CN115144522A - Control method for comprehensively controlling quality of gecko formula granules - Google Patents

Abstract

The invention discloses a control method for comprehensively controlling the quality of gecko formula granules. The invention establishes a group of fast, comprehensive and highly specific gecko formula particle quality detection methods. Quality control is carried out from a plurality of angles of thin-layer identification, characteristic spectrum and content measurement, and more powerful guarantee is provided for industrialization of the gecko formula granules. The method disclosed by the invention is simple to operate, and verified by methodology, the repeatability and the stability are good, the recovery rate is reliable, the detection cost is low, the detection efficiency is high, and an important guarantee is provided for standardizing the production of the gecko formula particles.

Description

Control method for comprehensively controlling quality of gecko formula granules

Technical Field

The invention relates to a method for controlling the quality of a medicament, in particular to a method for comprehensively controlling the quality of gecko formula granules.

Background

Gekko Swinhonis is dried whole body of Gekko Swinhoana Guenther of Gekko animal. Gecko contains various active ingredients, including wasp poison analogs, proteins, vitamins, histamine, nucleosides, amino acids, etc. (Liu Yanjie, wu Xiaodong, xu Hanmei. Gecko medicinal value research progress. Pharmaceutical progress [ J ],2015, 39 (12): 915-919). Its smell is slightly smelly and its taste is salty; has the effects of dispelling pathogenic wind, arresting convulsion, resolving hard mass and removing toxic substance; mainly treats stroke paralysis, wind-phlegm fright epilepsy and scrofula and malignant sore (Jiangsu province food and drug administration, jiangsu province Chinese medicinal material standard [ S ]. Jiangsu phoenix science and technology publishing company, 2016.

The modern pharmacological action and clinical application research shows that the gecko has better curative effects on tumors (Chen Jianxiang, zhang Hong, but not radiotherapy and chemotherapy combined with Jinlong capsules for treating malignant tumors and clinical curative effect observation [ J ]. Zhejiang medicine, 2007, 29 (7): 650-652), oral ulcer treatment (Liu Shunqin, liu Chang, gecko powder for treating oral ulcer and curative effect observation [ J ]. Chinese traditional medicine information journal, 2000, 7 (31): 53-54), hyperplasia of mammary glands (Peng Yixia, yang Puxuan, song Lixia. Mammary gland hyperplasia 78 cases [ J ]. Shanxi Chinese traditional medicine, 2001, 22 (12): 722), mammary gland fibroma (Zhang Changfu. Mammary gland fibroma 117 cases [ J ]. Shanxi, 2001, 22 (9): 522), tuberculosis and Jiang Weijian. The breast gland fibroma capsule for treating mammary gland fibroma [ J ] (2003, the southern part of tuberculosis, and the lung tuberculosis [ J ] (2003, 3234: J19, the lung tuberculosis).

The traditional Chinese medicine formula particle is prepared by carrying out water extraction, concentration, drying and granulation on single traditional Chinese medicine decoction pieces, is a novel form of decoction piece innovation, has the characteristics of convenient carrying and simple use compared with the traditional decoction piece decoction, and can meet the requirements of modern fast-paced work and life. In recent years, the traditional Chinese medicine formula granules are rapidly developed, and national standards of formula granules of a plurality of traditional Chinese medicine varieties are successively provided, so that great contribution is made to standardizing the production of the traditional Chinese medicine formula granules and promoting the modernization of traditional Chinese medicines. The gecko formula granules are prepared by gecko decoction pieces through extraction, concentration, drying and granulation by a modern process, retain the clinical effect of the gecko and simultaneously consider the portability of use.

Some quality control measures for geckos have been reported. Bao Huayin et al establish a gecko drug fingerprint analysis method, perform electrophoretic identification on a drug by using a PAGE method, perform photographic processing on an electrophoretic gel by using a gel imaging system, and analyze an electrophoretic band by using Genescope analysis software to obtain a PAGE fingerprint of the drug (Bao Huayin, a research on a traditional Chinese medicine gecko quality control key technology and a quality evaluation system [ D ]. Shandong university of traditional Chinese medicine, 2012). 3238 Zxft 3238, et al, establish the quality standards of Gekko Swinhonis, and respectively perform thin layer identification and analysis of water, water soluble extract, alcohol soluble extract, ash and total polysaccharide (3262 Zxft 3262. Gekko Swinhonis quality standard research [ J ] Asia-Tai traditional Chinese medicine, 2017, 13 (18): 25-27). Tian Yuqing et al established a method for measuring the content of nucleoside components in Gekko Swinhonis by using an HPLC method (Tian Yuqing, liu Yang, zhang Ning, etc.. HPLC simultaneously measures the content of 8 nucleoside components [ J ] in Gekko Swinhonis in different producing areas. Chinese medicinal materials 2020, 43 (9): 4). The existing literature data show that the quality control research of the gecko is mainly focused on medicinal materials, the research on the quality control of gecko formula granules is less, and as shown in the quality standard of gecko formula granules released by the drug administration in Shanghai, thin-layer identification, characteristic spectrum, extract, content measurement and other methods of the gecko are established, so that the quality control of the gecko formula granules is based.

Gecko is not currently recorded in the 2020 edition of the chinese pharmacopoeia, and although some literature on the quality control of gecko drugs has been studied, various problems exist. As described in section 2.1, in the aspect of fingerprint, researchers adopt a gel electrophoresis method to analyze gecko medicinal materials, and the operation is relatively complex; in the aspect of content measurement, whether total polysaccharide or nucleoside components exist, the specificity is not strong or the detection time cost is too high.

For the quality research of gecko formula granules, the research data is less at present, only the quality standard of gecko (also named "tianlong") formula granules issued by the drug administration in Shanghai can be referred to, and through patent search, the characteristic spectrum method related to the patent with the patent number of CN113960203A is consistent with the method in the quality standard of gecko formula granules in Shanghai. In addition, the thin layer identification method in the gecko formula particle standard disclosed in Shanghai city only uses the reference substance for identification and does not use the reference medicinal material, the content index is pterin-6-carboxylic acid, the content is low, the clinical effect of the index is unclear, and the rationality of the index selection is questionable.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a control method for comprehensively controlling the quality of gecko formula particles, which has the advantages of good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency.

