CN110857939A - Detection method of traditional Chinese medicine composition with sleep improvement function - Google Patents
Detection method of traditional Chinese medicine composition with sleep improvement function Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine detection, and discloses a detection method of a traditional Chinese medicine composition with a sleep improving function, which comprises the steps of identifying lucid ganoderma, multiflower knotweed stem and spina date seed by using a thin-layer chromatography and determining the content of spinosad by using a high performance liquid chromatography, wherein the lucid ganoderma and the multiflower knotweed stem are subjected to the same pretreatment method by using the method, and a specific developing agent system is selected, so that various components in the lucid ganoderma and the multiflower knotweed stem can be well separated, and the simultaneous identification of two medicinal materials is realized; in the chromatogram of each test sample, spots with the same color are displayed at the positions corresponding to the chromatograms of the positive reference substances, and no interference spots exist in the chromatograms of the negative reference substances, which shows that the method has stronger specificity. Meanwhile, the method simplifies the operation process, reduces the time required for identification, improves the working efficiency, reduces the detection cost, provides a new idea for the thin-layer chromatography identification of each component in the traditional Chinese medicine composition, and is beneficial to the improvement of the quality control standard of the traditional Chinese medicine.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a detection method of a traditional Chinese medicine composition with a sleep improving function.
Background
Adequate sleep, balanced diet and proper exercise are three health standards recognized by international society. During a person's lifetime, one third of the time spent in sleep is an indispensable component of health as a necessary process for life. Persistent sleep disorders can lead to physical and mental damage, socioeconomic losses, and other adverse consequences, including cardiovascular disease, mental illness, poor immunity, and even increased mortality, have become serious public health problems.
Therefore, chinese patent document CN102188610A discloses a traditional Chinese medicine composition with sleep improving function, which comprises extracts of ganoderma lucidum, arillus longan, semen ziziphi spinosae, semen boitae, caulis polygoni multiflori, rhizoma polygonati, fructus ziziphi jujubae, etc., but only introduces the raw material composition and the preparation method of the traditional Chinese medicine composition, does not provide a corresponding product detection method, and is not beneficial to the control of the internal quality of the composition in the production, circulation and use processes. In the prior art, many detection and analysis methods for ganoderma lucidum and vine of multiflower knotweed are available, for example, the study of 'allergic health particle quality control method' in the publication of China journal literature discloses a thin-layer chromatography identification method for vine of multiflower knotweed and ganoderma lucidum, and the method has the disadvantages that the pretreatment modes of vine of multiflower knotweed and ganoderma lucidum are different, the finally selected developing agent system is quite different, the developing agent used in the former is a benzene-ethanol system, and the developing agent used in the latter is a petroleum ether (60-90 ℃) -ethyl formate-formic acid system, so that the operation is quite inconvenient, and particularly when large-scale detection is needed, the workload is quite large, and the working efficiency is low.
Disclosure of Invention
Therefore, the invention aims to provide a detection method of the traditional Chinese medicine composition with the function of improving sleep.
Therefore, the invention provides a detection method of a traditional Chinese medicine composition, which comprises the step of identifying lucid ganoderma, tuber fleeceflower stem and spina date seed in the traditional Chinese medicine composition by using a thin-layer chromatography, wherein the thin-layer chromatography comprises the following steps:
(1) preparation of positive control solution and test solution
Respectively adding a first solvent into a ganoderma lucidum positive control, a polygonum multiflorum positive control and a spina date seed positive control, filtering, evaporating filtrate to dryness, and adding a second solvent to respectively serve as respective positive control solutions;
adding the first solvent into the Chinese medicinal composition, filtering, evaporating the filtrate, and adding the second solvent to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking the Ganoderma positive control solution, caulis Polygoni Multiflori positive control solution and sample solution, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent, taking out, air drying, placing under ultraviolet lamp for inspection and/or spraying with sulfuric acid ethanol solution, and oven drying until spots appear clearly; and the combination of (a) and (b),
sucking the positive control solution and the sample solution of semen Ziziphi Spinosae, respectively dropping on the same silica gel thin layer plate, developing with water saturated n-butanol as developing agent, taking out, air drying, and inspecting under ultraviolet lamp.
Further, the positive reference substance of the lucid ganoderma is selected from at least one of lucid ganoderma reference medicinal materials or lucid ganoderma extracts; the positive control of caulis Polygoni Multiflori is selected from at least one of caulis Polygoni Multiflori control medicinal materials or caulis Polygoni Multiflori extract; the positive control of the spina date seed is at least one of a spina date seed control medicinal material or a spina date seed extract.
Further, the first solvent is one or more of methanol, ethanol and propanol; the second solvent is one or more of methanol, ethanol and propanol.
