Characteristic spectrum, content determination and application of a kind of blood charcoal by high performance liquid chromatography

Abstract

The present invention provides a method for constructing a high-performance liquid phase characteristic spectrum of blood charcoal, the determination of its content and its application, comprising the following steps: A) hydrolyzing the blood charcoal raw material with hydrochloric acid, filtering, evaporating the filtrate, and dissolving the residue with hydrochloric acid to obtain a test solution; B) derivatizing the test solution and then determining it by high-performance liquid chromatography to obtain a high-performance liquid phase characteristic spectrum of blood charcoal; the chromatographic conditions include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.02% phosphoric acid solution, and gradient elution. The present invention adopts high-performance liquid chromatography, selects acetonitrile-0.02% phosphoric acid solution as the mobile phase for gradient elution, and uses alanine as a reference substance to establish the repeatability and precision of the characteristic spectrum of blood charcoal medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules. The method is stable and reliable, and the quality of blood charcoal medicinal materials, decoction pieces, standard decoctions, intermediates and their formula granules can be controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a high-performance liquid phase characteristic spectrum of blood charcoal, determination of its content and application. Background Art

[0002] Xueyu charcoal was first recorded in Shennong's Herbal Classic and is a traditional Chinese medicinal material. It is bitter and flat. It enters the liver and stomach meridians. It has the effects of astringency, hemostasis, blood stasis, and diuresis. It is used to treat vomiting blood, hemoptysis, epistaxis, hematuria, hematuria, hematochezia, metrorrhagia, traumatic bleeding, and dysuria. Human hair contains mainly keratin, in addition to fat and melanin and iron, zinc, copper, calcium, magnesium, etc. After charcoal making, the organic matter is destroyed and the ash mainly contains sodium, potassium, calcium, iron, copper, zinc, etc. At present, the basic research on the medicinal material of Xueyu charcoal in my country is relatively weak. There are reports on research on the processing of Xueyu charcoal, using its hemostatic effect as an evaluation indicator [1]. There are reports on quality control research using indicators such as inorganic ions, ash, and extracts [2-4]. However, the standard control indicators are simple and it is difficult to effectively control the quality of the medicinal material. As a result, the quality of the medicinal material and its finished drug preparations is uneven, which has long restricted the development and effective utilization of the medicinal resources of Xueyu charcoal. This study provides a basis for the quality evaluation and resource development and utilization of related preparations such as Xueyutan medicinal materials, decoction pieces, standard decoctions, extracts and formula granules.

[0003] Currently, there are few studies on the quality of Xueyutan medicinal materials and their compound preparations. There has never been any relevant report on high performance liquid chromatography analysis and content determination, and there is a lack of an effective means to control their quality. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a high-performance liquid phase characteristic spectrum, content determination and application of Xueyu charcoal. The invention aims to construct a high-performance liquid phase characteristic spectrum method and content determination method of Xueyu charcoal, which comprehensively reflects the quality level of Xueyu charcoal and its related preparations, and provides guidance for the quality control of Xueyu charcoal medicinal materials, decoction pieces and their related preparations.

[0005] The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of blood charcoal, comprising the following steps:

[0006] A) hydrolyzing the blood charcoal raw material with hydrochloric acid, filtering, evaporating the filtrate to dryness, and dissolving the residue with hydrochloric acid to obtain a test solution; the blood charcoal raw material is a blood charcoal medicinal material, decoction piece, standard decoction, intermediate or its formula granules;

[0007] B) Derivatizing the test solution and then determining it by high performance liquid chromatography to obtain a high performance liquid chromatography characteristic spectrum of the charcoal; the chromatographic conditions include: a C18 column, mobile phase A of acetonitrile, mobile phase B of 0.02% phosphoric acid solution, and gradient elution.

[0008] The present invention establishes for the first time a high-performance liquid chromatography characteristic spectrum method for the detection of Xueyu charcoal medicinal materials, herbal slices, standard decoctions, extracts, formula granules and related preparations; in the process of establishing the characteristic spectrum of Xueyu charcoal, the present invention confirms 7 common characteristic peaks, identifies 4 components, and studies their relative retention times and relative peak areas, thereby ensuring its chemical composition stability and safety of use.

[0009] The 0.02% phosphoric acid solution in the present invention is a volume fraction, specifically a 0.02% phosphoric acid aqueous solution.

[0010] In the specific embodiment of the present invention, octadecylsilane bonded silica gel is used as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm). In the specific embodiment, the chromatographic columns used are Kromasil100-5-C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 1); SHIMADZU Shim-pack GIST C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 2); Waters XBridge C18, 4.6 mm × 250 mm, 5 μm (chromatographic column 3);

[0011] The concentration of hydrochloric acid used in the hydrolysis of the present invention is 3 to 9 mol / L; when the hydrochloric acid concentration is 9 mol / L, the content of proline and alanine is higher and the hydrolysis is more complete, so the hydrochloric acid concentration of the hydrolysis in the embodiment is preferably 9 mol / L;

[0012] The hydrolysis temperature is 105-155° C., and the hydrolysis time is 1-4 hours. In a specific embodiment, the hydrolysis temperature is 150° C., and the hydrolysis time is 2 hours.

[0013] The mass of the blood charcoal and the volume of hydrochloric acid are (0.1~0.5)g:(10~20)mL; in a specific embodiment, when the amount of 9mol / L hydrolysis hydrochloric acid added is 10ml, proline and alanine can be fully hydrolyzed; and the chromatogram with a sample weight of 0.2g has a good peak shape and a moderate peak area, so the mass of the blood charcoal and the volume of 9mol / L hydrochloric acid are 0.2g:10mL.

[0014] Dissolve the residue in 0.1 mol / L hydrochloric acid. Dissolve the residue in a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, and shake well to obtain the product.

[0015] In a specific embodiment, the preparation process of the test solution in the method for constructing the characteristic spectrum of blood residue charcoal specifically includes:

[0016] After grinding the test sample raw material, take 0.2g, accurately weigh it, put it into a stoppered hydrolysis tube, accurately add 10ml of 9mol / 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 9mol / 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 the test sample solution is obtained.

[0017] The gradient elution described in the present invention is specifically:

[0018] 0-35min, phase A: 12-20%, phase B: 88-80%;

[0019] 35-40 min, phase A: 20-35%, phase B: 80-65%;

[0020] 40-55 min, phase A: 35%, phase B: 65%;

[0021] 55-65 min, phase A: 35-80%, phase B: 65-20%;

[0022] 65-73min, phase A: 80%, phase B: 20%.

