CN110794079A - Identification method of ephedra and aconitine preparation - Google Patents
Identification method of ephedra and aconitine preparation Download PDFInfo
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Abstract
The invention provides a method for identifying a ephedra and aconitine preparation, which adopts high performance liquid chromatography to identify monkshood and uses polar ether to connect phenyl bonded silica gel as a chromatographic column of a filling agent; the method adopts the thin-layer chromatography to identify the ephedra and the asarum, and can identify the ephedra-aconitine preparation with simplicity, accuracy, strong specificity and good durability.
Description
Technical Field
The invention relates to a medicine, in particular to an identification method of a ephedra, aconitum carmichaeli and ephedra, aconitum carmichaeli preparation, and belongs to the technical field of medicines.
Technical Field
The ephedra-aconite preparation is a preparation prepared from ephedra, monkshood and asarum, is short for preparation, consists of monkshood, ephedra and asarum and has the effects of relieving exterior syndrome, dispelling cold, consolidating body resistance and activating yang.
The herba Ephedrae and Aconiti lateralis Preparata preparation contains radix Aconiti lateralis Preparata. The appearance of the prepared preparation of the ephedra and the asarum is basically consistent with that of other traditional Chinese medicine preparations, and visual identification cannot be carried out.
Disclosure of Invention
The invention aims to provide an identification method of a ephedra aconite preparation, which is characterized in that monkshood in the ephedra preparation is identified by high performance liquid chromatography, asarum in the ephedra aconite preparation is identified by thin layer chromatography, and the ephedra in the ephedra aconite preparation is negative and free of interference, and has good specificity and durability.
The purpose of the invention is realized by the following technical scheme:
the identification method of the ephedra and aconitine preparation is characterized by comprising the following steps: identifying herba Ephedrae and herba asari in the preparation by thin layer chromatography, and identifying radix Aconiti lateralis Preparata by high performance liquid chromatography.
The identification method of the ephedra in the ephedra aconitine preparation is characterized by adopting the thin-layer chromatography to identify the ephedra: grinding the sample, weighing 0.5-2g, adding concentrated ammonia, adding chloroform 30ml, heating and refluxing for 60min, cooling, filtering, evaporating filtrate, and dissolving the residue with methanol 2ml to obtain sample solution; adding methanol into ephedrine hydrochloride reference to obtain 1mg solution per 1ml as reference solution; sucking the above two solutions 5-15 μ l each, dropping on silica gel G thin layer plate, spreading with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying ninhydrin solution, heating at 105 deg.C until spots appear clearly; in the chromatogram of the test solution, the same red spot appears at the position corresponding to the chromatogram of the reference solution, and the product is determined to be ephedra-containing ephedra herba preparation;
the identification method of the ephedra and aconite preparation is characterized by adopting the thin-layer chromatography to identify the asarum in the ephedra and aconite preparation: grinding the sample, weighing 2-6g, adding methanol 100ml, ultrasonic treating for 20min, filtering, evaporating filtrate, dissolving residue with water 30ml, shaking and extracting with diethyl ether for 3 times, each time 30ml, mixing diethyl ether solutions, volatilizing, and dissolving residue with ethyl acetate 1ml to obtain sample solution; preparing herba asari control 0.5g, and making into control solution by the same method; adding methanol into asaricin reference substance to obtain 1ml solution containing 1mg as reference substance solution; sucking 5-15 μ l of each of the three solutions, respectively dropping on silica gel G thin layer plate, developing with n-hexane-chloroform-ethyl acetate (16: 3: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and blowing with hot air until spots develop color clearly; in the chromatogram of the test solution, spots with the same color should be displayed at the corresponding positions of the chromatograms of the reference substance and the reference material, and the sample is judged to be the ephedra and aconite preparation containing asarum;
the identification method of the ephedra and aconite preparation is characterized by adopting a high performance liquid chromatography to identify monkshood in the ephedra and aconite preparation: the chromatographic condition and the system applicability test use polar ether connected phenyl bonded silica gel as a filling agent; acetonitrile-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) (21: 79) is used as a mobile phase; the detection wavelength is 222-242 nm; the number of theoretical plates is not less than 10000 calculated according to the benzoylmesaconine peak;
