A method for detecting a characteristic spectrum of a horsefly and its preparation and application thereof

Abstract

The application provides a kind of detection method and application of tabanus and its preparation characteristic map, method includes: A) the raw material of test sample is dissolved using solvent, extraction, obtain the liquid to be measured;B) the liquid to be measured is determined using high performance liquid chromatography, obtains tabanus and its preparation characteristic map;The chromatographic conditions of high performance liquid chromatography are as follows: column is C 18 column;Mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid solution, gradient elution.The application adopts high performance liquid chromatography, selects acetonitrile-0.05% acetic acid as mobile phase for gradient elution, with hypoxanthine as reference, establishes HPLC characteristic map of tabanus medicinal material, decoction piece, extract and its formula granule, reproducibility, precision is good, method is stable, reliable, can control the quality of tabanus medicinal material, decoction piece, extract and its formula granule.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for detecting the characteristic spectra of horseflies and their preparations, and its application. Background Technology

[0002] Tabanus is the dried body of the female adult of the following insects: *Arylotus bivittateinus* Takahasi, *Tabanus signatipennis* Portsch., *Tabanus yao Macquart.*, or *Tabanus trigeminus* Coquillett. It is listed in the *Chinese Medicinal Materials Standards* (1992 edition) and has the effects of dispelling blood stasis, breaking up accumulations, and promoting menstruation. It is used for abdominal masses, lower abdominal blood stasis, amenorrhea due to blood stasis, and blood stasis from injuries.

[0003] Currently, quality testing for horseflies is limited to their shape, and there is no comprehensive detection method that reflects and controls horseflies. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting the characteristic spectra of Tabanus and its preparations and its application. This method can be used for the quality detection of Tabanus medicinal materials, decoction pieces, extracts and formulation granules, and the results are reliable, providing a new method for the quality control of Tabanus.

[0005] This invention provides a method for detecting the characteristic spectra of horseflies and their preparations, comprising the following steps:

[0006] A) Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution;

[0007] B) The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatograms of the horsefly and its preparations;

[0008] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid solution, and gradient elution is used.

[0009] This invention involves dissolving and extracting the test sample raw material using a solvent to obtain the test solution. The test sample raw material is *Tanukiella* medicinal material, processed slices, extract, and its formulated granules. In this invention, the extraction solvent is 10% methanol; when 10% methanol is used as the extraction solvent, the chromatographic peak information content is larger, the peak shape is better, and the resolution is better.

[0010] In this invention, both the 10% methanol and 0.05% acetic acid solutions are volume fractions. Both the 10% methanol and 0.05% acetic acid solutions are aqueous solutions.

[0011] The extraction method is ultrasonic extraction; the ultrasonic extraction power is 580-620W, the ultrasonic extraction frequency is 35-45kHz, the ultrasonic extraction time is 20-40min, and the ratio of the mass g of the test sample raw material to the volume mL of the solvent is (0.5-1):(20-25).

[0012] Specifically, if the raw material for the test sample is horsefly medicinal material or processed medicinal slices, the preparation process of the test sample solution is as follows:

[0013] Take an appropriate amount of the powder (passed through a No. 3 sieve), about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0014] If the raw material for the test sample is horsefly formula granules or extract, the specific preparation process of the test sample solution is as follows:

[0015] Take an appropriate amount of this product, grind it into a fine powder, weigh about 0.5g accurately, place it in a stoppered conical flask, add 25ml of 10% methanol accurately, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0016] This invention uses high-performance liquid chromatography (HPLC) to determine the test solution and obtain characteristic chromatograms of the horsefly and its preparations; the flow rate of the mobile phase is 0.6-0.8 ml per minute; the column temperature is 20-30℃; in a specific embodiment, the flow rate of the mobile phase is 0.7 ml per minute; the column temperature is 25℃.

[0017] The detection wavelength is 240-260 nm; when the detection wavelength is 250 nm, the chromatographic peak information is greater and the chromatographic baseline is more stable. Therefore, the preferred detection wavelength of the present invention is 250 nm.

[0018] The theoretical plate number of this invention, calculated based on xanthine peaks, should be no less than 5000.

[0019] The injection volume of this invention is 5–10 μL; preferably 10 μL.

[0020] In this invention, the gradient elution specifically refers to:

[0021] 0–15 min, Phase A: 0%, Phase B: 100%;

[0022] 15–16 min, Phase A: 0–0.5%, Phase B: 100–99.5%;

[0023] 16–25 min, Phase A: 0.5%, Phase B: 99.5%;

[0024] 25–28 min, Phase A: 0.5–3%, Phase B: 99.5–97%;

[0025] 28–35 min, Phase A: 3%, Phase B: 97%.

