Construction method of characteristic chromatogram of Chinese patent medicine and characteristic chromatogram thereof

Abstract

The invention relates to a construction method of a characteristic chromatogram of a Chinese patent medicine and the characteristic chromatogram of the Chinese patent medicine, and the method adopts an HPLC (High Performance Liquid Chromatography) method to measure the content of imperatorin serving as an effective component in Zhengkang capsules and constructs the high performance liquid chromatography characteristic chromatogram of the Zhengkang capsules. 10 characteristic peaks are determined as characteristic spectrums and are respectively derived from dragon's blood, rheum officinale and radix angelicae, wherein the components derived from the dragon's blood comprise loureirin B (peak 2) and pterostilbene (peak 4); components from rheum officinale comprise a peak 1 (aloe-emodin), a peak 3, a peak 6 (emodin), a peak 9 and a peak 10; the components from the radix angelicae comprise imperatorin (peak 5), peak 7 and peak 8; main components such as polyphenols, anthraquinones and furocoumarins are covered, and a quality evaluation system of the Chinese patent medicine Zhikang capsule is constructed; the method is good in specificity, precision, repeatability and solution stability, and can be used for standardizing production of Chinese patent medicine Zhikang capsules and ensuring uniformity and stability of internal quality of products to provide comprehensive quality control.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicine preparations, and specifically relates to a method for constructing a characteristic spectrum of a traditional Chinese medicine and a characteristic spectrum thereof. Background Art

[0002] Zhikang Capsule is an exclusive product produced by Xi'an Qianhe Pharmaceutical Co., Ltd. It is composed of 14 medicinal herbs, including rhubarb, coptis root, notoginseng, angelica root, donkey-hide gelatin, dragon bone (calcined), bletilla striata, vinegar myrrh, cuttlebone, madder, dragon's blood, liquorice, pearl and borneol. It has the effects of clearing away heat, cooling blood and stopping bleeding, removing blood stasis, promoting tissue regeneration and relieving pain. It is mainly used clinically to treat various bleeding diseases such as traumatic hemorrhage, metrorrhagia, hematemesis and bloody stool. Zhikang Capsule has been on the market for many years because of its definite efficacy and high safety, and has been recognized and trusted by medical workers and patients. It has been included in many guidelines, pathways and textbooks such as "Guidelines for the Clinical Application of Chinese Patent Medicines", "International Guidelines for Clinical Practice of Traditional Chinese Medicine", "Guidelines for Chronic Disease Management of Peptic Ulcer in the Elderly", "Guidelines for the Integrated Diagnosis and Treatment of Abnormal Uterine Bleeding with Ovulatory Disorders with Traditional Chinese and Western Medicine", "Interpretation of Clinical Pathway Therapeutic Drugs", "Modern Anorectal Surgery", "Anorectal Surgical Nursing", and "Obstetrics and Gynecology Integrating Traditional Chinese and Western Medicine" and is recommended for clinical use; because Zhikang Capsules are safe and effective, its standards were included in the "Chinese Pharmacopoeia" in 2015, and the current implementation standard is the current edition of the "Chinese Pharmacopoeia".

[0003] Although the current quality standards of Zhikang Capsules have carried out microscopic identification of Bletilla striata in the prescription, thin layer identification of rhubarb, coptis chinensis, Panax notoginseng, and dragon's blood, limit inspection of rhein in rhubarb, content determination of rhein and chrysophanol in rhubarb, and content determination of volatile borneol, its quality standards are higher than those of other Chinese patent medicines, but these quality control indicators cannot fully reflect the overall quality of Zhikang Capsules. In recent years, the method of using fingerprints or characteristic spectra of traditional Chinese medicines to control their overall quality has been widely used in view of the multi-component, multi-target, and multi-pathway characteristics of traditional Chinese medicines. The use of fingerprints or characteristic spectra can, on the one hand, effectively identify the authenticity of samples through the characteristics of the spectra, and on the other hand, effectively control the quality of the product by controlling the area, proportion, or retention time of the main characteristic peaks of the spectra, ensuring the stability and consistency of the product quality, thereby further ensuring the quality and efficacy of the product. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for constructing a characteristic spectrum of Zhikang Capsules, a Chinese patent medicine, and its characteristic spectrum. The characteristic spectrum established by the method can more comprehensively and effectively perform intrinsic quality control on Zhikang Capsules.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for establishing a characteristic spectrum of a Chinese patent medicine for clearing heat, cooling blood, stopping bleeding, removing blood stasis, promoting tissue regeneration and relieving pain. The Chinese patent medicine is composed of rhubarb, coptis root, notoginseng, angelica dahurica, donkey-hide gelatin, dragon bone (calcined), bletilla striata, myrrh, cuttlebone, madder, dragon's blood, liquorice, pearl and borneol.

