Determination method for the contents of 8 components in Xiongju Shangqing tablets

Abstract

The present invention provides a method for simultaneously determining the contents of 8 components in Xiongju Shangqing Tablets. The HPLC method is used in the present invention to determine neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 1,5-O-dicaffeoylquinic acid. The HPLC method is adopted in the present invention to establish a method for simultaneously determining the contents of 8 components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 1,5-O-dicaffeoylquinic acid in Xiongju Shangqing Tablets. The method is simple and highly specific, providing a basis for the establishment of the quality standard of Xiongju Shangqing Tablets. The established method can play a role in process control to a certain extent and provide a reference for the quality control and quality evaluation of the product.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for determining the contents of 8 components in Xiongju Shangqing Tablets. Background of the Invention

[0002] Xiongju Shangqing Tablets are included in the Chinese Pharmacopoeia (2020 Edition), and are composed of 15 traditional Chinese medicines such as Ligusticum chuanxiong, Chrysanthemum morifolium, Scutellaria baicalensis, Gardenia jasminoides, Fructus Viticis stir-fried, Coptis chinensis, etc. In the formula, Chrysanthemum morifolium and Ligusticum chuanxiong are used together to clear heat and relieve the exterior, promote qi and activate blood circulation, dispel wind and relieve pain, and they are the monarch drugs; Forsythia suspensa, Mentha haplocalyx, and Fructus Viticis disperse wind-heat, clear the head and eyes, and dispel wind and relieve pain. Scutellaria baicalensis, Gardenia jasminoides, and Coptis chinensis clear heat and purge fire, detoxify and relieve pain, and assist the monarch drugs to clear heat and relieve the exterior, and dispel wind and relieve pain, and they are the ministerial drugs; The whole formula together exerts the effects of clearing heat and relieving the exterior, and dispersing wind and relieving pain. The current quality standard only includes the thin-layer identification for Chrysanthemum morifolium, Gardenia jasminoides, and Coptis chinensis, as well as the content determination of baicalin in Scutellaria baicalensis, and there is no content determination item for the monarch drug Chrysanthemum morifolium.

[0003] Although there are currently studies on simultaneously determining multiple components in Xiongju Shangqing Tablets by HPLC method, most of the determined components are baicalin, berberine hydrochloride, etc., and there are no relevant reports on the content determination method specifically for multiple components in Chrysanthemum morifolium.

[0004] Through literature review and experimental research, it can be known that although the main components in Chrysanthemum morifolium medicinal materials include chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid, etc., and the contents of neochlorogenic acid, cryptochlorogenic acid, and 1,3-O-dicaffeoylquinic acid, etc. are relatively low, in Xiongju Shangqing Tablets, it is found that it also contains relatively high contents of neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, etc. The most important essence of the quality of traditional Chinese medicine is the content of effective components, and these effective substances will also undergo chemical reactions and mutual conversions under the action of physical, chemical and other factors during the processing of medicinal materials or the production of Chinese patent medicines. Through a series of studies, it is found that during the preparation process of Xiongju Shangqing Tablets, chemical components in Chrysanthemum morifolium will undergo mutual conversions. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for determining the contents of 8 components in Xiongju Shangqing Tablets simultaneously. The present invention uses HPLC method to determine neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid.

[0006] To solve the above problems, the present invention adopts the following technical solution: A method for determining the contents of 8 components in Xiongju Shangqing Tablets, which uses HPLC method to establish a method for simultaneously determining the contents of 8 components in Xiongju Shangqing Tablets, and is characterized in that it includes the following steps:

[0007] a. Prepare a mixed reference solution;

[0008] b. Prepare a test solution;

[0009] c. Assay method: Accurately pipette 10 μl each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, determine the content by the external standard method, and you can get it;

[0010] Chromatographic conditions:

[0011] Chromatographic column: PICKERING C18 column (4.6×250 mm, 5 μm),

[0012] Column temperature: 30 °C,

[0013] Flow rate: 1 mL·min –1 ,

[0014] Detection wavelength: 328 nm,

[0015] Sample injection volume: 10 μl,

[0016] Use acetonitrile as mobile phase A and 0.4% phosphoric acid as mobile phase B. The gradient elution program is shown in the following table.

