Method for detecting content of multiple components in herba inulae granules for clearing arthralgia

Abstract

The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly discloses a method for detecting the content of multiple components in herba inulae and caulis lonicerae particles for clearing arthralgia. According to the content detection method, the content of 11 components including sinomenine, neochlorogenic acid, magnoflorine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide and ammonium glycyrrhizinate in the preparation is determined by adopting an HPLC (High Performance Liquid Chromatography) multi-wavelength switching method. The quality detection method disclosed by the invention is stable, reliable, high in specificity and good in reproducibility, can be used for comprehensively and effectively controlling the quality of the herba inulae arthralgia clearing granules, and is beneficial to stabilizing the product quality and ensuring the safety and effectiveness of clinical medication, so that the requirements of medical treatment and markets are better met.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of multiple components of Jinteng Qingbi granules, and in particular to a method for detecting the contents of 11 components of Jinteng Qingbi granules based on HPLC, belonging to the field of analysis of traditional Chinese medicine preparations. Background Art

[0002] Jinteng Qingbi Granules (National Medicine Standard Z20123065, produced by Lunan Houpu Pharmaceutical Co., Ltd.) are prepared from 11 Chinese medicinal herbs including honeysuckle, caulis spatholobi, herba scutellariae, radix rehmanniae, rhizoma scutellariae, herba scutellariae, licorice, angelica, white peony root, and centipede. The main effects are clearing away heat and detoxifying, promoting blood circulation and reducing swelling, and relieving pain. It is used for the active stage of rheumatoid arthritis, which belongs to the syndrome of internal toxic heat and damp-heat blocking the collaterals. Symptoms include swollen joints, pain that refuses to be pressed, morning stiffness, fever when touched, or red skin, fever, sweating, thirst, dry stool and yellow urine, red tongue, yellow and greasy fur, and slippery and rapid pulse.

[0003] The whole formula of Jinteng Qingbi Granules uses honeysuckle and sinomenine as the main drugs; Hedyotis diffusa, Trillium, Herba Lycopodii, Angelica sinensis, and Paeonia lactiflora as the assistant drugs; Scrophularia ningpoensis, Rehmannia glutinosa, and Licorice as the adjuvant drugs; and Centipede as the messenger drug. The drugs are matched to have the effects of clearing away heat and detoxifying, promoting blood circulation and reducing swelling, and relieving numbness and pain. Honeysuckle is rich in volatile oils, flavonoids, and organic acids. Experimental research results show that its decoction, oral solution, and injection have the effects of antipyretic, anti-inflammatory, and regulating the body's immune function. Sinomenine mainly contains active ingredients such as sinomenine and magnolia alkaloids, which have anti-inflammatory, analgesic, anti-rheumatic, and immunosuppressive effects. It is a commonly used Chinese medicine for the treatment of rheumatoid arthritis. Angelica, white peony root, Hedyotis diffusa, and Herba Deerbearingae can nourish blood and invigorate blood circulation, relieve acute pain, and strengthen tendons and bones; Scrophularia, Dry Rehmannia root, Licorice, and Tripterygium wilfordii can nourish yin and cool blood, and purge fire and detoxify; Centipede can invigorate blood circulation and eliminate blood stasis, attack toxins and dissipate stagnation, and dredge meridians and relieve pain. Pharmacological studies have shown that centipede can improve microcirculation and has strong analgesic and anti-inflammatory effects. All the medicines are used together to achieve the effects of clearing away heat and detoxification, invigorating blood circulation, eliminating blood stasis and swelling, and dredge meridians and relieve pain.

[0004] Jinteng Qingbi Granules are a compound Chinese medicine preparation containing multiple Chinese medicines with complex chemical components. Comprehensive and effective quality control is an important basis for ensuring clinical efficacy, but the quality control methods of Jinteng Qingbi Granules are rarely reported. At present, the analysis of Jinteng Qingbi Granules is mainly based on the quantitative analysis of single components of medicinal materials and the qualitative identification of some formula medicinal materials. Its active ingredients are still unclear, and there is a lack of more comprehensive and comprehensive quality control research. In order to ensure product quality, it is necessary to conduct a comprehensive study on its quality detection method.

