A method for constructing a fingerprint of a Qizhi Tongluo formula and a method for evaluating the quality of a capsule

Abstract

This invention provides a method for constructing a fingerprint spectrum of the Astragalus-Hirudo-Tongluo formula and a method for quality evaluation of its capsules, belonging to the field of traditional Chinese medicine analysis technology. This invention sequentially detects samples using an ultraviolet detector and an evaporative light scattering detector. Under the same chromatographic conditions, using two different detectors for the same sample allows for the simultaneous acquisition of fingerprint spectra of compounds with both strong and weak ultraviolet absorption. This method maximizes the detection of chemical components in the Astragalus-Hirudo-Tongluo formula while saving time and effort. The method is simple, easy to implement, highly sensitive, specific, and robust. Based on the fingerprint spectrum method, this invention identifies chromatographic peaks, thereby achieving better quality control of the Astragalus-Hirudo-Tongluo formula.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine drug analysis technology, specifically relating to a method for constructing a fingerprint spectrum of a formula containing Astragalus membranaceus and Hirudo medicinalis, and a method for evaluating the quality of capsules. Background Technology

[0002] The Qi-Zhi formula for unblocking collaterals consists of 26 Chinese medicinal herbs, including Astragalus membranaceus and Hirudo medicinalis. Its main functions are to invigorate Qi, promote blood circulation, and unblock collaterals. It is suitable for the adjunctive treatment of stroke sequelae such as hemiplegia, limb numbness, facial paralysis, speech impairment, and fatigue. It has significant clinical efficacy. However, a review of domestic and foreign literature revealed that its basic research is weak, especially in terms of quality control. Only research on identification and content determination methods was found. Although identification and content determination can control its quality to a certain extent, due to the complexity of the chemical components of Chinese herbal compound formulas, it is difficult to achieve comprehensive quality control by relying solely on identification and content determination.

[0003] The current quality standards for the Astragalus and Hirudo Detoxifying Formula only focus on the content determination of astragaloside A in addition to routine capsule testing. Furthermore, a review of relevant literature on the Astragalus and Hirudo Detoxifying Formula reveals that quality studies on this formula involve thin-layer chromatography identification and content determination. However, the Astragalus and Hirudo Detoxifying Formula is a large formula with complex components and numerous active ingredients. Currently, no quality control method has been found that can simultaneously detect multiple components of the Astragalus and Hirudo Detoxifying Formula. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing a fingerprint spectrum of the Astragalus and Hirudo formula for unblocking collaterals and a method for evaluating the quality of the capsules, so as to comprehensively detect multiple chemical components and construct a fingerprint spectrum to better achieve quality control of the Astragalus and Hirudo formula for unblocking collaterals.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for constructing a fingerprint spectrum of the Astragalus and Hirudo formula for unblocking collaterals, wherein an ultraviolet detector and an evaporative light scattering detector are used sequentially to perform chromatographic detection on the Astragalus and Hirudo formula for unblocking collaterals and the raw material single herb slices, respectively;

[0007] The chromatographic detection includes: using octadecylsilane-bonded silica gel as the packing material; and using acetonitrile-0 to 0.05% formic acid solution as the mobile phase for gradient elution.

[0008] Preferably, the spectral conditions are the same when performing chromatographic detection using an ultraviolet detector and an evaporative light scattering detector, respectively.

[0009] Preferably, the method further includes performing the chromatographic detection on the single herbal sample, and comparing the chromatograms of each sample with the chromatograms of the reference standard in sequence to confirm the substance represented by each chromatographic peak and / or the herbal material to which it belongs.

[0010] Preferably, the reference standards include: ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rf, ginsenoside Re, ginsenoside Rg1, gentiopicrin, ferulic acid, hydroxysaffron yellow A, paeoniflorin, stilbene glycoside, and purslane glycoside.

[0011] This invention also provides the application of fingerprint patterns constructed using the above-described method in evaluating the quality of the Astragalus and Hirudo formula for unblocking collaterals.

[0012] Preferably, the Astragalus and Hirudo formula includes Astragalus and Hirudo formulas in the same or different dosage forms.

[0013] Preferably, after constructing the fingerprint spectrum using the above construction method, the method further includes comparing the characteristic peaks.

[0014] Preferably, the characteristic peaks include peaks of chemical components that can be detected simultaneously by an ultraviolet detector and an evaporative light scattering detector: paeoniflorin, hydroxysaffron yellow A, imperatorin, and stilbene glycoside.

[0015] Preferably, the characteristic peaks also include peaks of chemical components that can only be detected by an ultraviolet detector: ferulic acid and gentianin.

[0016] The characteristic peaks also include peaks of chemical components that can only be detected by an evaporative light scattering detector: ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and astragaloside A.

