CN113917040A

CN113917040A - Classification and identification method of ginsenoside components in panax traditional Chinese medicine and application thereof

Info

Publication number: CN113917040A
Application number: CN202111284341.2A
Authority: CN
Inventors: 杨文志; 李雪; 胡莹; 王小岩; 刘美玉; 徐晓艳; 宓月光
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-01-11
Anticipated expiration: 2041-11-01
Also published as: CN113917040B

Abstract

The invention relates to the technical field of Chinese medicine component classification and identification, in particular to a classification and identification method of ginsenoside components in ginseng and application thereof. The invention adopts a high performance liquid chromatography/mass spectrometry combined analysis method, utilizes a Q-Trap 4500 triple quadrupole-linear ion Trap mass spectrometer, detects neutral saponin and malonyl saponin by an MRM-IDA-EPI mode, and detects oleanolic acid type ginsenoside by an MIM-IDA-EPI mode, thereby realizing the classification and identification of different ginsenosides in the ginseng traditional Chinese medicine, and further realizing the classification and identification of hundreds of ginsenosides. The classification and identification method can be used for identification of Panax Chinese medicines and characterization and identification of ginsenoside in Chinese patent medicine containing Panax Chinese medicines, and is helpful for promoting quality control of Panax Chinese medicines and Chinese patent medicine containing Panax Chinese medicines.

Description

Classification and identification method of ginsenoside components in panax traditional Chinese medicine and application thereof

Technical Field

The invention relates to the technical field of Chinese medicine component classification and identification, in particular to a classification and identification method of ginsenoside components in ginseng and application thereof.

Background

In recent years, ginseng has remarkable advantages in the development of medical treatment and health industry, and is widely researched, developed and utilized. In particular, different ginseng traditional Chinese medicines such as ginseng, red ginseng, American ginseng, pseudo-ginseng, panax japonicus, rhizoma panacis japonici and rhizoma panacis majoris have different drug properties, and the medicines can be administered according to symptoms only by precise compatibility in the practical application process, so that the identification of the medicinal materials is very important for the quality of the product containing the ginseng traditional Chinese medicines. After the traditional Chinese medicine decoction pieces are crushed and processed, the traditional Chinese medicine decoction pieces are difficult to distinguish by conventional character identification and microscopic identification; in addition, the physical and chemical identification of traditional Chinese medicines derived from Panax in Chinese pharmacopoeia mostly uses ginsenoside as index, but ginsenoside is the main active ingredient in traditional Chinese medicines derived from Panax, exists in multiple traditional Chinese medicines derived from Panax, and each traditional Chinese medicine derived from Panax often contains multiple subtypes of ginsenoside, so that different traditional Chinese medicines derived from Panax are difficult to accurately distinguish by using a single or a few types of ginsenoside. Therefore, exploring the method capable of accurately characterizing and identifying different subtype saponin components in the panax traditional Chinese medicine has important significance for accurately identifying the panax traditional Chinese medicine and products thereof.

Disclosure of Invention

Aiming at the technical problems, the invention provides a classification identification method of ginsenoside components in panax traditional Chinese medicines and application thereof, and 567 ginsenosides can be classified and identified by the classification identification method and can be used for characterization and identification of the panax traditional Chinese medicines and products containing the panax traditional Chinese medicines.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a method for classifying and identifying ginsenoside component in Panax traditional Chinese medicine comprises classifying and identifying ginsenoside in Panax traditional Chinese medicine by high performance liquid chromatography/mass spectrometry;

the mass spectrum is a triple quadrupole-linear ion trap mass spectrum; classifying and identifying ginsenoside by adopting information-dependent acquisition-enhanced product ion scanning technology, detecting neutral saponin and malonyl saponin by using MRM-IDA-EPI mode, and detecting oleanolic acid type ginsenoside by using MIM-IDA-EPI mode.

The classification identification method adopts information-dependent acquisition-enhanced product ion scanning (IDA-EPI) technology to perform classification identification on ginsenoside. For neutral saponin and malonylated saponin, the MRM acquisition mode has better sensitivity and ion response compared with NL and MIM modes, the effective EPI number obtained by the MRM-IDA-EPI method is higher than that obtained by NL-IDA-EPI, the secondary fragment abundance obtained by acquisition is better than that obtained by NL-IDA-EPI and MIM-IDA-EPI, and therefore MRM-IDA-EPI is selected for neutral saponin and malonylated saponin. However, experiments show that for OA (oleanolic acid) type components, MIM can capture secondary components far higher than MRM mode and capture more unknown components, so MIM-IDA-EPI method is selected for the characterization and identification of OA saponin.

Preferably, the neutral saponins include protopanaxatriol type ginsenoside, protopanaxadiol type ginsenoside, oktelong type ginsenoside and ginsengenin.

Preferably, the protopanaxatriol type ginsenoside comprises ginsenoside F1, 20(S) -ginsenoside Rh1, 20(R) -ginsenoside Rh1, ginsenoside F3, 20(S) -notoginsenoside A3, ginsenoside F5, notoginsenoside R2, 20(R) -notoginsenoside R2, ginsenoside 2, ginsenoside Rf, ginsenoside Rg1, notoginsenoside R1, notoginsenoside Fp1, ginsenoside Re, 20-O-glucosyl-ginsenoside Rf, ginsenoside Re2, ginsenoside Re3 and the like.

Preferably, the protopanaxadiol type ginsenoside includes ginsenoside Rh2, 20(R) -ginsenoside Rh2, ginsenoside K, ginsenoside F2, ginsenoside Rg3, 20(R) -ginsenoside Rg3, notoginsenoside Fe, ginsenoside Rd2, notoginsenoside K, ginsenoside Rd, gypenoside XVII, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Ra1, ginsenoside Ra2, notoginsenoside R4, notoginsenoside T and the like.

Preferably, the ocrtrone-type ginsenoside includes 24(R) -pseudoginsenoside Rt5 and 24(R) -pseudoginsenoside F11, and the like.

Preferably, the ginsengenin comprises 20(S) -protopanaxatriol, 20(S) -protopanaxadiol, oleanolic acid, 20(S),24(R) -ocristolone aglycone, etc.

Preferably, the ginsenoside further comprises ginsenoside Rk3, ginsenoside Rh4, notoginsenoside T5, ginsenoside Rg5, ginsenoside Rg6, ginsenoside F4, ginsenoside Rk1, 5, 6-dehydroginsenoside Rd, vietnam ginsenoside R8 and 5, 6-dehydroginsenoside Rb1, and the like.

