CN110794049B - Detection method of cold granules - Google Patents
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Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a detection method of cold granules. The detection method of the invention improves the liquid chromatography elution conditions of baicalin and phillyrin A in the prior art, measures the contents of the baicalin and the phillyrin A in the cold granules by using the chromatography elution conditions, and takes the contents of the baicalin and the phillyrin A as the quantitative indexes of the cold granules. The detection method has the advantages of good separation degree, high sensitivity, strong specificity and the like.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a detection method of cold granules.
Background
A Chinese medicinal granule (batch 20170905) for treating common cold in children, and syndrome of pathogenic heat hyperactivity comprises Scutellariae radix, fructus forsythiae, herba Moslae, herba Agastaches, caulis Lonicerae, flos Chrysanthemi Indici, herba Artemisiae Annuae, and thin Evodia, and has antiviral, antipyretic, analgesic, and antiinflammatory effects. The preparation method of the cold granules mainly comprises the steps of extracting with water, precipitating with ethanol, drying, crushing, adding a proper amount of auxiliary materials, and granulating, wherein the content of baicalin in the baical skullcap root and the content of forsythin A in weeping forsythia are quantitative indexes.
The cold treating granule contains eight kinds of medicine, each medicine contains different kinds of compounds, such as flavone compounds, anthraquinone compounds, organic acids, alkaloids, polysaccharides and the like, the compounds have wide polar distribution range, and the compounds range from water-soluble saccharides to medium-polar flavone compounds, weak organic acids, weak polar terpenes and the like, and are easy to cause poor separation due to the interference of various compounds in detection, so that the precision and the separation degree of the compounds are influenced.
Disclosure of Invention
In order to overcome the above problems in the prior art, the present invention aims to provide a method for detecting cold particles with good separation degree, high sensitivity and strong specificity, so as to solve the above defects.
The invention provides a method for detecting cold granules, which comprises the following steps:
a. taking baicalin reference substance, preparing serial baicalin reference substance solutions, and analyzing by high performance liquid chromatography to obtain baicalin standard curve; taking a forsythin A reference substance, preparing forsythin A reference substance solutions with series concentrations, and analyzing by high performance liquid chromatography to obtain a forsythin A standard curve;
b. preparing a sample S1, measuring by high performance liquid chromatography to obtain the chromatographic peak area of baicalin in the sample S1, and calculating the content of baicalin in the sample S1 according to the chromatographic peak area of the baicalin and the baicalin standard curve in the step a; preparing a sample S2, measuring by high performance liquid chromatography to obtain the chromatographic peak area of phillyrin A in the sample S2, and calculating the content of phillyrin A in the sample S2 according to the chromatographic peak area of phillyrin A and the standard curve of phillyrin A in the step a;
wherein, the chromatographic conditions for measuring the baicalin standard curve in the step a and the baicalin content in the sample S1 in the step b are as follows: a C18 chromatography column; the mobile phase is methanol and 0.2 volume percent phosphoric acid aqueous solution; gradient elution conditions: the volume fraction of the methanol is 43% in 0-25 min, 43-90% in 25-29 min, 90% in 29-35 min, 90-43% in 35-37 min, and 43% in 37-48 min; the flow rate is 0.8-1.0 mL/min;
the chromatographic conditions for measuring the forsythin A standard curve in the step a and the forsythin A content in the sample S2 in the step b are as follows: the mobile phase is acetonitrile and 0.4 volume percent acetic acid aqueous solution; gradient elution conditions: the volume fraction of acetonitrile is 14% in 0-15 min, the volume fraction of acetonitrile is 14-22% in 15-32 min, the volume fraction of acetonitrile is 22-80% in 32-34 min, the volume fraction of acetonitrile is 80% in 34-48 min, the volume fraction of acetonitrile is 80-14% in 48-50 min, and the volume fraction of acetonitrile is 14% in 50-60 min; the flow rate is 0.8-1.0 mL/min.
