HPLC (High Performance Liquid Chromatography) method for distinguishing leeches from leeches and related adulterants thereof and application of HPLC method

Abstract

The invention discloses a characteristic chromatogram construction method of leech medicinal materials and related adulterants of the leech medicinal materials. The construction method comprises the following steps: establishing characteristic spectrums of the leech medicinal material and the related counterfeit species thereof through a high performance liquid chromatography, wherein the characteristic spectrums comprise the steps of respectively extracting and preparing test sample solutions from the leech medicinal material and the related counterfeit species thereof; the method comprises the following steps: preparing a test solution, preparing a reference solution, and respectively detecting the test solution and the reference solution by high performance liquid chromatography to obtain a corresponding characteristic chromatogram, so that the leech medicinal material and the related adulterants thereof can be quickly distinguished. The leech reference medicinal material is selected from leeches or leeches in the leech family; the leech medicinal material related adulterants are selected from poecilobdella manillensis, poecilobdella manillensis or poecilobdella japonica. The method is simple and convenient, has the advantages of being simple to operate, good in separation degree, high in precision, good in repeatability, good in stability and the like, and can rapidly and accurately distinguish the leeches from the leeches and the related adulterants of the leeches and the leeches by utilizing HPLC (High Performance Liquid Chromatography).

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to an HPLC method for distinguishing leeches and their closely related counterfeit products and an application thereof. Background Art

[0002] The traditional Chinese medicine leech comes from the dried body of Whitmaniapigra Whitman, Hirudonipponica Whitman or Whitmania acranulata Whitman, which are animals of the Hirudinidae family. It has the effects of breaking blood, promoting menstruation, removing blood stasis and eliminating symptoms. Modern pharmacological studies have shown that leeches have multiple effects such as anticoagulation, antithrombosis, anti-atherosclerosis, antiplatelet aggregation, anti-tumor, anti-inflammatory, improving blood rheology, and protecting against cerebral ischemia-reperfusion injury. Although hirudin is widely considered to be the anticoagulant component of leeches, leeches also include chemical components such as protein peptides, pteridines, glycolipids, carboxylates, and free amino acids. In recent years, the antiplatelet properties of organosulfur compounds have attracted more and more attention. The pteridine compounds isolated and identified from leeches have similar sulfur structures to antiplatelet drugs including ticlopidine, clopidogrel and ticagrelor, and it is speculated that this type of compound is a potential active substance.

[0003] There are more than 680 species of leeches distributed all over the world, and more than 100 species in China. There are only three legal origins of Chinese medicinal leeches, namely leeches, leeches, and willow leaf leeches. In recent years, the market demand for Chinese medicinal leeches has increased, and its price has increased year by year and is in short supply, so counterfeit products are common. The main adulterated leeches include Philippine cattle leeches (Poecilobdella manillensis Lesson), smooth golden leeches (Whitmania laevis Baird), rod-patterned cattle leeches (Poecilobdellajavanica Wahlberg) and Japanese leeches (Mimobdellajaponica Blanchard), etc., resulting in uneven quality of commercially available leech medicinal materials, decoction pieces and preparations, which seriously affects clinical efficacy.

[0004] Although the original animals of leeches, leeches, willow-leaf leeches, and closely related counterfeit products such as Philippine cattle leeches, smooth golden line leeches, rod-patterned cattle leeches, and Japanese leeches can be distinguished morphologically, leeches in traditional Chinese medicine have a variety of medicinal forms, and the decoction pieces are dried leech segments, and leech preparations are mostly powders, which make it difficult to observe morphological characteristics and determine the origin. The thin-layer identification method included in the "Chinese Pharmacopoeia" (2020 edition) cannot effectively distinguish leeches and leeches and their closely related counterfeit products. In addition, some reports use molecular identification, but this method uses micro-sampling, samples are easily contaminated, and errors are prone to operation, which may result in false positive or false negative results.

[0005] The characteristic spectrum of traditional Chinese medicine is a method to characterize the chemical components of traditional Chinese medicine through modern analytical techniques (such as chromatography, spectroscopy, mass spectrometry, etc.). It can reflect the various characteristic chemical components in traditional Chinese medicine and provide comprehensive and rich information. Therefore, it is often used in the quality control research of traditional Chinese medicine. This method can be used to identify the authenticity of traditional Chinese medicines and traditional Chinese medicine preparations, and ensure the stability, safety and effectiveness of traditional Chinese medicine. In the early stage, there have been patents and literature reports on the use of characteristic spectra / fingerprints to control the quality of leech or leech medicinal materials, but the methods are complex, with high requirements for detection instruments and chromatographic columns, and the sample preparation steps are cumbersome, which is not conducive to the rapid and easy popularization of the detection method; in addition, some reports use non-specific indicator components such as xanthine, hypoxanthine, free amino acids, amber acid or sodium succinate as quality control indicators for leech medicinal materials. The methods lack specificity and are difficult to accurately and comprehensively reflect the quality of medicinal materials; in recent years, important indicator components of leech medicinal materials, hirudinamines and nucleosides, have received increasing attention. Although there are reports on the determination of related component contents and characteristic spectra, the lack of comparative studies on common closely related counterfeit products on the market has limited the identification effect of the method, and the established method has high requirements for instruments and equipment and lacks universality.

[0006] Therefore, there is an urgent need in this field to develop an HPLC detection method that can accurately distinguish the original species of leeches and leeches and their closely related mixed and counterfeit products, and is easy to operate, stable and reproducible, so as to strengthen the quality control of leech medicinal materials, prevent the large-scale circulation of mixed and counterfeit products, and ensure clinical efficacy. Summary of the invention

[0007] Based on this, the present invention provides a method for constructing a characteristic spectrum of leech medicinal materials and their closely related mixed and counterfeit products, and the construction method comprises the following steps:

[0008] Preparation of leech control medicinal material solution: weigh an appropriate amount of leech medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the leech control medicinal material solution; wherein the leech control medicinal material is selected from leech (Hirudonipponica Whitman) or leech (Whitmaniapigra Whitman) of Hirudinidae;

[0009] Preparation of the closely related mixed and counterfeit control medicinal material solution: weigh an appropriate amount of closely related mixed and counterfeit control medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the closely related mixed and counterfeit control medicinal material solution; wherein the closely related mixed and counterfeit control medicinal material is selected from the Hirudo philadelphica or Hirudo clavipes, or Hirudo japonica of the Hirudinaceae family;

[0010] Preparation of reference solution: weigh appropriate amount of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine reference substances, add solvent to prepare the reference solution with concentration of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine of 1 to 100 μg / mL, for example, 20 to 50 μg / mL;

[0011] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a standard characteristic spectrum of leech reference medicinal materials and closely related mixed and counterfeit reference medicinal materials is obtained;

[0012] The chromatographic conditions for HPLC detection were: Kromasil C 18 The chromatographic column has a mobile phase A selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is acid water, an alkaline aqueous solution and / or a buffered saline solution, and a gradient elution program is: 0-5 min, 5% A→9% A; 5-20 min, 9% A; 20-22 min, 9%→14% A; 22-40 min, 14% A; 40-42 min, 14% A→20% A; 42-80 min, 20% A; a flow rate of 0.5-1.5 mL / min, a column temperature of 20-40° C., a detection wavelength of 200-300 nm, and an injection volume of 5-15 μL.

[0013] Furthermore, the container is a conical flask, such as a conical flask with a stopper.

[0014] Furthermore, the solvent is an alcohol, such as methanol.