[0017] Beneficial Effects: This invention provides a method for constructing the fingerprint spectrum of the Astragalus and Hirudo formula for unblocking collaterals. It employs a tandem method using a high-performance ultraviolet (HPLC-UV) detector and an evaporative light scattering (HPLC-ELSD) detector. Under the same chromatographic conditions, using two different detectors—first UV detection followed by evaporative light scattering—allows for the simultaneous acquisition of fingerprint spectra of compounds with both strong and weak UV absorption. This method maximizes the detection of chemical components in the Astragalus and Hirudo formula while saving time and effort. The method is simple, easy to implement, highly sensitive, specific, and robust. In the embodiments of this invention, HPLC-UV identifies five chemical components: paeoniflorin, hydroxysaffron yellow A, ferulic acid, imperatorin, and stilbene glycoside; HPLC-ELSD identifies ten chemical components: hydroxysaffron yellow A, paeoniflorin, imperatorin, stilbene glycoside, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3.

[0018] Based on the fingerprint spectrum, this invention also constructs a low-quality evaluation method for the Astragalus and Hirudo formula, which utilizes a sufficient number of biomarkers for comprehensive evaluation, thereby better achieving quality control of the Astragalus and Hirudo formula. Attached Figure Description

[0019] Figure 1 HPLC-UV fingerprint of Astragalus and Hirudo Tongluo Capsules;

[0020] Figure 2 HPLC-ELSD fingerprint of Astragalus and Hirudo Tongluo Capsules;

[0021] Figure 3 The chromatograms are for elution conditions 1–8, with the upper chromatogram being HPLC-UV (220 nm) and the lower chromatogram being HPLC-ELSD.

[0022] Figure 4 The chromatograms are for elution condition 9, with the top chromatogram being HPLC-UV (220nm) and the bottom chromatogram being HPLC-ELSD.

[0023] Figure 5 HPLC-UV chromatograms at different detection wavelengths;

[0024] Figure 6 HPLC-UV chromatograms (220 nm) under different mobile phases;

[0025] Figure 7 HPLC-ELSD chromatograms at different nitrogen flow rates;

[0026] Figure 8 HPLC-ELSD chromatograms at different drift tube temperatures;

[0027] Figure 9 The chromatograms used to investigate sample pretreatment are shown in the top image (HPLC-UV) and the bottom image (HPLC-ELSD).

[0028] Figure 10 The images show chromatograms for different extraction solvents. The top image is an HPLC-UV chromatogram, and the bottom image is an HPLC-ELSD chromatogram.

[0029] Figure 11 The images show the chromatograms for different solvent dosages. The top image is the HPLC-UV chromatogram, and the bottom image is the HPLC-ELSD chromatogram.

[0030] Figure 12 The images show fingerprint chromatograms; the top image is an HPLC-UV chromatogram, and the bottom image is an HPLC-ELSD chromatogram.

[0031] Figure 13 The images show the fingerprint peak assignment results. The top image is the HPLC-UV chromatogram, and the bottom image is the HPLC-ELSD chromatogram.

[0032] Figure 14 Results for different flow rates (220 nm);

[0033] Figure 15 Results of observation at different flow rates (ELSD);

[0034] Figure 16 Results of observations at different column temperatures (220 nm);

[0035] Figure 17 Results of observation at different column temperatures (ELSD);

[0036] Figure 18 Results for different chromatographic columns (220 nm);

[0037] Figure 19 Results for different chromatographic columns (ELSD);

[0038] Figure 20 HPLC-UV fingerprints of 15 batches of Astragalus and Hirudo Tongluo Capsules;

[0039] Figure 21 The HPLC-ELSD fingerprint of 15 batches of Qizhi Tongluo Capsules. Detailed Implementation

[0040] This invention provides a method for constructing a fingerprint spectrum of the Astragalus and Hirudo formula for unblocking collaterals, wherein an ultraviolet detector and an evaporative light scattering detector are used sequentially to perform chromatographic detection on the Astragalus and Hirudo formula for unblocking collaterals and the raw material single herb slices, respectively;

[0041] The chromatographic detection includes: using octadecylsilane-bonded silica gel as the packing material; and using acetonitrile-0 to 0.05% formic acid solution as the mobile phase for gradient elution.

[0042] This invention does not specifically limit the dosage form of the Astragalus and Leech Collateral-Clearing Formula, preferably including capsules, tablets, or decoctions. The examples use Astragalus and Leech Collateral-Clearing Capsules as an example, but this should not be considered the entire scope of protection of this invention. The formula of the Astragalus and Leech Collateral-Clearing Capsules has been disclosed in Chinese Patent CN101411742A, specifically including Astragalus membranaceus, leech, ginseng, Ophiopogon japonicus, Schisandra chinensis, earthworm, Ilex pubescens, processed Polygonum multiflorum, Ligusticum chuanxiong, Angelica sinensis, Curcuma longa, Eupolyphaga sinensis, Salvia miltiorrhiza, Paeonia lactiflora, Spatholobus suberectus, Curcuma longa, Carthamus tinctorius, Lycopus lucidus, Bombyx mori, Buthus martensii, Gastrodia elata, Arisaema cum bile, Notopterygium incisum, Cinnamomum cassia, Gleditsia sinensis, and borneol.