Preferably, the oleanolic acid type ginsenoside comprises panax japonicus saponin IVa, panax japonicus saponin IV, pseudoginsenoside Rt1, ginsenoside Ro and the like.

Preferably, the malonylated saponin includes malonyl ginsenoside Re1, malonyl ginsenoside Rd, malonyl ginsenoside Rc, malonyl ginsenoside Rb2, malonyl ginsenoside Rb1, and the like.

Preferably, the chromatographic conditions of the high performance liquid chromatography are:

a chromatographic column: a polarity-modified octadecylsilane chemically bonded silica chromatographic column;

mobile phase a was acetonitrile and mobile phase B was 0.1% v/v formic acid-water solution, and the procedure of the linear gradient elution was as follows:

flow rate: 0.28-0.32 mL/min;

column temperature: 30-40 ℃.

The selection of the mobile phase in the liquid chromatogram has influence on the peak appearance and peak type of different components, the mobile phase and the linear elution program adopted by the invention can improve the separation degree of different ginsenosides and reduce the tailing phenomenon, and the peak area of each component under the mobile phase is larger, thus being beneficial to the effective classification and identification of each component.

In the above linear gradient elution procedure, if the volume percentage of mobile phase a and mobile phase B changes after different time, it means that the mobile phase changes linearly during the time period, e.g., the proportion of mobile phase a is 12% at 0min, the proportion of mobile phase a is 20% at 6min, it means that the mobile phase a increases linearly from 12% to 20% at 0 to 6 min. The same flow phase ratio for 45min and 41min indicates that the flow phase ratio is unchanged during this time period.

Preferably, the column is BEH Shield RP 18. Under the chromatographic conditions of the invention, better resolution, peak type and peak number of chromatographic peaks can be achieved by adopting the chromatographic column.

Preferably, the column temperature is 40 ℃. Under the chromatographic condition of the invention, more peak output numbers and better separation degree can be achieved at the column temperature of 40 ℃.

Preferably, the flow rate is 0.3 ml/min.

Preferably, the mass spectrometer is a Q-Trap 4500 triple quadrupole-linear ion Trap mass spectrometer.

Preferably, the ion source of the mass spectrum is an electrospray ion source, and data are collected in a negative ion mode; the parameters of the ion source are: the ionization voltage (Ionspray voltage) is-4500V, the Temperature (Temperature) is 550.0 ℃, the air Curtain Gas (Curtain Gas) is 35.0psi, the spray Gas 1(Gas 1) is 55.0psi, the auxiliary heating Gas 2(Gas 2) is 55.0psi, the Collision Gas (Collision Gas) is High, and the Collision cell exit voltage (Collision cell exit potential) is-13.0V.

Preferably, the collision energy (CE value) in the MIM-IDA-EPI mode is 70eV to 100eV, and the collision energy in the MRM-IDA-EPI mode is 70eV to 120 eV; the information-dependent acquisition response intensity is 1-2.

The declustering voltage (DP) can give certain resonance energy to ions, prevent the adsorption of solvent molecules and realize declustering. If the declustering voltage is too high, the ions will fragment due to resonance, resulting in-source fragmentation (CID). The Collision Energy (CE) plays an important role in analytical characterization and identification of compounds. Information dependent acquisition response strength may have an impact on EPI population. By optimizing key mass spectrum parameters, the sensitivity of the instrument can be improved, and the accurate analysis and identification of compound data are facilitated.

The invention also provides application of the classification identification method of the ginsenoside components in the panax traditional Chinese medicines in characterization and identification of ginsenoside. The method can identify various saponins in Panax Chinese medicine, and can be used for identifying Panax Chinese medicine and characterizing and identifying ginsenoside in Chinese patent medicine containing Panax Chinese medicine.

The invention has the beneficial effects that: the chromatographic peak obtained in the process of classifying and identifying the ginsenosides by the classification and identification method of the invention has good separation degree and larger peak area, and can classify and identify 567 ginsenosides, and the quality control of the ginsenosides and Chinese patent medicines containing the ginsenosides can be assisted by the characterization and identification of the ginsenosides.

Drawings

The invention will be further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a flowchart of the prediction of oleanolic acid type saponin in example 2 of the present invention;

FIG. 2 is a schematic diagram showing the cleavage of neutral saponin in example 2 of the present invention;

FIG. 3 is a schematic diagram showing the cleavage of malonylated saponin in example 2 of the present invention;

FIG. 4 is a schematic diagram showing the cleavage of oleanolic acid type saponin in example 2 of the present invention;

FIG. 5 is a graph comparing different mobile phase additives of comparative example 1 of the present invention; in the figure, the noto-R1, Re, Rb1, m-Rb2, p-F11, Ro, Chiku-IVa and m-floral-Re1 respectively represent notoginsenoside R1, ginsenoside Re, malonyl ginsenoside Rb1, 24(R) -pseudoginsenoside F11, ginsenoside Ro, panax japonicus IVa and malonyl ginsenoside Re 1;

FIG. 6 is a graph comparing the number of peaks separated in the SIM chromatograms of 10 reversed phase chromatography columns in comparative example 2 of the present invention;

FIG. 7 is a comparison of the column temperatures of the BEH Shield RP18 column in the chromatographic separation of comparative example 3 of the present invention;

FIG. 8 is a graph comparing different acquisition modes in comparative example 4 of the present invention with the MRM-IDA-EPI method.

