A method for constructing characteristic maps of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations

Abstract

The present invention belongs to the technical field of quality detection of traditional Chinese medicines. The present invention provides a method for constructing a characteristic spectrum of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations, comprising the following steps: preparation of a test solution: weighing a test sample, adding an extraction solvent for extraction, and preparing a test solution; determination: taking the test solution and detecting it by ultra-high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, mobile phase A is methanol, mobile phase B is an ammonium formate aqueous solution with a pH value of 3.9-4.1 and a concentration of 0.0095-0.0105 mol / L, and the elution mode is gradient elution. 10 or 12 common peaks are established, the peak separation is good, the baseline is stable, and the peak shape is symmetrical, which fully demonstrates the chemical composition characteristics of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations, and provides a fast and comprehensive detection means for the quality detection of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and in particular relates to a method for constructing a characteristic spectrum of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations. Background Art

[0002] Leeches are the dried whole body of Whitmania pigra Whitman, Hirudo nipponica Whitman or W.acranulata Whitman, which are animals of the Hirudinidae family. They are commonly used Chinese medicinal materials listed in the 2020 edition of the Chinese Pharmacopoeia. They have the effects of breaking blood and promoting menstruation, removing blood stasis and eliminating symptoms. They are mainly used clinically for blood stasis, amenorrhea, lumps and masses, hemiplegia caused by stroke, and injuries caused by falls.

[0003] At present, there are about 500 species of leeches in the world. The 2020 edition of the Chinese Pharmacopoeia stipulates three types of leeches, namely, Whitmania pigra Whitman, Hirudo nipponica Whitman or W.acranulata Whitman, which are leeches of the Hirudinidae family. These three leeches belong to the subclass of Hirudinidae and the order of Arhynchobdellida in taxonomy. Among them, W.pigra is also known as the wide-bodied golden leech, and W.acranulata is also known as the sharp golden leech, both of which belong to the Haemopidae family; H.nipponica is also known as the Japanese medicinal leech, which belongs to the Hirudinidae family. The present invention is based on the records of leeches belonging to the Hirudinidae family in the 2020 edition of the Chinese Pharmacopoeia.

[0004] Scalded leeches are processed products obtained by scalding leeches with talcum powder. Due to the differences in active ingredients between the raw and processed products after scalding, the study of the different treatments of raw and cooked leeches has practical significance in the quality control of traditional Chinese medicine.

[0005] There are many kinds of leeches on the market, and there are differences in active ingredients between leeches of different origins. According to the requirements of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules" (No. 16, 2021) issued by the State Food and Drug Administration, Chinese medicinal materials of different origins cannot be mixed with each other. Therefore, it is imperative to identify leeches of different origins. Therefore, the identification of different origins of the same variety is also of practical significance for monitoring the quality of traditional Chinese medicine. Summary of the invention

[0006] Therefore, the object of the present invention is to provide a method for constructing a characteristic spectrum of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations. The method can respectively construct characteristic spectra of leech medicinal materials, leech pieces, standard decoctions of leech pieces, leech formula granules, scalded leech pieces, standard decoctions of scalded leech pieces or scalded leech formula granules, which can not only be used to accurately identify leeches and their pharmaceutical preparations and scalded leeches and their pharmaceutical preparations, but also can be used to identify leeches and their pharmaceutical preparations from common counterfeits, as well as scalded leeches from common counterfeits, and can comprehensively and effectively identify leeches (leeches) and scalded leeches (leeches).

[0007] To this end, the present application provides a method for constructing a characteristic spectrum of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations, comprising the following steps:

[0008] Preparation of test solution: Weigh the test sample, add extraction solvent for extraction, and prepare the test solution;

[0009] Determination: The test solution is detected by ultra performance liquid chromatography, using octadecylsilane bonded silica gel as filler, mobile phase A is methanol, mobile phase B is an aqueous solution of ammonium formate with a pH value of 3.9-4.1 and a concentration of 0.0095-0.0105 mol / L, and the elution mode is gradient elution.

[0010] Further, the gradient elution includes the following procedures:

[0011] From 0 to 10 min, the volume fraction of mobile phase A increased from 15% to 31%; the volume fraction of mobile phase B decreased from 85% to 69%;

[0012] From 10 to 16 min, the volume fraction of mobile phase A increased from 31% to 35%, and the volume fraction of mobile phase B decreased from 69% to 65%;

[0013] From 16 to 25 min, the volume fraction of mobile phase A increased from 35% to 42%, and the volume fraction of mobile phase B decreased from 65% to 58%;

[0014] From 25 to 35 min, the volume fraction of mobile phase A increased from 42% to 45%, and the volume fraction of mobile phase B decreased from 58% to 55%;

[0015] From 35 to 46 minutes, the volume fraction of mobile phase A increased from 45% to 60%, and the volume fraction of mobile phase B decreased from 55% to 40%.

[0016] Furthermore, in the ultra-high performance liquid chromatography detection, the flow rate is 0.19-0.21 ml per minute, the column temperature is 28-32°C, the detection wavelength is 320nm-365nm, and the injection volume is 0.5-2μl; and / or, the extraction solvent is 30%-70% methanol; and / or, the gradient elution also includes the following procedures: 46-51min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; 51-52min, the volume fraction of mobile phase A changes from 60% to 15%, and the volume fraction of mobile phase B changes from 40% to 85%; 52-55min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0017] Furthermore, the method further comprises the steps of using a solvent to dissolve one or more of hirudinamine C, hirudinamine B, and vitamin B2 to prepare a reference solution, and using any of the above-mentioned ultra-high performance liquid chromatography methods to detect the reference solution to obtain a reference characteristic spectrum. Preferably, hirudinamine C, hirudinamine B, and vitamin B2 are used as reference substances and dissolved in 30%-70% methanol to prepare a reference solution.

[0018] Furthermore, the preparation method of the test solution includes weighing 0.5g to 1.5g (e.g., 0.8g to 1.2g) of the test sample, accurately adding 10ml to 50ml of 30-70% methanol, weighing the weight, ultrasonically treating for 15-60 minutes, cooling, weighing the weight again, supplementing the lost weight with 30-70% methanol, shaking well, filtering, and taking the filtrate to obtain.

[0019] Furthermore, the method also includes importing characteristic spectra of different batches of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations into a similarity evaluation system for chromatographic characteristic spectra of traditional Chinese medicine promulgated by the State Pharmacopoeia Committee to generate a comparison characteristic spectrum of leeches and their pharmaceutical preparations or scalded leeches and their pharmaceutical preparations, wherein the comparison characteristic spectrum of leeches and their pharmaceutical preparations has 10 characteristic peaks, and the comparison characteristic spectrum of scalded leeches and their pharmaceutical preparations has 12 common characteristic peaks, peak 1 is hirudinamine C, peak 2 is hirudinamine A, peak 4 is hirudinamine B, peak 5 is Whitmanine B, and peak 7 is vitamin B2.

[0020] Furthermore, the leeches and their pharmaceutical preparations are selected from leech medicinal materials, leech pieces, standard decoctions of leech pieces or leech formula granules; scalded leeches and their pharmaceutical preparations are selected from scalded leech pieces, standard decoctions of scalded leech pieces or scalded leech formula granules. Preferably, the leeches or scalded leeches are derived from leeches.