The technical scheme is as follows: the invention provides a method for comprehensively controlling the quality of gecko formula granules, which comprises the following steps:

(1) Content determination and characteristic spectrum detection to-be-tested sample solution preparation: taking 0.2 g-1.0 g of gecko formula particles, grinding, adding an organic solvent for extraction, and taking a subsequent filtrate to obtain a test solution;

(2) Content determination and preparation of a characteristic spectrum detection reference substance solution: 0.5 g-2 g of gecko reference medicinal material is taken, 30% -70% methanol is added for extraction, and subsequent filtrate is taken as reference substance solution of the reference medicinal material; dissolving appropriate amount of tyrosine control substance and tryptophan control substance in water or methanol water solution to obtain reference substance solution;

(3) Content determination and characteristic spectrum detection methodology verification: the analysis method is verified in the aspects of preparation method, linearity, precision density, accuracy, specificity and durability;

(4) The content determination and characteristic spectrum detection method comprises the following steps: respectively sucking reference substance solution and test solution 0.5-2 μ l, and measuring;

(5) Preparing a thin-layer identification test solution: taking 0.2 g-1.0 g of gecko formula particles, adding an organic solvent for extraction, and taking subsequent filtrate to obtain a test solution;

(6) Preparation of thin-layer identification reference solution: taking 0.5 g-2 g of gecko reference medicinal material, adding an organic solvent for extraction, and taking subsequent filtrate to obtain reference medicinal material solution;

(7) Verification of thin-layer identification methodology: the analysis method is verified in the aspects of preparation method, specificity and durability;

(8) The thin layer identification and detection method comprises the following steps: respectively sucking reference solution and sample solution, spotting on silica gel thin layer plate, developing, air drying, and developing.

Further, the extraction solvent is water, methanol or a methanol water solution with the volume concentration of 30-70%, and the extraction time is 10-45 minutes; the extraction volume is 10ml to 50ml.

Further, tyrosine: 20-60 mug/ml; tryptophan: 10-20 mu g/ml.

Furthermore, the chromatographic column takes octadecylsilane chemically bonded silica as a filler; acetonitrile is taken as a mobile phase A, 0.05-0.3% phosphoric acid solution is taken as a mobile phase B, and gradient elution is adopted; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 20-30 ℃; an ultraviolet detector is used.

Further, the gradient elution is as follows:

further, the liquid chromatography conditions were: the characteristic spectrum of the gecko formula particle shows 7 characteristic peaks in the chromatogram of a test solution and corresponds to the retention time of the 7 characteristic peaks in the chromatogram of a reference substance of a reference medicinal material, wherein the peaks 4 and 7 are consistent with the retention time of the reference substance peaks of a tyrosine reference substance and a tryptophan reference substance, the peak corresponding to the tyrosine reference substance is an S peak, the relative retention time of the peaks 1, 2, 3, 5, 6 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.48 (Peak 1), 0.56 (Peak 2), 0.74 (Peak 3), 1.37 (Peak 5), 1.50 (Peak 6).

Furthermore, the thin layer plate is silica gel G, the developing agent is n-butyl alcohol-glacial acetic acid-water (4: 1), and the color developing agent is ninhydrin test solution.

Furthermore, the method is suitable for gecko medicinal materials, decoction pieces, processed products, preparation intermediates or preparation finished products thereof.

The invention establishes a group of fast, comprehensive and highly specific gecko formula particle quality detection methods, which comprise a thin layer identification method and a liquid phase method for simultaneously measuring a characteristic spectrum and the contents of tyrosine and tryptophan. Compared with the Gecko formula granule standard disclosed in patent CN113960203A and Shanghai, the Gecko formula granule has the advantages that:

(1) The thin-layer identification method uses the reference medicinal material as reference, has more corresponding spots, and accords with the integral quality control characteristic of the traditional Chinese medicine.

(2) The invention adopts a set of chromatographic method to realize qualitative and quantitative control of geckos. Tyrosine and tryptophan are used as content index components, the gecko contrast medicinal material is used as a reference, a characteristic map containing 7 characteristic peaks is established, more than half of the characteristic peaks are identified, a multi-index control means meeting the gecko integrity requirement is established for the first time, and compared with the existing method, the method is quicker, more comprehensive and more effective, and is more beneficial to quality control of gecko formula particles. The characteristic spectrum defines 7 characteristic peaks which are more than 6 in the patent CN 113960203A; the number of the reference medicinal materials is 4, which exceeds 50% of the characteristic peak, and meanwhile, the reference medicinal materials are used as reference substances, so that the consistency of the formula granules and the medicinal materials can be reflected.

(3) The content determination indexes are tyrosine and tryptophan indexes, the content is high, the drug effect is clearer compared with pterin-6-carboxylic acid, and meanwhile, the quality standard of the gecko formula particles can be effectively controlled by the double indexes compared with a single index. The method provided by the invention considers the integrity of the traditional Chinese medicine, provides a technical control means for the quality of gecko raw materials, intermediates and formula granules, and provides a powerful guarantee for industrialization and quality control of the gecko raw materials, intermediates and formula granules.

(4) The method adopts the ultra-high liquid chromatography and the thin-layer chromatography, reasonably optimizes chromatographic conditions, establishes an organic combination of a characteristic spectrum, a content measurement method and a thin-layer identification method of the gecko formula particles, the characteristic spectrum and the thin-layer identification belong to qualitative methods, the content measurement belongs to a quantitative method, can quantitatively analyze main effective components, the thin-layer identification operation is convenient, equipment is simple, the cost is low, different polar components can be analyzed by optimizing a developing agent, the characteristic spectrum method depends on liquid phase equipment, the artificial interference is less, the detection sensitivity is high, the method is mainly suitable for analyzing medium-polarity and low-polarity components, the medium-polarity and low-polarity components are mutually complemented.

Has the advantages that: the method is more comprehensive, simple, rapid and effective, and the quality control method of the gecko formula granules is used for controlling the quality from a plurality of angles of thin-layer identification, characteristic spectrum and content measurement, so that more powerful guarantee is provided for industrialization of the gecko formula granules. The method disclosed by the invention is simple to operate, and verified by methodology, the repeatability and the stability are good, the recovery rate is reliable, the detection cost is low, the detection efficiency is high, and an important guarantee is provided for standardizing the production of the gecko formula particles.