preparing reference solution by precisely weighing appropriate amount of benzoylmesaconine reference substance, benzoylaconitine reference substance, and benzoylhypaconine reference substance, and adding acetonitrile to obtain mixed solution containing 50 μ g, 10 μ g, and 10 μ g of acetonitrile per 1ml as reference stock solution; precisely measuring 5ml of reference substance stock solution, placing into a 25ml measuring flask, adding 0.1% phosphoric acid solution to dilute to scale, and shaking;
preparation of test solution the sample is ground, 0.3-2 g is weighed, precisely weighed, placed in a conical flask with a plug, 0.lmol/L hydrochloric acid solution 25ml is precisely added, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 40kHz) is carried out for 40 minutes while shaking constantly, cooling is carried out, the weight is weighed again, 0.lmol/L hydrochloric acid solution is used for complementing the lost weight, shaking is carried out evenly, centrifugation (rotating speed is 4000 rpm) is carried out for 30 minutes, filtration is carried out, 10ml of filtrate is precisely weighed, added on a solid phase extraction column (taking a mixed type cation exchange reverse phase adsorbent as a filling agent, 150mg or 200mg and 6ml in capacity, and acetonitrile and water are used for elution respectively in sequence of 6 ml), 3ml of water, 5 → 100 of ammonia solution, water, methanol and acetonitrile are used for elution respectively of 5m L, after the elution liquid flow of elution is completed, the mixed solution of acetonitrile-concentrated ammonia solution (90: 10) is placed for 5 minutes, collecting eluate, recovering solvent at 40 deg.C or below under reduced pressure to dryness, adding the residue into mixed solution 5m l of acetonitrile-0.1% phosphoric acid solution (20: 80) for dissolving, and filtering to obtain filtrate;
the determination method comprises precisely sucking 10-30 μ 1 each of the reference solution and the sample solution, injecting into a liquid chromatograph, and determining;
in the chromatogram of the test sample, the chromatogram peak response is at the corresponding position of the chromatogram of benzoylmesaconine, benzoylaconine and benzoylhypaconine, and the product is determined to be the ephedrine preparation containing radix Aconiti lateralis.
For a better understanding of the present invention, the following further illustrates the beneficial effects of the present invention through studies of the methodology of the present invention. Methodology is intended to further illustrate the role of the invention and is not a limitation of the invention.
Firstly, preparing a sample
1. Preparing the ephedra and aconitine granules
Taking 10kg of monkshood, 7kg of ephedra and 3.5kg of asarum, adding water, decocting twice, adding 10 times of water for 2 hours for the first time, adding 8 times of water for 2 hours for the second time, mixing the two decoctions, filtering, concentrating into thick paste, drying under reduced pressure below 80 ℃, crushing, mixing with a proper amount of dextrin, preparing into granules, drying, finishing granules to obtain the ephedra-monkshood granules.
2. Preparation of negative granules without asarum
Taking 135g of monkshood and 90g of ephedra, and preparing the negative granules without asarum according to the preparation method of the ephedra-aconite granules.
3. Preparation of negative granules without monkshood
Taking 120g of ephedra herb and 60g of asarum to prepare the negative granules without asarum according to the preparation method of the ephedra-aconite granules.
4. Preparation of Ephedra-free negative granules
Taking 120g of monkshood and 40g of asarum, and preparing the negative granules without ephedra according to the preparation method of the ephedra-monkshood granules.
Secondly, identifying ephedra in ephedra drugs by adopting thin-layer chromatography
1. Specificity test
Weighing appropriate amount of the product, weighing 0.8g, adding several drops of concentrated ammonia solution, adding 30ml of chloroform, heating and refluxing for 60min, cooling, filtering, evaporating filtrate, and dissolving residue with 2ml of methanol to obtain test solution.
Preparing herba Ephedrae negative test solution by the same method.
The reference substance solution is prepared by adding methanol into ephedrine hydrochloride reference substance to obtain 1mg solution per 1 ml.
Sucking the three solutions, 10 μ l each of the reference solution, the test solution and the negative test solution, respectively dropping on silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying with ninhydrin solution, and heating at 105 deg.C until the spots are developed clearly.
The atlas is displayed on the corresponding position of the chromatogram of the reference substance, the solution of the test sample has the same red spots, and the solution of the negative test sample has no spots on the corresponding position, which proves that the method has good specificity and is suitable for identifying the ephedra in the ephedra monkshood granules.
