Construction method of characteristic chromatogram of jingcan and its preparation and content determination method

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and specifically provides a construction method and a content determination method of a characteristic chromatogram of Bombyx Batryticatus and its preparation, the construction method of the characteristic chromatogram comprising the following steps: (1) preparation of a test sample solution: the extraction solvent of the test sample solution is selected from water or a methanol aqueous solution with a volume percentage of not more than 30%; (2) the test sample solution is detected by using high performance liquid chromatography, octadecylsilane bonded silica gel is used as a filler, and an organic acid salt with a concentration of 1-10 mmol / L and acetonitrile with a pH value of 4.3-4.7 are used as mobile phases for gradient elution, so that the characteristic chromatogram of the test sample is obtained. The multiple common characteristic peaks are well separated, the obtained characteristic chromatogram has a smooth baseline, the detection time is short, the separation degree is high, and the characteristic peak shape is good, which provides a basis for quality detection and control of Bombyx Batryticatus and its preparation, and realizes overall component characterization of Bombyx Batryticatus and its preparation.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine detection technology, and specifically relates to a method for constructing a characteristic spectrum of a silkworm and its preparation and a method for determining its content. Background Art

[0002] Bombyx batryticatus refers to the dried, whole larvae of the Bombyx moth, an insect of the family Bombycidae, that have died from infection with Beauveria bassiana. The dead silkworms are collected, mixed with lime, and then dried in the sun or baked. They are cylindrical, often curved and wrinkled. They are approximately 2 to 5 cm long and 4 to 7 mm in diameter. Their surface is off-white or light brown, often covered with a white powdery frost. The head, legs, and segments are clearly visible. Silk clumps are often entangled on the outside of the body. The head is yellowish-brown and round. The legs are eight pairs, forming protrusions. They are hard and brittle, easily broken. The cross-section is flat, with varying shades of brown and black, often shiny. The outer layer is white and powdery, with four brown, bright circles inside. They have a slight rancid odor and a slightly salty taste. Straight, plump, firm, white, and with a smooth cross-section are preferred. They dispel wind, calm nerves, and resolve phlegm and resolve stagnation. Bombyx batryticatus can be used to prevent and treat convulsions, sore throat, submandibular lymphadenitis, facial nerve paralysis, itchy skin and other diseases.

[0003] The 2020 edition of the Chinese Pharmacopoeia only includes the identification of the medicinal properties of Bombyx batryticatus. So far, there is no clear chemical composition index to evaluate Bombyx batryticatus medicinal materials. The current literature reports mostly use simple determination of the content of one or several components, which can no longer fully reflect the quality of the medicinal materials. Chinese herbal formula granules and standard decoctions are common dosage forms of Bombyx batryticatus. Among them, Chinese herbal formula granules are extracted from Chinese herbal medicine slices with water and produced by extraction, drying, granulation and other processes. Their clinical efficacy should be consistent with that of decoctions. Standard decoctions are the material benchmark for measuring whether Chinese herbal formula granules are basically consistent with decoctions. However, whether it is a standard decoction or a Chinese herbal granule, the identification characteristics of the original medicinal material have been lost, that is, it is impossible to inspect and identify the medicinal material from the shape, size, texture, etc. Therefore, it is necessary to establish a method for rapid identification of the characteristic spectrum of Bombyx batryticatus and its preparations and the content determination of Beauveria bassiana toxins, to provide a basis for effective control and scientific evaluation of the quality of Bombyx batryticatus and its preparations. Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of silkworm pupa and its preparations and a method for content determination. This method establishes a characteristic spectrum of this variety based on the characteristics of silkworm pupa and its pharmaceutical preparations (formulated granules), achieves effective separation of each characteristic peak, and increases the number of characteristic peaks. The results show that this method is simple and easy to operate, has the advantages of good separation effect, high sensitivity, good repeatability, and short detection time. The content determination method has good specificity, providing a scientific basis for comprehensively and rapidly establishing quality control standards for silkworm pupa pharmaceutical preparations.

[0005] The present invention provides a method for constructing a characteristic spectrum of a silkworm and its preparation, comprising the following steps:

[0006] (1) Preparation of test solution: The extraction solvent of the test solution is selected from water or a methanol aqueous solution with a volume percentage not exceeding 30%;

[0007] (2) The sample solution was tested by high performance liquid chromatography using octadecylsilane bonded silica gel as a filler and organic acid salts with a concentration of 1-10 mmol / L and acetonitrile at a pH of 4.3-4.7 as a mobile phase for gradient elution to obtain a characteristic spectrum of the sample.

[0008] Furthermore, the gradient elution is performed according to the following procedure: 0 → 8-10 minutes → 35 minutes → 50 minutes, and the volume percentage of acetonitrile in the mobile phase is 0% → 0% → 3% → 8-10%; preferably, the gradient elution is performed according to the following procedure: 0 → 8 minutes → 35 minutes → 50 minutes, and the volume percentage of acetonitrile in the mobile phase is 0% → 0% → 3% → 8%. Preferably, the chromatographic conditions of the high performance liquid chromatography method in step (2) further include: a detection wavelength of 204-262 nm, a flow rate of 0.8-1.2 ml / min, a column temperature of 20-30° C., and an injection volume of 5-30 μl. More preferably, the detection wavelength is 258-262 nm.

[0009] Furthermore, step (1) is to take a Bombyx batryticatus test sample, add an extraction solvent to extract, obtain an extract, separate the extract into solid and liquid, and take the liquid, which is the test sample solution. Preferably, step (1) satisfies any one or more of the following AC:

[0010] A. In step 1), add 50-500 times the amount of extraction solvent as the Bombyx batryticatus test sample;

[0011] B. In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 15 min-1 h;

[0012] C. In step 2), the solid-liquid separation is independently selected from centrifugation or filtration.

[0013] Furthermore, the construction method also includes the steps of using at least one of uridine, uracil, uric acid, uridine, adenine, guanosine and adenosine as a reference substance, dissolving the reference substance in a solvent to prepare a reference substance solution, and detecting the reference substance solution by high performance liquid chromatography according to the construction method according to any one of claims 1 to 3 to obtain a reference spectrum of the reference substance. Preferably, the solvent is selected from water or a methanol aqueous solution with a volume percentage of not more than 30%. Preferably, each 1 mL of the reference substance solution contains 5-100 μg of each reference substance.

[0014] Furthermore, the chromatographic column is a Waters XSelect HSS T3 5μm 4.6mm*250mm column; and / or the organic acid salt is selected from at least one of sodium acetate, ammonium formate and ammonium acetate.

[0015] Furthermore, the characteristic spectrum of the silkworm and its preparation has 10 characteristic peaks, peak 4, peak 5, and peak 7 correspond to the retention times of the reference peaks of uridine, adenine, and guanosine, respectively, and the peak corresponding to the uridine reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, and the specified value is: 0.39 (peak 1), 0.58 (peak 2), 0.71 (peak 3), 1.36 (peak 6), 2.16 (peak 8), 2.27 (peak 9), and 2.59 (peak 10).

[0016] Furthermore, the relative peak area between peak 2 and peak S in the characteristic spectrum of the silkworm and its preparation is not less than 5.5.

[0017] The preparation method of the reference solution includes the following steps: the construction method also includes the steps of using the residue obtained by water extraction and drying of the silkworm reference medicinal material and then obtaining a reference medicinal material reference solution according to any of the above construction methods, and detecting the reference solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference medicinal material reference atlas.

[0018] The present invention also provides a method for determining the content of beauveria bassiana toxin in silkworm batryticatus and its preparation, comprising the following steps:

[0019] (1) Preparation of Bombyx batryticatus test solution and reference solution;

[0020] (2) The Bombyx batryticatus test sample solution and the reference sample solution were detected by high performance liquid chromatography-tandem mass spectrometry. The high performance liquid chromatography conditions were as follows: octadecylsilane bonded silica gel was used as the filler, acetonitrile containing 4.5-5.5 mmol / L ammonium formate, 0.08-0.12% formic acid and 4-6% water was used as the mobile phase A, and an aqueous solution containing 4.5-5.5 mmol / L ammonium formate and 0.08-0.12% formic acid was used as the mobile phase B. Gradient elution was performed to obtain a characteristic spectrum of the test sample.

