A method for detecting HPLC fingerprint of traditional Chinese medicine

Abstract

The application discloses a kind of HPLC fingerprint detection methods of traditional Chinese medicine, the traditional Chinese medicine is made of ten traditional Chinese medicinal materials such as radix codonopsis, atractylodes, poria, licorice, angelica, chuanxiong, white peony root, prepared rehmannia, astragalus, cassia, with the effect of tonifying qi and blood, the fingerprint established in the application includes 11 common characteristic peaks, respectively belong to ten medicinal materials, so it can identify all ten medicinal materials in traditional Chinese medicine, and also identifies 5 components therein, which are glycyrrhizin, ammonium glycyrrhizinate, verbascoside, calycosin-7-glucoside and astragaloside A respectively, so the application can more comprehensively control drug quality, solve the phenomenon of fake and inferior drugs, and ensure the safety and effectiveness of clinical use.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a HPLC fingerprint detection method for traditional Chinese medicines in Shiquandabugao. Background Art

[0002] The national drug standard (WS3-B-0471-91) includes a traditional Chinese medicine for warming and tonifying qi and blood and used for qi and blood deficiency, named "Shiquandabugao", which is a product of Jianmin Pharmaceutical Group Co., Ltd., with the approval number "国药准字Z42020618". This drug is made from a total of ten traditional Chinese medicinal materials, namely Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Glycyrrhiza glabra, Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Rehmannia glutinosa, Astragalus membranaceus, and Cinnamomum cassia, and is used to treat symptoms such as pale or sallow complexion caused by qi and blood deficiency, mental fatigue, palpitation, chest distress, shortness of breath, dizziness, spontaneous sweating, pale tongue, white coating, and weak and thready pulse.

[0003] In the existing national standard of this variety, there are only characters, inspection items, etc., without thin-layer chromatography identification items and component content determination. The standard requirements are relatively low, and the quality control of the ten medicinal materials in the prescription is not specifically carried out, making it difficult to truly reflect and monitor the product quality, and unable to fundamentally solve the problem of fake and inferior drugs, with relatively large deficiencies and limitations.

[0004] Fingerprint is a comprehensive and quantifiable method for detecting traditional Chinese medicinal materials and traditional Chinese medicine preparations, mainly used to evaluate the authenticity, superiority, and stability of the quality of traditional Chinese medicinal materials and semi-finished and finished preparations. It is a commonly used method for identifying the authenticity and evaluating the quality in the field of quality detection and analysis of traditional Chinese medicines. Fingerprint can comprehensively reflect the types and quantities of chemical components in traditional Chinese medicines. Especially in the current situation where the active components of traditional Chinese medicine compound preparations have not been fully elucidated, it can achieve a comprehensive evaluation of the internal quality of traditional Chinese medicines and effective control of their overall substances, and is currently the most effective means for quality control of traditional Chinese medicines and their preparations. The detection methods of traditional Chinese medicine fingerprint include chromatography, spectroscopy, wave spectroscopy, etc. Chromatography mainly includes thin-layer chromatography, high-performance liquid chromatography, gas chromatography, etc. Among them, high-performance liquid chromatography (HPLC) has the advantages of simplicity, sensitivity, high efficiency, good reproducibility, etc., and is the main method for the study of traditional Chinese medicine fingerprint.

[0005] After retrieval, no literature reports on the simultaneous fingerprint analysis and detection of the ten traditional Chinese medicinal materials in Shiquandabugao have been found. Summary of the Invention

[0006] Aiming at the deficiencies in the current quality detection method and evaluation system of Shiquandabugao, the purpose of the present invention is to establish a HPLC fingerprint detection and analysis method for Shiquandabugao, aiming to more comprehensively monitor and evaluate the drug quality as a whole and ensure the safety and effectiveness of the drug in clinical use.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] An HPLC fingerprint detection method for a traditional Chinese medicine, wherein the traditional Chinese medicine is made from ten herbs including Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Rehmannia glutinosa, Astragalus membranaceus, and Cinnamomum cassia, and has the effect of warming and tonifying qi and blood. The detection method includes the following steps:

[0009] (1) Extract the Chinese herbal medicine with methanol using ultrasound, filter it with a microporous membrane, and collect the filtrate to obtain the test solution;

[0010] (2) Weigh out astragaloside A, verbascoside, verbascoside, ammonium glycyrrhizate and glycyrrhizin reference standards respectively, and prepare solutions of each single reference standard in methanol.

