CN117783408B

CN117783408B - Quality control method for fig formula particles

Info

Publication number: CN117783408B
Application number: CN202311843208.5A
Authority: CN
Inventors: 吴美兰; 孟祥颖; 陈剑; 孙立恩
Original assignee: ZHEJIANG JINGYUETANG PHARMACEUTICAL CO Ltd
Current assignee: ZHEJIANG JINGYUETANG PHARMACEUTICAL CO Ltd
Priority date: 2023-12-28
Filing date: 2023-12-28
Publication date: 2024-05-14
Anticipated expiration: 2043-12-28
Also published as: CN117783408A

Abstract

The invention discloses a quality control method of fig formula particles, which comprises the steps of character, identification, characteristic spectrum, inspection, extract and rutin content determination; wherein the identification adopts thin layer chromatography, comprising the steps of: (1) preparation of a sample solution: taking 5g of fig formula particle sample, grinding, adding ethanol, carrying out ultrasonic treatment, filtering, adding potassium hydroxide-ethanol solution into filtrate, and saponifying at normal temperature; diluting the saponification liquid with water, extracting with diethyl ether for several times, collecting lower saponification water solution extracted each time, adjusting pH to 5.0, extracting with diethyl ether for several times, collecting diethyl ether solution extracted each time, evaporating to dryness, and dissolving residue with 1ml diethyl ether to obtain sample solution; (2) preparing a fig control medicinal material solution; (3) preparation of psoralen reference substance solution; (4) measurement. The method has better specificity, accuracy and precision, can realize effective control of the quality of compound particles, and ensures the medication safety.

Description

Quality control method for fig formula particles

Technical Field

The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality control method of fig formula particles.

Background

Regarding fig formula particles, a systematic quality detection method is not formed at present, and only the existing detection means are used for detecting the fig formula particles, so that the whole internal quality of the fig formula particles cannot be reflected, the quality control requirement of the traditional Chinese medicine formula particles cannot be met, and especially, the fig formula particles cannot be accurately identified due to poor specificity and durability when the existing detection means are used for carrying out thin-layer identification on the fig formula particles. Therefore, it is necessary to establish a quality control method for fig formula particles.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides the fig formula particle quality control method which has good specificity, accuracy and precision, can realize effective control of the quality of compound particles, and ensures the medication safety.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

A quality control method of fructus fici granule comprises the steps of character identification, characteristic spectrum detection, extract and rutin content determination; the extract is measured by a hot dipping method, and the characteristic spectrum and the rutin content are measured by a high performance liquid chromatography;

The identification adopts thin layer chromatography, comprising the following steps:

(1) Preparation of test solution: taking 5g of fig formula particle sample, grinding, adding ethanol, carrying out ultrasonic treatment, filtering, adding potassium hydroxide-ethanol solution into filtrate, and saponifying at normal temperature; diluting the saponification liquid with water, extracting with diethyl ether for several times, collecting lower saponification water solution extracted each time, adjusting pH to 5.0, extracting with diethyl ether for several times, collecting diethyl ether solution extracted each time, evaporating to dryness, and dissolving residue with 1ml diethyl ether to obtain sample solution;

(2) Preparation of control medicinal material solution: taking 1g of fig control medicinal material, and preparing a control medicinal material solution according to a preparation method of a sample solution;

(3) Preparation of a control solution: taking psoralen reference substance, adding ethyl acetate to prepare a solution containing 1mg per 1ml, and taking the solution as reference substance solution;

(4) And (3) measuring: sucking 10 μl of the sample solution, 5 μl of the control medicinal material solution and 5 μl of the control medicinal material solution, and respectively spotting on the same silica gel G thin layer plate according to the volume ratio of 9:1, using toluene-acetone as developing agent, developing, taking out, airing, and placing under 365nm ultraviolet lamp for inspection.

In the identification of the thin layer chromatography, the mass concentration of the potassium hydroxide-ethanol solution is 10%; the amount of potassium hydroxide-ethanol solution was 25ml.

The feature map determination comprises the following steps:

(1) Preparation of control drug reference solution: taking 0.5g of fig control medicine, placing the fig control medicine into a conical bottle with a plug, adding 25ml of 50v/v% methanol solution, sealing, weighing, refluxing in a water bath for 60 minutes, taking out, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, filtering, and taking the subsequent filtrate as a control medicine reference substance solution;

(2) Preparation of a reference solution for a control: adding methanol into chlorogenic acid reference substance to obtain solution containing 40 μg per 1ml, and taking the solution as chlorogenic acid reference substance solution; adding methanol into rutin reference substance to obtain solution containing 25 μg per 1ml, and taking the solution as rutin reference substance solution;

(3) Preparation of test solution: taking 1g of fig formula particles, grinding, placing into a conical flask with a plug, adding 25ml of 50v/v% methanol solution, weighing, performing ultrasonic treatment, cooling, weighing again, supplementing the reduced weight with 50v/v% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the fig formula particles;

(4) And (3) measuring: respectively taking reference substance solution of reference medicine, reference substance solution of chlorogenic acid and rutin, and reference substance solution of reference substance and test substance solution of 10 μl, and injecting into liquid chromatograph for measurement.

The chromatographic conditions for the feature map determination are as follows: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, and 0.1v/v% phosphoric acid is taken as a mobile phase B; gradient elution, gradient conditions are:

the flow rate is 0.8mL/min; the column temperature is 30 ℃; the detection wavelength was 354nm.

