A method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture

Abstract

The invention discloses a method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture, belongs to the technical field of drug detection, adopts high performance liquid chromatography to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture, the detection solution comprises a test solution, a reference solution and a Polygonum hydropiper negative solution, and the chromatographic conditions of the high performance liquid chromatography are limited: octadecylsilane bonded silica gel is used as a filler, acetonitrile solution is used as a mobile phase A, trifluoroacetic acid solution is used as a mobile phase B, gradient elution is performed, and the gradient elution time is 60 minutes. The content of Polygonum hydropiper can be accurately detected, and the detection method has high sensitivity and good separation.

Description

Technical Field

[0001] The invention relates to a method for detecting chemical substances, in particular to a method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture, and belongs to the technical field of drug detection. Background Art

[0002] Fengliao Changweikang Mixture has the effect of clearing away heat, removing dampness and removing stagnation. It is used for acute gastroenteritis, which belongs to the type of diarrhea caused by indigestion and the type of diarrhea caused by damp heat, with symptoms such as abdominal pain and diarrhea, foul-smelling diarrhea, nausea and vomiting, or fever, aversion to cold, yellow tongue coating, and rapid pulse. It can also be used for patients with stomach pain caused by food stagnation and symptoms such as epigastric pain, refusal to press, aversion to food, vomiting, belching, acid regurgitation, thick and greasy tongue coating or yellow and greasy tongue coating, and slippery and rapid pulse. The main ingredients of Fengliao Changweikang Mixture are Acer truncatum and Polygonum hydropiper. Polygonum hydropiper is the dried whole herb of Polygonum hydropiper or Polygonum hydropiper of the Polygonaceae family. It is dug when the flowers bloom in summer and autumn, and the impurities are removed and dried in the sun.

[0003] Polygonum hydropiper is an annual herb, 0.5-2.5m high, with an erect, branched stem, with many purple-red spots on the surface, covered with cotton wool, and slightly swollen nodes. The panicle is terminal or axillary, 2-6 cm long; the flowers are small, green-white or pink, densely grown; the perianth is 4-5-lobed, with veins and no glandular dots; the stamens are usually 6, the ovary is oval, flat, slightly concave in the middle of both sides, brown-black and shiny, and wrapped in the persistent perianth. The leaves are alternate, with short petioles or almost sessile; the leaves are lanceolate, with acuminate tips, cuneate bases, entire or wavy, dark green above, covered with sparse hairs, and densely covered with gray-white hairs below; the sheath is membranous and tubular. The extract of Polygonum hydropiper has a good inhibitory effect on a variety of pathogenic microorganisms such as Shigella dysenteriae, diphtheria, Salmonella typhimurium, Bacillus aeruginosa, Proteus, Escherichia coli, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Bacillus octaciformis. This is also an important reason why Polygonum hydropiper is used as a Chinese herbal medicine.

[0004] In the prior art, the quality of Fengliao Changweikang mixture is determined by the determination of rutin content, and both the Chinese yew and Polygonum hydropiper in the prescription of Fengliao Changweikang mixture contain rutin, which interferes with each other, resulting in the inability to effectively monitor the amount of Polygonum hydropiper. Therefore, it is necessary to conduct a separate content test on Polygonum hydropiper in Fengliao Changweikang mixture. At present, various detection methods for Polygonum hydropiper are still affected by interference from other substances, such as interference from rutin, which is also a component in Chinese yew, resulting in the inability to accurately and sensitively detect the content of Polygonum hydropiper in Fengliao Changweikang mixture. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture, which solves the interference problem in the process of determining the content of Polygonum hydropiper in Fengliao Changweikang mixture, and adopts high performance liquid chromatography to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture. The method of the present invention can accurately detect the content of Polygonum hydropiper in Fengliao Changweikang mixture, and has high sensitivity and good separation degree.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture comprises the following steps: high performance liquid chromatography is used to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture; a detection solution comprises a test solution, a reference solution and a Polygonum hydropiper negative solution; and detection conditions comprise the following chromatographic conditions: octadecylsilane bonded silica gel is used as a filler, acetonitrile solution is used as a mobile phase A, trifluoroacetic acid solution is used as a mobile phase B, a detection wavelength is 340 nm-360 nm, a column temperature is 30° C., gradient elution is performed, and the gradient elution time is 60 min.

[0008] Further, the gradient elution program was 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B.

[0009] Furthermore, the mobile phase B is a 0.1% trifluoroacetic acid solution.

