CN104267109A - Radix bupleuri medicinal material detection method - Google Patents
Radix bupleuri medicinal material detection method Download PDFInfo
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- CN104267109A CN104267109A CN201410374865.4A CN201410374865A CN104267109A CN 104267109 A CN104267109 A CN 104267109A CN 201410374865 A CN201410374865 A CN 201410374865A CN 104267109 A CN104267109 A CN 104267109A
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- medicinal material
- saikosaponin
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Landscapes
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Abstract
The invention belongs to the field of traditional Chinese medicine quality detection and relates to a radix bupleuri medicinal material detection method. The radix bupleuri medicinal material detection method comprises determining saikosides a and d in radix bupleuri and determining pesticide residue content of the radix bupleuri medicinal material. The radix bupleuri medicinal material detection method solves the technical problem of operation complexity, limitation and difficult quality control of the traditional physicochemical property discriminating method, has high accuracy, can be operated simply, has a low cost and has a good application prospect and economic benefits.
Description
Technical Field
The invention belongs to the field of quality detection of traditional Chinese medicinal materials, and particularly relates to a quality detection method of a radix bupleuri medicinal material.
Background
The bupleurum is a traditional Chinese medicine prepared by processing dried roots of the dianthus chinensis, is sweet in taste and slightly warm in nature, has the effects of entering liver and stomach channels, clearing deficiency heat, removing infantile malnutrition heat and the like, and is clinically used for treating yin deficiency fever, bone steaming fatigue heat, infantile malnutrition heat and other diseases. The bupleurum medicinal material has been on the market in China for many years, and the clinical effect is widely determined. The bupleuri radix contains multiple chemical components, mainly including saikosaponin (four kinds of saikosaponin a, b, c, d), sterol, volatile oil, fatty acid and polysaccharide, etc. Modern researches show that the components contained in bupleurum have wide physiological activity, wherein the contents of saikosaponin a and saikosaponin d are closely related to the antipyretic and anti-inflammatory effects of bupleurum, so that the contents of the saikosaponin a and saikosaponin d are often used as important indexes for controlling the quality of bupleurum medicinal materials.
In recent years, the market demand of bupleurum is continuously increased, wild resources are reduced year by year, and the development of artificial cultivation is slow, so that the storage amount and the yield of bupleurum resources are greatly reduced. Leading to the appearance of a plurality of counterfeit products in the market, wherein the pesticide residue of a plurality of counterfeit products exceeds the standard and explodes the table, bringing great impact to the quality, the medication safety and the effectiveness of the bupleurum medicinal materials, and becoming the bottleneck restricting the development of bupleurum series medicinal materials.
At present, the effective detection method of the quality bupleurum is the traditional identification method of morphological characteristics and physicochemical characteristics, has certain limitation, is not enough to control the quality of the bupleurum on the whole, and as a clinical common traditional Chinese medicine, the truth and the falseness of the bupleurum are influenced by a plurality of factors such as physicochemical indexes, large pesticide residue and the like, so a scientific quality detection method is necessary to be established for evaluating the quality of the bupleurum.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to solve the problems of complex detection method and difficult quality control of the bupleurum medicinal material in the prior art, and further provide a method for simply and quickly detecting the bupleurum medicinal material with high accuracy.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the invention provides a method for detecting bupleurum medicinal materials, which comprises the following steps of measuring the content of saikosaponin a and saikosaponin d and measuring the pesticide residue, wherein the method comprises the following steps of:
a method for detecting bupleurum medicinal material is characterized in that the method comprises the following steps of measuring the total amount of saikosaponin a and saikosaponin d and measuring the pesticide residue, wherein,
a: the method for measuring the total amount of the saikosaponin a and the saikosaponin d comprises the following steps:
(1) accurately weighing reference substances of picroside a and picroside d, and adding methanol to prepare a solution containing 0.4mg of picroside a and 0.5mg of picroside dL in each 1mL, namely the reference solution;
(2) accurately weighing 0.5g of bupleuri radix powder to be measured, adding 25mL of methanol solution containing 5% concentrated ammonia reagent, performing ultrasonic treatment at 30 deg.C, filtering, washing container and residue with 20mL of methanol for 2 times, mixing washing solution and filtrate, and recovering solvent to dry; dissolving the residue with methanol, diluting to 5mL, filtering, and collecting the filtrate to obtain sample solution;
(3) according to the high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and gradient elution is carried out according to the following procedures: from 0-50min, mobile phase a: the volume ratio of the mobile phase B is from 25%: 75% → 90%: 10 percent; from 50-55min, mobile phase a: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 210 nm; the theoretical plate number is not less than 10000 calculated according to the peak of saikosaponin a;
respectively and precisely weighing 20 μ l of reference solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and measuring;
b: the determination of the pesticide residue comprises the following steps:
(1) accurately weighing a proper amount of triphenyl phosphate, and adding acetone to prepare a solution containing 100 mu g of triphenyl phosphate per 1ml as an internal standard stock solution;
precisely measuring 1ml of each pesticide reference product storage solution and 1ml of the internal standard storage solution respectively, and adding acetone to a constant volume of 100ml to serve as a mixed reference product storage solution; precisely measuring appropriate amount, adding acetonitrile to constant volume to obtain 20-1000ng/ml solutions with different concentrations, and using as mixed reference solution;
dissolving appropriate amount of ribonolactone in acetonitrile to obtain a solution containing 20mg of ribonolactone per 1ml, dissolving appropriate amount of sorbitol in water to obtain a solution containing 10mg of sorbitol per 1 ml; precisely measuring the ribonolactone and sorbitol solution respectively by 1ml, mixing uniformly, adding acetonitrile to constant volume to 10ml, and using the mixture as an analysis protective agent;
(2) precisely weighing 10g of medicinal material fine powder to be measured, adding 1g of sodium chloride, uniformly mixing, precisely adding 100ml of acetone, carrying out ice bath ultrasonic treatment for 30 minutes, centrifuging, quickly transferring supernatant into a conical bottle with a plug containing 1g of anhydrous sodium sulfate, and standing for 30 minutes; then, precisely measuring 60ml of the solution, concentrating the solution under reduced pressure to be nearly dry, and adding a solvent with the volume ratio of 1:1, dissolving a sample in cyclohexane-ethyl acetate solution, fixing the volume to 10ml, filtering, taking filtrate, purifying by GPC gel permeation chromatography, and performing gel permeation chromatography on the filtrate in a volume ratio of 1: eluting with cyclohexane-ethyl acetate solution of 1 as mobile phase, collecting eluate, transferring into KD bottle, and concentrating under reduced pressure to near dryness;
adding the mixture into the sample in a volume ratio of 1: dissolving 5ml of the ethyl acetate-acetone mixed solution of 1, transferring the dissolved solution to a graphite carbon-amino mixed solid phase extraction column, and mixing the dissolved solution with the volume ratio of 1: eluting with 15ml of ethyl acetate-acetone mixed solution of 1, collecting eluent, blowing nitrogen to near dryness, adding 5 mu l of internal standard stock solution, adding acetonitrile to constant volume to 1ml, and using the volume as a test solution;
(3) precisely measuring 400 mu L of each of the mixed reference substance solution and the test substance solution with each concentration, respectively adding 100 mu L of the analysis protective agent, uniformly mixing, precisely absorbing 1 mu L of the analysis protective agent, and performing gas chromatography-mass spectrometry combined instrument measurement; wherein,
the gas chromatographic analysis conditions are as follows: taking an elastic quartz capillary column DB17ms with the specification of 30m multiplied by 0.25mm multiplied by 0.25um, taking high-purity helium as a carrier gas, the column flow rate is 1.3ml/min, the sample injection amount is 1 mu l, adopting high-pressure non-split-flow sample injection, setting the temperature of a sample injection port to be 230 ℃, and specifically adopting a temperature rise program as follows: the initial temperature was 60 ℃, ramped to 120 ℃ at 30 ℃/min, ramped to 200 ℃ at 10 ℃/min, ramped to 230 ℃ at 2 ℃/min, ramped to 300 ℃ at 30 ℃/min, and held for 7 minutes;
further, the EI source mass spectrometry conditions are as follows: the electron energy is set to 70eV, the ion source temperature is 230 ℃, and the interface temperature is 250 ℃.