[0021] Furthermore, the gradient elution program of the HPLC includes: 0→0.5 minutes→4 minutes→7 minutes, and the volume percentage of mobile phase A in the mobile phase is 80%→80%→100%→80%; and / or,

[0022] The column temperature of the HPLC is 25-40°C and the flow rate is 0.2-0.4 mL / min; and / or,

[0023] The mass spectrometry conditions were as follows: electrospray ionization positive mode, multiple reaction monitoring, mass-to-charge ratio of 804.1→244.0 was selected as the quantitative ion pair; mass-to-charge ratio of 804.1→262.0 was selected as the qualitative ion pair.

[0024] Furthermore, the preparation method of the Bombyx batryticatus test sample solution includes: taking a Bombyx batryticatus test sample, adding an extraction solvent for extraction to obtain an extract, separating the extract into solid and liquid, and taking the liquid, which is the test sample solution. Preferably, the extraction solvent is selected from at least one of water, methanol and ethanol; preferably, the extraction method is ultrasonic extraction or heating reflux extraction; preferably, the extraction time is 15-60 minutes; preferably, 50-500 times the amount of extraction solvent as the Bombyx batryticatus test sample is added.

[0025] The present invention also provides the use of any of the above-mentioned methods for constructing a characteristic spectrum of Bombyx batryticatus and its preparations or the method for determining the content of Beauveria bassiana toxin in Bombyx batryticatus and its preparations in the quality inspection of Bombyx batryticatus products.

[0026] The present invention also provides a quality detection method for silkworm pupa and its preparations, comprising constructing a characteristic spectrum of the silkworm pupa and / or silkworm preparation to be tested according to any of the above-mentioned methods for constructing a characteristic spectrum of the silkworm pupa and its preparations to obtain a characteristic spectrum, or determining the content of beauveria bassiana toxin in silkworm pupa and its preparations using any of the above-mentioned methods for determining the content of beauveria bassiana toxin.

[0027] The technical solution of the present invention has the following advantages:

[0028] 1. The method for constructing a characteristic spectrum of silkworm pupa and its preparation provided by the present invention uses water or a methanol aqueous solution with a volume percentage not higher than 30% as an extraction solvent, octadecylsilane bonded silica gel as a filler, and an organic acid salt with a concentration of 1-10 mmol / L and acetonitrile at a pH value of 4.3-4.7 as a mobile phase for gradient elution to obtain a characteristic spectrum of the test sample, achieves good separation of multiple common characteristic peaks, and the obtained characteristic spectrum has a stable baseline, short detection time, high separation, and good characteristic peak shape, providing a basis for quality detection and control of silkworm pupa and its preparation, and realizing the characterization of the overall components of silkworm pupa and its preparation.

[0029] By optimizing the gradient elution program, the types of organic acid salts in the mobile phase, the concentration of organic acid salts, pH value, flow rate, column temperature and other chromatographic conditions, the separation of common chromatographic peaks was further improved, so that uridine, uracil, uric acid, uridine, adenine, guanosine and adenosine were all well separated, and the characteristic properties of the characteristic spectrum were further improved, which enabled more comprehensive quality monitoring of the silkworm formula granules.

[0030] 2. The method for constructing the characteristic spectrum of Bombyx batryticatus and its preparations provided by the present invention has good peak shape and separation degree of characteristic peaks and good repeatability of characteristic peaks, and has better separation efficiency, peak capacity and sensitivity; this method has high working efficiency, and has the advantages of simplicity, stability, high precision and good repeatability. It can be applied to different types of Bombyx batryticatus preparations, can control the consistency of the quality of Bombyx batryticatus preparations, and quickly realize the quality control of different types of Bombyx batryticatus preparations.

[0031] The detection method of the silkworm preparation provided by the present invention can better reflect the characteristic peaks that characterize the types and numbers of the overall material components of the chemical components in the silkworm preparation by controlling the chromatographic conditions, thereby achieving quality control of the silkworm preparation according to the relative retention time of each characteristic peak. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is the chromatogram of the test sample of the characteristic spectrum of the Bombyx Batryticatus Formula Granules;

[0034] Figure 2 This is the characteristic chromatogram of Bombyx Batryticatus Formula Granules - detection wavelength 206nm;

[0035] Figure 3 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - gradient 2;

[0036] Figure 4 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - gradient 3;

[0037] Figure 5 This is the characteristic spectrum of Bombyx Batryticatus Formula Granules test sample chromatogram - 5mmol sodium acetate (pH 4.7);

[0038] Figure 6 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - 5mmol ammonium formate (pH 4.7);

[0039] Figure 7 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - 1mmol ammonium acetate (pH 4.7);

[0040] Figure 8 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - 5mmol ammonium acetate (pH 4.7);

[0041] Figure 9 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - 10mmol ammonium acetate (pH 4.7);

[0042] Figure 10 This is the chromatogram of the characteristic spectrum of the Bombyx Batryticatus Formula Granules - pH value 4.3;

[0043] Figure 11 This is the chromatogram of the test sample of the characteristic spectrum of the Bombyx Batryticatus Formula Granule - pH value 4.5;

[0044] Figure 12 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - flow rate 0.8ml / min;

[0045] Figure 13 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - flow rate 1.0ml / min;

[0046] Figure 14 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - flow rate 1.2ml / min;

[0047] Figure 15 This is the chromatogram of the characteristic spectrum of the Bombyx Batryticatus Formula Granules - column temperature 20℃;

[0048] Figure 16 This is the chromatogram of the characteristic spectrum of the Bombyx Batryticatus Formula Granules - column temperature 30℃;

[0049] Figure 17 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - Agilent chromatographic column;

[0050] Figure 18 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - JIER chromatographic column;

[0051] Figure 19 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules. The chromatogram of the test sample is a Waters column and an Agilent chromatograph.

[0052] Figure 20 This is the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample chromatogram - Waters chromatograph;

[0053] Figure 21 Characteristic spectra of 18 batches of Bombyx Batryticatus granules;

[0054] Figure 22 This is the chromatogram of the control medicinal material solution of Bombyx batryticatus;

[0055] Figure 23 This is the reference characteristic spectrum of Bombyx Batryticatus Formula Granules; Peak 1: uracil; Peak 2: uric acid; Peak 4 (S): uridine; Peak 5: adenine; Peak 7: guanosine; Peak 10: adenosine;

[0056] Figure 24 This is an overlay diagram of the positioning of different reference substances; Peak 1: uracil, Peak 2: uric acid, Peak 4: uridine, Peak 5: adenine, Peak 7: guanosine, Peak 10: adenosine;

[0057] Figure 25 is the blank solvent test sample chromatogram;

[0058] Figure 26 This is the chromatogram of the stir-fried silkworm mixed control solution;

[0059] Figure 27 This is the chromatogram of the sample of Bombyx Batryticatus Formula Granules;

[0060] Figure 28 This is the chromatogram of Beauveria bassiana toxin reference substance;

[0061] Figure 29 This is the chromatogram of the test sample of Bombyx Batryticatus Formula Granules;

[0062] Figure 30 This is the blank control chromatogram of Bombyx Batryticatus Formula Granules;

[0063] Figure 31 This is the chromatogram of the auxiliary materials of Bombyx Batryticatus formula granules. DETAILED DESCRIPTION

[0064] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0065] If specific experimental procedures or conditions are not specified in the examples, the procedures or conditions described in the literature in the field can be followed. Reagents or instruments used without manufacturer's information are commercially available. % Methanol refers to the volume percentage of methanol in the methanol-water solution.