Drawings

Fig. 1 is a characteristic spectrum of gecko formula granules, peak 2: uric acid; peak 4 (S): tyrosine; peak 6: pterin-6-carboxylic acid; peak 7: tryptophan;

fig. 2 is a gecko intermediate profile, peak 2: uric acid; peak 4 (S): tyrosine; peak 6: pterin-6-carboxylic acid; peak 7: tryptophan;

FIG. 3 is a characteristic spectrum of Gekko Swinhonis, peak 2: uric acid; peak 4 (S): tyrosine; peak 6: pterin-6-carboxylic acid; peak 7: tryptophan;

fig. 4 is a characteristic spectrum of gecko decoction pieces, peak 2: uric acid; peak 4 (S): tyrosine; peak 6: pterin-6-carboxylic acid; peak 7: tryptophan

FIG. 5 is a chromatogram of control and Gekko Swinhonis formulation particles;

FIG. 6 UPLC chromatogram of Gekko Swinhonis formulation particle at different detection wavelengths;

FIG. 7 tyrosine full wavelength scan;

FIG. 8 Tryptophan full wavelength scan;

FIG. 9 UPLC profile for different extraction solvent studies;

FIG. 10 UPLC plots for different extraction mode considerations;

FIG. 11 UPLC plots for different extraction time studies;

FIG. 12 UPLC plots for different extraction volume studies;

FIG. 13 Gekko Swinhonis formula granule feature spectrum chromatography column investigation;

FIG. 14 is a column temperature study of the characteristic spectrum of the gecko formulation granule;

FIG. 15 Gecko formula particle feature map flow rate investigation;

FIG. 16 is a linear plot of a tyrosine control;

FIG. 17 is a graph of the linear relationship of the tryptophan control;

fig. 18 TLC patterns for different spot sizes of gecko formula granules, wherein 1: 1 mul of test sample; 2: 3 mul of test sample; 3: 5 mul of test sample; 4: 8 mul of test sample; s1: 1 mul of reference medicinal material; s2: 3 mul of reference medicine; s3: 5 mul of reference medicine; s4: 8 mul of reference medicine;

fig. 19 TLC spectrum of gecko formulation granule specificity experiment, wherein 1: negative control; 2 to 4: gecko formula granules; s: gekko Swinhonis control medicinal materials;

fig. 20 TLC spectra of the rhizoma kadsurae formulations under different temperature conditions, wherein, 1-3: gecko formula granules; s: gekko Swinhonis control medicinal materials;

FIG. 21 TLC spectra of Gekko Swinhonis formulation granules under different humidity conditions, wherein 1-3: gecko formula granules (21110049); s: gekko Swinhonis control medicinal materials;

FIG. 22 is a TLC chromatogram of thin-layer plate gecko formula granules from different manufacturers, wherein 1-3: gecko formula granules; s: gekko Swinhonis control medicinal material

(Qingdao Kang Yexin pharmaceutical silica gel desiccant GmbH (left), qingdao maritime factory (middle), nicoti chemical industry research institute (right));

FIG. 23 thin layer chromatogram for identification of Gekko Swinhonis formulation particle samples from different batches, 1-3: gecko formula granules; s: gekko Swinhonis control;

FIG. 24 separation effect of different gradients;

FIG. 25 is a graph comparing the extraction efficiencies of different extraction solvents;

FIG. 26 is a graph showing the comparison of extraction efficiencies of different extraction methods;

FIG. 27 is a graph of the comparison of extraction efficiency at different extraction times;

FIG. 28 is a graph of the comparison of extraction efficiencies for different extraction volumes;

FIG. 29 Gecko formulation granule specificity test;

FIG. 30 Gecko formulation particle integrity test;

FIG. 31 Gecko formula granule specificity test;

fig. 32 TLC spectra of gecko formulation granules (different developing solvent studies), wherein, left: developing agent I; and (3) right: developer II, 1-2: a test article; s1 to S2: a reference medicinal material;

fig. 33 TLC spectra (different test article preparation methods) of gecko formulation granules, wherein, left: a first method for preparing a test sample; and (3) right: and a second sample preparation method, 1-2: a test article; s1 to S2: and (5) comparison medicinal materials.

Detailed Description

The embodiment provides a group of detection methods for comprehensively controlling the quality of gecko formula granules, which specifically comprise the following steps:

(1) Thin-layer identification: grinding Gekko Swinhonis granule 0.5g, adding water 10ml, treating with ultrasound for 30 min, and filtering to obtain filtrate as test solution. Adding water 20ml into another 1g of gecko control medicinal material, heating and refluxing for 30 min, shaking up, filtering, and taking the filtrate as the control medicinal material solution. Sucking the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed.

(2) Characteristic spectrum and content determination:

chromatographic condition parameters: octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); acetonitrile is taken as a mobile phase A, 0.08 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 220nm.

Preparation of reference solutions: taking 1g of gecko reference drug, placing into a conical flask with a plug, adding 25ml of 50% methanol, sealing, performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, filtering, and taking the subsequent filtrate as the reference solution of the reference drug. Taking a proper amount of a casein reference substance and a tryptophan reference substance, precisely weighing, and adding water to prepare solutions containing 40 mu g of tyrosine and 15 mu g of tryptophan in each 1ml serving as reference substance solutions of the reference substances.

Preparation of a test solution: precisely weighing about 0.5g of gecko formula granules, placing the gecko formula granules into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the gecko formula granules.

The determination method comprises the following steps: precisely sucking 1 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

The method provided by the present invention is described in detail below with reference to examples, and the specific experimental conditions and results thereof described in the examples are only for illustrating the present invention and should not be construed as limiting the present invention described in detail in the claims.

Example establishment of Gekko Swinhonis formula granule characteristic spectrum method and research on methodology thereof

1. Instruments and reagents

Agilent-1290 hplc, thermo hplc; KQ-250B ultrasonic cleaning machine (Kunshan ultrasonic Instrument Co., ltd.); electronic analytical balance (Mettler-Torledo instruments (Shanghai) Co., ltd.), temperature-controlled water bath (Nantong Huatai laboratory instruments Co., ltd.), HY-4 oscillator (King Tan Kexing apparatus factory), pure water system (Millipore Co., ltd.), TGL-16C type centrifuge (Shanghai Ann science apparatus factory), acetonitrile (chromatographic purity, thermo Fisher Co., ltd.), methanol (chromatographic purity, thermo Fisher Co., ltd.), water as ultrapure water, and other reagents as analytical purity.

Gekko Swinhonis control drug was purchased from Shanghai hong Yongsheng Biotech limited, no. 220041-202104.

Tyrosine (140609-201914) and tryptophan (140686-201303) control were purchased from the Chinese food and drug testing institute.

Gekko Swinhonis formulation granules are provided by Jiangyin Tianjiang pharmaceutical industry Co.

2. Determination of detection wavelength

Taking a proper amount of the product, grinding, taking about 0.5g, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 15 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product. Chromatograms of the samples at 220nm,254nm and 300nm were collected as shown in FIG. 7.

The result shows that the number of peaks is the largest under the wavelength of 220nm, the retained information is the largest, and the wavelength of 220nm is selected as the detection wavelength by referring to the gecko standard decoction characteristic spectrum.

3. Optimization of chromatographic conditions

The method comprises the following steps: octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out according to the specification of the following table; flow rate 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 220nm.

The method 2 comprises the following steps: octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out according to the specification of the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 220nm.

The method 3 comprises the following steps: octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); acetonitrile is taken as a mobile phase A, 0.08 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 220nm.

The results show (table, fig. 24) that method 3 has the best separation effect, symmetrical peak shape and moderate retention time, so that method 3 is selected for subsequent study.