[0011] (3) Accurately pipette the test solution or each single reference solution and perform detection using a high-performance liquid chromatograph (HPLC). The HPLC contains an ultraviolet (UV) detector with a set detection wavelength of 205-330 nm. The stationary phase of the HPLC is C2. 18 The chromatographic column uses methanol as mobile phase A, acetonitrile as mobile phase B, and acid solution as mobile phase C. Gradient elution is employed at a flow rate of 0.5-2.0 ml / min and a column temperature of 25-35℃. The theoretical plate number is not less than 3000.

[0012] (4) Record the chromatograms of the test solution and each single reference solution. Using the Chinese Pharmacopoeia Commission's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, import, perform multi-point correction and data matching on the chromatogram of the test solution to obtain the standard fingerprint spectrum of the Chinese medicine. Compare the fingerprint spectrum of the test sample with the chromatograms of each single reference solution and evaluate the similarity with the standard fingerprint spectrum.

[0013] In step (1) and (2), the methanol is 70% (by volume) methanol, and in step (3), the methanol is pure methanol.

[0014] The ultrasonic extraction was performed at a power of 300W, a frequency of 40kHz, and a duration of 30min.

[0015] The microporous filter membrane has a pore size of 0.45 μm.

[0016] The ultraviolet detector is set to a detection wavelength of 220 nm.

[0017] The mobile phase C is 0.3% (by volume) glacial acetic acid.

[0018] The gradient elution time is 75 minutes, divided into 9 segments. The mobile phase volume ratio for each time segment is as follows:

[0019]

[0020]

[0021] The column temperature is 25°C.

[0022] Each 1 ml of each single reference solution contains 25 μg of verbascoside, 0.6 mg of astragaloside A, 10 μg of glycyrrhizin, 0.2 mg of ammonium glycyrrhizate, and 15 μg of verbascoside, respectively.

[0023] The standard fingerprint chromatogram contains 11 common peaks. Peak 1 is attributed to Ligusticum chuanxiong, peak 2 to Atractylodes macrocephala, peak 3 to Poria cocos, peak 4 to Angelica sinensis and Cinnamomum cassia, peaks 5 and 6 to Astragalus membranaceus, peaks 7 and 10 to Glycyrrhiza uralensis, peak 8 to Rehmannia glutinosa, peak 9 to Paeonia lactiflora, and peak 11 to Codonopsis pilosula. Comparison with the chromatograms of individual reference solutions confirmed that peak 5 is astragaloside A, peak 6 is verbascoside, peak 7 is glycyrrhizin, peak 8 is verbascoside, and peak 10 is ammonium glycyrrhizate. Using the glycyrrhizin peak as a reference, the relative retention times of the other 10 common peaks were calculated, and the relative standard deviations of each common peak were all within 1.7%.

[0024] Using the Chinese herbal chromatographic fingerprint similarity evaluation system (2004A version), the chromatogram of the sample to be tested is compared with the fitted standard liquid phase fingerprint spectrum to obtain the similarity. The sample with a similarity greater than 0.900 is judged as qualified.