[0026] The present invention also includes the preparation of a reference solution: uracil, guanine, adenine, and hypoxanthine are dissolved in methanol to obtain a reference solution; the concentration of the reference solution is 40 μg / mL;

[0027] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the characteristic chromatograms of the horsefly and its preparations were identified based on the chromatogram of the reference.

[0028] Specifically, the preparation process of the reference solution is as follows:

[0029] Take appropriate amounts of uracil, guanine, adenine, and hypoxanthine reference standards, weigh them accurately, and add 10% methanol to prepare a solution containing 40 μg of each per ml.

[0030] Take about 1.0g of the Tabanus reference material and place it in a stoppered conical flask. Add 25ml of 10% methanol, weigh it, sonicate (600W power, 40kHz frequency) for 30 minutes, cool it, weigh it again, replenish the lost weight with 10% methanol, shake well, filter it, and take the filtrate as the reference solution for the reference material.

[0031] The beneficial effects of this invention are that, under liquid chromatography conditions, the material groups of the horsefly medicinal material, decoction pieces, extracts and their formulation particles can be controlled by characteristic chromatograms, and hypoxanthine can be used to locate the characteristic chromatograms; this can greatly reduce the cost of detection and achieve qualitative detection.

[0032] This invention uses a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity between Tabanus and its preparations, obtaining a standard HPLC characteristic chromatogram of Tabanus medicinal material consisting of 8 characteristic peaks, where the peak corresponding to the hypoxanthine reference is the S peak; in the standard characteristic chromatogram, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.27 (peak 1), 0.54 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.91 (peak 5), 1.25 (peak 7), and 1.30 (peak 8).

[0033] This invention employs a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity between Tabanus and its preparations, obtaining a standard HPLC characteristic chromatogram of Tabanus slices and extracts consisting of eight characteristic peaks, where the peak corresponding to the hypoxanthine reference is the S peak. In the standard characteristic chromatogram, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.27 (peak 1), 0.54 (peak 2), 0.71 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.25 (peak 7), and 1.30 (peak 8).

[0034] This invention uses a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity between Tabanus and its preparations, obtaining a standard HPLC characteristic chromatogram of Tabanus formulation granules consisting of 8 characteristic peaks, where the peak corresponding to the hypoxanthine reference is the S peak; in the standard characteristic chromatogram, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.26 (peak 1), 0.53 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.26 (peak 7), and 1.30 (peak 8).

[0035] Quality judgment criteria: Take samples of Tabanus medicinal materials, processed slices, extracts and their formulation granules, and operate according to the same method as above to obtain the characteristic chromatograms of Tabanus medicinal materials, processed slices, extracts and their formulation granules. Use the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the standard characteristic chromatograms of Tabanus medicinal materials, processed slices, standard decoctions and their formulation granules and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0036] The method provided by this invention can effectively monitor the quality of different batches of horsefly medicinal materials, decoction pieces, extracts and their formulation granules, ensuring their quality stability. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0037] The characteristic chromatograms of the horsefly medicinal material, processed slices, extracts and their formulation granules established in this invention use rosmarinic acid as a reference, focusing on the order of each characteristic peak and its correlation with the medicinal material and intermediate products. This method can comprehensively evaluate the overall quality characteristics of the products and is scientific and reliable.

[0038] This invention provides a method for identifying horsefly medicinal materials, processed slices, extracts, and their formulation granules, characterized in that the method described in the above technical solution is used for detection, and the detection results are analyzed.

[0039] This invention provides a method for detecting the characteristic chromatograms of Tabanus and its preparations, comprising the following steps: A) dissolving and extracting the test sample raw material using a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain the characteristic chromatograms of Tabanus and its preparations; the HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid solution, and gradient elution is used. This invention employs HPLC, using acetonitrile-0.05% acetic acid as the mobile phase for gradient elution, and hypoxanthine as a reference, to establish HPLC characteristic chromatograms of Tabanus medicinal materials, processed slices, extracts, and their formulation granules. The method exhibits good repeatability and precision, is stable and reliable, and can be used to control the quality of Tabanus medicinal materials, processed slices, extracts, and their formulation granules. Attached Figure Description

[0040] Figure 1 This is an investigation into the method for extracting horsefly medicinal material in Example 1 of the present invention;

[0041] Figure 2 This refers to the chromatographic peak identification in Example 1 of the present invention;

[0042] Figure 3 Characteristic atlases of 22 batches of horsefly medicinal materials;

[0043] Figure 4 The image shows the characteristic spectrum of the medicinal material of Tabanus, in which peak 2 is uracil, peak 3 is adenine, peak 4 is guanine, and peak 6 (S) is hypoxanthine.

