Detection method of ginsenoside component of nephritis-relieving tablet

Abstract

The invention relates to the technical field of detection, in particular to a method for detecting the ginsenoside component of a nephritis-relieving tablet, the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the nephritis-relieving tablet are simultaneously determined by adopting a liquid chromatography-mass spectrometry method, and the method comprises the following steps: (1) preparing a test solution; (2) setting liquid chromatography conditions; (3) setting mass spectrum conditions; the invention establishes a liquid chromatography-mass spectrometry method for simultaneously determining three ginsenoside components in the nephritis-relieving tablet, and the method has the advantages of simple and convenient sample treatment process, short analysis time (only 4.5 min), high sensitivity, high specificity and the like. Compared with the content determination item of the nephritis-relieving tablet in 2020 edition Chinese Pharmacopoeia, the content determination of the characteristic component ginsenoside Rb1 of ginseng is increased, and a reference can be provided for the overall quality control and evaluation of the nephritis-relieving tablet.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a method for detecting ginsenoside components of Shenyanshu tablets. Background Art

[0002] Chronic glomerulonephritis (CGN, abbreviated as chronic nephritis) is a long-term, slowly progressive disease with a complex etiology. Angiotensin receptor antagonists, immunosuppressants, and symptomatic hormonal drugs are commonly used clinically to improve patients' glomerular hypertension and proteinuria levels. However, the above-mentioned Western medicine treatments have adverse reactions such as poor efficacy, high recurrence rate, and infection.

[0003] In recent years, clinical practice has shown that the combination of traditional Chinese medicines and Western medicines for the treatment of chronic nephritis has significant clinical efficacy, improving symptoms such as hematuria and proteinuria. Shenyanshu Tablets are a traditional Chinese medicine composed of ten herbs: Atractylodes macrocephala, Poria cocos, Imperata cylindrica, Stephania tetrandra, Ginseng (without the stem), Polygonatum sibiricum, Cuscuta australis, Lycium barbarum, Honeysuckle, and Taraxacum mongolicum. They benefit the kidneys and spleen, promote diuresis, and reduce swelling. Clinically, they are often used in combination with other medications to treat various forms of chronic nephritis.

[0004] Currently, research on Shenyanshu tablets primarily focuses on their pharmacological effects and the clinical efficacy of combined medications, with limited research on their active ingredients and content determination. This preparation is complex and contains numerous active ingredients, including ginsenosides, the principal ingredient of which is ginsenoside Rg1 and Re, which are known to tonify the spleen and replenish qi. However, the current standard for Shenyanshu tablets only uses high-performance liquid chromatography (HPLC), a method with drawbacks such as complex sample preparation and lengthy analysis times.

[0005] In recent years, liquid chromatography-mass spectrometry (LC-MS) has been increasingly used in the analysis of traditional Chinese medicine compound preparations due to its advantages such as strong selectivity and high sensitivity. This study establishes an ultra-rapid liquid chromatography-tandem mass spectrometry method for the simultaneous determination of the three ginsenosides (ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1) in Shenyanshu tablets, providing a scientific basis for the study of the pharmacological basis and quality control methods for Shenyanshu tablets.

[0006] Based on this, the present invention provides a method for detecting the ginsenoside components of Shenyanshu tablets. Summary of the Invention

[0007] The present invention relates to a method for detecting ginsenoside components of Shenyanshu tablets.

[0008] The present invention provides the following technical solutions:

[0009] A method for detecting ginsenoside components in Shenyanshu tablets, using liquid chromatography-mass spectrometry to simultaneously determine the contents of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in Shenyanshu tablets, comprising the following steps:

[0010] (1) Preparation of test solution: Take 10 Shenyanshu tablets, remove the sugar coating and grind them into powder. Take 0.25 g and add 20 mL of chromatographic grade methanol. Ultrasonic extraction is carried out at a power of 250 W and a frequency of 50 kHz for 30 min. After cooling, make up the weight and centrifuge at 12000 rpm for 3 min. The supernatant is filtered through a 0.22 μm microporous membrane to obtain the test solution.