[0066] Example 1 Optimization of chromatographic conditions in the characteristic spectrum construction method

[0067] 1. Instruments and test drugs

[0068] (1) Instruments and equipment:

[0069] Chromatograph 1 (Waters): Waters 2695 chromatography system, including a quaternary gradient infusion pump (Alliance 2695), a 120-position high-performance autosampler, an imported column oven, a Waters 2996 DAD diode array detector, and an Empower 3 chromatography management system;

[0070] Chromatograph 2 (Agilent): Agilent 1260 Infinity II Prime LC chromatography system, including a quaternary gradient infusion pump (1260 Flexible Pump), a high-performance autosampler (1260 Vialsanpier), an imported column oven (1260MCT), a diode array detector (1260DAD WR), and an Empower 3 chromatography management system;

[0071] Electronic balance: Sartorius Scientific Instruments (Beijing) Co., Ltd. (BSA124S), Shimadzu Enterprise Management (China) Co., Ltd. (AUW120D)

[0072] Ultrasonic instrument: KQ-500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.

[0073] (2) Chromatographic column

[0074] Column 1: Waters Xselect HSS T3 5 μm 4.6 mm × 250 mm; SN: 01693127117727;

[0075] Column 2: GL InertSustain AQ-C18 5μm 4.6mm×250mm; SN: 19H0044004;

[0076] Chromatographic column 3: Agilent ZORBAX SB-Aq 5μm 4.6mm×250mm; SN:USAG019748.

[0077] (3) Reagents

[0078] Acetonitrile, methanol, glacial acetic acid, and ammonium acetate were of chromatographic grade, and water was ultrapure water.

[0079] (4) Drug testing

[0080] Uracil (batch number: 100469-201302, mass fraction 99.6%, China Food and Drug Administration);

[0081] Uric acid (batch number: 9602, mass fraction 98.0%, Shanghai Shidande Standard Technology Service Co., Ltd.);

[0082] Uridine (batch number: 110887-202104, mass fraction 99.6%, China Food and Drug Administration);

[0083] Adenine (batch number: 110886-201102, mass fraction 99.4%, China Food and Drug Administration);

[0084] Guanosine (batch number: 111977-201501, mass fraction 93.6%, China Food and Drug Administration);

[0085] Adenosine (batch number: 110879-201703, mass fraction 99.7%, China Food and Drug Administration);

[0086] Bombyx batryticatus formula granules are prepared by conventional methods in the field, for example, in the present invention by the following method. According to the provisions of the "Bombyx batryticatus" decoction piece [Processing] under the "Bombyx batryticatus" item of the 2020 edition of the "Chinese Pharmacopoeia", take the Bombyx batryticatus medicinal material, wash it and dry it to remove impurities. Process the Bombyx batryticatus decoction pieces that meet the requirements. Take the Bombyx batryticatus decoction pieces, add water and boil them twice. The first time, add 8 times the weight of water to soak for 30 minutes, boil for 30 minutes, filter with a 200-mesh filter cloth, add 7 times the weight of water for the second time, boil for 25 minutes, filter with a 200-mesh filter cloth, combine the filtrate, concentrate to a fluid paste with a relative density of 1.02~1.12g / mL (65±5℃), spray dry, add an appropriate amount of maltodextrin, mix well, dry granulate, and package.

[0087] 2. Investigation of chromatographic conditions

[0088] (1) Preliminary determination of the detection method

[0089] Preparation of reference solution: Take appropriate amount of uridine, adenine and guanosine reference substances, weigh accurately, add 10% methanol to make a mixed solution containing 15μg uridine, 5μg adenine and 10μg guanosine per 1ml, shake well, and obtain.

[0090] Preparation of test solution: Take about 0.2g of the powder of this product (Bombyx batryticatus formula granules), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, let it cool, weigh it, make up the loss with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0091] Liquid chromatography detection: Accurately pipette 20 μl of each reference solution and test solution and inject into high performance liquid chromatography. The chromatographic conditions are as follows: octadecyl bonded silica gel as the filler (chromatographic column: Waters Xselect HSS T3 column); acetonitrile as mobile phase A, 5 mmol ammonium acetate solution (adjusted to pH 4.70 with 10% glacial acetic acid solution) as mobile phase B, gradient elution according to the table below; wavelength, 260 nm; column temperature, 25°C; flow rate, 1.0 ml per minute.

[0092] Table 1 Gradient elution table - Gradient 1

[0093]

[0094] Table 2 Characteristic spectrum of Bombyx Batryticatus formula granules Chromatographic peak parameters of test sample solution

[0095]

[0096] See the table above and Figure 1 As shown, using acetonitrile-5mmol ammonium acetate (pH adjusted to 4.7 with 10% glacial acetic acid solution) as the mobile phase, the peak shape was good, with 12 chromatographic peaks having symmetry factors ranging from 0.84 to 1.15, good symmetry, and a relatively stable baseline. However, the separation between peaks 11 and 12 was poor. The chromatographic conditions were optimized using the following method for the test solution of the same batch of Bombyx Batryticatus Formula Granules.

[0097] (2) Selection of detection wavelength

[0098] The test solution prepared according to the method under item 2(1) of this embodiment was injected into a high performance liquid chromatograph, and the characteristic spectrum of the Bombyx Batryticatus Formula Granules was detected online in the ultraviolet region of 190-400 nm by a diode array detector. The other chromatographic conditions were the same as those described in item 2(1) of this embodiment. The maximum absorption wavelengths of the chromatographic peaks are shown as follows:

[0099] Table 3 Maximum absorption wavelength of each chromatographic peak

[0100]

[0101] The maximum absorption wavelengths of the above chromatographic peaks are mainly concentrated between 196nm and 286nm. Therefore, the chromatographic peak parameters of 206nm were extracted in the 3D channel and compared with the chromatographic peak parameters of 260nm in item 2(1). The results are as follows:

[0102] Table 4 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test solution - different wavelengths

[0103]

[0104]

[0105] From the above table and Figure 1 and 2 Comparison shows that the wavelength of 260nm has the largest number of peaks, the richest information, good response values, and uniform distribution of peaks, so the wavelength of 260nm is selected as the characteristic spectrum wavelength.

[0106] (3) Investigation of mobile phase gradient

[0107] The test solution prepared according to the method under item 2(1) of this example was injected into a high performance liquid chromatograph and eluted with different gradients using acetonitrile-5mmol ammonium acetate (pH adjusted to 4.70 with 10% glacial acetic acid solution) as the mobile phase. Elution was performed according to Gradient 2 and Gradient 3 in the table below. The remaining chromatographic conditions were the same as those described in item 2(1) of this example. Comparison and analysis were made with Gradient 1 in Table 1. The results are as follows:

[0108] Table 5 Characteristic spectrum of Bombyx batryticatus formula granules - gradient 2

[0109]

[0110] Table 6 Characteristic spectrum of Bombyx batryticatus formula granules - gradient 3

[0111]

[0112] Table 7 Characteristic spectrum of Bombyx batryticatus granules Chromatographic peak parameters of test solution - different gradients

[0113]

[0114]

[0115] From the above table and Figure 1 、 3 Comparison of the mobile phases of acetonitrile and 5 mmol ammonium acetate (pH 4.7) reveals that gradient 3 provides better separation and symmetry than gradients 1 and 2. Therefore, gradient elution was performed according to gradient 3 for the characteristic spectrum determination.

[0116] (4) Investigation of organic acid salt types in the mobile phase

[0117] The test solution prepared according to the method under item 2(1) of this example was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". 5 mmol / L sodium acetate and ammonium formate with a pH value of 4.7 were used as mobile phase B, respectively. The remaining chromatographic conditions were the same as those described in item 2(1) of this example. The mobile phase B was compared with the 5 mmol / L ammonium acetate with a pH value of 4.7 in item (3) above to investigate the effects of different types of organic acid salts on the system suitability parameters of each characteristic peak of the characteristic spectrum of the silkworm formula granules, and appropriate chromatographic parameters were selected based on the results.

[0118] Table 8 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test solution - different salt types

[0119]

[0120] From the above table and Figure 4-6In comparison, acetonitrile-5mmol ammonium acetate (pH 4.7) as the mobile phase had better characteristic peak separation and better system suitability parameters, so 5mmol ammonium acetate (pH 4.7) was preferred as the mobile phase salt type for subsequent studies.

