A method for constructing HPLC characteristic chromatogram of buffalo horn medicinal material, standard decoction and formula granules

Abstract

The application provides a kind of water buffalo horn medicinal materials, standard decoction and its formula granules HPLC characteristic chromatogram construction method, comprising: A) water buffalo horn raw materials are extracted using solvent, to obtain the test solution;B) after the test solution is subjected to pre-column derivatization reaction, using high performance liquid chromatography, to obtain the HPLC characteristic chromatogram of water buffalo horn raw materials;The high performance liquid chromatography chromatographic conditions are: the chromatographic column is C18 column;Mobile phase A is acetonitrile-methanol-100mmol / L sodium acetate aqueous solution, mobile phase B is tetrahydrofuran-11mmol / L sodium acetate aqueous solution, gradient elution.The application uses high performance liquid chromatography, selects the above mobile phase for gradient elution, with alanine as reference, establishes the HPLC characteristic chromatogram of water buffalo horn medicinal materials, standard decoction and its formula granules, with good repeatability, precision, stable and reliable method, can control the quality of water buffalo horn medicinal materials, standard decoction and its formula granules.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, in particular to a construction method of HPLC characteristic spectrum of buffalo horn medicinal material, standard decoction and formula granules thereof. BACKGROUND

[0002] Buffalo horn is a substitute for rhinoceros horn in traditional Chinese medicine, which has the functions of clearing heat, cooling blood, calming shock and detoxification. It is used to treat heat entering blood system, shock, restlessness, delirium, measles, jaundice, hematemesis, epistaxis, metrorrhagia, carbuncle and abscess.

[0003] Buffalo horn is the horn of Bubalus bubalis Linnaeus. After the horn is taken, it is boiled, the horn plug is removed, and the dried medicinal material is obtained. The buffalo horn standard decoction is a freeze-dried powder prepared by processing the medicinal material according to a fixed preparation process; the buffalo horn formula granule is a formula granule prepared by processing the buffalo horn medicinal material according to the main quality indicators of the standard decoction.

[0004] In order to ensure the uniformity and stability of the quality of buffalo horn medicinal material, standard decoction and formula granules, a new characteristic spectrum method needs to be established to control the quality, and it can be clearly identified from buffalo horn and its preparations. SUMMARY

[0005] Therefore, the present application aims to solve the technical problem by providing a construction method of HPLC characteristic spectrum of buffalo horn medicinal material, standard decoction and formula granules thereof. The HPLC characteristic spectrum method of buffalo horn medicinal material, standard decoction and formula granules thereof constructed by the present application is stable and reliable, and can control the quality of buffalo horn medicinal material, standard decoction and formula granules thereof.

[0006] The present application provides a construction method of HPLC characteristic spectrum of buffalo horn medicinal material, standard decoction and formula granules thereof, which comprises:

[0007] A) extracting the buffalo horn raw material with a solvent to obtain a test solution;

[0008] B) after pre-column derivatization reaction of the test solution, determining by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the buffalo horn raw material;

[0009] The high performance liquid chromatography method has the following chromatographic conditions: the chromatographic column is a C18 column; the mobile phase A is acetonitrile-methanol-100mmol / L sodium acetate aqueous solution, the mobile phase B is tetrahydrofuran-11mmol / L sodium acetate aqueous solution, and gradient elution is adopted;

[0010] The gradient elution is specifically as follows:

[0011] 0-10min, A phase: 2%→10%, B phase: 98%→90%;

[0012] 10-21 min, phase A: 10%→11%, phase B: 90%→89%;

[0013] 21-55 min, phase A: 11%→70%, phase B: 89%→30%.

[0014] The application provides a construction method of HPLC characteristic chromatogram of buffalo horn medicinal material, standard decoction and formula granules.

[0015] According to the application, the buffalo horn raw material is buffalo horn medicinal material, standard decoction and formula granules.

[0016] According to the application, the preparation of the buffalo horn medicinal material standard decoction / formula granule test solution is specifically as follows: the buffalo horn raw material is ultrasonically extracted by 25% methanol, cooled, shaken, filtered, and then obtained.

[0017] The ultrasonic power is 600 W, and the frequency is 40 kHz; the extraction time is 30-40 min; and more preferably, the extraction time is 30 min.

[0018] The ratio of the mass g of the buffalo horn raw material to the volume mL of the solvent is 1-2:25-50.

[0019] In one specific embodiment, the ratio of the mass g of the buffalo horn medicinal material to the volume mL of the solvent is 2:25.

[0020] In one specific embodiment, the ratio of the mass g of the buffalo horn standard decoction and formula granules to the volume mL of the solvent is 1:50.

[0021] The above extraction solvent chromatographic peak information is large, the extraction efficiency is high, and the effect is good; when the addition amount of the extraction solvent is the above ratio, the peak shape and the separation degree are good, and the peak size is appropriate.

[0022] The buffalo horn raw material in the application is buffalo horn medicinal material, standard decoction and formula granules. The application does not limit the above raw materials, and the above raw materials can be subjected to quality control and qualitative detection by the method of the application.

[0023] After the test solution is subjected to a pre-column derivatization reaction, the HPLC characteristic chromatogram of the buffalo horn raw material is obtained by high performance liquid chromatography.

[0024] The application also includes preparation of a control and a control medicinal material reference solution.

[0025] Glycine control and alanine control are respectively taken, dissolved by 25% methanol, and a control reference solution is obtained.

[0026] In some embodiments of the present application, the concentration of glycine and alanine reference substances is 16 μg / mL each.

[0027] The concentration of glycine reference substance is 16 μg / mL; and the concentration of alanine reference substance is 16 μg / mL.

[0028] The water buffalo horn reference medicinal material is dissolved in 25% methanol, ultrasonically treated, cooled, filtered, and a reference medicinal material solution is obtained.

[0029] After the reference substances and the reference medicinal material solution are subjected to pre-column derivatization reaction, high performance liquid chromatography is used for determination, and chromatograms of the reference substances and the reference medicinal material solution are obtained; and the components of the HPLC characteristic chromatogram of the water buffalo horn medicinal material, decoction piece, standard decoction and formula granules are qualitatively determined according to the chromatograms of the reference substances and the reference medicinal material solution.

[0030] The pre-column derivatization of the present application specifically includes: reaction with o-phthalaldehyde solution in a borate buffer solution with pH 10.2; the concentration of the borate buffer solution is 0.4 mol / L; the preparation of the o-phthalaldehyde solution includes: taking o-phthalaldehyde, dissolving it in a 0.4 mol / L borate buffer solution-acetonitrile-3-mercaptopropionic acid mixed solution, and preparing a solution containing 10 mg per 1 ml; the volume ratio of the borate buffer solution-acetonitrile-3-mercaptopropionic acid mixed solution is 7:1:0.125.

[0031] In some preferred embodiments, computer simulation pre-column derivatization determination is achieved by using o-phthalaldehyde (OPA) solution.

[0032] Note: 0.4 mol / L borate buffer solution (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, and adjust the pH value to 10.2 with 40% sodium hydroxide solution, and obtain (the liquid needs to be refrigerated).

[0033] O-phthalaldehyde (OPA) solution: take o-phthalaldehyde, dissolve it in a 0.4 mol / L borate buffer solution-acetonitrile-3-mercaptopropionic acid mixed solution (7:1:0.125), and prepare a solution containing 10 mg per 1 ml.

[0034] In some preferred embodiments, the pre-column derivatization reaction: the test sample solution and the reference substance solution are reacted with o-phthalaldehyde (OPA) solution in a borate buffer solution with pH 10.2.

[0035] In one of the preferred embodiments, the pre-column derivatization procedure (bottle 1-3 are OPA solution, water, borate buffer solution respectively): 2.5 μl (borate buffer solution) is taken from bottle 3; 1 μl of sample is taken; 0 μl (water, clean the needle) is taken from bottle 2; 3.5 μl is mixed in air for 5 times; 0.2 min is waited; 0.5 μl (OPA solution) is taken from bottle 1; 0 μl (water, clean the needle) is taken from bottle 2; 4 μl is mixed in air for 10 times; 0.2 min is waited; injection is performed.

[0036] The mobile phase A is acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution, wherein the volume ratio of acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution is 7:9:4, and the pH is adjusted to 7.2 by using 2% glacial acetic acid;

[0037] The mobile phase B is tetrahydrofuran-11 mmol / L sodium acetate aqueous solution, wherein the volume ratio of tetrahydrofuran-11 mmol / L sodium acetate aqueous solution is 3:500, 0.2 μl of triethylamine solution is added to every 1 ml of tetrahydrofuran-11 mmol / L sodium acetate aqueous solution, and the pH is adjusted to 7.2 by using 2% glacial acetic acid.

[0038] The inventors find that, by using the above mobile phase, the chromatogram baseline is stable, and the chromatographic peaks are more and the peak shape is better.

[0039] The gradient elution is preferably specifically as follows:

[0040] 0-10 min, A phase: 2%→10%, B phase: 98%→90%;

[0041] 10-21 min, A phase: 10%→11%, B phase: 90%→89%;

[0042] 21-55 min, A phase: 11%→70%, B phase: 89%→30%.

