CN102121927B - Method for processing Chinese herbal medicine sample containing several pesticide residues before determination - Google Patents
Method for processing Chinese herbal medicine sample containing several pesticide residues before determination Download PDFInfo
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Abstract
The invention discloses a method for processing a Chinese herbal medicine sample containing carbamate pesticide residues before determination, comprising the following steps: accurately weighing 2g of a smashed Chinese herbal medicine sample; adding 30ml of acetonitrile in a centrifuge tube; eddying for 2 minutes; centrifuging for 5 minutes at 4000 rmp; taking a supernate; repeatedly extracting residues by 10-20ml of acetonitrile once; eddying for 2 minutes; centrifuging for 5minutes at 4000 rmp; combining supernates; drying the extracting solution by evaporation at 40DEG C; dissolving the residues by 5ml of acetone-normal hexane at the volume ratio of 20:80; standing to be purified; activating a PSA (pressure swing adsorption) solid phase extraction column; connecting the PSA column withan injector with the volume of 25ml by an adapter of a solid phase extraction column; restoring the volume by 2ml of methyl alcohol and 0.1% methanoic acid water; performing sampling introduction after passing through a 0.22mu m filter membrane; and determining by an ultrahigh-performance liquid phase-mass spectrum/ mass spectrometer. The processing method which is disclosed by the invention has the advantages of high determination speed, simple equipment, low determination cost, and high accuracy, stability and reliability.
Description
Technical field:
The invention belongs to the determination techniques field of residues of pesticides, relate to the assay method of the Chinese herbal medicine sample that contains carbamate pesticide residue, the disposal route before the Chinese herbal medicine sample determination that contains 15 kinds of carbamate pesticide residues of saying so more specifically.
Background technology:
Residues of pesticides refer in agricultural production that a part of agricultural chemicals directly or indirectly remains in cereal, vegetables, fruit, livestock products, the aquatic products and the phenomenon in soil and the water body behind the applying pesticides.The residues of pesticides problem is produced along with agricultural chemicals is a large amount of and is widely used and produces.The agricultural chemicals that uses at present, can become innoxious substance some within a short period of time through biodegradation, and some organic chlorine agriculture chemicals are difficult to degraded, are the strong agricultural chemicals of residual.Vegetable pesticide residue exceeds standard, and can directly jeopardize the nervous system and the vitals such as liver, kidney of human body.Remains of pesticide is accumulated in human body simultaneously, after necessarily measuring, can cause some chronic diseases, like muscle numbness, cough etc., even can bring out vascular diseases, diabetes and cancer etc.Because residues of pesticides are very big to the mankind and biohazard, various countries all carry out strict management to using of agricultural chemicals, and the molten amount of being permitted of residues of pesticides in the food has been made regulation.
Traditional Chinese medicine is the rarity of our Chinese nation, and long history in several thousand is arranged.12807 kinds of the existing natural resources of Chinese medicinal materials of China, wherein medicinal plant is 11146 kinds, 1581 kinds of medicinal animals; 80 kinds of mineral drugs; Nearly 6,000,000 mu of Chinese crude drug cultivated area, output surpasses 350,000 tons, and the Chinese medicine production that these abundant natural medicine resources are China provides abundant raw.Yet the quality security problem that in Chinese medicine is produced, occurs has restricted the development of China's Chinese medicine.The quality safety direct relation people masses' of Chinese medicine daily life and life security concern that social harmony is stable.The people will grasp health in own hand, strengthens sense of self-protection.Surpass poisonous and harmful substances such as MIN heavy metal and arsenic salt, aflatoxins, residues of pesticides, microorganism, can cause huge injury the human organs such as immune system, nervous system, reproductive system, bone hematopoiesis function, blood vessel and digestive system of giving human body.Chinese crude drug is taken by patient and weakling as a kind of Special food, and Time of Administration is long, more is prone to cause accumulate poisoning.Contain multiple residual agricultural chemicals impurity such as carbamate chemicals for agriculture in the Chinese herbal medicine sample.
Carbamate chemicals for agriculture is one type of broad-spectrum pesticide with good insecticidal effect, is widely used in the control of disease and pest in the processes such as proportion of crop planting and Chinese crude drug cultivation both at home and abroad because of characteristics such as it is efficient, low toxicities.Meanwhile, water environment pollution, the residues of pesticides problem that exceeds standard of this type medicine generation has also caused people's extensive concern.
