Detection method of ginsenoside content and its application

Abstract

The present invention relates to the technical field of determination of ingredients in Chinese patent medicines, and in particular to a method for detecting ginsenoside content and an application thereof. The present invention utilizes a multiple heart-cutting two-dimensional liquid chromatography tandem charged aerosol detector method to detect the content of ginsenosides including notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3(S), and ginsenoside Rg3(R). The method has the advantages of accuracy, high sensitivity, and strong specificity, is suitable for determining the content of ginsenosides in different Chinese patent medicines containing Panax genus Chinese medicines, and can be used reliably, comprehensively, and accurately for quality control of ginsenosides in Chinese patent medicines containing Panax genus Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of determination of components of traditional Chinese medicines, and in particular to a method for detecting ginsenoside content and an application thereof. Background Art

[0002] Chinese patent medicines are widely used in clinical practice, but their chemical composition is relatively complex, which brings great challenges to the development of accurate and easy-to-use quality control methods. Taking Chinese patent medicines containing Panax genus Chinese medicine as an example, when the content of various ginsenosides is determined according to traditional high performance liquid chromatography (HPLC), the separation between different ginsenosides and between ginsenosides and other components is poor, and even co-elution with other components is achieved. In addition, when different ginsenosides are detected separately, it is usually necessary to use a complex sample preparation procedure, which inevitably leads to low analysis efficiency and component loss, and the obtained content results cannot accurately reflect the content of various ginsenosides in different Chinese patent medicines. Therefore, for Chinese patent medicines with the same or similar index components, it is difficult to accurately distinguish them using current pharmacopoeial standards or traditional high performance liquid chromatography, thereby being unable to identify the authenticity of these Panax genus Chinese medicines in Chinese patent medicines. In view of this, it is necessary to establish a new method for determining the content of various ginsenoside components in Chinese patent medicines containing Panax genus Chinese medicines to achieve a more comprehensive and accurate determination of the content of various ginsenoside components. Summary of the Invention

[0003] In response to the above technical problems, the present invention provides a method for detecting the content of ginsenosides and its application. The detection method can simultaneously determine the content of 16 ginsenosides in traditional Chinese medicines containing Panax genus traditional Chinese medicines, and can be used for quality control of ginsenosides in traditional Chinese medicines containing Panax genus traditional Chinese medicines.

[0004] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions:

[0005] A method for detecting the content of ginsenosides, wherein the ginsenosides include: notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3(S) and ginsenoside Rg3(R); the content of the ginsenosides is determined by multi-heart-cutting two-dimensional liquid chromatography connected to a charged aerosol detector.

[0006] This detection method adopts multiple heart-cutting two-dimensional liquid chromatography coupled with a charged aerosol detector (MHC-2DLC-CAD). By rationally selecting chromatographic separation conditions, it can simultaneously determine the contents of 16 ginsenosides in Panax genus medicinal materials or traditional Chinese medicines containing Panax genus Chinese medicines. This method has the advantages of accuracy, high sensitivity, and strong specificity. It is suitable for determining the content of ginsenosides in Panax genus medicinal materials, as well as for determining the content of ginsenosides in different traditional Chinese medicines containing Panax genus Chinese medicines. It can effectively solve the problems of poor applicability, low analytical efficiency, difficulty in achieving variety differentiation, few indicator components, weak specificity, and often difficulty in identifying adulteration of traditional Chinese medicines currently used for quality control of traditional Chinese medicines containing Panax genus Chinese medicines. Therefore, it can be used reliably, comprehensively, and accurately for the quality control of ginsenosides in traditional Chinese medicines containing Panax genus Chinese medicines, providing a new method for further improving the quality standards of traditional Chinese medicines containing Panax genus Chinese medicines.

[0007] Preferably, the detection method comprises the following steps:

[0008] preparing a reference solution of the ginsenoside; extracting the sample to be tested to obtain a sample solution to be tested;

[0009] After the reference solution and the test sample solution are injected into a two-dimensional liquid chromatograph, the ginsenoside component attribution of each chromatographic peak is determined by a charged aerosol detector, the chromatographic peak area of ​​each ginsenoside in the reference solution and the test sample solution is obtained, and the content of each ginsenoside in the test sample solution is calculated.

[0010] The detection method provided by the present invention can simultaneously detect the above-mentioned ginsenosides, so the reference solution can be prepared into a mixed reference solution to shorten the detection time.

[0011] Preferably, the solvent for preparing the reference solution is 60-80% v / v methanol aqueous solution, more preferably 70% v / v methanol aqueous solution.

[0012] Preferably, the sample to be tested is ultrasonically extracted with a 60-80% v / v methanol aqueous solution. The mass volume ratio of the sample to be tested to the methanol aqueous solution can be selected to be 1:(8-12) g / mL.

[0013] Preferably, the ultrasonic time is 0.5-1.5 h, the ultrasonic power is 300-500 W, and the extraction temperature is 20-38°C.

[0014] The detection conditions for the charged aerosol detector are preferably: atomization temperature: 35-45°C; data acquisition frequency: 2-10 Hz; filter constant: 2-5 s; power function: 0.98-1.02; and gain: 98-102 pA. More preferred parameters are: atomization temperature: 40°C; data acquisition frequency: 5 Hz; filter constant: 3.6 s; power function: 1.00; and gain: 100 pA.

[0015] The detection method of the present invention can calculate the content of each ginsenoside in the sample solution to be tested by the standard curve method. In order to obtain a more accurate standard curve, the reference solution should be prepared with at least 7 concentrations. The concentrations of the ginsenoside reference substances can be: notoginsenoside R1 concentration is 0.75-770 μg / mL, ginsenoside Rg1 concentration is 1-3220 μg / mL, ginsenoside Re concentration is 0.80-890 μg / mL, 24(R)-pseudoginsenoside F11 concentration is 0.62-79 μg / mL, ginsenoside Rf concentration is 0.83-106 μg / mL, ginsenoside Ra2 concentration is 0.56-72 μg / mL, ginsenoside Rb1 concentration is 0.76-1560 μg / mL, ginsenoside Rg2 concentration is 0.73-1 The concentration of ginsenosides was 88 μg / mL, the concentration of ginsenoside Rh1 was 0.63-324 μg / mL, the concentration of ginsenoside Rc was 0.43-950 μg / mL, the concentration of ginsenoside Ro was 0.56-143 μg / mL, the concentration of ginsenoside Rb2 was 0.41-210 μg / mL, the concentration of ginsenoside Rb3 was 0.32-82 μg / mL, the concentration of ginsenoside Rd was 1.04-530 μg / mL, the concentration of ginsenoside Rg3(S) was 0.61-78 μg / mL, and the concentration of ginsenoside Rg3(R) was 0.57-73 μg / mL. Within the above concentration range, each ginsenoside showed good linearity.

