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, eugenol, gallic acid, p-hydroxybenzoic acid, 1-caffeoyl-beta-D-glucose, 2-methoxybenzoic acid, 4-acetylbenzoic acid, 3,4-dihydroxyphenylpropionic acid, 3-O-feruloylquinic acid, 5-hydroxyferulic acid, 4-methoxycinnamic acid, methyl cinnamate, methyl caffeic acid; 46 flavonoids, which are rutin, isovitexin, liquiritin, hyperoside, isoquercitrin, luteolin, genistin, hesperidin, apigenin, isoliquiritin, daidzein, liquiritigenin, glycitein, calycosin, luteolin, naringenin, isoliquiritigenin, formononetin, lilac daphnetin, glabridin, glucooisoliquiitin apioside, genistein-7-O-geniposide, genistein-7,4 '-di-O-beta-D-glucopyranoside, lonicerin, vetivol-7-O-beta-D-glucoside, 6' '-O-acetylisoquin apioside, 7-Hydroxy-3' -acetoxy-4 '-methoxyisoflavone, butein-4' -O-beta-D-glucopyranoside, 4 '-Hydroxy-7-acetoxyflavanone, 6' '-O-acetylisoquinatin, chalcon caringenin-4-O-beta-D-glucopyranoside, 4',6,7-trihydroxy-2 '-methoxychalcone, 4', 7-dihydroxyflavone, licochalcone B, 2(s) -3',5', 7-trihydroxyflavanone, 4'-O-Acetyl-naringenin, equol, licoisoflavone B, licoflavonol, licochalcone A, glabridin glycosides, licoisoflavone A,3,4-didehydroglobin, licoflavone B, isoangustone A,4' -methylglabridin, 39 triterpenoids, platycodin L, desoxydiosgenin D, glycyrrhizic acid, hypobaric acid, betulinic acid, oleanolic acid, ursolic acid, platycodin G2, apigenin-removed Platycodin E, triterpen N1, beta-geniotriosylpolycodigenin, platycodin E, triterpen N2, apigenin-removed Platycodin D3, platycodin D2, platycodin H, triterpen N3, triterpen N4, deapi-2'' -O-Acetyl Platycodin D2, triterpen N5,3'' -O-Acetyl-polygalacin D2, platycodin C, platycodin V, platycodonic acid D, glycyrrhizin A3, triterpen N6, glycyrrhizin G2, yun Gandai K2, yun Gandai G2, glycyrrhizin E2, glycyrrhizin K2, 22-Acetyl Gan Caoquan, 3-O- (alpha-L-arabinopyranosyl (1-2) -beta-D-glucopyranosyloxy) glycyrrhetinic acid, urabin C, urabin W, glycyrrhizin J2,3'' -O-beta-D-glucopyranosyl Platycodin; 33 other compounds, forsythoside A, acteoside, arctigenin, dibutyl phthalate, lactose, uridine, uracil, forsythic acid-1 ' -O-B-D-glucoside, loganin acid, forsythoside E, forsythide, forsythoside C, secologenin acid, forsythenside A, akebia phenylethanoid B, forsythoside G, akebiaquinata phenylethanoid glycoside A, matairesinol glycoside, 7,2',4' -trihydroxy-5-methoxy-3-coumarin, forsydoliside A, suspenidoside D, suspenidoside B, forsythiaside, arctigenin E, arctigenin H, matairesinol, arctigenin A, arctigenin F, glycyronin I, glycycoumarin, glycyrol and stigmasterol;
wherein, the compounds Triterpen N1, triterpen N2, triterpen N3, triterpen N4, triterpen N5 and Triterpen N6 are 6 new compounds, the structural formula is shown in the following formula in sequence,
Triterpene N1：
Figure 112422DEST_PATH_IMAGE001
；
Triterpene N2：
Figure 780163DEST_PATH_IMAGE002
；
Triterpene N3：
Figure 917884DEST_PATH_IMAGE003
；
Triterpene N4：
Figure 380089DEST_PATH_IMAGE004
；
Triterpene N5：
Figure 603260DEST_PATH_IMAGE005
；
Triterpene N6：
Figure 809113DEST_PATH_IMAGE006
。
5. the separation and identification method according to claim 4, wherein 144 compounds except chlorogenic acid are first separated from compound XILINGJIEDU Capsule or FUFANGXILINGJIEDU tablet; the compound of the bergamol-7-O-beta-D-glucoside, the 7-Hydroxy-3 '-acetoxyl-4' -methoxyisoflavone, the 4'-Hydroxy-7-acetoxyflavone and the 4' -O-Acetyl-naringenin are firstly separated from the liquorice, and the compound of the Glabradine glycosides is firstly separated from the burdock.
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