A quality control method and application of fingerprint spectrum of compound ginkgo leaf dry paste powder

Abstract

The present invention belongs to the field of drug detection technology, and specifically relates to a quality control method and application of a fingerprint spectrum of a compound ginkgo leaf tablet dry paste powder. The method has the characteristics of good specificity, high precision and good repeatability; at the same time, the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder established has a large amount of information and strong characteristics, which can effectively reflect the overall quality of the compound ginkgo leaf tablet dry paste powder and ensure the stable uniformity, safety and effectiveness of the drug quality.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a quality control method and application of a fingerprint spectrum of a compound ginkgo leaf tablet dry paste powder. Background Art

[0002] Compound Ginkgo Leaf Tablets is derived from an empirical prescription and is suitable for patients with vascular dementia with phlegm and blood stasis blocking the collaterals and kidney essence deficiency. The formulation, drug selection, and prescription of Compound Ginkgo Leaf Tablets are in line with the theory of traditional Chinese medicine and the etiology and pathogenesis of the applicable disease, and have obvious characteristics among similar products. The clinical efficacy of Compound Ginkgo Leaf Tablets is to promote blood circulation and remove blood stasis, remove phlegm and dredge the collaterals, and invigorate the brain and improve intelligence, which is also the basis for its formulation; drug selection needs to have the following characteristics, namely, treating the symptoms and the root cause at the same time to mainly treat the symptoms, using both cold and heat to mainly balance, and regulating qi and blood to mainly treat blood; the prescription is selected according to the pathogenesis of vascular dementia with kidney deficiency as the root cause and phlegm and blood stasis as the symptoms, but the symptoms are more urgent.

[0003] The compound ginkgo leaf tablets contain two parts: ginkgo leaf extract and compound ginkgo leaf tablet dry paste powder, which are pressed into tablets through a suitable preparation molding process.

[0004] Among them, ginkgo leaf extract, as the main medicine in the compound ginkgo leaf formula, has the effects of promoting blood circulation, removing blood stasis, unblocking meridians and relieving pain. It works on cerebral blood stasis, the initial cause of the disease. It is a variety included in the 2020 edition of the "Chinese Pharmacopoeia". It has multiple indicators such as total ginkgolic acid content, total flavonol glycoside content, terpene lactone content, fingerprint spectrum, etc., which can ensure the uniformity and stability of the raw material quality.

[0005] In addition, the compound ginkgo leaf dry paste powder, as another important part of the compound ginkgo leaf, is a mixed extract including four herbs: epimedium, earthworm, chuanxiong, and polygala (processed). Among them, the ministerial herb epimedium warms the kidney and strengthens yang, replenishes essence and nourishes the brain; the ministerial herb earthworm clears away heat and calms the nerves, dredges the meridians, and matches the cold and warm properties of epimedium; the adjuvant herb chuanxiong promotes blood circulation and qi, dispels wind and relieves pain, and can assist the monarch herb ginkgo leaf extract, the ministerial herb epimedium and the ministerial herb earthworm in promoting blood circulation and removing blood stasis, and chuanxiong also enters the qi, and cooperates with the ginkgo leaf extract to enter the blood, treating both qi and blood; the adjuvant polygala (processed) removes phlegm and opens the orifices, calms the mind and improves intelligence, and connects the heart and kidney.

[0006] The composition of compound ginkgo leaf dry powder is more complex, and its quality becomes the key factor to determine the quality of compound ginkgo leaf. However, the overall composition characterization of the mixed extract of four herbs, epimedium, earthworm, ligusticum chuanxiong and polygala tenuifolia (processed), in compound ginkgo leaf dry powder has not been reported.

[0007] At present, existing studies have only reported on the characterization of the components of single herbs such as Epimedium, Pheretima, Chuanxiong, and Polygala. In the quality standard of dry paste powder we established, there are only two herbs, Chuanxiong and Polygala (processed), and the single ingredient content index of icariin.

[0008] By consulting patents and literature, no patents or literature on the establishment of fingerprint spectrum of compound ginkgo leaf tablet dry paste powder have been found. The research status of the mixed extracts in compound ginkgo leaf tablet dry paste powder: epimedium, earthworm, chuanxiong, and polygala (processed) is as follows:

[0009] ① Only one of the herbs in the compound ginkgo leaf dry paste powder was studied, such as the characterization of the ingredients of epimedium or polygala tenuifolia (processed):

[0010] Kang Shuai et al. from the China Food and Drug Inspection Institute used HPLC to establish the fingerprint and content determination method of flavonoid glycosides in Epimedium;

[0011] Jiang Yong et al. from Peking University used HPLC to establish the medicinal fingerprint of Polygala tenuifolia (processed);

[0012] ②Only the representative components of two herbs in the compound ginkgo leaf dry paste powder were studied, such as the content characterization of the two herbs, epimedium and ligusticum chuanxiong:

[0013] Wang Ling and others from Zhejiang Chinese Medical University established a method for determining the content of icariin ferulate in the two herbs Epimedium and Ligusticum chuanxiong.

[0014] In addition, ③ Chinese patent application CN102818861A established a fingerprint spectrum detection method for 10 drugs in Qingdu Anshen Capsule, including Astragalus, Epimedium, Rhubarb, Angelica, Chuanxiong, White Peony, Rehmannia, Panax Notoginseng, Poria, and Alisma. Although it contains two drugs, Epimedium and Chuanxiong, icariin is used as a reference. When this method is used to detect the compound ginkgo leaf dry paste powder, the overlap rate between the fingerprint peaks in the test results is high and the separation is poor, which cannot reflect the types and quantities of chemical components contained in the compound ginkgo leaf dry paste powder. Therefore, it is not suitable for fingerprint spectrum detection of compound ginkgo leaf dry paste powder.

[0015] Based on the above research progress, it can be seen that the evaluation method for compound ginkgo leaf dry paste powder is single and the information provided is little.

[0016] As a comprehensive and quantifiable identification method, the fingerprint of traditional Chinese medicine has the characteristics of large amount of information, strong characteristic, integrity, fuzziness, etc., which can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine. At present, the fingerprint of traditional Chinese medicine has become one of the quality control modes that conforms to the characteristics of traditional Chinese medicine, and its application in the quality control of traditional Chinese medicine preparations is becoming more and more extensive. Traditional Chinese medicine is a multi-component, multi-target synergistic system with complex ingredients. It is difficult to fully control the quality of the product by controlling a single indicator. In order to improve the quality of drugs and ensure the safety and effectiveness of clinical drug use, it is urgent to establish a fingerprint of traditional Chinese medicine.

[0017] The establishment of fingerprints is based on systematic chemical composition research and pharmacological research. Generally, a main active ingredient or index ingredient that is easily available and has a high content in the sample is selected as a reference, which helps to ensure the stability and reproducibility of the fingerprint. However, the compound ginkgo leaf dry paste powder is made from a mixed extract of four herbs: epimedium, earthworm, chuanxiong and polygala (processed). The composition of the compound ginkgo leaf dry paste powder is complex, and the peaks of each component are dense in the fingerprint of the prior art, and the response size is different, and the sensitivity is low; in addition, the reference of the fingerprint of the compound ginkgo leaf dry paste powder has not been studied, and it is impossible to describe and evaluate the quality of the compound ginkgo leaf dry paste powder as a whole, and it is difficult to effectively reflect the overall quality of the compound ginkgo leaf dry paste powder.