[0025] The beneficial effects of this invention are:

[0026] This invention successfully established a fingerprint spectrum detection and analysis method for the traditional Chinese medicine "Shi Quan Da Bu Gao" (Ten-Ingredient Tonic Paste). The established fingerprint spectrum includes 11 common characteristic peaks, belonging to ten medicinal materials: Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Rehmannia glutinosa, Astragalus membranaceus, and Cinnamomum cassia. Therefore, it can comprehensively identify the ten medicinal materials in the product. Furthermore, it identifies five components: astragaloside A, verbascoside, glycyrrhizin, verbascoside, and ammonium glycyrrhizate. Thus, this invention can more comprehensively control drug quality, solve the problem of counterfeit and substandard drugs, and ensure the safety and effectiveness of clinical use.

[0027] The fingerprint chromatogram established by this invention has good peak shape (large peak area and high resolution), and can complete the detection with one extraction and injection. The method is simple, quick, and has good reproducibility. It can monitor all the medicinal ingredients in the prescription and make up for the deficiencies of the current detection methods and standards for this product. Attached Figure Description

[0028] Figure 1 This is a liquid chromatogram prepared under optimal conditions.

[0029] Figure 2 This is a comparative chromatogram of the extraction solvent.

[0030] Figure 3 These are comparative chromatograms at different wavelengths.

[0031] Figure 4 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (1).

[0032] Figure 5 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (2).

[0033] Figure 6 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (3).

[0034] Figure 7 These are chromatograms comparing results obtained at different column temperatures.

[0035] Figure 8 It is a superimposed image of fingerprint spectra from 12 batches of samples.

[0036] Figure 9 It is the standard fingerprint spectrum of the Shi Quan Da Bu Gao (a traditional Chinese medicine tonic).

[0037] Figure 10 It is an identification diagram comparing the sample fingerprint spectrum with the chromatograms of each reference standard. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments.

[0039] Instruments and reagents: Shimadzu LC-2040C CN high-performance liquid chromatograph, including a diode array detector and LabSolutions workstation; PGJ-10 / 20-AS ultrapure water system (Wuhan Pinguan Instrument Equipment Co., Ltd.); Mettler ME203E, Mettler ME104E, and Mettler TOLEDO XPE105 electronic balances (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Agilent column (5μm, 250×4.6mm).

[0040] Astragaloside A reference standard (purity 95.0%, batch number 110781-202219), glycyrrhizin reference standard (purity 95.2%, batch number 111610-202209), ammonium glycyrrhizate reference standard (purity 94.4%, batch number 110731-202122), verbascoside reference standard (purity 95.2%, batch number 111530-201914), and verbascoside isoflavone glucoside reference standard (purity 96.8%, batch number 111920-201907) were all purchased from the National Institutes for Food and Drug Control. Acetonitrile and methanol were of chromatographic grade, n-butanol, ethyl acetate, glacial acetic acid, etc. were of analytical grade, and water was purified water.

[0041] The Shi Quan Da Bu Gao (Ten-Ingredient Tonic Paste) preparation is from Jianmin Pharmaceutical Group Co., Ltd., with batch numbers: 220901, 221001, 221002, 221101, 221201, 221202, 221203, 230102, 230201, 230202, 230301, 230302, designated as S1-S12. The formula and preparation method are as follows:

[0042] Codonopsis pilosula 80g, Atractylodes macrocephala (fried) 80g, Poria cocos 80g, Glycyrrhiza uralensis (honey-processed) 40g, Angelica sinensis 120g, Ligusticum chuanxiong 40g, Paeonia lactiflora (wine-fried) 80g, Rehmannia glutinosa (processed) 120g, Astragalus membranaceus (honey-processed) 80g, Cinnamomum cassia 20g.

[0043] The above ten ingredients are decocted three times with water. The first and second decoctions are each for two hours, and the third decoction is for one hour. The decoctions are combined, filtered, and the filtrate is allowed to stand for 24 hours. The supernatant is then concentrated under reduced pressure to obtain a clear extract with a relative density of 1.28 to 1.32 (85-90℃). 700g of refined honey is added to every 100g of the clear extract, mixed well, heated to boiling, and filtered to obtain the final product.