[0007] The present invention provides a method for constructing a characteristic spectrum of Zhikang Capsule, which specifically comprises the following steps:

[0008] 1) Chromatographic conditions and system suitability test

[0009] The chromatographic column is filled with octadecylsilane bonded silica gel; the mobile phase is acetonitrile as phase A and 0.1% by volume phosphoric acid aqueous solution as phase B, and gradient elution is performed; the column temperature is 35°C to 45°C; the ultraviolet detector has a detection wavelength of 220nm to 280nm; the flow rate is 0.8ml / min to 1.2ml / min; the theoretical plate number is calculated based on the imperatorin peak and should not be less than 10,000;

[0010] 2) Preparation of reference solution

[0011] Take an appropriate amount of imperatorin reference substance, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml as the reference substance solution;

[0012] 3) Preparation of test solution

[0013] Take about 0.5-1.5g of the contents of Zhikang capsules, accurately weigh, place in a stoppered conical bottle, accurately add 10-30ml of 70%-80% methanol, and ultrasonically treat for 15-45 minutes at an ultrasonic power of 300W and a frequency of 40kHz; cool, filter, and take the filtrate to obtain;

[0014] 4) Determination

[0015] Accurately pipette 2-10 μl of the reference solution and the test solution respectively, inject into the HPLC to determine the content of imperatorin, and construct the HPLC characteristic spectrum of Zhikang Capsules.

[0016] According to a further description of the present invention: the chromatographic column is Waters XBridge-C18 (4.6×250 mm, 5 μm); the flow rate is 1.0 mL / min; the column temperature is 40° C.; and the detection wavelength is 254 nm.

[0017] According to a further description of the present invention: the gradient elution procedure is as follows, wherein the mobile phase ratios are all volume percentages: 0-20 min, mobile phase A is 35%, mobile phase B is 65%;

[0018] 20-40 min, mobile phase A is 35%-45%, mobile phase B is 65%-55%;

[0019] 40-60 min, mobile phase A is 45%-70%, mobile phase B is 55%-30%;

[0020] 60-70 min, mobile phase A is 70%-35%, mobile phase B is 30%-55%.

[0021] According to a further description of the present invention: in step 3), about 1.0 g of the content of Zhikang Capsules is taken, accurately weighed, placed in a stoppered conical flask, 20 ml of 75% methanol is accurately added, and ultrasonic treatment is performed for 30 minutes with an ultrasonic power of 300 W and a frequency of 40 kHz; cooled, filtered, and the filtrate is obtained.

[0022] The present invention also provides a characteristic spectrum of Zhikang capsule obtained by the construction method. The present invention also provides the application of the characteristic spectrum of Zhikang capsule in evaluating the quality and content determination of Zhikang capsule.

[0023] According to a further description of the present invention: the characteristic spectrum should present 10 characteristic peaks, among which the retention time of peak 5 should correspond to the retention time of the imperatorin reference peak; taking peak 5 corresponding to the imperatorin reference peak as the reference peak S, the relative retention times of peaks 1 to 4, peaks 6 to 10 and peak S are calculated: the relative retention time of each characteristic peak should be within ±10% of the specified value, and the specified value is: 0.40 (peak 1), 0.69 (peak 2), 0.83 (peak 3), 0.92 (peak 4), 1.04 (peak 6), 1.12 (peak 7), 1.23 (peak 8), 1.37 (peak 9), and 1.50 (peak 10).

[0024] Furthermore, the characteristic spectrum determined the sources of 10 common characteristic peaks, which were respectively from dragon's blood, rhubarb, and angelica dahurica; among them, the components derived from dragon's blood included dragon's blood acid B (peak 2) and pterostilbene (peak 4); the components derived from rhubarb included peak 1 (aloe-emodin), peak 3 (unknown component), 6-emodin (rhubarb peak), peak 9 (unknown component), and peak 10 (unknown component); the components derived from angelica dahurica included imperatorin (peak 5), peak 7, (unknown component) and peak 8 (unknown component).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The method for constructing the characteristic spectrum of Zhikang capsule of the Chinese patent medicine of the present invention adopts high performance liquid chromatography for gradient elution, which can not only determine the content of imperatorin, but also construct the high performance liquid chromatography characteristic spectrum of Zhikang capsule. Ten characteristic peaks are determined as characteristic spectrum, and the components from dragon's blood are dragon's blood B (peak 2) and pterostilbene (peak 4); the components from rhubarb are peak 1 (aloe-emodin), peak 3, peak 6 (emodin), peak 9, peak 10; the components from angelica dahurica are imperatorin (peak 5), peak 7, peak 8; the main components such as polyphenols, anthraquinones and furanocoumarins are covered, and the quality evaluation system of Zhikang capsule of the Chinese patent medicine is constructed; the method has good specificity, precision, repeatability and solution stability, and can be used to standardize the production of Zhikang capsule and ensure the uniformity and stability of the intrinsic quality of the product to provide comprehensive quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings:

[0028] Figure 1 It is the liquid chromatogram of different mobile phase gradients;

[0029] Figure 2 Examine the chromatograms for different acid regulators;

[0030] Figure 3 Examine the chromatograms for different acid regulator concentrations;