[0018] Therefore, there is an urgent need to establish a method for fingerprinting of compound ginkgo leaf tablets dry paste powder, to conduct comprehensive quality control and evaluation of the compound ginkgo leaf tablets dry paste powder, to effectively identify the common fingerprint peaks, to effectively reflect the overall quality of the compound ginkgo leaf tablets dry paste powder, to comprehensively reflect the types and quantities of chemical components contained in the ginkgo leaf tablets dry paste powder, to focus on the overall appearance characteristics, and to ensure the stable uniformity, safety and effectiveness of the drug quality. Summary of the invention

[0019] In view of the above technical problems, the present invention provides a method for detecting the fingerprint spectrum of compound ginkgo leaf tablet dry paste powder. The method adopts high performance liquid chromatography (HPLC) to conduct comprehensive quality control and evaluation of compound ginkgo leaf tablet dry paste powder. ① For the first time, it is found that selecting icariin as a reference is conducive to the discrimination of each common fingerprint peak; ② On the basis of using icariin as a reference, the chromatographic detection conditions are controlled, which has the characteristics of good specificity, high precision and good repeatability; ③ In addition, the fingerprint spectrum of compound ginkgo leaf tablet dry paste powder established by controlling the reference and chromatographic detection conditions has the characteristics of large amount of information and strong characteristic. Through the selection of reference and the control of chromatographic conditions, the overall quality of compound ginkgo leaf tablet dry paste powder can be effectively reflected, the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder can be comprehensively reflected, and the overall appearance characteristics are emphasized to ensure the quality stability, uniformity, safety and effectiveness of the drug. The details are as follows:

[0020] 1. First, the present invention first discovered that using icariin as a reference is conducive to the discrimination of each common fingerprint peak: ① The fingerprint spectrum has 9 common fingerprint peaks, and each fingerprint peak can be separated and quantified; ② The reference icariin is derived from the ministerial drug epimedium, which is consistent with the functions and indications of the compound ginkgo leaf tablets; ③ Icariin is a main active ingredient or indicator ingredient that is easy to obtain and has a high content, which conforms to the general principle of being a reference. Therefore, using icariin as a reference is helpful to ensure the stability and reproducibility of the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder.

[0021] 2. At the same time, by controlling the chromatographic detection conditions, the detection method has the characteristics of good specificity, high precision and good repeatability: ① Good specificity: a total of 9 fingerprint peaks were determined, and the attribution of each chromatographic peak could be determined; ② High precision: the relative retention time and relative peak area RSD values ​​of each fingerprint peak were less than 0.24% and 0.87% respectively; the similarity between each fingerprint spectrum was 1.000; ③ Good repeatability: the relative retention time and relative peak area RSD values ​​of each fingerprint peak were less than 0.24% and 1.5% respectively; the similarity between each fingerprint spectrum was 1.000;

[0022] 3. In addition, by controlling the reference material and chromatographic detection conditions at the same time, the fingerprint of the compound ginkgo leaf dry paste powder was established, which has the characteristics of large amount of information and strong characteristic: ① The corresponding chromatographic peaks of the fingerprint components are retained intact (9 fingerprint peaks), and the separation effect between the chromatographic peaks is good (good peak shape); ② The solution has good stability: the relative retention time and relative peak area RSD values ​​of each fingerprint peak are less than 0.04% and 0.86%, respectively; the similarity between each fingerprint spectrum is 1.000.

[0023] In one aspect, the present invention provides a method for detecting a pharmaceutical preparation, comprising the following steps:

[0024] 1) Using HPLC method to determine the HPLC fingerprint of the drug preparation to obtain the fingerprint of the test sample;

[0025] 2) using HPLC to measure the HPLC spectrum of the icariin reference substance to obtain a reference fingerprint, and comparing it with the fingerprint of the test substance in step 1);

[0026] 3) The fingerprint of the test sample should show 9 common peaks, among which the chromatographic peak with the same retention time as the chromatographic peak of the icariin reference substance in step 2) is used as the reference peak. When the relative retention time and relative deviation of each common peak meet the following conditions, and the chromatographic peaks of 0 to 45 minutes are calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, and the similarity between the fingerprint of the test sample and the reference fingerprint is not less than 0.90, it is determined that the pharmaceutical preparation contains compound ginkgo leaf tablet dry extract powder:

[0027] Peak 1: relative retention time 0.199 ± 0.020, relative deviation 0-10%;

[0028] Peak 2: relative retention time 0.207±0.021, relative deviation 0-10%;

[0029] Peak 3: relative retention time 0.217±0.022, relative deviation 0-10%;

[0030] Peak 4: relative retention time 0.680±0.068, relative deviation 0-10%;

[0031] Peak 5: relative retention time 0.966±0.097, relative deviation 0-10%;

[0032] Peak 6: relative retention time 1.000, relative deviation 0%;

[0033] Peak 7: relative retention time 1.023 ± 0.102, relative deviation 0-10%;

[0034] Peak 8: relative retention time 1.048±0.148, relative deviation 0-10%;

[0035] Peak 9: relative retention time 1.393 ± 0.139, relative deviation 0-10%;

[0036] Wherein, the chromatographic conditions of the HPLC method include:

[0037] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0038] Mobile phase: acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B;

[0039] Elution mode: gradient elution.

[0040] In some embodiments, the model of the chromatographic column is GL Inertsil ODS-3 or Welch UltimateAQ C18.

[0041] In some embodiments, the gradient elution program is as follows:

[0042] Time / min Mobile phase A / % Mobile phase B / % 0 15 85 10 20 80 35 40 60 36 80 20 40 80 20 41 15 85 45 15 85 .

[0043] In some embodiments, the mobile phase B is a 0.3% to 0.5% phosphoric acid aqueous solution, preferably a 0.4% phosphoric acid aqueous solution.

[0044] In some embodiments, in the chromatographic conditions, the detection wavelength is 220-390 nm, preferably, the detection wavelength is 320 nm.

[0045] Another aspect of the present invention provides a method for detecting compound ginkgo leaf tablet dry paste powder, which comprises the following steps:

[0046] 1) Using high performance liquid chromatography (HPLC) to determine the HPLC fingerprint of the compound ginkgo leaf tablet dry powder, to obtain the fingerprint of the test sample;

[0047] 2) using HPLC to measure the HPLC spectrum of the icariin reference substance to obtain a reference fingerprint, and comparing it with the fingerprint of the test substance in step 1);

[0048] 3) The fingerprint of the test sample should show 9 common peaks, among which the chromatographic peak with the same retention time as the chromatographic peak of the icariin reference substance in step 2) is used as the reference peak. The relative retention time and relative deviation of each common peak meet the following conditions, and the chromatographic peaks of 0 to 45 minutes are calculated according to the similarity evaluation system of Chinese medicine chromatographic fingerprints. The similarity between the fingerprint of the test sample and the reference fingerprint is not less than 0.90.

[0049] Peak 1: relative retention time 0.199 ± 0.020, relative deviation 0-10%;

[0050] Peak 2: relative retention time 0.207±0.021, relative deviation 0-10%;

[0051] Peak 3: relative retention time 0.217±0.022, relative deviation 0-10%;

[0052] Peak 4: relative retention time 0.680±0.068, relative deviation 0-10%;

[0053] Peak 5: relative retention time 0.966±0.097, relative deviation 0-10%;

[0054] Peak 6: relative retention time 1.000, relative deviation 0%;

[0055] Peak 7: relative retention time 1.023 ± 0.102, relative deviation 0-10%;

[0056] Peak 8: relative retention time 1.048±0.148, relative deviation 0-10%;

[0057] Peak 9: relative retention time 1.393 ± 0.139, relative deviation 0-10%;

[0058] The chromatographic conditions of the HPLC method include:

[0059] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0060] Mobile phase: acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B;

[0061] Elution mode: gradient elution.

[0062] In some embodiments, in the chromatographic conditions, the chromatographic column is filled with octadecylsilane bonded silica gel; preferably, the chromatographic column model is GL Inertsil ODS-3 or Welch Ultimate AQ C18.