The rutin content detection specifically comprises the following steps:

(1) Preparation of a control solution: taking rutin reference substance, adding methanol to prepare a solution containing 25 mug per 1 ml;

(2) Preparation of test solution: taking 1g of fig formula particles, grinding, adding 25ml of 50v/v% methanol solution, weighing, carrying out ultrasonic treatment, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the fig formula particle;

(3) And (3) measuring: respectively sucking 10 μl of the reference solution and 10 μl of the sample solution, and injecting into a liquid chromatograph for measurement, measuring peak area, and calculating rutin content.

The chromatographic conditions for measuring the rutin content are as follows: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, and 0.1v/v% phosphoric acid is taken as a mobile phase B; gradient elution, gradient conditions are:

The checking method comprises the following steps: and (3) according to the rule of 0104 granules in the rule of 2020 edition of Chinese pharmacopoeia, checking the moisture, granularity and loading difference of the fig formula granule sample and the microbial limit.

The color and shape of the sample were visually observed at a bright place of the indoor light, and the sample was smelled and tasted.

The fig formula particle is prepared by the following steps: decocting fructus fici decoction pieces with water for 2 times, wherein the first time is added with 12 times of water, soaking for 30min, decocting for 30min, and the second time is added with 10 times of water, and decocting for 20 min; filtering, mixing the filtrates, concentrating under reduced pressure to relative density of 1.05-1.15, adding adjuvants, mixing, spray drying, adding adjuvants, mixing, and granulating.

The beneficial effects of the invention are as follows:

(1) The fig formula particles are saponified by the sodium hydroxide ethanol solution at normal temperature, so that the required test solution is prepared, and the thin-layer chromatography identification is performed, so that the defects of the existing identification method, such as specialization, weak temperature and humidity durability, are overcome, and the fig formula particles can be accurately identified.

(2) The quality control method for combining the characteristics, identifying, characteristic spectrum, checking, and measuring the contents of extract and rutin is constructed, has stronger specificity and precision, realizes the effective control of the quality of compound particles, and can ensure the safety and effectiveness of medication.

Drawings

FIG. 1 is an appearance of three batches of fig formula particle samples;

FIG. 2 is a chromatogram obtained to verify the specificity of the thin-layer identification method of fig formula particles;

FIG. 3 is a chromatogram obtained to verify durability of the fig formula particle thin layer identification method at 4 ℃;

FIG. 4 is a chromatogram obtained to verify the durability of the fig formula particle thin layer identification method at 30 ℃;

FIG. 5 is a chromatogram obtained to verify the durability of the fig formula particle thin layer identification method at 35% humidity;

FIG. 6 is a chromatogram obtained to verify the durability of the fig formula particle thin layer identification method at 75% humidity;

FIG. 7 is a chromatogram obtained from a thin layer panel from Qingdao ocean chemical Co., ltd for performing a thin layer identification test of fig formula particles;

FIG. 8 is a chromatogram obtained by performing a thin layer identification test of fig formula particles with a thin layer plate purchased from Qingdao silicon technologies Co., ltd;

FIG. 9 is a chromatogram obtained from a thin layer identification test of fig formula particles with a thin layer plate from Qingdan Yonglai silica gel limited;

FIG. 10 is a thin layer identification test chromatogram of a fig formula particle sample;

FIG. 11 is a liquid chromatogram of rutin control obtained by verifying the positioning and system applicability of the fig formula granule rutin content determination method;

FIG. 12 is a liquid chromatogram of a test sample obtained to verify the positioning and system applicability of the fig formula particle rutin content determination method;

FIG. 13 is a liquid chromatogram of a blank auxiliary material obtained by verifying the influence of the auxiliary material on the rutin content measurement of the fig formula particles;

FIG. 14 is a liquid chromatogram obtained by personnel a performing an intermediate precision test of the fig formula particle rutin content determination method;

FIG. 15 is a liquid chromatogram obtained by personnel b performing an intermediate precision test of the fig formula granule rutin content determination method;

FIG. 16 is a liquid chromatogram obtained by personnel c performing an intermediate precision test of the fig formula particle rutin content determination method;

FIG. 17 is a liquid chromatogram of a sample obtained by performing a chromatographic column durability test under the condition of measuring rutin content of fig formula particles at a column temperature of 30 ℃;

FIG. 18 is a liquid chromatogram of a sample obtained by performing a chromatographic column durability test under the condition of measuring rutin content of fig formula particles at a column temperature of 25 ℃;

FIG. 19 is a liquid chromatogram of a test sample obtained by performing a chromatographic column durability test under the condition of measuring rutin content of fig formula particles at a column temperature of 35 ℃;

FIG. 20 is a liquid chromatogram of a test sample obtained by performing a column durability test under the condition of measuring rutin content of fig formula particles at a flow rate of 0.7 ml/min;

FIG. 21 is a liquid chromatogram of a test sample obtained by performing a column durability test under the condition of measuring rutin content of fig formula particles at a flow rate of 0.9 ml/min;

FIG. 22 is a liquid chromatogram of a test sample obtained by performing a chromatographic column durability test under the condition of wavelength 349nm and measuring rutin content of fig formula particles;

FIG. 23 is a liquid chromatogram of a test sample obtained by performing a chromatographic column durability test under the condition of measuring rutin content of fig formula particles at a wavelength of 359 nm;

FIG. 24 is a liquid chromatogram of a test sample obtained by performing a chromatographic column durability test under the condition of IntertSustain C chromatographic column for measuring rutin content in fig formula particles;