[0031] Figure 4 It is the liquid chromatogram of different detection wavelengths;

[0032] Figure 5 The liquid chromatograms of different chromatographic columns;

[0033] Figure 6 The liquid chromatograms at different column temperatures;

[0034] Figure 7 The liquid chromatograms at different flow rates;

[0035] Figure 8 The liquid chromatograms with different injection volumes;

[0036] Fig. 9 Blank, reference, and sample chromatograms for system suitability testing;

[0037] Fig.10 To investigate the relevant spectra (Dragon Blood Drainage, Rhubarb) for specificity;

[0038] Fig.11 To investigate the relevant spectra (Bletilla striata, donkey-hide gelatin) for specificity;

[0039] Fig.12 To investigate the relevant spectra (Angelica Dahuricae, Rubia cordifolia) for specificity;

[0040] Fig.13 To investigate the relevant atlases for specificity (pearl, vinegar myrrh);

[0041] Fig.14 To investigate the relevant atlases for specificity (Cuttlebone, Borneol);

[0042] Fig.15 To investigate the relevant spectra for specificity (calcined dragon bone, Panax notoginseng);

[0043] Fig.16 To investigate the relevant spectra for specificity (Glycyrrhiza uralensis and Coptis chinensis);

[0044] Fig.17 It is the characteristic spectrum of the test solution of the present invention;

[0045] Fig.18 Determine the spectrum for multiple batches;

[0046] In the figure, peak 1: aloe-emodin; peak 2: dragon's blood acid B; peak 4: pterostilbene; peak 5: imperatorin (S); peak 6: emodin. DETAILED DESCRIPTION

[0047] The following embodiments may help those skilled in the art to more fully understand the present invention, but shall not limit the present invention in any way.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified; some of the instruments, reagents and drugs used are as follows:

[0049] Instruments: Agilent 1260 HPLC (Agilent Technologies Inc.), Waters 2695 HPLC (Waters Technologies Inc.), AB Sciex Triple 4600 high-resolution mass spectrometer (SCIEX), Waters XBridge-C18 (4.6×250mm, 5μm) chromatographic column, Agilent Eclipse XDB-C18 (4.6×250mm, 5μm) chromatographic column, Waters Atlantis TM T3 (4.6×250mm, 5μm) chromatographic column, one-tenth balance (Mettler-Toledo, ME104), one-millionth balance (Mettler-Toledo, XPR2), CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd., KQ-300DB), electric constant temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., HWS26), coolant circulation pump (Shanghai Dayan Instrument Equipment Co., Ltd., LTC-5 / 30), high-speed desktop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., H1650-W).

[0050] Reagents: methanol (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.); acetonitrile (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.); water (distilled water, Guangzhou Watsons Food and Beverage Co., Ltd.); formic acid (mass spectrometry grade, Shanghai Anpu Experiment Technology Co., Ltd.); phosphoric acid (chromatographic grade, Shanghai Anpu Experiment Technology Co., Ltd.).

[0051] Test drugs: Zhikang Capsules (Xi'an Qianhe Pharmaceutical Co., Ltd.), rheumatoid arthritis reference solution, aloe-rheumatoid arthritis reference solution, imperatorin reference solution, pterostilbene reference solution, dragon's blood A reference solution, and dragon's blood B reference solution, all from Shanghai Shidande Standard Technology Service Co., Ltd.

[0052] Example 1: Establishment of a characteristic spectrum method of Zhikang capsule contents by HPLC

[0053] Based on a large number of experimental explorations, a method for determining the content of imperatorin in Zhikang Capsules by HPLC was preliminarily established, and a characteristic spectrum for content determination in Zhikang Capsules was established.

[0054] 1.1 Choice of mobile phase

[0055] 1.1.1 Selection of different gradients in mobile phase:

[0056] Preparation of reference solution: Take 12.5 mg of imperatorin reference substance, place it in a 25 ml volumetric flask, add methanol to make a solution containing 0.5 mg per 1 ml, as the reference solution.

[0057] Preparation of the test solution: Take about 1.0 g of the content of Zhikang Capsule preparation, accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of 75% methanol, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain.

[0058] Chromatographic conditions and system suitability test:

[0059] Chromatographic column: Octadecylsilane bonded silica gel is used as filler, and the chromatographic column is: Waters XBridge-C18 (4.6×250mm, 5μm);

[0060] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution, flow rate: 1.0mL / min; column temperature: 40℃; detection wavelength: 254nm; theoretical plate number is calculated based on the imperatorin peak and should not be less than 10000;

[0061] Determination: Pipette 5 μl of the above-mentioned reference solution and Zhikang Capsule test solution and inject them into liquid chromatography for determination.

