CN114778739B - Method for detecting quality of fried chicken's gizzard-membrane standard decoction - Google Patents
Method for detecting quality of fried chicken's gizzard-membrane standard decoction Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a quality detection method of a fried chicken's gizzard-membrane standard decoction, which comprises the steps of determining the characters, dry extract yield, thin-layer identification, extract, characteristic spectrum and guanosine content of the fried chicken's gizzard-membrane standard decoction, limiting the content standard of the fried chicken's gizzard-membrane standard decoction to 0.2-1.1mg of guanosine in each 1g of the fried chicken's gizzard-membrane standard decoction, wherein the dry extract yield is determined by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the guanosine content are measured by liquid chromatography. The quality detection method of the fried chicken's gizzard-membrane standard decoction provided by the invention evaluates the quality of the fried chicken's gizzard-membrane standard decoction through multi-aspect measurement, lays a solid foundation for the stable quality of products, can establish feasible quality standards of the fried chicken's gizzard-membrane standard decoction, and realizes effective control of the quality of the fried chicken's gizzard-membrane standard decoction.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a method for detecting the quality of a standard decoction of fried chicken's gizzard-skin.
Background
The modern medicine needs to have three characteristics of stability, uniformity, safety and effectiveness, and the Chinese patent medicine is difficult to compare with western medicines in the aspects, so that the detection is more needed by adopting various means, and the reliability and the stability of the detection result are ensured. The chicken's gizzard membrane is the gizzard membrane of chicken of Phasianidae, and the fried chicken's gizzard membrane is the clean chicken's gizzard membrane, stir-fried according to the clear stir-frying or scalding method until the chicken's gizzard membrane is swelled, has sweet taste, is mainly used for invigorating stomach and promoting digestion, astringing essence and stopping enuresis, and is used for indigestion, vomiting and diarrhea, infantile malnutrition, enuresis and spermatorrhea. At present, a systematic quality detection method is not formed in the standard decoction of fried chicken's gizzard-membrane, and the traditional detection means are only adopted to detect the fried chicken's gizzard-membrane decoction, so that the quality control requirement of traditional Chinese medicine formula particles cannot be met. Therefore, it is necessary to establish a standard decoction quality detection method of fried chicken's gizzard-membrane for medicinal material quality control.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a method for detecting the quality of a standard decoction of fried chicken's gizzard-membrane so as to better control the quality of the decoction of fried chicken's gizzard-membrane, characterize the quality of the medicament and improve the stability of the medicament.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the invention provides a method for detecting the quality of a standard decoction of fried chicken's gizzard-membrane, which comprises the following detection methods,
determining the properties of the fried chicken's gizzard-membrane standard decoction, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the guanosine content, limiting the standard decoction content standard to 0.2-1.1mg of guanosine content per 1g, wherein the dry extract extraction rate is determined by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the guanosine content are measured by adopting a liquid chromatography;
the determination of the characteristic spectrum by liquid chromatography comprises: performing liquid chromatograph analysis, wherein the solution prepared from endothelium corneum Gigeriae Galli reference material is used as reference solution b, the solution prepared from guanosine reference material is used as reference solution b, the solution prepared from parched endothelium corneum Gigeriae Galli standard decoction sample is used as sample solution b, and the reference solution b, reference solution b and sample solution b are respectively and precisely absorbed, respectively injected into liquid chromatograph, and measured to obtain the final product; wherein the chromatographic conditions adopted are that: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um); mobile phase: acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table a;
Table a gradient elution procedure
Flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm.
In one embodiment, the decoction method comprises: soaking the fried chicken's gizzard-membrane decoction pieces in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid, concentrating, and drying to obtain dry extract powder of standard decoction of fried chicken's gizzard-membrane.
In one embodiment, the thin layer chromatography comprises the steps of:
(1) Preparing a test sample solution a: taking 1g of fried chicken's gizzard-membrane standard decoction sample, adding 20mL of ethanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1mL of a mixed solution of ethyl acetate and ethanol in a ratio of 2:1 into residues to dissolve to obtain a sample solution a;
(2) Preparing a control medicinal material solution a: taking 2g of chicken's gizzard-membrane reference medicine, adding 100mL of water, decocting and keeping micro boiling for 1h, cooling, filtering, evaporating filtrate to dryness, cooling, adding 20mL of ethanol into residues to dissolve, performing ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, adding 1mL of a mixed solution of ethyl acetate and ethanol in a ratio of 2:1 into residues to dissolve, and preparing a reference substance solution a;
(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: sucking 10 μl of the sample solution a and 5 μl of the control medicinal material solution a; developing agent: the volume ratio is 10:2:1 in chloroform-ethyl acetate-methanol; presaturation for 30min, and spreading distance of 11cm; and (5) checking: the image was taken at 365nm with an ultraviolet lamp.
In one embodiment, the hot dip method uses ethanol as a solvent and the range of the extract is determined by a hot dip method under the alcohol-soluble extract determination method.
In one embodiment, the determination of the characteristic spectrum by liquid chromatography further comprises the steps of:
(1) Preparation of reference solution b: taking 0.3g of chicken's gizzard-membrane reference medicine, adding 25mL of 10% ethanol, refluxing for 30min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as reference solution b;
(2) Preparing a reference substance solution b: taking a proper amount of guanosine reference substance, precisely weighing, adding 50% methanol for dissolving, and preparing a reference substance solution b with the concentration of 10 ug/mL;
(3) Preparing a test sample solution b: taking 0.3g of fried chicken's gizzard-membrane standard decoction sample, precisely weighing, placing into a conical flask with a plug, adding 25mL of precisely weighed 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.
In one embodiment, determining guanosine content using liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from guanosine reference substance as a reference substance solution c, taking a solution prepared from a fried chicken's gizzard-membrane standard decoction sample as a test substance solution c, precisely sucking the reference substance solution c and the test substance solution c respectively, injecting the reference substance solution c and the test substance solution c into the liquid chromatograph respectively, and measuring to obtain the medicine; wherein the chromatographic conditions adopted are that: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um); mobile phase: acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to a specified rule; flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm.
In one embodiment, the determination of guanosine content by liquid chromatography further comprises the steps of:
(1) Preparing a reference substance solution: taking a proper amount of guanosine reference substance, precisely weighing, adding 50% methanol to prepare a solution containing guanosine with the concentration of 10ug/ml, and taking the solution as a reference substance solution c;
(2) Preparing a test solution: taking about 0.3g of fried chicken's gizzard-membrane standard decoction sample, precisely weighing, placing the sample into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.
Compared with the prior art, the invention has the beneficial effects that:
(1) The quality of the fried chicken's gizzard-membrane standard decoction is evaluated through various measurements by researching the properties of the fried chicken's gizzard-membrane standard decoction, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the guanosine content measurement, a solid foundation is laid for the stable quality of the product, a feasible quality standard of the fried chicken's gizzard-membrane standard decoction can be established, the effective control of the quality of the fried chicken's gizzard-membrane standard decoction is realized, and in addition, by adopting the chromatographic conditions of the method, the liquid phase analysis is carried out, so that the chromatogram with better and clearer separation degree can be obtained.