[0121] (5) Investigation of organic acid salt concentration in mobile phase

[0122] The test solution prepared according to the method under item 2(1) of this example was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". The effects of different concentrations of ammonium acetate (1 mmol, 5 mmol, 10 mmol) on the system suitability parameters of each characteristic peak of the characteristic spectrum of the silkworm formula granules were investigated. The remaining chromatographic conditions were the same as those described in item 2(1) of this example, and appropriate chromatographic parameters were selected based on the results.

[0123] Table 9 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test solution - different salt concentrations

[0124]

[0125]

[0126] From the above table and Figure 7-9 In comparison, when the ammonium acetate concentration is 1 mmol, the peak shape of Peak 2 is poor. When the ammonium acetate concentration is 5 mmol and 10 mmol, the system applicability parameters of each characteristic peak are good. Combined with actual conditions, the ammonium acetate concentration of 5 mmol is preferred for subsequent research.

[0127] (6) Investigation of pH value in mobile phase B

[0128] The test solution prepared according to the method under item 2(1) of this embodiment was injected into the high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". Different pH values ​​(4.3, 4.5) were examined for comparison with the pH value of 4.7 in item (5) above. The other chromatographic conditions were the same as those described in item 2(1) of this embodiment. The influence of the pH value of the mobile phase on the system suitability parameters of each characteristic peak of the characteristic spectrum of the silkworm formula granules was examined, and appropriate chromatographic parameters were selected based on the results.

[0129] Table 10 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test solution - different column temperatures

[0130]

[0131] From the above table and Figure 8 、 10-11, when the pH value is 4.3, peak 1 is poorly separated from the adjacent impurity peaks; when the pH value is 4.7, peak 5 is poorly separated from peak 6; when the pH value is 4.5, the system applicability parameters of each characteristic peak are good. Combined with actual conditions, the mobile phase pH value of 4.5 is preferred for subsequent research.

[0132] (7) Investigation of flow rate

[0133] The test solution prepared according to the method under item 2(1) of this example was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". 5 mmol / L ammonium acetate (pH 4.5) was used as mobile phase B. The effects of different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) on the system suitability parameters (peak shape, separation, symmetry factor, etc.) of each characteristic peak of the characteristic spectrum of the silkworm formula granules were investigated. Appropriate chromatographic parameters were selected based on the results. The remaining chromatographic conditions were the same as those described in item 2(1) of this example.

[0134] Table 11 Characteristic spectrum of Bombyx batryticatus granules Chromatographic peak parameters of test solution - different flow rates

[0135]

[0136] From the above table and Figure 12-14 In comparison, peak 11 and peak 12 were poorly separated when the flow rate was 0.8 ml / min, and peak 1 was poorly separated when the flow rate was 1.2 ml / min, indicating that different flow rates have a great influence on the separation of chromatographic peaks. Combined with actual conditions, the preferred flow rate is 1.0 ml / min for subsequent research.

[0137] (8) Investigation of column temperature

[0138] The test solution prepared according to the method under item 2(1) of this embodiment was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". Different column temperatures (20°C, 30°C) were compared with the column temperature of 25°C in item (7) above. The influence of column temperature on the system suitability parameters of each characteristic peak of the characteristic spectrum of the silkworm formula granules (peak shape, separation, symmetry factor, etc.) was investigated, and appropriate chromatographic parameters were selected based on the results. The remaining chromatographic conditions were the same as those described in item 2(1) of this embodiment.

[0139] Table 12 Characteristic spectrum of Bombyx Batryticatus Formula Granules Chromatographic peak parameters of test sample solution - different column temperatures

[0140]

[0141]

[0142] From the above table and Figure 13 、 15-16, when the column temperature was 20℃, the separation of peak 1, peak 11 and peak 12 was poor, and when the column temperature was 30℃, the separation of peak 1 was poor. When the column temperature was 25℃, the system applicability parameters of each characteristic peak were good. Combined with the actual situation, the column temperature of 25℃ was selected for subsequent research.

[0143] (9) Selection of different chromatographic columns

[0144] The test solution prepared according to the method under item 2(1) of this embodiment was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". The effects of different chromatographic columns (Agilent, Technummer, Waters) on the system suitability parameters (peak shape, resolution, symmetry factor, etc.) of each characteristic peak of the characteristic spectrum of the silkworm formula granules were investigated, and appropriate chromatographic parameters were selected based on the results. The remaining chromatographic conditions were the same as those described in item 2(1) of this embodiment.

[0145] Table 13 Characteristic spectrum of Bombyx Batryticatus Formula Granules Chromatographic peak parameters of test solution - different chromatographic columns

[0146]

[0147] Note: Agilent column: Agilent ZORBAX SB-Aq 5μm 4.6mm*250mm; SN: USAG019748; Techtronic column: GL InertSustain AQ-C18 5μm 4.6mm*250mm; SN: 19H0044004; Waters column: WatersXSelect HSS T3 5μm 4.6mm*250mm; SN: 01693127117727;

[0148] From the above table and Figure 17-19 In comparison, the separation of chromatographic peaks by Agilent and Techel columns was significantly worse than that by Waters columns, so Waters (Waters XSelect HSS T3 5μm 4.6mm*250mm) was preferred as the chromatographic column for determining the characteristic spectrum of Bombyx batryticatus granules.

[0149] (10) Selection of different liquid chromatographs

[0150] The test solution prepared according to the method under item 2(1) of this embodiment was injected into a high performance liquid chromatograph and eluted according to the above-mentioned "gradient 3". The effects of the liquid chromatograph (Waters) and the Agilent chromatograph under item (9) on the system suitability parameters of each characteristic peak of the characteristic spectrum of the silkworm formula granules (peak shape, separation, symmetry factor, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results. The remaining chromatographic conditions were the same as those described in item 2(1) of this embodiment.

[0151] Table 14 Characteristic spectrum of Bombyx Batryticatus Formula Granules Chromatographic peak parameters of test sample solution - different chromatographs

[0152]

[0153] Note: Agilent liquid chromatograph: Agilent 1260 InfinityⅡ Prime LC;

[0154] Waters liquid chromatograph: Waters2695-2996

[0155] From the above table and Figure 19 、 20 Comparison showed that the system suitability parameters of each characteristic peak were better when using Agilent and Waters liquid chromatographs. After comprehensive consideration, Waters liquid chromatograph was selected as the liquid chromatograph for subsequent experiments.

[0156] (11) Optimal chromatographic conditions

[0157] Octadecyl bonded silica gel was used as the filler (the chromatographic column used was Waters XSelect HSS T3 5μm 4.6mm*250mm); acetonitrile was used as the mobile phase A, and 5mmol ammonium acetate solution (adjusted to pH 4.50 with 10% acetic acid) was used as the mobile phase B. Gradient elution was performed according to the table below; the wavelength was 260nm; the column temperature was 25°C; the flow rate was 1.0ml per minute; and the number of theoretical plates calculated based on the uridine peak should be no less than 5000.

[0158]

[0159] Example 2 Investigation of the preparation method of the test solution in the construction of the characteristic spectrum

[0160] 1. Instruments and test drugs

[0161] Same as Example 1.

[0162] 2. Investigation of extraction methods

[0163] The two extraction methods of heating reflux and ultrasonic extraction were compared, and the appropriate extraction method was determined based on the peak area, symmetry factor and separation degree of each characteristic peak in the characteristic spectrum.

[0164] Take about 0.2g of the powder of this product (Bombyx Batryticatus Formula Granules), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, weigh it, take reflux extraction for 30 minutes and ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes respectively, take it out, let it cool, weigh it, make up the weight loss with 10% methanol, mix it, filter it, and take the filtrate to obtain it. According to the optimal chromatographic conditions of Example 1, item (11), the influence of different extraction methods on the system suitability parameters of each characteristic peak of the characteristic spectrum of Bombyx Batryticatus Formula Granules was investigated, and the appropriate extraction method was selected according to the results.