About carbamate chemicals for agriculture, the detection method of report has vapor-phase chromatography (GC), GC-MS (GC-MS), high performance liquid chromatography (HPLC) and liquid chromatograph mass spectrography method (LC-MS) etc. at present.Since such medicine have polarity by force, characteristics such as not volatile, thermally labile, be not suitable for GC method and the detection of GC-MS method.Though HPLC post column derivatization method is a classical way, detect simultaneously various ingredients when residual the chromatographic resolution difficulty bigger, poor sensitivity, and can not carry out reliable qualitative analysis.And the LC-MS technology integrates high sensitivity, high selectivity and the extremely strong many advantages such as qualitative ability of the high score of HPLC from ability and MS; Especially Ultra Performance Liquid Chromatography-tandem mass spectrum coupling technique (UPLC-MS/MS); Have that analyst coverage is wide, separating power is strong, highly sensitive, characteristics such as analysis speed is fast, be highly suitable for the analysis of carbamate chemicals for agriculture.In recent years, UPLC-MS/MS is by the increasing mensuration that is applied to carbamate pesticide residue in the agricultural product, but its applied research in Chinese crude drug also seldom.At present, mostly the detection method of carbamate chemicals for agriculture is to adopt the HPLC method in the Chinese crude drug, and pre-treating method is loaded down with trivial details, and the medicinal material kind of detection is single.
Residual its key that whether can accurately detect trace pre-treating method that is sample, sample preparation good can reduce impurity and disturb, and reduces the pollution of impurity to instrument, improves the sensitivity and the accuracy of analysis result.The method that the sample-pretreating method that this patent is set up is developed for my unit voluntarily, domestic open source literature is not found relevant report.
Summary of the invention:
Shortcoming that the objective of the invention is to overcome prior art and the not enough disposal route that provides before a kind of Chinese herbal medicine sample determination that contains carbamate pesticide residue, undertaken by following step:
(1) accurately take by weighing the Chinese herbal medicine sample 2g of pulverizing, in centrifuge tube, add the 20-30mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm gets supernatant, and residue repeats to extract once with the 10-20mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm, merging supernatant;
(2) with extract at 40 ℃ of following evaporates to dryness, residue is with the dissolving of 20: 80 acetone of volume ratio-normal hexane 5mL, and is to be clean;
(3) the PSA solid-phase extraction column is earlier used 6mL acetone, 6mL acetone-normal hexane volume ratio activation in 20: 80, and the PSA post linked to each other with the 25mL syringe with the solid-phase extraction column adapter;
(4) sample liquid is transferred in the syringe, uses volume ratio again, 1/4 acetone/normal hexane washing sample bottle, each 5mL washes twice, and cleansing solution all is transferred in the syringe, collects all effluent; After treating that sample liquid all drains off, use the acetone/normal hexane 30mL wash-out of volume ratio 50/50 again, merge effluent; It behind 40 ℃ of following evaporates to dryness, with methyl alcohol/0.1% formic acid water 2mL redissolution of volume ratio 40/60, and is crossed 0.22 μ m filter membrane sample introduction, adopt ultra high efficiency liquid phase-mass spectrum/GC-MS to measure.
The present invention combines the solid phase extraction sample treatment technology, has overcome matrix interference problem complicated in the Chinese herbal medicine sample, has set up the UPLC-MS/MS detection method of 15 kinds of carbamates chemicals for agriculture in the Chinese crude drug first.Be applicable to that multiple carbamate chemicals for agriculture is measured simultaneously in the various Chinese crude drugs.Particularly Chinese crude drugs such as genseng, the Radix Astragali, feverfew, cassia seed can mensuration itself contain the micro-numerical value of carbamate chemicals for agriculture, for rationally correct use of Chinese crude drug provides strong foundation.
The present invention adds recovery research through 15 kinds of carbamates chemicals for agriculture, adopts ultra high efficiency liquid phase-mass spectrum/GC-MS (UPLC/MS/MS) method method, sets up simple and direct 15 kinds of feasible carbamate pesticide residue analytical approachs.
The retention time that the present invention measures is 2.38-10.04min, and quantitative limit 2～10 μ g/kg detect and are limited to 0.2-2.5 μ g/kg, and the recovery is 60%～95%.
More detailed disposal route of the present invention and condition determination are following:
1, instrument condition
1.1 chromatographic condition
Chromatographic column: Acquity BEH C18 (2.1mm * 100mm, 1.7 μ m); Sample size: 10 μ L; Column temperature: 30 ℃.Is that moving phase is carried out linear gradient elution with acetonitrile (A phase) with 0.1% aqueous formic acid (B mutually).Gradient elution program: 0～10min, the ratio of A phase is by 10% linear change to 90%, 10～12min, the ratio of A phase keeps 10%; Flow velocity is 0.2mL/min.