(2) The fried chicken's gizzard-membrane decoction pieces are decocted to obtain a fried chicken's gizzard-membrane decoction piece standard decoction, the average content of guanosine is 0.64mg/g, the measured content range is 0.420-0.861 mg/g, the SD (standard deviation) is 0.142, calculated according to the mean value +/-3 SD, the allowable content range of guanosine is 0.21-1.07 mg/g, so the guanosine content range of the standard decoction is calculated as follows: 0.2 mg/g-1.1 mg/g; the average guanosine transfer rate is 84.14%, the transfer rate range is 61.72% -95.11%, the SD is 9.56, according to the technical requirements of quality control and standard establishment of traditional Chinese medicine formula particles, the allowable guanosine content transfer rate range is 58.9% -109.4% calculated by 70% -130% of the transfer rate average value, and is 55.5% -112.8% calculated by +/-3 SD, so that the guanosine content transfer rate range of the standard decoction is proposed to be: 55.5-100%, and the result shows that the guanosine content and the transfer rate of the standard decoction of a plurality of batches are all within the allowable range, so the invention can provide reference for the quality standard research of fried chicken's gizzard-skin formula particles.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a sample application amount investigation TLC chart of a standard decoction of fried chicken's gizzard-membrane decoction pieces according to a thin layer chromatography in an embodiment of the invention; wherein, the sample injection amount of the control medicinal material solution of the 1 group is 3 mu L, the sample injection amount of the control medicinal material solution of the 2 group is 5 mu L, the sample injection amount of the control medicinal material solution of the 3 group is 10 mu L, the sample injection amount of the sample solution of the 4 group is 3 mu L, the sample injection amount of the sample solution of the 5 group is 5 mu L, and the sample injection amount of the sample solution of the 6 group is 10 mu L.
FIG. 2 is a thin-layer diagram of a standard decoction of 15 batches of fried chicken's gizzard-membrane decoction pieces in an embodiment of the invention; wherein, the A group of patterns are negative control sample thin layer patterns, the S group is chicken 'S gizzard-membrane control medicinal material solution thin layer patterns, 1-15 groups are chicken' S gizzard-membrane stir-fried test articles 15 batches of standard decoction thin layer patterns, and 15 batches are 220301PT, 220302PT, 220303PT, Y220204 PT-Y2202015 PT.
FIG. 3 is a graph showing the comparison of different detection wavelengths in detection wavelength investigation of standard decoction of fried chicken's gizzard-membrane decoction pieces by thin layer chromatography; wherein the detection wavelength of S1 is 230nm, the detection wavelength of S2 is 210nm, and the detection wavelength of S3 is 254nm.
FIG. 4 is a graph showing the comparison of different column temperatures in the column temperature investigation of the standard decoction of fried chicken's gizzard-membrane decoction pieces according to the thin layer chromatography; wherein the column temperature of S1 is 30 ℃, the column temperature of S2 is 35 ℃, and the column temperature of S3 is 25 ℃.
FIG. 5 is a graph showing the comparison of different flow rates in the flow rate investigation of the standard decoction of fried chicken's gizzard-membrane decoction pieces according to thin layer chromatography; wherein the flow rate of S1 is 0.8mL/min, the flow rate of S2 is 0.6mL/min, and the flow rate of S3 is 1.0mL/min.
FIG. 6 is a characteristic diagram of gradient 1, gradient 2 and gradient 3 in elution gradient investigation of standard decoction of fried chicken's gizzard-membrane decoction pieces according to thin layer chromatography.
FIG. 7 is a comparison chart of different extraction methods in the investigation of the extraction method of the present invention; s1 is reflux extraction of a characteristic spectrum of a sample solution; s2 is ultrasonic extraction of a characteristic spectrum of the sample solution.
FIG. 8 is a graph showing the comparison of different extraction times in the extraction time investigation of the present invention; wherein S1 is ultrasonic extraction for 40min to obtain a characteristic spectrum of the sample solution; s2, ultrasonically extracting a characteristic spectrum of the sample solution for 20 minutes; s3, ultrasonic extraction is carried out for 30min on the characteristic spectrum of the sample solution.
FIG. 9 is a graph showing the comparison of different extraction solvents in the investigation of the extraction solvents of the present invention; s1 is a characteristic spectrum of a sample solution prepared by extracting 30% ethanol; s2 is a characteristic spectrum of a sample solution prepared by 60% methanol extraction; s3 is a characteristic spectrum of the sample solution prepared by 10% ethanol extraction.
FIG. 10 is a graph showing the comparison of different solvent amounts in the investigation of the solvent amount extracted by the sample according to the present invention; wherein S1 is a characteristic spectrum of a sample solution with a solvent content of 35 ml; s2 is a characteristic spectrum of a sample solution with a solvent amount of 25 ml; s3 is a characteristic spectrum of a sample solution with a solvent content of 15 ml.
FIG. 11 is a graph of blank solvent comparison in a specificity study of the present invention; s1 is a reference substance solution characteristic map; s2 is a characteristic spectrum of the solution of the sample, and S3 is a characteristic spectrum of a blank solvent (10% ethanol).
FIG. 12 is a graph of the common peak superposition characteristics for the repeatability test of the present invention; s1 is a test sample solution common peak superposition characteristic spectrum under repeatability 1; s2, overlapping a characteristic spectrum by a common peak of the sample solution under the condition of the renaturation 2; s3 is a test sample solution shared peak superposition characteristic map under repeatability 3; s4 is a test sample solution shared peak superposition characteristic map under the condition of repeatability 4; s5, overlapping a characteristic spectrum by a common peak of the sample solution under the condition of the renaturation 5; s6, the sample solution under the condition of the renaturation 6 shares a peak superposition characteristic spectrum.
FIG. 13 is a graph of the common peak superposition characteristics for the precision test of the present invention; s1 is a test sample solution common peak superposition characteristic spectrum under the precision of 1; s2 is a test sample solution common peak superposition characteristic spectrum under the precision of 2; s3 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 3; s4 is a test sample solution common peak superposition characteristic spectrum under the precision of 4; s5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 5; s6 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 6.
FIG. 14 is a graph of the common peak superposition characteristics for stability testing in accordance with the present invention; s1 is a test sample solution common peak superposition characteristic spectrum measured in 0 h; s2 is a test sample solution common peak superposition characteristic spectrum measured in 2 h; s3, a common peak superposition characteristic spectrum of the sample solution measured in 4 hours; s4, a common peak superposition characteristic spectrum of the sample solution measured in 8 hours; s5, a test sample solution common peak superposition characteristic spectrum measured in 12 hours; s6, the characteristic spectrum of the common peak superposition of the test sample solution measured in 24 hours.
FIG. 15 is a characteristic spectrum of a different chromatographic column investigation of the invention; wherein S1 is the chromatographic column of the batch number PF-78, and S2 is the chromatographic column of the batch number PF-63.
