Construction method of leech characteristic chromatogram

Abstract

The invention provides a construction method of a leech characteristic chromatogram, and belongs to the technical field of quality analysis and detection of traditional Chinese medicines. The method comprises the following steps: preparing a reference solution; preparing a test solution; and precisely sucking the reference solution and the test solution respectively, and injecting into a liquid chromatograph for detection. According to the method, the chemical component characteristics of the leech can be fully revealed, the characteristic peak information is rich and has high specificity, and the quality information of the leech is comprehensively reflected, so that the comprehensive and effective control on the quality of the leech is realized. According to the method, the problem that the leech traditional Chinese medicine preparation is difficult to identify due to lack of appearance characters is solved, and a solid basis is provided for quality control and evaluation of the leech and the leech product, especially the traditional Chinese medicine preparation.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality analysis and testing technology, and in particular to a method for constructing a leech characteristic spectrum. Background Technology

[0002] Characteristic maps of traditional Chinese medicine (TCM) have become an effective method for controlling the quality of raw TCM materials and compound TCM preparations in recent years, providing a broader perspective for TCM quality control research. TCM characteristic maps are holistic and quantifiable, highlighting the complete picture of the complex chemical components of TCM. Relying on these maps, a comprehensive evaluation of the intrinsic chemical components of TCM and overall quality control can be achieved, making the quality of TCM materials more controllable.

[0003] Leeches possess the properties of breaking up blood stasis and promoting menstruation, as well as removing blood stasis and eliminating masses. They are used for amenorrhea due to blood stasis, abdominal masses, hemiplegia due to stroke, and injuries from falls. Their main components are proteins and polypeptides, along with some small molecule compounds and trace elements. However, current research on leeches largely focuses on the content determination of single components such as proteins, nucleosides, or pteridines. Due to the diversity and complexity of effective components in traditional Chinese medicine, the determination of the content of a single component is insufficient to explain the intrinsic quality of the medicinal material and lacks specificity.

[0004] Chinese invention patent application No. 202211271229.X discloses a method of eluting with acetonitrile as mobile phase A and formic acid as mobile phase B to establish a characteristic spectrum. However, the spectrum in this prior art has poor peak shape and poor separation.

[0005] Therefore, it is very important to establish a method for constructing a leech feature map with better peak shape and separation. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a leech feature spectrum, in order to solve the technical problems of poor peak shape and resolution in the spectrum in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for constructing a leech feature map, comprising the following steps:

[0009] 1) Prepare reference solutions; Dissolve uracil reference standard, hypoxanthine reference standard, xanthine reference standard, and hirudin C reference standard in methanol aqueous solution to obtain standard solutions, and then dissolve each standard solution in methanol aqueous solution to obtain reference solutions;

[0010] 2) Preparation of test solution: Dissolve the test sample in methanol aqueous solution and perform ultrasonic extraction. After filtration, the test solution is obtained.

[0011] 3) Accurately pipette the reference solution and the test solution separately and inject them into the liquid chromatograph for detection.

[0012] In step 3), the chromatographic conditions are as follows: column: C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1 mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.5–1 mL / min; injection volume: 5–15 μL; column temperature: 20–40 °C; wavelength: 250–260 nm.

[0013] Furthermore, in step 3), the gradient elution procedure in the liquid chromatography detection is as follows:

[0014] 0 min: 2% methanol;

[0015] 0–18 min, excluding 0 min: 2–3% methanol;

[0016] 18–19 min, excluding 18 min: 3–10% methanol;

[0017] 19–29 min, excluding 19 min: 10–20% methanol;

[0018] 29–43 min, excluding 29 min: 20–35% methanol;

[0019] 43–48 min, excluding 43 min: 35–56% methanol;

[0020] 48–58 min, excluding 48 min: 56–62% methanol;

[0021] 58–62 min, excluding 58 min: 62% methanol.

[0022] Furthermore, the pH of the 0.1 mol / L ammonium formate solution is 3 to 5.

