A method for constructing HPLC characteristic patterns of snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and their formula particles

Abstract

The present invention provides a kind of snake slough (Zephagnum moss) medicinal material, decoction piece, standard decoction and its formula granule HPLC characteristic spectrum construction method, including: A) snake slough raw material is extracted with solvent to obtain liquid to be measured;B) liquid to be measured is determined using high performance liquid chromatography, obtains the HPLC characteristic spectrum of snake slough (Zephagnum moss) raw material;Described high performance liquid chromatography chromatographic condition is:Chromatographic column is C18 column;Mobile phase A is acetonitrile, and mobile phase B is 0.05% glacial acetic acid, gradient elution.The present invention adopts high performance liquid chromatography, selects acetonitrile 0.05% glacial acetic acid as mobile phase to carry out gradient elution, with uric acid as reference substance, establishes snake slough (Zephagnum moss) snake slough (Zephagnum moss) medicinal material, decoction piece, standard decoction and its formula granule HPLC characteristic spectrum, repeatability, precision are good, method is stable, reliable, can control the quality of snake slough (Zephagnum moss) medicinal material, decoction piece, standard decoction and its formula granule.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for constructing an HPLC characteristic spectrum of a snake skin (Zephagnum davidianum) medicinal material, a decoction piece, a standard decoction and its formula particles. Background Art

[0002] Snake slough is the dried epidermal membrane shed by the colubrid Zaocys dhumnades (Cantor), which has the effects of dispelling wind, calming nerves, relieving cataracts, and detoxifying. Standard decoctions are freeze-dried powders made from the medicinal materials after processing according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of snake skin (Black-banded Snake), its medicinal pieces and its standard decoction, a new characteristic map method was established in this paper to control its quality. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC characteristic spectrum of snake slough (black-necked snake) medicinal materials, decoction pieces, standard decoctions and their formula granules. The HPLC characteristic spectrum method of snake slough (black-necked snake) medicinal materials, decoction pieces, standard decoctions and their formula granules constructed by the present invention is stable and reliable, and can control the quality of snake slough (black-necked snake) medicinal materials, decoction pieces, standard decoctions and their formula granules.

[0005] The present invention provides a method for constructing an HPLC characteristic spectrum of a snake skin (Zephagnum moss) medicinal material, a decoction piece, a standard decoction, and its formula particles, comprising:

[0006] A) extracting the snake skin material with a solvent to obtain a test solution;

[0007] B) the test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the snake slough (Zephagnum gargoyles) raw material;

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

[0009] The present invention provides a method for constructing an HPLC characteristic spectrum of a snake skin (Zodia davidiana) medicinal material, decoction pieces, standard decoction and its formula particles. First, the snake skin (Zodia davidiana) raw material is taken and solvent extracted to obtain a test solution. The solvent is preferably 10% methanol.

[0010] According to the present invention, the snake skin (Zodia davidiana) raw materials are snake skin (Zodia davidiana) medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

[0011] According to the present invention, the preparation of the test solution of snake slough (Dieselgut) medicinal materials / decoction pieces / standard decoction / formulated granules is specifically as follows: the snake slough raw material is ultrasonically extracted with 10% methanol, cooled, shaken, and filtered to obtain the product.

[0012] The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 30 to 40 minutes, more preferably 30 minutes.

[0013] The ratio of the mass g of the snake skin (black snake) raw material to the volume mL of the solvent is 0.5:45-55.

[0014] In a specific embodiment, the ratio of the mass g of the snake skin (Zephagnum gargoyles) raw material to the volume mL of the solvent is 0.5:50.

[0015] The present invention adopts the above extraction solvent, which has large chromatographic peak information and good effect.

[0016] The snake skin (Zephagnum davidianum) raw material of the present invention is a snake skin (Zephagnum davidianum) medicinal material, snake skin (Zephagnum davidianum) decoction piece or decoction. The present invention is not limited to it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0017] The present invention also includes preparing reference substances and reference medicinal material reference solutions:

[0018] Take uric acid reference substance, guanosine reference substance, and adenosine reference substance respectively, dissolve them in 10% methanol to obtain reference substance solutions;

[0019] In some specific embodiments of the present invention, the concentration of the uric acid reference substance is 80 μg / mL;

[0020] The concentration of the guanosine reference substance was 50 μg / mL; the concentration of the adenosine reference substance was 50 μg / mL.

