Method for removing matrix and simultaneous identification of alkaloids in compound fuyun effervescent suppository

Abstract

The application provides a method for removing matrix and simultaneously identifying alkaloids of compound fuyun effervescent suppositories, and belongs to the technical field of material component extraction, which comprises the following steps: taking the compound fuyun effervescent suppository, adding concentrated ammonia test solution, standing, making the compound fuyun effervescent suppository be fully wetted by the concentrated ammonia test solution, adding water and stirring, making the compound fuyun effervescent suppository be fully dissolved, adding a double-layer separation column, eluting, collecting the eluent, evaporating, dissolving the residue after evaporation with anhydrous ethanol, constant volume, shaking, filtering, taking the filtrate, and obtaining the test sample solution containing both matrine and berberine hydrochloride; the method for removing the matrix and non-tested components by using the double-layer separation column is effective, the removal rate of the matrix and non-tested components reaches 85%, and the enrichment effect of matrine and berberine hydrochloride is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of substance composition extraction, and relates to an extraction technology of effective components of Chinese patent medicines, in particular to a method for removing a matrix in detection of compound lotus effervescent suppositories and a method for simultaneously identifying alkaloids. BACKGROUND

[0002] The compound lotus effervescent suppository is a Chinese patent medicine for treating damp-heat type vaginal itching, including symptoms of trichomonas vaginitis, mold vaginitis and the like. The dosage form is a suppository preparation, which is composed of a matrix, a medicine powder and an effervescent agent. The matrix accounts for the largest proportion, more than 50% of the total amount of the suppository preparation, and is a mixture of polyoxyethylene stearate, monolipid and double ester formed by polyoxyethylene, stearic acid and palmitic acid. The effervescent agent is composed of sodium bicarbonate and adipic acid. The medicine powder is obtained by extraction, concentration and drying of sophora flavescens, phellodendron, cnidium, abelmoschus leaf, mugwort leaf and white alum according to the product preparation method, and mainly contains alkaloids, coumarins, flavonoids and inorganic salts (potassium aluminum sulfate) and the like.

[0003] Alkaloids are the effective components of sophora flavescens and phellodendron. The effective component of sophora flavescens is sophoramine, and the effective component of phellodendron is berberine hydrochloride. Both of them are important indicators for quality control of the compound lotus effervescent suppository. However, the matrix component contained in the compound lotus effervescent suppository will affect the detection of sophoramine and berberine hydrochloride. Therefore, in order to realize the qualitative and quantitative detection of sophoramine and berberine hydrochloride in the medicine powder, the matrix component in the compound lotus effervescent suppository needs to be removed to ensure that it does not interfere with the detection results.

[0004] Currently, three methods are mainly used to remove the matrix components and other non-tested components during the detection. The first method is solvent extraction, the process of which is as follows: taking the tested product, cutting it into thin slices, dissolving it in chloroform, filtering, drying the filter residue, adding methanol and heating to reflux extraction, filtering, and concentrating to obtain the test solution; the second method is acid-base method, the process of which is as follows: taking the tested product, cutting it into pieces, mixing, accurately weighing, placing it in a conical flask with a plug, adding hydrochloric acid solution, heating to reflux extraction, filtering, measuring the filtrate, placing it in a separatory funnel, adjusting the pH value with concentrated ammonia solution, extracting with chloroform, evaporating to dryness, dissolving the residue in the mobile phase, diluting to the mark, and shaking to obtain the test solution; the third method is dissolution liquid cooling method, the process of which is as follows: taking the tested product, cutting it into pieces, mixing, accurately weighing, placing it in a conical flask with a plug, adding water, heating in a water bath to dissolve, standing, allowing the matrix to coagulate and precipitate, filtering, measuring the filtrate, placing it in a separatory funnel, adding concentrated ammonia solution, extracting with chloroform, recovering the solvent to dryness, dissolving the residue in the mobile phase, diluting to the mark, and shaking to obtain the test solution. The first method uses chloroform to extract alkaloid components, and the polyoxyethylene stearate in the matrix is a good emulsifier and has a large molecular weight. In the preparation of the test solution, the filter paper is easily blocked, the filtering process is slow, and the filtered solution still contains polyoxyethylene stearate and other problems. In the second method, strong acid and strong base are used, which destroys the original chemical components, causes the loss of berberine hydrochloride which is also an alkaloid, and cannot be effectively quantified, affecting the quality control quality of the product. The third method is difficult to control in actual operation. The tested components are precipitated with the matrix, or wrapped in the precipitated matrix, causing deviation in the test results.

[0005] In summary, the existing methods all cause deviation in the detection results of alkaloids in the compound fuyun effervescent suppository, and cannot obtain the test solution for accurate detection of matrine and berberine hydrochloride. SUMMARY

[0006] In view of the problems that the existing methods all cause deviation in the detection results of alkaloids in the compound fuyun effervescent suppository, and cannot obtain the test solution for accurate detection of matrine and berberine hydrochloride, the present application provides a method for removing the matrix in the detection of compound fuyun effervescent suppository and a method for simultaneously identifying alkaloids.