FIG. 16 is a graph of a guanosine control in a standard decoction feature graph of the present invention.
FIG. 17 is a graph of the reference medicinal material of the fried chicken's gizzard-skin in the characteristic spectrum measurement of the standard decoction of the invention.
FIG. 18 shows the reference pattern of endothelium corneum Gigeriae Galli in the measurement of the characteristic pattern of the standard decoction of the present invention
FIG. 19 is a superposition spectrum of 15 batches of chicken's gizzard-skin traditional Chinese medicine materials in the measurement of the characteristic spectrum of the standard decoction of the invention; wherein, S1 (4) -S15 (4) respectively represent 220201-220215 of overlapping patterns of 15 batches of chicken' S gizzard-skin traditional Chinese medicine materials.
FIG. 20 shows a pattern of common peaks of 15 batches of chicken's gizzard-membrane Chinese medicinal materials in the measurement of the characteristic pattern of the standard decoction of the invention.
FIG. 21 is a superposition spectrum of 15 batches of fried chicken's gizzard-skin traditional Chinese medicine decoction pieces in the measurement of the characteristic spectrum of the standard decoction of the invention; wherein, S1 (4) -S15 (4) respectively represent the overlapping patterns of 15 batches of fried chicken' S gizzard-membrane traditional Chinese medicine decoction pieces of Y220301-Y220315.
FIG. 22 shows a common peak spectrum of 15 batches of fried chicken's gizzard-membrane traditional Chinese medicine decoction pieces in the measurement of the characteristic spectrum of the standard decoction of the invention.
FIG. 23 is a superposition of 15 batches of fried chicken's gizzard-membrane standard decoction patterns in the measurement of the standard decoction feature pattern of the invention; wherein, S1 (4) -S15 (4) represents the overlapping pattern of 15 batches of fried chicken' S gizzard-membrane standard decoction of T220201-T220215.
FIG. 24 is a graph showing a standard decoction fit of 15 batches of fried chicken's gizzard-membrane in the measurement of the characteristic spectrum of the standard decoction of the invention.
FIG. 25 is a graph showing a linear investigation of different concentrations of guanosine controls in a linear range assay according to the present invention.
FIG. 26 is a comparison chart of a specificity investigation in the determination of the guanosine content characteristic spectrum of the standard decoction according to the present invention; s1 is a reference substance solution characteristic map; s2 is a characteristic spectrum of the solution of the sample, and S3 is a characteristic spectrum of a blank solvent (10% ethanol).
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
The invention provides a quality detection method of a fried chicken's gizzard-membrane standard decoction, which comprises the following detection method, wherein the standard decoction content standard is limited to 0.2-1.1 mg of guanosine in each 1g by measuring the characters, dry extract extraction rate, thin-layer identification, extract, characteristic spectrum and guanosine content of the fried chicken's gizzard-membrane standard decoction, and the dry extract extraction rate is measured by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the guanosine content are measured by liquid chromatography.
In this embodiment:
preparing a fried chicken's gizzard-membrane standard decoction: referring to the decoction method in the medical institution Chinese medicine decoction room management Specification (Chinese medicine administration 2009 No. 3), 15 batches of fried chicken's gizzard-membrane decoction pieces are taken, water is added until the decoction pieces are about 4-5cm higher than the medicinal materials, the decoction pieces are soaked for 30-40min, the decoction is carried out twice, the first decoction time is 30-40min, the second decoction time is 25-30min, solid-liquid separation is carried out when the decoction pieces are hot, the filtrates are combined, concentrated and dried, and 15 batches of fried chicken's gizzard-membrane standard decoction dry paste powder are prepared.
1. Dry extract yield test
15 batches of fried chicken's gizzard-membrane decoction pieces are taken, 15 batches of standard decoction dry paste powder are prepared according to the preparation method, the dry extract yield is calculated by using the dry paste powder (see table 1), the average yield is calculated to be 3.63 percent, the allowable range of the paste yield is calculated according to the allowable range of the standard limit (average value 70% -130%), the allowable range of the paste yield is calculated to be 2.54% -4.72%, and the allowable range of the standard decoction paste yield of the fried chicken's gizzard-membrane decoction pieces is calculated to be 2.5% -4.5%.
Table 1: paste yield
The results show that the paste yield of 15 batches of standard decoction is 3.2-4.3%, and the paste yield accords with the range of 2.5-4.5% of the planned limit.
2. Property investigation
According to the physical characteristics of 15 batches of fried chicken's gizzard-membrane standard decoction, the chicken's gizzard-membrane standard decoction is described as light yellow to yellow powder; slight fishy smell and bitter taste.
3. Thin layer authentication
The product is a dried extract of single decoction piece fried chicken's gizzard-membrane, and is prepared by taking chicken's gizzard-membrane reference medicinal materials as a reference by referring to a method under the item of 'thin layer identification' of fried chicken's gizzard-membrane prescription granule in a second edit of traditional Chinese medicine prescription granule thin layer chromatography color atlas', and 15 batches of sample tests prove that the sample spots of the sample to be tested are clear and negative reference samples have no interference, so the sample is assumed to be the product [ identification ]. The test methods and results are as follows:
3.1 method fumbling, sample application amount investigation and inspection method investigation
Test method according to thin layer chromatography (China pharmacopoeia 2020 edition four-part rule 0502) test
Sample solution preparation: taking 1g of the product powder, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating the filtrate, and adding 1ml of a mixed solution of ethyl acetate and ethanol (2:1) into residues to dissolve the residues to obtain a sample solution.
Preparing a control medicinal material solution: taking 2g of chicken's gizzard-membrane reference medicine, adding 100ml of water, decocting and keeping micro-boiling for 1 hour, cooling, filtering, evaporating filtrate to dryness, cooling, adding 20ml of ethanol into residues to dissolve, and preparing the reference medicine solution by the same method (ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, adding 1ml of a mixed solution of ethyl acetate and ethanol in a ratio of 2:1 into residues to dissolve).
Thin layer chromatography conditions: thin layer plate: silica gel G thin layer plate; sample application amount: sucking 3 μl of the sample solution and the control medicinal solution, 5 μl and 10 μl; developing agent: chloroform-ethyl acetate-methanol (10:2:1); presaturation for 30min, and spreading distance of 11cm; and (5) checking: the image was taken under an ultraviolet lamp (365 nm).
Results: in the sample chromatogram, fluorescent spots with the same color appear at the positions corresponding to the control medicinal material chromatogram, the TLC map is shown in figure 1 by fumbling and sample application amount investigation of the fried chicken's gizzard membrane method, in the sample application amount investigation, the sample application effect of 10 mu L of the sample solution and 5 mu L of the control medicinal material solution is best, and the sample application amount of 10 mu L of the sample to be tested and the sample application amount of 5 mu L of the control medicinal material are determined.
3.2 thin layer identification of Standard decoction
The test method comprises the following steps: test by thin layer chromatography (rule 0502 of four parts of Chinese pharmacopoeia 2020 edition)
Sample solution preparation: taking 1g of the product powder, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating the filtrate, and adding 1ml of a mixed solution of ethyl acetate and ethanol (2:1) into residues to dissolve the residues to obtain a sample solution.
