Detection method for organophosphorus pesticide residues in traditional Chinese medicinal materials

Abstract

The invention discloses a detection method for organophosphorus pesticide residues in traditional Chinese medicinal materials. The detection method comprises the following steps: preparing a reference substance solution, detecting the reference substance solution, preparing a test solution, detecting the test solution and analyzing a detection result. According to the detection method, a thin-layer chromatography mode is adopted, compared with an existing chromatography separation detection mode, an expensive multifunctional processor does not need to be configured, the silica gel G thin-layer plate subjected to sample application is placed in developing solvent steam for pre-saturation, the silica gel G thin-layer plate subjected to sample application is expanded in a stable state, the reproducibility is good, and the detection accuracy is high. The method is simple in operation, convenient to detect, capable of meeting detection requirements of different organophosphorus pesticide residues, capable of providing theoretical basis for organophosphorus pesticide residue detection of traditional Chinese medicinal materials and providing technical support and reference methods for quality control of the traditional Chinese medicinal materials, and suitable for application and popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for detecting organophosphorus pesticide residues in traditional Chinese medicines. Background Art

[0002] Organophosphorus pesticides are a class of organic compounds containing phosphorus. There are many types. At present, there are at least more than 60 types that are mass-produced and used. According to their toxicity, they can be divided into four categories: highly toxic, highly toxic, moderately toxic, and low-toxic. Organophosphorus pesticides have the advantages of small dosage, high insecticide efficiency, strong selectivity, little damage to crops, and economic use. They are widely used because of their unstable nature, easy decomposition in nature, extremely short residual time in crops, and easy hydrolysis by enzymes in organisms, and no accumulation in the body. Since the planting cycle of Chinese medicinal materials is relatively long, various pesticides are inevitably used during their growth. Although they basically need to be dried or baked from harvest to spare raw materials, and undergo a long storage process, the organophosphorus pesticides used are basically degraded when used as raw materials, and the residual amount is low or even undetectable. However, given that some organophosphorus pesticides are highly toxic pesticides with strong acute toxicity to mammals, once contaminated by them, it is very easy to cause acute poisoning in humans and animals. Therefore, the "Chinese Pharmacopoeia" specifically stipulates the "Method for Determination of Organophosphorus Pesticide Residues in Medicinal Materials and Preparations", which shows that the determination of organophosphorus pesticide residues in Chinese medicinal materials is necessary and of practical significance.

[0003] The detection method specified in the Pharmacopoeia is to first add anhydrous sodium sulfate to the test sample, then use ethyl acetate as a molten medium for ultrasonic extraction twice, dilute the extract and place it on an activated carbon column, place it on a multifunctional vacuum sample processor, use n-hexane-ethyl acetate for elution and separation, and finally use chromatography to separate and detect a variety of organophosphorus pesticides. In the process of planting Chinese medicinal materials, in order to prevent losses caused by pests and diseases, farmers inevitably use pesticides to reduce risks. Therefore, excessive pesticide residues have seriously lagged behind the pace of innovation and development of Chinese medicinal materials. At present, the residual pesticides in Chinese medicinal materials are mainly organochlorine, organophosphorus, and pyrethroids. Organochlorine pesticides are stable in nature and have a long half-life, making them difficult to degrade in the soil. Organochlorine pesticides can still be detected in most Chinese medicinal materials. Organophosphorus pesticides are of many varieties and high efficacy, and most of them are highly toxic products. The determination method of organophosphorus pesticide residues specified in the "Chinese Pharmacopoeia" requires the configuration of an expensive multifunctional vacuum sample processor, and the second is that the operating cost of the instrument is increased and the repeatability is poor.

