A method for constructing HPLC characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and preparations thereof and its application

Abstract

The present invention provides a method and application for high-performance liquid chromatography (HPLC) characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts, and preparations thereof, comprising the following steps: hydrolyzing the dog whip raw material with hydrochloric acid, filtering, evaporating the filtrate, and dissolving the residue with hydrochloric acid to obtain a test solution; derivatizing the test solution and determining it by HPLC to obtain a HPLC characteristic spectrum of the dog whip medicinal materials, decoction pieces, extracts, and preparations thereof; the chromatographic conditions include: a C18 column, mobile phase A of acetonitrile, mobile phase B of 0.2% phosphoric acid solution, and gradient elution. The present invention uses mobile phase A of acetonitrile and mobile phase B of 0.2% phosphoric acid solution for gradient elution, and uses glycine and alanine as reference substances to establish a characteristic spectrum of the dog whip medicinal materials, decoction pieces, extracts, and preparations thereof with good repeatability and precision. The method is stable and reliable, and can control the quality of the dog whip medicinal materials, decoction pieces, extracts, and preparations thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a method for constructing an HPLC characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and preparations thereof, and application thereof. Background Art

[0002] Dog penis is the dried penis and testicles of the canine Canis familiaris Linnaeus. The dog is killed, the penis and testicles are removed, and the attached hair, skin, muscle, and fat are removed. The penis and testicles are then straightened and dried. It has the effects of warming the kidneys, strengthening yang, and improving essence.

[0003] The existing technology basically does not disclose the fingerprint-related technology of dog whip medicinal materials, decoction pieces, extracts, and preparations, but in order to ensure the uniformity of the quality of dog whip medicinal materials, decoction pieces, extracts and preparations, scientific technical means need to be provided. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing an HPLC characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and their preparations and its application. The invention aims to construct a high-performance liquid phase characteristic spectrum method for dog whip medicinal materials, decoction pieces, extracts and their preparations to comprehensively reflect the quality level of dog whip medicinal materials, decoction pieces, extracts and their preparations, and provide guidance for the quality control of dog whip medicinal materials, decoction pieces, extracts and their preparations.

[0005] The present invention provides a method for constructing an HPLC characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and preparations thereof, comprising the following steps:

[0006] The dog penis raw material is hydrolyzed with hydrochloric acid, filtered, the filtrate is evaporated to dryness, and the residue is dissolved with hydrochloric acid to obtain a test solution;

[0007] The test solution is derivatized and then determined by high performance liquid chromatography to obtain high performance liquid chromatography characteristic spectra of dog whip medicinal materials, decoction pieces, extracts and preparations thereof; the chromatographic conditions include: C18 column, mobile phase A is acetonitrile, mobile phase B is 0.2% phosphoric acid solution, and gradient elution.

[0008] The present invention hydrolyzes a raw material of dog penis with hydrochloric acid, filters it, evaporates the filtrate to dryness, and dissolves the residue with hydrochloric acid to obtain a test solution. The concentration of the hydrochloric acid used in the hydrolysis is 3 to 9 mol / L; the hydrolysis temperature is 145 to 155° C., and the hydrolysis time is 1 to 4 hours. In a specific embodiment, the concentration of the hydrochloric acid used in the hydrolysis is 6 mol / L, the hydrolysis temperature is 150° C., and the hydrolysis time is 2 hours.

[0009] The mass of the dog whip raw material and the volume of hydrochloric acid are (0.1-0.5) g: (10-20) mL; in a specific embodiment, the mass of the ditch edge raw material and the volume ratio of hydrochloric acid is 0.2 g: 10 mL.

[0010] The residue was dissolved in 0.1 mol / L hydrochloric acid.

[0011] The method comprises the following steps: derivatizing a test sample solution and then determining the solution by high performance liquid chromatography to obtain a high performance liquid phase characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and preparations thereof; the chromatographic conditions include: a C18 column, mobile phase A of acetonitrile, mobile phase B of 0.2% phosphoric acid solution, and gradient elution.

[0012] The gradient elution described in the present invention is specifically:

[0013] 0-25min, phase A: 12-30%, phase B: 88-70%;

[0014] 25-60min, phase A: 30-80%, phase B: 70-20%.

[0015] The flow rate of the mobile phase in the present invention is 0.8-1.2 mL / min, preferably 1.0 mL / min; the injection volume is 10 μL; the column temperature is 25-35°C, and in a specific embodiment, the column temperature is 25°C; the detection wavelength is 246 nm; and the number of theoretical plates calculated based on the amino acid peak is not less than 5000.

[0016] The derivatization reagents used in the derivatization of the present invention include phenyl isothiocyanate and triethylamine.

[0017] The process of derivatization of the test solution described in the present invention includes:

[0018] The test solution is mixed with an acetonitrile solution of phenyl isothiocyanate and an acetonitrile solution of triethylamine, and the mixture is allowed to stand at room temperature for 55 to 65 minutes to obtain a derivative product; the derivative product is mixed with n-hexane, shaken, allowed to stand, the lower layer solution is removed, filtered, and the filtrate is taken to obtain a derivatized test solution.

[0019] The present invention also includes the preparation of reference solution:

[0020] The reference medicinal material of dog whip was hydrolyzed with hydrochloric acid, filtered, the filtrate was evaporated to dryness, and the residue was dissolved with hydrochloric acid to obtain a reference medicinal material reference solution; glycine and alanine were dissolved with 0.1 mol / L hydrochloric acid to prepare a 300 μg / mL reference substance mixed solution.

[0021] The present invention also includes the derivatization of the reference solution:

[0022] The reference solution is mixed with an acetonitrile solution of phenyl isothiocyanate and an acetonitrile solution of triethylamine, and the mixture is allowed to stand at room temperature for 55 to 65 minutes to obtain a derivative product; the derivative product is mixed with n-hexane, shaken, allowed to stand, the lower layer solution is removed, filtered, and the filtrate is taken to obtain a derivatized reference solution.

[0023] The invention adopts a Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of a dog whip test sample, and obtains an HPLC standard characteristic spectrum of a dog whip extract, a medicinal material, a decoction piece and a preparation consisting of 8 characteristic peaks, which correspond to 8 characteristic retention times in a chromatogram of a control medicinal material reference substance, wherein peak 1 and peak 6 should correspond to the retention times of glycine and alanine reference substance peaks, respectively; the peak corresponding to the alanine reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of a specified value, and the specified value is: 0.82 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.94 (peak 5), 1.06 (peak 7) and 1.37 (peak 8).