Preparing a control medicinal material solution: decocting endothelium corneum Gigeriae Galli reference material 2g with water 100ml for 1 hr, cooling, filtering, evaporating filtrate, dissolving residue in ethanol 20ml, and making into reference medicinal solution.
Thin layer chromatography conditions: thin layer plate: silica gel G thin layer plate; sample application amount: sucking 10 μl of the sample solution and 5 μl of the control medicinal solution; developing agent: chloroform-ethyl acetate-methanol (10:2:1); presaturation for 30min, span 11cm, inspection: the image was taken under an ultraviolet lamp (365 nm).
Experimental conditions: in the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material, and 15 batches of standard decoction TLC patterns are shown in figure 2.
4. Determination of extract
The results of hot dipping method under the condition of 15 batches of standard decoction and ethanol as solvent according to the alcohol-soluble extract assay (general rule 2201 of Chinese pharmacopoeia 2020 edition) are shown in Table 2.
Table 2: extract measurement results
The result shows that the average value of 15 batches of standard decoction extract is 69.46 percent, and the lower limit of the allowable range (average value 70-130 percent) of the standard limit is referred to, so that the alcohol-soluble extract of the product is not less than 48.5 percent. The measurement results of 15 batches of standard decoction meet the requirements of the planned limit.
5. Feature profile testing
5.1 instruments, reagents and reagents
(1) Instrument: high performance liquid chromatograph (LC-2030 Plus (E01-625) shimadzu (japan)); the Shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um) accession number PF-63; thermostatic waterbath (HMTD-7000, yongguangming medical instruments Co., ltd., beijing); ultrasonic cleaners (KQ-300 DE, kunshan ultrasonic instruments Co., ltd.); one ten-thousandth balance (PX 224ZH, ohus instruments limited); parts per million flat (AWU 220D, japan shimadzu limited).
(2) Reagent: ethanol (Tianjin far chemical reagent Co., ltd.) and methanol (Tianjin Denko chemical reagent Co., ltd.) are chromatographically pure; acetonitrile (Tianjin chemical reagent Co., ltd.) was chromatographic pure, phosphoric acid (Tianjin chemical reagent manufacturing Co., ltd.) and water was ultrapure water (laboratory self-made).
(3) Control: guanosine (lot number: 111977-201501, content: 93.60%, national institute of food and drug testing), endothelium corneum Gigeriae Galli control (lot number: 121153-202003, national institute of food and drug testing), fried endothelium corneum Gigeriae Galli control (lot number: DST 20099-039, chengdu Lemeitian medical science and technology Co., ltd.).
5.2 test methods
5.2.1 determination of chromatographic conditions
(1) Determination of the optimal absorption wavelength
The chromatographic condition based on the method is combined with a DAD detector to perform maximum absorption spectrum scanning on the fried chicken's gizzard-membrane standard decoction sample, the scanning wavelength is 200-400nm, and the sample has stronger and more absorption near 200-300nm, so that detection wavelengths of 210nm, 230nm and 254nm are selected for research, and the optimal absorption wavelength is determined.
Taking a proper amount of fried chicken's gizzard-membrane standard decoction, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Chromatographic conditions: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 μm); mobile phase: acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed as specified in table 3; flow rate: 1.0ml per minute; column temperature: 25 ℃; detection wavelength: 210. 254, 230nm.
Table 3:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
| 55～65 | 0→0 | 100→100 |
The results show that by comparing the 3 detection wavelength chromatograms, as shown in fig. 3, when 254nm is selected as the detection wavelength, the response value of each characteristic peak is larger, the base line is stable, and the interference is smaller, so that 254nm is selected as the detection wavelength.
(2) Investigation of column temperature
The experiment is carried out by selecting 3 column temperatures at 25deg.C, 30deg.C and 35deg.C for comparison, and selecting proper column temperature.
Taking a proper amount of fried chicken's gizzard-membrane standard decoction, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Chromatographic conditions: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 μm); mobile phase: acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed as specified in table 4; flow rate: 1.0ml per minute; column temperature: 25 ℃, 30 ℃ and 35 ℃; detection wavelength: 254nm.
Table 4:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
| 55～65 | 0→0 | 100→100 |
The results show that by comparing chromatograms of 3 different column temperatures, as shown in fig. 4, the chromatographic peak information and peak shape differences of 3 mobile phases are not large, and when 30 ℃ is selected as the column temperature, the peak separation degree is better, so that 30 ℃ is selected as the column temperature.
(3) Investigation of flow Rate
The experiment selects 3 flow rates of 0.6ml/min, 0.8ml/min and 1.0ml/min for comparison, and selects a proper flow rate.
Taking a proper amount of fried chicken's gizzard-membrane standard decoction, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Chromatographic conditions: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 μm); mobile phase: gradient elution was performed as specified in table 5 with acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 0.6ml, 0.8ml, 1.0ml per minute; column temperature: 30 ℃; detection wavelength: 254nm.
Table 5:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
| 55～65 | 0→0 | 100→100 |
The results show that by comparing chromatograms of 3 different flow rates, as shown in fig. 5, the chromatographic peak information and peak shape difference of 3 mobile phases are not large, and when 0.8ml/min is selected as the flow rate, the peak separation degree is better, so that 0.8ml/min is selected as the flow rate.
(4) Gradient optimization
And (5) optimizing the elution gradient of the standard decoction feature map of the fried chicken's gizzard membrane, and determining the optimal gradient.
Taking a proper amount of fried chicken's gizzard-membrane standard decoction, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Chromatographic conditions: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 μm); mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the specifications in tables 6, 7 and 8; flow rate: 1.0ml per minute; column temperature: 30 ℃; detection wavelength: 254nm.
Table 6: gradient 1
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～20 | 2→5 | 98→95 |
| 20～25 | 5→20 | 95→80 |
| 25～30 | 20→20 | 80→80 |
| 30～50 | 20→30 | 80→70 |
| 50～55 | 30→95 | 70→5 |
| 55～60 | 95→3 | 5→97 |
| 60～70 | 3→3 | 97→97 |
Table 7: gradient 2
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～15 | 0→3 | 100→97 |
| 15～25 | 2→4 | 98→96 |
| 25～30 | 4→6 | 96→94 |
| 30～32 | 6→20 | 94→80 |
| 32～42 | 20→25 | 80→75 |
| 42～47 | 25→95 | 75→5 |
| 47～53 | 95→0 | 5→100 |
| 53～63 | 0→0 | 100→100 |
Table 8: gradient 3
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
Referring to fig. 6, the result shows that the gradient 3 with better separation degree and more peak shape information is finally determined as the elution gradient of the standard decoction feature map of the fried chicken's gizzard-skin by optimizing the elution gradient of the standard decoction feature map of the fried chicken's gizzard-skin.
5.2.2 chromatographic conditions
Chromatographic conditions: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um), accession number PF-63; mobile phase: gradient elution was performed as specified in table 9 with acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 0.8ml per minute; column temperature: 30 ℃; detection wavelength: 254nm.