[0004] Thin layer chromatography (TLC) is a trace, fast and simple separation and analysis method. It is to coat a suitable stationary phase on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After spotting and developing, the relative shift value (Rf) is compared with the relative shift value of the chromatogram obtained by the same method with a suitable reference substance, which is used for drug identification, impurity inspection or content determination. This method is a very important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. It has the characteristics of short development time, strong separation ability and high sensitivity. The UV-visible spectrophotometer is a commonly used spectral detection equipment with a wavelength range of 190-1100nm. Spectrophotometric analysis is an effective means to study the composition, structure and interaction of substances based on the absorption spectrum of the substance. It is a band spectrum that reflects the information of certain groups in the molecule. It can be qualitatively analyzed by combining standard optical spectra with other means. Therefore, in order to reduce costs and facilitate the detection of pesticide residues in Chinese medicinal materials, it is very necessary to provide a simple and accurate detection method. A clear chromatogram can be obtained by spotting, developing and inspecting, which can not only detect pesticide residues in Chinese medicinal materials, but also improve work efficiency to meet the detection needs of different organophosphorus pesticide residues. Summary of the invention

[0005] The technical problem to be solved by the present invention is to address the problems existing in the background technology, and to utilize the existing ultraviolet-visible spectrophotometer to provide a detection method that is easy to operate. Using this detection method, a clear chromatogram can be obtained by spotting, developing and inspecting. This can not only detect pesticide residues in traditional Chinese medicines, but also improve work efficiency to meet the detection needs of different organophosphorus pesticide residues. Specifically, it is a detection method for organophosphorus pesticide residues in traditional Chinese medicines.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for detecting organophosphorus pesticide residues in traditional Chinese medicines, the detection method comprising the following steps: S1. Preparation of reference solution: Accurately pipette 40ul of 1000ug / ml acephate and omethoate standard stock solutions and 20ul of each of dichlorvos, dimethoate, methamidophos, monocrotophos, phorate, profenofos, parathion, methyl parathion, diazinon, fenthion, methyl isofos, quinalphos, malathion, chlorpyrifos, chlorpyrifos and ethion standard stock solutions in a 10ml volumetric flask, dilute to the mark with acetone, mix thoroughly, and obtain a mixed solution of acephate and omethoate at a concentration of 4ug / ml and 2ug / ml of the other 16 standard stock solutions for use; S2. Test of reference solution Take 60ul of the above-mentioned organophosphorus reference substance mixed solution into a sample bottle, add 540ul of acetone, mix thoroughly, and obtain a reference substance mixed solution with a concentration of 0.4ug / ml for acephate and omethoate and a concentration of 0.2ug / ml for the other 16 reference substances, and spot them on the same silica gel G thin layer plate, first place the spotted silica gel G thin layer plate in the vapor of a developing agent for pre-saturation, and then place it in the developing agent for development, take it out and dry it after development, and then heat the dried silica gel G thin layer plate at a temperature not lower than 100°C, and then use a UV-visible spectrophotometer to inspect it to obtain a reference substance color spectrum; S3. Preparation of test solution Weigh the Chinese herbal medicine to be tested and crush it into powder, add the extractant to extract, filter and evaporate the filtrate after extraction to obtain the residue, dissolve the residue with a solvent to obtain the test solution, and test it; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value can be calculated by the corresponding peak area.

[0007] Further, using the method for detecting organophosphorus pesticide residues in Chinese medicinal materials described in the present invention, in the preparation process of the test solution described in step S3, the specific preparation method is: first, the Chinese medicinal materials to be detected are crushed into powder, 4-6g of the Chinese medicinal material powder is taken, 30-40ml of water is added and boiled three times, the first time is 2.5-3.5 hours, the second and third times are 1.5-2.5 hours each, filtered, the filtrate is combined, and evaporated to near dryness, and 30-50ml of 70-75% ethanol is used for ultrasonic treatment for 30-40 minutes. minutes, filter, extract the residue repeatedly, filter, combine the filtrate, evaporate to dryness, add 10-20 ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 40-60 ml each time, combine the extracts, evaporate to dryness, add 1-3 ml of water to the residue to dissolve it, add it to the polyamide column, elute with 80-120 ml of water, discard the washing liquid, and then elute with 40-60 ml of 70-75% ethanol, collect the eluate, evaporate to dryness, add 1-3 ml of methanol to dissolve the residue to prepare the test solution.