[0016] Preferably, the chromatographic conditions of the first dimension in the two-dimensional liquid chromatography are:

[0017] Chromatographic column: octadecylsilane bonded silica gel column;

[0018] Mobile phase A was 0.05-0.15% v / v formic acid in water, and mobile phase B was acetonitrile. Linear gradient elution was performed. The procedure of the linear gradient elution was as follows:

[0019]

[0020]

[0021] Flow rate: 0.2-0.4 mL / min;

[0022] Column temperature: 25-35℃.

[0023] The chromatographic conditions of the second dimension in the two-dimensional liquid chromatography are:

[0024] Chromatographic column: octadecylsilane bonded silica gel column;

[0025] Mobile phase A was water, and mobile phase B was acetonitrile containing 0.05-0.15% v / v formic acid, and linear gradient elution was performed. The procedure of the linear gradient elution was as follows:

[0026]

[0027]

[0028] Flow rate: 1.4-1.6 mL / min;

[0029] Column temperature: 25-35℃.

[0030] Preferably, the injection volume is 2-5 μL.

[0031] The valve switching program is as follows: 0-14.4 minutes, 10-1; 14.4-15.0 minutes, 1-2; 15.0-16.1 minutes, 10-1; 16.1-16.9 minutes, 1-2; 16.9-35.9 minutes, 10-1; 35.9-37.5 minutes, 1-2; 37.5-45.4 minutes, 10-1; 45.4-49.8 minutes, 1-2; 49.8- 53.3 minutes, 10-1; 53.3-56.8 minutes, 1-2; 56.8-59.4 minutes, 10-1; 59.4-63.0 minutes, 1-2; 63.0-67.0 minutes, 10-1; 67.0-67.8 minutes, 1-2; 67.8-75.5 minutes, 10-1; 75.5-76.6 minutes, 1-2; 76.6-95.0 minutes, 10-1.

[0032] The mobile phase and linear elution program adopted in the present invention can improve the separation effect of different ginsenosides, increase the number of peaks, and reduce the spectrum fluctuation and baseline noise caused by cutting, which is conducive to the accurate detection of various ginsenosides.

[0033] Preferably, the first-dimension chromatography column is a Poroshell 120 EC-C18, and the second-dimension chromatography column is an XBridge Shield RP18. Under the chromatographic conditions of the present invention, this column combination can achieve uniform distribution of the ginsenoside peaks, with the retention time of the next chromatographic peak after ginsenoside Re being later, thereby achieving better separation of the ginsenoside peaks.

[0034] Preferably, the Trap column is Acclaim RSLC 120 C18. When this column is used as the interface device, the fluctuation caused by cutting in the chromatogram is smaller and the baseline is more stable.

[0035] Furthermore, the present invention also provides the use of the above-mentioned ginsenoside content detection method in detecting the ginsenoside content in traditional Chinese medicines. The above-mentioned detection method can accurately detect the content of multiple ginsenosides in traditional Chinese medicines, enabling a more comprehensive and accurate determination of the content of multiple ginsenoside components in traditional Chinese medicines.

[0036] The present invention has the beneficial effect of providing a universal method for detecting multiple ginsenosides in Panax genus medicinal materials or various Chinese patent medicines containing Panax genus medicinal materials. This method is accurate, highly sensitive, and highly specific, and is suitable for determining the content of multiple ginsenosides in different Panax genus medicinal materials or Chinese patent medicines containing Panax genus medicinal materials. This method utilizes the same sample preparation technology and chromatographic separation conditions, enabling simultaneous control of the quality of ginsenosides in different Panax genus medicinal materials or Chinese patent medicines containing Panax genus medicinal materials, i.e., a "one method, multiple uses" strategy. The established method can obtain more comprehensive and accurate results on the content of ginsenoside components in Panax genus medicinal materials or Chinese patent medicines containing Panax genus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0038] Figure 1 The structural formulas of 16 ginsenoside standards; Glc is glucose (C6H 10 O5), Xyl is xylose (C5H8O4), Rha is rhamnose (C6H 10 O4), Ara is arabinose (C5H8O4), and GlurA is glucuronic acid (C6H8O6);

[0039] Figure 2 Schematic diagram of the switching of multiple heart-cutting valves in Example 1.

[0040] Figure 3 These are chromatograms of the blank solution, mixed reference solution, and mixed test sample solution of the specificity test in Example 1; wherein, Figure A is the chromatogram of the blank solution, Figure B is the chromatogram of the mixed reference solution, and Figure C is the chromatogram of the mixed test sample solution.

[0041] Figure 4 The chromatograms of the mixed ginsenoside solution detected using different chromatographic columns in Comparative Example 2 are shown.

[0042] Figure 5 The chromatograms of the mixed ginsenoside solution detected using different mobile phases in Comparative Example 3 are shown. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0044] When using traditional high-performance liquid chromatography to determine the content of multiple ginsenosides in traditional Chinese medicines containing Panax genus herbs, the separation between different ginsenosides and between ginsenosides and other components is poor, and some ginsenosides may even co-elute with other components. Complex sample preparation procedures are usually required, which can easily lead to low analysis efficiency and component loss. The obtained content results cannot accurately reflect the content of various ginsenosides in different traditional Chinese medicines, and thus cannot identify the authenticity of these Panax genus herbs in the traditional Chinese medicines.