[0023] In the present invention, the flow rate of the mobile phase is 0.8-1.2 mL / min, and the chromatogram peak shape and separation effect meet the requirements; in a specific embodiment, the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL;

[0024] The column temperature is 25-35°C, and the chromatogram peak shape and separation effect meet the requirements; in a specific embodiment, the column temperature is 25°C, 30°C or 35°C, preferably 30°C.

[0025] The present invention uses the maximum absorption wavelengths of proline and alanine in the spectrum and refers to the chromatogram. When the detection wavelength is 245nm, the index peak has a greater absorption and the chromatogram baseline is more stable, so the detection wavelength is 245nm.

[0026] The theoretical plate number calculated based on the amino acid peaks should not be less than 5000.

[0027] The mobile phase B is a 0.02% phosphoric acid solution, by volume; specifically, a 0.02% phosphoric acid aqueous solution.

[0028] The present invention also includes the preparation of reference solution:

[0029] Take the reference medicinal material of Xueyu Carbon and 9 mol / L hydrochloric acid, mix and hydrolyze, cool, filter, take the filtrate and evaporate to dryness, add 0.1 mol / L hydrochloric acid to dissolve the residue, and obtain the reference medicinal material solution;

[0030] Alanine and proline were dissolved in 0.1 mol / L hydrochloric acid to prepare 50 μg / mL reference substance solutions.

[0031] The volume ratio of the blood charcoal control medicinal material to 9 mol / L hydrochloric acid is 1 g: (9-11) mL. In a specific embodiment, the volume ratio of the blood charcoal control medicinal material to 9 mol / L hydrochloric acid is 1 g: 10 mL. The volume ratio of the subsequent filtrate to 0.1 mol / L hydrochloric acid is 1:5.

[0032] The present invention provides a method for determining the content of proline and alanine in blood charcoal, comprising the following steps:

[0033] S1. Hydrolyze the blood charcoal sample with hydrochloric acid, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.1 mol / L hydrochloric acid to obtain a test solution;

[0034] Dissolve alanine and proline separately in 0.1 mol / L hydrochloric acid to obtain reference substance solutions;

[0035] S2. Derivatizing the reference substance solution and the test solution to obtain a derivatized reference substance solution and a derivatized test solution;

[0036] S3. Injecting the derivatized reference substance solution and the test solution into a liquid chromatograph, and using high performance liquid chromatography to obtain chromatograms of the chemical components in the derivatized reference substance solution and the derivatized test solution under the same detection conditions;

[0037] According to the concentration of the reference substance solution, the peak area of ​​the reference substance solution in the chromatogram, and the area of ​​the components in the blood charcoal corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step S3, the contents of proline and alanine in the blood charcoal are calculated by the external standard method.

[0038] The present invention establishes a method for determining the content of blood charcoal, using the total amount of proline and alanine as an indicator, which can control the intrinsic quality of blood charcoal and related preparations from an overall and macroscopic perspective, ensure the efficacy of the medicine, and enable the medicinal materials and related preparations to be subjected to more formal quality control; the method has good stability, high precision, good reproducibility, and is convenient and easy to master.

[0039] In the present invention, the derivatization process of the control medicinal material reference solution, the reference substance reference solution and the test sample solution respectively includes:

[0040] Add phenyl isothiocyanate in acetonitrile and triethylamine in acetonitrile to the control medicinal material reference solution, the reference substance solution and the test sample solution, respectively, and let them stand to obtain a derivative product; mix the derivative product with n-hexane, shake, let it stand, take the lower layer solution, filter, and take the filtrate to obtain a derivatized control medicinal material reference solution, a derivatized reference substance solution and a derivatized test sample solution.

[0041] The volume ratio of the derivative product to n-hexane is 1:0.98-1.1, preferably 1:1.

[0042] The present invention uses proline solutions with concentrations of 8.1340 μg / mL, 10.1675 μg / mL, 20.3350 μg / mL, 40.6700 μg / mL, and 101.6750 μg / mL, accurately draws 10 μl, injects into a liquid chromatograph, measures, obtains the peak area, and draws a response curve Y=16822.3413X-13903.6133 with concentration (X, μg / mL) as the abscissa and peak area (Y) as the ordinate. 2 =0.9999. Therefore, the injection concentration of the proline solution in the present invention is 8.3140-203.3500 μg / mL;

[0043] The present invention uses alanine solutions with concentrations of 7.9860 μg / mL, 9.9825 μg / mL, 19.9650 μg / mL, 39.9300 μg / mL, and 99.8250 μg / mL, respectively. 10 μl is accurately drawn and injected into a liquid chromatograph for measurement to obtain the peak area. A response curve Y=15137.7776X-48353.7156 is drawn with concentration (X, μg / mL) as the abscissa and peak area (Y) as the ordinate. 2 =0.9994. Therefore, the injection concentration of the alanine solution in the present invention is 7.9860-199.6500 μg / mL.

[0044] The present invention adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of a Xueyu charcoal sample, and obtains a Xueyu charcoal HPLC standard characteristic spectrum consisting of 7 characteristic peaks, which correspond to 7 characteristic retention times in a chromatogram of a control medicinal material reference substance, wherein peak 3 and peak 4 should respectively correspond to the retention times of corresponding reference substance peaks; the peak corresponding to the alanine reference substance peak is the S peak, and in the standard characteristic spectrum, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of a specified value, and the specified values ​​are respectively: 0.45 (peak 1), 0.67 (peak 2: glycine), 1.09 (peak 5), 1.65 (peak 6: valine), and 1.85 (peak 7).

[0045] The present invention provides a method for identifying Xueyu charcoal medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules thereof, which is characterized in that the construction method described in the above technical solution is used for detection and the detection results are analyzed.