Further, the method for preparing the test sample S1 is as follows: taking the cold granules, grinding, sieving, weighing 0.35-0.7 g, adding 50-100 mL of methanol solution, carrying out ultrasonic treatment for 30-60 min, and filtering to obtain the medicine.
Further, the method for preparing the supply sample S2 is: grinding and sieving cold granules, weighing 0.7-1.4 g, adding 10-50 mL of methanol solution, carrying out ultrasonic treatment for 30-60 min, and filtering to obtain the medicine.
Further, in the chromatographic conditions for measuring the baicalin standard curve in the step a and the baicalin content in the sample supply S1 in the step b, the column temperature is 35-40 ℃, and the ultraviolet detection wavelength is 280 nm.
Further, in the chromatographic conditions for measuring the forsythin A standard curve in the step a and measuring the forsythin A content of the sample S2 in the step b, the column temperature is 35-40 ℃, and the ultraviolet detection wavelength is 330 nm.
Further, in the chromatographic conditions described in step a and step b, the amount of sample was 5. mu.L or 10. mu.L.
Further, in the step a, the standard curve is a linear equation between the peak area of the reference substance chromatographic peak and the reference substance concentration.
Therefore, compared with the prior art, the detection method has the following beneficial effects:
(1) according to the method, the elution condition in the baicalin separation process is improved, isocratic elution is improved into gradient elution, the separation degree, precision and stability of the target are improved, and the generation of negative interference is reduced.
(2) By improving the elution condition in the separation process of the forsythin A, the invention solves the problem of the deviation of the retention time of the target object caused by lack of the balance time after elution in the prior art, further improves the precision and the separation degree of the analysis method, and reduces the generation of negative interference.
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but within the scope of the invention, without departing from the basic idea of the invention.
Example 1
The invention provides a method for detecting cold granules, which comprises the following steps:
1.1, chromatographic conditions for measuring baicalin
A chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 1.0 mL/min;
sample injection amount: 5 mu L of the solution; column temperature: 35 ℃; measuring wavelength: 280 nm.
Mobile phase: methanol, 0.2 vol% phosphoric acid in water, gradient elution conditions (volume fraction) were:
TABLE 1 determination of gradient elution conditions of baicalin
1.2, determining chromatographic conditions of forsythin A
A chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 1.0 mL/min;
sample introduction amount: 5 mu L of the solution; column temperature: 35 ℃; measuring wavelength: 330 nm.
Mobile phase: acetonitrile, 0.4 vol% acetic acid aqueous solution, and gradient elution conditions (volume fraction):
TABLE 2 determination of gradient elution conditions for forsythin A
1.3 preparation of baicalin Standard Curve
Taking a proper amount of baicalin reference substance, precisely weighing, adding methanol to prepare a solution containing about 80 mu g per 1mL, preparing a standard stock solution of 80ug/mL, taking the standard stock solution with a corresponding volume, diluting with methanol to obtain standard solutions of 10, 20, 40, 60 and 80ug/mL, injecting into a liquid chromatograph under the chromatographic condition of 1.1, and performing linear regression by taking the concentration of the baicalin reference substance as a horizontal coordinate and the peak area integral value as a vertical coordinate.
1.4 preparation of Forsythiaside A Standard Curve
Taking a proper amount of a phillyrin A reference substance, precisely weighing, adding methanol to prepare a solution containing 0.2mg per 1mL, preparing a standard stock solution of 200ug/mL, taking the standard stock solution with a corresponding volume, accurately diluting the standard stock solution with methanol to a solution to be detected of 20, 50, 100, 150 and 200ug/mL, injecting the solution into a liquid chromatograph under the chromatographic condition of 1.2, and performing linear regression by taking the concentration of the phillyrin A reference substance as a horizontal coordinate and a peak area integral value as a vertical coordinate.