2. Stability test of solution
Preparing test solution and control solution according to the above method, standing the test solution at room temperature for 0, 4, and 8 hr respectively.
Sucking 10 μ l of each of the control solution and the sample solution at 0, 4, and 8 hr, respectively, dropping on silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying with ninhydrin solution, heating at 105 deg.C until the spots are clearly observed.
The result shows that the test solution is placed at room temperature for 8 hours, the method has no influence on the identification of the ephedra, and the test solution is stable
The qualitative was good.
3. Thin layer plate for different brands
Taking a sample under the solution stability test item, and carrying out identification test on the product herba Ephedrae by using different thin layer plates according to the previous method.
The results show that different thin layer plates have no influence on the identification of the ephedra.
4. Development temperature
Sampling samples under the solution stability test item, respectively spreading at 10 deg.C, 20 deg.C and 30 deg.C, and performing identification test on herba Ephedrae.
As a result, the identification of the Chinese ephedra can be effectively carried out within the normal temperature range.
The research result shows that the method is suitable for identifying the ephedra in the ephedra granule.
Thirdly, identifying the asarum in the ephedra and aconitine medicine by adopting thin-layer chromatography
Collecting 4g of the product, adding 100ml of methanol, performing ultrasonic treatment for 20min, filtering, evaporating the filtrate to dryness, dissolving the residue with 30ml of water, extracting with diethyl ether for 3 times (30 ml each time), mixing the diethyl ether solutions, volatilizing, and dissolving the residue with 1ml of ethyl acetate to obtain a sample solution.
The negative test solution is prepared by taking 7g of asarum negative preparation, and is prepared by the same method as the test solution.
Control solution: taking 0.5g of reference medicinal material, and preparing reference medicinal material solution by the same method.
Sucking 10 μ l of the above three solutions as reference substance solution, respectively dropping 10 μ l of each of the test solution and the reference medicinal material test solution on silica gel G thin layer plate, developing with n-hexane-chloroform-ethyl acetate (16: 3: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and blowing hot air to spot for clear color observation.
The spots of the test solution are clear, the spots with the same color appear at the corresponding positions of the asarum fine lignan and the reference medicinal material, and the negative test solution has no interference, and the result shows that the method has good specificity and is suitable for identifying the asarum in the ephedra and aconitum particles.
1. Thin layer plate for different brands
The product was tested for identity using different thin layer plates as before.
The results show that the use of different thin-layer plates has no influence on the identification of the asarum.
2. Development temperature
Spotting each solution, and developing at 15, 25 and 30 deg.C.
As a result, the asarum can be effectively identified within the normal temperature range.
Test results show that the method is suitable for identifying the asarum in the ephedra and aconitum particles.
Fourthly, identifying the aconite by adopting high performance liquid chromatography
1. Specificity test
The chromatographic condition and the system applicability test use polar ether connected phenyl bonded silica gel as a filling agent; acetonitrile-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) (21: 79) is used as a mobile phase; the detection wavelength was 232 nm. The number of theoretical plates is not less than 10000 calculated according to benzoylmesaconine peak.
Preparation of reference solution A proper amount of benzoylmesaconine reference substance, benzoylaconine reference substance, and benzoylhypaconine reference substance is precisely weighed, and acetonitrile is added to make into mixed solution containing 50 μ g, 10 μ g, and 10 μ g of acetonitrile per 1ml, and used as reference stock solution. Precisely measuring 5ml of the reference stock solution, placing in a 25ml measuring flask, adding 0.1% phosphoric acid solution to dilute to scale, and shaking up to obtain the final product.
Preparation of test solution about 0.7 g of the content of the product is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of 0.lmol/L hydrochloric acid solution, tightly plugged, weighed, ultrasonically treated (power 250W, frequency 40kHz) for 40 minutes, shaken at all times, cooled, weighed again, made up with 0.lmol/L hydrochloric acid solution to make up the lost weight, shaken evenly, centrifuged (rotation speed is 4000 revolutions per minute) for 30 minutes, filtered, precisely weighed 10ml of the subsequent filtrate, added on a solid phase extraction column (taking a mixed type cation exchange reversed phase adsorbent as a filler, 150mg or 200mg, with a capacity of 6ml, and eluted sequentially with acetonitrile and water each 6 ml), eluted sequentially with 3ml of water, 5 → 100 of ammonia solution, water, methanol and acetonitrile each 5m L, placed for 5 minutes after the elution liquid runs out, eluted with 10ml of mixed test solution of acetonitrile-concentrated ammonia solution (90: 10), collecting eluate, recovering solvent at 40 deg.C or below under reduced pressure to dry, dissolving the residue in mixed solution 5m l of acetonitrile-0.1% phosphoric acid solution (20: 80), metering to volume of 5ml, filtering, and collecting filtrate.