[0021] Furthermore, the established characteristic spectrum of leeches and their pharmaceutical preparations has 10 common characteristic peaks, wherein peak 1 corresponds to the retention time of the peak of the hirudinamine C reference substance, the peak corresponding to the hirudinamine C reference substance is taken as the S peak, and the relative retention times of peaks 2 to 5, peak 7, peak 9 to 12 and the S peak are calculated to be within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 3.24 (peak 7), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12); and / or,

[0022] The established characteristic spectrum of scalded leeches and their pharmaceutical preparations has 12 common characteristic peaks, wherein peak 1 corresponds to the retention time of the peak of a hirudinamine C reference substance, the corresponding peak of the hirudinamine C reference substance is taken as the S peak, the relative retention times of peaks 2 to 12 and peak 1 are calculated to be within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 2.93 (peak 6), 3.24 (peak 7), 4.11 (peak 8), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12); the corresponding peak of the hirudinamine C reference substance is taken as the S peak, the relative peak area of ​​the total peak area of ​​peaks 6 and 8 and the S peak is calculated to be within the range of the specified value, and the specified value is: not less than 0.030.

[0023] The present invention also provides a method for distinguishing leeches and their pharmaceutical preparations from scalded leeches and their pharmaceutical preparations, comprising:

[0024] Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to any of the construction methods described above;

[0025] The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by any of the above-mentioned construction methods, and identification is performed based on the comparison results.

[0026] The present invention also provides a method for distinguishing leeches and their pharmaceutical preparations from counterfeit products, comprising:

[0027] Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to any of the construction methods described above;

[0028] The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by any of the above-mentioned construction methods using leeches and / or leech pharmaceutical preparations derived from leeches as test samples, and identification is performed based on the comparison results. Preferably, the mixed and counterfeit product is a leech derived from willow leaf leech, Philippine cattle leech or Japanese medicinal leech.

[0029] The present invention also provides a method for distinguishing leeches derived from leeches and their pharmaceutical preparations from mixed and counterfeit products, comprising:

[0030] Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to any of the construction methods described above;

[0031] The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by any of the above-mentioned construction methods using scald leeches and / or scald leech pharmaceutical preparations derived from leeches as test samples, and identification is performed based on the comparison results. Preferably, the counterfeit product is a scald leech derived from willow leaf leeches, Philippine cattle leeches or Japanese medicinal leeches.

[0032] The present invention also provides a quality detection method for leeches and their pharmaceutical preparations and scalded leeches and their pharmaceutical preparations, comprising: using a product to be detected as a test sample and constructing a characteristic spectrum of the product to be detected according to any of the above-mentioned construction methods;

[0033] The obtained characteristic spectrum of the product to be tested is compared with the characteristic spectrum constructed by any of the construction methods described above, and the quality is evaluated based on the comparison results.

[0034] The technical solution of the present invention has the following advantages:

[0035] 1. The method for constructing the characteristic spectrum of the leech and its pharmaceutical preparation or the scalded leech and its pharmaceutical preparation described in the present invention. The present invention constructs the characteristic spectrum of the leech and its pharmaceutical preparation, and establishes 10 common peaks, with good peak separation, stable baseline, and good peak symmetry, which fully demonstrates the chemical composition characteristics of the leech and its pharmaceutical preparation, and provides a rapid and comprehensive detection method for the quality detection of the leech and its pharmaceutical preparation.

[0036] The invention constructs a characteristic spectrum of scald leeches and their pharmaceutical preparations, and establishes 12 common peaks, with good peak separation, stable baseline, and good peak symmetry, which fully demonstrates the chemical composition characteristics of scald leeches and their pharmaceutical preparations, and provides a rapid and comprehensive detection method for the quality detection of scald leeches and their pharmaceutical preparations.

[0037] 2. The characteristic spectrum method of leeches and their pharmaceutical preparations, scalded leeches and their pharmaceutical preparations constructed by the present invention is stable, highly precise and has good reproducibility.

[0038] 3. The method for identifying leeches and their pharmaceutical preparations from scalded leeches and their pharmaceutical preparations of the present invention has strong specificity and can quickly and accurately distinguish and identify leeches and their pharmaceutical preparations, scalded leeches and their pharmaceutical preparations and their mixed and counterfeit products. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 This is the chromatogram of the test sample of leech formula granules;

[0041] Figure 2 This is the chromatogram of the test sample of the scalded leech (leech) formula granules;

[0042] Figure 3 is the chromatogram of hirudinamine C reference substance;

[0043] Figure 4 is the chromatogram of hirudinamine B reference substance;

[0044] Figure 5 This is the chromatogram of vitamin B2 reference substance;

[0045] Figure 6 To optimize the gradient 1 chromatogram;

[0046] Figure 7 To optimize the gradient 2 chromatogram;

[0047] Figure 8 To optimize the gradient 3 chromatogram;

[0048] Fig. 9 It is the chromatogram of absorption wavelength at 230nm;

[0049] Fig.10 It is a chromatogram with absorption wavelength of 254nm;

[0050] Fig.11 It is a chromatogram with absorption wavelength of 275nm;

[0051] Fig.12 It is a chromatogram with absorption wavelength of 290nm;

[0052] Fig.13 It is the chromatogram of absorption wavelength at 320nm;

[0053] Fig.14 It is a chromatogram with absorption wavelength of 365nm;

[0054] Fig.15 This is the chromatogram of the methanol-0.01mol / L ammonium formate mobile phase system;

[0055] Fig.16 This is the chromatogram of the methanol-0.01 mol / L ammonium acetate mobile phase system;

[0056] Fig.17 This is the chromatogram of the test sample extracted from scalded leech (leech) with 30% methanol;

[0057] Fig.18 This is the chromatogram of the test sample extracted from scalded leech (leech) with 50% methanol;

[0058] Fig.19 This is the chromatogram of the test sample extracted from scalded leech (leech) with 70% methanol;

[0059] Fig. 20 The chromatogram is for a sample size of 0.5 g;

[0060] Fig.21 The chromatogram is for a sample size of 1.0 g;

[0061] Fig. 22 The chromatogram is for the sampling amount of 1.5 g;

[0062] Fig.23 This is the reference characteristic map of leeches;

[0063] Fig.24 This is a reference characteristic map of scalded leeches;

[0064] Fig.25 Chromatograms of multiple batches of leech formula particles;

[0065] Fig.26 This is a chromatogram of multiple batches of scalded leech (leech) formula particles;

[0066] Fig. 27 Reference product positioning chromatogram;

[0067] Fig.28 It is the chromatogram of negative particles;

[0068] Fig.29 This is a chromatogram comparison of leech (leech) and scalded leech (leech) formula particles;

[0069] Fig.30 This is a chromatogram comparison of leech (leech) and leech (willow leaf leech) formula particles;

[0070] Fig.31 This is a chromatogram comparison of leech (leech) and scalded leech (willow leaf leech) formula particles;

[0071] Fig.32 This is a chromatogram comparison of leech (leech) and leech (Philippine cattle leech) formula particles;

[0072] Fig.33 This is a chromatogram comparison of leech (leech) and scalded leech (Philippine cattle leech) formula particles;

[0073] Fig.34 This is a chromatogram comparison of leeches and leech (Japanese medical leech) formula particles;

[0074] Fig.35 This is a chromatogram comparison of leeches and scalded leeches (Japanese medical leeches) formula particles;

[0075] Fig.36 This is a chromatogram comparison of scalded leeches (leeches) and leeches (willow leaf leeches) formula particles;

[0076] Fig.37 This is a chromatogram comparison of scalded leeches (leeches) and scalded leeches (willow leaf leeches) formula particles;

[0077] Fig.38 This is a chromatogram comparison of the granules of the scalded leech (leech) and the leech (Philippine cattle leech);

[0078] Fig.39 This is a chromatogram comparison of scalded leeches (leeches) and scalded leeches (Philippine cattle leeches) formula particles;

[0079] Fig.40 This is a chromatogram comparison of scalded leeches (leeches) and leeches (Japanese medical leeches) formula particles;

[0080] Fig.41 This is a chromatogram comparison of the granules of scalded leeches (leeches) and scalded leeches (Japanese medical leeches). DETAILED DESCRIPTION

[0081] The following examples are provided to further understand the present invention better, are not limited to the best mode, and are not intended to limit the content and scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the enlightenment of the present invention or by combining the features of the present invention with other prior arts, all falls within the protection scope of the present invention. If no specific experimental steps or conditions are indicated in the examples, the operation or conditions of the conventional experimental steps described in the literature in this area can be carried out. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially. With reference to the "Technical Requirements for Quality Control and Standardization of Chinese Herbal Formula Granules", for different original varieties, or varieties that need to be distinguished from specific origins in clinical use, the Chinese name of the original variety or origin variety should be marked in brackets in the name, such as leeches and scalded leeches in the present invention. Leeches refer to leeches whose origin is the leech Whitmania pigra Whitman, and scalded leeches refer to scalded leeches whose origin is the leech Whitmania pigra Whitman.

