CN113640431B - Detection method of strong loquat dew - Google Patents

Abstract

The invention provides a detection method of strong loquat dew. The method comprises the steps of carrying out dilution and extraction treatment on a standard strong loquat dew product, adding a solubilizer to improve the extraction quantity of active ingredients in the medicament, reducing the emulsification phenomenon, selecting the standard strong loquat dew product as a standard spectrogram, avoiding illegal merchants from reaching the detection qualification only through adding a single active substance, and reducing the phenomenon of secondary filling. Before liquid chromatography, performing crude analysis by thin layer chromatography, preliminarily determining whether the product is qualified, and performing high performance liquid chromatography on eluent with reasonable design and elution sequence. Before liquid chromatography test, the test solution is filtered by using a modified filter membrane, so that impurities in the test solution are reduced, the detection speed and the detection accuracy are improved, and the service life of the chromatographic column is prolonged.

Description

Detection method of strong loquat dew

Technical Field

The invention relates to the technical field of medicine quality control, in particular to a detection method of strong loquat dew.

Background

The strong loquat dew is composed of seven traditional Chinese medicines of loquat She poppy shell, radix stemonae, cynanchum glaucescens and the like, and is a second volume-loaded variety of the standard Chinese medicine prescription preparation of the ministry of health; has effects of nourishing yin, astringing lung, relieving cough and eliminating phlegm: can be used for treating chronic cough, and bronchitis. Wherein the folium Eriobotryae is principal drug for treating cough, and plantula Papaveris is main drug for treating cough, and contains morphine, codeine, papaverine, and protopine. It is well known that the efficacy of Chinese patent medicines is not derived from any single active ingredient, but is the result of the combined action of multiple active part groups, and the quality standard of the powerful loquat dew is only controlled in identification and relative density. These two cannot characterize the quality of the strong loquat dew, and there is a quality risk. The fingerprint can more comprehensively represent the components of the strong loquat dew and can more strictly control the quality of the strong loquat dew.

So far, the existing quality standard of the strong loquat dew is too simple, the product quality cannot be effectively controlled, and the quality of the commercial strong loquat dew is uneven. Because the preparation contains narcotic poppy shell, if the dosage of the narcotic poppy shell is controlled improperly, the preparation is easy to be addicted after long-term administration. Modern chemical and pharmacological studies show that the main active ingredients of poppy shell are alkaloids, including papaverine, narcotine, thebaine, morphine and codeine, etc., which belong to the strong addictive ingredients. To ensure the safety and effectiveness of the formulation, its content must be controlled. The existing medicine quality standard only has the chemical reaction identification of alkaloid and flavone, has poor specificity and poor controllability, and has no content measurement item. At present, the content measurement research only reports that the content of morphine, codeine or papaverine in poppy shells in the strong loquat dew is measured by HPLC or gas chromatography. With the development of modern analysis technology, the liquid chromatography-mass spectrometry technology has gradually become an effective means for quantitative analysis of traditional Chinese medicine components by virtue of the advantages of high sensitivity, strong specificity, rapidness, high efficiency and the like. Therefore, the characteristic spectrogram special for the strong loquat dew is obtained by adopting the standard strong loquat dew solution through liquid chromatography, so that the quality control level of the strong loquat dew is improved, and the safety of products is ensured.

Disclosure of Invention

The invention mainly aims to provide a detection method of strong loquat dew.

The second purpose of the invention is to provide a fingerprint spectrum of standard strong loquat dew and a testing method thereof.

The invention is realized by the following scheme:

a method for detecting strong loquat dew.

The detection method of the strong loquat dew comprises the following steps:

establishment of standard powerful loquat dew product detection method

1) Taking 10 parts of standard strong loquat dew product, adding diluent into the standard strong loquat dew product for dilution, fixing the volume to 5-6 times of the volume of the original product, heating the original product to 50 ℃, preserving heat and standing for 1h for later use;

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 30-40 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 40-45 parts of diluent into the filter residues again, heating to 45 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 5.5-6.0 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

4) Taking 5ml of solution a, adding ethanol solution into the solution a to fix the volume to 500ml, taking 20ml, 20ml and 20ml, respectively placing the solution a, the 20ml and the 20ml into a 50ml volumetric flask, and adding ethanol into the solution a to fix the volume to scale marks; filtering the solution by a microporous filter membrane, and then placing the solution into a liquid chromatograph for test analysis to obtain data of absorption peaks within 0-35min, and introducing the data into Matlab software for drawing, wherein time is an abscissa, and taking an average value of absorption values of 4 groups of data as an ordinate to obtain a standard curve of the strong loquat dew;

two), detection of samples

1) And (3) processing the strong loquat dew product to be tested according to the method, and then obtaining a corresponding spectrogram through a liquid chromatograph, and judging the loquat dew product to be qualified when the relative peak area of the spectrogram and the standard spectrogram is within 2%. The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) The prepared solution m and the solubilizer are mixed according to the volume ratio of (100-105): mixing (0.3-0.5), adding petroleum ether 20-30 parts, heating the solution to 75deg.C at a rate of 1-2deg.C/min, stirring for 3-5 hr, and cooling to room temperature.

Preparation of the solubilizer:

a. putting 5-9 parts of tetraethylammonium bromide into a reactor, adding 3-5 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the mixture to 50 ℃ at a speed of 3-5 ℃/min to obtain a solution i, and standing and preserving heat for later use;

b. 2-4 parts of tetramethyl ammonium fluoride is put into a reactor, acetone solution is added, the temperature is raised to 90 ℃ at the speed of 1-3 ℃/min, 12-15 parts of solution i is added dropwise into the reactor at the speed of 15-18 drops/min, stirring is carried out while dropwise adding, the temperature is raised to 150 ℃ together, and the reaction is carried out for 4-5 hours under heat preservation, thus obtaining a reactant a;

c. adding 15-20 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at the speed of 1-3 ℃/min, and carrying out heat preservation reaction for 3-4 hours to obtain the catalyst.