[0008] Further, a method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention is adopted. In the preparation process of the test solution described in step S3, the specific preparation method is: take 5g of traditional Chinese medicine powder, add 30ml of water and boil three times, the first time for 3 hours, the second and third times for 2 hours each, filter, combine the filtrate, evaporate to near dryness, use 50ml of 75% ethanol, ultrasonically treat for 35 minutes, filter, repeat extraction of the filter residue once, filter, combine the filtrate, evaporate to dryness, add 10ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 40ml each time, combine the extracts, evaporate to dryness, add 2ml of water to the residue to dissolve it, add it to a polyamide column, elute with 100ml of water, discard the washing liquid, and then elute with 50ml of 75% ethanol, collect the eluate, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, and prepare the test solution.

[0009] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the developing agent is a mixture obtained by mixing toluene, ethyl acetate and formic acid in a volume ratio of (8-12): (6-10): 1.

[0010] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the developing agent is a mixture of toluene, ethyl acetate and formic acid in a volume ratio of 10:8:1.

[0011] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the temperature at which the dried silica gel G thin layer plate is heated is not less than 110°C.

[0012] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the temperature of the steam of the developing agent is 45-55°C.

[0013] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the spotted silica gel G thin layer plate is heated before being placed in the vapor of the developing agent for pre-saturation, and the pre-saturation heating conditions are: temperature of 75-85°C and time of 0.5-1.5 min.

[0014] Furthermore, in the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention, the silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate.

[0015] Furthermore, the method for detecting organophosphorus pesticide residues in traditional Chinese medicines described in the present invention is adopted, wherein the polyamide column has a mesh size of 100 to 200, an inner diameter of 1 to 1.5 cm, and a weight of 10 g, and is wet packed.

[0016] Compared with the prior art, the method for detecting organophosphorus pesticide residues in traditional Chinese medicinal materials disclosed in the present invention has the following beneficial effects: a thin layer chromatography method is adopted, and compared with the prior chromatography separation and detection method, no expensive multifunctional processor is required, and the spotted silica gel G thin layer plate is pre-saturated with developing agent steam so that the spotted silica gel G thin layer plate is developed in a stable state, and has good reproducibility and is convenient for detection, and can meet the detection requirements of different organophosphorus pesticide residues, and not only provides a theoretical basis for the detection of organophosphorus pesticide residues in traditional Chinese medicinal materials, but also provides technical support and reference methods for the quality control of traditional Chinese medicinal materials, and is suitable for popularization and use. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. It will be apparent to those skilled in the art that various modifications and changes may be made to the specific embodiments of the present specification without departing from the scope or spirit of the present invention. Other embodiments obtained from the specification of the present invention will be apparent to the technician. The present specification and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to. Example 1