[0005] The present invention discloses a HPLC wavelength switching method for quantitatively analyzing 11 components of Jinteng Qingbi granules, and comprehensively evaluating and controlling the quality of Jinteng Qingbi granules, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication. HPLC was used to simultaneously determine the 11 components in the Jinteng Qingbi granules preparation. The test results showed that the method has the characteristics of high separation efficiency, while saving analysis costs, reducing the amount of solvent used, and being more environmentally friendly. The separation method established in the test provides a useful reference for improving the quality standard of Jinteng Qingbi granules. Summary of the invention

[0006] The technical problem to be solved by the present invention is to further optimize the detection method on the basis of the existing technology, so as to realize the simultaneous determination of the contents of 11 ingredients in the five medicinal ingredients of Caulis Atractylodes macrocephala, Honeysuckle, Licorice, Angelica sinensis and Scrophularia ningpoensis in Jinteng Qingbi Granules through a single injection, thereby solving the drawbacks of the existing technology that the quality control indicator components are single and one-sided, and simplifying the operation method and improving the detection efficiency.

[0007] Through long-term exploration and repeated experiments, the inventors successfully combined the HPLC content detection of 11 ingredients in the preparation, including sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpaoside, ligustilide, and ammonium glycyrrhizinate, into one, providing a new method for detecting Jinteng Qingbi Granules in a more comprehensive and accurate manner.

[0008] Among them, sinomenine and magnoliavine are components of Sinomenium sinomenium, six types of chlorogenic acid series are components of honeysuckle, harpaoside is a component of Scrophularia ningpoensis, ammonium glycyrrhizinate is a component of licorice, and ligustilide is a component of angelica.

[0009] The present invention adopts the HPLC wavelength switching method to simultaneously determine the contents of sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpaoside, ligustilide and ammonium glycyrrhizinate in the preparation, so as to achieve a comprehensive evaluation and control of the quality of Jinteng Qingbi Granules, thereby providing an accurate basis for the authenticity identification and intrinsic quality detection of Jinteng Qingbi Granules.

[0010] A method for detecting the content of multiple components of Jinteng Qingbi granules, specifically comprising the following steps:

[0011] A. Preparation of test solution: Take Jinteng Qingbi granules, grind them into powder, weigh them accurately, put them into a volumetric flask, add an appropriate amount of 20%-50% methanol solution, treat them with ultrasound, take them out, cool them, add 20%-50% methanol solution to the same volume, shake them well, filter them, and use them as the test solution;

[0012] B. Preparation of reference solution: accurately weigh appropriate amount of sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate reference substances, place in a volumetric flask, add 20%-50% methanol solution to dissolve, make up to volume, shake well, and obtain;

[0013] C. HPLC chromatographic conditions: Chromatographic column is C 18 Chromatographic column, methanol: acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B, gradient elution; column temperature: 20-30°C; detection wavelength: wavelength switching 218-326nm;

[0014] D. Determination method: Accurately aspirate the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in step C.

[0015] Preferably, in step A, 40% methanol solution is added for extraction, and ultrasonic treatment is performed for 30 minutes; ultrasonic conditions: 300W, 40kHz.

[0016] Step B: Add 40% methanol solution to dissolve.

[0017] The chromatographic column in step C is Waters CSHC18 chromatographic column, specification is 4.6×250mm, 5μm.

[0018] In step C, the mobile phase A is methanol:acetonitrile in a volume ratio of 1:1-3, preferably, the volume ratio of methanol:acetonitrile is 1:1; the mobile phase B is 0.05-0.5% phosphoric acid solution, preferably 0.2% phosphoric acid solution.

