HPLC method for distinguishing leeches and their close relatives and application thereof

Abstract

The application discloses a characteristic spectrum construction method of medicinal leech and its close relative adulterants. The construction method comprises the following steps: a characteristic spectrum of medicinal leech and its close relative adulterants is established by high performance liquid chromatography, including extracting and preparing test sample solutions from medicinal leech and its close relative adulterants respectively; a control sample solution is prepared, and the test sample solution and the control sample solution are detected by high performance liquid chromatography, so that corresponding characteristic spectra are obtained, and medicinal leech and its close relative adulterants can be quickly distinguished. The medicinal leech control sample is selected from medicinal leech or leech of the Hirudinidae family; the close relative adulterants of the medicinal leech are selected from Limnatis humilis, Haemadipsa rills or Haemadipsa japonica. The method is simple and convenient, has the advantages of simple operation, good separation degree, high precision, good repeatability and good stability, and can realize rapid and accurate distinction of medicinal leech, leech and its close relative adulterants by HPLC.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an HPLC method for distinguishing leeches from bloodworms and their closely related adulterants, and its application. Background Technology

[0002] The traditional Chinese medicine leech is derived from the dried bodies of leeches (Whitmania pigra Whitman), Hirudonipponica Whitman, or Whitmania acranulata Whitman), and is believed to have the effects of breaking up blood stasis, promoting menstruation, and eliminating masses. Modern pharmacological studies have shown that leeches have various effects, including anticoagulation, antithrombosis, anti-atherosclerosis, antiplatelet aggregation, antitumor, 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 contain other chemical components such as proteins, polypeptides, pteridines, glycolipids, carboxylic acid esters, and free amino acids. In recent years, the antiplatelet properties of organosulfur compounds have attracted increasing attention. Pteridine compounds isolated and identified from leeches have similar sulfur structures to antiplatelet drugs such as ticlopidine, clopidogrel, and ticagrelor, suggesting that these compounds are potential active substances.

[0003] There are over 680 species of leeches distributed worldwide, with over 100 species found in China. The legally recognized sources of leeches used in traditional Chinese medicine are only three: *Hirudo medicinalis*, *Leech scutellaria*, and *Leech scutellaria var. willowensis*. In recent years, the market demand for medicinal leeches has been increasing, with prices rising annually and supply falling short of demand, leading to the prevalence of adulterants. Major adulterants include *Poecilobdella manillensis* Lesson, *Whitmania laevis* Baird, *Poecilobdella javanica* Wahlberg, and *Mimobdella japonica* Blanchard, resulting in inconsistent quality of commercially available leech raw materials, processed slices, and preparations, seriously affecting clinical efficacy.

[0004] Although the legally recognized medicinal materials of leeches, blood leeches, and willow-leaf leeches can be morphologically distinguished from closely related adulterants such as burdock leeches, golden-thread leeches, bar-shaped burdock leeches, and Japanese leeches, the medicinal form of leeches in traditional Chinese medicine varies. Decoction pieces are dried leech segments, and leech preparations are mostly powders, making it difficult to observe morphological characteristics and determine the original material. The thin-layer chromatography identification method included in the Chinese Pharmacopoeia (2020 edition) is not yet effective in distinguishing leeches from blood leeches and their closely related adulterants. Furthermore, some reports use molecular identification, but this method involves micro-samples, which are easily contaminated and prone to errors, potentially leading to false positives or false negatives.

[0005] Characteristic chromatograms of traditional Chinese medicine (TCM) are a method for characterizing the chemical components of TCM using modern analytical techniques (such as chromatography, spectroscopy, and mass spectrometry). They can reflect a variety of characteristic chemical components in TCM, providing comprehensive and rich information, and are therefore commonly used in quality control research of TCM. This method can be used to identify the authenticity of TCM materials and preparations, ensuring the stability, safety, and efficacy of TCM. Previous patents and literature have reported the use of characteristic chromatograms / fingerprint chromatograms for quality control of leeches or bloodsucking medicinal materials. However, the established methods are complex, requiring high-quality detection instruments and chromatographic columns, and the sample preparation procedures are cumbersome, which is not conducive to the rapid and convenient popularization of detection methods. In addition, some reports have used non-specific indicator components such as xanthine, hypoxanthine, free amino acids, succinic acid, or sodium succinate as quality control indicators for leeches. These methods lack specificity and cannot accurately and comprehensively reflect the quality of the medicinal materials. In recent years, important indicator components in leeches, such as hirudin and nucleoside components, have received increasing attention. Although there are reports on the determination of the content of related components and characteristic chromatograms, the lack of comparative studies on commonly found closely related adulterants in the market results in limited identification effectiveness of the methods. Furthermore, the established methods require high-quality instruments and equipment and lack universality.

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

[0007] Based on this, the present invention provides a method for constructing characteristic spectra of leech medicinal materials and their closely related adulterants, the method comprising the following steps:

[0008] Preparation of leech reference solution: Weigh an appropriate amount of leech powder, sieve it, place it in a container, add solvent, weigh it, extract for a period of time, cool it, weigh it again, replenish the lost weight with solvent, shake well, centrifuge, and take the supernatant to obtain the leech reference solution; wherein, the leech reference material is selected from Hirudonipponica Whitman or Whitmania pigra Whitman of the Hirudinaceae family;

[0009] Preparation of the reference medicinal material solution of closely related adulterants: Weigh an appropriate amount of powder of the reference medicinal material of closely related adulterants, sieve it, place it in a container, add solvent, weigh it, extract for a period of time, cool it, weigh it again, use solvent to replenish the lost weight, shake it well, centrifuge it, and take the supernatant to obtain the reference medicinal material solution of closely related adulterants; wherein, the reference medicinal material of closely related adulterants is selected from Hirudo medicinalis or Hirudo barbata, or Hirudo nipponiae of the Hirudo family;

[0010] Preparation of reference solution: Weigh appropriate amounts of hirudin A, hirudin B, hirudin C, SZ-1 and / or inosine reference standards, add solvent to prepare a reference solution with a concentration of hirudin A, hirudin B, hirudin C, SZ-1 and / or inosine of 1 to 100 μg / mL, for example 20 to 50 μg / mL;

[0011] Based on the results of high performance liquid chromatography detection of the test solution and the reference solution, standard characteristic chromatograms of leech reference medicinal material and its closely related adulterant reference medicinal material were obtained.