[0021] This embodiment provides a method for detecting organophosphorus pesticide residues in traditional Chinese medicines. The traditional Chinese medicine is Polygonatum sibiricum. The detection method includes preparation of a reference solution, detection of the reference solution, preparation of a test solution, detection of the test solution, and analysis of the detection results. The specific steps are as follows: S1. Preparation of reference solution: Accurately pipette 40ul of 1000ug / ml acephate and omethoate standard stock solutions and 20ul of each of dichlorvos, dimethoate, methamidophos, monocrotophos, phorate, profenofos, parathion, methyl parathion, diazinon, fenthion, methyl isofos, quinalphos, malathion, chlorpyrifos, chlorpyrifos and ethion standard stock solutions in a 10ml volumetric flask, dilute to the mark with acetone, mix thoroughly, and obtain a mixed solution of acephate and omethoate at a concentration of 4ug / ml and 2ug / ml of the other 16 standard stock solutions for use; S2. Test of reference solution 60ul of the mixed solution of the above-mentioned organophosphorus reference substance was taken into a sample bottle, and 540ul of acetone was added, and the mixture was thoroughly mixed to obtain a mixed solution of reference substances with a concentration of 0.4ug / ml for acephate and omethoate and a concentration of 0.2ug / ml for the other 16 reference substances, and the solution was spotted on the same silica gel G thin layer plate, and the spotted silica gel G thin layer plate was first placed in the vapor of a developing agent for pre-saturation, and then placed in the developing agent for development, and after development, it was taken out and dried, and then the dried silica gel G thin layer plate was heated at a temperature not lower than 100°C, and then examined by a UV-visible spectrophotometer to obtain a color spectrum of the reference substance; S3. Preparation of test solution Weigh the Chinese medicinal material Polygonatum sibiricum to be tested, crush it into powder, add an extractant to extract, filter and evaporate the filtrate after extraction to obtain a residue, dissolve the residue with a solvent to obtain a test solution, and test it; the specific preparation method is: first crush the Polygonatum sibiricum to be tested into powder, take 4g of the Chinese medicinal material powder, add 30ml of water and boil it three times, the first time for 2.5 hours, the second and third times for 1.5 hours each, filter, combine the filtrate, evaporate to near dryness, use 70% ethanol 30ml, ultrasonic treatment for 30 minutes, filter, repeat extraction of the filter residue once, filter, combine the filtrate, evaporate to dryness, add 10ml of water to the residue to dissolve it, shake and extract it with ethyl acetate twice, 40ml each time, combine the extracts, evaporate to dryness, add 1ml of water to the residue to dissolve it, add it to a polyamide column, elute with 80ml of water, discard the washing liquid, and then elute with 70% ethanol 40ml, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and prepare the test solution; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value is calculated by the corresponding peak area, that is, the conventional external standard method is used for calculation. In this way, the organophosphorus pesticide residue content can be determined based on the peak area of ​​the same characteristic peaks.

[0022] The detection method described in this embodiment is adopted, wherein the developing agent is a mixture of toluene, ethyl acetate and formic acid in a volume ratio of 8:6:1; the temperature of heating the dried silica gel G thin layer plate is 105°C; the temperature of the developing agent vapor is 45°C; before being placed in the developing agent vapor for pre-saturation, the spotted silica gel G thin layer plate is also heated before pre-saturation, and the pre-saturation heating conditions are: temperature 75°C, time 0.5min; the silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate; the polyamide column is 100 mesh, the column inner diameter is 1cm, 10g, and the column is wet packed. Example 2

[0023] This embodiment provides a method for detecting organophosphorus pesticide residues in traditional Chinese medicines. The traditional Chinese medicine is Astragalus. The detection method includes reference solution preparation, reference solution detection, test solution preparation, test solution detection and test result analysis. The specific steps are as follows: S1. Preparation of reference solution, the same as in Example 1; S2. The reference solution was tested in the same manner as in Example 1; S3. Preparation of test solution Weigh the Chinese herbal medicine Astragalus to be tested, crush it into powder, add the extractant to extract, filter and evaporate the filtrate after extraction to obtain the residue, dissolve the residue with a solvent to obtain a test solution, and test it; the specific preparation method is: first crush the Astragalus to be tested into powder, take 5g of the Chinese herbal medicine powder, add 30ml of water and boil it three times, the first time for 3 hours, the second and third times for 2 hours each, filter, combine the filtrate, evaporate to near dryness, use 50ml of 75% ethanol, ultrasonic treatment for 35 minutes, filter, repeat extraction once, filter, combine the filtrate, evaporate to dryness, add 10ml of water to the residue to dissolve it, shake and extract it with ethyl acetate twice, 40ml each time, combine the extracts, evaporate to dryness, add 2ml of water to the residue to dissolve it, add it to a polyamide column, elute with 100ml of water, discard the washing liquid, and then elute with 50ml of 75% ethanol, collect the eluate, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, and prepare the test solution; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value is calculated by the corresponding peak area, that is, the conventional external standard method is used for calculation. In this way, the organophosphorus pesticide residue content can be determined based on the peak area of ​​the same characteristic peaks.

