CN117990847A - Method for detecting quality of fried earthworm formula particles - Google Patents
Method for detecting quality of fried earthworm formula particles Download PDFInfo
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- G—PHYSICS
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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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Abstract
The invention provides a quality detection method of fried earthworm formula particles, which comprises the steps of carrying out shape investigation, thin layer identification, characteristic spectrum construction, inosine content measurement, extract detection and granule general term detection on the fried earthworm formula particles; wherein, the characteristic spectrum construction and the inosine content measurement are both measured by adopting a liquid chromatography, wherein octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, and 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase B in the characteristic spectrum measured by the liquid chromatography, and gradient elution is carried out according to a stipulation; the quality detection method of the fried earthworm formula particles can detect the fried earthworm formula particles through a plurality of indexes, can establish a feasible quality standard of the fried earthworm formula particles, realize effective control of the quality of the fried earthworm formula particles, and lay a solid foundation for the stability of the quality of the fried earthworm formula particles.
Description
Technical Field
The invention belongs to the technical field of quality control of traditional Chinese medicinal materials, and particularly relates to a quality detection method of fried earthworm formula particles.
Background
Modern medicines are required to have three characteristics of stability, uniformity, safety and effectiveness, and for Chinese patent medicines, various means are required to be adopted for detection, so that the reliability and stability of detection results are ensured. The traditional Chinese medicine formula granule is a single traditional Chinese medicine product prepared by adopting modern scientific technology and imitating the traditional Chinese medicine decoction decocting way, and refining the traditional Chinese medicine decoction pieces through the processes of leaching, concentrating, drying and the like. The product maintains the property, taste and efficacy of the traditional Chinese medicine decoction pieces, has stable quality, is applied to the formulation of clinical prescriptions of the traditional Chinese medicine, meets the requirements of dialectical treatment and prescription change, and has the advantages of no need of decoction, convenient administration, quick absorption, accurate dosage, safety, cleanness, convenient carrying and the like. The single medicinal materials are subjected to water extraction, concentration, drying, granulation and other procedures to prepare the formula particles, and compared with the traditional medicinal materials, the form characteristics of the formula particles are obviously changed, and the authenticity and quality of the product cannot be obtained by naked eye observation. Therefore, it is necessary to establish a quality evaluation system of the traditional Chinese medicine formula particles so as to comprehensively reflect the inherent quality of the formula particles.
The parched Lumbricus is a processed product of Lumbricus (Geosaurus), which is dry body of Pheretima aspergillum PHERETIMA ASPERGILLUM (E.Perrie), pheretima aspergillum PvulgarisChen, williams Lumbricus Pguillelmi (Michaelsen) or Pheretima pectinifera PPECTINIFERAMICHAELSEN of Legioptera. The former is known as "Guangdong earthworm", the last three are known as Lu (Lu mainly produced in Guangxi, guangdong and Fujian. Cold nature and salty taste. The fried earthworm is an important Chinese medicine for treating high fever unconsciousness, convulsion, arthralgia, limb numbness, oliguria edema, hypertension and other diseases, and no quality standard related to the prescription granule of the fried earthworm exists in China at present.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a method for detecting the quality of the fried earthworm formula particles, which comprehensively reflects the internal quality of the formula particles and better controls the quality of the formula particles.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
The quality detection method of the fried earthworm formula particles is characterized by comprising the following detection methods:
Carrying out thin layer identification, characteristic spectrum construction and inosine content measurement on the fried earthworm prescription granule, limiting the inosine content standard of the fried earthworm prescription granule to 4.6mg/g-15.6mg/g, wherein the thin layer identification adopts a thin layer chromatography for identification, and the characteristic spectrum construction and the inosine content measurement are both measured by adopting a liquid chromatography;
Wherein, the characteristic spectrum is determined by liquid chromatography, octadecylsilane chemically bonded silica is taken as a filler, acetonitrile is taken as a mobile phase A, 0.01mol/L potassium dihydrogen phosphate 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
The flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength is 210nm;
the step of determining the characteristic spectrum by liquid chromatography comprises the following steps:
S11: preparing a reference substance solution a of a control medicinal material: taking 1.0g of earthworm control medicine, adding 25mL of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, and taking a subsequent filtrate as a reference substance solution a of the control medicine; taking an appropriate amount of inosine reference substance, precisely weighing, adding methanol to prepare a solution containing 20 mug of inosine per 1ml of the solution as reference substance solution a;
S12: preparing a test sample solution a: precisely weighing 0.3g of ground fried earthworm formula particles, placing into a container, adding 25mL of 30% methanol, carrying out ultrasonic treatment for 20 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the finished product;
S13: respectively sucking reference solution a of reference material, reference solution a of reference material and sample solution a of sample 10 μl, and measuring with liquid chromatograph.
Further, the thin layer chromatography includes the steps of:
s21: taking 1.0g of fried earthworm prescription granule sample, adding 10ml of boiling water for dissolution, cooling, centrifuging, and taking supernatant as a sample solution b;
s22: taking L-lysine reference substance, leucine reference substance and valine reference substance, respectively adding water to prepare solutions containing 1mg, 1mg and 0.5mg of each 1ml of the solutions as reference substance solutions b;
S23: thin layer chromatography was performed, thin layer plate: silica gel G thin layer plate; sample application amount: sucking 3. Mu.l of the test solution b and 3. Mu.l of the reference solution b; developing agent: n-butanol-glacial acetic acid-water; color development: spraying ninhydrin test solution, heating at 105deg.C until the spots are clear, and inspecting under sunlight.