Table 9:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
| 55～65 | 0→0 | 100→100 |
5.2.3 preparation of reference and control solutions: taking 0.3g of reference medicinal material, adding 25mL of 10% ethanol, refluxing for 30min, cooling, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain reference solution; and (3) taking a proper amount of guanosine reference substance, precisely weighing, adding 50% methanol to prepare a solution containing guanosine 10ug per 1ml, and shaking uniformly to prepare the reference substance solution.
5.2.4 preparation of test solution: taking about 0.3g of the fried chicken's gizzard-membrane standard decoction sample powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
5.2.5 assay: precisely sucking 10 μl of each of the control solution and the sample solution, and measuring with a liquid chromatograph.
5.3 examination of the pretreatment method of the sample solution
5.3.1 investigation of extraction method: the test solutions were prepared by different extraction methods, including heat refluxing and ultrasonic treatment, respectively, and were measured according to the test method 5.2 described above. As shown in FIG. 7 and Table 10, the separation degree of the two extraction modes is high, the difference between the ultrasonic treatment total peak area/sampling amount and the heating reflux is small, and the sample extraction mode is selected to be ultrasonic treatment in consideration of the experiment simplicity.
Table 10:
5.3.2 investigation of extraction time: sample solutions were prepared at different times of ultrasonic extraction, and were measured according to the test method 5.2 described above. As shown in FIG. 8 and Table 11, the number of main peaks was the same, the samples were completely extracted, the extraction times were different, and the peak areas were different from each other, and the intermediate value was selected as compared with the comparative time, so that the extraction time was determined to be 30 minutes.
Table 11:
5.3.3 investigation of extraction solvent: the test solutions were prepared with different extraction solvents, and were measured according to the 5.2 test method. The results showed that the extraction was incomplete when the extraction solvent was high concentration methanol, as shown in fig. 9 and table 12, and was therefore excluded. Guanosine compounds are polar large substances, so 10% ethanol is easier to extract, and therefore 10% ethanol is determined as an extraction solvent.
Table 12: investigation of results with different extraction solvents
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5.3.4 investigation of extraction solvent amount: sample solutions were prepared by ultrasonic treatment with different amounts of sample solvents (15 ml, 25ml, 35 ml), and were measured by the 5.2 test method. As shown in FIG. 10 and Table 13, the number of main peaks was the same, the amounts of the solvents were 0.64-0.68, the Rsd was 3.13%, and the amounts of the solvents for the extracted samples were 25ml because the amounts of the solvents were 15ml, 25ml and 35ml were examined, and the differences were small.
Table 13: investigation results of different extraction solvent amounts
In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking about 0.3g of a standard decoction sample of the chicken's gizzard-membrane powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
5.4 feature map analysis method verification
5.4.1 specificity investigation: the sample was measured under the above chromatographic conditions of 5.2 items with 10ul of solvent 10% ethanol. Experiments show that the blank solvent has no interference as shown in fig. 11.
5.4.2 repeatability test: about 0.3g of the same batch of samples are taken, 6 parts are taken, the measurement is carried out according to the 5.2 chromatographic condition, the result shows that 4 common peaks exist in the 6 parts of sample characteristic patterns, a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) is adopted for evaluating the similarity of the designated 4 common characteristic peaks, and the RSD (reactive species) of the relative retention time and the retention time are all in a qualified range (see tables 14, 15 and fig. 12 for details), so that the method is good in reproducibility.
Table 14: relative retention time of characteristic patterns for repeatability test
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 1 | 0.401 | 0.401 | 0.401 | 0.401 | 0.401 | 0.401 | 0.00 |
| 2 | 0.644 | 0.644 | 0.644 | 0.644 | 0.644 | 0.644 | 0.00 |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | 0.00 |
| 4 | 1.056 | 1.056 | 1.056 | 1.056 | 1.056 | 1.056 | 0.00 |
Table 15: repetitive test of characteristic pattern relative peak area
5.4.3 precision test: taking about 0.3g of a batch of samples, measuring according to 5.2 chromatographic conditions, continuously injecting 6 needles for measuring, basically keeping the peak shape and the peak number consistent, and adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), evaluating the similarity of the designated 4 common characteristic peaks, wherein the relative retention time and the RSD of the retention time are in a qualified range (see tables 16, 17 and FIG. 13 for details), thus showing that the method has good precision.
Table 16: relative retention time of precision test characteristic spectrum
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 1 | 0.4 | 0.401 | 0.4 | 0.398 | 0.4 | 0.4 | 0.25 |
| 2 | 0.643 | 0.644 | 0.644 | 0.641 | 0.643 | 0.643 | 0.17 |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | 0.00 |
| 4 | 1.057 | 1.057 | 1.057 | 1.056 | 1.056 | 1.056 | 0.05 |
Table 17: relative peak area of characteristic spectrum for precision test
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | RSD(％) |
| 1 | 2.427 | 2.418 | 2.416 | 2.406 | 2.399 | 2.401 | 0.45 |
| 2 | 0.930 | 0.928 | 0.933 | 0.925 | 0.923 | 0.924 | 0.40 |
| 3(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 4 | 0.820 | 0.810 | 0.803 | 0.790 | 0.803 | 0.806 | 1.24 |
5.4.4 stability test: taking about 0.3g of the same batch of samples, measuring according to 5.2 chromatographic conditions, and respectively carrying out sample injection measurement at 0h, 2h, 4h, 8h, 12h and 24h, wherein the peak shape and the peak number of the characteristic spectrum are basically stable, and carrying out similarity evaluation on the designated 4 common characteristic peaks by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), wherein the RSD (reactive species) of the relative retention time and the retention time are in a qualified range (see tables 18, 19 and 14 for details), so that the solution of the sample to be tested is more stable within 24 hours.
Table 18: stability test characteristic pattern relative retention time
| Peak number | 0 | 2h | 4h | 8h | 12h | 24h | RSD(％) |
| 1 | 0.4 | 0.4 | 0.4 | 0.4 | 0.401 | 0.399 | 0.14 |
| 2 | 0.643 | 0.644 | 0.643 | 0.643 | 0.644 | 0.641 | 0.16 |
| 3(S) | 1 | 1 | 1 | 1 | 1 | 1 | 0.00 |
| 4 | 1.057 | 1.057 | 1.056 | 1.056 | 1.056 | 1.056 | 0.04 |
Table 19: stability test characteristic spectrum relative peak area
5.4.5 durability inspection
(1) Investigation of different chromatographic columns
In the process of optimizing the method, the chromatographic columns of different types have great influence on the separation degree of the standard decoction characteristic spectrum of the fried chicken's gizzard membrane, so that the Shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um) chromatographic column is selected. To ensure the reproducibility of the method using the same type of chromatographic column, the effect of the same type of 2 chromatographic columns on the durability was compared, namely, the Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um) PF-78, shim-pack GIST C18-AQ
(4.6 mmx250mm,5 um) PF-63, as shown in FIG. 15 and tables 20 and 21.