[0043] This invention employs a method of first using an ultraviolet detector and then an evaporative light scattering detector, cascading them together (referred to as HPLC-UV-ELSD detector) to simultaneously detect multiple chemical components in the Astragalus and Hirudo formula. During the chromatographic detection, the chromatographic column preferably uses octadecyl bonded silica, more preferably an Agilent ZORBAX Plus C18. In this embodiment, the Agilent ZORBAX Plus C18 column is preferably 4.6*250mm in diameter and 5μm in diameter. Furthermore, during detection, the evaporative light scattering detector is set with a drift tube temperature of 80–110°C and a nitrogen flow rate of 1.5–2.5 L / min; the ultraviolet detector is set with a detection wavelength of 220 nm and a column temperature of 25–35°C.

[0044] In this invention, the elution gradient preferably varies slightly depending on the mobile phase. When the value is 0 in the acetonitrile-0 to 0.05% formic acid solution, acetonitrile-water is used as the mobile phase. This invention demonstrates several concentration gradients based on different mobile phases, but these should not be considered as representing the entire scope of protection of this invention.

[0045] Table 1 Gradient elution conditions

[0046]

[0047]

[0048] Before performing the chromatographic detection, this invention preferably includes sample extraction. The extraction method is not particularly limited and can be performed using conventional extraction methods in the art. In this embodiment, ultrasonic extraction is used, but this should not be considered the entire scope of protection of this invention. The solvent for ultrasonic extraction preferably includes methanol, the ultrasonic extraction power is 250W, the frequency is 40kHz, and the ultrasonic extraction time is 30–60 min. After ultrasonic extraction, this invention preferably includes removing, cooling, filtering, and evaporating the filtrate to dryness; the residue is then dissolved again in methanol and transferred to a 10ml volumetric flask, diluted to the mark with methanol, and shaken well to obtain the sample for subsequent detection. During the detection, the flow rate is preferably 0.8–1.2 mL / min, more preferably 1.0 mL / min; and the column temperature is preferably 25–35℃, more preferably 30℃.

[0049] In this invention, it is preferable to further perform chromatographic detection on the reference standard, and sequentially compare the chromatograms obtained from each sample with the chromatogram of the reference standard to confirm the substances represented by each chromatographic peak and / or their classification as medicinal materials. The reference standard of this invention preferably includes: ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rf, ginsenoside Re, ginsenoside Rg1, gentianin, ferulic acid, hydroxysaffron yellow A, paeoniflorin, stilbene glycoside, and purslane.

[0050] The samples described in this invention preferably include samples of the same dosage form and / or samples of different dosage forms. The examples use samples of the same dosage form (capsules) as an example to illustrate the verification of samples from the same batch and / or different batches. The dosage forms described in this invention preferably include capsules, tablets, and / or decoctions. Using the detection method described in this invention, two chromatograms can be obtained: an HPLC-UV chromatogram and an HPLC-ELSD chromatogram. In this embodiment of the invention, for the HPLC-UV chromatogram: peak 4 was identified as belonging to Ligusticum chuanxiong; peaks 5, 15, and 29 to Ilex pubescens; peaks 16 and 17 (paeoniflorin) to Paeonia lactiflora; peaks 8 (hydroxysafflower yellow A) and 9 to Carthamus tinctorius; peak 19 (ferulic acid) to Angelica sinensis; peaks 26 (purslane), 32, 35, and 43 to Notopterygium incisum; peaks 6, 24 (stilbene glycoside), 34, and 37 to processed Polygonum multiflorum; and based on the reference standard information, five chromatographic peaks were identified as peak 24 (stilbene glycoside), peak 19 (ferulic acid), peak 26 (purslane), and peak 8 (hydroxysafflower yellow A). Peak 17 (paeoniflorin); For the HPLC-ELSD chromatogram: Peaks 4, 9, and 18 were assigned to Ilex pubescens; Peaks 7 (hydroxysafflower yellow A) and 8 were assigned to Carthamus tinctorius; Peaks 12, 33, and 37 were assigned to Astragalus membranaceus; Peaks 10 and 11 (paeoniflorin) were assigned to Paeonia lactiflora; Peak 15 (peucedanin) was assigned to Notopterygium incisum; Peak 13 (stilbene glycoside) was assigned to processed Polygonum multiflorum; Peaks 20 (ginsenoside Rg1), 21 (ginsenoside Re), 25, 26 (ginsenoside Rf), 27 (ginsenoside Rb1), 30 (ginsenoside Rb2), 31 (ginsenoside Rb3), and 32 were assigned to Panax ginseng.

[0051] This invention also provides the application of fingerprint patterns constructed using the above-described method in evaluating the quality of the Astragalus and Hirudo formula for unblocking collaterals.

[0052] Using the fingerprint spectrum described in this invention, fingerprint spectra of compounds with strong and weak ultraviolet absorption can be obtained simultaneously under the same chromatographic conditions. This method offers advantages in terms of time and effort savings, maximizing the detection of chemical components in the Astragalus-Hirudo-Tongluo formula. It is simple, easy to implement, highly sensitive, specific, and robust. In this invention, the fingerprint spectrum allows for a comprehensive evaluation using a sufficient number of markers, thereby better achieving quality control of the Astragalus-Hirudo-Tongluo formula. The Astragalus-Hirudo-Tongluo formula of this invention preferably comprises Astragalus-Hirudo-Tongluo formulas derived from the same or different batches.