Further, the thin layer chromatography includes the steps of:
S31: taking 1.0g of stir-fried earthworm prescription granule sample, adding 20ml of chloroform, carrying out ultrasonic treatment for 20min, filtering, evaporating filtrate to dryness, and dissolving residues with 1ml of chloroform to obtain a sample solution c;
S32: adding chloroform into 1.0g of parched Lumbricus control material, ultrasonic treating for 20min, filtering, evaporating filtrate, and dissolving residue with chloroform 1ml to obtain control material solution c;
S33: performing thin layer chromatography under the following conditions: silica gel G thin layer plate; sample application amount: 5uL of each test solution c and control medicinal solution c; developing agent: toluene-acetone was inspected under 365nm UV lamp.
Further, the step of measuring inosine content by liquid chromatography includes:
s41: taking a proper amount of inosine reference substance, precisely weighing, adding 70% methanol to prepare a solution containing 20 mug of inosine per 1ml, and taking the solution as reference substance solution d;
s42: weighing 0.3g of fried earthworm formula particles, placing the particles into a bottle, precisely adding 25mL of 30% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution d;
S43: precisely sucking 5 μl of the reference solution d and the sample solution d, respectively, and injecting into a liquid chromatograph for measurement;
Wherein, the liquid chromatography conditions adopt octadecylsilane chemically bonded silica as filler, and the volume ratio is 3: acetonitrile of 97: 0.01mol/L potassium dihydrogen phosphate solution is a mobile phase; the flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength was 210nm.
Further, in the step S11, 30% methanol by volume is added in the preparation of the reference solution a of the reference medicinal material; 70% methanol by volume is added in the preparation of the reference substance solution a.
Further, n-butanol in the step S23: glacial acetic acid: the volume ratio of water is 4:1:1.
Further, in the step S33, toluene: the volume ratio of acetone is 9:1.
Further, the method also comprises the following detection methods: and (3) carrying out character identification, general examination and extract examination on the fried earthworm prescription granules, wherein the extract is measured by adopting a hot dipping method.
Further, the hot dip method uses ethanol as a solvent, and the range of the extract is measured by adopting a hot dip method under the alcohol-soluble extract measuring method.
Further, the granule general term examination includes moisture, particle size, and solubility examination.
The invention has the beneficial effects that the characteristic investigation, the thin layer identification, the characteristic spectrum measurement, the general term inspection of the granule and the extract inspection and the content measurement are carried out on the fried earthworm formula particles, wherein the thin layer identification adopts the thin layer chromatography for identification; the general term examination of the granule comprises moisture, granularity and dissolubility examination; the extract is measured by a hot dipping method; the characteristic spectrum and the inosine content are measured by adopting a liquid chromatography; according to the quality detection method of the fried earthworm formula particles, disclosed by the invention, the quality of the fried earthworm formula particles is detected by indexes in multiple aspects, so that a feasible quality standard of the fried earthworm formula particles can be established, the effective control of the quality of the fried earthworm formula particles is realized, and a solid foundation is laid for the stability of the quality of the fried earthworm formula particles;
the chromatographic condition of the application is adopted to carry out liquid phase analysis, so that a characteristic map with better and clearer separation degree can be obtained, the method has strong specificity, better stability, high precision and better repeatability.
The content of inosine in the product is limited to 4.6-15.6 mg per 1 g.
Drawings
FIG. 1 (a) is a thin layer identification chart obtained by a method I of a test sample in the fried earthworm formula particle, wherein A is negative, S1 is L-lysine reference substance, S2 is leucine reference substance, S3 is valine reference substance, and 1-3 are test substances 220301 ～ 220303;
FIG. 1 (b) is a thin layer identification chart obtained by a second method for a test sample of the fried earthworm prescription granule, wherein A is negative, S is a earthworm control medicinal material, and 1-3 are test samples 220301 ～ 220303;
FIG. 2 is a comparison of different extraction methods;
FIG. 3 is a graph comparing different extraction solvents;
FIG. 4 is a graph of different extraction times;
FIG. 5 is a graph showing the comparison of different sampling amounts;
FIG. 6 is a blank solvent comparison graph of a feature map specificity investigation;
FIG. 7 is a repetitive test consensus peak overlay profile;
FIG. 8 is a plot of the common peak superposition characteristics for the precision test;
FIG. 9 is a stability test consensus peak overlay feature map;
FIG. 10 is a chromatogram of the common peak superposition profile of column tests;
FIG. 11 is a column temperature test consensus peak overlay feature map;
FIG. 12 is a inosine control profile;
FIG. 13 is a diagram showing inosine and test samples;
FIG. 14 is a graph of Lumbricus (limnodrilus) control medicinal material;
FIG. 15 is a superposition of characteristic maps of 3 pilot samples;
FIG. 16 is a graph showing the superposition of characteristic maps of 3 pilot samples and a control medicinal material;
FIG. 17 is a graph of a common peak fit for 3 pilot samples;
FIG. 18 is a plot of a common peak fit for 3 pilot samples and control;
FIG. 19 is a blank solvent comparison graph in assay specificity study;
FIG. 20 is a standard curve of inosine control.