1.5 preparation and detection of sample S1
Taking cold particles, grinding, weighing 0.35g, placing in a conical flask with a plug, adding 50mL of methanol solution, accurately weighing the total weight of a sample before ultrasonic treatment and the conical flask with the plug, performing ultrasonic extraction for 30min, performing ultrasonic extraction with the ultrasonic power of 300W and the frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample after ultrasonic treatment and the conical flask with the plug, complementing the weight loss by methanol, shaking up, filtering by a 0.45-micron filter membrane, injecting into a liquid chromatograph according to the chromatographic condition of 1.1, and obtaining the content of baicalin in a sample S1 according to the obtained peak area integral value and the baicalin standard curve obtained by 1.3.
1.6 preparation and detection of sample S2
Taking cold particles, grinding, weighing 0.7g, placing in a conical flask with a plug, adding 10mL of methanol solution, accurately weighing the total weight of a sample before ultrasonic treatment and the conical flask with the plug, performing ultrasonic extraction for 30min, performing ultrasonic extraction with the ultrasonic power of 300W and the frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample after ultrasonic treatment and the conical flask with the plug, complementing the weight loss by methanol, shaking up, filtering by a 0.45-micron filter membrane, injecting into a liquid chromatograph according to the chromatographic condition of 1.2, and obtaining the content of the forsythin A in a sample S2 according to the obtained peak area integral value and the forsythin A standard curve obtained by 1.4.
Example 2
The invention provides a method for detecting cold granules, which comprises the following steps:
2.1, determining the chromatographic conditions of baicalin
A chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 0.8 mL/min;
sample introduction amount: 10 mu L of the solution; column temperature: 40 ℃; measuring wavelength: 280 nm.
Mobile phase: methanol, 0.2 vol% phosphoric acid in water, and gradient elution conditions were the same as in example 1, table 1.
2.2 chromatographic conditions for measuring phillyrin A
And (3) chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 0.8 mL/min;
sample injection amount: 10 mu L of the solution; column temperature: 40 ℃; measuring wavelength: 330 nm.
Mobile phase: acetonitrile, 0.4 vol% acetic acid in water, and gradient elution conditions were the same as in example 1, table 2.
2.3 preparation of baicalin Standard Curve
The method for preparing the baicalin standard curve was the same as 1.3 in example 1, except that the chromatographic conditions were 2.1.
2.4 drawing of Forsythiaside A Standard Curve
The method for drawing a baicalin standard curve was the same as 1.4 in example 1, except that the chromatographic conditions were 2.2.
2.5 preparation and detection of sample S1
Taking cold particles, grinding, weighing 0.7g, placing into a conical flask with a plug, adding 100mL of methanol solution, weighing the total weight of the sample and the conical flask with the plug before accurate ultrasonic treatment, performing ultrasonic extraction for 60min, performing ultrasonic power of 300W and frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample and the conical flask with the plug after ultrasonic treatment, supplementing the weight loss by using methanol, shaking uniformly, filtering by using a 0.45-micron filter membrane, injecting into a liquid chromatograph according to 2.1 chromatographic conditions, and analyzing according to the obtained peak area integral value and the baicalin standard curve obtained by 2.3 to obtain the content of the baicalin in a sample S1.
2.6 preparation and detection of sample S2
Taking cold particles, grinding, weighing 1.4g, placing in a conical flask with a plug, adding 50mL of methanol solution, accurately weighing the total weight of a sample before ultrasonic treatment and the conical flask with the plug, performing ultrasonic extraction for 30min, performing ultrasonic treatment at the ultrasonic power of 300W and the frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample after ultrasonic treatment and the conical flask with the plug, supplementing the weight loss by using methanol, shaking uniformly, filtering by using a 0.45-micron filter membrane, injecting into a liquid chromatograph according to the chromatographic condition of 2.2, and obtaining the content of the forsythin A in a sample S2 according to the obtained peak area integral value and the standard curve of the forsythin A obtained by 2.4.