[0062] In order to investigate the effect of different mobile phase gradients on the characteristic spectrum of Zhikang Capsules, a gradient with better characteristic peak response and better separation effect was selected. Method 1: The gradient elution procedure is as follows, where the mobile phase ratios are all volume percentages:

[0063] 0-20min, mobile phase A is 35%, mobile phase B is 65%;

[0064] 20-40 min, mobile phase A is 35%-45%, mobile phase B is 65%-55%;

[0065] 40-60 min, mobile phase A is 45%-70%, mobile phase B is 55%-30%;

[0066] 60-70 min, mobile phase A is 70%-35%, mobile phase B is 30%-55%.

[0067] According to the above method 1, except for adjusting the gradient, the data were collected according to the elution gradient in Table 1. The test results are shown in Table 2 and Figure 1 .

[0068] Table 1: Gradient adjustment survey table

[0069]

[0070] Note: Phase A is acetonitrile.

[0071] Table 2: Gradient adjustment investigation results

[0072]

[0073] Note: The two values ​​of separation are the separation between the target peak and its preceding and succeeding peaks. NA means there are no peaks before or after the target peak.

[0074] The results showed that the retention time of some characteristic peaks in method 4 was too early, making them difficult to distinguish. In methods 2, 3 and 5, the retention time of peak 11 was around 60 minutes, close to the end of the effective elution time. Considering the different dead volumes of different instruments and equipment, the retention time of this peak may exceed the effective elution time. Therefore, after comprehensive consideration, the gradient elution conditions of method 1 were selected as the final conditions.

[0075] 1.1.2 Selection of different acid regulators in mobile phase:

[0076] The effects of different mobile phase acid regulators on the characteristic spectrum of Zhikang Capsules were investigated, and the acid regulator with better characteristic peak response and better separation effect was selected as the mobile phase additive. The test solution was prepared according to the same preparation method as "1.1.1 in Example 1", and the gradient elution condition was selected as method 1. Except for adjusting the mobile phase acid regulator, the rest of the data was collected according to the chromatographic conditions under "1.1.1 in Example 1"; the test results are shown in Table 3 and Figure 2 .

[0077] Table 3: Results of investigation on different acid regulators

[0078]

[0079] The results showed that there were obvious differences in the chromatographic behaviors under different acid regulators. When the acid regulator was formic acid, peak 4 was missing, so phosphoric acid was selected as the acid regulator.

[0080] The effect of different mobile phase acid regulator phosphoric acid concentrations on the characteristic spectrum of Zhikang Capsules was further investigated to select the concentration of phosphoric acid regulator with better characteristic peak response and better separation effect. The experimental results are shown in Table 4 and Figure 3 .

[0081] Table 4: Results of investigation of different acid regulator concentrations

[0082]

[0083] The results showed that when no phosphoric acid was added, peak 4 was missing, and there was no obvious difference in the chromatographic behavior as the concentration of the additive decreased. Considering the pH tolerance and service life of the chromatographic column, the concentration of additive phosphoric acid was selected to be 0.1%, that is, a phosphoric acid aqueous solution with a volume fraction of 0.1% was used as mobile phase B.

[0084] In summary, the optimal mobile phase and gradient system conditions are: the mobile phase is acetonitrile as phase A and a phosphoric acid aqueous solution with a volume fraction of 0.1% as phase B, and gradient elution is performed; the gradient elution program is as follows, wherein the mobile phase ratios are all volume percentages: 0-20 min, mobile phase A is 35%, and mobile phase B is 65%; 20-40 min, mobile phase A is 35%-45%, and mobile phase B is 65%-55%; 40-60 min, mobile phase A is 45%-70%, and mobile phase B is 55%-30%; 60-70 min, mobile phase A is 70%-35%, and mobile phase B is 30%-55%.

[0085] 1.2 Selection of detection wavelength

[0086] The test solution was prepared according to the same preparation method as in "Example 1, 1.1.1". Except for adjusting the detection wavelength, the data were collected according to the chromatographic conditions in "Example 1, 1.1.1". The test results are shown in Figure 4The results showed that the responses of the characteristic peaks at different detection wavelengths such as 220nm, 254nm, 280nm, 300nm, 330nm, and 360nm were quite different. Taking into account the responses of the characteristic peaks, the interference of impurities around the characteristic peaks, and the flatness of the baseline, 254nm was selected as the detection wavelength.

[0087] 1.3 Selection of different chromatographic columns

[0088] The effects of different chromatographic columns on the characteristic spectrum of Zhikang Capsules were investigated. Waters XBridge-C18 (4.6×250mm, 5μm, column number: column 1), Agilent Eclipse XDB-C18 (4.6×250mm, 5μm, column number: column 2), Waters Atlantis TM T3 (4.6×250mm, 5μm, number: column 3), the test solution was prepared according to the same preparation method as in "1.1.1 in Example 1". Except for adjusting the chromatographic column, the rest was carried out according to the chromatographic condition gradient elution under "1.1.1 in Example 1" and method 1 was used for data collection. The appropriate chromatographic column was selected by observing the tailing factor and separation degree of each characteristic peak in the spectrum; the test results are shown in Table 5 and Figure 5 .