FIG. 25 is a liquid chromatogram of a test sample obtained by performing a chromatographic column durability test under the condition of UltimateAQ C chromatographic column for measuring rutin content in fig formula particles;

FIG. 26 is a liquid chromatogram of a test sample obtained by performing a column durability test under the condition of measuring rutin content of Ficus carica formula particles by using a Shim-packVP-ODS column;

FIG. 27 is a liquid chromatogram of a fig control drug obtained by verifying the positioning and system applicability of the fig formulation particle characterization spectrogram determination method;

FIG. 28 is a liquid chromatogram of chlorogenic acid control obtained by verifying the positioning and system applicability of the fig formula particle feature spectrogram determination method;

FIG. 29 is a liquid chromatogram of rutin control obtained by verifying the positioning and system applicability of the fig formula particle feature spectrogram determination method;

FIG. 30 is a liquid chromatogram of a test sample obtained to verify the positioning and system applicability of the fig formula particle feature spectrogram determination method;

Fig. 31 is a liquid chromatogram of a blank adjuvant obtained by verifying the specificity of the fig formula particle feature spectrogram determination method.

Detailed Description

The invention is further described with reference to the drawings and detailed description which follow:

the preparation method of the fig formula granule comprises the steps of taking fig decoction pieces, adding water for decoction for 2 times (12 times of water is added for the first time, soaking is carried out for 30 minutes, decoction is carried out for 30 minutes, 10 times of water is added for the second time, decoction is carried out for 20 minutes), filtering, merging filtrate, concentrating under reduced pressure until the relative density is 1.05-1.15, adding auxiliary materials, uniformly mixing, spray-drying, adding auxiliary materials again, uniformly mixing, and granulating.

The preparation method comprises the following steps: (1) extraction and concentration: adding fig decoction pieces into an extraction tank, adding 12 times of water, soaking, starting hot steam heating, decocting for 30 minutes for the first time, adding 10 times of water for decocting for 20 minutes for the second time, filtering decoction by a 300-mesh filter screen, combining the two filtrates to obtain an extract, and concentrating the extract under reduced pressure until the relative density is 1.05-1.15 (measured at 55 ℃), thus obtaining an extract. (2) spray drying: weighing appropriate amount of silicon dioxide and maltodextrin, dissolving in water, and mixing the solution with the extract. Setting the air inlet temperature to 180 ℃, controlling the air inlet temperature to 170-190 ℃ and the air outlet temperature to 70-95 ℃, and performing spray drying to obtain extract powder. (3) total mixing: and weighing a proper amount of maltodextrin, and mixing with the extract powder to obtain total mixed powder. (4) granulating: and (3) taking the total mixed powder, and granulating by a dry method to obtain the fig formula granules. And (5) filling the prepared granules into a medicinal low-density polyethylene bag for sealing for later use.

Three batches of fig formula particles are prepared according to the method, and the batch numbers are YK20231001, YK20231002 and YK20231003 respectively. The first batch of fig formula particles comprises the following formula: 30.0kg of fig decoction pieces, 64.4g of silicon dioxide during spray drying, 1288.2g of maltodextrin and 2206.4g of maltodextrin during total mixing. The formula of the second batch of fig formula particles is as follows: 30.0kg of fig decoction pieces, 60.5g of silicon dioxide during spray drying, 1210g of maltodextrin and 2679.5g of maltodextrin during total mixing. The formula of the third batch of fig formula particles is as follows: 30.0kg of fig decoction pieces, 56.1g of silicon dioxide during spray drying, 1122.7g of maltodextrin and 3207.7g of maltodextrin during total mixing.

1. Determination of Properties

Taking one part of each of the three batches of fig formula particles, visually observing the color and shape of a sample at a bright indoor light place, smelling the smell of the sample, and taking a small amount of the smell. The property detection results of the three batches of samples are shown in table 1, and the particle appearance is shown in fig. 1. The fig standard decoction is brown to yellowish brown powder, has slight smell and slightly bitter taste, and is prepared into brown to yellowish brown fig formula particles according to the characteristic observation of three batches of samples and combining the research and detection results; light smell, sweet taste.

Table 1 fig formulation granule properties

2. Thin layer authentication

The thin-layer chromatography identification method of the fig formula particle comprises the following steps:

2.1 preparation of test solutions: taking 5g of fig formula particles, grinding, adding 25ml of ethanol, carrying out ultrasonic treatment for 30min, filtering, adding 25ml of potassium hydroxide-ethanol solution with the mass concentration of 10% into the filtrate, and saponifying at normal temperature. Diluting the saponification liquid with 30ml of water, extracting with diethyl ether three times, 20ml each time, collecting the lower saponification aqueous solution extracted each time, adjusting ph to 5.0 with dilute hydrochloric acid, extracting with diethyl ether twice, 30ml each time, collecting diethyl ether solution extracted each time, evaporating to dryness, and dissolving the residue with 1ml diethyl ether to obtain a sample solution.

2.2 Preparation of control drug solution: taking fig reference medicinal material (Shidand standard technical service Co., ltd., batch number: TCMR 009401) 1g, and preparing into reference medicinal material solution according to the preparation method of the sample solution.

2.3 Preparation of control solution: taking psoralen reference substance (batch No. 110739-201918, content 99.6%) and adding ethyl acetate to prepare 1mg solution per 1ml as reference substance solution.

2.4 Determination: according to thin layer chromatography (0502 of Chinese pharmacopoeia 2020 edition), sucking 10 μl of sample solution, 5 μl of control medicinal material solution and 5 μl of control solution, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone (volume ratio of 9:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the sample chromatogram, fluorescent spots with the same color should be displayed at the positions corresponding to the control chromatogram and the control chromatogram.

