Fingerprint detection method for nucleoside and amino acid components in centipede pharmaceutical preparations

Abstract

The present invention relates to the technical field of traditional Chinese medicine detection, and discloses a fingerprint detection method for nucleoside and amino acid components in centipede pharmaceutical preparations. Using the fingerprint detection method, a fingerprint of the centipede pharmaceutical preparation can be constructed. The resulting fingerprint is highly characteristic and rich in chromatographic information, fully demonstrating the chemical composition characteristics of the centipede pharmaceutical preparation. Furthermore, the detection method has good separation, high precision, good stability, and good repeatability, enabling comprehensive and rapid detection of nucleotide components, amino acid components, and their contents in the centipede pharmaceutical preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, in particular to a fingerprint spectrum detection method for nucleoside and amino acid components in centipede medicinal preparations, and further to a fingerprint spectrum detection method, a content determination method and a quality control method for nucleoside and amino acid components in centipede medicinal preparations. Background Art

[0002] Centipede Formula Granules are derived from the traditional Chinese medicine centipede, which is extracted, concentrated, and granulated. Centipede is the dried body of Scolopendra subspinipes mutilans L. Koch, a member of the Scolopendra family. It primarily contains various ingredients, including amino acids and nucleoside compounds, and is commonly used to calm wind and spasms, dredge meridians and relieve pain, and eliminate toxins and resolve stagnation.

[0003] The 2020 edition of the Chinese Pharmacopoeia stipulates that the quality control of centipede mainly includes the raw material variety, preparation of medicinal pieces, properties of medicinal pieces, and physical and chemical identification. Some literature also describes the chemical composition of centipede, pointing out that centipede contains nucleosides, amino acids, and other components. However, due to the differences in properties between medicinal pieces and pharmaceutical preparations, the quality control methods suitable for centipede medicinal pieces are not applicable to the quality control of centipede pharmaceutical preparations.

[0004] There are also quality detection methods for centipede drug preparations in the related art. However, these methods can usually only detect a single component, and it is difficult to quickly perform quality detection on centipede drug preparations as a whole. Summary of the Invention

[0005] In view of this, the present invention provides a fingerprint spectrum detection method, content determination method and quality control method for nucleoside and amino acid components in centipede pharmaceutical preparations, so as to solve the problem that it is difficult to quickly and comprehensively perform quality inspection on centipede pharmaceutical preparations using related technologies.

[0006] In a first aspect, the present invention provides a method for fingerprint detection of nucleoside and amino acid components in centipede pharmaceutical preparations, comprising the following steps:

[0007] The test solution was taken and tested by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography method include:

[0008] Octadecylsilane bonded silica gel was used as the filler, methanol was used as the mobile phase A, and water was used as the mobile phase B. Gradient elution was performed. The gradient elution procedure included:

[0009] 0-15 min, the volume percentage of methanol in the mobile phase was 1%;

[0010] 15-30 min, the volume percentage of methanol in the mobile phase is 1% → 12%;

[0011] From 30 to 45 minutes, the volume percentage of methanol in the mobile phase is 12% → 40%.

[0012] The present invention provides a fingerprint spectrum detection method for nucleoside and amino acid components in the centipede drug preparation. The method uses octadecylsilane bonded silica gel as a filler, acetonitrile-water as a mobile phase for gradient elution, and selects a specific elution procedure to detect and obtain a fingerprint spectrum containing 8 common characteristic peaks. This method not only significantly increases the number of common characteristic peaks, but also achieves good separation of these common characteristic peaks. The fingerprint spectrum detection method has a simple elution procedure, and the baseline of the obtained fingerprint spectrum is stable. The peak shapes of the characteristic peaks are good, and the separation between the characteristic peaks is high. The method can accurately locate the peak positions of the eight characteristic peaks, namely, uracil, tyrosine, hypoxanthine, xanthine, phenylalanine, inosine, guanosine and tryptophan. This method is beneficial to the comprehensive quality detection and overall quality control of the centipede drug preparation, thereby helping to improve the safety and stable uniformity of the drug, and providing a basis for the quality detection and control of the centipede drug preparation.

[0013] By using the above-mentioned fingerprint detection method, a fingerprint of the centipede drug preparation can be constructed, and the obtained fingerprint is highly characteristic and rich in chromatographic information, which can fully display the chemical component characteristics of the centipede drug preparation; in addition, the above-mentioned detection method has good separation, high precision, good stability and repeatability, and can comprehensively and quickly detect the nucleotide components, amino acid components and their contents in the centipede drug preparation.

[0014] In an optional embodiment, the chromatographic conditions of the high performance liquid chromatography method further include at least one of the following conditions:

[0015] 1) The detection wavelength is 205nm~215nm;

[0016] 2) Column temperature is 23°C to 27°C;

[0017] 3) Flow rate: 0.9 mL / min to 1.1 mL / min;

[0018] 4) The injection volume is 5 μL to 15 μL;

[0019] 5) The chromatographic column is a Dikma Platisil ODS-C18 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 5 μm.

[0020] In an optional embodiment, the gradient elution procedure further comprises:

[0021] 45-50 min, the volume percentage of methanol in the mobile phase is 40% → 45%;

[0022] 50-55 min, the volume percentage of methanol in the mobile phase is 45%;

[0023] 55-56 min, the volume percentage of methanol in the mobile phase is 45% → 1%;

[0024] 56-60 min, the volume percentage of methanol in the mobile phase is 1%.

[0025] In an optional embodiment, the preparation method of the test solution is as follows:

[0026] Take the test sample, add solvent, extract, separate solid and liquid, and take liquid to obtain the test sample solution;

[0027] Optionally, the solvent is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 20% to 40%;

[0028] Optionally, relative to 0.5 g of the test sample, the amount of the solvent added is 20 mL to 30 mL;

[0029] Optionally, the extraction is ultrasonic extraction, and the ultrasonic time is 15 minutes to 45 minutes.

[0030] In an optional embodiment, the fingerprint detection method further comprises the steps of preparing a reference solution using a hypoxanthine reference substance and a phenylalanine reference substance, and detecting the mixed reference solution using the high performance liquid chromatography method to obtain a reference spectrum of the reference substance;

[0031] And / or, the fingerprint detection method further comprises the steps of preparing a control medicinal material solution using centipede control medicinal material, and detecting the control medicinal material solution using the high performance liquid chromatography method to obtain a control medicinal material reference spectrum;

[0032] Optionally, the preparation process of the control medicinal material solution includes: taking centipede control medicinal material, adding water and heating to reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting, separating the solid and liquid, and taking the liquid.

[0033] In an optional embodiment, the centipede pharmaceutical preparation includes centipede formula granules.

[0034] In a second aspect, the present invention provides a method for determining the content of nucleosides and amino acids in a centipede pharmaceutical preparation, comprising the following steps:

[0035] Take the test solution and the reference solution and test them respectively using the high performance liquid chromatography method in the above fingerprint detection method;

[0036] Among them, the reference substances include hypoxanthine reference substance and phenylalanine reference substance;

[0037] Optionally, the detection wavelength of hypoxanthine is 249 nm, and the detection wavelength of phenylalanine is 210 nm.