[0046] The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Xueyu charcoal, comprising the following steps: A) hydrolyzing the Xueyu charcoal raw material with hydrochloric acid, filtering, evaporating the filtrate, and dissolving the residue with hydrochloric acid to obtain a test solution; B) derivatizing the test solution and determining it by high-performance liquid chromatography to obtain a high-performance liquid chromatography characteristic spectrum of Xueyu charcoal; the chromatographic conditions include: a C18 column, mobile phase A of acetonitrile, mobile phase B of 0.02% phosphoric acid solution, and gradient elution. The present invention uses high-performance liquid chromatography, selects acetonitrile-0.02% phosphoric acid solution as the mobile phase for gradient elution, and uses alanine as a reference substance to establish characteristic spectrums of Xueyu charcoal medicinal materials, decoction pieces, standard decoctions, intermediates, and their formulated granules, with good repeatability and precision. The method is stable and reliable, and can be used to control the quality of Xueyu charcoal medicinal materials, decoction pieces, standard decoctions, intermediates, and their formulated granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Select a graph for the mobile phase;

[0048] Figure 2 is the UV absorption spectrum of proline-reference substance;

[0049] Figure 3 is the UV absorption spectrum of proline-test sample;

[0050] Figure 4 is the ultraviolet absorption spectrum of alanine-reference substance;

[0051] Figure 5 is the ultraviolet absorption spectrum of alanine-test sample;

[0052] Figure 6 is the flow velocity investigation result;

[0053] Figure 7 is the column temperature investigation result;

[0054] Figure 8 To investigate the specificity of blood residue charcoal;

[0055] Figure 9 is the standard curve of proline;

[0056] Figure 10 is the standard curve of alanine;

[0057] Figure 11 Examining chromatograms for different instruments;

[0058] Figure 12 To investigate the concentration of hydrochloric acid for hydrolysis;

[0059] Figure 13 Weigh the sample for inspection;

[0060] Figure 14 To investigate the amount of hydrochloric acid added for hydrolysis;

[0061] Figure 15 To investigate the hydrolysis time;

[0062] Figure 16 Identify chromatographic peaks;

[0063] Figure 17 Examine the chromatogram for intermediate precision;

[0064] Figure 18 To investigate the durability of the chromatographic column;

[0065] Figure 19 This is the characteristic spectrum of Xueyu charcoal medicinal materials;

[0066] Figure 20 This is the characteristic spectrum of Xueyutan decoction pieces;

[0067] Figure 21 This is the characteristic spectrum of the standard decoction of Xueyutan;

[0068] Figure 22 This is the characteristic spectrum of the intermediate of blood residue carbon;

[0069] Figure 23 Verification diagram of characteristic spectra of three batches of Xueyu charcoal formula particles;

[0070] Figure 24 This is the reference characteristic spectrum of Xueyu charcoal medicinal materials;

[0071] Figure 25 This is the reference characteristic spectrum of Xueyutan decoction pieces;

[0072] Figure 26 This is the reference characteristic spectrum of the standard decoction of Xueyutan;

[0073] Figure 27 This is the reference characteristic spectrum of Xueyu charcoal extract;

[0074] Figure 28 This is the comparison characteristic spectrum of Xueyu charcoal formula particles. DETAILED DESCRIPTION

[0075] In order to further illustrate the present invention, the following detailed description of the high-performance liquid phase characteristic spectrum, content determination and application of a blood residue charcoal provided by the present invention is given in combination with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0076] Experimental instruments and materials

[0077] Liquid chromatograph: Waters e2695 high performance liquid chromatograph, Agilent 1260 high performance liquid chromatograph;

[0078] Electronic balance: ME204E, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0079] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0080] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0081] Xueyu charcoal control medicinal material (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS030306);

[0082] Glycine (China Food and Drug Inspection Institute, batch number: 140689-202006, purity: 100.0%);

[0083] Proline (China Food and Drug Inspection Institute, batch number: 140677-202109, purity: 100.0%);

[0084] Alanine (China Food and Drug Inspection Institute, batch number: 140680-202005, purity: 99.9%);

[0085] Valine (China Food and Drug Administration, batch number: 140681-201703, purity: 99.5%);

[0086] Leucine (China Food and Drug Administration, batch number: 140687-201905, purity: 99.9%);

[0087] Acetonitrile and phosphoric acid were of chromatographic grade; water was ultrapure water; other reagents were of analytical grade.

[0088] Xueyu charcoal medicinal material batch number: 23001, 23002, 23003, 23004, 23005, 23006, 23007, 23008, 23009, 23010, 23011, 23012, 23013, 23014, 23015, 23016;

[0089] Xueyu charcoal slices batch number: YP01, YP02, YP03, YP04, YP05, YP06, YP07, YP08, YP09, YP10, YP11, YP12, YP13, YP14, YP15, YP16;

[0090] Batch numbers of Xueyutan standard decoction: BT01, BT02, BT03, BT04, BT05, BT06, BT07, BT08, BT09, BT10, BT11, BT12, BT13, BT14, BT15, BT16;

[0091] Xueyu carbon intermediate batch number: TQW01, TQW02, TQW03;

[0092] Batch numbers of Xueyu charcoal formula granules: GSP01, PFKL01, PFKL02, PFKL03.

[0093] Example 1

[0094] 1.1 Experimental conditions

[0095] 1.1.1 Chromatographic conditions and system suitability test

[0096] Use 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, 0.02% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 245 nm. The number of theoretical plates calculated based on the alanine peak should be no less than 5000.

[0097] Table 1

[0098]

[0099] 1.1.2 Preparation of reference solution

[0100] Take 1 g of the reference medicinal material of blood charcoal, place it in a hydrolysis tube with a stopper, accurately add 10 ml of 9 mol / L hydrochloric acid solution, seal it, weigh it, hydrolyze it at 150℃ for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and use it as the reference solution of the reference medicinal material.

[0101] Take appropriate amount of alanine reference substance and proline reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution of 50 μg alanine and 50 μg proline per 1 ml, which is used as the reference substance solution.

[0102] 1.1.3 Preparation of test solution

[0103] Medicinal material test solution: Take about 1 g of the powder of this product (passed through No. 3 sieve), accurately weigh it, place it in a stoppered hydrolysis tube, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it tightly, weigh it, place it at 150℃ for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0104] For the test solution of the decoction piece, take about 1 g of the powder of this product (passed through No. 3 sieve), accurately weigh it, put it into a hydrolysis tube with a stopper, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it tightly, weigh it, put it at 150℃ for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0105] For the standard decoction test solution, take about 0.1 g of the product, accurately weigh it, place it in a hydrolysis tube with a stopper, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it tightly, weigh it, place it at 150°C for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0106] Intermediate test solution: Take about 0.2 g of the product, accurately weigh it, place it in a stoppered hydrolysis tube, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it, weigh it, place it at 150°C for hydrolysis for 2 hours, cool it, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0107] Formula granules test solution Take an appropriate amount of this product, grind it finely, take about 0.2g, accurately weigh it, put it in a hydrolysis tube with a stopper, accurately add 10ml of 9mol / L hydrochloric acid solution, stopper it tightly, weigh it, put it at 150℃ for hydrolysis for 2 hours, let it cool, weigh it again, make up the lost weight with 9mol / 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.

