CN104458931B - A kind of detection method of chromatogram of Radix Isatidis - Google Patents

A kind of detection method of chromatogram of Radix Isatidis Download PDF

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CN104458931B
CN104458931B CN201410374924.8A CN201410374924A CN104458931B CN 104458931 B CN104458931 B CN 104458931B CN 201410374924 A CN201410374924 A CN 201410374924A CN 104458931 B CN104458931 B CN 104458931B
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precisely
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isatis root
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CN104458931A (en
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宋平顺
李登鹏
陈杰
韩斌
李波
赵建邦
李士博
李冬华
张明童
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GANSU ZHONGTIAN PHARMACEUTICAL Co Ltd
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Abstract

The invention belongs to Chinese crude drug quality testing field, be specifically related to a kind of quality determining method of Radix Isatidis.The method comprises the mensuration of the uridine contained by Radix Isatidis, guanosine, adenosine and total acid content and the mensuration to Radix Isatidis persticide residue.The invention solves the complicated operation of the discrimination method of traditional physicochemical property and the technical matters of limitation and difficult quality regulation and control, high to provide a kind of accuracy, fast easy and simple to handle, detection method with low cost, has a good application prospect and economic benefit.

Description

Detection method of radix isatidis medicinal material
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 an isatis root medicinal material.
Background
Radix Isatidis, also called indigo root, indigo root or isatis root, is a Chinese medicinal material. All the places in China. The radix Isatidis is divided into radix Isatidis and radix Isatidis, and the radix Isatidis is derived from root of Isatis tinctoria and radix Isatidis of Brassicaceae; radix Isatidis is rhizome and root of herba Kalimeridis of Acanthaceae. Has effects of clearing away heat and toxic materials, cooling blood, relieving swelling, and relieving sore throat. Radix isatidis is a medicine which is extremely large in dosage and cannot be sold for a long time, and the sales of radix isatidis can be seen to increase rapidly in the history of the radix isatidis. The isatis root contains various chemical components, mainly contains uridine, guanosine, adenosine, total acid and the like. Modern researches show that the components contained in the isatis root have wide physiological activity, and the content of the components is closely related to the effects of clearing heat, removing toxicity and the like of the isatis root, so that the content of the components is often used as an important index for controlling the quality of the isatis root medicinal material.
In recent years, the market demand of the radix isatidis is continuously increased, wild resources are reduced year by year, and the artificial cultivation is slow in development, so that the storage amount and the yield of the radix isatidis 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 surface, bringing great impact to the quality, the safety and the effectiveness of the radix isatidis medicinal materials, and becoming the bottleneck restricting the development of radix isatidis series medicinal materials.
At present, the effective detection method of the genuine radix isatidis is the traditional identification method of morphological characteristics and physicochemical characteristics, has certain limitations, is not enough to control the quality of the radix isatidis on the whole, and as a common clinical traditional Chinese medicine, the authenticity and quality of the radix isatidis are influenced by a plurality of factors such as physicochemical indexes, large pesticide residue and the like, so that a scientific quality detection method is necessary to be established for evaluating the quality of the radix isatidis.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to solve the problems of complex detection method of the radix isatidis medicinal material and difficult quality control in the prior art, and further provide a method for simply and quickly detecting the radix isatidis 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 detection method of an isatis root medicinal material, which comprises the following steps of measuring the content of active ingredients and measuring the pesticide residue, wherein the steps of:
a: the method for measuring the content of uridine, guanosine and adenosine comprises the following steps:
(1) precisely weighing 3.56mg of guanosine control, 4.60mg of uridine control and 4.49mg of adenosine control respectively, and adding methanol to prepare mixed control solutions containing 22.78 μ g, 29.40 μ g and 28.74 μ g respectively per 1 mL;
(2) accurately weighing 0.5g of isatis root medicinal material powder, accurately adding 25mL of water, and carrying out ultrasonic treatment for 40 minutes; filtering the supernatant with 0.45 μm filter membrane, and collecting the filtrate to obtain sample solution;
(3) performing high performance liquid chromatography test by using an octadecylsilane chemically bonded silica chromatographic column, acetonitrile as a mobile phase A and water as a mobile phase B, and performing gradient elution according to the following procedures: 2-20min, A: b is 2%: 98% → 40%: 60 percent; the column temperature is controlled to be 30 ℃, the flow rate is 0.6mL/min, and the detection wavelengths are respectively as follows: 0min, 254 nm; 7min, 262 nm; 8min, 246 nm; for 10min, 259 nm; 12min, 254 nm;
respectively taking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;
b: the determination of the total acid content comprises the following steps:
(1) accurately weighing 5g of radix isatidis to be tested, adding 70% ethanol, placing the mixture on a water bath, heating, refluxing and extracting for 3 times, wherein 50ml of the mixture is obtained each time, and refluxing for 1.5 hours each time, filtering, concentrating the filtrate to the relative density of 1.05 at the temperature of 60 ℃, adjusting the pH value to 2-3 with concentrated hydrochloric acid, extracting with ethyl acetate, recovering the ethyl acetate under reduced pressure until the ethyl acetate is dry, adding 70% ethanol, dissolving and fixing the volume to 25ml, and obtaining a sample solution;
(2) the total organic acid is calculated by salicylic acid, and the content of the total organic acid is determined by a direct titration method;
c: 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 ℃.