Further, the thin layer chromatography comprises the following steps:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 1.5-3 g of lucid ganoderma contrast medicinal material, 0.2-0.3 g of tuber fleeceflower stem contrast medicinal material and 0.5-1.5 g of spina date seed contrast medicinal material, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the filtrate as respective contrast medicinal material solutions;
taking 0.5-1.5 g of lucid ganoderma extract, 0.5-1.5 g of tuber fleeceflower stem extract and 0.5-1.5 g of spina date seed extract, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the filtrate as respective extract solution;
taking 4.5-5.5 g of a negative control product without a lucid ganoderma extract, 4.5-5.5 g of a negative control product without a vine of multiflower knotweed extract and 4.5-5.5 g of a negative control product without a spina date seed extract, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the ethanol solution as a negative control product solution;
taking 4.5-5.5 g of the traditional Chinese medicine composition, adding 20-40 mL of ethanol into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5-1.5 mL of ethanol for dissolving to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking 4-6 muL of the lucid ganoderma contrast medicinal material solution, 4-6 muL of the multiflower knotweed vine contrast medicinal material solution, 4-6 muL of the lucid ganoderma extract solution, 4-6 muL of the multiflower knotweed vine extract solution, 4-6 muL of the negative contrast product solution without the lucid ganoderma extract, 4-6 muL of the negative contrast product solution without the multiflower knotweed vine extract and 4-6 muL of the test product solution, respectively dropping on the same silica gel thin layer plate, developing by taking cyclohexane-ethyl acetate-formic acid with the volume ratio of 5: 0.2 as a developing agent, taking out, airing, placing under an ultraviolet lamp of 366nm to inspect and/or spray sulfuric acid ethanol solution, and drying until spots are clear in color; and the combination of (a) and (b),
sucking the spina date seed reference medicinal material solution 4-6 muL, the spina date seed extract solution 4-6 muL, the negative reference substance solution 4-6 muL without the spina date seed extract and the sample solution 4-6 muL, respectively dropping on the same silica gel thin layer plate, developing by using water saturated n-butyl alcohol as a developing agent, taking out, airing, and inspecting under an ultraviolet lamp of 366 nm.
Further, the thin layer chromatography comprises the following steps:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 2g of lucid ganoderma reference medicinal material, 0.25g of tuber fleeceflower stem reference medicinal material and 1g of spina date seed reference medicinal material, respectively adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and then adding 1mL of ethanol for dissolving, wherein the ethanol is respectively used as respective reference medicinal material solutions;
respectively adding 30mL of ethanol into 1g of ganoderma lucidum extract, 1g of caulis polygoni multiflori extract and 1g of spina date seed extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain respective extract solutions;
taking 5g of a negative control product without a lucid ganoderma extract, 5g of a negative control product without a vine of multiflower knotweed extract and 5g of a negative control product without a spina date seed extract, respectively adding 30mL of ethanol into the negative control product, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 1mL of ethanol into the filtrate to dissolve the filtrate, and respectively taking the filtrate as respective negative control product solutions;
taking 5g of the traditional Chinese medicine composition, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution;
(2) testing by thin layer chromatography
Sucking the above Ganoderma contrast medicinal material solution 5 μ L, caulis Polygoni Multiflori contrast medicinal material solution 5 μ L, Ganoderma extract solution 5 μ L, caulis Polygoni Multiflori extract solution 5 μ L, negative control solution 5 μ L without Ganoderma extract, negative control solution 5 μ L without caulis Polygoni Multiflori extract and test sample solution 5 μ L, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid at volume ratio of 5: 0.2 as developing agent, taking out, air drying, inspecting and/or spraying with sulfuric acid ethanol solution under 366nm ultraviolet lamp, and drying until spots appear clearly; and the combination of (a) and (b),
sucking the above semen Ziziphi Spinosae control medicinal material solution 5 μ L, semen Ziziphi Spinosae extract solution 5 μ L, negative control solution 5 μ L without semen Ziziphi Spinosae extract and sample solution 5 μ L, respectively dropping on the same silica gel thin layer plate, developing with water saturated n-butanol as developing agent, taking out, air drying, and inspecting under 366nm ultraviolet lamp.
Further, the detection method also comprises the step of measuring the content of the spinosad in the traditional Chinese medicine composition by using a high performance liquid chromatography, wherein the high performance liquid chromatography comprises the following steps:
(1) preparation of standard solution of spinosin and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, and adding a third solvent to prepare a spinosyn standard solution;
and (3) adding a third solvent into the traditional Chinese medicine composition, and filtering to obtain filtrate, namely the sample solution of the traditional Chinese medicine composition.
(2) Absorbing the standard solution of the spinosyns, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
(3) and (3) sucking the sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
Further, the third solvent is methanol.
Further, the high performance liquid chromatography comprises the following steps:
(1) chromatographic condition and system adaptability test
Octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is not more than 2.5 microns; taking acetonitrile as a mobile phase A and water as a mobile phase B for gradient elution; the column temperature is 25-35 ℃; the detection wavelength is 330-340 nm; the number of theoretical plates is not less than 5000 calculated according to the spinosad; the gradient elution process is as follows:
0-10 min, mobile phase A%: 12 → 19, mobile phase B: 88 → 81;
10-12 min, mobile phase A%: 19 → 100, mobile phase B: 81 → 0;
12-15 min, mobile phase A%: 100, mobile phase B: 0;
15-16 min, mobile phase A%: 100 → 12, mobile phase B: 0 → 88;
(2) preparation of standard solution of spinosad and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, adding methanol and diluting into at least 3 spinosyn standard solutions with different concentrations;
taking 1g of the traditional Chinese medicine composition, adding 25mL of methanol, carrying out ultrasonic treatment for 30 minutes, and filtering by using a 0.22-micrometer organic filter membrane to obtain a filtrate, namely a sample solution of the traditional Chinese medicine composition;
(3) sucking 1 mu L of the standard solution of the spinosad, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
(4) and (3) sucking 1 mu L of the sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
In the invention, the traditional Chinese medicine composition has the function of improving sleep, and the raw materials of the traditional Chinese medicine composition comprise a lucid ganoderma extract, a spina date seed extract and a caulis polygoni multiflori extract.
