CN105784894B - Pesticide residue detection method for traditional Chinese medicine - Google Patents
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 abstract 1
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Abstract
The invention relates to a method for detecting pesticide residue in modern Chinese medicinal preparations, which establishes a GC-MS/MS detection method for detecting 103 pesticide residues comprising organic chlorine, organic phosphorus, dicarboximide, pyrethroid and the like, adopts ethyl acetate as an extraction solvent in a test, purifies an amino column, and determines by adding a standard solution to a Chinese medicinal substrate, and provides an effective and accurate quality control method for the safety of Chinese medicaments.
Description
The technical field is as follows:
the invention relates to a method for detecting pesticide residues in a medicine product, in particular to a method for detecting various pesticide residues in a modern Chinese medicine preparation.
Background art:
the safety problem of the traditional Chinese medicine frequently occurs in recent years, which brings great challenge to the traditional Chinese medicine which is always approved to be safe and effective. In addition, the excessive publicity and misleading of some media to the safety problem of traditional Chinese medicine make people pay attention to the safety of traditional Chinese medicine to an unprecedented extent, and the view of 'the world people do not doubt the efficacy of traditional Chinese medicine, but doubting the safety of traditional Chinese medicine' becomes the bottleneck restricting the development of traditional Chinese medicine. Also is the bottleneck restricting the internationalization of traditional Chinese medicine. Wherein pesticide residue is an important factor influencing the safety of traditional Chinese medicines.
So-called pesticide residue: refers to pesticide parent substance and special derivatives with toxic and pathological significance, such as degradation or transformation products, metabolites, industrial impurities and the like, which are remained in medicinal parts under the influence of growth environment or after pesticides are used in the processes of planting, processing and storing traditional Chinese medicines.
Traditional Chinese medicinal materials are easy to contact pesticides in the planting, production and processing processes, and the national registration system for pesticide application in the traditional Chinese medicinal materials is not complete, so that pesticide residues possibly existing in the traditional Chinese medicinal materials cannot be determined; therefore, unknown trace pesticide residues exist in the traditional Chinese medicine compound preparation prepared by extracting traditional Chinese medicinal materials. The refined traditional Chinese medicine compound preparation matrix contains a large amount of micromolecular substances, is complex and interferes the determination of trace pesticide residues. The invention provides a method for detecting trace pesticide residues in traditional Chinese medicines on the basis of the prior art.
The invention content is as follows:
the invention provides a method for measuring pesticide residues in traditional Chinese medicines, which adopts GC-MS/MS (gas chromatography-mass spectrometry), namely a triple quadrupole gas-mass spectrometry combined detection technology, to detect the pesticide residues in the traditional Chinese medicines, and comprises the following steps:
step 2, purifying the extracting solution,
step 3, injecting the purified solution into a GC-MS/MS chromatograph to obtain a chromatogram,
and 4, taking the matrix and the standard solution as a standard curve to eliminate the matrix effect, and calculating according to the chromatogram to obtain the content of pesticide residue in the traditional Chinese medicine.
Wherein the extraction in the step 1 comprises the following steps: pulverizing Chinese medicinal materials, sieving, collecting 1.0-3.0g of pulverized sample, precisely weighing, adding 5-15mL ethyl acetate, extracting for 20-40min, centrifuging for 2-4min, collecting supernatant, volatilizing to near dry, dissolving with 1-3mL eluate, and purifying.
Wherein the leacheate is selected from the following solutions:
dichloromethane to methanol at 100: 1 (volume ratio);
acetonitrile: toluene ═ 3: 1 (volume ratio);
toluene, and the like.
Wherein the extraction is selected from shaking extraction or ultrasonic extraction.
The volatile near-dry is selected from water bath rotary evaporation to near-dry or nitrogen blowing to near-dry.
The preferred extraction method is as follows: taking traditional Chinese medicines, crushing the traditional Chinese medicines, sieving the traditional Chinese medicines by a No. 3 sieve, taking 2.0g of the crushed samples, precisely weighing, adding 10mL of ethyl acetate into a 50mL polypropylene plastic centrifuge tube, shaking and extracting for 30min (135r/min), centrifuging for 3min at 5000r/min, taking 5mL of supernate into a 150mL chicken heart bottle, carrying out rotary evaporation in a water bath at 40 ℃ until the supernate is nearly dry, dissolving 2mL of leacheate (V (dichloromethane): V (methanol): 100: 1), and waiting for purification.
