CN110609098A - Method for detecting residual quantity of forchlorfenuron in grapes - Google Patents
Method for detecting residual quantity of forchlorfenuron in grapes Download PDFInfo
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- CN110609098A CN110609098A CN201910901265.1A CN201910901265A CN110609098A CN 110609098 A CN110609098 A CN 110609098A CN 201910901265 A CN201910901265 A CN 201910901265A CN 110609098 A CN110609098 A CN 110609098A
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- GPXLRLUVLMHHIK-UHFFFAOYSA-N forchlorfenuron Chemical compound C1=NC(Cl)=CC(NC(=O)NC=2C=CC=CC=2)=C1 GPXLRLUVLMHHIK-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 239000005979 Forchlorfenuron Substances 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 38
- 241000219094 Vitaceae Species 0.000 title claims abstract description 29
- 235000021021 grapes Nutrition 0.000 title claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 14
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 50
- 239000000523 sample Substances 0.000 claims description 32
- 150000002500 ions Chemical class 0.000 claims description 30
- 239000003463 adsorbent Substances 0.000 claims description 24
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 20
- 238000000605 extraction Methods 0.000 claims description 19
- 238000000746 purification Methods 0.000 claims description 19
- 239000012071 phase Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 8
- 238000004811 liquid chromatography Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 7
- 238000002390 rotary evaporation Methods 0.000 claims description 7
- 238000000889 atomisation Methods 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 238000004821 distillation Methods 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000002514 liquid chromatography mass spectrum Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 17
- 241000219095 Vitis Species 0.000 abstract description 14
- 235000009754 Vitis X bourquina Nutrition 0.000 abstract description 14
- 235000012333 Vitis X labruscana Nutrition 0.000 abstract description 14
- 235000014787 Vitis vinifera Nutrition 0.000 abstract description 14
- 235000013305 food Nutrition 0.000 abstract description 12
- 239000003640 drug residue Substances 0.000 abstract description 6
- 239000011159 matrix material Substances 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 235000013399 edible fruits Nutrition 0.000 description 6
- 241000219112 Cucumis Species 0.000 description 3
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000012113 quantitative test Methods 0.000 description 3
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000447 pesticide residue Substances 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- HXYXTCJDWHHCBW-UHFFFAOYSA-N acetonitrile;toluene Chemical compound CC#N.CC1=CC=CC=C1 HXYXTCJDWHHCBW-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- VLCQZHSMCYCDJL-UHFFFAOYSA-N tribenuron methyl Chemical compound COC(=O)C1=CC=CC=C1S(=O)(=O)NC(=O)N(C)C1=NC(C)=NC(OC)=N1 VLCQZHSMCYCDJL-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/143—Preparation by elimination of some components selective absorption
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the technical field of food detection, and particularly relates to a method for detecting residual quantity of forchlorfenuron in grapes. According to the method for detecting the residual quantity of the forchlorfenuron in the grapes, the recovery rate of the method in the grape matrix is 95.8% -99.2%, and the relative standard deviation is 1.5% -3.4% by adopting a high performance liquid chromatography-mass spectrometry combined method for detection, so that the defect that the forchlorfenuron drug residue in the grapes cannot be accurately measured by the existing method is overcome, and the forchlorfenuron abuse or excessive use on food is effectively prevented; the method has the characteristics of simple operation, high analysis speed, high sensitivity and good reproducibility, obviously reduces the labor intensity of testing personnel, improves the detection efficiency, and has important significance for quantitative detection of the forchlorfenuron drug residue in the grapes in the field of food detection.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for detecting residual quantity of forchlorfenuron in grapes.
Background
Forchlorfenuron (English name: Forchlorfenuron, CAS number: 68157-60-8; molecular formula: C)12H10ClN3O); the chemical name is 1- (2-chloro-4-pyridine) -3-phenylurea, which is a novel pesticide for regulating plant growth, has cytokinin activity, can promote cell division and protein synthesis, improves photosynthesis and enhances stress resistance. The plant growth promoter is widely applied to melon and fruit plants, has good effects of promoting flower bud differentiation, flower retention, fruit retention and fruit hypertrophy, and improves fruit setting rate and the like. However, abuse and overuse of forchlorfenuron pose serious challenges and threats to agricultural product safety.