[0017] Mobile phase gradient elution program

[0018]

[0019] Preferably, in step a, the preparation of the mixed reference solution: Weigh the reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid, and add 70% methanol to prepare a mixed reference solution with mass concentrations of 20 μg·mL- 1 , 30 μg·mL -1 , 20 μg·mL -1 , 20 μg·mL -1 , 20 μg·mL -1 , 30 μg·mL -1 , 30 μg·mL -1 , 10 μg·mL -1 respectively.

[0020] Preferably, in step b, the preparation of the test solution: Take 20 tablets of Xiongju Shangqing Tablets, remove the coating, weigh, grind them finely, take 0.6 g, weigh, place it in a stoppered conical flask, add 50 mL of 70% methanol, stopper tightly, weigh, ultrasonically treat for 40 minutes, take it out, let it cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate to obtain it.

[0021] The beneficial effects of the present invention are as follows:

[0022] The present invention uses the HPLC method to establish a method for simultaneously determining the contents of 8 components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid in Xiongju Shangqing Tablets. The method is simple and highly specific, providing a basis for the establishment of the quality standard of Xiongju Shangqing Tablets. The established method can play a role in process control to a certain extent and provide a reference for the quality control and quality evaluation of the product. Description of the Drawings

[0023] Figure 1 It is the HPLC chromatogram of the mixed reference substances of each component;

[0024] Figure 2 It is the HPLC chromatogram of the Xiongju Shangqing Tablet sample of each component;

[0025] Figure 3 It is the HPLC chromatogram of the negative sample of each component;

[0026] Among them: 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Cryptochlorogenic acid; 4. 1,3-O-dicaffeoylquinic acid; 5. Luteoloside; 6. 3,4-O-dicaffeoylquinic acid; 7. 3,5-O-dicaffeoylquinic acid; 8. 1,5-O-dicaffeoylquinic acid. Detailed Embodiments

[0027] The following further elaborates on the present application in combination with the embodiments. It can be understood that the specific embodiments described herein are only used to explain the relevant invention and do not limit the invention.

[0028] Embodiment

[0029] 1. Instruments and Reagents

[0030] 1.1 Instruments

[0031] High-performance liquid chromatograph equipped with a quaternary pump and a DAD detector (Thermo, USA); KQ-400KDE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Mettler XPE26 electronic balance (Mettler, Switzerland, 0.001 mg); Mettler XS105 electronic balance (Mettler, Switzerland, 0.01 mg); Milli-Q ultrapure water purification system (Millipore, USA).

[0032] 1.2 Reagents

[0033] Acetonitrile was of chromatographic purity, purchased from Sigma Company, USA; water was ultrapure water, self-made; other reagents were all of analytical purity; chlorogenic acid reference substance (batch number: 110753 - 202119, content calculated as 96.3%), 1,3 - O - dicaffeoylquinic acid reference substance (batch number: 111717 - 201402, content calculated as 94.5%), luteoloside reference substance (batch number: 111720 - 202111, content calculated as 96.6%), 3,5 - O - dicaffeoylquinic acid reference substance (batch number: 111782 - 202208, content calculated as 95.9%), the above 4 kinds of reference substances were purchased from the National Institutes for Food and Drug Control; neochlorogenic acid reference substance (batch number: DSTDX001504, content ≥ 98%), cryptochlorogenic acid reference substance (batch number: DSTDY003501, content ≥ 98%), 3,4 - O - dicaffeoylquinic acid reference substance (batch number: DSTDY003703, content ≥ 98%), 1,5 - O - dicaffeoylquinic acid reference substance (batch number: DSTDE000602, content ≥ 98%), the above 4 kinds of reference substances were purchased from Lemeitian Pharmaceutical DST Biotechnology Co., Ltd.; samples of Xiongju Shangqing Tablets were all purchased from pharmacies, and the detailed information is shown in Table 1.