[0165] Table 15 Characteristic spectrum of Bombyx Batryticatus Formula Granules Chromatographic peak parameters of test sample solution - different extraction methods

[0166]

[0167]

[0168] Chromatograms and data analysis showed that under fixed extraction time conditions, the difference in total peak area was small when using ultrasonic extraction and reflux extraction. Therefore, both ultrasonic extraction and heating reflux extraction were feasible. Taking all factors into consideration, ultrasonic extraction was the preferred extraction method.

[0169] 3. Extraction power inspection

[0170] Take about 0.2g of the powder of this product (Bombyx Batryticatus Formula Granules), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, weigh it, and ultrasonicate it at different ultrasonic powers (power 200W, power 300W, power 400W) for 30 minutes, take it out, let it cool, weigh it, make up the weight loss with 10% methanol, mix it, filter it, and take the filtrate to obtain it. According to the optimal chromatographic conditions of item (11) of Example 1, the influence of different ultrasonic powers on the system suitability parameters of each characteristic peak of the characteristic spectrum of Bombyx Batryticatus Formula Granules was investigated, and the appropriate extraction power was selected according to the results.

[0171] Table 16 Characteristic spectrum of Bombyx batryticatus granules Chromatographic peak parameters of test solution - different ultrasonic powers

[0172]

[0173]

[0174] The chromatogram and data analysis showed that under the condition of fixed extraction time, the total peak area difference was small when different ultrasonic powers were used for extraction, so 200W-400W were both acceptable. Taking all factors into consideration, 300W ultrasonic power was preferred for extraction.

[0175] 4. Investigation of extraction solvent

[0176] The extraction solvent types were investigated for the preparation of the characteristic spectrum of the Bombyx Batryticatus Formula Granules test sample. Methanol, anhydrous ethanol, water, 10% methanol, and 20% methanol were used as extraction solvents. The appropriate extraction solvent was determined based on the total peak area, symmetry factor, and separation of each characteristic peak of the characteristic spectrum. About 0.2 g of the powder of this product (Bombyx Batryticatus Formula Granules) was accurately weighed and placed in a stoppered conical flask. 25 ml of the above solvents were accurately added. Ultrasonic extraction (power 300 W, frequency 40 kHz) was performed for 30 minutes according to the above conditions. The product was taken out, cooled, weighed, and the corresponding solvent was added to make up for the weight loss. The product was mixed and filtered to obtain the product. The optimal chromatographic conditions of item (11) of Example 1 were determined to investigate the effects of different extraction solvent types on the system suitability parameters of each characteristic peak of the characteristic spectrum of the Bombyx Batryticatus Formula Granules, and the appropriate extraction solvent was selected based on the results.

[0177] Table 17 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test sample solution - different extraction solvent types

[0178]

[0179]

[0180] The results showed that when the sampling volume, extraction method and extraction time were fixed, the characteristic spectrum was detected by changing the extraction solvent. The total peak area when water, 10% methanol and 20% methanol were used as extraction solvents was greater than the total peak area when methanol and anhydrous ethanol were used as extraction solvents. Almost no components were extracted from anhydrous ethanol, and the extraction efficiency of methanol was very low, indicating that the water solubility of each characteristic peak was good. The extraction efficiency in aqueous alcohol decreased with the increase of alcohol concentration. Therefore, water or a methanol aqueous solution with a volume percentage of not more than 30% can be used as the extraction solvent. Combined with the comprehensive consideration of the chromatographic adaptability parameters, it is more reasonable to prefer water as the extraction solvent.

[0181] 5. Investigation of the amount of extraction solvent added

[0182] The amount of extraction solvent added to the characteristic spectrum of the Bombyx Batryticatus Formula Granules was investigated. About 0.2 g of the powder of this product (Bombyx Batryticatus Formula Granules) was taken and accurately weighed. The powder was placed in a stoppered conical flask and a measuring flask, and 15 ml, 25 ml, 50 ml, and 100 ml of water were accurately added, respectively, and the weight was weighed. The ultrasonic extraction (power 300 W, frequency 40 kHz) was carried out for 30 minutes according to the above-mentioned extraction method. The product was taken out, cooled, weighed, and the weight loss was supplemented with water. The product was mixed and filtered to obtain the product. The optimal chromatographic conditions of Example 1, item (11) were determined to investigate the effects of different amounts of extraction solvent added on the system suitability parameters (peak shape, resolution, symmetry factor, etc.) of each characteristic peak of the characteristic spectrum of the Bombyx Batryticatus Formula Granules, and the appropriate amount of solvent was selected according to the results.

[0183] Table 18 Characteristic spectrum of Bombyx batryticatus granules Chromatographic peak parameters of test sample solution - different solvent dosages

[0184]

[0185]

[0186] The results showed that with the increase of solvent dosage, the total peak area and the area of ​​each characteristic peak increased in corresponding proportion; the ratio of the total peak area to the sampling volume of the four was slightly different, and the four solvent dosages could all be used as the preparation amount of the test sample. Considering the durability of the detection method, the solvent dosage should have a certain fluctuation range, so the middle value of 25ml was proposed as the solvent dosage of the test sample.

[0187] 6. Investigation of extraction time

[0188] The extraction time of the characteristic spectrum of the Bombyx Batryticatus Formula Granule was investigated. About 0.2 g of the powder of this product (Bombyx Batryticatus Formula Granule) was taken, accurately weighed, and placed in a stoppered conical flask. 25 ml of water was accurately added and weighed. The sample was ultrasonically extracted (power 300 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. The sample was taken out, cooled, weighed, and the weight loss was supplemented with methanol. The sample was mixed and filtered. The optimal chromatographic conditions of Example 1, item (11) were used to determine the effect of different extraction times on the system suitability parameters of each characteristic peak of the characteristic spectrum of the Bombyx Batryticatus Formula Granule, and the appropriate extraction time was selected based on the results.

[0189] Table 19 Characteristic spectrum of Bombyx batryticatus formula granules Chromatographic peak parameters of test solution - different extraction times

[0190]

[0191]

[0192] Through the above data analysis, samples were prepared and characteristic spectra were measured by changing the extraction time while keeping other conditions unchanged. The results showed that the total peak area of ​​each characteristic peak of the test sample at each extraction time was not much different, and no other miscellaneous peaks appeared as time went on. Therefore, 15 minutes to 60 minutes can be used, and the preferred extraction time is 30 minutes.

[0193] 7. Sampling volume inspection

[0194] To investigate whether the characteristic peaks in the Bombyx Batryticatus Formula Granules were completely extracted under different sampling amounts, about 0.1, 0.2, and 0.4 g of the powder of this product (Bombyx Batryticatus Formula Granules) were taken respectively, accurately weighed, placed in a stoppered conical flask, accurately added with 25 ml of water, and ultrasonically extracted (power 300 W, frequency 40 kHz) for 30 minutes. The product was taken out, cooled, weighed, and water was added to make up for the loss in weight. The product was mixed and filtered to obtain the product. The product was measured according to the optimal chromatographic conditions of item (11) of Example 1. The influence of different sampling amounts on the system suitability parameters of the characteristic peaks of the characteristic spectrum of the Bombyx Batryticatus Formula Granules was investigated, and the appropriate sampling amount was selected according to the results.

[0195] The results showed that when the sampling volume was between 0.1 and 0.4 g, the total peak area and the area of ​​each characteristic peak increased in a corresponding proportion; the ratio of the total peak area to the sampling volume of the three was slightly different, and the three sampling volumes could all be used as the preparation volume of the test sample. After comprehensive consideration, the middle value of the sampling volume, 0.2 g, was selected for subsequent research.

[0196] 8. Sample volume inspection

[0197] The system applicability of each characteristic peak in the Bombyx Batryticatus Formula Granules under different injection volumes was investigated. Therefore, about 0.2 g of the powder of this product (Bombyx Batryticatus Formula Granules) was taken, accurately weighed, placed in a stoppered conical flask, 25 ml of water was accurately added, the weight was weighed, and ultrasonic extraction (power 300 W, frequency 40 kHz) was performed for 30 minutes. The product was taken out, cooled, weighed, water was added to make up for the loss in weight, mixed, and filtered. 5 μl, 10 μl, 15 μl, and 20 μl were injected for measurement respectively. The effects of different injection volumes on the system applicability parameters of each characteristic peak in the characteristic spectrum of the Bombyx Batryticatus Formula Granules were investigated, and the appropriate injection volume was selected according to the results.