[0043] The baseline separation is good, the separation degree of each peak is good, and the baseline is stable under the above elution gradient.

[0044] The C18 column has a specification of 2.7 μm, 2.1×150 mm, and the column temperature is 20℃.

[0045] The flow rate of the mobile phase is preferably 0.5 ml / min.

[0046] It is found that the separation of each chromatographic peak is better, and the peak shape is more symmetrical under the flow rate of 0.5 ml / min, which is the most preferred scheme.

[0047] The detection wavelength is preferably 334 nm.

[0048] The present inventors find that the chromatographic information is rich at 334 nm, each component has good absorption, the response value is moderate, the separation degree of each peak is good, and the baseline is smooth.

[0049] The injection amount of the present application is preferably 1 muL.

[0050] The present application has the beneficial effect that the similarity of the HPLC characteristic spectrum of the water buffalo horn medicinal material, the standard decoction and the formula granules is evaluated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the HPLC standard characteristic spectrum of the water buffalo horn medicinal material, the standard decoction and the formula granules is obtained, wherein peak 2 is glycine, peak 4 (S) is alanine, peak 5 is serine, peak 6 is aspartic acid, peak 7 is glutamic acid, peak 8 is threonine, and peak 9 is valine.

[0051] In the water buffalo horn standard decoction characteristic spectrum, alanine is taken as the reference peak S peak, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified value is 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8) and 1.71 (peak 9) respectively.

[0052] The quality judgment standard is that the water buffalo horn medicinal material, the standard decoction and the formula granules samples are taken, the above-mentioned same method is operated, the characteristic spectrum of the water buffalo horn medicinal material, the standard decoction and the formula granules is obtained, the national pharmacopoeia committee's traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition is used to analyze the standard characteristic spectrum and the sample characteristic spectrum of the water buffalo horn medicinal material, the standard decoction and the formula granules, and the similarity is greater than 0.90.

[0053] The method provided by the present application can effectively monitor the quality of different batches of water buffalo horn medicinal material, standard decoction and formula granules, so that the quality is stable, and the method has the characteristics of high precision and good reproducibility, and is beneficial to comprehensively monitor the quality of products.

[0054] The characteristic spectrum of the water buffalo horn medicinal material, the standard decoction and the formula granules established by the present application takes alanine as the reference, focuses on the order of each characteristic peak and the correlation with the medicinal material and the intermediate product, can comprehensively evaluate the overall quality characteristics of the product, and the method is scientific and reliable.

[0055] The newly established characteristic spectrum method of the present application can detect the components with relatively large polarity in the water buffalo horn and the standard decoction thereof, and the sample preparation method is simple and easy to operate, and the identified characteristic peaks are relatively more. The water buffalo horn and its preparation can be accurately and reliably detected by the characteristic spectrum. The authenticity and the consistency and stability of the quality of the water buffalo horn and its preparation can be effectively detected and controlled. The present application provides a basis for effectively controlling and more comprehensively evaluating the quality of the water buffalo horn standard decoction. The quality of the water buffalo horn and the standard decoction thereof is ensured to be uniform and stable.

[0056] This invention provides a method for constructing HPLC characteristic chromatograms of buffalo horn medicinal material, standard decoction, and its formulation granules, comprising: A) extracting buffalo horn raw material with a solvent to obtain a test solution; B) subjecting the test solution to a pre-column derivatization reaction, and then determining it by high-performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatogram of the buffalo horn raw material; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution, and mobile phase B is tetrahydrofuran-11 mmol / L sodium acetate aqueous solution, with gradient elution. This invention uses HPLC with the above-mentioned mobile phases for gradient elution, using alanine as a reference, to establish HPLC characteristic chromatograms of buffalo horn medicinal material, standard decoction, and its formulation granules. The method exhibits good repeatability and precision, is stable and reliable, and can control the quality of buffalo horn medicinal material, standard decoction, and its formulation granules. Attached Figure Description

[0057] Figure 1 Sample preparation method investigation;

[0058] Figure 2 Investigation of extraction solvents;

[0059] Figure 3 Investigation of solvent addition amount;

[0060] Figure 4 Investigation of solvent addition amount;

[0061] Figure 5 Chromatographic peak identification;

[0062] Figure 6 Investigations using different chromatographic columns;

[0063] Figure 7 Characteristic Atlas of Buffalo Horn Medicinal Materials - 1;

[0064] Figure 8 Characteristic Atlas of Buffalo Horn Medicinal Materials - 2;

[0065] Figure 9 Comparative characteristic atlas of medicinal materials;

[0066] Figure 10 Chromatographic peak identification;

[0067] Figure 11 Investigations using different chromatographic columns;

[0068] Figure 12 Characteristic spectrum of standard buffalo horn decoction;

[0069] Figure 13 Characteristic spectrum of standard buffalo horn decoction;

[0070] Figure 14 Compare with the characteristic maps;

[0071] Figure 15 Water buffalo horn formula granules of different wavelength chromatogram;

[0072] Figure 16 Mobile phase selection;

[0073] Figure 17 Column temperature investigation;

[0074] Figure 18 Flow rate investigation;

[0075] Figure 19 Delay investigation;

[0076] Figure 20 Sample preparation method investigation;

[0077] Figure 21 Extraction solvent investigation;

[0078] Figure 22 Solvent addition amount investigation;

[0079] Figure 23 Extraction time investigation;

[0080] Figure 24 Chromatographic peak identification;

[0081] Figure 25 Different chromatographic column investigation;

[0082] Figure 26 Water buffalo horn formula granules (SNJ-KL01) characteristic map;

[0083] Figure 27 Water buffalo horn formula granules (SNJ-KL02) characteristic map;

[0084] Figure 28 Water buffalo horn formula granules (SNJ-KL03) characteristic map;

[0085] Figure 29 Control characteristic map;

[0086] Figure 30 The chromatogram of Comparative Example 1;

[0087] Figure 31 The chromatogram of Comparative Example 2;

[0088] Figure 32 The superimposed chromatogram of the formula granules without negative interference in the present experimental study. DETAILED DESCRIPTION

[0089] The application provides a water buffalo horn medicinal material, a standard decoction and a preparation granule HPLC characteristic spectrum construction method. Those skilled in the art can refer to the content herein and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they all belong to the protection scope of the application. The method and application of the application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technical solution of the application.

[0090] In order to further illustrate the application, the application provides a water buffalo horn medicinal material, a standard decoction and a preparation granule HPLC characteristic spectrum construction method, which are described in detail below in combination with embodiments.

[0091] Example 1: Water buffalo horn medicinal material HPLC characteristic spectrum

[0092] 2.2.1.1 Instruments and materials

[0093] High liquid chromatograph: Agilent 1260 type liquid chromatograph;

[0094] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0095] Ultra-pure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0096] Ultrasonic cleaner: KQ600DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0097] Chromatographic column: chromatographic column 1 (ZORBAX Extend-C18 2.1*150mm, 2.7μm), chromatographic column 2 (ZORBAX SB-C18 2.1*150mm, 2.7μm), chromatographic column 3 (UHPLC XB-C18 2.1*150mm, 2.7μm); Chromatographic column: chromatographic column 1 (ZORBAX Extend-C18 2.1*150mm, 2.7μm), chromatographic column 2 (ZORBAX SB-C18 2.1*150mm, 2.7μm), chromatographic column 3 (UHPLC XB-C18 2.1*150mm, 2.7μm);

[0098] Acetonitrile, methanol, tetrahydrofuran, glacial acetic acid are chromatographically pure, water is ultra-pure water, and the rest of the reagents are analytically pure.

[0099] Glycine (China Food and Drug Inspection Research Institute, batch number: 140689-202207, purity: 100.0%)

[0100] Alanine (China Food and Drug Inspection Research Institute, batch number: 140680-202206, purity: 99.9%)

[0101] Buffalo horn control medicinal materials (batch number: ycwkq23042504, Sichuan Weikeqi Biotechnology Co., Ltd.)

[0102] Buffalo horn medicinal materials batch number: SNJ-YC-01, SNJ-YC-02, SNJ-YC-03, SNJ-YC-04, SNJ-YC-05, SNJ-YC-06, SNJ-YC-07, SNJ-YC-08, SNJ-YC-09, SNJ-YC-10, SNJ-YC-11, SNJ-YC-12, SNJ-YC-13, SNJ-YC-14, SNJ-YC-15, SNJ-YC-16, SNJ-YC-17, SNJ-YC-18, SNJ-YC-19.

[0103] According to the reference buffalo horn standard decoction characteristic chromatogram methodological investigation results, the characteristic chromatogram analysis method of buffalo horn medicinal materials is as follows:

[0104] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjust PH to 7.2 with 2% ice acetic acid) (7:9:4) as mobile phase A, tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (add 0.2 μl of triethylamine solution to 1 ml, mix, adjust PH to 7.2 with 2% ice acetic acid) (3:500) as mobile phase B, gradient elution according to the following table; the flow rate is 0.5 ml per minute; the column temperature is 20℃; the detection wavelength is 334 nm. The theoretical plate number should not be less than 5000 calculated by glycine peak.