[0034] Table 1 Information of 8 batches of Xiongju Shangqing Tablet samples

[0035]

[0036] 2. Methods and results

[0037] 2.1 Chromatographic conditions

[0038] Chromatographic column: PICKERING C18 column (4.6×250 mm, 5 μm); acetonitrile was used as mobile phase A, 0.4% phosphoric acid was used as mobile phase B, and the gradient elution program is shown in Table 2; column temperature: 30 °C, flow rate: 1 mL·min -1 ; Detection wavelength: 328 nm; injection volume: 10 μl.

[0039] Table 2 Mobile phase gradient elution program

[0040]

[0041] Precisely pipette 10 μl of the mixed reference substance solution and the test solution respectively, inject them into the liquid chromatograph, determine, and calculate the content by the external standard method to obtain.

[0042] 2.2 Preparation of solutions

[0043] 2.2.1 Mixed reference substance solution

[0044] Accurately weigh 5.003 mg of neochlorogenic acid, 7.509 mg of chlorogenic acid, 5.005 mg of cryptochlorogenic acid, 5.006 mg of 1,3-O-dicaffeoylquinic acid, 5.007 mg of luteoloside, 7.502 mg of 3,4-O-dicaffeoylquinic acid, 7.507 mg of 3,5-O-dicaffeoylquinic acid, and 3.003 mg of 1,5-O-dicaffeoylquinic acid and place them in the same 250-ml volumetric flask. Add 200 mL of 70% methanol, sonicate for 5 minutes to dissolve, take out, cool to room temperature, dilute to the mark with 70% methanol, and shake well to obtain the mixed reference substance solution.

[0045] 2.2.2 Preparation of the test solution

[0046] Take 20 tablets of Xiongju Shangqing Tablets (batch number 220301), remove the coating, accurately weigh, grind finely, accurately weigh 0.6008 g (equivalent to the amount of about two tablets), place in a stoppered conical flask, accurately add 50 mL of 70% methanol, tightly stopper, weigh, sonicate for 40 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the continuous filtrate to obtain the solution.

[0047] 2.2.3 Preparation of the negative sample solution

[0048] According to the prescription ratio and preparation process of Xiongju Shangqing Tablets, prepare a negative sample without chrysanthemum, and prepare the negative sample solution according to the method under "test solution".

[0049] 2.3 Specificity test

[0050] Respectively take the mixed reference substance solution, the test solution and the negative sample solution, and according to the chromatographic conditions under "2.1", inject 10 μl respectively for analysis, record the chromatogram, see the attached figure. In the sample chromatogram, chromatographic peaks with retention times consistent with those of the reference substances neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid are respectively shown. There are no corresponding chromatographic peaks in the negative sample chromatogram, indicating that other medicinal flavors except chrysanthemum do not interfere with the determination of the components to be measured, and the method has good specificity.

[0051] 2.4 Investigation of linear relationship

[0052] Prepare solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid with concentrations of 0.3988, 0.5902, 0.3828, 0.4278, 0.4128, 0.6418, 0.7752, and 0.2016 mg·mL in turn with 70% methanol solution-1 For the mixed reference substance stock solution, accurately pipette 0.5, 1, 5, 10, 20, and 50 mL respectively into 100-mL volumetric flasks, add 70% methanol to volume to the mark, shake well, and thus obtain a series of mixed reference substance solutions with different concentrations. Accurately pipette 10 μL of each of the above series of reference substance solutions and inject them into the high-performance liquid chromatograph respectively, and carry out sample injection and determination according to the chromatographic conditions under item "2.1", and record the peak areas. Using the injection amount of the reference substance (μg) as the abscissa (x) and the integrated peak area value as the ordinate (y), plot the standard curve. The results are shown in Table 3, indicating that the linear relationships of each component are good within their respective linear ranges.