[0023] Furthermore, in step 1), the mass concentration of uracil reference standard in the reference solution is 40–60 μg / mL, the mass concentration of hypoxanthine reference standard is 40–60 μg / mL, the mass concentration of xanthine reference standard is 40–60 μg / mL, and the mass concentration of hirudin C reference standard is 8–10 μg / mL.

[0024] Furthermore, in steps 1) and 2), the mass fraction of the methanol aqueous solution is independently 25% to 75%.

[0025] Furthermore, in step 2), the mass-to-volume ratio of the test sample to the methanol-water solution is 0.5–2 g: 20–40 mL.

[0026] Furthermore, in step 2), the ultrasonic extraction time is 20–40 min.

[0027] The beneficial effects of this invention are:

[0028] The method of this invention can fully reveal the chemical composition characteristics of leeches, providing rich and highly specific characteristic peak information, comprehensively reflecting the quality information of leeches, and thus achieving comprehensive and effective control over leech quality. This invention solves the problem of difficulty in identifying leeches in traditional Chinese medicine preparations due to the lack of visual characteristics, providing a solid basis for the quality control and evaluation of leeches and their products, especially traditional Chinese medicine preparations. Attached Figure Description

[0029] Figure 1 The characteristic chromatogram of the test solution in Example 1;

[0030] Figure 2 The chromatogram for Example 2 shows the extraction solvent used as a 25% methanol aqueous solution;

[0031] Figure 3 The chromatogram for Example 3 shows the extraction solvent used as a 75% methanol aqueous solution;

[0032] Figure 4 The chromatogram of mobile phase B in Comparative Example 1 was obtained using acetonitrile.

[0033] Figure 5 The chromatogram of mobile phase A in Comparative Example 2 using pure water is shown.

[0034] Figure 6 The chromatogram of mobile phase A in Comparative Example 3 using 0.1% formic acid water is shown.

[0035] Figure 7 The chromatogram of mobile phase A in Comparative Example 4 using 0.1% acetic acid water is shown.

[0036] Figure 8 The chromatogram of mobile phase A in Comparative Example 5 using a 0.02 mol / L formic acid ammonium solution (adjusted to pH 4) is shown below.

[0037] Figure 9 The chromatogram of mobile phase A in Comparative Example 6 using a 0.005 mol / L formic acid ammonium solution (adjusted to pH 4);

[0038] Figure 10 The chromatogram of mobile phase A in Comparative Example 7 using 0.01 mol / L formic acid ammonium solution (without pH adjustment) is shown below.

[0039] Figure 11 The chromatogram for Comparative Example 8, where pure water was used as the extraction solvent;

[0040] Figure 12The chromatogram of ethanol was used as the extraction solvent for Comparative Example 9.

[0041] Figure 13 The chromatogram for Comparative Example 10 is obtained using reflux extraction. Detailed Implementation

[0042] This invention provides a method for constructing a leech feature map, comprising the following steps:

[0043] 1) Prepare reference solutions; Dissolve uracil reference standard, hypoxanthine reference standard, xanthine reference standard, and hirudin C reference standard in methanol aqueous solution to obtain standard solutions, and then dissolve each standard solution in methanol aqueous solution to obtain reference solutions;

[0044] 2) Preparation of test solution: Dissolve the test sample in methanol aqueous solution and perform ultrasonic extraction. After filtration, the test solution is obtained.

[0045] 3) Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph for detection.

[0046] In this invention, in step 3), the chromatographic conditions are as follows: column: C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1 mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.5–1 mL / min; injection volume: 5–15 μL; column temperature: 20–40 °C; wavelength: 250–260 nm.

[0047] Preferably, in step 3), the chromatographic conditions are as follows: column: C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1 mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.6–0.9 mL / min; injection volume: 7–12 μL; column temperature: 25–35 °C; wavelength: 252–258 nm.

[0048] Further preferred, in step 3), the chromatographic conditions are as follows: column: C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1 mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.8 mL / min; injection volume: 10 μL; column temperature: 30 ℃; wavelength: 254~256 nm.