[0015] Furthermore, the concentration of the methanol is 10% to 90%, for example, about 50%.

[0016] Furthermore, the mass / volume (g / ml) ratio of the leech medicinal material to the solvent is 0.01-0.5, such as about 0.25, about 0.083, about 0.05 or about 0.022.

[0017] Furthermore, the sieve is a No. 3 sieve.

[0018] Furthermore, the extraction method is selected from one of the following: immersion, percolation, ultrasound or reflux.

[0019] Furthermore, the power of the ultrasound is 150-350W, for example, about 250W.

[0020] Furthermore, the frequency of the ultrasound is 20-60 kHz, for example, about 40 kHz.

[0021] Furthermore, the ultrasound duration is 10 to 60 minutes, for example, about 30 minutes.

[0022] Furthermore, the concentration of hirudinamine A in the reference solution is 10-50 μg / mL, for example, about 31.8 μg / mL.

[0023] Furthermore, the concentration of hirudinamine B in the reference solution is 10-50 μg / mL, for example, about 31.2 μg / mL.

[0024] Furthermore, the concentration of hirudinamine C in the reference solution is 10-50 μg / mL, for example, about 30.6 μg / mL.

[0025] Furthermore, the flow rate is 0.8-1.2 mL / min, for example, about 1.0 mL / min.

[0026] Furthermore, the column temperature is 25-35°C, for example, about 30°C.

[0027] Furthermore, the detection wavelength is 230-280 nm, for example, 245 nm.

[0028] Furthermore, the injection volume is 8 to 12 μL, for example, about 10 μL.

[0029] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

[0030] Furthermore, the mobile phase A is a mixed solution of methanol:acetonitrile=4:1.

[0031] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of organic acids and salts thereof, weak bases and salts thereof of different concentrations.

[0032] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0033] Furthermore, the acid aqueous solution is a 0.01% to 0.1% acid aqueous solution.

[0034] Furthermore, the acid aqueous solution is a 0.03% to 0.07% trifluoroacetic acid aqueous solution.

[0035] Furthermore, the acid aqueous solution is a 0.04% to 0.06% trifluoroacetic acid aqueous solution.

[0036] Furthermore, the acid aqueous solution is about 0.05% trifluoroacetic acid aqueous solution.

[0037] Furthermore, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.

[0038] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0039] Furthermore, when the detection wavelength is 245 nm, the characteristic spectrum of the leech includes 7 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak d is the chromatographic peak of SZ-1, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of mycloidine B.

[0040] Furthermore, when the detection wavelength is 245nm, the characteristic spectrum of the leech includes 6 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of hirudinine B.

[0041] According to another aspect of the present invention, there is provided an HPLC identification method for distinguishing leech medicinal materials and their closely related mixed products, the identification method comprising the following steps:

[0042] (1) Establishing a standard characteristic spectrum for reference medicinal materials of leech and its closely related mixed and counterfeit products according to the above characteristic spectrum construction method;

[0043] (2) taking a leech sample to be tested, preparing a test solution of the leech sample to be tested according to the above-mentioned characteristic spectrum construction method, and performing detection according to the chromatographic conditions in the above-mentioned characteristic spectrum construction method to obtain a spectrum of the leech sample to be tested; and

[0044] (3) comparing the leech sample spectrum obtained in step (2) with the standard characteristic spectrum of the Chinese medicinal material leech and its closely related mixed counterfeit products obtained in step (1); those that meet the requirements are leech medicinal materials, and those that do not meet the requirements are closely related mixed counterfeit products of leech medicinal materials.

[0045] Furthermore, the compliance requirement includes one of the following:

[0046] (1) When the detection wavelength is 245 nm, the spectrum of the leech sample to be tested shows 7 characteristic chromatographic peaks, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of SZ-1, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of leechline B; or

[0047] (2) When the detection wavelength is 245 nm, six characteristic chromatographic peaks are shown in the spectrum of the leech sample to be tested, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, an unknown chromatographic peak and the chromatographic peak of myristine B.

[0048] Furthermore, the leech sample to be tested is a leech (Hirudo nipponica Whitman) or a leech (Whitmaniapigra Whitman) of the Hirudinidae family.

[0049] Furthermore, the closely related counterfeit products include Philippine leeches, rod-shaped leeches and / or Japanese leeches.

[0050] Furthermore, when the detection wavelength is 245 nm, the spectrum of the leech sample shows five characteristic chromatographic peaks, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of inosine, the chromatographic peak of mycelium B and the chromatographic peak of Poecilobdellasulfide B.

[0051] Furthermore, when the detection wavelength is 245 nm, six characteristic chromatographic peaks are presented in the spectrum of the leech sample, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, the chromatographic peak of mycloidine B and the chromatographic peak of Poecilobdellasulfide B.

[0052] Furthermore, the chromatographic peak of the mycloidine B and the chromatographic peak of the poecilobdellasulfide B were identified by UPLC-QTOF-MS / MS technology.

[0053] Furthermore, when the detection wavelength was 245 nm, a characteristic chromatographic peak was present in the spectrum of the Japanese leech sample, which was the chromatographic peak of inosine.

[0054] According to another aspect of the present invention, there is provided a use of the above construction method or the above identification method in quality detection, quality evaluation or quality control of leech medicinal materials, or in distinguishing leeches and their closely related mixed products.

[0055] Beneficial effects of the present invention:

[0056] The method of the invention characterizes 8 chromatographic peaks respectively, and the baseline of HPLC detection is stable and the separation degree of the target object is good, so that HPLC can be used to quickly and accurately distinguish leeches and leeches and their closely related mixed products. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0058] Figure 1 The chromatograms of different species of leeches under the conditions of HPLC analysis method 1. Among them, b: leechamine B; c: leechamine C.

[0059] Figure 2 The chromatograms of different species of leeches under the conditions of HPLC analysis method 2. Among them, a: leechamine A; b: leechamine B; c: leechamine C.

[0060] Figure 3 The chromatograms of different species of leeches under HPLC analysis method 3. Among them, a: leechamine A; b: leechamine B; c: leechamine C.

[0061] Figure 4 The chromatograms of leech samples under HPLC analysis conditions 4 and 5. Wherein, a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0062] Figure 5 The chromatogram of the leech sample under the HPLC analysis method 6-9 conditions, wherein a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0063] Figure 6 The chromatogram of the leech sample under the HPLC analysis method 10-12 conditions, where a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0064] Figure 7 The chromatograms of leech samples under the conditions of HPLC analysis methods 13 and 14 are shown in Figure 1. Among them, a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0065] Figure 8 The chromatograms of leech samples under HPLC analysis conditions 15 and 16. Wherein, a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0066] Fig. 9 The chromatogram of the leech sample under the HPLC analysis method 17-21 conditions, where a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine.

[0067] Fig.10 The chromatograms of reference substance 1 in different acid waters (0.05% formic acid water, 0.05% acetic acid water and 0.05% trifluoroacetic acid water) under the conditions of HPLC analysis method 20 are shown in FIG. a: hirudinamine A; b: hirudinamine B; c: hirudinamine C.

[0068] Fig.11The chromatograms of different acid waters (0.05% formic acid water, 0.05% acetic acid water and 0.05% trifluoroacetic acid water) of leech samples under HPLC analysis method 20. Among them, a: hirudinamine A; b: hirudinamine B; c: hirudinamine C.