1.2 mass spectrum condition
Ionization pattern: ESI (+); Capillary voltage: 3.0Kv; Ion source temperature: 110 ℃; Desolventizing temperature degree: 350 ℃; Desolventizing airshed: 550L/h; Taper hole blowback air flow: 50L/h; Detection mode: multiple-reaction monitoring scan pattern (MRM); The mass spectrum acquisition parameter is seen table 1.
The Mass Spectrometer Method condition of 15 kinds of agricultural chemicals of table 1
2, sample-pretreating method
2.1 extract
Accurately take by weighing and pulverize sample 2g (being accurate to 0.01g) in 50mL acrylic plastering centrifuge tube, add the 20mL acetonitrile, vortex 2min on vortex mixer, the centrifugal 5min of 4000rpm gets supernatant in evaporative flask.Residue repeats to extract once with the 10mL acetonitrile, vortex 2min, and the centrifugal 5min of 4000rpm merges supernatant in above-mentioned evaporative flask.With extract under 40 ℃ on Rotary Evaporators evaporate to dryness, residue with 5mL acetone-normal hexane (20+80, V: V) dissolving, to be clean.
2.2 purify
The PSA solid-phase extraction column is used earlier 6mL acetone, 6mL acetone-normal hexane (20+80, V: V) activation, and the PSA post linked to each other with the 25mL syringe with the solid-phase extraction column adapter.Sample liquid is transferred in the syringe, and (20+80, V: V) washing sample bottle, each 5mL washes twice, and cleansing solution all is transferred in the syringe, collects all effluent to use acetone-normal hexane again.After treating that sample liquid all drains off, (50+50, V: V) wash-out merges effluent to use 30mL acetone-normal hexane again.With it behind 40 ℃ of following evaporates to dryness, with 2mL methyl alcohol-0.1% formic acid water (40+60, V: V) redissolve, and cross 0.22 μ m filter membrane.
If the back discovery liquid that redissolves is muddy, grease is more, pigment heavier (like ginkgo leaf, the red sage root etc.); The liquid that then will redissolve is transferred in the 5mL polypropylene centrifuge tube, adds the 2mL normal hexane again, hand mixing 1min; (attention action can not be violent, with anti-emulsion), the centrifugal 5min of 4000rpm.Take off layer liquid and cross 0.22 μ m filter membrane sample introduction.
3, the range of linearity, sensitivity, accuracy and precision
This patent is with matrix match-on criterion working solution drawing standard curve.Detectability and the quantitative limit of various agricultural chemicals in genseng, the Radix Astragali, feverfew and four kinds of Chinese crude drugs of cassia seed is respectively 0.2～2.5 μ g/kg and 2～10 μ g/kg.The detectability and the quantitative limit of various medicines see table 2 for details.
At LOQ, 5 times of LOQ and three concentration levels of 10 times of LOQ are done the interpolation recovery test, and each concentration is done 5 parallel sample, compare with blank sample simultaneously, extract according to the method described above, purify and measure.Add concentration level at these three, average recovery rate and the relative standard deviation (RSD) of various agricultural chemicals in genseng, the Radix Astragali, feverfew and four kinds of Chinese crude drugs of cassia seed all listed in the table 3.
Typical curve, detectability and the quantitative limit of 15 kinds of medicines of table 2
The recovery and the relative standard deviation of 15 kinds of carbamates chemicals for agriculture of table 3
The determination of residual amount method advantage and the characteristics of 15 kinds of carbamates chemicals for agriculture disclosed by the invention are: the pre-treatment process is simple, and matrix interference is little, and accuracy, stability and reliability are high.
Embodiment
Below in conjunction with embodiment the present invention is done further explanation, it is indicative that embodiment is merely, and means that never it limits scope of the present invention by any way.
