Method for simultaneously determining active ingredients of praeruptorin A, praeruptorin B and hesperidin in phlegm-eliminating cough-relieving pills

Abstract

The invention discloses a method for simultaneously determining active ingredients of praeruptorin A, praeruptorin B and hesperidin in a phlegm-eliminating cough-relieving pill, and belongs to the technical field of traditional Chinese medicine ingredient analysis. The method realizes simultaneous accurate determination of three key components by optimizing operation conditions of test solution preparation, reference solution preparation and high performance liquid chromatography, not only has high detection efficiency and excellent linear relationship, but also has excellent stability and repeatability, and ensures the reliability of detection results. By adding diatomite, the grinding efficiency is improved, and the quality of a test solution is improved; the hesperidin reference substance is dissolved by heating, so that the problem of turbidity caused by insolubility in methanol is solved; a specific chromatographic column and an optimized mobile phase are used for gradient elution, target components are effectively separated, interference is avoided, and good specificity is shown. Scientific basis is provided for quality control of the phlegm-eliminating cough-relieving pills, and methodological reference is provided for detection of similar components in other traditional Chinese medicine preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component analysis, and specifically relates to a method for simultaneously determining effective components of peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in Chutan Zhisou pills. Background Art

[0002] Chutanzhisou Pills are composed of 18 herbs, including peucedanum chinense, dried tangerine peel, atractylodes macrocephala, pinellia tuber, platycodon grandiflorum, scutellaria baicalensis, and liushenqu. They have the effects of clearing the lungs and reducing fire, removing phlegm and relieving cough. In the finished formula, peucedanum chinense and dried tangerine peel are both the main herbs. Peucedanum chinense has the effects of lowering qi and relieving cough, and dispersing lung heat. Peucedanum peucedanum A and Peucedanum peucedanum B are the main active ingredients of peucedanum, which have the effects of relieving cough and reducing phlegm, and anti-inflammatory. Dried tangerine peel has the effects of regulating qi and reducing phlegm. Hesperidin is derived from the active ingredients in dried tangerine peel and also has pharmacological activities such as anti-inflammatory and antioxidant. The current standard of this drug is the second volume of Chinese medicine finished formula preparations of the "Drug Standards of the Ministry of Health of the People's Republic of China" (WS3-B-0365-90). This quality standard only conducts the properties and microscopic identification of Chutanzhisou Pills, and does not conduct qualitative or quantitative determination of any ingredients in the drug, which is not conducive to monitoring and controlling the quality of medicinal materials and preparations. There is little research on improving the quality standard of Chutan Zhisou Pills in the existing literature, and there is no literature or patent reporting the simultaneous content determination of the index components of the main medicines Peucedanum chinense and dried tangerine peel in Chutan Zhisou Pills.

[0003] Traditional determination methods cannot determine multiple components at the same time. The determination process is cumbersome and time-consuming, and cannot meet the needs of fast and accurate quality control. In addition, the components of different medicinal materials interfere with each other, which increases the difficulty of determination. In Chutan Zhisou Pills, peucedanum chinense and dried orange peel are the main ingredients, which are the main medicinal materials for curative effect. In the quality research of Chinese medicine preparations, it is a challenging task to separate and purify specific active ingredients from complex Chinese medicine preparations. The existing determination methods mainly focus on the effective ingredients of peucedanum chinense A and peucedanum chinense B. Hesperidin is an important active ingredient of Chutan Zhisou Pills. Since hesperidin is slightly soluble in methanol, it is easy to make the solution turbid when preparing the reference solution, resulting in poor linearity and recovery rate. Adding other cosolvents is easy to cause solvent interference and affect the peak shape of the chromatographic peak. In addition, there are 18 Chinese medicines in Chutan Zhisou Pills, and the extraction of effective ingredients interferes with each other. Hesperidin has different polarity from other active ingredient compounds to be detected, so it is difficult to determine them at the same time. The simultaneous detection of peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin is of great significance for improving the quality control level of Chutan Zhisou Pills and ensuring the stability and safety of the drug's efficacy, and provides a reference for further improving the quality standards of Chutan Zhisou Pills. Summary of the invention