[0007] The present application utilizes the characteristics of the compound fuyun effervescent suppository dissolved in water, and effectively removes the matrix and non-tested components by using a double-layer separation column elution method, the removal rate of the matrix and non-tested components reaches more than 85%, and the enrichment effect of matrine and berberine hydrochloride is improved; the specific technical scheme is as follows:

[0008] The method for removing the matrix in the detection of compound fuyun effervescent suppository comprises the following steps:

[0009] Take compound lotus effervescent suppository, add concentrated ammonia test solution, stand, make compound lotus effervescent suppository be fully wetted by concentrated ammonia test solution, add water and stir, make compound lotus effervescent suppository fully dissolve, add double-layer separation column, elute, collect eluent, evaporate to dryness, dissolve the residue after evaporation to dryness with anhydrous ethanol, make up to volume, shake well, filter, take the filtrate, and obtain the test solution containing both matrine and berberine hydrochloride;

[0010] The double-layer separation column comprises upper D101 macroporous adsorption resin and lower 30-60 mesh polyamide resin.

[0011] Further limited, the steps are specifically:

[0012] Take 0.3g-1g of compound lotus effervescent suppository, add 0.5ml-2ml of concentrated ammonia test solution, stand, make compound lotus effervescent suppository be fully wetted by concentrated ammonia test solution, add 50ml-200ml of water and stir, make compound lotus effervescent suppository fully dissolve, add double-layer separation column, elute, collect eluent, evaporate to dryness, dissolve the residue after evaporation to dryness with anhydrous ethanol, make up to 10ml, shake well, filter, take the filtrate, and obtain the test solution containing both matrine and berberine hydrochloride.

[0013] Further limited, the elution process is: first elute with 3-5 column volumes of deionized water, then elute with 3-5 column volumes of ethanol with a volume concentration of 10%, discard the deionized water eluent and ethanol eluent, and finally elute with 3-5 column volumes of ethanol with a volume concentration of 50%.

[0014] Further limited, the column height of the double-layer separation column is 15cm-20cm, and the inner diameter of the double-layer separation column is 1.5cm-3cm.

[0015] Further limited, the ratio of the height of the upper D101 macroporous adsorption resin to the height of the lower 30-60 mesh polyamide resin is 2:1-1:1.

[0016] The method for simultaneously identifying alkaloids in compound lotus effervescent suppository comprises the following steps:

[0017] 1) Preparation of reference solution:

[0018] Take matrine and berberine hydrochloride, add anhydrous ethanol, and prepare matrine reference solution and berberine hydrochloride reference solution, respectively. Each milliliter of the matrine reference solution contains 60ug of matrine, and each milliliter of the berberine hydrochloride reference solution contains 20ug of berberine hydrochloride.

[0019] 2) Chromatographic conditions: chromatographic column with octadecylsilane bonded silica gel as filler, acetonitrile-0.1%-0.3% phosphoric acid solution as mobile phase, acetonitrile and phosphoric acid solution in a volume ratio of 25:75; detection wavelength of 190nm-230nm; column temperature of 20°C-30°C; flow rate of 0.8ml / min-1.2ml / min;

[0020] 3) Chromatographic peak determination, the matrine control solution, berberine hydrochloride control solution and test sample solution containing both matrine and berberine hydrochloride in step 1) are injected into the liquid chromatograph, and chromatograms are recorded under the chromatographic conditions in step 2) to realize the identification of matrine and berberine hydrochloride;

[0021] The alkaloids refer to matrine and berberine hydrochloride.

[0022] Further limitation, the volume concentration of the phosphoric acid solution is 0.1%.

[0023] Further limitation, the detection wavelength is 220nm.

[0024] Further limitation, the pH value of the phosphoric acid solution is adjusted to 8.0 by triethylamine.

[0025] The thin-layer identification method of alkaloids in compound fuyong effervescent suppository comprises the following steps:

[0026] Matrine identification: the above prepared test sample solution containing both matrine and berberine hydrochloride is taken; the matrine control sample is taken and added with methanol to prepare a control sample solution containing 1mg of matrine per milliliter; according to the thin-layer chromatography test, the test sample solution and the matrine control sample solution are respectively taken and spotted on the corresponding positions of the same silica gel G thin-layer plate, a mixed solution of toluene-acetone-ethyl acetate-concentrated ammonia solution in a volume ratio of 2:3:4:0.2 is used as the developing agent, and after development, removal, air-drying, and spraying with dilute bismuth potassium iodide solution, the test sample solution and the matrine control sample solution both show the same orange-red spots, which indicates that the test sample solution contains matrine;

[0027] Berberine hydrochloride identification: the above prepared test sample solution containing both matrine and berberine hydrochloride is taken; the berberine hydrochloride control sample is taken and added with methanol to prepare a control sample solution containing 0.5mg of berberine hydrochloride per milliliter; according to the thin-layer chromatography test, the test sample solution and the berberine hydrochloride control sample solution are respectively taken and spotted on the same silica gel G thin-layer plate, a mixed solution of ethyl acetate-butyronitrile-formic acid-water in a volume ratio of 10:7:0.5:0.5 is used as the developing agent, and after development, removal, air-drying, and inspection under a 365nm ultraviolet lamp, the test sample solution and the berberine hydrochloride control sample solution both show the same yellow spots, which indicates that the test sample solution contains berberine hydrochloride.

[0028] The present application has the following advantages compared with the prior art:

[0029] 1、The method for removing matrix in the compound fuyun effervescent suppository of the application, which utilizes the characteristics of the compound fuyun effervescent suppository dissolved in water, and adopts the method of double-layer separation column elution to effectively remove the matrix components and non-test components. The double-layer separation column is composed of the upper D101 macroporous adsorption resin and the lower 30-60 mesh polyamide resin, and the removal rate reaches more than 85%, which improves the enrichment effect of matrine and berberine hydrochloride, overcomes the problem of deviation of the detection results of alkaloids caused by the existing removal method, and the prepared test solution containing matrine and berberine hydrochloride at the same time can be used for accurate detection of matrine and berberine hydrochloride.