4. Preparation of test solution

4.1 examination of different extraction solvents

Taking a proper amount of the product, grinding, taking about 0.5g, paralleling 5 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of each of water, 30% methanol, 50% methanol, 70% methanol and methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W, frequency 40 kHz) for 15 minutes, cooling, weighing again, complementing the weight loss by using corresponding solvents, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. Precisely sucking 1 μ l of each test solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and calculating the ratio of characteristic peak area/sample weighing amount, the results are shown in Table 1, FIGS. 10 and 25.

TABLE 1 comparison of extraction efficiency (peak area/sample weight) for different extraction solvents

As can be seen from the above table: the extraction efficiency is relatively close by taking methanol with different concentrations as extraction solvents, and the extraction solvent is determined to be 50% methanol by comprehensively considering that the solvation effect of the methanol with the concentration of 70% or more is relatively obvious and the water as the solvent extraction solution is not clear and is difficult to filter.

4.2 examination of different extraction methods

Taking a proper amount of the product, grinding, taking about 0.5g, paralleling 3 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz), shaking for extraction, heating and refluxing for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking a subsequent filtrate. Precisely sucking 1 μ l of each test solution, injecting into liquid chromatograph, measuring according to the above chromatographic conditions, and calculating characteristic peak area/sample weighing amount, the result is shown in Table 2, FIG. 11, and FIG. 27.

TABLE 2 comparison of extraction efficiency (peak area/sample weight) for different extraction modes

As can be seen from the above table: the extraction efficiency of the ultrasonic extraction and the reflux extraction is close, and the extraction method is determined to be ultrasonic treatment in consideration of the simplicity of operation.

4.3 investigation of different extraction times

Taking a proper amount of the product, grinding, taking about 0.5g, paralleling 4 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W, frequency 40 kHz) for 10 minutes, 15 minutes, 30 minutes and 45 minutes, cooling, weighing again, complementing the loss weight with 50% methanol, shaking uniformly, filtering, and taking a subsequent filtrate. Separately and precisely sucking 1 μ l of each sample solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, and calculating the characteristic peak area/sample weighing amount, the results are shown in table 3, fig. 11 and fig. 27.

TABLE 3 comparison of extraction efficiency (peak area/sample weight) for different extraction times

As can be seen from the above table: the difference of characteristic peak area/sample weighing in the gecko formula particles is small when the gecko formula particles are subjected to ultrasonic treatment at different time, the extraction is sufficient after the gecko formula particles are subjected to ultrasonic treatment for 15 minutes, and the extraction time is determined to be 30 minutes in order to ensure the extraction efficiency.

4.4 investigation of different extraction volumes

Taking a proper amount of the product, grinding, taking about 0.5g, paralleling 3 groups, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml, 25ml and 50ml of 50% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 50% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product. Separately, 1 μ l of each sample solution was precisely aspirated, injected into a liquid chromatograph, measured under the above chromatographic conditions, and the characteristic peak area/sample weight extraction volume/25 was calculated, and the results are shown in table 4, fig. 12, and fig. 28.

Table 4 comparison of extraction efficiency for different extraction volumes (peak area/weight extracted volume/25)

As seen from the above table: when the extraction volume is 10ml, extraction is insufficient, and when the extraction volume is 25ml, the value of characteristic peak area/sample weight extraction volume/25 in the gecko formula granules is large, so that the extraction volume is determined to be 25ml.

4.5 determination of preparation method of test solution

According to the research result, the preparation method of the test solution for determining the characteristic spectrum of the gecko formula particles comprises the following steps:

grinding Gekko Swinhonis formula granules, precisely weighing about 0.5g, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, ultrasonically treating (250W power, 40kHz frequency) for 30 min, cooling, weighing again, supplementing the weight loss with 50% methanol, shaking up, filtering, and collecting the filtrate.

5. Study of the feature Pattern methodology

5.1 specialization examination

The test solution of Gekko Swinhonis formula granule and the negative solution of Gekko Swinhonis are injected into a liquid chromatograph, and the result is shown in FIG. 29.

The experimental results show that: the negative solution has no chromatographic peak at the retention time of each characteristic peak in the chromatogram of the test solution, which indicates that the solvent has no interference on the determination of the gecko formula particles, and the gecko formula particles determined by the method have specificity.

5.2 Overall examination

Under certain chromatographic conditions, the elution time is prolonged, and whether the residual impurity peak affects subsequent samples under a given chromatographic condition system is examined, and the result is shown in figure 30 below.

According to experimental results, the elution time is prolonged, no impurity peak exists, and the chromatographic condition basically meets the principle of maximum information content and has no influence on the analysis of subsequent samples.

5.3 precision investigation

Taking the same batch of samples, preparing a test solution according to the test solution preparation method, continuously injecting samples for 6 times, wherein each sample is 1 mu l, recording the retention time of characteristic peaks, and calculating the relative retention time according to text requirements, wherein the results are shown in a table 5.

TABLE 5 precision results (relative Retention time)

The results show that the RSD of the relative retention time of each characteristic peak is less than 1 percent, and the precision is good.

5.4 intermediate precision investigation

Taking the same batch of samples, preparing a test solution according to a test solution preparation method, processing 3 parts of the samples by two experimenters A, B according to the requirements under the text items, respectively injecting 1 mu l of samples on Agilent instruments and Thermo instruments, recording the retention time of characteristic peaks, calculating the relative retention time according to the text requirements, and obtaining a result table 6.

TABLE 6 intermediate precision investigation (relative retention time)

The results show that: the RSD of the characteristic peak of the sample relative to the retention time is less than 8 percent, which shows that different instruments have certain influence on the relative retention time, but the relative retention time is within the specified range, and the intermediate precision test is good.

5.5 stability Studies

Taking the same batch of samples, preparing test solutions according to the preparation method of the test solution, injecting 1 mul of samples in 0, 2, 4, 6, 8, 12, 18 and 24 hours respectively, recording the retention time of characteristic peaks, calculating the relative retention time according to text requirements, and obtaining the results shown in Table 7.

TABLE 7 stability Studies (relative Retention time)

The result shows that the RSD of the relative retention time of each characteristic peak is less than 1%, and the test solution has good stability within 24 hours.

5.6 repeatability test

Taking the same batch of samples, paralleling 6 groups, preparing test solutions according to the preparation method of the test solution, injecting 1 mul of the test solutions, recording the retention time of characteristic peaks, calculating the relative retention time according to the text requirements, and obtaining the results shown in the table 8.

TABLE 8 repeatability tests (relative retention time)

The results show that the repeatability test of the method is good.