[0038] In a third aspect, the present invention provides the application of the above-mentioned fingerprint detection method and / or content determination method in the quality control of nucleoside and amino acid components in centipede pharmaceutical preparations.

[0039] In a fourth aspect, the present invention provides a method for quality control of nucleoside and amino acid components in a centipede pharmaceutical preparation, comprising the steps of obtaining a fingerprint of the centipede pharmaceutical preparation to be tested according to the above-mentioned fingerprint detection method, and comparing the fingerprint with a control fingerprint;

[0040] Wherein, the control fingerprint is obtained by fitting the fingerprint obtained by the above-mentioned fingerprint detection method using at least one batch of standard products of centipede drug preparations through the average or median method;

[0041] And / or, it includes the step of determining the content of nucleosides and amino acids in the centipede pharmaceutical preparation to be tested according to the above-mentioned content determination method.

[0042] In an optional embodiment, the control fingerprint includes 8 common characteristic peaks, wherein peak 1 is a uracil peak, peak 2 is a tyrosine peak, peak 3 is a hypoxanthine peak, peak 4 is a xanthine peak, peak 5 is a phenylalanine peak, peak 6 is an inosine peak, peak 7 is a guanosine peak, and peak 8 is a tryptophan peak;

[0043] Taking the phenylalanine peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.29, the specified value corresponding to peak 2 is 0.43, the specified value corresponding to peak 3 is 0.55, the specified value corresponding to peak 4 is 0.68, the specified value corresponding to peak 6 is 1.19, the specified value corresponding to peak 7 is 1.24, and the specified value corresponding to peak 8 is 1.47;

[0044] Optionally, the similarity between the fingerprint of the centipede pharmaceutical preparation to be tested and the fingerprint of the control is greater than 0.90. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] Figure 1 is the fingerprint of 18 batches of centipede formula granule samples in Example 2 of the present invention;

[0047] Figure 2 This is the fingerprint after rearranging the chromatographic peaks in Example 2 of the present invention;

[0048] Figure 3 is the spectrum of the mixed reference substance in Example 2 of the present invention;

[0049] Figure 4 is the control characteristic spectrum determined in Example 2 of the present invention;

[0050] Figure 5 is the negative control chromatogram in Example 3 of the present invention;

[0051] Figure 6 This is the chromatogram of the test sample in Example 3 of the present invention;

[0052] Figure 7 This is the integrity verification chromatogram in Example 3 of the present invention;

[0053] Figure 8 This is the chromatogram obtained by using chromatographic column 1 in Example 3 of the present invention;

[0054] Figure 9 This is the chromatogram obtained by using chromatographic column 2 in Example 3 of the present invention;

[0055] Figure 10 This is a chromatogram obtained by using chromatographic column 3 in Example 3 of the present invention;

[0056] Figure 11 and 12 This is the chromatogram of the reference substance in Example 5 of the present invention;

[0057] Figure 13 and 14 This is a chromatogram of the test sample in Example 5 of the present invention;

[0058] Figure 15 This is the chromatogram obtained by using gradient 1 detection in Example 6 of the present invention;

[0059] Figure 16 This is a chromatogram obtained by using gradient 2 detection in Example 6 of the present invention;

[0060] Figure 17 This is the chromatogram obtained by using gradient 3 detection in Example 6 of the present invention;

[0061] Figure 18 This is a chromatogram obtained by detecting at 210 nm using a mobile phase of 0.1% phosphoric acid-methanol in Example 6 of the present invention;

[0062] Figure 19 This is a chromatogram obtained by detecting at 249 nm using a mobile phase of 0.1% phosphoric acid-methanol in Example 6 of the present invention;

[0063] Figure 20 This is a chromatogram obtained by detecting at 210 nm using a mobile phase of 0.1% formic acid-methanol in Example 6 of the present invention;

[0064] Figure 21 This is a chromatogram obtained by detecting at 249 nm using a mobile phase of 0.1% formic acid-methanol in Example 6 of the present invention;

[0065] Figure 22 This is a chromatogram obtained by detecting at 210 nm using a mobile phase of water-methanol in Example 6 of the present invention;

[0066] Figure 23 This is a chromatogram obtained by detecting at 249 nm using a mobile phase of water-methanol in Example 6 of the present invention;

[0067] Figure 24 This is the chromatogram of the test sample detected at a wavelength of 249 nm during the specificity verification in Example 7 of the present invention;

[0068] Figure 25 This is the chromatogram of the test sample detected at a wavelength of 210 nm during the specificity verification in Example 7 of the present invention;

[0069] Figure 26 This is the negative blank control chromatogram obtained by detecting at a wavelength of 249 nm during the specificity verification in Example 7 of the present invention;

[0070] Figure 27 This is the negative blank control chromatogram obtained by detecting at a wavelength of 210 nm during the specificity verification in Example 7 of the present invention;

[0071] Figure 28 This is the hypoxanthine linear standard curve when verifying the linear relationship in Example 7 of the present invention;

[0072] Figure 29 This is the linear standard curve of phenylalanine when verifying the linear relationship in Example 7 of the present invention. DETAILED DESCRIPTION

[0073] 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.

[0074] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0075] In order to solve the problems existing in the above-mentioned related art, according to a first aspect of the present invention, a fingerprint spectrum detection method for nucleoside and amino acid components in centipede pharmaceutical preparations is provided, comprising the following steps:

[0076] The test solution was taken and tested by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography method include:

[0077] Octadecylsilane bonded silica gel was used as the filler, methanol was used as the mobile phase A, and water was used as the mobile phase B. Gradient elution was performed. The gradient elution procedure included:

[0078] 0-15 min, the volume percentage of methanol in the mobile phase was 1%;

[0079] 15-30 min, the volume percentage of methanol in the mobile phase is 1% → 12%;

[0080] From 30 to 45 minutes, the volume percentage of methanol in the mobile phase is 12% → 40%.

[0081] The present invention provides a fingerprint spectrum detection method for nucleoside and amino acid components in the centipede drug preparation. The method uses octadecylsilane bonded silica gel as a filler, acetonitrile-water as a mobile phase for gradient elution, and selects a specific elution procedure to detect and obtain a fingerprint spectrum containing 8 common characteristic peaks. This method not only significantly increases the number of common characteristic peaks, but also achieves good separation of these common characteristic peaks. The fingerprint spectrum detection method has a simple elution procedure, and the baseline of the obtained fingerprint spectrum is stable. The peak shapes of the characteristic peaks are good, and the separation between the characteristic peaks is high. The method can accurately locate the peak positions of the eight characteristic peaks, namely, uracil, tyrosine, hypoxanthine, xanthine, phenylalanine, inosine, guanosine and tryptophan. This method is beneficial to the comprehensive quality detection and overall quality control of the centipede drug preparation, thereby helping to improve the safety and stable uniformity of the drug, and providing a basis for the quality detection and control of the centipede drug preparation.