Embodiment 1
(1) accurately takes by weighing the Chinese herb astragalus sample 2g (being accurate to 0.01g) of pulverizing, in centrifuge tube, add the 20mL acetonitrile, vortex 2min; The centrifugal 5min of 4000rpm gets supernatant, and residue repeats to extract once with the 20mL acetonitrile; Vortex 2min, the centrifugal 5min of 4000rpm merges supernatant;
(2) with extract at 40 ℃ of following evaporates to dryness, residue is with the dissolving of 20: 80 acetone of volume ratio-normal hexane 5mL, and is to be clean;
(3) the PSA solid-phase extraction column is earlier used 6mL acetone, 6mL acetone/normal hexane volume ratio 20/80 activation, and the PSA post linked to each other with the 25mL syringe with the solid-phase extraction column adapter;
(4) sample liquid is transferred in the syringe, again with acetone/normal hexane washing sample bottle of using volume ratio 1/4 again, each 5mL washes twice, and cleansing solution all is transferred in the syringe, collects all effluent; After treating that sample liquid all drains off, use the acetone/normal hexane 30mL wash-out of volume ratio 50/50 again, merge effluent; It behind 40 ℃ of following evaporates to dryness, with methyl alcohol/0.1% formic acid water 2mL redissolution of volume ratio 40/60, and is crossed 0.22 μ m filter membrane sample introduction, adopt ultra high efficiency liquid phase-mass spectrum/GC-MS to measure.
Embodiment 2
(1) accurately take by weighing the Chinese herbal medicine cassia seed sample 2g of pulverizing, in centrifuge tube, add the 250mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm gets supernatant, and residue repeats to extract once with the 15mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm, merging supernatant;
(2) with extract at 40 ℃ of following evaporates to dryness, residue is with the acetone of volume ratio 20/80/normal hexane 5mL dissolving, and is to be clean;
(3) the PSA solid-phase extraction column is earlier used 6mL acetone, 6mL acetone/normal hexane volume ratio 20/80 activation, and the PSA post linked to each other with the 25mL syringe with the solid-phase extraction column adapter;
(4) sample liquid is transferred in the syringe, uses the acetone/normal hexane washing sample bottle of volume ratio 1/4 again, each 5mL washes twice, and cleansing solution all is transferred in the syringe, collects all effluent; After treating that sample liquid all drains off, use the acetone/normal hexane 30mL wash-out of volume ratio 50/50 again, merge effluent; It behind 40 ℃ of following evaporates to dryness, with methyl alcohol/0.1% formic acid water 2mL redissolution of volume ratio 40/60, and is crossed 0.22 μ m filter membrane sample introduction, adopt ultra high efficiency liquid phase-mass spectrum/GC-MS to measure.
Claims (1)
1. the disposal route before the Chinese herbal medicine sample determination that contains carbamate pesticide residue is characterized in that being undertaken by following step:
(1) accurately take by weighing the Chinese herbal medicine sample 2g of pulverizing, in centrifuge tube, add the 20-30mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm gets supernatant, and residue repeats to extract once with the 10-20mL acetonitrile, vortex 2min, the centrifugal 5min of 4000rpm, merging supernatant;
(2) supernatant after will merging is at 40 ℃ of following evaporates to dryness, and residue obtains sample liquid with acetone/normal hexane 5mL dissolving of 5mL volume ratio 20/80;
(3) the PSA solid-phase extraction column is used earlier the 6mL acetone rinsing, and then with the acetone/normal hexane activation of 6mL volume ratio 20/80;
(4) sample liquid is transferred in the syringe, the acetone/normal hexane with volume ratio 1/4 washs again, and cleansing solution all is transferred in the syringe, collects all effluent; After treating that sample liquid all drains off, use 50: 50 acetone of volume ratio-normal hexane 30mL wash-out again, merge effluent; It behind 40 ℃ of following evaporates to dryness, is redissolved with 2mL volume ratio 40: 60 methyl alcohol-0.1% formic acid water, and cross 0.22 μ m filter membrane sample introduction, adopt ultra high efficiency liquid phase-mass spectrum/GC-MS to measure.
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| CN102520099A (en) * | 2011-12-08 | 2012-06-27 | 云南烟草科学研究院 | Method for detecting carbamate pesticide content in total particle matter in cigarette mainstream smoke |
| CN102621254A (en) * | 2012-04-09 | 2012-08-01 | 福州大学 | Pretreatment method for detection of acetamiprid in dry fruits and vegetable |
| CN106383192B (en) * | 2016-11-09 | 2018-05-11 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of detection method of Mobucin |
| CN112034068A (en) * | 2020-09-08 | 2020-12-04 | 河南诺玛科技有限公司 | Systemic pesticide detection method applied to fruits and vegetables |
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| 孙涛等.超高效液相色谱_串联质谱法快速测定蔬菜中7种氨基甲酸酯类农药残留.《分析实验室》.2010,第29卷(第4期),第87页至第91页. * |
| 蒋施等.超高效液相色谱_串联质谱法测定蔬菜和食用菌中19种氨基甲酸酯农药残留量.《理化检验-化学分册》.2010,第46卷(第6期),第593页至第597页. * |
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