In the determination of the pesticide residue, the GPC gel permeation chromatography purification step specifically comprises the following conditions: the filler is Bio-Beads S-X3200-400 mesh, the purification column is 2.5mm multiplied by 40cm, the specific elution parameters are purification and impurity removal 900S, the target is collected for 1200S, and the column is cleaned for 300S.
Further, in the measurement of the pesticide residue, the pesticide reference substance comprises dichlorvos, methamidophos, acephate, tetrachloronitrobenzene, hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, gamma-hexachlorocyclohexane, omethoate, diazinon, pentachlorophenyl, monocrotophos, disulfoton, phosphamidol I, dimethoate, heptachlor, pentachloroaniline, chlorothalonil, chlorpyrifos-methyl, aldrin, captan, phosphamidol II, methyl parathion, methyl pyrimidylphenyl sulfide, methyl pentachlorophenyl sulfide, metalaxyl, triadimefon, chlorpyrifos, malathion, fenitrothion, parathion, pendimethalin, cis epoxy heptachlor, trans epoxy heptachlor, triadimenol A, triadimenol B, trans chlordane, cis endosulfan, cis chlordane, trans endosulfan, PP ' -DDE, PP ' -DDD, DDT ' -DDT, PP' -DDT, dieldrin, methidathion, endrin, ethion, triphenyl phosphate, bifenthrin, endosulfan, iprodione, fenpropathrin, dicofol, cyhalothrin, methoxyDDT, dicofol, vothiofos, permethrin 1, permethrin 2, cypermethrin, fenvalerate, deltamethrin.
In the step A, in the content determination of the saikosaponin a and the saikosaponin d, the particle size of the medicinal material to be determined is 180-.
Furthermore, in the step B, in the determination of the pesticide residue, the particle size of the to-be-detected medicinal material is 180-.
In the step of measuring the total amount of the saikosaponin a and the saikosaponin d, the conditions of the step of ultrasonic treatment are as follows: the power is 200W, the frequency is 40KHz, and the ultrasonic treatment is carried out for 30 min.
The method of the invention determines the authenticity of the bupleurum medicinal material by measuring the content of saikosaponin a and saikosaponin d contained in the bupleurum medicinal material and detecting the pesticide residue of the bupleurum medicinal material. And the content of the saikosaponin a and the content of the saikosaponin d are determined by high performance liquid chromatography, and the method is not only accurate, but also simple and easy to implement. The method detects the residual pesticide amount of the medicinal materials in a gas chromatography-mass spectrometry combined mode, has high detection accuracy and simple and quick method, can effectively detect common pesticide types in the bupleurum medicinal materials at one time through specific screening of detection conditions, and has better accuracy and practical effect.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific examples thereof and with the accompanying drawings, in which:
FIG. 1 is a chart of a bupleuri radix mixed control in example 1 of the present invention;
FIG. 2 is a chart of a sample of bupleuri radix according to example 1 of the present invention;
FIG. 3 is a full scan gas chromatogram of a pesticide standard in example 2 of the present invention;
FIG. 4 is a 0-10 minute scanning gas chromatogram of the pesticide standard in example 2 of the present invention;
FIG. 5 is a 10-20 minute scanning gas chromatogram of the pesticide standard in example 2 of the present invention;
FIG. 6 is a 20-40 minute scanning gas chromatogram of the pesticide standard in example 2 of the present invention.
Detailed Description
Example 1 determination of the Total amount of saikosaponin a, d in Bupleurum chinense
1 Instrument and reagent
1.1 instruments
Shimadzu LC20-AT liquid chromatograph;
waters2695-2998 liquid chromatograph;
ultrasonic cleaner (250W, 40 kHz).