5.7 durability examination

(1) Chromatographic column investigation

Taking the same batch of samples, preparing a test sample solution according to the preparation method of the text test sample solution, and respectively adopting Acclaim RSLC 120 C18 (Thermo, 2.1mm multiplied by 100mm,2.2 mu m);

T3(Agilent，2.1mm×100mm， 1.6μm)；ACQUITY

HSS T3 (Waters, 2.1X 100mm,1.8 μm) three columns, 1 μ l each was injected and the corresponding chromatograms were recorded, the results are shown in FIG. 14.

The results show that: ACQUITY

The HSS T3 chromatographic column has good separation, symmetrical peak shape, moderate retention time and Acclaim ^TM RSLC 120 C18 column and

the T3 chromatographic column has the phenomenon of poor resolution or poor peak shape, and the differences of chromatograms of the 3 chromatographic columns are large, so that the gecko formula particle characteristic spectrum detection recommends using the chromatographic column as ACQUITY

HSS T3(Waters，2.1×100mm，1.8μm)。

(2) Investigation of column temperature

Taking the same batch of samples, preparing the test solution according to the text test solution preparation method, inspecting three temperatures of 20 ℃,25 ℃ and 30 ℃, injecting 1 mu l of sample respectively, recording the retention time of the characteristic peak, calculating the relative retention time according to the text requirements, and obtaining the results shown in figure 15 and table 9, and inspecting the column temperature (relative retention time) shown in table 9.

TABLE 9 column temperature investigation (relative Retention time)

The result shows that the column temperature is between 20 and 30 ℃, the measurement result of the sample is stable, the column temperature meets the retention time requirement specified by the method, and the durability is good

(3) Investigation of flow velocity

Taking the same batch of samples, preparing the sample solution according to the preparation method of the text sample solution, observing three flow rates of 0.25ml, 0.30ml and 0.35ml per minute, respectively injecting 1 mu l of sample, recording the retention time of the characteristic peak, and calculating the relative retention time according to the text requirements, wherein the results are shown in a figure 15 and a table 10.

TABLE 10 investigation of flow Rate (relative Retention time)

The result shows that the flow rate is between 0.25ml/min and 0.35ml/min, the measurement result of the sample is stable, the retention time requirements specified by the method are met, and the durability is good.

6. Sample assay

Taking 3 batches of gecko formula particle samples and 17 batches of gecko medicinal materials and decoction pieces, operating according to the text, and obtaining the measurement results shown in the table.

TABLE 11 sample measurement results (relative Retention time)

TABLE 12 Gekko Swinhonis drug sample assay results (relative retention time)

TABLE 13 Gekko Swinhonis decoction piece sample determination results (relative Retention time)

5.1 example establishment of a method for determining the content of Gekko Swinhonis formulation particles and methodological research

1 instruments and reagents

Thermo ultra high performance liquid chromatograph; agilent-1290 ultra performance liquid chromatograph; KQ-250B ultrasonic cleaning machine (Kunshan ultrasonic Instrument Co., ltd.); electronic analytical balance (mettler-toledo instruments (shanghai) ltd); temperature-controlled water bath (south-channel huatai laboratory instruments ltd); HY-4 oscillator (Kyoto instruments, jin Tan city); pure water system (Millipore corporation); AS165W type centrifuge (shikawa (shanghai) commercial limited); acetonitrile (chromatographically pure, thermo Fisher corporation); methanol (chromatographic purity, thermo Fisher corporation); phosphoric acid (chromatographically pure, aladin corporation); the water is ultrapure water; other reagents were analytically pure.

Tyrosine (140609-201914) and tryptophan (140686-201303) controls are purchased from China food and drug testing research institute;

gekko Swinhonis formulation granules are provided by Jiangyin Tianjiang pharmaceutical industry Co.

2 check the origin and purity of reference substance

Tyrosine is purchased from China food and drug testing research institute, with the number of 140609-201914, and is used for content determination, the content is 99.9%, and no treatment is needed before use.

Tryptophan is purchased from China food and drug testing institute, and is numbered 140686-201303 for content determination, the content is 99.9%, and no treatment is needed before use.

3 determination of the detection wavelength

The test is carried out by scanning tyrosine and tryptophan reference substance solution at full wavelength and recording ultraviolet absorption diagram, which is shown in figure 8 and figure 9.

As a result, since tyrosine and tryptophan each have a large absorption at a wavelength of about 220nm, 220nm was selected as a detection wavelength for measuring the content.

4 determination of chromatographic conditions

A method for determining the content of tyrosine and tryptophan in the gecko formula particles is established by referring to a gecko characteristic spectrum determination method, and the chromatographic conditions are as follows:

octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); acetonitrile is taken as a mobile phase A, 0.08 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.3ml per minute; the column temperature was 25 ℃; the detection wavelength was 220nm.

5 preparation of test solutions

5.1 investigation of different extraction solvents

Taking a proper amount of gecko formula particles, grinding, taking about 0.5g of gecko formula particles, paralleling 5 groups, paralleling 2 parts of each group, precisely weighing, placing in a conical flask with a plug, precisely adding water, 30% methanol, 50% methanol, 70% methanol and 25ml of methanol respectively, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with corresponding solvent, shaking up, filtering, and taking a subsequent filtrate to obtain the gecko formula particle. Precisely sucking 1 μ l of each test solution, injecting into liquid chromatograph, and calculating tyrosine and tryptophan contents, the results are shown in FIG. 9, table 14, and Table 15.

TABLE 14 comparison of different extraction solvents-tyrosine

TABLE 15 comparison of different extraction solvents-Tryptophan

As a result, it was found that: the extraction results of water, 30% methanol, 50% methanol and 70% methanol are relatively close, but considering that the high concentration organic phase is easy to generate solvation effect, the water extraction solution is not clear, so the extraction solvent is determined to be 50% methanol.

5.2 examination of different extraction methods

Taking a proper amount of gecko formula particles, grinding, taking about 0.5g of gecko formula particles, paralleling 3 groups, paralleling 2 parts of each group, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W, frequency 40 kHz), shaking for extraction, heating and refluxing for 30 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the gecko formula particle. Precisely sucking 1 μ l of each test solution, injecting into liquid chromatograph, and calculating tyrosine and tryptophan contents, the results are shown in FIG. 10, table 16, and Table 17.

TABLE 16 comparison of different extraction methods-tyrosine

TABLE 17 comparison of different extraction methods-Tryptophan

As a result, it was found that: the extraction efficiency of ultrasonic treatment, shaking and heating reflux is equivalent, and the extraction method is determined to be ultrasonic treatment in consideration of the convenience of operation.

5.3 investigation of different extraction times

Taking a proper amount of gecko formula particles, grinding, taking about 0.5g of gecko formula particles, paralleling 4 groups, paralleling 2 parts of each group, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W, frequency 40 kHz) for 10 minutes, 15 minutes, 30 minutes and 45 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the gecko formula particle. Precisely sucking 1 μ l of each test solution, injecting into liquid chromatograph, and calculating tyrosine and tryptophan contents, the results are shown in FIG. 11, table 18, and Table 19.