[0045] To address this issue, this study investigated a method for detecting ginsenoside content using multiple heart-cutting two-dimensional liquid chromatography coupled with a charged aerosol detector. This method was capable of simultaneously detecting multiple ginsenosides (structural formulas such as ginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3, and ginsenoside Rg3(R). Figure 1 The obtained method has the advantages of accuracy, high sensitivity and strong specificity, and is suitable for determining the content of various ginsenosides in different Panax genus herbs or Chinese patent medicines containing Panax genus herbs.

[0046] The reagents used in the following examples are:

[0047] Acetonitrile (HPLC grade): Thermo Fisher Scientific, USA;

[0048] Formic acid (HPLC grade): ACS, USA;

[0049] Deionized water was purified using a Milli-Q system (Millipore, USA).

[0050] Sixteen ginsenoside standards were purchased from Shanghai Shidande Biotechnology Co., Ltd. or Chengdu Dester Biotechnology Co., Ltd.

[0051] Twenty-eight Chinese patent medicines (TCMs) were purchased from various pharmacies. The following were: Naodesheng Tablets, Shenyang Hongyao Capsules, Xinkeshu Tablets, Jingtong Granules, Huoxuezhitong Powder, Danqi Tablets, Shuxiong Tablets, Sanqixueshangning Capsules, Sanqishangyao Tablets, Fufangxueshuantong Capsules, Gucining Capsules, Xiaoshuantongluo Capsules, Diedahuoxue Powder, Fufangdanshen Tablets, Fufangdanshen Dropping Pills, Naoan Capsules, Qipi Pills, Shenlingbaizhu Powder, Renshen Zaizao Pills, Renshen Lutong Pills, Renshen Yangrong Pills, Renshen Guipi Pills, Shiyiwei Shenqi Tablets, Mugua Pills, Yangshen Baofei Pills, Ershiqi Dingkun Pills, Dingkun Pills, and Guilingji. The batch numbers and manufacturer information are shown in Table 1.

[0052] Table 1 Batch information of Chinese patent medicine

[0053]

[0054]

[0055] The column information is shown in Table 2.

[0056] Table 2 Column information

[0057] serial number model Specification Manufacturer information 1 Poroshell 120 EC-C18 3.0×150mm,2.7μm Agilent 2 COSMOCORE PBr 2.1×100mm,2.6μm COSMOSIL 3 XSelect HSS T3 3.0×150mm,3.5μm Waters 4 Polaris C18-A 3.0×150mm,3.0μm Agilent 5 XBridge Shield RP18 4.6×150mm,3.5μm Waters 6 Kinetex XB-C18 2.1×100mm,2.6μm Philomon 7 COSMOCORE C18 2.1×100mm,2.6μm COSMOSIL 8 XCharge C18 3.0×100mm,3.0μm Acchrom 9 Poroshell 120 EC-C18 4.6×100mm,2.7μm Agilent 10 Exsil Plus 100 C18 PFP 4.6×150mm,3.0μm Exmere Ltd 11 Poroshell 120 SB-C18 4.6×100mm,2.7μm Agilent 12 Kinetex XB-C18 3.0×100mm,2.6μm Philomon 13 Poroshell 120 SB-Aq 4.6×100mm,2.7μm Agilent 14 Atlantis T3 3.0×150mm,3.0μm Waters 15 Poroshell 120 EC-C18 3.0×100mm,2.7μm Agilent 16 Exsil Mixed-Mode C18 / Cation 2.0×100mm,3.0μm Exmere Ltd

[0058] The analytical instruments used in the following examples are:

[0059] UltiMate 3000 ultra-high performance liquid chromatography system: Thermo Fisher Scientific, USA;

[0060] SB-4200DTS / P ultrasonic extractor: Ningbo Xinzhi Biotechnology Co., Ltd.;

[0061] Unless otherwise specified, the reagents and medicinal materials involved in the following examples can be obtained from commercial sources or by methods known in the art.

[0062] Example 1

[0063] The embodiment of the present invention provides a method for detecting the content of ginsenosides, wherein the ginsenosides to be detected include: notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3(S), ginsenoside Rg3(R). The content of each ginsenoside in the sample to be tested is determined by a multi-heart-cutting two-dimensional liquid chromatography coupled with a charged aerosol detector. The schematic diagram of the multi-heart-cutting valve switching is shown in FIG. Figure 2 shown.

[0064] 1. Chromatographic conditions

[0065] The trap column was Acclaim RSLC 120 C18 (3.0 × 33 mm, 3 μm);

[0066] The chromatographic conditions for the first dimension are:

[0067] Chromatographic column: Poroshell 120 EC-C18 (3.0×150, 2.7 μm);

[0068] Mobile phase A was 0.10% v / v formic acid in water, and mobile phase B was acetonitrile;

[0069] Flow rate 0.2 mL / min; right pump;

[0070] Column temperature: 30°C;

[0071] The chromatographic conditions for the second dimension were:

[0072] Chromatographic column: XBridge Shield RP18 (4.6×150, 3.5μm);

[0073] Mobile phase A was water, and mobile phase B was acetonitrile containing 0.10% v / v formic acid;

[0074] Flow rate 1.5mL / min; left pump;

[0075] Column temperature: 30°C;

[0076] The injection volume was 3 μL. The linear gradients of the first and second dimension chromatograms and valve switching are shown in Table 3.

[0077] The detection conditions of the charged aerosol detector were as follows: atomization temperature: 40°C; data acquisition frequency: 5 Hz; filter constant: 3.6 s; power function: 1.00; and gain: 100 pA.