Preparation of negative sample solution A negative sample of the coupon (black coupon) was prepared as above.
The determination method comprises precisely sucking control solution, test solution and negative test solution 20 μ 1 respectively, injecting into liquid chromatograph, and determining.
The result shows that the method has no interference in negative and good separation, and the method is used for determining the monoester alkaloid in the ephedra and aconitum chinese pekinense granules and has strong specificity.
2. The invention discloses a methodological verification for identifying monkshood in a ephedra and aconitine medicine by high performance liquid chromatography
2.1 System suitability test
And (5) continuously injecting a reference substance solution for determination, and inspecting the applicability of the system. The results are shown in the following table:
results of System suitability test
Note: the degree of separation is an index for evaluating the degree of separation between the component to be measured and the adjacent coexisting substance or hardly-separable substance, "- -" in the table indicates that no chromatographic peak is detected before the target peak, and therefore the degree of separation is not shown in the integral table.
The system applicability test result shows that the repeatability of each monoester alkaloid chromatographic peak, the number of theoretical plates, the separation degree and the tailing factor determined by the method all accord with corresponding regulations in the high performance liquid chromatography, and the method has good applicability.
2.2 stability of the solution to be tested
Standing the sample solution at room temperature, sucking 20 μ l at 0, 8, 16, and 24 hr, respectively, injecting into liquid chromatograph, and measuring.
Stability results of the solutions tested
| Time of standing | 0 hour | 8 hours | 16 hours | 24 hours | RSD% |
| Peak area of benzoylmesaconine | 637940.688 | 642634.938 | 636349.625 | 638929.813 | 0.42 |
| Area of benzoylaconine peak | 73126.203 | 73002.602 | 73256.000 | 720620102 | 0.75% |
| Peak area of benzoylhypaconine | 125136.000 | 125152.297 | 125698.000 | 125989.000 | 0.34% |
The results show that the test solution has good stability within 24 hours.
2.3 reproducibility
The content of the product is taken, measured 6 times respectively according to the previous method, and the measurement results are compared.
Repetitive results
| 1 | 2 | 3 | 4 | 5 | 6 | Average | RSD% | |
| Benzoylmesaconine (μ g/g) | 652.91 | 642.79 | 641.61 | 646.02 | 647.35 | 649.15 | 646.64 | 0.972 |
| Benzoylaconine (mug/g) | 90.82 | 86.46 | 90.45 | 89.07 | 89.04 | 88.74 | 89.10 | 1.763 |
| Benzoylhypaconine (μ g/g) | 142.34 | 139.50 | 140.77 | 141.52 | 140.47 | 139.53 | 140.69 | 0.798 |
| Total of three alkaloids (μ g/g) | 886.07 | 868.75 | 872.84 | 876.61 | 876.86 | 877.43 | 876.43 | 0.658 |
The result shows that the method for determining the monoester alkaloid in the ephedra and aconite granules has good repeatability.
2.4 durability test
Comparative example of different flow phases
The contents of the product were collected and measured according to the procedure described above using different flow ratios.
Results of different flow phase ratios
| Different proportions of mobile phase | 20.5:79.5 | 21:79 | 20:80 | RSD% |
| Benzoylmesaconine microgram/g | 637.24 | 629.37 | 634.81 | 0.64 |
| Mu g/g of benzoylaconitine | 87.63 | 88.15 | 88.08 | 0.32 |
| Mu g/g of benzoylhypaconine | 142.98 | 144.04 | 146.34 | 1.19 |
| The total of the three alkaloids is mu g/g | 867.84 | 869.23 | 861.56 | 0.471 |
The results show that the method has good durability when the ratio of the mobile phase is changed within a certain range.