[0010] Furthermore, the detection wavelength was 350 nm and the injection volume was 10 μl.

[0011] Further, the chromatographic column is ACE Excel 5C18-PFP, 4.6 mm×25 cm, 5 μm or ACE Excel 5C18-AR, 4.6 mm×25 cm, 5 μm or Hypersil GOLDTM PFP, 4.6 mm×25 cm, 5 μm.

[0012] Furthermore, the preparation of the negative solution of Polygonum hydropiper: accurately measure 10 ml of the negative mixture of Polygonum hydropiper, put it in a 25 ml volumetric flask, add solvent to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the negative solution of Polygonum hydropiper.

[0013] Furthermore, the preparation of the test solution: accurately measure 10 ml of Fengliao Changweikang mixture, put it into a 25 ml volumetric flask, add solvent to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution.

[0014] Furthermore, the reference solution is prepared by accurately weighing an appropriate amount of quercetin reference substance, dissolving it in a solvent and diluting it to prepare a solution containing about 20 μg of quercetin per 1 ml, thereby obtaining the reference solution.

[0015] Furthermore, the following detection operation steps are included:

[0016] S1. Take a blank solvent for testing and obtain a blank solvent chromatogram;

[0017] S2. Take the negative solution of Polygonum hydropiper for detection to obtain a chromatogram of the negative solution of Polygonum hydropiper;

[0018] S3. Take the reference solution for testing and obtain a chromatogram of the reference solution;

[0019] S4. Take the test solution for testing and obtain a chromatogram of the test solution;

[0020] S5. Calculate the content of the main component of Polygonum hydropiper in the test sample through the chromatogram data of the reference solution chromatogram and the test solution chromatogram.

[0021] Furthermore, the solvent is a methanol solution.

[0022] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0023] The method of the invention can accurately detect the content of Polygonum hydropiper in Fengliao Changweikang mixture, and has high sensitivity and good separation degree.

[0024] Firstly, the mobile phase A and B substances of the high performance liquid chromatography method can be combined to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture, with high sensitivity, baseline separation and good separation degree; if other mobile phase A and B substances are used for determination and analysis, the sensitivity is low, the chromatogram cannot be detected, and the method is not applicable.

[0025] Secondly, the present invention can better separate and detect the effective components of Polygonum hydropiper in the sample by limiting the gradient elution program in the spectral conditions, obtain the corresponding chromatogram, baseline separation, and good separation degree; if the gradient elution program of other chromatographic conditions is adopted, the effective substances in the sample cannot be well separated, and the corresponding chromatogram is detected, part of the baseline is not separated, and the separation degree is poor. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0027] Figure 1 is a chromatogram of the test solution of the present invention;

[0028] Figure 2 It is a chromatogram of the reference substance solution of the present invention;

[0029] Figure 3 is a chromatogram of a blank solvent of the present invention;

[0030] Figure 4 It is a chromatogram of the negative solution of Polygonum hydropiper of the present invention;

[0031] Figure 5 is a chromatogram of a reference solution in a systematic test of the present invention;

[0032] Figure 6 It is the superposition diagram of the chromatograms of each solution in the specificity test of the present invention;

[0033] Figure 7 It is a linear graph of the quercetin reference solution in the linearity and range test of the present invention;

[0034] Figure 8 is a chromatogram of the test solution in the intermediate precision test of the present invention;

[0035] Fig. 9 It is the chromatogram of the reference solution in the solution stability test of the present invention (standing for 12 hours);

[0036] Fig.10 It is the chromatogram of the test solution in the solution stability test of the present invention (standing for 12 hours);

[0037] Fig.11 is the chromatogram of 80% of the test solution in the accuracy test of the present invention;

[0038] Fig.12 is a chromatogram of a 100% test solution in the accuracy test of the present invention;

[0039] Fig.13 is a chromatogram of a 120% test solution in the accuracy test of the present invention;

[0040] In the accompanying drawings, Figures 1 to 6 , Figures 8 to 13 The vertical axis is mAU; the horizontal axis is time, min. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention and the related drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Below is a combination of the embodiments and Figures 1 to 13 The present invention is described in further detail:

[0043] A method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture, using high performance liquid chromatography to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture, the detection solution includes a test solution, a reference solution and a Polygonum hydropiper negative solution, and the detection conditions include the following chromatographic conditions:

[0044] Chromatographic column: Octadecylsilane bonded silica as filler; ACE Excel 5C18-PFP, 4.6mm×25cm, 5μm or ACE Excel 5C18-AR, 4.6mm×25cm, 5μm or Hypersil GOLDTM PFP, 4.6mm×25cm, 5μm or chromatographic column with equivalent performance;

[0045] Mobile phase A: acetonitrile solution;

[0046] Mobile phase B: trifluoroacetic acid solution;

[0047] Detection wavelength: 340nm-360nm;

[0048] Column temperature: 30°C;

[0049] Injection volume: 10 μl;

[0050] Gradient elution was performed with a gradient elution time of 60 min;

[0051] Elution program: 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B.