Before liquid chromatography, preliminary analysis is performed by thin layer chromatography, so that waste of detection cost is reduced, and the specific detection method comprises the following steps:

and (3) placing the solution a of the standard solution and the sample solution on a thin layer chromatography silica gel G plate in a spot plate mode, evaporating the liquid in the solution by natural airing, placing the solution in the chromatography solution, obliquely placing the solution until the chromatography solution climbs to 6/7 area of the silica gel plate, taking out the solution, airing, placing the solution under a 256mm ultraviolet lamp, and judging whether the positions of the observation points are the same or not, if so, carrying out the next analysis, and if not, judging that the solution is a non-qualified product, and eliminating the solution.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. 15-20 parts of cyclohexane is put into a reactor, 40-45 parts of ethyl acetate solution is added, 1-3 parts of propylene carbonate is added after the temperature is raised to 50 ℃ together, the temperature is raised to 70 ℃, and the reaction is carried out for 3 hours under the heat preservation, thus obtaining an intermediate product a;

b. adding 30-40 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5-6h with heat preservation, standing and preserving heat to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 25-28 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

Wherein, the step 4) also comprises modification of the microporous filter membrane, and the specific modification steps are as follows:

1) Soaking microporous membrane in ethanol solution, heating to 38-42deg.C at a rate of 1-2deg.C/min, performing microwave vibration, keeping temperature, oscillating for 2 hr, taking out, washing with methanol solution, and standing;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 3-5 ℃/min, and preserving heat and standing for 4-5 hours to obtain a primary modified filter membrane;

3) Soaking the preliminary modified filter membrane in methanol solution, controlling the temperature of the methanol solution to be 0-3 ℃, preserving heat, standing for 3-4h, taking out and airing to obtain the membrane.

The preparation method of the modifier comprises the following steps:

putting 1-3 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 42-45 ℃ at a speed of 1-3 ℃/min, adding 2-4 parts of N, N-dimethylformamide diethyl acetal into the reactor while the reactor is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3-4 hours to obtain a product a;

ii) adding 2-4 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 140-145 ℃ at the speed of 4-6 ℃/min, and carrying out heat preservation reaction for 4-6 hours to obtain a product b;

Iii) adding 4-6 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 80-85 ℃, and reacting for 4-5 hours at a temperature maintaining condition to obtain a product c;

iv) adding 1-2 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding toluene solution, heating to 153-157 ℃ at the speed of 2-4 ℃/min, simultaneously starting a condensation reflux device during heating, and carrying out heat preservation reaction for 5-6h to obtain the catalyst.

The invention has the following advantages:

the method provided by the invention is to dilute and extract the standard strong loquat dew product, extract the effective components in the standard strong loquat dew product and analyze the effective components, and the principle is to extract the effective components in the strong loquat dew; wherein the diluent contains a solubilizer, which improves the extraction rate of the effective components in the strong loquat dew. The effective components of the solubilizer can not only improve the extraction content in the medicament, but also reduce the emulsification phenomenon, improve the analysis precision and improve the repeatability of the detection result. The effective components in the solubilizer are suitable for a high performance liquid chromatograph, a standard powerful loquat dew product is selected as a standard spectrogram, the spectrogram obtained by testing various samples is detected and analyzed, the phenomenon that illegal merchants only add a single effective substance to reach detection qualification is avoided, and the phenomenon of secondary filling is reduced.

The effective components in the herbal medicine are extracted through the diluent, and after the effective components are obtained, the elution phase, the elution sequence and the elution time which are reasonable in design are needed, and meanwhile, the influence of the solubilizer is reduced, so that the analysis result is prevented from being influenced. Since methanol solution is selected in the elution process, triethylamine which is the same as organic matters is added into the methanol solution to adjust the pH, and meanwhile, the tailing condition of characteristic peaks in liquid chromatography is reduced; similarly, the corresponding buffering agent is added into the water phase, the pH value of the whole eluting phase is adjusted, and the phenomena of asymmetric peak, broad peak and split peak are reduced. Before liquid chromatography analysis, thin layer chromatography is adopted to perform crude analysis, whether the strong loquat dew product is qualified or not is preliminarily determined, the resolution efficiency is improved, and meanwhile, the influence effect of the solubilizer is further reduced by performing preliminary judgment through the thin layer chromatography.

Because the solubilizer component and the special elution sequence added in the diluent reduce the influence of the solubilizer on experimental results, but the phenomena of wide peak, split peak and impurity peak at blank appear in the test sample, the test sample needs to be filtered before liquid chromatography test, and the common filter membrane can not effectively remove impurities in the test solution during filtration, so that the filler in the chromatographic column can be damaged in the test process, the chromatographic column is blocked, the service life of the chromatographic column is shortened, and the filter membrane before entering the column needs to be modified before the sample solution is detected and analyzed; the influence of the solubilizer on the experimental result is further reduced by modifying the filter membrane, the influence factors in the test solution are filtered, the service life of the chromatographic column is prolonged, and the detection cost is reduced; the modified filter membrane can effectively reduce impurities in the test solution, and the detection speed and the detection accuracy are improved; through modifying the filter membrane, the solubilizer in the diluent, the elution speed and the sequence, the method can detect the quality problem of the strong loquat dew with low resistance, high speed and high efficiency.

Detailed description of the preferred embodiments

Example 1

A method for detecting strong loquat dew.

The detection method of the strong loquat dew comprises the following steps:

establishment of standard powerful loquat dew product detection method

1) Taking 10 parts of a standard strong loquat dew product, adding a diluent into the standard strong loquat dew product for dilution, fixing the volume to 5.5 times of the volume of the original product, heating the original product to 50 ℃, and preserving heat and standing for 1h for later use;

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 35 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 43 parts of diluent into the filter residues again, heating to 45 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 5.8 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

4) Taking 5ml of solution a, adding ethanol solution into the solution a to fix the volume to 500ml, taking 20ml, 20ml and 20ml, respectively placing the solution a, the 20ml and the 20ml into a 50ml volumetric flask, and adding ethanol into the solution a to fix the volume to scale marks; filtering the solution by a microporous filter membrane, and then placing the solution into a liquid chromatograph for test analysis to obtain data of absorption peaks within 0-35min, and introducing the data into Matlab software for drawing, wherein time is an abscissa, and taking an average value of absorption values of 4 groups of data as an ordinate to obtain a standard curve of the strong loquat dew;

Two), detection of samples

1) And (3) processing the strong loquat dew product to be tested according to the method, and then obtaining a corresponding spectrogram through a liquid chromatograph, and judging the loquat dew product to be qualified when the relative peak area of the spectrogram and the standard spectrogram is within 2%.