[0078] Table 3 Gradient elution program and valve switching information

[0079]

[0080]

[0081] 2. Preparation of sample solution

[0082] Preparation of mixed ginsenoside reference solution: Accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3(S), and ginsenoside Rg3(R), dissolve them in 70% methanol aqueous solution to prepare a mixed reference stock solution, and store at -20°C for later use. Among them, the concentration of notoginsenoside R1 was 770.00 μg / mL, the concentration of ginsenoside Rg1 was 3220.00 μg / mL, the concentration of ginsenoside Re was 890.00 μg / mL, the concentration of 24(R)-pseudoginsenoside F11 was 79.00 μg / mL, the concentration of ginsenoside Rf was 106.00 μg / mL, the concentration of ginsenoside Ra2 was 72.00 μg / mL, the concentration of ginsenoside Rb1 was 1560.00 μg / mL, and the concentration of ginsenoside Rg2 was 188.00 μg / mL. / mL, the concentration of ginsenoside Rh1 was 324.00 μg / mL, the concentration of ginsenoside Rc was 440.00 μg / mL, the concentration of ginsenoside Ro was 143.00 μg / mL, the concentration of ginsenoside Rb2 was 210.00 μg / mL, the concentration of ginsenoside Rb3 was 82.00 μg / mL, the concentration of ginsenoside Rd was 530.00 μg / mL, the concentration of ginsenoside Rg3(S) was 78.00 μg / mL, and the concentration of ginsenoside Rg3(R) was 73.00 μg / mL.

[0083] 3. Establish a standard curve:

[0084] The inventors found in their research that due to the nature of the CAD detector, the concentration is not proportional to the response. The present application uses a second-order polynomial regression equation to analyze and evaluate the linearity. The standard curve is y = ax 2 +bx+c, where y refers to the peak area, x refers to the concentration, and a, b, and c are constants.

[0085] Accurately measure the above-mentioned mixed reference substance stock solution, use 70% methanol aqueous solution as solvent, dilute in sequence to obtain mixed reference substance solutions of different concentrations, and store at -20°C for future use. The concentrations of each mixed reference substance solution are shown in Table 4. Take 3 μL of the mixed reference substance solution of each concentration, inject it, and analyze it according to the above-mentioned chromatographic conditions and detection conditions to obtain the liquid chromatogram of each reference substance at different concentrations. With the peak area (y) of the analyte as the vertical coordinate and the concentration (x) of the analyte as the horizontal coordinate, construct the standard curve of each ginsenoside component, obtain the regression equation and correlation coefficient of each ginsenoside component, and determine its linear range. The concentration of each reference substance when the S / N (signal-to-noise ratio) is 10 is used as the limit of quantification (LOQ), and the concentration of each reference substance when the S / N is 3 is used as the limit of detection (LOD). The results are shown in Table 5. The linear regression equation of each ginsenoside component, the correlation coefficient R 2 All of them are above 0.999, indicating that the linear relationship is good within the scope of investigation.

[0086] Table 4 Mixed reference solution (concentration unit: μg / mL)

[0087]

[0088]

[0089] Table 5 Detection limit, quantification limit and linearity results

[0090]

[0091] 4. Specificity test

[0092] Accurately pipette 3 μL of the mixed reference solution of concentration 4 in Table 4, and analyze the sample according to the above chromatographic conditions and detection conditions to obtain the chromatogram of the mixed reference solution, as shown in the figure below. Figure 3 As shown in Figure B.

[0093] Preparation of mixed test sample solution: Take Sanqi Shangyao Tablets SST-1, Ershiqi Dingkun Pills EDP-1, and Guilingji GLJ-1 samples, remove the coating of each, cut into pieces, take out the contents, take 200, 1000, and 800 mg in turn, combine to obtain a mixed Chinese patent medicine sample, accurately weigh 1000 mg of the mixed Chinese patent medicine sample into a 15 mL centrifuge tube, add 3 mL of 70% v / v methanol aqueous solution, and perform ultrasonic extraction twice, each time for 1 hour, extraction power 400 W, extraction temperature 25°C, centrifuge at 4000 rpm for 10 minutes, combine the two supernatants and transfer them to a 10 mL volumetric flask, dilute with 70% v / v methanol to the scale line, shake well, and let stand to obtain a mixed test sample solution.

[0094] Take 1 mL of the mixed sample solution and centrifuge it at 14000 rpm for 10 min. Accurately aspirate 3 μL of the supernatant and inject it into the sample for analysis according to the above chromatographic conditions and detection conditions to obtain the chromatogram of the mixed sample, as shown in the figure. Figure 3 As shown in Figure C.

[0095] Accurately pipette 3 μL of 70% v / v methanol aqueous solution of blank solution, inject and analyze according to the above chromatographic conditions and detection conditions, and obtain the chromatogram of blank solution, as shown in the figure below. Figure 3 As shown in Figure A.

[0096] according to Figure 3 It can be seen that the chromatographic peaks of the 16 ginsenosides in the mixed sample solution are well separated from the chromatographic peaks of other components, the baseline separation of the 16 ginsenosides is achieved, and no interference is observed in the chromatographic peak of the blank solution.

[0097] 5. Precision test

[0098] Intra-day precision: Mixed reference solutions at three levels, concentration 5, concentration 4, and concentration 2, as shown in Table 4, were sampled and analyzed according to the above-mentioned chromatographic and detection conditions. The samples were injected six times continuously on the same day, and the peak areas of the 16 ginsenosides were recorded. The standard curves of the ginsenosides in Table 5 were used to calculate the contents of the 16 ginsenosides according to the external standard method. The relative standard deviations (RSDs) were calculated. The results are shown in Table 6. The RSDs were 0.70%-4.25%.

[0099] Inter-day precision: The mixed reference solutions at concentrations of 5, 4, and 2, as listed in Table 4, were injected and analyzed under the aforementioned chromatographic and detection conditions. Samples were injected three times daily for three consecutive days. The peak areas of the 16 ginsenosides were recorded. The contents of the 16 ginsenosides were calculated using the standard curves for each ginsenoside in Table 5 using the external standard method. The RSDs were calculated and shown in Table 7. The RSDs ranged from 1.21% to 5.57%. This indicates good instrument precision.

[0100] Table 6 Precision test results

[0101]

[0102]

[0103] 6. Repeatability test

[0104] Six 1000 mg portions of the mixed Chinese patent medicine sample from the specificity test were accurately weighed. The mixed sample solutions were prepared according to the specificity test method and injected separately. The samples were analyzed under the above-described chromatographic and detection conditions. The peak areas of the ginsenosides were recorded. The content of each ginsenoside was calculated using the external standard method using the standard curves for each ginsenoside in Table 5. The RSD values ​​were calculated, as shown in Table 7. The RSDs ranged from 4.03% to 6.73%. The results demonstrate good reproducibility of this method.