[0052] The specific steps for preparing the solution are as follows:

[0053] (1) Preparation of the negative solution of Polygonum hydropiper: Accurately measure 10 ml of the negative mixture of Polygonum hydropiper, place it in a 25 ml volumetric flask, add methanol solution to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the negative solution of Polygonum hydropiper.

[0054] (2) Preparation of test solution: Accurately measure 10 ml of Fengliao Changweikang mixture and place it in a 25 ml volumetric flask. Add methanol solution to dilute to the mark, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution.

[0055] (3) Preparation of reference solution: Accurately weigh an appropriate amount of quercetin reference substance, dissolve it in methanol solution and dilute it to make a solution containing about 20 μg of quercetin per 1 ml to obtain the reference solution.

[0056] The detection operation steps are as follows:

[0057] S1. Take a blank solvent and use high performance liquid chromatography to detect it to obtain a blank solvent chromatogram; the blank solvent is a methanol solution. Using a methanol solution as a blank solvent for detection can eliminate the interference of the methanol solution and obtain better chromatogram data.

[0058] S2. Take the negative solution of Polygonum hydropiper and use high performance liquid chromatography to detect it, and obtain a chromatogram of the negative solution of Polygonum hydropiper; using the negative solution of Polygonum hydropiper as an interfering solvent for detection can eliminate the interference of the negative solution of Polygonum hydropiper and obtain better chromatogram data.

[0059] S3. Take the reference solution and use high performance liquid chromatography to detect it, and obtain a chromatogram of the reference solution; use the quercetin reference solution for detection, and use the standard chromatogram as a reference to clearly compare the content of the test substance.

[0060] S4. Take the test sample solution and use high performance liquid chromatography to detect it, and obtain a chromatogram of the test sample solution; by eliminating the interference of the blank solvent and the interference of the Polygonum hydropiper negative solution, and using the quercetin reference solution as a reference standard chromatogram, the substances that need to be detected in the test sample and the content of the detected substances can be clearly detected and compared.

[0061] S5. Calculate the quercetin content of Polygonum hydropiper in the test sample through the chromatographic data of the reference solution chromatogram and the test solution chromatogram.

[0062] By accurately measuring the content of quercetin in the test sample, the content of Polygonum hydropiper in Fengliao Changweikang mixture can be deduced, avoiding the interference of other substances and accurately measuring the medicinal material of Polygonum hydropiper. Furthermore, in the production process of Fengliao Changweikang mixture, the feeding amount of Polygonum hydropiper medicinal material can be accurately controlled, realizing the practical significance of the determination of the content of Polygonum hydropiper.

[0063] Example 1

[0064] The specific detection process of this embodiment 1 is as follows:

[0065] 1. Instruments and Equipment

[0066] Table 1 Instrument and equipment information

[0067] Instruments and Equipment Device Model High performance liquid chromatography 1260Ⅱ Chromatographic columns ACE Excel 5C18-PFP, 4.6mm×25cm, 5μm Analytical Balance MSE3.6P-0CE-DM, XS105DU

[0068] 2. Reagents and consumables

[0069] Purified water: first-class water

[0070] Methanol: HPLC grade

[0071] Acetonitrile: HPLC grade

[0072] Trifluoroacetic acid: HPLC grade

[0073] 3. Test and reference substances

[0074] Table 2 Information of test and reference substances

[0075] name supplier Fengliao Changweikang Mixture Hainan Wanzhou Green Pharmaceutical Co., Ltd. Polygonum hydropiper Yin-yang mixture Hainan Wanzhou Green Pharmaceutical Co., Ltd. Polygonum hydropiper Hainan Wanzhou Green Pharmaceutical Co., Ltd. Quercetin China Food and Drug Inspection Institute

[0076] Note: Polygonum hydropiper has been identified as a medicinal material by the China Food and Drug Inspection Institute.