The technical scheme of the invention has the following advantages:
1. the method comprises the steps of identifying the lucid ganoderma, the vine of multiflower knotweed and the spina date seed in the traditional Chinese medicine composition by using a thin-layer chromatography, and determining the content of the spinosad in the traditional Chinese medicine composition by using a high performance liquid chromatography. Meanwhile, the method simplifies the operation process, reduces the time required for identification, improves the working efficiency, reduces the detection cost, provides a new idea for the thin-layer chromatography identification of each component in the traditional Chinese medicine composition, and is beneficial to the improvement of the quality control standard of the traditional Chinese medicine.
2. When the method is used for identifying the spina date seed extract by using the thin-layer chromatography, the pretreatment conditions are the same as those of lucid ganoderma and tuber fleeceflower stem, so that the operation process is further simplified, and the working efficiency is improved.
3. The detection method of the traditional Chinese medicine composition provided by the invention has the characteristics of good stability, strong specificity, high accuracy, good repeatability and the like, and can effectively control the product quality of the traditional Chinese medicine composition in production, circulation, use and other links.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a thin-layer chromatogram for simultaneous identification of Ganoderma lucidum and caulis Polygoni Multiflori in example 1 of the present invention;
FIG. 2 is a thin layer chromatogram for identification of wild jujube seeds in example 2 of the present invention;
FIG. 3 is a chromatogram for determining the content of spinosad in the Chinese medicinal composition by high performance liquid chromatography;
FIG. 4 is a special investigation chromatogram for determining the content of spinosad in the Chinese medicinal composition by high performance liquid chromatography;
FIG. 5 is a linear relationship investigation diagram for measuring the content of spinosad in the Chinese medicinal composition by high performance liquid chromatography.
Description of reference numerals:
1-negative control containing no caulis Polygoni Multiflori extract; 2-caulis polygoni multiflori extract; 3-caulis polygoni multiflori contrast medicinal materials; 4-negative control containing no Ganoderma extract; 5-ganoderma lucidum extract; 6-ganoderma lucidum reference medicinal material; 7-9 test articles (20170204-1, 20170204-2, 20170204-3); 10-negative control without spina date seeds; 11-wild jujube seed extract; 12-semen Ziziphi Spinosae control drug.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the following examples, the traditional Chinese medicine composition for improving sleep function is provided by perfection (china) ltd; the negative control containing no caulis Polygoni Multiflori extract, the negative control containing no Ganoderma extract, and the negative control containing no semen Ziziphi Spinosae extract are provided by Perfect (Chinese) Limited company; ganoderma extract, caulis Polygoni Multiflori extract and semen Ziziphi Spinosae extract are provided by Guangdong pharmaceutical limited; the ganoderma lucidum reference medicinal material, the caulis polygoni multiflori reference medicinal material, the spina date seed reference medicinal material and the spinosin reference substance are purchased from China food and drug testing research institute.
In the following examples, methanol, ethanol and propanol were analytically pure, with a volume concentration of 95% or more; acetonitrile is chromatographic grade, and the volume concentration is more than or equal to 99.9 percent.
Example 1
The embodiment 1 provides a quality control method of a traditional Chinese medicine composition with a function of improving sleep, which comprises the following steps:
1. identifying Ganoderma and caulis Polygoni Multiflori in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, a negative control product without the vine of multiflower knotweed extract, 5g of each negative control product without the lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 1mL of ethanol to dissolve the filtrate to be respectively used as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 on the general rule of 2015 edition of pharmacopoeia of the people's republic of China, 5 mu L of each solution is absorbed, the solution is respectively spotted on the same silica gel G thin-layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), cyclohexane-ethyl acetate-formic acid (the volume ratio is 5: 0.2) is used as a developing agent, the developing solution is taken out, dried in the air, sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color development is clear. The results are shown in FIG. 1, the chromatogram of the test sample and the extract shows the main spots with the same color at the same position as the chromatogram of the control material, and the chromatogram of the negative control sample does not show the main spots with the same color at the same position as the chromatogram of the control material.
2. Identifying semen Ziziphi Spinosae in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, 5g of each negative reference substance without the spina date seed extract and 1g of the spina date seed extract, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate in a water bath, and adding 1mL of ethanol for dissolving to obtain a test solution, a negative reference substance solution and an extract solution. Taking 1g of semen Ziziphi Spinosae as reference material, and making into reference material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, 1 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), developed by taking water saturated n-butyl alcohol as a developing agent, taken out, dried and inspected under an ultraviolet lamp of 366 nm. The results are shown in FIG. 2, the chromatogram of the test solution and the extract solution showed the main spots of the same color at the same position as the chromatogram of the control solution, and the chromatogram of the negative control solution showed no main spots of the same color at the same position as the chromatogram of the control solution.