The method for identifying the fig formula particles by thin-layer chromatography is verified by the method, and the method is proved to have specificity and good durability. The specific test is as follows:

(a) Specificity test

A sample of the fig formula particle (lot number: YK 20231001) was taken and prepared as a test solution according to the method under item "2.1". Taking fig control medicinal material and controlling the solution according to the method of '2.2'. Taking psoralen reference substance and comparing the reference substance solution according to the method of item 2.3. Taking silicon dioxide and maltodextrin to prepare blank auxiliary material solution according to the method of '2.1'. Absorbing 10 μl of blank auxiliary material solution and test material solution, 5 μl of control medicinal material solution and 5 μl of control material solution, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone (9:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The result shows that fluorescent spots with the same color appear in the chromatogram of the test sample at the positions corresponding to the chromatograms of the control medicinal materials and the reference substance; the method has specificity as shown by no fluorescence spots in the blank auxiliary material chromatogram at the positions corresponding to the control medicinal material chromatogram and the control substance chromatogram. The related lamellar diagram is shown in figure 2 (in the figure, the sample YK20231001, blank auxiliary material, fig control medicinal material and psoralen control material are sequentially shown from left to right).

(B) Durability test

B1, temperature durability

Taking a fig formula particle sample (batch number: YK 20231001) and preparing a test solution according to the method under the item "2.1", taking fig control medicinal material and preparing a control medicinal material solution according to the method under the item "2.2", taking psoralen control material and preparing a control solution according to the method under the item "2.3". Respectively sucking 10 μl, 5 μl and 5 μl of the above three solutions, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone (9:1) as spreading agent at 4deg.C and 30deg.C, taking out, air drying, and inspecting with ultraviolet lamp (365 nm). The results show that fluorescent spots with the same color appear in the chromatogram of the test sample at positions corresponding to the chromatograms of the control medicinal materials and the reference substance. The related chromatograms are shown in figure 3 (YK 20231001, fig control drug and psoralen control in sequence from left to right in the figure), and figure 4 (YK 20231001, fig control drug and psoralen control in sequence from left to right in the figure).

B2, humidity durability

Taking the sample solution under the item b1, the fig control medicinal material solution and the psoralen control substance solution, respectively sucking 10 μl, 5 μl and 5 μl of the three solutions under the environments of 35% and 75% of humidity, respectively spotting on a same silica gel G thin layer plate, using toluene-acetone (9:1) as developing agent, developing, taking out, airing, and putting under an ultraviolet lamp (365 nm) for detection. The results show that fluorescent spots with the same color appear in the chromatogram of the test sample at positions corresponding to the chromatograms of the control medicinal materials and the reference substance. The related chromatograms are shown in figure 5 (YK 20231001, fig control medicine and psoralen control in sequence from left to right in the figure), and figure 6 (YK 20231001, fig control medicine and psoralen control in sequence from left to right in the figure).

B3, thin layer plate of different manufacturer

Taking the sample solution, the fig control medicinal material solution and the psoralen control substance solution under the item b1, respectively sucking 10 μl, 5 μl and 5 μl of the three solutions, respectively spotting on silica gel G thin layer plates of three different manufacturers, using toluene-acetone (9:1) as developing agent, developing, taking out, airing, and placing under an ultraviolet lamp (365 nm) for detection. The results show that fluorescent spots with the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the test sample chromatogram. The chromatograms are shown in FIG. 7 (YK 20231001, fig control medicine and psoralen control substance are sequentially purchased from Qingdao ocean chemical Co., ltd. From left to right in the drawing), FIG. 8 (YK 20231001, fig control medicine and psoralen control substance are sequentially purchased from left to right in the drawing), and FIG. 9 (YK 20231001, fig control medicine and psoralen control substance are sequentially purchased from left to right in the drawing), and the silica gel G thin layer plate is purchased from Qingdao Xin Yi Lai silica gel Co., ltd. From Qingdao).

Sample detection: according to the thin-layer chromatography identification method, three batches of fig formula particle samples are taken for identification, the result is shown in table 2, and the related chromatogram is shown in figure 10 (YK 20231001, YK20231002, YK20231003, fig control medicinal material and rutin control in sequence from left to right in the figure).

Table 2 fig formula particle identification

3. Inspection of

And (3) according to the rule of 0104 granules in the rule of the edition 2020 of Chinese pharmacopoeia, checking the moisture, granularity, loading difference and microorganism limit of 3 batches of fig formula granule samples. The test results are shown in Table 3.

Table 3 table of inspection results for fig formula particles

4. Determination of extract

Three batches of fig formula particles (batch numbers: YK20231001, YK20231002, YK 20231003) were measured according to the hot dipping method under the condition of alcohol-soluble extract measurement method (general rule 2201 of Chinese pharmacopoeia 2020 edition) and ethanol was used as a solvent, and the results are shown in Table 4.

Table 4 table of fig formula granule extract results

The preparation of the limit of the extract of the fig formula granule is based on the following steps: the extract limit of the fig standard decoction is 'calculated as decoction pieces, and is not less than 6.9 percent'. In order to ensure the consistency of the quality of the prescription granule and the quality of the standard decoction, the specification of the product is that each 1 gram of fig prescription granule is equivalent to 3.0 grams of decoction pieces, and the limit of the extract of the product is formulated as 'not less than 20.7 percent'. The detection results of the three batches of sample extracts all meet the regulations.