[0105] Table 7 Repeatability test results

[0106]

[0107]

[0108] 7. Stability test

[0109] The mixed sample solution used in the specificity test was injected at 0, 2, 4, 8, 12, 24, and 48 hours after preparation. The samples were analyzed according to the above chromatographic and detection conditions. The peak areas of the ginsenosides were recorded. The content of each ginsenoside was calculated using the external standard method using the standard curves for each ginsenoside in Table 5. The RSD values ​​were calculated, as shown in Table 8. The RSDs ranged from 2.51% to 5.63%. The results demonstrate that the mixed sample solution exhibited good stability within 48 hours of preparation.

[0110] Table 8 Stability test results

[0111] Element stability(%) Notoginsenoside R1 5.63 Ginsenoside Rg1 3.55 Ginsenoside Re 3.82 24(R)-Pseudoginsenoside F11 3.13 Ginsenoside Rf 3.60 Ginsenoside Ra2 / Ginsenoside Rb1 3.69 Ginsenoside Rg2 S 5.12 Ginsenoside Rh1 2.51 Ginsenoside Rc 3.65 Ginsenoside Ro 2.19 Ginsenoside Rb2 3.60 Ginsenoside Rb3 4.44 Ginsenoside Rd 5.45 Ginsenoside Rg3(S) 5.54 Ginsenoside Rg3(R) /

[0112] 8. Sample recovery test

[0113] Using the standard curves for each ginsenoside in Table 5, the external standard method was used to calculate the concentration of each ginsenoside in the mixed test sample solution used in the specificity test. A mixed standard stock solution of ginsenosides was prepared at a concentration five times the calculated concentration of each ginsenoside in the mixed test sample solution. Recovery tests were conducted at three levels, labeled 50%, 100%, and 150%, corresponding to the calculated concentrations of each ginsenoside in the mixed test sample solution. Nine 500 mg portions of the mixed Chinese patent medicine sample used in the specificity test were accurately weighed and spiked with the mixed standard stock solution at three levels. Mixed spiked test sample solutions were prepared according to the specificity test method, and the samples were injected and analyzed under the aforementioned chromatographic and detection conditions. The recovery rates were calculated and shown in Table 9, ranging from 93.76% to 111.14%. The results demonstrate the accuracy and reliability of this method.

[0114] Table 9 Sample recovery test results

[0115]

[0116] 9. Matrix effect experiment

[0117] The relative response value method was used for evaluation. The standard curves of the ginsenosides in Table 5 were used to calculate the concentration of each ginsenoside in the mixed sample solution in the specificity test according to the external standard method. A mixed reference stock solution of ginsenosides was prepared, wherein the concentration of notoginsenoside R1 was 229 μg / mL, the concentration of ginsenoside Rg1 was 569 μg / mL, the concentration of ginsenoside Re was 320 μg / mL, the concentration of 24(R)-pseudoginsenoside F11 was 89 μg / mL, the concentration of ginsenoside Rf was 70 μg / mL, the concentration of ginsenoside Rb1 was 453 μg / mL, and the concentration of ginsenoside Rg The concentration of ginsenoside R2 was 81 μg / mL, the concentration of ginsenoside Rh1 was 99 μg / mL, the concentration of ginsenoside Rc was 414 μg / mL, the concentration of ginsenoside Ro was 326 μg / mL, the concentration of ginsenoside Rb2 was 221 μg / mL, the concentration of ginsenoside Rb3 was 80 μg / mL, the concentration of ginsenoside Rd was 306 μg / mL, and the concentration of ginsenoside Rg3(S) was 71 μg / mL. For matrix effect analysis, 20 μL of the mixed ginsenoside solution was added to 180 μL of the mixed Chinese patent medicine extract sample to prepare solution A; 20 μL of the mixed ginsenoside solution was added to 180 μL of 70% aqueous methanol to prepare solution B; and 20 μL of 70% aqueous methanol was added to 180 μL of the mixed Chinese patent medicine extract sample to obtain solution C. Ginsenoside peak areas were recorded under the above chromatographic conditions. The contents of the analytes were calculated using the standard curve method and the RSD values ​​were calculated. Matrix effects were calculated to be between 92.82% and 107.62%. The results are shown in Table 10, indicating that the determination of each compound was not affected by matrix effects. For each analyte, matrix effect (%) = (peak area of ​​solution A - peak area of ​​solution C) / (peak area of ​​solution B) × 100%.

[0118] Table 10 Matrix effect experimental results

[0119] Element Matrix effect (%) Notoginsenoside R1 96.29±4.47 Ginsenoside Rg1 104.47±4.46 Ginsenoside Re 98.05±4.19 24(R)-Pseudoginsenoside F11 99.85±5.66 Ginsenoside Rf 107.62±5.30 Ginsenoside Ra2 / Ginsenoside Rb1 100.50±10.46 Ginsenoside Rg2 S 100.54±8.06 Ginsenoside Rh1 103.89±4.98 Ginsenoside Rc 102.09±5.22 Ginsenoside Ro 104.90±4.09 Ginsenoside Rb2 102.90±3.51 Ginsenoside Rb3 103.14±4.13 Ginsenoside Rd 109.52±3.73 Ginsenoside Rg3(S) 92.82±8.32 Ginsenoside Rg3(R) /

[0120] Example 2

[0121] This example provides the application of the detection method in Example 1 in detecting the content of ginsenoside components in different Chinese patent medicines containing Panax genus Chinese medicines.

[0122] Preparation of the test sample solution: Take an appropriate amount of the Naodesheng Tablets NDT-1 sample from Table 1, remove the coating, grind, and accurately weigh 1000 mg into a 15 mL centrifuge tube. Add 3 mL of 70% methanol aqueous solution and perform ultrasonic extraction twice, each for 1 hour at 400 W, 25° C, and centrifuge at 4000 rpm for 10 minutes. Combine the two supernatants and transfer them to a 10 mL volumetric flask. Dilute to the mark with 70% methanol, shake well, and let stand to obtain the Naodesheng Tablets NDT-1 sample test sample solution. The remaining 55 batches of test sample solutions listed in Table 1 were prepared using the same method. Samples were injected and analyzed according to the chromatographic and detection conditions of Example 1. The ginsenoside content of each batch of Chinese patent medicine sample was then calculated using the standard curves for ginsenosides in Table 5 of Example 1 using the external standard method. The results are shown in Table 11.