FIG. 9 is a comparison of the results of comparative example 5 of the present invention at different declustering voltages; in the figure, the noto-R1, Re, Rb1, m-Rb2, p-F11, Ro, Chiku-IVa and m-floral-Re1 respectively represent notoginsenoside R1, ginsenoside Re, malonyl ginsenoside Rb1, 24(R) -pseudoginsenoside F11, ginsenoside Ro, panax japonicus IVa and malonyl ginsenoside Re 1;

FIG. 10 is a comparison of the results for different values of collision energy in NL in the NL46-IDA-EPI mode in comparative example 5 of the present invention;

FIG. 11 is a comparison of the results of EPI with different values of collision energy in NL46-IDA-EPI mode in comparative example 5 of the present invention;

FIG. 12 is a comparison of the results for different values of collision energy in NL in the NL44-IDA-EPI mode in comparative example 5 of the present invention;

FIG. 13 is a comparison of the results of EPI with different values of collision energy in NL44-IDA-EPI mode in comparative example 5 of the present invention;

FIG. 14 is a graph comparing different collision energies in the MIM-IDA-EPI mode of comparative example 5 of the present invention;

FIG. 15 is a comparison of the results of different information-dependent acquisition response strengths in comparative example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Ginsenoside belongs to triterpenoid saponin, and can be roughly divided into neutral saponin, malonylated saponin and acidic saponin according to the chemical properties of ginsenoside, wherein the neutral saponin comprises PPD type (protopanaxadiol type), PPT type (protopanaxatriol type) and OT type (oktaralone type), the ginsenoside containing PPD type or PPT type aglycon is the most common ginsenoside, and can be combined with formic acid to form formic acid adduct ions together with OT type saponin; the malonylated saponin is a special saponin form, and CO is easily lost₂(44.01Da) and C₃H₂O₃(86.00Da), especially loss of CO₂A group; the acidic saponin including OA type saponin exists in Panax japonicus, and has the characteristic of no obvious formic acid adduct ion. Aiming at different chemical properties of ginsenoside, the embodiment of the invention is used for carrying out all-round classification characterization and identification on ginseng multi-source medicinal materials by combining various 'information dependent acquisition-enhanced product ion scanning (IDA-EPI)' technologies constructed on the basis of an ultra-efficient liquid phase and a triple quadrupole-linear ion trap mass spectrometer with a non-target and target scanning technology. Since the neutral saponin and malonylated saponin are easy to lose 46Da and 44Da, the neutral saponin is easy to add the formic acid group 46 when formic acid is added in the mobile phase, and the malonylated saponin is easy to lose CO₂The group 44, OA type saponin is acidic, is not easy to adduct with formic acid, has no obvious adduct form and has no obvious loss fragment, the embodiment of the invention respectively constructs an MRM-IDA-EPI method to collect neutral saponin and malonyl acid acylated saponin components, determines that the MIM-IDA-EPI method is used for capturing OA type saponin, has better complementarity and target property aiming at components with different properties, and systematically realizes the classified capture of different varieties of medicinal material components of panax.

Reagents and drugs used in the following examples:

acetonitrile (Fisher, Fair lawn, NJ, USA), methanol (Fisher, Fair lawn, NJ, USA), formic acid Fisher, Fair lawn, NJ, USA) are all LC-MS grades. Deionized water was purified by a Milli-Q system (Millipore, Bedford, MA, USA).

The 60 controls were mainly divided into:

18 protopanaxatriol-type ginsenosides (PPT): ginsenoside F1(1), 20(S) -ginsenoside Rh1(2), 20(R) -ginsenoside Rh1(3), ginsenoside F3(4), 20(S) -notoginsenoside A3(5), ginsenoside F5(6), notoginsenoside R2(7), 20(R) -notoginsenoside R2(8), ginsenoside Rg2(9), ginsenoside Rf (10), ginsenoside Rg1(11), notoginsenoside R1(12), notoginsenoside Fp1(13), ginsenoside Re (14), 20-O-glucosyl-ginsenoside Rf (15), ginsenoside Re2(16), ginsenoside Re3 (17);

22 protopanaxadiol-type ginsenosides (PPD): ginsenoside Rh2(19), 20(R) -ginsenoside Rh2(20), ginsenoside K (21), ginsenoside F2(22), ginsenoside Rg3(23), 20(R) -ginsenoside Rg3(24), notoginsenoside Fe (25), ginsenoside Rd2(26), notoginsenoside K (27), ginsenoside Rd (28), gypenoside XVII (29), ginsenoside Rb2(31), ginsenoside Rb3(32), ginsenoside Rc (33), ginsenoside Ra1(37), ginsenoside Ra2(38), notoginsenoside R4(39), notoginsenoside T (40);

2 oxtriptolone-type ginsenosides (OT): 24(R) -pseudoginsenoside Rt5(45), 24(R) -pseudoginsenoside F11 (46);

four ginsengenin: 20(S) -protopanaxatriol (57), 20(S) -protopanaxadiol (58), oleanolic acid (59), 20(S),24(R) -oxtriptolide (60);

other neutral saponins: ginsenoside Rk3(47), ginsenoside Rh4(48), notoginsenoside T5(49), ginsenoside Rg5(50), ginsenoside Rg6(51), ginsenoside F4(52), ginsenoside Rk1(53), 5, 6-dehydroginsenoside Rd (54), vietnam ginsenoside R8(55), 5, 6-dehydroginsenoside Rb1 (56);

4 oleanolic acid type ginsenosides (OA): chikusetsusaponin IVa (41), chikusetsusaponin IV (42), pseudoginsenoside Rt1(43), and ginsenoside Ro (44);

5 malonylated saponins: malonyl ginsenoside Re1(18), malonyl ginsenoside Rd (30), malonyl ginsenoside Rc (34), malonyl ginsenoside Rb2(35), malonyl ginsenoside Rb1 (36).

The above controls were all from Shanghai Shidan De Biotechnology, Inc. and Chengderster Biotechnology, Inc.

The Panax species are Panax ginseng (produced from Changbai mountain of Jilin province, 5-6 years old), Ginseng radix Rubri (produced from Changbai mountain of Jilin province, 5 years old), Panax quinquefolium (produced from Weizhou, America, David), Notoginseng radix (produced from Yunnan Wenshan, 20 heads), Panax japonicus (produced from Sichuan), and Panax ginseng leaf (produced from Changbai mountain of Jilin province).

The analytical instruments used in the following examples:

I-Class ultra high performance liquid phase system, Q-Trap 4500 triple quadrupole-linear ion Trap mass spectrometer (Applied Biosystems-SCIEX Scientific, Concord, Canada).

