HPLC content determination method of traditional Chinese medicine for relieving cough and reducing sputum

Abstract

The invention discloses an HPLC content determination method of a cough-relieving and sputum-reducing traditional Chinese medicine, the cough-relieving and sputum-reducing traditional Chinese medicine is prepared from eight traditional Chinese medicinal materials including scutellaria baicalensis, thunberg fritillary bulb, fructus forsythiae, houttuynia cordata, semen armeniacae amarae, radix stemonae, radix peucedani and liquorice, and the contents of six components including baicalin, forsythin, quercitrin, amygdalin, praeruptorin A and liquiritin are determined by the method. Chromatographic conditions are as follows: a C18 chromatographic column is adopted, a mobile phase A is a phosphoric acid aqueous solution, a mobile phase B is a methanol / acetonitrile mixed solution, gradient elution is performed, the elution flow rate is 0.5-2 ml / min, the column temperature is 25-40 DEG C, and the detection wavelengths are 220 nm and 254 nm. According to the method, the six medicinal materials and active ingredients are detected, the quality of traditional Chinese medicine is improved, fake and adulterated traditional Chinese medicine is prevented, and the effectiveness, safety and consistency of clinical medication are ensured. The method also has the advantages of high accuracy and sensitivity, short detection period, low cost and the like.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and specifically relates to an HPLC content determination method of a traditional Chinese medicine for relieving cough and reducing phlegm. Background Art

[0002] CN 108186954 A discloses a Chinese patent medicine for clearing away heat, promoting lung function, resolving phlegm and relieving cough. It is composed of eight Chinese medicinal materials, namely, scutellaria baicalensis, thunbergii bulb, forsythia suspensa, houttuynia cordata, bitter almond, radix stemonae, peucedanum chinense and licorice, and is prepared by modern technology according to traditional Chinese medicine theory. Scutellaria baicalensis is the monarch, thunbergii bulb and forsythia suspensa are the ministers, houttuynia cordata, bitter almond, radix stemonae and peucedanum chinense are the assistants, and licorice is the messenger. It has a certain effect on cough and sputum caused by respiratory tract infection, allergy, asthma and other chronic diseases. This product belongs to the national Class I new Chinese medicine, with the trade name "Qinbei cough relieving granules", and a multi-center phase III clinical study is being carried out.

[0003] At present, the quality inspection method of this traditional Chinese medicine is not perfect. Only the content of the main drug Scutellaria baicalensis is determined, and thin layer identification is also performed on individual medicinal materials. The quality control has the following main shortcomings: first, the content determination and thin layer identification require the use of different instruments, and the samples need to be treated differently, resulting in complex operations, long detection cycles, and high costs; second, thin layer identification is a qualitative test, which can only distinguish the presence or absence of medicinal materials, but cannot determine the amount of ingredients, resulting in the inability to ensure the consistency of drug quality; third, there are no detection indicators for most of the medicinal materials in the prescription, which provides an opportunity for adulteration and counterfeiting of clinical drugs.

[0004] As we all know, the quality of Chinese medicinal materials and the production and circulation of drugs will have an impact on the quality of Chinese medicine, and ultimately affect the clinical efficacy of Chinese medicine. At present, the country's requirements for clinical trials of new drugs are becoming increasingly stringent. In order to ensure the effectiveness, safety and consistency of clinical drugs, the most effective means is to develop testing methods that can comprehensively control the quality of Chinese medicine.

[0005] Based on this, the applicant started from the active ingredients and primary and secondary positions of traditional Chinese medicine, combined with the analysis of the physical and chemical properties of the ingredients and conducted a large number of experimental explorations, and finally developed a HPLC method that can simultaneously detect the content of six ingredients. These six ingredients are baicalin, forsythin, quercetin, amygdalin, peucedanum praeruptorum A, and liquiritin, which are the active ingredients of the six medicinal materials in Qinbei Cough Granules, namely Scutellaria baicalensis, Forsythia suspensa, Houttuynia cordata, Bitter Apricot kernel, Peucedanum praeruptorum, and Licorice. They encompass the monarch, minister, adjuvant, and messenger of traditional Chinese medicine, and cover a variety of natural medicinal ingredients such as coumarins, lignans, flavonoids, and cyanogenic glycosides. Studies have shown that most of these ingredients are closely related to the cough and phlegm-relieving effects of traditional Chinese medicine.