[0044] Example 1: Screening of chromatographic conditions

[0045] 1. Investigation of sample extraction solvent

[0046] The extraction effects of five different extraction solvents—70% (v / v) methanol, ethyl acetate, water-saturated n-butanol, pure methanol, and 50% (v / v) ethanol—were investigated, as detailed below:

[0047] Take an appropriate amount of this product (batch number: 230201) from the dosage section, accurately weigh 5 portions, each about 5g, and accurately add 25ml of 70% methanol, ethyl acetate, water-saturated n-butanol, pure methanol, and 50% ethanol respectively. Weigh the contents, sonicate (power 300W, frequency 40kHz) for 30min, cool, weigh again, replenish the lost weight with the corresponding extraction solvent, shake well, filter through a microporous membrane (0.45um), and collect the filtrate to obtain the final product.

[0048] The chromatographic conditions are as follows:

[0049] Column: Agilent (250mm × 4.6mm, 5µm);

[0050] Mobile phase: methanol, acetonitrile, 0.3% (v / v) glacial acetic acid;

[0051] Gradient elution procedure:

[0052]

[0053] The flow rate is 1 ml / min;

[0054] The column temperature is 25℃;

[0055] The injection volume was 10 μL;

[0056] Detection wavelength: 220nm;

[0057] The theoretical plate number should be no less than 3000. Chromatographic recording time is 75 minutes.

[0058] A comparison shows that ( Figure 2 Under the same detection conditions, the chromatogram of the test solution prepared by extraction with ethyl acetate had very few peaks, and none of the major chromatographic peaks were eluted, indicating that ethyl acetate could not effectively extract most of the components in this product. The chromatogram of the test solution prepared with 50% methanol was missing several characteristic peaks, including peaks 3, 4, 5, 6, and 7. The chromatogram of the test solution prepared by extraction with water-saturated n-butanol was missing characteristic peak 6, and the peak shapes of characteristic peaks 1, 2, and 5 were poor, and the overall responsiveness of the major chromatographic peaks was weak. The chromatograms of the test solutions prepared by extraction with 70% methanol and pure methanol were basically consistent, with good overall peak quantity and resolution of major chromatographic peaks. However, the peak shape of peak 2 in the chromatogram prepared by extraction with 70% methanol was better, and considering the cost of reagents, 70% methanol was preferred as the extraction solvent.

[0059] 2. Selection of detection wavelength

[0060] A sample of Shi Quan Da Bu Gao (a traditional Chinese medicine tonic) from batch 230201 was tested. The chromatographic effects at different wavelengths (205, 220, 235, 250, 270, 290, 310, and 330 nm) were examined. 70% methanol was used as the extraction solvent, and other chromatographic conditions were the same as in Example 1.

[0061] The test results show that ( Figure 3Generally, the smaller the wavelength, the more chromatographic peaks there are, and the stronger the peak signal. For example, at wavelengths of 290nm, 310nm, and 330nm, several chromatographic peaks, such as 4, 8, 10, and 11, are missing, and the overall responsiveness of other major chromatographic peaks is weak. At 270nm and 250nm, chromatographic peaks 3, 4, 8, and 11 are missing; at 235nm, peak 11 is missing, and the signal of peak 3 is relatively weak. The overall effect of the chromatograms at 205nm and 220nm is basically the same, with better peak quantity, peak shape, and resolution of major chromatographic peaks than at other wavelengths. However, compared to other wavelengths, the baseline fluctuation is larger at 205nm, and the responsiveness of peak 11 is weaker. Therefore, considering all factors, the optimal detection wavelength for fingerprint chromatograms is 220nm.

[0062] 3. Optimization of the mobile phase elution process

[0063] Gradient elution conditions were optimized for a mobile phase system consisting of methanol (A): acetonitrile (B): 0.3% glacial acetic acid aqueous solution (C). The following four gradient elution conditions were investigated:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Result: As Figure 4 , 5 As shown in Figures 6 and 1, under gradient elution conditions (1) and (2), the elution times of the chromatographic peaks are relatively concentrated, especially in the middle part of the chromatogram, and the resolution of some major peaks is poor. Under gradient elution condition (3), the resolution and peak shape of some peaks are poor, and the baseline is not flat. Under gradient elution condition (4), the elution time distribution of the major chromatographic peaks is suitable, the peak shape and peak resolution are good, and the detection time is short. Therefore, gradient elution condition (4) is preferred.