[0063] In some preferred embodiments, the gradient elution procedure is as follows:

[0064] Time / min Mobile phase A / % Mobile phase B / % 0 15 85 10 20 80 35 40 60 36 80 20 40 80 20 41 15 85 45 15 85

[0065] In some embodiments, in the chromatographic conditions, mobile phase B is a 0.3% to 0.5% phosphoric acid aqueous solution, preferably, mobile phase B is a 0.4% phosphoric acid aqueous solution.

[0066] In some embodiments, in the chromatographic conditions, the detection wavelength is 220-390 nm, preferably, the detection wavelength is 320 nm.

[0067] The present invention also provides a fingerprint spectrum of the compound ginkgo leaf dry paste powder, and the process of establishing the spectrum comprises the following steps:

[0068] (1) Solution preparation:

[0069] Test solution: weigh 0.35 g of dry paste powder, accurately add 20 mL of 80% methanol, seal, weigh, ultrasonically treat (power 250 w, frequency 40 kHz) for 10 min, cool, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate.

[0070] Reference solution: Weigh the icariin reference substance and use 80% methanol to prepare a solution containing 120 μg per 1 mL.

[0071] (2) Chromatographic conditions:

[0072] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0073] Mobile phase: acetonitrile as mobile phase A; 0.4% by volume phosphoric acid solution as mobile phase B;

[0074] Elution mode: gradient elution;

[0075] Flow rate: 1.0 mL / min;

[0076] Column temperature: 45°C;

[0077] Detection wavelength: 320nm.

[0078] (3) Detection:

[0079] Pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatograph, measure, and record the chromatogram within 45 min.

[0080] By using HPLC and comparing with the reference substance information, four active ingredients in the compound ginkgo leaf dry extract powder were identified, namely 3,6'-diesinapoylsucrose (peak 4), epimedin C (peak 5), icariin (peak 6) and ligustilide (peak 9). Among them, 3,6'-dienasinoylsucrose is derived from the adjuvant drug Polygala tenuifolia (processed). According to the principle of monarch, minister, assistant and envoy in the compatibility of traditional Chinese medicine, it is not a preferred option for the reference peak; ligustilide is derived from the adjuvant drug Chuanxiong. If it is used as a reference, peak 9 is the reference peak (S peak), and the relative retention times of the other 8 peaks are all less than 1.000, which is not conducive to the positioning and judgment of the remaining common peaks. In addition, ligustilide itself is a volatile component with poor stability, so it is not a preferred option for the reference peak; epimedin C and icariin are both derived from the adjuvant drug Epimedium, but existing literature reports that the pharmacological effects of epimedin C are mainly manifested in antioxidant, anti-inflammatory, anti-osteoporosis and anti-tumor activities, while icariin has the effects of increasing cardiovascular and cerebrovascular blood flow, promoting hematopoietic function, and also has It has the effects of tonifying the kidney and strengthening yang, anti-aging, warming the kidney and strengthening yang, replenishing essence and nourishing the brain, and plays a role in the main pathogenesis of kidney deficiency. The selection of icariin as a reference is more consistent with the functions and indications of compound ginkgo leaf tablets. On the other hand, the icariin peak is in the most intensive period of the chromatogram, which can provide an accurate reference for the positioning of peaks 5, 7 and 8 that are easily interfered and difficult to distinguish. In addition, the peak corresponding to icariin is the S peak, and the calculated relative retention time of each fingerprint peak is moderate, and its properties are stable. Obviously, compared with 3,6'-diesinapoylsucrose (peak 4), epimedin C (peak 5) and ligustilide (peak 9), icariin (peak 6) is more suitable as a reference peak to determine the HPLC fingerprint of compound ginkgo leaf tablet dry paste powder.

[0081] Therefore, the icariin peak was selected as the reference peak to determine the compound ginkgo leaf dry paste powder.

[0082] The HPLC fingerprint of the compound ginkgo leaf dry paste powder constructed by the present invention comprises 9 common main components, icariin is used as a reference, peak 6 is used as a reference peak (S peak), the fingerprint has 9 common fingerprint peaks, the peak corresponding to the reference peak is used as the S peak, the relative retention time of each fingerprint peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value. The specified values ​​are peak 1: 0.199, peak 2: 0.207, peak 3: 0.217, peak 4: 0.680, peak 5: 0.966, peak 6: 1.000 (S peak), peak 7: 1.023, peak 8: 1.048, peak 9: 1.393.

[0083] The above chromatographic peaks constitute the fingerprint characteristics of the compound ginkgo leaf dry paste powder.

[0084] The present invention has achieved at least the following beneficial effects:

[0085] The present invention provides a method for detecting the fingerprint spectrum of compound ginkgo leaf tablet dry paste powder, the method adopts high performance liquid chromatography (HPLC) to conduct comprehensive quality control and evaluation of the compound ginkgo leaf tablet dry paste powder, ① for the first time, it is found that selecting icariin as a reference is conducive to the discrimination of each common fingerprint peak; ② on the basis of using icariin as a reference, the chromatographic detection conditions are controlled, which has the characteristics of good specificity, high precision and good repeatability; ③ in addition, the fingerprint spectrum of compound ginkgo leaf tablet dry paste powder established by controlling the reference and chromatographic detection conditions has the characteristics of large amount of information and strong characteristic. Through the selection of reference and the control of chromatographic conditions, the overall quality of compound ginkgo leaf tablet dry paste powder can be effectively reflected, the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder can be comprehensively reflected, the overall appearance characteristics can be emphasized, the stability of the production process can be monitored, and the quality stability, uniformity, safety and effectiveness of the drug can be ensured. The details are as follows:

[0086] 1. Selection of reference objects:

[0087] 1) The present invention first discovered that using icariin as a reference is beneficial to the discrimination of each common fingerprint peak, and helps to ensure the stability and reproducibility of the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder, which is specifically manifested as follows:

[0088] ① The fingerprint spectrum has 9 common fingerprint peaks, each of which can be separated and quantified;

[0089] ② The reference substance icariin is derived from the ministerial drug Epimedium, which is consistent with the functions and indications of the compound ginkgo leaf tablets (including ginkgo leaf extract and compound ginkgo leaf tablet dry paste powder);

[0090] ③ Icariin is a major active ingredient or indicator ingredient that is easily available and has a high content, which conforms to the general principle of being used as a reference.

[0091] 2) On the contrary, the use of other reference substances (for example, ligustilide and epimedin C) is not conducive to the identification of the common fingerprint peaks, and the stability and reproducibility of the fingerprint spectrum of the compound ginkgo leaf dry paste powder cannot be guaranteed.

[0092] 2. On the basis of selecting icariin as the reference substance, the chromatographic detection conditions are controlled so that the detection method has the characteristics of good specificity, high precision and good repeatability:

[0093] For example, by selecting a specific chromatographic column (for example, a chromatographic column with octadecylsilane bonded silica gel as a filler), a specific chromatographic column gradient elution program, a concentration of the mobile phase B phosphoric acid solution between 0.3% and 0.5%, and a detection wavelength between 220 and 390 nm, the detection method has the characteristics of good specificity, high precision, and good repeatability, which is specifically manifested as follows:

[0094] ①Good specificity: A total of 9 fingerprint peaks were determined, and the attribution of each chromatographic peak can be determined;

[0095] ② High precision: the RSD values ​​of the relative retention time and relative peak area of ​​each fingerprint peak are less than 0.24% and 0.87% respectively; the similarity between each fingerprint spectrum is 1.000;

[0096] ③Good repeatability: The RSD values ​​of the relative retention time and relative peak area of ​​each fingerprint peak were less than 0.24% and 1.5%, respectively; the similarity between each fingerprint spectrum was 1.000.