[0012] The chromatographic conditions for high performance liquid chromatography (HPLC) detection were as follows: using Kromasil C 18 The chromatographic column, mobile phase A selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B being acidic water, alkaline aqueous solution and / or buffer salt aqueous solution, gradient elution program: 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; flow rate 0.5-1.5 mL / min, column temperature 20-40℃, detection wavelength 200-300 nm, injection volume 5-15 μL.

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

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

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

[0016] Furthermore, the mass / volume (g / ml) ratio between the leech medicinal material and the solvent is 0.01 to 0.5, for example, about 0.25, about 0.083, about 0.05 or about 0.022.

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

[0018] Furthermore, the extraction method is selected from one of the following: impregnation, percolation, ultrasonication, 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 duration of the ultrasound is 10–60 minutes, for example, about 30 minutes.

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

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

[0024] Furthermore, the concentration of hirudin 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 to 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 8–12 μL, for example, about 10 μL.

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

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

[0031] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more organic acids and their salts, weak bases and their salts of different concentrations.

[0032] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of 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.

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

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

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

[0036] Furthermore, the aqueous solution of the acid is an aqueous solution of approximately 0.05% trifluoroacetic acid.

[0037] Furthermore, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0038] Furthermore, the pH value of the buffer salt solution is no greater than 7.0.

[0039] Furthermore, at the detection wavelength of 245 nm, the characteristic spectrum of the leech includes 7 characteristic peaks: peak a is the chromatographic peak of hirudin A, peak b is the chromatographic peak of hirudin B, peak c is the chromatographic peak of hirudin 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 leech alkaloid B.

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

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

[0042] (1) Based on the above-mentioned method for constructing characteristic maps, establish the standard characteristic maps of Chinese medicinal materials, including leeches and their closely related adulterants;

[0043] (2) Take the leech sample to be tested, prepare the test solution of the leech sample according to the above-mentioned method for constructing the characteristic chromatogram, and detect it according to the chromatographic conditions in the above-mentioned method for constructing the characteristic chromatogram to obtain the chromatogram of the leech sample to be tested; and

[0044] (3) Compare the spectrum of the leech sample obtained in step (2) with the standard characteristic spectrum of the Chinese medicine leech and its closely related adulterants obtained in step (1). If the spectrum meets the requirements, it is a leech medicine; if it does not meet the requirements, it is a closely related adulterant of the leech medicine.

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

[0046] (1) At a detection wavelength of 245 nm, the chromatogram of the leech sample showed seven characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, hirudin C, SZ-1, inosine, an unknown chromatographic peak, and lepidocine B; or

[0047] (2) At the detection wavelength of 245 nm, the chromatogram of the leech sample to be tested showed 6 characteristic chromatographic peaks, namely the chromatographic peak of hirudin A, the chromatographic peak of hirudin B, the chromatographic peak of hirudin C, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of leech alkaloid B.

[0048] Furthermore, the leech sample to be tested was either a leech (Hirudo nipponica Whitman) or a blood leech (Whitmaniapigra Whitman).

[0049] Furthermore, the closely related adulterants include Hirudo nipponia, Hirudo barbatula, and / or Hirudo spp.

[0050] Furthermore, at a detection wavelength of 245 nm, the chromatogram of the leech sample showed five characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, inosine, salicylic acid B, and Poecilobdellasulfide B.

[0051] Furthermore, at a detection wavelength of 245 nm, the chromatogram of the *Hirudo medicinalis* sample showed six characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, hirudin C, inosine, salicylic acid B, and Poecilobdellasulfide B.

[0052] Furthermore, the chromatographic peaks of leech alkaloid B and Poecilobdellasulfide B were identified using UPLC-QTOF-MS / MS technology.

[0053] Furthermore, at a detection wavelength of 245 nm, the chromatogram of the Japanese leech sample showed a characteristic chromatographic peak, which was the chromatographic peak of inosine.

[0054] According to another aspect of the present invention, the above-described construction method or identification method is provided for use in the quality detection, quality evaluation or quality control of leech medicinal materials, or for use in distinguishing leeches from blood leeches and their closely related adulterants.

[0055] The beneficial effects of this invention are:

[0056] The method of the present invention characterized eight chromatographic peaks, and the baseline of HPLC detection was stable and the target analyte separation was good, thereby achieving rapid and accurate differentiation of leeches and blood leeches and their closely related adulterants using HPLC. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0058] Figure 1 Chromatograms of different leech species under HPLC analysis method 1 are shown. Wherein, b: hirudin B; c: hirudin C.

[0059] Figure 2 Chromatograms of different leech species under HPLC analysis method 2 are shown. Where a: hirudin A; b: hirudin B; c: hirudin C.

[0060] Figure 3 Chromatograms of different leech species under HPLC analysis method 3 are shown. Where a: hirudin A; b: hirudin B; c: hirudin C.

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

[0062] Figure 5 The chromatograms of leech samples under HPLC analysis conditions 6-9 are shown. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; e: inosine.

[0063] Figure 6 The image shows the chromatogram of a leech sample analyzed by HPLC under conditions 10-12. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; e: inosine.

[0064] Figure 7 The chromatograms of leech samples under HPLC analysis conditions 13 and 14 are shown. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; e: inosine.

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

[0066] Figure 9 The image shows the chromatogram of a leech sample under HPLC analysis conditions 17-21. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; e: inosine.

[0067] Figure 10 The chromatograms of reference standard 1 under different acid solutions (0.05% formic acid solution, 0.05% acetic acid solution, and 0.05% trifluoroacetic acid solution) under HPLC analysis method 20 are shown. Wherein, a: hirudin A; b: hirudin B; c: hirudin C.