[0077] 4. Testing conditions

[0078] Table 3 Instrument detection parameter information

[0079]

[0080] 5. Solution preparation

[0081] (1) Preparation of the negative solution of Polygonum hydropiper: Accurately measure 10 ml of the negative mixture of Polygonum hydropiper, place it in a 25 ml volumetric flask, add methanol solution to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the negative solution of Polygonum hydropiper.

[0082] (2) Preparation of test solution: Accurately measure 10 ml of Fengliao Changweikang mixture and place it in a 25 ml volumetric flask. Add methanol solution to dilute to the mark, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution.

[0083] (3) Preparation of reference solution: Accurately weigh an appropriate amount of quercetin reference substance, dissolve and dilute with methanol solution to prepare a solution containing approximately 20 μg of quercetin per 1 ml. This is the reference solution.

[0084] 6. Test operation and test results

[0085] S1. Take 10 μL of methanol solution and detect it by high performance liquid chromatography to obtain a blank solvent chromatogram; using methanol solution as a blank solvent for detection can eliminate the interference of methanol solution and obtain better chromatogram data.

[0086] S2. Take 10 μL of the negative solution of Polygonum hydropiper and use high performance liquid chromatography to detect and obtain a chromatogram of the negative solution of Polygonum hydropiper. Using the negative solution of Polygonum hydropiper for detection can eliminate the interference of the negative solution of Polygonum hydropiper and obtain better chromatogram data.

[0087] S3. Take 10 μL of the reference solution and detect it by high performance liquid chromatography to obtain a chromatogram of the reference solution; use the quercetin reference solution for detection, and use the standard chromatogram as a reference to clearly calculate the content of the test substance.

[0088] S4. Take 10 μL of the test solution and detect it by high performance liquid chromatography to obtain a chromatogram of the test solution; by eliminating the interference of the blank solvent and the interference of the Polygonum hydropiper negative solution, and using the quercetin reference solution as a reference standard chromatogram, the substances to be detected can be clearly detected and compared, and the content of the detected substances can be calculated.

[0089] S5. Calculate the quercetin content of Polygonum hydropiper in the test sample through the chromatographic data of the reference solution chromatogram and the test solution chromatogram.

[0090] Test results: See Figures 1 to 4 It can be seen that the quercetin content contained in Polygonum hydropiper was detected in the test solution of Fengliao Changweikang mixture.

[0091] Example 2

[0092] The specific detection operation process is consistent with that of Example 1, except that the chromatographic column of Example 2 is different from the chromatographic column of Example 1.

[0093] Chromatographic column: octadecylsilane bonded silica as filler; ACE Excel 5C18-AR, 4.6mm×25cm, 5μm;

[0094] The remaining chromatographic conditions were consistent with those in Example 1.

[0095] The results showed that the negative solution of Polygonum hydropiper did not interfere with the determination of quercetin. The content of quercetin, the main component of Polygonum hydropiper, was accurately detected in the test solution of Fengliao Changweikang mixture.

[0096] Example 3

[0097] The specific detection operation process is consistent with that of Example 1, except that the chromatographic column of Example 3 is different from the chromatographic column of Example 1.

[0098] Chromatographic column: Octadecylsilane bonded silica as filler; Hypersil GOLDTM PFP, 4.6mm×25cm, 5μm;

[0099] The remaining chromatographic conditions were consistent with those in Example 1.

[0100] The results showed that the negative solution of Polygonum hydropiper did not interfere with the determination of quercetin. The content of quercetin, the main component of Polygonum hydropiper, was accurately detected in the test solution of Fengliao Changweikang mixture.

[0101] As can be seen from the above, both Example 1 and Example 3 can accurately detect the content of quercetin contained in Polygonum hydropiper from the test solution. Therefore, the method of the present invention can accurately determine the content of quercetin contained in Polygonum hydropiper in the test sample, thereby calculating the content of Polygonum hydropiper in Fengliao Changweikang mixture, avoiding the interference of other components, and accurately determining the Polygonum hydropiper medicinal material. Furthermore, the amount of Polygonum hydropiper fed can be accurately controlled during the production process of Fengliao Changweikang mixture, realizing the practical significance of the determination of the content of Polygonum hydropiper.

[0102] 7. Method Validation

[0103] 7.1 System suitability test

[0104] Acceptance criteria: (1) The resolution between the quercetin peak and adjacent peaks in both the test solution and the reference solution should be greater than 1.5. (2) The theoretical plate number calculated based on the quercetin peak should be no less than 3000. (3) The RSD of the injection precision of the reference solution should be less than 2.0% (n=6). (4) The solvent should not interfere with the detection of the quercetin peak.

