CN104198600A - Method for detecting radix astragali - Google Patents
Method for detecting radix astragali Download PDFInfo
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- CN104198600A CN104198600A CN201410373774.9A CN201410373774A CN104198600A CN 104198600 A CN104198600 A CN 104198600A CN 201410373774 A CN201410373774 A CN 201410373774A CN 104198600 A CN104198600 A CN 104198600A
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Abstract
The invention belongs to the field of detection of the quality of traditional Chinese medicinal materials and in particular relates to a method for detecting the quality of radix astragali. The method comprises the steps of determining the content of astragaloside and calycosin contained in radix astragali and determining the pesticide residues of radix astragali. The method conduces to solving the technical problems that identification methods of traditional physicochemical properties are complex to operate and have limitation and the quality is difficult to regulate and control, has high degree of accuracy, is simple, convenient and fast to operate, is low in cost and has good application prospect and economic benefits.
Description
Technical field
The invention belongs to Chinese crude drug quality testing field, be specifically related to a kind of quality determining method of Milkvetch Root.
Background technology
The Radix Astragali, contain kind of a chemical composition, there are sucrose, glucuronic acid, Astragaloside IV, calycosin, onocerin, astragaloside II, astragaloside III, astragalus polyose A, B, C, D, lymphatic temperament, several amino acids, bitter principle, astragaloside, choline, folic acid, flavane compound and contain the various trace elements such as selenium, silicon, zinc, cobalt, copper, molybdenum.For invigorating qi for strengthening superficies, diuresis holder poison, apocenosis, expelling pus and promoting granulation.Milkvetch Root has gone on the market for many years in China, and clinical effectiveness is able to extensively certainly.The effective constituent that principal ingredient Astragaloside IV wherein, calycosin, onocerin, astragaloside II, astragaloside III are the Radix Astragali, modern study shows that it has physiological activity widely, antipyretic and anti-inflammatory effect of the content of these compositions and the Radix Astragali is closely related, therefore contained mentioned component the important indicator of number Chang Zuowei Milkvetch Root quality control.
The market demand of the Radix Astragali continued to increase in recent years, and wild resource reduces year by year, and artificial cultivation slower development is all declining to a great extent Radix Astragali resource reserves and output.Cause having occurred on market a lot of adulterants, wherein the persticide residue of the many adulterants quick-fried table that exceeds standard, has brought very large impact to the quality of Milkvetch Root, the safety and efficacy of medication, has become the bottleneck of restriction Radix Astragali series medicinal material exploitation.
At present, effective detection method of the certified products Radix Astragali is traditional morphological feature and the discrimination method of physicochemical property, there is certain limitation, be not enough to control on the whole the quality of the Radix Astragali, as clinical conventional Chinese medicine simply, its true and false quality is subject to the impact of the factors such as its physical and chemical index, persticide residue be many, is therefore necessary that the quality determining method of setting up a kind of science evaluates the quality of the Radix Astragali.
Summary of the invention
For this reason, technical matters to be solved by this invention is Milkvetch Root detection method complexity in prior art, the problem of difficult quality regulation and control, and then provide a kind of accuracy high, detect simply, fast the method for Milkvetch Root.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
The invention provides a kind of detection method of Milkvetch Root, comprise the determination step of following active constituent content and the determination step of persticide residue, wherein,
A: the mensuration of Astragaloside content comprises the steps:
(1) it is appropriate that precision takes Astragaloside IV reference substance, adds methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast;
(2) accurately weighed Milkvetch Root powder 4g to be measured, put in apparatus,Soxhlet's, add methyl alcohol 40ml, cold soaking, add again methyl alcohol appropriate, add hot reflux 4 hours, extract reclaims solvent and is concentrated into dry, the residue 10ml that adds water, low-grade fever makes to dissolve, with water saturated normal butyl alcohol jolting extraction 4 times, each 40ml, merge normal butyl alcohol liquid, fully wash 2 times with ammonia solution, each 40ml, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue add water 5ml make dissolve, let cool, adsorb by D101 type large pore resin absorption column, with water 50ml wash-out, discard water liquid, use again 40% ethanol 30ml wash-out, discard eluent, continue with 70% ethanol 80ml wash-out, collect eluent, evaporate to dryness, dissolve and be transferred in 5ml measuring bottle with methyl alcohol, add methyl alcohol to scale, shake up, as need testing solution,