[0082] By using the above-mentioned fingerprint detection method, a fingerprint of the centipede drug preparation can be constructed, and the obtained fingerprint is highly characteristic and rich in chromatographic information, which can fully display the chemical component characteristics of the centipede drug preparation; in addition, the above-mentioned detection method has good separation, high precision, good stability and repeatability, and can comprehensively and quickly detect the nucleotide components, amino acid components and their contents in the centipede drug preparation.

[0083] In an optional embodiment, the chromatographic conditions of the high performance liquid chromatography method further include at least one of the following conditions:

[0084] 1) The detection wavelength is 205nm~215nm;

[0085] 2) Column temperature is 23℃~27℃;

[0086] 3) Flow rate: 0.9 mL / min to 1.1 mL / min;

[0087] 4) The injection volume is 5 μL to 15 μL;

[0088] 5) The chromatographic column is a Dikma Platisil ODS-C18 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 5 μm.

[0089] Illustratively, the detection wavelength can be 210 nm, 205 nm, or 215 nm; the column temperature can be 25° C., 23° C., or 27° C.; the flow rate can be 1.0 mL / min, 0.9 mL / min, or 1.1 mL / min; and the injection volume can be 10 μL, 5 μL, or 15 μL.

[0090] In an optional embodiment, the gradient elution procedure further comprises:

[0091] 45-50 min, the volume percentage of methanol in the mobile phase is 40% → 45%;

[0092] 50-55 min, the volume percentage of methanol in the mobile phase is 45%;

[0093] 55-56 min, the volume percentage of methanol in the mobile phase is 45% → 1%;

[0094] 56-60 min, the volume percentage of methanol in the mobile phase is 1%.

[0095] In an optional embodiment, the preparation method of the test solution is as follows:

[0096] Take the test sample, add solvent, extract, separate solid and liquid, and take liquid to obtain the test sample solution;

[0097] Optionally, the solvent is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 20% to 40%;

[0098] Optionally, relative to 0.5 g of the test sample, the amount of the solvent added is 20 mL to 30 mL;

[0099] Optionally, the extraction is ultrasonic extraction, and the ultrasonic time is 15 minutes to 45 minutes.

[0100] Illustratively, the preparation method of the test solution is as follows: take an appropriate amount of the product, grind it into powder, accurately weigh it, accurately add the solvent, weigh it, ultrasonically treat it, make up the lost weight with the solvent, shake it well, filter it, and take the filtrate to obtain it.

[0101] In a particularly preferred embodiment, the preparation method of the test solution is as follows: take an appropriate amount of the product, grind it finely, take about 0.5 g, accurately weigh it, accurately add 25 mL of 30% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 53 KHz) for 30 minutes, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0102] In an optional embodiment, the fingerprint detection method further comprises the steps of preparing a reference solution using a hypoxanthine reference substance and a phenylalanine reference substance, and detecting the mixed reference solution using the high performance liquid chromatography method to obtain a reference spectrum of the reference substance;

[0103] And / or, the fingerprint detection method further comprises the steps of preparing a control medicinal material solution using centipede control medicinal material, and detecting the control medicinal material solution using the high performance liquid chromatography method to obtain a control medicinal material reference spectrum;

[0104] Optionally, the preparation process of the control medicinal material solution includes: taking centipede control medicinal material, adding water and heating to reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting, separating the solid and liquid, and taking the liquid.

[0105] Exemplarily, the preparation process of the control medicinal material solution includes: taking centipede control medicinal material, adding water, heating and refluxing, filtering, evaporating the filtrate to dryness, adding solvent to the residue, ultrasonic treatment, making up for weight loss with solvent, filtering, and taking the filtrate as the control medicinal material solution.

[0106] In a particularly preferred embodiment, the preparation process of the control medicinal material solution includes: taking 3.0 g of centipede control medicinal material, adding 50 mL of water, heating and reflux for 30 minutes, filtering, evaporating the filtrate, adding 25 mL of 30% methanol to the residue, weighing the weight, ultrasonically treating (power 250 W, frequency 53 KHz) for 30 minutes, taking it out, supplementing the weight loss with the extraction solvent, filtering, and taking the filtrate as the control medicinal material solution.

[0107] In an optional embodiment, the centipede pharmaceutical preparation includes centipede formula granules.

[0108] The centipede pharmaceutical preparation involved in the present invention can be prepared by the following method:

[0109] Take centipede medicinal materials, heat and reflux extract at least once, add 6 to 12 times the weight of water each time to extract for at least 0.5 hours, filter, combine the filtrates, concentrate the filtrates to a relative density of 1.05 to 1.10 g / mL at 60°C, add conventional excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, rapid-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. The pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix. The filler includes at least one of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, and sucrose; the disintegrant includes at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, and cross-linked sodium carboxymethyl cellulose; the lubricant includes magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; the suspending agent includes at least one of polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, and hydroxypropyl methylcellulose; the binder includes at least one of starch slurry, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose; the sweetener includes at least one of saccharin sodium, aspartame, sucrose, cyclamate, and glycyrrhetinic acid; the flavoring agent includes sweeteners and various flavors; the preservative includes at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, and eucalyptus oil; and the matrix includes at least one of PEG6000, PEG4000, and insect wax.

[0110] In a second aspect, the present invention provides a method for determining the content of nucleosides and amino acids in a centipede pharmaceutical preparation, comprising the following steps:

[0111] Take the test solution and the reference solution and test them respectively using the high performance liquid chromatography method in the above fingerprint detection method;

[0112] Among them, the reference substances include hypoxanthine reference substance and phenylalanine reference substance;

[0113] Optionally, the detection wavelength of hypoxanthine is 249 nm, and the detection wavelength of phenylalanine is 210 nm.

[0114] In a third aspect, the present invention provides the application of the above-mentioned fingerprint detection method and / or content determination method in the quality control of nucleoside and amino acid components in centipede pharmaceutical preparations.

[0115] In a fourth aspect, the present invention provides a method for quality control of nucleoside and amino acid components in a centipede pharmaceutical preparation, comprising the steps of obtaining a fingerprint of the centipede pharmaceutical preparation to be tested according to the above-mentioned fingerprint detection method, and comparing the fingerprint with a control fingerprint;

[0116] Wherein, the control fingerprint is obtained by fitting the fingerprint obtained by the above-mentioned fingerprint detection method using at least one batch of standard products of centipede drug preparations through the average or median method;

[0117] And / or, it includes the step of determining the content of nucleosides and amino acids in the centipede pharmaceutical preparation to be tested according to the above-mentioned content determination method.