3. Determining the content of spinosin in the Chinese medicinal composition by using a high performance liquid chromatography method
(1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is 2.5 microns; taking acetonitrile as a mobile phase A and water as a mobile phase B for gradient elution; the column temperature is 30 ℃; the flow rate is 1 mL/min; the detection wavelength is 335 nm; the number of theoretical plates is not less than 5000 calculated according to the spinosad; the gradient elution process comprises the following specific steps:
0-10 min, mobile phase A%: 12 → 19, mobile phase B: 88 → 81;
10-12 min, mobile phase A%: 19 → 100, mobile phase B: 81 → 0;
12-15 min, mobile phase A%: 100, mobile phase B: 0;
15-16 min, mobile phase A%: 100 → 12, mobile phase B: 0 → 88.
(2) Preparation of standard solution of spinosad and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, adding methanol to dissolve and dilute the spinosyn reference substance into a spinosyn standard solution with the concentration of 10.7 mu g/mL, 21.4 mu g/mL, 42.8 mu g/mL, 85.6 mu g/mL and 107 mu g/mL respectively;
taking 1g of the traditional Chinese medicine composition, placing the traditional Chinese medicine composition into a 25mL volumetric flask, adding about 20mL of methanol with the volume concentration of 70%, and fully shaking up. Putting the volumetric flask into an ultrasonic cleaner, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale by using methanol with the volume concentration of 70 percent, shaking up, and filtering by using a 0.22 mu m organic filter membrane to obtain filtrate, namely the sample solution of the traditional Chinese medicine composition.
(3) Measurement and calculation
Precisely absorbing 1 mu L of the standard solution of the spinosad, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
precisely sucking 1 mu L of sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
Example 2
The embodiment 2 provides a quality control method of a traditional Chinese medicine composition with a function of improving sleep, which comprises the following steps:
1. identifying Ganoderma and caulis Polygoni Multiflori in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, a negative control product without the vine of multiflower knotweed extract, 5g of each negative control product without the lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 20mL of methanol, carrying out ultrasonic treatment for 40 minutes, filtering, evaporating filtrate in a water bath to dryness, adding 1.5mL of propanol to dissolve the filtrate, and respectively taking the filtrate as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, the above solutions are respectively absorbed by 4 mu L, respectively dropped on the same silica gel G thin layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), and cyclohexane-ethyl acetate-formic acid (volume ratio is 5: 0.2) is used as a developing agent, developed, taken out, dried and inspected under an ultraviolet lamp (366 nm). The results are shown in fig. 5, the test sample and extract chromatogram showed the same color of the main spot at the same position as the control chromatogram, and the negative control chromatogram showed no main color spot at the same position as the control chromatogram.
2. Identifying semen Ziziphi Spinosae in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, 5g of each negative reference substance without the spina date seed extract and 1g of the spina date seed extract, adding 20mL of methanol, carrying out ultrasonic treatment for 40 minutes, filtering, evaporating the filtrate in a water bath, and adding 1.5mL of propanol for dissolving to obtain a test solution, a negative reference substance solution and an extract solution. Taking 1g of semen Ziziphi Spinosae as reference material, and making into reference material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, 1 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), developed by taking water saturated n-butyl alcohol as a developing agent, taken out, dried and inspected under an ultraviolet lamp of 366 nm. The result shows that the chromatogram of the test solution and the extract solution has main spots with the same color at the same position as the chromatogram of the control solution, and the chromatogram of the negative control solution has no main spots with the same color at the same position as the chromatogram of the control solution.
3. Determining the content of spinosin in the Chinese medicinal composition by using a high performance liquid chromatography method
(1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is 1.7 mu m; taking acetonitrile as a mobile phase A and water as a mobile phase B for gradient elution; the column temperature is 25 ℃; the flow rate is 0.5 mL/min; the detection wavelength is 340 nm; the number of theoretical plates is not less than 5000 calculated according to the spinosad; the gradient elution process comprises the following specific steps:
0-10 min, mobile phase A%: 12 → 19, mobile phase B: 88 → 81;
10-12 min, mobile phase A%: 19 → 100, mobile phase B: 81 → 0;
12-15 min, mobile phase A%: 100, mobile phase B: 0;
15-16 min, mobile phase A%: 100 → 12, mobile phase B: 0 → 88.
(2) Preparation of standard solution of spinosad and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, adding methanol to dissolve and dilute the spinosyn reference substance into a spinosyn standard solution with the concentration of 10.7 mu g/mL, 21.4 mu g/mL, 42.8 mu g/mL, 85.6 mu g/mL and 107 mu g/mL respectively;
taking 1g of the traditional Chinese medicine composition, placing the traditional Chinese medicine composition into a 25mL volumetric flask, adding about 20mL of methanol with the volume concentration of 70%, and fully shaking up. Putting the volumetric flask into an ultrasonic cleaner, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale by using methanol with the volume concentration of 70 percent, shaking up, and filtering by using a 0.22 mu m organic filter membrane to obtain filtrate, namely the sample solution of the traditional Chinese medicine composition.