[0049] In this invention, the gradient elution procedure in step 3) of liquid chromatography detection is as follows:

[0050] 0 min: 2% methanol;

[0051] 0–18 min, excluding 0 min: 2–3% methanol;

[0052] 18–19 min, excluding 18 min: 3–10% methanol;

[0053] 19–29 min, excluding 19 min: 10–20% methanol;

[0054] 29–43 min, excluding 29 min: 20–35% methanol;

[0055] 43–48 min, excluding 43 min: 35–56% methanol;

[0056] 48–58 min, excluding 48 min: 56–62% methanol;

[0057] 58–62 min, excluding 58 min: 62% methanol.

[0058] In this invention, the pH of the 0.1 mol / L ammonium formate solution is 3 to 5, preferably 3.5 to 4.5, and more preferably 4.

[0059] In this invention, in step 1), the mass concentration of uracil reference standard in the reference solution is 40–60 μg / mL, preferably 45–55 μg / mL, more preferably 50 μg / mL; the mass concentration of hypoxanthine reference standard is 40–60 μg / mL, preferably 45–55 μg / mL, more preferably 50 μg / mL; the mass concentration of xanthine reference standard is 40–60 μg / mL, preferably 45–55 μg / mL, more preferably 50 μg / mL; and the mass concentration of hirudin C reference standard is 8–10 μg / mL, preferably 8.2–9.5 μg / mL, more preferably 8.4–9 μg / mL.

[0060] In this invention, in steps 1) and 2), the mass fraction of the methanol aqueous solution is independently 25-75%, preferably 35-65%, and more preferably 40-60%.

[0061] In this invention, in step 2), the mass-to-volume ratio of the test sample to the methanol aqueous solution is 0.5-2g: 20-40mL, preferably 0.5-2g: 22-35mL, and more preferably 0.8-1.5g: 25-30mL.

[0062] In this invention, in step 2), the ultrasonic extraction time is 20-40 min, preferably 25-35 min, and more preferably 30 min.

[0063] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] The instruments used in this invention embodiment are: LC-20AT high performance liquid chromatograph (Shimadzu Corporation), KM-500DB ultrasonic cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.), AL204 0.01% electronic balance (Mettler-Toledo Instruments Co., Ltd.), XPE105 0.01% electronic balance (Mettler-Toledo Instruments Co., Ltd.), and DK-98-Ⅱ electric thermostatic water bath (Tianjin Test Instruments Co., Ltd.).

[0065] The reagents used in this invention are as follows: methanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number: 202310053), anhydrous ethanol (analytical grade, Hunan Huihong Reagent Co., Ltd., batch number: 202310062), methanol (chromatographic grade, American Tiandi Co., Ltd., batch number: 22065114), acetonitrile (chromatographic grade, American Tiandi Co., Ltd., batch number: 22035335), formic acid (chromatographic grade, Tianjin Kemio Chemical Reagent Co., Ltd., batch number: 20211228), acetic acid (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20220607), and purified water (China Resources Yibao Beverage Co., Ltd.). (China) Co., Ltd., Batch No.: 20220904), Ammonium Formate (Shanghai Maclean Biochemical Technology Co., Ltd.), Hirudin C (Chengdu Medsun Technology Co., Ltd., Purity: 99.86%, Batch No.: 1804964-28-0), Hypoxanthine (China National Institutes for Food and Drug Control, Purity: 99.4%, Batch No.: 140661-202005), Xanthine (China National Institutes for Drug Control, Purity: 100%, Batch No.: 140662-200802), Uracil (China National Institutes for Food and Drug Control, Purity: 99.60%, Batch No.: 100469-201302).

[0066] Example 1

[0067] Uracil reference standard, hypoxanthine reference standard, xanthine reference standard, and hirudin C reference standard were dissolved in 50% methanol aqueous solution to obtain standard solutions. Then, each standard solution was dissolved in 50% methanol aqueous solution to obtain reference solutions. In the reference solutions, the mass concentrations of uracil reference standard, hypoxanthine reference standard, xanthine reference standard, and hirudin C reference standard were 50 μg / mL, 50 μg / mL, and 8.4 μg / mL, respectively.