[0004] In view of the important role of hesperidin in Chutan Zhisou Pills, and the difficulty in simultaneous detection due to its poor solubility, complex interaction with other ingredients, and different polarities with peucedanum praeruptoside A and peucedanum peucedanum B, the existing detection methods are unable to achieve the technical status of the simultaneous determination of the key active ingredients peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in Chutan Zhisou Pills. The present invention aims to provide a method for the simultaneous determination of the active ingredients peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in Chutan Zhisou Pills. By simultaneously determining the three key pharmacological ingredients, the quality status of the drug can be more comprehensively understood, providing a reference for further improving the quality standard of Chutan Zhisou Pills. In addition, this method can also provide a useful reference and reference for the simultaneous determination of multiple active ingredients in other traditional Chinese medicine preparations.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for simultaneously determining effective ingredients of praeruptoside A, praeruptoside B and hesperidin in Chutan Zhisou Pills, which specifically comprises the following steps:

[0007] S1, take Chutan Zhisou Pills, add diatomaceous earth to grind, dissolve, and ultrasonically treat to obtain the test solution;

[0008] S2, peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin were used as reference substances, dissolved in methanol, and hesperidin was dissolved and heated in a water bath to prepare a mixed reference substance solution;

[0009] S3, performing high performance liquid chromatography analysis on the test solution and the reference solution under the same conditions, recording the corresponding chromatograms, and calculating the peak area using an external standard method to obtain the test results;

[0010] Octadecylsilane bonded silica gel was used as a filling column, acetonitrile was used as a mobile phase A, and a phosphoric acid aqueous solution with a mass concentration of 0.1% to 0.2% was used as an eluent for gradient elution. The gradient elution procedure was as follows:

[0011]

[0012] .

[0014] In S1, the added amount of diatomaceous earth is 10% to 20% of the mass of the Chutan Zhisou Pills, which is used to disperse the sample to prevent agglomeration during extraction, which is beneficial to extraction, and also has a filtering aid effect to facilitate filtration.

[0015] In S1, the dissolving solvent is any one of pure methanol, 50% methanol aqueous solution, 70% methanol aqueous solution and chloroform.

[0016] Furthermore, the dissolving solvent is pure methanol.

[0017] In S1, the ultrasonic treatment conditions are: power 450-550 W, frequency 30-60 KHz, 15-25 min.

[0018] Furthermore, the ultrasonic treatment conditions are: power 500 W, frequency 40 KHz, 20 min.

[0019] In S2, the water bath temperature is 50°C to 70°C, and the time is 1 min to 2 min.

[0020] Furthermore, the water bath temperature is 60° C. and the time is 1 min.

[0021] In S2, the mass concentration of peucedanum arborescens A in each 1 mL of the reference solution is 63.02 μg / mL, the mass concentration of peucedanum arborescens B is 32.52 μg / mL, and the mass concentration of hesperidin is 282.27 μg / mL.

[0022] In S3, the mass concentration of the phosphoric acid aqueous solution is 0.2%.

[0023] Furthermore, the chromatographic column used in the high performance liquid chromatography is a ZORBAXSB-C18 column (250 mm×4.6 mm, 5 μm).

[0024] In S3, the HPLC detection wavelength is 280-400 nm, the column temperature is 25-35° C., and the flow rate is 0.8-1.2 mL / min.

[0025] Furthermore, the column temperature is 30°C and the flow rate is 1.0 mL / min.