(3) according to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-water taking volume ratio as 32:68 is mobile phase; Evaporative light-scattering detector detects;
Accurate reference substance solution 10 μ l, the 20 μ l of drawing respectively, need testing solution 20 μ l, injection liquid chromatography, measures, and calculates with external standard two-point method logarithmic equation; Wherein, number of theoretical plate calculates and should be not less than 4000 with Astragaloside IV peak;
The mensuration of B calycosin glucoside content comprises the steps:
(1) get calycosin glucoside reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 50 μ g, product solution in contrast;
(2) accurately weighed Milkvetch Root powder 1g to be measured, precision adds methyl alcohol 50ml, weighed weight, add hot reflux 4 hours, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 25ml, reclaims solvent to dry, residue adds methyl alcohol and dissolves and be settled to 5ml, as need testing solution;
(3) according to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Taking acetonitrile as mobility A, taking 0.2% formic acid solution as Mobile phase B, carry out gradient elution according to following program: from 0-20min, mobile phase A: the volume ratio of Mobile phase B is by 20%:80% → 40%:60%; From 20-30min, mobile phase A: the volume ratio of Mobile phase B is 40%:60%; Controlling and detecting wavelength is 260nm;
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, note people liquid chromatograph, measures; Number of theoretical plate calculates and should be not less than 3000 by calycosin glucoside peak;
C: the mensuration of described persticide residue comprises the steps:
(1) to take triphenyl appropriate for precision, adds acetone and make the solution of every 1ml containing 100 μ g triphenyl, as interior mark stock solution;
Precision measures each agricultural chemicals reference substance stock solution 1ml and described interior mark stock solution 1ml respectively, adds acetone and is settled to 100ml, as mixing reference substance stock solution; Precision measures in right amount respectively, adds acetonitrile constant volume and make the solution of the variable concentrations of 20-1000ng/ml, as mixing reference substance solution;
Separately get ribose acid lactone appropriate, add acetonitrile dissolving and make the solution of every 1ml containing 20mg ribose acid lactone, separately get sorbierite appropriate, be dissolved in water and make the solution of every 1ml containing sorbierite 10mg; Precision measures above-mentioned ribose acid lactone respectively, the each 1ml of sorbitol solution mixes, and adds acetonitrile and is settled to 10ml, as analysis protectant;
(2) precision takes medicinal material fine powder 10g to be measured, and adds sodium chloride 1g to mix, and precision adds acetone 100ml, and ice-bath ultrasonic is processed 30 minutes, centrifugal, and supernatant is moved into and is equipped with in the tool plug conical flask of 1g anhydrous sodium sulfate rapidly, places 30 minutes; Precision measures above-mentioned solution 60ml and is evaporated near doing subsequently, and to add volume ratio be thiacyclohexane-ethyl acetate solution sample dissolution of 1:1 and be settled to 10ml, after filtration, getting filtrate purifies through GPC gel permeation chromatography, thiacyclohexane-ethyl acetate solution taking volume ratio as 1:1 is mobile phase wash-out, collect eluent, move in KD bottle, be evaporated near dry;
Be that ethyl acetate-acetone mixed solution 5ml of 1:1 dissolves to adding volume ratio in above-mentioned sample, and be transferred on graphitic carbon-amino mixing solid phase extraction column, ethyl acetate-acetone mixed solution 15ml wash-out taking volume ratio as 1:1, collect eluent, nitrogen blows near dry, add subsequently described interior mark stock solution 5 μ l, add acetonitrile and be settled to 1ml, as need testing solution;
(3) precision measures above-mentioned each concentration mixing reference substance solution and the each 400 μ L of need testing solution, and adds respectively described analysis protectant 100 μ L to mix, and precision is drawn 1 μ L respectively subsequently, carries out gas chromatograph-mass spectrometer (GCMS) mensuration; Wherein,
Described analytical conditions for gas chromatography is: getting specification is the fused-silica capillary column DB17ms of 30m × 0.25mm × 0.25um, taking high-purity helium as carrier gas, column flow rate 1.3ml/ minute, sample size 1 μ l, adopt high pressure Splitless injecting samples, it is 230 DEG C that injector temperature is set, and heating schedule is specially: 60 DEG C of initial temperatures, rise to 120 DEG C, rise to 200 DEG C, rise to 230 DEG C, rise to 300 DEG C with 30 DEG C/min with 2 DEG C/min with 10 DEG C/min with 30 DEG C/min, and keep 7 minutes;
The condition of described EI source mass spectroscopy is: electron energy 70eV is set, 230 DEG C of ion source temperatures, 250 DEG C of interface temperature.
Wherein, in the mensuration of described persticide residue, the condition of described GPC gel permeation chromatography purifying step is specially: filler is Bio-Beads S-X3 200-400 order, decontaminating column is 2.5mm × 40cm, concrete wash-out parameter is for purifying impurity elimination 900s, and object is collected 1200s, and pillar cleans 300s.