[0082] % methanol aqueous solution refers to the volume percentage of methanol in the methanol aqueous solution.

[0083] The pharmaceutical preparation of leeches or scalded leeches described in the present invention can be prepared by conventional methods, for example but not limited to the following methods:

[0084] Take leeches or scald leeches, heat and reflux for extraction at least once, add 6 to 12 times the weight of water each time, extract for at least 1.0h, filter, combine the filtrates, concentrate the filtrates to a relative density of 1.02-1.08g / ml at 60°C, add pharmaceutically acceptable excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, rapid-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. The pharmaceutically acceptable excipients are: fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include sweeteners and various flavors; preservatives include parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include PEG6000, PEG4000, insect wax, etc.

[0085] Take leech medicinal materials and prepare them into leech slices or scalded leech slices that meet the requirements according to the relevant provisions of the 2020 edition of the "Chinese Pharmacopoeia". Leech medicinal materials are washed, cut into sections, and dried to become leech slices. Leech slices are scalded with talc until they are slightly swollen to become scalded leeches. These two kinds of slices are used clinically and are two different forms of slices of the same medicinal material.

[0086] In the following embodiments, leech (leech) and scalded leech (leech) formula granules are prepared with leech (leech) decoction pieces and scalded leech (leech) decoction pieces as raw materials, respectively, according to the following method: take leech (leech) decoction pieces or scalded leech (leech) decoction pieces, heat and reflux extraction 3 times, add 10 times the weight of water for the first time to soak for 30 minutes, heat and reflux extraction for 1.5 hours, filter, add 8 times the weight of water for the second time to extract for 1.0 hours, filter, add 6 times the weight of water for the third time to extract for 1.0 hours, filter, combine the filtrate, concentrate the filtrate to 60°C with a relative density of 1.02g / ml~1.08g / ml, dry, add maltodextrin as an auxiliary material to the dry powder, mix well and then perform dry granulation to prepare leech (leech) formula granules or scalded leech (leech) formula granules.

[0087] Leech (willow leaf leech) formula granules, scalded leech (willow leaf leech) formula granules, leech (Philippine cattle leech) formula granules, scalded leech (Philippine cattle leech) formula granules, leech (Japanese medical leech) formula granules, scalded leech (Japanese medical leech) formula granules are made by using leech (willow leaf leech) decoction pieces, scalded leech (willow leaf leech) decoction pieces, leech (Philippine cattle leech) decoction pieces, scalded leech (Philippine cattle leech) decoction pieces, leech (Japanese medical leech) decoction pieces and scalded leech (Japanese medical leech) decoction pieces as raw materials respectively and are made in the same way.

[0088] Example 1

[0089] This embodiment provides a method for constructing a characteristic map of leeches and scalded leeches, comprising the following steps:

[0090] (1) Preparation of test solution: Take leech formula granules and scalded leech formula granules as test samples, respectively. Take about 1.0 g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0091] (2) Preparation of reference solution: Accurately weigh hirudinamine C reference substance, add 50% methanol to make a solution containing 0.0001 g per 1 ml, shake well, and use this as reference A solution;

[0092] Accurately weigh hirudinamine B reference substance, add 50% methanol to make a solution containing 0.0001g per 1ml, shake well, and use it as reference substance B solution;

[0093] Accurately weigh the vitamin B2 reference substance, add 50% methanol to make a solution containing 0.0001g per 1ml, shake well, and use it as the reference solution C;

[0094] (3) Liquid chromatography detection: 0.001 ml of the test solution and the reference solution were accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination, and the characteristic spectra of leeches, scalded leeches, reference A solution, reference B solution, and reference C solution were obtained respectively; wherein the liquid chromatography conditions are as follows: octadecylsilane bonded silica gel was used as the filler (Shim-pack GIST_HP C18-Aq, column length 150mm, inner diameter 2.1mm, particle size 1.9μm); methanol as mobile phase A, 0.01mol / L ammonium formate aqueous solution (acetic acid added to adjust pH to 4.0) as mobile phase B, gradient elution according to the provisions in the following table: 0-10min, the volume fraction of mobile phase A changes from 15 to 31%, and the volume fraction of mobile phase B changes from 85 to 69%; 10-16min, the volume fraction of mobile phase A changes from 31 to 35%, and the volume fraction of mobile phase B changes from 69 to 65%; 16-25min, the volume fraction of mobile phase A changes from 35 to 42%, and the volume fraction of mobile phase B changes from 65 to 58%; 25-3 5min, the volume fraction of mobile phase A changes to 42-45%, and the volume fraction of mobile phase B changes to 58-55%; 35-46min, the volume fraction of mobile phase A changes to 45-60%, and the volume fraction of mobile phase B changes to 55-40%; 46-51min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; 51-52min, the volume fraction of mobile phase A changes to 60-15%, and the volume fraction of mobile phase B changes to 40-85%; 52→55min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%; the column temperature is 30℃; the flow rate is 0.20ml per minute; the detection wavelength is 365nm. The injection volume is 1μl.

[0095] The results are shown in the attached figure of the specification. Figure 1 This is the characteristic spectrum of the leech formula granule test sample. Figure 2 This is the characteristic spectrum of the scalded leech (leech) formula granules test sample. Figure 3 is the chromatogram of hirudinamine C reference substance, Figure 4 is the chromatogram of hirudinamine B reference substance, Figure 5 This is the chromatogram of vitamin B2 reference substance.

[0096] Example 2 Investigation of the construction method

[0097] 1. Investigation of chromatographic conditions

[0098] (1) Elution gradient investigation

[0099] The scalded leech formula granules were used as the test sample, and the test sample solution prepared in Example 1 was detected by ultra-high performance liquid chromatography. The chromatographic conditions of Example 1 were used for detection, and 254 nm was used as the detection wavelength. The only difference was the different elution gradients. The different elution procedures were as follows.

[0100] Table 1 Optimization of gradient 1 to 3 elution program

[0101]

[0102] Depend on Figures 6 to 8 It can be seen that the main chromatographic peaks of the optimization gradient 1 and optimization gradient 2 methods are mainly nucleic acid components, which are common components in animal traditional Chinese medicine and have weak specificity. The chromatographic peaks of optimization gradient 3 have more information, and there are multiple chromatographic peaks that are specific components of scald leeches, such as hirudinamine C and hirudinamine B. This temporary mobile phase gradient is optimized gradient 3 for subsequent condition screening.

[0103] (2) Experimental study on absorption wavelength

[0104] The scalded leech (leech) formula granules were used as the test sample, and the test solution prepared in Example 1 was detected by ultra-high performance liquid chromatography. The chromatographic conditions of Example 1 were used for detection, and the detection wavelength was used as a variable to analyze the chromatograms at different absorption wavelengths of 230nm, 254nm, 275nm, 290nm, 320nm and 365nm. The separation effect of the chromatographic peaks was used as the judgment standard. The separation effect of 320nm and 365nm was better. The number of chromatographic peaks and peak height were used as indicators to determine 365nm as the detection wavelength. The results are shown in FIG. Figure 9-14 .