[0044] Figure 5 Characteristic atlases of 22 batches of horsefly slices;

[0045] Figure 6 The image shows the characteristic spectrum of Tabanus processed slices, where peak 2 is uracil, peak 3 is adenine, peak 4 is guanine, and peak 6 (S) is hypoxanthine.

[0046] Figure 7 This refers to the chromatographic peak identification in Example 3 of the present invention;

[0047] Figure 8 This is a superimposed image of the characteristics of horsefly extract;

[0048] Figure 9 The image shows the characteristic spectrum of the extract of Tabanus, where peak 2 is uracil, peak 3 is adenine, peak 4 is guanine, and peak 6 (S) is hypoxanthine.

[0049] Figure 10 Chromatograms of different wavelengths for the formulation granules of horsefly;

[0050] Figure 11 Column temperature study for Tabanus formulation granules;

[0051] Figure 12To investigate the flow rate of the formulation particles for horsefly;

[0052] Figure 13 Investigation of extraction solvents for Tabanus formulation granules;

[0053] Figure 14 An investigation into the extraction method of horsefly formulation granules;

[0054] Figure 15 The extraction time of the horsefly formula granules was investigated;

[0055] Figure 16 The amount of solvent added to the formulation granules for horsefly was investigated;

[0056] Figure 17 This is a specific overlay image;

[0057] Figure 18 These are the characteristic chromatograms for three batches of horsefly formulation granules for verification.

[0058] Figure 19 This is a characteristic spectrum of the formulation granules for horsefly control. Detailed Implementation

[0059] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for detecting the characteristic spectra of horseflies and their preparations provided by the present invention and its application, should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1: Detection of the characteristic spectrum of horsefly medicinal materials

[0061] The 2002 edition of the "Standards for Processing Traditional Chinese Medicine Slices in Sichuan Province" defines *Tabanus bivittateinus*, *Tabanus signatipennis* Portsch., *Tabanus yao Macquart.*, or *Tabanus trigeminus* Coquillett. as the dried bodies of female adult *Tabanus bivittateinus*, *Tabanus signatipennis* Portsch., *Tabanus yao Macquart.*, or *Tabanus trigeminus* Coquillett. as insects of the family Tabanidae. They are captured in summer and autumn, killed by boiling water or strung together with thread, and then dried. They are mainly produced in Shaanxi, Hebei, and Guizhou provinces.

[0062] To ensure the reliability of the established quality standards for horseflies, a total of 22 batches of horsefly medicinal materials from Shaanxi Province, Hebei Province, and Guizhou Province were collected. The specific origin information is shown in Table 1.

[0063] Table 1 Summary of Origin Information for the Medicinal Material *Protoceratops fasciatus*

[0064]

[0065]

[0066] 1.1 Instruments and Materials

[0067] High performance liquid chromatograph;

[0068] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0069] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0070] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0071] 1.2 Reagents and Test Chemicals

[0072] Acetonitrile and acetic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0073] Uracil (China National Institutes for Food and Drug Control, batch number: 100469-201302, content calculated as 99.6%)

[0074] Guanine (China National Institutes for Food and Drug Control, batch number: 140631-202008, content calculated as 98.9%)

[0075] Adenine (China National Institutes for Food and Drug Control, batch number: 110886-202203, content calculated as 99.8%)

[0076] Hypoxanthine (China National Institutes for Food and Drug Control, batch number: 140661-202005, content calculated as 99.4%)

[0077] Tabanus control material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 320074-202112).

[0078] 22 batches of horsefly medicinal materials (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: MC-01~MC-22)

[0079] 1.3 Feature Detection Methods

[0080] The method for characterizing the medicinal material *Tamarix chinensis* is as follows:

[0081] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.05% acetic acid solution as mobile phase B, with gradient elution as specified in Table 2; flow rate 0.7 mL / min; column temperature 25 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should be no less than 5000.

[0082] Table 2 Gradient elution program

[0083]

[0084] Preparation of the test solution: Take an appropriate amount of the powder (passed through a No. 3 sieve), about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0085] 1.4 Investigation of the preparation and extraction methods of the test sample

[0086] Take an appropriate amount of this product powder (batch number: MC-22), approximately 1.0 g, accurately weighed, and place it in a stoppered conical flask. When the extraction method is reflux and sonication (600 W power, 40 kHz frequency), add 25 ml of 10% methanol for 30 min. Cool, shake well, filter, and collect the filtrate. For water extraction, add 25 ml of water, boil for half an hour, filter, evaporate to dryness, add 25 ml of 10% methanol, sonicate (600 W power, 40 kHz frequency) for 30 min, cool, shake well, filter, and collect the filtrate. See [link to product description]. Figure 1 .