5. Content determination

The method for detecting the rutin content in the fig formula particle comprises the following steps:

5.1 preparation of control solution: taking rutin reference substance (supplied by Chinese food and drug verification institute, batch number 100080-202012, content 91.6%), adding methanol to make into solution containing 25 μg per 1 ml;

5.2 preparation of sample solution: taking 1g of fig formula particles, grinding, placing into a conical bottle with a plug, precisely adding 25ml of 50v/v% methanol solution, weighing, performing ultrasonic treatment (power is 250W, frequency is 50 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the fig beverage;

5.3 measurement: respectively sucking 10 μl of the reference solution and 10 μl of the sample solution, and injecting into a liquid chromatograph for measurement, measuring peak area, and calculating rutin content.

The chromatographic conditions were as follows: chromatographic column: inertSustain C18.6mm.times.250 mm,5 μm; mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, and eluting according to the gradient specified in Table 5; detection wavelength: 354nm; flow rate: 0.8ml/min; column temperature: 30 ℃; sample injection amount: 10 μl.

TABLE 5

The invention examines the influence of the test sample solutions prepared under the conditions of different extraction solvents, different feed-liquid ratios and different extraction modes on the detection result of the rutin content in the fig formula particles, and the result is shown in the table 7, and finally the optimal test sample solution preparation method as described in the item 5.2 is determined.

The method for preparing the sample solution under the condition of different extraction solvents comprises the following steps: grinding 3 parts of fig formula particles (batch number: YK 20231001) into 0.5g, precisely weighing, placing into a conical bottle with a plug, precisely adding water, 50v/v% methanol and 25ml of methanol respectively, weighing, performing ultrasonic treatment (power 250W and frequency 50 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with corresponding solvent, shaking, filtering, and collecting the subsequent filtrate.

The method for preparing the sample solution under the condition of different feed liquid ratios comprises the following steps: grinding the above materials into powder (batch number: YK 20231001) 0.25g, 0.5g and 1g, precisely weighing, placing into conical flask with plug, precisely adding 50v/v% methanol 25ml, weighing, ultrasonic treating (power 250W, frequency 50 kHz) for 30min, cooling, weighing, supplementing the lost weight with 50v/v% methanol, shaking, filtering, and collecting filtrate.

The method for preparing the sample solution under the conditions of different extraction modes comprises the following steps: taking 3 parts of ground powder (batch number: YK 20231001) 1g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 50v/v% methanol, weighing, respectively heating and refluxing for 30 minutes, performing ultrasonic treatment (power 250W, frequency 50 kHz) for 45 minutes, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, shaking, filtering, and collecting the subsequent filtrate.

TABLE 6 sample extraction conditions investigation results table

The invention also carries out methodological verification on the method for detecting the rutin content in the fig formula particles. The specific test is as follows:

(A) Positioning and system applicability test

A1, taking a rutin reference substance, precisely weighing 13.94mg, placing in a 50ml volumetric flask, adding a proper amount of methanol for ultrasonic dissolution, fixing the volume to a scale, and shaking uniformly to prepare a rutin reference substance mother solution (255.4 mug/ml). The flask was precisely removed from 1ml to 10ml, the volume was fixed to the scale with methanol, and the flask was shaken well to prepare a control solution (25.54. Mu.g/ml).

A2, precisely weighing 1g of fig formula particle sample (batch number: YK 20231001), and preparing a test solution according to the method of item 5.2.

A3, precisely sucking 10 mu l of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and measuring the peak area.

The result shows that the retention time of rutin in the control solution chromatogram (figure 11) is 43.762min, the peak area is 586849, the theoretical plate number is 103853, and the tailing factor is 1.138; rutin retention time in the sample solution chromatogram (figure 12) is 43.731min, peak area is 311318, theoretical plate number is 98179, and tailing factor is 1.094; the system has good applicability.

(B) Blank auxiliary material

Precisely weighing 0.01g of silicon dioxide and 0.5g of maltodextrin, and preparing a test solution into a blank auxiliary material solution according to the method of item 5.2. Precisely sucking 10 μl, injecting into high performance liquid chromatograph, recording chromatogram, and observing whether the adjuvant has influence. The result shows that the auxiliary materials have no influence on the content measurement, and the related chromatogram is shown in figure 13.

(C) Precision of sample injection

And (3) precisely removing 10 μl of the control solution under item "A1", injecting into a high performance liquid chromatograph, recording a chromatogram, and measuring the peak area. RSD% was calculated by repeating 6 times. The results are shown in Table 7. The result shows that the method has good precision.

TABLE 7 sample injection precision test results

(D) Stability test

1G of fig formula particles (batch number: YK 20231001) is taken, precisely weighed and prepared into a test solution according to the method described in item 5.2. And precisely sucking 10 μl of the sample solution to be tested, injecting into a liquid chromatograph, sequentially injecting samples at 0, 3, 6, 9, 12, 18 and 24 hours, recording a chromatogram, and calculating the average value of the peak area of rutin and the RSD value. The results are shown in Table 8. The results show that the test solution has good stability within 24 hours.

TABLE 8 determination of test solutions stability test results for content

(E) Repeatability test

6 Parts of the same batch of fig formula particle samples (batch number: YK 20231003) are taken, 1g is precisely weighed, and a test solution is prepared according to the method described in item 5.2. Precisely sucking 10 μl of the sample solution, and injecting into a liquid chromatograph to calculate rutin content. The results are shown in Table 9. The result shows that the method has good repeatability.

