CN114414672B - Method for determining and analyzing content of dioscin in Longxiang asthma relieving capsules - Google Patents
Method for determining and analyzing content of dioscin in Longxiang asthma relieving capsules Download PDFInfo
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- CN114414672B CN114414672B CN202111533613.8A CN202111533613A CN114414672B CN 114414672 B CN114414672 B CN 114414672B CN 202111533613 A CN202111533613 A CN 202111533613A CN 114414672 B CN114414672 B CN 114414672B
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
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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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- 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
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- 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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Abstract
The invention relates to a method for determining and analyzing the content of dioscin in Longxiang asthma-relieving capsules, which comprises the following steps: 1) Preparing a test solution: precisely weighing the contents of the dragon's fragrant asthma-relieving capsule, dissolving in methanol, filtering, taking filtrate, adding silica gel, stirring, evaporating to dryness, adding onto silica gel column, eluting with chloroform-methanol-water mixed solution as eluent, collecting eluate, evaporating to dryness, dissolving residue with methanol, filtering, and taking subsequent filtrate as sample solution; 2) Preparing a dioscin standard solution: precisely weighing dioscin standard substance, and adding methanol to obtain dioscin standard solution; 3) Detecting the sample solution and the dioscin standard solution by high performance liquid chromatography, and calculating the content of dioscin in the Longxiang antiasthmatic capsule by an external standard two-point logarithmic equation. The method can realize accurate detection of the dioscin content, and has the advantages of good linear relation, precision and stability, no interference, low detection limit and quantitative limit and high accuracy.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining and analyzing the content of dioscin in a Longxiang antiasthmatic capsule.
Background
Longxiang Pingchuan Capsule, named as Chinese patent medicine. It is prepared from Ningpo Yam rhizome, herba Cymbopogonis, cortex Magnolia officinalis, and herba Ephedrae. Has the functions of promoting blood circulation, eliminating phlegm, ventilating and smoothing lung and relieving asthma, and is used for treating bronchial asthma which belongs to the syndrome of phlegm stasis obstructing the lung, and the syndrome is shown as follows: chest oppression, wheezing, cough, expectoration, abdominal fullness and distention, and dark and gloomy complexion.
Further perfecting the quality standard of the Longxiang asthma-relieving capsule, ensuring the medicine quality, and establishing a simple, accurate and good-reproducibility method for detecting the content of dioscin in the Longxiang asthma-relieving capsule is very necessary.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the method for determining and analyzing the content of the dioscin in the Longxiang antiasthmatic capsule, which has the advantages of high precision, good repeatability, high accuracy, good stability and accurate measurement result, and accurately measures the content of the dioscin so as to ensure the stable quality of the medicine and the safety and effectiveness of clinical medication.
The invention is realized by the following technical scheme:
the method for measuring and analyzing the content of dioscin in the Longxiang asthma relieving capsule comprises the following steps:
1) Preparing a test solution: precisely weighing the contents of the Longxiang asthma-relieving capsule, dissolving in methanol, filtering, taking filtrate, adding silica gel, stirring, evaporating to dryness, adding onto silica gel column, eluting with chloroform-methanol-water mixed solution as eluent, collecting eluate, evaporating to dryness, dissolving residue in methanol, filtering, and taking subsequent filtrate as sample solution;
2) Preparing a dioscin standard solution: precisely weighing dioscin standard substance, and adding methanol to obtain dioscin standard solution;
3) Detecting the sample solution and the dioscin standard solution by high performance liquid chromatography, and calculating by using an external standard two-point method logarithmic equation to obtain the final product containing dioscin (in terms of dioscin) in each capsule, wherein the content of dioscin is not less than 5.0mg.
Further, in the step 1), the inner diameter of the column is 1cm, and the dosage of 200-300 meshes of silica gel is 10g.
Furthermore, silica gel is filled into the column by a dry method, and is balanced by eluent before use.
Further, in step 1), the volume ratio of chloroform-methanol-water is 70.
Further, in the step 1), the content of the Longxiang asthma-relieving capsule is placed in a methanol solution for ultrasonic treatment for 30min, the ultrasonic power is more than or equal to 200W, and the ultrasonic frequency is more than or equal to 20kHz.
Further, in the step 1), chloroform-methanol-water mixed solution is used as eluent, elution is carried out twice, the first 15ml is discarded, and the second 25ml is collected.
Furthermore, the elution speed is less than or equal to 0.5 drop/second.
Further, in the step 3), the conditions of the high performance liquid chromatography are as follows:
a chromatographic column: silica gel is used as a filling agent;
mobile phase: hexane-ethanol, both in a volume of 70;
sample introduction volume: 5 μ L.
Further, the detector is an evaporative light scattering detector.