[0053] In applying the fingerprint spectrum, after constructing the fingerprint spectrum using the above-described method, the present invention preferably further includes comparing characteristic peaks. These characteristic peaks preferably include peaks of chemical components that can be simultaneously detected by an ultraviolet detector and an evaporative light scattering detector: paeoniflorin, hydroxysaffron yellow A, imperatorin, and stilbene glycoside. In the present invention, the characteristic peaks preferably also include peaks of chemical components that can only be detected by a single detector, such as peaks of chemical components that can only be detected by an ultraviolet detector: ferulic acid and mangiferin; and peaks of chemical components that can only be detected by an evaporative light scattering detector: ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and astragaloside A.

[0054] In this invention, although the composition of some peaks could not be determined, the detection method provided by this invention was used to perform the same detection on 15 batches of Astragalus and Hirudo Tongluo Capsules, yielding a total of 45 HPLC-UV common peaks. Figure 1 The relative retention times of each peak were statistically analyzed, as shown in Table 2. The peak labels in Table 2 are the same as those in the HPLC-UV chromatograms above. A total of 39 common peaks were obtained in HPLC-ELSD. Figure 2 The relative retention times of each peak were statistically analyzed, as shown in Table 3. The peak labels in Table 3 are the same as those in the above HPLC-ELSD chromatogram.

[0055] Table 2. Relative retention times of HPLC-UV fingerprints

[0056]

[0057]

[0058] Table 3. Relative retention times of HPLC-ELSD fingerprints of Astragalus and Hirudo Tongluo Capsules

[0059] Common peak number Relative retention time Common peak number Relative retention time Common peak number Relative retention time 1 0.086～0.096 14 0.976～0.986 27 1.672～1.682 2 0.092～0.102 15 1.000 28 1.678～1.688 3 0.150～0.160 16 1.118～1.128 29 1.710～1.720 4 0.186～0.196 17 1.155～1.165 30 1.727～1.737 5 0.275～0.285 18 1.216～1.226 31 1.804～1.814 6 0.324～0.334 19 1.260～1.270 32 1.839～1.849 7 0.375～0.385 20 1.301～1.311 33 1.851～1.861 8 0.393～0.403 21 1.311～1.321 34 1.853～1.863 9 0.525～0.530 22 1.574～1.584 35 1.865～1.875 10 0.566～0.576 23 1.603～1.613 36 1.874～1.884 11 0.643～0.653 24 1.619～1.629 37 1.901～1.911 12 0.871～0.881 25 1.643～1.653 38 1.914～1.924 13 0.933～0.943 26 1.653～1.663 39 1.921～1.931

[0060] Before constructing the fingerprint spectrum, the sample processing method is preferably the same as described above, and will not be repeated here.

[0061] The following detailed description, in conjunction with embodiments, illustrates a method for constructing a fingerprint spectrum of an Astragalus and Hirudo formula for unblocking collaterals and a method for evaluating the quality of capsules provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] 1. Experiment Content

[0064] 1.1 Optimization of gradient elution conditions and investigation of the mobile phase system

[0065] 1.1.1 Optimization of Gradient Elution Conditions

[0066] Preparation of the test solution: Accurately weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder into three portions, place them in a stoppered conical flask, accurately add 50ml of methanol, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, filter, and evaporate the filtrate to dryness. Dissolve the residue in methanol and transfer to a 10ml volumetric flask. Add methanol to the mark and mix well to obtain the test solution.

[0067] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-water and acetonitrile-formic acid were used as the mobile phases, with gradient elution performed according to the specifications in Table 1; a UV detector and an evaporative light scattering detector (AllChrom ELSD6000) were used; the drift tube temperature was 110℃, the nitrogen flow rate was 2.5 L / min; and the column temperature was 30℃. A Waterse2695 HPLC-ELSD-UV system was used for liquid chromatography; an Agilent ZORBAX Plus C18 column (4.6*250 mm, 5 μm) was used. The detection wavelength of 220 nm and the ELSD signal chromatogram were recorded.

[0068] The results of the above 9 gradient elutions are as follows: Figure 3 and Figure 4 As shown, the gradient elution conditions for the HPLC-UV-ELSD fingerprinting method were optimized, and method 8 was selected for gradient elution. Under this condition, the chromatographic signal was abundant and the response value was high in the UV 220 nm and evaporative light channels, and the peak shape and resolution of each chromatographic peak were good. Experimental results for gradient elution condition 9 showed that, under both UV and evaporative light detector conditions, no chromatographic peaks with high response values ​​appeared in the chromatogram after an analysis time of 130 minutes; therefore, the analysis time was 130 minutes.

[0069] 1.1.2 Investigation of detection wavelength under ultraviolet conditions

[0070] Comparison of different detection wavelengths (210nm, 220nm, 240nm, 260nm, 280nm, 300nm, 320nm, and 380nm) yields the following results: Figure 5As shown, when the detection wavelength is selected as 220nm, there are more signal peaks in the chromatogram and the response values ​​of each chromatographic peak are higher. Therefore, the detection wavelength is selected as 220nm under ultraviolet conditions.