[0118] In an optional embodiment, the control fingerprint includes 8 common characteristic peaks, wherein peak 1 is a uracil peak, peak 2 is a tyrosine peak, peak 3 is a hypoxanthine peak, peak 4 is a xanthine peak, peak 5 is a phenylalanine peak, peak 6 is an inosine peak, peak 7 is a guanosine peak, and peak 8 is a tryptophan peak;

[0119] Taking the phenylalanine peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.29, the specified value corresponding to peak 2 is 0.43, the specified value corresponding to peak 3 is 0.55, the specified value corresponding to peak 4 is 0.68, the specified value corresponding to peak 6 is 1.19, the specified value corresponding to peak 7 is 1.24, and the specified value corresponding to peak 8 is 1.47;

[0120] Optionally, the similarity between the fingerprint of the centipede pharmaceutical preparation to be tested and the fingerprint of the control is greater than 0.90.

[0121] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0122] The instruments, reagents and reagents involved in the examples are as follows:

[0123] 1. The preparation method of centipede formula granules is:

[0124] Take centipede, heat reflux extraction twice, add 10 times the weight of water for the first time, soak for 30 minutes, heat reflux extraction for 1.0 hour, filter, add 8 times the weight of water for the second time, extract for 1.0 hour, filter, combine the two filtrates, concentrate the filtrate to a relative density of 1.05 g / mL at 60°C, spray dry, add maltodextrin as an auxiliary material to the dry powder, mix well, and then dry granulate to make granules.

[0125] 2. Instruments and equipment:

[0126] High-performance liquid chromatograph 1: Waters e2695 chromatography system, including a quaternary gradient infusion pump (ACQUITY Arc model), a 120-position high-performance autosampler, an imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system;

[0127] High-performance liquid chromatograph 2: Thermo ULtimate 3000, including Pump: LPG-3400SD; CoLumCompartment: TCC-3000RS; AutosumpLer: WPS-3000SL; Photometer: DAD-3000;

[0128] High performance liquid chromatograph 3: Shimadzu LC-20AT chromatography system, including CTO-20AC quaternary pump, SIL-20AC autosampler, SPD M20A detector, and chromatography workstation;

[0129] Other instruments: 1 / 10,000 electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., FA1004); 1 / 100,000 electronic balance (Sartorius Scientific Instrument Co., Ltd., SQP); ultrasonic cleaning machine: WB400US Shanghai Wangbiao Instrument Co., Ltd.

[0130] Column 1: Agilent ZORBAX SB-AQ C18 (4.6 mm × 250 mm, 5 μm);

[0131] Column 2: DIKMA platisil 5 μm ODS C18 (4.6 mm × 250 mm, 5 μm);

[0132] Column 3: Welch AQ-C18 (4.6 mm × 250 mm, 5 μm);

[0133] 3. Drug testing:

[0134] Uracil reference substance (batch number: 100469-201302, purity: 99.6%, purchased from China Food and Drug Inspection Institute);

[0135] Tyrosine reference substance (batch number: 140609-201914, purity: 99.9%, purchased from China Food and Drug Inspection Institute);

[0136] Hypoxanthine reference substance (batch number: 140661-202005, purity: 99.4%, purchased from China Food and Drug Inspection Institute);

[0137] Xanthine reference substance (batch number: 140662-200802, purity: 100.0%, purchased from China Food and Drug Administration)

[0138] Phenylalanine reference substance (batch number: 140676-201706, purity: 100.0%, purchased from China Food and Drug Inspection Institute);

[0139] Inosine reference substance (batch number: 140669-202007, purity: 99.2%, purchased from China Food and Drug Inspection Institute);

[0140] Guanosine reference substance (batch number: 111977-201501, purity: 93.6) was purchased from the China Food and Drug Administration;

[0141] Tryptophan reference substance (batch number: 140686-202108, purity: 99.9%, purchased from China Food and Drug Inspection Institute);

[0142] Centipede control medicinal material (batch number: 400015-202111, purchased from Shanghai Hongyong Biotechnology Co., Ltd.);

[0143] Centipede formula granules (Batch numbers: 1905001W, 1907001S, 1908001W, 1912002W, 2001004S, 2001002W, 2003001W, 2108001W, 2011003S, 2102003S, 2106002S, 2109002W, 2111001S, 2111002S, 2202002W, 2205001W, 2210002S, 2212002S, source: China Resources Sanjiu Pharmaceutical Co., Ltd., China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd.);

[0144] 4. Reagents: Methanol was of chromatographic grade, water was ultrapure water; other reagents were of analytical grade.

[0145] Example 1

[0146] This embodiment provides a fingerprint detection method for centipede formula granules (batch number: 1905001W):

[0147] (1) Solution preparation:

[0148] Preparation of control medicinal material solution: Take 3.0 g of centipede control medicinal material, add 50 mL of water, reflux for 30 minutes, filter, evaporate the filtrate to dryness, accurately add 25 mL of 30% methanol, weigh, sonicate (power 250 W, frequency 53 kHz) for 30 minutes, remove, make up the weight loss with extraction solvent, filter, and take the filtrate to prepare the control medicinal material solution;

[0149] Preparation of reference solution: Take uracil reference, tyrosine reference, hypoxanthine reference, xanthine reference, phenylalanine reference, inosine reference, guanosine reference, and tryptophan reference, add 30% methanol, and prepare a mixed reference solution containing 0.1 mg of each reference substance per 1 mL;

[0150] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, about 0.5g, accurately weigh it, accurately add 25mL of 30% methanol, weigh it, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, make up the weight loss with the extraction solvent, filter it, and take the filtrate to obtain it.

[0151] (2) Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 General Chapter 0512):

[0152] Chromatographic conditions: octadecylsilane bonded silica gel as the filler (column length 4.6 mm × 250 mm, particle size 5 μm); methanol as mobile phase A, aqueous solution as mobile phase B, gradient elution as specified in Table 1; flow rate 1.0 mL / min, column temperature 25°C; detection wavelength 210 nm, theoretical plate number calculated based on the hypoxanthine peak should be no less than 5000.

[0153] Table 1 Gradient elution program used in Example 1

[0154]

[0155] Assay: Accurately pipette 10 μl of each of the reference herbal solution, mixed reference solution, and test solution into a high-performance liquid chromatograph for determination. The resulting chromatograms show the basic information for each peak in the mixed reference, reference herbal, and test sample chromatograms as shown in Table 2.

[0156] Table 2 Test results of mixed reference substances, reference medicinal materials and test substances

[0157]

[0158] As can be seen in Table 2, the test sample chromatogram exhibits eight characteristic peaks, which correspond to the retention times of the eight characteristic peaks in the mixed reference sample chromatogram and the reference medicinal material chromatogram. Among the eight characteristic peaks, peak 1 is the uracil peak, peak 2 is the tyrosine peak, peak 3 is the hypoxanthine peak, peak 4 is the xanthine peak, peak 5 is the phenylalanine peak, peak 6 is the inosine peak, peak 7 is the guanosine peak, and peak 8 is the tryptophan peak. Taking the phenylalanine peak as the S peak, the relative retention times of each characteristic peak and the S peak are within ±10% of the specified values, where the specified values ​​are: 0.29 (peak 1), 0.43 (peak 2), 0.55 (peak 3), 0.68 (peak 4), 1.19 (peak 6), 1.24 (peak 7), and 1.47 (peak 8).

