CN104198599A - Method for detecting glycyrrhiza uralensis - Google Patents
Method for detecting glycyrrhiza uralensis Download PDFInfo
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- CN104198599A CN104198599A CN201410373582.8A CN201410373582A CN104198599A CN 104198599 A CN104198599 A CN 104198599A CN 201410373582 A CN201410373582 A CN 201410373582A CN 104198599 A CN104198599 A CN 104198599A
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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 glycyrrhiza uralensis. The method comprises the steps of determining the total quantity of active ingredients of liquiritin and determining the pesticide residues. 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 licorice medicinal materials.
Background technology
Radix Glycyrrhizae is the root and rhizome of per nnial herb Radix Glycyrrhizae (Glycyrrhiza urlensis Fisch.), and nature and flavor are sweet flat, and the thoughts of returning home, lung, spleen, stomach warp, be one of medicinal material of commonly using clinically, has the laudatory title of " ten side's nine grass ".Pharmacological research show Radix Glycyrrhizae have invigorate the spleen and benefit qi, moisten the lung and relieve the cough, qualcomm meridian, sharp qi and blood, the effect that clearing heat and detoxicating, moistening lung is eliminated the phlegm in hemostasis, be widely used in clinically treating the diseases such as gastric ulcer and duodenal ulcer, abscess of throat, bronchitis, cough, arthritis, allergy and for protecting the liver, reducing blood lipid, anticancer, anti-interferon induce and strengthen cellular immunity adjusting etc.Radix Glycyrrhizae contains number of chemical composition, and principal ingredient has glycyrrhizic acid, liquiritin etc.Modern study shows that composition glycyrrhizic acid that Radix Glycyrrhizae contains and liquiritin are that Radix Glycyrrhizae has and invigorates the spleen and benefits qi, clearing heat and detoxicating, expelling phlegm and arresting coughing, relieving spasm to stop pain, the multiple pharmacologically active such as coordinating the drug actions of a prescription.There is physiological activity widely, therefore the important indicator of the number Chang Zuowei Radix Glycyrrhizae quality control of contained glycyrrhizic acid, liquiritin content.
The market demand of Radix Glycyrrhizae 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 Licorice 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 Radix Glycyrrhizae, the safety and efficacy of medication, has become the bottleneck of restriction Radix Glycyrrhizae series medicinal material exploitation.
At present, effective detection method of certified products Radix Glycyrrhizae 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 Radix Glycyrrhizae, 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 Radix Glycyrrhizae.
Summary of the invention
For this reason, technical matters to be solved by this invention is Radix Glycyrrhizae 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 Radix Glycyrrhizae.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
The invention provides the detection method of a kind of Radix Glycyrrhizae, the method comprises the assay step of following glycyrrhizic acid, liquiritin and the determination step of persticide residue, wherein:
A detection method for Radix Glycyrrhizae, is characterized in that, the method comprises the determination step of following liquiritin effective constituent total amount and the determination step of persticide residue, wherein,
A: the mensuration of described glycyrrhizic acid, liquiritin total amount comprises the steps:
(1) precision takes liquiritin reference substance, ammonium glycyrrhetate reference substance, adds 70% ethanol and makes respectively the solution of every 1ml containing liquiritin 20 μ g, ammonium glycyrrhetate 0.2mg, is reference substance solution;
(2) precision takes licorice medicinal materials powder 0.2g to be measured, and precision adds 70% ethanol 100ml, and weighed weight ultrasonic processing, let cool weighed weight again, supplies the weight of less loss with 70% ethanol, filters and get subsequent filtrate, obtains need testing solution;
(3) according to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, taking acetonitrile as mobile phase A, the phosphoric acid solution taking 0.05% is Mobile phase B, carry out gradient elution according to following program: from 0-8min, mobile phase A: the volume ratio of Mobile phase B is 19%:81%; From 8-35min, mobile phase A: the volume ratio of Mobile phase B is by 19%:81% → 50%:50%; From 35-36min, mobile phase A: the volume ratio of Mobile phase B is by 50%:50% → 100%:0; From 36-40min, mobile phase A: the volume ratio of Mobile phase B is by 100%:0 → 19%:81%; Detection wavelength is 237nm;
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures; Number of theoretical plate calculates and should be not less than 5000 by liquiritin peak;
B: 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;
Further, 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.
In the mensuration of wherein said persticide residue, the condition of described GPC gel permeation chromatography purifying step is specially: filler is Bio-Beads S-X3200-400 order, and decontaminating column is 2.5mm × 40cm, and concrete wash-out parameter is for purifying impurity elimination 900s, object is collected 1200s, and pillar cleans 300s.