Example 3
The invention provides a method for detecting cold granules, which comprises the following steps:
3.1, determining the chromatographic conditions of baicalin
A chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 0.9 mL/min;
sample introduction amount: 5 mu L of the solution; column temperature: 38 ℃; measuring wavelength: 280 nm.
Mobile phase: methanol, 0.2 vol% phosphoric acid in water, and gradient elution conditions were the same as in Table 1 of example 1.
3.2 chromatographic conditions for determining forsythin A
A chromatographic column: welch Ultimate C18 column (4.6 mm. times.250 mm, 5 μm); flow rate: 0.9 mL/min;
sample injection amount: 5 mu L of the solution; column temperature: 38 ℃; measuring wavelength: 330 nm.
Mobile phase: acetonitrile, 0.4 vol% acetic acid in water, and gradient elution conditions were the same as in example 1, table 2.
3.3 drawing of Standard Curve of baicalin
The method for drawing the baicalin standard curve was the same as 1.3 in example 1, except that the chromatographic conditions were 3.1.
3.4 drawing of Forsythiaside A Standard Curve
The method for drawing the baicalin standard curve was the same as 1.4 in example 1, except that the chromatographic conditions were 3.2.
3.5 preparation and detection of sample S1
Taking cold particles, grinding, weighing 0.5g, placing into a conical flask with a plug, adding 50mL of methanol solution, accurately weighing the total weight of a sample before ultrasonic treatment and the conical flask with the plug, performing ultrasonic extraction for 60min, performing ultrasonic treatment at the ultrasonic power of 300W and the frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample after ultrasonic treatment and the conical flask with the plug, supplementing the weight loss by using methanol, shaking uniformly, filtering by using a 0.45-micron filter membrane, injecting into a liquid chromatograph according to the chromatographic condition of 3.1, and analyzing according to the obtained peak area integral value and the baicalin standard curve obtained by 3.3 to obtain the content of the baicalin in a sample S1.
3.6 preparation and detection of sample S2
Taking cold particles, grinding, sieving, weighing 0.8g, placing in a conical flask with a stopper, adding 25mL of methanol solution, accurately weighing the total weight of a sample before ultrasonic treatment and the conical flask with the stopper, performing ultrasonic extraction for 30min, performing ultrasonic treatment at the ultrasonic power of 300W and the frequency of 40kHz, taking out, cooling, accurately weighing the total weight of the sample after ultrasonic treatment and the conical flask with the stopper, supplementing the weight loss by using methanol, shaking uniformly, filtering by using a 0.45-micron filter membrane, injecting into a liquid chromatograph according to the chromatographic condition of 3.2, and obtaining the content of the forsythin A in a sample S2 according to the obtained peak area integral value and the forsythin A standard curve obtained by 3.4.
Comparative example 1
According to the chromatographic conditions in the content determination method under the item of scutellaria baicalensis in the section of China pharmacopoeia of 2015 edition, the method adopts an isocratic determination method, takes methanol-water-phosphoric acid as a mobile phase, and has the volume ratio of 47: 53: 0.2; other chromatographic conditions, standard curve preparation, sample preparation and analysis methods were the same as in example 1 except for the above.
Phillyrin a was measured with reference to chromatographic conditions in a method for measuring the content of forsythia suspense under the item in "chinese pharmacopoeia", 2015 edition, using methanol-water as a mobile phase (the proportion of the mobile phase is expressed by volume fraction) by a gradient condition measuring method shown in table 3 below; other chromatographic conditions, standard curve preparation, sample preparation and analysis methods were the same as in example 1.