[0006] Table 1 Structural classification of the components to be tested

[0007] Summary of the invention

[0008] The purpose of the present invention is to construct an HPLC content determination method for a cough-relieving and phlegm-resolving Chinese medicine, which uses one system to detect the content of multiple different types of chemical components and can be better used to control product quality and ensure the effectiveness, safety and consistency of the Chinese medicine.

[0009] The above purpose is achieved through the following technical solutions:

[0010] A HPLC content determination method for a cough-relieving and phlegm-resolving Chinese medicine, wherein the cough-relieving and phlegm-resolving Chinese medicine is made of eight Chinese medicinal materials, namely, scutellaria baicalensis, fritillaria thunbergii, forsythia suspensa, houttuynia cordata, apricot kernel, radix stemonae, peucedanum peucedanum and licorice. The method determines the contents of six components, namely, baicalin, forsythin, quercetin, amygdalin, praeruptorin A and liquiritin. Among them, baicalin, liquiritin and quercetin belong to flavonoids, forsythin belongs to lignans, amygdalin belongs to cyanogenic glycosides, and praeruptorin A belongs to coumarins. The content determination method comprises the following steps:

[0011] 1) preparing a mixed reference solution with the standard substances of the six components;

[0012] 2) The sample to be tested is extracted with methanol, and the extract is applied to a macroporous adsorption resin column. After adsorption equilibrium, it is first eluted with water and then with methanol, and the methanol eluate is collected and prepared into a sample solution to be tested;

[0013] 3) The mixed reference solution and the sample solution to be tested are respectively detected by a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, and the detection wavelengths of the ultraviolet detector are set to 220 nm and 254 nm, wherein 220 nm is used to detect liquiritin, amygdalin, forsythiaside, and praeruptorin A, and 254 nm is used to detect quercetin and baicalin. The stationary phase of the high performance liquid chromatograph is a C18 chromatographic column, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is a methanol / acetonitrile mixed solution, and gradient elution is adopted, the flow rate is 0.5-2 ml / min, and the column temperature is 25-40°C.

[0014] Preferably, the resin model of the macroporous adsorption resin column is AB-8.

[0015] Preferably, the concentration of the phosphoric acid aqueous solution is 0.02% (volume), and the volume ratio of the methanol / acetonitrile mixed solution is 3:7.

[0016] Preferably, the gradient elution procedure is:

[0017] Time min Mobile phase A% Mobile phase B% 0 90 10 5 90 10 30 80 20 35 80 20 60 55 45 70 15 85 75 15 85 77 90 10 80 90 10

[0018] Preferably, the model of the C18 chromatographic column is Waters C18 4.6*250mm,5μm.

[0019] Preferably, the mixed reference solution contains 20 μg / ml baicalin, 7 μg / ml forsythin, 4 μg / ml quercetin, 70 μg / ml amygdalin, 0.5 μg / ml praeruptoside A, and 15 μg / ml liquiritin.

[0020] Preferably, the injection volume of the sample solution to be tested is 5-20 μl.

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

[0022] The present invention establishes for the first time a multi-index component quality detection method for Qinbei cough granules, which can detect 6 compounds belonging to four major categories of natural drug ingredients. Compared with the existing detection methods of the product, the present invention has the following obvious advantages:

[0023] (1) Convenience. The present invention can analyze the contents of multiple active ingredients by processing the sample once and injecting it once under the same chromatographic conditions, which has the advantages of high detection efficiency, short cycle, and low cost.

[0024] (2) Comprehensiveness. Quantitative testing is more conducive to ensuring the consistency of the quality of traditional Chinese medicine than qualitative testing. At the same time, the present invention realizes the detection of 6 kinds of medicinal materials and active ingredients, which is conducive to improving the quality of traditional Chinese medicine, preventing counterfeiting and adulteration, and ensuring the effectiveness and safety of clinical medication.