Wherein the purification in the step 2 comprises the following steps: before the amino column is loaded, pre-washing the column with 2-4mL of eluent, when the liquid level of the pre-washing liquid reaches the top of the column packing, quickly moving the sample solution into the column, and receiving the eluent by using a flat-bottomed flask; washing the heart-shaped bottle with 10-20ml of total leacheate for 2-4 times, transferring the leacheate into the column when the liquid level of the last leacheate is close to the top of the column packing, collecting all the effluent, performing rotary concentration in a water bath at 35-45 ℃ until the effluent is nearly dry, adding 1-3ml of acetone or acetonitrile, mixing uniformly, and performing gas chromatography-mass spectrometry.
The preferred purification method is: before loading the amino column, pre-washing the column with 3mL of eluent, quickly moving the sample solution into the column when the liquid level of the pre-washing solution reaches the top of the column packing, and receiving the eluent by using a 150mL flat-bottomed flask; washing the chicken heart bottle with 3ml, 5ml and 5ml of leacheate for 3 times, transferring the leacheate into the column when the liquid level of the last leacheate is close to the top of the column packing, and collecting all effluent. Concentrating in 40 deg.C water bath until it is nearly dry, adding 1ml acetone, and mixing.
Wherein the leacheate is the same as the leacheate in the step 1.
Wherein the chromatographic conditions of step 3 are as follows:
gas chromatography: the chromatographic column is selected from: rtx-5MS, HP-5MS or DB-5MS columns. The initial temperature is 50-70 deg.C, and the holding time is 0-5 min; the temperature rise rate of the first stage is in the range of 15-35 ℃ min-1Heating to 85-150 ℃; the temperature rise rate of the second stage is in the range of 8-15 ℃ min-1The maximum temperature is 300-350 ℃, and the holding time is 5-20 min. Preferably, the column temperature-raising program: maintaining at 50 deg.C for 1min at 25 deg.C for min-1The temperature is raised to 125 ℃ at a speed, and then 10 ℃ min-1Heating to 300 deg.C, and maintaining for 10 min;
mass spectrum conditions: an ionization mode: EI, ionization energy: 70 eV; ion source temperature: 200 ℃, transmission line temperature: 250 ℃; solvent retardation: 2.8 min; carrier gas: high purity helium (purity: 99.999%); column flow rate: 1.69 mL/min-1(ii) a Sample inlet temperature: injecting samples at 250 ℃ without shunting; sample introduction amount: 1 μ L.
And (3) in a full scanning mode, wherein the scanning mass range is 40-500, and the retention time of each compound in the sample is obtained.
And (3) selective ion detection: according to the peak-appearing sequence, parameters of each compound are obtained.
The preparation method of the standard curve in the step 4 is as follows:
taking a proper amount of traditional Chinese medicine samples, and preparing a sample matrix solution by operating according to a sample pretreatment mode; and preparing the mixed reference substance stock solution and the sample-added substance matrix solution into matrix mixed standard working solutions with different concentrations for making a standard working curve. 2-500 ng/mL of each pesticide is prepared-1Respectively and precisely absorbing 1 mu L of the series of mixed standard solutions, injecting the mixed standard solutions into a gas chromatography-mass spectrometer, and measuring and calculating. And drawing a standard curve by taking the peak area as a vertical coordinate and the mass concentration as a horizontal coordinate.
Wherein the matrix is: and (3) extracting and purifying the traditional Chinese medicine sample according to the steps 1 and 2 to obtain a solution.
The preparation method of the mixed reference stock solution comprises respectively weighing 10.00mg of each reference, precisely weighing, placing in 1000mL measuring flask, adding acetone to dissolve, and metering to desired volume to obtain 10.0 mg. L－1The standard stock solution of (4) was stored at-20 ℃.
The preparation method of the matrix labeling solution comprises the following steps: a series of standard solutions are diluted step by step with a matrix solution prepared from Chinese medicinal samples.
Wherein 2-500 ng/mL of each pesticide is prepared-1The preparation method of the series of mixed standard solutions comprises the following steps: placing 0.5mL of the mixed reference stock solution in a 10mL measuring flask, adding the matrix solution to dissolve, and fixing the volume to a scale to obtain a solution with a concentration of 500 ng/mL-1The mixed standard solution of (1) is diluted with the base solution to give a standard solution having a concentration of 200, 100, 50, 20, 5, 2 ng.mL-1The series of mixed standard solutions.
The chromatographic conditions were the same as in step 3.