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 meshes, the purification column is 2.5mm multiplied by 40cm, and the specific elution parameters are as follows: the impurities are removed by purification for 900s, the target is collected for 1200s, and the column is washed for 300 s.
Further, 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, pentachlorophenyl, monocrotophos, fonofos, phosphamidol I, dimethoate, heptachlor, pentachloroaniline, chlorothalonil, chlorpyrifos-methyl, aldrin, captan, phosphamidol II, methyl parathion, methyl pyrimidylphenyl sulfide, metalaxyl, triadimefon, chlorpyrifos, malathion, fenitrothion, parathion, pendimethalin, cis-epoxy heptachlor, trans-epoxy heptachlor, triadimenol A, triadimenol B, trans-chlordane, cis-endosulfan, 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 B, the total acid content is determined by the following specific steps:
taking 5ml of a sample solution, placing the sample solution in a triangular flask, adding 2 drops of phenolphthalein indicator, titrating the solution by using a calibrated 0.1mol/L sodium hydroxide titration solution until the solution is pink, and taking the titration solution as an end point, wherein each 1ml of the sodium hydroxide titration solution is equivalent to 14.088mg of salicylic acid.
In the step A, in the determination of the contents of isatis root methyl glycoside, calycosin, formononetin, isatis root saponin II and isatis root saponin III, the particle size of the medicinal material to be determined is 180-2000 mu m.
In the step C, in the determination of the pesticide residue, the particle size of the medicinal material to be detected is 180-2000 μm.
The method of the invention determines the authenticity and quality of the radix isatidis medicinal material by measuring the content of the effective components of the radix isatidis medicinal material and detecting the pesticide residue of the radix isatidis medicinal material. And the content of the effective components of the isatis root is 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 radix isatidis 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 an HPLC chromatogram of a control of example 1 of the present invention (1-guanosine, 2-uridine, 3-adenosine);
FIG. 2 is an HPLC chromatogram of a sample of example 1 of the present invention (1-guanosine, 2-uridine, 3-adenosine);
FIG. 3 is a full scan gas chromatogram of a pesticide standard in example 3 of the present invention;
FIG. 4 is a 0-10 minute scanning gas chromatogram of the pesticide standard in example 3 of the present invention;
FIG. 5 is a 10-20 minute scanning gas chromatogram of the pesticide standard in example 3 of the present invention;
FIG. 6 is a 20-40 minute scanning gas chromatogram of the pesticide standard in example 3 of the present invention.
Detailed Description
Example 1 determination of contents of Isatis root methyl glycoside, Calycosin, Formononetin, Isatis root saponin II, and Isatis root saponin III in Isatis root medicinal materials
1. Instrument and reagent
1.1 instruments
waters2695-2998 HPLC, ultrasonic cleaner (250W, 40 kHz).
1.2 reagent
Guanosine control (A0778, purity 98.4%, Tianjin Mark biology Co., Ltd.), uridine control (887-.