[0026] Furthermore, the detection wavelengths of the praeruptorin A and praeruptorin B are 321 nm, and the detection wavelength of the hesperidin is 284 nm.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The method provided by the present invention for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in the phlegm-killing and cough-stopping pills aims at the problem of simultaneously determining the key effective ingredients praeruptorin A, praeruptorin B and hesperidin in the phlegm-killing and cough-stopping pills. In the dissolution of hesperidin, dissolution in a water bath at 60°C for 1 min can effectively solve the problems of turbidity, difficulty in dissolving and poor linearity of the standard curve in the preparation of the reference substance; the addition of diatomaceous earth helps to improve the grinding effect of the phlegm-killing and cough-stopping pills, making it easier to dissolve with methanol and prevent agglomeration during extraction, and at the same time has a filtering effect, which is convenient for filtration, thereby improving the extraction quality of the test solution and providing a good basis for subsequent high performance liquid chromatography analysis; the mobile phase (acetonitrile-phosphoric acid solution) is used for gradient elution, which can effectively separate and detect the target components, avoid the interference of other components, and embody good specificity. The improvement of the reference solution and the conditions of high performance liquid chromatography (HPLC) realizes the simultaneous determination of the test solution and the detection of the target components. The three effective ingredients of peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in Chutan Zhisou Pills were determined with high detection efficiency. The mass concentrations of the three ingredients showed a good linear relationship with the chromatographic peak area, and the correlation coefficients were not less than 0.9995. The relative standard deviation of the determination results was 0.44%-0.90% (n=6). The method had good stability and repeatability, and could ensure the reliability of each test result. The average recovery rate of the samples was 96.90%-102.98%, indicating that the method had a high recovery rate and could accurately reflect the true content of the target components in the samples. The method can provide strong technical support for the quality control of Chutan Zhisou Pills, ensure the stable and effective product quality, and help to enhance the market competitiveness of the products. It is not only suitable for the quality control of Chutan Zhisou Pills, but also can provide methodological reference for the detection of similar ingredients in other traditional Chinese medicine preparations, and promote the modernization and standardization of traditional Chinese medicine.

[0029] Furthermore, a specific chromatographic column (such as Agilent ZORBAX SB-C18) and an optimized mobile phase (acetonitrile-0.2% phosphoric acid solution) are used for gradient elution, which can effectively separate and detect the target components, avoid interference from other components, embody good specificity, and can more effectively separate and detect the target components, improve the accuracy and sensitivity of detection. The gradient elution program gradually adjusts the composition of the mobile phase according to the retention time differences of different components on the chromatographic column, so as to achieve continuous and effective separation of the three components and improve the accuracy and stability of detection; the detection wavelengths are 321nm (peucedanum praeruptoside A, peucedanum praeruptoside B) and 284nm (hesperidin), respectively, which can ensure high-sensitivity detection of the target components; gradient elution is performed by using a specific chromatographic column filler and an eluent composed of a mobile phase, which can achieve effective separation and detection of the three components, avoid interference from other components, and embody good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1It is a chromatogram of the test solution of the present invention, wherein 1 is hesperidin, 2 is peucedanum praeruptoside A, and 3 is peucedanum praeruptoside B;

[0031] Figure 2 is a chromatogram of the mixed reference substance solution of the present invention, wherein 1 is hesperidin, 2 is peucedanum praeruptoside A, and 3 is peucedanum praeruptoside B;

[0032] Figure 3 It is the chromatogram of negative sample solution of the present invention. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, 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 should fall within the scope of protection of the present invention.

[0034] Main instruments and reagents used in the present invention

[0035] High performance liquid chromatograph: 1260 Infinity II, diode array detector, Agilent Technologies, USA.

[0036] Electronic balance: BSA224S, sensitivity 0.1 mg, Sartorius Instruments Co., Ltd., Germany.

[0037] Electronic balance: XPR105DR, sensitivity 0.01 mg, Mettler-Toledo Instrument Co., Ltd., Switzerland.

[0038] Ultrasonic cleaner: Model KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.

[0039] Ultrapure water preparation system: Milli-Q IQ7000, Millipore Instrument Co., Ltd., USA.

[0040] Peucedanum praeruptorum A reference substance: mass fraction is 99.4%, batch number is 111711-201904, China Food and Drug Inspection Institutes.

[0041] Peucedanum peucedanum reference substance: mass fraction is 99.7%, batch number is 111904-202105, China Food and Drug Inspection Institutes.

[0042] Hesperidin reference substance: mass fraction is 97.2%, batch number is 110721-202220, China Food and Drug Inspection Institutes;

[0043] Methanol and acetonitrile: both were chromatographically pure and were purchased from Sigma Chemical Company, USA.

[0044] Phosphoric acid: chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd.

[0045] Samples of Chutan Zhisou Pills: batch numbers are 20010706, 20010566, and 20010110, from Tong Ren Tang Pharmaceutical Factory, Beijing Tong Ren Tang Co., Ltd.

[0046] Negative sample of Chutan Zhisou Pills: Tongrentang Pharmaceutical Factory, Beijing Tongrentang Co., Ltd.