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.
In this embodiment:
The product is a formulation granule prepared by processing dried body of limnodrilus PHERETIMA ASPERGILLUM (E.Perrier) of limnodrilus of Equidae and processing according to main quality index of standard decoction.
The specific preparation method of the formula granule comprises the following steps: decocting 5000g of fried earthworm decoction pieces in water, filtering, concentrating filtrate into clear paste (the paste yield of the dry extract is 10% -18%), adding an appropriate amount of auxiliary materials, drying (or drying and crushing), adding an appropriate amount of auxiliary materials, uniformly mixing, granulating and preparing 1000 g.
The specific preparation method of the standard decoction comprises the following steps: decocting 100g of fried earthworm decoction pieces in water, filtering, concentrating the filtrate into clear paste (the paste yield of the dry extract is 10% -18%), and drying (or drying and crushing) to obtain the finished product.
Paste yield: 15 batches of fried earthworm (limnodrilus) decoction pieces are taken, 15 batches of standard decoction dry paste powder are prepared according to the preparation method, dry extract yield is calculated according to the dry paste powder (see the table below), average yield is calculated to be 14.49%, the allowable range of paste yield is calculated according to the standard limit (average value 70% -130%), the allowable range of paste yield is calculated to be 10.14% -18.84%, and the allowable range of paste yield of the standard decoction of the fried earthworm (limnodrilus) decoction pieces is calculated to be 10% -18%.
Surface b stir-fried earthworm (limnodrilus) decoction piece standard decoction extract rate
The results show that the paste yield of 15 batches of standard decoction is 12.2-15.8%, and the paste yield accords with the range of 10-18% of the planned limit.
1. Property investigation
Traits: taking one sample of three batches of fried earthworm (limnodrilus) formula particles, and observing and recording the shape, color and smell of the samples. The product is granule with light brown yellow to yellowish brown color; fishy smell, slightly salty taste.
2. Thin layer authentication
The test was performed by thin layer chromatography (rule 0502 of four parts of chinese pharmacopoeia 2020).
The method comprises the following steps:
sample solution preparation: taking 1g of the product, adding 10ml of boiling water for dissolution, cooling, centrifuging, and taking the supernatant as a sample solution.
Control solution: taking L-lysine reference substance, leucine reference substance and valine reference substance, respectively adding water to obtain solutions containing 1mg, 1mg and 0.5mg of each 1ml of the solutions as reference substance solutions.
Thin layer chromatography conditions: thin layer plate: silica gel G thin layer plate; sample application amount: sucking 3 μl of the sample solution and 3 μl of the control medicinal solution; developing agent: n-butanol-glacial acetic acid-water (4:1:1); color development: spraying ninhydrin test solution, heating at 105deg.C until the spots are clear, and inspecting under sunlight. As shown in FIG. 1 (a), the results showed that spots of the same color appear in the sample chromatogram at positions corresponding to the control chromatogram and the control chromatogram.
The second method is as follows:
Taking 1G of the powder, adding 20ml of chloroform, carrying out ultrasonic treatment for 20min, filtering, evaporating filtrate, adding trichloro (1 ml of methane is dissolved to be used as a sample solution), taking 1G of earthworm reference medicine, preparing the reference medicine solution by the same method, carrying out thin layer chromatography (rule 0502 of the fourth edition of Chinese pharmacopoeia 2020), respectively sucking 5 μl of the reference medicine solution and 10 μl of the sample solution, respectively, spotting on the same silica gel G thin layer plate, using toluene-acetone (9:1) as a developing agent, developing, taking out, airing, and placing under an ultraviolet lamp (365 nm) to be detected, wherein the result shows that fluorescent spots with the same color are displayed on the positions corresponding to the chromatogram of the reference medicine in the chromatogram of the sample.
3. Feature map
3.1 Feature map measuring method
3.1.1 Experimental conditions
(1) Instrument: thermo ultra-high performance liquid chromatograph (U3000, sammer femto-tech (china) limited); shimadzu high performance liquid chromatograph (LC-2030 puls, shimadzu corporation, japan); shimadzu Shim-PACK GIST C-AQ (250 mmx4.6mm,5 μm), mid-spectrum red RD-C18 (250 mmx4.6mm,5 μm); 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, water was ultrapure water (laboratory self-made), and monopotassium phosphate (Tianjin chemical reagent Co., ltd.).
(3) Reagent: inosine (lot number: 140669-202007, content: 99.20%, national food and drug verification institute), lumbricus (Pheretima aspergillum) control medicinal material (lot number: 120987-201508, national food and drug verification institute).
(4) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); acetonitrile is taken as a mobile phase A, 0.01mol/L potassium dihydrogen phosphate solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength was 210nm. The theoretical plate number should be not less than 3000 calculated as inosine peak.
TABLE 1 gradient elution procedure
3.1.2 Preparation of reference solution
Taking 1.0g of earthworm (limnodrilus) reference medicinal material, adding 25ml of 30% methanol, weighing, performing ultrasonic treatment for 30 minutes, cooling, filtering, and taking the subsequent filtrate as reference substance solution of the reference medicinal material. And (3) taking a proper amount of inosine reference substance, precisely weighing, and adding 70% methanol to prepare a solution containing 20 mug of inosine per 1 ml.
