CN117571855A - Analysis method for detecting brassinolide in microbial agent based on nanomaterial QuEChERS-UPLC-MSMS - Google Patents
Analysis method for detecting brassinolide in microbial agent based on nanomaterial QuEChERS-UPLC-MSMS Download PDFInfo
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- CN117571855A CN117571855A CN202311526738.7A CN202311526738A CN117571855A CN 117571855 A CN117571855 A CN 117571855A CN 202311526738 A CN202311526738 A CN 202311526738A CN 117571855 A CN117571855 A CN 117571855A
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 56
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 title claims abstract description 47
- IXVMHGVQKLDRKH-KNBKMWSGSA-N brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-KNBKMWSGSA-N 0.000 title claims abstract description 47
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 6
- 238000004458 analytical method Methods 0.000 title claims description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 33
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000002048 multi walled nanotube Substances 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 17
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000019253 formic acid Nutrition 0.000 claims abstract description 13
- 239000012629 purifying agent Substances 0.000 claims abstract description 13
- 238000010828 elution Methods 0.000 claims abstract description 9
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 150000001875 compounds Chemical class 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 15
- 150000002500 ions Chemical class 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims description 10
- 238000000132 electrospray ionisation Methods 0.000 claims description 8
- 230000010355 oscillation Effects 0.000 claims description 8
- IXVMHGVQKLDRKH-QHBHMFGVSA-N 24-Epibrassinolide Natural products C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-QHBHMFGVSA-N 0.000 claims description 7
- 239000003599 detergent Substances 0.000 claims description 7
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- 239000000047 product Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- HJIKODJJEORHMZ-DNVPGCTHSA-N isohomobrassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@H](O)[C@@H](O)[C@H](C(C)C)CC)CC[C@H]3[C@@H]21 HJIKODJJEORHMZ-DNVPGCTHSA-N 0.000 claims description 4
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- 238000005516 engineering process Methods 0.000 description 7
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- 150000002596 lactones Chemical class 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 239000005648 plant growth regulator Substances 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 239000003337 fertilizer Substances 0.000 description 4
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- 150000001646 brassinolides Chemical class 0.000 description 3
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- 241000196324 Embryophyta Species 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 239000002068 microbial inoculum Substances 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241001629511 Litchi Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010074268 Reproductive toxicity Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
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- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- CWFOCCVIPCEQCK-UHFFFAOYSA-N chlorfenapyr Chemical compound BrC1=C(C(F)(F)F)N(COCC)C(C=2C=CC(Cl)=CC=2)=C1C#N CWFOCCVIPCEQCK-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 1
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- 231100000372 reproductive toxicity Toxicity 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- UNAANXDKBXWMLN-UHFFFAOYSA-N sibutramine Chemical compound C=1C=C(Cl)C=CC=1C1(C(N(C)C)CC(C)C)CCC1 UNAANXDKBXWMLN-UHFFFAOYSA-N 0.000 description 1
- 229960004425 sibutramine Drugs 0.000 description 1
- -1 sibutramine in food Chemical class 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- 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
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention provides a method for detecting brassinolide in a microbial agent based on a nanomaterial QuEChERS-UPLC-MSMS, which adopts nano-ZrO with specific content 2 、PSA、C 18 MWCNTs are used as a purifying agent combination, the samples are subjected to pretreatment, 0.1% formic acid aqueous solution and methanol are used as mobile phases, and the content of brassinolide in the microbial agent samples can be accurately analyzed under a specific gradient elution condition. The method has the advantages of high sensitivity, strong universality, high accuracy, good stability, simple and convenient operation and the like, and provides technical support for detecting the microbial agent plant source pesticide.
Description
Technical Field
The invention relates to the technical field of botanical pesticide detection, in particular to a method for detecting botanical pesticide residues in microbial agents based on a nanomaterial QuEChERS technology.
Background
With the development of agricultural planting, the problems of soil-borne diseases, soil hardening and the like caused by continuous cropping and excessive use of chemical fertilizers not only cause the quality reduction of agricultural products and the yield reduction, but also cause ecological environment pollution, and the development of the agricultural product industry is seriously affected. Therefore, changing this current situation is an urgent problem to be solved in agricultural development.
The microbial agent is a low-carbon, purely natural, nontoxic, harmless and pollution-free organic microbial agent. The fertilizer has the effects of regulating the nutrition balance in plants, improving the fertilizer utilization rate, improving soil and the like, and has been widely applied to agricultural production in recent years.