[0011] (2) Liquid chromatography conditions: A Shim-Pack XR-ODS column, 75 mm × 3.0 mm, 2.2 μm, was used. 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program was from 30% to 57% of mobile phase B within 0 to 4.5 min. The equilibrium time was 5 min, and the flow rate was 0.5 mL min. -1 , column temperature was 35 °C, and injection volume was 5 μL;

[0012] (3) Mass spectrometry conditions: Electrospray ionization source, positive ion mode detection, nebulizer gas pressure 50 psi, auxiliary gas pressure 50 psi, curtain gas pressure 20 psi, monitoring mode selected ion monitoring, where the monitoring ion of ginsenoside Rg1 is m / z 823.7 [M+Na] + The monitoring ion of ginsenoside Re is m / z969.9[M+Na] + The monitoring ion of ginsenoside Rb1 is m / z1132.4[M+Na] + .

[0013] As a further technical solution, the weight supplementation in step (1) refers to supplementing the lost weight with chromatographically pure methanol.

[0014] As a further technical solution, the preparation of the test solution in step (1) further includes: taking 1 mL of the supernatant and placing it in a 10 mL volumetric flask, adding methanol to the scale and then filtering.

[0015] As a further technical solution, the initial ratio of the mobile phase for the gradient elution in step (2) is A:B=70:30.

[0016] As a further technical solution, the response value of the selected ion monitoring in step (3) is a linear relationship between the peak area and the mass concentration.

[0017] As a further technical solution, the linear range of ginsenoside Rg1 is 0.05-2.0 μg·mL -1 The linear range of ginsenoside Re is 0.05~2.0μg·mL -1 The linear range of ginsenoside Rb1 was 0.1~4.0μg·mL -1 .

[0018] As a further technical solution, the detection limit of ginsenoside Rg1 was 0.024 μg mL -1 The limit of quantification was 0.081 μg·mL -1 The detection limit of ginsenoside Re was 0.065 μg·mL -1 The limit of quantification was 0.219 μg·mL -1 The detection limit of ginsenoside Rb1 was 0.002 μg·mL -1 The limit of quantification was 0.007 μg·mL -1 .

[0019] As a further technical solution, the preparation of reference solution is also included: 5 mg of ginsenoside Rg1, Re, and Rb1 reference substances are accurately weighed and placed in a 5 mL volumetric flask, methanol is added to the scale, and shaken to prepare a concentration of 1 mg mL -1 A single reference solution.

[0020] As a further technical solution, in the precision test, the concentrations of ginsenosides Rg1, Re, and Rb1 in the mixed reference solution were 0.2 μg·mL -1 , 0.2 μg·mL -1 , 0.4 μg·mL -1 .

[0021] As a further technical solution, in the stability test, the test solution is measured 0, 2, 4, 8, 12, and 24 hours after preparation:

[0022] Beneficial effects of the present invention:

[0023] The liquid chromatography-mass spectrometry method established in the present invention achieves the simultaneous, rapid and accurate determination of ginsenosides Rg1, Re and Rb1 in Shenyanshu tablets by optimizing the key parameters of sample preparation, chromatographic separation and mass spectrometry detection.

[0024] The core advantages of the method of the present invention are:

[0025] High efficiency: sample processing is simple (ultrasound 30 minutes), analysis time is short (4.5 minutes), which solves the problems of complex operation and time-consuming traditional methods;

[0026] High specificity and sensitivity: using SIM mode and [M+Na] + The high stability of ions eliminates matrix interference, has a low detection limit, and can accurately determine low-content components;

[0027] Reliability: The method met the requirements for linearity, precision, stability, and recovery, providing a scientific basis for the quality control of Shenyanshu tablets. Compared with the current pharmacopoeia standard, the newly added determination of ginsenoside Rb1 more comprehensively reflects the quality of the preparation.

[0028] In summary, the method of the present invention significantly improves the efficiency and accuracy of the quality control of Shenyanshu tablets, and is of great significance for ensuring the clinical efficacy of Chinese patent medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the positive ion scan of ginsenoside Rg1(A);

[0030] Figure 2 This is the positive ion scan of ginsenoside Re (B);

[0031] Figure 3 This is the positive ion scan of ginsenoside Rb1(C);

[0032] Figure 4 SIM mass spectrum of ginsenoside Rg1 reference substance (1);

[0033] Figure 5 SIM mass spectrum of ginsenoside Rg1 sample (2);

[0034] Figure 6 SIM mass spectrum of ginsenoside Re reference substance (1);

[0035] Figure 7 SIM mass spectrum of ginsenoside Re sample (2);

[0036] Figure 8 SIM mass spectrum of ginsenoside Rb1 reference substance (1);

[0037] Figure 9 This is the SIM mass spectrum of ginsenoside Rb1 sample (2). DETAILED DESCRIPTION

[0038] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0039] The present invention provides a method for detecting ginsenoside components of Shenyanshu tablets, which is specifically as follows:

[0040] 1. Instruments and reagents

[0041] 1.1 Instrument

[0042] 3200QTRAP mass spectrometer (ABSciex, USA), ProminenceUPLC ultra-high speed liquid chromatograph (Shimadzu, Japan), KQ-250DE ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.).