[0068] Figure 11Chromatograms of leech samples under different acidic solutions (0.05% formic acid solution, 0.05% acetic acid solution, and 0.05% trifluoroacetic acid solution) under HPLC analysis method 20. Wherein, a: hirudin A; b: hirudin B; c: hirudin C.

[0069] Figure 12 The HPLC chromatograms are for 12 batches of leech samples. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; d: SZ-1; e: inosine; f: unknown; g: leech alkaloid B.

[0070] Figure 13 The HPLC chromatograms are for 11 batches of leech samples. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; e: inosine; f: unknown; g: leech alkaloid B.

[0071] Figure 14 The HPLC chromatogram of *Hirudo medicinalis* sample is shown below. a: hirudin A; b: hirudin B; c: hirudin C; e: inosine; g: salicylic acid B; h: Poecilobdellasulfide B.

[0072] Figure 15 The image shows the HPLC chromatogram of a *Hirudo medicinalis* sample. a: hirudin A; b: hirudin B; e: inosine; g: salicylic acid B; h: Poecilobdellasulfide B.

[0073] Figure 16 The images show the HPLC chromatograms of two batches of Japanese hirudin samples. In this chromatogram, e represents inosine.

[0074] Figure 17 This is a high-resolution mass spectrometry (UPLC) chromatogram of leeches. The top image is the UPLC chromatogram, and the bottom image is the base peak ion spectrum in positive ion mode. g: leech alkaloid B.

[0075] Figure 18 This is a high-resolution mass spectrometry (UPLC) chromatogram of leeches. The top image is the UPLC chromatogram, and the bottom image is the base peak ion spectrum in positive ion mode. g: leech alkaloid B.

[0076] Figure 19 This is a high-resolution mass spectrometry (HPLC) chromatogram of *Hirudo medicinalis*. The top image is the UPLC chromatogram, and the bottom image is the base peak ion chromatogram in positive ion mode, h: *Poecilobdellasulfide* B.

[0077] Figure 20 This is a high-resolution mass spectrometry (HPLC) chromatogram of *Poecilobdella sulfide*. The top image is the UPLC chromatogram, and the bottom image is the base peak ion spectrum in positive ion mode, h: *Poecilobdella sulfide* B.

[0078] Figure 21This is a high-resolution mass spectrometry (HPLC) chromatogram of *Hirudo medicinalis* from Japan. The top image is the UPLC chromatogram, and the bottom image is the base peak ion chromatogram in positive ion mode.

[0079] Figure 22 This is the mass spectrum of Poecilobdellasulfide B in leeches under positive ion mode, as described in the literature (Song Wanli. Study on non-peptide chemical components of Hirudo medicinalis [D]. Shenzhen University, 2018).

[0080] Figure 23 This is the mass spectrum of peak h (Poecilobdellasulfide B, retention time 13.72 min) in the base ion spectrum of *Hirudo medicinalis* in positive ion mode. The top image is the secondary mass spectrum, and the bottom image is the primary mass spectrum.

[0081] Figure 24 This is the mass spectrum of peak h (Poecilobdellasulfide B, retention time 13.71 min) in the base ion spectrum of *Hirudo medicinalis* in positive ion mode. The top image is the secondary mass spectrum, and the bottom image is the primary mass spectrum.

[0082] Figure 25 This is the mass spectrum of leech alkaloid B in positive ion mode from the literature (Li Tao. Study on the chemical composition of broad-bodied golden thread leech [D]. Jinan University, 2013.).

[0083] Figure 26 This is the mass spectrum of peak g (estropine B, retention time 20.44 min) in the ion spectrum of leeches in positive ion mode. The upper spectrum is the secondary mass spectrum, and the lower spectrum is the primary mass spectrum.

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

[0085] Figure 28 To extract the quasi-molecular ion peak [M+H] of leech alkaloid B from the base peak ion diagram of *Hirudo medicinalis* in positive ion mode. + The mass spectrum of (355.01). Wherein, g: lepidoline B.

[0086] Figure 29 To extract the quasi-molecular ion peak [M+H] of leech alkaloid B from the base peak ion diagram of *Hirudo medicinalis* in positive ion mode. + The mass spectrum of chromatographic peak g (epimethine B, retention time 20.43 min) in the mass spectrum of (355.01) is shown in the figure. The upper figure is the secondary mass spectrum and the lower figure is the primary mass spectrum.

[0087] Figure 30 To extract the quasi-molecular ion peak [M+H] of leech alkaloid B from the base peak ion diagram of *Hirudo medicinalis* in positive ion mode. + The mass spectrum of (355.01). Wherein, g: lepidoline B.

[0088] Figure 31 To extract the quasi-molecular ion peak [M+H] of leech alkaloid B from the base peak ion diagram of *Hirudo medicinalis* in positive ion mode. + The mass spectrum of chromatographic peak g (epimethine B, retention time 20.41 min) in the mass spectrum of (355.01) is shown in the figure. The upper figure is the secondary mass spectrum and the lower figure is the primary mass spectrum.