[0123] Table 11 Determination of ginsenoside content in Chinese patent medicines (mg / g)

[0124]

[0125]

[0126] From the above results, it can be seen that the method for detecting the content of ginsenosides provided by the present invention is capable of simultaneously detecting the contents of multiple ginsenosides in Chinese patent medicines containing Panax genus Chinese medicines.

[0127] Comparative Example 1

[0128] This comparative example provides detection results under other interface conditions.

[0129] Chromatographic conditions: Trap columns were selected as Acclaim RSLC 120 C18 (3.0×33 mm, 3 μm), Accucore RP-MS (4.6×50 mm, 2.6 μm), and HYPERCARB (4.6×50 mm, 3 μm).

[0130] First-dimensional chromatographic column: XCharge C18 (3.0×100, 3 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile; column temperature: 30°C; flow rate: 0.2 mL / min; gradient elution: 0-7 minutes, 15%-30% B; 7-50 minutes, 30% B; 50-60 minutes, 30-45% B; 60-75 minutes, 45% B; 75-82 minutes, 45-60% B; 82-90 minutes, 60-75% B; 90-95 minutes, 75-98% B; 95-99 minutes, 98% B.

[0131] Two-dimensional chromatography column: Poroshell 120 SB-Aq (4.6×100, 2.7 μm); mobile phase: phase A: 0.1% formic acid in water, phase B: acetonitrile; column temperature: 30°C; flow rate: 1.5 mL / min; gradient elution program: 0-15.0 min, 5% B; 15-15.5 min, 5-25% B; 15.5-18.5 min, 25-35% B; 18.5-18.6 min, 35-5% B; 18.6-19.6 min, 5% B; 19.6-19.7 min, 5-20% B; 19.7-22 min, 20-40% B; 22-22.1 min, 40-5% B; 22.1-23.2 min, 5% B. 23.2-23.3 minutes, 5-20% B; 23.3-24.8 minutes, 20-40% B; 24.8-24.9 minutes, 40-5% B; 24.9-25.9 minutes, 5% B; 25.9-26.0 minutes, 5-20% B; 26-30.3 minutes, 20-40% B; 30.3-30.4 minutes, 40-5% B; 30.4-35.9 minutes, 5% B; 35.9-36 minutes, 5-35% B; 36-40 minutes, 35% B; 40-40.1 minutes, 35-5% B; 40.1-42.4 minutes, 5% B; 42.4-42.5 minutes, 5-15% B; 4 2.5-46.5 minutes, 15-45% B; 46.5-46.6 minutes, 45-5% B; 46.6-48.9 minutes, 5% B; 48.9-49 minutes, 5-25% B; 49-50.4 minutes, 25-50% B; 50.4-50.5 minutes, 50-5% B; 50.5-52.9 minutes, 5% B; 52.9-53.0 minutes, 5-30% B; 53-61 minutes, 30-50% B; 61-61.1 minutes, 50-5% B; 61.1-61.9 minutes, 5% B; 61.9-62 minutes, 5-40% B; 62-65.3 minutes, 40-50% B; 65 .3-65.4 minutes, 50-5% B; 65.4-66.9 minutes, 5% B; 66.9-67 minutes, 5-40% B; 67-68 minutes, 40-60% B; 68-68.1 minutes, 60-5% B; 68.1-68.9 minutes, 5% B; 68.9-69 minutes, 5-30% B; 69-72 minutes, 30-60% B; 72-72.1 minutes, 60-5% B; 72.1-76 minutes, 5% B; 76-76.5 minutes, 5-45% B; 76.5-85 minutes, 45-70% B; 85-85.5 minutes, 70-5% B; 85.5-107 minutes, 5% B.

[0132] The valve switching program is as follows: 0-13.5 minutes, 10-1; 13.5-15.5 minutes, 1-2; 15.5-18.5 minutes, 10-1; 18.5-19.7 minutes, 1-2; 19.7-22.0 minutes, 10-1; 22-23.3 minutes, 1-2; 23.3-24.8 minutes, 10-1; 24.8-26 minutes, 1-2; 26-30.4 minutes, 10-1; 30.4-36.0 minutes, 1-2; 36.0-40.0 minutes, 10-1; 40-42.5 minutes, 1-2; 42.5-46.5 minutes, 10-1; 46.5-49.0 minutes, 1-2; 49.0-50.5 minutes, 10-1; 50.5-53.0 minutes, 1-2; 53.0-61.0 minutes, 10-1; 61.0-62.0 minutes, 1-2; 62.0-65.3 minutes, 10-1; 65.3-67.0 minutes, 1-2; 67.0-68.0 minutes, 10-1; 68.0-69.0 minutes, 1-2; 69.0-72.0 minutes, 10-1; 72.0-76.0 minutes, 1-2; 76.0-107.0 minutes, 10-1.

[0133] The above conditions are only used to compare different interface devices and are not the final conditions to be adopted.

[0134] The injection volume and detection conditions of the charged aerosol detector are the same as those in Example 1.

[0135] Preparation of mixed ginsenoside reference solution: Preparation of mixed ginsenoside solution: Accurately weigh 1 mg each of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, Vietnamese ginsenoside R4, 24(R)-pseudoginsenoside F11, ginsenoside Rf, ginsenoside F3, ginsenoside Ra2, ginsenoside Ra1, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rg2(R), ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, japonicus saponin IV, 24(R)-pseudoginsenoside RT5, ginsenoside Rd, japonicus saponin IVa, ginsenoside Rg3(S), and ginsenoside Rg3(R) reference substances, and dissolve them in 70% methanol aqueous solution to prepare a mixed ginsenoside stock solution with a concentration of 1 mg / mL. Accurately pipette 50 μL of each solution and mix them to prepare a mixed ginsenoside solution with a concentration of 43.48 μg / mL.