[0024] The present invention also provides a method for identifying dog whip medicinal materials, decoction pieces, extracts and preparations thereof, which uses the construction method described in the above technical solution to perform detection and analyze the detection results.

[0025] The present invention aims to establish a high-performance liquid chromatography characteristic spectrum method for dog whip medicinal materials, decoction pieces, extracts, and preparations thereof, comprehensively reflecting the quality level of dog whip medicinal materials, decoction pieces, extracts, and preparations thereof, and providing guidance for the quality control of dog whip medicinal materials, decoction pieces, extracts, and preparations thereof. The present invention uses acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B for gradient elution, and uses glycine and alanine as reference substances. The method establishes a characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts, and preparations thereof with good repeatability and precision. The method is stable and reliable, and can be used to control the quality of dog whip medicinal materials, decoction pieces, extracts, and preparations thereof.

[0026] The present invention is characterized by strong operability, convenient analysis, and stability. The present invention utilizes high-performance liquid chromatography fingerprint technology to control the quality of dog whip extracts and their preparations, effectively controlling product quality. The present invention can analyze differences and variations in dog whip extracts and their preparations, facilitating an overall evaluation of the scientificity and rationality of dog whip-related process steps. Furthermore, the present invention can comprehensively control the intrinsic quality of dog whip extracts and their preparations, thereby ensuring the clinical efficacy of dog whip formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the chromatogram of different wavelengths of dog penis extract made from wine;

[0028] Figure 2 Investigation of different column temperatures for wine-made dog penis extract;

[0029] Figure 3 For wine-based dog whip extract different flow rate investigation;

[0030] Figure 4 Identification of chromatographic peaks of characteristic spectrum of dog penis extract made from wine;

[0031] Figure 5 Investigation of different instruments for making dog penis extract for wine;

[0032] Figure 6 Investigation of different chromatographic columns for wine-made dog whip extract;

[0033] Figure 7 This is the characteristic spectrum of the dog penis extract made from wine;

[0034] Figure 8 is the extract control characteristic spectrum;

[0035] Figure 9 For the hydrolysis temperature investigation;

[0036] Figure 10 To investigate the hydrolysis time;

[0037] Figure 11 For sample weighing inspection;

[0038] Figure 12 Identify the chromatographic peaks of the characteristic spectrum of dog whip medicinal material;

[0039] Figure 13 To investigate different instruments for dog whip medicinal materials;

[0040] Figure 14 To investigate the durability of different chromatographic columns for dog whip medicinal materials;

[0041] Figure 15 Verification diagram of characteristic spectra of 16 batches of dog penis medicinal materials;

[0042] Figure 16 This is the reference characteristic spectrum of the medicinal material Goubian;

[0043] Figure 17 This is the characteristic spectrum of the wine-processed dog penis decoction piece;

[0044] Figure 18 This is the reference characteristic spectrum of wine-processed dog penis slices;

[0045] Figure 19 This is the chromatogram of different wavelengths of the wine-made dog whip formula granules;

[0046] Figure 20 To investigate the chromatogram for column temperature;

[0047] Figure 21 Investigate the chromatogram for flow rate;

[0048] Figure 22For the hydrolysis temperature investigation;

[0049] Figure 23 To investigate the hydrolysis time;

[0050] Figure 24 For sample weighing inspection;

[0051] Figure 25 Identify the chromatographic peaks of the characteristic spectrum of the finished product of wine-made dog whip;

[0052] Figure 26 Comparison of characteristic spectra of different instruments for finished wine-made dog whip products;

[0053] Figure 27 To investigate the durability of the chromatographic column;

[0054] Figure 28 Verification diagram of characteristic spectra of three batches of wine-made dog whip granules;

[0055] Figure 29 This is the reference characteristic spectrum of the wine-made dog whip formula granules. DETAILED DESCRIPTION

[0056] To further illustrate the present invention, the following detailed description of the method for constructing the HPLC characteristic spectrum of a dog whip medicinal material, decoction pieces, extract and preparation thereof provided by the present invention and its application is given in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0057] Table 1 Summary of the origin of dog whip medicinal materials

[0058]

[0059]

[0060] Preparation of wine-processed dog penis slices

[0061] Take clean dog penis medicinal material, cut into sections and slices, place them in a wire sieve, and simmer over low heat until crispy. Once the dog penis is hot, remove from heat, spray with an appropriate amount of white wine, and simmer again over low heat until crispy. Remove from heat and spray with an appropriate amount of white wine again. Repeat this process several times until crispy. Let cool. Sixteen batches of dog penis medicinal material were processed into 16 batches of wine-processed dog penis slices. See Table 2 for specific information.

[0062] Table 2 Corresponding table of 16 batches of wine-made dog firecrackers

[0063]

[0064] Preparation of Dog Whip Extract Made from Wine

[0065] Take 100g of wine-processed dog penis slices, add water and decoct twice, add 10 times the water for the first decoction, soak for 30 minutes, boil, keep boiling for 60 minutes, filter with a 200-mesh sieve, and immediately cool to room temperature; add 8 times the water for the second decoction, boil, keep boiling for 40 minutes, filter with a 200-mesh sieve, combine the decoctions, immediately cool to room temperature, concentrate, freeze-dry, and package to obtain the product.

[0066] 16 batches of wine-processed dog penis extracts were prepared from 16 batches of wine-processed dog penis slices. The corresponding information is shown in Table 3 below.

[0067] Table 3 Preparation of wine-processed dog whip extract

[0068]

[0069]

[0070] Example 1 Method for constructing HPLC characteristic spectrum of wine-processed dog whip extract:

[0071] 1. Feature map:

[0072] 1.1 Experimental instruments and materials

[0073] Instruments: 2 high performance liquid chromatographs of different brands;

[0074] Electronic balances: ME-04E, ME204E, XPE26 (Mettler-Toledo Instruments Co., Ltd.);

[0075] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0076] Electric constant temperature water bath: KQ-600DB (1500W, 50Hz; Shanghai Yilin Scientific Instrument Co., Ltd.);

[0077] Chromatographic columns: 3 different types of chromatographic columns.

[0078] Phosphoric acid, acetonitrile (chromatographic grade); water was ultrapure water, and other reagents were of analytical grade.