Further, in the mensuration of described persticide residue, described agricultural chemicals reference substance comprises DDVP, acephatemet, orthene, tecnazene, hexachloro-benzene, α-benzene hexachloride, β-benzene hexachloride, γ-benzene hexachloride, δ-benzene hexachloride, flolimat, diazinon, pentachloronitrobenzene, Azodrin, Fonofos, phosphamidon I, Rogor, heptachlor, pentachloroaniline, Bravo, chlorpyrifos-methyl, drinox, captan, phosphamidon II, parathion-methyl, pirimiphos-methyl, methyl five chlorophenyl thioether, metalaxyl, triazolone, chlopyrifos, malathion, fenifrothion, parathion, pendimethalin, cis Heptachlor epoxide, trans Heptachlor epoxide, Triadimenol A, Triadimenol B, trans Niran, cis 5a,6,9,9a-hexahydro-6,9-methano-2,4, cis Niran, trans 5a,6,9,9a-hexahydro-6,9-methano-2,4, pp'-DDE, PP'-DDD, OP'-DDT, PP'-DDT, dieldrite, methidathion, endrin, Ethodan, triphenyl, Biphenthrin, 5a,6,9,9a-hexahydro-6,9-methano-2,4 sulfuric ester, iprodione, Fenpropathrin, dicofol, lambda-cyhalothrin, methoxy DDT, tetradiphon, Phosalone, Permethrin 1, Permethrin 2, cypermethrin, fenvalerate, decis.
In the mensuration of described Astragaloside IV, calycosin content, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
In the determination step of described Astragaloside content, the internal diameter of described D101 type large pore resin absorption column is 1.5cm, long 12cm.
In the mensuration of described persticide residue, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
The method of the invention, by the mensuration to the contained Astragaloside content of Milkvetch Root and the detection to Milkvetch Root persticide residue, is carried out the criterion of Milkvetch Root true and false quality.And by high effective liquid chromatography for measuring Astragaloside content, described method is not only accurate and simple.The method of the invention detects the remains of pesticide amount of medicinal material by the mode of gas chromatography-mass spectrography, the accuracy not only detecting is high, method is simple, quick, and by the particular screen to testing conditions, can disposable pesticide variety common in Milkvetch Root be carried out to disposable effective detection, described method accuracy rate and practical function are all better.
Brief description of the drawings
For content of the present invention is more likely to be clearly understood, according to specific experiment example of the present invention also by reference to the accompanying drawings, the present invention is further detailed explanation below, wherein:
Fig. 1 is the full scan gas chromatogram of the embodiment of the present invention 3 Pesticides standard items;
Fig. 2 is 0-10 minute scanning gas chromatogram of the embodiment of the present invention 3 Pesticides standard items;
Fig. 3 is 10-20 minute scanning gas chromatogram of the embodiment of the present invention 3 Pesticides standard items;
Fig. 4 is 20-40 minute scanning gas chromatogram of the embodiment of the present invention 3 Pesticides standard items.
Embodiment
The mensuration of embodiment 1 Astragaloside content
1. instrument and reagent
1.1 instrument Shimadzu LC-20AT type high performance liquid chromatographs, Shimadzu SPD-M10A VP type diode array detector, plum Teller AL-204 type electronic balance, D101 type large pore resin absorption column, apparatus,Soxhlet's.
1.2 reagent Astragaloside IVs, calycosin glucoside standard items, purchased from Chinese food medicine qualification institute, acetonitrile, methyl alcohol (chromatographic grade); Formic acid, ammonia solution, normal butyl alcohol, purified water, is analytical reagent.
1.3 medicinal material sample collections various places, Gansu Radix Astragali samples, be selected from respectively for the reagent material place of production batch: 2 batches of Longxi County, Gansu, be labeled as respectively 2 batches of the first positive Dong Jiabao of 1-2, the west of Gansu Province, Gansu, the west of Gansu Province, Gansu first Yang Caiziping, Weiyuan, Gansu Hui Chuan, Lintao, Gansu, Weiyuan, Gansu, be labeled as respectively 3 batches, 1-2, Lian Feng Zhan Po village, Weiyuan, be labeled as respectively 1-3, Gansu commodity.
2. method
Chromatographic condition and system suitability are taking octadecylsilane chemically bonded silica as filling agent; Taking acetonitrile-water (32:68) as mobile phase; Evaporative light-scattering detector.Number of theoretical plate calculates and should be not less than 4000 with Astragaloside IV peak.
It is appropriate that the preparation precision of reference substance solution takes Astragaloside IV reference substance, adds methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product.