[0097] Description of the drawings

[0098] Figure 1 The HPLC chromatogram of the compound ginkgo leaf dry paste powder test solution of Example 1;

[0099] Figure 2 The HPLC chromatogram of the reference substance of the compound ginkgo leaf tablet dry paste powder with icariin as the reference substance in Example 3;

[0100] Figure 3 It is a fingerprint superposition spectrum of 10 batches of compound ginkgo leaf tablet dry paste powder of Example 13;

[0101] Figure 4 It is a reference spectrum of the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder of Example 13;

[0102] Figure 5 This is the fingerprint spectrum of the typical test solution of the compound ginkgo leaf dry paste powder measured by the present invention in Example 13. DETAILED DESCRIPTION

[0103] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. In the embodiments of the present invention, "%" refers to volume percentage unless otherwise specified.

[0104] Example 1

[0105] A method for detecting a fingerprint spectrum of a compound ginkgo leaf tablet dry paste powder, the method comprising the following steps and conditions:

[0106] 1-1. Solution preparation:

[0107] Reference solution: Take 5 mg of ligustilide reference substance, place it in a 100 mL volumetric flask, dissolve it in 80% methanol and dilute to the scale, shake well, and obtain.

[0108] Test solution: accurately weigh 0.35 g of dry paste powder, accurately add 20 mL of 80% methanol, seal, ultrasonically treat (power 250 w, frequency 40 kHz) for 10 min, shake well, filter, and obtain the filtrate.

[0109] 1-2. Test conditions:

[0110] Chromatographic column: model GL Inertsil ODS-3, specification 4.6*250mm, 5μm;

[0111] Mobile phase: Mobile phase A is acetonitrile; Mobile phase B is 0.4% phosphoric acid solution;

[0112] Elution method: gradient elution;

[0113] Gradient elution program:

[0114] Time / min Mobile phase A / % Mobile phase B / % 0 15 85 10 20 80 35 40 60 36 80 20 40 80 20 41 15 85 45 15 85

[0115] Detection wavelength: 320nm;

[0116] Flow rate: 1.0 mL / min;

[0117] Column temperature: 45℃.

[0118] 1-3. Experimental steps and conclusions:

[0119] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0120] Table 1 Relative retention time and relative peak area using ligustilide as reference

[0121]

[0122]

[0123] in conclusion:

[0124] Using ligustilide as a reference, the HPLC fingerprint of the compound ginkgo leaf dry paste powder ( Figure 1), there are 9 common fingerprint peaks in the fingerprint spectrum (Table 2): Taking the No. 9 peak corresponding to the reference substance ligustilide as the reference peak (S peak), the relative retention time and relative peak area of ​​each fingerprint peak and the S peak are calculated. Since the reference peak (S peak) is at the end of the spectrum, the relative retention times of the other 8 peaks before the No. 9 peak are all less than 1.000, the peak distribution is relatively concentrated, and the relative retention time differences between peaks 5, 6, 7, and 8 are extremely small, which is not conducive to the discrimination of common peaks;

[0125] In addition, ligustilide is derived from the adjuvant Ligusticum chuanxiong, which is a volatile component with poor stability;

[0126] The above results show that ligustilide cannot be used as a preferred reference for determining the fingerprint of compound ginkgo leaf dry paste powder.

[0127] Example 2

[0128] Referring to Example 1, the type of reference substance was changed. The difference from Example 1 was that the preparation of the reference solution was replaced by "reference solution: take 5 mg of ligustilide reference substance, put it in a 100 mL volumetric flask, dissolve and dilute to the scale with 80% volume of methanol, shake well, and obtain.": "reference solution: take 5 mg of epimedin C reference substance, put it in a 100 mL volumetric flask, dissolve and dilute to the scale with 80% volume of methanol, shake well, and obtain." The other solution preparations, test conditions and experimental procedures were the same as in Example 1.

[0129] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0130] Table 2 Relative retention time and relative peak area using epimedin C as reference

[0131] Chromatographic peak number Relative retention time Relative peak area Peak 1 0.206 0.243 Peak 2 0.214 0.370 Peak 3 0.225 1.202 Peak 4 0.705 1.861 Peak 5(S) 1.000 1.000 Peak 6 1.035 2.553 Peak 7 1.059 0.514 Peak 8 1.085 0.803 Peak 9 1.443 1.469

[0132] in conclusion:

[0133] Using epimedin C as reference, the HPLC fingerprint of the compound ginkgo leaf dry paste powder ( Figure 1 ), there are 9 common fingerprint peaks in the fingerprint (Table 3), with the corresponding peak No. 5 of the reference as the reference peak and the S peak as the reference peak, the relative retention time and relative peak area of ​​each fingerprint peak and the S peak are calculated, and the relative retention time is distributed between 0.206 and 1.443, while the relative retention time with ligustilide as the reference is distributed between 0.143 and 0.734. Obviously, with epimedin C as the reference, the relative retention time range is wider, which is more conducive to peak discrimination, and is significantly better than the HPLC fingerprint of the compound ginkgo leaf tablet dry paste powder obtained when ligustilide is used as the reference in Example 1;

[0134] However, existing literature reports (Li Songlin et al.'s study "Discussion on the Quantitative Index of Epimedium in the Chinese Pharmacopoeia"; Li Han et al.'s study "Comparison of the in vitro pharmacological activities of the main components of four flavonoid glycosides of Epimedium"; Zhou Kun et al.'s study "Study on the in vivo anti-osteoporosis mechanism of Epimedium C") show that the pharmacological effects of Epimedium C are mainly manifested in antioxidant, anti-inflammatory, anti-osteoporosis and anti-tumor activities, and have no direct correlation with the clinical efficacy of compound Ginkgo leaf tablets (promoting blood circulation and removing blood stasis, removing phlegm and unblocking collaterals, and strengthening the brain and improving intelligence);

[0135] The above results show that epimedin C cannot be used as a preferred reference for determining the fingerprint of compound ginkgo leaf dry extract powder.

[0136] Example 3

[0137] Referring to Example 1, the type of reference substance was changed. The difference from Example 1 was that the preparation of the reference solution was replaced by "reference solution: take 5 mg of ligustilide reference substance, put it in a 100 mL volumetric flask, dissolve and dilute to the scale with 80% volume of methanol, shake well, and obtain.": "reference solution: take 3 mg of icariin reference substance, put it in a 25 mL volumetric flask, dissolve and dilute to the scale with 80% volume of methanol, shake well, and obtain." Other solution preparations, test conditions and experimental procedures are the same as in Example 1.

[0138] Take 10 μL of the reference solution and the test solution, inject them into the HPLC, measure, and record the chromatogram within 45 minutes. Figure 2 , and the results are shown in the table below.