[0069] Fig.12 The HPLC chromatograms of 12 batches of leech samples, where a: leechamine A; b: leechamine B; c: leechamine C; d: SZ-1; e: inosine; f: unknown; g: leechine B.

[0070] Fig.13 The HPLC chromatograms of 11 batches of leech samples are shown below: a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; e: inosine; f: unknown; g: hirudinine B.

[0071] Fig.14 The HPLC chromatogram of the sample of Hirudinium corymbiformis. a: Hirudinamine A; b: Hirudinamine B; c: Hirudinamine C; e: Inosine; g: Mylodella B; h: Poecilobdellasulfide B.

[0072] Fig.15 The HPLC chromatogram of the leech sample. a: Hirudin A; b: Hirudin B; e: Inosine; g: Mylodella B; h: Poecilobdellasulfide B.

[0073] Fig.16 The HPLC chromatograms of two batches of Japanese leech samples are shown below: e: inosine.

[0074] Fig.17 This is a high-resolution mass spectrometry analysis of leeches. The upper figure is the UPLC chromatogram, and the lower figure is the base peak ion diagram in positive ion mode, g: leechline B.

[0075] Fig.18 This is a high-resolution mass spectrometry analysis of leeches. The upper figure is the UPLC chromatogram, and the lower figure is the base peak ion diagram in positive ion mode, g: leechline B.

[0076] Fig.19 This is a high-resolution mass spectrometry analysis of the rod-shaped leech. The upper figure is the UPLC chromatogram, and the lower figure is the base peak ion diagram in positive ion mode, h: Poecilobdellasulfide B.

[0077] Fig. 20 This is a high-resolution mass spectrometry analysis of Poecilobdella philadelphica. The upper figure is the UPLC chromatogram, and the lower figure is the base peak ion diagram in positive ion mode, h: Poecilobdellasulfide B.

[0078] Fig.21This is a high-resolution mass spectrometry analysis of Japanese leech. The upper figure is the UPLC chromatogram, and the lower figure is the base peak ion diagram in positive ion mode.

[0079] Fig. 22 This is the mass spectrum of Poecilobdellasulfide B in Philippine leech in positive ion mode in the literature (Song Wanli. Research on non-peptide chemical components of Philippine leech [D]. Shenzhen University, 2018).

[0080] Fig.23 This is the mass spectrum of the chromatographic peak h (Poecilobdellasulfide B, retention time 13.72min) in the base peak ion diagram of the bar-patterned leech in the positive ion mode. The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0081] Fig.24 This is the mass spectrum of the chromatographic peak h (Poecilobdellasulfide B, retention time 13.71min) in the base peak ion diagram of the leech in positive ion mode. The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0082] Fig.25 This is the mass spectrum of schizone B in leeches in positive ion mode in the literature (Li Tao. Research on the chemical composition of wide-bodied golden line leech [D]. Jinan University, 2013.).

[0083] Fig.26 This is the mass spectrum of the chromatographic peak g (cotyledonine B, retention time 20.44min) in the base peak ion diagram of leech in positive ion mode. The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0084] Fig. 27 This is the mass spectrum of the chromatographic peak g (cotyledonine B, retention time 20.29min) in the base peak ion diagram of leech in positive ion mode. The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0085] Fig.28 Extract the quasi-molecular ion peak [M+H] of hylospermuminosae B from the base peak ion diagram of Hirudo sphaerocephala in positive ion mode + The mass spectrum of (355.01). Wherein, g: myloquine B.

[0086] Fig.29 Extract the quasi-molecular ion peak of mycorrhizine B from the base peak ion diagram of Hirudo clavipes in positive ion mode [M+H] + The mass spectrum of the chromatographic peak g (mycine B, retention time 20.43min) in the mass spectrum of (355.01). The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0087] Fig.30 Extract the quasi-molecular ion peak [M+H] of hyoscyamine B from the base peak ion diagram of leech in positive ion mode + The mass spectrum of (355.01). Wherein, g: myloquine B.

[0088] Fig.31 Extract the quasi-molecular ion peak of mycorrhizal B from the base peak ion diagram of leech in positive ion mode [M+H] + The mass spectrum of the chromatographic peak g (mycine B, retention time 20.41min) in the mass spectrum of (355.01). The upper figure is the secondary mass spectrum, and the lower figure is the primary mass spectrum.

[0089] Fig.32 The HPLC chromatograms of different origin leech medicinal materials and mixed products. Among them, leech: HPLC chromatogram of sample No. 9; leech: HPLC chromatogram of sample No. 15; bar-grained leech: HPLC chromatogram of sample No. 24; Philippine leech: HPLC chromatogram of sample No. 25; Japanese leech: HPLC chromatogram of sample No. 26. Among them, a: hirudinamine A; b: hirudinamine B; c: hirudinamine C; d: SZ-1; e: inosine; f: unknown; g: leechline B. DETAILED DESCRIPTION

[0090] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0091] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0092] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0093] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0094] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0095] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0096] As described in the background technology section, there is an urgent need in the art to provide a fast, stable, and well-resolved HPLC identification method. In order to solve the above problem, the present invention provides a method for constructing a characteristic spectrum of leech medicinal materials and their closely related mixed products, and the construction method comprises the following steps:

[0097] Preparation of leech control medicinal material solution: weigh an appropriate amount of leech medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the leech control medicinal material solution; wherein the leech control medicinal material is selected from leech (Hirudonipponica Whitman) or leech (Whitmaniapigra Whitman) of Hirudinidae;

[0098] Preparation of the closely related mixed and counterfeit control medicinal material solution: weigh an appropriate amount of closely related mixed and counterfeit control medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the closely related mixed and counterfeit control medicinal material solution; wherein the closely related mixed and counterfeit control medicinal material is selected from the Hirudo philadelphica or Hirudo clavipes, or Hirudo japonica of the Hirudinaceae family;

[0099] Preparation of reference solution: weigh appropriate amount of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine reference substances, add solvent to prepare the reference solution with concentration of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine of 1 to 100 μg / mL, for example, 20 to 50 μg / mL;

[0100] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a standard characteristic spectrum of leech reference medicinal materials and closely related mixed and counterfeit reference medicinal materials is obtained;

[0101] The chromatographic conditions for HPLC detection were: Kromasil C 18 The chromatographic column has a mobile phase A selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is acid water, an alkaline aqueous solution and / or a buffered saline solution, and a gradient elution program is: 0-5 min, 5% A→9% A; 5-20 min, 9% A; 20-22 min, 9%→14% A; 22-40 min, 14% A; 40-42 min, 14% A→20% A; 42-80 min, 20% A; a flow rate of 0.5-1.5 mL / min, a column temperature of 20-40° C., a detection wavelength of 200-300 nm, and an injection volume of 5-15 μL.

[0102] In the present invention, when time, temperature, wavelength, injection volume, concentration, ratio, power, frequency, flow rate, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "20-40" is disclosed, the described range should be interpreted as including the range "20-40", "20-35", "20-30", "20-25", "25-40", "25-35", "25-30", "30-40", "30-35", "35-40", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0103] In a preferred embodiment, the container is a conical flask, such as a conical flask with a stopper.

[0104] In a preferred embodiment, the solvent is an alcohol, such as methanol.

[0105] Compared with other solvents, methanol is more effective in extracting leech and leech components.

[0106] In a preferred embodiment, the concentration of methanol is 10% to 90%, such as about 50%.

[0107] Compared with other concentrations of methanol, about 50% methanol has a better extraction effect on the organic matter contained in leeches and leeches.

[0108] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%.