TABLE 9 results of repeatability test of content determination method

(F) Sample addition recovery test

6 Conical flasks with plugs are taken, 0.6ml of reference substance mother liquor (255.4 mu g/ml) under the item "A1" is respectively and precisely removed, the mixture is added into the conical flasks, volatilized, six parts of fig formula particle samples (batch number: YK20231001, content of 0.34 mg/g) are precisely weighed, each part of fig formula particle samples is 0.5g, 25ml of 50v/v% methanol is precisely added into the conical flasks with plugs, the mixture is closely plugged, the mixture is weighed, the mixture is subjected to ultrasonic treatment for 30 minutes, cooled and weighed, the reduced weight is complemented by 50v/v% methanol, the mixture is uniformly shaken, filtered, and subsequent filtrate is taken as a sample solution.

10 Μl of each of the above sample solutions was precisely sucked and injected into a high performance liquid chromatograph, the chromatogram was recorded, the peak area was measured, and the recovery rate was calculated according to the following formula, and the results are shown in Table 10. The results show that the sample recovery rate of the six tests is between 90 and 108 percent, the average value is 101.5 percent, and the RSD is 1.43 percent, which shows that the recovery rate test results are good.

Table 10 results of rutin recovery test

(G) Intermediate precision test

The same batch of fig granule samples (batch number: YK 20231001) were tested by different personnel, different dates and different instruments, processed according to the law, and the content thereof was measured, and the results are shown in Table 11. The results show that the intermediate precision result of the method is good. The relevant chromatograms are shown in fig. 14-16.

TABLE 11 results of intermediate precision test

(H) Durability test

H1 column temperature durability

According to the prescribed chromatographic conditions, changing column temperature + -5deg.C, taking fig granule sample (batch number: YK 20231001), preparing into test solution according to the method described in item "5.2", and measuring rutin content. The results are shown in Table 12 below.

Table 12 results of column temperature durability test of content measuring method

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H2 flow rate durability

According to the prescribed chromatographic conditions, the flow rate is changed to + -0.1 ml/min, the sample (batch number: YK 20231001) is prepared into a sample solution according to the method under item "5.2", and the rutin content is measured. The results are shown in Table 13 below.

Table 13 results of flow durability test for content determination method

H3 wavelength durability

According to the prescribed chromatographic conditions, the wavelength was changed to + -5 nm, the sample (lot number: YK 20231001) was prepared into a sample solution according to the method under item "5.2", and the rutin content was measured. The results are shown in Table 14 below.

Table 14 results of wavelength durability test of content measuring method

H4 column durability

Selecting different chromatographic columns according to specified chromatographic conditions, sampling (batch number: YK 20231001), preparing into sample solution according to the method under item "5.2", and measuring rutin content. The results are shown in Table 15 below.

TABLE 15 chromatographic column test results

The results of the durability test show that the column temperature, the flow rate, the wavelength and the durability of the chromatographic column of the method all meet the requirements of content measurement. The relevant chromatograms are shown in figures 17-26.

Sample detection: according to the method for detecting the rutin content by the high performance liquid chromatography, 3 batches of fig formula particles (YK 20231001, YK20231002 and YK 20231003) are taken to detect the rutin content, and the results are shown in Table 16. The limit of the rutin content in the fig standard decoction is 0.07-0.25 mg of rutin in the standard decoction corresponding to 1g of decoction pieces. In order to ensure the consistency of the quality of the prescription granule and the quality of the standard decoction, the specification of the product is that each 1 gram of fig prescription granule is equivalent to 3.0 grams of decoction pieces, and the content limit of the product is formulated as that the rutin content of each 1 gram of the product is 0.21 mg-0.75 mg.

Table 16 rutin content determination results of Ficus carica formula particles

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6. Feature map

The characteristic spectrum measuring method of the fig formula particle comprises the following steps:

6.1 preparation of control drug reference solution: taking 0.5g of fig control medicinal material (Shiadad standard technical service Co., ltd., TCMR 009401), placing into a conical flask with a plug, adding 25ml of 50v/v% methanol, sealing, weighing, refluxing in water bath for 60 minutes, taking out, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, filtering, and taking the subsequent filtrate as reference substance solution of the control medicinal material.

6.2 Preparation of reference solution for reference: taking chlorogenic acid reference substance (batch No. 110753-202119, content 96.3%) from Chinese food and drug inspection institute, precisely weighing, adding methanol to obtain solution containing 40 μg per 1ml, and taking the solution as chlorogenic acid reference substance solution; the rutin reference substance solution is prepared according to the preparation method of the reference substance solution under the item of 5 and rutin content.

6.3 Preparation of test sample solution: grinding Ficus carica formula particles into fine powder 1g, precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of 50v/v% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 50 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, shaking uniformly, filtering, and collecting subsequent filtrate.

6.4 Measurement: precisely sucking 10 μl of reference substance solution of reference substance of chlorogenic acid, reference substance solution of reference substance of rutin and reference substance solution of test substance, and injecting into liquid chromatograph for measurement. The chromatographic conditions were as follows:

Chromatographic column: inertSustain C18.6mm.times.250 mm,5 μm; octadecylsilane chemically bonded silica is used as a filler; detection wavelength: 354nm; flow rate: 0.8ml/min; column temperature: 30 ℃; sample injection amount: 10 μl; mobile phase: elution was performed with acetonitrile as mobile phase A and 0.1v/v% phosphoric acid as mobile phase B, with a gradient as specified in Table 17.