The methanol and the acetonitrile are chromatographically pure, the water is purified water, and the ethanol is analytically pure.
1.3 samples of medicinal materials
Collecting radix Isatidis samples of different regions, wherein the production region of the test medicinal materials is selected from: the batches of Gansu Shouyang 2 are respectively marked as 1-2 batches, the batches of Gansu Shouyang Dongfu, Gansu Weiyuanchuan Chuan, Gansu Lin 27950 and Gansu Weiyuan 2 are respectively marked as 1-2 batches and Weiyuan Lotus peak blossom slope village 3 batches, and are respectively marked as 1-3 batches and Gansu commodities.
2. Method of producing a composite material
2.1 preparation of control solutions
Precisely weighing guanosine control sample 3.56mg, uridine control sample 4.60mg and adenosine control sample 4.49mg, respectively, adding methanol to obtain mixed control solutions containing 22.78 μ g, 29.40 μ g and 28.74 μ g, respectively, per 1mL, to obtain control solutions.
2.2 preparation of test solutions
Pulverizing radix Isatidis (sieving with a fourth sieve) about 0.5g, precisely weighing, precisely adding 25mL water, and performing ultrasonic treatment for 40 min. Filtering the supernatant with 0.45 μm filter membrane, and collecting the filtrate to obtain the sample solution.
2.3 chromatographic conditions
Octadecylsilane chemically bonded silica is used as a filling agent; gradient elution was performed according to the following table using acetonitrile (a) -water (B) as mobile phase (see table 1); the detection wavelength adopts multi-wavelength detection (see table 2); the column temperature is 30 ℃; the flow rate was 0.6 mL/min.
TABLE 1 assay elution gradient
TABLE 2 wavelength of content determination
2.4 reference Linear relationship examination
Precisely sucking 2, 5, 10, 15, 20 and 25 μ l of the reference substance solution, injecting into a liquid chromatograph, recording chromatogram, performing linear regression with the sample injection mass number (x) of each reference substance as abscissa and peak area as ordinate (y), and obtaining HPLC chromatogram of the reference substance solution as shown in figure 1.
Guanosine, wherein y is 7E-06x-0.0033, r is 0.9995, and the linear range is 45.56-569.5 mu g;
uridine, y is 1E-06x +0.0326, r is 0.9999, and the linear range is 58.8-735 mu g;
and the linear range of the adenosine is 57.5-713.4 mu g, wherein y is 5E-07x +0.0221, and r is 0.9999.
2.5 precision investigation
A sample of Isatis root No. 3 was prepared as described under item 2.2, and the sample was continuously injected 5 times, so that the RSD of the peak areas of guanosine, uridine and adenosine injected each time were 0.8%, 0.5% and 0.3%, respectively, and the HPLC chromatogram of the sample is shown in FIG. 2.
2.6 reproducibility examination
The sample preparation No. 3 isatis root sample was prepared in 5 parts of the test solution by the method under item 2.2, and the RSD of the peak areas of guanosine, uridine and adenosine in the 5 parts of sample were 2.9%, 3.6% and 2.3%, respectively, as a result of separate sample injection.
2.7 stability Studies
A test sample solution of Isatis indigotica fort was prepared according to the method under item 2.2, and the peak areas of guanosine, uridine and adenosine were measured at 0, 2, 4, 6, 8, 10, 12, and 16 hours, respectively, and the RSD of the peak areas of guanosine, uridine, and adenosine were 2.6%, 2.8%, and 3.8%, respectively.
2.8 sample recovery test
5 parts of a sample of the same lot, 0.25g (containing 0.1065% of guanosine, 0.0821% of uridine and 0.0502% of adenosine), of which the content is known, were precisely weighed, and 1mL (0.2561mg/mL) of guanosine, 2mL (0.1146mg/mL) of uridine and 1mL (0.1365mg/mL) of adenosine, which were control stock solutions, respectively, were precisely added, and prepared according to the method for preparing the test sample solution, and the average recovery rate was measured and calculated. 102.37%, 103.06% and 102.46%, respectively, and RSD 0.59%, 2.33% and 2.24%, respectively.
2.9 assay
Respectively sucking 10 μ l of the test solution, injecting into a liquid chromatograph, measuring, and recording chromatogram.