[0109] In a preferred embodiment, the mass / volume (g / ml) ratio of the leech material to the solvent is 0.01-0.5, such as about 0.25, about 0.083, about 0.05 or about 0.022.

[0110] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.05" includes ±5% of 0.05, or from 0.0475 to 0.0525.

[0111] In a preferred embodiment, the sieve is a No. 3 sieve.

[0112] In a preferred embodiment, the extraction method is selected from the following: immersion, percolation, ultrasound or reflux.

[0113] Compared with other extraction methods, ultrasound is more capable of fully extracting the organic matter contained in leeches and leeches.

[0114] In a preferred embodiment, the power of the ultrasound is 150-350W, for example, about 250W.

[0115] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0116] In a preferred embodiment, the frequency of the ultrasound is 20-60 kHz, such as about 40 kHz.

[0117] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0118] In a preferred embodiment, the ultrasonication time is 10 to 60 min, such as about 30 min.

[0119] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0120] In a preferred embodiment, the concentration of hirudinamine A in the reference solution is 10-50 μg / ml, for example, about 31.8 μg / ml.

[0121] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 31.8" includes ±5% of 31.8, or from 30.21 to 33.39.

[0122] In a preferred embodiment, the concentration of hirudinamine B in the reference solution is 10-50 μg / ml, for example, about 31.2 μg / ml.

[0123] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 31.2" includes ±5% of 31.2, or from 29.64 to 32.76.

[0124] In a preferred embodiment, the concentration of hirudinamine C in the reference solution is 10-50 μg / ml, for example, about 30.6 μg / ml.

[0125] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30.6" includes ±5% of 30.6, or from 29.07 to 32.13.

[0126] In a preferred embodiment, the flow rate is 0.8-1.2 ml / min, such as about 1.0 ml / min.

[0127] Compared with other flow rates, a flow rate of about 1.0 ml / min makes the sample peak faster.

[0128] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0129] In a preferred embodiment, the column temperature is 25-35°C, such as about 30°C.

[0130] Compared with other temperatures, a temperature of about 30°C significantly improves the tailing phenomenon of the chromatographic peaks, and the peak shape and the separation between peaks are better.

[0131] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0132] In a preferred embodiment, the detection wavelength is 230-280 nm, for example 245 nm.

[0133] In a preferred embodiment, the injection volume is 8-12 μl, such as about 10 μl.

[0134] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0135] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0136] Using the specifications of this chromatographic column, the separation between the peaks of the sample can meet the requirements of qualitative identification.

[0137] In a preferred embodiment, the mobile phase A is a mixed solution of methanol:acetonitrile=4:1.

[0138] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of organic acids and their salts, weak bases and their salts of different concentrations.

[0139] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0140] In a preferred embodiment, the acid aqueous solution is a 0.01% to 0.1% acid aqueous solution.

[0141] In a preferred embodiment, the acid aqueous solution is a 0.03% to 0.07% trifluoroacetic acid aqueous solution.

[0142] In a preferred embodiment, the acid aqueous solution is a 0.04% to 0.06% trifluoroacetic acid aqueous solution.

[0143] In a preferred embodiment, the aqueous acid solution is about 0.05% trifluoroacetic acid in water.

[0144] Compared with other organic acids at other concentrations, about 0.05% trifluoroacetic acid aqueous solution makes the chromatographic peak of the analyte better in shape and has a better separation effect from impurity peaks.

[0145] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.05%" includes ±5% of 0.05%, or from 0.0475% to 0.0525%.

[0146] In a preferred embodiment, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.

[0147] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.

[0148] In a preferred embodiment, when the detection wavelength is 245 nm, the characteristic spectrum of the leech includes 7 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak d is the chromatographic peak of SZ-1, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of mycloidine B.

[0149] In a preferred embodiment, when the detection wavelength is 245 nm, the characteristic spectrum of the leech includes 6 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of hirudinine B.

[0150] According to another aspect of the present invention, there is provided an HPLC identification method for distinguishing leech medicinal materials and their closely related mixed products, the identification method comprising the following steps:

[0151] (1) Establishing a standard characteristic spectrum for reference medicinal materials of leech and its closely related mixed and counterfeit products according to the above characteristic spectrum construction method;

[0152] (2) taking a leech sample to be tested, preparing a test solution of the leech sample to be tested according to the above-mentioned characteristic spectrum construction method, and performing detection according to the chromatographic conditions in the above-mentioned characteristic spectrum construction method to obtain a spectrum of the leech sample to be tested; and

[0153] (3) comparing the leech sample spectrum obtained in step (2) with the standard characteristic spectrum of the Chinese medicinal material leech and its closely related mixed counterfeit products obtained in step (1); those that meet the requirements are leech medicinal materials, and those that do not meet the requirements are closely related mixed counterfeit products of leech medicinal materials.

[0154] In a preferred embodiment, the compliance requirement includes one of the following:

[0155] (1) When the detection wavelength is 245 nm, the spectrum of the leech sample to be tested shows 7 characteristic chromatographic peaks, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of SZ-1, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of leechline B; or

[0156] (2) When the detection wavelength is 245 nm, six characteristic chromatographic peaks are shown in the spectrum of the leech sample to be tested, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, an unknown chromatographic peak and the chromatographic peak of myristine B.

[0157] In a preferred embodiment, the leech sample to be tested is a leech (Hirudo nipponica Whitman) or a leech (Whitmania pigra Whitman) of the family Hirudinidae.

[0158] In a preferred embodiment, the closely related counterfeit products include Philippine leeches, rod-shaped leeches and / or Japanese leeches.

[0159] In a preferred embodiment, when the detection wavelength is 245 nm, the spectrum of the leech sample shows five characteristic chromatographic peaks, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of inosine, the chromatographic peak of mycloidine B and the chromatographic peak of Poecilobdellasulfide B.

[0160] In a preferred embodiment, when the detection wavelength is 245 nm, the bar-lined leech sample spectrum presents 6 characteristic chromatographic peaks, namely the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, the chromatographic peak of mycloidine B and the chromatographic peak of Poecilobdellasulfide B.

[0161] In a preferred embodiment, the chromatographic peak of the scutellarine B and the chromatographic peak of the poecilobdellasulfide B are identified by UPLC-QTOF-MS / MS technology.

[0162] In a preferred embodiment, when the detection wavelength is 245 nm, the Japanese leech sample spectrum shows a characteristic chromatographic peak, which is the chromatographic peak of inosine.

[0163] According to another aspect of the present invention, there is provided a use of the above construction method or the above identification method in quality detection, quality evaluation or quality control of leech medicinal materials, or in distinguishing leeches and their closely related mixed products.

[0164] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed according to conventional conditions or conditions recommended by the manufacturer. If specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specification. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially, such as those that can be purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0165] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0166] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0167] Example 1

[0168] The method of the present invention is used to analyze different varieties of leeches to distinguish leeches and leeches and their closely related mixed products, as follows:

[0169] 1. Materials

[0170] 1.1 Experimental instruments

[0171] Shimadzu LC 20A high performance liquid chromatograph (Shimadzu Corporation, Japan), Kromasil C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm).

[0172] 1.2 Reagents and medicinal materials

[0173] Hirudin A (DSTDS047901) and hirudin B (DSTDS047501) were purchased from Chengdu Dester Biotechnology Co., Ltd. with a purity of ≥98%; hirudin C (14713) was purchased from Shanghai Standard Standard Technology Service Co., Ltd. with a purity of ≥98%. Methanol and acetonitrile were purchased from Thermo Fisher Scientific (China) Co., Ltd., Wahaha purified water, and other reagents were analytical grade.