The latest revision edition of the maximum limit of pesticide residues in national food standards for food safety (GB 2763-. And the purification step in the pretreatment process in GB 20770-. In the current national food safety supervision spot inspection implementation rules (2019), forchlorfenuron is already brought into the melon and fruit risk monitoring range, so that a rapid detection method for forchlorfenuron pesticide residues in melons and fruits is provided according to the objective condition.
Disclosure of Invention
The invention provides a method for detecting residual quantity of forchlorfenuron in grapes, aiming at preventing the forchlorfenuron from being abused or excessively used on food and solving the problems of complicated steps, low accuracy and the like of the existing forchlorfenuron detection method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a method for detecting residual quantity of forchlorfenuron in grapes adopts a high performance liquid chromatography-mass spectrometry combined method for detection; comprises the step of pre-treating a sample to be detected; wherein, the extraction reagents used for the pretreatment of the sample to be detected are acetonitrile and dSPE extraction bags; the volume ratio of the acetonitrile to the dSPE extraction bag is 10:1-5 in mL/g; and purifying the extracting solution of the sample to be detected by adopting an adsorbent and a solid phase extraction column.
Further, the pretreatment steps of the sample to be detected are as follows:
step one, collection: crushing a sample to be tested, sieving, homogenizing, sealing and storing;
step two, extraction: accurately weighing 5.00g of sample (accurate to 0.001g) in a 50mL centrifuge tube, adding 10mL acetonitrile, and 1 package of SPE extraction package; performing vortex extraction at 2000r/min for 20-35min, centrifuging at 4500r/min for 3-8min to obtain first extractive solution, and transferring into a purification tube; adding 10mL acetonitrile into the centrifugal tube, homogenizing at high speed at 4500r/min for 1-4min, centrifuging at 4500r/min for 3-8min to obtain a second extractive solution, and mixing with the first extractive solution for purification;
step three, adsorption purification: adding adsorbent into the purification tube, shaking for 1-3min, and centrifuging at 4500r/min for 3-8min to obtain first purified solution; taking 10mL of the centrifuged purification solution, placing the solution in a 50mL distillation flask, performing rotary evaporation to 1mL in a water bath at 60 ℃, and purifying by using a solid phase extraction column;
step four, solid phase extraction and purification: activating and leaching the solid phase extraction column by eluent in advance; transferring the rotary evaporation concentrated solution to a solid phase extraction column, eluting the column by using eluent, receiving the eluent by using a receiving bottle, and carrying out rotary evaporation to dryness in a water bath at 40 ℃ after the elution is finished; then adding 2mL of 10% acetonitrile solution to a constant volume of 1mL, and filtering through a 0.45-micrometer microporous filter membrane to obtain a test sample solution for determination by a liquid chromatography-mass spectrometer.
Further, the adsorbent comprises one or more of a PSA adsorbent, a C18 adsorbent and an anhydrous magnesium sulfate adsorbent.
Furthermore, the weight ratio of the PSA adsorbent to the C18 adsorbent to the anhydrous magnesium sulfate adsorbent is 1:1-3: 2-4.
Further, the solid phase extraction column comprises an amino solid phase extraction column, wherein the eluent comprises acetonitrile and toluene, and the volume ratio of the acetonitrile to the toluene is 1-3: 1.
Further, the dSPE extraction package comprises MgSO4NaCl, said MgSO4The mass ratio of the NaCl to the NaCl is 3-5: 1.
Further, the conditions of the liquid chromatography in the high performance liquid chromatography-mass spectrometry combined method are as follows:
mobile phase: phase A, water; phase B, acetonitrile;
gradient elution was performed using the following conditions:
| time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0.00-1.00 | 60 | 40 |
| 1.00-1.50 | 60-10 | 40-90 |
| 1.50-3.00 | 10 | 90 |
| 3.00-3.10 | 10-60 | 90-40 |
| 3.10-7.00 | 60 | 40 |
Further, the liquid chromatography adopts a C18 chromatographic column, and/or the column temperature is 40 ℃, and/or the flow rate is 0.3mL/min, and/or the sample injection amount is 5 mu L.