3. Determination of content limits
3.1 the methodology investigation of the method for simultaneously determining guanosine, uridine and adenosine established shows that the method is accurate, sensitive, simple, convenient and rapid, and can be used for quality control of isatis root medicinal materials, decoction pieces and preparations thereof.
3.2 Limit the total amount of guanosine, uridine and adenosine in the 14 samples tested was between 0.086% and 0.465%, averaged at 0.211%, and the sum of the amounts of guanosine, uridine and adenosine must not be less than 0.15% when 2 samples were rejected.
4. The results are shown in Table 3 below.
TABLE 3 Isatis root sample sources, specifications and assay (%)
Example 2 determination of Total acid content of Isatis root
Collecting radix Isatidis samples of different regions, wherein the production region of the test medicinal materials is selected from: the batches of Gansu Shouyang 2 are respectively marked as 1-2 batches, the batches of Gansu Shouyang Dongfu, Gansu Weiyuanchuan Chuan, Gansu Lin 27950 and Gansu Weiyuan 2 are respectively marked as 1-2 batches and Weiyuan Lotus peak blossom slope village 3 batches, and are respectively marked as 1-3 batches and Gansu commodities.
Precisely weighing 5g of isatis root, placing the isatis root into a triangular flask, adding 70% ethanol, placing the mixture on a water bath, heating and refluxing for 3 times, 50ml each time and 1.5 hours each time, filtering, concentrating the filtrate to about relative density of 1.05(60 ℃), adjusting the pH value to 2-3 with concentrated hydrochloric acid, extracting for 3 times with ethyl acetate, recovering the ethyl acetate under reduced pressure until the ethyl acetate is dry, adding 70% ethanol, dissolving and dissolving in a 25ml volumetric flask, and obtaining the isatis root extract.
The total organic acid is calculated by salicylic acid, and the content of the total organic acid is determined by a direct titration method. Taking 5ml of a test solution, putting 2 drops of phenolphthalein indicator into the test solution, titrating with a calibrated sodium hydroxide titration solution (0.1mol/L), taking an end point when the solution is pink, wherein each 1ml of the sodium hydroxide titration solution is equivalent to 14.088mg of salicylic acid, and the measurement results are shown in Table 4.
TABLE 4 Isatis root sample origin, Specification and assay (%)
5. The method for simultaneously measuring the contents of 6 components, namely cinnamic acid, salicylic acid, syringic acid, benzoic acid and caffeic acid, by using the efficient capillary electrophoresis method established in the project for setting the total acid limit is characterized in that the contents are trace and cannot be quantitatively analyzed, and the total organic acid component contents are adopted for limit control. The total organic acid content of 18 samples is measured to be 2.09-48.6%, the average is 11.28%, only 5 samples are measured when the organic acid content is more than 10.0%, the content is not lower than 3.0%, and 2 samples are not qualified.
Example 3 determination of pesticide residue amount of Isatis root medicinal Material
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 indigowoad root and other genuine batch samples purchased from the production place or the Lanzhou Huanghe 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: the ethyl acetate (1:1) solution dissolves the sample and quantitatively dilutes it to 10ml, mixes it well, filters it, purifies the filtrate by Gel Permeation Chromatography (GPC), 5ml is injected, (GPC main parameters: packing Bio-Beads S-X3200-400 mesh, purifying column 2.5mm × 40cm, elution parameters: prerun900s, mainrun1200s, tailrun300s), and the ratio of 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 5, 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 5GC-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 6.
Precision test of Table 664 pesticides
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 down and detecting according to a selected method, deducting the background to calculate the recovery rate of various pesticides in the samples, and the result is shown in Table 7.
Sample application recovery test for 764 pesticides in table
2.6.3 detection Limit determination
The pesticide standard solutions were diluted to different concentrations and the minimum amounts of the components were determined, the results are shown in table 8.
Table 864 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 9.
TABLE 964 Linear equation 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 100 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 tables 10 and 11.