[0105]

[0106] Preparation of reference solution: take 2g of buffalo horn control medicinal materials, put it in a conical flask with a plug, add 25% methanol 25ml, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, take the filtrate, as the reference solution of control medicinal materials. Take appropriate amount of glycine and alanine reference substance, accurately weigh, add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml, as the reference solution of control substance.

[0107] Preparation of test solution: take about 2g of buffalo horn medicinal material powder (pass through No. 3 sieve), accurately weigh, put it in a conical flask with a plug, accurately add 25% methanol 25ml, tightly plug, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with 25% methanol, shake well, filter, take the filtrate, which is obtained.

[0108] Inject 1 μl of the sample solution and 1 μl of the reference solution into the liquid chromatograph, and determine the content of the sample by calculating the relative retention time of each characteristic peak in the sample chromatogram with respect to the S peak in the reference chromatogram.

[0109] Note: 0.4 mol / L borate buffer (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, and adjust the pH to 10.2 with 40% sodium hydroxide solution to obtain the solution (the solution needs to be refrigerated).

[0110] OPA solution: take o-phenylenediamine, dissolve in a 0.4 mol / L borate buffer-acetonitrile-3-mercaptopropionic acid mixed solution (7:1:0.125) to prepare a solution containing 10 mg per 1 ml.

[0111] Pre-column derivatization reaction: the sample solution and the reference solution are reacted and derivatized with the o-phenylenediamine (OPA) solution in the borate buffer solution at pH 10.2.

[0112] Pre-column derivatization program (bottle 1~bottle 3 are OPA solution, water, and borate buffer solution respectively): take 2.5 μl (borate buffer solution) from bottle 3; take 1 μl of sample; take 0 μl of water (clean the needle) from bottle 2; mix 3.5 μl in the air for 5 times; wait for 0.2 min; take 0.5 μl of OPA solution from bottle 1; take 0 μl of water (clean the needle) from bottle 2; mix 4 μl in the air for 10 times; wait for 0.2 min; inject the sample.

[0113] The sample chromatogram should exhibit 9 characteristic peaks, which should correspond to the retention times of the 9 characteristic peaks in the reference chromatogram of the control drug material, wherein peak 2 and peak 4 should correspond to the retention times of the control reference peaks, and the peak corresponding to the alanine control reference peak is the S peak. The relative retention times of the other characteristic peaks with respect to the S peak should be within ±10% of the specified values. The specified values are: 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8), and 1.71 (peak 9).

[0114] 2.2.1.2 Wavelength, column temperature, and flow rate selection

[0115] The characteristic chromatographic conditions and system suitability of the buffalo horn medicinal material are consistent with the characteristic chromatogram method of the buffalo horn standard decoction and buffalo horn formula granules.

[0116] The column was packed with octadecylsilane-bonded silica gel. Mobile phase A consisted of acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjusted to pH 7.2 with 2% glacial acetic acid) (7:9:4), and mobile phase B consisted of tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (0.2 μl triethylamine solution was added per 1 ml, mixed, and adjusted to pH 7.2 with 2% glacial acetic acid) (3:500). Gradient elution was performed according to the specifications in the table below. The flow rate was 0.5 ml / min; the column temperature was 20 °C; and the detection wavelength was 334 nm. The theoretical plate number, calculated based on the glycine peak, should be no less than 5000.

[0117]

[0118] 2.2.1.3 Investigation into the preparation of the test solution

[0119] 2.2.1.3.1 Examination of Sample Preparation Methods

[0120] Take approximately 2.0 g of buffalo horn powder (passed through a No. 3 sieve) (batch number: SNJ-YC-01), place it in a stoppered conical flask, add 25 ml of 25% methanol, seal tightly, and investigate the extraction methods for reflux and ultrasonication. Extraction time is 30 minutes. Cool, shake well, filter, and collect the filtrate. See [link to sample preparation]. Figure 1 . Figure 1 The sample preparation method was investigated.

[0121] The results showed that the chromatographic peaks of ultrasonic extraction and reflux extraction were consistent, therefore, ultrasonic extraction was determined to be the extraction method for the test sample.

[0122] 2.2.1.3.2 Investigation of Extraction Solvents

[0123] Take 2g of buffalo horn powder (batch number: SNJ-YC-01), place it in a stoppered conical flask, add 25ml each of methanol, 50% methanol, 25% methanol, and water, and sonicate (600W power, 40kHz frequency) for 30 minutes. Shake well, filter, and collect the filtrate. See Figure 2 . Figure 2 Extraction solvent investigation; the results showed that the extraction efficiency was higher when the extraction solvent was 25% methanol, and the extraction solvent was tentatively set as 25% methanol.

[0124] 2.2.1.3.3 Investigation of Solvent Addition Amount

[0125] Take 2g of this product powder (batch number: SNJ-YC-01), place it in a stoppered conical flask, add 25ml, 50ml, and 100ml of 25% methanol respectively, sonicate (600W power, 40kHz frequency) for 30 minutes, shake well, filter, and collect the filtrate to obtain the product. See [link to product description]. Figure 3 . Figure 3 Investigation of the amount of solvent added.

[0126] The results show that when the amount of extraction solvent is 25 ml, the peak shape and separation degree are good, and the peak size is appropriate, so the amount of solvent is selected as 25 ml.

[0127] 2.2.1.3.4 Extraction time investigation

[0128] Take 2 g of water buffalo horn medicinal material powder (batch number: SNJ-YC-01) and place it in a conical flask with a plug. Add 25% methanol 25 ml and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes and 60 minutes respectively. Shake well, filter, and take the filtrate, which is obtained. See Figure 4 . Figure 4 Solvent amount investigation. The results show that when the extraction time is 30 minutes, the extraction is complete. Therefore, the extraction time is determined as 30 minutes.

[0129] In summary, the preparation method of the test solution of the characteristic spectrum of water buffalo horn medicinal material is determined as follows: take 2 g of the powder (pass through a No. 3 sieve) of the product, place it in a conical flask with a plug, add 25% methanol 25 ml, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate, which is obtained.

[0130] 2.2.1.4 Methodology investigation

[0131] 2.2.1.4.1 Chromatographic peak identification

[0132] Preparation of test solution: prepare the test solution of water buffalo horn medicinal material according to the above proposed experimental conditions.

[0133] Preparation of reference solution: take 2 g of water buffalo horn control medicinal material, place it in a conical flask with a plug, add 25% methanol 25 ml, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the control medicinal material. Take appropriate amounts of glycine reference substance and alanine reference substance, accurately weigh, add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml as the reference solution of the reference substance.

[0134] Preparation of negative control solution: prepare the negative control solution of water buffalo horn medicinal material according to the above proposed experimental conditions.

[0135] Positioning of the characteristic spectrum peaks of water buffalo horn medicinal material. See Figure 5 . Figure 5 Chromatographic peak identification. The results show that peak 2 is glycine and peak 4 is alanine. In the following methodology investigation, 9 characteristic peaks in the sample were investigated.

[0136] 2.2.1.4.2 Precision test

[0137] Take the test solution of water buffalo horn medicinal material (SNJ-YC-01), according to the proposed experimental method, continuously sample 6 times, 1 μl each time, and calculate the retention time of each characteristic peak. See Table 1.

[0138] Table 1 Precision investigation - retention time

[0139]

[0140] The results show that the retention time RSD of each characteristic peak of the sample is 0.04% to 0.47%, and the instrument precision is good.

[0141] 2.2.1.4.3 Reproducibility investigation

[0142] Precisely weigh 6 portions of water buffalo horn medicinal material (SNJ-YC-01), and prepare and determine according to the proposed experimental method. See Table 2.

[0143] Table 2 Reproducibility investigation - relative retention time

[0144]

[0145] The results show that the relative retention time RSD of the 6 samples is 0.00% to 2.06%, indicating that the method has good reproducibility.

[0146] 2.2.1.4.4 Intermediate precision investigation

[0147] 4.4.1 Investigation of different personnel and different times

[0148] On the basis of the above proposed experimental conditions, different personnel (A, B) at different times (T1, T2) precisely weighed 1 portion of water buffalo horn medicinal material (SNJ-YC-01) each, prepared the test sample, and determined. See Table 3.

[0149] Table 3 Personnel and time investigation - relative retention time

[0150]

[0151] The results show that the relative retention time RSD of each characteristic peak is 0.00% to 2.78% when the same sample is determined by different personnel at different times, and the method has good stability.

[0152] 2.2.1.4.5 Column durability investigation

[0153] On the basis of the above proposed experimental conditions, column 1, column 2, and column 3 were used for analysis investigation, and the results are shown in Table 4. Figure 6 . Figure 6 Different column investigation.

[0154] The results showed that when using the three chromatographic columns to detect the samples, column 2 did not produce any peaks, and peaks 3 and 4 of column 1 were not separated. This method exhibits poor column robustness, while column 3 showed better resolution. Column 3 is recommended for use. UHPLCXB-C182.1*150mm, 2.7μm).

[0155] 2.2.1.4.6 Stability Assessment

[0156] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 6h, 12h, 18h, and 24h. See Table 4.