3.1.3 Preparation of sample solutions
Grinding the fried earthworm formula particles, precisely weighing about 0.3g, placing the ground earthworm formula particles into a conical bottle with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment for 20 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the earthworm composite.
3.1.4 Assays
Respectively precisely sucking 10 μl of reference solution and sample solution, and injecting into liquid chromatograph for measurement.
3.2 Methodology investigation and validation of feature atlas determination
The method for pre-treating the sample of the characteristic spectrum of the fried earthworm formula particles is examined, and the influence of the extraction mode, the extraction solvent, the extraction time and the sampling amount on the characteristic spectrum of the fried earthworm formula particles is mainly examined.
3.2.1 Investigation of the extraction method
The test sample solutions are prepared by different extraction methods respectively, and the result of the determination of the map by the experimental method of 3.1 shows that the number of main peaks is consistent, and the peak shapes of different extraction modes have little difference (see figure 2). The calculation result shows that the RSD value of the ratio of the total peak area to the sampling amount is 0.03% (see table 2 below) which is less than 3.0%, so that the sample extraction mode is selected as ultrasonic for saving time and facilitating operation.
TABLE 2 comparison of extraction methods
3.2.2 Investigation of extraction solvent
Test solutions were prepared with different extraction solvents, respectively, and the measurement was performed according to the method described under item "3.1" above. The graph result shows that the number of main peaks is consistent, and the peak shape difference is large in different extraction times (see figure 3), so that 30% ethanol is removed. The calculation result shows that the RSD value of the ratio of the total peak area to the sampling amount is 9.04% (see table 3 below), and the "total peak area/sampling amount" is the maximum when the extraction solvent is 30% methanol, and the separation degree is high, so that 30% methanol is determined as the extraction solvent.
TABLE 3 comparison of extraction solvents
3.2.3 Extraction time investigation
Test solutions were prepared at different times, and the measurement was performed according to the method described under "3.1" above. As shown in fig. 4 and table 4, the results showed that the number of main peaks was the same and the peak shape difference at different extraction times was not large, so that 20 minutes was determined as the extraction time. The calculation result shows that the RSD value of the ratio of the total peak area to the sampling amount is 2.26% and less than 3.0%, so that the extraction time is 20 minutes.
TABLE 4 comparison of extraction times
3.2.4 Investigation of sample size
Test solutions were prepared in different amounts and were measured as described under "3.1" above. As shown in FIG. 5 and Table 5, the results of the spectra showed that the number of main peaks was consistent, the peak shape difference was small with different sampling amounts, the RSD value of the ratio of the total peak area to the sampling amount was 3.09%, more than 3.0%, and there was a significant difference. Therefore, 0.3g of a sample having a large value (total peak area/sample) was selected as the sample for the sample solution preparation.
TABLE 5 comparison of sample volumes
In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking a proper amount of the product, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment for 20 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
3.3 Feature map analysis method verification
3.3.1 Investigation of specificity
The blank solvent was taken and assayed as described in "3.1" above. The test shows that: the blank solvent was undisturbed (see figure 6 below) and the process was well specific.
3.3.2 Repeatability test
About 0.3g of the same batch of samples were taken and 6 parts were taken, and the measurement was performed according to the method of item 3.1 above, and a sample was introduced for 6-needle measurement. The measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number (see fig. 7 below). The characteristic spectrum has 4 common peaks, inosine is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation results showed that the relative peak area RSD value, relative retention time RSD value, were less than 2.0%, 5.0%, respectively, all within the acceptable ranges (see table 6 and table 7 below). Experiments show that the method has good reproducibility.
TABLE 6 relative retention time of the characteristic patterns of the repeatability test
TABLE 7 repeatability test characteristic spectra versus peak area
3.3.3 Precision test
A batch of about 0.3g of sample was taken and tested by the test method under item "3.1" with 6 needles continuously introduced. The measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number (see fig. 8 below). The characteristic spectrum has 4 common peaks, inosine is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation results showed that the relative peak area RSD value, relative retention time RSD value, were less than 2.0%, 5.0%, respectively, all within the acceptable ranges (see tables 8 and 9 below). Experiments show that the method has good precision.
TABLE 8 relative retention time of the characteristic spectra for precision test
TABLE 9 precision test characteristic spectrum relative peak area
3.3.4 Stability test
Taking about 0.3g of the fried earthworm formula particle sample, and respectively carrying out sample injection measurement at 0h, 2h, 4h, 8h, 12h and 24h according to the test method under the item "3.1". The measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number (see fig. 9 below). The characteristic spectrum has 4 common peaks, inosine is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation results showed that the relative peak area RSD value, relative retention time RSD value, respectively, was less than 2.0%, 5.0%, all within the acceptable range (see table 10 and table 11 below). The test shows that the test solution is stable within 24 hours.