1.2 reagents
Methanol, acetonitrile (chromatically pure), drochen distilled water;
the saikosaponin a (110777-.
1.3 samples of medicinal materials
Collecting Bupleurum samples from different places in Gansu province, wherein the test medicinal material production places are respectively selected from: gansu Xixi county, Gansu Linxia county, Gansu Zhang county, Gansu Li county, Gansu Xiong county in 2 batches (respectively marked as 1-2), Gansu Lingshi county, Gansu Zhang county, Gansu Wudu district Anhua town, Gansu Taishan county, Gansu Ping Shuliang city mountain Kongtong district, Gansu Wushan county in 2 batches (respectively marked as 1-2), Gansu Xixi county Yunnan town, Gansu Xiong county Tong Yiyi town in 3 batches (respectively marked as 1-3). The identification result of each test drug is Bupleurum chinense DC.
2. Method of producing a composite material
Accurately weighing picroside a and picroside d reference substances, and adding methanol to prepare a solution containing 0.4mg of picroside a and 0.5mg of picroside d per 1mL, namely the reference substance solution;
accurately weighing 0.5g of bupleuri radix powder to be measured, adding 25mL of methanol solution containing 5% concentrated ammonia reagent, performing ultrasonic treatment at 30 deg.C, filtering, washing container and residue with 20mL of methanol for 2 times, mixing washing solution and filtrate, and recovering solvent to dry; dissolving the residue with methanol, diluting to 5mL, filtering, and collecting the filtrate to obtain sample solution;
according to the high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and gradient elution is carried out according to the following procedures: from 0-50min, mobile phase a: the volume ratio of the mobile phase B is from 25%: 75% → 90%: 10 percent; from 50-55min, mobile phase a: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 210 nm; the theoretical plate number is not less than 10000 calculated according to the peak of saikosaponin a;
respectively and precisely weighing 20 μ l of reference solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and measuring;
3. results
The chromatogram of 19 batches of test drugs is shown in figures 1 and 2, and the detection results are shown in table 1 below.
TABLE 1 Collection and assay of Bupleurum samples (%)
Example 2 determination of amount of pesticide residue in Bupleurum chinense
1 Instrument and reagent
1.1 instruments
Clarus600 gas chromatography-mass spectrometer (PE company, USA), HS10260D ultrasonic extractor (Kunshan ultrasonic instrument Co., Ltd.), RE-52A rotary evaporator (Shanghai Yangrong Biochemical instruments factory).
1.2 reagent
Dichlorvos, methamidophos, acephate, tetrachloronitrobenzene, hexachlorobenzene, alpha-hexachlorohexa, beta-hexachlorohexa, gamma-hexachlorohexa, omethoate, diazinon, pentachloronitrobenzene, monocrotophos, chlorfenapyr, phosphamidol I, dimethoate, heptachlor, pentachloranil, chlorothalonil, chlorpyrifos-methyl, aldrin, captan, phosphamidol II, methyl parathion, pirimiphos-methyl, pentachlorophenyl sulfide, metalaxyl, triadimefon, chlorpyrifos, malathion, fenitrothion, parathion, pendimethalin, cis-epoxy heptachlor, trans-epoxy heptachlor, triadimenol A, triadimenol B, trans-chlordane, cis-chlordane, trans-endosulfan, PP '-DDD, PP' -DDT, Dietschafos, ethion, thion, chlorpyrifos, chlorp, 64 kinds of pesticide reference substance storage solutions such as triphenyl phosphate, bifenthrin, endosulfan, iprodione, fenpropathrin, dicofol, cyhalothrin, methoxy DDT, trichlorfone, fenthion, permethrin 1, permethrin 2, cypermethrin, fenvalerate, deltamethrin and the like are provided by China food and drug testing institute.
Triphenyl phosphate, acetone, acetonitrile, cyclohexane and ethyl acetate are imported pesticide residue grade; ribonolactone, sorbitol, sodium chloride and anhydrous sodium sulfate are superior pure.