[0024] The detection method described in this embodiment is adopted, wherein the developing agent is a mixture of toluene, ethyl acetate and formic acid in a volume ratio of 10:8:1; the temperature of heating the dried silica gel G thin layer plate is not less than 120°C; the temperature of the developing agent vapor is 48°C; before being placed in the developing agent vapor for pre-saturation, the spotted silica gel G thin layer plate is also heated before pre-saturation, and the pre-saturation heating conditions are: temperature 80°C, time 1.5min; the silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate; the polyamide column is 150 mesh, the column inner diameter is 1cm, 10g, and the column is wet packed. Example 3

[0025] This embodiment provides a method for detecting organophosphorus pesticide residues in traditional Chinese medicines. The traditional Chinese medicine is Angelica sinensis. The detection method includes preparation of a reference solution, detection of the reference solution, preparation of a test solution, detection of the test solution, and analysis of the detection results. The specific steps are as follows: S1. Preparation of reference solution, the same as in Example 1; S2. The reference solution was tested in the same manner as in Example 1; S3. Preparation of test solution Weigh the Chinese medicinal material angelica to be tested, crush it into powder, add an extractant to extract, filter and evaporate the filtrate after extraction to obtain a residue, dissolve the residue with a solvent to obtain a test solution, and test it; the specific preparation method is: first crush the angelica to be tested into powder, take 6g of angelica powder, add 40ml of water and boil it three times, the first time for 3.5 hours, the second and third times for 2.5 hours each, filter, combine the filtrate, evaporate to near dryness, use 50ml of 75% ethanol, ultrasonic treatment for 40 minutes, filter, repeat extraction of the filter residue once, filter, combine the filtrate, evaporate to dryness, add 20ml of water to the residue to dissolve it, shake and extract it with ethyl acetate twice, 60ml each time, combine the extracts, evaporate to dryness, add 1-3ml of water to the residue to dissolve it, add it to a polyamide column, elute with 120ml of water, discard the washing liquid, and then elute with 60ml of 75% ethanol, collect the eluate, evaporate to dryness, add 3ml of methanol to the residue to dissolve it, and prepare the test solution; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value is calculated by the corresponding peak area, that is, the conventional external standard method is used for calculation. In this way, the organophosphorus pesticide residue content can be determined based on the peak area of ​​the same characteristic peaks.

[0026] The detection method described in this embodiment is adopted, wherein the developing agent is a mixture of toluene, ethyl acetate and formic acid in a volume ratio of 12:10:1; the temperature of heating the dried silica gel G thin layer plate is not less than 130°C; the temperature of the developing agent vapor is 50°C; before being placed in the developing agent vapor for pre-saturation, the spotted silica gel G thin layer plate is also heated before pre-saturation, and the pre-saturation heating conditions are: temperature 82°C, time 1.5min; the silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate; the polyamide column is 200 mesh, the column inner diameter is 1.5cm, 10g, and the column is wet packed. Example 4

[0027] This embodiment provides a method for detecting organophosphorus pesticide residues in traditional Chinese medicines, wherein the traditional Chinese medicine is Gastrodia elata, and the detection method includes preparation of a reference solution, detection of the reference solution, preparation of a test solution, detection of the test solution, and analysis of the detection results, and the specific steps are as follows: S1. Preparation of reference solution, the same as in Example 1; S2. The reference solution was tested in the same manner as in Example 1; S3. Preparation of test solution Weigh the Chinese herbal medicine to be tested, crush it into powder, add the extractant to extract, filter and evaporate the filtrate to obtain the residue after extraction, dissolve the residue with a solvent to obtain the test solution, and test it; the specific preparation method is: weigh the Chinese herbal medicine Gastrodia elata to be tested, crush it into powder, add the extractant to extract, filter and evaporate the filtrate to obtain the residue after extraction, dissolve the residue with a solvent to obtain the test solution, and test it; the specific preparation method is: first crush the Gastrodia elata to be tested into powder, take 5g of the Chinese herbal medicine powder, add 35ml of water and boil it three times, the first time for 3 hours, the second and third times for 2. 5 hours, filter, combine the filtrate, evaporate to near dryness, use 72% ethanol 35ml, ultrasonic treatment for 35 minutes, filter, repeat extraction once, filter, combine the filtrate, evaporate to dryness, add 15ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 50ml each time, combine the extracts, evaporate to dryness, add 2ml of water to the residue to dissolve it, add it to the polyamide column, elute with 100ml of water, discard the washing liquid, and continue to elute with 72% ethanol 50ml, collect the eluate, evaporate to dryness, add 2ml of methanol to dissolve the residue to prepare the test solution; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value is calculated by the corresponding peak area, that is, the conventional external standard method is used for calculation. In this way, the organophosphorus pesticide residue content can be determined based on the peak area of ​​the same characteristic peaks.