[0198] The results showed that when the injection volume increased between 5μl and 20μl, the total peak area and the area of ​​each characteristic peak increased in a corresponding proportion; the ratio of the total peak area to the injection volume of the four was relatively small; considering that the response values ​​of some chromatographic peaks were small, an injection volume of 20μl was selected for subsequent research.

[0199] 9. Best preparation method

[0200] Take the test sample, grind it into powder, take about 0.2g, weigh it accurately, put it into a stoppered conical flask, add 25ml of water accurately, weigh it, and treat it ultrasonically (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh it, make up the loss with water, shake it well, filter it, and take the filtrate to obtain it.

[0201] Example 3 Construction of the control characteristic spectrum

[0202] 1. Instruments and test drugs

[0203] Same as Example 1.

[0204] 2. Construction of control feature map

[0205] 18 batches of Bombyx Batryticatus decoction pieces were taken and prepared into test solution according to the method of item 9 of Example 2, and Bombyx Batryticatus control medicinal material was taken and prepared into control medicinal material reference solution according to the following method. The samples were tested under the optimal chromatographic conditions of item 2 (11) of Example 1 to obtain characteristic spectra. Figure 21-22 The reference characteristic spectrum was generated by using the characteristic spectrum similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee. Figure 23 The measurement results show that the relative retention time of each characteristic peak of the test sample is within ±10% of the specified value.

[0206] Take 0.8 g of Bombyx batryticatus reference medicinal material, place it in a stoppered conical flask, accurately add 50 ml of water, weigh the weight, heat and reflux for 30 minutes, cool, weigh, make up the lost weight with water, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0207] Table 20 Results of relative retention time determination of characteristic spectra of multiple batches of Bombyx batryticatus samples

[0208]

[0209]

[0210] Table 21 Results of relative peak area determination of characteristic spectra of multiple batches of Bombyx batryticatus test products

[0211]

[0212]

[0213] 3. Identification of characteristic peaks

[0214] Preparation of each reference solution: Take appropriate amount of uridine, adenine, and guanosine reference substances, accurately weigh them, add 10% methanol to make a mixed solution containing 15μg uridine, 5μg adenine, and 10μg guanosine per 1ml, shake well, and use it as the reference solution;

[0215] The above-mentioned reference solution and test solution (prepared by the method of "Preparation of test solution" in item 2(1) of Example 1) were respectively aspirated, and a characteristic spectrum was obtained under the optimal chromatographic conditions in item 2(11) of Example 1.

[0216] Feature maps such as Figure 24 As shown, by comparing with the spectrum of the reference solution, it was determined that peak 1 was uracil; peak 2 was uric acid; peak 4 was uridine; peak 5 was adenine; peak 7 was guanosine; and peak 10 was adenosine.

[0217] 4. Specified value

[0218] The relative retention time of the characteristic spectrum is determined based on research results: the characteristic spectrum of the silkworm formula granule has 10 characteristic peaks, which should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 4, 5, and 7 should correspond to the retention times of the uridine, adenine, and guanosine reference substances, respectively; the peak corresponding to the uridine reference 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, which is: 0.39 (peak 1), 0.58 (peak 2), 0.71 (peak 3), 1.35 (peak 6), 2.16 (peak 8), 2.26 (peak 9), and 2.59 (peak 10). In addition, the relative peak area of ​​Peak 2 (uric acid) is specified, and its relative peak area shall not be less than 5.5.

[0219] Table 22 Relative retention time of the comparison chart of Bombyx Batryticatus Formula Granule

[0220]

[0221] Table 23 Relative peak areas of the comparison spectrum of Bombyx Batryticatus Formula Granules

[0222]

[0223] Table 24 Relative retention time of characteristic spectra of Bombyx batryticatus reference medicinal materials

[0224]

[0225]

[0226] Table 25 Relative peak areas of characteristic spectra of Bombyx batryticatus control medicinal materials

[0227]

[0228] Example 4 Methodological Verification of the Characteristic Spectrum Construction Method

[0229] 1. Precision experiment

[0230] (1) Instrument precision

[0231] Take about 0.2 g of the same batch of Bombyx batryticatus granules, weigh accurately, and prepare 1 test solution according to the method of Example 2, item 9. Inject the sample 6 times continuously under the optimal chromatographic conditions of Example 1, item 2 (11), record the chromatogram, and calculate the relative retention time and relative peak area RSD% of each characteristic peak.

[0232] The results showed that when the same test solution was injected six times continuously, the relative retention time RSD% of the 10 characteristic peaks were all less than 2.0, and the relative peak area RSD% were all less than 5.0, indicating that the instrument had good precision.

[0233] (2) Repeatability test

[0234] Take about 0.2 g of the same batch of Bombyx Batryticatus granules, weigh 6 portions in parallel, accurately weigh, and prepare 6 test solutions in parallel according to the method of Example 2, item 9. Samples were injected and analyzed under the optimal chromatographic conditions of Example 1, item 2 (11), and the chromatograms were recorded. The relative retention time and relative peak area RSD% of each characteristic peak were calculated.

[0235] The results showed that: 6 test sample solutions were prepared in parallel and injected for detection. The relative retention time RSD% of the 10 characteristic peaks in its characteristic spectrum were all less than 2.0%, and the relative peak area RSD% were all less than 5.0% except for peaks 5 and 8, which had a small proportion of the total peak area and a larger RSD% (6.87% and 6.55%). The instrument precision was good.

[0236] (3) Intermediate precision (different operators)

[0237] The same batch of Bombyx batryticatus formula granules was taken and independently operated by experimenters A, B, and C. The test solution was prepared according to the method of item 9 of Example 2. The sample was injected and analyzed under the optimal chromatographic conditions of item 2 (11) of Example 1. The chromatogram was recorded and the relative retention time and relative peak area RSD% of each characteristic peak were calculated.

[0238] The results showed that three different experimenters prepared the test solution according to the test sample preparation method, and the sample was tested. The RSD% of the relative retention time of the 10 characteristic peaks was less than 2.0%, and the relative peak area was less than 5.0%. The method had good precision among different experimenters.

[0239] (4) Intermediate precision (different liquid chromatographs)

[0240] The test solution prepared according to the method of item 9 of Example 2 was tested on an Agilent high performance liquid chromatograph (Agilent 1260) and a Waters high performance liquid chromatograph (Waters 2695), respectively. The chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated.

[0241] The results showed that the RSD% of the relative retention time of each characteristic peak was less than 2.0%, and the relative peak area of ​​peak 5 was less than 5.0%, except for peak 5 which had a small proportion of the total peak area and a large RSD% (5.52%). The intermediate precision of different instruments of this method was good.

[0242] 2. Exclusivity

[0243] Preparation of mixed control solution: Take appropriate amount of uridine, adenine and guanosine reference substances, weigh accurately, add 10% methanol to make a mixed solution containing 15μg uridine, 5μg adenine and 10μg guanosine per 1ml, shake well, and use it as the reference solution.

[0244] The test solution was prepared according to the method of Example 2, item 9, and the negative sample of Bombyx Batryticatus Formula Granule (solvent blank, i.e., water) was investigated to see whether it would cause interference. Under the optimal chromatographic conditions of Example 1, item 2 (11), the blank solvent (water), the mixed control solution, and the Bombyx Batryticatus Formula Granule test solution were analyzed by HPLC, and the chromatograms were recorded. The results are shown in FIG. Figure 25-27 As shown, there is no interference in the negative test and the method has good specificity.

[0245] 3. Stability inspection

[0246] Take the same sample solution prepared in Example 2, item 9, and inject it at 0, 2, 4, 8, 12, 18, and 24 hours after preparation under the optimal chromatographic conditions of Example 1, item 2(11). Record the chromatogram, and calculate the relative retention time and relative peak area RSD% of each characteristic peak to investigate the stability of the sample solution.