[0068] Add 1g of leech powder (batch number 20231009SZ010) to 25mL of 50% methanol, weigh it, extract it by ultrasound for 30min, make up the weight loss with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0069] Precisely pipette the reference solution and the test solution separately, inject them into the liquid chromatograph for detection;

[0070] The chromatographic conditions were as follows: column: Agilent ZORBAX SBAq C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1 mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.8 mL / min; injection volume: 10 μL; column temperature: 30 ℃; wavelength: 254 nm.

[0071] The gradient elution procedure for liquid chromatography detection is shown in Table 1:

[0072] Time (min) 0.01 mol / L ammonium formate (%) Methanol (%) 0 98 2 18 97 3 19 90 10 29 80 20 43 65 35 48 44 56 58 38 62 62 38 62

[0073] Reference standards were used to identify each chromatographic peak in the fingerprint spectrum. The preparation of the reference standard solution is described in the preparation of the reference solution. The peaks were injected separately according to the optimal chromatographic conditions and identified by the reference standard.

[0074] The fingerprint chromatogram of the test sample should show 9 characteristic peaks, which should correspond to the 4 characteristic peaks in the chromatogram of the reference sample. Test results are as follows: Figure 1 As shown.

[0075] Depend on Figure 1 It can be seen that the characteristic spectrum includes 9 common peaks, of which peak 1 is uracil, peak 3 (S1) is hypoxanthine, peak 4 is xanthine, and peak 6 is hirudin C.

[0076] Repeatability test: Take about 1g of sample (batch number 20231009SZ010), accurately weigh it, prepare 6 parallel samples, inject them separately, record the chromatograms and integrate them, and calculate the contents of uracil, hypoxanthine, xanthine and hirudin C as reference peaks; calculate the relative retention time and relative peak area with peak 3 (hypoxanthine) as reference peak. The results are shown in Tables 2 to 4.

[0077] Table 2. Content detection results of leech samples in repeatability tests.

[0078]

[0079]

[0080] Table 3. Results of Relative Retention Time Detection in Repeatability Tests of Leech Samples

[0081] Sample number 1 2 3 4 5 6 RSD (%) Peak 1 0.42 0.42 0.42 0.42 0.42 0.42 0.62 Peak 2 0.51 0.51 0.50 0.50 0.51 0.51 0.52 Peak 3 (S1) 1.00 1.00 1.00 1.00 1.00 1.00 0 Peak 4 1.07 1.07 1.07 1.07 1.07 1.07 0.03 Peak 5 1.20 1.19 1.19 1.19 1.19 1.19 0.15 Peak 6 1.87 1.86 1.85 1.85 1.86 1.87 0.62 Peak 7 2.23 2.23 2.20 2.21 2.22 2.24 0.59 Peak 8 2.55 2.51 2.50 2.50 2.52 2.54 0.77 Peak 9 2.71 2.69 2.66 2.69 2.69 2.71 0.76

[0082] Table 4. Results of relative peak area detection in repeatability tests of leech samples.

[0083] Sample number 1 2 3 4 5 6 RSD (%) Peak 1 0.34 0.34 0.34 0.35 0.34 0.34 1.27 Peak 2 0.15 0.15 0.15 0.15 0.15 0.15 0.59 Peak 3 (S1) 1.00 1.00 1.00 1.00 1.00 1.00 0 Peak 4 0.16 0.16 0.16 0.16 0.16 0.16 1.34 Peak 5 0.13 0.13 0.13 0.13 0.13 0.13 1.28 Peak 6 0.09 0.09 0.09 0.09 0.09 0.09 3.02 Peak 7 0.09 0.10 0.10 0.09 0.10 0.09 6.20 Peak 8 0.12 0.12 0.12 0.12 0.12 0.12 0.62 Peak 9 0.06 0.06 0.06 0.06 0.06 0.06 2.98

[0084] As shown in Tables 2-4, the content, relative retention time, and relative peak area RSD are all less than 5%, indicating that the method of the present invention has good repeatability.