TABLE 17

The invention verifies the characteristic spectrum measuring method of the fig formula particle, and the specific test is as follows:

s1, positioning and system applicability

1) Taking 0.5g of fig control medicine, placing the fig control medicine into a conical bottle with a plug, adding 25ml of 50v/v% methanol, sealing, weighing, refluxing in a water bath for 60 minutes, taking out, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, filtering, and taking the subsequent filtrate as a control medicine reference substance solution.

2) Taking chlorogenic acid reference substance, precisely weighing 23.04mg, placing in a 50ml volumetric flask, adding a proper amount of methanol, carrying out ultrasonic dissolution, fixing the volume to a scale, shaking uniformly, preparing into chlorogenic acid reference substance mother liquor (443.8 mug/ml), precisely transferring chlorogenic acid mother liquor 1ml to 10ml volumetric flask, fixing the volume to the scale with methanol, shaking uniformly, preparing into chlorogenic acid reference substance solution (44.38 ug/ml), and preparing rutin reference substance solution (25.54 mug/ml) according to the method of A1 under the determination of rutin content, wherein the rutin reference substance solution is used as a rutin reference substance solution.

3) Accurately weighing fig formula particle samples (batch number: YK 20231001) 1g, placing into a conical flask with a plug, precisely adding 25ml of 50v/v% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 50 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50v/v% methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution.

4) Respectively precisely sucking reference solution of reference material, chlorogenic acid reference solution, rutin reference solution and test solution of 10 μl, respectively, and measuring by high performance liquid chromatograph, and recording chromatogram.

The result shows that chlorogenic acid retention time in the chromatogram of the reference solution of the reference medicinal material is 19.332min, theoretical plate number is 26128, and tailing factor is 1.230; rutin retention time is 43.667min, theoretical plate number is 108564, and tailing factor is 1.011. Chlorogenic acid retention time in the reference substance solution chromatogram of the reference substance is 19.286min, theoretical plate number is 26476, and tailing factor is 1.163; the retention time of rutin is 43.684min, the theoretical plate number is 112909, and the tailing factor is 1.045. Chlorogenic acid retention time in the chromatogram of the sample solution is 19.324min, theoretical plate number is 22345, and tailing factor is 1.253; rutin retention time is 43.660min, theoretical plate number is 106528, and tailing factor is 1.020. The system has good applicability, and the related chromatograms are shown in figures 27-30.

S2, specificity

Precisely weighing 0.01g of silicon dioxide and 0.5g of maltodextrin, and preparing a blank auxiliary material solution according to the method of preparing a 6.3 sample solution. Precisely sucking 10 μl, injecting into high performance liquid chromatograph, recording chromatogram, and observing whether the adjuvant has influence. The result shows that the auxiliary materials have no influence on the characteristic spectrum measurement. The relevant chromatogram is shown in FIG. 31.

S3, precision test

1G of a sample (batch number: YK 20231001) was precisely weighed, and a sample solution was prepared according to the method of "preparation of a 6.3 sample solution". And precisely sucking 10 μl of the sample solution, injecting into a high performance liquid chromatograph, repeating for 6 times, taking the peak corresponding to the rutin reference peak as an S peak, and calculating the relative retention time and RSD value of the peak 1, the peak 2, the peak 3, the peak 5, the peak 6 and the S peak, wherein the result is shown in Table 18.

Table 18 precision versus retention time results table

The result shows that the method has good precision.

S4, stability test

1G of a sample (batch number: YK 20231001) was precisely weighed, and a sample solution was prepared according to the method of "preparation of a 6.3 sample solution". 10 mu l of the sample solution is precisely sucked at 0, 3, 6, 9, 12, 18 and 24 hours, the sample solution is respectively injected into a high performance liquid chromatograph, the peak corresponding to the rutin reference peak is taken as an S peak, and the relative retention time and RSD value of the peak 1, the peak 2, the peak 3, the peak 5, the peak 6 and the S peak are calculated, and the result is shown in a table 19.

Table 19 stability versus retention time results table

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

S5, repeatability test

6 Parts of the same sample (batch number: YK 20231001) were precisely weighed, and a sample solution was prepared according to the method "preparation of 6.3 sample solution". And precisely sucking 10 μl of the sample solution, injecting into a high performance liquid chromatograph, recording the retention time of the common peak, taking the peak corresponding to the rutin reference peak as an S peak, and calculating the relative retention time and RSD value of the peak 1, the peak 2, the peak 3, the peak 5, the peak 6 and the S peak, wherein the result is shown in Table 20.

Table 20 table of repeatability versus retention time results

The results show that the method has good repeatability.

S6, intermediate precision test

The same batch of samples (batch number: YK 20231001) was tested by different personnel, different dates and different instruments, and specific experimental parameters are shown in Table 21. The characteristic spectrum of the sample was examined, and the relative retention time of each peak was calculated, and the results are shown in Table 22.

Table 21 intermediate precision experimental parameters

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Table 22 relative retention time of intermediate precision test feature patterns

The results show that the intermediate precision result of the method is good. The relevant chromatograms are shown in fig. 14-16.

S7, durability test

A) Column temperature durability

According to the prescribed chromatographic conditions, column temperature.+ -. 5 ℃ was changed, samples (lot number: YK 20231001) were taken, the sample solution was prepared according to the method under "preparation of 6.3 sample solution", 10. Mu.l of the sample solution was precisely sucked up, and the samples were injected into a high performance liquid chromatograph, the total peak retention time was recorded, and the relative retention time and RSD values of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak were calculated with respect to the peak of rutin reference as the S peak, and the results are shown in Table 23.