[0021] Take snake skin reference medicinal material and dissolve it in 10% methanol, sonicate, cool, and filter to obtain a reference medicinal material solution;

[0022] The reference substance and the reference medicinal material reference solution are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference, respectively; and the components of the HPLC characteristic spectra of the snake skin medicinal material, decoction pieces, standard decoction and its formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal material reference.

[0023] The mobile phase A of the present invention is acetonitrile, the mobile phase B is 0.05% glacial acetic acid solution, and the elution is performed in a gradient manner.

[0024] The gradient elution of the present invention is preferably specifically:

[0025] 0-10 min, phase A: 1%, phase B: 99%;

[0026] 10-22 min, phase A: 1%-10%, phase B: 99%-90%;

[0027] 22-27 min, phase A: 10%-25%, phase B: 90%-75%;

[0028] 27-35min, phase A: 25%, phase B: 75%.

[0029] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0030] C18 column, size 3 μm, 4.6×150 mm; column temperature 25°C.

[0031] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above-mentioned 25°C condition.

[0032] The mobile phase flow rate is preferably 0.7 ml / min.

[0033] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.7 ml / min, which is the most preferred solution.

[0034] The detection wavelength of the present invention is preferably 250 nm.

[0035] The inventors found that at 250 nm, the chromatographic information was rich, each component had good absorption, the response value was moderate, the peaks were well separated, and the baseline was stable.

[0036] The injection volume of the present invention is preferably 10 μL.

[0037] The beneficial effect of the present invention is that, under one liquid chromatography condition, the substance group of snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and their formula granules are controlled by fingerprints, and uric acid, guanosine and adenosine are used to locate the fingerprints; the cost of detection can be greatly reduced, and qualitative detection can be achieved.

[0038] The similarity of the HPLC characteristic spectra of Snake (Black-banded Snake) medicinal materials, decoction pieces, standard decoctions and their formula granules was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of Snake (Black-banded Snake) medicinal materials, decoction pieces, standard decoctions and their formula granules were obtained, which consisted of 8 characteristic peaks, including Peak 1: uracil; Peak 2: guanine; Peak 3 (S): uric acid; Peak 4: hypoxanthine; Peak 5: xanthine, Peak 6 is uridine, Peak 7 is guanosine, and Peak 8 is adenosine.

[0039] In the characteristic spectrum of the snake skin (black-banded snake) standard decoction, uric acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), and 2.20 (peak 8).

[0040] Quality judgment standard: Take samples of snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain the characteristic spectra of snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and their formula granules. The standard characteristic spectra and sample characteristic spectra of snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and their formula granules are analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Chinese Medicine" (2012 edition) of the National Pharmacopoeia Committee, and the similarity is greater than 0.90.

[0041] The method provided by the present invention can effectively monitor the quality of different batches of snake skin (black-banded snake) snake skin (black-banded snake) medicinal materials, decoction pieces, standard decoctions and formula granules thereof, so that the quality is stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0042] The characteristic maps of snake skin (grass snake) medicinal materials, decoction pieces, standard decoctions and their formula granules established by the present invention use uric acid, guanosine and adenosine as reference substances, focus on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, and can comprehensively evaluate the overall quality features of the products. The method is scientific and reliable.

[0043] The newly developed characteristic spectrum method of the present invention can detect components with high polarity in snake skin (Zephaga davidiana) and its standard decoction. Furthermore, the sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on snake skin (Zephaga davidiana) and its preparations. The authenticity, quality consistency, and stability of snake skin (Zephaga davidiana) and its preparations can be effectively detected and controlled. This provides a basis for effectively controlling and comprehensively evaluating the quality of snake skin (Zephaga davidiana) standard decoctions, ensuring the uniformity and stability of the quality of snake skin (Zephaga davidiana) and its standard decoctions.