The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) The prepared solution m and the solubilizer are mixed according to the volume ratio of 103: mixing at a ratio of 0.4, adding 25 parts of petroleum ether, heating the solution to 75 ℃ at a speed of 1.5 ℃/min, preserving heat, stirring for 4 hours, and cooling to room temperature to obtain the product.

Preparation of the solubilizer:

a. putting 7 parts of tetraethylammonium bromide into a reactor, adding 4 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the toluene solution to 50 ℃ at a speed of 4 ℃/min to obtain a solution i, and standing and preserving heat for later use;

b. 3 parts of tetramethyl ammonium fluoride is put into a reactor, acetone solution is added, the temperature is raised to 90 ℃ at the speed of 2 ℃/min, 13 parts of solution i is dropwise added into the reactor in a dropwise manner, the dropwise speed is 17 drops/min, the mixture is stirred while being dropwise added, the mixture is heated to 150 ℃ together, and the mixture is subjected to thermal insulation reaction for 4.5 hours to obtain a reactant a;

c. And (3) adding 18 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at a speed of 2 ℃/min, and carrying out heat preservation reaction for 3.5 hours to obtain the catalyst.

Before liquid chromatography, preliminary analysis is performed by thin layer chromatography, so that waste of detection cost is reduced, and the specific detection method comprises the following steps:

and (3) placing the solution a of the standard solution and the sample solution on a thin layer chromatography silica gel G plate in a spot plate mode, evaporating the liquid in the solution by natural airing, placing the solution in the chromatography solution, obliquely placing the solution until the chromatography solution climbs to 6/7 area of the silica gel plate, taking out the solution, airing, placing the solution under a 256mm ultraviolet lamp, and judging whether the positions of the observation points are the same or not, if so, carrying out the next analysis, and if not, judging that the solution is a non-qualified product, and eliminating the solution.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. adding 18 parts of cyclohexane into a reactor, adding 43 parts of ethyl acetate solution, heating to 50 ℃ together, adding 2 parts of propylene carbonate, heating to 70 ℃, and reacting for 3 hours at a constant temperature to obtain an intermediate product a;

b. adding 35 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5.5 hours at a constant temperature, and standing and preserving the temperature to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

Wherein, the step 4) also comprises modification of the microporous filter membrane, and the specific modification steps are as follows:

1) Soaking the microporous filter membrane in ethanol solution, heating to 40 ℃ at a speed of 1.5 ℃/min, simultaneously carrying out microwave vibration, preserving heat and oscillating for 2 hours, taking out, washing with methanol solution, and standing for later use;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 4 ℃/min, and preserving heat and standing for 4.5 hours to obtain a primary modified filter membrane;

3) And (3) soaking the preliminary modified filter membrane in a methanol solution, controlling the temperature of the methanol solution to be 1 ℃, preserving heat, standing for 3.5h, taking out and airing to obtain the membrane.

The preparation method of the modifier comprises the following steps:

putting 2 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 2 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

Example 2

A method for detecting strong loquat dew.

The verification method of the strong loquat dew comprises the following steps:

establishment of standard powerful loquat dew product detection method

1) Taking 10 parts of a standard strong loquat dew product, adding a diluent into the standard strong loquat dew product for dilution, fixing the volume to 5 times of the volume of the original product, heating the original product to 50 ℃, preserving heat and standing for 1h for later use;

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 40 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 40 parts of diluent into the filter residues again, heating to 45 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 5.5 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

4) Taking 5ml of solution a, adding ethanol solution into the solution a to fix the volume to 500ml, taking 20ml, 20ml and 20ml, respectively placing the solution a, the 20ml and the 20ml into a 50ml volumetric flask, and adding ethanol into the solution a to fix the volume to scale marks; filtering the solution by a microporous filter membrane, and then placing the solution into a liquid chromatograph for test analysis to obtain data of absorption peaks within 0-35min, and introducing the data into Matlab software for drawing, wherein time is an abscissa, and taking an average value of absorption values of 4 groups of data as an ordinate to obtain a standard curve of the strong loquat dew;

Two), detection of samples

1) And (3) processing the strong loquat dew product to be tested according to the method, and then obtaining a corresponding spectrogram through a liquid chromatograph, and judging the loquat dew product to be qualified when the relative peak area of the spectrogram and the standard spectrogram is within 2%.

The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) The prepared solution m and the solubilizer are mixed according to the volume ratio of 100: mixing at a ratio of 0.5, adding 20 parts of petroleum ether, heating the solution to 75 ℃ at a speed of 2 ℃/min, preserving heat, stirring for 3 hours, and cooling to room temperature to obtain the product.

Preparation of the solubilizer:

a. putting 5 parts of tetraethylammonium bromide into a reactor, adding 5 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the toluene solution to 50 ℃ at a speed of 3 ℃/min to obtain a solution i, and standing and preserving heat for later use;

b. putting 4 parts of tetramethyl ammonium fluoride into a reactor, adding an acetone solution, heating to 90 ℃ at a speed of 1 ℃/min, dropwise adding 15 parts of solution i into the reactor in a dropwise manner, stirring while dropwise adding the solution at a speed of 15 drops/min, heating to 150 ℃ together, and carrying out heat preservation reaction for 5 hours to obtain a reactant a;

c. Adding 15 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at the speed of 3 ℃/min, and carrying out heat preservation reaction for 3 hours to obtain the catalyst.

Before liquid chromatography, preliminary analysis is performed by thin layer chromatography, so that waste of detection cost is reduced, and the specific detection method comprises the following steps:

and (3) placing the solution a of the standard solution and the sample solution on a thin layer chromatography silica gel G plate in a spot plate mode, evaporating the liquid in the solution by natural airing, placing the solution in the chromatography solution, obliquely placing the solution until the chromatography solution climbs to 6/7 area of the silica gel plate, taking out the solution, airing, placing the solution under a 256mm ultraviolet lamp, and judging whether the positions of the observation points are the same or not, if so, carrying out the next analysis, and if not, judging that the solution is a non-qualified product, and eliminating the solution.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. putting 20 parts of cyclohexane into a reactor, adding 40 parts of ethyl acetate solution, heating to 50 ℃ together, adding 1 part of propylene carbonate, heating to 70 ℃, and reacting for 3 hours at a constant temperature to obtain an intermediate product a;

b. adding 40 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5 hours at a constant temperature, and standing and preserving the temperature to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 25 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

Wherein, the step 4) also comprises modification of the microporous filter membrane, and the specific modification steps are as follows:

1) Soaking the microporous filter membrane in ethanol solution, heating to 42 ℃ at a speed of 1 ℃/min, simultaneously carrying out microwave vibration, preserving heat and oscillating for 2 hours, taking out, flushing with methanol solution, and standing for later use;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 3 ℃/min, and preserving heat and standing for 5 hours to obtain a preliminary modified filter membrane;

3) And (3) soaking the preliminary modified filter membrane in methanol solution, controlling the temperature of the methanol solution to be 0 ℃, preserving heat, standing for 4 hours, taking out and airing to obtain the membrane.