The invention has the beneficial effects that:
in the quality standard of the proto-dragon fragrance asthma-relieving capsule, the detected component of the dioscorea nipponica is diosgenin which is a degradation product, and the quality of a finished medicine (the dragon fragrance asthma-relieving capsule) and the quantity of the medicinal materials used for production cannot be faithfully and accurately reflected. The quality control component of the dioscorea nipponica traditional Chinese medicinal material is dioscin which is not uniform with patent medicine, so that the phenomenon that the quality of the raw material is inconsistent with the quality of the patent medicine is caused. The analysis method can solve the problems, under the condition of stable production process, the content of the patent medicine can be accurately inferred through the content of the dioscin in the dioscorea nipponica makino traditional Chinese medicinal materials, and the quality condition of the used raw materials can also be judged through the content data of the patent medicine.
Drawings
FIG. 1 is a high performance liquid chromatogram of a solution containing 0.1mg of dioscin per lml and 0.3mg of dioscin per 1ml in example 1 of the present invention.
FIG. 2 is a high performance liquid chromatogram of the first test sample liquid in example 1 of the present invention.
FIG. 3 is a high performance liquid chromatogram of a second sample solution in example 1 of the present invention.
FIG. 4 is a high performance liquid chromatogram of the third test sample liquid in example 1 of the present invention.
FIG. 5 is a linear graph of dioscin in linear relationship test according to the present invention.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
A method for determining and analyzing the content of dioscin in Longxiang asthma relieving capsules comprises the following steps:
1) Preparing a test solution: precisely weighing 0.4g of the content of the Longxiang asthma-relieving capsule, putting the content into a 50ml measuring flask, adding about 40ml of methanol, carrying out ultrasonic treatment for 30min (power 200W and frequency 20 kHz), adding the methanol to scale, shaking up, and filtering; taking 10ml of subsequent filtrate, adding 1.0g of silica gel, stirring uniformly, evaporating to dryness, and adding onto a silica gel column, wherein: silica gel is loaded into a column by adopting a dry method with 200-300 meshes, 10g and 1cm inner diameter, before use, the column is balanced by eluent, chloroform-methanol-water mixed solution with the volume ratio of 70; continuously eluting, collecting 25ml eluate, evaporating to dryness, dissolving residue with methanol, transferring to 10ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution.
2) Preparing a dioscin standard solution: weighing Dioscorea opposita Thunb saponin standard substance precisely, and adding methanol to make into solution containing 0.1mg per lml and 0.3mg per 1ml respectively.
3) Precisely measuring the sample solution and the standard solution by 5 μ L respectively, injecting into a liquid chromatograph,
the detector is an evaporative light scattering detector. Wherein: the conditions of the high performance liquid chromatography are as follows: a chromatographic column: silica gel is used as a filling agent; mobile phase: hexane-ethanol, both in a volume of 70; sample introduction volume: 5 μ L. The theoretical plate number of the high performance liquid chromatography system is not less than 3000 calculated according to dioscin peak.
Measuring according to the chromatographic conditions, recording a chromatogram, and calculating the content of dioscin in the Longxiang antiasthmatic capsule by using an external standard two-point method logarithmic equation.
The determination of the dioscin content is carried out on 3 batches of products according to the steps of the method, and the result is as follows:
| |
1 | 2 | 3 |
| Content of dioscin/mg | 9.1 | 9.0 | 8.1 |
According to the results, the results of the dioscin content determination of 3 batches of products are all larger than 5.0mg, so that the determination and analysis method is accurate.
The content determination and analysis method of dioscin in the longxiang asthma-relieving capsule is verified below
1. Verifying used drugs, reference substances, reagents, instruments and participants
Reagent and medicine
Reference substance
| Name of reference substance | Batch number | Source |
| Dioscorea opposita saponin | 111707-201402 | China Institute for food and drug control |
Instrument for measuring the position of a moving object
The verification is based on the Chinese pharmacopoeia four-part general rule 9101-an analysis method verification guiding principle.
Verifying item authentication content and acceptance criteria
2. The analysis method verifies that:
preparation of sample solution:
solution A: mobile phase, hexane-ethanol (70.
Solution B: precisely weighing dioscin (about 31 mg) as reference substance (about 1.25 mg/ml), placing in 25ml measuring flask, dissolving in methanol, diluting to scale, and mixing.
Solution C: and precisely measuring 4ml of the solution B by using a reference substance solution (about 0.5 mg/ml) for the linear test, putting the solution B into a 10ml measuring flask, adding methanol to dilute the solution to a scale, and uniformly mixing the solution to obtain the test reagent.
Solution D: the linear test is carried out by precisely measuring 1ml of the solution C with a reference substance solution (about 0.05 mg/ml), putting the solution C into a 10ml measuring flask, adding methanol to dilute the solution to the scale, and mixing the solution C and the solution C uniformly.