[0044] The present invention provides a method for constructing an HPLC characteristic spectrum of a snake slough (black-banded snake) medicinal material, a decoction piece, a standard decoction, and a formula granule thereof, comprising: A) extracting the snake slough raw material with a solvent to obtain a test liquid; B) determining the test liquid with a high-performance liquid chromatography method to obtain an HPLC characteristic spectrum of the snake slough (black-banded snake) raw material; the chromatographic conditions of the high-performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.05% glacial acetic acid, and gradient elution. The present invention adopts high-performance liquid chromatography, selects acetonitrile-0.05% glacial acetic acid as the mobile phase for gradient elution, and uses uric acid, guanosine, and adenosine as reference substances to establish an HPLC characteristic spectrum of the snake slough (black-banded snake) medicinal material, a decoction piece, a standard decoction, and a formula granule thereof. The method has good repeatability and precision, is stable, and is reliable, and can control the quality of the snake slough (black-banded snake) medicinal material, a decoction piece, a standard decoction, and a formula granule thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Chromatogram of snake slough granules;

[0046] Figure 2 Investigation of different column temperatures for snake skin formula granules;

[0047] Figure 3 Flow rate investigation;

[0048] Figure 4 Investigation on the delayed effect of snake skin formula granules;

[0049] Figure 5 Investigation of extraction solvents;

[0050] Figure 6 Investigation of extraction methods;

[0051] Figure 7 Extraction time investigation;

[0052] Figure 8 Investigation of solvent addition amount;

[0053] Figure 9 Chromatographic peak identification;

[0054] Figure 10 Different instruments;

[0055] Figure 11 Different chromatographic columns;

[0056] Figure 12 Comparative characteristic spectrum of snake skin formula granules;

[0057] Figure 13 Verification diagram of characteristic spectrum of Snake She formula granules;

[0058] Figure 14 Comparative characteristic spectrum of snake slough medicinal materials;

[0059] Figure 15 Verification diagram of the medicinal material characteristic spectrum of snake skin (black-banded snake);

[0060] Figure 16 Comparative characteristic spectrum of snake skin decoction pieces;

[0061] Figure 17 Verification diagram of the characteristic spectrum of snake slough medicinal pieces.

[0062] Figure 18 Comparative characteristic spectrum of snake slough standard decoction;

[0063] Figure 19 Verification diagram of the characteristic spectrum of snake slough standard decoction;

[0064] Figure 20 This is a picture of snake skin (black-necked snake) particles. DETAILED DESCRIPTION

[0065] The present invention provides a method for constructing an HPLC characteristic spectrum of a snake skin (Against the Graves' Snake) medicinal material, a decoction piece, a standard decoction, and its formula granules. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit, and scope of the present invention to realize and apply the technology of the present invention.

[0066] To further illustrate the present invention, the following describes in detail a method for constructing an HPLC characteristic spectrum of a snake skin (Zephagnum davidianum) medicinal material, a decoction piece, a standard decoction, and its formula granules provided by the present invention in combination with the examples.

[0067] High performance liquid chromatograph: Instrument 1 Agilent 1200, Instrument 2 Thermo Fisher, Instrument 3 Shimadzu;

[0068] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0069] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0070] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0071] Chromatographic columns: Chromatographic column 1 C18-AQ 150×4.6mm 3.0μm; Chromatographic column 2 Shim-pack Scepter C18-120 4.6×150mm 3.0μm; Chromatographic column 3 Inertsil ODS-31 50×4.6mm 3.0μm;

[0072] Acetonitrile, methanol, and glacial acetic acid were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade.

[0073] Uric acid reference substance (Shanghai Shidande Standard Technology Service Co., Ltd., batch number: 9602, purity: 100%);

[0074] Snake skin (Zephagnum gargoyles) control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 380140-202301);

[0075] Snake slough formula granules ST-01, Snake slough formula granules ST-02, ST-03, ST-04;

[0076] Snake slough medicinal materials: ST-YC-01, ST-YC-02, ST-YC-03, ST-YC-04, ST-YC-05ST-YC-06, ST-YC-07, ST-YC-08, ST -YC-09, ST-YC-10, ST-YC-11, ST-YC-12, ST-YC-13, ST-YC-14, ST-YC-15, ST-YC-16, ST-YC-17;

[0077] Snake slough pieces: ST-YP-01, ST-YP-02, ST-YP-03, ST-YP-04, ST-YP-05ST-YP-06, ST-YP-07, ST-YP-08, ST -YP-09, ST-YP-10, ST-YP-11, ST-YP-12, ST-YP-13, ST-YP-14, ST-YP-15, ST-YP-16, ST-YP-17;

[0078] Snake skin standard decoction: ST-BT-01, ST-BT-02, ST-BT-03, ST-BT-04, ST-BT-05ST-BT-06, ST-BT-07, ST-BT-08, ST-BT-09, ST-BT-10, ST-BT-11, ST-BT-12, ST-BT-13, ST-BT-14, ST-BT-15, ST-BT-16, ST-BT-17.