[0089] Figure 32 HPLC chromatograms of different leech medicinal materials and adulterants are shown. Specifically: leech: HPLC chromatogram of sample 9; leech vesicle: HPLC chromatogram of sample 15; *Hirudo medicinalis*: HPLC chromatogram of sample 24; *Hirudo medicinalis*: HPLC chromatogram of sample 25; *Hirudo nipponia*: HPLC chromatogram of sample 26. Wherein, a: hirudin A; b: hirudin B; c: hirudin C; d: SZ-1; e: inosine; f: unknown; g: leech alkaloid B. Detailed Implementation

[0090] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0092] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0093] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0094] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0095] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0096] As described in the background section, there is an urgent need in the art for a rapid, stable, and well-separated HPLC identification method. To address this problem, the present invention provides a method for constructing characteristic chromatograms of leech medicinal materials and their closely related adulterants, the method comprising the following steps:

[0097] Preparation of leech reference solution: Weigh an appropriate amount of leech powder, sieve it, place it in a container, add solvent, weigh it, extract for a period of time, cool it, weigh it again, replenish the lost weight with solvent, shake well, centrifuge, and take the supernatant to obtain the leech reference solution; wherein, the leech reference material is selected from Hirudonipponica Whitman or Whitmania pigra Whitman of the Hirudinaceae family;

[0098] Preparation of the reference medicinal material solution of closely related adulterants: Weigh an appropriate amount of powder of the reference medicinal material of closely related adulterants, sieve it, place it in a container, add solvent, weigh it, extract for a period of time, cool it, weigh it again, use solvent to replenish the lost weight, shake it well, centrifuge it, and take the supernatant to obtain the reference medicinal material solution of closely related adulterants; wherein, the reference medicinal material of closely related adulterants is selected from Hirudo medicinalis or Hirudo barbata, or Hirudo nipponiae of the Hirudo family;

[0099] Preparation of reference solution: Weigh appropriate amounts of hirudin A, hirudin B, hirudin C, SZ-1 and / or inosine reference standards, add solvent to prepare a reference solution with a concentration of hirudin A, hirudin B, hirudin C, SZ-1 and / or inosine of 1 to 100 μg / mL, for example 20 to 50 μg / mL;

[0100] Based on the results of high performance liquid chromatography detection of the test solution and the reference solution, standard characteristic chromatograms of leech reference medicinal material and its closely related adulterant reference medicinal material were obtained.

[0101] The chromatographic conditions for high performance liquid chromatography (HPLC) detection were as follows: using Kromasil C 18 The chromatographic column, mobile phase A selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B being acidic water, alkaline aqueous solution and / or buffer salt aqueous solution, gradient elution program: 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; flow rate 0.5-1.5 mL / min, column temperature 20-40℃, detection wavelength 200-300 nm, injection volume 5-15 μL.

[0102] In this 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 a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “20–40” is disclosed, the described range should be interpreted as including ranges “20–40”, “20–35”, “20–30”, “20–25”, “25–40”, “25–35”, “25–30”, “30–40”, “30–35”, “35–40”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

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

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

[0105] Compared to other solvents, methanol is more effective at extracting components from leeches and bloodsuckers.

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

[0107] Compared to other concentrations of methanol, approximately 50% methanol is more effective at extracting organic matter from leeches and bloodsuckers.

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

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

[0110] In this 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 one of the following: impregnation, percolation, ultrasonication, or reflux.

[0113] Compared to other extraction methods, ultrasound is more effective at extracting the organic matter contained in leeches and bloodsuckers.

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

[0115] In this 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 to 60 kHz, for example, about 40 kHz.

[0117] In this 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 ultrasound duration is 10 to 60 minutes, for example, about 30 minutes.

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

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

[0121] In this 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 hirudin B in the reference solution is 10–50 μg / ml, for example, about 31.2 μg / ml.

[0123] In this 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 hirudin C in the reference solution is 10–50 μg / ml, for example, about 30.6 μg / ml.

[0125] In this 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 to 1.2 ml / min, for example, about 1.0 ml / min.

[0127] Compared to other flow rates, a flow rate of approximately 1.0 ml / min results in a faster sample peak elution rate.

[0128] In this 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, for example, about 30°C.

[0130] Compared to other temperatures, a temperature of approximately 30°C significantly improves peak tailing in chromatography, resulting in better peak shape and separation between peaks.

[0131] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, 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 to 12 μl, for example, about 10 μl.

[0134] In this 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 chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

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

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

[0138] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous 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 acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from different concentrations of 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.

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

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

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

[0143] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.05% trifluoroacetic acid.

[0144] Compared to other organic acids at other concentrations, an aqueous solution of approximately 0.05% trifluoroacetic acid produces better peak shapes for the analyte and better separation from impurity peaks.

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

[0146] In a preferred embodiment, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0147] In a preferred embodiment, the pH value of the buffer salt solution is not 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 hirudin A, peak b is the chromatographic peak of hirudin B, peak c is the chromatographic peak of hirudin 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 leech alkaloid 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 hirudin A, peak b is the chromatographic peak of hirudin B, peak c is the chromatographic peak of hirudin C, peak e is the chromatographic peak of inosine, peak f is an unknown chromatographic peak, and peak g is the chromatographic peak of leech alkaloid B.

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

[0151] (1) Based on the above-mentioned method for constructing characteristic maps, establish the standard characteristic maps of Chinese medicinal materials, including leeches and their closely related adulterants;

[0152] (2) Take the leech sample to be tested, prepare the test solution of the leech sample according to the above-mentioned method for constructing the characteristic chromatogram, and detect it according to the chromatographic conditions in the above-mentioned method for constructing the characteristic chromatogram to obtain the chromatogram of the leech sample to be tested; and

[0153] (3) Compare the spectrum of the leech sample obtained in step (2) with the standard characteristic spectrum of the Chinese medicine leech and its closely related adulterants obtained in step (1). If the spectrum meets the requirements, it is a leech medicine; if it does not meet the requirements, it is a closely related adulterant of the leech medicine.

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

[0155] (1) At a detection wavelength of 245 nm, the chromatogram of the leech sample showed seven characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, hirudin C, SZ-1, inosine, an unknown chromatographic peak, and lepidocine B; or

[0156] (2) At the detection wavelength of 245 nm, the chromatogram of the leech sample to be tested showed 6 characteristic chromatographic peaks, namely the chromatographic peak of hirudin A, the chromatographic peak of hirudin B, the chromatographic peak of hirudin C, the chromatographic peak of inosine, the unknown chromatographic peak and the chromatographic peak of leech alkaloid B.

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

[0158] In a preferred embodiment, the closely related adulterants include Hirudo nipponia, Hirudo barbatula, and / or Hirudo spp.