Table 23 results of column temperature durability test by the feature map method

B) Flow rate durability

According to the prescribed chromatographic conditions, the flow rate.+ -. 0.1ml/min was changed, the sample (lot number: YK 20231001), the sample solution was prepared according to the method under "preparation of 6.3 sample solution", 10. Mu.l of the sample solution was precisely sucked up, and the sample solution was injected into a high performance liquid chromatograph, the retention time of the common peak was recorded, and the relative retention time and RSD values of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak were calculated with respect to the peak of rutin reference as the S peak, and the results are shown in Table 24.

Table 24 results of the characteristic spectrum method flow velocity durability test

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C) Wavelength durability

According to the prescribed chromatographic conditions, the wavelength was changed to.+ -.5 nm, samples (lot number: YK 20231001), the sample solution was prepared according to the method under "preparation of sample solution 6.3", 10. Mu.l of the sample solution was precisely sucked up, and the sample solution was injected into a high performance liquid chromatograph, the total peak retention time was recorded, and the relative retention time and RSD values of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak were calculated with respect to the peak of rutin reference as the S peak, and the results are shown in Table 25.

Table 25 results of the characteristic spectrum method wavelength durability test

D) Column durability

According to the prescribed chromatographic conditions, different types of chromatographic columns are selected, samples (batch number: YK 20231001) are taken, a sample solution is prepared according to the method under the item of 'preparation of 6.3 sample solution', 10 μl of the sample solution is precisely sucked, the sample solution is injected into a high performance liquid chromatograph, the total peak retention time is recorded, the peak corresponding to the rutin reference peak is taken as an S peak, and the relative retention time and RSD value of the peak 1, the peak 2, the peak 3, the peak 5, the peak 6 and the S peak are calculated, and the result is shown in a table 26.

Table 26 characterization method column durability test results

The results of the durability test show that the flow rate, the wavelength and the durability of the chromatographic column of the method all meet the requirements of characteristic spectrum research; in the durability at column temperature, peak 2 and peak 3 in the chromatogram at 25℃are combined, so it is recommended that the column temperature be 30 ℃. The relevant chromatograms are shown in figures 17-26.

Sample detection:

The relative retention time of each characteristic peak of the characteristic spectrum according to the specified fig formula particle is within +/-10% of the specified value. Calculating the relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak by taking the peak corresponding to the rutin reference object peak as the S peak, wherein the relative retention time specified values of the characteristic peaks are respectively as follows: 0.45 (Peak 1), 0.74 (Peak 2), 0.76 (Peak 3), 1.07 (Peak 5), 1.20 (Peak 6).

3 Batches of fig formula particles (batch numbers: YK20231001, YK20231002 and YK 20231003) were taken, and the characteristic spectrum was measured according to the above method, and the results are shown in Table 27.

Table 27 relative retention time of fig formula particle profile

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A quality control method of fig formula particles is characterized by comprising a method for determining characters, identification, characteristic patterns, inspection, extract and rutin content; the extract is measured by a hot dipping method, and the characteristic spectrum and the rutin content are measured by a high performance liquid chromatography;

(4) And (3) measuring: sucking 10 μl of the sample solution, 5 μl of the control medicinal material solution and 5 μl of the control medicinal material solution, and respectively spotting on the same silica gel G thin layer plate according to the volume ratio of 9:1, using toluene-acetone as developing agent, developing, taking out, airing, and placing under 365nm ultraviolet lamp for inspection;

The feature map determination comprises the following steps:

(4) And (3) measuring: respectively taking reference substance solution of reference medicine, reference substance solution of chlorogenic acid and rutin, and reference substance solution of reference substance and sample solution of sample, respectively 10 μl, and injecting into liquid chromatograph for measurement;

the chromatographic conditions for the feature map determination are as follows: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, and 0.1v/v% phosphoric acid is taken as a mobile phase B; gradient elution;

The gradient conditions are:

the flow rate is 0.8mL/min; the column temperature is 30 ℃; the detection wavelength is 354nm;

The rutin content detection specifically comprises the following steps:

(3) And (3) measuring: respectively sucking 10 μl of the reference substance solution and 10 μl of the sample solution, injecting into a liquid chromatograph for measurement, measuring peak area, and calculating rutin content;

2. The quality control method of fig formula particles according to claim 1, wherein in the identification by thin layer chromatography, the mass concentration of potassium hydroxide-ethanol solution is 10%; the amount of potassium hydroxide-ethanol solution was 25ml.

3. The fig granule quality control method according to claim 1, wherein the inspection method comprises the following steps: and (3) according to the rule of 0104 granules in the rule of 2020 edition of Chinese pharmacopoeia, checking the moisture, granularity and loading difference of the fig formula granule sample and the microbial limit.

4. The fig granule quality control method according to claim 1, wherein the character detection step comprises: the color and shape of the sample were visually observed at a bright place of the indoor light, and the sample was smelled and tasted.

5. The fig formula particle quality control method according to claim 1, wherein the fig formula particle is prepared by the following steps: decocting fructus fici decoction pieces with water for 2 times, wherein the first time is added with 12 times of water, soaking for 30min, decocting for 30min, and the second time is added with 10 times of water, and decocting for 20 min; filtering, mixing the filtrates, concentrating under reduced pressure to relative density of 1.05-1.15, adding adjuvants, mixing, spray drying, adding adjuvants, mixing, and granulating.