[0174] 27 batches of samples were collected from different regions of China, including 12 batches of leeches, 11 batches of leeches, 1 batch of bar-patterned leeches, 1 batch of Philippine leeches and 2 batches of Japanese leeches. The specific information is as follows:

[0175] Sample 1 leech, produced in Jining County, Jining City, Shandong Province;

[0176] Sample 2 leech, produced in Sucheng District, Suqian City, Jiangsu Province;

[0177] Sample 3: leech, produced in Bamencheng Town, Baodi District, Tianjin;

[0178] Sample 4 leech, originating from Xihua County, Zhoukou City, Henan Province;

[0179] Sample 5 leech origin: Jining County, Jining City, Shandong Province;

[0180] Sample 6 leech origin: Sucheng District, Suqian City, Jiangsu Province;

[0181] Sample 7 leech origin: De'an County, Jiujiang City, Jiangxi Province;

[0182] Sample 8 leech origin: Yuanjiang City, Yiyang City, Hunan Province;

[0183] Sample 9 leech origin: Jining County, Jining City, Shandong Province;

[0184] Sample 10 leech origin: Gong'an County, Jingzhou City, Hubei Province;

[0185] Sample 11 leech origin: Gong'an County, Jingzhou City, Hubei Province;

[0186] Sample 12 leech origin: Bamencheng Town, Baodi District, Tianjin;

[0187] Sample 13 leech origin: Shandong (purchased at Anguo medicinal materials market);

[0188] Sample 14 leech origin: Jining County, Jining City, Shandong Province;

[0189] Sample 15 leech origin: Sucheng District, Suqian City, Jiangsu Province;

[0190] Sample 16 leech origin: Bamencheng Town, Baodi District, Tianjin;

[0191] Sample 17 leech origin: Xihua County, Zhoukou City, Henan Province;

[0192] Sample 18 leech origin: Jining County, Jining City, Shandong Province;

[0193] Sample 19 leech origin: Sucheng District, Suqian City, Jiangsu Province;

[0194] Sample 20 leech origin: De'an County, Jiujiang City, Jiangxi Province;

[0195] Sample 21 leech origin: Yuanjiang City, Yiyang City, Hunan Province;

[0196] Sample 22 leech origin: Gong'an County, Jingzhou City, Hubei Province;

[0197] Sample 23 leech origin: Gong'an County, Jingzhou City, Hubei Province;

[0198] Sample 24, Leech clavata, was produced in Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region;

[0199] Sample 25 Philippine cattle leech originates from Zhongshan City, Guangdong Province;

[0200] Sample 26 Japanese leech origin: Xihua County, Zhoukou City, Henan Province;

[0201] Sample 27 Japanese leech is produced in Xinhui District, Jiangmen City, Guangdong Province.

[0202] 2.1. Preparation of test solution

[0203] Preparation method of test sample 1: Take about 0.5g of leech powder (passed through No. 3 sieve), weigh accurately, place in a 50mL stoppered conical flask, add 22.5mL of 50% methanol, stopper, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30min, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain.

[0204] Sample preparation method 2: Take about 0.5 g of leech medicinal material sample powder (passed through No. 3 sieve), accurately weigh, place in a 50 mL stoppered conical flask, add 2 mL of 50% methanol, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant.

[0205] Sample preparation method 3: Take about 0.5 g of leech medicinal material sample powder (passed through No. 3 sieve), accurately weigh, place in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant.

[0206] 2.2 Preparation of reference solution

[0207] Reference solution 1: Accurately weigh appropriate amounts of hirudinamine A, hirudinamine B, and hirudinamine C, dilute with 50% methanol to make a mixed reference solution containing 31.8 μg of hirudinamine A, 31.2 μg of hirudinamine B, and 30.6 μg of hirudinamine C per 1 mL, respectively, and place at 4°C for use in optimizing chromatographic conditions.

[0208] Reference solution 2: Accurately weigh appropriate amounts of SZ-1, hirudinamine A, hirudinamine B, hirudinamine C, and inosine, make up to volume with 50% methanol and dilute to make a mixed reference solution containing 31.0 μg of SZ-1, 31.2 μg of hirudinamine A, 30.6 μg of hirudinamine B, 30.0 μg of hirudinamine C, and 33.6 μg of inosine per 1 mL, respectively. Keep at 4°C for use in the detection of multiple batches of samples.

[0209] The structural formulas of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1, inosine, mylodellasulfide B, and Poecilobdellasulfide B are as follows:

[0210]

[0211] SZ-1 is a green solid. HR-ESI-MS gives a quasi-molecular ion peak of m / z 357.0328 [M+H]+, with a calculated value of 357.0327. The inferred molecular formula is C 12 H 12 N4O5S2, degree of unsaturation is 9.

[0212] exist 1 In the H-NMR (DMSO-d6, 600MHz) spectrum, two active hydrogen signal peaks are shown [δ H 6.27 (1H, brs), δ H 5.11 (1H, brs)], a hydrogen signal of an oxygen-containing methine [δ H 5.68 (1H, t, J = 5.8 Hz, 13-OH], a hydrogen signal of an oxygen-containing methylene group [δ H 3.65 (1H, d, J = 5.7 Hz, 14-OH)], and two methyl characteristic signal peaks [δ H 3.26(3H,s,H-10), 3.15(3H,s,H-12)].

[0213] exist 13 There are 12 carbon signals in the C-NMR (DMSO-d6, 150 MHz) spectrum. Combined with HSQC spectrum analysis, [δ C 161.9(C-2),δ C 153.7 (C-4)] are two keto carbonyl signal peaks, [125.3 (C-4a), 137.5 (C-5a), 128.8 (C-6), 153.2 (C-7), 141.6 (C-8a), δ C 157.7 (C-9a)] are 6 unsaturated carbon signals. The signals of 2 hydroxyl-substituted methine carbons and methylene carbons are [δ C 67.1(C-13), δ C 66.9 (C-14)]. The remaining two carbon signals are attributed to two methyl groups [δ C 27.8 (C-10), 40.2 (C-12)], these hydrogen and carbon signals are assigned in Table 1. The above information is similar to the compound (R)-hirudonucleodisulfide B in the literature, which is speculated to be a pteridine derivative. The main difference between them is that the nitrogen atom at the 3rd position of this compound is substituted by a methyl group, and the sulfur atom at the 11th position is a sulfinyl group. The chemical shifts of the two methyl groups substituted at the 3rd and 11th positions [δ H 3.26 (3H, s, H-10), 3.15 (3H, s, H-12)] also indirectly confirmed this.

[0214] 2D-NMR spectroscopy also provided some evidence for the structure of the compound. 1 H- 1 The crossover signals H-13 / H2-14 in the H COSY spectrum showed the presence of C13-C14 (vicinal diol) structure fragments. The related signals of H-10 / C-2 / C-4, H-12 / C-6, H-13 / C-6, and H-14 / C-7 in the HMBC spectrum verified the methyl substitution of N-3, the sulfinyl substitution of C-6, and the vicinal diol substitution of C-7, respectively.

[0215] In addition, the characteristic signal peaks in the IR spectrum such as amino (3239cm -1 ), methyl (2921cm -1 ), carbonyl (1728cm -1 ), carbon-nitrogen double bond (1668cm -1 ), sulfinyl (1594cm -1 ), double bond (1545cm -1 ) was similar to Whitmanine B and (R)-hirudonucleodisulfide B. Therefore, the structure of the compound was determined and named SZ-1.