[0079] The decoction pieces, standard soups and granules are all produced by Sichuan Xinlv; the medicinal materials are purchased from Sichuan, Chongqing, Hubei and Guangxi.

[0080] Example 1

[0081] 1.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the packing (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile was used as mobile phase A, and 0.05% glacial acetic acid solution was used as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0082]

[0083] Preparation of reference solution: Take 1 g of snake skin (Black Band Snake) reference medicinal material, place it in a stoppered conical flask, add 25 ml of 10% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Separately, take appropriate amounts of uric acid reference substance, guanosine reference substance, and adenosine reference substance, accurately weigh them, add 10% methanol to make a mixed solution containing 80 μg of uric acid, 50 μg of guanosine, and 50 μg of adenosine per 1 ml, which is used as the reference substance reference solution. Preparation of test solution: Take appropriate amount of snake skin formula granules, grind them finely, take 0.5 g, place it in a stoppered conical flask, add 50 ml of 10% methanol, stopper it, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate.

[0084] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. 1.2

[0086] 1.2.1 Determination of detection wavelength

[0087] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 230nm, 250nm, 270nm, 280nm, 290nm, and 310nm were extracted respectively. Figure 1 . Figure 1 Chromatogram of snake skin granules; from bottom to top are 250nm, 310nm, 290nm, 280nm, 270nm, and 230nm.

[0088] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 250 nm, so the detection wavelength was determined to be 250 nm.

[0089] 1.2.2 Column temperature investigation

[0090] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 2 As shown in Table 1.

[0091] Table 1 Column temperature investigation - characteristic peak relative retention time ratio

[0092]

[0093] Figure 2 Different column temperatures of snake slough formula granules were investigated; the results of the column temperature investigation showed that when the column temperature was 25℃, the chromatographic peaks were relatively symmetrical and the separation was good, so 25℃ was finally determined as the column temperature for the characteristic spectrum method of snake slough formula granules.

[0094] 1.2.3 Flow rate investigation

[0095] Based on the experimental conditions proposed above, the flow rates of 0.7ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 3 As shown in Table 2.

[0096] Table 2 Flow rate-relative retention time

[0097]

[0098] Figure 3 Flow rate investigation: The results showed that when the flow rate was 0.7 ml / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 0.7 ml / min.

[0099] 1.2.4 Delay test

[0100] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 70 minutes. Figure 4 shown. Figure 4 The delayed properties of snake slough formula granules were investigated; the results showed that there was basically no chromatographic peak after 35 minutes, so the sample detection time was set at 35 minutes.

[0101] 1.2.5 Extraction solvent investigation

[0102] Take about 0.5g of snake skin formula granules (ST-01) and place it in a stoppered conical flask. Add 25ml of 10% methanol, 30% methanol, 50% methanol, 70% methanol, methanol, water, 10% ethanol, 1% sodium hydroxide, 0.5% ammonia water, 1% ammonia water, and 2% ammonia water, respectively. Seal the flask tightly and sonicate (power 600W, frequency 40kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate to obtain, such as Figure 5 shown. Figure 5 Investigation of extraction solvents.

[0103] The results showed that the chromatographic peak information content was large when 10% methanol was used as the extraction solvent, so the extraction solvent for the test sample was determined to be 10% methanol.

[0104] 1.2.6 Investigation of extraction methods

[0105] Take about 0.5g of snake skin formula granules, place them in a stoppered conical flask, add 25ml of 10% methanol, and investigate the extraction methods of the test sample when they are refluxed and ultrasonically. The extraction time is 30 minutes, cool, shake well, filter, and take the filtrate to obtain, such as Figure 6 shown. Figure 6 Investigation of extraction methods.

[0106] The results showed that ultrasonic extraction and reflux extraction had the same effect on the test sample. Because ultrasonic extraction is more convenient, ultrasonic extraction was determined to be the extraction method for the test sample.