[0070] 4. Column temperature investigation

[0071] 230201 batches of Shiquan Dabu Gao (a traditional Chinese medicine tonic) samples were tested, and the overall effect of the chromatograms measured at column temperatures of 25℃, 30℃, and 35℃ was compared. Figure 7 The results showed that column temperature had little effect on the overall chromatogram. Considering that at a column temperature of 30℃, there were many impurity peaks near peak 4 and the signal of peak 11 was weak; while at a column temperature of 35℃, peak 7 contained impurity peaks and the signal of peak 10 was weak, the preferred column temperature was 25℃.

[0072] Example 2 Methodological Investigation

[0073] 1. Repeatability test

[0074] Six test solutions were prepared from the same batch of Shi Quan Da Bu Gao (batch number: 230201) under optimized conditions, and their repeatability was assessed. Using glycyrrhizin peak No. 7 as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the relative standard deviation of the relative retention time of each chromatographic peak was less than 2.0%, and the relative standard deviation of the relative peak area of ​​each chromatographic peak was less than 4.2%, indicating good repeatability. (See Tables 1 and 2).

[0075] Table 1. Relative retention times of each peak in the repeatability test of Shi Quan Da Bu Gao (a traditional Chinese medicine tonic).

[0076]

[0077]

[0078] Table 2. Relative peak areas of each peak in the repeatability test of Shi Quan Da Bu Gao (a traditional Chinese medicine tonic).

[0079] Peak 1 2 3 4 5 6 average value RSD 1 3.063 2.893 3.051 2.889 2.927 3.047 2.978 2.81％ 2 4.252 4.471 4.246 4.305 4.116 4.327 4.258 1.84％ 3 0.434 0.457 0.434 0.441 0.421 0.442 0.438 2.71％ 4 0.388 0.408 0.388 0.394 0.377 0.395 0.396 3.57％ 5 1.320 1.392 1.319 1.333 1.286 1.342 1.333 2.61％ 6 0.315 0.320 0.313 0.331 0.306 0.315 0.318 2.94％ 7(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 8 0.642 0.657 0.649 0.650 0.622 0.648 0.645 1.88％ 9 1.309 1.371 1.311 1.323 1.266 1.330 1.318 2.59％ 10 0.144 0.140 0.144 0.141 0.141 0.139 0.142 1.47％ 11 0.217 0.237 0.217 0.229 0.211 0.231 0.224 4.09％

[0080] 2. Precision test

[0081] A sample solution of the same herbal tonic paste (batch number: 230201) was injected six times consecutively to assess the instrument's precision. Using the glycyrrhizin peak (No. 7) as a reference peak, the relative retention times and relative peak areas of each common peak were calculated (RSD). The results showed that the relative standard deviations of the relative retention times of each common peak were less than 1.2%, and the relative standard deviations of the relative peak areas were less than 2.0%, indicating good precision. (See Tables 3 and 4.)

[0082] Table 3. Relative retention times of each peak in the precision test of the Shi Quan Da Bu Gao (a traditional Chinese tonic paste).

[0083]

[0084]

[0085] Table 4. Relative peak areas of each peak in the precision test of the Shi Quan Da Bu Gao (a traditional Chinese tonic paste).