[0159] Example 2

[0160] This example is used to illustrate the establishment of a control fingerprint of a centipede pharmaceutical preparation:

[0161] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a control fingerprint using the fingerprints of 18 batches of representative centipede formula granule samples, and reference substances such as hypoxanthine and phenylalanine were used to locate and identify them. The fingerprints of each batch of centipede formula granule samples were detected according to the method of Example 1. The fingerprints of the 18 batches of centipede formula granule samples are as follows: Figure 1 As shown, Figure 1 , the batch numbers of S1 to 18 are 1905001W, 1907001S, 1908001W, 1912002W, 2001004S, 2001002W, 2003001W, 2108001W, 2011003S, 2102003S, 2106002S, 2109002W, 2111001S, 2111002S, 2202002W, 2205001W, 2210002S, and 2212002S; R is the reference medicinal material atlas.

[0162] (1) Confirmation and identification of characteristic peaks in characteristic spectra

[0163] By analyzing the test results of the fingerprints of multiple batches of test samples, a reference characteristic spectrum was generated using the fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee. By identifying and specifying the characteristic peaks, the HPLC characteristic spectrum of the centipede formula granules obtained had a total of 8 chromatographic peaks. The remaining chromatographic peaks were all chromatographic peaks with small responses or poor separation. Therefore, the 8 chromatographic peaks with large responses were selected as common characteristic peaks, and the chromatographic peaks were rearranged according to the order of the chromatographic peaks, such as Figure 2 shown.

[0164] According to literature reports, the chemical components of centipedes mainly include centipede venom (proteins and peptides), special small molecules (quinoline alkaloids), nucleosides, amino acids, histamine, and total phospholipids. Among them, centipede venom, special small molecules (quinoline alkaloids), nucleosides, and amino acids are all active ingredients. Considering that centipede venom is mostly composed of proteins and peptides, which have poor water solubility and easily lose activity after heating and boiling, while special small molecules (quinoline alkaloids), nucleosides, and amino acids have better water solubility, the small molecules (quinoline alkaloids), nucleosides, and amino acids were selected for analysis and research. After analyzing the special small molecule compounds (quinoline alkaloids), nucleosides, and amino acid components of the centipede formula granules, it was found that the special small molecule compound (quinoline alkaloid) component 3,8-dihydroxyquinoline, the nucleoside component hypoxanthine, and the amino acid component phenylalanine were all detected. Therefore, quinoline components, nucleoside components, and amino acid components were selected as the main development components of the characteristic spectrum of the centipede formula granules.

[0165] According to the results of peak identification and reference positioning, 8 known peaks were confirmed, namely peak 1 (uracil), peak 2 (tyrosine), peak 3 (hypoxanthine), peak 4 (xanthine), peak 5 (phenylalanine), peak 6 (inosine), peak 7 (guanosine), and peak 8 (tryptophan). Among them, tyrosine, phenylalanine, and tryptophan are amino acid components, and uracil, hypoxanthine, xanthine, inosine, and guanosine are nucleoside components, all of which are one of the main active ingredients of centipede. The characteristic spectrum contains the main chemical components of centipede.

[0166] (2) Selection basis of characteristic peak S peak of characteristic spectrum

[0167] According to the peak identification and peak selection results of the characteristic peaks in the centipede formula granules, in the characteristic spectrum of the centipede formula granules, hypoxanthine has the highest response, followed by phenylalanine, both of which are one of the main active ingredients of centipede. Considering that the detection time of the centipede nucleoside and amino acid characteristic spectrum is 45 minutes, the time interval between each chromatographic peak in the chromatogram is large, and the peak elution time of the phenylalanine chromatographic peak is moderate (retention time 25 minutes), and the peak separation is good, in order to ensure the durability of the relative retention time of each characteristic peak, phenylalanine is used as the S peak of the characteristic spectrum to calculate the relative retention time of each characteristic peak.

[0168] (3) Confirmation of characteristic peaks in characteristic spectra

[0169] Through HPLC reference product positioning research, in the HPLC chromatogram of centipede formula granules, 8 peaks are known chromatographic peaks, namely peak 1 (uracil), peak 2 (tyrosine), peak 3 (hypoxanthine), peak 4 (xanthine), peak 5 (phenylalanine), peak 6 (inosine), peak 7 (guanosine), and peak 8 (tryptophan). Among them, the mixed reference product spectrum is shown in Figure 3 The LC / MS / MS analysis results are shown in Table 3.

[0170] Table 3 LC / MS / MS analysis results of centipede formula granules

[0171]

[0172] (4) Characteristic peak specification value of characteristic spectrum

[0173] The relative retention time of the characteristic spectrum is determined based on the research results: the characteristic spectrum of centipede formula granules should show 8 characteristic peaks, which should correspond to the retention time of the corresponding characteristic peaks of the reference medicinal materials. The final reference characteristic spectrum is as follows: Figure 4 The relative retention time of each characteristic peak and S peak in the reference characteristic spectrum is shown in Table 4, and the relative peak area of ​​each characteristic peak and S peak is shown in Table 5.

[0174] Table 4 Relative retention time of each characteristic peak and S peak in the characteristic spectrum

[0175]

[0176] Table 5 Comparison of the relative peak areas of the characteristic peaks and S peaks in the characteristic spectrum

[0177]

[0178]

[0179] Example 3

[0180] This example is used to perform methodological verification on the detection method in Example 1. In this example, unless otherwise specified, the preparation method of each solution and the high performance liquid chromatography method used are the same as those in Example 1, and will not be repeated in this example.

[0181] (1) Specificity verification

[0182] Prepare the test solution (batch number: 1905001W) and use 30% methanol as the negative control solution for HPLC analysis. The results are as follows Figure 5 and Figure 6 As shown. Among them, Figure 5 is the negative control chromatogram, Figure 6 is the chromatogram of the test product, Figure 5 and Figure 6 It can be seen that the negative has no interference.

[0183] (2) Integrity Verification

[0184] Prepare the test solution (batch number: 1905001W) and perform HPLC analysis. The determination is still carried out after the peak appears. The determination time is twice that of the fingerprint determination time. Figure 7 As shown, it can be seen that after 60 minutes, there is basically no chromatographic peak in the fingerprint, indicating that this method can already determine the main chromatographic peaks of the centipede formula granule fingerprint, indicating that the method is relatively complete.

[0185] (3) Instrument precision verification

[0186] Six replicate injections of the same centipede formula granules (lot number 1905001W) were performed. The relative standard deviations (RSDs) of the retention times of each characteristic peak relative to the S peak were calculated. The results are shown in Table 6, where t represents the retention time and t / ts represents the relative retention time. As can be seen in Table 6, the RSDs of the relative retention times of each characteristic peak relative to the S peak were all less than 2.0%, indicating good instrumental precision for this method.