Further, the mass spectrum conditions in the high performance liquid chromatography-mass spectrometry combined method are as follows:
furthermore, the mass spectrum adopts an ion source negative ion scanning mode (ESI), the monitoring mode is a multi-reaction monitoring mode (MRM), the temperature of the capillary tube is 270 ℃, the atomization voltage is 3500V, the temperature of the atomization gas is 300 ℃, the sheath gas is 45psi, and the auxiliary gas is 15 arb.
The invention provides a method for detecting residual quantity of forchlorfenuron in grapes, which detects by adopting a high performance liquid chromatography-mass spectrometry combination method, the recovery rate of the detection method in grape matrix reaches 95.8-99.2%, and the relative standard deviation is between 1.5-3.4%, thereby solving the defect that the prior method can not accurately detect the forchlorfenuron drug residue in grapes and effectively preventing the forchlorfenuron from being abused or excessively used on food; the method has the characteristics of simple operation, high analysis speed, high sensitivity and good reproducibility, obviously reduces the labor intensity of testing personnel, improves the detection efficiency, and has important significance for quantitative detection of the forchlorfenuron drug residue in the grapes in the field of food detection.
Drawings
FIG. 1 is a TIC ion flow chromatogram of a forchlorfenuron standard;
FIG. 2 is a TIC ion flow chromatogram of a forchlorfenuron blank matrix in grapes;
FIG. 3 is a TIC ion flow chromatogram of forchlorfenuron in grapes;
FIG. 4 is a forchlorfenuron correction graph;
FIG. 5 is a TIC ion flow chromatogram of part A solution;
FIG. 6 is a TIC ion flow chromatogram of part B solution;
FIG. 7 is a TIC ion flow chromatogram of solution C;
FIG. 8 is a TIC ion flow chromatogram of part D of the solution;
FIG. 9 is a TIC ion flow chromatogram of E parts solution;
FIG. 10 is a TIC ion flow chromatogram of F solutions.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Unless otherwise specified, the following instruments and reagents were used:
the main apparatus is as follows: thermo TSQ QUANTUM ULTRA;
the main reagents are as follows: standard forchlorfenuron (purity is more than or equal to 96%); acetonitrile (chromatographically pure); water (chromatographically pure); dSPE extract package (assay pure); PSA adsorbents (N-propylethylenediamine, precursor secondary amine) (40-63 μm); c18 adsorbent (40-63 μm); anhydrous magnesium sulfate adsorbent (40-63 μm); acetonitrile (analytical grade); toluene (analytically pure).
Example one
A method for detecting residual quantity of forchlorfenuron in grapes adopts a high performance liquid chromatography-mass spectrometry combined method for detection; the method comprises the step of pretreating a sample to be detected, and the steps are as follows:
step one, collection: crushing a grape sample in a crusher, sieving all the samples with a 20-target standard mesh sieve, uniformly mixing the prepared samples, dividing the samples into two parts, attaching sample labels, and sealing and storing;
step two, extraction: accurately weighing 5.00g sample (accurate to 0.001g) in a 50mL centrifuge tube, adding 10mL acetonitrile, and 1 packet dSPE extraction packet comprising MgSO 14NaCl, 1-pack dSPE extract contained 4g MgSO41g of NaCl; performing vortex extraction at 2000r/min for 30min, centrifuging at 4500r/min for 5min to obtain first extractive solution, and transferring into a purification tube; adding 10mL of acetonitrile into the centrifugal tube, homogenizing at a high speed of 4500r/min for 2min, centrifuging at 4500r/min for 5min to obtain a second extracting solution, combining the second extracting solution with the first extracting solution to be purified, and extracting once again by utilizing high-speed homogenization, so that the extraction effect is increased, the amount of a sample is increased, and the accuracy of subsequent tests is more convenient;
step three, adsorption purification: adding 50mg of PSA adsorbent, 100mg of C18 adsorbent and 150mg of anhydrous magnesium sulfate adsorbent into a purification tube, violently shaking for 1min, centrifuging at 4500r/min for 5min to obtain a first purification solution, and effectively removing natural pigment and partial impurities in a sample matrix through the step; taking 10mL of the centrifuged purification solution, placing the solution in a 50mL distillation flask, performing rotary evaporation to 1mL in a water bath at 60 ℃, and purifying by using a solid phase extraction column;