[0107] 1.2.7 Extraction time investigation

[0108] Take about 0.5g of snake skin formula granules, place them in a stoppered conical flask, add 25ml of 10% methanol, and observe the ultrasonic extraction time of the test sample for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake well, filter, and take the filtrate to obtain, such as Figure 7 shown. Figure 7 Extraction time investigation.

[0109] The results showed that the extraction was sufficient when the extraction time was 30 minutes. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0110] 1.2.8 Investigation of solvent addition amount

[0111] Take about 0.5g of snake skin formula granules, grind them into powder, take about 0.1g, place it in a stoppered conical flask, add 15ml, 25ml, and 50ml of 10% methanol respectively, stopper it tightly, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product. Figure 8 . Figure 8 Investigation of solvent addition amount.

[0112] The results showed that when the amount of solvent added was 50 ml, the characteristic spectrum chromatographic peak area was moderate. Therefore, the amount of solvent added for the test sample was determined to be 50 ml.

[0113] 1.2.9 Determine the test sample preparation method

[0114] Take an appropriate amount of snake skin formula granules (batch number: ST-01), grind it into powder, take 0.5g, accurately weigh it, place it in a stoppered conical flask, add 50ml of 10% methanol, stopper it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0115] 2.1 Methodological Investigation

[0116] 2.1.1 Chromatographic peak identification

[0117] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of snake slough formula granules.

[0118] Preparation of control medicinal material and reference solution: Take 1 g of snake skin (Black-banded Snake) control medicinal material, add 25 ml of water, boil for 30 minutes, filter, evaporate to dryness, add 25 ml of 10% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0119] Take appropriate amount of uric acid, uracil, guanine, hypoxanthine and xanthine reference substances, weigh accurately, add 10% methanol to make a solution containing 50 μg per 1 ml, which serves as the reference substance solution.

[0120] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of snake slough formula granules.

[0121] Position the characteristic spectrum peaks of the snake skin formula granules. Figure 9 shown. Figure 9 Chromatographic peak identification.

[0122] 2.1.2 Precision test

[0123] The test solution of She Tu Formula Granules (Batch No.: ST-01) was injected six times in succession according to the proposed experimental method, with 10 μL injected each time. The retention time of each characteristic peak was calculated as shown in Table 3.

[0124] Table 3 Precision investigation-retention time

[0125]

[0126] The results showed that the RSD of each characteristic peak retention time was 0.10%-1.45%. This method has good precision.

[0127] 2.1.3 Repeatability study

[0128] Six portions of snake slough granules (batch number: ST-01) were accurately weighed and prepared and tested according to the proposed experimental method, as shown in Table 4.

[0129] Table 4 Repeatability study - relative retention time

[0130]

[0131]

[0132] The results showed that the RSD of the relative retention time of each characteristic peak was 0.12%-1.09%. The method has good reproducibility.

[0133] 2.1.4 Intermediate precision study

[0134] 2.1.4.1 Investigation of different instruments

[0135] Based on the experimental conditions proposed above, two portions of snake slough formula granules (batch number: ST-01) were accurately weighed to prepare the test solution, which was then measured on instrument 1 Agilent 1200, instrument 2 Thermo Fisher, and instrument 3 Shimadzu. Figure 10 , as shown in Table 5.

[0136] Table 5 Instrument durability investigation - relative retention time ratio

[0137]

[0138] Figure 10 Different instruments. The results showed that when the three instruments were used to detect the sample, the RSD of the relative retention times of the characteristic peaks ranged from 0.31% to 1.80%, indicating that different instruments had little effect on the relative retention times of this method. This method has good instrument durability.

[0139] 2.1.4.2 Inspection by different personnel and time

[0140] Based on the experimental conditions proposed above, different individuals (A and B) accurately weighed two portions of Snake Slough Formula Granules (Batch No.: ST-01) at different times (T1 and T2) to prepare test samples for measurement, as shown in Table 6.

[0141] Table 6 Personnel and time inspection-relative retention time

[0142]

[0143] The results showed that the method was stable when different people measured the same sample at different times.