[0139] Table 3 Relative retention time and relative peak area using icariin as reference

[0140] Chromatographic peak number Relative retention time specification Relative retention time Relative peak area Peak 1 0.199 0.199 0.095 Peak 2 0.207 0.207 0.145 Peak 3 0.217 0.217 0.471 Peak 4 0.680 0.680 0.729 Peak 5 0.966 0.966 0.392 Peak 6(S) 1.000 1.000 1.000 Peak 7 1.023 1.023 0.201 Peak 8 1.048 1.048 0.314 Peak 9 1.393 1.393 0.576

[0141] in conclusion:

[0142] Using icariin as a reference, the HPLC fingerprint of the compound ginkgo leaf dry paste powder ( Figure 1 ), there are 9 common fingerprint peaks in the fingerprint spectrum, with the corresponding peak No. 6 of the reference as the reference peak (S peak), and the peak No. 6 (S peak) is in the most intensive period of the chromatogram, which can provide an accurate reference for the positioning of the peaks No. 5, No. 7 and No. 8 that are easily interfered and difficult to distinguish, so that the peaks No. 5, No. 7 and No. 8 are easy to separate; the relative retention time and relative peak area of ​​each fingerprint peak and the S peak are calculated, and the relative retention time is within ±10% of the specified value, and the relative peak area is distributed between 0.095 and 1.000, which can be quantitative;

[0143] The reference substance icariin is derived from the ministerial drug Epimedium, which can increase cardiovascular and cerebrovascular blood flow, promote hematopoietic function, and also has the effects of tonifying the kidney and strengthening yang, anti-aging, etc. It warms the kidney and strengthens yang, replenishes essence and nourishes the brain, and works on the main pathogenesis of kidney deficiency, which is consistent with the clinical efficacy of compound ginkgo leaf tablets (promoting blood circulation and removing blood stasis, removing phlegm and unblocking meridians, and strengthening the brain and improving intelligence);

[0144] Therefore, icariin complies with the general principle of being used as a reference (generally, a major active ingredient or index ingredient that is easily available and has a high content in the sample is selected as a reference, which helps to ensure the stability and reproducibility of the fingerprint), and icariin is preferably selected as a reference to determine the fingerprint of the compound ginkgo leaf dry paste powder.

[0145] summary:

[0146] The results of Examples 1 to 3 show that, compared with Example 1 using ligustilide (peak 9) as a reference and Example 2 using epimedin C (peak 5) as a reference, only Example 3 using icariin (peak 6) as a reference can effectively reflect the overall quality of the compound ginkgo leaf tablet dry paste powder, and is more suitable as a reference peak to determine the HPLC fingerprint of the compound ginkgo leaf tablet dry paste powder, which is specifically manifested as follows:

[0147] i. It has 9 common fingerprint peaks, and the fingerprint peaks in the spectrum can be separated and quantified;

[0148] ii. The reference substance icariin is derived from the ministerial drug Epimedium, which is consistent with the functions and indications of the compound Ginkgo leaf tablets;

[0149] iii. Icariin is a major active ingredient or indicator ingredient that is easily available and has a high content, which complies with the general principle of being used as a reference;

[0150] Therefore, icariin was selected as a reference to determine the fingerprint of compound ginkgo leaf dry paste powder.

[0151] The above results show that the HPLC fingerprint of the compound ginkgo leaf tablet dry paste powder obtained by using the reference icariin has 9 common fingerprint peaks, and the fingerprint peaks in the spectrum can be separated and quantified; at the same time, the reference icariin is derived from the ministerial drug epimedium, which is consistent with the functions and indications of the compound ginkgo leaf tablet; in addition, icariin is a main active ingredient or indicator ingredient that is easy to obtain and has a high content, which conforms to the general principle of being a reference. Therefore, using icariin as a reference helps to ensure the stability and reproducibility of the fingerprint. The following examples all use icariin as a reference to determine the fingerprint of the compound ginkgo leaf tablet dry paste powder for methodological verification.

[0152] Example 4 Specificity Test

[0153] Solution preparation:

[0154] Blank solvent: 80% methanol solution.

[0155] Reference solution: Take 3 mg of icariin reference substance, place it in a 25 mL volumetric flask, dissolve it in 80% methanol and dilute to the scale, shake well, and obtain.

[0156] Single herb extract solution (peak attribution): Take 0.15g of Polygala tenuifolia (processed) extract sample, 0.15g of Pheretima japonica extract sample, 0.12g of Ligusticum chuanxiong extract sample, and 0.12g of Epimedium brevicornum extract sample, accurately weigh, put them into stoppered conical flasks respectively, accurately add 20mL of 80% methanol, ultrasonically treat (power 250w, frequency 40kHz) for 10min, filter, and take the filtrate to obtain.

[0157] Test solution: accurately weigh 0.35 g of dry paste powder, accurately add 20 mL of 80% methanol, seal, ultrasonically treat (power 250 w, frequency 40 kHz) for 10 min, shake well, filter, and obtain the filtrate.

[0158] Positioning reference substance solution: Accurately weigh appropriate amounts of epimedin C, icariin, 3,6'-diesinaroylsucrose, and ligustilide reference substances, and use 80% methanol to prepare solutions containing 50 μg, 120 μg, 60 μg, and 50 μg per 1 mL, respectively.

[0159] Other steps and test conditions are the same as in Example 3.

[0160] Take 10 μL of each of the above solutions, inject into high performance liquid chromatograph, measure, record the chromatogram within 45 min, and the results are shown in the table below.

[0161] Table 4 Specificity - Chromatographic Peak Attribution and Identification

[0162]

[0163] in conclusion:

[0164] The blank solvent had no interference in the fingerprint detection of compound ginkgo leaf dry powder;

[0165] Chromatographic peak attribution: Compared with single herbal medicine, it was confirmed that peaks 1, 2, 4, 7 and 8 were attributed to Polygala tenuifolia, peaks 3, 5 and 6 were attributed to Epimedium, and peak 9 was attributed to Ligusticum chuanxiong;

[0166] Comparison with the positioning reference solution confirmed that peak 4 was 3,6'-diesinapoylsucrose, peak 5 was epimedin C, peak 6 was icariin, and peak 9 was ligustilide;

[0167] Therefore, in the HPLC fingerprint with icariin as the reference peak, a total of 9 fingerprint peaks were identified, and the attribution of each chromatographic peak could be determined.

[0168] Example 5 Selection of chromatographic column

[0169] The solution was prepared as in Example 3.

[0170] Change the chromatographic conditions Column type:

[0171] The chromatographic column of Example 1: model GL Inertsil ODS-3, specification 4.6*250mm, 5μm;

[0172] Replace with,

[0173] Method 1: Chromatographic column: Welch Ultimate AQ C18, specification 4.6*250mm, 5μm;

[0174] Method 2: Chromatographic column: model Welch Ultimate LP C8, specification 4.6*250mm, 5μm;

[0175] Other steps and test conditions are the same as in Example 3.

[0176] Take 10 μL of the reference solution and the test solution mentioned above, inject them into the high performance liquid chromatograph for measurement, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0177]

[0178] in conclusion:

[0179] The chromatographic column of Example 3 uses octadecylsilane bonded silica gel as a filler (for example, model GL Inertsil ODS-3, specification 4.6*250 mm, 5 μm), and 9 fingerprint peaks can be obtained with good peak shape;

[0180] The chromatographic column of method 1 of Example 5 uses octadecylsilane bonded silica gel as a filler (for example, model Welch Ultimate AQ C18, specification 4.6*250mm, 5μm), and 9 fingerprint peaks can be obtained with good peak shape;

[0181] The chromatographic column of method 2 of Example 5 uses octylsilane bonded silica gel as a filler (e.g., model Welch Ultimate LP C8, specification 4.6*250 mm, 5 μm), and only 6 fingerprint peaks are attributed, and the peak shape is poor;

[0182] summary:

[0183] 1). The chromatographic column using the octadecylsilane bonded silica gel of the present invention as a filler has a large amount of information and strong characteristics in the fingerprint spectrum of the compound ginkgo leaf dry paste powder, which can effectively reflect the overall quality of the compound ginkgo leaf dry paste powder:

[0184] For example, Example 3 uses a chromatographic column model GL Inertsil ODS-3, specification 4.6*250mm, 5μm and Example 5 Method 1 uses a chromatographic column model Welch Ultimate AQ C18, specification 4.6*250mm, 5μm. The fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder has a large amount of information and strong characteristics, which can effectively reflect the overall quality of the compound ginkgo leaf tablet dry paste powder, specifically as follows:

[0185] i. 9 fingerprint peaks were obtained, and the corresponding chromatographic peaks of the fingerprint components were kept intact;

[0186] ii. The peak shape is good and the separation effect between the chromatographic peaks is good.