[0136] A loop and trap column are commonly used as interface devices. However, considering that the volume of the first-dimensional eluate from the cleaved ginsenosides would exceed the loop volume (500 μL), a 1000 μL loop could result in significant diffusion. Therefore, a trap column was selected for this experiment. One mL of the mixed ginsenoside solution was centrifuged at 14,000 rpm for 10 minutes. Three μL of the supernatant was injected and analyzed under the aforementioned chromatographic and detection conditions to obtain a chromatogram of the mixed ginsenoside solution. Three trap columns, Acclaim RSLC 120 C18 (3.0×33, 3μm), Accucore RP-MS (4.6×50, 2.6μm), and HYPERCARB (4.6×50, 3μm), were used for detection. Ginsenosides were almost not retained when connected to the HYPERCARB (4.6×50, 3μm) column. Compared with the Accucore RP-MS (4.6×50, 2.6μm), the chromatogram fluctuation caused by cutting was smaller and the baseline was more stable when connected to the Acclaim RSLC 120 C18 (3.0×33, 3μm) column. Therefore, the trap column Acclaim RSLC 120 C18 (3.0×33, 3μm) was the optimal choice for the interface device.

[0137] Comparative Example 2

[0138] This comparative example provides detection results using other chromatographic columns.

[0139] The chromatographic columns selected are 16 chromatographic columns shown in Table 2.

[0140] First-dimension chromatographic conditions: mobile phase A was 0.1% formic acid in water, mobile phase B was methanol; column temperature: 30°C; flow rate: 0.2 mL / min; injection volume: 3 μL; gradient elution: 0-4 min, 46-60% B; 4-15 min, 60-63% B; 15-25 min, 63% B; 25-27 min, 63-65% B; 27-37 min, 65% B; 37-50 min, 65-75% B; 50-62 min, 75-80% B; 62-75 min, 80% B; 75-82 min, 80-90% B; 82-87 min, 90-98% B; 87-90 min, 98% B;

[0141] Second-dimension chromatographic conditions: mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile; column temperature: 30°C; flow rate: 0.2 mL / min; injection volume: 3 μL; gradient elution: 0-7 min, 20% B; 7-9 min, 20-24% B; 9-32 min, 24-26% B; 32-50 min, 26% B; 50-60 min, 26-32% B; 60-80 min, 32% B; 80-95 min, 32-35% B; 95-105 min, 35-50% B; 105-112 min, 50-65% B; 112-115 min, 65-98% B; 115-119 min, 98% B.

[0142] The above conditions are only used to compare different chromatographic columns and are not the final conditions to be used.

[0143] The injection volume and detection conditions of the charged aerosol detector are the same as those in Example 1.

[0144] 1 mL of the mixed ginsenoside solution from Comparative Example 1 was centrifuged at 14,000 rpm for 10 minutes. 3 μL of the supernatant was sampled and analyzed using the above-described chromatographic and detection conditions to obtain a chromatogram of the mixed ginsenoside solution. The mixed ginsenoside solution was analyzed using the various octadecylsilane bonded silica gel columns listed in Table 2, using the above-described chromatographic conditions.

[0145] When methanol was used as mobile phase B, ginsenosides in the mixed ginsenoside solution were separated to varying degrees on 16 chromatographic columns, but an ideal chromatographic column with uniform distribution of ginsenoside peaks could not be found.

[0146] The chromatogram of the mixed ginsenoside solution detected using acetonitrile as mobile phase B is as follows: Figure 4 shown.

[0147] First, regarding the selection of the first-dimensional column, considering undersampling, a certain interval between the retention times of first-dimensional chromatographic peaks is desirable to allow sufficient time for second-dimensional elution. Ginsenoside Rg1 and ginsenoside Re typically require a longer separation time. Experiments have shown that using the Poroshell 120 EC-C18 (3.0×150, 2.7μm) and XSelectHSS T3 (3.0×150, 3.5μm) columns results in a uniform distribution of the ginsenosides, with the next peak after ginsenoside Re having a later retention time.

[0148] Regarding the choice of two-dimensional chromatography columns, the two-dimensional chromatography columns XBridge Shield RP18 (4.6×150, 3.5μm) and Poroshell 120 SB-Aq (4.6×100, 2.7μm) have larger particle sizes and can better separate and retain ginsenosides.

[0149] By optimizing the elution gradient, ginsenosides were better separated when Poroshell 120 EC-C18 (3.0×150, 2.7μm) was selected as the first-dimensional column and XBridge Shield RP18 (4.6×150, 3.5μm) was selected as the second-dimensional column. This combination became the best choice of chromatographic columns.

[0150] Comparative Example 3

[0151] This comparative example provides detection results using other mobile phases.

[0152] Chromatographic conditions: The mobile phases for the first and second dimensions were acetonitrile-water, acetonitrile (0.1% v / v formic acid)-water, acetonitrile-water (0.1% v / v formic acid), and acetonitrile (0.1% v / v formic acid)-water (0.1% v / v formic acid), respectively, in pairs.

[0153] First-dimensional column: Poroshell 120 EC-C18 (3.0×150, 2.7μm); flow rate: 0.2mL / min; first-dimensional gradient elution: 0-5 minutes, 15% B-25% B; 5-25 minutes, 25-27% B; 25-40 minutes, 27-30% B; 40-60 minutes, 30-32% B; 60-85 minutes, 32-35% B; 85-90 minutes, 35-50% B; 90-100 minutes, 35-50% B; 100-110 minutes, 50-65% B; 110-118 minutes, 65-98% B; 118-122 minutes, 98% B.