[0108] 1.1.4 Determination method

[0109] Accurately measure 5 ml each of the reference solution and the test solution into 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 to each flask. Shake well. After standing at room temperature for 1 hour, add 50% acetonitrile to the mark and shake well. Take 10 ml and add 10 ml of n-hexane, shake, and stand for 10 minutes. Remove the lower layer, filter, and obtain the filtrate. Accurately pipette 10 μl each of the derivatized reference solution and the test solution into a liquid chromatograph for determination.

[0110] 1.2 Establishment of content determination method

[0111] 1.2.1 Chromatographic conditions and system suitability test

[0112] 1.2.1.1 Mobile phase selection

[0113] Based on the experimental conditions of the above-mentioned formulated particle test solution, the separation effects of two different mobile phases were investigated: acetonitrile-0.02% formic acid and acetonitrile-0.02% phosphoric acid. Figure 1 and Table 2:

[0114] Table 2 Analysis results of different mobile phases

[0115]

[0116] The results showed that when acetonitrile-0.02% phosphoric acid was used as the mobile phase, the peak shape and separation effect of the target peak were better, so acetonitrile-0.02% phosphoric acid solution was selected as the mobile phase for subsequent investigations.

[0117] 1.2.1.2 Wavelength Selection

[0118] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning on the proline reference solution, alanine reference solution and test solution. Figures 2 to 5 By analyzing the maximum absorption wavelengths of proline and alanine in the spectrum and referring to the chromatogram, the index peak has greater absorption when the detection wavelength is 245nm, and the chromatogram baseline is more stable, so the detection wavelength is determined to be 245nm.

[0119] 1.2.1.3 Flow rate investigation

[0120] According to the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min and 1.2ml / min were investigated respectively. Figure 6 and Table 3:

[0121] Table 3 Analysis results of different flow rates

[0122]

[0123] The results showed that when the flow rates were 0.8ml / min, 1.0ml / min and 1.2ml / min, the chromatogram peak shape and separation effect met the requirements, and the proposed flow rate was 1.0ml / min.

[0124] 1.2.1.3 Column temperature investigation

[0125] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 7 , Table 4.

[0126] Table 4 Analysis results of different column temperatures

[0127]

[0128] The results showed that when the column temperature was 25℃, 30℃ and 35℃, the chromatogram peak shape and separation effect met the requirements, and 30℃ was proposed as the detection column temperature.

[0129] In summary, the chromatographic conditions for the characteristic spectrum of blood charcoal and the system usability test are tentatively determined as follows: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.02% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1 above; flow rate 1.0 ml / min; column temperature 30°C; detection wavelength 245 nm. The number of theoretical plates calculated based on the alanine peak should be no less than 5000.

[0130] 1.3 Preparation of test solution

[0131] 1.3.1 Investigation of hydrochloric acid concentration for hydrolysis

[0132] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.5g, accurately weigh it, place it in a stoppered hydrolysis tube, accurately add 10ml of 3mol / L, 6mol / L, and 9mol / L hydrochloric acid solution, respectively, stopper it, weigh it, place it at 150℃ for 1 hour, let it cool, weigh it again, make up the lost weight with the corresponding 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 are done. Accurately aspirate 10μl of each test solution and inject it into the liquid chromatograph to calculate the total amount of proline and alanine at different hydrochloric acid concentrations. The results are shown in Table 5:

[0133] Table 5 Investigation of hydrolysis hydrochloric acid concentration

[0134]

[0135] The results showed that when the hydrolysis hydrochloric acid concentration was 9 mol / L, the contents of proline and alanine were higher and the hydrolysis was more complete, so the hydrolysis hydrochloric acid concentration was temporarily set at 9 mol / L.

[0136] 1.3.2 Sample weighing inspection

[0137] Approximately 0.1g, 0.2g, and 0.5g of this product (Batch No.: GSP01) were accurately weighed and placed in stoppered hydrolysis tubes. 10ml of 9mol / L hydrochloric acid solution was accurately added, the tubes were sealed, and the weight was determined. Hydrolysis was performed at 150°C for 1 hour. The tubes were cooled and weighed again. The weight loss was made up with 9mol / L hydrochloric acid solution. The tubes were shaken and filtered. Accurately measure 5ml of the filtrate into an evaporating dish and evaporated to dryness. The residue was dissolved in 0.1mol / L hydrochloric acid solution. The solution was transferred to a 25ml volumetric flask and 0.1mol / L hydrochloric acid solution was added to the mark. The solution was shaken and the total amount of proline and alanine was calculated for each sample solution. The results are shown in Table 6.

[0138] Table 6 Results of sample weighing

[0139]

[0140] From the above, it can be seen that there is no obvious difference in the extraction effect when the sample weight is 0.1g, 0.2g, and 0.5g. Since the chromatogram peak shape of the sample weight of 0.2g is better and the peak area is moderate, the sample weight is temporarily set to 0.2g.

[0141] 1.3.3 Investigation of the amount of hydrochloric acid added for hydrolysis

[0142] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered hydrolysis tube, accurately add 10ml, 15ml, and 20ml of 9mol / L hydrochloric acid solution respectively, stopper it, weigh it, place it at 150℃ for hydrolysis for 1 hour, let it cool, weigh it again, make up the lost weight with 9mol / 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 are done. Accurately aspirate 10μl of each test solution and inject it into the liquid chromatograph to calculate the total amount of proline and alanine under different amounts of hydrochloric acid added. The results are shown in Table 7:

[0143] Table 7 Investigation of the amount of hydrochloric acid added for hydrolysis

[0144]

[0145] The results showed that when the amount of hydrolysis hydrochloric acid added was 10 ml, proline and alanine could be fully hydrolyzed, so the amount of hydrolysis hydrochloric acid added was temporarily set at 10 ml.

[0146] 1.3.4 Investigation of hydrolysis time

[0147] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered hydrolysis tube, accurately add 10ml of 9mol / L hydrochloric acid solution, seal it, weigh it, and hydrolyze it at 150℃ for 2 hours, 3 hours, and 4 hours respectively. Let it cool, weigh it again, make up the lost weight with 9mol / 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 are done. Accurately aspirate 10μl of each test solution and inject it into the liquid chromatograph. Calculate the total amount of proline and alanine at different hydrolysis times. The results are shown in Table 8:

[0148] Table 8 Hydrolysis time investigation

[0149]

[0150] The results showed that the extraction was complete within 2 hours of hydrolysis, so the hydrolysis time was tentatively set at 2 hours.