[0079] Glycine (China Food and Drug Inspection Institute, batch number: 140689-202207, content is 100.0%),

[0080] Alanine (China Food and Drug Inspection Institute, batch number: 140680-202206, content is 99.9%),

[0081] Threonine (China Food and Drug Inspection Institute, batch number: 140682-202103, content is 99.9%),

[0082] Glutamic acid (China Food and Drug Inspection Institute, batch number: 140690-202305, content is 99.9%),

[0083] L-Proline amino acid (Shanghai Yuanye Biotechnology Co., Ltd., batch number: Z12O11H127142, content is 99.4%),

[0084] Aspartic acid (Shanghai Yuanye Biotechnology Co., Ltd., batch number: N27GB169220, content is 99.4%),

[0085] Gou Jian reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 270106-202305),

[0086] Lyophilized powder of wine-processed dog penis extract (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: JZGB-BT-230601, JZGB-BT-230602, JZGB-BT-230603, JZGB-BT-230604, JZGB-BT-230605, JZGB-BT-230606, JZGB-BT-230607, JZGB-BT-230608, JZGB-BT-230609, JZGB-BT-230610, JZGB-BT-230611, JZGB-BT-230612, JZGB-BT-230613, JZGB-BT-230614, JZGB-BT-230615, JZGB-BT-230616).

[0087] Chromatographic conditions and system suitability test

[0088] Use octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; flow rate: 1.0 ml / min; column temperature: 25°C; detection wavelength: 246 nm. The theoretical plate number, calculated based on the alanine peak, should be no less than 5000.

[0089]

[0090] Preparation of Reference Solution: Take 0.2 g of the reference medicinal material Goubian and place it in a stoppered hydrolysis tube. Add 10 ml of 6 mol / L hydrochloric acid solution, stopper tightly, and hydrolyze at 150°C for 3 hours. Cool, filter, and transfer the filtrate to an evaporating dish. Wash the hydrolysis tube and the filter residue with 10 ml of water in batches, filter, and combine the filtrate in an evaporating dish and evaporate to dryness. Dissolve the residue in 25 ml of 0.1 mol / L hydrochloric acid solution to prepare the reference medicinal material solution. Take appropriate amounts of glycine and alanine reference substances, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per ml. This is the reference substance mixed solution.

[0091] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.1g, accurately weigh it, put it into a hydrolysis tube with a stopper, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it tightly, weigh it, put it into 150℃ for hydrolysis for 3 hours, let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0092] Accurately measure 5 ml each of the reference solution and the test solution into 25 ml volumetric flasks. Add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile. Shake well. After standing at room temperature for 1 hour, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution and add 10 ml of n-hexane. Shake well and stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0093] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0094] 1.2 Chromatographic conditions and system suitability test

[0095] 1.2.1 Wavelength Selection

[0096] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of glycine, glutamic acid, aspartic acid, threonine, L-proline, and alanine solutions, and the chromatograms of the test solution at wavelengths of 210 nm, 230 nm, 246 nm, 250 nm, 270 nm, and 290 nm were extracted, respectively. Figure 1 . Figure 1 The results showed that when the detection wavelength was 246 nm, the chromatographic peak had a larger amount of information and the chromatogram baseline was more stable, so the detection wavelength was determined to be 246 nm.

[0097] 1.2.2 Column temperature investigation

[0098] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 2 , Figure 2 The results showed that at 25℃, the chromatogram peak shape was more symmetrical and the retention time was more appropriate, so the column temperature was selected to be 25℃.

[0099] 1.2.3 Flow rate investigation

[0100] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min and 1.2ml / min were investigated respectively. Figure 3 , Figure 3 It shows that when the flow rate is 1.0 ml / min, the chromatogram peak shape is relatively symmetrical and the retention time is appropriate, so the flow rate is selected as 1.0 ml / min.

[0101] In summary, the chromatographic conditions and system suitability tests for the characteristic spectrum of the wine-processed dog penis extract were determined as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table above; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0102] 1.3 Methodological Investigation

[0103] 1.3.1 Chromatographic peak identification

[0104] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of dog penis extract made with wine.

[0105] Preparation of reference solution: Take appropriate amount of glycine, glutamic acid, aspartic acid, threonine, L-proline and alanine reference substances, accurately weigh them, add 0.1 mol / L hydrochloric acid solution to make a solution containing 60 μg of each per 1 ml, and obtain the reference substance mixed solution.

[0106] Preparation of reference solution of dog whip control medicinal material: Take 0.2 g of dog whip control medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, combine the filtrate into an evaporating dish, evaporate it to dryness, add 25 ml of 0.1 mol / L hydrochloric acid solution to the residue to dissolve it, and use it as the reference solution of control medicinal material.

[0107] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of dog penis extract without alcohol was prepared.

[0108] The characteristic peaks of the wine-processed dog whip extract were located. Figure 4 The results show that the method has good specificity. In the following methodological test, eight characteristic peaks in the sample were examined.

[0109] 1.3.2 Precision test

[0110] Take the test solution of the dog penis extract made with wine and inject 10 μl of it 6 times continuously according to the proposed experimental method. Calculate the retention time of each characteristic peak. See Table 4:

[0111] Table 4 Precision investigation-retention time

[0112]

[0113] It can be seen from Table 1 that the RSD of the retention time of each characteristic peak is 0.26% to 1.09%, and the precision of the instrument is good.

[0114] 1.3.2 Repeatability Study

[0115] Six portions of wine-processed dog whip extract (batch number: JZGB-BT-230607) were accurately weighed and prepared and assayed according to the proposed experimental method. See Table 5:

[0116] Table 5 Repeatability study - characteristic peak relative retention time ratio

[0117]

[0118] Table 2 shows that the relative retention times of the characteristic peaks are consistent, and the RSD of the relative retention time is 0.00%. This method has good reproducibility.

[0119] 1.3.4 Intermediate precision study

[0120] 1.3.4.1 Investigation of different instruments

[0121] Based on the experimental conditions proposed above, 12 portions of wine-processed dog whip extract were accurately weighed to prepare test solutions, which were then measured on HPLC 1 (serial numbers 1 to 6) and HPLC 2 (serial numbers 7 to 12). Figure 5 and Table 6:

[0122] Table 6 Different instruments investigated - relative retention time ratios of characteristic peaks

[0123]

[0124]

[0125] Table 6 shows that the intermediate precision of this method is good.