The preparation method of the modifier comprises the following steps:

putting 3 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 45 ℃ at a speed of 1 ℃/min, adding 2 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 4 hours to obtain a product a;

ii) adding 2 parts of 2, 3-glycidoxypropyl trimethoxy silane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 140 ℃ at a speed of 6 ℃/min, and carrying out heat preservation reaction for 6 hours to obtain a product b;

iii) adding 4 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 85 ℃, and reacting for 4 hours at a constant temperature to obtain a product c;

iv) adding 2 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 157 ℃ at a speed of 2 ℃/min, simultaneously starting a condensation reflux device during heating, and carrying out heat preservation reaction for 5 hours to obtain the catalyst.

Example 3

A method for detecting strong loquat dew.

The verification method of the strong loquat dew comprises the following steps:

establishment of standard powerful loquat dew product detection method

1) Taking 10 parts of a standard strong loquat dew product, adding a diluent into the standard strong loquat dew product for dilution, fixing the volume to 6 times of the volume of the original product, heating the original product to 50 ℃, preserving heat and standing for 1h for later use;

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 30 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 45 parts of diluent into the filter residues again, heating to 45 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 6.0 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

4) Taking 5ml of solution a, adding ethanol solution into the solution a to fix the volume to 500ml, taking 20ml, 20ml and 20ml, respectively placing the solution a, the 20ml and the 20ml into a 50ml volumetric flask, and adding ethanol into the solution a to fix the volume to scale marks; filtering the solution by a microporous filter membrane, and then placing the solution into a liquid chromatograph for test analysis to obtain data of absorption peaks within 0-35min, and introducing the data into Matlab software for drawing, wherein time is an abscissa, and taking an average value of absorption values of 4 groups of data as an ordinate to obtain a standard curve of the strong loquat dew;

Two), detection of samples

1) And (3) processing the strong loquat dew product to be tested according to the method, and then obtaining a corresponding spectrogram through a liquid chromatograph, and judging the loquat dew product to be qualified when the relative peak area of the spectrogram and the standard spectrogram is within 2%.

The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) The prepared solution m and the solubilizer are mixed according to the volume ratio of 105: mixing at a ratio of 0.3, adding 30 parts of petroleum ether, heating the solution to 75 ℃ at a speed of 1 ℃/min, preserving heat, stirring for 5 hours, and cooling to room temperature to obtain the product.

Preparation of the solubilizer:

a. putting 9 parts of tetraethylammonium bromide into a reactor, adding 3 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the toluene solution to 50 ℃ at a speed of 5 ℃/min to obtain a solution i, and standing and preserving heat for later use;

b. 2 parts of tetramethyl ammonium fluoride is put into a reactor, an acetone solution is added, the temperature is raised to 90 ℃ at the speed of 3 ℃/min, 12 parts of solution i is dropwise added into the reactor in a dropwise manner, the dropwise speed is 18 drops/min, the mixture is stirred while being dropwise added, the mixture is heated to 150 ℃ together, and the mixture is subjected to thermal insulation reaction for 4 hours to obtain a reactant a;

c. Adding 20 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at the speed of 1 ℃/min, and carrying out heat preservation reaction for 4 hours to obtain the catalyst.

Before liquid chromatography, preliminary analysis is performed by thin layer chromatography, so that waste of detection cost is reduced, and the specific detection method comprises the following steps:

and (3) placing the solution a of the standard solution and the sample solution on a thin layer chromatography silica gel G plate in a spot plate mode, evaporating the liquid in the solution by natural airing, placing the solution in the chromatography solution, obliquely placing the solution until the chromatography solution climbs to 6/7 area of the silica gel plate, taking out the solution, airing, placing the solution under a 256mm ultraviolet lamp, and judging whether the positions of the observation points are the same or not, if so, carrying out the next analysis, and if not, judging that the solution is a non-qualified product, and eliminating the solution.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. 15 parts of cyclohexane is put into a reactor, 45 parts of ethyl acetate solution is added, 3 parts of propylene carbonate is added after the temperature is raised to 50 ℃ together, the temperature is raised to 70 ℃, and the reaction is carried out for 3 hours under the heat preservation, thus obtaining an intermediate product a;

b. adding 30 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 6 hours at a constant temperature, and standing and preserving the temperature to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 28 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

Wherein, the step 4) also comprises modification of the microporous filter membrane, and the specific modification steps are as follows:

1) Soaking the microporous filter membrane in ethanol solution, heating to 38deg.C at a rate of 2deg.C/min, performing microwave vibration, maintaining the temperature, oscillating for 2 hr, taking out, washing with methanol solution, and standing for use;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 5 ℃/min, and preserving heat and standing for 4 hours to obtain a preliminary modified filter membrane;

3) And (3) soaking the preliminary modified filter membrane in a methanol solution, controlling the temperature of the methanol solution to be 3 ℃, preserving heat, standing for 3 hours, taking out and airing to obtain the membrane.

The preparation method of the modifier comprises the following steps:

putting 1 part of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 42 ℃ at a speed of 3 ℃/min, adding 4 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3 hours to obtain a product a;

ii) adding 4 parts of 2, 3-glycidoxypropyl trimethoxy silane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 145 ℃ at a speed of 4 ℃/min, and carrying out heat preservation reaction for 4 hours to obtain a product b;

iii) adding 6 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 80 ℃, and carrying out heat preservation reaction for 5 hours to obtain a product c;

iv) adding 1 part of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 153 ℃ at a speed of 4 ℃/min, starting a condensing reflux device at the same time when heating, and carrying out heat preservation reaction for 6 hours to obtain the catalyst.