[0157] Table 4 Stability Study - Retention Time

[0158]

[0159] The results showed that the RSD of the retention time of the characteristic peak was between 0.64% and 3.42%, and the sample solution was stable within 24 hours.

[0160] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above nine characteristic peaks will be included in subsequent investigations.

[0161] 2.2.1.4.7 Determination of characteristic peaks and establishment of reference spectra

[0162] Using this method, characteristic spectral analysis was performed on 19 batches of buffalo horn medicinal material samples, and the relative retention time and relative peak area ratio were calculated. (See...) Figure 7-8 Table 5. Figure 7 Characteristic spectrum of buffalo horn medicinal material-1; Peak 2: glycine; Peak 4 (S): alanine (from bottom to top S1-S10 are: SNJ-YC-01, SNJ-YC-02, SNJ-YC-03, SNJ-YC-04, SNJ-YC-05, SNJ-YC-06, SNJ-YC-07, SNJ-YC-08, SNJ-YC-09, SNJ-YC-10); Figure 8 Characteristic spectrum of buffalo horn medicinal material - 2; Peak 2: glycine; Peak 4 (S): alanine. (From bottom to top, S1-S9 are: SNJ-YC-11, SNJ-YC-12, SNJ-YC-13, SNJ-YC-14, SNJ-YC-15, SNJ-YC-16, SNJ-YC-17, SNJ-YC-18, SNJ-YC-19)

[0163] Table 5. Relative retention time of medicinal material characteristic maps

[0164]

[0165] According to the principle of relative retention time stability and each batch sample can be detected and the peak is relatively high, a total of 9 peaks with better durability are selected as characteristic peaks. According to the results of methodological investigation and 19 batches of verification results, the theoretical plate number is tentatively determined to be not less than 5000 according to the glycine peak.

[0166] Finally, the final provision is that the test sample chromatogram should present 9 characteristic peaks, which should correspond to the retention time of the 9 characteristic peaks in the reference material chromatogram of the control drug, among which peak 2 and peak 4 should correspond to the retention time of the corresponding control reference peaks. The peak corresponding to the alanine control reference peak is the S peak, and the relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value. The specified value is: 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.71 (peak 9).

[0167] The 19 batches of water buffalo horn medicinal materials were synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of water buffalo horn medicinal material characteristic chromatogram was established. See Figure 9 . Figure 9 Control characteristic chromatogram; peak 2: glycine; peak 4 (S): alanine

[0168] In summary, the characteristic chromatogram method is:

[0169] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjusted to pH 7.2 with 2% ice acetic acid) (7:9:4) as mobile phase A, tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (0.2 μl of triethylamine solution was added to 1 ml, mixed, and the pH was adjusted to 7.2 with 2% ice acetic acid) (3:500) as mobile phase B, gradient elution was carried out according to the following table; the flow rate was 0.5 ml per minute; the column temperature was 20℃; the detection wavelength was 334 nm. The theoretical plate number should be not less than 5000 according to the glycine peak.

[0170]

[0171] Preparation of reference solution: take 2 g of water buffalo horn control drug, put it in a conical flask with plug, add 25% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, take the filtrate as the reference solution of the control drug. Take appropriate amount of glycine reference substance and alanine reference substance, accurately weigh, add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml as the reference solution of the control substance.

[0172] The test solution was prepared by precisely adding 25% methanol 25 ml to 2 g of the powder (passed through a No. 3 sieve) in a conical flask with a stopper, tightly stoppering, weighing, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling, re-weighing, making up the weight loss with 25% methanol, shaking, filtering, and taking the filtrate to obtain the test solution.

[0173] The determination method was as follows: 1 μl of the control solution and the test solution was precisely taken and injected into the liquid chromatograph, and the pre-column derivatization was determined by using o-phthalaldehyde (OPA) solution and computer simulation.

[0174] Note: 0.4 mol / L borate buffer (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, and adjust the pH to 10.2 with 40% sodium hydroxide solution to obtain the solution (the solution needs to be refrigerated).

[0175] O-phthalaldehyde (OPA) solution: take o-phthalaldehyde, dissolve in a 0.4 mol / L borate buffer-acetonitrile-3-mercapto propionic acid mixed solution (7:1:0.125) to prepare a solution containing 10 mg per 1 ml.

[0176] Pre-column derivatization reaction: the test solution and the control solution were reacted and derivatized with o-phthalaldehyde (OPA) solution in a borate buffer at pH 10.2.

[0177] Pre-column derivatization program (bottle 1~bottle 3 are OPA solution, water, and borate buffer, respectively): 2.5 μl (borate buffer) was taken from bottle 3; 1 μl of sample was taken; 0 μl (water, needle cleaning) was taken from bottle 2; 3.5 μl was mixed in the air for 5 times; 0.2 min was waited; 0.5 μl (OPA solution) was taken from bottle 1; 0 μl (water, needle cleaning) was taken from bottle 2; 4 μl was mixed in the air for 10 times; 0.2 min was waited; and the sample was injected.

[0178] Nine characteristic peaks should be present in the test chromatogram, and should correspond to the retention times of the nine characteristic peaks in the control drug reference chromatogram, among which peaks 2 and 4 should correspond to the retention times of the control reference peaks, and the peak corresponding to the alanine control reference peak is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, which should be within ±10% of the specified value. The specified value is: 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8), and 1.71 (peak 9). See Figure 9 .

[0179] Example 2 Standard Water Buffalo Horn Decoction HPLC Characteristic Chromatogram

[0180] 2.2.2.1 Experimental instruments and materials

[0181] Buffalo horn standard decoction (SNJ-BT01, SNJ-BT02, SNJ-BT03, SNJ-BT04, SNJ-BT05, SNJ-BT06, SNJ-BT07, SNJ-BT08, SNJ-BT09, SNJ-BT10, SNJ-BT11, SNJ-BT12, SNJ-BT13, SNJ-BT14, SNJ-BT15, SNJ-BT16, SNJ-BT17, SNJ-BT18, SNJ-BT19).

[0182] The rest is the same as in Example 1

[0183] Chromatographic conditions and system suitability test with octadecylsilane bonded silica gel as the filler; with acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (pH 7.2 adjusted with 2% glacial acetic acid) (7:9:4) as mobile phase A, and tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (0.2 μl of triethylamine solution was added per 1 ml, mixed, and the pH was adjusted to 7.2 with 2% glacial acetic acid) (3:500) as mobile phase B, gradient elution was carried out according to the following table; the flow rate was 0.5 ml per minute; the column temperature was 20°C; the detection wavelength was 334 nm. The theoretical plate number should not be less than 5000 calculated by glycine peak.

[0184]

[0185] Preparation of reference solution: Take 2 g of buffalo horn control medicinal material, place it in a conical flask with a plug, add 25% methanol 25 ml, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, take the filtrate, as the reference solution of the control medicinal material. Take appropriate amount of glycine and alanine reference substance, accurately weigh, add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml, as the reference solution of the control substance.

[0186] Preparation of test solution: Take 1 g of the product, place it in a conical flask with a plug, add 25% methanol 50 ml, tightly plug, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, and it is obtained.

[0187] Determination method: accurately pipette 1 μl of the control solution and the test solution respectively, inject into the liquid chromatograph, and use o-phenylenediamine (OPA) solution to calculate the pre-column derivatization by computer simulation.

[0188] Note: 0.4 mol / L borate buffer (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, adjust the pH value to 10.2 with 40% sodium hydroxide solution, and it is obtained (this solution needs to be refrigerated).

[0189] O-Phthalaldehyde (OPA) solution: Take O-Phthalaldehyde, dissolve in 0.4 mol / L borate buffer-acetonitrile-3-mercaptopropionic acid mixed solution (7:1:0.125), prepare a solution containing 10 mg per 1 ml.

[0190] Pre-column derivatization reaction: The test solution and the control solution were reacted with O-Phthalaldehyde (OPA) solution in borate buffer solution at pH 10.2.

[0191] Pre-column derivatization procedure (bottle 1~bottle 3 are OPA solution, water, borate buffer solution respectively): Take 2.5 μl (borate buffer solution) from bottle 3; take 1 μl of sample; take 0 μl (water, clean the needle) from bottle 2; mix 3.5 μl in air, 5 times; wait for 0.2 min; take 0.5 μl (OPA solution) from bottle 1; take 0 μl (water, clean the needle) from bottle 2; mix 4 μl in air, 10 times; wait for 0.2 min; inject.

[0192] 2.2.2.2 Chromatographic conditions and system suitability test

[0193] The substance composition of the standard decoction and the finished granules is consistent, and the characteristic chromatographic conditions and system suitability and the granule characteristic spectrum method are consistent.

[0194] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjust pH to 7.2 with 2% glacial acetic acid) (7:9:4) as mobile phase A, tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (add 0.2 μl of triethylamine solution per 1 ml, mix well, adjust pH to 7.2 with 2% glacial acetic acid) (3:500) as mobile phase B, gradient elution according to the following table; flow rate is 0.5 ml per minute; column temperature is 20℃; detection wavelength is 334 nm. The theoretical plate number calculated according to the glycine peak should not be less than 5000.