[0126] 1.3.5 Durability inspection

[0127] 1.3.5.1 Column durability assessment

[0128] Based on the experimental conditions proposed above, different brands of chromatographic columns 1 (Shim-pack GIST C18), chromatographic column 2 (Shim-pack Scepter C18), chromatographic column 3 ( C18) was investigated. The results are shown in Figure 6and Table 7:

[0129] Table 7 Column durability investigation - characteristic peak relative retention time ratio

[0130]

[0131] The results showed that the RSDs of the relative retention times of the characteristic peaks of different brands of chromatographic columns ranged from 0.00 to 2.88%.

[0132] 1.3.5.2 Stability investigation

[0133] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h. See Table 8:

[0134] Table 8 Stability Study-Retention Time

[0135]

[0136]

[0137] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.01% and 0.04%, and the sample solution was relatively stable within 24 hours.

[0138] In summary, the RSDs of the retention time / relative retention time of each characteristic peak met the requirements in all the above investigations, and this method is good.

[0139] 1.4 Determination of characteristic peaks and establishment of reference maps

[0140] 1.4.1 Verification results of 16 batches of wine-processed dog penis extract

[0141] Using this method, characteristic spectrum analysis was performed on 16 batches of samples and relative retention times were calculated. Figure 7 and Table 9, Figure 13 The batch numbers from bottom to top are: JZGB-BT-230601, JZGB-BT-230602, JZGB-BT-230603, JZGB-BT-230604, JZGB-BT-230605, JZGB-BT-230606, JZGB-BT-230607, JZGB-BT-230608, JZGB-BT-230609, JZGB-BT-230610, JZGB-BT-230611, WFG-BT-210912, JZGB-BT-230613, JZGB-BT-230614, JZGB-BT-230615, JZGB-BT-230616:

[0142] Table 9 Relative retention time ratios of wine-processed dog whip extracts

[0143]

[0144] Based on the principle of stable relative retention times and the ability to detect peaks at relatively high levels across all batches of samples, a total of eight peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 6 was used as the S peak, the RSD of the relative retention times of Peaks 1 to 7 was 0.00%.

[0145] 1.4.2 Establishment of relative retention time limits

[0146] The summary of the methodology inspection items and verification results is shown in Table 10:

[0147] Table 10 Summary of RSD (%) results of each item of the methodology - relative retention time

[0148]

[0149]

[0150] The results showed that, with the exception of column durability, the RSD values ​​of the retention times or relative retention times of the characteristic peaks met the requirements in all the above-mentioned tests, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigation.

[0151] Final regulations: It is tentatively stipulated that 8 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance. Among them, Peak 1 and Peak 6 should correspond to the retention times of the glycine and alanine reference substance peaks respectively. The peak corresponding to the alanine reference substance peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.82 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.94 (peak 5), 1.06 (peak 7), and 1.37 (peak 8).

[0152] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 16 batches of dog whip extracts, and a reference spectrum of the characteristic spectrum of the wine-processed dog whip extract was established. Figure 8 , among which, Peak 1: glycine; Peak 2: glutamic acid; Peak 3: aspartic acid; Peak 4: threonine; Peak 5: L-proline; Peak 6 (S): alanine.

[0153] 1.5 Determination of the characteristic spectrum of wine-processed dog whip extract

[0154] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 1.0 ml / min; column temperature, 25°C; detection wavelength, 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0155]

[0156] Preparation of Reference Solution: Take 0.2 g of the reference medicinal material Goubian and place it in a stoppered hydrolysis tube. Add 10 ml of 6 mol / L hydrochloric acid solution, stopper tightly, and hydrolyze at 150°C for 2 hours. Cool, filter, and transfer the filtrate to an evaporating dish. Wash the hydrolysis tube and the filter residue with 10 ml of water in batches, filter, and combine the filtrate in an evaporating dish and evaporate to dryness. Dissolve the residue in 25 ml of 0.1 mol / L hydrochloric acid solution to prepare the reference medicinal material solution. Take appropriate amounts of glycine and alanine reference substances, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per ml to obtain the reference substance solution.

[0157] Preparation of test solution: Take an appropriate amount of the product, take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper, weigh the weight, place at 150℃ for hydrolysis for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0158] Accurately measure 5 ml each of the reference solution and the test solution into 25 ml volumetric flasks. Add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile. Shake well. After standing at room temperature for 1 hour, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution and add 10 ml of n-hexane. Shake well and stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0159] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0160] Example 2 Construction of HPLC Characteristic Spectra of Dog Whip Medicinal Material and Wine-Processed Dog Whip Pieces

[0161] 2.1 Experimental instruments and materials

[0162] Instruments: 2 high performance liquid chromatographs of different brands;

[0163] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0164] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0165] Electric constant temperature water bath: KQ-600DB (1500W, 50Hz; Shanghai Yilin Scientific Instrument Co., Ltd.);

[0166] Chromatographic columns: 3 different types of chromatographic columns;

[0167] 2.2 Reagents and test drugs

[0168] Dog whip medicinal materials (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: GB01, GB02, GB03, GB04, GB05, GB06, GB07, GB08, GB09, GB10, GB11, GB12, GB13, GB14, GB15, GB16).

[0169] 2.3 Chromatographic conditions

[0170] Determined by high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0171] Chromatographic Conditions and System Suitability: Use octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, and gradient elution as specified in the table below; flow rate: 1.0 ml / min; column temperature: 25°C; detection wavelength: 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0172]

[0173] Preparation of reference substance solution: Take appropriate amount of glycine reference substance and alanine reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per 1 ml, and obtain the reference substance mixed solution.

[0174] Preparation of reference solution of control medicinal material: Take 0.2 g of dog whip control medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, combine the filtrate into an evaporating dish, evaporate it to dryness, add 25 ml of 0.1 mol / L hydrochloric acid solution to the residue to dissolve it, and use it as the reference solution of control medicinal material.

[0175] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper, weigh the weight, place at 150℃ for hydrolysis for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0176] Accurately measure 5 ml of the reference solution and the test solution, place them in separate 25 ml volumetric flasks, add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile, and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile, shake well, and let stand at room temperature for 1 hour. Then, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution, add 10 ml of n-hexane, shake, and let stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0177] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0178] 2.4 Preparation of test solution

[0179] 2.4.1 Hydrolysis temperature investigation

[0180] Take an appropriate amount of the powder of this product (passed through a No. 3 sieve) (batch number: GB02), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, seal, weigh, and hydrolyze at 120℃, 150℃, and 180℃ for 3 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain. Figure 9 , Figure 9 The results showed that when the hydrolysis temperature was 120℃, 150℃ and 180℃, the chromatogram effects were basically the same. In order to ensure sufficient extraction, the hydrolysis temperature of the test sample was determined to be 150℃.