1.3 samples
The genuine batch samples of radix bupleuri and the like purchased from the production place or the Lanzhou yellow river medicinal material market are all raw medicinal materials, and are numbered and detected according to different batches.
2 methods and results
2.1 preparation of internal standard stock solution and analytical protectant
Accurately weighing proper amount of triphenyl phosphate, and adding acetone to prepare solution containing 100 mu g of acetone per 1 ml.
Preparation of an analytical protective agent: taking a proper amount of ribonolactone, adding acetonitrile to dissolve the ribonolactone, and preparing a solution containing 20mg of ribonolactone per 1 ml; in addition, a proper amount of sorbitol is taken, and water is added for dissolving to prepare a solution containing 10mg of sorbitol per 1 ml. Precisely measuring the ribonolactone and sorbitol solutions by 1ml respectively, adding into the same 10ml measuring flask, diluting with acetonitrile to scale, and shaking.
2.2 preparation of Mixed control solutions
Precisely measuring 1ml of each pesticide reference stock solution and 1ml of internal standard stock solution respectively, placing into a 100ml measuring flask, diluting with acetone to scale, shaking up, and using as a mixed reference stock solution (1 microgram/ml). Accurately weighing appropriate amount, respectively, and adding acetonitrile to obtain 20-1000ng/ml mixed reference solutions of different concentration series.
2.3 preparation of test solutions
Precisely weighing 10g of each batch of test sample fine powder, placing the fine powder in a 100ml conical flask, adding 1g of sodium chloride, precisely adding 100ml of acetone, carrying out ultrasonic treatment in ice bath for 30 minutes, centrifuging, quickly transferring supernatant into the conical flask with the plug containing 1g of anhydrous sodium sulfate, and standing for 30 minutes. Precisely measuring 60ml, concentrating under reduced pressure to near dryness, adding a small amount of cyclohexane: dissolving a sample with ethyl acetate (1:1) solution, quantitatively diluting to 10ml, mixing uniformly, filtering, purifying the filtrate by Gel Permeation Chromatography (GPC), injecting 5ml, (GPC main parameters: packing Bio-Beads S-X3200-400 meshes, purifying column 2.5mm × 40cm, elution parameters: pre run 900S, main run 1200S, tail run 300S), purifying with cyclohexane: eluting with ethyl acetate (1:1) as mobile phase, collecting eluate, transferring into KD bottle, and concentrating under reduced pressure to near dryness.
2.4 purification of the sample solution
Adding 5ml of ethyl acetate-acetone (1:1) mixed solution for dissolving, quantitatively transferring the dissolved solution to a graphite carbon (500mg) -amino (500mg) mixed solid phase extraction column, eluting the column with 15ml of ethyl acetate-acetone (1:1) mixed solution, collecting eluent, blowing nitrogen to the near-dry state, adding acetonitrile into residues for dissolving, adding 5 mu l of internal standard stock solution, and fixing the volume of the acetonitrile to 1ml to obtain the product.
2.5 gas-mass spectrometry
Gas chromatography analysis conditions elastic quartz capillary column (30m × 0.25mm × 0.25um) DB17ms, carrier gas is high-purity helium, and column flow rate is 1.3 ml/min; the injection port temperature is 230 ℃, the injection amount is 1 mu l, and high-pressure non-split injection is carried out.
Specific temperature rise procedures are as follows: the initial temperature is 60 deg.C, 30 deg.C/min to 120 deg.C, 10 deg.C/min to 200 deg.C, 2 deg.C/min to 230 deg.C, 30 deg.C/min to 300 deg.C, and is maintained for 7 min.
Mass spectrometry (EI source) measurement conditions: 70eV, the ion source temperature 230 ℃ and the interface temperature 250 ℃.
The SIM conditions of each sample to be tested are shown in Table 3, and the full-scan gas chromatogram and the 0-10 min, 10-20 min and 20-40 min scan gas chromatograms of each pesticide standard are respectively shown in figures 3, 4, 5 and 6.