Comparative example 1

A method for testing strong loquat dew.

The method for establishing the standard strong loquat dew product detection method in the strong loquat dew detection method comprises the following steps of:

1) Taking 10 parts of a standard strong loquat dew product, adding a diluent into the standard strong loquat dew product for dilution, and standing for 1h at normal temperature for standby after the volume is fixed to 5.5 times of the volume of the original product;

The procedure is as in example 1.

Comparative example 2

A method for testing strong loquat dew.

The building step 2) of the standard strong loquat dew product detection method in the strong loquat dew detection method is as follows:

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 35 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 43 parts of diluent into the filter residues again, heating to 40 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

the procedure is as in example 1.

Comparative example 3

A method for testing strong loquat dew.

The building step 2) of the standard strong loquat dew product detection method in the strong loquat dew detection method is as follows:

2) Filtering the prepared solution by adopting a microporous molecular film, collecting filtrate, adding 35 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, and uniformly mixing the filtrate and the filtrate a for later use;

the procedure is as in example 1.

Comparative example 4

A method for testing strong loquat dew.

The method for establishing the standard strong loquat dew product detection method in the strong loquat dew detection method comprises the following step 3) of:

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 5.3 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

the procedure is as in example 1.

Comparative example 5

A method for testing strong loquat dew.

Wherein the diluent is n-butanol solution.

The procedure is as in example 1.

Comparative example 6

A method for testing strong loquat dew.

The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) Adding 25 parts of petroleum ether into the prepared solution m, heating the solution to 75 ℃ at the speed of 1.5 ℃/min, preserving heat and stirring for 4 hours, and cooling to room temperature to obtain the product.

The procedure is as in example 1.

Comparative example 7

A method for testing strong loquat dew.

The preparation method of the diluent comprises the following steps:

mixing isopropanol 50 parts, toluene 30 parts, solubilizer 0.4 parts and petroleum ether 25 parts uniformly to obtain the final product.

The procedure is as in example 1.

Comparative example 8

A method for testing strong loquat dew.

The preparation method of the diluent comprises the following steps:

1) 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

2) The prepared solution m and the solubilizer are mixed according to the volume ratio of 103: mixing at a ratio of 0.7, adding 25 parts of petroleum ether, heating the solution to 75 ℃ at a speed of 1.5 ℃/min, preserving heat, stirring for 4 hours, and cooling to room temperature to obtain the product.

The procedure is as in example 1.

Comparative example 9

A method for testing strong loquat dew.

Wherein, step a. in the preparation of the solubilizer is:

a. putting 11 parts of tetraethylammonium bromide into a reactor, adding 4 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the toluene solution to 50 ℃ at a speed of 4 ℃/min to obtain a solution i, and standing and preserving heat for later use;

the procedure is as in example 1.

Comparative example 10

A method for testing strong loquat dew.

Wherein, step a. in the preparation of the solubilizer is:

a. 7 parts of tetraethylammonium bromide is put into a reactor, 2 parts of tetrabutylammonium bisulfate is added into the reactor, toluene solution is added into the reactor to be completely dissolved, the temperature is raised to 50 ℃ at the speed of 4 ℃/min, so as to obtain solution i, and the solution i is kept stand and kept warm for standby;

The procedure is as in example 1.

Comparative example 11

A method for testing strong loquat dew.

Wherein, step a. in the preparation of the solubilizer is:

a. 7 parts of tetraethylammonium bromide is put into a reactor, 4 parts of tetrabutylammonium bisulfate is added into the reactor, toluene solution is added into the reactor to be completely dissolved, the temperature is raised to 50 ℃ at the speed of 6 ℃/min, so as to obtain solution i, and the solution i is kept stand and kept warm for standby;

the procedure is as in example 1.

Comparative example 12

A method for testing strong loquat dew.

Wherein, step b. in the preparation of the solubilizer is:

b. adding 6 parts of tetramethyl ammonium fluoride into a reactor, adding an acetone solution, heating to 90 ℃ at a speed of 2 ℃/min, dropwise adding 13 parts of solution i into the reactor in a dropwise manner, stirring while dropwise adding at a speed of 17 drops/min, heating to 150 ℃ together, and reacting for 4.5 hours at a temperature maintaining to obtain a reactant a;

the procedure is as in example 1.

Comparative example 13

A method for testing strong loquat dew.

Wherein, step b. in the preparation of the solubilizer is:

b. 3 parts of tetramethyl ammonium fluoride is put into a reactor, acetone solution is added, 13 parts of solution i is added dropwise into the reactor in a dropwise manner, the dropping speed is 17 drops/min, stirring is carried out while dropping, the temperature is increased to 150 ℃ at the speed of 2 ℃/min, and the reaction is carried out for 4.5 hours under heat preservation, thus obtaining a reactant a;

The procedure is as in example 1.

Comparative example 14

A method for testing strong loquat dew.

Wherein, step c. in the preparation of the solubilizer is:

c. and (3) adding 18 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 3.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 15

A method for testing strong loquat dew.

Wherein, the preparation of the solubilizer comprises the following steps:

a. and (3) putting 7 parts of tetraethylammonium bromide into a reactor, adding 4 parts of tetrabutylammonium bisulfate, 3 parts of tetramethylammonium fluoride and 18 parts of ethylene glycol dimethyl ether into the reactor, adding a toluene solution to dissolve the materials completely, heating the materials to 120 ℃ at a speed of 4 ℃/min, and carrying out heat preservation reaction for 3-4 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 16

A method for testing strong loquat dew.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. putting 22 parts of cyclohexane into a reactor, adding 43 parts of ethyl acetate solution, heating to 50 ℃ together, adding 2 parts of propylene carbonate, heating to 70 ℃, and reacting for 3 hours at a constant temperature to obtain an intermediate product a;

b. adding 35 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5.5 hours at a constant temperature, and standing and preserving the temperature to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.

The procedure is as in example 1.

Comparative example 17

A method for testing strong loquat dew.