[0030] 2、The test solution containing matrine and berberine hydrochloride at the same time obtained by the double-layer column chromatography method of the application, after thin-layer differential chromatography development, the matrix does not interfere with the determination of the chromatography results; the separation degree of berberine hydrochloride and other impurities in the thin-layer chromatogram is obviously better than that of the acid-base method in the existing quality standard, and the double-layer column chromatography method can not only prepare the test solution of matrine or berberine hydrochloride at one time, but also can improve the chromatography effect.

[0031] 3、The test solution containing matrine and berberine hydrochloride at the same time prepared by the application can be used for the determination of matrine and berberine hydrochloride at the same time, realizes one measurement for multiple evaluations of the product, and improves the quality control standard of the product.

[0032] 4、The method for removing matrix in the compound fuyun effervescent suppository of the application uses deionized water and ethanol as test reagents, avoids the use of strong acid and strong base and toxic reagents, and ensures the safety and environmental protection of the sample preparation process.

[0033] 5、The elution process of the application is as follows: first, deionized water is used for elution to remove the matrix and inorganic salts; then, 10% ethanol by volume is used for elution to increase the elution capacity and further remove the non-test components with large polarity and molecular weight; finally, 50% ethanol by volume is used for elution to enrich matrine and berberine hydrochloride. After removing the matrix, inorganic salts, non-test components with large polarity and molecular weight, matrine and berberine hydrochloride can be more accurately enriched, and the influence of the above-mentioned substances on the enrichment process is avoided.

[0034] 6、The ratio of the height of the upper D101 macroporous adsorption resin to the height of the lower 30-60 mesh polyamide resin is 2:1-1:1, which can obtain better enrichment effect.

[0035] 7. The simultaneous identification method of alkaloids in the compound fuyun effervescent suppository, provides a liquid phase identification method for simultaneously determining matrine and berberine hydrochloride in the compound fuyun effervescent suppository, the liquid chromatography conditions adopted do not contain ion pair reagent, and the defect that the retention time is backwardly shifted due to the use of ion pair reagent in the liquid phase determination of berberine hydrochloride is overcome.

[0036] 8. The present application also discloses a thin layer identification method of alkaloids in the compound fuyun effervescent suppository, by preparing matrine control solution and berberine hydrochloride control solution, and comparing the color development with the test solution, the qualitative detection of matrine and berberine hydrochloride in the test solution is realized. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The chromatogram of the test solution in step 3) of Example 10;

[0038] Figure 2 The chromatogram of the matrine control solution in step 1) of Example 10;

[0039] Figure 3 The chromatogram of the berberine hydrochloride control solution in step 1) of Example 10;

[0040] Figure 4 The chromatogram of the first batch of matrine and berberine hydrochloride;

[0041] Figure 5 The chromatogram of the second batch of matrine and berberine hydrochloride;

[0042] Figure 6 The chromatogram of the third batch of matrine and berberine hydrochloride;

[0043] Figure 7 The chromatogram of the matrine control prepared in the chromatographic measurement of the three batches;

[0044] Figure 8 The chromatogram of the berberine hydrochloride control prepared in the chromatographic measurement of the three batches;

[0045] Figure 9 The thin layer identification chromatogram of matrine in Example 11;

[0046] Figure 10 The thin layer identification chromatogram of berberine hydrochloride in Example 11. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be further explained and described below in combination with the drawings and examples, but the present application is not limited to the following described embodiments.

[0048] Example 1

[0049] The method for removing the matrix in the detection of the compound fuyun effervescent suppository of the present example comprises the following steps:

[0050] Take the compound fuyun effervescent suppository, add concentrated ammonia test solution, and stand still. The compound fuyun effervescent suppository is fully wetted by the concentrated ammonia test solution. Add water and stir to fully dissolve the compound fuyun effervescent suppository in water to form a dissolved solution. Add the dissolved solution to the double-layer separation column, elute, collect the eluent, evaporate, dissolve the residue after evaporation with anhydrous ethanol, make up the volume, shake well, filter, take the filtrate, and obtain the test solution containing both matrine and berberine hydrochloride.

[0051] The double-layer separation column in the present example comprises the upper layer of D101 macroporous adsorption resin and the lower layer of 30-60 mesh polyamide resin.

[0052] Example 2

[0053] The method for removing the matrix in the detection of the compound fuyun effervescent suppository of the present example comprises the following steps:

[0054] Take the compound fuyun effervescent suppository, cut it into thin slices, take 0.5 g, and place it in a conical flask with a stopper. Add 1 ml of concentrated ammonia test solution and stand still for 30 minutes. The compound fuyun effervescent suppository is fully wetted by the concentrated ammonia test solution. Add 100 ml of water and stir to fully dissolve the compound fuyun effervescent suppository in water to form a dissolved solution. Add the dissolved solution to the double-layer separation column, elute, collect the eluent, evaporate, dissolve the residue after evaporation with anhydrous ethanol, transfer to a 10 ml volumetric flask, add anhydrous ethanol to make up the volume to 10 ml, shake well, filter, take the filtrate, and obtain the test solution containing both matrine and berberine hydrochloride.

[0055] The specific process of elution in the present example is as follows: first, elute with 4 column volumes of deionized water, then elute with 4 column volumes of ethanol with a volume concentration of 10%, and discard the eluent of deionized water and ethanol. Finally, elute with 4 column volumes of ethanol with a volume concentration of 50%.