[0047] The experimental water was ultrapure water;

[0048] Chromatographic column: ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA);

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0050] Example 1

[0051] See attached Figure 1 The embodiment provides a method for simultaneously determining the effective ingredients of peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in Chutan Zhisou Pills. The specific preparation method is as follows:

[0052] 1. Solution Preparation

[0053] (1) Mixed reference solution

[0054] Stock solutions of peucedanum praeruptorin A and peucedanum praeruptorin B reference substances: Take an appropriate amount of 31.70 mg of peucedanum praeruptorin A and 16.31 mg of peucedanum praeruptorin B reference substances, accurately weigh them, place them in 10 mL volumetric flasks respectively, dilute them to the mark with methanol, shake well, and obtain stock solutions of peucedanum praeruptorin A and peucedanum praeruptorin B reference substances respectively.

[0055] Reference solutions of peucedanum praeruptorin A, peucedanum praeruptorin B, and hesperidin: 14.52 mg of hesperidin reference substance was accurately weighed and placed in a 50 mL volumetric flask, and placed in a 60°C water bath for 1 min to accelerate dissolution; then 1 mL of peucedanum praeruptorin A and 1 mL of peucedanum praeruptorin B reference substance stock solutions were respectively placed in the same 50 mL volumetric flask, and methanol was added to dilute to the mark to prepare mixed reference substance solutions with mass concentrations of 63.02 μg / mL, 32.52 μg / mL, and 282.27 μg / mL for peucedanum praeruptorin A, respectively.

[0056] (2) Test solution

[0057] Take 10 pills of Chutan Zhisou Pills sample, cut into small pieces, accurately weigh 3 g, add 0.5 g of diatomaceous earth and grind it into fine pieces, put it into a stoppered conical flask, accurately add 25 mL of pure methanol, stopper it and weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 20 min, let it cool, weigh it again, make up the weight with methanol, shake well, filter it, and use it as the test solution. Prepare the negative sample solution without Peucedanum chinense and Citrus reticulatae in the same way.

[0058] 2. Determination

[0059] The mixed reference solution, sample solution and negative sample solution were taken separately, and the samples were injected for determination. The chromatographic peak area of ​​each solution was recorded, and the standard working curve was drawn. The contents of peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin in the samples were calculated according to the external standard method.

[0060] Chromatographic column: ZORBAXSB-C 18 Column (250mm×4.6mm, 5μm, Agilent Technologies, USA); column temperature: 30℃; detection wavelength: 321nm for peucedanum praeruptoside A and peucedanum praeruptoside B, 284nm for hesperidin; injection volume: 5μL; mobile phase: phase A is acetonitrile, phase B is 0.2% phosphoric acid aqueous solution, flow rate is 1.0mL / min; elution mode: gradient elution, the gradient elution program is shown in Table 1.

[0061] Table 1: Gradient elution program

[0062]

[0063] Example 2

[0064] This example optimizes the parameters of the method for simultaneously determining the effective ingredients of praeruptoside A, praeruptoside B and hesperidin in Chutan Zhisou Pills of the present invention.

[0065] 1. Chromatographic column

[0066] In this experiment, acetonitrile-0.2% phosphoric acid aqueous solution was used as the mobile phase, and the separation effect and chromatographic peak shape of the three index components by Waters Symmentry-C18 column (250mm×4.6mm, 5μm) and ZORBAXSB-C18 column (250mm×4.6mm, 5μm) were investigated respectively. The results showed that when the ZORBAXSB-C18 column was used, the baseline was stable, the separation was good, the chromatographic peak shape was symmetrical, and the retention time was appropriate. Therefore, the ZORBAXSB-C18 column (250mm×4.6mm, 5μm) was selected as the analytical column.

[0067] 2. Detection wavelength

[0068] Based on experiment (1), this experiment used a diode array detector to scan the mixed reference solution in the range of 200-400 nm. The scanning spectrum showed that the two components, peucedanum argentein A and peucedanum argentein B, had maximum absorption at 321 nm, and hesperidin had maximum absorption at 284 nm. Taking into account factors such as separation, chromatographic peak shape, and interference from other peaks, the detection wavelengths of peucedanum argentein A and peucedanum argentein B were set at 321 nm, and the detection wavelength of hesperidin was set at 284 nm.