Example 1

The embodiment of the invention provides a classification identification method of ginsenoside components in a ginseng traditional Chinese medicine, which is used for classifying and identifying the ginsenoside in a ginseng traditional Chinese medicine sample (ginseng, red ginseng, American ginseng, pseudo-ginseng, panax japonicus, rhizoma panacis majoris and ginseng leaves) by using a high performance liquid chromatography/mass spectrometry combined analysis method;

preparation of sample solution:

respectively taking 25mg of each of 7 batches of samples (ginseng, red ginseng, American ginseng, pseudo-ginseng, panax japonicus and ginseng leaves) of the traditional Chinese medicines of the genus Panax, which are collected and carried by pharmacopoeia, adding 5mL of 70% methanol-water solution (V/V) for dissolving, carrying out vortex oscillation for 2min, carrying out ultrasonic extraction for 1h, cooling an extracting solution, complementing weight loss by using 70% methanol-water solution, centrifuging at 14000rpm (11481g) for 10min, taking supernatant, filtering by using a 0.22 mu m microporous filter membrane to obtain a sample solution of the traditional Chinese medicines of the genus Panax with the concentration of 5mg/mL, and taking 100 mu L of filtrate for LC-MS analysis.

The high performance liquid chromatography analysis is completed in an I-Class ultra performance liquid system, and the chromatographic conditions are as follows:

a chromatographic column: BEH Shield RP18 (2.1X 100mm, 1.7 μm);

mobile phase: acetonitrile (a), 0.1% v/v formic acid-water solution (B);

column temperature: 40 ℃;

flow rate: 0.3 mL/min;

sample introduction amount: 3 mu L of the solution;

a linear gradient elution was performed, the procedure of which was as follows:

the Q-Trap 4500 triple quadrupole-linear ion Trap mass spectrum is used as an analysis detector, and the mass spectrum conditions are as follows:

the ion source is an electrospray ion source, the ionization voltage is 4500V, the temperature is 550.0 ℃, the gas curtain gas is 35.0psi, the spray gas 1 is 55.0psi, the auxiliary heating gas 2 is 55.0psi, the collision gas is High, and the outlet voltage of the collision chamber is 13.0V.

By adopting the parameters and setting the sample volume to be 3 mu L, the seven ginseng traditional Chinese medicine samples are subjected to target collection of two collection methods of three mother ion lists, neutral saponin and malonyl saponin are detected by using MRM-IDA-EPI mode, and oleanolic acid type ginsenoside is detected by using MIM-IDA-EPI mode. CE values in the MIM-IDA-EPI mode are 70eV to 100eV, and CE values in the MRM-IDA-EPI mode are 70eV to 120 eV; the information-dependent acquisition response intensity is 1-2. Data analysis was performed using Analyst (1.6.3) software.

Example 2

The embodiment of the invention provides application of the classification and identification method of the ginsenoside components in the panax traditional Chinese medicine in the embodiment 1 in characterization and identification of the ginsenoside components in the panax traditional Chinese medicine.

Classifying and identifying the saponin components of the ginseng traditional Chinese medicine according to the liquid chromatography condition and the mass spectrum condition of the example 1, adopting the information-dependent acquisition-enhanced product ion scanning (IDA-EPI) technology to characterize and identify the ginsenoside, detecting neutral saponin and malonylated saponin by using an MRM-IDA-EPI mode, and detecting oleanolic acid type ginsenoside by using an MIM-IDA-EPI mode. CE values in the MIM-IDA-EPI mode are 70eV to 100eV, and CE values in the MRM-IDA-EPI mode are 70eV to 120 eV; the information-dependent acquisition response intensity is 1-2.

1. Construction of ion pairs

(1) Ginsenoside mother ion list construction

Firstly, predicting possible structures (aglycon + glycosyl + substituent) according to the known structural characteristics of the OA type saponin, and constructing an OA type saponin database. According to the known reported OA type aglycone structure, the characteristics of the saponin are totally counted as follows; the OA type saponin has been reported to be 44 in total, as shown in Table 1, the number of sugar substrates is 5, and the substituent group is 4 (-CH)₂/-C₂H₄/-C₃H₆/-C₄H₈)。

Table 1 has reported information table of oleanolic acid type saponins

According to the above rules, the invention summarizes the number of the glycosyl groups and the number of the substituent groups contained in the structures of 44 saponins, and predicts the glycosyl groups and the substituent groups on the basis of the known original structure of oleanolic acid type saponin: setting a molecular design rule that the number of 5 glycosyl groups connected to each oleanolic acid type aglycone in the prediction process is at least 1 and at most 6, the number of non-glycosyl groups is at most 2 and at least 1, and finally, the predicted number of glycosyl groups and the number of substituent groups both contain the existing number in the known saponin structure and meet the condition of the rule.

According to the rule, the OA type saponin database is expanded and predicted, 8905 molecular formulas are constructed in total, wherein the molecular formulas comprise known oleanolic acid type saponin, all predicted mass numbers are subjected to de-duplication, and 338 different mass numbers are finally obtained and used as a list of OA type ginsenoside prediction parent ions input into an MIM method and used for targeted collection of traditional Chinese medicine data from panax. A flow chart for molecular prediction of type OA is shown in FIG. 1.

(2) MRM ion pair construction

And (3) carrying out condition selection on the parent ions transmitted by the Q1 in an IDA mode, sending the parent ions into the Q3 to obtain daughter ions, confirming the components by matching the parent ions with the daughter ions, and sending the components into an ion trap for cracking. The neutral saponin and malonylated saponin ion pair is confirmed, the unified standard is that the data of seven ginseng source traditional Chinese medicine samples collected under NL (46/44) -IDA-EPI mode are considered, and the mass number in the list is found to be [ M-H [ -H ]]^－Peaks, as parent ions for the MRM mode, and each component corresponding to the secondary fragment that responds most strongly as daughter ions, construct ion pairs for MRM-IDA-EPI mode acquisition. Finally, the neutral saponin obtains 106 ion pairs, the malonic acid acylation totally comprises 23 ion pairs, and secondary fragments can be captured by molecular prediction in an MIM-IDA-EPI modeThe mass number of the components is 150, and the method is used for data acquisition and identification of ginseng multi-variety source medicinal materials.