TABLE 10 stability test characteristic spectra versus retention time
TABLE 11 stability test characteristic spectra versus peak area
3.3.5 Durability inspection
(1) Investigation of different chromatographic columns
A batch of 0.3g of samples was taken and tested by the test method under item "6.2" using 2 chromatographic columns (250 mm x4.6mm,5 μm) of different models from different manufacturers (medium-spectrum red RD-C18, shimadzu GIST AQ-C18, respectively) by sample injection. The measurement results show that the characteristic patterns of the test samples have inconsistent peak shapes and peak numbers (see figure 10 below). The characteristic spectrum has 4 common peaks, inosine is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation results showed that the relative retention time RSD value, the relative peak area RSD value, were greater than 3.0% and greater than 5.0%, respectively, all within the reject ranges (see table 12 and table 13 below). Experiments show that chromatographic columns of different models of different manufacturers have great influence on the characteristic spectrum measurement, namely, the characteristic spectrum measurement can only be carried out by using chromatographic columns of specified models.
TABLE 12 chromatographic column test characteristic pattern relative retention time
TABLE 13 chromatographic column test characteristic patterns versus peak area
(2) Investigation of different column temperatures
A batch of about 0.3g of the sample was taken and assayed by the test method under item "3.1" at a column temperature of 18℃and 20℃and 22℃respectively. The measurement results showed that the characteristic patterns of the test samples were substantially identical in peak shape and peak number (see FIG. 11 below). The characteristic spectrum has 4 common peaks, inosine is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD value and the relative peak area RSD value are respectively smaller than 3.0% and 5.0%, and are all in the acceptable range (see the table below). The test shows that the column temperature changes by +/-2 ℃, the influence on the characteristic spectrum measurement is small, namely the durability of different column temperatures is good.
TABLE 14 column temperature test characteristic spectra relative retention time
TABLE 15 column temperature test characteristic spectra relative peak area
3.3 Knots
The characteristic spectrum method meets the requirements through the verification of specificity, precision, repeatability and stability and the investigation of durability, and the results show that the established method can be well used for the characteristic spectrum measurement of the fried earthworm (limnodrilus).
3.4 Determination of three batches of finished product characteristic maps
3.4.1 Determination of three batches of finished product characteristic maps
According to the proposed characteristic spectrum analysis method, measuring characteristic spectrums of 3 batches of fried earthworms (limnodrilus ginseng), and positioning through inosine, the result shows that 4 common peaks exist in the characteristic chromatograms of three batches of the fried earthworms (limnodrilus ginseng), and the retention time of the 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material corresponds to that of the corresponding 4 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein the peak corresponding to the reference substance of the inosine is peak 3, and the characteristic spectrums of the common peaks; see in detail figures 12-18.
3.4.2 Determination of the characteristic Pattern relative Retention time
According to the above-mentioned characteristic spectrum analysis method, 3 batches of fried earthworm (limnodrilus) pilot sample characteristic spectrum are measured. The results showed that there were 4 common peaks in the characteristic spectra, the relative retention time of the common peak and the S peak was calculated with the peak (3) corresponding to the inosine reference peak as the reference peak S, the relative retention time and the range thereof (see table 16 below), and the relative retention time of the characteristic peaks peak 1, peak 2, and peak 4 with the peak corresponding to the inosine reference as the S peak, the relative retention time and the range thereof were calculated.
Table 163 batch pilot sample characteristic pattern relative retention time
In summary, the standard decoction feature spectrum measurement method established by adopting the high performance liquid chromatography is adopted, and the established method is subjected to precision, repeatability, stability verification and durability investigation according to the four-part analysis method verification guiding principle (general rule 9101) of the Chinese pharmacopoeia 2020 edition, and meets the requirements. And determining the characteristic spectrum of 3 batches of pilot samples according to the drawn characteristic spectrum analysis method. The results showed that 4 common characteristic peaks were calibrated, with peak 3 being the inosine reference characteristic peak. Calculating the relative retention time of peak 1, peak 2, peak 4 and S peak by taking peak 3 corresponding to the inosine reference as S peak, and setting the average value of the relative retention time of characteristic peaks of 3 batches of pilot samples as a specified value, wherein the specific values are respectively as follows: 0.72 (Peak 1), 0.79 (Peak 2), 1.46 (Peak 4), combined with standard decoction, pilot and pilot test results and test errors to determine the relative retention time of each peak allowed range of + -10%.
4 Extract
4.1 Test methods
The extraction solvent of the traditional Chinese medicine formula granule is water for pharmacy, ethanol is used as solvent according to the technical requirement, and the solvent is measured according to a hot dipping method under the condition of alcohol-soluble extract measurement method (2201 in the general rule of the year 2020 of Chinese pharmacopoeia).
4.2 Investigation of extract
15 Batches of standard decoction are taken, ethanol is used as a solvent according to the technical requirement, and the result is determined according to a hot dipping method under the condition of alcohol-soluble extract determination method (2201 in the general rule of Chinese pharmacopoeia 2020 edition), and the result is shown in the following table 17 (a).
Table 17 (a) extract measurement results
The result shows that the average value of 15 batches of standard decoction extract is 36.47%, and the lower limit of the allowable range of the reference standard limit (average value 70% -130%) is not less than 25.5%. The measurement results of 15 batches of standard decoction meet the requirements of the planned limit.