[0195]

[0196] 2.2.2.3 Preparation of test solution

[0197] The substance composition of the standard decoction and the finished granules is consistent, and the test solution preparation method is consistent with the finished granules. The test solution preparation method is as follows: take 1 g of water buffalo horn standard decoction, place it in a conical flask with a plug, add 50 ml of 25% methanol, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, which is obtained.

[0198] 2.2.2.4 Methodology review

[0199] 2.2.2.4.1 Chromatographic Peak Identification

[0200] Preparation of the test solution: Prepare the buffalo horn standard decoction test solution according to the experimental conditions proposed above.

[0201] Preparation of reference solution: Take 2g of buffalo horn reference material, place it in a stoppered conical flask, add 25ml of 25% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of glycine reference standard and alanine reference standard, accurately weigh them, and add 25% methanol to prepare a mixed solution containing 16μg of each per ml, as the reference solution.

[0202] Preparation of negative control solution: Prepare negative control solution of water-deficient ox horn standard decoction according to the experimental conditions proposed above.

[0203] The characteristic peaks of the standard buffalo horn decoction were located. (See...) Figure 10 . Figure 10 Chromatographic peak identification. Results showed that peak 2 was glycine and peak 4(S) was alanine. Nine characteristic peaks in the sample were investigated in the following methodological studies.

[0204] 2.2.2.4.2 Precision Test

[0205] Take the standard decoction of buffalo horn (batch number: SNJ-BT01) as the test sample solution, and inject it 6 times consecutively according to the proposed experimental method, 1 μl each time, and calculate the retention time of each characteristic peak. See Table 6.

[0206] Table 6 Precision Examination - Retention Time

[0207]

[0208] The results showed that the retention time RSD of each characteristic peak was 0.05% to 0.29%, indicating that the instrument has good precision.

[0209] 2.2.2.4.3 Repeatability Test

[0210] Accurately weigh 6 portions of the standard buffalo horn decoction (batch number: SNJ-BT01), and prepare and determine the solution according to the proposed experimental method. See Table 7.

[0211] Table 7 Repeatability Tests - Relative Retention Time

[0212]

[0213]

[0214] The results showed that the relative retention time RSD of 6 samples was 0.00%~2.04%, indicating that the method had good repeatability.

[0215] 2.2.2.4.4 Intermediate precision investigation

[0216] 4.4.1 Different personnel and time investigation

[0217] On the basis of the above experimental conditions, different personnel (A, B) at different times (T1, T2) respectively precision weighing of buffalo horn standard decoction (batch number: SNJ-BT01) 1, preparation of test sample, determination. See Table 8.

[0218] Table 8 Personnel and time investigation- relative retention time

[0219]

[0220] The results showed that the determination of the same sample by different personnel at different times, the RSD of the relative retention time of each characteristic peak was 0.44%~2.78%, and the method had good stability.

[0221] 2.2.2.4.5 Column durability investigation

[0222] On the basis of the above experimental conditions, the analysis investigation was carried out by using chromatographic column 1, chromatographic column 2 and chromatographic column 3 respectively, and the results were shown in Table 10. Figure 11 . Figure 11 Different chromatographic column investigation; the results showed that the detection of samples by using the above three chromatographic columns, chromatographic column 2 did not peak, chromatographic column 1 peak 3 and peak 4 were not separated, the method had poor chromatographic column durability, and chromatographic column 3 had good separation degree; it was recommended to use chromatographic column 3 UHPLC XB-C18 2.1*150mm, 2.7μm.

[0223] 2.2.2.4.6 Stability investigation

[0224] On the basis of the above experimental conditions, the same test sample solution was taken and determined at 0h, 4h, 8h, 12h, 16h and 24h respectively. See Table 9.

[0225] Table 9 Stability investigation- retention time

[0226]

[0227] The results showed that the RSD of the retention time of the characteristic peak was 0.74%~3.31%, and the sample solution was stable within 24 hours.

[0228] In summary, the RSD of each characteristic peak relative to the retention time meets the requirements in the above investigations, and the method is good. The above 9 characteristic peaks are included in the subsequent investigation.

[0229] 2.2.2.4.7 Determination of characteristic peaks and establishment of reference chromatogram

[0230] 4.7.1 Validation results of 19 batches of buffalo horn standard decoction

[0231] Using this method, 19 batches of samples were analyzed by characteristic chromatogram, and the relative retention time and relative peak area were calculated. See Table 10. Figure 12-13 Figure 12 Characteristic chromatogram of buffalo horn standard decoction; Peak 2: glycine; Peak 4 (S): alanine (S1-S10 from bottom to top: SNJ-BT01, SNJ-BT02, SNJ-BT03, SNJ-BT04, SNJ-BT05, SNJ-BT06, SNJ-BT07, SNJ-BT08, SNJ-BT09, SNJ-BT10); Figure 13 Characteristic chromatogram of buffalo horn standard decoction; Peak 2: glycine; Peak 4 (S): alanine; (S1-S9 from bottom to top: SNJ-BT11, SNJ-BT12, SNJ-BT13, SNJ-BT14, SNJ-BT15, SNJ-BT16, SNJ-BT17, SNJ-BT18, SNJ-BT19)

[0232] Table 10 Relative retention time of buffalo horn standard decoction

[0233]

[0234]

[0235] According to the principle of stable relative retention time and detectable peaks in each batch of samples, a total of 9 peaks with good durability were selected as characteristic peaks. According to the results of methodological investigation and validation of 19 batches of standard decoction, the theoretical plate number was tentatively determined to be not less than 5000 calculated by the glycine peak.

[0236] Finally, it is specified that 9 characteristic peaks should be present in the test sample chromatogram, and should correspond to the retention time of the 9 characteristic peaks in the reference material chromatogram of the control drug, among which peak 2 and peak 4 should correspond to the retention time of the corresponding control reference peak. The peak corresponding to the alanine control reference peak is the S peak, and the relative retention time of the remaining characteristic peaks to the S peak should be within ±10% of the specified value. The specified value is: 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8), 1.71 (peak 9). ​

[0237] The 19 batches of buffalo horn standard decoction were synthesized by using Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and the control chromatogram of buffalo horn standard decoction characteristic chromatogram was established. See Figure 14 . Figure 14 Control characteristic chromatogram; peak 2: glycine; peak 4 (S): alanine

[0238] In summary, the characteristic chromatogram method is:

[0239] Chromatographic conditions and system suitability test With octadecylsilane bonded silica gel as the filler; with acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjust pH to 7.2 with 2% ice acetic acid) (7:9:4) as mobile phase A, and tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (add 0.2 μl of triethylamine solution to 1 ml, mix well, and adjust pH to 7.2 with 2% ice acetic acid) (3:500) as mobile phase B, gradient elution was carried out according to the following table; the flow rate was 0.5 ml per minute; the column temperature was 20°C; the detection wavelength was 334 nm. The theoretical plate number should not be less than 5000 calculated by the glycine peak.

[0240]

[0241] Preparation of reference solution Take 2 g of buffalo horn control medicinal materials, place them in a conical flask with a plug, add 25% methanol 25 ml, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, take the filtrate, and use it as the reference solution of the control medicinal materials. Take appropriate amount of glycine and alanine reference substances, accurately weigh, add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml, and use it as the reference solution of the control substances.

[0242] Preparation of test solution Take about 1 g of the product, accurately weigh, place it in a conical flask with a plug, accurately add 25% methanol 50 ml, tightly plug, weigh, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 25% methanol, shake well, filter, and take the filtrate.

[0243] Determination method: accurately take 1 μl of the control substance solution and the test solution, respectively, inject into the liquid chromatograph, and use o-phthaldehyde (OPA) solution for computer simulation of pre-column derivatization determination.

[0244] Note: 0.4 mol / L borate buffer (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, adjust the pH value to 10.2 with 40% sodium hydroxide solution, and obtain (the solution needs to be refrigerated).

[0245] O-Phthalaldehyde (OPA) solution: Take O-Phthalaldehyde, dissolve in 0.4 mol / L borate buffer-acetonitrile-3-mercaptopropionic acid mixed solution (7:1:0.125), prepare a solution containing 10 mg per 1 ml.

[0246] Pre-column derivatization reaction: The test sample solution and the control sample solution were reacted with O-Phthalaldehyde (OPA) solution in borate buffer solution at pH 10.2 for derivatization.

[0247] Pre-column derivatization procedure (bottle 1~bottle 3 are OPA solution, water, borate buffer solution respectively): Take 2.5 μl (borate buffer solution) from bottle 3; take 1 μl of sample; take 0 μl (water, clean the needle) from bottle 2; mix 3.5 μl in the air, 5 times; wait for 0.2 min; take 0.5 μl (OPA solution) from bottle 1; take 0 μl (water, clean the needle) from bottle 2; mix 4 μl in the air, 10 times; wait for 0.2 min; inject. See the chromatogram in Figure 14 .