step four, solid phase extraction and purification: taking an amino solid phase extraction column (150mg, 3mL), and activating and leaching the extraction column with 4mL of acetonitrile-toluene (v/v, 3:1) in advance;
when the liquid level reached the top of the packing, the rotary evaporated concentrate was transferred to a solid phase extraction column and the eluate was collected in a 50mL pear-shaped flask, followed by 3mL acetonitrile: the distillation flask was washed 3 times with toluene (v/v, 3:1) so that the target in the flask was completely transferred to the solid phase extraction column;
then 10mL of acetonitrile: toluene (v/v, 3:1) elution column;
after the elution is finished, the solution is rotated and evaporated to dryness in a water bath at 40 ℃;
adding 2mL of 10% acetonitrile solution, diluting to 1mL, filtering with a 0.45-micrometer microporous membrane to obtain a test sample solution for determination by a liquid chromatography-mass spectrometer, and further purifying organic macromolecular impurities such as sugar, amino acid and the like contained in the sample by using a solid phase extraction column, so that the subsequent measured substances are more accurate, and the interference of the impurities is reduced.
In this embodiment, before detecting a test sample solution, a known forchlorfenuron standard solution is detected under the following liquid chromatography conditions and mass spectrometry conditions, and a TIC ion flow chromatogram of a forchlorfenuron standard is obtained, as shown in fig. 1;
the conditions of the liquid chromatography were as follows:
the liquid chromatography adopts a chromatographic column: Waters/C181.6 μm 2.1 × 100mm, column temperature 40 deg.C, flow rate 0.3mL/min, sample volume 5 μ L;
mobile phase: phase A, water; phase B, acetonitrile; gradient elution was performed using the conditions of table 1:
TABLE 1
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0.00-1.00 | 60 | 40 |
| 1.00-1.50 | 60-10 | 40-90 |
| 1.50-3.00 | 10 | 90 |
| 3.00-3.10 | 10-60 | 90-40 |
| 3.10-7.00 | 60 | 40 |
The mass spectrometry conditions were as follows:
the mass spectrum adopts an ion source negative ion scanning mode (ESI), the monitoring mode is a multi-reaction monitoring mode (MRM), the temperature of a capillary tube is 270 ℃, the atomization voltage is 3500V, the temperature of atomization gas is 300 ℃, the sheath gas is 45psi, the auxiliary gas is 15arb, and the parent ions, the daughter ions and the collision energy are shown in a table 2:
TABLE 2
Note: label x is the quantification ion.
After the known standard liquid of forchlorfenuron is detected according to the liquid chromatography condition and the mass spectrum condition, the test sample liquid of the grapes is detected, which comprises the following specific steps:
1) qualitative testing
Injecting a proper amount of the treated grape test sample liquid into the adjusted liquid chromatography-mass spectrometer to obtain a TIC ion flow chromatogram of a forchlorfenuron blank matrix in the grape, as shown in figure 2;
injecting known standard liquid of forchlorfenuron into the grape, and injecting the standard liquid into the adjusted liquid chromatogram-mass spectrometer to obtain a TIC ion flow chromatogram of forchlorfenuron in the grape, as shown in FIG. 3;
response energy by chromatogram 2 of 5.98X 102And response energy of chromatogram 3 8.51 × 104It is known that forchlorfenuron is not present in the grapes;
2) standard curve of forchlorfenuron
Preparing 6 groups of forchlorfenuron with different concentrations by using standard forchlorfenuron, wherein the forchlorfenuron comprises 50 mu g/L, 100 mu g/L, 150 mu g/L, 200 mu g/L, 250 mu g/L and 300 mu g/L, sequentially injecting into the adjusted liquid chromatogram-mass spectrometer, measuring corresponding peak areas, and comparing the peak areas with the concentrationsThe calibration curve of forchlorfenuron is obtained by using the least square method calculation, wherein Y is 6892.65X +93885.9, and R is20.9976, as shown in fig. 4. Wherein Y represents a response value and X represents a concentration.