[0086] Peak 1 2 3 4 5 6 average value RSD 1 3.586 3.605 3.584 3.576 3.607 3.573 3.589 0.40％ 2 4.120 4.208 4.152 4.226 4.171 4.206 4.181 0.96％ 3 0.440 0.431 0.434 0.435 0.422 0.432 0.432 1.38％ 4 0.392 0.401 0.408 0.394 0.399 0.396 0.398 1.44％ 5 2.109 2.074 2.103 2.067 2.085 2.093 2.089 0.78％ 6 0.365 0.363 0.357 0.364 0.359 0.353 0.360 1.30％ 7(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 8 0.667 0.67 0.656 0.673 0.666 0.669 0.667 0.88％ 9 0.689 0.697 0.694 0.703 0.696 0.684 0.694 0.95％ 10 0.144 0.142 0.139 0.138 0.143 0.141 0.141 1.64％ 11 0.241 0.24 0.236 0.241 0.238 0.236 0.239 0.98％

[0087] 3. Stability test

[0088] A sample solution of the same Shi Quan Da Bu Gao (batch number: 230201) was taken and placed at room temperature. It was then injected into the liquid chromatograph at 0h, 4h, 8h, 12h, and 24h, using the glycyrrhizin peak (No. 7) as the reference peak. The relative retention times and relative peak areas of each common peak were examined for their RSDs. The results showed that the relative standard deviations of the relative retention times of each chromatographic peak were all less than 1.3%, and the relative standard deviations of the relative peak areas of each chromatographic peak were all less than 3.0%, indicating good stability of the sample solution at room temperature. (See Tables 5 and 6.)

[0089] Table 5. Relative retention times of each peak in the stability test of Shi Quan Da Bu Gao (a traditional Chinese medicine tonic).

[0090] Peak 0h 2h 4h 8h 12h 24h average value RSD 1 0.204 0.197 0.200 0.199 0.201 0.202 0.201 1.21％ 2 0.285 0.277 0.280 0.278 0.280 0.282 0.280 1.03％ 3 0.484 0.479 0.480 0.479 0.481 0.482 0.481 0.40％ 4 0.783 0.782 0.799 0.780 0.781 0.783 0.785 0.91％ 5 0.837 0.840 0.841 0.844 0.838 0.842 0.840 0.31％ 6 0.951 0.946 0.953 0.947 0.955 0.954 0.951 0.39％ 7(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 8 1.057 1.056 1.046 1.048 1.055 1.052 1.052 0.43％ 9 1.238 1.247 1.246 1.240 1.235 1.245 1.242 0.39％ 10 1.555 1.553 1.549 1.551 1.547 1.553 1.551 0.19％ 11 1.820 1.818 1.812 1.816 1.815 1.808 1.815 0.24％

[0091] Table 6. Relative peak areas of each peak in the stability test of Shi Quan Da Bu Gao (a traditional Chinese medicine tonic).

[0092] Peak 0h 2h 4h 8h 12h 24h average value RSD 1 3.060 2.968 3.018 3.014 2.989 3.005 3.009 1.03％ 2 4.166 4.232 4.324 4.176 4.360 4.156 4.236 2.06％ 3 0.441 0.457 0.434 0.451 0.422 0.442 0.441 2.81％ 4 0.390 0.418 0.408 0.394 0.387 0.396 0.399 2.97％ 5 1.333 1.337 1.372 1.339 1.346 1.362 1.348 1.15％ 6 0.341 0.336 0.340 0.341 0.344 0.334 0.339 1.08％ 7(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 8 0.762 0.752 0.754 0.762 0.758 0.756 0.757 0.55％ 9 0.673 0.699 0.704 0.673 0.709 0.711 0.695 2.51％ 10 0.148 0.156 0.146 0.148 0.152 0.153 0.151 2.51％ 11 0.249 0.247 0.243 0.246 0.248 0.241 0.246 1.25％

[0093] Example 3: Establishment and Analysis of Fingerprint Patterns

[0094] 1. Establishment of standard fingerprint spectrum and determination of common peaks