[0105] (3) Investigation of mobile phase system

[0106] The scalded leech (leech) formula granules were used as the test sample, and the test sample solution prepared in Example 1 was detected by ultra-high performance liquid chromatography. The chromatographic conditions of Example 1 were used for detection, and methanol-0.01 mol / L ammonium formate aqueous solution and methanol-0.01 mol / L ammonium acetate aqueous solution were used as the mobile phase system, respectively. The separation effect of the chromatographic peak was used as the evaluation standard. The results are shown in Figure 15-16 When the mobile phase system was methanol-0.01 mol / L ammonium formate aqueous solution, the separation effect of the chromatographic peaks was better, so this mobile phase system was selected for subsequent condition screening.

[0107] 2. Preparation of test solution

[0108] (1) Investigation of extraction solvents

[0109] The scalded leech (leech) formula granules were used as the test sample, and 30% methanol aqueous solution, 50% methanol aqueous solution and 70% methanol aqueous solution were used as extraction solvents. The test sample solution was prepared according to the method in Example 1, and the chromatographic conditions of Example 1 were used for detection. The results are shown in Figure 17-19 There was no significant difference in the chromatographic separation effects of the test samples prepared using the above three extraction solvents, but after comparing and analyzing the system suitability parameters of each peak, 50% methanol aqueous solution was recommended as the extraction solvent.

[0110] (2) Investigation of the sampling quantity of the test product

[0111] The scalded leech (leech) formula granules were used as the test sample, and 0.5g, 1g and 1.5g of the test sample were weighed respectively. The test sample solution was prepared according to the method in Example 1, and the chromatographic conditions of Example 1 were used for detection. The results are shown in Figure 20-22 There was no significant difference in the chromatographic separation effects of the test samples prepared with the above three sampling amounts, but some samples were not fully extracted when the sampling amount was 1.5g, and the samples were completely dissolved when the sampling amount was 0.5g, but the peak response of the chromatographic peak was relatively low. After comprehensive analysis, it is recommended that the sampling amount be set at 1g.

[0112] Example 3 Construction of the control characteristic spectrum

[0113] (1) Construction of reference feature map

[0114] 15 batches of leech (leech) formula granules and 15 batches of scalded leech (leech) formula granules were used as test samples, and test sample solutions were prepared and tested according to the method of Example 1 to obtain characteristic spectra of 15 batches of leech (leech) formula granules and 15 batches of scalded leech (leech) formula granules.

[0115] Using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" software recommended by the Chinese Pharmacopoeia Committee, the similarity of the obtained characteristic spectra was compared (the reference characteristic spectrum was established by the average), and the reference characteristic spectra of leeches and scalded leeches were generated. The results are shown in Figures 23-24 .

[0116] The test results of the characteristic graphs of leeches and scalded leeches were analyzed and compared, and 10 chromatographic peaks with good separation in the UPLC characteristic graph of leeches and 12 chromatographic peaks with good separation in the UPLC characteristic graph of scalded leeches were confirmed. Among them, the chromatographic graph of leeches lacked peaks 6 and 8 in the chromatographic graph of scalded leeches. The chromatographic peak with the same retention time as the hirudinamine C reference was taken as the S peak, and the relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated. The results are shown in Tables 2 to 5. The results are as follows Figure 25-26 As shown, Fig.25In the figure, S1(10) to S15(10) are characteristic spectra of 15 batches of leech formula granules, and R(10) is the control characteristic spectrum. Fig.26 Among them, S1(12)~S15(12) are characteristic spectra of 15 batches of scalded leech formula granules, and R(12) is the control characteristic spectrum.

[0117] Table 2 Determination results of relative retention time of characteristic spectra of 15 batches of leech formula granules

[0118]

[0119]

[0120] Table 3 Results of relative peak area determination of characteristic spectra of 15 batches of leech formula granules

[0121]

[0122] Table 4 Determination results of relative retention time of characteristic spectra of 15 batches of scalded leech formula granules

[0123]

[0124] Table 5 Results of relative peak area determination of characteristic spectra of 15 batches of scalded leech formula granules

[0125]

[0126]

[0127] The results showed that the characteristic spectra of 15 batches of leech formula granules all had 10 common characteristic peaks, among which peak 1 had the same retention time as the hirudinamine C reference substance, the chromatographic peak corresponding to the hirudinamine C reference substance peak was taken as the S peak, and the relative retention times of peaks 2 to 5, peak 7, peak 9 to 12 and the S peak were all within the range of ±10% of the specified values, and the specified values ​​were: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 3.24 (peak 7), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), and 8.69 (peak 12).

[0128] The characteristic spectra of 15 batches of scalded leech formula granules all have 12 common characteristic peaks, among which peak 1 has the same retention time as the hirudinamine C reference substance. The chromatographic peak corresponding to the hirudinamine C reference substance peak is taken as the S peak, and the calculated relative retention times of peaks 2 to 10 with the S peak are all within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 2.93 (peak 6), 3.24 (peak 7), 4.11 (peak 8), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), and 8.69 (peak 12); the chromatographic peak corresponding to the hirudinamine C reference substance peak is taken as the S peak, and the calculated relative peak area of ​​the total peak area of ​​peaks 6 and 8 with the S peak are all within the range of the specified value, and the specified value is: not less than 0.030.

[0129] The method can quickly and intuitively distinguish leeches and scalded leeches, and provides a more scientific basis for the quality control of leeches and scalded leeches.

[0130] (2) Determination of common characteristic peaks of leeches and scalded leeches:

[0131] A total of 5 compounds were identified and assigned to characteristic peaks by UPLC-HRMS. The results are shown in Table 6.

[0132] Table 6 UPLC-HRMS analysis results of scalded leeches

[0133]

[0134] (3) Characteristic spectral peak identification

[0135] Preparation of reference solution: Accurately weigh hirudinamine C reference substance, add 50% methanol to make a solution containing 0.0001g per 1ml, shake well, and use it as reference A solution; accurately weigh hirudinamine B reference substance, add 50% methanol to make a solution containing 0.0001g per 1ml, shake well, and use it as reference B solution; accurately weigh vitamin B2 reference substance, add 50% methanol to make a solution containing 0.0001g per 1ml, shake well, and use it as reference C solution.

[0136] According to the preparation method of Example 1, a leech formula granule test solution and a scalded leech formula granule test solution were prepared. The reference solution and the leech formula granule test solution and the scalded leech formula granule test solution were tested by high performance liquid chromatography according to the method of Example 1 and then compared. The results are as follows: Fig. 27 Among them, S1 is a scalded leech formula granule, S2 is a leech formula granule, S3 is a reference A, S4 is a reference B, and S5 is a reference C.

[0137] Through the chromatographic peak retention time of the compounds and comparison with the reference substances, a total of three compounds were identified, namely hirudinamine C (peak 1), hirudinamine B (peak 4), and vitamin B2 (peak 7).

[0138] (4) Similarity analysis

[0139] The similarity of leech formula granules and scalded leech formula granules was calculated by comparing the similarity of the obtained characteristic spectra (using the average to establish the reference characteristic spectrum) using the software "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" recommended by the Chinese Pharmacopoeia Committee. The results are shown in Table 7. The similarities between 15 batches of leech formula granules and the leech reference spectrum were all greater than 0.90, and the similarities between 15 batches of scalded leech formula granules and the scalded leech reference spectrum were also greater than 0.90, indicating that the main material basis of leeches and scalded leeches between different batches is basically the same, and they are similar as a whole.

[0140] Table 7 Similarity analysis results of leech and scalded leech granules

[0141]

[0142]

[0143] (5) Feature Spectrum Analysis

[0144] Leech formula particles should show 10 characteristic peaks in the UPLC characteristic spectrum, of which peak 1 has the same retention time as the hirudinamine C reference substance, and the chromatographic peak corresponding to the hirudinamine C reference substance peak is the S peak. The relative retention times of peaks 2 to 5, peak 7, peak 9 to 12 and S peak are calculated to be within the range of ±10% of the specified value, and the specified values ​​are: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 3.24 (peak 7), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12). Distinguishing points from scalded leeches: Peaks 6 and 8 should not appear in the UPLC characteristic spectrum.