[0056] The height of the double-layer separation column in the present example is 15 cm, and the inner diameter of the double-layer separation column is 2.0 cm.

[0057] The double-layer separation column in the present example comprises the upper layer of D101 macroporous adsorption resin and the lower layer of 30-60 mesh polyamide resin. The ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 1:1, i.e. the height of the upper layer of D101 macroporous adsorption resin is 7.5 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 7.5 cm.

[0058] Example 3

[0059] The removal method of the matrix in the detection of the compound fuyun effervescent suppository of the present example is based on the example 1, and the steps are specifically as follows:

[0060] The compound fuyun effervescent suppository is taken, cut into thin slices, and 0.3 g of the thin slices is placed in a conical flask with a stopper. 0.5 ml of concentrated ammonia test solution is added, and the compound fuyun effervescent suppository is allowed to stand for 30 minutes to be fully wetted by the concentrated ammonia test solution. 50 ml of water is added to stir the compound fuyun effervescent suppository to be fully dissolved in the water to form a dissolution solution. The dissolution solution is added to the double-layer separation column for elution. The eluate is collected, evaporated, and dissolved in an appropriate amount of anhydrous ethanol. The solution is transferred to a 10 ml volumetric flask, diluted with anhydrous ethanol to 10 ml, shaken, filtered, and the filtrate is collected to obtain a test solution containing both matrine and berberine hydrochloride.

[0061] The specific process of the elution in the present example is as follows: 3 column volumes of deionized water are used for elution, and then 3 column volumes of ethanol with a concentration of 10% are used for elution. The eluate of deionized water and the eluate of ethanol are discarded. Finally, 3 column volumes of ethanol with a concentration of 50% are used for elution.

[0062] The height of the double-layer separation column in the present example is 15 cm, and the inner diameter of the double-layer separation column is 1.5 cm.

[0063] The double-layer separation column in the present example includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin. The ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 2:1, i.e., the height of the upper layer of D101 macroporous adsorption resin is 10 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 5 cm.

[0064] Example 4

[0065] The removal method of the matrix in the detection of the compound fuyun effervescent suppository of the present example is based on the example 1, and the steps are specifically as follows:

[0066] The compound fuyun effervescent suppository is taken, cut into thin slices, and 0.3 g of the thin slices is placed in a conical flask with a stopper. 0.5 ml of concentrated ammonia test solution is added, and the compound fuyun effervescent suppository is allowed to stand for 30 minutes to be fully wetted by the concentrated ammonia test solution. 50 ml of water is added to stir the compound fuyun effervescent suppository to be fully dissolved in the water to form a dissolution solution. The dissolution solution is added to the double-layer separation column for elution. The eluate is collected, evaporated, and dissolved in an appropriate amount of anhydrous ethanol. The solution is transferred to a 10 ml volumetric flask, diluted with anhydrous ethanol to 10 ml, shaken, filtered, and the filtrate is collected to obtain a test solution containing both matrine and berberine hydrochloride.

[0067] The specific process of elution in this embodiment is: first eluting with 4 column volumes of deionized water, then eluting with 4 column volumes of 10% ethanol by volume, discarding the deionized water eluate and the ethanol eluate; finally eluting with 4 column volumes of 50% ethanol by volume.

[0068] The height of the double-layer separation column in this embodiment is 16 cm, and the inner diameter of the double-layer separation column is 1.6 cm.

[0069] The double-layer separation column in this embodiment includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 1:1, i.e. the height of the upper layer of D101 macroporous adsorption resin is 8 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 8 cm.

[0070] Example 5

[0071] The removal method of the matrix in the detection of the compound fuyun effervescent suppository in this embodiment is based on Example 1, and the steps are specifically as follows:

[0072] Take the compound fuyun effervescent suppository, cut it into thin slices, take 0.6 g, and place it in a conical flask with a stopper. Add 1.2 ml of concentrated ammonia test solution, stand for 30 minutes, so that the compound fuyun effervescent suppository is fully wetted by the concentrated ammonia test solution. Add 100 ml of water and stir to make the compound fuyun effervescent suppository fully dissolved in the water to form a dissolution solution. Add the dissolution solution to the double-layer separation column, elute, collect the eluate, evaporate to dryness, dissolve the residue with an appropriate amount of anhydrous ethanol, transfer to a 10 ml volumetric flask, add anhydrous ethanol to 10 ml, shake well, filter, and take the filtrate to obtain a test solution containing both matrine and berberine hydrochloride.

[0073] The specific process of moderate elution in this embodiment is: first eluting with 4 column volumes of deionized water, then eluting with 4 column volumes of 10% ethanol by volume, discarding the deionized water eluate and the ethanol eluate; finally eluting with 4 column volumes of 50% ethanol by volume.

[0074] The height of the double-layer separation column in this embodiment is 18 cm, and the inner diameter of the double-layer separation column is 1.8 cm.

[0075] The double-layer separation column in this embodiment includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 2:1, i.e. the height of the upper layer of D101 macroporous adsorption resin is 12 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 6 cm.

[0076] Example 6

[0077] The method for removing the matrix in the detection of the compound fuyun effervescent suppository of the present example is based on the example 1, and the steps are specifically as follows:

[0078] The compound fuyun effervescent suppository is cut into thin slices, 0.7 g of which is placed in a conical flask with a stopper, 1.4 ml of concentrated ammonia test solution is added, and it is left to stand for 30 minutes to allow the compound fuyun effervescent suppository to be fully wetted by the concentrated ammonia test solution. Then, 120 ml of water is added to stir the compound fuyun effervescent suppository to fully dissolve in the water to form a dissolved solution. The dissolved solution is added to the double-layer separation column for elution, and the eluate is collected, dried, dissolved in an appropriate amount of anhydrous ethanol, transferred to a 10 ml volumetric flask, added with anhydrous ethanol to a constant volume of 10 ml, shaken, filtered, and the filtrate is collected to obtain the test solution containing both matrine and berberine hydrochloride.