[0025] (3) Accuracy and sensitivity. The present invention improves the HPLC chromatographic peak separation and peak area by exploring and optimizing the detection conditions. The detection result has high accuracy and sensitivity, and can detect a minimum of about 0.005% of praeruptorin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Chromatogram of amygdalin reference substance (220nm).

[0027] Figure 2 : Chromatogram of liquiritin reference substance (220nm).

[0028] Figure 3 : Chromatogram of Forsythiaside reference substance (220nm).

[0029] Figure 4 : Chromatogram of praeruptorin A reference substance (220nm).

[0030] Figure 5 : Chromatogram of quercetin reference substance (254nm).

[0031] Figure 6 : Chromatogram of baicalin reference substance (254nm).

[0032] Figure 7 : Chromatogram of mixed reference solution (220nm).

[0033] Figure 8 : Chromatogram of mixed reference solution (254nm).

[0034] Fig. 9 : Chromatogram of test solution (220nm).

[0035] Fig.10 : Chromatogram of test solution (254nm).

[0036] Fig.11 : Spectrum of negative control sample lacking Houttuynia cordata. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with specific embodiments.

[0038] Instruments and materials used in the examples:

[0039] Waters e2695-2489 high performance liquid chromatograph, quaternary pump, online vacuum degassing system, autosampler, column oven, UV detector; Direct-Q3 ultrapure water instrument (Millpore); KQ-500B ultrasonic cleaner (power 500w, frequency 40kHz, Kunshan Ultrasonic Instrument Co., Ltd.); XSE205DU (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); MSA3.6P-OCE-DM (Sartorius).

[0040] Amygdalin reference substance (purity 93.1%, batch number 110820-202109), baicalin reference substance (purity 97.2%, batch number 110715-202223), quercetin reference substance (purity 93.5%, batch number 111538-202007), forsythin reference substance (purity 95.8%, batch number 110821-202318), praeruptorin A reference substance (purity 99.4%, batch number 111711-201904), and liquiritin reference substance (purity 95.2%, batch number 11610-202209) were all purchased from the China Food and Drug Inspection Institute; methanol and acetonitrile were chromatographic grade, water was purified water, and the rest were chemical grade. The traditional Chinese medicine Qinbei cough granules were produced by Hubei Li Shizhen Pharmaceutical Research Co., Ltd. The prescription and preparation process are as follows:

[0041] Prescription: 400g of Scutellaria baicalensis, 400g of Fritillaria thunbergii, 600g of Houttuynia cordata, 400g of Bitter Apricot, 400g of Stemona, 400g of Peucedanum, 480g of Forsythia suspensa, 240g of Licorice

[0042] Preparation method: For the above eight medicinal materials, volatile oils are extracted from Forsythia suspensa, Houttuynia cordata, and Peucedanum chinense by steam distillation, and the liquid is collected in another container, and the volatile oil is reserved; Fritillaria thunbergii and Stemona tuberosa are refluxed once with 70% ethanol, and the refluxed liquid is reserved; the above medicinal residues and the other three medicinal materials such as Scutellaria baicalensis are decocted with water twice. The decoction and the liquid extracted with volatile oil are combined, filtered, and the filtrate is concentrated to a relative density of 1.15-1.20, ethanol is added to make the alcohol content 65%, stirred, and allowed to stand for 24 hours, filtered, the filtrate is combined with the refluxed liquid, and the ethanol is recovered and concentrated to a relative density of 1.30-1.35, dried at low temperature, crushed, dextrin and stevioside are added in appropriate amounts, mixed, granulated, and a total of 1000g is made, and finally the volatile oil is sprayed and packaged.

[0043] Example 1 Detection method and verification thereof

[0044] 1. Detection method

[0045] Preparation of reference solution: Weigh a small amount of baicalin, forsythiain, quercetin, amygdalin, praeruptoside A, and liquiritin reference substances into 6 injection vials, add methanol to dissolve completely, and prepare a single reference solution containing baicalin, forsythiain, quercetin, amygdalin, praeruptoside A, and liquiritin as the reference solution.

[0046] Preparation of mixed reference solution: Weigh appropriate amounts of baicalin, forsythiaside, quercetin, amygdalin, praeruptoside A, and liquiritin reference substances, accurately weigh, dissolve in methanol and dilute to make a mixed reference solution containing approximately 20 μg baicalin, 7 μg forsythiaside, 4 μg quercetin, 70 μg amygdalin, 0.5 μg praeruptoside A, and 15 μg liquiritin per 1 ml.