[0028] The detection method described in this embodiment is adopted, wherein the developing agent is a mixture of toluene, ethyl acetate and formic acid in a volume ratio of 10:6:1; the temperature for heating the dried silica gel G thin layer plate is 125°C; the temperature of the developing agent vapor is 55°C; before being placed in the developing agent vapor for pre-saturation, the spotted silica gel G thin layer plate is also heated before pre-saturation, and the pre-saturation heating conditions are: temperature 85°C, time 1 min; the silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate; the polyamide column is 100 mesh, the column inner diameter is 1.5 cm, 10 g, and the column is wet packed.

[0029] The detection method described in Examples 1 to 4 is adopted, and is respectively illustrated by taking Polygonatum sibiricum, Astragalus membranaceus, Angelica sinensis and Gastrodia elata as examples. In the specific implementation process, the spotted silica gel G thin layer plate is first placed in the developing agent vapor for pre-saturation, so that the spotted silica gel G thin layer plate is developed in a stable state of solid phase, gas phase and liquid phase, so that the appearance of multi-solvent front in the development process is avoided during the identification process, and the spots in the development process are ensured to be rounded, thereby improving the accuracy of identification. Then, the dried silica gel G thin layer plate after development is heated (not less than 100° C.) to ensure that the residual developing agent components on the thin layer plate are completely removed, and the masking of the developing agent on the spots of the Chinese medicinal materials is reduced, thereby greatly improving the detection accuracy.

[0030] It can be seen that by adopting the detection method described in the present application, a clear chromatogram can be obtained through one spotting, one development, and one inspection, which greatly improves the one-time success rate of thin-layer identification of Chinese medicinal materials, meets the efficiency of identification of organophosphorus pesticide residues in Chinese medicinal materials in actual production, and not only provides a theoretical basis for the detection of organophosphorus pesticide residues in Chinese medicinal materials, but also provides technical support and reference methods for the quality control of Chinese medicinal materials, and is suitable for popularization and use.

[0031] Other aspects of the present invention that are not described in detail are all conventional techniques known to those skilled in the art.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting organophosphorus pesticide residues in traditional Chinese medicine, characterized in that: The detection method comprises the following steps: S1. Preparation of reference solution: Accurately pipette 40ul of 1000ug / ml acephate and omethoate standard stock solutions and 20ul of each of dichlorvos, dimethoate, methamidophos, monocrotophos, phorate, profenofos, parathion, methyl parathion, diazinon, fenthion, methyl isofos, quinalphos, malathion, chlorpyrifos, chlorpyrifos and ethion standard stock solutions in a 10ml volumetric flask, dilute to the mark with acetone, mix thoroughly, and obtain a mixed solution of acephate and omethoate at a concentration of 4ug / ml and 2ug / ml of the other 16 standard stock solutions for use; S2. Test of reference solution Take 60ul of the above-mentioned organophosphorus reference substance mixed solution into a sample bottle, add 540ul of acetone, mix thoroughly, and obtain a reference substance mixed solution with a concentration of 0.4ug / ml for acephate and omethoate and a concentration of 0.2ug / ml for the other 16 reference substances, and spot them on the same silica gel G thin layer plate, first place the spotted silica gel G thin layer plate in the vapor of a developing agent for pre-saturation, and then place it in the developing agent for development, take it out and dry it after development, and then heat the dried silica gel G thin layer plate at a temperature not lower than 100°C, and then use a UV-visible spectrophotometer to inspect it to obtain a reference substance color spectrum; S3. Preparation of test solution Weigh the Chinese herbal medicine to be tested and crush it into powder, add the extractant to extract, filter and evaporate the filtrate to obtain the residue after extraction, dissolve the residue with a solvent to obtain the test solution, and test it; S4. Testing of test solution Take 1 ml of the sample solution to be tested, spot it on the same silica gel G thin layer plate, place the spotted silica gel G thin layer plate in the vapor of the developing agent for pre-saturation, then place it in the developing agent for development, take it out and dry it after development, then heat the dried silica gel G thin layer plate at a temperature not lower than 100° C., use a UV-visible spectrophotometer, sample and inspect under the same conditions to obtain the sample spectrum; S5. Analysis of test results The chromatogram of the test sample is compared with the chromatogram of the reference sample. If the same characteristic peaks are found, it means that the organophosphorus pesticide remains in the test sample, and the content value can be calculated by the corresponding peak area.