[0079] The specific process of elution in the present example is as follows: first, 4 column volumes of deionized water are used for elution, and then 4 column volumes of ethanol with a concentration of 10% are used for elution. The eluate of deionized water and the eluate of ethanol are discarded. Finally, 4 column volumes of ethanol with a concentration of 50% are used for elution.

[0080] The height of the double-layer separation column in the present example is 20 cm, and the inner diameter of the double-layer separation column is 2.0 cm.

[0081] The double-layer separation column in the present example includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin. The ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 1.5:1, i.e. the height of the upper layer of D101 macroporous adsorption resin is 12 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 8 cm.

[0082] Example 7

[0083] The method for removing the matrix in the detection of the compound fuyun effervescent suppository of the present example is based on the example 1, and the steps are specifically as follows:

[0084] The compound fuyun effervescent suppository is cut into thin slices, 0.7 g of which is placed in a conical flask with a stopper, 1.4 ml of concentrated ammonia test solution is added, and it is left to stand for 30 minutes to allow the compound fuyun effervescent suppository to be fully wetted by the concentrated ammonia test solution. Then, 120 ml of water is added to stir the compound fuyun effervescent suppository to fully dissolve in the water to form a dissolved solution. The dissolved solution is added to the double-layer separation column for elution, and the eluate is collected, dried, dissolved in an appropriate amount of anhydrous ethanol, transferred to a 10 ml volumetric flask, added with anhydrous ethanol to a constant volume of 10 ml, shaken, filtered, and the filtrate is collected to obtain the test solution containing both matrine and berberine hydrochloride.

[0085] The specific process of the moderate elution in this embodiment is: first eluting with 5 column volumes of deionized water, then eluting with 5 column volumes of ethanol with a volume concentration of 10%, discarding the deionized water eluate and the ethanol eluate; finally eluting with 5 column volumes of ethanol with a volume concentration of 50%.

[0086] The height of the double-layer separation column in this embodiment is 15 cm, and the inner diameter of the double-layer separation column is 2.2 cm.

[0087] The double-layer separation column in this embodiment includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 2:1, that is, the height of the upper layer of D101 macroporous adsorption resin is 10 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 5 cm.

[0088] Example 8

[0089] The removal method of the matrix in the detection of the compound fuyun effervescent suppository in this embodiment is based on Example 1, and the steps are specifically as follows:

[0090] Take the compound fuyun effervescent suppository, cut it into thin slices, take 0.9 g, and place it in a conical flask with a stopper. Add 1.8 ml of concentrated ammonia test solution, stand for 30 minutes, and make the compound fuyun effervescent suppository fully wetted by the concentrated ammonia test solution. Add 180 ml of water and stir to make the compound fuyun effervescent suppository fully dissolved in the water to form a dissolution solution. Add the dissolution solution to the double-layer separation column, elute, collect the eluate, evaporate to dryness, dissolve the residue with an appropriate amount of anhydrous ethanol, transfer to a 10 ml volumetric flask, add anhydrous ethanol to 10 ml, shake well, filter, and take the filtrate to obtain a test solution containing both matrine and berberine hydrochloride.

[0091] The specific process of the moderate elution in this embodiment is: first eluting with 5 column volumes of deionized water, then eluting with 5 column volumes of ethanol with a volume concentration of 10%, discarding the deionized water eluate and the ethanol eluate; finally eluting with 5 column volumes of ethanol with a volume concentration of 50%.

[0092] The height of the double-layer separation column in this embodiment is 17 cm, and the inner diameter of the double-layer separation column is 2.5 cm.

[0093] The double-layer separation column in this embodiment includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 1.43:1, that is, the height of the upper layer of D101 macroporous adsorption resin is 10 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 7 cm.

[0094] Example 9

[0095] The removing method of the matrix in the compound fuyun effervescent suppository in this example is based on the example 1, and the steps are specifically as follows:

[0096] Take the compound fuyun effervescent suppository, cut into thin slices, take 1 g, put it in a conical flask with a plug, add 2 ml of concentrated ammonia test solution, stand for 30 minutes, so that the compound fuyun effervescent suppository is fully wetted by the concentrated ammonia test solution, add 200 ml of water and stir, so that the compound fuyun effervescent suppository is fully dissolved in water to form a dissolution solution, add the dissolution solution to a double-layer separation column, elute, collect the eluate, evaporate to dryness, dissolve the residue after evaporation to dryness with an appropriate amount of anhydrous ethanol, transfer to a 10 ml volumetric flask, add anhydrous ethanol to 10 ml, shake well, filter, take the filtrate, and obtain the test solution containing both matrine and berberine hydrochloride.

[0097] The specific process of moderate elution in this example is as follows: first, elute with 5 column volumes of deionized water, then elute with 5 column volumes of ethanol with a concentration of 10%, and discard the eluate of deionized water and ethanol; finally, elute with 5 column volumes of ethanol with a concentration of 50%.

[0098] The height of the double-layer separation column in this example is 19 cm, and the inner diameter of the double-layer separation column is 3.0 cm.