[0087] The results showed that there was essentially no difference between ultrasonic extraction and water extraction of the test samples, as indicated in the figure above, suggesting that ultrasonic treatment is more convenient. Therefore, ultrasonic extraction was selected as the extraction method for the horsefly medicinal material in this experiment.

[0088] 1.5 Methodological Examination

[0089] 1.5.1 Chromatographic Peak Identification

[0090] Preparation of the test solution: Prepare the test solution of the horsefly extract according to the experimental conditions proposed above.

[0091] Preparation of reference solution: Take appropriate amounts of uracil, guanine, adenine and hypoxanthine reference standards, weigh them accurately, and add 10% methanol to prepare a solution containing 40 μg of each per ml.

[0092] Take about 1.0g of the Tabanus reference material and place it in a stoppered conical flask. Add 25ml of 10% methanol, weigh it, sonicate (600W power, 40kHz frequency) for 30 minutes, cool it, weigh it again, replenish the lost weight with 10% methanol, shake well, filter it, and take the filtrate as the reference solution for the reference material.

[0093] Preparation of negative control solution: Prepare negative control solution of Tabanus molluscioides according to the experimental conditions proposed above.

[0094] Identification of characteristic spectral peaks in the medicinal material *Tamarix chinensis*. (See...) Figure 2 .

[0095] The results showed that the negative solution had no interference, and the method has good specificity.

[0096] 1.5.2 Precision Test

[0097] Take the test solution of horsefly slices and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 3.

[0098] Table 3 Precision Examination - Retention Time

[0099]

[0100] The results showed that the retention time RSD of each characteristic peak was 0.07%–0.41%. The instrument has good precision.

[0101] 1.5.3 Repeatability Test

[0102] Six portions of the horsefly medicinal material (batch number: MC-22) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 4.

[0103] Table 4 Repeatability Tests - Relative Retention Time Ratio

[0104]

[0105]

[0106] The results show that the relative retention RSD of each characteristic peak is 0%-0.57%. The method has good repeatability.

[0107] 1.5.4 Stability Assessment

[0108] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. See Table 5.

[0109] Table 5 Stability Study - Retention Time

[0110]

[0111] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.16% and 0.42%, and the sample solution was relatively stable within 24 hours.

[0112] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that this method is effective.

[0113] 1.5.5 Determination of characteristic peaks and establishment of reference spectra

[0114] 1.5.5.1 Establishment of Limits for Relative Retention Time

[0115] Table 6 summarizes the methodology, the examination items, and the verification results.

[0116] Table 6 Summary of RSD% for Methodological Results - Standard - Retention Time - Relative Retention Time

[0117]

[0118] The results show that the relative retention times of each characteristic peak are relatively stable, with the RSD range of peaks 1 to 8 being 0.00%–0.57%. The relative retention time of each peak is tentatively set at ±10%.

[0119] The final specification stipulates that the characteristic spectrum of the test sample should show 8 characteristic peaks, among which the peak corresponding to the reference peak of hypoxanthine is the S peak.

[0120] 1.5.5.1.2 Verification results of 22 batches of horsefly medicinal materials

[0121] Using this method, characteristic spectral analysis was performed on 22 batches of samples, and relative retention times were calculated. See Figure 3 (The batch numbers represented by each graph from bottom to top are S1:MC-01~MC-22, see Table 7.)

[0122] Table 7. Relative retention time of 22 batches of horsefly medicinal materials

[0123]

[0124] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the chromatogram of the test sample should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the hypoxanthine reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.54 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.91 (peak 5), 1.25 (peak 7), and 1.30 (peak 8).

[0125] The above samples were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference characteristic chromatogram of the horsefly medicinal material was established. (See attached image.) Figure 4 .

[0126] Example 2: Detection of the characteristic map of horsefly slices

[0127] 2.1 Experimental Materials

[0128] Tabanus slices (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: MC-230101; MC-230102; MC-230103; MC-230104; MC-230105; MC-230106; MC-230107; MC-230108; MC-230109; MC-230110; MC-230111; MC-23012; MC-23013; MC-23014; MC-23015; MC-23016; MC-230117; MC-230118; MC-230119; MC-230120; MC-230121; MC-230122).

[0129] 2.2 Chromatographic conditions

[0130] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.05% acetic acid solution as mobile phase B, with gradient elution as specified in Table 2 above; flow rate 0.7 mL / min; column temperature 25 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should be no less than 5000.