[0105] Specific operation: Prepare the test solution and reference solution according to the above solution preparation method, and use methanol solution as the solvent, conduct on-machine testing, and record the test results.

[0106] The results showed that the retention time of the main component quercetin peak of the reference solution was about 43 minutes. (1) The separation between the quercetin peak and the adjacent peaks of the test solution and the reference solution was greater than 1.5. (2) The number of theoretical plates calculated based on the quercetin peak was greater than 3000. (3) The RSD of the injection precision of the reference solution was 0.29%, less than 2.0% (n=6). (4) The solvent did not interfere with the detection of the quercetin peak. The system suitability test met the requirements, and the specific test results are shown in the following table:

[0107] Table 4 System suitability test results (1)

[0108] name Retention time (min) Separation Theoretical plate number Solvents N / A N / A N / A Reference solution 43.377 N / A 17224 Test solution 43.440 2.939 / 2.802 18191

[0109] Table 5 System suitability test results (2)

[0110]

[0111] 7.2 Specificity test

[0112] Acceptance criteria: (1) The resolution between the quercetin peak and adjacent peaks in both the test solution and the reference solution should be greater than 1.5. (2) The theoretical plate number calculated based on the quercetin peak should be no less than 3000. (3) The negative solution of Polygonum hydropiper should not interfere with the detection of the quercetin peak. (4) The solvent should not interfere with the detection of the quercetin peak.

[0113] Specific operation: According to the preparation method of the above solution, prepare the Polygonum hydropiper negative solution, reference solution and test solution, use methanol solution as solvent, conduct on-machine test, and record the test results.

[0114] The results showed that the retention time of the main component quercetin peak of the reference solution was about 43 minutes. (1) The separation between the quercetin peak and the adjacent peaks of the test solution and the reference solution was greater than 1.5. (2) The theoretical plate number calculated based on the quercetin peak was greater than 3000. (3) The negative solution of Polygonum hydropiper did not interfere with the detection of the quercetin peak. (4) The solvent did not interfere with the detection of the quercetin peak. The specificity test met the requirements, and the specific results are shown in the following table:

[0115] Table 6 Specificity test results

[0116] name Retention time (min) Separation Theoretical plate number Solvents N / A N / A N / A Polygonum hydropiper negative solution N / A N / A N / A Reference solution 43.377 N / A 17224 Test solution 43.440 2.939 / 2.802 18191

[0117] 7.3 Linearity and range tests

[0118] Acceptance criteria: regression equation correlation coefficient r≥0.999.

[0119] Specific operation: Take an appropriate amount of quercetin reference substance, add methanol solution to prepare reference substance solutions of different concentrations of 4μg / ml, 10μg / ml, 20μg / ml, 50μg / ml, and 100μg / ml, accurately measure 10μl of each of the above reference substance solutions, inject them into the liquid chromatograph, record the chromatogram, draw a standard curve with concentration as the horizontal axis X and the peak area as the vertical axis Y, and calculate the regression equation and correlation coefficient.

[0120] The results showed that the quercetin reference substance was in the range of 4μg / ml-100μg / ml, the linear equation was: y=21.232x+0.7304, the correlation coefficient r=1.0000, and the linear relationship was good. The linearity and range test met the requirements, and the specific results are shown in the following table:

[0121] Table 7 Linear relationship test results of reference substances

[0122]

[0123] 7.4 Intermediate precision test

[0124] Acceptance criteria: The RSD of the intermediate precision test content should be less than 5.0% (n=12).

[0125] Specific operation: According to the above solution preparation method, prepare 6 test solution and 2 reference solution respectively, and test them on the machine. Then another person will perform the above operation at different time.

[0126] Calculate the contents of 12 test solutions and find the RSD value.

[0127] The results showed that the average intermediate precision was 49.5 μg / ml, the RSD was 2.5% (n=12), and the RSD was less than 5.0% (n=12). The intermediate precision test met the requirements, and the specific results are shown in the following table:

[0128] Table 8 Intermediate precision test results (n = 12)

[0129]

[0130] 7.5 Solution stability test

[0131] Acceptance criteria: When the reference solution and the test solution are placed at room temperature for 12 hours, the ratio of the main component peak area of ​​the injected solution at each time period to the peak area of ​​the 0h solution should be within 98.0%-102.0%.

[0132] Specific operation: Prepare reference solution and test solution according to the above solution preparation method, place them at room temperature, test them on the machine at 0, 2, 4, 6, 8, and 12 hours, and record the test results.