The about 4g of powder in this product is got in the preparation of need testing solution, accurately weighed, put in apparatus,Soxhlet's, add methyl alcohol 40ml, cold soaking spends the night, add again methyl alcohol appropriate, add hot reflux 4 hours, extract reclaims solvent and is concentrated into dry, the residue 10ml that adds water, low-grade fever makes to dissolve, with water saturated normal butyl alcohol jolting extraction 4 times, each 40ml, merge normal butyl alcohol liquid, fully wash 2 times with ammonia solution, each 40ml, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue add water 5ml make dissolve, let cool, by D101 type large pore resin absorption column (internal diameter 1.5cm, long 12cm), with water 50ml wash-out, discard water liquid, use again 40% ethanol 30ml wash-out, discard eluent, continue with 70% ethanol 80ml wash-out, collect eluent, evaporate to dryness, dissolve and be transferred in 5ml measuring bottle with methyl alcohol, add methyl alcohol to scale, shake up, obtain.
Accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l, injection liquid chromatography, measures, and calculates with external standard two-point method logarithmic equation, to obtain final product.
This product is pressed dry product and is calculated, containing Astragaloside IV (C
41h
68o
14) must not be less than 0.040%.
3. the qualification result of the Astragaloside content of pair above-mentioned medicinal material is as follows in table 1.
The content of 2 compositions (%) in table 1 Milkvetch Root
According to measurement result, continue to use one of Chinese Pharmacopoeia version in 2010 to Radix Astragali Astragaloside IV, calycosin glucoside content limit.
Embodiment 2 calycosin glucoside content:
1. instrument and reagent
1.1 instrument Shimadzu LC-20AT type high performance liquid chromatographs, Shimadzu SPD-M10A VP type diode array detector, plum Teller AL-204 type electronic balance, D101 type large pore resin absorption column, apparatus,Soxhlet's.
1.2 reagent Astragaloside IVs, calycosin glucoside standard items, purchased from Chinese food medicine qualification institute, acetonitrile, methyl alcohol (chromatographic grade); Formic acid, ammonia solution, normal butyl alcohol, purified water is analytical reagent.
1.3 medicinal material sample collections various places, Gansu Radix Astragali samples, be selected from respectively for the reagent material place of production batch: 2 batches of Longxi County, Gansu, be labeled as respectively 2 batches of the first positive Dong Jiabao of 1-2, the west of Gansu Province, Gansu, the west of Gansu Province, Gansu first Yang Caiziping, Weiyuan, Gansu Hui Chuan, Lintao, Gansu, Weiyuan, Gansu, be labeled as respectively 3 batches, 1-2, Lian Feng Zhan Po village, Weiyuan, be labeled as respectively 1-3, Gansu commodity.
2. method
Chromatographic condition and system suitability are taking octadecylsilane chemically bonded silica as filling agent; Taking acetonitrile as mobility A, taking 0.2% formic acid solution as Mobile phase B, the program according to the form below is carried out gradient elution; Detection wavelength is 260nm; Number of theoretical plate calculates and should be not less than 3000 by calycosin glucoside peak.
It is appropriate, accurately weighed that calycosin glucoside reference substance is got in the preparation of reference substance solution, adds methyl alcohol and make the solution of every 1ml containing 50 μ g, to obtain final product.
The about 1g of above-mentioned each sample powder (crossing sieve No. four) is got in the preparation of need testing solution, accurately weighed, puts in round-bottomed flask, precision adds methyl alcohol 50ml, and weighed weight adds hot reflux 4 hours, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 25ml, reclaim solvent to dry, residue adds methyl alcohol and dissolves, and is transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, to obtain final product.
Determination method is accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, and note people liquid chromatograph, measures, and to obtain final product.
This product is pressed dry product and is calculated, containing calycosin glucoside (C
22h
22o
10) must not be less than 0.020%.
3. the qualification result of the calycosin glucoside content of pair above-mentioned each medicinal material sample is shown in as shown in table 2 below.
The content of 2 compositions (%) in table 2 Milkvetch Root
According to measurement result, continue to use one of Chinese Pharmacopoeia version in 2010 to Radix Astragali Astragaloside IV, calycosin glucoside content limit.
Embodiment 3 Milkvetch Root residues of pesticides quantitative determinations
1 instrument and reagent
1.1 instrument
Clarus600 gas chromatograph-mass spectrometer (GCMS) (PE company of the U.S.), the ultrasonic extraction apparatus of HS10260D (Kunshan Ultrasonic Instruments Co., Ltd.), RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant).