[0019] Preferably, the mobile phase gradient elution in step C is performed according to the following procedure:

[0020]

[0021] Preferably, the wavelength switching gradient table in step C is:

[0022]

[0023] Preferably, the detection column temperature in step C is 25° C. and the sample injection volume is 5 μL.

[0024] Compared with the prior art, the method for detecting the multi-component content of Jinteng Qingbi granules of the present invention has achieved the following beneficial technical effects:

[0025] 1) The present invention establishes an upgraded quality detection method for Jinteng Qingbi Granules, and adopts the HPLC wavelength switching method to quantitatively determine the contents of 11 ingredients in the preparation, including sinomenine, neochlorogenic acid, magnolia pine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpaoside, ligustilide, and ammonium glycyrrhizinate, which can further effectively control the quality of Jinteng Qingbi Granules and achieve a comprehensive evaluation of the preparation quality.

[0026] 2) The present invention simultaneously detects 11 components in Jinteng Qingbi Granules through wavelength switching HPLC, thereby achieving synchronous determination of multiple components, simplifying the operation method, improving detection efficiency, saving consumables, and saving labor costs.

[0027] 3) The present invention successfully combines the HPLC content detection of 11 components in the preparation, namely, sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpaoside, ligustilide, and ammonium glycyrrhizinate, through a gradient elution procedure, providing a new method for detecting and evaluating the stability of Jinteng Qingbi Granules in a more comprehensive and accurate manner.

[0028] 4) Compared with the existing quality detection method of Jinteng Qingbi Granules, the method of the present invention has the advantages of being rapid, sensitive, and repeatable. It can accurately determine the 11 components in Jinteng Qingbi Granules, has strong specificity and good reproducibility, and can comprehensively and effectively control the quality of Jinteng Qingbi Granules, which is beneficial to stabilizing the quality of the product, ensuring the safety and effectiveness of clinical medication, and at the same time better meeting medical and market needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the HPLC chromatogram of the mixed reference substance;

[0030] Figure 2 This is the HPLC chromatogram of the Jinteng Qingbi granules test sample;

[0031] Figure 3 It is a negative HPLC chromatogram lacking Sinomenium sinomenium;

[0032] Figure 4 This is the negative control HPLC chromatogram lacking honeysuckle;

[0033] Figure 5 This is the HPLC chromatogram of the negative control lacking licorice;

[0034] Figure 6 This is the negative control HPLC chromatogram lacking Angelica sinensis;

[0035] Figure 7 is the negative control HPLC chromatogram lacking Scrophularia ningpoensis;

[0036] Note: The chromatographic peaks 1-11 in the attached figure represent sinomenine, neochlorogenic acid, magnoliavine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate, respectively. DETAILED DESCRIPTION

[0037] In order to facilitate a full understanding of the implementation of the present invention, the present invention is further described below through typical implementation cases. At the same time, since the following examples are only preferred implementations of the present invention, their descriptions are relatively specific and detailed, so they cannot be understood as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles and concepts of the present invention, several modifications and improvements can also be made, which should also belong to the scope of protection of the present invention.

[0038] Example 1 Study on HPLC Determination Methodology of 11 Components of Jinteng Qingbi Granules

[0039] 1. Drugs and reagents

[0040] 1.1 Reference substances and reagents:

[0041] Table 1 Reference Material Information

[0042]

[0043]

[0044] Methanol and acetonitrile were of chromatographic grade, water was double distilled water, and the other reagents were of analytical grade.

[0045] 1.2 Drug testing

[0046] Jinteng Qingbi Granules were provided by Lunan Houpu Pharmaceutical Co., Ltd. The sample batch number is shown in Table 1.

[0047] Table 2 Batch number of Jinteng Qingbi granules test samples

[0048]

[0049] 2 Instrument: Thermo Vanquish ultra-high performance liquid chromatograph (USA): diode array detector;

[0050] 3Chromatographic conditions: Column: Waters CSH C18 chromatographic column (4.6x150mm, 2.5μm); methanol:acetonitrile (1:1) as mobile phase A, 0.2% phosphoric acid as mobile phase B for gradient elution; column temperature: 25°C; detection wavelength: wavelength switching 218-326nm; injection volume 5μL.