[0144] 2.1.4.3 Durability inspection

[0145] 2.1.4.3.1 Column durability assessment

[0146] Based on the experimental conditions proposed above, the chromatographic columns are: chromatographic column 1C18-AQ

[0147] 150×4.6mm 3.0μm; Column 2 Shim-pack Scepter C18-120 4.6×150mm 3.0μm; Column 3 Inertsil ODS-31 50×4.6mm 3.0μm

[0148] Conduct an investigation. Figure 11 , as shown in Table 7.

[0149] Table 7 Column durability investigation - relative retention time

[0150]

[0151] Figure 11 Different chromatographic columns. Figure 11 It can be seen that the chromatographic peak separation of each column is good and the peaks are clear, and the chromatographic columns have good durability.

[0152] 2.1.4.3.2 Stability investigation

[0153] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h, respectively.

[0154] Table 8 Stability Study-Retention Time

[0155]

[0156] The results showed that the RSD of the corresponding characteristic peak retention time was stable within 24 hours in the range of 0.08% to 1.10% of the sample solution.

[0157] The RSDs of the remaining eight characteristic peaks relative to their retention times met the requirements in all the above investigations, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigations.

[0158] Example 2 Characteristic Spectrum and Verification Results of Snake Skin Formula Granules

[0159] 2.1 Characteristic Spectrum of Snake Slough Formula Granules

[0160] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile as mobile phase A, and 0.05% glacial acetic acid solution as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0161]

[0162] Preparation of reference solution Take 1g of snake skin (Dark-banded snake) reference medicinal material, place it in a stoppered conical flask, add 25ml of 10% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Separately, take appropriate amounts of uric acid reference substance, guanosine reference substance, and adenosine reference substance, accurately weigh them, add 10% methanol to make a mixed solution containing 80μg of uric acid, 50μg of guanosine, and 50μg of adenosine per 1ml, which is used as the reference substance solution.

[0163] Preparation of the test solution: Take an appropriate amount of snake skin formula granules, grind them into powder, take 0.5g, place it in a stoppered conical flask, add 50ml of 10% methanol, stopper it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the solution.

[0164] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0165] There should be 8 characteristic peaks in the characteristic spectrum of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to uric acid is the S peak. The relative retention time of the remaining characteristic peaks and the S peak should be calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), 2.20 (peak 8). The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 3 batches of snake skin formula granules, and a reference spectrum of the characteristic spectrum of snake skin formula granules was established. Figure 12 shown. Figure 12 Comparative characteristic spectrum of snake slough formula granules; Peak 1: uracil; Peak 2: guanine; Peak 3 (S): uric acid; Peak 4: hypoxanthine; Peak 5: xanthine, Peak 6 is uridine, Peak 7 is guanosine, and Peak 8 is adenosine.

[0166] 2.23 batches of snake slough formula granules verification results

[0167] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time. Figure 12 , as shown in Table 9.

[0168] Table 9 Relative retention time of three batches of snake skin formula granules

[0169]

[0170]

[0171] Figure 13 Validation of the characteristic spectrum of She Tu Formula Granules from the first batch; based on the principle of stable relative retention times and the fact that peaks were detectable across all batches at relatively high levels, eight peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 3 was used as the S peak, the relative retention time RSDs of the characteristic peaks for all three batches of She Tu Formula Granules were less than 1%.

[0172] 2.3 Establishment of relative retention time limit values

[0173] The summary of the methodology inspection items and verification results is shown in Table 10:

[0174] Table 10 Summary of RSD% results for each item of the methodology - relative retention time

[0175]

[0176] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0177] Final Rule: If the RSDs of the relative retention times of each characteristic peak meet the requirements in all of the above considerations, the method is considered good. The test sample's characteristic spectrum should show eight characteristic peaks, of which the peak corresponding to the uric acid reference is the S peak.

[0178] Example 3 Snake skin medicinal material characteristic spectrum and verification results 3.1

[0180] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile as mobile phase A, and 0.05% glacial acetic acid solution as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0181]

[0182]

[0183] Preparation of reference solution Take 1g of snake skin (Dark-banded snake) reference medicinal material, place it in a stoppered conical flask, add 25ml of 10% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Separately, take appropriate amounts of uric acid reference substance, guanosine reference substance, and adenosine reference substance, accurately weigh them, add 10% methanol to make a mixed solution containing 80μg of uric acid, 50μg of guanosine, and 50μg of adenosine per 1ml, which is used as the reference substance solution.