TABLE 10 background determination of pesticide residue of Isatis root (mg/kg)
TABLE 10 background determination (mg/kg) of pesticide residue of radix Isatidis
TABLE 11 report of pesticide residue detection of Isatis root (mg/kg)
3. Analysis of measurement results
3.1 results of pesticide residue measurement 17 different pesticide residues were detected in 15 batches of samples, the detection rate was 100%, and the current situation of pesticide residue in radix Isatidis was relatively common. The pesticide residue batches include 3 batches of phosphamidon, 2 batches of metalaxyl, 6 batches of parathion, 2 batches of dicofol, 1 batch of fenpropathrin, 9 batches of dicofol, 2 batches of cyhalothrin, 2 batches of chlorothalonil, 2 batches of methyl parathion, 1 batch of pendimethalin, 1 batch of fenitrothion, 1 batch of dieldrin, 1 batch of hexachlorobenzene, 1 batch of ethion and 2 batches of fluthion.
3.2 the pesticide residue number in the isatis root medicinal material is different, the few varieties are 1 pesticide residue, the majority is 2-3 pesticide residues, and the pesticide residue of each individual variety reaches 5 pesticide residues. 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 8 forbidden pesticides including methyl parathion, fenitrothion, thiothion, phosphamidon, metalaxyl, dichlorodiphenyl trichloroethane and Dieldrin are detected from the banned pesticide radix isatidis. Other pesticides to be detected belong to insecticides and pesticide pesticides which can be used in production.
3.4 the other pesticide superscripts are analyzed by adopting the maximum pesticide residue limit regulation in the food of the national standard GB2763-2005, and the pesticide superscripts exceed the food residue standard, namely that parathion exceeds 2-5 times, dicofol exceeds 11 times, dicofol exceeds 1-3 times, methyl parathion exceeds 3 times, fluoxathion exceeds 12-16 times, phosphamidon exceeds 5-10 times, and DDT exceeds 6 times.
In the total analysis, the detection rate of the pesticides of the isatis root sample is 100%, 7 forbidden pesticides are detected, 6 pesticides in 13 batches of samples exceed the standard, and the sample exceeding rate is 86.6%.
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.
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 (4)

1. A method for detecting isatis root medicinal material is characterized by comprising the following steps of measuring the content of active ingredients, measuring the pesticide residue and measuring the pesticide residue, wherein,
a: the method for measuring the content of uridine, guanosine and adenosine comprises the following steps:
(1) precisely weighing 3.56mg of guanosine control, 4.60mg of uridine control and 4.49mg of adenosine control respectively, and adding methanol to prepare mixed control solutions containing 22.78 μ g, 29.40 μ g and 28.74 μ g respectively per 1 mL;
(2) accurately weighing 0.5g of isatis root medicinal material powder, accurately adding 25mL of water, and carrying out ultrasonic treatment for 40 minutes; filtering the supernatant with 0.45 μm filter membrane, and collecting the filtrate to obtain sample solution;
(3) performing high performance liquid chromatography test by using an octadecylsilane chemically bonded silica chromatographic column, acetonitrile as a mobile phase A and water as a mobile phase B, and performing gradient elution according to the following procedures: 0-20min, A: b is 2%: 98% → 40%: 60 percent; the column temperature is controlled to be 30 ℃, the flow rate is 0.6mL/min, and the detection wavelengths are respectively as follows: 0min, 254 nm; 7min, 262 nm; 8min, 246 nm; for 10min, 259 nm; 12min, 254 nm;
respectively taking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;
b: the determination of the total acid content comprises the following steps:
(1) accurately weighing 5g of radix isatidis to be tested, adding 70% ethanol, placing the mixture on a water bath, heating, refluxing and extracting for 3 times, wherein 50ml of the mixture is obtained each time, and refluxing for 1.5 hours each time, filtering, concentrating the filtrate to the relative density of 1.05 at the temperature of 60 ℃, adjusting the pH value to 2-3 with concentrated hydrochloric acid, extracting with ethyl acetate, recovering the ethyl acetate under reduced pressure until the ethyl acetate is dry, adding 70% ethanol, dissolving and fixing the volume to 25ml, and obtaining a sample solution;