[0099] The double-layer separation column in this example includes an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the ratio of the height of the upper layer of D101 macroporous adsorption resin to the height of the lower layer of 30-60 mesh polyamide resin is 1.11:1, i.e. the height of the upper layer of D101 macroporous adsorption resin is 10 cm, and the height of the lower layer of 30-60 mesh polyamide resin is 9 cm.

[0100] Example 10

[0101] The simultaneous identification method of alkaloids in the compound fuyun effervescent suppository in this example includes the following steps:

[0102] 1) Preparation of reference solution:

[0103] Take matrine and berberine hydrochloride, add anhydrous ethanol, and prepare matrine reference solution and berberine hydrochloride reference solution, respectively. Each milliliter of the matrine reference solution contains 60 ug of matrine, and each milliliter of the berberine hydrochloride reference solution contains 20 ug of berberine hydrochloride.

[0104] 2) Chromatographic conditions: the chromatographic column is filled with octadecylsilane-bonded silica gel, the mobile phase is acetonitrile-0.1% phosphoric acid solution by volume, the volume ratio of acetonitrile and phosphoric acid solution is 25:75, the pH of the mobile phase is adjusted to 8.0 by triethylamine, the detection wavelength is 220 nm, the column temperature is 20-30°C, and the flow rate is 0.8-1.2 ml / min. Specifically, the column temperature can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30°C; specifically, the flow rate can be 0.8, 0.9, 1.0, 1.1 or 1.2 ml / min.

[0105] 3) Chromatographic peak determination: 10 μl of the matrine reference solution, the berberine hydrochloride reference solution and the test solution containing both matrine and berberine hydrochloride prepared in Example 2 in step 1) are injected into the liquid chromatograph, respectively, and the chromatogram is recorded under the chromatographic conditions in step 2), see Figure 1 、 Figure 2 and Figure 3 , to realize the identification and quantitative detection of matrine and berberine hydrochloride.

[0106] It should be noted that the detection wavelength in this embodiment is 190-230 nm, which can be one of 190, 200, 210, 220 or 230 nm.

[0107] It should be noted that the volume concentration of the phosphoric acid solution in this embodiment is 0.1-0.3%, which can be one of 0.1%, 0.2% or 0.3%.

[0108] It should be noted that the test solution containing both matrine and berberine hydrochloride in this embodiment, in addition to that prepared in the example, can also be the test solution containing both matrine and berberine hydrochloride prepared in any one of Examples 3-8.

[0109] Example 11

[0110] The thin-layer identification method of alkaloids in compound fuyong effervescent suppositories includes the following steps:

[0111] Matrine identification: the prepared solution containing both matrine and berberine hydrochloride in Example 2 was taken as the test solution; another matrine reference substance was taken, methanol was added, and a reference solution containing 1 mg of matrine per milliliter was prepared; according to the thin layer chromatography method (Chinese Pharmacopoeia General 0502), 5 μl of the test solution and the matrine reference solution were respectively taken and spotted on the same silica gel G thin layer plate, a mixture liquid with a volume ratio of 2:3:4:0.2 of toluene-acetone-ethyl acetate-concentrated ammonia solution was used as the developing agent, and after development, removal, and air drying, a diluted bismuth potassium iodide solution was sprayed; the test solution and the matrine reference solution both showed the same orange-red spots, which indicated that the test solution contained matrine; otherwise, it indicated that the test solution did not contain matrine.

[0112] Berberine hydrochloride identification: the prepared solution containing both matrine and berberine hydrochloride in Example 2 was taken as the test solution; another berberine hydrochloride reference substance was taken, methanol was added, and a reference solution containing 0.5 mg of berberine hydrochloride per milliliter was prepared; according to the thin layer chromatography method (Chinese Pharmacopoeia General 0502), 5 μl of the test solution and the berberine hydrochloride reference solution were respectively taken and spotted on the same silica gel G thin layer plate, a mixture liquid with a volume ratio of 10:7:0.5:0.5 of ethyl acetate-methyl ketone-formic acid-water was used as the developing agent, and after development, removal, and air drying, the thin layer plate was placed under a 365 nm ultraviolet lamp for inspection; the test solution and the berberine hydrochloride reference solution both showed the same yellow spots, which indicated that the test solution contained berberine hydrochloride; otherwise, it indicated that the test solution did not contain berberine hydrochloride.

[0113] Verification test

[0114] Experiment 1: screening experiment of double-layer separation column in the matrix removal method of the application

[0115] Referring to Table 1, 10 cm D101 macroporous adsorption resin + 5 cm polyamide column, 15 cm D101 macroporous adsorption resin column, 10 cm polyamide column + 5 cm D101 macroporous adsorption resin, and 15 cm polyamide column were respectively used to perform adsorption and elution according to the method in Example 2 of the application, the enrichment contents of matrine and berberine hydrochloride and the removal rates of non-tested components under different separation columns were determined by liquid chromatography, and the types of upper and lower separation columns were determined.

[0116] Table 1: adsorption and elution of matrine and berberine hydrochloride under different chromatographic columns

[0117]

[0118]

[0119] Referring to Table 1, only the macroporous adsorption resin in the upper layer and the polyamide resin in the lower layer can realize successful column loading, achieve the effect of removing 85% of non-target components while enriching more matrine and berberine hydrochloride.

[0120] The upper layer macroporous adsorption resin and the lower layer polyamide double-layer chromatography column with a total height of 15 cm were used, the height ratio of the upper layer to the lower layer was 1:3, 1:2, 1:1, 1.5:1, 2:1 and 3:1 respectively, adsorption and elution were carried out according to the method in Example 2 of the present application, the enrichment content of matrine and berberine hydrochloride and the removal rate of non-target components in different separation columns were determined by liquid chromatography, and the height ratio of the upper layer to the lower layer of the separation column was determined.