[0047] Sample treatment: Take an appropriate amount of Qinbei cough-relieving granules, grind them into powder, accurately weigh about 0.2g and place it in a stoppered conical flask, accurately add 50ml of methanol, ultrasonicate for 30min, cool to room temperature, filter, accurately measure 25ml of the filtrate, and apply it to an AB-8 macroporous adsorption resin column (filling amount 2g, diameter 1cm). After adsorption equilibrium, first elute with 50ml of water, then elute with 50ml of methanol, collect the methanol eluate, evaporate to dryness in a constant temperature water bath at 40℃, dissolve the residue with methanol and transfer it to a 10ml volumetric flask, add methanol to the scale, shake well, filter with a 0.45μm filter membrane, and take the filtrate.

[0048] Chromatographic column: Waters C18 4.6*250mm,5μm

[0049] Detection wavelength: 220nm (amygdalin, liquiritin, forsythin, praeruptoside A), 254nm (quercetin, baicalin)

[0050] Mobile phase: A: 0.02% phosphoric acid solution B: methanol: acetonitrile (3:7)

[0051] Flow rate: 1.0ml / min

[0052] Injection volume: 10 μl

[0053] Column temperature: 30°C

[0054] Gradient elution:

[0055] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 30 80 20 35 80 20 60 55 45 70 15 85 75 15 85 77 90 10 80 90 10

[0056] 2. Methodological investigation

[0057] 2.1. Specificity test

[0058] Take blank solvent, mixed reference solution, each component positioning solution, and test solution and inject them into the liquid chromatograph respectively, and record the chromatogram. The blank solvent is required to have no interference; the retention time of each component in the positioning solution of each component is consistent with that in the mixed reference solution, and the separation degree between each component and the adjacent peak in the mixed reference solution and the test solution should be no less than 1.5.

[0059] Table 2 Specificity test results

[0060]

[0061]

[0062] Conclusion: The blank solution does not interfere with the detection of each component. The separation between each component and the adjacent peak of the reference solution is not less than 1.5. The peak elution time of the reference solution and the positioning solution is consistent, indicating that this method has good specificity.

[0063] The HPLC chromatograms of each sample are shown in Figure 1-10 shown.

[0064] 2.2. Linearity and range

[0065] Six concentration points were selected in the range of quantitation limit concentration to 500% of the index concentration. Linear relationship was plotted as a function of the measured corresponding signal (peak area) versus the concentration of the analyte, and linear regression was performed using the least squares method. Correlation coefficients were reported to confirm a good linear relationship.

[0066] Table 3 Linear equations

[0067]

[0068] Conclusion: The correlation coefficients of the six principal component linear equations are all greater than 0.999, indicating that the linear relationships are good.

[0069] 2.3. Accuracy

[0070] By adding reference solutions of each component with known amounts of 100% limit concentration to this product, and then determining the ratio of the measured value of each component in the added sample to the theoretical value as the recovery rate, prepare 6 sample solutions in parallel, and the recovery rates are required to be between 90% and 108%, and the RSD value of the recovery rate shall not exceed 10%.

[0071] Table 4 Recovery

[0072]

[0073] Conclusion: The average recoveries of the six sample solutions were between 90% and 108%, and the RSD values ​​of the recoveries were less than 10%, indicating good accuracy.

[0074] Durability

[0075] By changing the chromatographic conditions, the ratio of the amount of each component under different chromatographic conditions to the amount measured under standard conditions is between 98% and 102%.

[0076] Table 5 Durability

[0077]

[0078] Conclusion: The ratio of the amount of each component under different chromatographic conditions to the amount measured under standard conditions is between 98% and 102%.

[0079] 2.5. Stability

[0080] Place the reference solution and the test solution at room temperature, detect the peak area of ​​each component at different time points, and calculate the RSD. The RSD of the peak area of ​​each component is required to be no greater than 2%.