[0187] Table 6 Relative retention time results of instrument precision test

[0188]

[0189] (4) Repeatability verification

[0190] Six parallel sample solutions of the test sample (batch number: 1905001W) were prepared from the same centipede formula granules and subjected to HPLC analysis. The RSD values ​​of the relative retention times of each characteristic peak and the S peak were calculated. The results are shown in Table 7. As can be seen from Table 7, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were all less than 2.0%, indicating good reproducibility of the method.

[0191] Table 7 Relative retention time results of repeatability test

[0192]

[0193] (5) Intermediate precision (personnel) verification

[0194] Three researchers prepared test solutions of the same centipede formula granules (batch number: 1905001W) and performed HPLC analysis. The RSD values ​​of the relative retention times of each characteristic peak and the S peak were calculated. The results are shown in Table 8. As can be seen from Table 8, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were all less than 2.0%, indicating that the intermediate precision of this method is good.

[0195] Table 8 Intermediate precision (personnel) relative retention time

[0196]

[0197] (6) Stability investigation

[0198] The test solution of centipede formula granules (1905001W) was taken and HPLC analysis was performed at 0h, 4h, 8h, 12h, 16h, and 24h after preparation. The RSD values ​​of the relative retention times of each characteristic peak and the S peak were calculated. The results are shown in Table 9. As can be seen from Table 9, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were all less than 2.0%, indicating that the test solution was stable within 24h and met the measurement requirements.

[0199] Table 9 Relative retention time results of stability test

[0200]

[0201] (7) Investigation of different column temperatures

[0202] The same sample solution (1905001W) was taken and the column temperature was set at 23℃, 25℃, and 27℃ respectively for HPLC determination. The effect of different column temperatures on the relative retention time of each characteristic peak in the centipede formula granule sample spectrum was investigated. The results are shown in Table 10.

[0203] Table 10 Relative retention time results at different column temperatures

[0204]

[0205] (8) Investigation of different flow rates

[0206] The same sample solution (1905001W) was taken and the flow rates were set to 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively, for HPLC determination to investigate the effect of different flow rates on the relative retention time of each characteristic peak in the centipede formula granule sample spectrum. The results are shown in Table 11.

[0207] Table 11 Relative retention time results at different flow rates

[0208]

[0209] (9) Investigation of different chromatographic columns

[0210] The same centipede formula granules (1905001W) test solution was taken and measured using chromatographic columns produced by different manufacturers (chromatographic column 1: Agilent ZORBAX SB-AQ C18; chromatographic column 2: DIKMA platisil 5um ODS; chromatographic column 3: WelchAQ-C18). The results are as follows: Figure 8-10 As shown. Among them, Figure 8 This is the chromatogram obtained by the detection of chromatographic column 1. Figure 9 This is the chromatogram obtained by the chromatographic column 2 detection. Figure 10 This is the chromatogram obtained by detection with chromatographic column 3.

[0211] Depend on Figure 8-10 It can be seen that the hydrophilic chromatographic columns of Agilent ZORBAX SB-AQ C18 and Welch AQ-C18 brands have problems with missing characteristic peaks and poor peak separation, while the detection results of DIKMA platisil 5um ODS C18 column are good.

[0212] (10) Inspection of different liquid phase equipment

[0213] The same sample solution (1905001W) was analyzed by HPLC using a Waters Arc, a Thermo Fisher U3000, and a Shimadzu LC-20AT, respectively. The effects of different HPLC instruments on the relative retention times of characteristic peaks in the centipede formula granule sample chromatogram were investigated. The results are shown in Table 12. As can be seen from Table 12, the relative retention times of some characteristic peaks in the characteristic chromatograms of each brand of instrument were within ±10% of the specified values, indicating that this method has good robustness across different instruments.

[0214] Table 12 Relative retention time results of different brands of instruments

[0215]

[0216] Example 4

[0217] According to the method of Example 1, the liquid phase fingerprints of 18 batches of centipede formula granules were measured, and the measurement results are shown in Table 13. As can be seen from Table 13, the characteristic spectra of all 18 batches of centipede formula granules showed 8 characteristic peaks, and the relative retention times of peaks 1, 2, 3, 4, 6, 7, 8 and S peak were within ±10% of the specified value.

[0218] Table 13 Relative retention time results of 18 batches of centipede formula granules

[0219]

[0220] Example 5

[0221] This example provides a method for determining the contents of hypoxanthine and phenylalanine in centipede formula granules (batch number: 1905001W):

[0222] (1) Solution preparation:

[0223] Preparation of reference solution: Take appropriate amount of hypoxanthine reference substance and phenylalanine reference substance, add 30% methanol to make reference solution containing 0.1 mg of each reference substance per 1 mL;

[0224] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, about 0.5g, accurately weigh it, accurately add 25mL of 30% methanol, weigh it, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, make up the weight loss with the extraction solvent, filter it, and take the filtrate to obtain it.

[0225] (2) Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 General Chapter 0512):

[0226] Chromatographic conditions: octadecylsilane bonded silica gel as the filler (column length 4.6 mm × 250 mm, particle size 5 μm); methanol as mobile phase A, aqueous solution as mobile phase B, gradient elution as specified in Table 14; flow rate 1.0 mL / min, column temperature 25°C; detection wavelengths 210 nm (phenylalanine) and 249 nm (hypoxanthine), theoretical plate numbers calculated for phenylalanine and hypoxanthine peaks should be no less than 3000.

[0227] Table 14 Gradient elution program used in Example 5

[0228]

[0229] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into high performance liquid chromatography instrument, and determine.

[0230] The chromatogram of the reference substance was determined as Figures 11-12 As shown, the chromatogram of the test sample is as Figures 13-14 As shown, the concentration of hypoxanthine in the reference solution is 0.095 mg / mL, and the concentration of phenylalanine is 0.095 mg / mL; the peak area of ​​hypoxanthine in the reference solution chromatogram is 4603659, and the peak area of ​​phenylalanine is 2632491; the peak area of ​​hypoxanthine in the test solution chromatogram is 7035633, and the peak area of ​​phenylalanine is 4453594. After calculation, the content of hypoxanthine in the test solution is 3.62 mg / g, and the content of phenylalanine is 4.00 mg / g.

[0231] Example 6

[0232] This example is used to illustrate the screening process of detection conditions. In this example, except for the elution gradient, the other detection conditions are the same as those in Example 5.

[0233] (1) Screening of elution gradient

[0234] Take the same centipede formula granules (batch number: 1905001W) test solution and perform HPLC detection according to the gradient 1-3 shown in Tables 15-17. The separation degree and system suitability parameters of hypoxanthine, phenylalanine, etc. in the chromatogram obtained under each elution program are investigated. The results are shown in Figure 15-17 And Tables 18-20. Among them, Figure 15 is the chromatogram obtained using gradient 1 detection, Figure 16 is the chromatogram obtained using gradient 2 detection, Figure 17 is the chromatogram obtained by using gradient 3 detection. Figure 15-17 As can be seen from Tables 18-20, when gradient 3 is used, the separation is better and the system suitability parameters are more ideal.