[0071] 1.1.3 Investigation of the mobile phase

[0072] The mobile phase systems selected were acetonitrile-water, acetonitrile-0.05% formic acid solution, acetonitrile (containing 0.05% formic acid)-0.05% formic acid solution, acetonitrile-0.02% formic acid solution, and acetonitrile-0.01% formic acid solution, and the gradient elution condition was investigated according to step 8. Chromatograms at 220 nm were recorded. Results are as follows... Figure 6 As shown, when a high concentration of formic acid is used in the aqueous phase, the baseline drift is severe. When appropriate amounts of formic acid are added to both the organic and aqueous phases, the baseline also drifts significantly. Therefore, it was ultimately decided to add an appropriate amount of formic acid only to the aqueous phase. When a 0.01% formic acid solution is used in the aqueous phase, the resolution of some chromatographic peaks is poor. Considering both the chromatographic peak resolution and the baseline drift, the mobile phase system was ultimately selected as acetonitrile-0.02% formic acid solution.

[0073] 1.1.4 Examination of Evaporative Photodetector Parameters

[0074] The preparation of the test solution, chromatographic conditions, system suitability, and assay method are the same as those described above.

[0075] With the drift tube temperature at 110℃ and nitrogen flow rate at 2.0 L·min -1 2.5L·min -1 3.0L·min -1 At that time, the chromatogram of the evaporative light detector channel is recorded as follows: Figure 7 As shown, a lower nitrogen flow rate results in a higher chromatographic peak response value, but the peak baseline is more erratic. Considering both the response value and baseline characteristics, a nitrogen flow rate of 2.5 L / min was selected. -1 Nitrogen flow rate is 2.5 L / min -1 When the drift tube temperature is 105℃, 110℃, and 115℃ respectively, the chromatograms of the evaporative light detector channel are recorded as follows: Figure 8 As shown, at higher temperatures, the response values ​​of each signal peak in the chromatogram are higher, but the baseline drifts more. Therefore, considering both the response values ​​of the chromatographic peaks and the baseline of the chromatogram, an appropriate drift tube temperature is selected, and finally, a drift tube temperature of 110℃ is chosen.

[0076] 1.2 Investigation on the preparation of the test solution

[0077] Three samples were first prepared, and the differences in their chromatographic peaks under UV conditions at 220 nm and under evaporative light were compared to select an appropriate method for preparing the test solution. Then, different extraction solvents and solvent volumes were investigated.

[0078] 1.2.1 Simplified Pre-experimentation of Sample Pretreatment

[0079] Sample 1: Weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder accurately, place it in a stoppered conical flask, add 50ml of methanol accurately, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, transfer to a 10ml volumetric flask, add methanol to the mark, shake well, and the sample is ready.

[0080] Sample 2: Weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder accurately, place it in a stoppered conical flask, add 50ml of methanol accurately, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, filter, and evaporate the filtrate to dryness.

[0081] Dissolve the residue in 20 ml of water. Extract with ethyl acetate five times, 30 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 10 ml volumetric flask, and dilute to the mark with methanol. Shake well to obtain the final product.

[0082] Sample 3: Take the aqueous layer of Sample 2, extract it 5 times with water-saturated n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, transfer it to a 10 ml volumetric flask, add methanol to the mark, shake well, and the sample is ready.

[0083] Experimental results are as follows Figure 9 As shown, when using an evaporative light detector, the number of signal peaks and absorption intensities in samples 1, 2, and 3 (direct ultrasonic extraction with methanol) and sample 3 (extraction with ethyl acetate followed by water-saturated n-butanol) were basically the same, while sample 2 (extraction with ethyl acetate) had fewer signal peaks. Considering the simplicity of operation, the same preparation method as sample 1 was selected for the test solution under evaporative light conditions. Simultaneously, experimental results showed that when using a UV detector, the number of signal peaks and relative absorption intensities in samples 1 (direct ultrasonic extraction with methanol) and 2 (extraction with ethyl acetate) were basically the same, while sample 3 (extraction with ethyl acetate followed by water-saturated n-butanol) had fewer signal peaks than samples 1 and 3, and no signal peaks appeared after 70 minutes of analysis. Considering the simplicity of operation and the number of signal peaks in the chromatogram, the same preparation method as sample 1 was selected for the test solution under UV conditions.

[0084] 1.2.2 Investigation on the preparation of test solution

[0085] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.02% formic acid solution was used as the mobile phase, and gradient elution was performed according to Table 1; evaporative light scattering detector (drift tube temperature 110℃, nitrogen flow rate 2.5-2.5 L / min); UV detector detection wavelength 220 nm; column temperature 30℃, injection volume 10 μL. Waters e2695 HPLC was used; the column used was an Agilent ZORBAX Plus C18 (4.6*250 mm, 5 μm).