(2) the total organic acid is calculated by salicylic acid, and the content of the total organic acid is determined by a direct titration method;
c: 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;
the GPC gel permeation chromatography purification step conditions are specifically as follows: the filler is Bio-Beads S-X3200-400 meshes, the purification column is 2.5mm multiplied by 40cm, and the specific elution parameters are as follows: purifying and removing impurities for 900s, collecting the target for 1200s, and cleaning the column for 300 s;
(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: setting electron energy at 70eV, ion source temperature at 230 ℃ and interface temperature at 250 ℃;
the pesticide reference substance comprises dichlorvos, methamidophos, acephate, tetrachloronitrobenzene, hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, gamma-hexachlorocyclohexane, omethoate, diazinon, pentachloronitrobenzene, monocrotophos, chlorfenapyr, phosphamidol I, dimethoate, heptachlor, pentachloranilide, chlorothalonil, chlorpyrifos-methyl, aldrin, captan, phosphamidol II, methyl parathion, pirimiphos-methyl, pentachlorophenyl thioether-methyl, metalaxyl, triadimefon, chlorpyrifos, malathion, fenitrothion, parathion, pendimethalin, cis epoxy heptachlor, trans epoxy heptachlor, triadimenol A, triadimenol B, trans-chlordane, cis-chlordane, trans-endosulfan, PP '-DDE, PP' -DDD, OP '-DDT, PP' -DDT, Dieldrin, methidathion, chlorpyrifos, chlorpyri, Endrin, ethion, triphenyl phosphate, bifenthrin, endosulfate, iprodione, fenpropathrin, dicofol, cyhalothrin, methoxychlor-DDT, dicofol, vofenthion, permethrin 1, permethrin 2, cypermethrin, fenvalerate and deltamethrin.
2. The method for detecting the isatis root medicinal material according to claim 1, wherein the determination of the total acid content specifically comprises the following steps:
taking 5ml of a sample solution, placing the sample solution in a triangular flask, adding 2 drops of phenolphthalein indicator, titrating the solution by using a calibrated 0.1mol/L sodium hydroxide titration solution until the solution is pink, and taking the titration solution as an end point, wherein each 1ml of the sodium hydroxide titration solution is equivalent to 14.088mg of salicylic acid.
3. The method for detecting isatis root according to claim 2, wherein in the determination of the content of calycosin, formononetin, isatis root saponin II and isatis root saponin III, the particle size of the medicinal material to be detected is 180-2000 μm.
4. The method for detecting isatis root drug according to any one of claims 1-3, wherein in the determination of the pesticide residue, the particle size of the drug to be detected is 180-2000 μm.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1591007A (en) * 2004-02-19 2005-03-09 广州白云山制药股份有限公司广州白云山中药厂 Finger print allas used for controlling isatidis root, its preparation quality and medicine effect and its making method
CN102288666A (en) * 2010-08-04 2011-12-21 年四辉 Method for measuring content of total organic acid
CN102331467A (en) * 2011-07-28 2012-01-25 江西普正制药有限公司 Method for detecting quality of south isatis root granules
CN102507832A (en) * 2011-10-26 2012-06-20 上海中医药大学 Method for measuring high performance liquid chromatography characteristic fingerprint of isatis root and isatis root preparation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1591007A (en) * 2004-02-19 2005-03-09 广州白云山制药股份有限公司广州白云山中药厂 Finger print allas used for controlling isatidis root, its preparation quality and medicine effect and its making method
CN102288666A (en) * 2010-08-04 2011-12-21 年四辉 Method for measuring content of total organic acid
CN102331467A (en) * 2011-07-28 2012-01-25 江西普正制药有限公司 Method for detecting quality of south isatis root granules
CN102507832A (en) * 2011-10-26 2012-06-20 上海中医药大学 Method for measuring high performance liquid chromatography characteristic fingerprint of isatis root and isatis root preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HPLC 波长切换技术同时测定板蓝根中4种活性成分含量;徐桂花 等;《中国实验方剂学杂志》;20120930;第18卷(第18期);70-73 *
中药材板蓝根中20种农药的多残留快速分析方法;高倩 等;《安徽农业大学学报》;20100131;第37卷(第1期);97-102 *
板蓝根提取物中总有机酸和水杨酸含量测定方法研究;马莉 等;《中国中药杂志》;20060531;第31卷(第10期);804-806 *

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