[0081] Table 6 Stability test results of reference solution

[0082] time Amygdalin Licorice glycoside Forsythiaside Purpura chinensis Quercetin Baicalin 0 385872 526837 116859 23148 190233 300949 6h 386705 527756 118113 23403 192980 301658 27h 385464 523522 116766 23055 190234 300950 50h 398984 536062 119172 23007 197415 303204 average value 389256 528544 117728 23153 192716 301690 RSD 1.67％ 1.01％ 0.97％ 0.76％ 1.76％ 0.35％

[0083] Table 7 Stability test results of the test solution

[0084]

[0085]

[0086] Conclusion: The RSD values ​​of the peak areas of the components of the reference solution measured within 50 hours were all less than 2%; the RSD values ​​of the peak areas of the components of the test solution measured within 71 hours were all less than 2%. The prepared solutions were stable and suitable for detection.

[0087] 2.6. Testing of samples from different batches

[0088] Three batches of samples were measured according to the constructed method, and the results are shown in the following table:

[0089] Table 8 Test results of three batches of samples

[0090]

[0091] Conclusion: The content differences among the three batches of samples were small and the quality consistency was high.

[0092] Example 2 Detection Condition Screening Test

[0093] 1. Screening test of sample pretreatment methods

[0094] Traditional Chinese medicine has complex ingredients, including test ingredients and a large number of impurities, and there are impurities whose physical and chemical properties are very similar to those of the test ingredients. If these impurities are not removed by appropriate means, they will inevitably interfere with the peak of the test ingredients in the chromatogram, resulting in abnormal separation and peak area, and thus reducing the accuracy of the test. For this reason, this experiment uses macroporous adsorption resin to purify the sample and screen the resin types. The test method is as follows:

[0095] 1) Sample processing

[0096] Referring to the sample processing method in Example 1, different types of macroporous resins were used to purify the extracted samples to obtain different test sample solutions.

[0097] 2) Chromatographic conditions

[0098] The chromatographic conditions are the same as in Example 1.

[0099] 3) Test results and analysis:

[0100] In this experiment, a variety of macroporous resins were selected, including AB-8 (weak polarity), D101 (non-polarity), HPD-100 (non-polarity), H103 (non-polarity), and DM130 (weak polarity). After treating the samples with different types of resins, the separation and peak areas of the six components are shown in Tables 9 and 10, respectively.

[0101] Table 9 Separation of test solution after purification by different types of macroporous resins

[0102] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis AB-8 / 1.56 8.22 9.67 2.49 19.96 D101 / 1.24 0.63 1.82 0.53 2.46 HPD-100 / 1.39 1.77 1.93 0.62 2.36 H103 / 1.30 1.35 1.37 1.08 1.14 DM130 / 1.05 0.82 2.01 1.51 /

[0103] Table 10 Peak area of ​​the test solution after purification by different types of macroporous resins

[0104] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis AB-8 407212 426830 167655 1083723 153861 34096 D101 380278 429599 105125 1002532 98513 27526 HPD-100 405683 471413 154325 823584 103199 28298 H103 355507 371084 142531 985265 105267 20425 DM130 389136 313743 123982 1053687 90430 /

[0105] From the perspective of separation, amygdalin is the first peak, and the separation from the front peak is not reflected. After the DM130 macroporous resin is treated, the separation between quercetin and the front peak is less than 1.0, and the effective separation of the chromatographic peak of praeruptorin A is not achieved; the separation of the quercetin and forsythin chromatographic peaks of the D101 macroporous resin is less than 1.0; the separation of the forsythin chromatographic peak and the front peak of the HPD-100 macroporous resin is less than 1.0; the separation of each chromatographic peak of the AB-8 macroporous resin is greater than 1.5. From the perspective of peak area, the peak areas of each chromatographic peak of the AB-8 macroporous resin are relatively high and balanced, especially the detection peak area of ​​praeruptorin A is at a higher level among all types of macroporous resins.

[0106] Separation and peak area are the two most critical indicators for evaluating HPLC detection methods. The greater the separation, the higher the detection accuracy, while the higher the peak area, the greater the detection sensitivity. Through the above experiments, the present invention preliminarily screened out AB-8 type macroporous adsorption resin and purified the test solution.

[0107] 2. Mobile phase screening test

[0108] The other detection conditions were the same as those in Example 1, and the test solution was detected using the following 4 mobile phases.