II different chromatographic columns
The contents of the product were collected and measured by the previous method using a column chromatography. Determination by different chromatographic columns
Results of different chromatographic columns
The result shows that the method has good durability to chromatographic columns of different brands.
Column III temperature
Taking the content of the product, and performing the measurement by adopting the column temperatures of 25 ℃, 30 ℃ and 35 ℃ respectively according to the previous method.
Results of different column temperatures
| Different column temperatures | Column temperature 25 deg.C | Column temperature 30 deg.C | Column temperature 35 deg.C | RSD% |
| Benzoylmesaconine microgram/g | 637.07 | 626.97 | 640.88 | 1.13 |
| Mu g/g of benzoylaconitine | 87.76 | 87.04 | 89.44 | 1.240 |
| Mu g/g of benzoylhypaconine | 144.97 | 145.19 | 145.80 | 0.30 |
| The total of the three alkaloids is mu g/g | 869.80 | 859.19 | 872.56 | 0.99 |
The result shows that the method has good durability when the column temperature is 25-35 ℃.
IV flow Rate
The contents of the product were collected and measured as before using flow rates of 0.8, 1.0, and 1.2ml/min, respectively.
Results of different flow rate measurements
| Different flow rates | Flow rate 0.8ml/min | Flow rate 1.0ml/min | Flow rate 1.2ml/min | RSD% |
| Benzoylmesaconine microgram/g | 638.04 | 642.59 | 633.80 | 0.69 |
| Mu g/g of benzoylaconitine | 87.83 | 84.78 | 86.35 | 1.77 |
| Mu g/g of benzoylhypaconine | 145.79 | 144.84 | 145.49 | 0.33 |
| The total of the three alkaloids is mu g/g | 871.66 | 872.21 | 865.64 | 0.42 |
The result shows that the method has good durability when the flow rate is 0.8-1.2 ml/min.
V different wavelengths
The content of the product is measured by the wavelength of 230nm, 232nm and 234nm according to the previous method.
Measurement results at different wavelengths
| Different wavelengths | 230nm | 232nm | 234nm | RSD% |
| Benzoylmesaconine microgram/g | 649.25 | 641.99 | 636.98 | 0.96 |
| Mu g/g of benzoylaconitine | 91.38 | 90.48 | 89.33 | 1.20 |
| Mu g/g of benzoylhypaconine | 147.19 | 150.68 | 142.43 | 2.68 |
| The total of the three alkaloids is mu g/g | 887.82 | 883.14 | 869.03 | 1.11 |
The results show that the method has good durability when the wavelength fluctuates within +/-2.
The test result shows that the method is suitable for identifying the monkshood in the ephedra and aconitine granules.
Examples
The invention is further illustrated by the following specific examples.
Example 1: identification of ephedra and aconitine capsules
(1) Preparing capsules:
taking 12kg of asarum, 8kg of ephedra herb and 4kg of asarum, adding water for decocting twice, adding 8 times of water for decocting for 2 hours for the first time, adding 8 times of water for decocting for 2 hours for the second time, combining the two decoctions, filtering, concentrating into thick paste, drying under reduced pressure below 80 ℃, crushing, mixing with a proper amount of starch, and encapsulating to obtain the macleyin capsule.
(2) Identification test
I identification of aconite
The chromatographic condition and the system applicability test use polar ether connected phenyl bonded silica gel as a filling agent; acetonitrile-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) (21: 79) is used as a mobile phase; the detection wavelength was 222 nm. The number of theoretical plates is not less than 10000 calculated according to benzoylmesaconine peak.
Preparation of reference solution A proper amount of benzoylmesaconine reference substance, benzoylaconine reference substance, and benzoylhypaconine reference substance is precisely weighed, and acetonitrile is added to make into mixed solution containing 50 μ g, 10 μ g, and 10 μ g of acetonitrile per 1ml, and used as reference stock solution. Precisely measuring 5ml of the reference stock solution, placing in a 25ml measuring flask, adding 0.1% phosphoric acid solution to dilute to scale, and shaking up to obtain the final product.