TABLE 3 gradient elution conditions for forsythin A in comparative example 1
Test example 1 method for measuring precision and separation degree of baicalin and forsythin A in the detection method of the present invention
The test is carried out according to the detection methods of the examples 1-3 and the comparative example 1, the standard curve of baicalin and forsythin A in each example/comparative example is only measured once, and the correlation coefficient is more than 99.5%; in each example/comparative example, the same experimenter repeats the test 6 times in the process from the preparation of the sample to the analysis of the upper liquid chromatograph, the peak areas, retention time values and peak separation degrees of baicalin and forsythin a chromatogram of each test are recorded, and the relative standard deviation (RSD values) and the peak separation degree mean values of the peak areas and the retention times are calculated, namely the method precision and the separation degree of examples 1-3 and comparative example 1, and the results are shown in table 4.
TABLE 4 methods precision and resolution test
Tests show that the correlation coefficients of the baicalin and phillyrin A standard curves in examples 1-3 and comparative example 1 are all over 99.8%, and the linearity is good. As can be seen from Table 4, when the detection method of the invention is used for determining baicalin and forsythin A in the cold granules, the relative standard deviation of the peak area and the retention time is lower than that of the comparative example 1, which shows that the detection method of the invention has better method precision and stronger reproducibility. In addition, it can be understood from table 4 that the relative standard deviation of the retention time of phillyrin a measured using the elution conditions of table 3 of comparative example 1 is high because the elution gradient of phillyrin a of comparative example 1 is not reasonably set, and after the gradient elution of the first sample, the second sample starts to be injected without equilibration, which causes the retention time shift of the sample injected later, affecting the precision and the resolution, and secondly, the mobile phase used in comparative example 1 is methanol and water, which improves the peak pattern not as effectively as methanol and 0.4 vol% acetic acid aqueous solution as the mobile phase, and also causes the reduction of the resolution.
Test example 2 influence of different extraction methods on baicalin and Forsythiaside A in sample
Taking 0.35g of finished powder of cold granules (batch number 20170905), 2 groups, parallelly preparing 2 parts of each group, precisely weighing, respectively placing into a conical flask, precisely adding 50ml of methanol into each group, weighing, ultrasonically extracting one group for 30min, reflux extracting the other group for 60min, cooling, weighing again, complementing the lost weight, shaking up, and filtering to obtain the test solution for measuring the baicalin content. Taking 0.7g of finished powder of the cold granules (batch number 20170905), preparing 2 groups of the powder, preparing 2 parts of the powder in parallel, precisely weighing, respectively placing the powder in a conical flask, precisely adding 25ml of methanol into each group, weighing the weight, ultrasonically extracting one group for 30min, performing reflux extraction on the other group for 60min, cooling, weighing the weight again, complementing the weight loss, shaking up, and filtering to obtain a test solution for determining the content of forsythiaside A. The contents of baicalin and forsythiaside A in different extraction methods were measured by 0.45um membrane filtration using the chromatographic conditions of 1.1 and 1.2 in example 1 and the standard curves obtained in 1.3 and 1.4, respectively, and the results are shown in Table 5.
TABLE 5 influence of different extraction methods on the determination of baicalin and forsythiaside A content
The results in table 5 show that the ultrasonic extraction and reflux extraction results are similar, but the ultrasonic extraction operation is simple, and the sample is not easy to lose in the transferring process.
Test example 3 Effect of different extraction time on baicalin and Forsythiaside A in sample
Taking 0.35g of finished powder of the same batch, weighing 3 groups, parallelly weighing 2 parts of each group, precisely weighing, respectively placing in a conical flask, precisely adding 50ml of methanol into each group, weighing, ultrasonically extracting for different times, taking out, cooling, weighing again, complementing the lost weight, shaking up, and filtering to obtain the test solution for measuring the baicalin content. Taking about 0.7g of finished powder of the same batch, weighing 2 parts of the finished powder in parallel for 3 groups, precisely weighing, respectively placing the groups in conical flasks, precisely adding 25ml of methanol into each group, weighing, ultrasonically extracting for different times, taking out, cooling, weighing, complementing the weight loss, shaking up, and filtering to obtain the test solution for determining the content of forsythiaside A. The contents of baicalin and forsythiaside a were determined using the chromatographic conditions of 1.1 and 1.2 in example 1 and the standard curves obtained in 1.3 and 1.4, respectively, at different extraction times, and the results are shown in table 6.