[0159] In a preferred embodiment, when the detection wavelength is 245 nm, the chromatogram of the leech sample shows five characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, inosine, salicylic acid B, and Poecilobdellasulfide B.

[0160] In a preferred embodiment, when the detection wavelength is 245 nm, the chromatogram of the *Hirudo medicinalis* sample shows six characteristic chromatographic peaks, namely the chromatographic peaks of hirudin A, hirudin B, hirudin C, inosine, salicylic acid B, and Poecilobdellasulfide B.

[0161] In a preferred embodiment, the chromatographic peaks of leech alkaloid B and Poecilobdellasulfide B are identified using UPLC-QTOF-MS / MS technology.

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

[0163] According to another aspect of the present invention, the above-described construction method or identification method is provided for use in the quality detection, quality evaluation or quality control of leech medicinal materials, or for use in distinguishing leeches from blood leeches and their closely related adulterants.

[0164] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used without specified manufacturers are all commercially available conventional products, such as those purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0166] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0167] Example 1

[0168] The method of this invention is used to analyze different varieties of leeches to distinguish them from bloodsuckers and their closely related adulterants, as detailed below:

[0169] 1. Materials

[0170] 1.1 Experimental Apparatus

[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 ≥98%; Hirudin C (14713) was purchased from Shanghai Shidander Standard Technical Service Co., Ltd., with a purity ≥98%. Methanol and acetonitrile were purchased from Thermo Fisher Scientific (China) Co., Ltd., and Wahaha purified water was used. All other reagents were of analytical grade.

[0174] Twenty-seven batches of samples were collected from different regions of China, including 12 batches of leeches, 11 batches of blood leeches, 1 batch of bar-shaped leeches, 1 batch of horny leeches, and 2 batches of Japanese leeches. Specific information is as follows:

[0175] Sample 1: Leech, originating from Jining County, Jining City, Shandong Province;

[0176] Sample 2, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0177] Sample 3, leeches, originated from Bamen Town, Baodi District, Tianjin.

[0178] Sample 4, leeches, originated from Xihua County, Zhoukou City, Henan Province;

[0179] Sample 5 leeches originated from Jining County, Jining City, Shandong Province;

[0180] Sample 6 leeches originated from Sucheng District, Suqian City, Jiangsu Province;

[0181] Sample 7 leeches originated from De'an County, Jiujiang City, Jiangxi Province;

[0182] Sample 8, leeches, originated from Yuanjiang City, Yiyang City, Hunan Province;

[0183] Sample 9 leeches originated from Jining County, Jining City, Shandong Province;

[0184] Sample 10, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0185] Sample 11, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0186] Sample 12, leeches, originated from Bamen Town, Baodi District, Tianjin.

[0187] Sample 13: Leeches, originating from Shandong (purchased from Anguo Medicinal Materials Market);

[0188] Sample 14, leeches, originated from Jining County, Jining City, Shandong Province;

[0189] Sample 15: Leeches, originating from Sucheng District, Suqian City, Jiangsu Province;

[0190] Sample 16, leeches, originated from Bamen Town, Baodi District, Tianjin.

[0191] Sample 17, leeches, originated from Xihua County, Zhoukou City, Henan Province;

[0192] Sample 18, leeches, originated from Jining County, Jining City, Shandong Province;

[0193] Sample 19, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0194] Sample 20: Leeches originating from De'an County, Jiujiang City, Jiangxi Province;

[0195] Sample 21, leeches, originated from Yuanjiang City, Yiyang City, Hunan Province;

[0196] Sample 22, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0197] Sample 23, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0198] Sample 24, a barbed leech, originated from Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region;

[0199] Sample 25, *Hirudo medicinalis*, originated in Zhongshan City, Guangdong Province;

[0200] Sample 26, Japanese leech, originated from Xihua County, Zhoukou City, Henan Province;

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

[0202] 2.1 Preparation of the test solution

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

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

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

[0206] 2.2 Preparation of the reference solution

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

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

[0209] The structural formulas of hirudin A, hirudin B, hirudin C, SZ-1, inosine, leech alkaloid B, and Poecilobdellasulfide B are shown below:

[0210]

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

[0212] exist 1 In the 1H-NMR (DMSO-d6, 600MHz) spectrum, two signal peaks of active hydrogen are observed [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 oxymethylene group [δ] H 3.65 (1H, d, J = 5.7 Hz, 14-OH)], and two characteristic methyl signal peaks [δ H 3.26(3H,s,H-10), 3.15(3H,s,H-12)].

[0213] exist 13 The C-NMR (DMSO-d6, 150MHz) spectrum contained 12 carbon signals. Combined with HSQC spectral analysis, [δ C 161.9(C-2), δ C [153.7(C-4)] represents two ketone 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)] represents the signals of 6 unsaturated carbons. The signals of the two methine carbons and methylene carbons substituted with hydroxyl groups 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 information mentioned above is similar to that of compound (R)-hirudonucleodisulfide B in the literature, presumably a pteridine derivative. Their main difference is that the nitrogen atom at position 3 is replaced by a methyl group, and the sulfur atom at position 11 is a thionyl group. The chemical shifts of the two methyl groups at positions 3 and 11 [δ] are also shown. H The values ​​of 3.26(3H, s, H-10) and 3.15(3H, s, H-12) also indirectly confirm this.

[0214] 2D-NMR spectroscopy also provides some evidence for the structure of the compound. 1 H- 1 The cross signals H-13 / H2-14 in the H COSY spectrum indicate the presence of a C13-C14 (vicinal diol) structural fragment. The cross signals H-10 / C-2 / C-4, H-12 / C-6, H-13 / C-6, and H-14 / C-7 in the HMBC spectrum confirm the methyl substitution at N-3, the thionyl substitution at C-6, and the vicinal diol substitution at C-7, respectively.

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

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

[0217]

[0218]

[0219] 2.3 Preparation of the control herbal solution

[0220] Take approximately 0.5 g of leech powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the leech control solution.