Preparation of test solution A test sample is finely ground, 0.3g is weighed, placed in a conical flask with a plug, 0.lmol/L hydrochloric acid solution 25ml is precisely added, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 40kHz) is carried out for 40 minutes, shaking is carried out at all times, cooling is carried out, the weight is weighed again, 0.lmol/L hydrochloric acid solution is used for complementing the lost weight, shaking is carried out evenly, centrifugation (rotating speed is 4000 revolutions per minute) is carried out for 30 minutes, filtration is carried out, 10ml of subsequent filtrate is precisely weighed, the subsequent filtrate is added to a solid phase extraction column (taking a mixed type cation exchange reversed phase adsorbent as a filling agent, 150mg or 200mg, the capacity is 6ml, acetonitrile and 6ml of water are used for elution in advance in sequence), 3ml of water, 5 → 100 of ammonia solution, water, methanol and 5m L of acetonitrile are used for elution in sequence, after the eluent is completely flowed, the subsequent elution is carried out by 10ml of mixed solution of acetonitrile-concentrated ammonia solution (90: 10), collecting eluate, recovering solvent at 40 deg.C or below under reduced pressure to dry, dissolving the residue in 5ml of mixed solution of acetonitrile-0.1% phosphoric acid solution (20: 80), diluting to 5ml, filtering, and collecting filtrate.
The determination method comprises precisely sucking 10 μ 1 each of the reference solution, the test solution and the negative test solution, injecting into a liquid chromatograph, and determining.
As a result: in the chromatogram of the test sample, there is a peak response at the corresponding position of the chromatogram with benzoylmesaconine, benzoylaconine and benzoylhypaconine.
And (4) conclusion: the capsule contains radix Aconiti lateralis Preparata.
II identification of Chinese ephedra
Weighing appropriate amount of the product, weighing 0.5g, adding several drops of concentrated ammonia solution, adding 30ml of chloroform, heating and refluxing for 60min, cooling, filtering, evaporating filtrate, and dissolving residue with 2ml of methanol to obtain test solution.
The reference substance solution is prepared by adding methanol into ephedrine hydrochloride reference substance to obtain 1mg solution per 1 ml.
Sucking the above solutions, respectively dropping 5 μ l of each of the reference solution, the test solution and the test solution on silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying with ninhydrin solution, heating at 105 deg.C until spots appear clearly. The same red spot should appear on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
As a result: the same red spot appears on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
And (4) conclusion: the capsule contains herba Ephedrae.
III Asarum identification
Collecting 2g of the product, adding 100ml of methanol, performing ultrasonic treatment for 20min, filtering, evaporating the filtrate to dryness, dissolving the residue with 30ml of water, extracting with diethyl ether for 3 times (30 ml each time), mixing the diethyl ether solutions, volatilizing, and dissolving the residue with 1ml of ethyl acetate to obtain a sample solution.
Control solution: taking 0.5g of reference medicinal material, and preparing reference medicinal material solution by the same method.
Sucking 10 μ l of the above three solutions as control solution, respectively dropping 5 μ l of each of the test solution and the control solution on silica gel G thin layer plate, developing with n-hexane-chloroform-ethyl acetate (16: 3: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and blowing hot air to spot for clear color observation. In the chromatogram of the test solution, spots of the same color should appear at the corresponding positions of the chromatograms of the reference solution and the reference medicinal material.
As a result: in the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatograms of the reference solution and the reference medicinal material.
And (4) conclusion: the capsule contains herba asari.
Example 2: identification of ephedra, aconite and asarum tablets
(1) Preparing tablets:
taking 13kg of asarum, 7kg of ephedra and 5kg of asarum, adding water for decocting twice, adding 10 times of water for decocting for 2 hours for the first time, adding 8 times of water for decocting for 1 hour for the second time, merging the filtrate, concentrating into thick paste, drying under reduced pressure below 80 ℃, crushing, mixing with a proper amount of starch, granulating by using 70% ethanol as a wetting agent, adding a proper amount of magnesium stearate, mixing uniformly, and tabletting to obtain the ephedra-aconitine tablet.
(2) Identification test
I identification of aconite
The chromatographic condition and the system applicability test use polar ether connected phenyl bonded silica gel as a filling agent; acetonitrile-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) (21: 79) is used as a mobile phase; the detection wavelength was 242 nm. The number of theoretical plates is not less than 10000 calculated according to benzoylmesaconine peak.