[0086] 1.2.2.1 Investigation of Extraction Solvents

[0087] Preparation of the test solution: Accurately weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder into four portions, place them in stoppered conical flasks, and accurately add water, 70% methanol, 50% methanol, and 50ml of methanol respectively. Seal the flasks tightly and sonicate (250W, 40kHz) for 30 minutes. Remove the flasks, cool, filter, and evaporate the filtrate to dryness. Dissolve the residue in methanol and transfer it to a 10ml volumetric flask. Dilute to the mark with methanol and mix well. Results are as follows: Figure 10 As shown, when the extraction solvent is water, there are fewer signal peaks in the chromatogram, the response value is lower, and the baseline is relatively drifting. When the extraction solvent is 70% methanol, 50% methanol, or methanol, the signal peaks in the chromatogram are basically consistent, but the baseline is best when the extraction solvent is methanol. Therefore, methanol is chosen as the extraction solvent.

[0088] 1.2.2.2 Investigation of Solvent Usage

[0089] The preparation of the test solution was the same as in 1.2.2.1, except that 25 mL, 50 mL, and 100 mL of methanol were used as the solvent. The results are as follows: Figure 11 As shown, when the test solution was extracted with 25 mL, 50 mL, and 100 mL of methanol, the extraction effect of each chromatographic peak in the chromatogram was basically the same. Therefore, for the sake of simplicity, 50 mL of methanol was chosen as the extraction solvent.

[0090] 1.2.2.3 Determined method for preparing the test solution and chromatographic conditions

[0091] Preparation of the test solution: Accurately weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder into three portions, place them in a stoppered conical flask, accurately add 50mL of methanol, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, filter, and evaporate the filtrate to dryness. Dissolve the residue in methanol and transfer to a 10mL volumetric flask. Add methanol to the mark and mix well to obtain the test solution.

[0092] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.02% formic acid solution was used as the mobile phase, and gradient elution was performed according to Table 1; evaporative light scattering detector (drift tube temperature 110℃, nitrogen flow rate 2.5 L / min); UV detector detection wavelength 220 nm; column temperature 30℃, injection volume 10 μL. Waters e2695 HPLC was used; the column used was an Agilent ZORBAX Plus C18 (4.6*250 mm, 5 μm).

[0093] The assay involves precisely pipetting 10 μL each of the test solution and the reference solution into a liquid chromatograph and measuring the results.

[0094] Experimental results are as follows Figure 12 As shown, there are 47 chromatographic peaks with high absorption intensity in both the UV detector and the evaporative light detector, of which 16 are shared by both detectors.

[0095] 1.2.2.4 Chromatographic Peak Assignment

[0096] The chromatographic conditions, preparation of the test solution, preparation of the reference solution, and determination method are the same as in 1.2.2.3.

[0097] Preparation of test solution for 21 single-herb medicinal slices in the prescription: Astragalus membranaceus, Ginseng, Ophiopogon japonicus, Schisandra chinensis, Angelica sinensis, Ligusticum chuanxiong, Ilex pubescens, Paeonia lactiflora, Spatholobus suberectus, Salvia miltiorrhiza, processed Polygonum multiflorum, Carthamus tinctorius, Lycopus lucidus, Curcuma longa, Curcuma longa, Gastrodia elata, Cinnamomum cassia, Notopterygium incisum, Gleditsia sinensis, Arisaema cum bile, and Borneol.

[0098] Accurately weigh approximately 2g of each individual herb powder from the prescription, place them in a stoppered conical flask, and precisely add 25mL of methanol to each. Seal the flask tightly and sonicate (250W power, 40kHz frequency) for 30 minutes. Remove the flask, cool it, filter it, and evaporate the filtrate to dryness. Dissolve the residue in methanol and transfer it to a 10ml volumetric flask. Add methanol to the mark and mix well to obtain the final product.

[0099] Peak attribution experimental results are as follows Figure 13 As shown, 47 peaks with high absorption intensity were detected by both the UV detector and the evaporative light detector, and 16 peaks were shared by both detectors. These peaks were found in Astragalus membranaceus (5 peaks), Ginseng (6 peaks), Ilex pubescens (3 peaks), Paeonia lactiflora (2 peaks), processed Polygonum multiflorum (4 peaks), Notopterygium incisum (4 peaks), Carthamus tinctorius (2 peaks), Schisandra chinensis (1 peak), and Angelica sinensis (1 peak). Thirteen of these peaks were identified using reference standards (ginsenosides Rb1, Rb2, Rb3, Rf, Re, Rg1, gentianin, ferulic acid, hydroxysaffron yellow A, paeoniflorin, stilbene glycoside, and purslane).

[0100] 1.3 Methodological Validation

[0101] 1.3.1 Precision Experiment

[0102] The chromatographic conditions, preparation method of the test solution, and determination method are the same as in 1.2.2.3.

[0103] The assay involves precisely pipetting 10 μL of the test solution and injecting it into the liquid chromatograph. Six consecutive injections are performed, and the result is obtained. At 220 nm, the results showed that 45 chromatographic peaks had a relative retention time RSD < 1% and a relative peak area RSD < 10.0%. The evaporative light scattering detection channel showed that 39 chromatographic peaks had a relative retention time RSD < 1% and a relative peak area RSD < 10.0%, indicating that the method has good precision.

[0104] 1.3.2 Repeatability Experiment

[0105] Preparation of the test solution: Accurately weigh approximately 2g of Astragalus and Hirudo Tongluo Capsule powder into 6 portions, place them in stoppered conical flasks, accurately add 50ml of methanol to each flask, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, filter, and evaporate the filtrate to dryness. Dissolve the residue in methanol and transfer to a 10ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the test solution.