[0051] Gradient elution schedule:

[0052]

[0053] Wavelength switching schedule:

[0054]

[0055] 4. Investigation of content detection methodology

[0056] 4.1 Preparation of test solution

[0057] Take Jinteng Qingbi granules, grind them into powder, take about 1g, weigh accurately, put into a 25mL volumetric flask, add an appropriate amount of 40% methanol solution, ultrasonically treat (300W, 40kHz) for 30 minutes, take out, cool, add 40% methanol solution to the scale, shake well, filter, and use as the test solution;

[0058] 4.2 Preparation of mixed reference solution

[0059] Accurately weigh appropriate amounts of sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpaoside, ligustilide, and ammonium glycyrrhizinate reference substances, and add 40% methanol to make a mixed solution containing 0.1436 mg, 0.0771 mg, 0.0918 mg, 0.1071 mg, 0.1288 mg, 0.0520 mg, 0.0518 mg, 0.0540 mg, 0.0262 mg, 0.0631 mg, and 0.1113 mg respectively per 1 mL.

[0060] 4.3 Preparation of negative control solution

[0061] Take the negative control lacking Sinomenium sinomenium, the negative control lacking honeysuckle, the negative control lacking angelica, the negative control lacking liquorice, and the negative control lacking Scrophularia ningpoensis respectively, and follow the same method as in item "4.1" to prepare negative control solutions. Figure 3-7 It can be seen that there is no interference at the positions of sinomenine, neochlorogenic acid, magnolia pine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate.

[0062] 4.4 Investigation of linear relationship

[0063] Accurately pipette the mixed reference solution under "4.2", add 40% methanol to the following concentrations, measure the peak area under the above chromatographic conditions, and inject 5 μL of each. Use the measured peak area as the ordinate and the reference concentration (μg / mL) as the abscissa to draw the standard curves of the 11 components, and obtain the regression equation and determination coefficient (R 2) and linear range, the results are shown in Table 3. It shows that sinomenine, neochlorogenic acid, magnolia pine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate have a good linear relationship between concentration and peak area within the linear range.

[0064] Table 3 Results of linear relationship investigation of 11 components of Jinteng Qingbi granules (n=6)

[0065]

[0066]

[0067]

[0068] 4.5 Precision test

[0069] Accurately pipette the mixed reference solution under "4.2", repeat the injection 6 times, 5 μL each time, and record the retention time and peak area of ​​each chromatographic peak. The results show that the RSD (n=6) of the retention time and peak area of ​​each component is less than 2.0%, indicating that the instrument has good precision. The results are shown in Tables 4 and 5.

[0070] Table 4 Precision test results (retention time)

[0071]

[0072] Table 5 Precision test results (peak area)

[0073]

[0074] 4.6 Stability test

[0075] Take the same batch number S0 (28200081) and prepare the test solution according to the method under "4.1". Measure at 0, 3, 6, 9, 12, and 24 hours after preparation and record the peak area. The RSD (n=6) of the peak area of ​​each component is less than 2.0%. The results show that the sample is stable within 24 hours. The results are shown in Table 6.

[0076] Table 6 Stability test results

[0077]

[0078] 4.7 Repeatability test

[0079] Take the same batch number S0 (28200081), prepare the test solution according to the method under "4.1", prepare 6 copies in parallel, and analyze them separately. The average contents of sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate in the samples are calculated to be 2.38, 1.34, 1.83, 1.69, 2.83, 0.73, 0.42, 1.00, 0.13, 0.98, and 1.17 mg / g, respectively, and their RSD (n=6) are all less than 2.0%. This shows that the method has good repeatability. The results are shown in Table 7.