[0181] 2.4.2 Investigation of hydrolysis time

[0182] Take an appropriate amount of the powder of this product (passed through a No. 3 sieve) (batch number: GB02), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, seal, weigh, place at 150℃ for 1h, 2h, and 3h, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain. Figure 10 , Figure 10 The results showed that when the hydrolysis time was 1h, 2h and 3h, the chromatogram effects were basically the same. In order to ensure sufficient extraction, the hydrolysis time of the test sample was determined to be 2h.

[0183] 2.4.3 Sample weighing inspection

[0184] Take an appropriate amount of the powder of this product (passed through a No. 3 sieve) (batch number: GB02), take about 0.1g, 0.2g, and 0.5g respectively, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, seal, weigh, place at 150℃ for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain. Figure 11 , Figure 11 The results show that when the sample weight is 0.2g, the characteristic spectrum peak area is moderate. Therefore, the sample weight is determined to be 0.2g.

[0185] 2.4.4 Determine the test sample preparation method

[0186] Take an appropriate amount of the powder of this product (passed through No. 3 sieve), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper, weigh the weight, place at 150℃ for hydrolysis for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0187] Accurately measure 5 ml of the reference solution and the test solution, place them in separate 25 ml volumetric flasks, add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile, and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile, shake well, and let stand at room temperature for 1 hour. Then, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution, add 10 ml of n-hexane, shake, and let stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0188] 2.4.5 Feature Graph Method

[0189] Determined by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0190] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 1.0 ml / min; column temperature, 25°C; detection wavelength, 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0191]

[0192] Preparation of reference solution: Take 0.2 g of dog whip reference medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, and combine the filtrate into an evaporating dish, evaporate it to dryness, and dissolve the residue in 25 ml of 0.1 mol / L hydrochloric acid solution to use as the reference solution of the control medicinal material.

[0193] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper, weigh the weight, place at 150℃ for hydrolysis for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0194] Accurately measure 5 ml of the reference solution and the test solution, place them in separate 25 ml volumetric flasks, add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile, and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile, shake well, and let stand at room temperature for 1 hour. Then, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution, add 10 ml of n-hexane, shake, and let stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0195] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0196] 2.5 Methodological Review

[0197] 2.5.1 Chromatographic peak identification

[0198] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of dog whip medicinal material.

[0199] Preparation of reference substance solution: Take appropriate amount of glycine, glutamic acid, aspartic acid, threonine, L-proline and alanine reference substances, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a solution containing 60 μg of each substance per 1 ml.

[0200] Preparation of reference solution of dog whip control medicinal material: Take 0.2 g of dog whip control medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, combine the filtrate into an evaporating dish, evaporate it to dryness, add 25 ml of 0.1 mol / L hydrochloric acid solution to the residue to dissolve it, and use it as the reference solution of control medicinal material.

[0201] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking dog whip medicinal material was prepared.

[0202] Position the characteristic spectrum peaks of Goubian medicinal materials. Figure 12 In summary, the eight characteristic peaks in the medicinal materials were included in the subsequent investigation and methodological research was carried out.

[0203] 2.5.2 Precision test

[0204] Take the test solution of the dog whip medicinal material and inject it continuously 6 times according to the proposed experimental method, 10μl each time, and calculate the retention time of each characteristic peak. See Table 11:

[0205] Table 11 Precision investigation-characteristic peak retention time

[0206]

[0207] The results showed that the RSD of the retention time of each characteristic peak was 0.66%-1.55%, and the precision of the instrument was good.

[0208] 2.5.3 Repeatability Study

[0209] 6 portions of Goubian medicinal material (batch number: XXLS202305379) were accurately weighed and prepared and tested according to the proposed experimental method. See Table 12:

[0210] Table 12 Repeatability study - relative retention time of characteristic peaks

[0211]

[0212]

[0213] The results showed that the relative retention RSD of each characteristic peak was 0.00%, indicating that the method had good repeatability.

[0214] 2.5.4 Intermediate precision study

[0215] Based on the experimental conditions proposed above, 12 portions of dog whip medicinal materials were accurately weighed to prepare the test solution, which was then measured on HPLC 1 and HPLC 2 respectively. Figure 13 , Table 13:

[0216] Table 13 Intermediate precision-relative retention time ratio

[0217]

[0218] The results showed that the method had good intermediate precision.

[0219] 2.5.5 Durability inspection

[0220] 2.5.5.1 Column durability assessment

[0221] Based on the experimental conditions proposed above, different brands of chromatographic columns 1 (Shim-pack GIST C18), chromatographic column 2 (Shim-pack Scepter C18), chromatographic column 3 ( C18) for investigation. Figure 14 , see Table 14:

[0222] Table 14 Column durability investigation - relative retention time of characteristic peaks

[0223]

[0224] The results show that the RSD values ​​of the relative retention times vary greatly when using different chromatographic columns. Column 1 is recommended.

[0225] 2.5.5.2 Stability investigation

[0226] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 4h, 8h, 12h, 16h and 24h respectively. See Table 15:

[0227] Table 15 24-hour stability study - characteristic peak retention time

[0228]

[0229] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.07%, and the sample solution was relatively stable within 24 hours.

[0230] In summary, the RSDs of the retention time / relative retention time of each characteristic peak met the requirements in all the above investigations, and this method is good.

[0231] 2.6 Determination of characteristic peaks and establishment of reference maps

[0232] 2.6.1 Verification results of 16 batches of dog penis medicinal materials

[0233] The proposed method was used to determine the characteristic spectra of 16 batches of samples of this product and calculate the relative retention time. Figure 15 (The batch numbers represented by each spectrum are S1: GB01; S2: GB02; S3: GB03; S4: GB04; S5: GB05; S6: GB06; S7: GB07; S8: GB08; S9: GB09; S10: GB10; S11: GB11; S12: GB12; S13: GB13; S14: GB14; S15: GB15; S16: GB16) and Table 16:

[0234] Table 16 Relative retention time of 16 batches of dog whip medicinal materials

[0235]

[0236]

[0237] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0238] 2.6.2 Establishment of relative retention time limit values

[0239] The summary of the methodology inspection items and verification results is shown in Table 17:

[0240] Table 17 Summary of RSD% results for each item of methodology - relative retention time (retention time)

[0241]

[0242] The results showed that the RSD values ​​of the retention time or relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. The above 8 characteristic peaks were included in the subsequent investigation.