[0248] Example 3 HPLC characteristic chromatogram of water buffalo horn formula granules

[0249] 2.2.3.1 Experimental instruments and materials

[0250] Water buffalo horn formula granules (Sichuan Xinglve Pharmaceutical Science and Technology Development Co., Ltd., batch numbers SNJ-KL01, SNJ-KL02, SNJ-KL03)

[0251] The rest is the same as in Example 1

[0252] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (adjust pH to 7.2 with 2% glacial acetic acid) (7:9:4) as mobile phase A, tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (add 0.2 μl of triethylamine solution per 1 ml, mix well, adjust pH to 7.2 with 2% glacial acetic acid) (3:500) as mobile phase B, gradient elution according to the following table; the flow rate was 0.5 ml per minute; the column temperature was 20℃; the detection wavelength was 334 nm. The theoretical plate number should not be less than 5000 calculated by the glycine peak.

[0253]

[0254] Preparation of reference solution: Take the water buffalo horn medicinal material 2 g, and put it in a conical flask with a plug. Add 25% methanol 25 ml, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Shake well, filter, and take the filtrate as the reference solution of the control medicinal material. Take glycine and alanine control substances, accurately weigh, and add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml, which is used as the reference solution of the control substance.

[0255] Preparation of test solution: Take the product, grind finely, and take 1 g. Put it in a conical flask with a plug, add 25% methanol 50 ml, tightly plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0256] Determination method: accurately take 1 μl of the control substance solution and the test solution, respectively, and inject into the liquid chromatograph. Use the o-phthalaldehyde (OPA) solution to determine by computer simulation of pre-column derivatization.

[0257] Note: 0.4 mol / L borate buffer solution (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, and adjust the pH value to 10.2 with 40% sodium hydroxide solution, which is obtained (the solution needs to be refrigerated).

[0258] O-phthalaldehyde (OPA) solution: take o-phthalaldehyde, dissolve in a 0.4 mol / L borate buffer solution-acetonitrile-3-mercapto propionic acid mixed solution (7:1:0.125), and prepare a solution containing 10 mg per 1 ml.

[0259] Pre-column derivatization reaction: the test solution and the control substance solution are reacted and derivatized with the o-phthalaldehyde (OPA) solution in the borate buffer solution at pH 10.2.

[0260] Pre-column derivatization program (bottle 1~bottle 3 are OPA solution, water, and borate buffer solution, respectively): take 2.5 μl (borate buffer solution) from bottle 3; take 1 μl of sample; take 0 μl (water, clean the needle) from bottle 2; mix 3.5 μl in the air for 5 times; wait for 0.2 min; take 0.5 μl (OPA solution) from bottle 1; take 0 μl (water, clean the needle) from bottle 2; mix 4 μl in the air for 10 times; wait for 0.2 min; inject.

[0261] 2.2.3.2 wavelength, column temperature, flow rate, mobile phase selection and delay test

[0262] 2.2.3.2.1 wavelength selection

[0263] On the basis of the experimental conditions, the diode array detector was used to scan the full waveband of the test solution, and the chromatogram of the test solution at 250 nm, 270 nm, 290 nm, 310 nm, and 334 nm was extracted, respectively. See Figure 15 . Figure 15 Water buffalo horn formula granules at different wavelengths chromatogram.

[0264] The results showed that the information amount of the chromatographic peak was larger at the detection wavelength of 334 nm, and the baseline of the chromatogram was more stable, so the detection wavelength was determined as 334 nm.

[0265] 2.2.3.2.2 Mobile phase selection

[0266] On the basis of the experimental conditions, the diode array detector was used to scan the full waveband of the test solution, and the chromatogram of the test solution at 250 nm, 270 nm, 290 nm, 310 nm, and 334 nm was extracted, respectively. See Figure 16 . Figure 16 Mobile phase selection; the results showed that using mobile phase 3 for gradient elution, the baseline of the chromatogram was more stable under this condition, the chromatographic peak was more, and the peak shape was good, so the mobile phase A was acetonitrile-methanol-100 mmol / L sodium acetate solution (adjusted to pH 7.2 with 2% ice acetic acid) (7:9:4), and the mobile phase B was tetrahydrofuran-11 mmol / L sodium acetate solution (0.2 μl of triethylamine solution was added per 1 ml, mixed, and adjusted to pH 7.2 with 2% ice acetic acid) (3:500) for gradient elution as the mobile phase of the determination method.

[0267] 2.2.3.2.3 Column temperature investigation

[0268] Based on the above experimental conditions, the column temperature was investigated at 20°C, 25°C, 30°C and 35°C, with glycine and alanine separation, theoretical plate number and other evaluation indexes. The results are shown in Table 11. Figure 17 Figure 17 Column temperature investigation.

[0269] Table 11 Column temperature investigation - relative retention time

[0270]

[0271] The results show that at a column temperature of 20°C-35°C, glycine and alanine can be effectively detected. The detection column temperature is set at 20°C.

[0272] 2.2.3.2.4 Flow rate investigation

[0273] Based on the above experimental conditions, the flow rate was investigated at 0.4 ml / min, 0.5 ml / min and 0.6 ml / min, with glycine and alanine separation, glycine theoretical plate number and other evaluation indexes. The results are shown in Table 12. Figure 18 Figure 18 Flow rate investigation.

[0274] Table 12 Flow rate investigation - relative retention time

[0275]

[0276] The results show that at a flow rate of 0.4 ml / min-0.6 ml / min, glycine and alanine can be effectively detected. The flow rate is set at 0.5 ml / min.

[0277] 2.2.3.2.5 Delayed investigation

[0278] Based on the above experimental conditions, the delayed test was carried out. The results are shown in Table 13. Figure 19 Figure 19 Delayed investigation; the results show that the sample has no chromatographic peak after 55 minutes, so the sample detection time is set at 55 minutes.

[0279] ​​​Based on the above, the water buffalo horn formula granules characteristic map chromatographic conditions and system adaptability test is determined as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution (with 2% ice acetic acid to adjust PH to 7.2) (7:9:4) as mobile phase A, tetrahydrofuran-11 mmol / L sodium acetate aqueous solution (add 0.2 μl triethylamine solution to 1 ml, mix, adjust PH to 7.2 with 2% ice acetic acid) (3:500) as mobile phase B, gradient elution according to the following table; the flow rate is 0.5 ml per minute; the column temperature is 20°C; the detection wavelength is 334 nm. The theoretical plate number calculated by glycine peak should not be less than 5000.

[0280]

[0281] 2.2.3.3. Investigation of preparation of test solution

[0282] 2.2.3.3.1 Investigation of extraction method

[0283] Take water buffalo horn formula granules (batch number: SNJ-KL01) in an appropriate amount, grind finely, take 1 g, put it in a conical flask with a plug, add 25% methanol 50 ml, tightly plug, and investigate the test product extraction method by reflux and ultrasonic extraction, respectively. The extraction time is 30 minutes, cool down, shake well, filter, and take the filtrate, which is obtained. See Figure 20 . Figure 20 Investigation of sample preparation method. The results show that there is little difference in the effect of reflux and ultrasonic extraction of the test product, and the ultrasonic method is fast and simple, so the test product extraction method is determined as ultrasonic extraction.

[0284] 2.2.3.3.2 Investigation of extraction solvent

[0285] Take water buffalo horn formula granules (batch number: SNJ-KL01) in an appropriate amount, grind finely, take 1 g, put it in a conical flask with a plug, and investigate the test product extraction solvent by adding methanol, 25% methanol, 50% methanol and water respectively, each 50 ml, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate, which is obtained. See Figure 21 . Figure 21 Investigation of extraction solvent; the results show that when the extraction solvent is 25% methanol, the extraction efficiency is higher, and the extraction solvent is temporarily determined as 25% methanol.

[0286] 2.2.3.3.3 Investigation of solvent addition amount

[0287] Take water buffalo horn formula granules (batch number: SNJ-KL01) appropriate amount, grind fine, take 1 g, put in the conical flask with plug, add 25% methanol 25 ml, 50 ml for observation, tightly, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, cool, shake, filter, take the filtrate, that is. See Figure 22 . Figure 22 The solvent addition amount observation; the results show that the extraction solvent addition amount is 50 ml, each chromatographic peak peak shape and separation degree is good, the peak size is appropriate, so the solvent amount is selected 50 ml.

[0288] 2.2.3.3.4 extraction time observation

[0289] Take water buffalo horn formula granules (batch number: SNJ-KL01) appropriate amount, grind fine, take 1.0 g, put in the conical flask with plug, add 25% methanol 50 ml, tightly, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, respectively, the test product extraction time is 30 minutes, 60 minutes for observation, cool, shake, filter, take the filtrate, that is. See Figure 23 . Figure 23 Extraction time observation; the results show that the extraction time is 30 minutes, has been extracted completely. Therefore, the extraction time is determined as 30 minutes.

[0290] In summary, the preparation method of water buffalo horn formula granules test solution is determined as follows: take the product appropriate amount, grind fine, take 1 g, put in the conical flask with plug, add 25% methanol 50 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, cool, shake, filter, take the filtrate, that is.

[0291] 2.2.3.4 methodological investigation

[0292] 2.2.3.4.1 chromatographic peak identification

[0293] Preparation of test solution: according to the above experimental conditions, preparation of water buffalo horn formula granules test solution.