1.2 reagent
DDVP, acephatemet, orthene, tecnazene, hexachloro-benzene, α-benzene hexachloride, β-benzene hexachloride, γ-benzene hexachloride, δ-benzene hexachloride, flolimat, diazinon, pentachloronitrobenzene, Azodrin, Fonofos, phosphamidon I, Rogor, heptachlor, pentachloroaniline, Bravo, chlorpyrifos-methyl, drinox, captan, phosphamidon II, parathion-methyl, pirimiphos-methyl, methyl five chlorophenyl thioether, metalaxyl, triazolone, chlopyrifos, malathion, fenifrothion, parathion, pendimethalin, cis Heptachlor epoxide, trans Heptachlor epoxide, Triadimenol A, Triadimenol B, trans Niran, cis 5a,6,9,9a-hexahydro-6,9-methano-2,4, cis Niran, trans 5a,6,9,9a-hexahydro-6,9-methano-2,4, pp'-DDE, PP'-DDD, OP'-DDT, PP'-DDT, dieldrite, methidathion, endrin, Ethodan, triphenyl, Biphenthrin, 5a,6,9,9a-hexahydro-6,9-methano-2,4 sulfuric ester, iprodione, Fenpropathrin, dicofol, lambda-cyhalothrin, methoxy DDT, tetradiphon, Phosalone, Permethrin 1, Permethrin 2, cypermethrin, fenvalerate, 64 kinds of agricultural chemicals reference substance sterling stock solutions such as decis, provide purchased from National Institute for Food and Drugs Control.
Triphenyl, acetone, acetonitrile, thiacyclohexane, ethyl acetate are the residual level of import agriculture; Ribose acid lactone, sorbierite, sodium chloride, anhydrous sodium sulfate are that top grade is pure.
1.3 sample
The genuine batch sample such as the Radix Astragali is bought in the place of production or is purchased from medicinal material market, the Yellow River, Lanzhou, is crude drug, below is numbered detection by different batches.
2 methods and result
The preparation of 2.1 interior mark stock solutions and analysis protectant
It is appropriate that precision takes triphenyl, adds acetone and make the solution of every 1ml containing 100 μ g, to obtain final product.
The preparation of analysis protectant: get ribose acid lactone appropriate, add acetonitrile and dissolve and make every 1ml containing the solution of 20mg; Separately get sorbierite appropriate, be dissolved in water and make the solution of every 1ml containing 10mg.Precision measures above-mentioned ribose acid lactone, the each 1ml of sorbitol solution respectively, to same 10ml measuring bottle, adds dilution in acetonitrile to scale, shakes up, and to obtain final product.
2.2 mix the preparation of reference substance solution
Precision measures each agricultural chemicals reference substance stock solution 1ml and interior mark stock solution 1ml respectively, puts in 100ml measuring bottle, to scale, shakes up, as mixing reference substance stock solution (1 μ g/ml) by acetone diluted.Precision measures in right amount respectively, adds acetonitrile and make the mixing reference substance solution of 20-1000ng/ml variable concentrations series, to obtain final product.
The preparation of 2.3 need testing solutions
Precision takes each batch of test sample fine powder 10g, puts in 100ml conical flask, adds 1g sodium chloride, and precision adds acetone 100ml, and ice-bath ultrasonic is processed 30 minutes, centrifugal, and supernatant is moved into and is equipped with in the tool plug conical flask of 1g anhydrous sodium sulfate rapidly, places 30 minutes.Precision measures 60ml and is evaporated near dry, add a small amount of thiacyclohexane: ethyl acetate (1:1) solution sample dissolution is also quantitatively diluted to 10ml, mix, filter, filtrate purifies through gel permeation chromatography (GPC), sample introduction 5ml, (GPC major parameter: filler Bio-Beads S-X3 200--400 order, decontaminating column 2.5mm × 40cm, wash-out parameter: purify impurity elimination 900s, object is collected 1200s, and pillar cleans 300s., taking thiacyclohexane: ethyl acetate (1:1), as mobile phase wash-out, is collected eluent, moves in KD bottle, be evaporated near dry.
The purification of 2.4 need testing solutions
Adding ethyl acetate-acetone (1:1) mixed solution 5ml dissolves and is quantitatively transferred on graphitic carbon (500mg)-amino (500mg) mixing solid phase extraction column, with ethyl acetate-acetone (1:1) mixed solution 15ml wash-out, collect eluent, nitrogen blows near dry, residue adds acetonitrile and dissolves, and adding interior mark stock solution 5 μ l, acetonitrile is settled to 1ml, to obtain final product.
2.5 gas-matter combination analysis
Analytical conditions for gas chromatography fused-silica capillary column (30m × 0.25mm × 0.25um) DB17ms, carrier gas is high-purity helium, column flow rate 1.3ml/min; Injector temperature is 230 DEG C, sample size 1 μ l, high pressure Splitless injecting samples.