[0145] The scalded leech formula granules should show 12 common characteristic peaks in the UPLC characteristic spectrum, among which peak 1 has the same retention time as the hirudinamine C reference substance, the chromatographic peak corresponding to the hirudinamine C reference substance peak is taken as the S peak, and the relative retention times of peaks 2 to 10 and the S peak are calculated to be within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 2.93 (peak 6), 3.24 (peak 7), 4.11 (peak 8), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12); the chromatographic peak corresponding to the hirudinamine C reference substance peak is taken as the S peak, and the relative peak area of ​​the total peak area of ​​peaks 6 and 8 and the S peak are calculated to be within the range of the specified value, and the specified value is: not less than 0.030. Distinguishing points for scalded leeches: In the UPLC characteristic spectrum, peaks 6 and 8 should appear stably, and the relative peak area of ​​the total peak area of ​​peaks 6 and 8 to the peak S should not be less than 0.030.

[0146] Example 4 Methodology Verification

[0147] (1) Instrument precision test

[0148] Take the same portion of leech formula granules, prepare a leech formula granule test solution according to the test sample preparation method in Example 1, inject 6 times, and examine the consistency of the relative retention time of the characteristic peak. Take the same portion of scalded leech formula granules, prepare a scalded leech formula granule test solution according to the test sample preparation method in Example 1, inject 6 times, and examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak.

[0149] The leech formula granules test samples all showed 10 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak, and the relative retention time of the remaining 9 common characteristic peaks and the S peak was calculated, and the RSD value of the relative retention time of each peak was less than 2%; the scalded leech formula granules test samples all showed 12 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak, and the relative retention time of the remaining 11 common characteristic peaks and the S peak was calculated, and the relative peak area of ​​the total peak area of ​​peak 6 and peak 8 and the S peak was calculated. The RSD of the relative retention time and relative peak area of ​​each peak was less than 2%. The results show that the instrument precision of this method is good.

[0150] (2) Method repeatability test

[0151] Take 6 portions of the same batch of leech formula granules for test, prepare leech (leech) test solution according to the test sample preparation method of Example 1, inject sample for analysis, and examine the consistency of the relative retention time of characteristic peaks. Take 6 portions of the same batch of scalded leech formula granules for test, prepare scalded leech (leech) formula granules for test according to the test sample preparation method of Example 1, inject sample for analysis, and examine the consistency of the relative retention time and relative peak area of ​​characteristic peaks.

[0152] The results showed that the leech formula granules presented 10 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 9 common characteristic peaks and the S peak was calculated, and the RSD value of the relative retention time of each peak was less than 2%; the scalded leech formula granules presented 12 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peak 6 and peak 8 and the relative peak area of ​​the S peak were calculated, and the RSD of the relative retention time and relative peak area of ​​each peak was less than 2%. This shows that the method has good repeatability.

[0153] (3) Intermediate precision (different operators)

[0154] The same batch of leech formula granules were taken, and three inspectors prepared leech (leech) test solution by the test sample preparation method in Example 1 using the same equipment at different times, and the sample was analyzed to examine the consistency of the relative retention time of the characteristic peak. The same batch of scalded leech formula granules were taken, and three inspectors prepared scalded leech (leech) formula granule test solution by the test sample preparation method in Example 1 using the same equipment at different times, and the sample was analyzed to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak.

[0155] The results showed that the leech formula granules presented 10 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 9 common characteristic peaks and the S peak was calculated. The RSD value of the relative retention time of each peak was in the range of 0%-0.47%, all less than 2%; the scalded leech formula granules presented 12 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peak 6 and peak 8 and the relative peak area of ​​the S peak were calculated. The RSD range of the relative retention time and relative peak area of ​​each peak was 0%-0.65%, all less than 2%. This shows that the intermediate precision of this method is good.

[0156] (4) Specificity inspection

[0157] Take negative granules (prepared by dry granulation of maltodextrin) and prepare negative granule test solution according to Example 1. Sample analysis is carried out and compared with the characteristic spectrum of leech and scalded leech formula granules constructed in Example 1. Figure 1 , 2 , 28 It can be seen that this method has good specificity and no negative interference.

[0158] (5) Stability test

[0159] Take the same portion of leech formula granules, prepare a leech formula granule test solution according to the test sample preparation method of Example 1, and inject and analyze at 0, 2, 4, 8, 12, and 24 hours to examine the consistency of the relative retention time of the characteristic peak. Take the same portion of scalded leech formula granules, prepare a scalded leech formula granule test solution according to the test sample preparation method of Example 1, and inject and analyze at 0, 2, 4, 8, 12, and 24 hours to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak.

[0160] The results showed that the leech formula granule test samples all presented 10 characteristic peaks, the chromatographic peak consistent with the retention time of the hirudinamine C reference peak was taken as the S peak, and the relative retention times of the remaining 9 common characteristic peaks and the S peak were calculated. The RSD values ​​of the relative retention times of each peak were in the range of 0%-0.29%, all less than 2%; the scalded leech formula granule test samples all presented 12 characteristic peaks, the chromatographic peak consistent with the retention time of the hirudinamine C reference peak was taken as the S peak, and the relative retention times of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peak 6 and peak 8 and the relative peak area of ​​the S peak, the RSD range of the relative retention time and relative peak area of ​​each peak was 0%-1.12%, all less than 2%, indicating that the test solution had good stability within 24 hours.

[0161] (6) Tests at different flow rates

[0162] Take the same portion of leech formula granules, prepare a leech formula granule test solution according to the test sample preparation method of Example 1, and analyze the sample at flow rates of 0.19ml / min, 0.20ml / min, and 0.21ml / min to examine the consistency of the relative retention time of the characteristic peak. Take the same portion of scalded leech formula granules, prepare a scalded leech formula granule test solution according to the test sample preparation method of Example 1, and analyze the sample at flow rates of 0.19ml / min, 0.20ml / min, and 0.21ml / min to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak.

[0163] The results showed that all the leech formula granules tested presented 10 characteristic peaks, with the chromatographic peak consistent with the retention time of the reference peak of hirudinamine C as the S peak, and the relative retention times of the remaining 9 common characteristic peaks and the S peak were calculated, and the RSD values ​​of the relative retention times of each peak were in the range of 0%-3.86%; all the scalded leech formula granules tested presented 12 characteristic peaks, with the chromatographic peak consistent with the retention time of the reference peak of hirudinamine C as the S peak, and the relative retention times of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peaks 6 and 8 and the relative peak area of ​​the S peak, and the RSD values ​​of the relative retention times and relative peak areas of each peak were in the range of 0%-4.63%, indicating that when the flow rate changes slightly, it has a certain influence on the relative retention time and relative peak area of ​​the characteristic peaks.

[0164] (7) Experiments at different column temperatures

[0165] Take the same portion of leech formula granules, prepare a leech formula granule test solution according to the test sample preparation method of Example 1, and analyze the sample at column temperatures of 28°C, 30°C, and 32°C to examine the consistency of the relative retention time of the characteristic peak. Take the same portion of scalded leech formula granules, prepare a scalded leech formula granule test solution according to the test sample preparation method of Example 1, and analyze the sample at column temperatures of 28°C, 30°C, and 32°C to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak.

[0166] The results showed that all the leech formula granules tested presented 10 characteristic peaks, with the chromatographic peak consistent with the retention time of the hirudinamine C reference peak as the S peak, and the relative retention times of the remaining 9 common characteristic peaks and the S peak were calculated, and the RSD values ​​of the relative retention times of each peak were in the range of 0%-5.89%; all the scalded leech formula granules tested presented 12 characteristic peaks, with the chromatographic peak consistent with the retention time of the hirudinamine C reference peak as the S peak, and the relative retention times of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peaks 6 and 8 and the relative peak area of ​​the S peak, and the RSD values ​​of the relative retention times and relative peak areas of each peak were in the range of 0%-7.70%, indicating that different column temperatures have a certain influence on the relative retention time and relative peak area of ​​each characteristic peak.