[0221] Take approximately 0.5 g of leech powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the leech control solution.

[0222] Take approximately 0.5 g of Hirudo medicinal material sample powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the Hirudo medicinal material control solution.

[0223] Take approximately 0.5 g of Hirudo medicinal material sample powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the Hirudo medicinal material reference solution.

[0224] Take approximately 0.5 g of Japanese leech-like medicinal material sample powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the Japanese leech-like medicinal material solution.

[0225] 2.3 Investigation of HPLC chromatographic conditions

[0226] Reference solution 1 and representative samples (1, 14, 24, 25, 26) of different leech species were analyzed under different HPLC chromatographic conditions (as shown in Table 2). Method 1 is the preliminary method, and methods 2 to 21 are the 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 mobile phases of acidic water:

[0235] The test solution prepared by method 3 and the reference solution 1 were examined by method 20 for different acidic waters of the mobile phase, mainly 0.05% formic acid water, 0.05% acetic acid water and 0.05% trifluoroacetic acid water.

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

[0237] The test sample was prepared according to preparation method 1, and isocratic elution was performed under HPLC chromatographic conditions 1. No chromatographic peak of hirudin A was observed within the analytical time, and the peak response value was low. Detection results for different leech varieties are as follows: Figure 1 As shown. Therefore, using sample preparation method 2 and extending the analysis time (method 2), peaks of hirudin A, B, and C were all detected. However, hirudin C had a retention time that was too early (5 min), making it susceptible to the influence of solvent peaks. The detection results for different leech varieties are as follows. Figure 2 As shown. Therefore, gradient elution was adjusted, and method 3 was used for analysis. The response values ​​of hirudin A, B, and C were low, which was presumably due to an unsuitable detection wavelength. The detection results for different leech species are shown below. Figure 3 As shown.

[0238] Methods 4 and 5 both used gradient elution, and the detection wavelength was changed to the maximum UV absorption wavelength of the reference standard, 245 nm. The results showed that when the organic phase ratio was 5%, the chromatographic peaks of inosine and hirudin C were poorly separated, and there were few chromatographic peaks. The HPLC chromatogram is shown below. Figure 4 As shown. Therefore, the mobile phase A was changed to acetonitrile, and the extraction solvent of the test sample was changed, i.e., test sample preparation method 3 and chromatographic analysis method 6 were used for detection. The chromatographic peak of hirudin B was not effectively separated from the adjacent chromatographic peak. The HPLC results of leech liquid under different methods are shown below. Figure 5 As shown. Adjusting the organic phase ratio in the mobile phase, i.e., using method 7-9, still did not separate the chromatographic peak of hirudin B from the adjacent chromatographic peaks, and the inosine chromatographic peak was interfered with by the solvent peak, so mobile phase A needs to be replaced.

[0239] When mobile phase A was changed to methanol:acetonitrile (1:1), and analytical method 10-12 was used for detection, the leech amine B peak was still not effectively separated from adjacent peaks, and the inosine peak showed poor resolution. The HPLC results for leeches under different methods are as follows: Figure 6 As shown.

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

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

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

[0243] Investigation of different acidic mobile phases: This study analyzed the reference solution 1 and the test solution prepared by method 3 using method 20. Analysis of reference solution 1 with different acidic mobile phases revealed that when the mobile phase was 0.05% acetic acid, the peaks of leechamine C and leechamine B exhibited tailing, as shown in the following description. Figure 10 As shown. Analysis of the leech sample solution using different acid solutions revealed that when the mobile phase was 0.05% formic acid, the resolution between the leech amine B peak and adjacent peaks was less than 1.5; when the mobile phase was 0.05% acetic acid, the leech amine C and leech amine B peaks showed tailing; and when the mobile phase was 0.05% trifluoroacetic acid, the peak shape and resolution of the main chromatographic peaks were good, such as... Figure 11 As shown, the final determined acidic aqueous phase of the mobile phase was 0.05% trifluoroacetic acid water.

[0244] In summary, the optimal method for preparing the test sample is as follows: Take approximately 0.5 g of leech powder (passed through a No. 3 sieve), accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with 50% methanol, shake well, centrifuge, and collect the supernatant to obtain the test sample.

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

[0246] Example 2

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

[0248] 1. Sample preparation

[0249] Take approximately 0.5g of powdered leeches, blood leeches, bark leeches, leeches, and Japanese leeches (passed through a No. 3 sieve), accurately weigh them, and place them in a 50mL stoppered conical flask. Add 10mL of 50% methanol, seal tightly, weigh, and sonicate (power 250W, frequency 40kHz) for 30min. Let cool, weigh again, and replenish the lost weight with 50% methanol. Shake well, centrifuge, and collect the supernatant to obtain the final product.

[0250] 2. Characteristic Atlas of Leech Medicinal Materials and Their Closely Related Adulterants

[0251] The above sample solution was analyzed under optimal HPLC detection conditions to obtain the corresponding characteristic chromatograms:

[0252] 2.1 Detection of leech samples

[0253] Constructing the feature map of leeches, such as Figure 12 As shown, there are 7 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and these relative retention times should be within ±10% of the specified values. 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), and 7.750 (peak a).

[0254] 2.2 Detection of leech samples

[0255] Constructing the feature map of leeches, such as Figure 13 As shown, there are 6 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and these relative retention times should be within ±10% of the specified values. 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), and 7.757 (peak a).

[0256] 2.3 Detection of *Hirudo medicinalis* samples

[0257] Constructing the characteristic map of *Hirudo medicinalis* as follows: Figure 14As shown, there are a total of 6 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. 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), and 7.837 (peak a).

[0258] 2.4 Detection of Hirudo medicinalis samples

[0259] Constructing the feature map of Hirudo nipponia, as follows Figure 15 As shown, there are 5 characteristic peaks, among which the peak corresponding to the inosine reference is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.000 (peak e, S), 4.312 (peak b), 4.897 (peak h), 7.011 (peak g), and 7.955 (peak a).