Wherein, the preparation of the chromatographic liquid comprises the following steps:

a. adding 18 parts of cyclohexane into a reactor, adding 43 parts of ethyl acetate solution, heating to 50 ℃ together, adding 2 parts of propylene carbonate, heating to 70 ℃, and reacting for 3 hours at a constant temperature to obtain an intermediate product a;

b. adding 35 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5.5 hours at a constant temperature, and standing and preserving the temperature to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 6:1 to obtain the chromatographic liquid.

The procedure is as in example 1.

Comparative example 18

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-14min, the phase A is 95-90% and the phase B is 5-10%;

when 14-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 19

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-84%, and the phase B is 10-16%;

when 25-31min, phase A is 84-81%, and phase B is 16-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 20

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-75%, and phase B is 19-25%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm;

Wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 21

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 0.5ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 22

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

Wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 33 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 23

A method for testing strong loquat dew.

Wherein, the elution sequence in the liquid chromatography of the step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

when 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

a phosphoric acid solution with water content of 0.45% and a flow rate of 1.0ml/min; the column temperature is 26 ℃, and the detection wavelength is 213mm; the column was Agilent HC-C18X 4.6mm 5 μm.

The procedure is as in example 1.

Comparative example 24

A method for testing strong loquat dew.

Wherein, the microporous filter membrane of the step 4) is not modified.

The procedure is as in example 1.

Comparative example 25

A method for testing strong loquat dew.

Wherein, the step 4) further comprises modification of the microporous filter membrane, and the step 1) in the specific modification step is as follows:

1) Soaking the microporous filter membrane in ethanol solution, performing microwave vibration on the microporous filter membrane at the same time, oscillating for 2 hours at normal temperature, taking out, washing with methanol solution, and standing for later use;

the procedure is as in example 1.

Comparative example 26

A method for testing strong loquat dew.

Wherein, the step 4) also comprises modification of the microporous filter membrane.

1) Soaking the microporous filter membrane in ethanol solution, heating to 40 ℃ at a speed of 1.5 ℃/min, simultaneously carrying out microwave vibration, preserving heat and oscillating for 2 hours, taking out, washing with methanol solution, and standing for later use;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 75 ℃ at the speed of 4 ℃/min, and preserving heat and standing for 4.5 hours to obtain a primary modified filter membrane;

3) And (3) soaking the preliminary modified filter membrane in a methanol solution, controlling the temperature of the methanol solution to be 1 ℃, preserving heat, standing for 3.5h, taking out and airing to obtain the membrane.

The procedure is as in example 1.

Comparative example 27

A method for testing strong loquat dew.

Wherein, the step 4) also comprises modification of the microporous filter membrane.

1) Soaking the microporous filter membrane in ethanol solution, heating to 40 ℃ at a speed of 1.5 ℃/min, simultaneously carrying out microwave vibration, preserving heat and oscillating for 2 hours, taking out, washing with methanol solution, and standing for later use;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 4 ℃/min, and preserving heat and standing for 4.5 hours to obtain a primary modified filter membrane;

3) And (3) soaking the preliminary modified filter membrane in methanol solution, standing for 3.5 hours at normal temperature, taking out and airing to obtain the membrane.

The procedure is as in example 1.

Comparative example 28

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

putting 5 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 2 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and reacting for 3.5 hours at a temperature maintaining speed to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 29

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

putting 2 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 5 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 30

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

Putting 2 parts of sulfonated polyether-ether-ketone and 3 parts of N, N-dimethylformamide diethyl acetal into a reactor, adding a methanol solution into the reactor, heating to 60 ℃ at a speed of 2 ℃/min, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 31

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

putting 2 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 2 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

Ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxy silane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 130 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 32

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

putting 2 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 2 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

Iii) adding 3 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

iv) adding 1.5 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, simultaneously starting a condensing reflux device during heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 33

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

putting 2 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 43 ℃ at a speed of 2 ℃/min, adding 3 parts of N, N-dimethylformamide diethyl acetal into the reactor while the mixture is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3.5 hours to obtain a product a;

ii) adding 3 parts of 2, 3-glycidoxypropyl trimethoxysilane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 143 ℃ at a speed of 5 ℃/min, and carrying out heat preservation reaction for 5 hours to obtain a product b;

iii) adding 5 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 83 ℃, and reacting for 4-5 hours at a temperature maintaining to obtain a product c;

Iv) adding 4 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding a toluene solution, heating to 155 ℃ at a speed of 3 ℃/min, starting a condensing reflux device at the same time when heating, and carrying out heat preservation reaction for 5.5 hours to obtain the catalyst.

The procedure is as in example 1.

Comparative example 34

A method for testing strong loquat dew.

The preparation method of the modifier comprises the following steps:

and i) 2 parts of sulfonated polyether-ether-ketone, 3 parts of 2, 3-glycidoxypropyl trimethoxy silane, 5 parts of N, N-dimethoxy amide and 1.5 parts of 3, 3-diethoxy ethyl propionate are put into a reactor, a methanol solution is added into the reactor, the temperature is raised to 43 ℃ at the speed of 2 ℃/min, 3 parts of N, N-dimethyl formamide diethyl acetal is added into the reactor while the mixture is hot, the mixture is heated to 60 ℃, and the mixture is subjected to thermal insulation reaction for 8 hours, so that the catalyst is obtained.

The procedure is as in example 1.

Comparative example 35

The detection method in CN 106198832B, a production quality control method of the strong loquat dew, is adopted to detect the strong loquat dew product.

Experimental example 1 reproducibility of Standard fingerprint of Standard Strong loquat dew

The high performance liquid fingerprint spectra of the standard loquat dew obtained by the examples and the comparative examples are subjected to reproducibility analysis test, the relative peak area RSD is detected and analyzed, and the specific test analysis results are shown in table 1:

Table 1 reproducibility of liquid chromatography

As can be seen from the data in table 1, the fingerprint patterns of the standard strong loquat dew obtained in examples 1 to 3 have excellent reproducibility, the relative peak areas are all within 1%, the RSD of the comparative example is all above 1%, the spectrogram repeatability of the characteristic fingerprint is poor, and when the data of the RSD is more than 3%, it cannot be used as a judgment fingerprint pattern of the standard strong loquat dew for detecting samples. Wherein, the comparative examples 1-4 are changed to the process in the process of detecting the standard strong loquat dew sample, and the standard curve of the test is changed, so the repeatability is reduced, but the standard loquat dew can still be used as the standard judgment curve. Comparative examples 5-8 change the diluent or change the preparation process and active ingredients of the diluent, resulting in a decrease in the stability of the data, but still some test standard curves are available; comparative examples 9-15 change the formulation or preparation method of the solubilizing agent, resulting in a change of the active ingredient entering the liquid chromatography column, so that reproducibility is reduced; comparative examples 18-23 changed the formulation of the eluent or the elution order of the eluent in the detection process, thereby resulting in a decrease in the reproducibility of the characteristic fingerprint pattern; comparative examples 24-34 change the modification process of the microporous filter membrane, and thus also affect the RSD of the fingerprint profile.