3) Quantitative test
Taking two parts A and B of the treated grape test sample liquid, adding 5 mu g/kg of forchlorfenuron into the solution A and the solution B, mixing uniformly, injecting into the adjusted liquid chromatogram-mass spectrometer, and obtaining a TIC ion current chromatogram of the forchlorfenuron in the grape, wherein as shown in figures 5 and 6, figure 5 is the TIC ion current chromatogram of the solution A; FIG. 6 is a TIC ion flow chromatogram of part B solution; by measuring the corresponding peak areas, the concentration of forchlorfenuron in the solution A was calculated from the standard curve to be 4.955. mu.g/kg, and the concentration of forchlorfenuron in the solution B was calculated to be 4.791. mu.g/kg, and the recovery rate and the Relative Standard Deviation (RSD) thereof were calculated, as shown in Table 3 below.
The forchlorfenuron content in the sample is calculated according to the following formula:
X＝C×V/m；
in the formula:
x-the forchlorfenuron content in the sample to be detected, wherein the unit is microgram per kilogram (mu g/kg);
c, calculating the concentration of forchlorfenuron in the sample liquid to be tested by using a standard curve;
v, final constant volume of the sample liquid to be tested;
m is the weight of the sample to be measured.
Example two
The difference between this embodiment and the first embodiment is: quantitative test
Taking two parts of C and D of the treated grape test sample liquid, adding 15 mu g/kg of forchlorfenuron into the solution C and D, mixing uniformly, injecting into the adjusted liquid chromatogram-mass spectrometer, and obtaining a TIC ion current chromatogram of the forchlorfenuron in the grape, wherein as shown in figures 7 and 8, figure 7 is the TIC ion current chromatogram of the solution C; FIG. 8 is a TIC ion flow chromatogram of part D of the solution; by measuring the corresponding peak areas, the concentration of forchlorfenuron in the solution C was 15.085. mu.g/kg and the concentration of forchlorfenuron in the solution D was 14.864. mu.g/kg were calculated from the standard curves, and the recovery rates and Relative Standard Deviations (RSD) thereof were calculated, as shown in Table 3 below.
EXAMPLE III
The difference between this embodiment and the first embodiment is: quantitative test
Taking two parts of processed grape test sample liquid E and F, adding 25 mu g/kg of forchlorfenuron into the solution E and F, mixing uniformly, injecting into the adjusted liquid chromatography-mass spectrometer, and obtaining a TIC ion current chromatogram of the forchlorfenuron in the grape, wherein as shown in figures 9 and 10, figure 9 is the TIC ion current chromatogram of the solution E; FIG. 10 is a TIC ion flow chromatogram of F solutions; the concentration of forchlorfenuron in the E solution was 24.020. mu.g/kg and the concentration of forchlorfenuron in the F solution was 24.814. mu.g/kg by measuring the corresponding peak areas from the standard curves, and the recovery rates and Relative Standard Deviations (RSD) thereof were calculated as shown in Table 3 below.
TABLE 3
According to the first, second and third embodiments, the recovery rate of the forchlorfenuron in the grape matrix reaches 95.8-99.2%, and the relative standard deviation is between 1.5-3.4%, so that the defect that the forchlorfenuron drug residue in the grapes cannot be accurately measured by the existing method is overcome, and the forchlorfenuron abuse or overuse on food is effectively prevented; the method has the characteristics of simple operation, high analysis speed, high sensitivity and good reproducibility, obviously reduces the labor intensity of testing personnel, improves the detection efficiency, and has important significance for quantitative detection of the forchlorfenuron drug residue in the grapes in the field of food detection.
The above description is only a preferred embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing on the protection scope of the present invention.
Claims (10)
1. A method for detecting residual quantity of forchlorfenuron in grapes is characterized by comprising the following steps: detecting by adopting a high performance liquid chromatography-mass spectrometry combined method; comprises the step of pre-treating a sample to be detected; wherein, the extraction reagents used for the pretreatment of the sample to be detected are acetonitrile and dSPE extraction bags; the volume ratio of the acetonitrile to the dSPE extraction bag is 10:1-5 in mL/g; and purifying the extracting solution of the sample to be detected by adopting an adsorbent and a solid phase extraction column.