[0235] Table 15 Gradient 1

[0236]

[0237] Table 16 Gradient 2

[0238]

[0239] Table 17 Gradient 3

[0240]

[0241] Table 18 Detection results using gradient 1

[0242]

[0243] Table 19 Detection results using gradient 2

[0244]

[0245] Table 20 Detection results using gradient 3

[0246]

[0247]

[0248] (2) Screening of different mobile phases

[0249] Take the same centipede formula granules (batch number: 1905001W) test solution and perform HPLC detection using 0.1% phosphoric acid-methanol, 0.1% formic acid-methanol, and water-methanol mobile phases to investigate the elution and separation effects of each mobile phase. Figure 18-23 and Tables 21-23, in which: Figure 18 This is a chromatogram obtained by using the mobile phase 0.1% phosphoric acid-methanol at 210 nm. Figure 19This is a chromatogram obtained by detecting at 249 nm using the mobile phase 0.1% phosphoric acid-methanol. Figure 20 The chromatogram is obtained by using the mobile phase 0.1% formic acid-methanol at 210 nm. Figure 21 The chromatogram is obtained by using the mobile phase 0.1% formic acid-methanol at 249 nm. Figure 22 The chromatogram is obtained by using the mobile phase water-methanol at 210 nm. Figure 23 The chromatogram is obtained using the mobile phase of water-methanol and detected at 249 nm.

[0250] Depend on Figure 18-23 As can be seen from Tables 21-23, when water-methanol is used as the mobile phase, the chromatographic separation is better, the baseline is stable, the peak shape is better, and the system suitability parameters are ideal.

[0251] Table 21 Detection results using mobile phase 0.1% phosphoric acid-methanol

[0252]

[0253] Table 22 Detection results using mobile phase 0.1% formic acid-methanol

[0254]

[0255] Table 23 Detection results using mobile phase water-methanol

[0256]

[0257] Example 7

[0258] This example is used to perform methodological verification on the content determination method in Example 5. In this example, unless otherwise specified, the preparation method of each solution and the high performance liquid chromatography method used are the same as those in Example 5, and will not be repeated in this example.

[0259] (1) Specificity inspection

[0260] Prepare the test solution and negative blank control solution, perform HPLC analysis according to the chromatographic conditions described under the hypoxanthine and phenylalanine content, and record the chromatogram, such as Figures 24-27 As shown, Figure 24 This is the chromatogram of the test sample detected at a wavelength of 249 nm. Figure 25 This is the chromatogram of the test sample detected at a wavelength of 210 nm. Figure 26 This is the negative blank control chromatogram detected at a wavelength of 249 nm. Figure 27 The negative blank control chromatogram is obtained by detecting at a wavelength of 210 nm. Figures 24-27 It can be seen that there is no interference in the negative control experiment.

[0261] (2) Linear relationship verification

[0262] Take an appropriate amount of hypoxanthine reference substance and add 50% methanol to prepare linear verification solutions containing 0.00436 mg, 0.00872 mg, 0.0174 mg, 0.0349 mg, 0.174 mg, and 1.74 mg of hypoxanthine reference substance per 1 mL. Accurately pipette 10 μl of each of the above linear verification solutions with different concentrations into the liquid chromatograph and measure the peak area, as shown in Table 24. Use the concentration of the reference substance as the horizontal axis and the peak area as the vertical axis to draw a standard curve, as shown in Table 24. Figure 28 As shown, the regression equation of hypoxanthine is: y = 30348033.58x + 374657.42, R 2 =0.9998. The experimental results showed that the linear relationship of hypoxanthine was good in the concentration range of 0.00436mg / ml to 1.74mg / ml.

[0263] Table 24 Linear relationship results of xanthine

[0264]

[0265] Take an appropriate amount of phenylalanine reference substance and add 50% methanol to prepare linear verification solutions containing 0.00300 mg, 0.0060 mg, 0.0120, 0.0241 mg, 0.120 mg, and 1.20 mg of phenylalanine reference substance per 1 mL. Accurately pipette 10 μl of each of the above linear verification solutions with different concentrations into the liquid chromatograph and measure the peak area, as shown in Table 25. Use the concentration of the reference substance as the horizontal axis and the peak area as the vertical axis to draw a standard curve, as shown in Table 25. Figure 29 As shown, the regression equation of phenylalanine is: y=25860179.10x+27413.41, R 2 =1.0000. The experimental results showed that the linear relationship of phenylalanine was good in the mass concentration range of 0.00301mg / ml to 1.20mg / ml.

[0266] Table 25 Phenylalanine linear relationship results

[0267]

[0268] (3) Precision test

[0269] ①Instrument precision test

[0270] The same centipede formula granules (1905001W) test solution was taken and injected 6 times, and the peak areas of hypoxanthine and phenylalanine were measured. As shown in Table 26, the results showed that the RSD values ​​of the hypoxanthine and phenylalanine peak areas were 0.23% and 0.73%, respectively, indicating that the instrument precision was good.

[0271] Table 26 Precision test results of hypoxanthine and phenylalanine

[0272]

[0273] ②Intermediate precision test (personnel)

[0274] Three experimenters, A, B, and C, used the same batch of centipede formula granules (1905001W) to prepare the test solution according to the method for the determination of hypoxanthine and phenylalanine content, and calculated the hypoxanthine and phenylalanine contents, as shown in Table 27. The results showed that the intermediate precision of the method was good.

[0275] Table 27 Intermediate precision test results of hypoxanthine and phenylalanine

[0276]

[0277] (4) Repeatability test

[0278] Six samples of the same batch of centipede formula granules (1905001W) were prepared according to the preparation method of the test solution. The contents of hypoxanthine and phenylalanine were determined, and the RSD of the content results was calculated. The results are shown in Table 28. The results showed that the RSD of hypoxanthine and phenylalanine were 1.4% and 1.2%, respectively, indicating that the method has good reproducibility.

[0279] Table 28 Repeatability test results of hypoxanthine and phenylalanine methods

[0280]

[0281] (5) Accuracy test

[0282] In the sample addition recovery test, 9 portions of centipede formula granules (1905001W, hypoxanthine content of 3.63mg / g) were accurately weighed, each approximately 0.25g, and placed in measuring flasks. Three portions of the sample were grouped together, and each group of sample was accurately added with 1ml of a reference solution containing 0.725mg / ml, 0.921mg / ml, and 1.110mg / ml of hypoxanthine. The sample was processed according to the sample preparation method and the content was determined. The hypoxanthine recovery was calculated, and the recovery results are shown in Table 29. The results showed that the average recovery of hypoxanthine was 99.9%, and the RSD values ​​were 2.8%, respectively, indicating that the method was accurate.

[0283] Table 29 Hypoxanthine accuracy test results

[0284]

[0285] Nine portions of the centipede formula granules (1905001W, phenylalanine content of 4.08 mg / g) were accurately weighed, approximately 0.25 g each, and placed in volumetric flasks. Three portions of the test sample were grouped together, and 1 ml of a reference solution containing 0.819 mg / ml, 1.016 mg / ml, or 1.231 mg / ml of phenylalanine was precisely added to each group of test samples. The samples were processed according to the test sample preparation method and the content was determined. The phenylalanine recovery was calculated, and the recovery results are shown in Table 30. The average phenylalanine recovery was 98.8%, with an RSD of 2.2%, indicating good accuracy of the method.