TABLE 6 influence of different extraction time on the determination of baicalin and forsythiaside A content (n ═ 2)
The results in table 6 show that the extraction time has no significant influence on the contents of baicalin and forsythiaside A, but the ultrasonic extraction content is slightly higher than 30 min.
Test example 4 Effect of different extraction solvents on baicalin and Forsythiaside A in samples
Taking 0.35g of finished powder of the same batch, preparing 4 groups of finished powder, preparing 2 parts of the finished powder in parallel for each group, precisely weighing, respectively placing the groups in a conical flask, precisely adding 50ml of methanol, 50% of methanol, 70% of methanol and 70% of ethanol into each group, weighing, ultrasonically extracting for 30min, cooling, weighing again, respectively complementing the weight loss by using the solvent, shaking up, and filtering to obtain a test solution for measuring the baicalin content. Taking about 0.7g of finished product powder of the same batch, carrying out 2 parts in parallel on 4 groups, precisely weighing, respectively placing in a conical flask, precisely adding 25ml of methanol, 50 vol% of methanol, 70 vol% of methanol and 70 vol% of ethanol into each group, weighing, carrying out ultrasonic extraction for 30min, cooling, weighing again, respectively supplementing the weight loss by using the above solvents, shaking uniformly, and filtering to obtain the sample solution for measuring the content of forsythiaside A. The contents of baicalin and forsythiaside a were determined using the chromatographic conditions of 1.1 and 1.2 in example 1 and the standard curves obtained in 1.3 and 1.4, respectively, for different extraction methods, and the results are shown in table 7.
TABLE 7 influence of different extraction solvents on the determination of baicalin and forsythiaside A content
The results in Table 7 show that methanol extraction is more effective than other solvents, and therefore methanol was selected as the extraction solvent in the present invention.
Test example 5 stability and sample recovery ratio of the detection method of the present invention
And (3) stability test: samples S1 and S2 prepared in example 1 and comparative example 1 are respectively taken and injected for 5 mul 0, 2, 8, 12 and 24h after preparation, the peak areas of chromatographic peaks of baicalin and forsythin A after each injection are recorded, and RSD values of the samples are calculated.
Sample application recovery rate test: and (4) measuring the sample recovery rate by a sample recovery method. Grinding the cold granules (batch No. 20170905), precisely weighing 0.175g, paralleling 6 parts, placing into a conical flask with a stopper, adding baicalin reference substance at a ratio of 100%, injecting 5 μ l sample according to preparation and chromatographic conditions of baicalin test solution of example 1 and comparative example 1, and determining the peak area of baicalin; grinding the cold granules (batch No. 20170905), precisely weighing 0.355g of parallel 6 parts, placing in a conical flask with a plug, adding phillyrin A reference substance according to 100%, injecting 5 μ l of phillyrin A according to the preparation and chromatographic conditions of the test solutions of phillyrin A in example 1 and comparative example 1, and measuring the peak area of phillyrin A; the recovery rate was calculated from the respective peak areas according to the following formula.
Recovery rate (content after addition of control-sample content)/amount of added control × 100%
TABLE 8 Process stability and recovery on spiking
The results in table 8 show that example 1 has better stability of baicalin and forsythoside a than comparative example 1, and has better RSD value; the average sample adding recovery rate of baicalin and forsythiaside A meets the requirement.