[0243] Final regulations: It is tentatively stipulated that 8 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance. Among them, Peak 1 and Peak 6 should correspond to the retention times of the glycine and alanine reference substance peaks respectively. The peak corresponding to the alanine reference substance peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.82 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.94 (peak 5), 1.06 (peak 7), and 1.37 (peak 8).

[0244] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to evaluate the similarity of 16 batches of dog penis.

[0245] The characteristic spectrum of medicinal materials was synthesized and a comparison spectrum of the characteristic spectrum of dog whip medicinal materials was established. Figure 16 .

[0246] 2.6.3 Verification of the Characteristic Spectrum of Wine-processed Dog Penis Pieces

[0247] According to the results of "2.4.4 Preparation of test solution", 16 batches of test solution of dog penis slices made with wine were prepared, and the samples were injected, measured and analyzed according to the results of "2.4.5 Chromatographic conditions and system suitability test", and the relative retention time was calculated. Figure 17 (The batch numbers represented by each spectrum are S1: JZGB-230601; S2: JZGB-230602; S3: JZGB-230603; S4: JZGB-230604; S5: JZGB-230605; S6: JZGB-230606; S7: JZGB-230607; S8: JZGB-230608; S9: JZGB-230609; S10: JZGB-230610; S11: JZGB-230611; S12: JZGB-230612; S13: JZGB-230613; S14: JZGB-230614; S15: JZGB-230615; S16: JZGB-230616) and Table 18:

[0248] Table 18 Relative retention times of characteristic peaks of 16 batches of wine-processed dog penis slices

[0249]

[0250]

[0251] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0252] Final regulations: It is tentatively stipulated that 8 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance. Among them, Peak 1 and Peak 6 should correspond to the retention times of the glycine and alanine reference substance peaks respectively. The peak corresponding to the alanine reference substance peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.82 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.94 (peak 5), 1.06 (peak 7), and 1.37 (peak 8).

[0253] The characteristic spectra of 16 batches of wine-processed dog penis slices were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectra of wine-processed dog penis slices was established. Figure 19 Among them, peak 1: glycine; peak 2: glutamic acid; peak 3: aspartic acid; peak 4: threonine; peak 5: L-proline; peak 6 (S): alanine.

[0254] 2.7 Determination of characteristic patterns of dog whip medicinal materials and decoction pieces

[0255] Chromatographic Conditions and System Suitability: Octadecylsilane bonded silica gel was used as the packing material (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 ml / min; the column temperature was 25°C; and the detection wavelength was 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0256]

[0257] Preparation of reference substance solution: Take appropriate amount of glycine reference substance and alanine reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per 1 ml, and obtain the reference substance mixed solution.

[0258] Preparation of reference solution of control medicinal material: Take 0.2 g of dog whip control medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, combine the filtrate into an evaporating dish, evaporate it to dryness, add 25 ml of 0.1 mol / L hydrochloric acid solution to the residue to dissolve it, and use it as the reference solution of control medicinal material.

[0259] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 0.2g, accurately weigh, place in a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper, weigh the weight, place at 150℃ for hydrolysis for 2 hours, cool, weigh again, make up the lost weight with 6mol / L hydrochloric acid solution, shake well, filter, accurately measure 5ml of the filtrate into an evaporating dish, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0260] Accurately measure 5 ml of the reference solution and the test solution, place them in separate 25 ml volumetric flasks, add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile, and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile, shake well, and let stand at room temperature for 1 hour. Then, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution, add 10 ml of n-hexane, shake, and let stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0261] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0262] Example 3 Construction of HPLC Characteristic Spectrum of Wine-processed Dog Whip Formula Granules

[0263] 3.1 Experimental instruments and materials

[0264] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.).

[0265] 3.2 Reagents and test drugs

[0266] Wine-processed dog penis formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 18120183, 2305031, 2305032, 2305033).

[0267] 3.3 Chromatographic conditions

[0268] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the table below.

[0269]

[0270] Preparation of Reference Solution: Take 0.2 g of the reference medicinal material Goubian and place it in a stoppered hydrolysis tube. Add 10 ml of 6 mol / L hydrochloric acid solution, stopper tightly, and hydrolyze at 150°C for 3 hours. Cool, filter, and transfer the filtrate to an evaporating dish. Wash the hydrolysis tube and the filter residue with 10 ml of water in batches, filter, and combine the filtrate in an evaporating dish and evaporate to dryness. Dissolve the residue in 25 ml of 0.1 mol / L hydrochloric acid solution to prepare the reference medicinal material solution. Take appropriate amounts of glycine reference substance and alanine reference substance, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per ml. This is the reference medicinal material mixed solution.

[0271] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.1g, accurately weigh it, put it into a hydrolysis tube with a stopper, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it tightly, weigh it, put it into 150℃ for hydrolysis for 3 hours, let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0272] Accurately measure 5 ml each of the reference solution and the test solution into 25 ml volumetric flasks. Add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile. Shake well. After standing at room temperature for 1 hour, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution and add 10 ml of n-hexane. Shake well and stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0273] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0274] 3.4 Chromatographic conditions and system suitability test

[0275] 3.4.1 Wavelength Selection

[0276] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of glycine, glutamic acid, aspartic acid, threonine, L-proline, and alanine solutions, and the chromatograms of the test solution at wavelengths of 210 nm, 230 nm, 246 nm, 250 nm, 270 nm, and 290 nm were extracted, respectively. Figure 19 The results showed that when the detection wavelength was 246 nm, the chromatographic peak had a larger amount of information and the chromatogram baseline was more stable, so the detection wavelength was determined to be 246 nm.

[0277] 3.4.2 Column temperature investigation

[0278] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 20 The results showed that at 25℃, the chromatogram peak shape was more symmetrical and the retention time was more appropriate, so the column temperature was selected as 25℃.