Concrete heating schedule: 60 DEG C of initial temperatures, rise to 120 DEG C with 30 DEG C/min, rise to 200 DEG C with 10 DEG C/min, rise to 230 DEG C with 2 DEG C/min, rise to 300 DEG C with 30 DEG C/min, keep 7min.
Mass spectrum (EI source) condition determination: 70eV, 230 DEG C of ion source temperatures, 250 DEG C of interface temperature.
Each product to be tested SIM condition is in table 3, and the full scan gas chromatogram of each standard sample of pesticide and 0-10 minute, 10-20 minute, a 20-40 minute scanning gas chromatogram are seen respectively shown in Fig. 1,2,3,4.
Table 3 GC-MS detects index and SIM condition determination
2.6 methodology checkings
2.6.1 precision test
The accurate rear mixed standard solution 1 μ L of dilution that draws, repeats sample introduction 5 times, calculates RSD value, in table 4.
The precision test of 64 kinds of agricultural chemicals of table 4
2.6.2 average recovery test
In sample, add respectively certain mixed standard solution, form 3 kinds of interpolation level 3 duplicate samples, extract earning and detect by method for selecting, deduction background calculates the recovery of various agricultural chemicals in sample, the results are shown in Table 5.
The average recovery test of 64 kinds of agricultural chemicals of table 5
2.6.3 detection limit is measured
Standard sample of pesticide solution dilution is become to variable concentrations, measure the limit of identification of component, the results are shown in Table 6.
The detection limit of 64 kinds of agricultural chemicals of table 6
2.6.4 linear relationship
Standard sample of pesticide solution dilution is become to variable concentrations, measure the range of linearity of component, the results are shown in Table 7.
The linear equation of 64 kinds of agricultural chemicals of table 7
2.7 determination method
Precision measures and mixes reference substance solution, the each 400 μ L of need testing solution respectively, adds analysis protectant 100 μ L, mix, and the accurate 1 μ L that draws respectively, injection gas chromatography GC-MS, measures, and calculates, and to obtain final product, and the results are shown in Table 8.
Table 8 Radix Astragali residues of pesticides background determinations (mg/kg)
3, measurement result analysis
3.1 pesticide residue determination results detect 17 kinds of different residues of pesticides in 15 batch samples, and recall rate is 100%, and Radix Astragali Pesticide Residues present situation is commonplace.Concrete residues of pesticides batch have: 3 batches of phosphamidons, 2 batches of metalaxyls, 6 batches of parathion, 2 batches of dicofols, 1 batch of Fenpropathrin, 9 batches of tetradiphons, 2 batches of lambda-cyhalothrins, 2 batches of Bravos, 2 batches of parathion-methyls, 1 batch of pendimethalin, 1 batch of fenifrothion, 1 batch of dieldrite, 1 batch of hexachloro-benzene, 1 batch of Ethodan, 2 batches of Phosalones.
It is a kind of residues of pesticides that 3.2 Milkvetch Root Pesticide Residues numbers have difference minority kind, and majority is 2～3 kinds of residues of pesticides, and indivedual kinds reach 5 kinds of residues of pesticides.Be subject to the factor impacts such as environment, planting technology, processing and storage, cause the reason more complicated of Chinese crude drug residues of pesticides.
Parathion-methyl, parathion, fenifrothion, Phosalone, phosphamidon, metalaxyl, D.D.T. (dichloro-diphenyl-trichloroethane) and dieldrite totally 8 kinds of banned pesticides in the 3.3 agricultural chemicals Radixs Astragali that ban use of, are detected.Other detect in agricultural chemicals, belong to operable pesticide, pesticide in production.
3.4 other agricultural chemicals exceed standard and adopt standard GB/T 2763-2005 Pesticide maximum residue limit regulation to analyze, exceed food residue standard: parathion exceeds standard 2～5 times, dicofol exceeds standard 11 times, tetradiphon exceeds standard 1～3 times, parathion-methyl exceeds standard 3 times, Phosalone exceeds standard 12～16 times, and phosphamidon exceeds standard 5～10 times, and D.D.T. (dichloro-diphenyl-trichloroethane) exceeds standard 6 times.
Total upper analysis, the agricultural chemicals recall rate 100% of Radix Astragali sample, detects 7 kinds of banned pesticides, and in 13 batch samples, 6 kinds of agricultural chemicals exceed standard, and sample exceeding standard rate is 86.6%.
3.5 according to this measurement result, suggestion China Chinese crude drug GAP is produced in the agricultural chemicals that bans use of, and in producing, allow to use and the agricultural chemicals that easily exceeds standard carries out limit examine.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And the apparent variation of being extended out thus or variation are still among the protection domain in the invention.