[0216] Table 1 SZ-1 1 H NMR and 13 C NMR data

[0217]

[0218]

[0219] 2.3. Preparation of control medicinal material solution

[0220] Take about 0.5 g of leech medicinal material sample powder (passed through No. 3 sieve), accurately weigh it, put it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper it, weigh it, and 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, centrifuge it, and take the supernatant to obtain the leech control medicinal material solution.

[0221] Take about 0.5 g of leech medicinal material sample powder (passed through No. 3 sieve), accurately weigh, place in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, take the supernatant, and obtain the leech control medicinal material solution.

[0222] Take about 0.5 g of the powder of the medicinal material sample of Hirudo clavipes (passed through No. 3 sieve), accurately weigh it, put it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper it, weigh it, and 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, centrifuge it, and take the supernatant to obtain the control medicinal material solution of Hirudo clavipes.

[0223] Take about 0.5 g of the powder of the medicinal material sample of Hirudo vomica (passed through No. 3 sieve), accurately weigh it, put it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper it, weigh it, and 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, centrifuge it, and take the supernatant to obtain the Hirudo vomica control medicinal material solution.

[0224] Take about 0.5 g of Japanese leech-like medicinal material sample powder (passed through No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper it, weigh it, ultrasonically treat it (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, centrifuge it, and take the supernatant to obtain the Japanese leech-like medicinal material control solution.

[0225] 2.3. Investigation of HPLC chromatographic conditions

[0226] The reference solution 1 and representative samples of different species of leeches (1, 14, 24, 25, 26) were analyzed by different HPLC chromatographic conditions (as shown in Table 2). Method 1 was a preliminary method, and methods 2 to 21 were optimized methods.

[0227] Column: Kromasil C 18 (4.6mm×250mm, 5μm), flow rate: 1.0mL / min, injection volume: 10μL.

[0228] Table 2 Mobile phase composition and elution method

[0229]

[0230]

[0231]

[0232]

[0233]

[0234] Investigation of different acid water as mobile phase:

[0235] The reference solution 1 and the test solution prepared by method 3 were used to investigate different acid waters in the mobile phase by method 20, mainly 0.05% formic acid water, 0.05% acetic acid water and 0.05% trifluoroacetic acid water.

[0236] 3. Results of investigation on HPLC chromatographic conditions

[0237] The test sample was prepared according to Preparation Method 1 and eluted isocratically according to HPLC chromatographic conditions Method 1. It was found that there was no chromatographic peak of hirudinamine A within the analysis time, and the response value of the chromatographic peak was low. Figure 1 Therefore, the sample preparation method 2 was used and the analysis time was extended (method 2) for detection. Hirudinamine A, B, and C all showed peaks, but the retention time of hirudinamine C was too early (5min) and was easily affected by the solvent peak. The detection results of different varieties of leeches are shown in Figure 2 Therefore, the gradient elution was adjusted and the analysis was performed using method 3. The response values ​​of hirudinamine A, B, and C were relatively low, which was presumed to be due to the inappropriate detection wavelength. The detection results of different leech species are shown in Figure 2. Figure 3 shown.

[0238] Method 4 and method 5 are both gradient elution, and the detection wavelength is changed to the maximum ultraviolet absorption wavelength of the reference substance, 245nm. The results show that when the organic phase ratio is 5%, the chromatographic peak separation of inosine and hirudinamine C is poor, and the chromatographic peaks are less. The HPLC chromatogram is as follows Figure 4 As shown. Therefore, the mobile phase A was changed to acetonitrile, and the extraction solvent of the test sample was changed, that is, the test sample preparation method 3 and the chromatographic analysis method 6 were used for detection. The chromatographic peak of hirudinamine B was not effectively separated from the adjacent chromatographic peaks. The HPLC results of leech fluid under different methods are shown as follows Figure 5 Adjust the organic phase ratio in the mobile phase, that is, use method 7-9. The chromatographic peak of hirudinamine B is still not separated from the adjacent chromatographic peaks, and the chromatographic peak of inosine is interfered by the solvent peak. The mobile phase A needs to be replaced.

[0239] When the mobile phase A was changed to methanol:acetonitrile (1:1), the chromatographic peak of hirudinamine B was still not effectively separated from the adjacent chromatographic peaks, and the separation of the chromatographic peak of inosine was poor. The HPLC results of leech under different methods are as follows: Figure 6 shown.

[0240] When the mobile phase A was changed to methanol:acetonitrile (2:1), the analytical method 13-14 was used for detection. The separation of the inosine chromatographic peak was poor, and the chromatographic peak of hirudinamine A was not detected. The HPLC results of leech under different methods are as follows: Figure 7 shown.

[0241] When the mobile phase A was changed to methanol:acetonitrile (3:1), the analytical method 15-16 was used for detection. The separation of the inosine chromatographic peaks was poor, and the chromatographic peak of hirudinamine C was not detected. The HPLC results of leeches under different methods are as follows: Figure 8 shown.

[0242] The mobile phase A was changed to methanol:acetonitrile (4:1) and the analytical method 17 was used for detection. The resolution of the inosine chromatographic peak was >1.5, but the resolution of hirudinamine B was poor. The HPLC results of leech under different methods are as follows: Fig. 9 As shown. When using analytical method 18 for detection, the chromatographic peak of hirudinamine A was not detected, and the separation of hirudinamine B was poor. When using analytical method 19 for detection, the separation of inosine, hirudinamine A, B, and C was >1.5, but the time was long (90min). The detection time was adjusted and analytical method 20 was used for detection. The target compounds were all detected and the separation was >1.5. Further attempts were made to increase the column temperature and use analytical method 21 for detection. It was found that the separation of hirudinamine B and C was less than 1.5 (see method 21).

[0243] Investigation of different acid waters in mobile phase: In this study, the reference solution 1 and the test solution prepared by method 3 were analyzed by method 20. The analysis of the reference solution 1 with different acid waters found that when the mobile phase was 0.05% acetic acid water, the chromatographic peaks of hirudinamine C and hirudinamine B were tailing, such as Fig.10 As shown. The analysis of the leech test solution with different acid waters found that when the mobile phase was 0.05% formic acid water, the separation degree between the chromatographic peak of hirudinamine B and the adjacent chromatographic peaks was less than 1.5; when the mobile phase was 0.05% acetic acid water, the chromatographic peaks of hirudinamine C and hirudinamine B were tailing; when the mobile phase was 0.05% trifluoroacetic acid water, the peak shape and separation degree of the main chromatographic peaks were good, as shown in Figure 1. Fig.11 As shown, the acid aqueous phase of the mobile phase was finally determined to be 0.05% trifluoroacetic acid water.

[0244] In summary, the best method for preparing the test sample is: take about 0.5 g of leech medicinal material sample powder (passed through No. 3 sieve), accurately weigh it, put it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, centrifuge it, and take the supernatant.

[0245] The optimal HPLC detection conditions are: Kromasil C 18 (250mm×4.6mm, 5μm) chromatographic column, methanol:acetonitrile (4:1) (A): 0.05% trifluoroacetic acid water (B) as mobile phase, conditions are 0-5min mobile phase A 5%→9%; 5-20min mobile phase A 9%→9%; 20-22min mobile phase A 9%→14%; 22-40min mobile phase A 14%→14%; 40-42min mobile phase A 14%→20%; 42-80min mobile phase A 20%→20%, detection wavelength is 245nm, flow rate is 1.0mL / min, column temperature is 30℃.