3 Pilot samples, each about 2g, were taken and ground and assayed as indicated in Table 17 (b) below.
Table 17 (b) extract investigation results
The average extract of three pilot samples was 20.6%. According to the specification of the product (each 1g of formula particle is equivalent to 5g of decoction pieces), the allowable range of the extract is calculated to be not less than 12.8% by referring to the lower limit of 15 batches of standard decoction extract limit (25.5%) and the allowable range of the extract yield (10% -18%). The alcohol-soluble extract limit of the finished granule was formulated to be 17.8% based on 70% of the standard soup extract limit (25.5%), and the 70% combined with the mean value of the pilot sample extract was formulated to be 14.5%, so that the alcohol-soluble extract of the product was formulated to be not less than 14.5%.
5 Content determination
5.1 Content of the ingredients to be measured
According to other provincial standards, the content detection index of this herb is inosine.
5.2 Test methods
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as filler (column length of 250mm, inner diameter of
4.6Mm, particle size 5 μm); acetonitrile-0.01 mol/L potassium dihydrogen phosphate solution (3:97) is taken as a mobile phase; the flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength was 210nm. The theoretical plate number should be not less than 3000 calculated as inosine peak.
Preparation of a control solution: taking a proper amount of inosine reference substance, precisely weighing, and adding 70% methanol to prepare a solution containing 20 mug of inosine per 1 ml.
Preparation of test solution: taking a proper amount of the product, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
5.3 Content determination methodology investigation
5.3.1 Investigation of the extraction method
Sample solutions were prepared in different extraction modes, and were measured according to the test method "5.2" described above. The results indicated that the samples were sonicated with the highest content (see table 18 below), so the sample extraction mode was chosen to be sonication.
Table 18 comparison of different extraction methods
5.3.2 Investigation of extraction time
According to the method described in item "5.2", samples were extracted at different times, and test solutions were prepared and measured according to the law. The results indicated that the sample was sonicated for 30 minutes with the highest content (see table 19 below), so the sample sonication time was chosen to be 30 minutes.
TABLE 19 comparison of different extraction times
5.3.3 Investigation of extraction solvent
According to the method described in item "5.2", the sample solutions were prepared by extracting the samples with different solvents, respectively, and the measurement was carried out according to the law. The results indicated that the solvent was 30% methanol with the highest content (see table 20 below), so the sample solvent was chosen to be 30% methanol.
TABLE 20 comparison of different extraction solvents
5.3.4 Take-up investigation
According to the method described under item "5.2", test solutions were prepared at different sampling amounts (0.3 g, 0.5g, 0.7 g), and the result showed that the sampling amount was 0.3g and the content was the highest (see Table 21 below), so that the sampling amount of the sample was 0.3g.
Table 21 comparison of different sample amounts
In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking a proper amount of the product, grinding, taking about 0.3g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
5.4 Content determination analysis method verification
5.4.1 Specificity test
The blank solvent was taken and tested according to the test method described above under "5.2", which showed that: the blank solvent was undisturbed (see figure 19 below) and the process was well specific.
5.4.2 Repeatability test
About 0.3g of standard decoction samples of the same batch are taken, 6 parts of standard decoction samples are taken, the measurement is carried out according to the test method of 5.2, the average value of inosine content in the measured samples is 13.5692mg/g, the RSD value is 0.66%, and the test shows that the repeatability of the method is good (see table 22 below).
Table 22 repeatability test
5.4.3 Precision test
A batch of standard decoction sample of about 0.3g was taken, 6 needles were continuously introduced according to the above-mentioned "5.2", the peak area was measured, the RSD value of the inosine peak area in the sample was calculated to be 0.16%, and the test showed that the instrument precision was good (see Table 23 below).
Table 23 precision test
5.4.4 Stability test
About 0.3g of a batch of decoction samples are taken, and samples are respectively taken at 0h, 2h, 4h, 8h, 12h and 24h according to the test method of '5.2', the peak areas are measured, the RSD value of the inosine peak areas in the samples is calculated to be 0.57%, and the test shows that the test sample solution is stable within 24 hours (see table 24 below).
Table 24 stability test
5.4.5 Linear Range test
Inosine control solution was taken at a concentration μg/ml:1.0029, 10.0290, 20.0580, 50.1450, 100.2900, 200.5800 were determined under chromatographic conditions under item "5.2". Drawing a standard curve by taking inosine concentration as an abscissa and peak area as an ordinate, performing linear regression,
The regression equation is: y= 34135x-24666R 2 =0.9999
It can be seen that inosine has a good linear relationship with its peak area in the range of 1.0029. Mu.g/ml to 200.5800. Mu.g/ml (see Table 25 below and FIG. 20 below).
Table 25 content Standard linearity investigation results
5.4.6 Durability inspection
(1) Investigation of different chromatographic columns
A batch of samples of about 0.3g was taken and assayed by the "5.2" test method using 3 columns of different types (250 mm x4.6mm,5 μm) (Agilent TC-C18 (2), medium spectrum red RD-C18, shimadzu GIST AQ-C18, respectively) from different manufacturers. The calculation showed that the measured RSD value of the content was 11.71%, more than 3.0%, in the reject range (see table 26 below). Experiments show that the chromatographic columns of different models of different manufacturers have great influence on the content measurement, namely, the chromatographic columns of specified models can only be used for the content measurement.