[0080] Table 7 Repeatability test results

[0081]

[0082]

[0083]

[0084] 4.8 Recovery test

[0085] Take Jinteng Qingbi Granules (batch number: S0 (28200081), the contents of sinomenine, neochlorogenic acid, magnolia alkaloids, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate in the sample are 2.38, 1.34, 1.83, 1.69, 2.83, 0.73, 0.42, 1.00, 0.13, 0.98, and 1.17 mg / g, respectively. Grind it into powder, take about 0.5 g, weigh it accurately, put it in a 25 mL volumetric flask, add appropriate amounts of sinomenine, neochlorogenic acid, Acid, magnolia alkaloids, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, ammonium glycyrrhizinate reference substances were prepared in parallel according to the test solution preparation method under "4.1", and the samples were injected and analyzed respectively. The average recovery rates of sinomenine, neochlorogenic acid, magnolia alkaloids, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate were calculated to be 97%-101%, and their RSDs were all less than 3.0%, indicating that the method has high accuracy. The results are shown in Table 8.

[0086] Table 8 Recovery test results

[0087]

[0088]

[0089]

[0090] 4.9 Content detection

[0091] Take 20 batches of Jinteng Qingbi Granules (batch numbers see Table 2), grind to about 1 g, weigh accurately, prepare the test solution according to the method under "4.1", inject and analyze separately, calculate the content of 11 components in 20 batches of Jinteng Qingbi Granules according to the regression equation in "Table 3", and the results are shown in Table 9.

[0092] Table 9 Contents of 11 ingredients in batch 20 of Jinteng Qingbi Granules

[0093]

[0094]

Claims

1. A method for detecting the content of multiple components of Jinteng Qingbi granules, characterized in that: High performance liquid chromatography was used with methanol-acetonitrile-phosphoric acid solution as the mobile phase and wavelength switching method was used to simultaneously determine the contents of sinomenine, neochlorogenic acid, magnoliavine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide and ammonium glycyrrhizinate in the preparation.

2. The method according to claim 1, characterized in that The following steps are involved: A. Preparation of test solution: Take Jinteng Qingbi granules, grind them into powder, weigh them accurately, put them into a volumetric flask, add an appropriate amount of 20%-50% methanol solution, treat them with ultrasound, take them out, cool them, add 20%-50% methanol solution to the same volume, shake them well, filter them, and use them as the test solution; B. Preparation of reference solution: accurately weigh appropriate amount of sinomenine, neochlorogenic acid, magnolamine, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizinate reference substances, place in a volumetric flask, add 20%-50% methanol solution to dissolve, make up to volume, shake well, and obtain; C. HPLC chromatographic conditions: Chromatographic column is C 18 Chromatographic column, methanol: acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B, gradient elution; column temperature: 20-30°C; detection wavelength: wavelength switching 218-326nm; D. Determination method: Accurately aspirate the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in step C.

3. The method according to claim 2, characterized in that Step A: Add 40% methanol solution for extraction and perform ultrasonic treatment for 30 minutes.

4. The method according to claim 2, characterized in that: Step B: Add 40% methanol solution to dissolve.

5. The method according to claim 2, characterized in that: The chromatographic column in step C is Waters CSHC18 chromatographic column, specification is 4.6×150mm, 2.5μm.

6. The method according to claim 2, characterized in that In step C, the volume ratio of mobile phase A methanol:acetonitrile is 1:1-3, and mobile phase B is 0.05-0.5% phosphoric acid solution.

7. The method according to claim 6, characterized in that In step C, the mobile phase A is methanol:acetonitrile in a volume ratio of 1:1, and the mobile phase B is 0.2% phosphoric acid solution.

8. The method according to claim 2, characterized in that: The mobile phase gradient elution step in step C is carried out according to the following conditions:

9. The method according to claim 2, characterized in that: The gradient table of wavelength switching in step C is:

10. The method according to claim 2, characterized in that The detection column temperature in step C is 25° C. and the sample injection volume is 5 μL.

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