TABLE 18 comparison of different extraction times-tyrosine

TABLE 19 comparison of different extraction times-Tryptophan

As a result, it was found that: the extraction time was determined to be 30 minutes, considering that the extraction was complete and time-saving, since the different extraction times had no significant effect on the tyrosine and tryptophan contents.

5.4 examination of different extraction volumes

Taking a proper amount of gecko formula particles, grinding, taking about 0.5g of gecko formula particles, paralleling 3 groups, paralleling 2 parts of each group, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml, 25ml and 50ml of 50% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the gecko formula particle. Precisely sucking 1 μ l of each test solution, injecting into liquid chromatograph, and calculating tyrosine and tryptophan contents, the results are shown in tables 20 and 21.

TABLE 20 comparison of different extraction volumes-tyrosine

TABLE 21 comparison of different extraction volumes-Tryptophan

As a result, it was found that: when the extraction volume is 10ml, the extraction is insufficient, when the extraction volume is 25ml, 50ml, the extraction efficiency is equivalent, and the extraction volume is determined to be 25ml from the comprehensive view point of sufficient extraction and solvent saving.

Through investigation, the preparation method of the test sample for finally determining the content measurement of the gecko particles comprises the following steps:

taking a proper amount of gecko formula particles, grinding, taking about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (with the power of 250W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the gecko formula particle.

6 methodological validation

6.1 Linear

The regression equation of tryptophan and tyrosine was determined by precisely aspirating 0.1. Mu.l, 0.2. Mu.l, 0.5. Mu.l, 1.0. Mu.l, 1.5. Mu.l, and 2.0. Mu.l of the mixed solutions of tyrosine and tryptophan control substances (concentration 41.50. Mu.g/mL and 13.19. Mu.g/mL), measuring the samples by the above chromatographic conditions, plotting a standard curve with the peak area integrated value as the ordinate and the sample amount (. Mu.g) as the abscissa, and finding the results in Table 22, table 23, FIG. 16, and FIG. 17.

TABLE 22 regression equation

TABLE 23 Tryptophan control sample weight to peak area relationship

The results show that: the tyrosine sample amount is 0.0041500 mug-0.0830000 mug, the tryptophan sample amount is 0.00131900 mug-0.0263800 mug, and the sample amount and the peak area value have good linear relation.

6.2 precision test

6.2.1 instrumental precision test

Precisely absorbing a sample solution, injecting the sample solution into a liquid chromatograph, measuring according to the chromatographic conditions, continuously injecting samples for 6 times, recording the peak area of the sample solution, and calculating the relative standard deviation, wherein the result is shown in the table below.

TABLE 24 Instrument precision test

The results show that: the instrument precision test is good (RSD percent is less than or equal to 2.0 percent).

6.2.2 repeatability tests

Taking a proper amount of gecko formula particles, grinding, taking about 0.5g of gecko formula particles, precisely weighing and paralleling 6 parts, preparing the sample solutions according to the preparation methods of the sample solutions respectively, injecting 1 mu l of the sample solutions respectively, measuring the peak areas of tryptophan and tyrosine, and calculating the content and RSD, wherein the results are shown in a table 25.

TABLE 25 repeatability test results for samples-tyrosine

TABLE 26 repeatability test results for samples-Tryptophan

The results show that: the repeatability test is good (RSD percent is less than or equal to 2.0 percent).

6.2.3 intermediate precision test

An appropriate amount of gecko formula particles are taken, ground into fine powder, about 0.5g of gecko formula particles are taken, 3 parts of gecko formula particles are respectively processed by two experimenters A, B according to the requirements, 1 mu l of samples are respectively injected on Thermo-UPLC and Agilent-UPLC, the peak area values of tryptophan and tyrosine are measured, the content and RSD are calculated, and the results are shown in Table 27.

TABLE 27 intermediate precision test-tyrosine

TABLE 28 intermediate precision test-Tryptophan

The results show that: the intermediate precision is good.

6.3 accuracy test

Taking three groups of samples with known contents (the tyrosine content is 2.16mg/g, the tryptophan content is 0.85 mg/g), grinding, taking about 0.25g, weighing in parallel 3 parts, accurately weighing, and adding reference substances which are 50%, 100% and 150% of each component contained in 0.25g of the samples respectively. The sample recovery test solutions were prepared by the above test solution preparation method, and 1. Mu.l of each sample was injected, and the recovery rate, the average recovery rate, and RSD were calculated according to the following formulas, and the results are shown in tables 29 and 30.

TABLE 29 tyrosine accuracy results

TABLE 30 Tryptophan accuracy results

The results show that: the recovery rate of tyrosine is between 99.14 and 104.61 percent, the recovery rate of tryptophan is between 98.09 and 100.58 percent, and the accuracy test result is good.

6.4 specificity test

The auxiliary materials added in the gecko formula granules are lactose, silicon dioxide and magnesium stearate. The experiment investigates the influence of the negative sample of the gecko lacking on the content determination of the gecko formula particles. Preparing a negative sample solution from the negative sample of the gecko lacking sample according to the preparation method of the test sample.

Injecting the Gekko Swinhonis formula granule test solution, the negative control solution and the tyrosine and tryptophan control solution into a liquid chromatograph.

The experimental results show (fig. 1): the negative chromatogram has no chromatographic peak at the retention time corresponding to the reference substance, which indicates that the auxiliary materials and the solvent have no interference to the determination of tyrosine and tryptophan, and the determination of the content of tyrosine and tryptophan in the gecko formula particles by the method has specificity.

6.5 integrity test

On the determined chromatographic conditions, the elution gradient at the highest acetonitrile ratio is maintained, the elution time is doubled, and whether the influence of residual impurity peaks on subsequent samples is detected under the given chromatographic condition system is examined, and the result is shown in fig. 31.

According to experimental results, the elution time is prolonged by one time, and no impurity peak exists, so that the chromatographic condition basically meets the principle of maximum information content, and the analysis of subsequent samples is not influenced.

6.6 durability test

6.6.1 stability test

Preparing the test solution according to the preparation method of the test solution from the Gekko Swinhonis formula particles, injecting 1 μ l of the sample at 0, 2, 4, 6, 8, 12, 18 and 24 hours respectively, measuring peak area value, and calculating RSD.

TABLE 31 stability test results

The results show that: the sample test solution has good stability within 24 hours.

6.6.2 investigation of different flow rates

The test solution was prepared by the above method for preparing test solution, and the tyrosine and tryptophan contents were observed at flow rates of 0.25ml/min, 0.30ml/min, and 0.35ml/min, and the results are shown in FIG. 16, table 32, and Table 33.