[0279] 3.4.3 Flow rate investigation

[0280] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 21 The results showed that when the flow rate was 1.0 ml / min, the chromatogram peak shape was relatively symmetrical and the retention time was appropriate, so the flow rate was selected as 1.0 ml / min.

[0281] In summary, the chromatographic conditions and system suitability testing for the characteristic spectrum of wine-processed dog whip granules are as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 246 nm. The number of theoretical plates calculated based on the alanine peak should be no less than 5000.

[0282]

[0283]

[0284] 3.5 Preparation of test solution

[0285] 3.5.1 Hydrolysis temperature investigation

[0286] Take an appropriate amount of this product (batch number: 18120183), grind it into powder, take about 0.2g, accurately weigh it, put it into a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it, weigh it, and hydrolyze it at 120℃, 150℃, and 180℃ for 3 hours respectively. Let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and you have it. Figure 22 The results showed that when the hydrolysis temperature was 120℃, 150℃ and 180℃, the chromatogram effects were basically the same. In order to ensure sufficient hydrolysis, the hydrolysis temperature of the test sample was determined to be 150℃.

[0287] 3.5.2 Investigation of hydrolysis time

[0288] Take an appropriate amount of this product (batch number: 18120183), grind it into powder, take about 0.2g, weigh it accurately, put it into a hydrolysis tube with a stopper, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it tightly, weigh it, and hydrolyze it at 150℃ for 1h, 2h, and 3h respectively. Let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and you have it. Figure 23 The results showed that the chromatographic effects were basically the same when the hydrolysis time was 1h, 2h, and 3h. In order to ensure sufficient hydrolysis, the hydrolysis time was determined to be 2h.

[0289] 3.5.3 Sample weighing inspection

[0290] Take an appropriate amount of this product (batch number: 18120183), grind it into powder, take about 0.1g, 0.2g, and 0.5g respectively, weigh accurately, put it into a stoppered hydrolysis tube, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it tightly, weigh it, put it into 150℃ hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and you have it. Figure 24 The results showed that when the sample weight was 0.2 g, the characteristic chromatographic peak area was moderate. Therefore, the sample weight was determined to be 0.2 g.

[0291] In summary, the preparation method of the test solution of the characteristic spectrum of the wine-made dog whip formula granules is determined as follows: take an appropriate amount of this product (batch number: 18120183), grind it into powder, take about 0.2g, accurately weigh it, put it in a hydrolysis tube with a stopper, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it, weigh it, put it at 150℃ for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0292] 3.6 Methodological Review

[0293] 3.6.1 Chromatographic peak identification

[0294] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of wine-made dog whip formula granules.

[0295] Preparation of reference solution: Take appropriate amount of glycine, glutamic acid, aspartic acid, threonine, L-proline and alanine reference substances, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a solution containing 60 μg of each substance per 1 ml.

[0296] Preparation of dog whip control medicinal material solution: Take 0.2 g of dog whip control medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 6 mol / L hydrochloric acid solution, stopper it, place it at 150°C for hydrolysis for 2 hours, cool it, filter it, transfer the filtrate to an evaporating dish, wash the hydrolysis tube and the filter residue with 10 ml of water in several times, filter it, combine the filtrate into an evaporating dish, evaporate it to dryness, add 25 ml of 0.1 mol / L hydrochloric acid solution to the residue to dissolve it, and use it as the control medicinal material reference solution.

[0297] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of dog whip formula granules without wine was prepared.

[0298] Position the characteristic spectrum peaks of the wine-made dog whip formula granules. Figure 25 .

[0299] 3.6.2 Precision test

[0300] Take the test solution of the wine-made dog whip formula granules and inject 10 μl of the sample 6 times continuously according to the proposed experimental method. Calculate the retention time of each characteristic peak. See Table 19:

[0301] Table 19 Precision investigation-characteristic peak retention time

[0302]

[0303] The results showed that the RSD values ​​of the retention times of the characteristic peaks were 0.38% to 0.99%, indicating that the instrument had good precision.

[0304] 3.6.3 Repeatability Study

[0305] Six portions of wine-processed dog whip granules were accurately weighed and prepared and assayed according to the proposed experimental method. See Table 20.

[0306] Table 20 Repeatability study - relative retention time of characteristic peaks

[0307]

[0308] The results showed that the relative retention times of the characteristic peaks were consistent, and the method had good repeatability.

[0309] 3.6.4 Intermediate precision study

[0310] Based on the experimental conditions proposed above, 12 portions of wine-made dog whip granules were accurately weighed to prepare test solutions, which were measured on HPLC 1 and HPLC 2, respectively. Figure 26 and Table 21:

[0311] Table 21 Intermediate precision-relative retention time ratio

[0312]

[0313]

[0314] The results showed that the method had good intermediate precision.

[0315] 3.6.5 Durability assessment

[0316] 3.6.5.1 Column Durability Assessment

[0317] Based on the experimental conditions proposed above, different brands of chromatographic columns 1 (Shim-pack GIST C18), chromatographic column 2 (Shim-pack Scepter C18), chromatographic column 3 ( C18) for investigation. Figure 27 and Table 22:

[0318] Table 22 Chromatographic column durability investigation - relative retention time of characteristic peaks

[0319]

[0320] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.00% to 2.88%, indicating that the chromatographic durability of this method was good.

[0321] 3.6.5.2 Stability assessment

[0322] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 4h, 8h, 12h, 16h, and 24h respectively. See Table 23:

[0323] Table 23 Stability Study-Characteristic Peak Retention Time

[0324]

[0325] The results showed that the RSD value of the corresponding characteristic peak retention time was 0.01% to 0.05%, and the sample solution was relatively stable within 24 hours.

[0326] In summary, the RSDs of the relative retention times of the characteristic peaks 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.

[0327] 3.7 Determination of characteristic peaks and establishment of reference spectrum

[0328] 3.7.1 Verification Results of Three Batches of Wine-Processed Dog Penis Granules

[0329] 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 28 and Table 24:

[0330] Table 24 Relative retention time of three batches of wine-processed dog whip granules

[0331]

[0332] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0333] 3.7.2 Establishment of relative retention time limit values

[0334] The summary of the methodology inspection items and verification results is shown in Table 25:

[0335] Table 25 Summary of RSD% results for each item of the methodology - relative retention time

[0336]

[0337] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0338] The test sample chromatogram 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 1 and Peak 6 should correspond to the retention times of the glycine and alanine reference material peaks, respectively. The peak corresponding to the alanine reference material peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.82 (Peak 2), 0.85 (Peak 3), 0.88 (Peak 4), 0.94 (Peak 5), 1.06 (Peak 7), and 1.37 (Peak 8).