[0260] 2.5 Detection of Japanese Hirudo medicinalis samples

[0261] Constructing the characteristic map of Japanese leeches, such as Figure 16 As shown, only one characteristic peak corresponds to the inosine reference peak, designated as the S peak. The relative retention times of the characteristic peak and the S peak are calculated, and these relative retention times 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 using reference standards.

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

[0265] 2. Identifying Feature Peaks Based on UPLC-QTOF-MS / MS

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

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

[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 Conical orifice backflush air flow rate 50 L·h -1 Using MS E Acquisition mode, collision voltage 25~50eV, scan range m / z 100~1200.

[0269] 2.3 Results Analysis

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

[0271] Table 3. Chromatographic peak identification of Poecilobdellasulfide B and leech alkaloid B

[0272]

[0273] Note: (a) leech; (b) blood leech; (c) leech; (d) bar-shaped leech; (e) Japanese leech.

[0274] 3. Identification results of 8 characteristic peaks

[0275] By comparing with reference standards and identifying peaks in UPLC-QTOF-MS / MS chromatographic data, eight characteristic peaks that can distinguish leech medicinal materials and their closely related adulterants were identified, as shown in Table 4.

[0276] Table 4. Characteristic components of different leech varieties

[0277]

[0278] In summary, this invention establishes a method for identifying the characteristic chromatograms of leeches and their closely related adulterants through HPLC analysis. Eight characteristic peaks were detected in the chromatograms, and this method was used to differentiate between leeches, blood leeches, bar-striped leeches, pheasant-like leeches, and Japanese leeches. See below:

[0279] Characteristic atlas of leech medicinal materials, as follows Figure 32 As shown, it has 7 characteristic peaks, namely, chromatographic peak a is the peak of hirudin A, chromatographic peak b is the peak of hirudin B, chromatographic peak c is the peak of hirudin C, chromatographic peak d is the peak of SZ-1, chromatographic peak e is the peak of inosine, chromatographic peak f is an unknown peak, and chromatographic peak g is the peak of salicyline B.

[0280] Characteristic atlas of leech medicinal materials, as shown below Figure 32 As shown, it has 6 characteristic peaks: chromatographic peak a is the peak of hirudin A, chromatographic peak b is the peak of hirudin B, chromatographic peak c is the peak of hirudin C, chromatographic peak e is the peak of inosine, chromatographic peak f is an unknown peak, and chromatographic peak g is the peak of lepidoamine B.

[0281] Characteristic atlas of the medicinal material *Hirudo medicinalis* (as shown in the image) Figure 32 As shown, it has 6 characteristic peaks: chromatographic peak a is the peak of hirudin A, chromatographic peak b is the peak of hirudin B, chromatographic peak c is the peak of hirudin C, chromatographic peak e is the peak of inosine, chromatographic peak g is the peak of leech alkaloid B, and chromatographic peak h is the peak of Poecilobdellasulfide B.

[0282] Characteristic atlas of Hirudo medicinal materials as follows: Figure 32 As shown, it has 5 characteristic peaks: chromatographic peak a is the peak of hirudin A, chromatographic peak b is the peak of hirudin B, chromatographic peak e is the peak of inosine, chromatographic peak g is the peak of leech alkaloid B, and chromatographic peak h is the peak of Poecilobdellasulfide B.

[0283] Characteristic atlas of Japanese Hirudo medicinal materials, such as Figure 32 As shown, it has one characteristic peak, namely chromatographic peak e, which is inosine.

[0284] In summary, the method of the present invention can quickly and accurately distinguish between leeches and blood leeches and their closely related adulterants.

[0285] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for constructing characteristic chromatograms of medicinal materials of Hirudo nipponica Whitman, Whitmania pigra Whitman, and medical leeches of LEECHES Hirudo, LEECHES Leptogaster and LEECHES Whitmania, characterized in that, The construction method comprises the following steps: Preparation of a control medicinal material solution of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae: a proper amount of medicinal material powder of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae is weighed, placed in a container, 47.5-52.5% methanol is added, the weight is determined, and after ultrasonic extraction for a period of time, the container is cooled, the weight is determined again, the lost weight is supplemented with solvent, and the solution is shaken, centrifuged, and the supernatant is taken to obtain the control medicinal material solution of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae; wherein the mass / volume ratio between the medicinal material of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae and the 47.5-52.5% methanol is 0.0475-0.0525, and the unit is g / ml; Preparation of a control medicinal material solution of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae: a proper amount of medicinal material powder of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae is weighed, placed in a container, 47.5-52.5% methanol is added, the weight is determined, and after ultrasonic extraction for a period of time, the container is cooled, the weight is determined again, the lost weight is supplemented with solvent, and the solution is shaken, centrifuged, and the supernatant is taken to obtain the control medicinal material solution of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae; wherein the mass / volume ratio between the medicinal material of Hirudo nipponica Whitman or Whitmania pigra Whitman of the Hirudinidae and the 47.5-52.5% methanol is 0.0475-0.0525, and the unit is g / ml; According to the results of the control medicinal material solution and the control solution detected by high performance liquid chromatography, a standard characteristic chromatogram of the control medicinal material of Hirudo nipponica Whitman, Whitmania pigra Whitman of the Hirudinidae, and the control medicinal material of Hirudo nipponica Whitman, Whitmania pigra Whitman of the Hirudinidae, and the control medicinal material of Hirudo nipponica Whitman, Whitmania pigra Whitman of the Hirudinidae is obtained; Chromatographic conditions for high performance liquid detection: using Kromasil C 18 Chromatographic column, mobile phase A is a mixture of methanol: acetonitrile = 4:1, mobile phase B is 0.0475%~0.0525% trifluoroacetic acid aqueous solution, 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; flow rate is 0.95~1.05 mL / min, column temperature is 28.5~31.5℃, detection wavelength is 245 nm, injection volume is 5~15 μL.