[0085] Precision test: Take about 1g of sample (batch number 20231009SZ010), weigh accurately, process, inject continuously 6 times and record the chromatogram. Use uracil, hypoxanthine, xanthine and hirudin C as reference peaks and calculate the content respectively. Use peak 3 (hypoxanthine) as reference peak and calculate the relative retention time and relative peak area. The results are shown in Tables 5 to 7.

[0086] Table 5. Content detection results of leech samples in precision test.

[0087] Sample number Hypoxanthine (mg / g) Xanthine (mg / g) Uracil (mg / g) Hirudin C (mg / g) 1 0.865 1.231 0.311 0.102 2 0.869 1.232 0.310 0.104 3 0.870 1.219 0.306 0.102 4 0.869 1.166 0.305 0.105 5 0.873 1.248 0.310 0.103 6 0.878 1.254 0.308 0.103 RSD (%) 0.49 2.58 0.69 0.97

[0088] Table 6. Results of relative retention time detection in precision tests of leech samples.

[0089] Number of injections 1 2 3 4 5 6 RSD (%) Peak 1 0.43 0.43 0.43 0.43 0.43 0.43 0.32 Peak 2 0.52 0.52 0.52 0.52 0.52 0.52 0.32 Peak 3 (S1) 1.00 1.00 1.00 1.00 1.00 1.00 0 Peak 4 1.07 1.07 1.07 1.07 1.07 1.07 0.05 Peak 5 1.15 1.15 1.15 1.15 1.15 1.15 0.08 Peak 6 1.96 1.95 1.96 1.94 1.95 1.96 0.44 Peak 7 2.38 2.36 2.38 2.35 2.36 2.37 0.48 Peak 8 2.64 2.62 2.63 2.60 2.62 2.63 0.50 Peak 9 2.85 2.82 2.84 2.81 2.83 2.84 0.50

[0090] Table 7. Detection results of relative peak area in precision tests of leech samples.

[0091]

[0092]

[0093] As shown in Tables 5-7, the RSD values ​​of content, relative retention time, and relative peak area are less than 5%, indicating that the method of the present invention has good precision.

[0094] Stability test: Take about 1g of sample (batch number 20231009SZ010), accurately weigh it, process it, and inject it at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, and 24h respectively. Record the chromatogram and integrate it. Use uracil, hypoxanthine, xanthine, and hirudin C as reference peaks and calculate their contents respectively. Use peak 3 (hypoxanthine) as reference peak and calculate the relative retention time and relative peak area. The results are shown in Tables 8-10.

[0095] Table 8. Content detection results of leech samples in stability test.

[0096] Injection time Hypoxanthine (mg / g) Xanthine (mg / g) Uracil (mg / g) Hirudin C (mg / g) 0h 0.865 1.231 0.311 0.102 2h 0.869 1.232 0.310 0.104 4h 0.870 1.219 0.306 0.102 6h 0.869 1.166 0.305 0.105 8h 0.873 1.248 0.310 0.103 12h 0.878 1.254 0.308 0.103 24h 0.873 1.305 0.277 0.114 RSD (%) 0.46 3.37 3.98 4.09

[0097] Table 9. Results of relative retention time in stability tests of leech samples.

[0098]

[0099]

[0100] Table 10. Results of relative peak area detection in stability tests of leech samples.

[0101] Injection time 0h 2h 4h 6h 8h 12h 24h RSD (%) Peak 1 0.39 0.39 0.38 0.38 0.38 0.38 0.34 4.12 Peak 2 0.18 0.18 0.18 0.18 0.18 0.18 0.16 3.96 Peak 3 (S1) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0 Peak 4 0.14 0.14 0.14 0.14 0.14 0.14 0.15 2.13 Peak 5 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.46 Peak 6 0.10 0.10 0.10 0.10 0.10 0.10 0.11 3.99 Peak 7 0.12 0.12 0.12 0.12 0.12 0.12 0.10 0.86 Peak 8 0.13 0.14 0.14 0.14 0.14 0.14 0.14 2.74 Peak 9 0.07 0.07 0.07 0.07 0.07 0.07 0.05 2.03

[0102] As shown in Tables 8-10, the content, relative retention time, and relative peak area RSD are less than 10%. The test results show that the stability of the test solution is good within 24 hours.