[0167] (8) Tests on different types of chromatographic columns

[0168] The same portion of leech formula granules was taken and prepared into a leech formula granule test solution according to the test sample preparation method in Example 1, and the samples were injected and analyzed using chromatographic columns of model Shim-pack GIST_HP C18-Aq (2.1 mm×150 mm, 1.9 μm) and ACQUITY UPLC HSS T3 (2.1 mm×150 mm, 1.8 μm) to examine the consistency of the relative retention time of the characteristic peaks. The same portion of scald leech formula granules was taken and prepared into a scald leech formula granule test solution according to the test sample preparation method in Example 1, and the samples were injected and analyzed using chromatographic columns with models Shim-pack GIST_HP C18-Aq (2.1 mm×150 mm, 1.9 μm) and ACQUITY UPLC HSS T3 (2.1 mm×150 mm, 1.8 μm) to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks.

[0169] The results showed that the leech formula granules presented 10 characteristic peaks, and the chromatographic peak consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 9 common characteristic peaks and the S peak was calculated, and the RSD value of the relative retention time of each peak was in the range of 0%-7.30%; the scalded leech formula granules presented 12 characteristic peaks, and the chromatographic peak consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peak 6 and peak 8 and the relative peak area of ​​the S peak were calculated, and the RSD of the relative retention time and relative peak area of ​​each peak was in the range of 0%-8.58%. The RSD values ​​of some chromatographic peaks were too large, but within a reasonable range, this method can be performed on the above two chromatographic columns.

[0170] (9) Tests on different types of chromatographs

[0171] Take the same portion of leech formula granules, prepare a leech formula granule test solution according to the test sample preparation method in Example 1, and use Waters H-CLASS, Agilent 1290, and Thermo Vanquish chromatographs for sample analysis to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks. Take the same portion of scalded leech formula granules, prepare a scalded leech formula granule test solution according to the test sample preparation method in Example 1, and use Waters H-CLASS, Agilent 1290, and Thermo Vanquish chromatographs for sample analysis to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks.

[0172] The results showed that the leech formula granules presented 10 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 9 common characteristic peaks and the S peak was calculated, and the RSD value of the relative retention time of each peak was in the range of 0%-5.18%; the scalded leech formula granules presented 12 characteristic peaks, and the chromatographic peak that was consistent with the retention time of the hirudinamine C reference peak was taken as the S peak. The relative retention time of the remaining 11 common characteristic peaks and the S peak, the total peak area of ​​peak 6 and peak 8 and the relative peak area of ​​the S peak were calculated, and the RSD of the relative retention time and relative peak area of ​​each peak was in the range of 0%-7.07%. The RSD values ​​of some chromatographic peaks were too large, but within a reasonable range, this method can be performed on the above three chromatographs.

[0173] Example 5

[0174] The present embodiment provides a method for distinguishing leeches from scalded leeches, comprising the following steps:

[0175] (1) Preparation of test solution: Take 1.0 g of leech formula granules (test sample 1) and scalded leech formula granules (test sample 16), accurately add 25 ml of 50% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain leech formula granule test solution and scalded leech formula granule test solution.

[0176] (2) Liquid chromatography detection: The sample solution was accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination, using octadecylsilane bonded silica gel as the filler (Shim-pack GIST_HP C18-Aq, column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm), methanol as the mobile phase A, and an aqueous solution containing 0.01 mol / L ammonium formate (adjusted to pH 4.0 with acetic acid) as the mobile phase B. Gradient elution was performed with a flow rate of 0.20 ml per minute, a column temperature of 30°C, a detection wavelength of 365 nm, and an injection volume of 1 μl. The gradient elution conditions are as follows: from 0 to 10 min, the volume fraction of mobile phase A changes to 15 to 31%, and the volume fraction of mobile phase B changes to 85 to 69%; from 10 to 16 min, the volume fraction of mobile phase A changes to 31 to 35%, and the volume fraction of mobile phase B changes to 69 to 65%; from 16 to 25 min, the volume fraction of mobile phase A changes to 35 to 42%, and the volume fraction of mobile phase B changes to 65 to 58%; from 25 to 35 min, the volume fraction of mobile phase A changes to 42 to 45%, and the volume fraction of mobile phase B changes to 65 to 58%. The integral fraction changes from 58 to 55%; from 35 to 46 min, the volume fraction of mobile phase A changes from 45 to 60%, and the volume fraction of mobile phase B changes from 55 to 40%; from 46 to 51 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; from 51 to 52 min, the volume fraction of mobile phase A changes from 60 to 15%, and the volume fraction of mobile phase B changes from 40 to 85%; from 52 to 55 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0177] like Fig.29 As shown, the characteristic spectrum of sample 1 (S1 in the attached figure) is consistent with the characteristic spectrum of leech formula granules, with 10 common characteristic peaks, and is a leech formula granule; the characteristic spectrum of sample 16 (S2 in the attached figure) is consistent with the characteristic spectrum of scalded leech formula granules, with 12 common characteristic peaks, and the relative peak area of ​​the total peak area of ​​peak 6 and peak 8 to the peak S is not less than 0.030, which is a scalded leech formula granule. The similarity evaluation system of Chinese medicine chromatographic characteristic spectrum 2012 version was used to evaluate the similarity between the test sample 1 and the control characteristic spectrum of leech formula granules, and the similarity was greater than 0.90; the similarity evaluation system of Chinese medicine chromatographic characteristic spectrum 2012 version was used to evaluate the similarity between the test sample 16 and the control characteristic spectrum of scalded leech formula granules, and the similarity was greater than 0.90.

[0178] The scalded leech (leech) formula granules have more peaks 6 and 8 than the leech (leech) formula granules. The leech (leech) formula granules also have a chromatographic peak at a position close to the retention time of peak 8 of the scalded leech (leech) formula granules. However, analysis of the absorption wavelength of the chromatographic peak found that the absorption wavelength of the chromatographic peak (253.9nm, 300.0nm, 367.7nm) is similar to that of the scalded leech (leech) formula granules. ) The absorption wavelengths of peak 8 in the sample (246.5nm, 364.0nm) are inconsistent, but consistent with the absorption wavelengths of the chromatographic peaks before peak 8 of the scalded leech (leech) sample (253.3nm, 300.7nm, 367.7nm), so this component may be consistent with the component of the chromatographic peak before peak 8 of the scalded leech (leech), so peak 8 in the scalded leech (leech) is a new component of the scalded leech (leech). Therefore, peaks 6 and 8 are the identification points of leeches and scalded leeches.