Solution E: and (3) precisely measuring a reference substance solution (about 0.2 mg/ml) for the linear test, precisely measuring the solution B1.6ml, putting the solution into a 10ml measuring flask, adding methanol to dilute to a scale, and uniformly mixing to obtain the product.
Solution F: measuring reference substance solution for linear test and reference substance solution (about 0.3 mg/ml) for content measurement, precisely measuring solution C3ml, placing in 5ml measuring flask, adding methanol to dilute to scale, and mixing.
Solution G: measuring the reference substance solution for linear test and the reference substance solution (about 0.1 mg/ml) for content measurement, precisely measuring the solution E5ml, placing in a 10ml measuring flask, adding methanol to dilute to scale, and mixing well to obtain the final product.
Solution H: negative sample solution. Taking 75g of crude magnolia officinalis particles, adding 70% ethanol, soaking for 12 hours, heating, refluxing and extracting for three times, wherein each time lasts for 1.5 hours, combining filtrates, recovering ethanol under reduced pressure until no ethanol smell exists, and concentrating into a thick paste; taking 51g of minced remote lemongrass herb, adding water to extract volatile oil for 4 hours, and adsorbing the volatile oil by using a proper amount of microcrystalline cellulose for later use; filtering herba Cymbopogonis extract, decocting the residue in water for 1 hr, filtering, mixing the filtrates, and concentrating to obtain fluid extract; cutting ephedra, adding acid water solution with pH value of 3-4, decocting for three times, each time for 1.5 hours, filtering, combining the three filtrates, concentrating under reduced pressure to obtain clear paste, mixing the clear paste, drying, and uniformly mixing with microcrystalline cellulose adsorbing the volatile oil of the lemongrass and gastric-soluble film coating powder to obtain a negative sample. Taking a negative sample, accurately weighing about 0.4g, placing in a 50ml measuring flask, adding about 40ml of methanol, performing ultrasonic treatment for 30min (power is more than or equal to 200W and frequency is more than or equal to 20 kHz), adding methanol to scale, mixing uniformly, and filtering; taking 10ml of subsequent filtrate, adding 1.0g of silica gel, uniformly stirring, evaporating to dryness, adding to a silica gel column [ 200-300 meshes, 10g, 1cm of inner diameter, loading the column by a dry method, balancing by using an eluent before use ], taking a chloroform-methanol-water (70: 30) mixed solution as the eluent, eluting (the elution speed is less than or equal to 0.5 drops/second) by 15ml, and discarding; continuously eluting, collecting 25ml of eluent, evaporating to dryness, dissolving the residue with methanol, transferring to 10ml measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate.
Solution I: mixing the negative sample with the control solution, collecting solution H1ml, adding solution E1ml, and mixing.
Solution J, K, L, M, N, O and solution P, Q, R, S, T, U: and (3) precisely testing the test solution, wherein two operators respectively take about 0.4g of each of 6 samples of the contents of the Longxiang asthma-relieving capsule at different dates, precisely weighing, and respectively preparing 6 test solutions according to the preparation methods of the test solutions by using a content measurement method.
Solution V, W, X, Y, Z, AA: taking about 0.2g of the content of the Longxiang asthma-relieving capsule (batch number: 17030501), adding about 2.5ml of solution, putting the mixture into a 50ml measuring flask, adding about 40ml of methanol, and preparing a test solution according to a content measurement method to obtain the Longxiang asthma-relieving capsule. 6 samples were prepared in the same manner.
Solution AB: the test solution is prepared according to the method for measuring the content of the dioscin.
3. Methodology validation
3.1, specificity test: precisely absorbing 5 mu L of each of the solutions H and I under the chromatographic condition for measuring the content of the dioscin, injecting the solution H and the solution I into a liquid chromatograph, and recording a chromatogram.
Measurement results
Dioscin showed a peak at 7.066min, and the yam negative sample showed no peak at this time.
And (4) conclusion: the specificity of the method for measuring the content of the yam in the Longxiang asthma-relieving capsule is good.
3.2 Linear relationship test
Under the chromatographic condition of dioscin content determination, precisely absorbing 5 mu L of each of solutions C, D, E, F and G, injecting the solution into a liquid chromatograph, recording the peak area of the chromatogram, drawing a line by taking the logarithm of the concentration as a horizontal coordinate and the logarithm of the peak area as a vertical coordinate, listing a linear equation, and calculating a regression coefficient.
Regression equation and correlation coefficient
The linear graph of dioscin is shown in fig. 5.
And (4) conclusion: the concentration of the dioscin is in a range of 46.56-465.61 mu g/ml, the concentration and the logarithm of peak area form a good linear relation, and the correlation coefficient r of a linear regression equation is =0.999.