[0121] Table 2: Adsorption and elution of matrine and berberine hydrochloride under different height ratios of the upper layer to the lower layer

[0122]

[0123] Referring to Table 2, different height ratios will bring changes in the adsorption and desorption effect of the target components. With the increase of the eluent concentration, the macroporous resin will drift upwards, and the matrix removal effect will become poor. The test results show that when the height ratio of the upper layer D101 macroporous adsorption resin to the lower layer 30-60 mesh polyamide resin is 2:1-1:1, the matrix removal effect is the best, and the enrichment effect of matrine and berberine hydrochloride is good.

[0124] Experiment 2: Removal effect of the matrix removal method of the present application and influence on alkaloid enrichment

[0125] The sample solution prepared by the existing solvent extraction method, acid-base method and dissolution solution cooling method was compared with the sample solution prepared by the method of Example 2 which contains matrine and berberine hydrochloride at the same time, and liquid phase identification of matrine and berberine hydrochloride was carried out respectively, and the identification results are shown in the following Table 3:

[0126] Table 3: Identification results of matrine and berberine hydrochloride in sample solutions prepared by solvent extraction method, acid-base method and dissolution solution cooling method and the method of Example 2

[0127]

[0128] Note: The solid content is the weight of the sample solution after evaporation;

[0129] Matrix and non-target component removal rate (%) = 100-(solid content / sample amount*100)

[0130] Referring to Table 3, the test sample solution containing matrine and berberine hydrochloride prepared by the matrix removal method of Example 2 does not affect the content of the components to be tested (matrine and berberine hydrochloride) compared with the test sample solution prepared by the existing method, and the removal rate of the matrix and non-components to be tested is greater than 85%, the removal efficiency of the matrix and non-components to be tested is high, the subsequent detection result of alkaloids in the compound Furong effervescent suppository is small; at the same time, the matrix removal method of Example 2 can also effectively reduce the solid content.

[0131] The matrine content obtained by the double-layer column chromatography method of the present application is basically consistent with that obtained by the acid-base method in the existing quality standard; however, the matrix removal method of the present application can enrich more berberine hydrochloride after removing the matrix, and therefore the enrichment effect of the present application on matrine and berberine hydrochloride is obviously better than that of the existing standard method. Referring to Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 8 , the liquid phase conditions of the present application can simultaneously realize the quantitative detection of matrine and berberine hydrochloride.

[0132] Experiment 3: Stability experiment of the matrix removal method of the present application:

[0133] The test sample solution containing matrine and berberine hydrochloride prepared in Example 2 was divided into three batches, and the content of matrine, the content of berberine hydrochloride, and the removal rate of the matrix and non-components to be tested were detected, and the detection results are shown in Table 4 below:

[0134] Table 4: Detection results of the content of matrine, the content of berberine hydrochloride, and the removal rate of the matrix and non-components to be tested in three batches

[0135]

[0136]

[0137] Referring to Table 4, it is further proved that the matrix removal method in the detection of the compound Furong effervescent suppository of the present application has a removal rate of the matrix and non-components to be tested of greater than 85%, and has good removal effect, and the method is stable and can be reproduced.

[0138] Experiment 4: Influence of the matrix removal method of the present application on thin layer identification:

[0139] 10 ml of the test sample solution containing matrine and berberine hydrochloride prepared in Example 2 was taken, and thin layer identification of matrine and berberine hydrochloride was carried out, and the identification results are as follows Figure 9 and Figure 10 After the development of the thin layer identification chromatography, the matrix does not interfere with the determination of the chromatography results; Figure 10The separation degree of berberine hydrochloride from other impurities in the thin layer chromatogram is obviously superior to that of the acid-base method in the current quality standard, and the double-layer column chromatography method can definitely improve the chromatographic effect.

[0140] Experiment 5: Influence of the matrix removal method of the application on the column efficiency of the alkaloid liquid phase identification

[0141] The influence of the test sample solution obtained by the double-layer column chromatography method of the application on the column efficiency was determined according to the method of Example 10, and the results are shown in Table 5 below:

[0142] Table 5: Influence of the double-layer column chromatography method on the column efficiency

[0143]

[0144] Referring to Table 5, the determination results of the alkaloids in the compound Furong effervescent suppository of the application show that the theoretical column numbers of matrine and berberine hydrochloride remain very high under the condition of continuous sampling for 6 times, which indicates that the adopted matrix removal method is effective and overcomes the damage of the matrix to the column efficiency of the chromatographic column.

[0145] Experiment 6: Influence of the chromatographic conditions of the application on the retention time drift of berberine hydrochloride

[0146] The influence of the chromatographic conditions on the retention time drift of berberine hydrochloride was determined by using the liquid phase identification method of Example 10, and the results are shown in Table 6 below:

[0147] Table 6: Influence of the chromatographic conditions on the retention time drift of berberine hydrochloride

[0148]

[0149] In the common determination methods of berberine hydrochloride, the mobile phase usually contains ion pair reagent (sodium dodecyl sulfate, etc.), which is easy to cause the retention time to drift backward during the identification process, and the determination results have poor repeatability. In the simultaneous identification method of the alkaloids in the compound Furong effervescent suppository of the application, the chromatographic conditions do not contain ion pair reagent, as shown in Table 6, under the condition of continuous sampling for 6 times, the retention time of berberine hydrochloride has little fluctuation, which overcomes the defect that the retention time of berberine hydrochloride drifts backward due to the use of ion pair reagent in the liquid phase determination.