[0184] Preparation of the test solution: Take 0.5 g of snake skin medicinal material powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of 10% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0185] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0186] The test sample's characteristic spectrum should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to uric acid is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), and 2.20 (peak 8).

[0187] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 19 batches of snake skin medicinal materials, and a comparison map of the characteristic spectrum of snake skin medicinal materials was established. Figure 14 shown. Figure 14 Comparative characteristic spectrum of snake skin medicinal materials; Peak 1: uracil; Peak 2: guanine; Peak 3 (S): uric acid; Peak 4: hypoxanthine; Peak 5: xanthine, Peak 6 is uridine, Peak 7 is guanosine, and Peak 8 is adenosine.

[0188] 3.2 Verification results of 17 batches of snake slough medicinal materials

[0189] The proposed method was used to determine the characteristic spectra of 17 batches of samples of this product and calculate the relative retention time. Figure 15 , as shown in Table 11. Figure 15 Verification diagram of the medicinal characteristic spectrum of snake skin (black-banded snake).

[0190] Table 11 shows the relative retention time of 17 batches of snake skin medicinal materials

[0191]

[0192]

[0193] Example 4 Snake Slough Pieces Characteristic Spectrum and Verification Results

[0194] 4.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the packing (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile was used as mobile phase A, and 0.05% glacial acetic acid solution was used as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0195]

[0196] Preparation of Reference Solution: 1 g of the reference medicinal material, Snake Skin (Dark-necked Snake), was placed in a stoppered conical flask. 25 ml of 10% methanol was added and ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes. The solution was cooled, shaken, and filtered. The filtrate was used as the reference medicinal material solution. Separately, appropriate amounts of uric acid reference substance, guanosine reference substance, and adenosine reference substance were accurately weighed and added with 10% methanol to prepare a mixed solution containing 80 μg of uric acid, 50 μg of guanosine, and 50 μg of adenosine per 1 ml. This was used as the reference solution.

[0197] Preparation of test solution

[0198] Take 0.5 g of snake skin decoction powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of 10% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0199] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0200] There should be 8 characteristic peaks in the characteristic spectrum of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to uric acid is the S peak. The relative retention time of the remaining characteristic peaks and the S peak should be calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), 2.20 (peak 8). The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 19 batches of snake skin decoction pieces, and a reference spectrum of the characteristic spectrum of snake skin decoction pieces was established. Figure 16 shown. Figure 16 Comparative characteristic spectrum of snake skin decoction pieces; Peak 1: uracil; Peak 2: guanine; Peak 3 (S): uric acid; Peak 4: hypoxanthine; Peak 5: xanthine, Peak 6 is uridine, Peak 7 is guanosine, and Peak 8 is adenosine.

[0201] 4.217 batches of snake skin decoction pieces certification results

[0202] The proposed method was used to determine the characteristic spectra of 17 batches of samples of this product and calculate the relative retention time. Figure 17 , as shown in Table 12. Figure 17 Verification diagram of the characteristic spectrum of snake slough medicinal pieces.

[0203] Table 1217 Relative retention time of snake skin slices

[0204]

[0205]

[0206] Example 5 Characteristic Spectrum and Verification Results of Snake Slough Standard Decoction

[0207] 5.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile was used as mobile phase A, and 0.05% glacial acetic acid solution was used as mobile phase B. Gradient elution was performed according to the table below; the flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0208]

[0209] Preparation of reference solution Take 1g of snake skin (Dark-banded snake) reference medicinal material, place it in a stoppered conical flask, add 25ml of 10% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Separately, take appropriate amounts of uric acid reference substance, guanosine reference substance, and adenosine reference substance, accurately weigh them, add 10% methanol to make a mixed solution containing 80μg of uric acid, 50μg of guanosine, and 50μg of adenosine per 1ml, which is used as the reference substance solution.

[0210] Preparation of test solution: Take 0.1 g of the product powder, place it in a stoppered conical flask, add 50 ml of 10% methanol, stopper it tightly, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0211] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0212] The test sample's characteristic spectrum should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to uric acid is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), and 2.20 (peak 8).