Experimental example 2 detection efficiency of Strong loquat dew

The spectrograms of the standard strong loquat dew obtained by the examples and the comparative examples are used for detecting 90 parts of samples of the strong loquat dew produced in the same batch, and simultaneously detecting 10 parts of interference components distributed by pure effective components in the strong loquat dew, wherein the preparation method of the interference component solution is as follows:

precisely weighing papaverine hydrochloride reference 13.74mg, narcotine reference 11.64mg, thebaine reference 15.50mg, morphine reference 11.57mg and codeine phosphate reference 14.52mg, respectively placing into 20mL measuring flask, adding appropriate amount of 0.5% formic acid in methanol solution, ultrasonically dissolving, fixing volume to scale, and shaking to obtain reference stock solution. Precisely weighing 30uL of papaverine control stock solution, 40uL of narcotine control stock solution, 15uL of thebaine control stock solution, 1400uL of morphine control stock solution and 300uL of codeine control stock solution, placing into a 10mL measuring flask, and fixing volume with methanol to obtain mixed control solution. Accurate absorption of morphine-D ₃ codeine-D ₃ And (3) placing 1.0mL of each internal standard solution into a 20mL measuring flask, and adding methanol to dilute to a scale to obtain the mixed internal standard solution.

Precisely measuring 1.0mL of a sample, placing the sample into a 50mL polytetrafluoroethylene centrifuge tube with a plug, adding 200uL of mixed internal standard working solution, adding 5mL of water, shaking uniformly, precisely adding 15.0mL of acetonitrile, swirling for 1min, adding a QuEChERS AOAC extraction salt package, rapidly shaking, swirling for 1min, centrifuging for 5min at 10000r/min, transferring 10mL of supernatant into a 15mL QuEChERS AOAC dispersion solid phase extraction tube, covering a bottle cap, shaking for 2min with force swirling, centrifuging for 5min at 5000 r/min, taking supernatant, passing through a 0.22um organic microporous filter membrane, and taking filtrate to be measured.

The detection efficiency is determined by the detection time and the detection accuracy, wherein the calculation formula of the detection efficiency is that

η=(（T _max -T _i ）×100%/T _max )*0.55+β*0.45

Wherein η (%) is the detection efficiency;

T _max (min) is the conventional longest detection time for a single sample;

T _i (min) is the detection time of the ith sample;

beta (%) is the accuracy;

the specific addition results are shown in Table 2:

TABLE 2 efficiency of detection

As can be seen from Table 2, the accuracy of examples 1-3 was 100%, and the relative detection time was the largest among all comparative examples, indicating that the detection time of examples 1-3 was the shortest in the overall detection time, so that the detection efficiency was the highest; the comparative example 36 uses the prior art for detection, but because of the interference group, the comparative example 36 can only judge whether the sample is the strong loquat dew according to the existing maximum component detection in the sample, and ignores the existence of other tiny components in the strong loquat dew, so that the accuracy is reduced, and the discrimination capability of a spectrogram is weak. Comparative examples 1 to 4 were modified in the pretreatment process of the strong loquat dew, and comparative examples 5 to 8 were modified in the active ingredient in the diluent, and these modifications resulted in a decrease in the extraction rate of the active ingredient in the product, so that the efficiency of detecting the product was decreased; comparative examples 9 to 15 were modified in the preparation of the solubilizing agent in the diluent, wherein comparative examples 9 to 10 and comparative example 12 were modified in the proportion of the raw material components in the solubilizing agent, and comparative examples 11 and comparative examples 13 to 15 were modified in the process of preparing the solubilizing agent, which resulted in modification of the effective components in the solubilizing agent and reduction of the extraction rate of the solubilizing agent, thus resulting in reduction of the detection efficiency in the detection process; comparative examples 16 to 17, in which the composition of the chromatographic liquid was changed, resulted in deterioration of the chromatographic effect, and on the contrary, the detection time was prolonged, further lowering the detection efficiency.

Claims (5)

1. A detection method of strong loquat dew is characterized in that: the detection method of the strong loquat dew comprises the following steps:

establishment of standard powerful loquat dew product detection method

1) Taking 10 parts of standard strong loquat dew product, adding diluent into the standard strong loquat dew product for dilution, fixing the volume to 5-6 times of the volume of the original product, heating the original product to 50 ℃, preserving heat and standing for 1h for later use;

2) Filtering the solution prepared in the step 1) by adopting a microporous molecular film, collecting filtrate, adding 30-40 parts of diluent into filter residues, heating to 35 ℃, preserving heat and standing for 30min, filtering again to obtain filtrate a and filter residues, adding 40-45 parts of diluent into the filter residues again, heating to 45 ℃, preserving heat and standing for 45min, filtering to obtain filtrate b and filter residues, and uniformly mixing the filtrate a and the filtrate b for later use;

3) The combined filtrate is put into a rotary evaporation dryer for evaporation treatment, and residues after rotary evaporation and drying are obtained; taking 0.5 part of residue, adding 10 parts of ethanol solution into the residue, and then adjusting the pH to 5.5-6.0 through 0.001mol/L HCl solution until the residue is completely dissolved to obtain a solution a;

4) Taking 5ml of solution a, adding ethanol solution into the solution a to fix the volume to 500ml, respectively putting 20ml, 20ml and 20ml into a 50ml volumetric flask, and adding ethanol to fix the volume to scale marks; filtering the solution by a microporous filter membrane, and then placing the solution into a liquid chromatograph for test analysis to obtain data of absorption peaks within 0-35min, and introducing the data into Matlab software for drawing, wherein time is an abscissa, and taking an average value of absorption values of 4 groups of data as an ordinate to obtain a standard curve of the strong loquat dew;

The preparation method of the diluent comprises the following steps:

A. 50 parts of isopropanol and 30 parts of toluene are heated to 60 ℃, and then uniformly mixed to obtain a solution m, and the solution m is kept warm and stands for later use;

B. the prepared solution m and the solubilizer are mixed according to the volume ratio of (100-105): mixing (0.3-0.5), adding petroleum ether 20-30 parts, heating the solution to 75deg.C at a rate of 1-2deg.C/min, stirring for 3-5 hr, and cooling to room temperature;

preparation of solubilizer:

a. putting 5-9 parts of tetraethylammonium bromide into a reactor, adding 3-5 parts of tetrabutylammonium bisulfate into the reactor, adding a toluene solution to completely dissolve the tetrabutylammonium bisulfate, heating the mixture to 50 ℃ at a speed of 3-5 ℃/min to obtain a solution i, and standing and preserving heat for later use;

b. 2-4 parts of tetramethyl ammonium fluoride is put into a reactor, acetone solution is added, the temperature is raised to 90 ℃ at the speed of 1-3 ℃/min, 12-15 parts of solution i is added dropwise into the reactor at the speed of 15-18 drops/min, stirring is carried out while dropwise adding, the temperature is raised to 150 ℃ together, and the reaction is carried out for 4-5 hours under heat preservation, thus obtaining a reactant a;

c. adding 15-20 parts of ethylene glycol dimethyl ether into the reactant a, heating to 50 ℃ at the speed of 1-3 ℃/min, and carrying out heat preservation reaction for 3-4 hours to obtain the catalyst;

Two), detection of samples

1) And (3) processing the strong loquat dew product to be tested according to a standard strong loquat dew product detection method, obtaining a corresponding spectrogram through a liquid chromatograph, and judging the strong loquat dew product to be qualified when the RSD of the relative peak area of the spectrogram and the standard spectrogram is within 2%.

2. The method for detecting the strong loquat dew according to claim 1, which is characterized in that: before liquid chromatography, preliminary analysis is performed by thin layer chromatography, so that waste of detection cost is reduced, and the specific detection method comprises the following steps:

and (3) placing the solution a of the standard solution and the sample solution on a thin layer chromatography silica gel G plate in a spot plate mode, evaporating the liquid in the solution by natural airing, placing the solution in the chromatography solution, obliquely placing the solution until the chromatography solution climbs to 6/7 area of the silica gel plate, taking out the solution, airing, placing the solution under a 256mm ultraviolet lamp, and judging whether the positions of the observation points are the same or not, if so, carrying out the next analysis, and if not, judging that the solution is a non-qualified product, and eliminating the solution.

3. The method for detecting the strong loquat dew according to claim 1, which is characterized in that: the elution order in the liquid chromatography of step 4) is as follows:

0-5 min, 95% of phase A and 5% of phase B;

When 5-12min, the phase A is 95-90% and the phase B is 5-10%;

when 12-25min, the phase A is 90-83% and the phase B is 10-17%;

when 25-31min, the phase A is 83-81%, and the phase B is 17-19%;

at 31-35min, phase A is 81-80%, and phase B is 19-20%;

wherein the organic phase is methanol solution;

the water phase is 0.45% phosphoric acid solution, and the flow rate is 1.0ml/min; the column temperature is 25-28 ℃, and the detection wavelength is 213mm;

wherein, 0.2% triethylamine solution is added into the organic phase; the column was Agilent HC-C18X 4.6mm 5 μm.

4. The method for detecting the strong loquat dew according to claim 1, which is characterized in that: the step 4) also comprises modification of the microporous filter membrane, and the specific modification steps are as follows:

1) Soaking microporous membrane in ethanol solution, heating to 38-42deg.C at a rate of 1-2deg.C/min, performing microwave vibration, keeping temperature, oscillating for 2 hr, taking out, washing with methanol solution, and standing;

2) Putting the filter membrane prepared in the step into dichloromethane solution, adding a modifier, heating to 80 ℃ at the speed of 3-5 ℃/min, and preserving heat and standing for 4-5 hours to obtain a primary modified filter membrane;

3) Soaking the preliminary modified filter membrane in methanol solution, controlling the temperature of the methanol solution to be 0-3 ℃, preserving heat, standing for 3-4 hours, taking out and airing to obtain the membrane;

The preparation method of the modifier comprises the following steps:

putting 1-3 parts of sulfonated polyether-ether-ketone into a reactor, adding a methanol solution into the reactor, heating to 42-45 ℃ at a speed of 1-3 ℃/min, adding 2-4 parts of N, N-dimethylformamide diethyl acetal into the reactor while the reactor is hot, heating to 60 ℃, and carrying out heat preservation reaction for 3-4 hours to obtain a product a;

ii) adding 2-4 parts of 2, 3-glycidoxypropyl trimethoxy silane into the product a, then adding a dichloromethane solution into the mixture, starting a condensing reflux device, heating to 140-145 ℃ at the speed of 4-6 ℃/min, and carrying out heat preservation reaction for 4-6 hours to obtain a product b;

iii) adding 4-6 parts of N, N-dimethoxy amide into the product b, then adding dimethyl sulfoxide solution, heating to 80-85 ℃, and reacting for 4-5 hours at a temperature maintaining condition to obtain a product c;

iv) adding 1-2 parts of 3, 3-diethoxy ethyl propionate into the product c, then adding toluene solution, heating to 153-157 ℃ at the speed of 2-4 ℃/min, simultaneously starting a condensation reflux device during heating, and carrying out heat preservation reaction for 5-6h to obtain the catalyst.

5. The method for detecting the strong loquat dew according to claim 2, which is characterized in that: the preparation of the chromatographic liquid comprises the following steps:

a. 15-20 parts of cyclohexane is put into a reactor, 40-45 parts of ethyl acetate solution is added, 1-3 parts of propylene carbonate is added after the temperature is raised to 50 ℃ together, the temperature is raised to 70 ℃, and the reaction is carried out for 3 hours under the heat preservation, thus obtaining an intermediate product a;

b. Adding 30-40 parts of petroleum ether into the intermediate product a, heating to 80 ℃, reacting for 5-6h with heat preservation, standing and preserving heat to obtain an intermediate product b;

c. and uniformly mixing the intermediate product b and ethanol according to the volume ratio of 7:1 to obtain the chromatographic liquid.