[0069] 3. Mobile phase

[0070] Based on Experiment (1) and Experiment (2), this experiment investigated the effects of different mobile phase systems and different elution methods on the test results. Four mobile phase systems, methanol-water, methanol-0.1% formic acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution and acetonitrile-0.2% phosphoric acid aqueous solution, were used to determine the contents of three index components in the sample by isocratic or gradient elution. The results showed that the peak shape and separation of the chromatographic peaks using the above four mobile phases were poor, and the retention time was long. According to the molecular polarity of the three index components, through the optimization of the gradient elution program, it was finally determined that acetonitrile-0.2% phosphoric acid aqueous solution was used as the mobile phase for gradient elution. The chromatogram baseline was stable, the three components were well separated, and the retention time was moderate.

[0071] 4. Sample handling

[0072] (1) Extraction solvent

[0073] This experiment investigated the effects of different extraction solvents used to treat the test samples to prepare the test solution on the test results. 25 mL of methanol, 50% methanol aqueous solution, 70% methanol aqueous solution, and chloroform were used as extraction solvents, and ultrasonic treatment for 30 minutes had an effect on the extraction results of peucedanum argentein, peucedanum argentein B, and hesperidin. The test results are shown in Table 2.

[0074] Table 2: Determination results of three components in different extraction solvents

[0075]

[0076] As shown in Table 2, when methanol is used as the extraction solvent, the extraction efficiency is the highest, followed by 70% methanol extraction efficiency, and chloroform cannot effectively extract the three components, so methanol is selected as the extraction solvent.

[0077] (2) Extraction method

[0078] Based on experiment (1), this experiment used methanol as the extraction solvent to investigate the extraction effects of ultrasonic extraction and heating reflux on the three components, peucedanum arborescens A, peucedanum arborescens B, and hesperidin. The results are shown in Table 3, indicating that the two extraction methods had no significant effect on the determination results of the three components in Chutan Zhisou Pills. Considering the simple operation and fewer impurity peaks, ultrasonic extraction was selected as the sample processing method.

[0079] Table 3: Determination results of three components by different extraction methods

[0080]

[0081] (3) Ultrasonic treatment time

[0082] Using methanol as the extraction solvent, the extraction effects of 3 components, peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin, were investigated at different ultrasonic times (20, 30, 45 and 60 min). The results are shown in Table 4.

[0083] Table 4: Determination results of three components at different ultrasonic times

[0084]

[0085] As shown in Table 4, the three components can be effectively extracted by ultrasound for 20 min. Considering the time cost and extraction efficiency, the ultrasound (500 W, 40 kHz) time was selected as 20 min.

[0086] Example 3

[0087] Based on Examples 1 and 2, this example adopts the optimal conditions of Example 2 to prepare the test solution conditions and the optimal detection conditions, and analyzes the detection results of the detection method of the present invention.

[0088] 1. Applicability and specificity test

[0089] 5 μL of reference solution, sample solution, and negative sample solution were accurately drawn, and the chromatographic conditions of Example 2 were used for injection and determination. The chromatograms of the sample solution, mixed reference solution, and negative sample solution were as shown in FIG. Figures 1 to 3 shown.

[0090] By the attached Figures 1 to 3The data show that the chromatograms of the test solution, mixed reference solution and negative sample solution of the present invention have good separation of the chromatographic peaks, indicating that the conditions of the determination method provided by the present invention can effectively separate different components, thereby accurately determining the content of each component; in the chromatogram of the negative sample solution, no chromatographic peaks were detected at the corresponding chromatographic peak positions of pekinenol A, pekinenol B and hesperidin reference substances. This indicates that the negative sample solution does not contain these three components, further verifying the specificity of the chromatographic conditions. This shows that the method system of the present invention has good applicability and can accurately and reliably determine the content of pekinenol A, pekinenol B and hesperidin in the sample.