Table 26 investigation results of different chromatographic columns
(2) Investigation of different column temperatures
A batch of about 0.3g of the sample was taken and assayed by the test method under item "5.2" at a column temperature of 18℃and at a temperature of 20℃and at a temperature of 22℃respectively. Experiments show that the method has good durability to small changes of column temperature.
Table 27 examination results of different column temperatures
(3) Investigation of different flow rates
A batch of about 0.3g of the sample was sampled and assayed by the test method under item "5.2" at a flow rate of 0.6ml/min, 0.8ml/min, 1.0ml/min, respectively. The calculated results show that the RSD value of the measured content is 1.20% and less than 3.0% (see table 28 below), and the test shows that the method has good durability against small variations in flow rate.
Table 28 investigation results of different flow rates
(4) Investigation of the proportion of different mobile phases
A batch of about 0.3g of sample was taken and assayed as described under "7.2" at organic to aqueous ratios of 3:97, 2:98, 1:99, respectively. The calculation shows that the RSD value of the measured content is 1.57% and less than 3.0% (see table 29 below), and the test shows that the method has good durability against small variations in flow rate.
Table 29 examination results of different mobile phase ratios
In conclusion, the whole analysis method meets the requirements of specificity, precision, repeatability, stability, linearity and durability inspection, and the established method can be well used for content measurement of content standard substances.
5.5 Three batch product content determination
According to the above-mentioned formulated content analysis method, the inosine content of 15 batches of standard decoction of fried earthworm (limnodrilus) and 15 batches of standard decoction of fried earthworm (limnodrilus) used for preparation thereof were measured, and the results are shown in Table 30 below.
Table 3015 measurement of standard decoction of stir-fried Pheretima (limnodrilus)
The average inosine content in the standard decoction of the product is 13.32mg/g, the measured content range is 11.230-14.423 mg/g, and the SD is 1.27; the allowable range of the inosine content is 9.32-17.32 mg/g calculated by the mean value of +/-30 percent. Therefore, the inosine content range of the standard decoction is assumed to be: 9.3 mg/g-17.3 mg/g. The results show that inosine and the transfer rate thereof in 15 batches of standard decoction are all within the allowable range, and can provide reference for the quality research of the fried earthworm (limnodrilus ginseng) formula particles.
The inosine content of the 3 batches of finished products was measured according to the above-described proposed content analysis method, and the results are shown in Table 31 below.
Table 31 results of three pilot plant product content measurements
According to the content range, the paste yield and the specification of the standard decoction of the fried earthworm (the limnodrilus), the content range of the standard decoction of the fried earthworm (the limnodrilus) is as follows: 9.3-17.3 mg/g, and the allowable range of the standard decoction plaster yield is 10-18%. The specification of the product is that each 1g of granule is equivalent to 5g of decoction pieces, and the dosage range of the auxiliary materials is 1-10% by 5=50%, 1-18% by 5=10% according to the calculation. Namely, the dosage range of the auxiliary materials is 10-50%, so the content range of the calculated formula particles is as follows: 9.3 (1-50%) =4.65 mg/g,17.3 (1-10%) =15.6 mg/g, i.e. the acceptable content range of the content standard substance is 4.6 mg/g-15.6 mg/g, so the content range of the product is 4.6 mg/g-15.6 mg/g.
The result of the measurement of the pilot sample according to the method is 10.97-11.40 mg/g. The average content was 11.17mg/g. Are within the limits.
6 Test item test
6.1 General term examination of granules: according to the related requirements under the rule 0104 granule item of the fourth edition of the Chinese pharmacopoeia 2020, the samples (batch numbers: 220301, 220302 and 220303) are subjected to granularity moisture and solubility examination, and the results are shown in the following table, so that the related requirements under the rule 0104 granule item of the fourth edition of the Chinese pharmacopoeia 2020 are met.
Table 32 examination of sample in the preparation of Lumbricus formulation
| Inspection item | 220301 | 220302 | 220303 |
| Moisture (less than or equal to 8.0 percent) | 5.5％ | 5.5％ | 5.2％ |
| Particle size (less than or equal to 15 percent) | 6％ | 5％ | 6％ |
| Solubility of | Meets the regulations | Meets the regulations | Meets the regulations |
6.2 Exogenous toxic and harmful substances detection: the medicinal materials and decoction pieces are collected in the first part of the 2020 edition of Chinese pharmacopoeia, and the standard requirements for controlling related harmful substances are that the granule preparation is not researched in related projects.
7. Specification of: the preparation method is formulated according to the feeding amount of the preparation method and the preparation amount of the process study, namely each 1g of formula particle is equivalent to 5g of decoction pieces.
8. And (3) storage: and (5) formulating according to the basic requirements of variety storage.
Those of ordinary skill 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 protection of the application 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 application, the steps may be implemented in any order and there are many other variations of the different aspects of one or more embodiments of the application as described above, which are not provided in detail for the sake of brevity.
One or more embodiments of the present application are intended to embrace all such alternatives, modifications and variations as fall within the broad scope of the present application. Accordingly, any omissions, modifications, equivalents, improvements and others which are within the spirit and principles of the one or more embodiments of the application are intended to be included within the scope of the application.