[0131] Preparation of the reference solution: Take approximately 1.0 g of Tabanus reference material and place it in a stoppered conical flask. Add 25 ml of 10% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of hypoxanthine reference standard, accurately weigh it, and add 10% methanol to prepare a solution containing 40 μg per ml, as the reference solution.

[0132] Preparation of the test solution: Take an appropriate amount of the powder (passed through a No. 3 sieve), about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0133] The determination method involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0134] 2.3 Verification results of 22 batches of horsefly slices

[0135] Using this method, characteristic spectral analysis was performed on 22 batches of samples, and relative retention times were calculated. See Figure 5 Table 8.

[0136] Table 8. Relative retention times of 22 batches of horsefly slices

[0137]

[0138]

[0139] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the test sample chromatogram should show eight characteristic peaks, corresponding to the retention times of the eight characteristic peaks in the reference herb chromatogram. The peak corresponding to the hypoxanthine reference standard peak is designated as 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 0.27 (peak 1), 0.54 (peak 2), 0.71 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.25 (peak 7), and 1.30 (peak 8).

[0140] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize the characteristic chromatograms of 22 batches of Tabanus larvae slices, and a reference chromatogram for the characteristic chromatograms of Tabanus larvae slices was established. (See attached image.) Figure 6 .

[0141] Example 3: Characteristic spectrum detection of horsefly extract

[0142] 3.1 Experimental Materials

[0143] Tabanus extract (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: MC-BT-230101; MC-BT-230102; MC-BT-230103; MC-BT-230104; MC-BT-230105; MC-BT-230106; MC-BT-230107; MC-BT-230108; MC-BT-230109; MC-BT-230110) ; MC-BT-230111; MC-BT-230112; MC-BT-230113; MC-BT-230114; MC-BT-230115; MC-BT-230116; MC-BT-230117; MC-BT-230118; MC-BT-230119; MC-BT-230120; MC-BT-230121; MC-BT-230122).

[0144] 3.2 Feature Methods

[0145] The characteristic methods for horsefly extract are as follows:

[0146] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.05% acetic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 2 above; the flow rate was 0.7 mL / min; the column temperature was 25 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should not be less than 5000.

[0147] Preparation of the test solution: Take an appropriate amount of this product, about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0148] 3.3 Methodological Examination

[0149] 3.3.1 Chromatographic Peak Identification

[0150] Preparation of the test solution: Prepare the test solution of the horsefly extract according to the experimental conditions proposed above.

[0151] Preparation of reference solution: Take appropriate amounts of uracil, guanine, adenine and hypoxanthine reference standards, weigh them accurately, and add 10% methanol to prepare a solution containing 40 μg of each per ml.

[0152] Take about 1.0g of the Tabanus reference material and place it in a stoppered conical flask. Add 25ml of 10% methanol, weigh it, sonicate (600W power, 40kHz frequency) for 30 minutes, cool it, weigh it again, replenish the lost weight with 10% methanol, shake well, filter it, and take the filtrate as the reference solution for the reference material.

[0153] Preparation of negative control solution: Prepare negative control solution of Tabanus extract according to the experimental conditions proposed above.

[0154] Identification of characteristic spectral peaks in the extract of the horsefly. See [link / reference needed]. Figure 7 The results showed that the negative solution did not cause interference, and the method has good specificity.

[0155] 3.3.2 Precision Test

[0156] Take the test solution of Tabanus extract (batch number: MC-BT-230101), inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 9.

[0157] Table 9 Precision Examination - Retention Time

[0158]

[0159]

[0160] The results showed that the retention time RSD of each characteristic peak was 0.06%–0.58%. The instrument has good precision.

[0161] 3.3.3 Repeatability Test

[0162] Six portions of Tabanus extract (batch number: MC-BT-230101) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 10.

[0163] Table 10 Repeatability Tests - Relative Retention Time

[0164]

[0165] The results show that the relative retention times of each characteristic peak are consistent, with an RSD of 0.00%–0.77%. This method exhibits good repeatability.

[0166] 3.3.4 Stability Assessment

[0167] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. See Table 11.

[0168] Table 11 Stability Study - Retention Time

[0169]

[0170]

[0171] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.16% and 0.36%, and the sample solution was relatively stable within 24 hours.

[0172] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that this method is effective.

[0173] 3.3.5 Determination of characteristic peaks and establishment of reference spectra

[0174] 3.3.5.1 Establishment of Limits for Relative Retention Time

[0175] Table 12 summarizes the methodology, the examination items, and the verification results.