[0089] Table 5: Results of investigations on different chromatographic columns

[0090]

[0091] The results showed that different types of filler columns had a certain influence on the characteristic spectra. By comparing the peak area, peak height, and spectra of each chromatographic peak to locate the characteristic peaks, it was found that when column 2 was used, peak 4 was missing; when column 3 was used, peaks 1 to 3 were all missing; when column 1 was used, the characteristic peaks were evenly distributed, the peak shape was good, and the spectrum was more beautiful. Considering the subsequent optimization process, Waters X-Bridge C18 (4.6×250mm, 5μm, number: column 1) was selected as the chromatographic column for this method.

[0092] 1.4 Different column temperature selection

[0093] The influence of different column temperatures on the characteristic spectrum of Zhikang Capsules was investigated to select the flow rate with better characteristic peak response and better separation effect. The test solution was prepared according to the same preparation method as in "1.1.1 in Example 1". Except for adjusting the column temperature, the rest of the data was collected according to the chromatographic conditions of method 1 for gradient elution under item 1.1.1 in "1.1.1 in Example 1". The test results are shown in Table 6 and Figure 6 .

[0094] Table 6: Results of investigation at different column temperatures

[0095]

[0096] The results showed that there were certain differences in chromatographic behavior at different column temperatures. As the column temperature increased, the retention time of each characteristic peak shortened. From an overall perspective, when the column temperature was 40°C, there was less interference from impurities around the characteristic peaks, so the column temperature was selected to be 40°C.

[0097] 1.5 Different flow rate options

[0098] The effects of different flow rates on the characteristic spectrum of Zhikang Capsules were investigated to select the flow rate with better characteristic peak response and better separation effect.

[0099] The test solution was prepared according to the same preparation method as in Example 1. Except for adjusting the flow rate, the rest of the data was collected according to the chromatographic conditions of Method 1 for gradient elution under 1.1.1 in Example 1. The test results are shown in Table 7 and Figure 7 .

[0100] Table 7: Results of investigation at different flow rates

[0101]

[0102] The results showed that there were certain differences in chromatographic behavior at different flow rates. As the flow rate increased, the retention time of each characteristic peak shortened. When the flow rate was 0.8 ml / min, there was obvious interference between peaks 4 and 5; when the flow rate was 1.2 ml / min, there was certain interference with peak 1, so the flow rate was selected to be 1.0 ml / min.

[0103] 1.6 Different injection volume selection

[0104] The influence of different injection volumes on the characteristic spectrum of Zhikang Capsules was investigated to select the injection volume with better characteristic peak response and better separation effect. The test solution was prepared according to the same preparation method as in "1.1.1 in Example 1". Except for adjusting the injection volume, the rest of the data was collected according to the chromatographic conditions of method 1 for gradient elution under item 1.1.1 in "1.1.1 in Example 1". The test results are shown in Table 8 and Figure 8 .

[0105] Table 8: Results of different injection volumes

[0106]

[0107] The results showed that there was no significant difference in chromatographic behavior with different injection volumes, and an injection volume of 5 μl was comprehensively selected for subsequent investigation.

[0108] Example 2: Investigation of the preparation method of the test solution

[0109] The extraction solvent, extraction method, extraction time and solid-liquid ratio used in the preparation of Zhikang Capsule test solution were investigated to determine the optimal preparation method of Zhikang Capsule test solution.

[0110] 2.1 Investigation of extraction solvent

[0111] The effects of different extraction solvents on the characteristic spectrum of this product were investigated. 25%, 50% and 75% methanol were used as extraction solvents respectively. The peak shape and separation of the characteristic peaks were observed and the "total peak area / sample weight" of the characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectrum of this product and select the best extraction solvent.

[0112] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 25% methanol, 50% methanol, 75% methanol 20ml, ultrasonic treatment (300W, 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain. According to the gradient elution under "1.1.1 in Example 1", the chromatographic conditions of method 1 are used for data collection; the test results are shown in Table 9.

[0113] Table 9: Investigation of different extraction solvents

[0114]

[0115] The results showed that as the proportion of methanol increased, the "total peak area / sample weight" increased accordingly, and the extraction effect of each characteristic peak was significantly improved, so 75% methanol was selected as the extraction solvent.

[0116] 2.2 Different extraction times

[0117] The effects of different extraction times on the characteristic spectrum of this product were investigated. The extraction times were set as 15 minutes, 30 minutes, and 45 minutes respectively. The peak shape and separation of the characteristic peaks were observed and the “total peak area / sample weight” of the characteristic peaks was calculated to compare the effects of different extraction times on the characteristic spectrum of this product and select the optimal extraction time.

[0118] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 20ml of 75% methanol, ultrasonically treat (300W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, cool, filter, and take the filtrate to obtain. Data collection is carried out according to the chromatographic conditions of method 1 for gradient elution under item 1.1.1 of "Example 1"; the test results are shown in Table 10.

[0119] Table 10: Investigation of different extraction times

[0120]

[0121] The results showed that as the extraction time increased, the "total peak area / sample weight" tended to be stable, and the influence of different extraction times on the extraction effect was not obvious, so a more moderate 30 minutes was selected as the extraction time.