[0151] In summary, the preparation method of the test sample for the determination of the content of residual carbon is tentatively as follows: take an appropriate amount of this product (GSP01 granules), grind it into powder, take about 0.2g, accurately weigh it, put it in a hydrolysis tube with a stopper, accurately add 10ml of 9mol / L hydrochloric acid solution, stopper it, weigh it, hydrolyze it at 150℃ for 2 hours, let it cool, weigh it again, make up the lost weight with 9mol / 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.

[0152] 1.3.5 Determination of blood charcoal content

[0153] 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, 0.02% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate, 1.0 ml / min; column temperature, 30°C; detection wavelength, 245 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0154] Preparation of reference solution: Take appropriate amount of proline reference substance and alanine reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 50 μg of proline and 50 μg of alanine per 1 ml.

[0155] Preparation of test solution: Take an appropriate amount of the product (GSP01 granules), grind it into powder, take about 0.2 g, accurately weigh it, place it in a hydrolysis tube with a stopper, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it tightly, weigh it, place it at 150 ° C for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0156] Accurately measure 5 ml each of the reference solution and the test solution into 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, respectively. Shake well. After standing at room temperature for 1 hour, add 50 vol% acetonitrile aqueous solution to the mark and shake well. Take 10 ml of the solution, add 10 ml of n-hexane, shake, and stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0157] 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.

[0158] Example 2 Methodological Investigation

[0159] 2.1.1 Specificity Investigation

[0160] Preparation of reference solution: Take appropriate amount of proline reference substance and alanine reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 50 μg proline and 50 μg alanine per 1 ml, which is used as the reference solution.

[0161] Preparation of test solution: Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh it, put it into a hydrolysis tube with a stopper, accurately add 10ml of 9mol / 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 9mol / 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.

[0162] Preparation of negative control solution: According to the above method for preparing the test solution, prepare a negative control solution lacking blood residual carbon. Figure 8 ;Depend on Figure 8 It can be seen that the chromatographic peak that appears in the negative solution is the derivatization reagent peak, and the negative solution has no interference with the determination of the peak to be tested, indicating that the method has good specificity.

[0163] 2.1.2 Precision investigation

[0164] Take the mixed solution of proline reference substance and alanine reference substance and inject it continuously 5 times, record the peak area, and calculate the RSD value. The results are shown in Table 9:

[0165] Table 9

[0166]

[0167] From the above, we can see that in the precision study, the RSD value of the peak area of ​​proline is 0.23%, and the RSD value of the peak area of ​​alanine is 0.13%, indicating that the precision of the instrument is good.

[0168] 2.1.3 Linear Relationship

[0169] Take an appropriate amount of proline reference substance (100.0% purity) and place it in a 20ml volumetric flask. Dissolve it in 0.1% hydrochloric acid solution to prepare a solution containing 203.35μg of proline per 1ml. Dilute it to concentrations of 8.1340μg / ml, 10.1675μg / ml, 20.3350μg / ml, 40.6700μg / ml, and 101.6750μg / ml. Take an appropriate amount of proline reference substance (100.0% purity) and place it in a 20ml volumetric flask. Dissolve it in 0.1% hydrochloric acid solution to prepare a solution containing 199.65μg of alanine per 1ml. Dilute it to concentrations of 7.9860μg / ml, 9.9825μg / ml, 19.9650μg / ml, 39.9300μg / ml, and 99.8250μg / ml. 10 μl of each solution was accurately drawn and injected into the liquid chromatograph for determination to obtain the peak area. The response curve was drawn with the concentration (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Tables 10 and 11. Figure 9 、 Figure 10 ;

[0170] Table 10 Proline standard curve analysis results

[0171]

[0172] Table 11 Alanine standard curve analysis results

[0173]

[0174] The results showed that when the proline injection concentration was 8.3140-203.3500 μg / ml, the linear relationship was y=16822.3413x-13903.6133, R 2 =0.9999, indicating that the proline concentration is in the range of 8.3140 to 203.3500 μg / ml, and a good linear relationship is shown. When the alanine concentration is in the range of 7.9860 to 199.6500 μg / ml, the linear relationship is y=15137.7776x-48353.7156, R 2 =0.9994, indicating that alanine showed a good linear relationship when the injection concentration was 7.9860-199.6500 μg / ml.

[0175] 2.1.4 Repeatability

[0176] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh 6 portions, and the same operator prepares the test solution according to the proposed experimental method. Accurately pipette 10μl of the test solution respectively, inject it into the liquid chromatograph, and calculate the total amount of proline and alanine in the 6 samples. The results are shown in Table 12.

[0177] Table 12 Repeatability test results

[0178]

[0179] The results showed that the RSD value of proline content in 6 repeatability test samples was 0.44%, and the RSD value of alanine content was 1.37%, indicating that this method has good repeatability.

[0180] 2.1.5 Intermediate precision

[0181] Different personnel (A1, A2) prepared the test sample (lot number: GSP01) solution according to the proposed method on different instruments (C1: Waters e2695, C2: Agilent 1260) at different times (T1, T2). 10 μl of the test sample solution was accurately aspirated and injected into the liquid chromatograph. The total amount of proline and alanine in the sample was calculated. The results are shown in Table 13 and Figure 11 ,Depend on Figure 11 It can be seen that different personnel performed the test on different instruments at different times, and the RSD value of the sample content determination results was 1.27%, and the intermediate precision of this method was good.

[0182] Table 13 Intermediate precision inspection results

[0183]

[0184] 2.1.6 Sample recovery

[0185] About 0.1 g of a test sample with a known content (batch number: GSP01, proline content of 9.5 mg / g, alanine content of 4.6 mg / g) was accurately weighed and divided into 6 portions. A certain amount of proline and alanine reference substances were accurately added to each portion. The test solution was prepared and measured according to the proposed method. The recovery rate was calculated. The results are shown in Table 14. The calculation formula is as follows:

[0186]

[0187] Table 14 Sample recovery test results

[0188]

[0189] From the above, we can see that the average recovery rate of proline is 100.2%, and the average recovery rate of alanine is 100.0%, and the accuracy of this method is good.

[0190] 2.1.7 Column durability assessment

[0191] The same sample (lot number GSP01) was tested using three different C18 columns: Kromasil100-5-C18, 4.6 mm × 250 mm, 5 μm (column 1); SHIMADZU Shim-pack GIST C18, 4.6 mm × 250 mm, 5 μm (column 2); and Waters XBridge C18, 4.6 mm × 250 mm, 5 μm (column 3). 10 μl of the sample solution was accurately aspirated and injected into the liquid chromatograph. The total amount of proline and alanine in the sample was calculated. The results are shown in Table 15.