[0176] Table 12 Summary of RSD% of Methodological Results - Standard - Retention Time - Relative Retention Time

[0177]

[0178] The results show that the relative retention times of each characteristic peak are relatively stable, with the RSD range of peaks 1 to 8 being 0.00%–0.77%. The relative retention time of each peak is tentatively set at ±10%.

[0179] The final specification stipulates that the characteristic spectrum of the test sample should show 8 characteristic peaks, among which the peak corresponding to the hypoxanthine reference is the S peak.

[0180] 3.3.6 Validation results of 22 batches of horsefly extract

[0181] Using this method, characteristic spectral analysis was performed on 22 batches of samples, and relative retention times were calculated. See Figure 8 (The batch numbers from bottom to top are: MC-BT-230101; MC-BT-230102; MC-BT-230103; MC-BT-230104; MC-BT-230105; MC-BT-230106; MC-BT-230107; MC-BT-230108; MC-BT-230109; MC-BT-230110; MC-BT-2301) 11; MC-BT-230112; MC-BT-230113; MC-BT-230114; MC-BT-230115; MC-BT-230116; MC-BT-2 30117; MC-BT-230118; MC-BT-230119; MC-BT-230120; MC-BT-230121; MC-BT-230122) and Table 13.

[0182] Table 13 Relative retention times of 22 batches of horsefly extract

[0183]

[0184]

[0185] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the test sample chromatogram should show eight characteristic peaks, corresponding to the retention times of the eight characteristic peaks in the reference herb chromatogram. The peak corresponding to the hypoxanthine reference is designated as 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 0.27 (peak 1), 0.54 (peak 2), 0.71 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.25 (peak 7), and 1.30 (peak 8).

[0186] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize 22 batches of Tabanus extracts, and a reference chromatogram of the characteristic chromatograms of Tabanus extracts was established. (See attached image.) Figure 9 .

[0187] Example 4: Detection of the characteristic spectrum of horsefly formulation granules

[0188] 4.1 Experimental Materials

[0189] Tabanus Formula Granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: S1, S2, S3).

[0190] 4.2 Chromatographic conditions

[0191] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.05% acetic acid solution as mobile phase B, with gradient elution as specified in Table 2 above; the detection wavelength was 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should be no less than 5000.

[0192] 4.2.1 Wavelength Selection

[0193] Based on the above-planned experimental conditions, a diode array detector was used to perform full-band scanning of uracil, guanine, adenine, hypoxanthine, and the test solution, and chromatograms of the test solution were extracted at wavelengths of 210 nm, 230 nm, 250 nm, 254 nm, 270 nm, and 290 nm, respectively. (See attached images.) Figure 10 The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable at a detection wavelength of 250 nm, therefore the detection wavelength was determined to be 250 nm.

[0194] 4.2.2 Column Temperature Investigation

[0195] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 20℃, 25℃, and 30℃, respectively. (See attached figures.) Figure 11 .

[0196] The column temperature study results showed that at a column temperature of 25℃, the chromatogram peaks had good separation and a flat baseline, so a column temperature of 25℃ was selected.

[0197] 4.2.3 Flow velocity investigation

[0198] Based on the above-specified experimental conditions, the flow rates of 0.6 ml / min, 0.7 ml / min, and 0.8 ml / min were investigated respectively. (See attached figures.) Figure 12 .

[0199] The results showed that at a flow rate of 0.7 ml / min, the chromatogram exhibited good peak separation, a flat baseline, and a moderate retention time. Therefore, a flow rate of 0.7 ml / min was selected.

[0200] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of the horsefly formulation granules were determined as follows: Octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.05% acetic acid solution as mobile phase B, with gradient elution as specified in Table 2 above; flow rate of 0.7 ml / min; column temperature of 25℃; and detection wavelength of 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should not be less than 5000.

[0201] 4.3 Test Sample Preparation Investigation

[0202] 4.3.1 Investigation of Extraction Solvents

[0203] Take an appropriate amount of this product (batch number: S1), grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml each of water, 10% methanol, 50% methanol, methanol, and 50% ethanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 13 The results showed that when 10% methanol was used as the extraction solvent, the chromatographic peak information was greater, the peak shape was better, and the separation was better. Therefore, 10% methanol was determined to be the extraction solvent for the test sample.

[0204] 4.3.2 Examination of Extraction Methods

[0205] Take an appropriate amount of this product (batch number: S1), grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 10% methanol, seal tightly, and test the extraction methods of reflux and ultrasound (power 600W, frequency 40kHz) for 30min. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 14 The results showed that the chromatographic effects were basically the same when using ultrasonic extraction and reflux extraction. Ultrasonic extraction was chosen as the extraction method for the test sample in this experiment.