Table 32 different flow rate investigation

Table 33 different flow rate investigation

The results show that: under three flow rates, the separation degrees of the chromatographic peaks of tyrosine and tryptophan are good, the contents are similar, and the durability is good.

Inspection of 6.6.3 for different column temperatures

Taking Gekko Swinhonis formula granule, preparing the test solution according to the above preparation method, and examining the tyrosine and tryptophan contents at 20 deg.C, 25 deg.C and 30 deg.C, the results are shown in FIG. 15, table 34 and Table 35.

TABLE 34 investigation of different column temperatures-tyrosine

TABLE 35 investigation of different column temperatures-Tryptophan

The results show that: at the temperature of the three columns, the separation degrees of the chromatographic peaks of tyrosine and tryptophan are good, the contents are similar, and the durability is good.

6.6.4 column inspection

Preparing the Gekko Swinhonis formula particles into a test solution according to the preparation method of the test solution, and respectively inspecting Acclaim RSLC 120 C18 (Thermo, 2.1mm x 100mm,2.2 μm);

T3(Waters，2.1mm×100mm， 1.6μm)；ACQUITY

HSS T3 (Waters, 2.1X 100mm,1.8 μm) tyrosine, and tyrosine in Gekko Swinhonis formula granules under different chromatographic column conditions,The tryptophan content results are shown in FIG. 14, table 36 and Table 37.

TABLE 36 comparison of different chromatography columns-tyrosine

TABLE 37 comparison of different chromatography columns-Tryptophan

The results show that: different chromatographic columns have little influence on the determination of the content of tyrosine and tryptophan, and considering that the same method is used for the determination of the content of the gecko and the characteristic spectrum, the determination of the content of the gecko formula granules recommends using the chromatographic column as ACQUITY

HSS T3 (Waters，2.1×100mm，1.8μm).

7. Sample assay

Taking 3 batches of gecko formula granules, 17 batches of gecko medicinal materials and decoction pieces, preparing a test solution of the test solution according to the preparation method of the test solution, and measuring the content of tyrosine and tryptophan in the sample, wherein the experimental results are shown in tables 38, 39 and 40.

TABLE 38 determination of tyrosine, tryptophan content in Gekko Swinhonis formulation granules

TABLE 39 determination of tyrosine and tryptophan content in Gekko Swinhonis

TABLE 40 determination of tyrosine and tryptophan contents in Gekko Swinhonis decoction pieces

5.3 example establishment of the method for identifying the thin layer of the triple wall tiger formulation particle and the methodological study

1 Instrument and reagent

The instrument comprises the following steps: the device comprises a thin-layer automatic imager (CAMAG TLC VIUALIZER), an ME204E ten thousandth balance (Mettler-Torlo), a KQ-250E type ultrasonic cleaner (Kunshan ultrasonic electronic Co., ltd.), an electronic temperature-adjusting electric furnace (Baoli scientific research instruments Co., ltd. Of Jiangyun city), and a GKC114 temperature-controlling water bath (Nantong Huatai laboratory instruments Co., ltd.).

Reagent preparation: methanol (national chemical group chemical reagent limited), absolute ethanol (national chemical group chemical reagent limited), n-butanol (national chemical group chemical reagent limited), and glacial acetic acid (national chemical group chemical reagent limited); silica gel G thin-layer plate (Qingdao oceanic chemical plant, qingdao Kang Yexin medicinal silica gel desiccant Co., ltd., nicoti City chemical industry research institute).

Gekko Swinhonis reference drug (lot number: 220041-202104) was purchased from Shanghai hong Yongsheng Biotech Co., ltd.

Gekko Swinhonis formulation granules are provided by Jiangyin Tianjiang pharmaceutical industry Co.

2 thin layer identification conditions

A first developing agent: adopting silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed.

And (2) developing agent II: developing with silica gel G thin layer plate and anhydrous alcohol-water (6: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed.

3 preparation of the solution

3.1 preparation of test solutions

The method comprises the following steps: taking 0.5g of the product, grinding, adding 10ml of water, and carrying out ultrasonic treatment for 30 minutes to obtain a test solution.

The second method comprises the following steps: taking 0.5g of the product, grinding, adding 10ml of 30% methanol, and carrying out ultrasonic treatment for 30 minutes to obtain a test solution.

3.2 preparation of control solutions

The method comprises the following steps: adding water 20ml into Gekko Swinhonis control 1g, heating under reflux for 30 min, and filtering to obtain filtrate as control solution.

The second method comprises the following steps: collecting Gekko Swinhonis control material 1g, adding 30% methanol 20ml, heating under reflux for 30 min, filtering, and collecting filtrate as control medicinal solution.

3.3 preparation of negative control solution

0.5g of negative sample of Gekko Swinhonis granules was sampled and prepared into a negative control solution in the same manner as in "3.1 method one".

4 determination of thin layer identification method

4.1 investigation of different spreading Agents

Preparing Gekko Swinhonis formula granule into test solution according to the first preparation method, preparing Gekko Swinhonis control solution into control solution according to the first preparation method, respectively spotting on silica gel G thin layer plate, respectively spreading the two developing agents, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed. The results are shown in FIG. 32.

As can be seen from the figure, by adopting the developing agent I, the spots are clear and the separation effect is better; while the chromatographic spot of developer two is tailing. By comprehensive consideration, the developing agent I is selected as the developing agent of the thin-layer chromatography of the gecko formula particles.

4.2 examination of methods for preparing test solution and control solution

Preparing Gekko Swinhonis formula granule into test solution according to the above two preparation methods, preparing corresponding reference medicinal material solution from Gekko Swinhonis reference medicinal material according to the above two preparation methods, respectively spotting on silica gel G thin layer plate, developing with the above determined thin layer chromatography developer, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed. The result is shown in FIG. 33.

As can be seen from the figure, the spots of the test solution of the two test treatment methods are clear, and the safety problem of the solvent is also considered, so the first method is selected as the preparation method of the test, and the chromatographic spots of the control solution prepared by the first method are clear and correspond to the chromatographic main spots of the test one by one, so the first method is selected as the control treatment method of the thin-layer identification.

4.3 examination of the amount of different spots

Preparing Gekko Swinhonis formula granule and Gekko Swinhonis control medicinal material into test solution and control solution, respectively, spotting on the same silica gel G thin layer plate according to text thin layer chromatography conditions at different sample amount, respectively, developing with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. The results are shown in FIG. 19.

As can be seen from the figure, when the sample amount of the sample solution is 1 to 8 mul and the sample amount of the reference medicinal material solution is 3 to 8 mul, the fluorescence spots on the corresponding positions of the sample chromatogram and the reference medicinal material chromatogram are clearly corresponding without other interference, so the difference between samples in different batches is considered, and the sample application amount of the sample solution and the reference medicinal material solution is 5 mul.