[0095] Following the established preparation method and liquid chromatography detection conditions for the Shi Quan Da Bu Gao (a traditional Chinese medicine supplement) test solution, 12 batches of Shi Quan Da Bu Gao samples were taken for analysis. The superimposed fingerprint chromatograms of the 12 batches of samples are shown in the figure. Figure 8 The main chromatographic peaks detected all appeared within 75 minutes. The obtained fingerprint chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004A)" software for processing. The median method was used, the time window width was set to 0.2, and a multi-point correction method was employed to perform full peak matching of the chromatograms, fitting and generating a standard control fingerprint chromatogram. See [link to relevant documentation]. Figure 9 Eleven common peaks were identified, and the relative retention times of other common peaks in the 12 batches of samples were calculated using the glycyrrhizin peak No. 7 as the reference peak (see Table 7). The average relative retention times (peak numbers) were 0.197 (1), 0.276 (2), 0.479 (3), 0.785 (4), 0.840 (5), 0.952 (6), 1.053 (8), 1.247 (9), 1.552 (10), and 1.818 (11), respectively, and their relative deviations were all within 1.7%.

[0096] Table 7-1 Relative retention times of common peaks in 12 batches of Shi Quan Da Bu Gao samples

[0097]

[0098] Table 7-2 Relative retention times of common peaks in 12 batches of Shi Quan Da Bu Gao samples

[0099]

[0100]

[0101] 2. Identification of common peaks

[0102] Preparation of reference solutions: Accurately weigh astragaloside A, verbascoside, verbascoside, ammonium glycyrrhizate, and glycyrrhizin reference standards, and prepare individual reference solutions containing 25 μg of verbascoside A, 0.6 mg of astragaloside A, 10 μg of glycyrrhizin, 0.2 mg of ammonium glycyrrhizate, and 15 μg of verbascoside per 1 ml using methanol.

[0103] The chromatograms of the reference solutions of astragaloside A, verbascoside, verbascoside, ammonium glycyrrhizate, and glycyrrhizin were compared with the sample fingerprint chromatograms. The chromatographic peaks at the same retention times as the reference solutions were identified. (See attached image.) Figure 10 The results showed that peaks 5, 6, 7, 8, and 10 were identified as astragaloside A, verbascoside glucoside, glycyrrhizin, verbascoside, and glycyrrhizic acid ammonium, respectively. Peak 7, glycyrrhizin, was located in the middle position and had a suitable size and shape; therefore, peak 7 was selected as the reference peak.

[0104] 3. Correlation between the fingerprint spectra of medicinal materials and finished products

[0105] Preparation of the test solution of medicinal materials: Each medicinal material in the prescription was extracted according to the preparation process of this product to obtain an extract. The chromatographic peaks were assigned according to the established fingerprint detection method. The results are shown in Table 8.

[0106] Table 8. Correlation map between the Ten-Ingredient Tonic Paste and medicinal materials.

[0107] Peak Codonopsis pilosula Atractylodes macrocephala Poria licorice Angelica sinensis Chuanxiong White peony root Rehmannia glutinosa Astragalus Cinnamon 1 + 2 + 3 + 4 + + 5 + 6 + 7 + 8 + 9 + 10 + 11 +

[0108] The 11 common peaks were classified as medicinal materials, and ten medicinal materials in the prescription were identified. Peak 1 was classified as Chuanxiong, peak 2 as Baizhu, peak 3 as Fuling, peak 4 as Danggui and Rougui, peaks 5 and 6 as Huangqi, peaks 7 and 10 as Gancao, peak 8 as Shudihuang, peak 9 as Baishao, and peak 11 as Dangshen.

[0109] 4. Similarity evaluation and sample qualification determination

[0110] The chromatograms of 12 batches of samples were compared with the standard fingerprint chromatograms. Similarity evaluation was performed according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2004A version), as shown in Table 9. The similarity scores of the 12 batches were 0.957, 0.973, 0.989, 0.975, 0.993, 0.978, 0.962, 0.958, 0.942, 0.939, 0.919, and 0.922, respectively. All 12 batches had similarity scores above 0.91, indicating good quality stability and uniformity among batches. Considering all factors, samples with a similarity score greater than 0.900 were selected as qualified products.