[0109] Mobile phase: (1): A: water; B: acetonitrile

[0110] (2): A: 0.02% phosphoric acid solution; B: acetonitrile

[0111] (3): A: 0.1% phosphoric acid solution; B: acetonitrile

[0112] (4): A: 0.2% phosphoric acid solution; B: acetonitrile

[0113] (5): A: 0.02% phosphoric acid solution; B: methanol: acetonitrile (3:7)

[0114] Test results and analysis:

[0115] Five different mobile phases were selected for this experiment. After comparison with the chromatograms of the mixed reference solution, the separation and peak areas of the six components were confirmed as shown in Tables 11 and 12, respectively.

[0116] Table 11 Separation of test solution with different mobile phases

[0117] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis Mobile phase(1) / 0.89 7.80 8.31 1..86 18.73 Mobile phase (2) / 1.01 5.19 8.41 1.42 17.18 Mobile phase (3) / 1.20 6.99 9.77 1.08 12.11 Mobile phase (4) / 0.82 8.55 9.24 1.88 12.90 Mobile phase(5) / 1.51 8.13 10.62 2.35 20.01

[0118] Table 12 Peak areas of test solutions with different mobile phases

[0119] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis Mobile phase(1) 375368 329837 147055 1065232 152964 29828 Mobile phase (2) 400750 385906 159256 1038295 131246 31848 Mobile phase (3) 367182 417155 155250 1085880 142097 32503 Mobile phase(4) 383010 298632 164859 1073581 156635 33621 Mobile phase(5) 407125 425999 166003 1089325 153967 32561

[0120] The results showed that the separation degree of mobile phase (5) for each component was greater than 1.5, and the peak area of ​​most components was higher than that of other mobile phases. Therefore, 0.02% phosphoric acid solution and methanol:acetonitrile (3:7) were determined as the optimal mobile phases.

[0121] 3. Gradient elution program screening test

[0122] The other detection conditions were the same as those in Example 1, and the test solution was detected using the following four gradient elution programs.

[0123] Elution procedure: (1)

[0124] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 50 10 90 50.1 90 10 60 90 10 (2)

[0126] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 80 20 35 55 45 50 10 90 50.1 80 20 60 80 20 (3)

[0128] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 90 10 30 60 40 40 60 40 50 10 90 60 10 90 61 90 10 70 90 10 (4)

[0130] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 30 80 20 35 80 20 60 55 45 70 15 85 75 15 85 77 90 10 80 90 10

[0131] Test results and analysis:

[0132] Four different elution procedures were screened in this experiment. After comparison with the chromatogram of the mixed reference solution, the separation and peak areas of the six components were confirmed as shown in Table 13 and Table 14, respectively.

[0133] Table 13 Separation of test solution with different elution procedures

[0134] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis Programs (1) / 0.75 3.38 9.67 1.88 10.74 Programs (2) / 0.62 9.41 9.48 2.14 11.80 Programs (3) / 1.05 7.43 8.94 4.35 17.34 Programs (4) / 1.55 8.24 10.36 2.50 19.89

[0135] Table 14 Peak areas of test solution with different elution procedures

[0136] Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis Programs (1) 393561 376088 150089 1016563 142314 32354 Programs (2) 391764 295421 169981 1032354 154221 32689 Programs (3) 405968 404543 158746 1056822 154239 33002 Programs (4) 406825 425088 166914 1087652 155821 33683

[0137] Result analysis: The separation degree between liquiritin and adjacent peaks in elution procedures (1) to (3) was less than 1.5, while under the elution gradient of procedure (4), all detected components could be effectively separated, so the gradient procedure (4) was finally selected.

[0138] 4. Detection wavelength screening test

[0139] The other detection conditions are the same as those in Example 1, and the following four wavelengths are used for detection.

[0140] Detection wavelength: 220nm, 254nm, 277nm, 310nm

[0141] Test results and analysis:

[0142] This test compared four detection wavelengths and, by comparison with the chromatogram of the mixed reference solution, confirmed the peak areas of the six components as shown in Table 15.