[0246] Example 2

[0247] This embodiment relates to the establishment of characteristic maps of leech medicinal materials and their closely related mixed and counterfeit products.

[0248] 1. Sample preparation

[0249] Take about 0.5g of powder of leech, leech, bar-patterned leech, Philippine leech, and Japanese leech medicinal material sample (passed through No. 3 sieve), accurately weigh, place in a 50mL stoppered conical flask, add 10mL of 50% methanol, stopper, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30min, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant.

[0250] 2. Characteristic map of leech medicinal materials and their closely related counterfeit products

[0251] The above sample solution was tested according to the optimal HPLC detection conditions to obtain the corresponding characteristic spectrum:

[0252] 2.1. Detection of leech samples

[0253] Construct the characteristic map of leeches such as Fig.12 As shown, there are 7 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.000 (peak e, S), 1.984 (peak c), 3.601 (peak d), 4.202 (peak b), 4.445 (peak f), 6.788 (peak g), 7.750 (peak a).

[0254] 2.2. Detection of leech samples

[0255] Construct the characteristic map of leeches such as Fig.13 As shown, there are 6 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.000 (peak e, S), 2.004 (peak c), 4.189 (peak b), 4.468 (peak f), 6.797 (peak g), 7.757 (peak a).

[0256] 2.3. Testing of samples of Holothuria clavata

[0257] Construct the characteristic map of the rod-shaped leech Fig.14As shown, there are 6 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.000 (peak e, S), 2.017 (peak c), 4.211 (peak b), 4.769 (peak h), 6.892 (peak g), 7.837 (peak a).

[0258] 2.4. Testing of samples of Philippine leech

[0259] Construct the characteristic map of Philippine leech Fig.15 As shown, there are 5 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 1.000 (peak e, S), 4.312 (peak b), 4.897 (peak h), 7.011 (peak g), 7.955 (peak a).

[0260] 2.5. Testing of Japanese leech samples

[0261] Constructing the characteristic map of Japanese leech Fig.16 As shown, only one characteristic peak is the inosine reference and the corresponding peak is peak S. Calculate the relative retention time of the characteristic peak and peak S, and the relative retention time should be within ±10% of the specified value. The specified value is: 1.000 (peak e, S).

[0262] Example 3

[0263] 1. Identify characteristic peaks through reference substances

[0264] The reference solution and the test solution were tested together, and characteristic peak a was identified as hirudinamine A, characteristic peak b as hirudinamine B, characteristic peak c as hirudinamine C, characteristic peak d as SZ-1, and characteristic peak e as inosine (S).

[0265] 2. Identification of characteristic peaks based on UPLC-QTOF-MS / MS

[0266] Representative samples of different species of leeches (1, 14, 24, 25, 26) were prepared according to the test solution method 3 and analyzed by the following UPLC-QTOF-MS / MS conditions.

[0267] 2.1UPLC conditions: Waters ACQUITY UPLC BEH C 18(2.1mm×100mm, 1.7μm) chromatographic column, methanol:acetonitrile (4:1) (A):0.1% formic acid water (B) as mobile phase, conditions are 0-2min mobile phase A 5%→9%; 2-8min mobile phase A 9%→9%; 8-9min mobile phase A 9%→14%; 9-16min mobile phase A14%→14%; 16-17min mobile phase A14%→20%; 17-32min mobile phase A20%→20%, detection wavelength is 245nm, flow rate is 0.2mL / min, column temperature is 30℃.

[0268] 2.2 Mass spectrometry conditions: electrospray ionization (ESI) source, positive ion mode scanning, capillary voltage 2.2 kV, cone voltage 40 V, compensation voltage 80 V, ion source temperature 120 °C, desolvation gas temperature 450 °C, desolvation gas flow rate 800 L·h -1 , cone hole back-blowing gas flow rate 50L·h -1 Using MS E Acquisition mode, collision voltage 25-50 eV, scan range m / z 100-1200.

[0269] 2.3 Results Analysis

[0270] The test fluids of different species of leeches were subjected to high-resolution mass spectrometry analysis under the above conditions. The results are as follows: Figures 17 to 21 As shown. By comparing the liquid chromatography retention time, mass spectrometry behavior, fragment ions and other characteristics of characteristic peak g with those in the literature, characteristic peak g was identified as mylodipine B; by comparing the liquid chromatography retention time, mass spectrometry behavior, fragment ions and other characteristics of characteristic peak g with those in the literature, characteristic peak h was identified as Poecilobdellasulfide B. The mass spectrometry results are shown in Table 3. Figures 22 to 31 shown.

[0271] Table 3 Chromatographic peak identification of Poecilobdellasulfide B and Mycium B

[0272]

[0273] Note: (a) leech; (b) leech; (c) Philippine cattle leech; (d) club-shaped cattle leech; (e) Japanese leech.

[0274] 3. 8 characteristic peak identification results

[0275] By comparing with reference materials and identifying UPLC-QTOF-MS / MS chromatographic peaks, eight characteristic peaks that can distinguish leech medicinal materials and their closely related mixed products were identified. The results are shown in Table 4.

[0276] Table 4 Characteristic components of different leech species

[0277]

[0278] In summary, the present invention has established a method for characteristic spectrum of leeches and their closely related counterfeit products by analyzing HPLC spectrum, and a total of 8 characteristic peaks are detected in the chromatogram, and the differences between leeches, leeches, bar-patterned leeches, Philippine leeches, and Japanese leeches are identified by this method. As follows:

[0279] Leech medicinal material characteristic spectrum Fig.32 As shown, there are 7 characteristic peaks, namely, chromatographic peak a is hirudinamine A peak, chromatographic peak b is hirudinamine B peak, chromatographic peak c is hirudinamine C peak, chromatographic peak d is SZ-1 peak, chromatographic peak e is inosine peak, chromatographic peak f is unknown peak, and chromatographic peak g is mycloidine B peak.

[0280] Leech medicinal material characteristic spectrum Fig.32 As shown, there are 6 characteristic peaks, namely, chromatographic peak a is hirudinamine A peak, chromatographic peak b is hirudinamine B peak, chromatographic peak c is hirudinamine C peak, chromatographic peak e is inosine peak, chromatographic peak f is unknown peak, and chromatographic peak g is hirudinamine B peak.

[0281] The characteristic spectrum of the medicinal material of Hirudo clavipes Fig.32 As shown, there are 6 characteristic peaks, namely, chromatographic peak a is hirudinamine A peak, chromatographic peak b is hirudinamine B peak, chromatographic peak c is hirudinamine C peak, chromatographic peak e is inosine peak, chromatographic peak g is mycloidine B peak, and chromatographic peak h is Poecilobdellasulfide B.

[0282] The characteristic spectrum of Philippine leech medicinal materials is as follows Fig.32 As shown, there are 5 characteristic peaks, namely, chromatographic peak a is hirudinamine A peak, chromatographic peak b is hirudinamine B peak, chromatographic peak e is inosine peak, chromatographic peak g is mycloidine B peak, and chromatographic peak h is Poecilobdellasulfide B.

[0283] Characteristic spectrum of Japanese leech-like medicinal materials Fig.32 As shown, there is one characteristic peak, namely, chromatographic peak e is inosine.

[0284] In summary, the method of the present invention can be used to quickly and accurately distinguish leeches and leeches and their closely related counterfeits.