[0192] Table 15 Results of the chromatographic column durability test

[0193]

[0194]

[0195] From the above, we can see that the RSD value of the sample content determination results when tested with different types of chromatographic columns is 1.91%, indicating that the chromatographic column of this method has good durability.

[0196] 2.1.8 Stability test

[0197] According to the experimental conditions proposed above, a test solution was prepared and the peak area of ​​uridine was measured at 0h, 3h, 7h, 11h, 17h, and 24h. The results are shown in Table 16:

[0198] Table 16 Stability test results

[0199]

[0200] From the above, it can be seen that the RSD value of the proline peak area in the sample solution within 24 hours is 0.40%, and the RSD value of the alanine peak area is 1.39%, indicating that the test solution has good stability within 24 hours.

[0201] 2.1.9 Sample content determination verification

[0202] According to the proposed method, 16 batches of medicinal materials, 16 batches of decoction pieces, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules of Xueyu Tan were tested. The results are shown in Tables 17-21:

[0203] Table 17 Results of content determination of 16 batches of Xueyu charcoal medicinal materials

[0204]

[0205] Table 18 Content determination results of 16 batches of Xueyu charcoal slices

[0206]

[0207]

[0208] Table 19 Content determination results of 16 batches of Xueyutan standard decoction

[0209]

[0210] Table 20 Content determination results of 3 batches of blood charcoal extracts

[0211]

[0212] Table 21 Test results of content of three batches of blood charcoal formula particles

[0213]

[0214] From the above, it can be seen that the content determination method of Xueyutan can effectively detect Xueyutan medicinal materials, decoction pieces, standard decoctions, extracts, and formula granule samples, making this method stable and feasible.

[0215] Example 3

[0216] The characteristic spectrum method was established based on the chromatographic conditions for content determination in Example 2.

[0217] 3.1 Preparation of test solution

[0218] 3.1.1 Investigation of hydrochloric acid concentration for hydrolysis

[0219] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.5g, accurately weigh it, put it into a stoppered hydrolysis tube, accurately add 10ml of 3mol / L, 6mol / L, and 9mol / L hydrochloric acid solution respectively, stopper it, weigh it, put it at 150℃ for hydrolysis for 1 hour, let it cool, weigh it again, make up the lost weight with the corresponding 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. See the results. Figure 12 The results showed that when the concentration of hydrolysis hydrochloric acid was 9 mol / L, the peak shapes of the characteristic peaks were good and the separation was moderate. In order to be consistent with the preparation of the test samples for content determination, the concentration of hydrolysis hydrochloric acid was temporarily set at 9 mol / L.

[0220] 3.1.2 Inspection of sample weight

[0221] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, and accurately weigh about 0.1g, 0.2g, and 0.5g respectively. Place it in a stoppered hydrolysis tube, accurately add 10ml of 9mol / L hydrochloric acid solution, seal it, weigh it, place it at 150℃ for 1 hour, let it cool, weigh it again, make up the lost weight with 9mol / 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. See the results. Figure 13 The results showed that when the sample weight was 0.2 g, the chromatogram peak shape and separation were better. In order to keep consistent with the preparation of the sample for content determination, the sample weight was determined to be 0.2 g.

[0222] 3.1.3 Investigation of the amount of hydrochloric acid added for hydrolysis

[0223] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh it, put it into a stoppered hydrolysis tube, accurately add 10ml, 15ml, and 20ml of 9mol / L hydrochloric acid solution respectively, stopper it, weigh it, put it at 150℃ for hydrolysis for 1 hour, let it cool, weigh it again, make up the lost weight with 9mol / 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. See the results. Figure 14 The results showed that when the amount of hydrolysis hydrochloric acid added was 10 ml, the peak shapes of the characteristic peaks were good and the separation was moderate. In order to be consistent with the preparation of the test samples for content determination, the amount of hydrolysis hydrochloric acid added was temporarily set at 10 ml.

[0224] 3.1.4 Investigation of hydrolysis time

[0225] Take an appropriate amount of this product (batch number: GSP01), grind it into powder, take about 0.2g, accurately weigh it, put it into a stoppered hydrolysis tube, accurately add 10ml of 9mol / L hydrochloric acid solution, seal it, weigh it, and hydrolyze it at 150℃ for 1 hour, 2 hours, 3 hours, and 4 hours respectively. Let it cool, weigh it again, make up the lost weight with 9mol / 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. See the results. Figure 15 The results showed that when the hydrolysis time was 2 h, the chromatogram peak shape and separation were better. In order to keep consistent with the preparation of the test sample for content determination, the hydrolysis time was determined to be 2 h.

[0226] In summary, the preparation method of the test solution of the characteristic spectrum of blood charcoal is determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.2g, accurately weigh it, put it in a hydrolysis tube with a stopper, accurately add 10ml of 9mol / 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 9mol / 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.

[0227] 3.1.5 Feature Mapping Method

[0228] 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, 0.02% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1 above, flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 245 nm. The number of theoretical plates, calculated based on the alanine peak, should be no less than 5000.

[0229] Preparation of reference solution: Take 1 g of blood charcoal reference medicinal material, place it in a stoppered hydrolysis tube, add 10 ml of 9 mol / L hydrochloric acid solution, stopper it, hydrolyze it at 150°C for 2 hours, let it cool, shake it well, filter it, take 5 ml of the filtrate, evaporate it to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and use it as the reference solution of the control medicinal material.

[0230] Take appropriate amount of alanine reference substance and proline reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution of 50 μg alanine and 50 μg proline per 1 ml, which is used as the reference substance solution.

[0231] Preparation of test solution: Take an appropriate amount of the product (GSP01 granules), grind it into powder, take about 0.2 g, accurately weigh it, place it in a hydrolysis tube with a stopper, accurately add 10 ml of 9 mol / L hydrochloric acid solution, stopper it tightly, weigh it, place it at 150 ° C for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it.

[0232] 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, respectively. 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.

[0233] 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.

[0234] 3.1.6 Methodological Investigation

[0235] 3.1.6.1 Chromatographic peak identification

[0236] Preparation of test solution According to the experimental conditions proposed above, the blood charcoal test solution was prepared.