[0247] The results showed that when the same test solution was sampled and tested within 24 hours, the relative retention time RSD% of its 10 characteristic peaks were all less than 2.0, and the relative peak areas were all less than 5.0%. Therefore, the test solution was stable within 24 hours, and the test solution of this method had good stability.

[0248] Example 5 Content Test Method

[0249] This embodiment provides a method for determining the content of Beauveria bassiana toxin in Bombyx batryticatus and its preparations, comprising the following steps:

[0250] Preparation of reference solution: Accurately weigh an appropriate amount of Beauveria bassiana toxin reference solution and add methanol to prepare a solution containing 10 ng of Beauveria bassiana toxin per 1 mL.

[0251] Preparation of the test solution: Take about 0.3 g of the test sample powder (in this example, the silkworm formula granules), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, weigh it, and ultrasonically treat it for 30 minutes (power 250 W, frequency 40 kHz). Let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0252] The reference solution and the test solution were determined by high performance liquid chromatography-tandem mass spectrometry. The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile containing 5 mmol / ammonium formate, 0.1% formic acid, and 5% water was used as the mobile phase A, and 5 mmol / ammonium formate, 0.1% formic acid was used as the mobile phase B. Gradient elution was performed as specified in the table below. The column temperature was 30°C, the flow rate was 0.3 mL per minute, and the number of theoretical plates calculated based on the Beauveria bassiana toxin should be no less than 5000.

[0253]

[0254] A mass spectrometer detector was used in electrospray positive ion mode (ESI+) for multiple reaction monitoring (MRM). The monitored ion pairs are shown in the following table:

[0255]

[0256] The results are shown in the table below and Figures 28-29 shown.

[0257]

[0258] Determination of the preparation method of the test solution in Example 6 content test method

[0259] 1. Selection of extraction solvent concentration

[0260] The extraction effects of different concentrations of methanol and ethanol on the toxin of Beauveria bassiana in the silkworm formula granules were investigated. About 0.3 g of the powder of this product was taken, accurately weighed, and placed in a stoppered conical flask. 25 mL of 50% methanol, 80% methanol, 100% methanol, 50% ethanol, 80% ethanol, and 100% ethanol were accurately added, weighed, and ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes. The mixture was cooled and weighed again. The lost weight was supplemented with the corresponding solvent, shaken, filtered, and the filtrate was taken to obtain the test solution, which was then determined by high performance liquid chromatography-tandem mass spectrometry in Example 5. Each group of parallel experiments were conducted four times. The experimental results showed that after four measurements of 50% methanol, 80% methanol, 100% methanol, 50% ethanol, 80% ethanol, and 100% ethanol, the average values ​​of Beauveria bassiana toxin content were as follows: 1.09μg / g, 1.12μg / g, 1.10μg / g, 1.12μg / g, 1.28μg / g, and 0.41μg / g, and the relative average deviations / % were 1.10%, 2.53%, 1.62%, 0.83%, 0.75%, and 0.39%, respectively.

[0261] The results showed that under different methanol extraction conditions, the measured values ​​of 50% methanol, 80% methanol, 100% methanol, 50% ethanol, and 80% ethanol were significantly greater than those of 100% ethanol. Under the condition of 80% methanol, the sample dissolution effect was also better. Therefore, 80% methanol is the preferred extraction solvent for the extraction of mycobacterium serovar in the silkworm formula granules.

[0262] 2. Selection of extraction method

[0263] Approximately 0.3 g of Bombyx batryticatus granules were accurately weighed and placed in a stoppered conical flask. 25 mL of 80% methanol was accurately added and weighed. The mixture was heated to reflux and ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes, respectively. The mixture was allowed to cool and weighed again. The weight loss was made up with 80% methanol, shaken well, filtered, and the filtrate was collected to obtain the test solution. The solution was then assayed using the high performance liquid chromatography-tandem mass spectrometry method described in Example 5. Four parallel experiments were performed in each group. The average values ​​of the beauveria toxin content measured by ultrasonic treatment and heating reflux extraction were 1.09 μg / g and 1.15 μg / g, respectively, with RSD values ​​of 1.28% and 0.61%, respectively.

[0264] Two different extraction methods were used, and the results showed that the content of the ultrasonically extracted sample was similar to that of the heated reflux extraction. In order to facilitate subsequent experiments, ultrasound was temporarily selected as the extraction method for Beauveria bassiana toxin in Bombyx batryticatus formula granules.

[0265] 3. Investigation of extraction time

[0266] In order to ensure the complete extraction of the target components, ultrasonic treatment times of 15, 30, and 45 minutes were tested respectively, and the optimal ultrasonic treatment time was determined according to the content of the target components.

[0267] Take about 0.3g of Bombyx batryticatus formula granules, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of 80% methanol, weigh them, and ultrasonically treat them (power 250W, frequency 40KHz) for 15, 30, and 45 minutes respectively. Let them cool, weigh them again, make up the lost weight with methanol, shake them well, filter them, take the filtrate, and obtain the test solution. Then, use the high performance liquid chromatography-tandem mass spectrometry method of Example 5 to determine the solution. Each group of parallel experiments was repeated 4 times. The average values ​​of the beauveria toxin content measured at 15, 30, and 45 minutes were 1.10μg / g, 1.09μg / g, and 1.09μg / g, respectively, and the RSD values ​​were 0.87%, 1.59%, and 0.80%, respectively.

[0268] Three different extraction times were used, and the results showed that all test samples could be completely extracted, and the contents of index components were comparable. In order to ensure complete extraction, the optimal ultrasonic treatment time was 30 minutes after comprehensive consideration.

[0269] 4. Selection of sampling volume

[0270] Take about 0.2, 0.3, and 0.5 g of the silkworm formula granules respectively, accurately weigh them, place them in a stoppered conical flask, accurately add 25 mL of methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let cool, weigh the weight again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain the test solution, which is then determined by high performance liquid chromatography-tandem mass spectrometry in Example 5. The average values ​​of Beauveria bassiana toxin content measured in the three groups of 0.2, 0.3 and 0.5g were 1.08μg / g, 1.09μg / g and 1.06μg / g, respectively, and the RSD values ​​were 0.47%, 0.86% and 0.42%, respectively. The results showed that under the conditions of different sampling volumes, the content of the index components did not differ significantly, indicating that within the sampling volume range of 0.2-0.5g, the index components in the formula granules can be completely extracted. Taking comprehensive consideration and combining the peak area of ​​the sample and the reference, the sampling volume of the test sample was determined to be 0.3g.

[0271] Example 6 Methodology Verification in Content Determination

[0272] 1. Specificity inspection

[0273] Take a blank sample solution (80% methanol) and a test solution of the excipient (maltodextrin) of the silkworm formula granules prepared according to the method for preparing the test solution in Example 5, and perform liquid chromatography-mass spectrometry analysis according to the chromatographic conditions of the high performance liquid chromatography-tandem mass spectrometry method in Example 5, and record the chromatogram. Figure 30-31 , the results showed that there was no interference in the negative control test.

[0274] 2. Limit of detection and limit of quantification

[0275] (1) Detection limit

[0276] The limit of detection (LOD) was determined according to the guidelines for validation of analytical methods in the 2020 edition of the Chinese Pharmacopoeia (CP). The signal from a test sample of known concentration was compared with that from a blank sample, achieving a signal-to-noise ratio of 3:1. The limit of detection for Beauveria bassiana toxin was 0.00862524 ng / mL.

[0277] (2) Limit of quantification

[0278] The limit of quantification (LOQ) for Beauveria bassiana toxin was determined according to the guidelines for validation of analytical methods in the 2020 edition of the Chinese Pharmacopoeia (Chinese Pharmacopoeia). The signal-to-noise ratio was 10:1. The limit of quantification for Beauveria bassiana toxin was 0.00431262 ng / mL.

[0279] The method of the present invention has low detection limit and quantitative limit and wide detection range.