Preparation of reference solution A proper amount of benzoylmesaconine reference substance, benzoylaconine reference substance, and benzoylhypaconine reference substance is precisely weighed, and acetonitrile is added to make into mixed solution containing 50 μ g, 10 μ g, and 10 μ g of acetonitrile per 1ml, and used as reference stock solution. Precisely measuring 5ml of the reference stock solution, placing in a 25ml measuring flask, adding 0.1% phosphoric acid solution to dilute to scale, and shaking up to obtain the final product.
Preparation of test solution the sample is ground, 2g is weighed, placed in a conical flask with a plug, 0.lmol/L hydrochloric acid solution 25ml is added precisely, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 40kHz) is carried out for 40 minutes, shaking is carried out constantly, cooling is carried out, the weight is weighed again, 0.lmol/L hydrochloric acid solution is used for complementing the lost weight, shaking is carried out evenly, centrifugation (rotating speed is 4000 revolutions per minute) is carried out for 30 minutes, filtration is carried out, 10ml of filtrate is taken precisely, the filtrate is added on a solid phase extraction column (taking mixed cation exchange reversed phase adsorbent as a filling agent, 150mg or 200mg, the capacity is 6ml, acetonitrile and 6ml of water are used for elution in advance in turn), 3ml of water, 5 → 100 of ammonia solution, water, methanol and 5m L of acetonitrile are used for elution respectively, after elution of elution liquid flow is carried out, the solution is placed for 5 minutes, 10ml of mixed solution of acetonitrile-concentrated ammonia solution (90: 10) is used for elution, collecting eluate, recovering solvent at 40 deg.C or below under reduced pressure to dry, dissolving the residue in mixed solution 5m l of acetonitrile-0.1% phosphoric acid solution (20: 80), metering to volume of 5ml, filtering, and collecting filtrate.
The determination method comprises precisely sucking control solution, test solution and negative test solution 30 μ 1 respectively, injecting into liquid chromatograph, and determining.
As a result: in the chromatogram of the test sample, there is a peak response at the corresponding position of the chromatogram with benzoylmesaconine, benzoylaconine and benzoylhypaconine.
And (4) conclusion: the test product of MAFUXIN tablet contains radix Aconiti lateralis Preparata.
II identification of Chinese ephedra
Grinding the sample, weighing 2g, adding several drops of concentrated ammonia solution, adding 30ml of chloroform, heating and refluxing for 60min, cooling, filtering, evaporating the filtrate, and dissolving the residue with 2ml of methanol to obtain the sample solution.
The reference substance solution is prepared by adding methanol into ephedrine hydrochloride reference substance to obtain 1mg solution per 1 ml.
Sucking the above solutions, respectively dropping 15 μ l of each of the reference solution, the test solution and the test solution on silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying with ninhydrin solution, heating at 105 deg.C until spots appear clearly. The same red spot should appear on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
As a result: the same red spot appears on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
And (4) conclusion: the tablet contains herba Ephedrae.
III Asarum identification
Grinding the sample, weighing 6g, adding methanol 100ml, ultrasonic treating for 20min, filtering, evaporating the filtrate to dryness, dissolving the residue with water 30ml, shaking and extracting with diethyl ether for 3 times, each time 30ml, mixing the diethyl ether solutions, volatilizing, and dissolving the residue with ethyl acetate 1ml to obtain the sample solution.
Control solution: taking 0.5g of reference medicinal material, and preparing reference medicinal material solution by the same method.
Sucking the three solutions 10 μ l and 15 μ l each, respectively, dropping on silica gel G thin layer plate, developing with n-hexane-chloroform-ethyl acetate (16: 3: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and blowing with hot air until spots appear clearly. In the chromatogram of the test solution, spots of the same color should appear at the corresponding positions of the chromatograms of the reference solution and the reference medicinal material.
As a result: in the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatograms of the reference solution and the reference medicinal material.
And (4) conclusion: the tablet contains herba Ephedrae.
Claims (4)
1. The identification method of the ephedra and aconitine preparation is characterized by comprising the following steps: identifying herba Ephedrae and herba asari in the preparation by thin layer chromatography, and identifying radix Aconiti lateralis Preparata by high performance liquid chromatography.