TABLE 3GC-MS detection index and SIM determination conditions
2.6 methodological validation
2.6.1 precision test
The diluted mixed standard solution was precisely aspirated by 1. mu.L, and the sample injection was repeated 5 times to calculate the RSD value, as shown in Table 4.
Precision test of 464 pesticides in table
2.6.2 sample recovery test
Adding certain mixed standard solution into the samples respectively to form 3 samples with 3 addition levels, extracting and breaking loose and detecting according to a selected method, and calculating the recovery rate of various pesticides in the samples by deducting the background, wherein the results are shown in Table 5.
Sample application recovery test for 564 pesticides in table
2.6.3 detection Limit determination
The pesticide standard solution was diluted to different concentrations and the minimum amount of components detected was determined, the results are shown in table 6.
TABLE 664 detection limits of pesticides
2.6.4 Linear relationship
The pesticide standard solutions were diluted to different concentrations and the linear range of the components was determined and the results are shown in table 7.
TABLE 764 Linear equations for pesticides
2.7 assay method
Respectively and precisely measuring 400 mu L of each of the mixed reference solution and the test solution, adding 00 mu L of the analysis protective agent, uniformly mixing, respectively and precisely absorbing 1 mu L of the mixture, injecting the mixture into a gas chromatography-mass spectrometer, measuring and calculating to obtain the test solution, wherein the results are shown in Table 8.
TABLE 8 background determination of pesticide residue in bupleuri radix (mg/kg)
3. Measurement results and analysis
3.1 pesticide residue determination results 7 batches of samples all detected pesticide residues, the detection rate was 100%, and the current situation of pesticide residues in radix bupleuri was relatively common. Relates to 14 different pesticides of organic chlorine, organic phosphorus and pyrethroid. The pesticide residue batches include 1 batch of hexachlorobenzene, 1 batch of chlorothalonil, 4 batches of methyl parathion, 1 batch of pirimiphos-methyl, 2 batches of parathion, 3 batches of triazolone, 1 batch of dicofol, 1 batch of pendimethalin, 1 batch of dieldrin, 1 batch of ethion, 1 batch of bifenthrin, 4 batches of methoxyDDT 2 and dicofol.
3.2 the Chinese thorowax root medicinal materials have different pesticide residues, wherein the pesticide residues of 2 varieties are different, and the pesticide residues of 2-3 varieties are different. The causes of pesticide residue in the traditional Chinese medicinal materials are complicated due to the influences of factors such as environment, planting technology, processing and storage.
3.3 in the production of Chinese medicinal material GAP, there are 54 kinds of forbidden pesticides. 4 kinds of forbidden pesticides including parathion, methyl parathion, Dieldrin and DDT are detected from the bupleurum. Other detected pesticides belong to pesticides which can be used in production.
3.4 the maximum residual limit of pesticide in the food is regulated and analyzed by adopting the national standard GB2763-2005, and although detected, the maximum residual limit of pesticide in the food is not exceeded: chlorothalonil, pirimiphos-methyl, dicofol, bifenthrin, pendimethalin, dieldrin and ethion. The food residue standard is exceeded, namely methyl parathion (0.1 mg/kg of grains and 0.02mg/kg of vegetables and fruits) exceeds the standard by 4-10 times; parathion (0.1 mg/kg of grains and 0.01mg/kg of vegetables and fruits) exceeds 2-12 times, triazolone (0.5 mg/kg of paddy and 0.05mg/kg of vegetables) exceeds 1-4 times, and dicofol (2 mg/kg of fruits) exceeds 0.4-0.9 time.
In the general analysis, the pesticide detection rate of the bupleurum samples is 100 percent, the 7 samples have pesticide exceeding standards, and the sample exceeding rate is 100 percent; 4 forbidden pesticides are detected, and the forbidden pesticides in 6 batches of samples exceed the standard, with the standard exceeding rate of 85.7%.