Claims (10)
1. The quality detection method of the fried earthworm formula particles is characterized by comprising the following detection methods:
Carrying out thin layer identification, characteristic spectrum construction and inosine content measurement on the fried earthworm prescription granule, limiting the inosine content standard of the fried earthworm prescription granule to 4.6mg/g-15.6mg/g, wherein the thin layer identification adopts a thin layer chromatography for identification, and the characteristic spectrum construction and the inosine content measurement are both measured by adopting a liquid chromatography;
Wherein, the characteristic spectrum is determined by liquid chromatography, octadecylsilane chemically bonded silica is taken as a filler, acetonitrile is taken as a mobile phase A, 0.01mol/L potassium dihydrogen phosphate 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
The flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength is 210nm;
the step of determining the characteristic spectrum by liquid chromatography comprises the following steps:
S11: preparing a reference substance solution a of a control medicinal material: taking 1.0g of earthworm control medicine, adding 25mL of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, and taking a subsequent filtrate as a reference substance solution a of the control medicine; taking an appropriate amount of inosine reference substance, precisely weighing, adding methanol to prepare a solution containing 20 mug of inosine per 1ml of the solution as reference substance solution a;
S12: preparing a test sample solution a: precisely weighing 0.3g of ground fried earthworm formula particles, placing into a container, adding 25mL of 30% methanol, carrying out ultrasonic treatment for 20 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the finished product;
S13: respectively sucking reference solution a of reference material, reference solution a of reference material and sample solution a of sample 10 μl, and measuring with liquid chromatograph.
2. The method for detecting the quality of the fried earthworm prescription granule as claimed in claim 1, wherein the thin layer chromatography comprises the following steps:
s21: taking 1.0g of fried earthworm prescription granule sample, adding 10ml of boiling water for dissolution, cooling, centrifuging, and taking supernatant as a sample solution b;
s22: taking L-lysine reference substance, leucine reference substance and valine reference substance, respectively adding water to prepare solutions containing 1mg, 1mg and 0.5mg of each 1ml of the solutions as reference substance solutions b;
S23: thin layer chromatography was performed, thin layer plate: silica gel G thin layer plate; sample application amount: sucking 3. Mu.l of the test solution b and 3. Mu.l of the reference solution b; developing agent: n-butanol-glacial acetic acid-water; color development: spraying ninhydrin test solution, heating at 105deg.C until the spots are clear, and inspecting under sunlight.
3. The method for detecting the quality of the fried earthworm prescription granule as claimed in claim 1, wherein the thin layer chromatography comprises the following steps:
S31: taking 1.0g of stir-fried earthworm prescription granule sample, adding 20ml of chloroform, carrying out ultrasonic treatment for 20min, filtering, evaporating filtrate to dryness, and dissolving residues with 1ml of chloroform to obtain a sample solution c;
S32: adding chloroform into 1.0g of parched Lumbricus control material, ultrasonic treating for 20min, filtering, evaporating filtrate, and dissolving residue with chloroform 1ml to obtain control material solution c;
S33: performing thin layer chromatography under the following conditions: silica gel G thin layer plate; sample application amount: 5uL of each test solution c and control medicinal solution c; developing agent: toluene-acetone was inspected under 365nm UV lamp.
4. The method for detecting the quality of the fried earthworm formula particles as claimed in claim 1, wherein the step of measuring the inosine content by liquid chromatography comprises the following steps:
s41: taking a proper amount of inosine reference substance, precisely weighing, adding 70% methanol to prepare a solution containing 20 mug of inosine per 1ml, and taking the solution as reference substance solution d;
s42: weighing 0.3g of fried earthworm formula particles, placing the particles into a bottle, precisely adding 25mL of 30% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution d;
S43: precisely sucking 5 μl of the reference solution d and the sample solution d, respectively, and injecting into a liquid chromatograph for measurement;
Wherein, the liquid chromatography conditions adopt octadecylsilane chemically bonded silica as filler, and the volume ratio is 3: acetonitrile of 97: 0.01mol/L potassium dihydrogen phosphate solution is a mobile phase; the flow rate is 0.8ml per minute; the column temperature is 20 ℃; the detection wavelength was 210nm.
5. The method for detecting the quality of the fried earthworm formula particles as claimed in claim 1, wherein 30% of methanol is added in the preparation of the reference solution a of the reference medicinal material in the step S11; 70% methanol by volume is added in the preparation of the reference substance solution a.
6. The method for detecting the quality of the fried earthworm formula particles as claimed in claim 2, wherein in the step S23, n-butanol is used: glacial acetic acid: the volume ratio of water is 4:1:1.
7. The method for detecting the quality of the fried earthworm formulation particles as claimed in claim 3, wherein in the step S33, toluene: the volume ratio of acetone is 9:1.
8. The method for detecting the quality of the fried earthworm prescription granule as in any one of claims 1 to 7, which is characterized by further comprising the following detection method: and (3) carrying out character identification, general examination and extract examination on the fried earthworm prescription granules, wherein the extract is measured by adopting a hot dipping method.
9. The method for detecting the quality of the fried earthworm formula particles as claimed in claim 8, wherein the hot dipping method uses ethanol as a solvent, and the range of the extract is measured by adopting a hot dipping method under the alcohol-soluble extract measuring method.
10. The method for detecting the quality of the fried earthworm formula particles as claimed in claim 8, wherein the general term inspection of the particles comprises inspection of moisture, granularity and dissolubility.
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