[0133] The results show that when the reference solution and the test solution are placed at room temperature for 12 hours, the peak area ratio of the main component of the injection solution at each time period to the peak area of ​​the 0h solution is within 98.0%-102.0%. The solution stability test meets the requirements, and the specific results are shown in the following table:

[0134] Table 9 Solution stability test results

[0135]

[0136] 7.6 Accuracy test

[0137] Acceptance criteria: The recovery limit of the accuracy test should be between 85% and 110.0%, and the recovery RSD should not exceed 5.0% (n=9).

[0138] Specific operations:

[0139] (1) Preparation of reference substance stock solution: Accurately weigh an appropriate amount of quercetin reference substance, dissolve it in methanol and dilute it to make a 1 ml solution containing approximately 50 μg of quercetin.

[0140] (2) Preparation of 80% test sample solution: Accurately weigh 5 ml of the test sample solution and place it in a 25 ml volumetric flask. Accurately add 3.0 ml of the reference sample stock solution to the flask, dilute to the mark with methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the 80% test sample solution.

[0141] (3) Preparation of 100% test sample solution: Accurately weigh 5 ml of the test sample solution and place it in a 25 ml volumetric flask. Accurately add 5.0 ml of the reference sample stock solution to the volumetric flask, dilute to the mark with methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the 100% test sample solution.

[0142] (4) Preparation of 120% test sample solution: Accurately weigh 5 ml of the test sample solution and place it in a 25 ml volumetric flask. Accurately add 7.0 ml of the reference sample stock solution to the volumetric flask, dilute to the mark with methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the 120% test sample solution.

[0143] (5) Prepare the reference solution according to the above solution preparation method, and prepare three portions of 80% test solution, 100% test solution, and 120% test solution respectively, conduct on-machine testing, and record the test results.

[0144] The results show that the average recovery rate of the content is 100.6%, the RSD is 2.5% (n=9), the average recovery rate is within 85%-110%, and the RSD is less than 5.0%. The accuracy test meets the requirements, and the specific results are shown in the following table:

[0145] Table 10 Accuracy (recovery rate) test results

[0146]

[0147] 7.6 Verification conclusion

[0148] Based on the above tests, it can be known that: the system adaptability meets the requirements, so the system used in the method of the present invention is effective and applicable; the specificity test meets the requirements, indicating that the method of the present invention can accurately and selectively analyze and identify the main component substances of Polygonum hydropiper to prevent interference with the detection of the main component peak; the linearity and range test meet the requirements, indicating that the detection concentration range of the method of the present invention is reasonably set and can meet the detection of the test solution; the intermediate precision test meets the requirements, indicating that the reproducibility and measurement precision of the method of the present invention are good; the solution stability test meets the requirements, indicating that the solution of the method of the present invention meets the determination requirements within 12 hours; the accuracy test meets the requirements, indicating that the recovery rate of the present invention can meet the detection requirements. All of the above verification items meet the requirements, and the method of this application can be used to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture.

[0149] Comparative Example 1

[0150] Take 10 μL of the reference solution in Example 1 and use a liquid chromatograph to perform determination and analysis. The detection conditions are:

[0151] Chromatographic column: octadecylsilane bonded silica as filler; ACE Excel 5C18-PFP, 4.6mm×25cm, 5μm;

[0152] Mobile phase A: acetonitrile solution;

[0153] Mobile phase B: 0.1% trifluoroacetic acid solution;

[0154] Detection wavelength: 350nm;

[0155] Column temperature: 30°C;

[0156] Gradient elution program: 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B.

[0157] Conclusion: Comparative Example 1 is a comparative test of Example 1, and liquid chromatograph and high performance liquid chromatograph are different detection instruments, and the detection methods used are also different. Therefore, the detection methods used in Comparative Example 1 and Example 1 are different, that is, under the same chromatographic detection conditions, different detection methods and corresponding detection instruments are used for detection, and the detection results obtained are different. According to the comparison of the detection results of Comparative Example 1 with Example 1, it can be known that under the same chromatographic detection conditions, different instruments are used, and the compounds that can be detected are different, and the effective substances of Polygonum hydropiper in Feng Liao Chang Wei Kang mixture cannot be detected. In contrast, the detection method and corresponding detection instrument used in Example 1 can accurately detect the main component substances of Polygonum hydropiper in Feng Liao Chang Wei Kang mixture, obtain the corresponding chromatogram, baseline separation, and good separation degree.