2. The construction method of claim 1, wherein, The container is an erlenmeyer flask.

3. The construction method of claim 2, wherein, The container is an erlenmeyer flask with a stopper.

4. The construction method of claim 1, wherein, The sieve is a No. 3 sieve.

5. The construction method of claim 1, wherein, The power of the ultrasonic extraction is 150-350 W.

6. The construction method of claim 5, wherein, The power of the ultrasonic extraction is 237.5-262.5 W.

7. The construction method of claim 1, wherein, The frequency of the ultrasonic extraction is 20-60 kHz.

8. The construction method of claim 7, wherein, The frequency of the ultrasonic extraction is 38-42 kHz.

9. The construction method of claim 1, wherein, The ultrasonic extraction time is 10-60 min.

10. The construction method of claim 9, wherein, The ultrasonic extraction time is 28.5-31.5 min.

11. The construction method of claim 1, wherein, The concentration of the control solution is 20-50 μg / mL.

12. The method of construction of claim 1, wherein, The concentration of the water leech amine A in the control solution is 10-50 μg / mL.

13. The construction method of claim 12, wherein, The concentration of hirudin A in the control solution is 30.21-33.39 μg / mL.

14. The method of construction of claim 1, wherein, The concentration of hirudin B in the control solution is 10-50 μg / mL.

15. The method of construction of claim 14, wherein, The concentration of hirudin B in the control solution is 29.64-32.76 μg / mL.

16. The method of construction of claim 1, wherein, The concentration of hirudin C in the control solution is 10-50 μg / mL.

17. The method of construction of claim 16, wherein, The concentration of hirudin C in the control solution is 29.07-32.13 μg / mL.

18. The method of construction of claim 1, wherein, The injection volume is 8-12 μL.

19. The method of construction of claim 18, wherein, The injection volume is 9.5-10.5 μL.

20. The method of construction of claim 1 wherein, The specification of the chromatographic column is: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

21. The method of construction of claim 1 wherein, The characteristic spectrum of the Hirudinidae Hirudo nipponica Whitman at the detection wavelength of 245 nm includes seven characteristic peaks, the a peak is the chromatographic peak of hirudin A, the b peak is the chromatographic peak of hirudin B, the c peak is the chromatographic peak of hirudin C, the d peak is the chromatographic peak of SZ-1, the e peak is the chromatographic peak of inosine, the f peak is an unknown chromatographic peak, and the g peak is the chromatographic peak of Whitmanin B.

22. The method of construction of claim 1, wherein, The characteristic spectrum of the Whitmania pigra Whitman at the detection wavelength of 245 nm includes six characteristic peaks, the a peak is the chromatographic peak of hirudin A, the b peak is the chromatographic peak of hirudin B, the c peak is the chromatographic peak of hirudin C, the e peak is the chromatographic peak of inosine, the f peak is an unknown chromatographic peak, and the g peak is the chromatographic peak of Whitmanin B.

23. An HPLC method for distinguishing between medicinal materials of Hirudo nipponica Whitman, medicinal materials of Whitmania pigra Whitman, medicinal materials of LEECHES of the family Hirudinidae, medicinal materials of LEECHES of the family Haemopidae, and medicinal materials of LEECHES of the family Erginidae, characterized in that, The method comprises the following steps: The method for establishing the characteristic spectrum according to any one of claims 1 to 22 establishes the standard characteristic spectrum of the medicinal material of the Hirudinidae Hirudo nipponica Whitman, the medicinal material of the Whitmania pigra Whitman, and the control medicinal material of the Haemopota spp., the Haemopis sowerbyi Moore, and the Sanguinaria japonica Whitman; wherein, at the detection wavelength of 245 nm, the characteristic spectrum of the Hirudinidae Hirudo nipponica Whitman presents seven characteristic chromatographic peaks, which are the chromatographic peak of hirudin A, the chromatographic peak of hirudin B, the chromatographic peak of hirudin C, the chromatographic peak of SZ-1, the chromatographic peak of inosine, an unknown chromatographic peak, and the chromatographic peak of Whitmanin B, respectively; wherein, at the detection wavelength of 245 nm, the characteristic spectrum of the Whitmania pigra Whitman presents six characteristic chromatographic peaks, which are the chromatographic peak of hirudin A, the chromatographic peak of hirudin B, the chromatographic peak of hirudin C, the chromatographic peak of inosine, an unknown chromatographic peak, and the chromatographic peak of Whitmanin B, respectively; wherein, at the detection wavelength of 245 nm, the characteristic spectrum of the Haemopis sowerbyi Moore presents five characteristic chromatographic peaks, which are the chromatographic peak of hirudin A, the chromatographic peak of hirudin B, the chromatographic peak of inosine, the chromatographic peak of Whitmanin B, and the chromatographic peak of Poecilobdellasulfide B, respectively; The sample chromatogram of the Haemadipsa ruffipes presents 6 characteristic chromatographic peaks at the detection wavelength of 245 nm, which are the chromatographic peak of haematin A, the chromatographic peak of haematin B, the chromatographic peak of haematin C, the chromatographic peak of inosine, the chromatographic peak of Whitmania pigra alkaloid B and the chromatographic peak of Poecilobdella sulfide B respectively. The sample chromatogram of the Whitmania pigra presents 1 characteristic chromatographic peak at the detection wavelength of 245 nm, which is the chromatographic peak of inosine.

24. The method of claim 23, wherein, The chromatographic peak of Whitmania pigra alkaloid B and the chromatographic peak of Poecilobdella sulfide B are identified by UPLC-QTOF-MS / MS technology.

25. Use of the construction method according to any one of claims 1 to 22 or the method according to claim 23 or 24 in quality detection or quality evaluation or quality control of Hirudo nipponica Whitman or Whitmania pigra Whitman, or in distinguishing Hirudo nipponica Whitman medicinal materials and Whitmania pigra Whitman medicinal materials, and in distinguishing Haemadipsa ruffipes, Haemadipsa japonica and Haemadipsa zebra.

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