[0106] The chromatographic conditions and determination methods are the same as in 1.2.2.3. At 220 nm, the results showed that the relative retention time RSD of 45 chromatographic peaks in the 6 samples was <1%, and the relative peak area RSD was <10.0%. The results of the evaporative light scattering detection channel showed that the relative retention time RSD of 39 chromatographic peaks was <1%, and the relative peak area RSD was <10.0%, indicating that the method has good repeatability.

[0107] 1.3.3 Stability Test

[0108] The chromatographic conditions, preparation method of the test solution, and determination method are the same as in 1.2.2.3.

[0109] For the assay, the same test solution was injected in 10 μL at 0 h, 5 h, 10 h, 15 h, 25 h, and 30 h for analysis. The results at 220 nm showed that the relative retention time RSD of 45 chromatographic peaks at different time points was <1%, and the relative peak area RSD was <10.0%. The evaporative light scattering detection channel showed that the relative retention time RSD of 39 chromatographic peaks was <1%, and the relative peak area RSD was <10.0%, indicating good stability of the solution within 24 hours.

[0110] 1.3.4 Durability Test

[0111] The tolerance of the detection method to minor changes in chromatographic conditions was investigated. This experiment examined different column temperatures (25℃, 30℃, 35℃), different flow rates (0.9 ml / min, 1.0 ml / min, 1.1 ml / min), different mobile phase systems (acetonitrile-water; acetonitrile-0.1% formic acid solution), and different chromatographic columns (Agilent ZORBAX Plus C18, Agilent TC C18, Waters Xselect Hss T3 C18). Gradient elution conditions were as shown in Table 1.

[0112] 1.3.4.1 Flow velocity investigation

[0113] The preparation of the test solution, chromatographic conditions, and determination method are the same as in 1.2.2.3. Results at 220 nm are shown below. Figure 14 The results of the evaporative light scattering detection channel are shown below. Figure 15 .

[0114] 1.3.4.2 Column Temperature Investigation

[0115] The preparation of the test solution, chromatographic conditions, and determination method are the same as in 1.2.2.3. Results at 220 nm are shown below. Figure 16 The results of the evaporative light scattering detection channel are shown below. Figure 17 .

[0116] 1.3.4.3 Chromatographic column investigation

[0117] The preparation of the test solution, chromatographic conditions, and determination method are the same as in 1.2.2.3. Results at 220 nm are shown below. Figure 18 The results of the evaporative light scattering detection channel are shown below. Figure 19 .

[0118] The robustness test results show that the detection method has good tolerance to minor changes in chromatographic conditions such as column temperature, flow rate, and column.

[0119] 1.4 Testing of different batches of samples

[0120] Fifteen batches of Qizhi Tongluo Capsules were tested using the above method. The batch numbers were 20200902(S1), 20200903(S2), 20200904(S3), 20200905(S4), 20200906(S5), 20200907(S6), 20200908(S7), 20200910(S8), and 20201002(S9). For samples 20201003(S10), 20201004(S11), 20201005(S12), 20201006(S13), 20201007(S14), and 20201008(S15), the preparation of the test solution, chromatographic conditions, and determination methods are the same as in 1.2.2.3. The results of the 15 batches of tests indicate that, under a detection condition of 220 nm, the S peak is purpuricin. The relative retention times of the 45 chromatographic peaks were 0.090, 0.096, 0.154, 0.192, 0.210, 0.279, 0.328, 0.379, 0.397, 0.406, 0.415, 0.426, 0.440, 0.469, 0.528, 0.569, 0.647, 0.704, 0.764, 0.793, 0.831, and 0.831, respectively. 0.842, 0.875, 0.936, 0.983, 1.00, 1.159, 1.193, 1.220, 1.263, 1.331, 1.348, 1.416, 1.517, 1.533, 1.577, 1.631, 1.744, 1.784, 1.828, 1.894, 1.909, 1.925, 1.947, 1.991. The RSDs for relative retention times are all less than 1%, while the RSDs for relative peak areas vary considerably. Under evaporative light scattering detector conditions, with purslane as the S peak, the relative retention times of the 39 chromatographic peaks were 0.091, 0.097, 0.155, 0.191, 0.280, 0.329, 0.380, 0.398, 0.530, 0.571, 0.648, 0.876, 0.938, 0.981, 1.00, 1.123, 1.160, 1.221, and 1.26, respectively. 5. The RSDs of the relative retention times (1.306, 1.316, 1.579, 1.608, 1.624, 1.648, 1.658, 1.677, 1.683, 1.715, 1.732, 1.809, 1.844, 1.856, 1.858, 1.87, 1.879, 1.906, 1.919, and 1.926) are all less than 1%, while the RSDs of the relative peak areas vary considerably. This indicates that the chemical composition of the raw materials used in Qizhi Tongluo Capsules differs between different batches.