[0091] 2. Linear relationship and detection limit

[0092] 1 μL, 2 μL, 3 μL, 5 μL and 8 μL of the mixed reference solution were accurately pipetted respectively, and the measurement was carried out under the optimal chromatographic conditions of Example 2. The injection volume (x) was used as the abscissa and the peak area (y) was used as the ordinate to draw the standard working curve to obtain the linear equation; the mixed reference solution was diluted in sequence, and the mass concentration corresponding to 3 times the signal-to-noise ratio was used as the detection limit, and the mass concentration corresponding to 10 times the signal-to-noise ratio was used as the quantitative limit; the linear range, linear equation, correlation coefficient, detection limit and quantitative limit of the mass concentrations of the three components are shown in Table 4.

[0093] Table 5: Mass concentration range, linear equation, correlation coefficient, detection limit and quantification limit of the three components

[0094]

[0095] It can be seen from the data in Table 5 that for pecupine A, its mass concentration range is between 0.063μg and 0.504μg, the linear equation is y=2077.1x+2.1897, and the correlation coefficient is as high as 0.9999, indicating that there is an excellent linear relationship between the peak area of ​​pecupine A and the injection amount; the mass concentration range of pecupine B is between 0.032 and 0.256μg, the linear equation is y=1705.1x+0.9964, and the correlation coefficient is also as high as 0.9999, indicating that there is also a good linear relationship between the peak area of ​​pecupine B and the injection amount; the mass concentration range of hesperidin is between 0.282 and 2.256μg, the linear equation is y=1819.3x+58.741, and the correlation coefficient is 0.9995, indicating that there is a strong linear relationship between the peak area of ​​hesperidin and the injection amount.

[0096] The detection limit of praeruptorin A was 0.007 μg·μL -1 ; The detection limit of pecuplantin was 0.012μg·μL -1 ; The detection limit of hesperidin is 0.015μg·μL -1, indicating that the detection sensitivity of peucedanum argentein A, peucedanum argentein B and hesperidin is relatively high.

[0097] The limit of quantification of praeruptorin A was 0.016 μg·μL -1 The limit of quantification of pecuplantin was 0.027 μg·μL -1 ; The limit of quantification of hesperidin was 0.056 μg·μL -1 , indicating that peucedanum argentein A, peucedanum argentein B and hesperidin can also be accurately quantified at lower concentrations.

[0098] In summary, the three components have good linear relationships within their respective mass concentration ranges and have high limits of detection and quantification, which provides a reliable basis for subsequent quantitative analysis.

[0099] 3. Precision test

[0100] Six samples of the same batch of Chutan Zhisou Pills (batch number 20010706) were taken and tested according to the test sample solution preparation method and optimal chromatographic conditions in Example 2. The average chromatographic peak area and relative standard deviation (RSD) of the tested components in the six samples were calculated. The results are shown in Table 6.

[0101] Table 6: Precision test results

[0102]

[0103] It can be seen from the data in Table 6 that the method of the present invention has good precision for determining the content of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills. The RSDs of the peak areas of praeruptorin A, praeruptorin B and hesperidin are 0.99%, 1.05% and 1.01% (n=6), respectively, indicating that the chromatographic peak area measurement values ​​of the key components of praeruptorin A, praeruptorin B and hesperidin in the 6 Chutan Zhisou Pills samples are very close, and the method has high precision.

[0104] 4. Stability test

[0105] A batch of newly prepared sample solutions (batch number 20010706) were taken and stability tests were carried out under the chromatographic conditions of Example 2 at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h. The chromatographic peak area of ​​each component was recorded, and the relative standard deviation of the chromatographic peak area measured at different time points was calculated. The results of the stability test are shown in Table 7.

[0106] Table 7: Stability test results

[0107]

[0108]

[0109] It can be seen from the data in Table 7 that for purpurogenol A, purpurogenol B and hesperidin, the chromatographic peak areas fluctuated slightly at different time points (0-24 hours), indicating that the concentrations of these components remained stable in the solution during the 24-hour observation period; the RSDs of the peak areas of purpurogenol A, purpurogenol B and hesperidin were 0.33%, 0.42% and 0.52%, respectively, and the RSD values ​​of the chromatographic peak areas of purpurogenol A, purpurogenol B and hesperidin were all low, indicating that their concentrations remained basically unchanged in the solution.