(3) Measurement and calculation
Precisely absorbing 1 mu L of the standard solution of the spinosad, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
precisely sucking 1 mu L of sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
Example 3
The embodiment 3 provides a quality control method of a traditional Chinese medicine composition with a function of improving sleep, which comprises the following steps:
1. identifying Ganoderma and caulis Polygoni Multiflori in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, a negative control product without the vine of multiflower knotweed extract, 5g of each negative control product without the lucid ganoderma, 1g of lucid ganoderma extract and 1g of vine of multiflower knotweed extract, adding 40mL of propanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating filtrate in a water bath to dryness, and adding 0.5mL of methanol for dissolving, wherein the filtrate is respectively used as a test solution, a negative control solution and an extract solution. Preparing 2g of lucid ganoderma reference medicinal material and about 0.25g of multiflower knotweed stem reference medicinal material into a reference medicinal material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015, 6 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin-layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), cyclohexane-ethyl acetate-formic acid (volume ratio is 5: 0.2) is used as a developing agent, developed, taken out, dried, inspected under an ultraviolet lamp (366nm), sprayed with 10% sulfuric acid ethanol test solution, and dried at 105 ℃ until the color is clear. The results are shown in FIG. 5, the chromatogram of the test sample and the extract showed the same color of the main spot at the same position as the chromatogram of the control material, the chromatogram of the negative control material showed no main spot at the same position as the chromatogram of the control material, and the results of the 365nm UV light inspection were consistent with the results of the 10% sulphuric acid ethanol reagent spraying and drying at 105 ℃.
2. Identifying semen Ziziphi Spinosae in the Chinese medicinal composition by thin layer chromatography
Taking the traditional Chinese medicine composition for improving the sleep function, 5g of each negative reference substance without the spina date seed extract and 1g of the spina date seed extract, adding 40mL of propanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the filtrate in a water bath, and adding 0.5mL of propanol for dissolving to obtain a test solution, a negative reference substance solution and an extract solution. Taking 1g of semen Ziziphi Spinosae as reference material, and making into reference material solution by the same method.
According to the test of 0502 of the general rules of the national pharmacopoeia of the people's republic of China 2015 edition, 1 mu L of each solution is absorbed, respectively dropped on the same silica gel G thin layer plate (20cm multiplied by 10cm, Qingdao sea wave silica gel desiccant Co., Ltd.), developed by taking water saturated n-butyl alcohol as a developing agent, taken out, dried and inspected under an ultraviolet lamp of 366 nm. The result shows that the chromatogram of the test solution and the extract solution has main spots with the same color at the same position as the chromatogram of the control solution, and the chromatogram of the negative control solution has no main spots with the same color at the same position as the chromatogram of the control solution.
3. Determining the content of spinosin in the Chinese medicinal composition by using a high performance liquid chromatography method
(1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is 2.2 microns; taking acetonitrile as a mobile phase A and water as a mobile phase B for gradient elution; the column temperature is 35 ℃; the flow rate is 1 mL/min; the detection wavelength is 330 nm; the number of theoretical plates is not less than 5000 calculated according to the spinosad; the gradient elution process comprises the following specific steps:
0-10 min, mobile phase A%: 12 → 19, mobile phase B: 88 → 81;
10-12 min, mobile phase A%: 19 → 100, mobile phase B: 81 → 0;
12-15 min, mobile phase A%: 100, mobile phase B: 0;
15-16 min, mobile phase A%: 100 → 12, mobile phase B: 0 → 88.
(2) Preparation of standard solution of spinosad and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, adding methanol to dissolve and dilute the spinosyn reference substance into a spinosyn standard solution with the concentration of 10.7 mu g/mL, 21.4 mu g/mL, 42.8 mu g/mL, 85.6 mu g/mL and 107 mu g/mL respectively;
taking 1g of the traditional Chinese medicine composition, placing the traditional Chinese medicine composition into a 25mL volumetric flask, adding about 20mL of methanol with the volume concentration of 70%, and fully shaking up. Putting the volumetric flask into an ultrasonic cleaner, carrying out ultrasonic treatment for 30 minutes, cooling, fixing the volume to a scale by using methanol with the volume concentration of 70 percent, shaking up, and filtering by using a 0.22 mu m organic filter membrane to obtain filtrate, namely the sample solution of the traditional Chinese medicine composition.
(3) Measurement and calculation
Precisely absorbing 1 mu L of the standard solution of the spinosad, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
precisely sucking 1 mu L of sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
Experimental example 1 examination of the applicability of the method
The sample solution was prepared according to the method for preparing the sample solution of the Chinese medicinal composition in the quality control method of the present invention, 1 μ L of the sample solution was aspirated and injected into the high performance liquid chromatograph, and the chromatogram was recorded according to the high performance liquid chromatography conditions of example 1 of the present invention, as shown in fig. 3. The systematic applicability of the chromatographic peaks of the spinosyns was calculated using chromatographic data processing software and the results are shown in table 1 below.
TABLE 1 systematic applicability study results of the Sprenosin chromatogram peaks
As can be seen from the above Table 1, the systematic applicability investigation result of the chromatographic peak of the spinosin meets the judgment standard, which indicates that the spinosin determination method of the invention has very good systematic applicability.
Experimental example 2 Special examination of the method
According to the method for preparing the sample solution of the traditional Chinese medicine composition in the quality control method, the negative reference substance solution without the spina date seeds and the sample solution of the traditional Chinese medicine composition are respectively prepared, 1 mu L of the negative reference substance solution and the sample solution of the traditional Chinese medicine composition are respectively sucked and injected into a high performance liquid chromatograph, and chromatograms are recorded, as shown in figure 4. The result shows that no chromatographic peak with the same retention time as that of the spinosad in the chromatogram of the sample solution of the traditional Chinese medicine composition is found in the chromatogram of the negative control solution without the spina date seeds, which proves that the negative control has no interference.