[0103] Examples 2-3

[0104] Compared with Example 1, the difference lies in the extraction solvent used in Examples 2 and 3, which were 25% methanol aqueous solution and 75% methanol aqueous solution, respectively. The test results are as follows: Figures 2-3 As shown.

[0105] Comparative Example 1

[0106] The difference between this example and Example 1 is that the mobile phase B in Comparative Example 1 is acetonitrile.

[0107] Tests were performed on Example 1 and Comparative Example 1, and the test results are as follows: Figure 4 As shown, the results indicate that, compared to acetonitrile, methanol provides better separation when the organic phase in the mobile phase is methanol. Therefore, methanol was selected as the mobile phase B in this invention.

[0108] Comparative Examples 2-4

[0109] Compared with Example 1, the difference is that in Comparative Examples 2 to 4, mobile phase A was pure water, 0.1% formic acid solution, and 0.1% acetic acid solution, respectively.

[0110] Examples 1 and Comparative Examples 2-4 were tested, and the results are as follows: Figures 5-7 As shown, the results indicate that the 0.01 mol / L formic acid ammonium solution has a better peak shape, while the fingerprint spectra of 0.1% formic acid water, 0.1% acetic acid water, and pure water have poor peak shapes and poor separation. Therefore, this invention selects 0.01 mol / L formic acid ammonium solution as mobile phase A.

[0111] Comparative Examples 5-7

[0112] Compared with Example 1, the difference is that in Comparative Examples 5 to 7, mobile phase A was 0.02 mol / L formic acid ammonium solution (adjusted to pH 4), 0.005 mol / L formic acid ammonium solution (adjusted to pH 4), and 0.01 mol / L formic acid ammonium solution (without pH adjustment), respectively.

[0113] Examples 1 and Comparative Examples 5-7 were tested, and the test results are as follows: Figures 8-10As shown, the results indicate that the fingerprint peaks of 0.005 mol / L formate solution (adjusted to pH 4) and 0.01 mol / L formate solution (without pH adjustment) are poor and the separation is not good. Therefore, this invention selects 0.01 mol / L formate solution (adjusted to pH 4) as mobile phase A.

[0114] Comparative Examples 8-9

[0115] Compared with Example 1, the difference is that the extraction solvents in Comparative Examples 8-9 are pure water and ethanol, respectively.

[0116] Examples 1-3 and Comparative Examples 8-9 were tested, and the test results are as follows: Figures 11-12 As shown, the results indicate that pure water and ethanol have low extraction efficiency and fewer chromatographic peaks; the peak area is larger when the solvent is 50% methanol. Therefore, this invention selects 50% ethanol as the extraction solvent.

[0117] Comparative Example 10

[0118] The difference between this example and Example 1 is that reflux extraction was used in Comparative Example 10.

[0119] Tests were performed on Example 1 and Comparative Example 10, and the results are as follows: Figure 13 As shown, the results indicate that when the retention time for reflux and ultrasonic extraction is 17.6 min, the peak areas are 2027239 and 2197922, respectively. Ultrasonic extraction has better efficiency and the peak shape is better than that of reflux. Therefore, ultrasonic extraction is selected as the extraction method in this invention.

[0120] The HPLC characteristic chromatograms were evaluated as follows: 15 batches of leech samples were treated as test samples to prepare test solutions. The batch numbers of the 15 batches of leech samples are shown in Table 11. The samples were injected according to the chromatographic conditions under the optimal chromatographic conditions section, and the HPLC characteristic chromatograms of the 15 batches of leech samples were recorded. The HPLC characteristic chromatograms of the 15 batches of leech samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2010 version)". Using S1 as a reference, the median method was used to generate a control chromatogram. The fingerprint similarity results are shown in Table 12.