[0237] Preparation of control medicinal material solution: Take 1 g of blood charcoal control medicinal material, place it in a stoppered hydrolysis tube, accurately add 10 ml of 9 mol / L hydrochloric acid solution, seal it, weigh it, hydrolyze it at 150℃ for 2 hours, let it cool, weigh it again, make up the lost weight with 9 mol / L hydrochloric acid solution, shake it well, filter it, accurately measure 5 ml of the filtrate into an evaporating dish, evaporate it to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer it to a 25 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake it well, and obtain it as the control medicinal material reference solution.

[0238] Preparation of reference solution: Take appropriate amount of glycine reference substance, proline reference substance, alanine reference substance, tyrosine reference substance, valine reference substance, and leucine reference substance, weigh accurately, and add 0.1 mol / L hydrochloric acid solution to make a solution containing 50 μg of each per 1 ml as the reference solution.

[0239] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of ischemic charcoal particles.

[0240] Position the characteristic peaks of the blood charcoal spectrum, see Figure 16 The results showed that among the 9 characteristic peaks of the test sample, peak 2 was glycine, peak 3 was proline, peak 4 was alanine, peak 6 was tyrosine, peak 7 was valine, and peak 9 was leucine. Therefore, in the following methodological investigation, the 9 characteristic peaks in the test sample were investigated.

[0241] 3.1.6.2 Precision test

[0242] Prepare 1 aliquot of the test solution according to the proposed experimental method, inject the sample 6 times continuously, and calculate the retention time and peak area of ​​each characteristic peak. See Table 22:

[0243] Table 22 Precision Investigation-Retention Time

[0244]

[0245] The results showed that the RSD of the retention time of each characteristic peak of the sample ranged from 0.02% to 0.75%, indicating that the instrumental precision of this method was good.

[0246] 3.1.6.3 Repeatability Study

[0247] Prepare six test sample solutions according to the proposed experimental method, measure, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 23.

[0248] Table 23 Repeatability study - relative retention time ratio

[0249]

[0250] The results showed that the RSD range of the relative retention time of the six samples was 0.00% to 0.06%, indicating that the method had good repeatability.

[0251] At time T1, personnel A1 prepared two test solutions according to the proposed experimental method and measured them on the C1 (Waterse2695) machine; at time T2, personnel A2 prepared two test solutions according to the proposed experimental method and measured them on the C2 (Agilent 1260) machine; calculate the relative retention time and relative peak area of ​​each characteristic peak. Figure 17 Table 24:

[0252] Table 24 Intermediate precision investigation - relative retention time ratio

[0253]

[0254] The results showed that when different people, at different times and using different instruments measured the same sample, the RSDs of the relative retention times of the characteristic peaks were 0.10% to 0.65%, indicating that the method had good intermediate precision.

[0255] 3.1.6.4 Column durability assessment

[0256] The same sample (lot number GSP01) was tested using three different C18 columns: Kromasil 100-5-C18, 4.6 mm × 250 mm, 5 μm (column 1); SHIMADZU Shim-pack GIST C18, 4.6 mm × 250 mm, 5 μm (column 2); and Waters XBridge C18, 4.6 mm × 250 mm, 5 μm (column 3). See Table 25. Figure 18 .

[0257] Table 25 Column durability investigation - relative retention time

[0258]

[0259] As can be seen from the figure above, 9 common peaks appear in the three different chromatographic columns, indicating that the chromatographic columns of this method have good durability.

[0260] 3.1.6.5 Stability

[0261] According to the experimental conditions proposed above, prepare a sample solution and measure it at 0h, 3h, 7h, 11h, 17h, and 24h respectively. See Table 26:

[0262] Table 26 Stability Study-Retention Time

[0263]

[0264] As shown in Table 26, the RSD of the characteristic peak retention time was 0.09% to 0.31%, and the sample solution was stable within 24 hours.

[0265] 3.1.6.6 Determination of characteristic peaks and establishment of reference maps

[0266] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, the remaining seven peaks with good reproducibility were ultimately selected as characteristic peaks. Peak 6 had a too-small peak area, and Peak 9 had poor peak shape and resolution. The RSD values ​​for the relative retention times of each characteristic peak were small and within ±10% of the mean. However, the RSD values ​​for the relative peak areas of each characteristic peak were larger. Therefore, only the specified values ​​for the relative retention times were considered, and the specified range for the relative retention times of each peak was tentatively set to ±10%.

[0267] 3.2 Sample characteristic spectrum verification

[0268] The proposed method was used to determine the characteristic spectra of 16 batches of Xueyutan medicinal materials, 16 batches of decoction pieces, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules, and the relative retention time was calculated. Figures 19 to 23 , Tables 27 to 30:

[0269] Figure 19 This is the characteristic spectrum of Xueyu charcoal medicinal materials, among which S1~S16 are: 23001, 23002, 23003, 23004, 23005, 23006, 23007, 23008, 23009, 23010, 23011, 23012, 23013, 23014, 23015, 23016;

[0270] Figure 20 Characteristic spectrum of Xueyu charcoal slices, S1 to S16 are: YP01, YP02, YP03, YP04, YP05, YP06, YP07, YP08, YP09, YP010, YP011, YP012, YP013, YP014, YP015, YP016;

[0271] Figure 21 Characteristic spectrum of the standard decoction of Xueyutan, where S1 to S16 are: BT01, BT02, BT03, BT04, BT05, BT06, BT07, BT08, BT09, BT10, BT11, BT12, BT13, BT14, BT115, BT16;

[0272] Figure 22 This is the characteristic spectrum of the intermediate of blood residue carbon;

[0273] Figure 23 This is the verification diagram of the characteristic spectrum of three batches of Xueyu charcoal formula particles;

[0274] Table 27 Relative retention time of characteristic spectrum of Xueyu charcoal medicinal materials

[0275]

[0276]

[0277] Table 28 Relative retention time of characteristic spectra of Xueyu charcoal slices

[0278]

[0279] Table 29 Relative retention time of 16 batches of standard decoction of Xueyu charcoal

[0280]

[0281]

[0282] Table 30 Relative retention time of three batches of blood residue carbon intermediates

[0283]

[0284] Table 31 Relative retention time of three batches of blood charcoal granules

[0285]

[0286] The results showed that the RSDs of the relative retention times of the seven characteristic peaks of 16 batches of medicinal materials, 16 batches of decoction pieces, 16 batches of standard decoctions, 3 batches of extracts and 3 batches of formula granules of Xueyu charcoal were all less than 2.0%.