5 determination of thin layer identification method

According to the research results, the thin layer identification method for determining the gecko formula particles comprises the following steps:

taking 0.5g of the product, grinding, adding 10ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, and taking the filtrate as a test solution. Adding water 20ml into another 1g of Gekko Swinhonis control material, heating and refluxing for 30 min, shaking, filtering, and collecting filtrate as control solution. Sucking 5ul of each of the above two solutions, respectively dropping on the same silica gel G thin layer plate, spreading with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at positions corresponding to those on the chromatogram of the control solution.

6 methodological validation

6.1 specificity test of samples

Sampling Gekko Swinhonis formula granule sample solution, control solution, and negative control solution on the same silica gel G thin layer plate, respectively, developing with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. The results are shown.

As can be seen from the figure, the chromatogram of the test sample of the gecko formula particle and the chromatogram of the reference medicinal material show the spots with the same color at the corresponding positions, and the negative control has no interference, which indicates that the thin-layer identification method has good specificity.

6.2 durability test

6.2.1 investigation of different temperatures

Collecting test solution and control solution, spreading on the same silica gel G thin layer plate at normal temperature, low temperature and high temperature respectively with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. The results are shown in FIG. 21.

As can be seen, spots with the same color appear on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference medicinal material of the gecko formula particle under different temperature conditions, and the separation effect is better. The experimental result shows that the temperature has no influence on the thin layer identification of the gecko formula particles, which indicates that the thin layer identification method has good durability to different temperatures.

6.2.2 investigation of different humidities

Collecting test solution and control solution, spreading on the same silica gel G thin layer plate under different humidity conditions with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. The results are shown in FIG. 22.

As can be seen, the chromatography of the test sample and the chromatography of the reference drug of the gecko formula granules show spots with the same color at corresponding positions under different humidity conditions, and the spots are basically separated. The experimental result shows that the humidity has no influence on the identification of the gecko square particle thin layer, which indicates that the thin layer identification method has good durability to different humidities.

6.2.3 inspection of different brands of thin layer sheets

Taking the sample solution and the control solution, spotting on different brands of silica gel G thin layer plates, respectively, developing with n-butanol-glacial acetic acid-water (4: 1) as developer, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. The results are shown in FIG. 23.

As can be seen, in different brands of silica gel G plates, spots with the same color are displayed on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference drug of the gecko formula particle, and the spot separation degree is better. Experimental results show that silica gel G plates of different brands have no influence on the thin layer identification result of gecko formula particles, and the thin layer identification method is good in durability of different thin layer plates.

7 sample detection

According to the determined thin-layer chromatography conditions, preparing the Gekko Swinhonis formula granules in different batches into test solution according to the preparation method of the test solution, respectively spotting the test solution on the same silica gel G thin-layer plate, developing with n-butanol-glacial acetic acid-water (4: 1) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. The results are shown in FIG. 24.

As can be seen, the chromatography of the gecko formula granules and the chromatography of the reference drug show spots with the same color at corresponding positions.

Claims (8)

1. A control method for comprehensively controlling the quality of gecko formula granules is characterized by comprising the following steps: the method comprises the following steps:

(1) Content determination and characteristic spectrum detection for preparing a test solution: taking 0.2 g-1.0 g of gecko formula particles, grinding, adding an organic solvent for extraction, and taking a subsequent filtrate to obtain a test solution;

(2) Content determination and preparation of a characteristic spectrum detection reference substance solution: 0.5 g-2 g of gecko reference medicinal material is taken, 30% -70% methanol is added for extraction, and subsequent filtrate is taken as reference substance solution of the reference medicinal material; dissolving appropriate amount of tyrosine control substance and tryptophan control substance in water or methanol water solution to obtain reference substance solution;

(3) Content determination and characteristic spectrum detection methodology verification: the analysis method is verified in the aspects of preparation method, linearity, precision, accuracy, specificity and durability;

(4) The content determination and characteristic spectrum detection method comprises the following steps: respectively sucking reference substance solution and test solution 0.5-2 μ l, and measuring;

(5) Preparing a thin-layer identification test solution: taking 0.2 g-1.0 g of gecko formula granules, adding an organic solvent for extraction, and taking subsequent filtrate to obtain a test solution;

(6) Preparation of thin-layer identification reference solution: taking 0.5 g-2 g of gecko reference medicinal material, adding an organic solvent for extraction, and taking subsequent filtrate to obtain reference solution of the reference medicinal material;

(7) Verification of thin-layer identification methodology: verifying the preparation method, specificity and durability of the analysis method;

(8) The thin layer identification and detection method comprises the following steps: respectively sucking reference substance solution and sample solution, spotting on silica gel thin layer plate, developing, air drying, and developing.

2. The method for controlling the quality of gecko formulation in a comprehensive manner as claimed in claim 1, wherein: the extraction solvent is water, methanol or 30-70% methanol water solution, and the extraction time is 10-45 minutes; the extraction volume is 10ml to 50ml.

3. The method for controlling the quality of gecko formulations according to claim 1, wherein: the reference substances and the concentrations are as follows: tyrosine: 20-60 mug/ml; tryptophan (tryptophan): 10-20 mu g/ml.

4. The method for controlling the quality of gecko formulations according to claim 1, wherein: the liquid phase chromatographic conditions are as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filler; acetonitrile is taken as a mobile phase A, 0.05-0.3% phosphoric acid solution is taken as a mobile phase B, and gradient elution is adopted; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 20-30 ℃; an ultraviolet detector is used.

5. The method for controlling the quality of gecko formulations according to claim 1, wherein: the gradient elutes as follows:

6. the method for controlling the quality of gecko formulation in a comprehensive manner as claimed in claim 1, wherein: the liquid chromatography conditions were: the gecko formula particle characteristic spectrum is characterized in that 7 characteristic peaks are presented in a chromatogram of a test solution and correspond to retention time of 7 characteristic peaks in a chromatogram of a reference substance of a reference medicinal material, wherein peaks 4 and 7 are consistent with retention time of reference substances of a tyrosine reference substance and a tryptophan reference substance, a peak corresponding to the tyrosine reference substance is an S peak, relative retention time of peaks 1, 2, 3, 5 and 6 and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.48 (Peak 1), 0.56 (Peak 2), 0.74 (Peak 3), 1.37 (Peak 5), 1.50 (Peak 6).

7. The method for controlling the quality of gecko formulation in a comprehensive manner as claimed in claim 1, wherein: the thin layer plate is silica gel G, the developing agent is n-butanol-glacial acetic acid-water (4: 1), and the color-developing agent is ninhydrin test solution.

8. The method for controlling the quality of gecko formulation in a comprehensive manner as claimed in claim 1, wherein: the method is suitable for gecko medicinal materials, decoction pieces, processed products, preparation intermediates or preparation finished products thereof.

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