Table 20: investigating characteristic spectrum relative retention time by different chromatographic columns
| Peak number | PF-78 | PF-63 | RSD(％) |
| 1 | 0.388 | 0.392 | 0.725 |
| 2 | 0.628 | 0.634 | 0.672 |
| 3(S) | 1 | 1 | 0.000 |
| 4 | 1.058 | 1.057 | 0.067 |
Table 21: different column temperatures are used for examining the relative peak areas of characteristic patterns
| Peak number | PF-78 | PF-63 | RSD(％) |
| 1 | 1.890 | 1.704 | 7.308 |
| 2 | 1.104 | 1.091 | 0.882 |
| 3(S) | 1.000 | 1.000 | 0.000 |
| 4 | 1.242 | 1.236 | 0.377 |
(2) Investigation of different column temperatures
Taking the sample solution under the 'chromatographic column durability investigation' item, and carrying out sample injection analysis under the condition that the chromatographic conditions are unchanged except that the column temperature is changed to 28 ℃, 30 ℃ and 32 ℃ respectively. The guanosine was used as reference peak S, the relative retention time and relative peak area of each characteristic peak and S peak were calculated, and RSD values were calculated, see tables 22 and 23, and the results showed that the relative retention time was less than 3%, indicating that the effect of column temperature was small and the durability was good at different column temperatures.
Table 22: different column temperatures examine the relative retention time of characteristic patterns
| Peak number | 28℃ | 30℃ | 32℃ | RSD(％) |
| 1 | 0.389 | 0.398 | 0.387 | 1.497 |
| 2 | 0.638 | 0.646 | 0.631 | 1.176 |
| 3(S) | 1 | 1 | 1 | 0.000 |
| 4 | 1.059 | 1.06 | 1.06 | 0.054 |
Table 23: different column temperatures are used for examining the relative peak areas of characteristic patterns
| Peak number | 28℃ | 30℃ | 32℃ | RSD(％) |
| 1 | 2.820 | 2.765 | 2.817 | 1.097 |
| 2 | 1.540 | 1.508 | 1.544 | 1.261 |
| 3(S) | 1.000 | 1.000 | 1.000 | 0.000 |
| 4 | 1.969 | 1.784 | 1.754 | 6.338 |
The characteristic spectrum method is proved by specificity, precision, repeatability and stability, meets the regulations, and is relatively stable in retention time after durability investigation.
5.5 Standard decoction characteristic Spectrum characterization analysis
5.5.1 Standard decoction feature Spectrum measurement
According to the proposed characteristic spectrum analysis method, 15 batches of fried chicken's gizzard-membrane standard decoction and 15 batches of traditional Chinese medicine decoction pieces characteristic spectrums used for preparation of the same are measured, and the result shows that 4 common peaks exist in the standard decoction and the traditional Chinese medicine decoction pieces characteristic spectrums used for preparation of the same and correspond to the retention time of 4 characteristic peaks in the chromatogram of the reference substance of the reference medicine, wherein the peak corresponding to the reference substance of guanosine is peak 2, and the common peak characteristic spectrums are shown in figures 16 to 24 in detail.
5.5.2 evaluation of characteristic chromatograms relative retention time
The similarity evaluation system (2012 edition) of the traditional Chinese medicine chromatographic fingerprint image is adopted to evaluate the similarity of the selected 4 common characteristic peaks, and the result shows that the similarity of the characteristic chromatograms of the standard decoction of 15 batches of fried chicken's gizzard-skin decoction pieces is above 0.9, which indicates that the quality of the standard decoction is relatively stable. The relative retention time of the common peak and the S peak was calculated using peak (3) corresponding to the guanosine reference peak as the S peak, and the relative retention time and the range thereof are shown in table 24.
Table 24: peak relative retention time of 15 batches of standard decoction
In summary, the standard decoction feature spectrum measurement method established by adopting the high performance liquid chromatography is adopted, and the established method is verified for precision, repeatability and stability according to the verification guiding principle (general rule 9101) of the four parts of analysis method in the edition 2020 of Chinese pharmacopoeia. And (3) performing similarity evaluation on the characteristic patterns of 15 batches of standard decoction samples by adopting a traditional Chinese medicine chromatographic fingerprint pattern similarity evaluation system (2012 edition), and calibrating 4 common characteristic peaks, wherein the peak 3 is guanosine. Calculating the relative retention time of the other 3 characteristic peaks by taking the peak corresponding to the guanosine reference as an S peak, and respectively setting the average value of the relative retention time of 15 batches of sample peaks as a specified value to be: 0.41 (Peak 1), 0.65 (Peak 2), 1.06 (Peak 4), the relative retention time allowable range was assumed to be + -10% taking into consideration multi-factor errors of test operation, instrument, reagent, etc.
6. Content determination
6.1 test method
The fried chicken's gizzard-membrane formula granule selects guanosine as the measurement component.
Chromatographic conditions: chromatographic column: chromatographic column: shimadzu Shim-pack GIST C18-AQ (4.6 mmx250mm,5 um) 63; mobile phase: gradient elution was performed as specified in table 25 with acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 0.8ml per minute; column temperature: 30 ℃; detection wavelength: 254nm.
Table 25:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～6 | 0→0 | 100→100 |
| 6～26 | 0→2 | 100→98 |
| 26～46 | 2→2 | 98→98 |
| 46～50 | 2→95 | 98→5 |
| 50～55 | 95→0 | 5→100 |
| 55～65 | 0→0 | 100→100 |
Preparing a reference substance solution: taking appropriate amount of guanosine reference substance, precisely weighing, adding 50% methanol to obtain solution containing guanosine 10ug per 1ml, and shaking.
Preparing a test solution: taking the powder of the product, about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Assay: precisely sucking 10 μl of each of the control solution and the sample solution, and measuring with a liquid chromatograph.
6.2 examination of the pretreatment method of the sample solution
6.2.1 investigation of extraction method: sample solutions were prepared by different extraction methods, and were measured according to the test method 6.1 described above. The results showed that the sample content of the reflux was not much different from that of the ultrasound, and the RSD was 1.31% (see table 26 for details), so the sample extraction mode was selected as the ultrasound treatment.
Table 26: comparison of different extraction methods
6.2.2 investigation of extraction time: sample solutions were prepared at different extraction times, and were measured according to the test method 6.1 described above. The results showed a difference between the extraction times (see Table 27 for details) and a RSD of 6.42% so the sample time was chosen to be 30 minutes.
Table 27: comparison of different extraction times
Investigation of 6.2.3 extraction solvent: sample solutions were prepared with different extraction solvents, and the measurement was performed according to the test method 6.1 described above. As a result, when the extraction solvent was 10% ethanol, guanosine content was high (see Table 28 for details) and RSD was 0.5%, and in the experiment, the extraction solvent was high in water content and good in peak shape, so that it was confirmed that 10% ethanol was used as the extraction solvent.