2. The method for identifying ephedra in ephedra aconite preparation according to claim 1, wherein the identification is carried out by thin layer chromatography: grinding the sample, weighing 0.5-2g, adding concentrated ammonia, adding chloroform 30ml, heating and refluxing for 60min, cooling, filtering, evaporating filtrate, and dissolving the residue with methanol 2ml to obtain sample solution; adding methanol into ephedrine hydrochloride reference to obtain 1mg solution per 1ml as reference solution; sucking the above two solutions 5-15 μ l each, dropping on silica gel G thin layer plate, spreading with chloroform-methanol-concentrated ammonia solution (20: 5: 0.5) as developing agent, taking out, air drying, spraying ninhydrin solution, heating at 105 deg.C until spots appear clearly; the test sample shows the same red spot at the position corresponding to the control sample chromatogram, and the product is determined to be ephedrine preparation containing herba Ephedrae.
3. The method for identifying ephedra and aconite preparation according to claim 1, wherein the asarum in the ephedra and aconite preparation is identified by thin layer chromatography: grinding the sample, weighing 2-6g, adding methanol 100ml, ultrasonic treating for 20min, filtering, evaporating filtrate, dissolving residue with water 30ml, shaking and extracting with diethyl ether for 3 times, each time 30ml, mixing diethyl ether solutions, volatilizing, and dissolving residue with ethyl acetate 1ml to obtain sample solution; preparing herba asari control 0.5g, and making into control solution by the same method; adding methanol into asaricin reference substance to obtain 1ml solution containing 1mg as reference substance solution; sucking 5-15 μ l of each of the three solutions, respectively dropping on silica gel G thin layer plate, developing with n-hexane-chloroform-ethyl acetate (16: 3: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and blowing with hot air until spots develop color clearly; in the chromatogram of the test solution, spots of the same color should appear at the corresponding positions of the chromatograms of the reference substance and the reference material, and the product is judged to be the herba asari-containing herba Ephedrae and Aconiti lateralis Preparata preparation.
4. The method for identifying a macbecine preparation according to claim 1, characterized in that the high performance liquid chromatography is adopted to identify monkshood in the macbecine preparation: the chromatographic condition and the system applicability test use polar ether connected phenyl bonded silica gel as a filling agent; acetonitrile-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) (21: 79) is used as a mobile phase; the detection wavelength is 222-242 nm; the number of theoretical plates is not less than 10000 calculated according to the benzoylmesaconine peak;
preparing reference solution by precisely weighing appropriate amount of benzoylmesaconine reference substance, benzoylaconitine reference substance, and benzoylhypaconine reference substance, and adding acetonitrile to obtain mixed solution containing 50 μ g, 10 μ g, and 10 μ g of acetonitrile per 1ml as reference stock solution; precisely measuring 5ml of reference substance stock solution, placing into a 25ml measuring flask, adding 0.1% phosphoric acid solution to dilute to scale, and shaking;
preparation of test solution the sample is ground, 0.3-2 g is weighed, precisely weighed, placed in a conical flask with a plug, 0.lmol/L hydrochloric acid solution 25ml is precisely added, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 40kHz) is carried out for 40 minutes while shaking constantly, cooling is carried out, the weight is weighed again, 0.lmol/L hydrochloric acid solution is used for complementing the lost weight, shaking is carried out evenly, centrifugation (rotating speed is 4000 rpm) is carried out for 30 minutes, filtration is carried out, 10ml of filtrate is precisely weighed, added on a solid phase extraction column (taking a mixed type cation exchange reverse phase adsorbent as a filling agent, 150mg or 200mg and 6ml in capacity, and acetonitrile and water are used for elution respectively in sequence of 6 ml), 3ml of water, 5 → 100 of ammonia solution, water, methanol and acetonitrile are used for elution respectively of 5m L, after the elution liquid flow of elution is completed, the mixed solution of acetonitrile-concentrated ammonia solution (90: 10) is placed for 5 minutes, collecting eluate, recovering solvent at 40 deg.C or below under reduced pressure to dryness, adding the residue into mixed solution 5m l of acetonitrile-0.1% phosphoric acid solution (20: 80) for dissolving, and filtering to obtain filtrate;
the determination method comprises precisely sucking 10-30 μ 1 each of the reference solution and the sample solution, injecting into a liquid chromatograph, and determining;
in the chromatogram of the test sample, the chromatogram peak response is at the corresponding position of the chromatogram of benzoylmesaconine, benzoylaconine and benzoylhypaconine, and the product is determined to be the ephedrine preparation containing radix Aconiti lateralis.
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