[0179] Example 6

[0180] This embodiment provides a method for identifying leeches and counterfeit products, comprising the following steps:

[0181] (1) Preparation of test solution: Take 1.0 g of leech (willow leaf leech) granules, scalded leech (willow leaf leech) granules, leech (Philippine cattle leech) granules, scalded leech (Philippine cattle leech) granules, leech (Japanese medical leech) granules, and scalded leech (Japanese medical leech) granules, respectively, accurately add 25 ml of 50% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0182] (2) Liquid chromatography detection: Accurately pipette the sample solutions respectively and inject them into ultra-high performance liquid chromatography for determination. Octadecylsilane bonded silica gel was used as filler (Shim-pack GIST_HP C18-Aq, column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm), methanol was used as mobile phase A, and aqueous solution containing 0.01 mol / L ammonium formate (with acetic acid added to adjust the pH to 4.0) was used as mobile phase B. Gradient elution was performed with a flow rate of 0.20 ml per minute, a column temperature of 30°C, a detection wavelength of 365 nm, and an injection volume of 1 μl. The gradient elution conditions are as follows: from 0 to 10 min, the volume fraction of mobile phase A changes to 15 to 31%, and the volume fraction of mobile phase B changes to 85 to 69%; from 10 to 16 min, the volume fraction of mobile phase A changes to 31 to 35%, and the volume fraction of mobile phase B changes to 69 to 65%; from 16 to 25 min, the volume fraction of mobile phase A changes to 35 to 42%, and the volume fraction of mobile phase B changes to 65 to 58%; from 25 to 35 min, the volume fraction of mobile phase A changes to 42 to 45%, and the volume fraction of mobile phase B changes to 65 to 58%. The integral fraction changes from 58 to 55%; from 35 to 46 min, the volume fraction of mobile phase A changes from 45 to 60%, and the volume fraction of mobile phase B changes from 55 to 40%; from 46 to 51 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; from 51 to 52 min, the volume fraction of mobile phase A changes from 60 to 15%, and the volume fraction of mobile phase B changes from 40 to 85%; from 52 to 55 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0183] like Figure 30-35The characteristic spectrum (S1 in the accompanying drawings) constructed by the above method is compared with the leech control characteristic spectrum constructed in Example 3. The characteristic spectrum of the leech (willow leaf leech) formula granules does not show peaks 10, 11 and 12, the characteristic spectrum of the scalded leech (willow leaf leech) formula granules does not show peaks 5, 7, 11 and 12, the characteristic spectrum of the leech (Philippine cattle leech) formula granules only shows peak 4, the characteristic spectrum of the scalded leech (Philippine cattle leech) formula granules only shows peaks 1 to 4, and the characteristic spectrum of the leech (Japanese medical leech) formula granules The spectrum does not show peaks 7, 9 and 10, and the characteristic spectrum of the scalded leech (Japanese medical leech) formula granules does not show peaks 7, 9 and 10. Therefore, these samples to be identified are not leech (leech) formula granules. This method can be used to distinguish leech (leech) formula granules and these counterfeit products (leech (willow leaf leech) formula granules, scalded leech (willow leaf leech) formula granules, leech (Philippine cattle leech) formula granules, scalded leech (Philippine cattle leech) formula granules, leech (Japanese medical leech) formula granules, scalded leech (Japanese medical leech) formula granules). The results of leech (willow leaf leech) formula granules, scalded leech (willow leaf leech) formula granules, leech (Philippine cattle leech) formula granules, scalded leech (Philippine cattle leech) formula granules, leech (Japanese medical leech) formula granules, scalded leech (Japanese medical leech), and scalded leech (leech) formula granules were respectively introduced into the "Chinese medicine chromatographic characteristic spectrum similarity evaluation system" (2012 version) and evaluated for similarity with the constructed leech (leech) control characteristic spectrum. The similarities were all less than 0.90.

[0184] Example 7

[0185] This embodiment provides a method for distinguishing scalded leeches from counterfeit products, comprising the following steps:

[0186] (1) Preparation of test solution: Take 1.0 g of leech (willow leaf leech) granules, scalded leech (willow leaf leech) granules, leech (Philippine cattle leech) granules, scalded leech (Philippine cattle leech) granules, leech (Japanese medical leech) granules, and scalded leech (Japanese medical leech) granules, respectively, accurately add 25 ml of 50% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0187] (2) Liquid chromatography detection: Accurately pipette the sample solutions respectively and inject them into ultra-high performance liquid chromatography for determination. Octadecylsilane bonded silica gel was used as filler (Shim-pack GIST_HP C18-Aq, column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm), methanol was used as mobile phase A, and aqueous solution containing 0.01 mol / L ammonium formate (with acetic acid added to adjust the pH to 4.0) was used as mobile phase B. Gradient elution was performed with a flow rate of 0.20 ml per minute, a column temperature of 30°C, a detection wavelength of 365 nm, and an injection volume of 1 μl. The gradient elution conditions are as follows: from 0 to 10 min, the volume fraction of mobile phase A changes to 15 to 31%, and the volume fraction of mobile phase B changes to 85 to 69%; from 10 to 16 min, the volume fraction of mobile phase A changes to 31 to 35%, and the volume fraction of mobile phase B changes to 69 to 65%; from 16 to 25 min, the volume fraction of mobile phase A changes to 35 to 42%, and the volume fraction of mobile phase B changes to 65 to 58%; from 25 to 35 min, the volume fraction of mobile phase A changes to 42 to 45%, and the volume fraction of mobile phase B changes to 65 to 58%. The integral fraction changes from 58 to 55%; from 35 to 46 min, the volume fraction of mobile phase A changes from 45 to 60%, and the volume fraction of mobile phase B changes from 55 to 40%; from 46 to 51 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; from 51 to 52 min, the volume fraction of mobile phase A changes from 60 to 15%, and the volume fraction of mobile phase B changes from 40 to 85%; from 52 to 55 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0188] like Figure 36-41As shown, the characteristic spectrum (S1 in the accompanying drawings) constructed by the above method is compared with the characteristic spectrum of the scalded leech (leech) control constructed in Example 3. The characteristic spectrum of the leech (willow leaf leech) formula granules does not show peaks 6, 8, 10, 11 and 12, the characteristic spectrum of the scalded leech (willow leaf leech) formula granules does not show peaks 5, 6, 7, 8, 11 and 12, the characteristic spectrum of the leech (Philippine cattle leech) formula granules only shows peak 4, the characteristic spectrum of the scalded leech (Philippine cattle leech) formula granules only shows peaks 1-4, and the characteristic spectrum of the leech (Japanese medical leech) formula granules does not show peaks There are peaks 6, 7, 8, 9 and 10, and the characteristic spectrum of the scalded leech (Japanese medical leech) formula granules does not show peaks 6, 7, 9 and 10. Therefore, these samples to be identified are not leech (leech) formula granules and / or scalded leech (leech) formula granules. This method can be used to distinguish the scalded leech (leech) formula granules and these counterfeit products (leech (willow leaf leech) formula granules, scalded leech (willow leaf leech) formula granules, leech (Philippine cattle leech) formula granules, scalded leech (Philippine cattle leech) formula granules, leech (Japanese medical leech) formula granules, scalded leech (Japanese medical leech) formula granules). The results of leech (willow leaf leech) formula granules, scalded leech (willow leaf leech) formula granules, leech (Philippine cattle leech) formula granules, scalded leech (Philippine cattle leech) formula granules, leech (Japanese medical leech) formula granules, scalded leech (Japanese medical leech), and scalded leech (leech) formula granules were respectively imported into the "Chinese medicine chromatographic characteristic spectrum similarity evaluation system" (2012 version), and the similarity was evaluated with the constructed scalded leech (leech) control characteristic spectrum. The similarities were all less than 0.90.