[0280] 3. Investigation of the linear relationship of Beauveria bassiana toxins:

[0281] Take an appropriate amount of Beauveria bassiana toxin, accurately weigh it, and add methanol to make solutions containing 1.0310ng, 2.0621ng, 5.1552ng, 10.3103ng, 25.7758ng, 51.5516ng, and 103.1031ng per 1mL. Accurately pipette 1μL of the seven Beauveria bassiana toxin reference solutions of different concentrations into a liquid chromatograph and measure the peak area. Plot a standard curve with Beauveria bassiana toxin concentration as the abscissa and the peak area integral as the ordinate. Regression equation: y = 5,806.2194x + 2,640.6620, R 2 =0.9999. The experimental results showed that the linear relationship of Beauveria bassiana toxin was good in the range of 1.0310 ng / mL to 103.1031 ng / mL.

[0282] 4. Precision test

[0283] (1) Instrument precision test

[0284] The same sample solution of Bombyx batryticatus formula granules was taken, and the conditions were determined according to the high performance liquid chromatography-tandem mass spectrometry method in Example 5. The sample was repeated 6 times to determine the peak area of ​​Beauveria bassiana toxin. The result showed that the peak area RSD was 1.41%, which was less than 3%, indicating that the instrument had good precision.

[0285] (2) Investigation of the intermediate precision of different personnel

[0286] The same batch of silkworm formula granules was independently operated by experimenters A and B, and processed according to the method of Example 5 to determine the content of Beauveria bassiana toxin. According to the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the test component in the sample is 1μg / g~10μg / g, the intermediate precision RSD is 2.83%, which meets the limit requirement of <11%. The results show that the intermediate precision of this method is good when operated by different operators and different chromatographs.

[0287] 5. Repeatability inspection

[0288] Six portions of the same batch of Bombyx Batryticatus Formula Granules were used to prepare test solutions and assay the contents and calculate the RSD. The results showed an RSD of 0.88%, less than 6%, indicating good repeatability of the method.

[0289] 6. Accuracy inspection

[0290] In a sample recovery test, approximately 0.15 g of nine batches of Bombyx batryticatus formula granules (B. bassiana toxin content 1.290 μg / g) were placed in conical flasks. A B. bassiana toxin reference substance was added at a ratio of 1:0.5, 1:1, and 1:1.5 to the sample B. bassiana toxin content. The steps were as follows: Standard solutions containing 7.59294 ng / mL, 15.18588 ng / mL, and 22.77882 ng / mL of B. bassiana toxin were prepared from the B. bassiana toxin reference substance. 12.5 mL of the same concentration of standard solution and 12.5 mL of 80% methanol were added to each of the three batches. The samples were then treated according to the sample preparation method in Example 5. The recovery rate was calculated using the accompanying sample content, and the recovery rate results are shown. According to the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the component to be tested in the sample is 1μg / g~10μg / g, the recovery limit is 80%~115%. The experimental results show that the recovery rate is 81.58%-87.58%, the average recovery rate is 84.79%, the RSD value is 2.70%, and the method has good accuracy.

[0291] 7. Durability inspection

[0292] (1) Solution stability

[0293] The same sample solution of Bombyx Batryticatus Formula Granules was injected at 0, 2, 4, 6, 8, and 12 hours after preparation. The content was determined by HPLC-MS / MS as described in Example 5 to investigate the stability of the solution. The results showed an RSD value of 2.94%, less than 3%, indicating that the sample solution was essentially stable within 12 hours and met the requirements of the assay.

[0294] (2) Durability study at different flow rates

[0295] About 0.3 g of the silkworm formula granules were taken and accurately weighed. A test solution was prepared according to the "Preparation of the Test Solution" in Example 5. The high performance liquid chromatography-tandem mass spectrometry method in Example 5 was used to test the content of Beauveria bassiana toxin at different flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. The results showed that the RSD value was 0.73%, which was less than 3%, indicating that different flow rates had little effect on the content of the index component.

[0296] (3) Investigation of different column temperatures

[0297] About 0.3 g of the silkworm formula granules were taken and accurately weighed. A test solution was prepared according to the "Preparation of the Test Solution" in Example 5. The high performance liquid chromatography-tandem mass spectrometry method in Example 5 was used to test the content of Beauveria bassiana toxin at different column temperatures of 28°C, 30°C, and 32°C. The results showed that the RSD value was 1.01%, which was less than 3%, indicating that different column temperatures had no significant effect on the content of the index component.

[0298] (4) Investigation of different chromatographic columns

[0299] About 0.3 g of the Bombyx batryticatus formula granules were accurately weighed and a test solution was prepared according to "Preparation of Test Solution" in Example 5. The test was performed on different chromatographic columns: Agilent ZORBAX Eclipse Plus C18, Waters ACQUITY UPLC HSST3, and Agilent ZORBAX SB-Aq. The rest were determined by high-performance liquid chromatography-tandem mass spectrometry in Example 5. The content of beauveria bassiana toxin was determined. The results showed that the RSD value was 1.96%, which was less than 3%, indicating that different chromatographic columns had little effect on the content of the Bombyx batryticatus formula granules. This method has good durability for chromatographic columns of different brands and different fillers.

[0300] The above methodological research results show that this method meets the requirements of content determination and can be used to determine Beauveria bassiana toxin in Bombyx batryticatus formula granules.

[0301] The content of beauveria bassiana toxin in each batch of formula granules is shown in the following table.

[0302] Table 26 Content of Beauveria bassiana toxin in 18 batches of Bombyx batryticatus granules

[0303]

[0304] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Bombyx Batryticatus Formula Granules, characterized in that: The following steps are included: (1) Preparation of test solution: The extraction solvent of the test solution is selected from water or a methanol aqueous solution with a volume percentage not higher than 30%; Preparation method of reference solution: Uridine, uracil, uric acid, adenine, guanosine and adenosine are used as reference substances, and the reference substances are dissolved in a solvent to prepare the reference solution; the solvent is selected from water or a methanol aqueous solution with a volume percentage not higher than 30%; (2) The test sample solution and the reference sample solution were respectively detected by high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler, ammonium acetate at a pH of 4.5 and a concentration of 5-10 mmol / L and acetonitrile as the mobile phase for gradient elution to obtain the characteristic spectrum of the test sample. The gradient elution was performed according to the following procedure: 0 → 8 minutes → 35 minutes → 50 minutes, the volume percentage of acetonitrile in the mobile phase was 0% → 0% → 3% → 8%, the flow rate was 1 ml / min, the column temperature was 25 °C, the chromatographic column was Waters XSelect HSS T3 5μm 4.6mm*250mm column, and the detection wavelength was 204-262nm.

2. The construction method according to claim 1, characterized in that The chromatographic conditions of the high performance liquid chromatography in step (2) also include: an injection volume of 5-30 μl.

3. The construction method according to claim 1, characterized in that The detection wavelength is 258-262nm.

4. The construction method according to claim 1, characterized in that Step (1) is to take a test sample of the Bombyx Batryticatus formula granules, add an extraction solvent to extract, obtain an extract, separate the extract into solid and liquid, and take the liquid, which is the test sample solution.

5. The construction method according to claim 4, characterized in that Step (1) satisfies any one or more of the following AC: A. In step (1), add 50-500 times the amount of extraction solvent as the test sample of Bombyx Batryticatus Formula Granules; B. In step (1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 15 min-1 h; C. In step (1), the solid-liquid separation is independently selected from centrifugation or filtration.

6. The construction method according to any one of claims 1 to 4, characterized in that: Each 1 mL of reference solution contains 5-100 μg of each reference substance.

7. Use of the method for constructing a characteristic spectrum of the Bombyx Batryticatus Formula Granule according to any one of claims 1 to 6 in quality inspection of Bombyx Batryticatus Formula Granule products.

8. A quality inspection method for Bombyx Batryticatus Formula Granules, characterized in that: The method comprises constructing a characteristic spectrum of the Bombyx batryticatus formula granules to be tested according to the method for constructing a characteristic spectrum of the Bombyx batryticatus formula granules according to any one of claims 1 to 6.

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