Experimental example 3 examination of the Linear relationship of the methods
mu.L of the standard solutions of spinosyns at concentrations of 10.7. mu.g/mL, 21.4. mu.g/mL, 42.8. mu.g/mL, 85.6. mu.g/mL, and 107. mu.g/mL, respectively, were taken and injected into a high performance liquid chromatograph, and chromatograms were recorded under the chromatographic conditions of example 1. Determining the nature by retention time, and measuring the peak area; the results of linear regression calculation using the standard solution concentration x as the abscissa and the peak area y as the ordinate are shown in table 2 and fig. 5 below.
TABLE 2 examination of the Linear relationship
As can be seen from Table 2 above, the linear relationship of spinosyns is good in the concentration range of 10.7-107.0. mu.g/mL.
Experimental example 4 repeatability test of the method
Precisely weighing 6 parts of the traditional Chinese medicine composition of the same batch number, wherein each part is 1.0g (accurate to 0.001g), preparing 6 parts of sample solution according to the method for preparing the sample solution of the traditional Chinese medicine composition in the quality control method, repeatedly injecting the sample for 6 times, and recording a chromatogram according to the chromatographic conditions of the example 1. The amount of spinosyns in the sample was calculated using the external standard method or formula (1) in the chromatographic data processing software, and the results are shown in table 3 below.
In the formula: the content of the spinosyns in the X-sample, g/100 g;
c, searching the concentration of the spinosad in the sample solution to be determined by a standard curve, wherein the concentration is mu g/mL;
v-the final constant volume (mL) of the sample liquid to be measured;
m-mass of sample, g.
TABLE 3 results of repeated investigation
As can be seen from the above Table 3, the repeatability investigation result of the invention meets the judgment standard, which indicates that the method of the invention has good repeatability.
Precision examination of the method of Experimental example 5
1.0407g of the traditional Chinese medicine composition of the same batch is precisely weighed, the constant volume is 25mL, 6 sample solutions are prepared according to the method for preparing the sample solution of the traditional Chinese medicine composition in the quality control method, the sample introduction is repeated for 6 times, and the chromatographic peak area is recorded, and the result is shown in the following table 4.
TABLE 4 results of precision examination
| 1 | 2 | 3 | 4 | 5 | 6 | RSD(％) | |
| Peak area | 581854 | 580775 | 577947 | 580542 | 579019 | 580485 | 0.24 |
As is clear from Table 4 above, the method of the present invention is excellent in precision.
Experimental example 6 accuracy examination of the method
According to the data requirement on accuracy in the verification guideline of the quality standard analysis method of 9101 medicine in the four parts of pharmacopoeia of the people's republic of China 2015 edition, 3 different concentrations are adopted in the experiment, 3 sample solutions are respectively prepared for each concentration and are measured, and the preparation of the sample solutions is carried out according to the method for preparing the sample solutions of the traditional Chinese medicine composition in the quality control method. The evaluation was carried out using the results of the measurement of 9 samples, i.e., the ratio of the added mass of the high, medium and low concentration control to the mass of the component to be measured in the sample was controlled to about 0.8: 1, 1: 1, 1.2: 1.
9 parts of the traditional Chinese medicine composition (batch number: 20180612-1, content: 0.114g/100g) with known spinosin content are precisely weighed, and each part is 0.5g (precisely to 0.001 g). Wherein, three portions of the spinosyn reference substance solution with the precise addition concentration of 1.07mg/mL are 0.40mL, namely the addition amount of the spinosyn is 428 mug; taking another three parts, precisely adding 0.5mL of the spinosyn reference substance solution, namely adding 535 mug of the spinosyn; and finally, precisely adding 0.6mL of the spinosyn control solution into three parts, namely adding 642 mu g of the spinosyn. The sample solutions prepared according to the method for preparing the sample solution of the Chinese medicinal composition in the quality control method of the present invention were sequentially measured for their contents of spinosyns according to the chromatographic conditions of example 1, and the recovery rate was calculated according to the formula (2), with the results as shown in table 5 below.
In the formula: a-the content of spinosin in the test sample, μ g;
b-amount of added spinosad, μ g;
c-measurement of Spiromone in solution with Spiromone sample added, μ g.
TABLE 5 results of accuracy examination
As can be seen from Table 5 above, the recovery rates of the present invention are all between 95% and 105%, indicating that the method of the present invention has good accuracy.
Experimental example 7 examination of detection limits of the methods
According to a determination method of a detection limit in the verification guideline of the four 9101 medicine quality standard analysis method of the pharmacopoeia of the people' S republic of China 2015 edition, a signal-to-noise ratio method is selected to determine the detection limit of the experiment, namely the Detection Limit (DL) of the analysis method is calculated by a signal-to-noise ratio (S/N), and the DL is defined as the corresponding analyte concentration when the S/N is 3. The method specifically comprises the following steps:
1.0407g of traditional Chinese medicine composition with known spinosin content is precisely weighed, wherein the spinosin content is 0.114g/100g, the sample solution is prepared according to the method for preparing the sample solution of the traditional Chinese medicine composition in the quality control method, 0.05mL of the sample solution is placed in a 25mL volumetric flask, the volume is fixed to a scale by using 70% methanol, the volume is evenly shaken, a 0.22-micrometer organic filter membrane is used for filtering, the filtrate is taken and injected into an ultra-high performance liquid chromatograph according to the chromatographic conditions of the embodiment 1, the chromatogram is recorded, the signal-to-noise ratio is calculated to be 3.6 through workstation chromatographic software, and the detection limit of the analysis method is 0.095 ug/mL.