Table 28: comparison of different extraction solvents
6.2.4 extraction of sample solvent quantity investigation: test solutions were prepared in different amounts (15 ml, 25ml, 35 ml) of the solvent, and the measurement was performed according to the test method 6.1 described above. The results show that the samples can be extracted from low solvent amount to high solvent amount, and the solvent amount is determined to be 25ml considering simple experimental selection and space (see Table 29 for details).
Table 29: comparison of different solvent amounts
Determination of 6.2.5 test sample solution preparation method
In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking the powder of the product, about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate.
6.3 methodological validation of content determination
6.3.1 repeatability test: about 0.3g of standard decoction samples in the same batch are taken, 6 parts of standard decoction samples are measured according to the test method 6.1, the average value of guanosine content in the measured samples is 0.640434mg/g, the RSD value is 0.75%, and the test shows that the method has good reproducibility (see Table 30 for details).
Table 30:
6.3.2 precision test: the sample solution shown in 6.1 is continuously sampled for 6 needles, the peak area is measured according to the test method of 6.1, and the RSD value of the guanosine peak area in the sample is calculated to be 0.31%, which shows that the instrument precision is good (see Table 31 for details).
Table 31:
6.3.3 stability test: taking about 0.3g of a batch of standard decoction samples, injecting the samples at 0h, 2h, 4h, 8h, 12h and 24h according to the test method 6.1, measuring the peak area, calculating the RSD value of the peak area to be 0.41%, and testing the sample solution to be tested to be stable within 24 hours (see Table 32 for details).
Table 32:
6.3.4 linear range test: taking guanosine reference substance solutions (the concentration is 0.02148 mg/ml), and respectively taking 5ml to 10ml volumetric flasks with the concentration of 0.01074mg/ml; taking a volumetric flask from 5ml to 10ml of 0.00537mg/ml solution, namely 0.002685mg/ml concentration, and so on; a concentration of 0.0013425mg/ml; the concentration of 0.00013425mg/ml is measured according to the chromatographic condition under 6.1 items, the guanosine peak area is taken as an ordinate, the sample injection amount is taken as an abscissa, a standard curve is drawn, and the linear regression is carried out
The regression equation is: y= 32592917.7812x-2926.7258, r 2 Guanosine was found to have a good linear relationship with its peak area in the range 0.00013425mg/ml to 0.02148mg/ml (see table 33 and fig. 25 for details).
Table 33: guanosine linear relationship investigation results
| Test solution | Peak area | Concentration of |
| Linearity 1 | 697446 | 0.02148 |
| Linearity 2 | 347955 | 0.01074 |
| Linearity 3 | 170275 | 0.00537 |
| Linearity 4 | 82311 | 0.002685 |
| Linearity 5 | 41317 | 0.0013425 |
| Linearity 6 | 3947 | 0.00013425 |
6.3.5 sample recovery test: about 0.3g of the sample (guanosine content: 0.65043 mg/g) was precisely weighed into 6 parts, 3ml of guanosine control solutions (0.02148 mg/ml) of known concentrations were added, test sample solutions were prepared by the method described in 6.1, and the measurement was performed under the chromatographic conditions described in 6.1, whereby the guanosine average recovery rate was 92.8411659% and the RSD was 4.59% (see table 34 for details).
Table 34: guanosine sample recovery test results
6.3.6 specificity investigation: the measurement was performed under the chromatographic conditions under the above item "6.1". The test shows that: the blank solvent has no interference, and the method has good specificity as shown in figure 26.
6.3.7 durability inspection
(1) Investigation of different chromatographic columns
The effect of 2 columns (4.6 mmx250mm,5 μm) (Shimadzu GIST AQ-C18 (PF-78), shimadzu GIST AQ-C18 (PF-63)) of the same manufacturer and different batches on the assay was compared. As shown in Table 35, the measured RSD value of the content is 0.0002% and less than 3.0%, which indicates that the analytical method has good durability in chromatographic columns of different batches of the same model.
Table 35:
(2) Investigation of different column temperatures
Taking the sample solution under the section of 'different chromatographic column inspection', adjusting the column temperature inspection at 25 ℃, 30 ℃ and 35 ℃, keeping other chromatographic conditions unchanged, and calculating the content and RSD value. As shown in Table 36, the RSD value of the content was measured to be 0.87% or less than 3.0%, indicating that the method was excellent in durability against small variations in column temperature.
Table 36:
(3) Investigation of different flow rates
Taking the sample solution under the section of 'different chromatographic column inspection', and calculating the content and RSD value under the condition of unchanged chromatographic conditions except that the flow rate is inspected for 0.6ml/min, 0.8ml/min and 1.0 ml/min. The results are shown in Table 37, and the measured RSD values of the contents are 3.95% and less than 5.0%, indicating that the method is excellent in durability against small variations in flow rate.
Table 37:
in conclusion, the guanosine separation degree and the chromatographic peak purity meet the quantitative requirements, and the whole analysis method meets the requirements through investigation of specificity, precision, repeatability, stability, sample addition recovery and drug resistance, so that the established method can be well used for measuring the guanosine content.
6.4 standard decoction and Chinese medicinal herb content determination
The fried chicken's gizzard-membrane medicinal material is firstly processed into segment tablets by a production place, and then processed into fried chicken's gizzard-membrane decoction pieces.
6.4.1 according to the above-mentioned content analysis method, 15 batches of fried chicken's gizzard-membrane standard decoction and 15 batches of fried chicken's gizzard-membrane decoction pieces and guanosine contents of medicinal materials used for preparation thereof are measured, and the results are shown in tables 38, 39 and 40 in detail.
Table 38:15 batches of chicken's gizzard-membrane medicinal materials measurement results
Table 39:15 batches of fried chicken's gizzard-membrane decoction pieces measurement results
Table 40: guanosine determination result of 15 batches of fried chicken's gizzard-membrane standard decoction
6.4.1 guanosine content transfer rate: according to the detection method determined by standard decoction methodology research, the guanosine content transfer rate is calculated for 15 batches of standard decoction and the measurement results of the prepared and used traditional Chinese medicine decoction pieces, the mass transfer condition is mastered, and a basis is provided for formulating the material internal control standard and the allowable range of the characterization parameters. The standard decoction of the fried chicken's gizzard-membrane decoction pieces is prepared by decocting the fried chicken's gizzard-membrane decoction pieces with water for 2 times, concentrating the filtrate, and freeze-drying. The guanosine content transfer rate is shown in Table 41.
Table 41: guanosine content transfer rate of 15 batches of fried chicken's gizzard-membrane standard decoction
From the data, the fried chicken's gizzard-membrane decoction pieces are decocted according to a scheme to prepare the fried chicken's gizzard-membrane decoction piece standard decoction, the guanosine average transfer rate of the fried chicken's gizzard-membrane decoction pieces is 84.14%, the measured transfer rate ranges from 61.72% to 95.11%, and the SD is 9.56. According to the technical requirements of quality control and standard formulation of traditional Chinese medicine formula particles, the allowable range of guanosine content transfer rate is 58.9-109.4% calculated according to 70-130% of the average value of the transfer rate; 55.5 to 112.8% by.+ -. 3 SD. Therefore, the guanosine content transfer rate range of the standard decoction is assumed to be: 55.5-100%. The results show that the guanosine transfer rates in 15 batches of standard decoction are all within the allowable range of +/-3 SD.