2. The method for detecting the residual quantity of forchlorfenuron in grapes according to claim 1, characterized in that the pretreatment steps of the sample to be detected are as follows:
step one, collection: crushing a sample to be tested, sieving, homogenizing, sealing and storing;
step two, extraction: accurately weighing 5.00g of sample (accurate to 0.001g) in a 50mL centrifuge tube, adding 10mL acetonitrile, and 1 package of SPE extraction package; performing vortex extraction at 2000r/min for 20-35min, centrifuging at 4500r/min for 3-8min to obtain first extractive solution, and transferring into a purification tube; adding 10mL acetonitrile into the centrifugal tube, homogenizing at high speed at 4500r/min for 1-4min, centrifuging at 4500r/min for 3-8min to obtain a second extractive solution, and mixing with the first extractive solution for purification;
step three, adsorption purification: adding adsorbent into the purification tube, shaking for 1-3min, and centrifuging at 4500r/min for 3-8min to obtain first purified solution; taking 10mL of the centrifuged purification solution, placing the solution in a 50mL distillation flask, performing rotary evaporation to 1mL in a water bath at 60 ℃, and purifying by using a solid phase extraction column;
step four, solid phase extraction and purification: activating and leaching the solid phase extraction column by eluent in advance; transferring the rotary evaporation concentrated solution to a solid phase extraction column, eluting the column by using eluent, receiving the eluent by using a receiving bottle, and carrying out rotary evaporation to dryness in a water bath at 40 ℃ after the elution is finished; then adding 2mL of 10% acetonitrile solution to a constant volume of 1mL, and filtering through a 0.45-micrometer microporous filter membrane to obtain a test sample solution for determination by a liquid chromatography-mass spectrometer.
3. The method for detecting the residual quantity of forchlorfenuron in grapes according to claim 2, characterized in that: the adsorbent comprises one or more of PSA adsorbent, C18 adsorbent and anhydrous magnesium sulfate adsorbent.
4. The method for detecting the residual amount of forchlorfenuron in grapes according to claim 3, characterized in that: the weight ratio of the PSA adsorbent to the C18 adsorbent to the anhydrous magnesium sulfate adsorbent is 1:1-3: 2-4.
5. The method for detecting the residual quantity of forchlorfenuron in grapes according to claim 2, characterized in that: the solid phase extraction column comprises an amino solid phase extraction column, wherein the eluent comprises acetonitrile and toluene, and the volume ratio of the acetonitrile to the toluene is 1-3: 1.
6. The method for detecting the residual quantity of forchlorfenuron in grapes according to claim 2, characterized in that: the dSPE extraction packet comprises MgSO4NaCl, said MgSO4The mass ratio of the NaCl to the NaCl is 3-5: 1.
7. The method for detecting the residual quantity of forchlorfenuron in grapes according to claim 1, characterized in that the conditions of the liquid chromatography in the high performance liquid chromatography-mass spectrometry are as follows:
mobile phase: phase A, water; phase B, acetonitrile;
gradient elution was performed using the following conditions:
8. The method for detecting the residual amount of forchlorfenuron in grapes according to claim 7, characterized in that: the liquid chromatography adopts a C18 chromatographic column, and/or the column temperature is 40 ℃, and/or the flow rate is 0.3mL/min, and/or the sample injection amount is 5 mu L.
9. The method for detecting the residual amount of forchlorfenuron in grapes according to claim 1, characterized in that: the mass spectrum conditions in the high performance liquid chromatography-mass spectrum combined method are as follows:
10. the method for detecting the residual amount of forchlorfenuron in grapes according to claim 9, characterized in that: the mass spectrum adopts an ion source negative ion scanning mode (ESI), the monitoring mode is a multi-reaction monitoring mode (MRM), the temperature of a capillary tube is 270 ℃, the atomization voltage is 3500V, the temperature of atomization gas is 300 ℃, the sheath gas is 45psi, and the auxiliary gas is 15 arb.
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