[0158] Comparative Example 2

[0159] Take 10 μL of the reference solution in Example 1 and perform analysis by high performance liquid chromatography. The detection conditions are as follows:

[0160] Chromatographic column: octadecylsilane bonded silica as filler; Sunniest PFP&C18, 4.6mm×25cm, 5μm;

[0161] Mobile phase A: acetonitrile solution;

[0162] Mobile phase B: 0.1% trifluoroacetic acid solution;

[0163] Detection wavelength: 350nm;

[0164] Column temperature: 30°C;

[0165] Gradient elution program: 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B.

[0166] Conclusion: Comparative Example 2 is a comparative test of Example 1. The method of Comparative Example 2 is to use high performance liquid chromatography for determination and analysis, except that the chromatographic column used is different from that of Example 1. Result: When determining, the separation degree between the main component and the adjacent impurity peak does not meet the content determination requirements, and the chromatographic column is not applicable. The method of Example 1 is to use high performance liquid chromatography, and the effective substances of Polygonum hydropiper in Fengliao Changweikang mixture are detected by a specific chromatographic column. According to the test results of Comparative Example 2 and Example 1, it can be seen that: using the same detection method, using different chromatographic columns, the chromatogram of the effective substances of Polygonum hydropiper cannot be detected, and there will be interference problems of the chromatographic column itself during the detection process. Different chromatographic columns have different interferences, and the interference problem cannot be eliminated, so the chromatographic column can only be replaced. In contrast, the chromatographic column used in Example 1 can accurately detect the content of the effective substances of Polygonum hydropiper in Fengliao Changweikang mixture, and the chromatographic column has a good retention time, high detection sensitivity, baseline separation, and good separation.

[0167] Comparative Example 3

[0168] Take 10 μL of the reference solution in Example 1 and perform analysis by high performance liquid chromatography. The detection conditions are as follows:

[0169] Chromatographic column: octadecylsilane bonded silica as filler; ACE Excel 5C18-PFP, 4.6mm×25cm, 5μm;

[0170] Mobile phase A: formic acid aqueous solution;

[0171] Mobile phase B: 0.1% formic acid in acetonitrile;

[0172] Detection wavelength: 350nm;

[0173] Column temperature: 30°C;

[0174] Gradient elution program: 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B.

[0175] Conclusion: Comparative Example 3 is a comparative test of Example 1. The detection methods used in Comparative Example 3 and Example 1 are the same, except that the components of mobile phase A and mobile phase B in the detection conditions are different. According to the comparison of the detection results of Comparative Example 3 with those of Example 1, it can be seen that in the same detection method, the substances of mobile phase A and mobile phase B are changed, and the substances detected are obviously different. In contrast, the substances of mobile phase A and mobile phase B used in Example 1 can better dissolve and separate the effective ingredients of Polygonum hydropiper in Fengliao Changweikang mixture, accurately detect the effective ingredients of Polygonum hydropiper, obtain the corresponding chromatogram, baseline separation, and good separation degree.

[0176] Comparative Example 4

[0177] Take 10 μL of the reference solution in Example 1 and perform analysis by high performance liquid chromatography. The detection conditions are as follows:

[0178] Chromatographic column: octadecylsilane bonded silica as filler; ACE Excel 5C18-PFP, 4.6mm×25cm, 5μm;

[0179] Mobile phase A: acetonitrile solution;

[0180] Mobile phase B: 0.1% trifluoroacetic acid solution;

[0181] Detection wavelength: 350nm;

[0182] Column temperature: 30°C;

[0183] Gradient elution program: 0 min, 2% A, 98% B; 20 min, 20% A, 80% B; 30 min, 55% A, 45% B; 40 min, 60% A, 40% B; 50 min, 30% A, 70% B; 60 min, 10% A, 90% B.

[0184] Conclusion: Comparative Example 4 is a comparative test of Example 1. The detection methods used in Comparative Example 4 and Example 1 are the same, except that the gradient elution procedures in the detection conditions are different. According to the comparison of the detection results of Comparative Example 4 with those of Example 1, it can be seen that in the same detection method, the substances detected are different when the gradient elution procedure is changed. In contrast, the gradient elution procedure used in Example 1 can better elute and separate the effective ingredients of Polygonum hydropiper in Fengliao Changweikang mixture, accurately detect the effective ingredients of Polygonum hydropiper, obtain the corresponding chromatogram, and have good baseline separation and separation.