[0122] 2.3 Investigation of different extraction methods

[0123] 2.3.1 Investigation of different extraction methods

[0124] The effects of different extraction methods on the characteristic spectrum of this product were investigated. Ultrasound and reflux were used as the extraction methods respectively. The peak shape and separation degree of the characteristic peaks were observed and the “total peak area / sample weight” of the characteristic peaks was calculated to compare the effects of different extraction methods on the characteristic spectrum of this product and select the best extraction method.

[0125] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 2 portions, accurately add 20ml of 75% methanol, respectively ultrasonically treat (300W, 40kHz), reflux for 30 minutes, cool, filter, and take the filtrate to obtain. The rest is carried out according to the gradient elution under "1.1.1 in Example 1" and the chromatographic conditions of method 1 for data collection; the test results are shown in Table 11.

[0126] Table 11: Investigation of different extraction methods

[0127]

[0128] The results showed that different extraction methods had little effect on the extraction efficiency of each component, and the chromatographic behavior had no significant change. For ease of operation, ultrasonic extraction was selected.

[0129] 2.3.2 Investigation of different extraction material-liquid ratios

[0130] The effects of different extraction material-liquid ratios on the characteristic spectrum of this product were investigated, with ratios of 1:10, 1:20 and 1:50 respectively. The peak shapes and separations of the characteristic peaks were observed, and the “total peak area / sample weight × extraction solution volume” of the characteristic peaks was calculated to compare the effects of different extraction material-liquid ratios on the characteristic spectrum of this product, and the optimal extraction material-liquid ratio was selected.

[0131] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 75% methanol 10ml, 20ml, 50ml respectively, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain. The rest is carried out according to the gradient elution under "1.1.1 in Example 1" and the chromatographic conditions of method 1 for data collection; the test results are shown in Table 12.

[0132] Table 12: Investigation of different extraction solid-liquid ratios

[0133]

[0134] The results showed that as the solid-liquid ratio decreased, "total peak area / sample weight × extraction solution volume" was relatively stable, indicating that the effect of the extraction solid-liquid ratio on the extraction efficiency was not significant, and 1:20 was comprehensively selected as the extraction solid-liquid ratio.

[0135] Example 3: System suitability test and specificity investigation

[0136] As can be seen from Examples 1-2, the optimal HPLC method for determining the characteristic spectrum of Zhikang Capsules specifically comprises the following steps:

[0137] 1) Chromatographic conditions and system suitability test

[0138] The chromatographic column is Waters XBridge-C18 (4.6×250mm, 5μm); the column temperature is 40°C; the UV detector has a detection wavelength of 254nm; the flow rate is 1.0ml / min; the theoretical plate number is calculated based on the imperatorin peak and should not be less than 10000;

[0139] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a 0.1% by volume phosphoric acid aqueous solution, and gradient elution is performed. The gradient elution procedure is as follows, wherein the mobile phase ratios are all volume percentages:

[0140] 0-20min, mobile phase A is 35%, mobile phase B is 65%;

[0141] 20-40 min, mobile phase A is 35%-45%, mobile phase B is 65%-55%;

[0142] 40-60 min, mobile phase A is 45%-70%, mobile phase B is 55%-30%;

[0143] 60-70 min, mobile phase A is 70%-35%, mobile phase B is 30%-55%;

[0144] 2) Preparation of reference solution

[0145] Take an appropriate amount of imperatorin reference substance, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml as the reference substance solution;

[0146] 3) Preparation of test solution

[0147] Take about 1.0g of the contents of Zhikang Capsules, weigh accurately, place in a stoppered conical flask, accurately add 20ml of 75% methanol, and treat with ultrasound for 30 minutes at an ultrasonic power of 300W and a frequency of 40kHz; cool, filter, and take the filtrate to obtain;

[0148] 4) Determination

[0149] Accurately pipette 2 to 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, determine the content of imperatorin, and construct the high performance liquid chromatographic characteristic spectrum of Zhikang Capsules.

[0150] 3.1 System suitability test

[0151] The test sample solution and the reference solution were prepared according to the law, and the analysis was carried out according to the chromatographic conditions under "Example 3", the chromatographic conditions and system suitability of the characteristic spectrum of Zhikang Capsules were examined, and whether the blank solvent would cause interference was examined.

[0152] The results showed that the blank solvent had no interference with the sample, and the retention time of the reference substance in the reference solution was consistent with the retention time of the corresponding component in the test sample. Fig. 9 .

[0153] 3.2 Exclusivity Investigation

[0154] Prepare the test solution and reference solution, medicinal material slices and negative solution according to the law, and analyze them according to the chromatographic conditions under "Example 3" to verify the source of each characteristic peak in the Zhikang Capsule test solution. Figure 10-16 . According to the results, the 10 characteristic peaks displayed in the collected characteristic spectrum are from dragon's blood, rhubarb, and angelica dahurica. Among them, the components derived from dragon's blood are dragon's blood B (peak 2) and pterostilbene (peak 4); the components derived from rhubarb are aloe-emodin (peak 1), peak 3, rhein (peak 6), peak 9, and peak 10; the components derived from angelica dahurica are imperatorin (peak 5), peak 7, and peak 8.