The natural brassinolide is a novel sterol substance with extremely strong activity, is internationally recognized as a high-efficiency and low-toxicity plant growth regulator, and is a novel substance with the most liveliness and vitality for developing high-yield, high-quality, high-efficiency agriculture and ecological agriculture in China. The main functions of the microbial inoculum are to promote cell division, enhance stress resistance and photosynthesis of crops, regulate nutrient distribution in plants, improve crop yield, improve crop quality and the like, and the microbial inoculum has no antagonism and synergistic effect, and can be used in a mixed mode.
At present, the commercial microbial agents are numerous in variety and uneven in quality, and the effective component types and the content of the plant-source pesticide brassinolide are unreasonably added by illegal sales enterprises to reduce cost and benefit, so that crop yield reduction and quality reduction are caused, and serious economic loss is caused for farmers. At the same time, the legal interests of the regular manufacturer are seriously impaired. In addition, as the dosage of brassinolide plant growth regulator pesticides increases and the application range expands increasingly, the safety of brassinolide plant growth regulator is attracting more attention. Research shows that different plant growth regulators have reproductive toxicity, and part of the plant growth regulators can also increase tumor risk, influence myocardial function and the like. Therefore, the development of a rapid, simple, sensitive and accurate analysis method for detecting the variety and the content of the brassinolide active ingredient in the microbial agent is urgent, and the method has great significance for guaranteeing the quality of microbial agent products and promoting the agricultural development.
The QuEChERS technology is a novel pretreatment method developed on the basis of a dispersion solid phase extraction technology, has the advantages of rapidness, simplicity, economy, high efficiency, reliability, safety and the like, and can achieve the optimal purification effect by changing the types and the filling amount of adsorbents for different types of samples. The QuEChERS technology is widely applied to the field of fruit and vegetable sample detection at present, but the application of pesticide residue detection in microbial agents is very few. The ultra-high performance chromatography-tandem mass spectrometry (UPLC-MSMS) has the advantages of wide liquid chromatography analysis range and strong selectivity, and can accurately, qualitatively and quantitatively analyze complex samples by acquiring primary and secondary ion fragments of components to be detected through a triple quadrupole mass spectrometer, and is widely adopted by related standards (national drug inspection and supplement inspection method inspection project approval parts 2006004 and 2012005, BJS 201701, determination of compounds such as sibutramine in food, and the like).
Zhang Junjie et al (pesticide science and administration, 2021,42 (6)) discloses a method for detecting residues of chlorfenapyr and its metabolites on folium Allii tuberosi and rhizoma Zingiberis recens by gas chromatography-tandem mass spectrometry, wherein the combination of purifying agent is 40mg PSA, 10mg C 18 5mg MWCNT. Wu Xuejin et al (southern agricultural journal, 2020, 51 (10): 2532-2539) disclose a QuEChERS decontaminationMethod for synchronously measuring 10 plant growth regulator residues in litchi by using ultra-high performance liquid chromatography-tandem mass spectrometry, wherein methanol-5 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid, v/v) buffer solution is adopted as mobile phase, and 150mg MgSO is adopted 4 、50mg PSA、50mg C 18 As a purification composition.
The current report on residue detection research of brassinolide is relatively few, the variety of detected active ingredients is also few, and the detection of various brassinolide active ingredients in microbial agents is not yet reported. In conclusion, the detection method of 3 brassinolide active ingredients in the microbial agent is established, and the requirements are urgent and have profound significance.
Disclosure of Invention
The invention adopts QuEChERS-UPLC-MSMS to develop a method with the advantages of simple operation, low cost, rapid analysis, high recovery rate and the like, and realizes the detection of trace 3 brassinolide active ingredients in the microbial agent so as to meet the market demand.
Therefore, the invention provides a method for detecting brassinolide in a microbial agent based on a nanomaterial QuEChERS-UPLC-MSMS, which comprises the following steps:
(1) Sample collection
And collecting microbial agent products to be detected, marking, and sealing and storing in a refrigerator at-18 ℃ for later use.
(2) QuEChERS sample pretreatment
Sufficiently grinding a frozen microbial agent sample by adopting a clean mortar, accurately weighing 2-4g of the ground sample into a 50mL centrifuge tube, adding 8-15mL of acetonitrile, rapidly shaking uniformly, adding 0.5-2g of sodium chloride and 3-6g of anhydrous magnesium sulfate, carrying out vortex oscillation extraction at 2500rpm for 7-14min, centrifuging at 5000rpm for 3-6min, and discarding the precipitate to obtain supernatant;
taking 1.5mL of the supernatant into a 2mL centrifuge tube, pre-loading a purifying agent combination and anhydrous magnesium sulfate into the centrifuge tube, carrying out vortex oscillation at 2500rpm for 3-8min, centrifuging at 10000rpm for 2-4min, and passing the supernatant through a 0.22 mu m organic filter membrane for detection.