[0213] Seventeen batches of snake slough standard decoctions were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison of the characteristic spectra of snake slough standard decoctions was established. Figure 18 shown. Figure 18 Comparative characteristic spectrum of snake slough standard decoction; Peak 1: uracil; Peak 2: guanine; Peak 3 (S): uric acid; Peak 4: hypoxanthine; Peak 5: xanthine, Peak 6: uridine, Peak 7: guanosine, Peak 8: adenosine.

[0214] 5.2 Verification results of 17 batches of snake slough standard decoction

[0215] The proposed method was used to determine the characteristic spectra of 17 batches of samples of this product and calculate the relative retention time. Figure 19 , as shown in Table 13. Figure 19 Verification diagram of the characteristic spectrum of snake slough standard decoction.

[0216] Table 13 Relative retention time of 17 batches of snake slough standard decoction

[0217]

[0218] Comparative Example 1

[0219] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); methanol as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate solution as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.7 ml / min; the column temperature was 25°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the uric acid peak, should be no less than 5000.

[0220]

[0221] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take 0.25 g, place it in a stoppered conical flask, add 50 ml of 70% methanol, stopper it tightly, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0222] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0223] This method was used to test the snake slough formula granules. The results were as follows: Figure 20 As shown, Figure 20 For the snake skin (black snake) particle map; Figure 20 It can be seen that using this method, the peak shape of the snake slough (Black-banded Snake) spectrum is poor, the peak separation is poor, the number of peaks is small, and the peak baseline is unstable. This method is not suitable for the identification of the snake slough characteristic spectrum of the present invention.

[0224] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC characteristic spectrum of snake skin medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising: A) extracting the snake skin material with 10% methanol by ultrasonic extraction, cooling, shaking, and filtering to obtain the product; The snake slough is the dried epidermal membrane shed by the black-banded snake; B) the liquid to be tested is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the snake skin raw material; The similarity of HPLC characteristic spectra of snake skin medicinal materials, decoction pieces, standard decoction and its formula granules was evaluated by using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint. The HPLC standard characteristic spectra of snake skin medicinal materials, decoction pieces, standard decoction and its formula granules were obtained, which consisted of 8 characteristic peaks, including peak 1: uracil; peak 2: guanine; peak 3: uric acid; peak 4: hypoxanthine; peak 5: xanthine, peak 6: uridine, peak 7: guanosine, peak 8: adenosine; uric acid was used as the reference peak S; The HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile; mobile phase B is 0.05% glacial acetic acid; gradient elution; The gradient elution is specifically as follows: 0-10 min, phase A: 1%, phase B: 99%; 10-22 min, phase A: 1%-10%, phase B: 99%-90%; 22-27 min, phase A: 10%-25%, phase B: 90%-75%; 27-35min, phase A: 25%, phase B: 75%.

2. The method according to claim 1, characterized in that It also includes the preparation of reference substances and reference medicinal material solutions: Take uric acid reference substance, guanosine reference substance, and adenosine reference substance respectively, dissolve them in 10% methanol to obtain reference substance solutions; Take snake skin reference medicinal material and dissolve it in 10% methanol, sonicate, cool, and filter to obtain a reference medicinal material solution; The reference substance and the reference medicinal material reference solution are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference, respectively; and the components of the HPLC characteristic spectra of the snake skin medicinal material, decoction pieces, standard decoction and its formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal material reference.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with a specification of 3.0 μm and 4.6×150 mm; the column temperature is 25° C.

4. The method according to claim 3, characterized in that The flow rate of the mobile phase is 0.7 mL / min; the detection wavelength is 250 nm; the theoretical plate number calculated based on the uric acid peak should be no less than 5000, and the injection volume is 10 μL.

5. The method according to claim 1, wherein In the characteristic spectrum of the snake slough standard decoction, uric acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.73 (peak 1), 0.89 (peak 2), 1.13 (peak 4), 1.36 (peak 5), 1.55 (peak 6), 2.06 (peak 7), and 2.20 (peak 8).

6. The method according to claim 1, characterized in that The snake skin raw materials in step A) are snake skin medicinal materials, decoction pieces, standard decoctions and formula granules thereof.

7. The method according to claim 1, characterized in that The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 30 to 40 minutes; The ratio of the mass g of the snake slough raw material to the volume mL of the solvent is 0.5:45-55.

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