[0187] 2). On the contrary, when other types of chromatographic columns are used (for example, the chromatographic column model Welch Ultimate LP C8 of Method 2 in Example 5, specification 4.6*250mm, 5μm), the fingerprint of the obtained compound ginkgo leaf tablet dry paste powder has less information and poor characteristic, and cannot reflect the overall quality of the compound ginkgo leaf tablet dry paste powder, which is specifically manifested as follows:

[0188] i. The number of fingerprint peaks is small, and the corresponding chromatographic peaks of fingerprint components are not completely retained;

[0189] ii. Poor peak shape and poor separation between chromatographic peaks.

[0190] Example 6: Selection of gradient elution program of mobile phase

[0191] The solution was prepared as in Example 3.

[0192] The gradient elution program of the mobile phase was changed from

[0193] Embodiment 1:

[0194] Time / min Mobile phase A / % Mobile phase B / % 0 15 85 10 20 80 35 40 60 36 80 20 40 80 20 41 15 85 45 15 85

[0195] Replace with:

[0196] Method 1:

[0197] Time (min) Mobile phase A / % Mobile phase B / % 0 10 90 10 20 80 35 40 60 36 80 20 40 80 20 41 15 85 45 15 85

[0198] Method 2:

[0199]

[0200]

[0201] Other steps and test conditions are the same as in Example 3.

[0202] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0203]

[0204] in conclusion:

[0205] The elution procedure of Example 3 can obtain 9 fingerprint peaks in the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder, with good peak shape, large amount of information and strong characteristic, which can comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0206] The elution procedure of Method 1 in Example 6 can obtain 9 fingerprint peaks in the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder. The separation effect of peaks 5 to 8 is not good, and the separation effect is poor. The obtained spectrum has a large amount of information, but the characteristic is not strong, and it cannot comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0207] The elution procedure of Method 2 in Example 6 can obtain 7 fingerprint peaks in the fingerprint spectrum of the compound ginkgo leaf tablet dry paste powder. Peaks 1 to 2 overlap with the miscellaneous peaks and cannot be separated. The separation effect is poor and cannot fully reflect the types and quantities of the chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0208] summary:

[0209] 1). Only by adopting the elution procedure of the present invention (e.g., Example 3), the fingerprint of the obtained compound ginkgo leaf tablet dry powder has a large amount of information and strong characteristics, which can effectively reflect the overall quality of the compound ginkgo leaf tablet dry powder, which is specifically manifested as follows:

[0210] i. 9 fingerprint peaks were obtained, and the corresponding chromatographic peaks of the fingerprint components were kept intact;

[0211] ii. The peak shape is good and the separation effect between the chromatographic peaks is good.

[0212] 2). On the contrary, by using other elution procedures (e.g., Methods 1 to 2 in Example 6), the fingerprint of the obtained compound ginkgo leaf tablet dry powder has less information and poor characteristics, and cannot reflect the overall quality of the compound ginkgo leaf tablet dry powder, which is specifically manifested as follows:

[0213] i. The fingerprint peaks are reduced, and the chromatographic peaks corresponding to the fingerprint components retain incomplete spectra with little information;

[0214] ii. Poor peak shape and poor separation between chromatographic peaks.

[0215] Example 7 Selection of Mobile Phase B Concentration

[0216] The solution was prepared as in Example 3.

[0217] The concentration of mobile phase B was changed from

[0218] Example 1: Mobile phase B is 0.4% phosphoric acid solution,

[0219] Replace with,

[0220] Method 1: Mobile phase B is 0.3% phosphoric acid solution;

[0221] Method 2: Mobile phase B is 0.5% phosphoric acid solution;

[0222] Other steps and test conditions are the same as in Example 3.

[0223] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0224]

[0225] in conclusion:

[0226] Example 1 and Example 7 Methods 1 to 2 use 0.4% phosphoric acid solution, 0.3% phosphoric acid solution, and 0.5% phosphoric acid solution, respectively. The fingerprints of the obtained compound ginkgo leaf tablet dry paste powder can comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder, and 9 fingerprint peaks can be obtained. The corresponding chromatographic peaks of the fingerprint components are intact; the peak shape is good, and the separation effect between the chromatographic peaks is good.

[0227] summary:

[0228] The mobile phase B uses a phosphoric acid solution with a concentration of 0.3% to 0.5% (for example, methods 1 to 2 of Example 3 and Example 7). The fingerprint of the obtained compound ginkgo leaf tablet dry powder has a large amount of information and strong characteristics, which can effectively reflect the overall quality of the compound ginkgo leaf tablet dry powder, specifically:

[0229] i. 9 fingerprint peaks were obtained, and the corresponding chromatographic peaks of the fingerprint components were kept intact;

[0230] ii. The peak shape is good and the separation effect between the chromatographic peaks is good.

[0231] Example 8: Selection of detection wavelength

[0232] The solution was prepared as in Example 3.

[0233] The gradient elution program of the mobile phase was changed from

[0234] Example 1: Detection wavelength: 320nm;

[0235] Replace with:

[0236] Method 1: Detection wavelength: 210nm;

[0237] Method 2: Detection wavelength: 220nm;

[0238] Method 3: Detection wavelength: 390nm;

[0239] Method 4: Detection wavelength: 400nm;

[0240] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 45 minutes. The results are shown in the table below.

[0241]

[0242]

[0243] in conclusion:

[0244] The detection wavelength of Example 3 is 320nm, and the fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder can obtain 9 fingerprint peaks with good peak shape. The obtained spectrum has a large amount of information and strong characteristics, and can comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0245] The detection wavelength of method 1 in Example 8 is 210 nm, and the fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder can obtain 9 fingerprint peaks, the baseline noise is large, the separation effect is poor, the obtained spectrum has a large amount of information, but the characteristic is not strong, and it cannot fully reflect the number of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0246] The detection wavelength of method 2 in Example 8 is 220nm, and the fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder can obtain 9 fingerprint peaks with good peak shape. The obtained spectrum has a large amount of information and strong characteristics, and can comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0247] The detection wavelength of method 3 in Example 8 is 390nm, and the fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder can obtain 9 fingerprint peaks with good peak shape. The obtained spectrum has a large amount of information and strong characteristics, and can comprehensively reflect the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0248] The detection wavelength of method 4 in Example 8 is 400nm, and the fingerprint spectrum of the obtained compound ginkgo leaf tablet dry paste powder can obtain 9 fingerprint peaks. The peaks 1 to 3, peak 7, and peak 8 are relatively small, and the separation effect is poor, which cannot fully reflect the number of chemical components contained in the compound ginkgo leaf tablet dry paste powder.

[0249] summary:

[0250] 1). Only when the chromatographic detection wavelength is between 220 and 390 nm (for example, methods 2 to 3 of Example 3 and Example 8), the fingerprint of the obtained compound ginkgo leaf tablet dry paste powder has a large amount of information and strong characteristics, which effectively reflects the overall quality of the compound ginkgo leaf tablet dry paste powder, which is specifically manifested as follows:

[0251] i. 9 fingerprint peaks were obtained, and the corresponding chromatographic peaks of the fingerprint components were kept intact;

[0252] ii. Good peak shape and good separation between chromatographic peaks;

[0253] 2) Among them, when the chromatographic detection wavelength is 320nm (for example, Example 3), the fingerprint spectrum of the compound ginkgo leaf tablet dry powder has a large amount of information and the strongest characteristics, which effectively reflects the overall quality of the compound ginkgo leaf tablet dry powder, which is specifically manifested as follows:

[0254] i. 9 fingerprint peaks were obtained, and the corresponding chromatographic peaks of the fingerprint components were kept intact;

[0255] ii. The peak shape is good, the peak response is the strongest, the baseline is stable, and the separation effect between the chromatographic peaks is the best.