[0154] Two-dimensional chromatography column: XBridge Shield RP18 (4.6×150, 3.5μm); flow rate: 1.5mL / min; two-dimensional gradient elution program: 0-15.0 minutes, 5% B; 15-16.5 minutes, 5-25% B; 16.5-17.9 minutes, 25-35% B; 17.9-18 minutes, 35-5% B; 18-18.4 minutes, 5% B; 18.4-19 minutes, 5-20% B; 19-27 minutes, 20% B; 27-32 minutes, 20-28% B; 32-32.4 minutes, 28%-5% B; 32.4-33.1 minutes minutes, 5% B; 33.1-33.2 minutes, 5-32% B; 33.2-40 minutes, 32-38% B; 40-40.1 minutes, 38-5% B; 40.1-42 minutes, 5% B; 42-42.6 minutes, 5-30% B; 42.6-54 minutes, 30-35% B; 54-55 minutes, 35-5% B; 55-62 minutes, 5% B; 62-62.8 minutes, 5-30% B; 62.8-67.7 minutes, 30-31% B; 67.7-68 minutes, 31-5% B; 68-71 minutes, 5% B; 71-71.4 minutes, 5-30% B; 71.4-76.6 minutes, 30-34% B; 76.6-77 minutes, 34-5% B; 77-78 minutes, 5% B; 78-78.5 minutes, 5-35% B; 78.5-80.6 minutes, 35-45% B; 80.6-81 minutes, 45-5% B; 81-82 minutes, 5% B; 82-82.9 minutes, 5-35% B; 82.9-84 minutes, 35-43% B; 84-84.1 minutes, 43-5% B; 84.1-86 minutes, 5% B; 86-86.1 minutes, 5-35% B; 86.1-94.6 minutes, 35-38% B; 94.6-95 minutes, 38-5% B; 95-98 minutes, 5% B; 98-98.2 minutes, 5-40% B; 98.2-105 minutes, 40-48% B; 105-109 minutes, 48-5% B; 109-111 minutes, 5% B; 111-112 minutes, 5-40% B; 112-120 minutes, 40-48% B; 120-121 minutes, 48-98% B; 121-124 minutes, 98% B.

[0155] The valve switching program is as follows: 0-16.1 minutes, 10-1; 16.1-16.5 minutes, 1-2; 16.5-17.9 minutes, 10-1; 17.9-18.4 minutes, 1-2; 18.4-32.4 minutes, 10-1; 32.4-33.2 minutes, 1-2; 33.2-41.2 minutes, 10-1; 41.2-42.6 minutes, 1-2; 42.6-54 minutes, 10-1; 54-62.8 minutes, 1-2; 62.8-67.7 minutes, 10-1 ; 67.7-71.4 minutes, 1-2; 71.4-76.6 minutes, 10-1; 76.6-78.5 minutes, 1-2; 78.5-80.6 minutes, 10-1; 80.6-82.9 minutes, 1-2; 82.9-84.0 minutes, 10-1; 84-86.1 minutes, 1-2; 86.1-94.6 minutes, 10-1; 94.6-98.2 minutes, 1-2; 98.2-110.25 minutes, 10-1; 110.25-111.25 minutes, 1-2.

[0156] The above conditions are only used to compare different mobile phases and are not the final conditions to be adopted.

[0157] The injection volume and detection conditions of the charged aerosol detector are the same as those in Example 1.

[0158] 1 mL of the mixed ginsenoside solution in Comparative Example 1 was centrifuged at 14,000 rpm for 10 min, and 3 μL of the supernatant was injected and analyzed according to the above chromatographic conditions and detection conditions to obtain a chromatogram of the mixed ginsenoside solution. The experimental results show that when the one-dimensional mobile phase and the two-dimensional mobile phase do not contain formic acid, the OA-type saponins ginsenoside Ro, ginsenoside chiku-IV, and ginsenoside chiku-IVa do not show peaks in the spectrum, as shown in FIG. Figure 5 In addition, the spectral noise increases with the addition of formic acid in the mobile phase, which is particularly obvious when formic acid is added to both the one-dimensional and two-dimensional phases.

[0159] It can be seen from the test results that using the mobile phase in Example 1 of the present invention can obtain more peaks, and the spectrum fluctuation and baseline noise caused by cutting are smaller.

[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting ginsenoside content, characterized in that: The ginsenosides include: notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, 24( R )-Pseudo-ginsenoside F11, ginsenoside Rf, ginsenoside Ra2, ginsenoside Rb1, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rc, ginsenoside Ro, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg3( S ) and ginsenoside Rg3( R ); using multiple heart-cutting two-dimensional liquid chromatography coupled with a charged aerosol detector to determine the content of the ginsenosides; The chromatographic conditions of the first dimension in the two-dimensional liquid chromatography are: Column: Poroshell 120 EC-C18, 3.0 × 150, 2.7 µm; Mobile phase A was 0.05-0.15% v / v formic acid aqueous solution, and mobile phase B was acetonitrile, and linear gradient elution was performed. The procedure of the linear gradient elution was as follows: Flow rate: 0.2-0.4 mL / min; Column temperature: 25-35°C; The chromatographic conditions of the second dimension in the two-dimensional liquid chromatography are: Column: XBridge Shield RP18, 4.6 × 150, 3.5 µm; Mobile phase A was water, and mobile phase B was acetonitrile containing 0.05-0.15% v / v formic acid, and linear gradient elution was performed. The procedure of the linear gradient elution was as follows: Flow rate: 1.4-1.6 mL / min; Column temperature: 25-35°C; The trap column was Acclaim RSLC 120 C18, with a size of 3.0 × 33 mm and a column size of 3 μm.

2. The method for detecting ginsenoside content according to claim 1, wherein: The detection method comprises the following steps: preparing a reference solution of the ginsenoside; extracting the sample to be tested to obtain a sample solution to be tested; After the reference solution and the test sample solution are injected into a two-dimensional liquid chromatograph, the ginsenoside component attribution of each chromatographic peak is determined by a charged aerosol detector, the chromatographic peak area of ​​each ginsenoside in the reference solution and the test sample solution is obtained, and the content of each ginsenoside in the test sample solution is calculated.

3. The method for detecting ginsenoside content according to claim 2, wherein: The solvent for preparing the reference solution is 60-80% v / v methanol in water; and / or The samples were ultrasonically extracted with 60-80% v / v methanol aqueous solution.

4. The method for detecting ginsenoside content according to any one of claims 1 to 3, wherein: The detection conditions of the charged aerosol detector are: atomization temperature: 35-45°C; data acquisition frequency: 2-10Hz; filtration constant: 2-5s; Power function: 0.98-1.02; Gain: 98-102pA.

5. Use of the method for detecting ginsenoside content according to any one of claims 1 to 4 in detecting the ginsenoside content in Chinese patent medicines.

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