It will be appreciated by those skilled in the art that the above embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (7)
1. The detection method of the cold granules is characterized by adopting an external standard method for detection, and comprises the following steps:
a. taking baicalin reference substance, preparing serial baicalin reference substance solutions, and analyzing by high performance liquid chromatography to obtain baicalin standard curve; taking forsythoside A reference substance, preparing forsythoside A reference substance solutions with series concentrations, and analyzing by high performance liquid chromatography to obtain forsythoside A standard curve;
b. preparing a sample S1, measuring by high performance liquid chromatography to obtain the chromatographic peak area of baicalin in the sample S1, and calculating the content of baicalin in the sample S1 according to the chromatographic peak area of baicalin and the baicalin standard curve in the step a; preparing a supply sample S2, measuring by using high performance liquid chromatography to obtain the forsythoside A chromatographic peak area in a supply sample S2, and calculating the content of the forsythoside A in the supply sample S2 according to the forsythoside A chromatographic peak area and the forsythoside A standard curve in the step a;
the chromatographic conditions for measuring the baicalin standard curve in the step a and the baicalin content in the sample S1 in the step b are as follows: a C18 chromatography column; the mobile phase is methanol and 0.2 volume percent phosphoric acid aqueous solution; gradient elution conditions: the volume fraction of the methanol is 43% in 0-25 min, 43-90% in 25-29 min, 90% in 29-35 min, 90-43% in 35-37 min, and 43% in 37-48 min; the flow rate is 0.8-1.0 mL/min;
the chromatographic conditions used for measuring the forsythiaside a standard curve in step a and the forsythiaside a content in sample S2 in step b are as follows: the mobile phase is acetonitrile and 0.4 volume percent acetic acid aqueous solution; gradient elution conditions: the volume fraction of acetonitrile is 14% in 0-15 min, the volume fraction of acetonitrile is 14-22% in 15-32 min, the volume fraction of acetonitrile is 22-80% in 32-34 min, the volume fraction of acetonitrile is 80% in 34-48 min, the volume fraction of acetonitrile is 80-14% in 48-50 min, and the volume fraction of acetonitrile is 14% in 50-60 min; the flow rate is 0.8-1.0 mL/min;
the cold granules are composed of eight medicines of scutellaria baicalensis, forsythia suspensa, elsholtzia, pogostemon cablin, honeysuckle stem, wild chrysanthemum, sweet wormwood and thin evodia herb;
the extraction solvent used for the preparation of sample S1 and sample S2 was methanol.
2. The method for detecting cold granules according to claim 1, wherein the method for preparing the test sample S1 is: grinding the cold granules into fine powder, weighing 0.35-0.7 g of the cold granules, adding 50-100 mL of methanol, carrying out ultrasonic treatment for 30-60 min, and filtering to obtain the traditional Chinese medicine composition.
3. The method for detecting cold granules according to claim 1, wherein the method for preparing the test sample S2 is: grinding the cold granules, weighing 0.7-1.4 g of the cold granules, adding 10-50 mL of methanol, carrying out ultrasonic treatment for 30-60 min, and filtering to obtain the traditional Chinese medicine composition.
4. The method for detecting cold granules according to claim 1, wherein in the chromatographic conditions for measuring the baicalin standard curve in the step a and the baicalin content in the sample S1 in the step b, the column temperature is 35-40 ℃ and the ultraviolet detection wavelength is 280 nm.
5. The method for detecting cold granules according to claim 1, wherein in the chromatographic conditions for measuring the forsythiaside A standard curve in the step a and the content of the forsythiaside A in the sample S2 in the step b, the column temperature is 35-40 ℃, and the ultraviolet detection wavelength is 330 nm.
6. The method for detecting cold particles according to claim 1, wherein the sample size is 5 μ L or 10 μ L.
7. The method for detecting cold granules according to claim 1, wherein in the step a, the standard curve is a linear equation between the peak area of the reference substance chromatographic peak and the concentration of the reference substance.
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| CN104483411A (en) * | 2014-12-29 | 2015-04-01 | 黑龙江珍宝岛药业股份有限公司 | Detection method for Forsythia suspensa and product containing Forsythia suspensa |
| CN106290646A (en) * | 2016-08-29 | 2017-01-04 | 河北国金药业有限责任公司 | The detection method of Compound Jinyinhua Granules |
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