[0256] 3). On the contrary, when other chromatographic detection wavelengths are used (for example, 210 nm in Method 1 of Example 8 and 400 nm in Method 4 of Example 8), the fingerprint of the obtained compound ginkgo leaf tablet dry paste powder has less information and poor characteristics, and cannot reflect the overall quality of the compound ginkgo leaf tablet dry paste powder, which is specifically manifested as follows:

[0257] Nine fingerprint peaks were obtained, the baseline noise was large, the corresponding chromatographic peaks of the fingerprint components were incomplete, and the separation effect was poor.

[0258] Example 9

[0259] The compound ginkgo leaf dry paste powder of the present application was determined by referring to the analytical method described in the specific embodiment of Chinese patent CN102818861A.

[0260] 9-1. Solution preparation:

[0261] Reference solution: Take 5 mg of icariin reference substance, place it in a 100 mL volumetric flask, dissolve it in 80% methanol and dilute to the scale, shake well, and obtain.

[0262] Test solution: accurately weigh 0.35 g of dry paste powder, accurately add 20 mL of 80% methanol, seal, ultrasonically treat (power 250 w, frequency 40 kHz) for 10 min, shake well, filter, and obtain the filtrate.

[0263] 9-2. Testing conditions:

[0264] Chromatographic column: model Ultimate XB-C18, specification 3.0*150mm, 3μm;

[0265] Mobile phase: Mobile phase A is acetonitrile; Mobile phase B is 0.04% phosphoric acid solution;

[0266] Elution mode: gradient elution;

[0267] Gradient elution program:

[0268] Time / min Mobile phase A / % Mobile phase B / % 0 2 98 10 18 82 20 18 82 55 30 70 90 88 12

[0269] Detection wavelength: 203nm;

[0270] Detection wavelength: 260nm;

[0271] Detection wavelength: 320nm;

[0272] Flow rate: 0.6 mL / min;

[0273] 9-3. Experimental steps and conclusions:

[0274] Take 10 μL of the reference solution and the test solution, inject them into the high performance liquid chromatograph, measure, and record the chromatogram within 90 minutes. The results are shown in the table below.

[0275]

[0276]

[0277] in conclusion:

[0278] In Example 9, the chromatographic conditions and analytical methods described in the specific embodiments of Chinese patent CN102818861A were used to determine the compound ginkgo leaf tablet dry paste powder of the present application. In the obtained compound ginkgo leaf tablet dry paste powder fingerprint spectrum, when the same detection wavelength (203 and 260nm) as CN102818861A was used, the fingerprint peaks overlapped, the separation was poor, and the components and content of the test solution could not be accurately located; when the detection wavelength was 320nm, 6 fingerprint peaks were obtained in the spectrum, among which peaks 7 to 8 belonging to Polygala tenuifolia overlapped with the reference Epimedium peak, and the method specificity was poor, and the types and quantities of chemical components contained in the compound ginkgo leaf tablet dry paste powder could not be reflected.

[0279] Example 10 Repeatability Test

[0280] The solution preparation was the same as in Example 3, wherein 6 portions of the test solution were prepared, denoted as S1 to S6.

[0281] The experimental steps and test conditions are the same as those in Example 3.

[0282] The retention time and peak area of ​​each common peak were recorded, and the relative retention time and relative peak area of ​​each common peak were calculated with reference to the retention time and peak area of ​​icariin peak. The results are shown in Tables 5 and 6. The 6 test sample spectra were imported into the 2012 edition of “Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine”, and the evaluation similarity results are shown in Table 7.

[0283] Table 5 Repeatability test results - relative retention time

[0284]

[0285]

[0286] Table 6 Repeatability test results - relative peak area

[0287]

[0288] Table 7 Repeatability test results - similarity

[0289]

[0290]

[0291] in conclusion:

[0292] The fingerprint of the obtained compound ginkgo leaf dry paste powder is:

[0293] The relative retention time RSD values ​​of each fingerprint peak were less than 0.24%, which was much smaller than the relative retention time RSD limit standard (≤3.0%);

[0294] The relative peak area RSD values ​​of each fingerprint peak were less than 1.5%, which was much smaller than the relative peak area RSD limit standard (≤5.0%);

[0295] The similarity between each fingerprint spectrum is 1.000, which is much greater than the similarity limit standard (>0.900);

[0296] The above results show that the method of the present invention has good repeatability.

[0297] Example 11 Precision Test

[0298] Solution preparation, experimental steps and test conditions are the same as those in Example 3. Different experimenters, on different dates, using different instruments, took the same sample solution, and injected it 6 times (recorded as S1 to S6) continuously according to the chromatographic conditions in the fingerprint construction method described in Example 1 above, detected the fingerprint spectrum, recorded the retention time and peak area of ​​each common peak, and calculated the relative retention time and relative peak area of ​​each common peak with the retention time and peak area of ​​icariin peak as reference. The results are shown in Tables 8 to 9; the 6 sample spectra were introduced into the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the similarity evaluation results are shown in Table 10.

[0299] Table 8 Precision test results - relative retention time

[0300]

[0301]

[0302] Table 9 Precision test results - relative peak area

[0303]

[0304] Table 10 Precision test results - similarity

[0305] sample Similarity (comparison) S1 1.000 S2 1.000 S3 1.000 S4 1.000 S5 1.000 S6 1.000

[0306] in conclusion:

[0307] The fingerprint of the obtained compound ginkgo leaf dry paste powder is:

[0308] The relative retention time RSD values ​​of each fingerprint peak were less than 0.24%, which was much smaller than the relative retention time RSD limit standard (≤3.0%);

[0309] The relative peak area RSD values ​​of each fingerprint peak were less than 0.87%, which was much smaller than the relative peak area RSD limit standard (≤5.0%);

[0310] The similarity between each fingerprint spectrum is 1.000, which is much greater than the similarity limit standard (>0.900);

[0311] The above results show that the method of the present invention has good precision.

[0312] Example 12 Solution Stability

[0313] Solution preparation, experimental steps and test conditions are the same as those in Example 3. Take the same sample solution, and inject at 0h, 3h, 6h, 12h, 24h and 48h respectively according to the chromatographic conditions in the fingerprint construction method described in Example 1 above, detect the fingerprint spectrum, record the retention time and peak area of ​​each common peak, and calculate the relative retention time and relative peak area of ​​each common peak with the retention time and peak area of ​​icariin peak as reference. The results are shown in Tables 11 and 12; import the 6 sample spectra into the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the evaluation similarity results are shown in Table 13.

[0314] Table 11 Stability test results - relative retention time

[0315]

[0316] Table 12 Stability test results - relative peak area

[0317]

[0318] Table 13 Stability test results - similarity

[0319] sample Similarity (comparison) S1 1.000 S2 1.000 S3 1.000 S4 1.000 S5 1.000 S6 1.000

[0320] in conclusion:

[0321] The fingerprint of the obtained compound ginkgo leaf dry paste powder is:

[0322] The relative retention time RSD values ​​of each fingerprint peak were less than 0.04%, which was much smaller than the relative retention time RSD limit standard (≤3.0%);

[0323] The relative peak area RSD values ​​of each fingerprint peak were less than 0.86%, which was much smaller than the relative peak area RSD limit standard (≤5.0%);

[0324] The similarity between each fingerprint spectrum is 1.000, which is much greater than the similarity limit standard (>0.900);

[0325] The above results show that the test solution of the method of the present invention has good stability.