[0339] The characteristic spectra of three batches of wine-made dog penis formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectra of wine-made dog penis formula granules was established. Figure 29 , where: Peak 1: glycine; Peak 2: glutamic acid; Peak 3: aspartic acid; Peak 4: threonine; Peak 5: L-proline; Peak 6 (S): alanine.

[0340] 3.8 Determination of the characteristic spectrum of wine-made dog whip formula granules

[0341] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 1.0 ml / min; column temperature, 25°C; detection wavelength, 246 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0342]

[0343] Preparation of Reference Solution: Take 0.2 g of the reference medicinal material Goubian and place it in a stoppered hydrolysis tube. Add 10 ml of 6 mol / L hydrochloric acid solution, stopper tightly, and hydrolyze at 150°C for 3 hours. Cool, filter, and transfer the filtrate to an evaporating dish. Wash the hydrolysis tube and the filter residue with 10 ml of water in batches, filter, and combine the filtrate in an evaporating dish and evaporate to dryness. Dissolve the residue in 25 ml of 0.1 mol / L hydrochloric acid solution to prepare the reference medicinal material solution. Take appropriate amounts of glycine and alanine reference substances, accurately weigh them, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 300 μg of each per ml. This is the reference substance mixed solution.

[0344] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2g, accurately weigh it, put it into a hydrolysis tube with a stopper, accurately add 10ml of 6mol / L hydrochloric acid solution, stopper it, weigh it, put it at 150℃ for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 6mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0345] Accurately measure 5 ml each of the reference solution and the test solution into 25 ml volumetric flasks. Add 2.5 ml of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile and 2.5 ml of a 1 mol / L triethylamine solution in acetonitrile. Shake well. After standing at room temperature for 1 hour, add 50% acetonitrile to the mark and shake well. Take 10 ml of the solution and add 10 ml of n-hexane. Shake well and stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0346] Determination method: Accurately aspirate 10 μl of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0347] As can be seen from the above examples, the present invention aims to construct a high-performance liquid chromatography characteristic spectrum method for dog whip medicinal materials, decoction pieces, extracts, and their formulated granules, which comprehensively reflects the quality level of dog whip medicinal materials, decoction pieces, extracts, and their formulated granules, and provides guidance for the quality control of dog whip medicinal materials, decoction pieces, extracts, and their formulated granules. The present invention uses acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B for gradient elution, and uses glycine and alanine as reference substances to establish a characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts, and their formulated granules with good repeatability and precision. The method is stable and reliable, and can control the quality of dog whip medicinal materials, decoction pieces, extracts, and their formulated granules.

[0348] 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 HPLC characteristic patterns of dog whip medicinal materials, decoction pieces, extracts and preparations thereof, comprising the following steps: The dog penis raw material is hydrolyzed with hydrochloric acid, filtered, the filtrate is evaporated to dryness, and the residue is dissolved with 0.1 mol / L hydrochloric acid to obtain a test solution; the concentration of the hydrochloric acid used for hydrolysis is 3-9 mol / L; the hydrolysis temperature is 145-155° C., and the hydrolysis time is 1-4 h; the mass of the dog penis raw material and the volume of hydrochloric acid are (0.1-0.5) g: (10-20) mL; The test solution was derivatized and then determined by high performance liquid chromatography to obtain a high performance liquid chromatography characteristic spectrum of dog whip medicinal materials, decoction pieces, extracts and preparations thereof; the chromatographic conditions included: a C18 column, mobile phase A was acetonitrile, mobile phase B was 0.2% phosphoric acid solution, and gradient elution; The gradient elution is specifically as follows: 0-25min, phase A: 12-30%, phase B: 88-70%; 25-60 min, phase A: 30-80%, phase B: 70-20%; Also includes the preparation of reference solutions: The control medicinal material of dog whip is hydrolyzed with hydrochloric acid, filtered, the filtrate is evaporated to dryness, and the residue is dissolved with hydrochloric acid to obtain a control medicinal material reference solution; Separately, glycine and alanine were dissolved in 0.1 mol / L hydrochloric acid to prepare a 300 μg / mL reference substance mixed solution.

2. The construction method according to claim 1, characterized in that The flow rate of the mobile phase was 0.8–1.2 mL / min, and the injection volume was 10 μL; Column temperature is 25-35°C; The detection wavelength is 246 nm; The theoretical plate number calculated based on the amino acid peaks should not be less than 5000.

3. The construction method according to claim 1, characterized in that The derivatization reagents used included phenyl isothiocyanate and triethylamine.

4. The construction method according to claim 3, characterized in that The process of derivatization of the test solution comprises: The test solution is mixed with an acetonitrile solution of phenyl isothiocyanate and an acetonitrile solution of triethylamine, and the mixture is allowed to stand at room temperature for 55 to 65 minutes to obtain a derivative product; the derivative product is mixed with n-hexane, shaken, allowed to stand, the lower layer solution is removed, filtered, and the filtrate is taken to obtain a derivatized test solution.

5. The construction method according to claim 4, characterized in that: Derivatization of reference solutions is also included: The reference solution is mixed with an acetonitrile solution of phenyl isothiocyanate and an acetonitrile solution of triethylamine, and the mixture is allowed to stand at room temperature for 55 to 65 minutes to obtain a derivative product; the derivative product is mixed with n-hexane, shaken, allowed to stand, the lower layer solution is removed, filtered, and the filtrate is taken to obtain a derivatized reference solution.

6. The construction method according to claim 1, characterized in that The similarity of the dog whip test sample was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the dog whip extract, medicinal materials, decoction pieces and preparations consisting of 8 characteristic peaks was obtained, which corresponded to the 8 characteristic retention times in the chromatogram of the control medicinal material reference material. Among them, peak 1 and peak 6 should correspond to the retention times of the glycine and alanine reference material reference material peaks, respectively; the peak corresponding to the alanine reference material peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.82 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.94 (peak 5), 1.06 (peak 7), and 1.37 (peak 8).

7. A method for identifying dog whip medicinal materials, decoction pieces, extracts and preparations thereof, characterized in that: The method of any one of claims 1 to 6 is used for detection, and the detection results are analyzed.

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