[0043] 1.2 Drug testing

[0044] Reference substances ginsenoside Rg1 (batch number 110703-202235), ginsenoside Re (batch number 110754-202330), and ginsenoside Rb1 (batch number 110704-202331) were purchased from the China Food and Drug Administration (CFDA) with a purity greater than 98%. Chromatographically grade acetonitrile and methanol were purchased from ThermoFisher, and purified water was purchased from Watsons.

[0045] 2 Methods and Results

[0046] 2.1 Chromatographic and mass spectrometry conditions

[0047] 2.1.1 Chromatographic conditions

[0048] A Shim-PackXR-ODS column (75 mm × 3.0 mm, 2.2 μm) was used with 0.1% formic acid water (A)-acetonitrile (B) as the mobile phase, gradient elution (0-4.5 min, 30% → 57% B), equilibrium time 5 min, and flow rate 0.5 mL min -1 , column temperature 35℃, injection volume 5μL.

[0049] 2.1.2 Mass spectrometry conditions

[0050] An electrospray ionization source (ESI source) was used in positive ion mode, with a nebulizer gas pressure of 50 psi, an auxiliary gas pressure of 50 psi, a curtain gas pressure of 20 psi, and a detection mode of selected ion monitoring (SIM). Ions used for quantification: Ginsenoside Rg1: 823.7 [M+Na] + , Ginsenoside Re:969.9[M+Na] + , ginsenoside Rb1:1132.4[M+Na] + .

[0051] 2.2 Solution preparation

[0052] 2.2.1 Preparation of reference solution

[0053] Accurately weigh 5 mg of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 reference substances, place them in a 5 mL volumetric flask, add methanol to the mark, and shake well to prepare 1 mg mL of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1. -1 A single reference solution.

[0054] 2.2.2 Preparation of test solution

[0055] Take 10 tablets of Shenyanshu tablets, remove the sugar coating from the sugar-coated tablets, grind them into powder, take 0.25g, accurately weigh, place in a 50mL stoppered conical flask, add 20mL of methanol, stopper, weigh, and extract by ultrasonic for 30min (power 250W, frequency 50kHz), let cool, weigh again, make up the lost weight with methanol, shake well, filter, take the filtrate, centrifuge at 12000rpm for 3min, take 1mL of the supernatant and place in a 10mL volumetric flask, add methanol to the scale, shake well, and filter through a 0.22μm microporous membrane before injection.

[0056] 2.3 Linear relationship investigation

[0057] Take appropriate amount of each reference solution in item "2.2.1" and use methanol as solvent to prepare a series of mixed reference solutions, in which the mass concentrations of ginsenoside Rg1 are 0.05, 0.1, 0.2, 0.4, 1.0 and 2.0 μg mL -1 The mass concentrations of ginsenoside Re were 0.05, 0.1, 0.2, 0.4, 1.0 and 2.0 μg·mL -1 The mass concentrations of ginsenoside Rb1 were 0.1, 0.2, 0.4, 0.8, 2.0 and 4.0 μg·mL -1 Take the above series of mixed reference solution and inject them into the liquid chromatography-mass spectrometer according to the conditions of "2.1" respectively, and record the peak area. The horizontal axis is the mass concentration of the reference substance (X, μg·mL -1 ), the ordinate is the integral value of the peak area (Y), and linear regression calculation is performed. The results are shown in Table 1:

[0058] Table 1 Linear relationship of ginsenosides in Shenyanshu tablets

[0059]

[0060] As can be seen from Table 1, the mass concentrations of the above three components are 0.05-2.0, 0.05-2.0 and 0.1-4.0 μg·mL, respectively. -1 There is a good linear relationship between the peak area and the peak area.

[0061] 2.4 Determination of detection limit and quantification limit

[0062] An appropriate amount of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 reference solution was diluted gradually with methanol and injected. The detection limit was investigated at a signal-to-noise ratio of 3, and the quantification limit was investigated at a signal-to-noise ratio of 10. The results are shown in Table 1.