Claims

1. A method for removing the matrix in the detection of compound hibiscus effervescent suppositories, characterized in that, Includes the following steps: Take compound hibiscus effervescent suppositories, add concentrated ammonia test solution, let stand to allow the suppositories to be fully wetted by the concentrated ammonia test solution, add water and stir to fully dissolve the suppositories, add to a double-layer separation column, elute, collect the eluent, evaporate to dryness, dissolve the residue after evaporation in anhydrous ethanol, make up to volume, shake well, filter, and collect the filtrate to obtain a test solution containing both matrine and berberine hydrochloride; the double-layer separation column has a column height of 15cm-20cm and an inner diameter of 1.5cm-3.0cm; the elution process is as follows: first elute with 3-5 column volumes of deionized water, then elute with 3-5 column volumes of 10% ethanol, discard the deionized water and ethanol eluents, and finally elute with 3-5 column volumes of 50% ethanol. The double-layer separation column comprises an upper layer of D101 macroporous adsorption resin and a lower layer of 30-60 mesh polyamide resin, wherein the height ratio of the upper layer of D101 macroporous adsorption resin to the lower layer of 30-60 mesh polyamide resin is 2:1 to 1:

1.

2. The method for removing the matrix in the detection of compound hibiscus effervescent suppositories as described in claim 1, characterized in that, The specific steps are as follows: Take 0.3g-1g of compound hibiscus effervescent suppositories, add 0.5ml-2ml of concentrated ammonia test solution, let stand to fully wet the suppositories with concentrated ammonia test solution, add 50ml-200ml of water and stir to fully dissolve the suppositories, add to a double-layer separation column, elute, collect the eluent, evaporate to dryness, dissolve the residue after evaporation in anhydrous ethanol, make up to 10ml, shake well, filter, and collect the filtrate to obtain a test solution containing both matrine and berberine hydrochloride.

3. A method for simultaneous identification of alkaloids in compound hibiscus effervescent suppositories, characterized in that, Includes the following steps: 1) Preparation of reference solution: Take matrine and berberine hydrochloride, add anhydrous ethanol, and prepare matrine reference solution and berberine hydrochloride reference solution respectively. Each milliliter of the matrine reference solution contains 60 μg of matrine, and each milliliter of the berberine hydrochloride reference solution contains 20 μg of berberine hydrochloride. 2) Chromatographic conditions: The column was packed with octadecylsilane-bonded silica gel, and the mobile phase was acetonitrile-phosphoric acid solution with a volume concentration of 0.1%-0.3% (v / v), with a volume ratio of acetonitrile to phosphoric acid solution of 25:75; the detection wavelength was 190nm-230nm; the column temperature was 20℃-30℃; and the flow rate was 0.8ml / min-1.2ml / min. 3) Chromatographic peak determination: The matrine reference solution, berberine hydrochloride reference solution and the test solution containing both matrine and berberine hydrochloride prepared in the matrix removal method for compound effervescent suppositories as described in claim 1 or claim 2 are injected into the liquid chromatograph respectively. The chromatograms are measured and recorded according to the chromatographic conditions in step 2) to achieve the identification of matrine and berberine hydrochloride. The alkaloids mentioned refer to matrine and berberine hydrochloride.

4. The method for simultaneous identification of alkaloids in compound hibiscus effervescent suppositories as described in claim 3, characterized in that, The phosphoric acid solution has a volume concentration of 0.1%.

5. The method for simultaneous identification of alkaloids in compound hibiscus effervescent suppositories as described in claim 3, characterized in that, The detection wavelength is 220nm.

6. The method for simultaneous identification of alkaloids in compound hibiscus effervescent suppositories as described in claim 3, characterized in that, Step 2) also includes adjusting the pH of the phosphoric acid solution to 8.0 using triethylamine.

7. A thin-layer chromatography method for identifying alkaloids in compound hibiscus effervescent suppositories, characterized in that, Includes the following steps: Identification of matrine: Take the test solution containing both matrine and berberine hydrochloride prepared in the matrix removal method for compound effervescent suppositories as described in claim 1 or claim 2; separately take matrine reference standard, add methanol to prepare a reference solution containing 1 mg matrine per milliliter; according to the thin-layer chromatography method, take the test solution and the matrine reference solution respectively, and spot them on the corresponding positions of the same silica gel G thin-layer plate, using a mixture of toluene-acetone-ethyl acetate-concentrated ammonia solution with a volume ratio of 2:3:4:0.2 as the developing solvent, develop, remove, dry, and spray with dilute bismuth potassium iodide solution. If both the test solution and the matrine reference solution show the same orange-red spots, it indicates that the test solution contains matrine. Identification of berberine hydrochloride: The test solution containing both matrine and berberine hydrochloride, prepared using the matrix removal method described in claim 1 or claim 2 for the detection of compound effervescent suppositories, is used as the test solution. A separate reference solution of berberine hydrochloride is prepared by adding methanol to a solution containing 0.5 mg of berberine hydrochloride per milliliter. Using thin-layer chromatography, the test solution and the reference solution of berberine hydrochloride are spotted onto the same silica gel G plate. A mixture of ethyl acetate, butanone, formic acid, and water (volume ratio 10:7:0.5:0.5) is used as the developing solvent. The plate is developed, removed, dried, and examined under a 365 nm UV lamp. If both the test solution and the reference solution of berberine hydrochloride show the same yellow spots, it indicates that the test solution contains berberine hydrochloride.

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