[0287] Final regulations: The test sample chromatogram should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3 and 4 should correspond to the retention times of the corresponding reference material peaks. The peak corresponding to the alanine reference material peak is the S peak, and the relative retention times of the remaining characteristic peaks to the S peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.45 (peak 1), 0.67 (peak 2), 1.09 (peak 5), 1.65 (peak 6), and 1.85 (peak 7).

[0288] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 16 batches of medicinal materials, 16 batches of decoction pieces, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules of Xueyu Carbon, and a reference spectrum of the corresponding characteristic spectra was established. Figures 24 to 28 , among which, Peak 2: glycine; Peak 3: proline; Peak 4 (S): alanine; Peak 6: valine.

[0289] It can be seen from the above embodiments that the present invention discloses the construction and application of a high-performance liquid phase characteristic spectrum and content determination detection method for Xueyu charcoal. The method confirms that there are 7 characteristic peaks in total, specifies their relative retention times, establishes a control characteristic spectrum, and includes a content determination method with the total amount of proline and alanine as indicators, which fully demonstrates the chemical composition characteristics of Xueyu charcoal and comprehensively reflects the quality information of Xueyu charcoal, thereby achieving comprehensive and effective control of the quality of Xueyu charcoal medicinal materials, decoction pieces and related preparations.

[0290] 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 a high-performance liquid chromatography characteristic spectrum of blood charcoal, comprising the following steps: A) hydrolyzing the blood charcoal raw material with hydrochloric acid, filtering, evaporating the filtrate to dryness, and dissolving the residue with hydrochloric acid to obtain a test solution; the blood charcoal raw material is a blood charcoal medicinal material, decoction piece, standard decoction, intermediate or its formula granules; B) derivatizing the test solution and then determining it by high performance liquid chromatography to obtain a high performance liquid chromatography characteristic spectrum of the charcoal; The process of derivatizing the test solution includes: Adding an acetonitrile solution of phenyl isothiocyanate and an acetonitrile solution of triethylamine to the test solution, allowing the mixture to stand to obtain a derivative product; mixing the derivative product with n-hexane, shaking, allowing the mixture to stand, removing the lower layer solution, filtering, and collecting the filtrate to obtain a derivatized test solution; Chromatographic conditions included: C18 column, mobile phase A: acetonitrile, mobile phase B: 0.02% phosphoric acid solution, gradient elution; injection volume: 10 μL; detection wavelength: 245 nm; theoretical plate number calculated based on amino acid peaks: not less than 5000; The gradient elution is specifically as follows: 0-35 min, phase A: 12-20%, phase B: 88-80%; 35-40 min, phase A: 20-35%, phase B: 80-65%; 40-55 min, phase A: 35%, phase B: 65%; 55-65 min, phase A: 35-80%, phase B: 65-20%; 65-73 min, phase A: 80%, phase B: 20%; Also includes the preparation of reference solutions: Alanine and proline were dissolved in 0.1 mol / L hydrochloric acid to prepare 50 μg / mL reference solutions.

2. The construction method according to claim 1, characterized in that The concentration of hydrochloric acid used for hydrolysis is 3~9 mol / L; the hydrolysis temperature is 105~155℃, and the hydrolysis time is 1~4h; The mass of the blood charcoal and the volume of hydrochloric acid are (0.1-0.5) g: (10-20) mL; The residue was dissolved in 0.1 mol / L hydrochloric acid.

3. The construction method according to claim 1, characterized in that The flow rate of the mobile phase was 0.8–1.2 mL / min; The column temperature is 25~35℃.

4. The construction method according to claim 1, characterized in that The similarity of the Xueyu charcoal sample was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a Xueyu charcoal HPLC standard characteristic spectrum consisting of 7 characteristic peaks was obtained, which corresponded to the 7 characteristic retention times in the chromatogram of the control medicinal material reference material. Among them, peak 3 and peak 4 should correspond to the retention times of the corresponding reference material peaks, respectively; the peak corresponding to the alanine reference material peak is the S peak. In the standard characteristic spectrum, the relative retention times of the remaining characteristic peaks and the S peak were calculated, and the relative retention times were within ±10% of the specified values, which were: 0.45 (peak 1), 0.67 (peak 2: glycine), 1.09 (peak 5), 1.65 (peak 6: valine), and 1.85 (peak 7).

5. A method for determining the content of proline and alanine in blood charcoal, comprising the following steps: S1. Hydrolyze the blood charcoal sample with hydrochloric acid, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.1 mol / L hydrochloric acid to obtain a test solution; Dissolve alanine and proline separately in 0.1 mol / L hydrochloric acid to obtain 50 μg / mL reference solution; S2. Derivatizing the reference substance solution and the test solution to obtain a derivatized reference substance solution and a derivatized test solution; The derivatization process of the reference substance solution and the test solution includes: Adding phenyl isothiocyanate in acetonitrile and triethylamine in acetonitrile to the reference substance solution and the test solution, respectively, and allowing to stand to obtain a derivative product; mixing the derivative product with n-hexane, shaking, allowing to stand, taking the lower layer solution, filtering, and taking the filtrate to obtain a derivatized reference substance solution and a derivatized test solution; S3. Injecting the derivatized reference substance solution and the test solution into a liquid chromatograph, and using high performance liquid chromatography to obtain chromatograms of the chemical components in the derivatized reference substance solution and the derivatized test solution under the same detection conditions; Chromatographic conditions included: C18 column, mobile phase A: acetonitrile, mobile phase B: 0.02% phosphoric acid solution, gradient elution; injection volume: 10 μL; detection wavelength: 245 nm; theoretical plate number calculated based on amino acid peaks: not less than 5000; The gradient elution is specifically as follows: 0-35 min, phase A: 12-20%, phase B: 88-80%; 35-40 min, phase A: 20-35%, phase B: 80-65%; 40-55 min, phase A: 35%, phase B: 65%; 55-65 min, phase A: 35-80%, phase B: 65-20%; 65-73 min, phase A: 80%, phase B: 20%; According to the concentration of the reference substance solution, the peak area of ​​the reference substance solution in the chromatogram, and the area of ​​the components in the blood charcoal corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step S3, the contents of proline and alanine in the blood charcoal are calculated by the external standard method.

6. The measuring method according to claim 5, characterized in that The injection concentration of the proline solution is 8.3140~203.3500μg / mL, and the proline regression equation is Y= 16822.3413X - 13903.6133, R 2 = 0.9999; The injection concentration of alanine solution is 7.9860~199.6500μg / mL, and the alanine regression equation is Y=15137.7776X-48353.7156, R 2 = 0.9994.

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