Example 8 durability test of the method
Three batches of samples of the traditional Chinese medicine composition are taken, 3 parts of sample solution is prepared respectively according to the method for preparing the sample solution of the traditional Chinese medicine composition in the quality control method, the content is measured respectively according to the chromatographic method under the item of spina date seeds in Chinese pharmacopoeia 2015 edition and the high performance liquid chromatography method, and the result is shown in the following table 6.
TABLE 6 durability examination results
As can be seen from Table 6 above, the relative deviation of the amounts of spinosyns measured under different chromatographic conditions is less than 5%, indicating that the measurement method of the present invention has good durability.
Experimental example 9 stability examination of samples
A sample solution of the same composition was injected at 0, 4, 12 and 24h under the same chromatographic conditions as in example 1, and the results are shown in Table 9 below.
Table 7 stability test results
As can be seen from Table 7 above, the RSD of the peak area of the chromatographic peak of the spinosin is 0.62%, indicating that the sample solution of the composition of the present invention is substantially stable within 24 h.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It is not necessary or necessary to exhaustively enumerate all embodiments herein, and obvious variations or modifications can be made without departing from the scope of the invention.
Claims (8)
1. A detection method of a traditional Chinese medicine composition comprises the step of identifying lucid ganoderma, caulis polygoni multiflori and spina date seeds in the traditional Chinese medicine composition by using a thin-layer chromatography, and is characterized in that the thin-layer chromatography comprises the following steps:
(1) preparation of positive control solution and test solution
Respectively adding a first solvent into a ganoderma lucidum positive control, a polygonum multiflorum positive control and a spina date seed positive control, filtering, evaporating filtrate to dryness, and adding a second solvent to respectively serve as respective positive control solutions;
adding the first solvent into the Chinese medicinal composition, filtering, evaporating the filtrate, and adding the second solvent to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking the Ganoderma positive control solution, caulis Polygoni Multiflori positive control solution and sample solution, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent, taking out, air drying, placing under ultraviolet lamp for inspection and/or spraying with sulfuric acid ethanol solution, and oven drying until spots appear clearly; and the combination of (a) and (b),
sucking the positive control solution and the sample solution of semen Ziziphi Spinosae, respectively dropping on the same silica gel thin layer plate, developing with water saturated n-butanol as developing agent, taking out, air drying, and inspecting under ultraviolet lamp.
2. The detection method according to claim 1, wherein the positive control of Ganoderma lucidum is selected from at least one of Ganoderma lucidum control medicinal material or Ganoderma lucidum extract; the positive control of caulis Polygoni Multiflori is selected from at least one of caulis Polygoni Multiflori control medicinal materials or caulis Polygoni Multiflori extract; the positive control of the spina date seed is at least one of a spina date seed control medicinal material or a spina date seed extract.
3. The detection method according to claim 1 or 2, wherein the first solvent is one or more of methanol, ethanol and propanol; the second solvent is one or more of methanol, ethanol and propanol.
4. The detection method according to any one of claims 1 to 3, wherein the thin layer chromatography comprises the steps of:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 1.5-3 g of lucid ganoderma contrast medicinal material, 0.2-0.3 g of tuber fleeceflower stem contrast medicinal material and 0.5-1.5 g of spina date seed contrast medicinal material, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the filtrate as respective contrast medicinal material solutions;
taking 0.5-1.5 g of lucid ganoderma extract, 0.5-1.5 g of tuber fleeceflower stem extract and 0.5-1.5 g of spina date seed extract, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the filtrate as respective extract solution;
taking 4.5-5.5 g of a negative control product without a lucid ganoderma extract, 4.5-5.5 g of a negative control product without a vine of multiflower knotweed extract and 4.5-5.5 g of a negative control product without a spina date seed extract, respectively adding 20-40 mL of ethanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, adding 0.5-1.5 mL of ethanol for dissolving, and respectively taking the ethanol solution as a negative control product solution;
taking 4.5-5.5 g of the traditional Chinese medicine composition, adding 20-40 mL of ethanol into the traditional Chinese medicine composition, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5-1.5 mL of ethanol for dissolving to obtain a sample solution;
(2) testing by thin layer chromatography
Sucking 4-6 muL of the lucid ganoderma contrast medicinal material solution, 4-6 muL of the multiflower knotweed vine contrast medicinal material solution, 4-6 muL of the lucid ganoderma extract solution, 4-6 muL of the multiflower knotweed vine extract solution, 4-6 muL of the negative contrast product solution without the lucid ganoderma extract, 4-6 muL of the negative contrast product solution without the multiflower knotweed vine extract and 4-6 muL of the test product solution, respectively dropping on the same silica gel thin layer plate, developing by taking cyclohexane-ethyl acetate-formic acid with the volume ratio of 5: 0.2 as a developing agent, taking out, airing, placing under an ultraviolet lamp of 366nm to inspect and/or spray sulfuric acid ethanol solution, and drying until spots are clear in color; and the combination of (a) and (b),
sucking the spina date seed reference medicinal material solution 4-6 muL, the spina date seed extract solution 4-6 muL, the negative reference substance solution 4-6 muL without the spina date seed extract and the sample solution 4-6 muL, respectively dropping on the same silica gel thin layer plate, developing by using water saturated n-butyl alcohol as a developing agent, taking out, airing, and inspecting under an ultraviolet lamp of 366 nm.