[0155] Example 4: Methodological Verification

[0156] 4.1 Repeatability

[0157] Take the same batch of Zhikang capsule samples, prepare 6 test solutions in parallel according to the test solution preparation method under Example 3, and collect data according to the specified chromatographic conditions, calculate the relative retention time and relative peak area of ​​each characteristic peak, and the results show that the RSD value of the relative retention time of each characteristic peak is less than 2%, and the RSD value of the relative peak area is less than 5%, indicating that the method has good repeatability. Detailed results are shown in Tables 13-16.

[0158] Table 13: Results of repeatability study on retention time of each characteristic peak

[0159]

[0160] Table 14: Results of repeatability test on peak areas of characteristic peaks

[0161]

[0162] Table 15: Results of the relative retention time of each characteristic peak in the repeatability study

[0163]

[0164] Table 16: Results of the relative peak areas of each characteristic peak in the repeatability study

[0165]

[0166] 4.2 Investigation of the intermediate precision of different personnel

[0167] Two experimenters, A and B, each took samples of Zhikang Capsules from the same batch, and prepared 6 test solutions in parallel according to the test solution preparation method under Example 3. Data were collected on the same instrument under the specified chromatographic conditions, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. Detailed results are shown in Tables 17-22.

[0168] Table 17: Results of the retention time of each characteristic peak in the intermediate precision study of different personnel

[0169]

[0170] Note: This table is the relevant results of person B. The relevant results of person A can be found in the relevant table under the repeatability test, the same below.

[0171] Table 18: Results of the peak areas of each characteristic peak in the intermediate precision study of different personnel

[0172]

[0173] Table 19: Results of relative retention time of characteristic peaks in the intermediate precision study of different personnel

[0174]

[0175] Table 20: Results of relative peak areas of characteristic peaks in the intermediate precision study of different personnel

[0176]

[0177] Table 21: Results of relative retention time of characteristic peaks in the intermediate precision study by different personnel (12 samples)

[0178]

[0179] Table 22: Results of relative peak areas of characteristic peaks investigated by different personnel for intermediate precision (12 samples)

[0180]

[0181] The results showed that the RSD values ​​of the relative retention time of each characteristic peak were less than 2%, and the RSD values ​​of the relative peak area were less than 5%, indicating that the method had good intermediate precision among different personnel.

[0182] Example 5, multiple batch determination

[0183] 17 batches of Zhikang capsules were prepared into test solutions according to the method of Example 3, injected into a high performance liquid chromatograph, and measured according to the chromatographic conditions of Example 3. The results are shown in Tables 23-26 and Fig.18 .

[0184] Table 23: Retention time results of multiple batches of assays

[0185]

[0186] Table 24: Peak area results of multiple batches of tests

[0187]

[0188] Table 25: Relative retention time results of multiple batches of determination tests

[0189]

[0190] Table 26: Relative peak area results of multiple batches of determination tests

[0191]

[0192] Through the detection of multiple batches of Zhikang capsules, 10 characteristic peaks were found in the chromatograms of each batch of test samples, among which the retention time of peak 5 corresponds to the retention time of the imperatorin reference peak. The peak corresponding to the imperatorin reference peak is the S peak, and the relative retention times of peaks 1 to 4, 6 to 10 and the S peak are calculated. The relative retention time of each characteristic peak is within ±10% of the specified value, and the specified value is: 0.40 (peak 1), 0.69 (peak 2), 0.83 (peak 3), 0.92 (peak 4), 1.04 (peak 6), 1.12 (peak 7), 1.23 (peak 8), 1.37 (peak 9), 1.50 (peak 10). From Tables 23-26 and Fig.18 It can be seen that there are 10 relatively obvious common peaks in the superposition diagram of the characteristic spectrum of Zhikang Capsules, and the relative retention time of the characteristic peaks in the above 17 batches of samples is very stable, with RSD less than 0.5%, far lower than the 3% specified in the standard; the relative peak area is also relatively stable, with RSD less than 4.0%, lower than the 5% specified in the standard. There is no significant difference in the relative retention time of each characteristic peak, but the relative peak area of ​​some characteristic peaks is quite different, so the retention time can be included in the standard for control.

[0193] Example 6: Accelerated stability study

[0194] The same batch (batch 20240101) of Zhikang capsules was taken for accelerated stability study at a temperature of 40°C ± 2°C and a relative humidity of 75 ± 5%. During the test period, samples were taken at the 0th month, 3rd month, and 6th month, and the relative retention time of each characteristic peak and the content of its characteristic component, imperatorin, were calculated according to the same preparation method and chromatographic conditions as in Example 3. The test results are shown in Tables 27-28.