Claims (6)
1. a detection method for Milkvetch Root, is characterized in that, the method comprises the determination step of following active constituent content and the determination step of persticide residue, wherein,
A: the mensuration of Astragaloside content comprises the steps:
(1) it is appropriate that precision takes Astragaloside IV reference substance, adds methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast;
(2) accurately weighed Milkvetch Root powder 4g to be measured, put in apparatus,Soxhlet's, add methyl alcohol 40ml, cold soaking, add again methyl alcohol appropriate, add hot reflux 4 hours, extract reclaims solvent and is concentrated into dry, the residue 10ml that adds water, low-grade fever makes to dissolve, with water saturated normal butyl alcohol jolting extraction 4 times, each 40ml, merge normal butyl alcohol liquid, fully wash 2 times with ammonia solution, each 40ml, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue add water 5ml make dissolve, let cool, adsorb by D101 type large pore resin absorption column, with water 50ml wash-out, discard water liquid, use again 40% ethanol 30ml wash-out, discard eluent, continue with 70% ethanol 80ml wash-out, collect eluent, evaporate to dryness, dissolve and be transferred in 5ml measuring bottle with methyl alcohol, add methyl alcohol to scale, shake up, as need testing solution,
(3) according to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-water taking volume ratio as 32:68 is mobile phase; Evaporative light-scattering detector detects;
Accurate reference substance solution 10 μ l, the 20 μ l of drawing respectively, need testing solution 20 μ l, injection liquid chromatography, measures, and calculates with external standard two-point method logarithmic equation; Wherein, number of theoretical plate calculates and should be not less than 4000 with Astragaloside IV peak;
The mensuration of B calycosin glucoside content comprises the steps:
(1) get calycosin glucoside reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 50 μ g, product solution in contrast;
(2) accurately weighed Milkvetch Root powder 1g to be measured, precision adds methyl alcohol 50ml, weighed weight, add hot reflux 4 hours, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 25ml, reclaims solvent to dry, residue adds methyl alcohol and dissolves and be settled to 5ml, as need testing solution;
(3) according to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Taking acetonitrile as mobility A, taking 0.2% formic acid solution as Mobile phase B, carry out gradient elution according to following program: from 0-20min, mobile phase A: the volume ratio of Mobile phase B is by 20%:80% → 40%:60%; From 20-30min, mobile phase A: the volume ratio of Mobile phase B is 40%:60%; Controlling and detecting wavelength is 260nm;
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, note people liquid chromatograph, measures; Number of theoretical plate calculates and should be not less than 3000 by calycosin glucoside peak;
C: the mensuration of described persticide residue comprises the steps:
(1) to take triphenyl appropriate for precision, adds acetone and make the solution of every 1ml containing 100 μ g triphenyl, as interior mark stock solution;
Precision measures each agricultural chemicals reference substance stock solution 1ml and described interior mark stock solution 1ml respectively, adds acetone and is settled to 100ml, as mixing reference substance stock solution; Precision measures in right amount respectively, adds acetonitrile constant volume and make the solution of the variable concentrations of 20-1000ng/ml, as mixing reference substance solution;
Separately get ribose acid lactone appropriate, add acetonitrile dissolving and make the solution of every 1ml containing 20mg ribose acid lactone, separately get sorbierite appropriate, be dissolved in water and make the solution of every 1ml containing sorbierite 10mg; Precision measures above-mentioned ribose acid lactone respectively, the each 1ml of sorbitol solution mixes, and adds acetonitrile and is settled to 10ml, as analysis protectant;
(2) precision takes medicinal material fine powder 10g to be measured, and adds sodium chloride 1g to mix, and precision adds acetone 100ml, and ice-bath ultrasonic is processed 30 minutes, centrifugal, and supernatant is moved into and is equipped with in the tool plug conical flask of 1g anhydrous sodium sulfate rapidly, places 30 minutes; Precision measures above-mentioned solution 60ml and is evaporated near doing subsequently, and to add volume ratio be thiacyclohexane-ethyl acetate solution sample dissolution of 1:1 and be settled to 10ml, after filtration, getting filtrate purifies through GPC gel permeation chromatography, thiacyclohexane-ethyl acetate solution taking volume ratio as 1:1 is mobile phase wash-out, collect eluent, move in KD bottle, be evaporated near dry;
Be that ethyl acetate-acetone mixed solution 5ml of 1:1 dissolves to adding volume ratio in above-mentioned sample, and be transferred on graphitic carbon-amino mixing solid phase extraction column, ethyl acetate-acetone mixed solution 15ml wash-out taking volume ratio as 1:1, collect eluent, nitrogen blows near dry, add subsequently described interior mark stock solution 5 μ l, add acetonitrile and be settled to 1ml, as need testing solution;
(3) precision measures above-mentioned each concentration mixing reference substance solution and the each 400 μ L of need testing solution, and adds respectively described analysis protectant 100 μ L to mix, and precision is drawn 1 μ L respectively subsequently, carries out gas chromatograph-mass spectrometer (GCMS) mensuration; Wherein,
Described analytical conditions for gas chromatography is: getting specification is the fused-silica capillary column DB17ms of 30m × 0.25mm × 0.25um, taking high-purity helium as carrier gas, column flow rate 1.3ml/ minute, sample size 1 μ l, adopt high pressure Splitless injecting samples, it is 230 DEG C that injector temperature is set, and heating schedule is specially: 60 DEG C of initial temperatures, rise to 120 DEG C, rise to 200 DEG C, rise to 230 DEG C, rise to 300 DEG C with 30 DEG C/min with 2 DEG C/min with 10 DEG C/min with 30 DEG C/min, and keep 7 minutes;
The condition of described EI source mass spectroscopy is: electron energy 70eV is set, 230 DEG C of ion source temperatures, 250 DEG C of interface temperature.