3.5 according to the measurement result, the pesticide prohibited in the production of the traditional Chinese medicine GAP and the pesticide which is allowed to be used but is easy to exceed the standard in the production are recommended to be checked in a limited way. The proposed methyl parathion is not more than 0.1mg/kg, and parathion is not more than 0.1 mg/kg.
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. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (6)
1. A method for detecting bupleurum medicinal material is characterized in that the method comprises the following steps of measuring the total amount of saikosaponin a and saikosaponin d and measuring the pesticide residue, wherein,
a: the method for measuring the total amount of the saikosaponin a and the saikosaponin d comprises the following steps:
(1) accurately weighing reference substances of picroside a and picroside d, and adding methanol to prepare a solution containing 0.4mg of picroside a and 0.5mg of picroside dL in each 1mL, namely the reference solution;
(2) accurately weighing 0.5g of bupleuri radix powder to be measured, adding 25mL of methanol solution containing 5% concentrated ammonia reagent, performing ultrasonic treatment at 30 deg.C, filtering, washing container and residue with 20mL of methanol for 2 times, mixing washing solution and filtrate, and recovering solvent to dry; dissolving the residue with methanol, diluting to 5mL, filtering, and collecting the filtrate to obtain sample solution;
(3) according to the high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and gradient elution is carried out according to the following procedures: from 0-50min, mobile phase a: the volume ratio of the mobile phase B is from 25%: 75% → 90%: 10 percent; from 50-55min, mobile phase a: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 210 nm; the theoretical plate number is not less than 10000 calculated according to the peak of saikosaponin a;
respectively and precisely weighing 20 μ l of reference solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and measuring;
b: the determination of the pesticide residue comprises the following steps:
(1) accurately weighing a proper amount of triphenyl phosphate, and adding acetone to prepare a solution containing 100 mu g of triphenyl phosphate per 1ml as an internal standard stock solution;
precisely measuring 1ml of each pesticide reference product storage solution and 1ml of the internal standard storage solution respectively, and adding acetone to a constant volume of 100ml to serve as a mixed reference product storage solution; precisely measuring appropriate amount, adding acetonitrile to constant volume to obtain 20-1000ng/ml solutions with different concentrations, and using as mixed reference solution;
dissolving appropriate amount of ribonolactone in acetonitrile to obtain a solution containing 20mg of ribonolactone per 1ml, dissolving appropriate amount of sorbitol in water to obtain a solution containing 10mg of sorbitol per 1 ml; precisely measuring the ribonolactone and sorbitol solution respectively by 1ml, mixing uniformly, adding acetonitrile to constant volume to 10ml, and using the mixture as an analysis protective agent;
(2) precisely weighing 10g of medicinal material fine powder to be measured, adding 1g of sodium chloride, uniformly mixing, precisely adding 100ml of acetone, carrying out ice bath ultrasonic treatment for 30 minutes, centrifuging, quickly transferring supernatant into a conical bottle with a plug containing 1g of anhydrous sodium sulfate, and standing for 30 minutes; then, precisely measuring 60ml of the solution, concentrating the solution under reduced pressure to be nearly dry, and adding a solvent with the volume ratio of 1:1, dissolving a sample in cyclohexane-ethyl acetate solution, fixing the volume to 10ml, filtering, taking filtrate, purifying by GPC gel permeation chromatography, and performing gel permeation chromatography on the filtrate in a volume ratio of 1: eluting with cyclohexane-ethyl acetate solution of 1 as mobile phase, collecting eluate, transferring into KD bottle, and concentrating under reduced pressure to near dryness;
adding the mixture into the sample in a volume ratio of 1: dissolving 5ml of the ethyl acetate-acetone mixed solution of 1, transferring the dissolved solution to a graphite carbon-amino mixed solid phase extraction column, and mixing the dissolved solution with the volume ratio of 1: eluting with 15ml of ethyl acetate-acetone mixed solution of 1, collecting eluent, blowing nitrogen to near dryness, adding 5 mu l of internal standard stock solution, adding acetonitrile to constant volume to 1ml, and using the volume as a test solution;
(3) precisely measuring 400 mu L of each of the mixed reference substance solution and the test substance solution with each concentration, respectively adding 100 mu L of the analysis protective agent, uniformly mixing, precisely absorbing 1 mu L of the analysis protective agent, and performing gas chromatography-mass spectrometry combined instrument measurement; wherein,
the gas chromatographic analysis conditions are as follows: taking an elastic quartz capillary column DB17ms with the specification of 30m multiplied by 0.25mm multiplied by 0.25um, taking high-purity helium as a carrier gas, the column flow rate is 1.3ml/min, the sample injection amount is 1 mu l, adopting high-pressure non-split-flow sample injection, setting the temperature of a sample injection port to be 230 ℃, and specifically adopting a temperature rise program as follows: the initial temperature was 60 ℃, ramped to 120 ℃ at 30 ℃/min, ramped to 200 ℃ at 10 ℃/min, ramped to 230 ℃ at 2 ℃/min, ramped to 300 ℃ at 30 ℃/min, and held for 7 minutes;
the EI source mass spectrometry conditions are as follows: the electron energy is set to 70eV, the ion source temperature is 230 ℃, and the interface temperature is 250 ℃.