2. Characterization and identification of ginsenoside

(1) Characterization and identification of neutral ginsenoside based on MRM-IDA-EPI data

Comparing the identified data with laboratory standard to determine some ginsenoside components. Neutral saponin contains three types of PPT type, PPD type and OT type, and formate ions are easy to be added to form [ M-H + HCOOH ]]^-In this example, representative components are selected from the results of identification of seven ginseng-derived traditional Chinese medicines, and the identification results are described. The cleavage behavior of neutral saponin is illustrated by taking compound #275 as an example, the secondary cleavage process is shown in FIG. 2, the compound removes one formic acid molecule to obtain a molecular ion peak M/z 945.5, and loses one molecular glucose to obtain M/z 783.4([ M-H-Glc- ] -M]^-) Further discarding a rhamnose molecule to obtain a fragment M/z 637.3([ M-H-Glc)]^-) Then continuously losing a glucose molecule to obtain PPT type characteristic aglycone fragment m/z 475.3([ PPT-H ]]^-) (ii) a This compound #275 was finally identified as ginsenoside Re after comparison to standards for retention time and secondary debris, etc. According to the identification process of the compound #275, the unknown component 305 is continuously identified according to the cracking rule, as shown in fig. 2, a fragment of m/z 797.4 is obtained after a molecular ion peak m/z 959.3 loses one rhamnose, a fragment of 637.2 is obtained after a glucuronic acid molecule continues to lose, an aglycon fragment m/z 475.4 is obtained finally after a glucose molecule loses one glucuronic acid molecule, and the compound #305 is finally identified as protopanaxatriol-glucosyl-glucuronic acid-rhamnoside (PPT-Glc-glcA-Rha) according to the cracking result of the secondary fragment.

(2) Characterization and identification of malonylated ginsenosides based on MRM-IDA-EPI data

And identifying the malonylated saponin in the ginseng source traditional Chinese medicine by adopting an MRM-IDA-EPI method based on the established malonylated saponin ion pair list. The malonylated ginsenoside has the characteristic of easily losing CO in the cracking process₂And C₃H₂O₃Thereby obtaining molecular ion peaks. The cleavage behavior of malonylated saponins is illustrated by taking compound #499 as an example, the secondary cleavage process is shown in fig. 3, the compound m/z 1163.6 loses one malonyl group to obtain 1077.6, fragments m/z 945.6, 783.4, 621.5, 459.5 and 375.4 are obtained, one xylose and three glucose are respectively lost according to the estimation of mass number, the fragments generated by malonyl ginsenoside Rc are basically the same, and finally, the compound #499 is compared with a standard product to be identified as the malonyl ginsenoside Rc. For unknown Compound #348(1017.4-637.4), the molecular ion peak of the neutral moiety after 86Da loss was M/z 931.4([ M-H-Mal ] as shown in FIG. 3]^－) Continuing to lose one xylose molecule, the fragment M/z 799.4([ M-H-Xyl-Mal) was obtained]^-) (ii) a Then two glucose molecules are lost to obtain PPT type sapogenin fragment m/z 475.3([ PPT-H)]^－) And 391.2, compound #348 was identified as malonyl protopanaxatriol-diglucosyl-xyloside (PPT-2 Glc-Xyl-Mal) depending on cleavage events and debris.

(3) Characterization and identification of OA type ginsenoside based on MIM-IDA-EPI data

According to the structural characteristics of aglycone, the ginseng genus also has a class of saponin which is OA type saponin, and the panax japonicus and the rhizoma panacis majoris are rich in the saponin and are characterized in that no obvious adduct formic acid parent ion exists, so the component is captured by adopting an MIM-IDA-EPI method in the embodiment of the invention. The cracking behavior of the OA-type saponin is illustrated by taking the compound #167 as an example, as shown in fig. 4, the compound #167(m/z 925.5, 925.5-569.4) has obvious secondary fragments m/z 793.3, 631.3 and 455.3 in the cracking process, which respectively represent that one xylose, one glucose and one glucuronic acid molecule are lost in the cracking process according to the calculation of mass number, and the component chikusetsusaponin IV is determined by comparing the structure reported in the literature with the standard product. According to the cleavage characteristics of known components of OA type, unknown Compound #271(m/z 981.5, 981.5-793.3) was resolved from the secondary fragments. As shown in FIG. 4, it can be seen that a characteristic fragment m/z 455.3 of OA type appears at the low-mass end, and the component can be temporarily considered as an OA type saponin; fragment m/z 793.3 from m/z 981.5, missing 188Da, presumably corresponding to one rhamnose and one acetyl group; in addition, the secondary fragments m/z 793.3, 613.3 and the aglycone fragment m/z 455.3 presumably drop one glucose and one glucuronic acid molecule. Compound #271 was therefore finally identified as acetylated oleanolic acid-glucuronyl-glucosyl-rhamnoside (OA-GlcA-Glc-Rha-Ac).

In this embodiment, seven ginseng-derived traditional Chinese medicines are characterized and identified by combining the above negative ion mode with various "IDA-EPI" modes, and neutral saponins are identified (or derived) by using the MRM-IDA-EPI method, and the types and amounts of the neutral saponins contained in each ginseng-derived traditional Chinese medicine sample are identified as follows: 68 kinds of ginseng, 59 kinds of red ginseng, 44 kinds of American ginseng, 65 kinds of pseudo-ginseng, 62 kinds of panax japonicus, 56 kinds of rhizoma panacis majoris and 63 kinds of ginseng leaves; identifying the types and the quantity of the malonylated saponins contained in each ginseng traditional Chinese medicine sample as follows: 16 ginseng species, 10 red ginseng species, 14 American ginseng species, 15 pseudo-ginseng species, 14 panax japonicus species, 16 panax japonicus species and 25 ginseng leaves; identifying oleanolic acid type saponin by adopting an MIM-IDA-EPI method, and identifying that the types and the quantity of the oleanolic acid type saponin contained in each ginseng traditional Chinese medicine sample are as follows: 23 ginseng, 16 red ginseng, 25 American ginseng, 20 pseudo-ginseng, 36 panax japonicus, 29 panax japonicus and 20 ginseng leaves.

Comparative example 1

This comparative example illustrates the effect of different flow on ginsenoside detection: the mobile phase A is acetonitrile, and the mobile phase B adopts 0.1% v/v formic acid-water solution, 0.1% v/v acetic acid-water solution, 3mM/L ammonium formate-water solution and 3mM/L ammonium acetate-water solution respectively. Other chromatographic conditions were the same as in example 1.

In the comparative example, 8 standard substances (notoginsenoside R1, ginsenoside Re, malonyl ginsenoside Rb1, 24(R) -pseudoginsenoside F11, ginsenoside Ro, panax japonicus saponin IVa, malonyl ginsenoside Re1 and malonyl ginsenoside Rb2) are selected from neutral saponin, OA-type saponin and malonyl saponin, and are used as index components to evaluate the influence of the above mobile phase on the ginsenoside detection result, and the peak areas of the compounds in different mobile phases are counted, as shown in Table 2.