[0110] 5. Spike recovery test

[0111] Accurately weigh 6 samples of the same batch (batch number: 20010706) with known contents of each component, add 10 mL of mixed reference solution, ensure that the added reference solution contains sufficient amounts of pecupine A, pecupine B and hesperidin, so as to carry out spike recovery test, prepare the test solution and determine the content according to the method of Example 2, inject and determine according to the chromatographic conditions of Example 2, record and save the chromatogram and the corresponding peak area or peak height data, calculate the content of pecupine A, pecupine B and hesperidin in each sample according to the measured peak area or peak height data, and calculate the recovery rate using the formula: recovery rate = (measured amount - background value) / spiked amount × 100%; wherein, the measured amount is the content of the component measured in the sample after spiked, the background value is the content of the component in the original sample, and the spiked amount is the content of the component in the added reference solution. For each component, the average recovery rate and relative standard deviation (RSD) of all samples are calculated, and the results are shown in Table 8.

[0112] Table 8: Spike recovery test results

[0113]

[0114]

[0115] It can be seen from the data in Table 8 that the average recoveries of praeruptorin A, praeruptorin B and hesperidin are 96.90%, 102.98% and 101.75%, respectively, and the recoveries are close to 100%, indicating that the method of the present invention has high accuracy in determining these components; the RSDs of praeruptorin A, praeruptorin B and hesperidin are 0.85%, 0.44% and 0.90% (n=6), respectively, and the RSD values ​​are low, indicating that the determination results between different samples have high consistency and stability.

[0116] In summary, the results of the spike recovery test verified the reliability and accuracy of the method of the present invention. The method can accurately determine the content of peucedanum argentein A, peucedanum argentein B and hesperidin in the sample, and the determination results are stable and reliable.

[0117] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for simultaneously determining the effective ingredients of praeruptoside A, praeruptoside B and hesperidin in Chutan Zhisou Pills, characterized in that: The specific steps include: S1, take Chutan Zhisou Pills, grind them with diatomaceous earth, dissolve them, and ultrasonically treat them to obtain the test solution; S2, peucedanum praeruptoside A, peucedanum praeruptoside B and hesperidin were used as reference substances, dissolved in methanol, and hesperidin was dissolved and heated in a water bath to prepare a mixed reference substance solution; S3, performing high performance liquid chromatography analysis on the test solution and the reference solution under the same conditions, recording the corresponding chromatograms, and calculating the peak area using an external standard method to obtain the test results; Octadecylsilane bonded silica gel was used as a filling column, acetonitrile was used as a mobile phase A, and a phosphoric acid aqueous solution with a mass concentration of 0.1% to 0.2% was used as an eluent for gradient elution. The gradient elution procedure was as follows:

2. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S1, the added amount of the diatomaceous earth is 10% to 20% of the mass of the Chutan Zhisou Pills.

3. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S1, the dissolving solvent is any one of pure methanol, 50% methanol aqueous solution, 70% methanol aqueous solution and chloroform.

4. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S1, the ultrasonic treatment conditions are: power 450-550 W, frequency 30-60 KHz, 15-25 min.

5. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S2, the water bath temperature is 50°C to 70°C, and the time is 1 min to 2 min.

6. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S2, the mixed reference solution has a mass concentration of 63.02 μg / mL of peucedanum apigenin A, a mass concentration of 32.52 μg / mL of peucedanum apigenin B, and a mass concentration of 282.27 μg / mL of hesperidin per 1 mL of the mixed reference solution.

7. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S3, the mass concentration of the phosphoric acid aqueous solution is 0.2%.

8. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 1, characterized in that: In S3, the HPLC detection wavelength is 280-400 nm, the column temperature is 25-35° C., and the flow rate is 0.8-1.2 mL / min.

9. The method for simultaneously determining the effective ingredients of praeruptoside A, praeruptoside B and hesperidin in Chutan Zhisou Pills according to claim 8, characterized in that: The column temperature was 30°C and the flow rate was 1.0 mL / min.

10. The method for simultaneously determining the effective ingredients of praeruptorin A, praeruptorin B and hesperidin in Chutan Zhisou Pills according to claim 8, characterized in that: The detection wavelengths of the praeruptorin A and praeruptorin B are 321 nm, and the detection wavelength of the hesperidin is 284 nm.

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