[0111] Table 9. Similarity Evaluation of 12 Batches of Shi Quan Da Bu Gao Samples

[0112] batch number Similarity batch number Similarity batch number Similarity 220901 0.957 221201 0.993 230201 0.942 221001 0.973 221202 0.978 230202 0.939 221002 0.989 221203 0.962 230301 0.919 221101 0.975 230102 0.958 230302 0.922

Claims

1. An HPLC fingerprint detection method for a traditional Chinese medicine, wherein the traditional Chinese medicine is made from ten herbs: Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Rehmannia glutinosa, Astragalus membranaceus, and Cinnamomum cassia, and has the effect of warming and tonifying qi and blood, characterized in that... The detection method includes the following steps: (1) Extract the Chinese herbal medicine with 70% methanol by ultrasonication, filter it with a microporous membrane, and take the filtrate to obtain the test solution; (2) Weigh out astragaloside A, verbascoside, verbascoside, ammonium glycyrrhizate and glycyrrhizin reference standards respectively, and prepare solutions of each single reference standard in 70% methanol. (3) Accurately pipette the test solution or each single reference solution and perform detection using a high-performance liquid chromatograph (HPLC). The HPLC contains an ultraviolet detector with a set detection wavelength of 220 nm. The stationary phase of the HPLC is C2. 18 The chromatographic column used mobile phase A as methanol, mobile phase B as acetonitrile, and mobile phase C as 0.3% glacial acetic acid. Gradient elution was employed at a flow rate of 0.5-2.0 ml / min and a column temperature of 25℃. The theoretical plate number was not less than 3000. (4) Record the chromatograms of the test solution and each single reference solution. Using the Chinese Pharmacopoeia Commission's Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, import, perform multi-point correction and data matching on the chromatogram of the test solution to obtain the standard fingerprint chromatogram of the traditional Chinese medicine. Compare the fingerprint chromatogram of the test sample with the chromatograms of each single reference solution and evaluate the similarity with the standard fingerprint chromatogram. The gradient elution time is 75 min, divided into 9 segments. The mobile phase volume for each time segment is as follows:

2. The HPLC fingerprint detection method for traditional Chinese medicine as described in claim 1, characterized in that: The ultrasonic extraction was performed at a power of 300W, a frequency of 40kHz, and a duration of 30min.

3. The HPLC fingerprint detection method for traditional Chinese medicine as described in claim 1, characterized in that: The pore size of the microporous filter membrane is 0.45 μm.

4. The HPLC fingerprint detection method for traditional Chinese medicine as described in claim 1, characterized in that: Each 1 ml of each single reference solution contains 25 μg of verbascoside, 0.6 mg of astragaloside A, 10 μg of glycyrrhizin, 0.2 mg of ammonium glycyrrhizate, and 15 μg of verbascoside, respectively.

5. The HPLC fingerprint detection method for traditional Chinese medicine as described in claim 1, characterized in that: The standard fingerprint chromatogram contained 11 common peaks. Peak 1 was assigned to Ligusticum chuanxiong, peak 2 to Atractylodes macrocephala, peak 3 to Poria cocos, peak 4 to Angelica sinensis and Cinnamomum cassia, peaks 5 and 6 to Astragalus membranaceus, peaks 7 and 10 to Glycyrrhiza uralensis, peak 8 to Rehmannia glutinosa, peak 9 to Paeonia lactiflora, and peak 11 to Codonopsis pilosula. Comparison with the chromatograms of individual reference solutions confirmed that peak 5 was astragaloside A, peak 6 was verbascoside glucoside, peak 7 was glycyrrhizin, peak 8 was verbascoside, and peak 10 was ammonium glycyrrhizate. Using the glycyrrhizin peak as a reference, the relative retention times of the other 10 common peaks were calculated, and the relative standard deviations of each common peak were all within 1.7%.

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