2. A method for detecting organophosphorus pesticide residues in traditional Chinese medicine according to claim 1, characterized in that: In the preparation process of the test solution described in step S3, the specific preparation method is: first crush the Chinese medicinal materials to be tested into powder, take 4-6g of the Chinese medicinal materials powder, add 30-40ml of water and boil three times, the first time for 2.5-3.5 hours, the second and third times for 1.5-2.5 hours respectively, filter, combine the filtrate, evaporate to near dryness, use 70-75% ethanol 30-50ml, ultrasonic treatment for 30-40 minutes, filter, repeat the extraction of the filter residue once, filter, combine the filtrate, evaporate to dryness, add 10-20ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, each time 40-60ml, combine the extracts, evaporate to dryness, add 1-3ml of water to the residue to dissolve it, add it to the polyamide column, elute with 80-120ml of water, discard the washing liquid, and then elute with 40-60ml of 70-75% ethanol, collect the eluate, evaporate to dryness, add 1-3ml of methanol to the residue to dissolve it, and prepare the test solution.

3. A method for detecting organophosphorus pesticide residues in traditional Chinese medicine according to claim 2, characterized in that: In the preparation process of the test solution described in step S3, the specific preparation method is: take 5g of Chinese medicinal material powder, add 30ml of water and boil three times, the first time for 3 hours, the second and third times for 2 hours each, filter, combine the filtrate, evaporate to near dryness, use 50ml of 75% ethanol, ultrasonic treatment for 35 minutes, filter, repeat extraction of the filter residue once, filter, combine the filtrate, evaporate to dryness, add 10ml of water to the residue to dissolve it, shake and extract with ethyl acetate twice, 40ml each time, combine the extracts, evaporate to dryness, add 2ml of water to the residue to dissolve it, add it to the polyamide column, elute with 100ml of water, discard the washing liquid, and then elute with 50ml of 75% ethanol, collect the eluate, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, and prepare the test solution.

4. A method for detecting organophosphorus pesticide residues in traditional Chinese medicine according to claim 1, characterized in that: The developing agent is a mixture obtained by mixing toluene, ethyl acetate and formic acid in a volume ratio of (8-12):(6-10):

1.

5. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 4, characterized in that: The developing agent is a mixture obtained by mixing toluene, ethyl acetate and formic acid in a volume ratio of 10:8:

1.

6. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 1, characterized in that: The temperature of heating the dried silica gel G thin layer plate is not less than 110°C.

7. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 1, characterized in that: The temperature of the steam of the developing agent is 45-55°C.

8. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 1, characterized in that: Before being pre-saturated in the steam of the developing agent, the spotted silica gel G thin layer plate was also heated before pre-saturation. The heating conditions before pre-saturation were: temperature of 75-85° C. and time of 0.5-1.5 min.

9. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 1, characterized in that: The silica gel G thin layer plate is a high-efficiency silica gel GF254 thin layer plate.

10. A method for detecting organophosphorus pesticide residues in traditional Chinese medicines according to claim 1, characterized in that: The polyamide column has 100-200 meshes, an inner diameter of 1-1.5 cm, 10 g, and is packed by wet method.