[0206] 4.3.3 Examination of extraction time

[0207] Take an appropriate amount of this product (batch number: S1), grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 10% methanol, seal tightly, and sonicate (power 600W, frequency 40kHz). Examine the extraction time of the sample at 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product description]. Figure 15 The results showed that the chromatograms were basically consistent under different extraction time conditions. To ensure sufficient extraction, the extraction time for the test sample was determined to be 30 minutes.

[0208] 4.3.4 Investigation of Solvent Addition Amount

[0209] Take an appropriate amount of this product (batch number: S1), grind it into a fine powder, and take about 0.5g. Place it in a stoppered conical flask, and add 10ml, 25ml, and 50ml of 10% methanol respectively. Seal the flasks tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to product details]. Figure 16 The results showed that the peak areas of the characteristic chromatograms were moderate when the solvent volume was 25 ml. Therefore, the solvent volume for the test sample was determined to be 25 ml.

[0210] 4.3.5 Determine the method for preparing the test sample

[0211] Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 10% methanol solution, seal tightly, extract by sonication (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0212] 4.3.6 Feature Mapping Method

[0213] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0214] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.05% acetic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 2 above; the flow rate was 0.7 mL / min; the column temperature was 25 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the hypoxanthine peak, should not be less than 5000.

[0215] Preparation of reference solution: Take approximately 1.0 g of Tabanus reference material and place it in a stoppered conical flask. Add 25 ml of 10% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Take an appropriate amount of hypoxanthine reference standard, accurately weigh it, and add 10% methanol to prepare a solution containing 40 μg per ml as the reference solution.

[0216] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, weigh about 0.5g accurately, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0217] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0218] 4.4 Methodological Examination

[0219] 4.4.1 Chromatographic Peak Identification

[0220] Preparation of the test solution: Prepare the test solution of the horsefly formula granules according to the experimental conditions proposed above.

[0221] Preparation of reference solution: Take appropriate amounts of uracil, guanine, adenine and hypoxanthine reference standards, weigh them accurately, and add 10% methanol to prepare a solution containing 40 μg of each per ml.

[0222] Take about 1.0g of the Tabanus reference material and place it in a stoppered conical flask. Add 25ml of 10% methanol, weigh it, sonicate (600W power, 40kHz frequency) for 30 minutes, cool it, weigh it again, replenish the lost weight with 10% methanol, shake well, filter it, and take the filtrate as the reference solution for the reference material.

[0223] Preparation of negative control solution: Prepare negative control solution of Tabanus formula granules according to the experimental conditions proposed above.

[0224] The characteristic spectral peaks of the horsefly formula granules were located, see [link / reference]. Figure 17 The results showed that the negative solution had no interference, indicating that the method has good specificity.

[0225] 4.4.2 Precision Test

[0226] Take the test solution of the horsefly formula granules and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 14.

[0227] Table 14 Precision Examination - Retention Time

[0228]

[0229] The results showed that the RSD values ​​of the retention times of each characteristic peak were 0.13%–0.55%. The instrument has good precision.

[0230] 4.4.3 Repeatability Test

[0231] Six portions of the horsefly formula granules were accurately weighed and prepared and measured according to the proposed experimental method, as shown in Table 15.

[0232] Table 15 Repeatability Tests - Relative Retention Time Ratios

[0233]

[0234] The results show that the relative retention time RSD values ​​of each characteristic peak are 0.00% to 0.52%, indicating that the method has good repeatability.

[0235] 4.4.6 Stability

[0236] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 12h, 16h, and 24h. See Table 16.

[0237] Table 16 Stability Study - Retention Time

[0238]

[0239] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.15%–0.39%, and the sample solution was relatively stable within 24 hours.

[0240] 4.4.7 Methodological Summary

[0241] When peak 6 is selected as the S peak, the RSD of the retention time or relative retention time of each stage is shown in Table 17.

[0242] Table 17 Summary of RSD values ​​when peak 6 is an S-peak

[0243]

[0244] The results show that the retention times or RSD values ​​of the relative retention times of each characteristic peak meet the requirements in all the above investigations, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0245] 4.4.8 Determination of characteristic peaks and establishment of reference spectra

[0246] 4.4.8.1 Validation results of three batches of horsefly formulation granules

[0247] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times were calculated. See [link / reference]. Figure 18 Table 18.