[0294] Reference solution preparation: take water buffalo horn control drug 2 g, put in the conical flask with plug, add 25% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, shake, filter, take the filtrate, as control drug reference solution. Another glycine reference substance, alanine reference substance appropriate amount, accurately weighed, add 25% methanol to prepare a mixture solution containing 16 μg per 1 ml, as reference substance reference solution.

[0295] Preparation of negative control solution: according to the above experimental conditions, preparation of water buffalo horn formula granules negative control solution.

[0296] The characteristic peak of buffalo horn formula granules was located. See Figure 24 . Figure 24 Chromatographic peak assignment; the results showed that peak 2 was glycine and peak 4 (S) was alanine. In the following methodological investigations, 9 characteristic peaks in the sample were investigated.

[0297] 2.2.3.4.2 Precision test

[0298] Buffalo horn formula granules (batch number: SNJ-KL01) were taken for the test sample solution, 6 times of continuous sample injection were carried out according to the proposed experimental method, 1 μl each time, and the retention time and peak area of each characteristic peak were calculated. See Table 13.

[0299] Table 13 Precision investigation - retention time

[0300]

[0301] The results showed that the relative retention time RSD of each characteristic peak of the sample was in the range of 0.03% to 0.20%, indicating that the sample injection precision of the method was good.

[0302] 2.2.3.4.3 Reproducibility investigation

[0303] Buffalo horn formula granules (batch number: SNJ-KL01) were precisely weighed in 6 portions, and prepared and determined according to the proposed experimental method. See Table 14.

[0304] Table 14 Reproducibility investigation - relative retention time

[0305]

[0306] The results showed that the relative retention time RSD of the 6 samples was in the range of 0.84% to 1.32%, indicating that the method had good reproducibility.

[0307] 2.2.3.4.4 Intermediate precision investigation

[0308] 2.2.3.4.4.1 Different personnel and time investigation

[0309] Under the above proposed experimental conditions, 1 portion of buffalo horn formula granules (batch number: SNJ-KL01) was precisely weighed by different personnel (A, B) at different times (T1, T2), the test sample was prepared, and determined. See Table 15.

[0310] Table 15 Personnel and time investigation - relative retention time

[0311]

[0312] The results showed that the RSD of relative retention time of each characteristic peak was 0.45%~2.78% when the same sample was determined by different personnel at different times, indicating that the intermediate precision of the method was good.

[0313] 2.2.3.4.5 Durability of the chromatographic column

[0314] Based on the above experimental conditions, chromatographic column 1, chromatographic column 2 and chromatographic column 3 were used for analysis, respectively, and the results are shown in Table 6. Figure 25 . Figure 25 Investigation of different chromatographic columns.

[0315] The results showed that the chromatographic column 2 did not produce peaks, and the peaks 3 and 4 of the chromatographic column 1 were not separated, indicating that the method had poor durability of the chromatographic column. The chromatographic column 3 had good separation degree. It was recommended to use the chromatographic column 3 (UHPLC XB-C18 2.1*150mm, 2.7μm).

[0316] 2.2.3.4.6 Stability investigation

[0317] Based on the above experimental conditions, the same test solution was taken and determined at 0h, 4h, 8h, 12h, 16h and 24h. See Table 16.

[0318] Table 16 Stability investigation - retention time

[0319]

[0320] The results showed that the RSD of the retention time of the characteristic peaks was 0.59%~3.04%, indicating that the sample solution was stable within 24 hours.

[0321] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, and the method was good. The above 9 characteristic peaks were included in the subsequent investigation.

[0322] 2.2.3.4.7 Determination of characteristic peaks and establishment of control chromatogram

[0323] 2.2.3.4.7.1 Verification results of 3 batches of buffalo horn formula granules

[0324] Using this method, 3 batches of samples were analyzed for characteristic chromatogram, and the relative retention time and relative peak area were calculated. See Table 17. Figure 26-28 . Figure 26 Characteristic chromatogram of buffalo horn formula granules (SNJ-KL01); peak 2: glycine; peak 4 (S): alanine; Figure 27 ​Buffalo horn formula granules (SNJ-KL02) characteristic map, peak 2: glycine; peak 4 (S): alanine; Figure 28 Buffalo horn formula granules (SNJ-KL03) characteristic map, peak 2: glycine; peak 4 (S): alanine.

[0325] Table 17 Relative retention time of buffalo horn formula granules

[0326]

[0327] According to the principle of relative retention time stability and the detection of each batch sample, 9 peaks were selected as characteristic peaks. According to the results of methodological investigation and 3 batches of granules verification, the theoretical plate number was tentatively calculated as not less than 5000 based on the glycine peak.

[0328] Finally, the test sample chromatogram should present 9 characteristic peaks, which should correspond to the relative retention time of the 9 characteristic peaks in the reference material chromatogram of the control drug, among which peak 2 and peak 4 should correspond to the reference peak retention time of the control, and the peak corresponding to the alanine control reference peak is the S peak. The relative retention time of the remaining characteristic peaks to the S peak should be within ±10% of the specified value. The specified value is: 0.36 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.62 (peak 6), 1.64 (peak 7), 1.70 (peak 8), 1.71 (peak 9).

[0329] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 3 batches of buffalo horn formula granules, and the control chromatogram of buffalo horn formula granules characteristic chromatogram was established. See Figure 29 . Figure 29 Control characteristic map; peak 2: glycine; peak 4 (S): alanine

[0330] In summary, the characteristic map method is:

[0331] Chromatographic conditions and system suitability test With octadecylsilane bonded silica gel as the filler; with acetonitrile-methanol-100 mmol / L sodium acetate solution (adjusted to PH 7.2 with 2% glacial acetic acid) (7:9:4) as mobile phase A, and tetrahydrofuran-11 mmol / L sodium acetate solution (add 0.2 μl of triethylamine solution to 1 ml, mix well, adjust PH to 7.2 with 2% glacial acetic acid) (3:500) as mobile phase B, gradient elution according to the following table; flow rate is 0.5 ml per minute; column temperature is 20℃; detection wavelength is 334 nm. The theoretical plate number should be not less than 5000 based on the glycine peak.

[0332]

[0333] Preparation of reference solution: Take 2 g of the control medicinal material of shui-niujiao, and put it into a conical flask with a stopper. Add 25% methanol 25 ml, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Shake well, filter, and take the filtrate as the reference solution of the control medicinal material. Take the glycine control product and alanine control product, accurately weigh, and add 25% methanol to prepare a mixed solution containing 16 μg per 1 ml, which is used as the reference solution of the control product.

[0334] Preparation of test solution: Take shui-niujiao formula granules, grind finely, and accurately weigh about 1 g. Put it into a conical flask with a stopper, accurately add 25% methanol 50 ml, tightly seal, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0335] Determination method: accurately take 1 μl of the control product solution and the test solution respectively, inject into the liquid chromatograph, and use o-phthalaldehyde (OPA) solution to determine by computer simulation of pre-column derivatization.

[0336] Note: 0.4 mol / L borate buffer (pH 10.2): take boric acid, prepare a 0.4 mol / L solution, and adjust the pH value to 10.2 with 40% sodium hydroxide solution, which is obtained (the solution needs to be refrigerated).

[0337] O-phthalaldehyde (OPA) solution: take o-phthalaldehyde, dissolve in a mixed solution of 0.4 mol / L borate buffer-acetonitrile-3-mercapto propionic acid (7:1:0.125), and prepare a solution containing 10 mg per 1 ml.

[0338] Pre-column derivatization reaction: the test solution and the control product solution are reacted and derivatized with o-phthalaldehyde (OPA) solution in borate buffer with pH 10.2.

[0339] Pre-column derivatization program (bottle 1~bottle 3 are OPA solution, water, and borate buffer respectively): take 2.5 μl (borate buffer) from bottle 3; take 1 μl of sample; take 0 μl (water, clean the needle) from bottle 2; mix 3.5 μl in the air for 5 times; wait for 0.2 min; take 0.5 μl (OPA solution) from bottle 1; take 0 μl (water, clean the needle) from bottle 2; mix 4 μl in the air for 10 times; wait for 0.2 min; inject.

[0340] The test sample chromatogram should show 9 characteristic peaks, which should correspond to the retention time of the 9 characteristic peaks in the reference chromatogram of the control drug. Peaks 2 and 4 should correspond to the retention time of the corresponding control reference peaks, respectively. The peak corresponding to the alanine control reference peak is the S peak. The relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value. The specified values are: 0.36 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.62 (peak 6), 1.64 (peak 7), 1.70 (peak 8), and 1.71 (peak 9). See the chromatogram in Figure 29 .

[0341] Comparative Example 1

[0342] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (column length 125 mm, inner diameter 4.6 mm, particle size 5 μm); 8.0 mmol / L phosphate buffer (pH 7.2) containing tetrahydrofuran as mobile phase A, and 8.0 mmol / L phosphate buffer (pH 7.2) + methanol + acetonitrile (50 + 35 + 15) as mobile phase B, gradient elution according to the following table; flow rate 1.0 ml / min; column temperature 40°C; detection wavelength 340 nm. The theoretical plate number should not be less than 5000, calculated based on the glycine peak.