TABLE 2 Peak area tables for different ingredients with different mobile phase additives

The peak area of each compound in the four mobile phases can be seen visually from the table above, except that the peak areas of the panax japonicus IVa and the malonyl ginsenoside Rb2 are the highest when 0.1% v/v acetic acid-water solution is used as the mobile phase B, the peak areas of the other 6 components are the highest when 0.1% v/v formic acid-water solution is used as the mobile phase B, and the peak areas of most neutral, OA-type and malonyl-type saponins are superior to those of the other three mobile phases. When the target components in different mobile phases were observed from the TIC chart, the mass numbers of 8 compounds were extracted and integrated, and the peak areas of the respective compounds were recorded, as shown in fig. 5, it can be seen that the peak area value was the highest for the PPT type and the PPD type when 0.1% v/v formic acid-water solution was used as the mobile phase B, and adduct ions were most easily generated; for the OT type, the difference is slightly better than 0.1% v/v formic acid-water solution at 3mM/L ammonium formate-water solution, but not very much; for OA type, ginsenoside Ro is best when 0.1% v/v formic acid-water solution is used as mobile phase B, and panax japonicus saponin IVa is best when 0.1% v/v formic acid-water solution is used as mobile phase B; as for malonylated saponins, malonyl ginsenoside Re1 is most preferred when 0.1% v/v formic acid-water solution is used as mobile phase B, and malonyl ginsenoside Rb2 is most preferred when 0.1% v/v acetic acid-water solution is used as mobile phase B. The results of 9 target components were combined, and the peak area values of 6 components in total were the highest when the mobile phase B was a 0.1% v/v formic acid-water solution, and the peak area values of the remaining three components were also not low, so that the mobile phase used in the example of the present invention was the optimum mobile phase.

Comparative example 2

The comparative example lists the effects of different chromatographic columns on ginsenoside detection: the influence of the different reversed phase columns on the ginsenoside component is evaluated by comparing the separation degree, the peak type and the number of peaks generated by chromatographic peaks of the component to be detected through the SIM mode. The information for each column is as follows:

BEH C18：2.1×100mm，1.8μm；Waters；

BEH Shield RP18：2.1×100mm，1.8μm；Waters；

HSS T3：2.1×100mm，1.8μm；Waters；

CSH C18：2.1×100mm，1.8μm；Waters；

Zorbax Eclipse Plus C18：2.1×100mm，1.8μm；Agilent；

Zorbax SB-Aq：2.1×100mm，1.8μm；Agilent；

HSS C18 SB：2.1×100mm，1.8μm；Waters；

Cortecs UPLC C18⁺：2.1×100mm，1.6μm；Waters；

Zorbax Extend C18：2.1×100mm，1.8μm；Agilent；

Zorbax SB-C18：2.1×100mm，1.8μm；Agilent。

other chromatographic conditions were the same as in example 1.

This comparative example was compared by selecting 23 mass numbers (505.2, 521.2, 665.5, 667.3, 683.5, 699.5, 793.6, 811.6, 815.5, 829.5, 845.5, 925.6, 955.5, 977.3, 991.6, 1007.6, 1031.6, 1163.8, 1117.7, 1123.7, 1153.7, 1193.7, 1285.7, 1387.7, 1417.8) capable of covering ginsenoside subtypes (including PPT-type and PPD-type, OA-type saponins, and malonylated saponins among neutral saponins) as reference indices.

Firstly, SIM spectrograms obtained by the 10 chromatographic columns are compared by a segmentation method, and the separation degrees of BEH Shield RP18, CSH C18 and HSS C18 SB in the first section (0-15min) are good; the degree of separation of BEH Shield RP18, HSS C18 SB and Zorbax extended C18 in the second stage (15-30min) was good; the BEH Shield RP18, HSS T3, Zorbax SB-C18 and Zorbax SB-C18 in the third section (30-45min) were better separated. The result of the separation degree of three sections is combined, so that the separation degree of BEH Shield RP18 is optimal;

according to response value 0.5e⁶The number of peaks was found to be 51 HSST 3, Cortecs UPLC C18 ⁺55, 49 BEH C18, 46 CSH C18, 51 Zorbax Eclipse Plus C18, 49 Zorbax extended C18, 48 Zorbax SB-C18, Zorbax S47B-Aq, 47 HSS C18 SB and 59 BEH Shield RP 18. The results are shown in FIG. 6. From the results, it was found that the number of peaks was the largest in the BEH Shield RP18 column.

The chromatographic columns can obtain good peak shapes.

The results are combined to show that BEH Shield RP18 is superior to other chromatographic columns in terms of separation degree, peak type and peak output number of chromatographic peaks.

Comparative example 3

The comparative example compares the effects of different column temperatures on ginsenoside detection: the column temperatures were 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively, and other chromatographic conditions were the same as in example 1. Examine the response values 0.5e respectively⁶As a result of the above-mentioned number of peaks, as shown in FIG. 7, the number of peaks at 25 ℃ was 50, the number of peaks at 30 ℃ was 56, the number of peaks at 35 ℃ was 53, and the number of peaks at 40 ℃ was 59. The peak number is the most when the column temperature is 40 ℃, the separation degree is the best, the separation degree is poor for PPD and PPT type saponin at 25 ℃, the separation degree is poor for OA type and PPT type saponin at 30 ℃, the separation degree is poor for malonic acidylated saponin and OA type saponin at 35 ℃, 1031 and 793 are not well separated at 40 ℃, but other components are well separated, and the peak number is increased by 9 after optimization. The results are combined to show that more peak output numbers and better separation degree can be achieved at the column temperature of 40 ℃.

Comparative example 4

The comparative example investigates the influence of different acquisition modes on the detection of ginsenoside:

the list of neutral saponins collected by MRM-IDA-EPI is M1, the list of malonylated saponins collected by MRM-IDA-EPI is M2, and the list of OA-type saponins collected by MRM-IDA-EPI is M3. Comparing the MRM-IDA-EPI method with M1 as the parent ion list with NL46-IDA-EPI, the total number of EPIs and the number of effective EPIs obtained by the MRM-IDA-EPI method were found to be more; compared with the NL44-IDA-EPI method, the MRM-IDA-EPI method using M2 as the parent ion list has a higher number of effective EPIs than the NL-IDA-EPI method, although the number of EPIs obtained by the NL44-IDA-EPI method is far higher. Therefore, MRM-IDA-EPI is more advantageous to be selected for neutral saponin and malonylated saponin.