2. the detection method of Milkvetch Root according to claim 1, it is characterized in that, in the mensuration of described persticide residue, the condition of described GPC gel permeation chromatography purifying step is specially: filler is Bio-Beads S-X3 200-400 order, decontaminating column is 2.5mm × 40cm, concrete wash-out parameter is for purifying impurity elimination 900s, and object is collected 1200s, and pillar cleans 300s.
3. the detection method of Milkvetch Root according to claim 1 and 2, is characterized in that, in the mensuration of described persticide residue, described agricultural chemicals reference substance comprises DDVP, acephatemet, orthene, tecnazene, hexachloro-benzene, α-benzene hexachloride, β-benzene hexachloride, γ-benzene hexachloride, δ-benzene hexachloride, flolimat, diazinon, pentachloronitrobenzene, Azodrin, Fonofos, phosphamidon I, Rogor, heptachlor, pentachloroaniline, Bravo, chlorpyrifos-methyl, drinox, captan, phosphamidon II, parathion-methyl, pirimiphos-methyl, methyl five chlorophenyl thioether, metalaxyl, triazolone, chlopyrifos, malathion, fenifrothion, parathion, pendimethalin, cis Heptachlor epoxide, trans Heptachlor epoxide, Triadimenol A, Triadimenol B, trans Niran, cis 5a,6,9,9a-hexahydro-6,9-methano-2,4, cis Niran, trans 5a,6,9,9a-hexahydro-6,9-methano-2,4, pp'-DDE, PP'-DDD, OP'-DDT, PP'-DDT, dieldrite, methidathion, endrin, Ethodan, triphenyl, Biphenthrin, 5a,6,9,9a-hexahydro-6,9-methano-2,4 sulfuric ester, iprodione, Fenpropathrin, dicofol, lambda-cyhalothrin, methoxy DDT, tetradiphon, Phosalone, Permethrin 1, Permethrin 2, cypermethrin, fenvalerate, decis.
4. according to the detection method of the arbitrary described Milkvetch Root of claim 1-3, it is characterized in that, in the mensuration of described Astragaloside IV, calycosin content, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
5. according to the detection method of the arbitrary described Milkvetch Root of claim 1-4, it is characterized in that, in the determination step of described Astragaloside content, the internal diameter of described D101 type large pore resin absorption column is 1.5cm, long 12cm.
6. according to the detection method of the arbitrary described Milkvetch Root of claim 1-5, it is characterized in that, in the mensuration of described persticide residue, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
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| CN112683612A (en) * | 2020-12-01 | 2021-04-20 | 颜妮 | Detection device and detection method for pesticide residue content of fruits |
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| CN105784894A (en) * | 2014-12-26 | 2016-07-20 | 天士力制药集团股份有限公司 | Pesticide residue detection method for traditional Chinese medicine |
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| CN104569199B (en) * | 2014-12-30 | 2016-08-24 | 上海现代中医药股份有限公司 | A kind of assay method of Radix Astragali finger printing |
| CN104597188A (en) * | 2014-12-31 | 2015-05-06 | 北京三元食品股份有限公司 | Method for detecting residual quantity of Dieldrin in dairy products |
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| CN107045030A (en) * | 2017-04-17 | 2017-08-15 | 广西壮族自治区梧州食品药品检验所 | A kind of method of calycosin glucoside in ASE methods extraction Radix Astragali |
| CN111024871A (en) * | 2019-12-06 | 2020-04-17 | 中国药科大学 | Radix astragali general cargo and goods selection grading marker, detection kit and application thereof |
| CN112683612A (en) * | 2020-12-01 | 2021-04-20 | 颜妮 | Detection device and detection method for pesticide residue content of fruits |
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