The average guanosine content in the standard decoction is 0.64mg/g, the measured content range is 0.420 mg/g-0.861 mg/g, and the SD is 0.142; the guanosine content allowable range is 0.21 mg/g-1.07 mg/g calculated by mean value + -3 SD. Therefore, the guanosine content range of the standard decoction is assumed to be: 0.2 mg/g-1.1 mg/g. The result shows that guanosine and the transfer rate in 15 batches of standard decoction are all within the allowable range, and can provide reference basis for the quality research of fried chicken's gizzard-skin formula particles.
According to the method for detecting the quality of the fried chicken's gizzard-membrane standard decoction, the characteristics, the dry extract yield, the thin-layer identification, the extract, the characteristic spectrum and the guanosine content of the fried chicken's gizzard-membrane standard decoction are researched, the quality of the fried chicken's gizzard-membrane standard decoction is assessed through multi-aspect measurement, a solid foundation is laid for the stability of the quality of a product, a feasible quality standard of the fried chicken's gizzard-membrane decoction can be established, the effective control of the quality of the fried chicken's gizzard-membrane standard decoction is realized, and a chromatographic condition of the method is adopted for carrying out liquid phase analysis, so that a chromatogram with better and clearer separation degree can be obtained; the fried chicken's gizzard-membrane decoction pieces are decocted to obtain a fried chicken's gizzard-membrane decoction piece standard decoction, the average content of guanosine is 0.64mg/g, the measured content range is 0.420-0.861 mg/g, the SD (standard deviation) is 0.142, calculated according to the mean value +/-3 SD, the allowable content range of guanosine is 0.21-1.07 mg/g, so the guanosine content range of the standard decoction is calculated as follows: 0.2 mg/g-1.1 mg/g; the average guanosine transfer rate is 84.14%, the transfer rate range is 61.72% -95.11%, the SD is 9.56, according to the technical requirements of quality control and standard establishment of traditional Chinese medicine formula particles, the allowable guanosine content transfer rate range is 58.9% -109.4% calculated by 70% -130% of the transfer rate average value, and is 55.5% -112.8% calculated by +/-3 SD, so that the guanosine content transfer rate range of the standard decoction is proposed to be: 55.5-100%, and the result shows that the guanosine content and the transfer rate of the standard decoction of a plurality of batches are all within the allowable range, so the invention can provide reference for the quality standard research of fried chicken's gizzard-skin formula particles.
Those skilled in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
Claims (4)
1. A method for detecting the quality of a standard decoction of fried chicken's gizzard-membrane is characterized by comprising the following detection methods,
determining the properties of the fried chicken's gizzard-membrane standard decoction, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the guanosine content, limiting the standard decoction content standard to 0.2-1.1mg of guanosine content per 1g, wherein the dry extract extraction rate is determined by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and the guanosine content are measured by adopting a liquid chromatography;
The determination of the characteristic spectrum by liquid chromatography comprises: performing liquid chromatograph analysis, wherein the solution prepared from endothelium corneum Gigeriae Galli reference material is used as reference solution b, the solution prepared from guanosine reference material is used as reference solution b, the solution prepared from parched endothelium corneum Gigeriae Galli standard decoction sample is used as sample solution b, and the reference solution b, reference solution b and sample solution b are respectively and precisely absorbed, respectively injected into liquid chromatograph, and measured to obtain the final product; wherein the chromatographic conditions adopted are that: shimadzu Shim-pack GIST C18-AQ, column length of 4.6mm, inner diameter of 250mm, and particle size of 5um; mobile phase: acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table a;
table a gradient elution procedure
Flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm;
the thin layer chromatography comprises the following steps:
s11: preparing a test sample solution a: taking 1g of fried chicken's gizzard-membrane standard decoction sample, adding 20mL of ethanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1mL of a mixed solution of ethyl acetate and ethanol in a ratio of 2:1 into residues to dissolve to obtain a sample solution a;
s12: preparing a control medicinal material solution a: taking 2g of chicken's gizzard-membrane reference medicine, adding 100mL of water, decocting and keeping micro boiling for 1h, cooling, filtering, evaporating filtrate to dryness, cooling, adding 20mL of ethanol into residues to dissolve, performing ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, adding 1mL of a mixed solution of ethyl acetate and ethanol in a ratio of 2:1 into residues to dissolve, and preparing a reference substance solution a;
S13: thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: sucking 10 μl of the sample solution a and 5 μl of the control medicinal material solution a; developing agent: the volume ratio is 10:2:1 in chloroform-ethyl acetate-methanol; presaturation for 30min, and spreading distance of 11cm; and (5) checking: looking at the ultraviolet lamp at 365 nm;
the determination of guanosine content by liquid chromatography comprises: performing liquid chromatograph analysis, taking a proper amount of guanosine reference substance, precisely weighing, adding 50% methanol to prepare a solution with guanosine concentration of 10ug/mL as a reference substance solution c, taking 0.3g of fried chicken's gizzard-membrane standard decoction sample, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, taking the subsequent filtrate as a sample solution c, precisely sucking the reference substance solution c and the sample solution c respectively, injecting the reference substance solution c and the sample solution c into a liquid chromatograph respectively, and measuring to obtain the guanosine reference substance; wherein the chromatographic conditions adopted are that: shimadzu Shim-pack GIST C18-AQ, column length of 4.6mm, inner diameter of 250mm, and particle size of 5um; mobile phase: acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table B;
Table b:
flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 254nm.
2. The method for detecting the quality of a standard decoction of fried chicken's gizzard-skin according to claim 1, wherein the decoction method comprises: soaking the fried chicken's gizzard-membrane decoction pieces in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid, concentrating, and drying to obtain dry extract powder of standard decoction of fried chicken's gizzard-membrane.
3. The method for detecting the quality of a standard decoction of fried chicken's gizzard-skin according to claim 1, wherein the hot-dip method uses ethanol as a solvent and adopts a hot-dip method under the alcohol-soluble extract detection method to detect the extract range.
4. The method for detecting the quality of a standard decoction of fried chicken's gizzard-skin according to claim 1, wherein the characteristic spectrum is measured by liquid chromatography, further comprising the steps of:
s21: preparation of reference solution b: taking 0.3g of chicken's gizzard-membrane reference medicine, adding 25mL of 10% ethanol, refluxing for 30min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as reference solution b;
s22: preparing a reference substance solution b: taking a proper amount of guanosine reference substance, precisely weighing, adding 50% methanol for dissolving, and preparing a reference substance solution b with the concentration of 10 ug/mL;
S23: preparing a test sample solution b: taking 0.3g of fried chicken's gizzard-membrane standard decoction sample, precisely weighing, placing into a conical flask with a plug, adding 25mL of precisely weighed 10% ethanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with 10% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.
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