5. The detection method according to any one of claims 1 to 4, wherein the thin layer chromatography comprises the steps of:
(1) preparation of reference medicinal material solution, extract solution, negative reference substance solution and test solution
Taking 2g of lucid ganoderma reference medicinal material, 0.25g of tuber fleeceflower stem reference medicinal material and 1g of spina date seed reference medicinal material, respectively adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and then adding 1mL of ethanol for dissolving, wherein the ethanol is respectively used as respective reference medicinal material solutions;
respectively adding 30mL of ethanol into 1g of ganoderma lucidum extract, 1g of caulis polygoni multiflori extract and 1g of spina date seed extract, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain respective extract solutions;
taking 5g of a negative control product without a lucid ganoderma extract, 5g of a negative control product without a vine of multiflower knotweed extract and 5g of a negative control product without a spina date seed extract, respectively adding 30mL of ethanol into the negative control product, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 1mL of ethanol into the filtrate to dissolve the filtrate, and respectively taking the filtrate as respective negative control product solutions;
taking 5g of the traditional Chinese medicine composition, adding 30mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1mL of ethanol for dissolving to obtain a test solution;
(2) testing by thin layer chromatography
Sucking the above Ganoderma contrast medicinal material solution 5 μ L, caulis Polygoni Multiflori contrast medicinal material solution 5 μ L, Ganoderma extract solution 5 μ L, caulis Polygoni Multiflori extract solution 5 μ L, negative control solution 5 μ L without Ganoderma extract, negative control solution 5 μ L without caulis Polygoni Multiflori extract and test sample solution 5 μ L, respectively dropping on the same silica gel thin layer plate, developing with cyclohexane-ethyl acetate-formic acid at volume ratio of 5: 0.2 as developing agent, taking out, air drying, inspecting and/or spraying with sulfuric acid ethanol solution under 366nm ultraviolet lamp, and drying until spots appear clearly; and the combination of (a) and (b),
sucking the above semen Ziziphi Spinosae control medicinal material solution 5 μ L, semen Ziziphi Spinosae extract solution 5 μ L, negative control solution 5 μ L without semen Ziziphi Spinosae extract and sample solution 5 μ L, respectively dropping on the same silica gel thin layer plate, developing with water saturated n-butanol as developing agent, taking out, air drying, and inspecting under 366nm ultraviolet lamp.
6. The detection method according to any one of claims 1 to 5, further comprising measuring the amount of spinosin in the Chinese medicinal composition by high performance liquid chromatography, wherein the high performance liquid chromatography comprises the following steps:
(1) preparation of standard solution of spinosin and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, and adding a third solvent to prepare a spinosyn standard solution;
and (3) adding a third solvent into the traditional Chinese medicine composition, and filtering to obtain filtrate, namely the sample solution of the traditional Chinese medicine composition.
(2) Absorbing the standard solution of the spinosyns, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
(3) and (3) sucking the sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
7. The detection method according to claim 6, wherein the third solvent is methanol.
8. The detection method according to claim 6 or 7, wherein the high performance liquid chromatography comprises the steps of:
(1) chromatographic condition and system adaptability test
Octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is not more than 2.5 microns; taking acetonitrile as a mobile phase A and water as a mobile phase B for gradient elution; the column temperature is 25-35 ℃; the detection wavelength is 330-340 nm; the number of theoretical plates is not less than 5000 calculated according to the spinosad; the gradient elution process is as follows:
0-10 min, mobile phase A%: 12 → 19, mobile phase B: 88 → 81;
10-12 min, mobile phase A%: 19 → 100, mobile phase B: 81 → 0;
12-15 min, mobile phase A%: 100, mobile phase B: 0;
15-16 min, mobile phase A%: 100 → 12, mobile phase B: 0 → 88;
(2) preparation of standard solution of spinosad and sample solution of traditional Chinese medicine composition
Taking a spinosyn reference substance, adding methanol and diluting into at least 3 spinosyn standard solutions with different concentrations;
taking 1g of the traditional Chinese medicine composition, adding 25mL of methanol, carrying out ultrasonic treatment for 30 minutes, and filtering by using a 0.22-micrometer organic filter membrane to obtain a filtrate, namely a sample solution of the traditional Chinese medicine composition;
(3) sucking 1 mu L of the standard solution of the spinosad, injecting the solution into a high performance liquid chromatograph, and drawing a peak area-solution concentration standard curve chart according to a measurement result;
(4) and (3) sucking 1 mu L of the sample solution of the traditional Chinese medicine composition, injecting the sample solution into a high performance liquid chromatograph, and calculating the content of the spinosad in the traditional Chinese medicine composition according to the peak area of the sample and a standard curve graph.
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