[0195] Table 27: Analysis of results of each peak during the investigation period

[0196]

[0197] Table 28: Summary of test results

[0198] Inspection period Imperatorin content results (μg / capsule) 0 month 2.75 Accelerate March 2.76 Accelerate June 2.72

[0199] The results showed that the relative retention time of each characteristic peak was less than 2%, and each capsule contained 2.75 μg of imperatorin at 0 month, 2.76 μg of imperatorin at 3 months, and 2.72 μg of imperatorin at 6 months, and the test results were consistent. The specific spectra and results are as follows. The experiment shows that this method is suitable for the quality control of Zhikang Capsules.

[0200] The above embodiments are preferred examples for implementing the present invention, and the present invention is not limited to the above embodiments. Any non-essential additions and substitutions made by those skilled in the art based on the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a Chinese patent medicine, characterized in that: The Chinese patent medicine is Zhikang Capsule, which specifically comprises the following steps: 1) Chromatographic conditions and system suitability test The chromatographic column is filled with octadecylsilane bonded silica gel; the mobile phase is acetonitrile as phase A and 0.1% by volume phosphoric acid aqueous solution as phase B, and gradient elution is performed; the column temperature is 35°C to 45°C; the ultraviolet detector has a detection wavelength of 220nm to 280nm; the flow rate is 0.8ml / min to 1.2ml / min; the theoretical plate number is calculated based on the imperatorin peak and should not be less than 10,000; 2) Preparation of reference solution Take an appropriate amount of imperatorin reference substance, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml as the reference substance solution; 3) Preparation of test solution Take about 0.5-1.5g of the contents of Zhikang capsules, accurately weigh, place in a stoppered conical bottle, accurately add 10-30ml of 70%-80% methanol, and ultrasonically treat for 15-45 minutes at an ultrasonic power of 300W and a frequency of 40kHz; cool, filter, and take the filtrate to obtain; 4) Determination Accurately pipette 2-10 μl of the reference solution and the test solution respectively, inject into the HPLC to determine the content of imperatorin, and construct the HPLC characteristic spectrum of Zhikang Capsules.

2. The method for constructing a characteristic spectrum of a Chinese patent medicine according to claim 1, characterized in that: The chromatographic column is Waters XBridge-C18, 4.6×250 mm, 5 μm; the flow rate is 1.0 mL / min; the column temperature is 40° C.; and the detection wavelength is 254 nm.

3. The method for constructing a characteristic spectrum of a Chinese patent medicine according to claim 1, characterized in that: The gradient elution procedure is as follows, wherein the mobile phase ratios are all in volume percentage: 0-20min, mobile phase A is 35%, mobile phase B is 65%; 20-40 min, mobile phase A is 35%-45%, mobile phase B is 65%-55%; 40-60 min, mobile phase A is 45%-70%, mobile phase B is 55%-30%; 60-70 min, mobile phase A is 70%-35%, mobile phase B is 30%-55%.

4. The method for constructing a characteristic spectrum of a Chinese patent medicine according to claim 1, characterized in that: In step 3), about 1.0 g of the contents of Zhikang Capsules are taken, accurately weighed, placed in a stoppered conical flask, 20 ml of 75% methanol is accurately added, and ultrasonic treatment is performed for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz; cooled, filtered, and the filtrate is obtained.

5. The method for constructing a characteristic spectrum of a Chinese patent medicine according to claim 1, characterized in that: The characteristic spectrum should present 10 characteristic peaks, among which the retention time of peak 5 should correspond to the retention time of the imperatorin reference peak; taking peak 5 corresponding to the imperatorin reference peak as the reference peak S, the relative retention times of peaks 1 to 4, peaks 6 to 10 and peak S are calculated: the relative retention time of peak 1 is 0.40±10%, the relative retention time of peak 2 is 0.69±10%, the relative retention time of peak 3 is 0.83±10%, the relative retention time of peak 4 is 0.92±10%, the relative retention time of peak 6 is 1.04±10%, the relative retention time of peak 7 is 1.12±10%, the relative retention time of peak 8 is 1.23±10%, the relative retention time of peak 9 is 1.37±10%, and the relative retention time of peak 10 is 1.50±10%.

6. The method for constructing a characteristic spectrum of a Chinese patent medicine according to claim 1, characterized in that: The characteristic spectrum determined the sources of 10 common characteristic peaks, which were respectively from dragon's blood, rhubarb, and angelica dahurica; among them, the components derived from dragon's blood include dragon's blood B in peak 2 and pterostilbene in peak 4; the components derived from rhubarb include aloe-emodin in peak 1, an unknown component in peak 3, rhein in peak 6, an unknown component in peak 9, and an unknown component in peak 10; the components derived from angelica dahurica include imperatorin in peak 5, an unknown component in peak 7, and an unknown component in peak 8.

7. The characteristic spectrum of Zhikang Capsules obtained according to the construction method described in any one of claims 1-6.

8. The characteristic spectrum of Zhikang Capsules according to claim 7 is used in evaluating the quality and content determination of Zhikang Capsules.

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