The traditional Chinese medicine is a traditional Chinese medicine preparation prepared by processing and extracting.
Determination of recovery, precision and limit of quantitation:
0.01, 0.05, 0.1 mg/kg of the total weight of the sample was added-1Mixing 3 levels of reference substance solution, extracting, purifying, measuring for 5 times, and calculating recoveryRate and method repeatability. The precision of the instrument is 50 ng/mL-1The mixed reference substance solution is continuously injected for 6 times, and the precision of each substance to be measured is less than 3 percent. And (4) quantitative limit: precisely weighing 2.0g of Chinese medicinal sample powder, adding a certain amount of reference substance mixed solution with a certain concentration, and performing quantitative limit experiment with standard concentration of 3 levels of 0.001, 0.002, and 0.005 mg/kg-1And each horizontal parallel measurement is carried out for 3 times, the selective recovery rate is between 60 and 120 percent, the RSD is less than 20 percent, and the signal-to-noise ratio obtained by adding the mass concentration of the standard solution is not less than 10: the lowest addition level of 1, as the limit of quantitation of this material, results are shown in table 1. The chromatogram is shown in FIG. 1.
Quantitative method:
for a single substance to be detected, a standard curve method or an external standard point method can be adopted for quantification, and for the same type pesticide residue substance to be detected containing isomers (such as permethrin, including permethrin-1 and permethrin-2), the total amount of the substance can be calculated by adopting a standard curve of permethrin.
The method has high sensitivity and good accuracy, can effectively detect the pesticide residues in the Chinese medicinal preparation, and is obtained by screening the operation method, wherein the screening process comprises the following steps:
the extraction method comprises the following steps:
a traditional Chinese medicine matrix contains a large amount of small molecular substances, a traditional ① water immersion method, an acetonitrile extraction and salting-out method are adopted, so that too many impurities are extracted, a solid phase extraction small column or a gel chromatographic column is easy to block during further purification, ② acetonitrile directly extracted has more impurities, single purification cannot meet detection requirements, solid phase extraction is combined with GPC (GPC) for serial purification, and a ③ acetone direct extraction method is adopted, so that solid phase extraction purification is independently used, matrix interference is serious during low-concentration addition, and the detection sensitivity of the method is slightly poor.
The invention adopts ethyl acetate as an extraction solvent, and can eliminate the interference of water-soluble and alcohol-soluble micromolecule components to the maximum extent. After extraction, the amino column is purified by one step to meet the sensitivity of 0.01 mg/kg－1And the accuracy is 70-110% (the low concentration is 60-120%), and the like.
The effect is as follows: 103 substances to be tested at addition level of 0.001-0.01 mg/kg－1The quantitative detection can be carried out in the middle. The extraction reagent ethyl acetate is friendly to the environment, and the reagent and time cost for further purification are saved.
Influence of matrix effects on the process:
the pesticide residue in food is detected, and the matrix is simple, so that the interference is small; traditional Chinese medicinal materials are much more complex than food; the matrix effect of the Chinese medicinal preparation prepared from the compound Chinese medicinal materials is more obvious. Complex matrices can interfere with assay results, causing false positives or false negatives.
The method establishes a multi-channel GC-MS/MS method, the mass spectrometry is used for detecting pesticide residues, false positive results can be well eliminated, and for each substance to be detected, the method sets a group of quantitative ions and two groups of qualitative ions (including the quantitative ions) for eliminating interference caused by complex matrixes. And all reference substance solutions are labeled by adopting blank matrixes, so that the inaccuracy of results caused by the matrix effect of mass spectrometry in the process of quantification is avoided. The method for determining the limit of quantitation adopts the steps of adding a sample, extracting and purifying the sample, then performing on-machine analysis, and selecting the lowest concentration which can meet the accuracy and the repeatability as the detection limit, wherein the detection limit is different from the detection limit of the traditional method of using a reference substance solution to perform signal-to-noise ratio. The detection limit reflects the ability of the extraction method and the instrument sensitivity to detect the substances to be detected.
The invention aims to solve the technical problem of providing a detection method for detecting multiple pesticide residues in a traditional Chinese medicine sample, which has the advantages of high sensitivity, high stability, simplicity in operation and cost saving, aiming at the problems of the traditional Chinese medicine preparation multiple pesticide residue analysis technology in the prior art.