[0063] 2.5 Precision test

[0064] Accurately pipette the mixed reference solution prepared in Item 2.3 (the concentrations of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 are 0.2, 0.2, and 0.4 μg mL, respectively) -1 ) were injected 6 times continuously. The RSDs of the peak areas of the three components were 1.5%, 1.9% and 0.6%, respectively, indicating good instrument precision.

[0065] 2.6 Stability test

[0066] The same sample of Shenyanshu was injected and tested at room temperature at 0, 2, 4, 8, 12, and 24 hours after solution preparation. The RSDs of the peak areas for ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were 1.5%, 1.0%, and 1.3%, respectively, indicating good stability of the test solution within 24 hours.

[0067] 2.7 Repeatability test

[0068] Six samples of Shenyanshu tablets from the same batch were used to prepare test solutions according to the method in "2.2.2." The samples were injected and analyzed, and the peak areas were recorded and the contents of the components to be tested were calculated. The results showed that the average percentages (n=6) of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were 0.027%, 0.080%, and 0.076%, respectively. The RSDs for the contents were 1.7%, 1.1%, and 1.8%, respectively, indicating good reproducibility of the method.

[0069] 2.8 Sample recovery test

[0070] Take 9 samples of Shenyanshu tablets from the same batch with known content of the tested components (the content of ginsenoside Rg1, Re and Rb1 is 0.027%, 0.0801% and 0.076% respectively), each 0.125g, and add appropriate amount of reference solution equivalent to 80%, 100% and 120% of the content of each tested component in the sample, respectively. Prepare the test solution according to the method of "2.2.2". Sample injection and determination are carried out. Calculate the recovery rate and RSD of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1. The results are shown in Table 2:

[0071] Table 2 Recovery rates of ginsenosides Rg1, Re, and Rb1 in Shenyanshu tablets (n=3)

[0072]

[0073] 2.9 Sample content determination

[0074] Six batches of Shenyanshu tablets were weighed and the test solution was prepared according to "2.2.2". The samples were injected and analyzed, and the contents of the three ginsenoside components to be tested were calculated by substituting them into the regression equation. The results are shown in Table 3. The total content of ginsenoside Rg1 and ginsenoside Re in the samples averaged 0.214 mg / tablet. -1 The results meet the requirements of the 2020 edition of the "Chinese Pharmacopoeia" (the total amount of ginsenoside Rg1 and ginsenoside Re in each tablet must not be less than 0.14 mg).

[0075] Table 3 Contents of ginsenosides Rg1, Re and Rb1 in Shenyanshu tablets

[0076]

[0077]

[0078] 3 Discussions

[0079] 3.1 Selection of extraction solvent and extraction method

[0080] At present, the main extraction methods for the saponin chemical components in ginseng are ultrasound and heating reflux. The present invention analyzes and compares the two extraction methods of ultrasound (0.25h, 0.5h, 1h) and heating reflux (1h, 2h, 2.5h). The results show that the extraction rate of ginsenosides is similar between ultrasound 0.5h and heating reflux 2h. In view of the simplicity of operation, ultrasound is selected as the extraction method of the present invention. The present invention also investigates the extraction solvent and uses five extraction solvents including 30% methanol, 50% methanol, 70% methanol, 100% methanol and water-saturated n-butanol. It is found that the extraction rate of ginsenosides in the sample is the highest when 100% methanol is used as the solvent. Therefore, the present invention uses 100% methanol as the extraction solvent. Because the Shenyanshu tablets contain many kinds of medicinal materials and complex ingredients, there are certain requirements for the sample processing method. The sample extraction method adopted by the present invention is simple. After the sample is ultrasonically extracted, it is directly diluted and injected, which simplifies the sample pre-treatment process.

[0081] 3.2 Selection of chromatographic conditions

[0082] The present invention investigated the separation effects of different mobile phase systems, such as acetonitrile-water, acetonitrile-0.1% formic acid aqueous solution, methanol-water, and methanol-0.1% formic acid aqueous solution, on ginsenoside compounds in Shenyanshu tablets under the same gradient conditions. The results showed that when acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase, the peak shape of the three ginsenosides was optimal and the separation effect was the best. In addition, the present invention optimized the flow rate and column temperature, and finally determined the flow rate to be 0.5 mL min -1 , the column temperature was 35°C.