[0285] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing a characteristic spectrum of leech medicinal materials and their closely related mixed products, characterized in that: The construction method comprises the following steps: Preparation of leech control medicinal material solution: weigh an appropriate amount of leech medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the leech control medicinal material solution; wherein the leech control medicinal material is selected from leech (Hirudonipponica Whitman) or leech (Whitmaniapigra Whitman) of Hirudinidae; Preparation of the closely related mixed and counterfeit control medicinal material solution: weigh an appropriate amount of closely related mixed and counterfeit control medicinal material powder, sieve it, place it in a container, add a solvent, weigh it, extract it for a period of time, let it cool, weigh it again, use the solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant, and obtain the closely related mixed and counterfeit control medicinal material solution; wherein the closely related mixed and counterfeit control medicinal material is selected from the leech of Hirudophyta or Hirudo clavata, or Hirudo japonica of Hirudoidae; Preparation of reference solution: weigh appropriate amount of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine reference substances, add solvent to prepare the reference solution with concentration of hirudinamine A, hirudinamine B, hirudinamine C, SZ-1 and / or inosine of 1 to 100 μg / mL, for example, 20 to 50 μg / mL; According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a standard characteristic spectrum of leech reference medicinal materials and closely related mixed and counterfeit reference medicinal materials is obtained; The chromatographic conditions for HPLC detection were: Kromasil C 18 The chromatographic column has a mobile phase A selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is acid water, an alkaline aqueous solution and / or a buffered saline solution, and a gradient elution program is: 0-5 min, 5% A→9% A; 5-20 min, 9% A; 20-22 min, 9%→14% A; 22-40 min, 14% A; 40-42 min, 14% A→20% A; 42-80 min, 20% A; a flow rate of 0.5-1.5 mL / min, a column temperature of 20-40° C., a detection wavelength of 200-300 nm, and an injection volume of 5-15 μL.

2. The construction method according to claim 1, characterized in that: The container is a conical flask, such as a conical flask with a stopper; Preferably, the solvent is an alcohol, such as methanol; Preferably, the concentration of methanol is 10% to 90%, for example about 50%; More preferably, the mass / volume (g / ml) ratio between the leech medicinal material and the solvent is 0.01 to 0.5, such as about 0.25, about 0.083, about 0.05 or about 0.022; More preferably, the sieve is a No. 3 sieve; Preferably, the extraction method is selected from one of the following: immersion, percolation, ultrasound or reflux; Preferably, the power of the ultrasound is 150-350W, for example, about 250W; Preferably, the frequency of the ultrasound is 20 to 60 kHz, for example, about 40 kHz; Preferably, the ultrasonication time is 10 to 60 min, such as about 30 min.

3. The construction method according to claim 1, characterized in that: The concentration of hirudinamine A in the reference solution is 10 to 50 μg / mL, for example, about 31.8 μg / mL; Preferably, the concentration of hirudinamine B in the reference solution is 10-50 μg / mL, for example, about 31.2 μg / mL; Preferably, the concentration of hirudinamine C in the reference solution is 10-50 μg / mL, for example, about 30.6 μg / mL.

4. The construction method according to claim 1, characterized in that: The flow rate is 0.8 to 1.2 ml / min, for example, about 1.0 ml / min; Preferably, the column temperature is 25-35°C, for example about 30°C; More preferably, the detection wavelength is 230-280 nm, for example 245 nm; More preferably, the injection volume is 8 to 12 μL, for example, about 10 μL; Preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

5. The construction method according to claim 1, characterized in that: The mobile phase A is a mixed solution of methanol:acetonitrile = 4:1; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of organic acids and their salts, weak bases and their salts of different concentrations; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations; More preferably, the acid aqueous solution is a 0.01% to 0.1% acid aqueous solution; More preferably, the acid aqueous solution is a 0.03% to 0.07% trifluoroacetic acid aqueous solution; More preferably, the acid aqueous solution is a 0.04% to 0.06% trifluoroacetic acid aqueous solution; More preferably, the aqueous acid solution is about 0.05% trifluoroacetic acid aqueous solution; More preferably, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution; More preferably, the pH value of the buffered saline solution is no greater than 7.

0.

6. The construction method according to claim 1, characterized in that: When the detection wavelength is 245 nm, the characteristic spectrum of the leech includes 7 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak d is the chromatographic peak of SZ-1, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of mycloidine B.

7. The construction method according to claim 1, characterized in that: When the detection wavelength is 245nm, the characteristic spectrum of the leech includes 6 characteristic peaks, peak a is the chromatographic peak of hirudinamine A, peak b is the chromatographic peak of hirudinamine B, peak c is the chromatographic peak of hirudinamine C, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of hirudinine B.

8. A HPLC identification method for distinguishing leech medicinal materials and their closely related counterfeit products, characterized in that: The identification method comprises the following steps: (1) Establishing a standard characteristic spectrum of Chinese medicinal materials for leeches and their closely related mixed and counterfeit products according to the characteristic spectrum construction method described in any one of claims 1 to 7; (2) taking a leech sample to be tested, preparing a test solution of the leech sample to be tested according to the method for constructing a characteristic spectrum according to claim 1 or 2, and performing detection according to the chromatographic conditions in the method for constructing a characteristic spectrum according to any one of claims 1 to 7 to obtain a spectrum of the leech sample to be tested; as well as (3) Comparing the leech sample spectrum obtained in step (2) with the standard characteristic spectrum of the Chinese medicinal material leech and its closely related mixed counterfeit products obtained in step (1), and those that meet the requirements are leech medicinal materials, and those that do not meet the requirements are closely related mixed counterfeit products of leech medicinal materials.

9. The identification method according to claim 8, characterized in that: The compliance requirement includes one of the following: (1) When the detection wavelength is 245 nm, the chromatogram of the leech sample to be tested shows 7 characteristic chromatographic peaks, which are the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of SZ-1, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of leechline B; or (2) When the detection wavelength is 245 nm, the chromatogram of the leech sample to be tested shows 6 characteristic chromatographic peaks, namely, the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of hyoscyamine B; Preferably, the leech sample to be tested is a leech (Hirudo nipponica Whitman) or a leech (Whitmaniapigra Whitman) of the Hirudinidae family; Preferably, the closely related counterfeit products include Philippine leeches, bar-patterned leeches and / or Japanese leeches; More preferably, when the detection wavelength is 245 nm, the chromatogram of the leech sample presents 5 characteristic chromatographic peaks, which are the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of inosine, the chromatographic peak of mylobarbital B and the chromatographic peak of Poecilobdellasulfide B; More preferably, when the detection wavelength is 245 nm, the chromatogram of the leech sample presents 6 characteristic chromatographic peaks, which are the chromatographic peak of hirudinamine A, the chromatographic peak of hirudinamine B, the chromatographic peak of hirudinamine C, the chromatographic peak of inosine, the chromatographic peak of mylobarine B and the chromatographic peak of Poecilobdellasulfide B; More preferably, the chromatographic peak of the scutellarine B and the chromatographic peak of the poecilobdellasulfide B are identified by UPLC-QTOF-MS / MS technology; More preferably, when the detection wavelength is 245 nm, the Japanese leech sample spectrum shows one characteristic chromatographic peak, which is the chromatographic peak of inosine.

10. Use of the construction method according to any one of claims 1 to 7 or the identification method according to claim 8 or 9 in quality detection, quality evaluation or quality control of leech medicinal materials, or in distinguishing leeches and their closely related counterfeits.

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