[0121] Table 11 Batch numbers of 15 leech samples

[0122] Serial Number Origin batch number 1 Hubei Tianmen-1 20231009SZ001 2 Hubei Tianmen-2 20231009SZ002 3 Qianjiang, Hubei 20231009SZ003 4 Shandong Weifang-1 20231009SZ004 5 Shandong Weifang-2 20231009SZ005 6 Linyi, Shandong 20231009SZ006 7 Jining, Shandong 20231009SZ007 8 Suzhou, Jiangsu -1 20231009SZ008 9 Suzhou, Jiangsu -2 20231009SZ009 10 Suzhou, Jiangsu -3 20231009SZ010 11 Suzhou, Jiangsu -4 20231009SZ011 12 Suzhou, Jiangsu -5 20231009SZ012 13 Suzhou, Jiangsu - 6 20231009SZ013 14 Suzhou, Jiangsu -7 20231009SZ014 15 Qingpu District, Shanghai 20231009SZ015

[0123] Table 12 Results of fingerprint similarity test

[0124]

[0125]

[0126]

[0127] As can be seen from Table 12, the similarity of the 15 batches of leech samples is above 0.9.

[0128] As shown in the above embodiments, this invention provides a method for constructing a characteristic spectrum of leeches, comprising the following steps: preparing a reference solution; preparing a test solution; and accurately injecting the reference solution and the test solution into a liquid chromatograph for detection. The method of this invention can fully reveal the chemical composition characteristics of leeches, providing rich and highly specific characteristic peak information, comprehensively reflecting the quality information of leeches, thereby achieving comprehensive and effective control over leech quality. This invention solves the problem of difficulty in identifying leeches in traditional Chinese medicine preparations due to the lack of visual characteristics, providing a solid basis for the quality control and evaluation of leeches and their products, especially traditional Chinese medicine preparations.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic map of leeches, characterized in that, The method comprises the following steps: 1) preparing a reference solution; dissolving the uracil control sample, the hypoxanthine control sample, the xanthine control sample and the hirudin C control sample in methanol water solution respectively to obtain standard sample solutions, and then dissolving the standard sample solutions in methanol water solution to obtain the reference solution; 2) preparing a test sample solution; dissolving the test sample in methanol water solution for ultrasonic extraction, and then filtering to obtain the test sample solution; 3) precisely pipetting the reference solution and the test sample solution respectively, and then injecting into a liquid chromatograph for detection; In step 3), the chromatographic conditions are as follows: a C18 chromatographic column with a size of 250mm*4.6mm and a thickness of 5um; mobile phase A: 0.1mol / L ammonium formate solution; mobile phase B: methanol; flow rate: 0.5-1mL / min; injection volume: 5-15ul; column temperature: 20-40 DEG C; wavelength: 250-260nm.

2. The method according to claim 1, wherein, In step 3), the gradient elution program in the liquid chromatography detection is as follows: 0min: 2% methanol; 0-18min, excluding 0min: 2-3% methanol; 18-19min, excluding 18min: 3-10% methanol; 19-29min, excluding 19min: 10-20% methanol; 29-43min, excluding 29min: 20-35% methanol; 43-48min, excluding 43min: 35-56% methanol; 48-58min, excluding 48min: 56-62% methanol; 58-62min, excluding 58min: 62% methanol.

3. The method according to claim 1 or 2, wherein, The pH of the 0.1mol / L ammonium formate solution is 3-5.

4. The method according to claim 3, wherein, In step 1), in the reference solution, the mass concentration of the uracil control sample is 40-60ug / mL, the mass concentration of the hypoxanthine control sample is 40-60ug / mL, the mass concentration of the xanthine control sample is 40-60ug / mL, and the mass concentration of the hirudin C control sample is 8-10ug / mL.

5. The method according to claim 4, wherein, In steps 1) and 2), the mass fraction of the methanol water solution is independently 25-75%.

6. The method according to claim 5, wherein, In step 2), the mass / volume ratio of the test sample and the methanol water solution is 0.5-2g:20-40mL.

7. The method for constructing a leech feature map according to claim 6, characterized in that, In step 2), the ultrasonic extraction time is 20-40min.

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