The secondary spectrogram quality and the fragment abundance are combined for analysis, the same components under the same retention time in different methods are selected, the secondary spectrogram quality and the fragment abundance are analyzed, and the result is shown in fig. 8. In the figure, the MRM-IDA-EPI method is respectively compared with the NL-IDA-EPI method adopted by neutral saponin and malonated saponin and the MIM-IDA-EPI method adopted by OA type saponin in parallel, and the results show that the richness of secondary fragments acquired by the MRM-IDA-EPI method in the three types of saponin is superior to that of NL-IDA-EPI and MIM-IDA-EPI; although the MRM-IDA-EPI method has a higher secondary response to OA-type saponins, the MIM-IDA-EPI method can capture more unknown components from the analysis that can trigger EPI number.

The results of comparing the different acquisition modes with the MRM-IDA-EPI method are shown in Table 3.

TABLE 3 comparison of different acquisition modes with the MRM-IDA-EPI method

Comparative example 5

The comparative example investigates the influence of different mass spectrum parameters on the detection of ginsenoside:

1. a mixed standard sample (notoginsenoside R1, ginsenoside Re, ginsenoside Rb1, 24(R) -pseudoginsenoside F11, ginsenoside Ro, panax japonicus saponin IVa, malonyl ginsenoside Re1 and malonyl ginsenoside Rb2) of representative components is selected, and the peak areas and the highest response values of the adduct ions EIC at DP values of 20, 40, 60, 80, 100 and 120 are examined. Taking 3-needle data, and calculating the average value and RSD value of the two index components. The three scanning methods aim at different structural subtypes and obtain the optimal DP value according to the classification evaluation of neutral, acidic and malonyl saponins. As shown in fig. 9.

And (4) conclusion: aiming at neutral saponin (PPD/PPT/OT) components, the corresponding strength of the adducted ions is the highest when the DP value of the notoginsenoside R1, the ginsenoside Re and the ginsenoside Rb1 is 20, the corresponding strength of the malonyl ginsenoside Rb2 is the highest when the DP value is 40, but the difference between the malonyl ginsenoside Rb2 and the DP value is small, and the corresponding strength of the 24(R) -pseudoginsenoside F11 is the highest when the DP value is 20; for OA type saponin, the corresponding strength of the ginsenoside Ro as a target component is the best when the DP value is 60 and 80, the difference is very small, and the corresponding strength of the panax japonicus saponin IVa is the best when the DP value is 60; for malonylated saponins, malonyl ginsenoside Re1 is preferred at DP 20, and malonyl ginsenoside Rb2 is preferred at DP 60. In summary, the optimal DP for NL46-IDA-EPI mode is 20eV, and for NL44-IDA-EPI and MIM-IDA-EPI modes is 60 eV.

2. Consider for CE values in NL and EPI:

(1) CE values in NL46-IDA-EPI mode

1 needle was collected at each CE value (18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60), and the number of parent ions of neutral saponins that can cause EPI was examined. As a result, as shown in fig. 10, 699.5, 683.5, 815.5, 829.5, 845.5, 977.3, 991.6, 1007.6, 1033.4, 1165.5, 1123.7, 1153.7, 1255.5, 1285.7 were selected as target mass numbers for evaluating CE values, and the CE value of NL was found to be 54eV as the optimum value, depending on the number of EPI parent ions that can be triggered.

(2) CE value of EPI in NL46-IDA-EPI mode

And (4) optimizing secondary collision energy by adopting a two-step method. Firstly, injecting 1 needle of neutral saponin under each CE value (40, 60, 70, 80, 90, 100, 120 and 140) by adopting an NL-IDA-EPI scanning mode, then setting a dynamic CE value, selecting a better CE value range, inspecting a secondary mass spectrum from molecular ions to aglycone ions in a more balanced way according to the mass of the secondary mass spectrum, determining more proper energy ranges to be 70, 80, 90, 100, 110 and 120 in the second step, continuously setting the dynamic energy range according to the mass of the secondary mass spectrum, and inspecting the number of parent ions of EPI which can be caused in the neutral saponin. As shown in FIG. 11, the two preferred energy ranges are 80eV to 120eV and 60eV to 120eV in the case of the secondary fragment, and the 60eV to 120eV energy is preferred for the saponin of 1 saccharide, and the fragment mass is capable of expressing sapogenin, the secondary fragment and the parent ion, but the saponin fragments having 4 and 5 saccharide numbers are poor in the case of the fragmentation, while the saponin fragments having 4 and 5 saccharide numbers in 80eV to 120eV are preferred in the case of the saponin fragments having 1 saccharide and a weaker saponin having a saccharide number in the total structure of ginsenoside but a large number of saponins having saccharide numbers in the total structure of ginsenoside, and thus 80eV to 120eV is more preferred.

(3) CE values in NL44-IDA-EPI mode

1 needle was collected at each CE value (18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60) respectively, and the number of parent ions that could trigger EPI was examined for malonylated saponins. 1325, 1295, 1193, 1179, 1177, 1163, 1047, 1031, 1017, 959, 885, 869 were selected as target mass numbers for evaluating CE values, and depending on the number of EPI parent ions that could be triggered, the CE value for the best NL was 54eV as shown in fig. 12.

(4) CE value of EPI in NL44-IDA-EPI mode

Respectively collecting 1 needle under each CE value (70, 80, 90, 100, 110 and 120), continuously setting a dynamic energy range according to the condition of the secondary fragment, and inspecting the quality of a secondary mass spectrum (structural subtypes are not distinguished, and all the subtypes are applicable): the result of the primary optimization of the secondary mass spectrum from molecular ions to aglycone ions according to single energy shows that the cracking conditions of-80 eV and-90 eV are slightly better, the dynamic CE values are set to be 80 eV-120 eV, 70 eV-110 eV, 80 eV-100 eV, 90 eV-110 eV and 70 eV-100 eV, as shown in FIG. 13, according to the secondary fragment condition, the better energy range is 70 eV-110 eV, compared with other energy ranges, the saponin secondary spectrum with different sugar numbers in the energy range can display parent ions, fragments and sapogenins, the intensity is more uniform, the fragments display more, and therefore, the more preferable CE value of EPI is 70 eV-110 eV.