[0248] Table 18. Relative retention times of three batches of horsefly granule formulations

[0249]

[0250]

[0251] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 6 was designated as the S peak, the relative retention time RSD of all eight characteristic peaks in the three batches of Tabanus granules was less than 2%.

[0252] 4.4.8.2 Establishment of Limits for Relative Retention Time

[0253] Therefore, the final stipulation is that the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 6 should correspond to the retention time of the reference peak, and the peak corresponding to the hypoxanthine reference peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.26 (peak 1), 0.53 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.26 (peak 7), and 1.30 (peak 8).

[0254] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize three batches of Tabanus granules formula, and a reference chromatogram of the characteristic chromatogram of Tabanus granules formula was established. (See attached image.) Figure 19 .

[0255] As can be seen from the above embodiments, the present invention provides a method for detecting the characteristic chromatograms of Tabanus and its preparations, comprising the following steps: A) dissolving and extracting the test sample raw material in a solvent to obtain the test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain the characteristic chromatograms of Tabanus and its preparations; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid solution, and gradient elution is used. The present invention uses HPLC, selecting acetonitrile-0.05% acetic acid as the mobile phase for gradient elution, and using hypoxanthine as a reference, to establish HPLC characteristic chromatograms of Tabanus medicinal materials, processed slices, extracts, and their formulation granules. The method exhibits good repeatability and precision, is stable and reliable, and can control the quality of Tabanus medicinal materials, processed slices, standard decoctions, and their formulation granules.

[0256] 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 detecting the characteristic spectrum of horseflies and their preparations, comprising the following steps: A) Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution; B) The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatograms of the horsefly and its preparations; The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid solution, and gradient elution is used. The gradient elution specifically refers to: 0~15min, Phase A: 0%, Phase B: 100%; 15-16 min, Phase A: 0-0.5%, Phase B: 100-99.5%; 16~25min, Phase A: 0.5%, Phase B: 99.5%; 25~28min, Phase A: 0.5~3%, Phase B: 99.5~97%; 28~35min, Phase A: 3%, Phase B: 97%.

2. The detection method according to claim 1, characterized in that, The method also includes preparing a reference solution: uracil, guanine, adenine, and hypoxanthine are dissolved in methanol to obtain a reference solution; the concentration of the reference solution is 40 μg / mL. The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the characteristic chromatograms of the horsefly and its preparations were identified based on the chromatogram of the reference.

3. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.6~0.8 ml per minute; the column temperature is 20~30℃; the detection wavelength is 240~260 nm; and the theoretical plate number, calculated based on xanthine peaks, should not be less than 5000.

4. The detection method according to claim 1, characterized in that, The solvent used for extraction is 10% methanol; the extraction method is ultrasonic extraction. The ultrasonic extraction power is 580~620W, the ultrasonic extraction frequency is 35~45kHz, and the ultrasonic extraction time is 20~40min. The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is (0.5~1):(20~25).

5. The detection method according to claim 1, characterized in that, The similarity between *Taenia solium* and its preparations was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. An HPLC standard characteristic chromatogram of *Taenia solium* consisting of eight characteristic peaks was obtained, with the peak corresponding to the hypoxanthine reference being the S peak. In the standard characteristic chromatogram, the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time should be within ±10% of a specified value. The specified value is: 0.27 (peak 1), 0.54 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.91 (peak 5), 1.25 (peak 7), 1.30 (peak 8).

6. The detection method according to claim 1, characterized in that, The similarity between *Taenia solium* and its preparations was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. This yielded a standard HPLC characteristic chromatogram of *Taenia solium* slices and extracts, consisting of eight characteristic peaks, with the peak corresponding to the hypoxanthine reference being the S peak. The relative retention times of each characteristic peak and the S peak were calculated in the standard characteristic chromatograms. These relative retention times should be within ±10% of a specified value. The specified value is: 0.27 (peak 1), 0.54 (peak 2), 0.71 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.25 (peak 7), 1.30 (peak 8).

7. The detection method according to claim 1, characterized in that, The similarity between *Taenia solium* and its preparations was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. An HPLC standard characteristic chromatogram of *Taenia solium* formulation granules, consisting of eight characteristic peaks, was obtained, with the peak corresponding to the hypoxanthine reference being the S peak. In the standard characteristic chromatogram, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of a specified value. The specified value is: 0.26 (peak 1), 0.53 (peak 2), 0.72 (peak 3), 0.78 (peak 4), 0.92 (peak 5), 1.26 (peak 7), 1.30 (peak 8).

8. A method for identifying the medicinal material, processed slices, extracts, and formulation granules of the horsefly, characterized in that, The detection is performed using the method described in any one of claims 1 to 7, and the detection results are analyzed.

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