[0143] Table 15 Peak areas of test solution at different detection wavelengths

[0144] wavelength Amygdalin Licorice glycoside Quercetin Baicalin Forsythiaside Purpura chinensis 220nm 406911 426584 135126 2546352 155783 32565 254nm / 116182 168823 1096905 28798 / 277nm / 254627 74473 2708015 42882 / 310nm / 126566 79119 1648690 / /

[0145] Conclusion: The above six components all have strong absorption at the near-terminal absorption wavelength of 220nm, but there are some differences in the absorption between the components: amygdalin, liquiritin, forsythiaside, and praeruptorin A have the strongest absorption at a wavelength of 220nm; quercetin has the strongest absorption at a wavelength of 254nm; and baicalin has the strongest absorption at a wavelength of 277nm.

[0146] The specificity of negative control samples of each component was studied, and it was found that at a wavelength of 220nm, the negative control sample lacking Houttuynia cordata had an obvious interference absorption peak at the retention time of the quercetin peak, which interfered with the detection of quercetin; while at a wavelength of 254nm, there was no obvious interference absorption peak. That is, at a wavelength of 220nm, the detection specificity of quercetin was poor, and the test results were inaccurate. At a wavelength of 254nm, the detection specificity of quercetin was better.

[0147] In addition, the content and response of each component were evaluated, and it was found that the content of baicalin in Qinbei cough granules was relatively high. At wavelengths of 220nm and 277nm, the peak areas exceeded 2 million, which is relatively large, more than 5 times that of other detected components; and at a wavelength of 254nm, the peak area of ​​baicalin was about 1 million, which is more suitable for the content determination of multiple index components.

[0148] Finally, amygdalin, liquiritin, forsythiaside, and praeruptoside A selected 220nm as the absorption wavelength, and quercetin and baicalin selected 254nm as the absorption wavelength.

Claims

1. A HPLC content determination method for a cough-relieving and phlegm-resolving Chinese medicine, wherein the cough-relieving and phlegm-resolving Chinese medicine is made of eight Chinese medicinal materials, namely, scutellaria baicalensis, fritillaria thunbergii, forsythia suspensa, houttuynia cordata, bitter almond, radix stemonae, peucedanum peucedanum, and liquorice, and characterized in that: The method determines the contents of six components, namely baicalin, forsythin, quercetin, amygdalin, praeruptorin A and liquiritin, wherein liquiritin, quercetin and baicalin belong to flavonoids, forsythin belongs to lignans, amygdalin belongs to cyanogenic glycosides, and praeruptorin A belongs to coumarins. The determination method comprises the following steps: 1) preparing a mixed reference solution with the standard substances of the six components; 2) The sample to be tested is extracted with methanol, and the extract is applied to a macroporous adsorption resin column. After adsorption equilibrium, it is first eluted with water and then with methanol, and the methanol eluate is collected and prepared into a sample solution to be tested; 3) The mixed reference solution and the sample solution to be tested are respectively detected on a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, and the detection wavelengths of the ultraviolet detector are set to 220 nm and 254 nm, wherein 220 nm is used to detect amygdalin, liquiritin, forsythiaside and praeruptorin A, and 254 nm is used to detect quercetin and baicalin, the stationary phase of the high performance liquid chromatograph is a C18 chromatographic column, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is a methanol / acetonitrile mixed solution, the gradient elution, the elution flow rate is 0.5-2 ml / min, and the column temperature is 25-40°C.

2. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The resin model of the macroporous adsorption resin column is AB-8.

3. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The concentration of the phosphoric acid aqueous solution is 0.02%, and the volume ratio of the methanol / acetonitrile mixed solution is 3:

7.

4. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The gradient elution procedure is:

5. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The model of the C18 chromatographic column is Waters HSS C18 4.6*250mm,5μm.

6. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The mixed reference solution contains 20 μg / ml of baicalin, 7 μg / ml of forsythiaside, 4 μg / ml of quercetin, 70 μg / ml of amygdalin, 0.5 μg / ml of praeruptoside A, and 15 μg / ml of liquiritin.

7. The HPLC content determination method of the cough-relieving and phlegm-resolving Chinese medicine as claimed in claim 1, characterized in that: The injection volume of the sample solution to be tested is 5-20 μl.

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