[0343]

[0344] Preparation of test sample solution: accurately weigh 0.5 g of water buffalo horn powder into a hydrolysis bottle, add 10 ml of 6 mol / L hydrochloric acid and 5 drops of freshly distilled phenol, and use high-purity nitrogen to remove the air in the hydrolysis bottle. Sinter the bottle mouth, and hydrolyze in a constant-temperature drying box at 110°C for 24 h. After cooling, filter the hydrolysis solution, transfer the filtrate to a 100 ml volumetric flask, dilute to the mark with water, shake well, accurately take 1 ml into a 10 ml volumetric flask, dilute to the mark with methanol, shake well, filter, and take the filtrate as the test sample solution.

[0345] Determination method: accurately take the test solution (pre-column automatic derivatization program: set the automatic sampler program to sequentially take 1 μl of OPA reagent, 5 μl of 0.1 mol / L pH 10.4 borate buffer solution, and 1 μl of sample. Set different solutions for each injection, wash the needle with distilled water before each injection, and mix online for 5 times. Wait for 1.5 min), inject into the chromatograph, and determine, i.e. the result is obtained.

[0346] The results are shown in Figure 30 , Figure 30 The chromatogram of Comparative Example 1 is shown in Figure 1. Figure 30 From bottom to top, they are the medicinal material, the marker soup, and the granules. Figure 30 As can be seen, the detection method has low reproducibility.

[0347] Comparative Example 2

[0348] Preparation of test solution: 10 g of water buffalo horn medicinal material was weighed, 100 mL of water was added, and reflux extraction was performed for 10 h. Filtration was performed, and the residue was extracted with 100 mL of water by reflux extraction for 10 h. The two extraction solutions were combined, concentrated under reduced pressure to about 100 mL, and placed at 4°C overnight. After filtration, the volume was adjusted to 100 mL to obtain the test solution. Prevail-C18 column (250 mm x 4.6 mm, 5 mm); mobile phase A: 0.025 mg / mL of sodium dihydrogen phosphate solution (5 mL / L of tetrahydrofuran, 40% sodium hydroxide solution was used to adjust the pH to 7.2), and mobile phase B: 0.025 mg / mL of sodium acetate solution-methanol-acetonitrile (500:350:150) (pH 7.2); column temperature: 25°C; detection wavelength: 338 nm for 0-51 min, and 262 nm after 51 min; automatic pre-column derivatization injection program of Agilent 1200 series liquid chromatograph was used, and the automatic derivatization program was as follows: (1) the injection needle absorbed 0 mL of water from bottle 1; (2) 5 mL of borate buffer was absorbed from bottle 3; (3) 0 mL of water was absorbed from bottle 1; (4) 1 mL of OPA was absorbed from bottle 4; (5) 0 mL of water was absorbed from bottle 1; (6) 1 mL of sample was absorbed; (7) 0 mL of water was absorbed from bottle 1; (8) 7 mL was mixed in air for 15 times; (9) 1 mL of FMOC was absorbed from bottle 5; (10) 0 mL of water was absorbed from bottle 1; (11) 8 mL was mixed in air for 20 times; (12) 32 mL of water was absorbed from bottle 2; (13) 20 mL was mixed in air at the maximum speed for 2 times; and (14) injection was performed. Elution gradient and volume flow rate. -1 -1 sodium acetate solution-methanol-acetonitrile (500:350:150) (pH 7.2); column temperature: 25°C; detection wavelength: 338 nm for 0-51 min, and 262 nm after 51 min; automatic pre-column derivatization injection program of Agilent 1200 series liquid chromatograph was used, and the automatic derivatization program was as follows: (1) the injection needle absorbed 0 mL of water from bottle 1; (2) 5 mL of borate buffer was absorbed from bottle 3; (3) 0 mL of water was absorbed from bottle 1; (4) 1 mL of OPA was absorbed from bottle 4; (5) 0 mL of water was absorbed from bottle 1; (6) 1 mL of sample was absorbed; (7) 0 mL of water was absorbed from bottle 1; (8) 7 mL was mixed in air for 15 times; (9) 1 mL of FMOC was absorbed from bottle 5; (10) 0 mL of water was absorbed from bottle 1; (11) 8 mL was mixed in air for 20 times; (12) 32 mL of water was absorbed from bottle 2; (13) 20 mL was mixed in air at the maximum speed for 2 times; and (14) injection was performed. Elution gradient and volume flow rate.

[0349]

[0350] The results are shown in Figure 31 , Figure 31 The chromatogram of Comparative Example 2 is shown in Figure 31 It can be seen that the target peak has negative interference, and the reproducibility is poor, and the column is damaged. However, the method of the present application has no negative interference, as shown in Figure 32 , Figure 32 The chromatogram of the formula granules without negative interference in the present experimental study is shown in the figure. From bottom to top in the figure, the formula granules, the mixed control, and the blank are shown.

[0351] The above only describes the preferred embodiments of the present application, and it should be noted that, for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.​

Claims

1. A method for constructing HPLC characteristic chromatograms of buffalo horn medicinal material, standard decoction, and its formulation granules, comprising: A) The buffalo horn raw material was extracted with solvent to obtain the test solution; the preparation of the test solution of buffalo horn medicine / standard decoction / formulation granules is as follows: the buffalo horn raw material was extracted with 25% methanol by ultrasonic extraction, cooled, shaken, and filtered to obtain the solution; B) After the test solution was subjected to pre-column derivatization reaction, the HPLC characteristic chromatogram of the buffalo horn raw material was obtained by high performance liquid chromatography. The pre-column derivatization specifically includes: derivatization by reacting with phthalaldehyde solution in a borate buffer solution at pH 10.2; The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile-methanol-100 mmol / L sodium acetate aqueous solution with a volume ratio of 7:9:4, and the pH is adjusted to 7.2 with 2% glacial acetic acid; The volume ratio of tetrahydrofuran-11 mmol / L sodium acetate aqueous solution in mobile phase B is 3:

500. Add 0.2 μl of triethylamine solution to each 1 ml of tetrahydrofuran-11 mmol / L sodium acetate aqueous solution, mix well, and adjust the pH to 7.2 with 2% glacial acetic acid. Gradient elution; The gradient elution specifically refers to: 0–10 min, Phase A: 2% → 10%, Phase B: 98% → 90%; 10–21 min, Phase A: 10%–11%, Phase B: 90%–89%; 21–55 min, Phase A: 11%–70%, Phase B: 89%–30%.

2. The method according to claim 1, characterized in that, This also includes the preparation of reference standards and reference drug solutions: Take glycine reference standard and alanine reference standard respectively, and dissolve them in 25% methanol to obtain reference standard solutions; The buffalo horn reference material was dissolved in 25% methanol, sonicated, cooled, and filtered to obtain a reference solution. After pre-column derivatization of the reference standard and reference medicinal material solutions, high-performance liquid chromatography (HPLC) was used to determine the chromatograms of the reference standard and reference medicinal material, respectively. Based on the chromatograms of the reference standard and reference medicinal material, the components of the buffalo horn medicinal material, decoction pieces, standard decoction and its formula granules were qualitatively determined.

3. The method according to claim 1, characterized in that, The chromatographic column is a C18 column with dimensions of 2.7 μm and 2.1 × 150 mm; the column temperature is 20 °C.

4. The method according to claim 3, characterized in that, The mobile phase flow rate is 0.5 mL / min; the detection wavelength is 334 nm; the theoretical plate number, calculated based on the glycine peak, should be no less than 5000; and the injection volume is 1 μL.

5. The method according to claim 3, characterized in that, The concentration of the borate buffer solution is 0.4 mol / L; the preparation of the phthalaldehyde solution includes: dissolving phthalaldehyde in a 0.4 mol / L borate buffer-acetonitrile-3-mercaptopropionic acid mixed solution to prepare a solution containing 10 mg per 1 ml; the volume ratio of the borate buffer solution-acetonitrile-3-mercaptopropionic acid mixed solution is 7:1:0.

125.

6. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of buffalo horn medicinal material, standard decoction and its formulation granules was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The HPLC standard characteristic chromatograms of buffalo horn medicinal material, standard decoction and its formulation granules, consisting of 9 characteristic peaks, were obtained, where peak 2 represents glycine and peak 4 represents alanine. In the characteristic spectrum of the buffalo horn standard decoction, alanine is used as the reference peak S. The relative retention time of each characteristic peak and peak S is calculated. The relative retention time is within ±10% of the specified value. The specified values ​​are: 0.35 (peak 1), 0.63 (peak 3), 1.30 (peak 5), 1.61 (peak 6), 1.63 (peak 7), 1.69 (peak 8), and 1.71 (peak 9).

7. The method according to claim 1, characterized in that, Step A) The buffalo horn raw material is buffalo horn medicinal material, standard decoction and its formula granules.

8. The method according to claim 1, characterized in that, The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 30~40min. The ratio of the mass (g) of the buffalo horn raw material to the volume (mL) of the solvent is 1~2:25~50.

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