At present, a method for detecting various pesticide residues aiming at a traditional Chinese medicine extract preparation does not exist, and a simple and feasible pretreatment method is developed, so that the method can be applied to detection of pesticide residue substances in the traditional Chinese medicine preparation or can be used as a control means of pesticide residue safety indexes of the traditional Chinese medicine preparation. And meanwhile, 103 pesticide residues are measured, so that the time cost and the labor cost are saved, the method is quick and accurate, and the quality of the preparation can be more comprehensively controlled.
Description of the drawings:
FIG. 1 is a chromatogram obtained by combining gas chromatography with mass spectrometry of 103 pesticide residues
Detailed Description
The invention is further illustrated by the following examples:
example 1 detection of multiple pesticide residues of pill of six ingredients with rehmannia
In this example, a certain brand of commercially available Liuwei Dihuang Wan was selected for detection of pesticide residues. The specific method comprises the following steps:
(1) sample pretreatment:
extraction:
taking a Liuwei Dihuang pill sample, crushing, sieving by a No. 3 sieve, taking 2.0g of the crushed sample, precisely weighing, adding 10mL of ethyl acetate into a 50mL polypropylene plastic centrifuge tube, shaking for extraction for 30min (135r/min), centrifuging for 3min at 5000r/min, taking 5mL of supernatant into a 150mL chicken heart bottle, carrying out rotary evaporation in a water bath at 40 ℃ until the supernatant is nearly dry, dissolving by 2mL of eluent (V (dichloromethane): V (methanol) ═ 100: 1), and waiting for purification.
Purifying:
before loading the amino column, pre-washing the column with 3mL of eluent, quickly moving the sample solution into the column when the liquid level of the pre-washing solution reaches the top of the column packing, and receiving the eluent by using a 150mL flat-bottomed flask; washing the chicken heart bottle with 3ml, 5ml and 5ml of leacheate respectively, transferring the leacheate into the column when the liquid level of the leacheate is close to the top of the column packing, and collecting all effluent. Concentrating in 40 deg.C water bath, adding 1ml acetone, mixing, and gas chromatography-mass spectrometry.
(2) And (2) quantitatively analyzing the sample pretreated in the step (1) by using GC-MS/MS, and eliminating the matrix effect according to a standard curve formed by adding the matrix and the standard solution to obtain the content of the multiple pesticide residues in the sample.
Chromatographic conditions are as follows:
the instrument adopts: triple quadrupole mass spectrometer (Shimadzu GCMS-TQ 803); rtx-5MS (30 m.times.0.24 mm, 0.25 um); a capillary column; column temperature program: maintaining at 50 deg.C for 1min at 25 deg.C for min-1The temperature is raised to 125 ℃ at a speed, and then 10 ℃ min-1Heating to 300 deg.C, and maintaining for 10 min; an ionization mode: EI, ionization energy: 70 eV; ion source temperature: 200 ℃, transmission line temperature: 250 ℃; solvent retardation: 2.8 min; carrier gas: high purity helium (purity: 99.999%); column flow rate: 1.69 mL/min-1(ii) a Sample inlet temperature: injecting samples at 250 ℃ without shunting; sample introduction amount: 1 mu L of the solution; and (4) selecting ion detection.
And (3) in a full scanning mode, wherein the scanning mass range is 40-500, and the retention time of each compound in the sample is obtained.
And (3) selective ion detection: according to the peak-appearing sequence, parameters of each compound are obtained.