2. The method for detecting the bupleurum medicinal material according to claim 1, wherein in the determination of the pesticide residue, the GPC gel permeation chromatography purification step specifically comprises the following conditions: the filler is Bio-Beads S-X3200-400 mesh, the purification column is 2.5mm multiplied by 40cm, the specific elution parameters are purification and impurity removal 900S, the target is collected for 1200S, and the column is cleaned for 300S.
3. The method for detecting the bupleurum medicinal material according to claim 1 or 2, wherein in the determination of the pesticide residue, the pesticide reference substance comprises dichlorvos, methamidophos, acephate, tetrachloronitrobenzene, hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, gamma-hexachlorocyclohexane, omethoate, diazinon, quintozene, monocrotophos, fonofos, phosphamidol, dimethoate, heptachlor, pentachloranilide, chlorothalonil, chlorpyrifos-methyl, aldrin, captan, phosphamidol II, methyl parathion, pirimiphos-methyl, pentachlorophenyl sulfide, metalaxyl, triadimefon, chlorpyrifos, malathion, fenitrothion, parathion, pendimethalin, cis epoxy heptachlor, trans epoxy heptachlor, triadimenol A, triadimenol B, trans-chlordane, cis endosulfan, cis chlordane, and, Trans-endosulfan, PP '-DDE, PP' -DDD, OP '-DDT, PP' -DDT, dieldrin, methidathion, endrin, ethion, triphenyl phosphate, bifenthrin, endosulfan, iprodione, fenpropathrin, dicofol, cyhalothrin, methoxyDDT, dicofol, vothiofos, permethrin 1, permethrin 2, cypermethrin, fenvalerate, deltamethrin.
4. The method for detecting bupleurum medicinal material according to any one of claims 1 to 3, wherein the particle size of the medicinal material to be detected is 180-2000 μm in the content determination of the saikosaponin a and d.
5. The method for detecting the bupleurum medicinal material as claimed in any one of claims 1 to 4, wherein the particle size of the medicinal material to be detected in the determination of the pesticide residue is 180-2000 μm.
6. The method for detecting the bupleurum medicinal material according to any one of claims 1 to 5, wherein in the step of measuring the total amount of the saikosaponin a and the saikosaponin d, the conditions of the step of ultrasonic processing are as follows: the power is 200W, the frequency is 40KHz, and the ultrasonic treatment is carried out for 30 min.
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| CN111089913A (en) * | 2019-10-16 | 2020-05-01 | 四川恒通动保生物科技有限公司 | Quality control method of bupleurum chinense injection |
| CN113030340A (en) * | 2021-04-09 | 2021-06-25 | 北京市药品检验所 | Method for measuring content of characteristic components of radix bupleuri in radix bupleuri and preparation thereof by liquid phase coupling high-resolution mass spectrometry |
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