[0083] 3.3 Optimization of mass spectrometry conditions

[0084] Most ginsenosides are tetracyclic triterpenoid compounds with hydroxyl groups in their structures. They are easy to form [M+Na] + Ions have a higher response value than negative ion scanning mode, so the present invention adopts positive ion scanning mode. The present invention previously used MRM mode for monitoring, which has low sensitivity and poor reproducibility of measurement results, which may be related to the [M+Na] + The parent ion is not easy to produce stable daughter ions. + The ions have strong stability and high response, and the SIM detection mode has more advantages in quantitative analysis than the MRM detection mode. Therefore, the present invention ultimately uses the SIM mode for quantitative analysis of these three ginsenosides.

[0085] 4 Conclusion

[0086] This study establishes a liquid chromatography-mass spectrometry method for the simultaneous determination of three ginsenosides in Shenyanshu tablets. This method simplifies sample preparation, reduces analysis time to 4.5 minutes, and exhibits high sensitivity and specificity. Compared to the Shenyanshu tablet content determination item in the 2020 edition of the Chinese Pharmacopoeia, the addition of ginsenoside Rb1, a characteristic ginseng component, provides a reference for overall quality control and evaluation of Shenyanshu tablets.

[0087] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification.

Claims

1. A method for detecting ginsenoside components in Shenyanshu tablets, characterized in that: The liquid chromatography-mass spectrometry method is used to simultaneously determine the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in Shenyanshu tablets, comprising the following steps: (1) Preparation of test solution: Take 10 Shenyanshu tablets, remove the sugar coating and grind them into powder. Take 0.25 g and add 20 mL of chromatographic grade methanol. Ultrasonic extraction is carried out at a power of 250 W and a frequency of 50 kHz for 30 min. After cooling, make up the weight and centrifuge at 12000 rpm for 3 min. The supernatant is filtered through a 0.22 μm microporous membrane to obtain the test solution. (2) Liquid chromatography conditions: A Shim-Pack XR-ODS column, 75 mm × 3.0 mm, 2.2 μm, was used. 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program was from 30% to 57% of mobile phase B within 0 to 4.5 min. The equilibrium time was 5 min, and the flow rate was 0.5 mL min. -1 , column temperature was 35 °C, and injection volume was 5 μL; (3) Mass spectrometry conditions: Electrospray ionization source, positive ion mode detection, nebulizer gas pressure 50 psi, auxiliary gas pressure 50 psi, curtain gas pressure 20 psi, monitoring mode selected ion monitoring, where the monitoring ion of ginsenoside Rg1 is m / z 823.7 [M+Na] + The monitoring ion of ginsenoside Re is m / z969.9[M+Na] + The monitoring ion of ginsenoside Rb1 is m / z1132.4[M+Na] + .

2. The detection method according to claim 1, wherein The weight replenishment described in step (1) refers to replenishing the lost weight with chromatographically pure methanol.

3. The detection method according to claim 1, wherein The preparation of the test solution in step (1) further includes: taking 1 mL of the supernatant and placing it in a 10 mL volumetric flask, adding methanol to the mark and then filtering.

4. The detection method according to claim 1, wherein The initial ratio of the mobile phase for the gradient elution in step (2) is A:B=70:

30.

5. The detection method according to claim 1, wherein The response value of the selected ion monitoring in step (3) is a linear relationship between the peak area and the mass concentration.

6. The detection method according to claim 5, characterized in that The linear range of ginsenoside Rg1 is 0.05~2.0μg·mL -1 The linear range of ginsenoside Re is 0.05~2.0μg·mL -1 The linear range of ginsenoside Rb1 was 0.1~4.0μg·mL -1 .

7. The detection method according to claim 6, characterized in that The detection limit of ginsenoside Rg1 was 0.024 μg·mL -1 The limit of quantification was 0.081 μg·mL -1 The detection limit of ginsenoside Re was 0.065 μg·mL -1 The limit of quantification was 0.219 μg·mL -1 The detection limit of ginsenoside Rb1 was 0.002 μg·mL -1 The limit of quantification was 0.007 μg·mL -1 .

8. The detection method according to claim 1, wherein The preparation of reference solution is also included: 5 mg of ginsenoside Rg1, Re, and Rb1 reference substances are accurately weighed and placed in a 5 mL volumetric flask. Methanol is added to the mark and shaken to prepare a concentration of 1 mg mL -1 A single reference solution.

9. The detection method according to claim 1, wherein In the precision test, the concentrations of ginsenosides Rg1, Re, and Rb1 in the mixed reference solution were 0.2 μg·mL -1 , 0.2 μg·mL -1 , 0.4 μg·mL -1 .

10. The detection method according to claim 1, characterized in that In the stability test, the test solution was measured 0, 2, 4, 8, 12, and 24 hours after preparation.

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