[0121] 1.5 Similarity Evaluation and Chromatographic Peak Assignment

[0122] The fingerprint chromatograms of 15 batches of Astragalus and Hirudo Tongluo Capsules (samples S1-S15) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (2012 version). The reference chromatogram was set, and the median method was used. The time window width was set to 0.1 min. Multi-point correction was performed and a control fingerprint chromatogram (R) was generated. The similarity was calculated.

[0123] The results showed that under UV chromatographic conditions at 220 nm, 45 common peaks were identified in 15 batches of Astragalus and Hirudo Tongluo Capsules, with relative retention times of 0.988, 0.973, 0.970, 0.995, 0.993, 0.990, 0.995, 0.996, 0.981, 0.996, 0.986, 0.995, 0.996, 0.994, and 0.995, respectively. The chromatograms of the samples were compared with those of single-herb medicinal slices and some reference standards. Peak 4 was assigned to *Ligusticum chuanxiong*; peaks 5, 15, and 29 to *Ilex pubescens*; peaks 16 and 17 (paeoniflorin) to *Paeonia lactiflora*; peaks 8 (hydroxysafflower yellow A) and 9 to *Carthamus tinctorius*; peak 19 (ferulic acid) to *Angelica sinensis*; peaks 26 (peucedanin), 32, 35, and 43 to *Notopterygium incisum*; and peaks 6, 24 (stilbene glycoside), 34, and 37 to processed *Polygonum multiflorum*. Based on the reference standard information, five chromatographic peaks were identified: peak 24 (stilbene glycoside), peak 19 (ferulic acid), peak 26 (peucedanin), peak 8 (hydroxysafflower yellow A), and peak 17 (paeoniflorin). Figure 1 and Figure 20 ).

[0124] Under evaporative light scattering detector (ELSD) chromatographic conditions, 39 common peaks were identified in 15 batches of Astragalus membranaceus and Hirudo medicinalis capsules. The similarity of the HPLC-ELSD fingerprint chromatograms of the 15 batches of samples were 0.978, 0.939, 0.937, 0.991, 0.991, 0.972, 0.992, 0.996, 0.959, 0.987, 0.953, 0.992, 0.988, 0.986, and 0.983, respectively, indicating that the differences between the batches of samples were small and the preparation process was stable. The chromatograms of the samples were compared with those of single-herb medicinal slices and some reference chromatograms. Peaks 4, 9, and 18 were assigned to *Ilex pubescens*; peaks 7 (hydroxysafflower yellow A) and 8 to *Carthamus tinctorius*; peaks 12, 33, and 37 to *Astragalus membranaceus*; peaks 10 and 11 (paeoniflorin) to *Paeonia lactiflora*; peak 15 (peucedanin) to *Notopterygium incisum*; peak 13 (stilbene glycoside) to *Polygonum multiflorum*; and peaks 20 (ginsenoside Rg1), 21 (ginsenoside Re), 25, 26 (ginsenoside Rf), 27 (ginsenoside Rb1), 30 (ginsenoside Rb2), 31 (ginsenoside Rb3), and 32 to ginseng. Ten peaks were identified in the reference standard. Figure 2 and Figure 21 ).

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a formula containing Astragalus membranaceus and Hirudo medicinalis, characterized in that, The chromatographic analysis of the Astragalus and Hirudo formula and the single-ingredient raw material was performed sequentially using an ultraviolet detector and an evaporative light scattering detector, respectively. Before the chromatographic analysis, the sample was extracted using 50%–100% methanol as the extraction solvent. The chromatographic detection includes: using octadecylsilane-bonded silica gel as the stationary phase and acetonitrile-0.02% formic acid solution as the mobile phase, performing gradient elution as shown below; The chromatographic detection includes the use of an ultraviolet detector and an evaporative light scattering detector, wherein the detection wavelength of the ultraviolet detector is 220 nm. It also includes performing the chromatographic detection on single medicinal material samples, and sequentially comparing the chromatograms of each sample with those of the reference standards to confirm the substances represented by each chromatographic peak and / or the medicinal materials to which they belong. The reference standards include: ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rf, ginsenoside Re, ginsenoside Rg1, gentianin, ferulic acid, hydroxysaffron yellow A, paeoniflorin, stilbene glycoside, and purslane.

2. The application of the fingerprint spectrum constructed using the construction method described in claim 1 in evaluating the quality of the Astragalus and Hirudo formula for unblocking collaterals.

3. The application according to claim 2, characterized in that, The Astragalus and Hirudo formula includes Astragalus and Hirudo formulas in the same or different dosage forms.

4. The application according to claim 2 or 3, characterized in that, After constructing the fingerprint spectrum using the construction method described in claim 1, the method further includes comparing the characteristic peaks.

5. The application according to claim 4, characterized in that, The characteristic peaks include peaks of chemical components that can be detected simultaneously by an ultraviolet detector and an evaporative light scattering detector: paeoniflorin, hydroxysafflower yellow A, purslane, and stilbene glycoside.

6. The application according to claim 4, characterized in that, The characteristic peaks also include peaks of chemical components that can only be detected by ultraviolet detectors: ferulic acid and gentianin; The characteristic peaks also include peaks of chemical components that are more easily detected by an evaporative light scattering detector: ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and astragaloside A.

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