3.3 degree of precision
3.3.1 repeatability test
Under the chromatographic condition of dioscin content measurement, respectively and precisely absorbing 5 mu L of solutions F and G and solutions J, K, L, M, N and O, injecting the solutions into a liquid chromatograph, recording a chromatogram, and calculating the content according to an external standard two-point method.
Test results
And (4) conclusion: the method for measuring the content of the dioscin in the Longxiang asthma relieving capsule has good repeatability, and the RSD value of the Ningpo yam rhizome after repeated measurement for six times is 2.15%.
3.3.2 intermediate precision
Under the chromatographic condition for measuring the content of the dioscin, another operator precisely sucks 5 mu l of each of the solutions F and G and the solutions P, Q, R, S, T and U respectively and injects the sucked solutions into another liquid chromatograph, records a chromatogram, and calculates the content according to an external standard two-point method.
Test results
And (4) conclusion: the intermediate precision of the determination method of the content of the dioscin in the Longxiang asthma relieving capsule is good, and the RSD value of the Ningpo yam rhizome (calculated by dioscin) which is repeatedly determined for twelve times is 1.88%.
3.4 recovery test
Under a dioscin content determination chromatographic system, respectively and precisely measuring 5 mul of solutions F and G and solutions V, W, X, Y, Z and AA, injecting the solutions into a liquid chromatograph, recording a chromatogram, and calculating the sample measurement amount and the recovery rate according to an external standard two-point method.
Method recovery test results
And (4) conclusion: the recovery rate of dioscin in the longxiang asthma relieving capsule is 92-105%, and the recovery rate is good.
3.5 stability test of the solution
Under the chromatographic condition of dioscin content determination, precisely absorbing 5 mu l of solution AB at 0, 2, 4, 6 and 8 hours after the sample solution is prepared, injecting the solution AB into a liquid chromatograph, recording a chromatogram, and calculating the content according to an external standard two-point method.
Test results
And (4) conclusion: the content of rhizoma Discoreae Nipponicae (calculated as dioscin) in the Longxiang asthma relieving capsule is stable within 8 hours, and the content RSD is 1.87%.
4. And (4) conclusion:
results of the experiment
And (4) conclusion: the method is suitable for measuring the content of Ningpo Yam rhizome (calculated as dioscin) in Longxiang asthma relieving capsule.
Of course, the above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, and will not be described herein again; the above embodiments and drawings are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that changes, modifications, additions or substitutions within the spirit and scope of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and shall also fall within the scope of the claims of the present invention.
Claims (7)
1. A method for measuring and analyzing the content of dioscin in Longxiang asthma relieving capsules is characterized by comprising the following steps: the method comprises the following steps:
1) Preparing a test solution: precisely weighing the contents of the Longxiang asthma-relieving capsule, dissolving in methanol, filtering, taking filtrate, adding silica gel, stirring, evaporating to dryness, adding onto silica gel column, eluting with chloroform-methanol-water mixed solution as eluent, collecting eluate, evaporating to dryness, dissolving residue in methanol, filtering, and taking subsequent filtrate as sample solution;
2) Preparing a dioscin standard solution: precisely weighing dioscin standard substance, and adding methanol to obtain dioscin standard solution;
3) The determination method comprises the following steps: detecting the sample solution and the dioscin standard solution by adopting a high performance liquid chromatography respectively, wherein the conditions of the high performance liquid chromatography are as follows: a chromatographic column: silica gel is used as a filling agent; mobile phase: hexane-ethanol, both in a volume of 70; sample introduction volume: 5 microliter, the detector adopts an evaporative light scattering detector; calculating by logarithmic equation of external standard two-point method to obtain the final product containing rhizoma Dioscoreae Nipponicae not less than 5.0mg in terms of dioscin.
2. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 1, wherein the method comprises the following steps: in the step 1), the inner diameter of the column is 1cm, and the dosage of 200-300-mesh silica gel is 10g.
3. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 1 or 2, which is characterized in that: silica gel is filled into the column by a dry method, and is balanced by eluent before use.
4. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 1, wherein the method comprises the following steps: in step 1), the volume ratio of chloroform-methanol-water is 70.
5. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 1, wherein the method comprises the following steps: in the step 1), the content of the Longxiang asthma-relieving capsule is placed in a methanol solution for ultrasonic treatment for 30min, the ultrasonic power is more than or equal to 200W, and the ultrasonic frequency is more than or equal to 20kHz.
6. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 1, wherein the method comprises the following steps: in the step 1), the chloroform-methanol-water mixed solution is used as eluent to elute twice, the first 15ml is discarded, and the second 25ml is collected.
7. The method for determining and analyzing the content of dioscin in the Longxiang Pingchuan capsule according to claim 6, wherein the method comprises the following steps: the elution speed is less than or equal to 0.5 drop/second.
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