[0326] Example 13 Similarity Comparison

[0327] Solution preparation, experimental steps and test conditions were the same as those in Example 3. The HPLC spectra of the collected 10 batches of compound ginkgo leaf tablet dry paste powder (recorded as S1 to S10) were sequentially imported into the 2012 edition of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" in AIA format. The results are shown in Figure 3The fingerprint of one of the test solution was used as the reference, the time window width was set to 0.1min, and after multi-point calibration and data matching, the mean method was used to generate the HPLC fingerprint comparison map (R) of the compound ginkgo leaf tablet dry paste powder. The similarity results are shown in Table 14, and the comparison map is shown in Figure 4 .

[0328] Table 14 Similarity results of HPLC spectra of 10 batches of compound ginkgo leaf tablet dry powder

[0329] sample Similarity (comparison) S1 0.999 S2 0.998 S3 0.998 S4 0.998 S5 0.998 S6 0.998 S7 0.999 S8 0.998 S9 1.000 S10 1.000

[0330] The HPLC spectrum of the typical test solution (clinical sample, batch number: 220101) collected was imported into the 2012 edition of the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” in AIA format. Figure 5 , and the similarity analysis was performed with the control spectrum (R). The similarity results are shown in Table 15.

[0331] Table 15 Similarity results of typical test products of compound ginkgo leaf dry paste powder

[0332]

[0333]

[0334] in conclusion:

[0335] The fingerprints of the obtained 10 batches of compound ginkgo leaf dry paste powder are as follows:

[0336] The fingerprints of compound ginkgo leaf dry paste powder all showed 9 fingerprint peaks ( Figure 4 ), the similarity is 0 and is greater than 0.90 (similarity standard / limit);

[0337] Typical test solution also shows 9 fingerprint peaks ( Figure 5 ), and the generated fingerprint map ( Figure 4 ), the similarity is 0.936, which is high and greater than 0.90 (similarity standard / limit), with high consistency.

[0338] In summary, the present invention provides a method for detecting the fingerprint spectrum of compound ginkgo leaf tablet dry powder. It is found for the first time that using icariin as a reference is conducive to distinguishing the common fingerprint peaks. The method has the characteristics of good specificity, high precision and good repeatability; the fingerprint spectrum of compound ginkgo leaf tablet dry powder obtained has a large amount of information and strong characteristics, which can effectively reflect the overall quality of compound ginkgo leaf tablet dry powder and ensure the quality uniformity and safety and effectiveness of the drug.

Claims

1. A method for detecting a pharmaceutical preparation, comprising the following steps: 1) Use HPLC method to determine the HPLC fingerprint of the drug preparation to obtain the fingerprint of the test sample; 2) Determine the HPLC spectrum of the icariin reference substance by HPLC method to obtain the reference fingerprint spectrum, and compare it with the fingerprint spectrum of the test substance in step 1); 3) The fingerprint of the test sample should show 9 common peaks, among which the chromatographic peak with the same retention time as the chromatographic peak of the icariin reference substance in step 2) is used as the reference peak. When the relative retention time and relative deviation of each common peak meet the following conditions, and the chromatographic peaks of 0-45min are calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, and the similarity between the fingerprint of the test sample and the reference fingerprint is not less than 0.90, it is determined that the pharmaceutical preparation contains compound ginkgo leaf tablet dry extract powder: Peak 1: relative retention time 0.199±0.020, relative deviation 0~10%; Peak 2: relative retention time 0.207±0.021, relative deviation 0~10%; Peak 3: relative retention time 0.217±0.022, relative deviation 0~10%; Peak 4: relative retention time 0.680±0.068, relative deviation 0~10%; Peak 5: relative retention time 0.966±0.097, relative deviation 0~10%; Peak 6: relative retention time 1.000, relative deviation 0%; Peak 7: relative retention time 1.023±0.102, relative deviation 0~10%; Peak 8: relative retention time 1.048±0.148, relative deviation 0~10%; Peak 9: relative retention time 1.393±0.139, relative deviation 0~10%; Wherein, the chromatographic conditions of the HPLC method include: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B; Elution method: Gradient elution. The gradient elution program is as follows: 。 2. The method according to claim 1, wherein: The model of the chromatographic column is GL Inertsil ODS-3 or Welch Ultimate AQ C18.

3. The method according to claim 1, wherein: Mobile phase B is 0.3%~0.5% phosphoric acid aqueous solution.

4. The method according to claim 3, wherein: Mobile phase B was 0.4% phosphoric acid in water.

5. The method according to claim 1, wherein: In the chromatographic conditions, the detection wavelength is 220~390nm.

6. The method according to claim 5, wherein: In the chromatographic conditions, the detection wavelength is 320 nm.

7. A method for detecting compound ginkgo leaf tablet dry paste powder, comprising the following steps: 1) Use HPLC method to determine the HPLC fingerprint of compound ginkgo leaf tablet dry powder to obtain the fingerprint of the test sample; 2) Determine the HPLC spectrum of the icariin reference substance by HPLC method to obtain the reference fingerprint spectrum, and compare it with the fingerprint spectrum of the test substance in step 1); 3) The fingerprint of the test sample should show 9 common peaks, among which the chromatographic peak with the same retention time as the chromatographic peak of the icariin reference substance in step 2) is used as the reference peak. The relative retention time and relative deviation of each common peak meet the following conditions, and the similarity between the chromatographic peak of 0~45min calculated by the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint is not less than 0.90: Peak 1: relative retention time 0.199±0.020, relative deviation 0~10%; Peak 2: relative retention time 0.207±0.021, relative deviation 0~10%; Peak 3: relative retention time 0.217±0.022, relative deviation 0~10%; Peak 4: relative retention time 0.680±0.068, relative deviation 0~10%; Peak 5: relative retention time 0.966±0.097, relative deviation 0~10%; Peak 6: relative retention time 1.000, relative deviation 0%; Peak 7: relative retention time 1.023±0.102, relative deviation 0~10%; Peak 8: relative retention time 1.048±0.148, relative deviation 0~10%; Peak 9: relative retention time 1.393±0.139, relative deviation 0~10%; Wherein, the chromatographic conditions of the HPLC method include: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B; Elution method: Gradient elution. The gradient elution program is as follows: 。 8. The method according to claim 7, wherein: The model of the chromatographic column is GL Inertsil ODS-3 or Welch Ultimate AQ C18.

9. The method according to claim 7, wherein: Mobile phase B is 0.3%~0.5% phosphoric acid aqueous solution.

10. The method according to claim 9, wherein: Mobile phase B was 0.4% phosphoric acid in water.

11. The method according to claim 7, wherein: In the chromatographic conditions, the detection wavelength is 220~390nm.

12. The method according to claim 11, wherein: In the chromatographic conditions, the detection wavelength is 320 nm.

13. A method for establishing a fingerprint spectrum of compound ginkgo leaf tablet dry paste powder, comprising the following steps: (1) Solution preparation: Test solution: weigh 0.35g of compound ginkgo leaf dry powder, accurately add 20mL of 80% methanol, seal, weigh, ultrasonically treat at 250w and 40kHz for 10min, cool, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain; Reference solution: Weigh the icariin reference substance and prepare a solution containing 120 µg per 1 mL with 80% methanol; (2) Chromatographic conditions: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: acetonitrile as mobile phase A; 0.4% volume fraction phosphoric acid solution as mobile phase B; Elution method: Gradient elution. The gradient elution program is as follows: ； Flow rate: 1.0 mL / min; Column temperature: 45°C; Detection wavelength: 320nm; (3) Detection: Pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatograph, measure, and record the chromatogram within 45 min.

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