Further, in the mensuration of described persticide residue, 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 steps A, in the mensuration of described glycyrrhizic acid, liquiritin content, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
Further, in step B, in the mensuration of described persticide residue, the particle diameter of described medicinal material to be measured is 180-2000 μ m.
In the determination step of described glycyrrhizic acid, liquiritin total amount, the condition of described ultrasonic processing is: power 250W, frequency 40kHz, ultrasonic processing 30 minutes.
The method of the invention, by the mensuration to the contained glycyrrhizic acid of Radix Glycyrrhizae, liquiritin content and the detection to Radix Glycyrrhizae persticide residue, is carried out the criterion of Radix Glycyrrhizae true and false quality.And by high effective liquid chromatography for measuring glycyrrhizic acid, liquiritin 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 Radix Glycyrrhizae 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 2 Pesticides standard items;
Fig. 2 is 0-10 minute scanning gas chromatogram of the embodiment of the present invention 2 Pesticides standard items;
Fig. 3 is 10-20 minute scanning gas chromatogram of the embodiment of the present invention 2 Pesticides standard items;
Fig. 4 is 20-40 minute scanning gas chromatogram of the embodiment of the present invention 2 Pesticides standard items.
Embodiment
The mensuration of embodiment 1 Radix Glycyrrhizae acid, liquiritin total amount
Medicinal material sample collection various places Radix Glycyrrhizae samples, be selected from respectively for the reagent material place of production batch: folk music Xing Zhai township, Shuan Wan township, Jinchang, Jinchang Hexibao, San Cha township, Jinchang, the life in 3 years of Minqin dam, the life in 4 years of Minqin dam, the life in 2 years of Changning, Minqin, the life in 3 years of Changning, Minqin, the life in 4 years of Changning, Minqin, Minqin Shi Feng life in 4 years, Minqin Dong Ba life in 3 years, quintar life in 1 year, quintar life in 2 years, quintar life in 3 years, 3 batches of quintars, be labeled as respectively 1-3, Jingtai, Dunhuang life in 3 years, respectful south, folk music, Changning, Minqin, Qingyang, Zhuanglang, Lanzhou, Gu Lang, the Beishan Mountain, Yuzhong, the Beishan Mountain, Yuzhong, Yongdeng Qin Wangchuan, clear water, Dangchang, Minxian County, vegetable seeds town, the west of Gansu Province, Dingxi, long-pending tor, Huan County, Yongjing, Lintao.
2. the research of this project of method adopts HPLC gradient wavelength method to measure the content of Radix Glycyrrhizae acid, liquiritin effective constituent simultaneously, and concrete steps are as follows:
Precision takes liquiritin reference substance, ammonium glycyrrhetate reference substance, adds 70% ethanol and makes respectively the solution of every 1ml containing liquiritin 20 μ g, ammonium glycyrrhetate 0.2mg, is reference substance solution;
Precision takes licorice medicinal materials powder 0.2g to be measured, and precision adds 70% ethanol 100ml, and weighed weight ultrasonic processing, let cool weighed weight again, supplies the weight of less loss with 70% ethanol, filters and get subsequent filtrate, obtains need testing solution;
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, taking acetonitrile as mobile phase A, the phosphoric acid solution taking 0.05% is Mobile phase B, carry out gradient elution according to following program: from 0-8min, mobile phase A: the volume ratio of Mobile phase B is 19%:81%; From 8-35min, mobile phase A: the volume ratio of Mobile phase B is by 19%:81% → 50%:50%; From 35-36min, mobile phase A: the volume ratio of Mobile phase B is by 50%:50% → 100%:0; From 36-40min, mobile phase A: the volume ratio of Mobile phase B is by 100%:0 → 19%:81%; Detection wavelength is 237nm;
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures; Number of theoretical plate calculates and should be not less than 5000 by liquiritin peak;
3. result is measured 42 batches, and concrete outcome is as follows in table 1.
Table 1 Radix Glycyrrhizae specification and assay (%)
Glycyrrhizic acid, liquiritin content limit are drafted: regulation of Chinese Pharmacopoeia version in 2010, Radix Glycyrrhizae acid content must not be less than 2.0%, liquiritin content must not be less than 0.50%, by this limit, in above-mentioned 42 batch samples, liquiritin content is all qualified, glycyrrhizic acid content have 2 batches defective, disqualification rate 5%.
In recent years, the quality of licorice medicinal materials increases, and considers the genunie medicinal materials standard in market, Gansu, drafts containing liquiritin (C
21h
22o
9) must not be less than 1.0%, glycyrrhizic acid (C
42h
62o
16) must not be less than 2.5%, by this regulation, in 13 batch samples, glycyrrhizic acid does not reach requirement, and 5 batch sample liquiritins do not reach requirement, account for respectively 35.1% and 13.5%.