(5) CE value in MIM-IDA-EPI mode

Mass numbers 1189.6, 1599.9, 1157.5, 955.5, 925.5, 793.4 were selected for energy optimization, as shown in fig. 14 for a second order fragmentation case for the mass number of the target component. Data at CE values of 50, 60, 70, 80, 90, 100, 110, 120 are collected, and the dynamic CE value range is set. The energies of 70eV and 80eV for high mass numbers are slightly smaller, the energies of 90eV and 100eV for low mass numbers are slightly higher, there is no parent ion fragment, when a dynamic energy range of 70eV to 100eV is set as the collision energy, the mass and integrity of the secondary fragment are better, the parent ion, the secondary fragment and the aglycone can be clearly seen, and therefore, the optimum CE value range is 70eV to 100 eV.

The MRM-IDA-EPI mode is a method based on two-part NL (46/44) -IDA-EPI acquisition results, so the optimal CE value of the MRM-IDA-EPI acquisition method is 70eV to 120 eV.

3. Information dependent acquisition response strength

The information-dependent acquisition response strength is respectively set to be 1-2, 1-3 and 1-5, and the reference standard is that the total number of EPIs (indicating MS) which can be triggered and are captured in the acquired parent ion list is considered and the effective EPI number in the total number is²Spectra can be used for identification, with secondary fragments clearly obtained), statistical results are shown in fig. 15. The total number of EPIs obtained when the number is set to be 1-2 is 203, and the number of effective EPIs is 177; 1-3, the total number of EPIs obtained is 182, and the effective EPIs are 170; the total number of EPIs that can be obtained is 201, and the effective EPIs are 173. By integrating the data, the optimal information dependence acquisition response intensity is 1-2.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A classification identification method of ginsenoside components in traditional Chinese medicines of Panax is characterized in that the ginsenoside components in the traditional Chinese medicines of Panax are classified and identified by a high performance liquid chromatography/mass spectrometry combined analysis method;

the mass spectrum is a triple quadrupole-linear ion trap mass spectrum; detecting ginsenoside by information-dependent acquisition-enhanced product ion scanning technology, detecting neutral saponin and malonyl saponin in MRM-IDA-EPI mode, and detecting oleanolic acid type ginsenoside in MIM-IDA-EPI mode.

2. The method according to claim 1, wherein the neutral saponins include protopanaxatriol type ginsenoside, protopanaxadiol type ginsenoside, ocotillone type ginsenoside and ginsengenin.

3. The method of classifying and identifying ginsenoside components in Panax as claimed in claim 2, wherein the protopanaxatriol-type ginsenoside comprises ginsenoside F1, 20(S) -ginsenoside Rh1, 20(R) -ginsenoside Rh1, ginsenoside F3, 20(S) -notoginsenoside A3, ginsenoside F5, notoginsenoside R2, 20(R) -notoginsenoside R2, ginsenoside Rg2, ginsenoside Rf, ginsenoside Rg1, notoginsenoside R1, notoginsenoside Fp1, ginsenoside Re, 20-O-glucosyl-ginsenoside Rf, ginsenoside Re2 and ginsenoside Re 3; and/or

The protopanaxadiol type ginsenoside comprises ginsenoside Rh2, 20(R) -ginsenoside Rh2, ginsenoside K, ginsenoside F2, ginsenoside Rg3, 20(R) -ginsenoside Rg3, notoginsenoside Fe, ginsenoside Rd2, notoginsenoside K, ginsenoside Rd, gypenoside XVII, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Ra1, ginsenoside Ra2, notoginsenoside R4 and notoginsenoside T; and/or

The ocrtron ginsenoside comprises 24(R) -pseudoginsenoside Rt5 and 24(R) -pseudoginsenoside F11; and/or

The ginsengenin comprises 20(S) -protopanaxatriol, 20(S) -protopanaxadiol, oleanolic acid and 20(S),24(R) -oxtriptologenin; and/or

The neutral saponin also comprises ginsenoside Rk3, ginsenoside Rh4, notoginsenoside T5, ginsenoside Rg5, ginsenoside Rg6, ginsenoside F4, ginsenoside Rk1, 5, 6-dehydroginsenoside Rd, vietnam ginsenoside R8 and 5, 6-dehydroginsenoside Rb 1.

4. The method for classifying and identifying ginsenoside components in ginseng as claimed in claim 1, wherein the oleanolic acid type ginsenosides comprise panax japonicus saponin IVa, panax japonicus saponin IV, pseudoginsenoside Rt1 and ginsenoside Ro; and/or

The malonyl saponins include malonyl ginsenoside Re1, malonyl ginsenoside Rd, malonyl ginsenoside Rc, malonyl ginsenoside Rb2 and malonyl ginsenoside Rb 1.

5. The method for classifying and identifying ginsenoside components in Panax as claimed in claim 1, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:

mobile phase a was acetonitrile and mobile phase B was 0.1% v/v formic acid-water solution, and the procedure for linear gradient elution was as follows:

flow rate: 0.28-0.32 mL/min;

column temperature: 30-40 ℃.

6. The method for classifying and identifying ginsenoside components in Panax as claimed in claim 5, wherein the chromatographic column is BEH Shield RP 18; and/or

The column temperature is 40 ℃; and/or

The flow rate was 0.3 ml/min.

7. The method for classifying and identifying ginsenoside components in Panax genus according to any one of claims 1-6, wherein the mass spectrum is a Q-Trap 4500 triple quadrupole-linear ion Trap mass spectrometer.

8. The method for classifying and identifying ginsenoside components in Panax as claimed in claim 7, wherein the ion source of mass spectrometry is an electrospray ion source, and data are collected in a negative ion mode; the parameters of the ion source are: the ionization voltage is-4500V, the temperature is 550.0 ℃, the gas curtain gas is 35.0psi, the spray gas 1 is 55.0psi, the auxiliary heating gas 2 is 55.0psi, the collision gas is High, and the outlet voltage of the collision chamber is-13.0V.

9. The method of classifying and identifying ginsenoside components in Panax as claimed in claim 8, wherein the collision energy in MIM-IDA-EPI mode is 70 eV-100 eV, and the collision energy in MRM-IDA-EPI mode is 70 eV-120 eV; the information-dependent acquisition response intensity is 1-2.

10. The use of the method for the classification and identification of ginsenoside components in Panax as claimed in any one of claims 1-9 in the characterization and identification of ginsenoside.