[0053] Table 3 Linear relationships of each component

[0054]

[0055] 2.5 Stability experiment

[0056] Accurately pipette 10 μL of each of the test solution prepared in item "2.2.2" and inject them into the high-performance liquid chromatograph respectively at 0, 2, 4, 8, 12, 18, and 24 h after preparation according to the chromatographic conditions under item "2.3.1", record the integrated peak area values of each component, calculate the RSD. The RSD values of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid are 0.2%, 0.2%, 0.2%, 0.3%, 0.9%, 0.5%, 0.7%, and 0.6% respectively. The results indicate that the test solution prepared in the experiment is stable within 24 h.

[0057] 2.6 Repeatability test

[0058] Take about 0.3, 0.6, and 0.9 g of the powder of Xiongju Shangqing Tablets (batch number 220301), take 3 portions of each, accurately weigh them, prepare the test solution according to the method under item "2.2.2", carry out sample injection and determination according to the chromatographic conditions under item "2.1", record the integrated peak area values of each component, calculate the contents and RSD of 8 index components. The results are shown in Table 4, indicating that the method has good repeatability.

[0059] Table 4 Results of repeatability test

[0060]

[0061] Note: Compounds 1-8 are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid in sequence.

[0062] 2.7 Recovery test

[0063] Take the powder of Xiongju Shangqing Tablets (batch number 220301), accurately weigh 9 portions, each portion being about 0.3 g. Divide every three portions into a group, and add 50 mL of mixed reference substance solutions with low, medium, and high concentrations prepared with 70% methanol solution respectively. Prepare the test sample solutions according to the method under "2.2.2.", inject samples for determination under the chromatographic conditions in "2.1.", calculate the recovery rate of sample addition. The results are shown in Table 5, indicating that the recovery rate of this method is good.

[0064] Table 5 Results of the recovery rate test of sample addition (n = 9)

[0065]

[0066]

[0067]

[0068]

[0069] 2.8 Determination of samples

[0070] Use the established method to determine 8 batches of collected Xiongju Shangqing Tablets samples, record the chromatograms, and calculate the contents of 8 index components. The results are shown in Table 6, indicating that there are certain differences in the contents of the components to be measured in samples of different batches. The established method has certain significance for the quality control of Xiongju Shangqing Tablets.

[0071] Table 6 Contents of index components in Xiongju Shangqing Tablets (mg·tablet -1 , n = 2)

[0072]

[0073] Note: Compounds 1 - 8 are in sequence: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid

[0074] The above description is only the preferred embodiments of this application and the explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for determining the contents of 8 components in Xiongju Shangqing Tablets. An HPLC method was used to establish a method for simultaneously determining the contents of 8 components in Xiongju Shangqing Tablets, which is characterized in that: It includes the following steps: a. Preparation of a mixed reference substance solution: Weigh reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, luteoloside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid, and add 70% methanol to prepare a mixed reference substance solution with mass concentrations of 20 μg·mL -1 , 30 μg·mL -1 , 20 μg·mL -1 , 20 μg·mL -1 , 20 μg·mL -1 , 30 μg·mL -1 , 30 μg·mL -1 , 10 μg·mL -1 respectively; b. Preparation of the test solution: Take 20 Xiongju Shangqing Tablets, remove the coating, weigh, grind finely, take 0.6 g, weigh, place in a stoppered conical flask, add 50 mL of 70% methanol, stopper tightly, weigh, ultrasonically treat for 40 minutes, take out, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution; c. Determination method: Precisely pipette 10 μl each of the mixed reference solution and the test solution, inject into the liquid chromatograph, determine, and calculate the content by the external standard method to obtain the result; Chromatographic conditions: Chromatographic column: PICKERING C18 column 4.6×250 mm, 5 μm, Column temperature: 30 °C, Flow rate: 1 mL·min –1 , Detection wavelength: 328 nm, Sample injection volume: 10 μl, Using acetonitrile as mobile phase A and 0.4% phosphoric acid as mobile phase B, the gradient elution program is as follows,

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