[0185] From the above comparative examples 1-4, it can be seen that:

[0186] First, under the same chromatographic detection conditions, different detection methods and corresponding instruments can detect different substances. The present application uses high performance liquid chromatography to detect the main component of Polygonum hydropiper medicinal material quercetin in Fengliao Changweikang mixture, thereby obtaining the corresponding chromatogram.

[0187] Second, in the same detection method, changing the chromatographic column can detect different substances. The chromatographic column used in this application can detect the main component of Polygonum multiflorum in Fengliao Changweikang mixture with higher detection sensitivity, thereby obtaining the corresponding chromatogram with baseline separation and good resolution.

[0188] Third, in the same detection method, by changing the substances and combinations of mobile phase A and mobile phase B, different substances can be detected. The mobile phase A and mobile phase B used in this application can dissolve and separate the main components of Polygonum multiflorum in Fengliao Changweikang mixture, and accurately detect the main components of Polygonum multiflorum in Fengliao Changweikang mixture, thereby obtaining the corresponding chromatogram with baseline separation and good separation degree.

[0189] Fourth, in the same detection method, changing the gradient elution program can detect different substances. The gradient elution program used in this application can better elute and separate the main components of Polygonum multiflorum in Fengliao Changweikang mixture, detect the content of its main components, and thus obtain the corresponding chromatogram with baseline separation and good separation degree.

[0190] In summary, the present application example proves that the content of the main component quercetin contained in Polygonum hydropiper can be detected by high performance liquid chromatography by limiting the chromatographic conditions, including the selection of chromatographic columns, the combination of mobile phase A and mobile phase B substances and the substance concentration, controlling the gradient elution time and elution program, so as to accurately detect and calculate the content of Polygonum hydropiper in Fengliao Changweikang mixture, and the detection method has high sensitivity and good separation. The interference problem of Polygonum hydropiper content in the determination process is solved.

[0191] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and all of them belong to the protection scope of the present application.

Claims

1. A method for detecting the content of Polygonum hydropiper in Fengliao Changweikang mixture, characterized in that: High performance liquid chromatography was used to detect the content of Polygonum hydropiper in Fengliao Changweikang mixture. The detection solution included a test solution, a reference solution and a Polygonum hydropiper negative solution. The detection conditions included the following chromatographic conditions: octadecylsilane bonded silica gel was used as a filler, acetonitrile solution was used as a mobile phase A, 0.1% trifluoroacetic acid solution was used as a mobile phase B, the detection wavelength was 340nm-360nm, the column temperature was 30℃, gradient elution was performed, and the gradient elution time was 60min; The gradient elution program was as follows: 0 min, 16% A, 84% B; 45 min, 16% A, 84% B; 46 min, 90% A, 10% B; 51 min, 90% A, 10% B; 52 min, 16% A, 84% B; 60 min, 16% A, 84% B; Preparation of the negative solution of Polygonum hydropiper: accurately measure 10 ml of the negative mixture of Polygonum hydropiper, put it into a 25 ml volumetric bottle, add solvent to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the negative solution of Polygonum hydropiper; Preparation of test solution: accurately measure 10 ml of Fengliao Changweikang mixture, place it in a 25 ml volumetric flask, add solvent to dilute to the scale, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution; Preparation of reference solution: Accurately weigh an appropriate amount of quercetin reference substance, dissolve it in a solvent and dilute it to make a solution containing about 20 μg of quercetin per 1 ml, thus obtaining the reference solution; The solvent is methanol solution.

2. The method for detecting the content of Polygonum hydropiper according to claim 1, characterized in that: The detection wavelength was 350 nm and the injection volume was 10 μl.

3. The method for detecting the content of Polygonum hydropiper according to claim 1, characterized in that: The chromatographic column was ACE Excel 5 C18-PFP, 4.6 mm × 25 cm, 5 μm or ACE Excel 5 C18-AR, 4.6 mm × 25 cm, 5 μm or Hypersil GOLDTM PFP, 4.6 mm × 25 cm, 5 μm.

4. The method for detecting the content of Polygonum hydropiper according to claim 1, characterized in that: The detection steps include the following: S1. Take a blank solvent for testing and obtain a blank solvent chromatogram; S2. Take the negative solution of Polygonum hydropiper for detection to obtain a chromatogram of the negative solution of Polygonum hydropiper; S3. Take the reference solution for testing and obtain a chromatogram of the reference solution; S4. Take the test solution for testing and obtain a chromatogram of the test solution; S5. Calculate the content of the main component of Polygonum hydropiper in the test sample through the chromatogram data of the reference solution chromatogram and the test solution chromatogram.

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