The parameters for each compound, in order of appearance, were as follows:
TABLE 2103 scanning time, scanning mode, quantitative and qualitative ion of pesticides
Claims (4)
1. A method for detecting pesticide residues in a traditional Chinese medicine preparation is characterized in that a gas phase-mass spectrometry combined detection technology is adopted to detect the pesticide residues in the traditional Chinese medicine preparation, and the method comprises the following steps:
step 1, extracting the components in the traditional Chinese medicine preparation,
step 2, purifying the extracting solution,
step 3, injecting the purified solution into a GC-MS/MS chromatograph to obtain a chromatogram,
and 4, taking the matrix and standard solution as a standard curve to eliminate matrix effect, and calculating the content of pesticide residue in the Chinese medicinal preparation according to a chromatogram, wherein the extraction in the step 1 comprises the following steps: pulverizing the Chinese medicinal preparation, sieving, collecting 1.0-3.0g of pulverized sample, precisely weighing, adding 5-15mL ethyl acetate, extracting for 20-40min, centrifuging for 2-4min, collecting appropriate amount of supernatant, volatilizing to near dry, dissolving with 1-3mL eluate, and purifying;
wherein the purification in the step 2 comprises the following steps: before the amino column is loaded, pre-washing the column with 2-4mL of eluent, and when the liquid level of the pre-washing liquid reaches the top of the column packing, transferring the sample solution into the column to receive the eluent; washing the heart-shaped bottle with 10-20ml of total leacheate for 2-4 times, transferring the leacheate into the column when the liquid level of the last leacheate is close to the top of the column packing, collecting all effluent liquid, performing rotary concentration in a water bath at 35-45 ℃ until the effluent liquid is nearly dry, adding 1-3ml of acetone or acetonitrile, uniformly mixing, and performing gas chromatography-mass spectrometry;
wherein the chromatographic conditions of step 3 are as follows:
gas chromatography: the chromatographic column is selected from: rtx-5MS, HP-5MS or DB-5MS column, with initial temperature of 50-70 deg.C, and holding time of 0-5 min; the temperature rise rate of the first stage is in the range of 15-35 ℃ min-1Heating to 85-150 ℃; the temperature rise rate of the second stage is in the range of 8-15 ℃ min-1The maximum temperature is 300-;
mass spectrum conditions: an ionization mode: EI, ionization energy: 70 eV; ion source temperature: 200 ℃, transmission line temperature: 250 ℃; solvent retardation: 2.8 min; carrier gas: purity: 99.999% high purity helium; column flow rate: 1.69 mL/min-1(ii) a Sample inlet temperature: injecting samples at 250 ℃ without shunting; sample introduction amount: 1 mu L of the mixture is added into the solution,
wherein the traditional Chinese medicine preparation is a pill of six ingredients with rehmannia,
wherein the leacheate is a mixed solution of dichloromethane and methanol with the volume ratio of 100: 1.
2. The detection method according to claim 1, wherein the extraction is selected from shaking extraction or ultrasonic extraction; the volatile near-dry is selected from water bath rotary evaporation to near-dry or nitrogen blowing to near-dry.
3. The detection method according to claim 1, wherein the purification method is: before the amino column is loaded, pre-washing the column with 3mL of eluent, and when the liquid level of the pre-washing liquid reaches the top of the column packing, quickly moving the sample solution into the column to receive the eluent; washing the chicken heart bottle with 3ml, 5ml and 5ml of leacheate respectively, transferring the leacheate into the column when the liquid level of the last leacheate is close to the top of the column packing, collecting all effluent liquid, concentrating the effluent liquid to be nearly dry by rotating in a water bath at 40 ℃, adding 1ml of acetone and mixing uniformly.
4. The detection method according to claim 1, wherein the gas chromatography: column temperature program: maintaining at 50 deg.C for 1min at 25 deg.C for min-1The temperature is raised to 125 ℃ at a speed, and then 10 ℃ min-1The temperature is raised to 300 ℃ and kept for 10 min.
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| CN106290692A (en) * | 2016-11-02 | 2017-01-04 | 百奥森（江苏）食品安全科技有限公司 | A kind of pesticide method |
| CN106442507A (en) * | 2016-11-03 | 2017-02-22 | 百奥森（江苏）食品安全科技有限公司 | Detection test paper for pyrethroid pesticide residues in foods |
| CN108072727B (en) * | 2016-11-05 | 2021-05-11 | 天士力医药集团股份有限公司 | Method for simultaneously determining residual quantity of 23 organochlorine pesticides in pseudo-ginseng |
| CN110470765A (en) * | 2019-08-26 | 2019-11-19 | 谱尼测试集团吉林有限公司 | A kind of method of 179 persticide residues in measurement fruits and vegetables |
| CN112433013A (en) * | 2020-11-26 | 2021-03-02 | 湖北世友中药饮片有限公司 | Method for detecting pesticide residue content in traditional Chinese medicine decoction pieces |
| CN116718709A (en) * | 2023-07-31 | 2023-09-08 | 广州南沙明曦检测服务有限公司 | Mobile phase and pesticide residue detection method for improving pesticide residue detection efficiency of high performance liquid chromatography-mass spectrometer |
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| CN101598707B (en) * | 2009-05-19 | 2012-05-02 | 烟台杰科检测服务有限公司 | Method for detecting various pesticide residue in oil-containing food |
| CN103529153B (en) * | 2013-07-26 | 2015-07-08 | 宁波检验检疫科学技术研究院 | Common rapid detection method for various pesticide residues in soybeans |
| CN104198600A (en) * | 2014-07-31 | 2014-12-10 | 甘肃中天药业有限责任公司 | Method for detecting radix astragali |
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