[0286] Table 30 Phenylalanine accuracy test results

[0287]

[0288] (6) Durability inspection

[0289] ①Stability test

[0290] The same centipede granule formula (1905001W) test solution was injected at 0, 6, 8, 12, and 24 hours after preparation. The peak areas of hypoxanthine and phenylalanine were measured. The test results are shown in Table 31. The results showed that the test solution was stable within 24 hours, with RSD values ​​of 0.22% and 0.46%, respectively, meeting the measurement requirements.

[0291] Table 31 Stability test results of hypoxanthine and phenylalanine in test solution

[0292]

[0293] ② Investigation of different column temperatures

[0294] The effects of column temperatures of 23°C, 25°C, and 27°C on the hypoxanthine and phenylalanine contents in Centipede Formula Granules (1905001W) were compared. The test results are shown in Table 32. These results demonstrate that the analytical method exhibits good robustness across different column temperatures, indicating that small changes in column temperature can meet system adaptability requirements.

[0295] Table 32 Durability test results of hypoxanthine and phenylalanine at different column temperatures

[0296]

[0297] ③ Investigation of different flow rates

[0298] The effects of different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min) on the hypoxanthine and phenylalanine contents in Centipede Formula Granules (1905001W) were compared. The test results are shown in Table 33. These results demonstrate the robustness of this analytical method across various flow rates, indicating that small changes in flow rate can meet system adaptability requirements.

[0299] Table 33 Durability test results of hypoxanthine and phenylalanine at different flow rates

[0300]

[0301] Example 8

[0302] According to the method of Example 5, the contents of hypoxanthine and phenylalanine in 18 batches of centipede formula granules were respectively determined. The results are shown in Table 34.

[0303] Table 34 Transfer rate and range of hypoxanthine content in 18 batches of centipede formula granules

[0304]

[0305]

[0306] 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 fingerprint detection method for nucleoside and amino acid components in centipede pharmaceutical preparations, characterized in that: The steps include: Take the test solution and the reference solution and detect them by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography include: Methanol was used as mobile phase A and water was used as mobile phase B for gradient elution. The gradient elution procedure included: From 0 to 15 min, the volume percentage of methanol in the mobile phase was 1%; 15-30 min, the volume percentage of methanol in the mobile phase is 1%→12%; 30-45 min, the volume percentage of methanol in the mobile phase is 12%→40%; The chromatographic column was a Dikma Platisil ODS-C18 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 5 μm; The detection wavelength is 205 nm to 215 nm; The preparation method of the test solution is as follows: take the test sample, add a solvent, extract, separate the solid and liquid, and take the liquid to obtain the test solution; the solvent is a methanol aqueous solution; The reference substances include hypoxanthine reference substance and phenylalanine reference substance.

2. The fingerprint detection method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method further include at least one of the following conditions: 1) Column temperature is 23℃~27℃; 2) Flow rate: 0.9 mL / min to 1.1 mL / min; 3) The injection volume is 5 µL to 15 µL.

3. The fingerprint detection method according to claim 1, characterized in that: The gradient elution procedure also includes: 45-50 min, the volume percentage of methanol in the mobile phase is 40%→45%; 50-55 min, the volume percentage of methanol in the mobile phase was 45%; 55-56 min, the volume percentage of methanol in the mobile phase is 45% → 1%; 56-60 min, the volume percentage of methanol in the mobile phase is 1%.

4. The fingerprint detection method according to claim 1, characterized in that: The volume percentage of methanol in the methanol aqueous solution is 20% to 40%.

5. The fingerprint detection method according to claim 1, characterized in that: The amount of the solvent added is 20 mL to 30 mL relative to 0.5 g of the test sample.

6. The fingerprint detection method according to claim 1, characterized in that: The extraction is ultrasonic extraction, and the ultrasonic time is 15 min to 45 min.

7. The fingerprint detection method according to claim 1, characterized in that: The fingerprint detection method further comprises the steps of preparing a reference solution using a hypoxanthine reference substance and a phenylalanine reference substance; And / or, the fingerprint detection method further includes the steps of preparing a control medicinal material solution using centipede control medicinal materials, and detecting the control medicinal material solution using the high performance liquid chromatography method to obtain a control medicinal material reference spectrum.

8. The fingerprint detection method according to claim 7, characterized in that: The preparation process of the control medicinal material solution includes: taking centipede control medicinal material, adding water and heating under reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting, separating the solid and liquid, and taking the liquid.

9. The fingerprint detection method according to any one of claims 1 to 8, characterized in that: The centipede pharmaceutical preparation comprises centipede formula granules.

10. A method for determining the content of nucleosides and amino acids in centipede pharmaceutical preparations, characterized in that: The steps include: Take the test solution and the reference solution, and respectively detect them by the high performance liquid chromatography method in any one of claims 1 to 9; Among them, the reference substances include hypoxanthine reference substance and phenylalanine reference substance.

11. The content determination method according to claim 10, characterized in that: The detection wavelength of phenylalanine is 210 nm.

12. Use of the fingerprint detection method according to any one of claims 1 to 9 and / or the content determination method according to claim 10 or 11 in the quality control of nucleoside and amino acid components in centipede pharmaceutical preparations.

13. A method for quality control of nucleoside and amino acid components in centipede pharmaceutical preparations, characterized in that: The method comprises the steps of obtaining a fingerprint of the centipede medicinal preparation to be tested according to the fingerprint detection method according to any one of claims 1 to 9, and comparing the fingerprint with a control fingerprint; Wherein, the control fingerprint is obtained by fitting the fingerprint obtained by the fingerprint detection method according to any one of claims 1 to 9 using at least one batch of standard products of centipede drug preparations through the average or median method; And / or, comprising the step of determining the content of nucleosides and amino acids in the centipede pharmaceutical preparation according to the content determination method according to claim 10 or 11.

14. The quality control method according to claim 13, characterized in that: The control fingerprint spectrum includes 8 common characteristic peaks, wherein peak 1 is uracil peak, peak 2 is tyrosine peak, peak 3 is hypoxanthine peak, peak 4 is xanthine peak, peak 5 is phenylalanine peak, peak 6 is inosine peak, peak 7 is guanosine peak, and peak 8 is tryptophan peak; Taking the phenylalanine peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.29, the specified value corresponding to peak 2 is 0.43, the specified value corresponding to peak 3 is 0.55, the specified value corresponding to peak 4 is 0.68, the specified value corresponding to peak 6 is 1.19, the specified value corresponding to peak 7 is 1.24, and the specified value corresponding to peak 8 is 1.

47.

15. The quality control method according to claim 14, characterized in that: The similarity between the fingerprint of the centipede pharmaceutical preparation to be tested and the fingerprint of the control is greater than 0.90.

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