[0189] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of leeches and their pharmaceutical preparations, characterized in that it comprises the following steps: Preparation of test solution: weigh 0.5g-1.5g of the test sample, accurately add 10ml-50ml of 30-70% methanol, weigh the weight, ultrasonically treat for 15-60 minutes, cool, weigh again, make up the lost weight with 30-70% methanol, shake well, filter, and take the filtrate to obtain; Determination: The test solution is tested by ultra-high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, mobile phase A is methanol, mobile phase B is an aqueous solution of ammonium formate with a pH value of 3.9-4.1 and a concentration of 0.0095-0.0105 mol / L, and the elution method is gradient elution, and the gradient elution includes the following procedures: From 0 to 10 min, the volume fraction of mobile phase A increased from 15% to 31%; the volume fraction of mobile phase B decreased from 85% to 69%; From 10 to 16 min, the volume fraction of mobile phase A increased from 31% to 35%, and the volume fraction of mobile phase B decreased from 69% to 65%; From 16 to 25 min, the volume fraction of mobile phase A increased from 35% to 42%, and the volume fraction of mobile phase B decreased from 65% to 58%; From 25 to 35 min, the volume fraction of mobile phase A increased from 42% to 45%, and the volume fraction of mobile phase B decreased from 58% to 55%; 35-46min, the volume fraction of mobile phase A changes from 45% to 60%, and the volume fraction of mobile phase B changes from 55% to 40%; it also includes the steps of using a solvent to dissolve one or more of hirudinamine C, hirudinamine B, and vitamin B2 to prepare a reference solution, and detecting the reference solution to obtain a reference characteristic spectrum, respectively using hirudinamine C, hirudinamine B, and vitamin B2 as reference substances and adding 30%-70% methanol to dissolve to prepare a reference solution; the established characteristic spectrum of leeches and their pharmaceutical preparations has 10 common characteristic peaks, wherein peak 1 corresponds to the retention time of the hirudinamine C reference substance peak, the corresponding peak of the hirudinamine C reference substance is taken as the S peak, and the relative retention times of peaks 2-5, peak 7, peak 9-12 and the S peak are calculated to be within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 3.24 (peak 7), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12).

2. The construction method according to claim 1, characterized in that: In the ultra-high performance liquid chromatography detection, the flow rate is 0.19-0.21 ml per minute, the column temperature is 28-32° C., the detection wavelength is 320 nm-365 nm, and the injection volume is 0.5-2 μl; and / or, the gradient elution also includes the following procedures: 46-51 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; 51-52 min, the volume fraction of mobile phase A decreases from 60% to 15%, and the volume fraction of mobile phase B increases from 40% to 85%; 52-55 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

3. The construction method according to claim 1, characterized in that: The leech and its pharmaceutical preparation are selected from leech medicinal materials, leech pieces, standard decoction of leech pieces or leech formula granules.

4. The construction method according to claim 1, characterized in that: The leech is originally a leech.

5. A method for constructing a characteristic spectrum of scalded leeches and their pharmaceutical preparations, characterized in that: The following steps are involved: Preparation of test solution: weigh 0.5g-1.5g of the test sample, accurately add 10ml-50ml of 30-70% methanol, weigh the weight, ultrasonically treat for 15-60 minutes, cool, weigh again, make up the lost weight with 30-70% methanol, shake well, filter, and take the filtrate to obtain; Determination: The test solution is tested by ultra-high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, mobile phase A is methanol, mobile phase B is an aqueous solution of ammonium formate with a pH value of 3.9-4.1 and a concentration of 0.0095-0.0105 mol / L, and the elution method is gradient elution, and the gradient elution includes the following procedures: From 0 to 10 min, the volume fraction of mobile phase A increased from 15% to 31%; the volume fraction of mobile phase B decreased from 85% to 69%; From 10 to 16 min, the volume fraction of mobile phase A increased from 31% to 35%, and the volume fraction of mobile phase B decreased from 69% to 65%; From 16 to 25 min, the volume fraction of mobile phase A increased from 35% to 42%, and the volume fraction of mobile phase B decreased from 65% to 58%; From 25 to 35 min, the volume fraction of mobile phase A increased from 42% to 45%, and the volume fraction of mobile phase B decreased from 58% to 55%; 35-46min, the volume fraction of mobile phase A changes from 45% to 60%, and the volume fraction of mobile phase B changes from 55% to 40%; it also includes the steps of using a solvent to dissolve one or more of hirudinamine C, hirudinamine B, and vitamin B2 to prepare a reference solution, and detecting the reference solution to obtain a reference characteristic spectrum, respectively using hirudinamine C, hirudinamine B, and vitamin B2 as reference substances and adding 30%-70% methanol to dissolve to prepare a reference solution; the established characteristic spectrum of scalded leeches and their pharmaceutical preparations has 12 common characteristic peaks, wherein peak 1 corresponds to the retention time of the hirudinamine C reference substance peak, the corresponding peak of the hirudinamine C reference substance is taken as S peak, and the relative retention time of peaks 2-12 and peak 1 is calculated to be within the range of ±10% of the specified value, and the specified value is: 1.66 (peak 2), 1.80 (peak 3), 2.30 (peak 4), 2.64 (peak 5), 2.93 (peak 6), 3.24 (peak 7), 4.11 (peak 8), 6.45 (peak 9), 6.97 (peak 10), 8.40 (peak 11), 8.69 (peak 12); taking the corresponding peak of hirudinamine C reference substance as S peak, the relative peak area of ​​the total peak area of ​​Peak 6 and Peak 8 and the peak area of ​​S peak are calculated within the specified value range, and the specified value is: not less than 0.

030.

6. The construction method according to claim 5, characterized in that: In the ultra-high performance liquid chromatography detection, the flow rate is 0.19-0.21 ml per minute, the column temperature is 28-32° C., the detection wavelength is 320 nm-365 nm, and the injection volume is 0.5-2 μl; and / or, the gradient elution also includes the following procedures: 46-51 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; 51-52 min, the volume fraction of mobile phase A decreases from 60% to 15%, and the volume fraction of mobile phase B increases from 40% to 85%; 52-55 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

7. The construction method according to claim 5, characterized in that: The scalded leech and its pharmaceutical preparation are selected from scalded leech pieces, standard decoction of scalded leech pieces or scalded leech formula granules.

8. The construction method according to claim 5, characterized in that: The scald leech is originally a leech.

9. A method for identifying leeches and their pharmaceutical preparations, characterized in that: include: Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to the construction method described in any one of claims 1 to 4; The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by the construction method described in any one of claims 1 to 4, and identification is performed based on the comparison result.

10. A method for identifying scalded leeches and their pharmaceutical preparations, characterized in that: include: Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to the construction method described in any one of claims 5 to 8; The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by the construction method described in any one of claims 5-8, and identification is performed based on the comparison result.

11. A method for distinguishing leeches and their pharmaceutical preparations from counterfeit products, characterized in that: include: Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to the construction method described in any one of claims 1 to 4; The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by the construction method described in any one of claims 1 to 4 using leeches and / or leech drug preparations derived from leeches as test samples, and identification is performed based on the comparison results.

12. The method for distinguishing leeches and their pharmaceutical preparations from counterfeit products according to claim 11, characterized in that: The counterfeit products are leeches originally from willow leaf leeches, Philippine cattle leeches or Japanese medicinal leeches.

13. A method for distinguishing scalded leeches and their pharmaceutical preparations from counterfeit and fake products, characterized in that: include: Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to the construction method described in any one of claims 5 to 8; The obtained characteristic spectrum of the product to be identified is compared with the characteristic spectrum constructed by the construction method described in any one of claims 5 to 8 using scalded leeches and / or scalded leech pharmaceutical preparations as test samples, and identification is performed based on the comparison results.

14. The method for distinguishing the scalded leech derived from leeches and its pharmaceutical preparation from mixed and counterfeit products according to claim 13, characterized in that: The counterfeit products are scald leeches whose origin is willow leaf leech, Philippine cattle leech or Japanese medicinal leech.

15. A method for quality inspection of leeches and their pharmaceutical preparations, characterized in that: include: Using the product to be detected as a test sample and constructing a characteristic spectrum of the product to be detected according to any one of the construction methods described in claims 1 to 4; The obtained characteristic spectrum of the product to be detected is compared with the characteristic spectrum constructed by the construction method described in any one of claims 1 to 4, and the quality evaluation is performed based on the comparison result.

16. A method for quality inspection of scalded leeches and their pharmaceutical preparations, characterized in that: include: Using the product to be detected as a test sample and constructing a characteristic spectrum of the product to be detected according to any one of the construction methods described in claims 5-8; The obtained characteristic spectrum of the product to be detected is compared with the characteristic spectrum constructed by the construction method described in any one of claims 5-8, and the quality evaluation is performed based on the comparison result.

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