Embodiment 2 Radix Glycyrrhizae residues of pesticides quantitative determinations
1 instrument and reagent
1.1 instrument Clarus600 gas chromatograph-mass spectrometer (GCMS)s (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 stock solutions such as decis are provided by 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 radix bupleuri is bought in the place of production or is purchased from medicinal material market, 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-X3200--400 order, decontaminating column 2.5mm × 40cm, wash-out parameter: pre run 900s, main run 1200s, tail run 300s), taking thiacyclohexane: ethyl acetate (1:1) is as mobile phase wash-out, collect eluent, move 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 2, 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 2 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 3.
The precision test of 64 kinds of agricultural chemicals of table 3
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 4.
The average recovery test of 64 kinds of agricultural chemicals of table 4
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 5.
The detection limit of 64 kinds of agricultural chemicals of table 5
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 6.
The linear equation of 64 kinds of agricultural chemicals of table 6
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 7.
Table 7 Radix Glycyrrhizae Detecting Pesticide report the test (mg/kg)
Table 7 Radix Glycyrrhizae Detecting Pesticide report the test (mg/kg) continued
3, measurement result analysis
3.19 batches of Radix Glycyrrhizae samples detect 24 kinds of different agricultural chemicals, and agricultural chemicals recall rate 86.2% wherein detects 5 kinds of banned pesticides, organochlorine 2 samples that exceed standard, exceeding standard rate 6.8%; The agricultural chemicals specifying by food and residual measurement, other 7 kinds of agricultural chemicals exceed standard, 14 batches, sample, exceeding standard rate is 48.2%.In conjunction with above-mentioned two kinds of agricultural chemicals, total exceeding standard rate 55%, Radix Glycyrrhizae Pesticide Residues present situation is commonplace.
During 3.2 agricultural chemicals service condition China of China Chinese crude drug GAP produce, the agricultural chemicals banning use of has 54 kinds of 13 classes.Fonofos, phosphamidon, acephatemet, D.D.T. (dichloro-diphenyl-trichloroethane), benzene hexachloride totally 5 kinds of banned pesticides in Radix Glycyrrhizae, are detected.Other detect in agricultural chemicals, and orthene, chlorpyrifos-methyl, lambda-cyhalothrin, Fenpropathrin, metalaxyl etc. belong to operable agricultural chemicals in production, but food standard is carried out running water.
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 Radix Glycyrrhizae, is characterized in that, the method comprises following glycyrrhizic acid, the determination step of liquiritin effective constituent total amount and the determination step of persticide residue, wherein,
A: the mensuration of described glycyrrhizic acid, liquiritin total amount comprises the steps:
(1) precision takes liquiritin reference substance, ammonium glycyrrhetate reference substance, adds 70% ethanol and makes respectively the solution of every 1ml containing liquiritin 20 μ g, ammonium glycyrrhetate 0.2mg, is reference substance solution;
(2) precision takes licorice medicinal materials powder 0.2g to be measured, and precision adds 70% ethanol 100 ml, and weighed weight ultrasonic processing, let cool weighed weight again, supplies the weight of less loss with 70% ethanol, filters and get subsequent filtrate, obtains need testing solution;
(3) according to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, taking acetonitrile as mobile phase A, the phosphoric acid solution taking 0.05% is Mobile phase B, carry out gradient elution according to following program: from 0-8min, mobile phase A: the volume ratio of Mobile phase B is 19%:81%; From 8-35min, mobile phase A: the volume ratio of Mobile phase B is by 19%:81% → 50%:50%; From 35-36min, mobile phase A: the volume ratio of Mobile phase B is by 50%:50% → 100%:0; From 36-40min, mobile phase A: the volume ratio of Mobile phase B is by 100%:0 → 19%:81%; Detection wavelength is 237nm;
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures; Number of theoretical plate calculates and should be not less than 5000 by liquiritin peak;
B: 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 Radix Glycyrrhizae 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-X3200-400 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 Radix Glycyrrhizae 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 Radix Glycyrrhizae of claim 1-3, it is characterized in that, in the mensuration of described glycyrrhizic acid, liquiritin 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 Radix Glycyrrhizae of claim 1-4, 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.
6. according to the detection method of the arbitrary described Radix Glycyrrhizae of claim 1-4, it is characterized in that, in the determination step of described glycyrrhizic acid, liquiritin total amount, the condition of described ultrasonic processing is: power 250W, frequency 40kHz, ultrasonic processing 30 minutes.
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Application publication date: 20141210 |