(3) UPLC-MSMS analysis and detection
And detecting the treated sample to be detected by adopting an UPLC-MSMS (ultra performance liquid chromatography-tandem mass spectrometry) method, and detecting brassinolide and the content thereof in the sample.
In one embodiment of the invention, the brassinolide comprises one or more of 28-epi-homobrassinolide, 28-Gao Yuntai-epi-brassinolide, 22,23, 24-epi-brassinolide.
In one embodiment of the present invention, the step "(2) sample pretreatment" is preferably:
fully grinding a microbial agent sample in a frozen state by adopting a clean mortar, accurately weighing 2.5g of the ground sample in a 50mL centrifuge tube, adding 10mL of acetonitrile, shaking uniformly rapidly, adding 1g of sodium chloride and 4g of anhydrous magnesium sulfate, performing vortex oscillation extraction for 10min at 2500rpm, centrifuging at 5000rpm for 5min, and discarding the precipitate to obtain a supernatant;
taking 1.5mL of the supernatant into a 2mL centrifuge tube, pre-loading a purifying agent combination and anhydrous magnesium sulfate into the centrifuge tube, carrying out vortex oscillation at 2500rpm for 5min, centrifuging at 10000rpm for 3min, and passing the supernatant through a 0.22 mu m organic filter membrane for detection.
In one embodiment of the invention, in the step "(2) sample pretreatment", the detergent combination is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 3-8mg MWCNTs (i.e.nano-ZrO 2 /PSA/C 18 MWCNTs=30/20/30/3-8 mg). Preferably, the combination of the purifying agents is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 3mg MWCNTs (i.e.nano-ZrO 2 /PSA/C 18 MWCNTs=30/20/30/3 mg). The addition amount of the anhydrous magnesium sulfate is 200mg.
Wherein nano-ZrO 2 Refers to nano zirconia; PSA refers to ethylenediamine-N-propyl silanized silica gel adsorbent; c (C) 18 Octadecylsilane chemically amplified (octadecylsilane chemically amplified) (50 μm); MWCNTs refer to multi-walled carbon nanotubes.
In one embodiment of the present invention, in the step "(3) UPLC-MSMS analytical test":
the ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is C 18 (2.6μm.2.1 mm 50 mm); separating the target compound by adopting a gradient elution mode by taking a formic acid aqueous solution (A phase) containing 0.1% and methanol (B phase) as mobile phases, wherein the gradient elution program is as follows: 0min 90%A+10%B,4.0min 5%A+95%B,5.5min5%A+95%B,5.6min 90%A+10%B,7.0min 90%A+10%B; flow rate: 0.40mL/min; the sample volume was 2. Mu.L and the column temperature was 35 ℃.
The mass spectrum conditions are as follows: electrospray ionization (ESI); a positive ion scanning mode, wherein the scanning mode is multi-reaction monitoring (MRM), the temperature is 450 ℃, and the voltage is 5.5kV; the atomizing gas GS1 pressure is 40psi; the atomizing gas GS2 pressure was 40psi.
Another aspect of the invention relates to the use of a detergent combination for cleaning a microbial inoculant sample when detecting the brassinolide content in the microbial inoculant by the QuEChERS-UPLC-MSMS method, wherein the detergent combination is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 3-8mg MWCNTs. Preferably, the combination of the purifying agents is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 、3mg MWCNTs。
The invention uses nano-ZrO 2 、PSA、C 18 And MWCNTs are used as purifying agents, and a method for analyzing trace 28-apparent brassinolide, 28-Gao Yuntai-apparent brassinolide and 22,23, 24-mixed brassinolide in the microbial agent is established based on the QuEChERS-UPLC-MSMS technology.
By adopting the technical scheme, the invention has the following beneficial effects:
the method has the advantages of simple operation, low cost, rapid analysis, high recovery rate and the like, and can realize the detection of trace 3 brassinolide active ingredients in the microbial agent.
In addition, the method can detect three brassinolide components in the microbial agent, namely 28-epi-homobrassinolide, 28-Gao Yuntai brassinolide and 22,23, 24-epi-brassinolide.
Drawings
Fig. 1: total ion flow chromatogram of the target compound when the mobile phase is 0.1% formic acid aqueous solution-methanol (II);
fig. 2: response intensity of 3 brassinolides in mobile phase system of 0.05% formic acid aqueous solution-methanol (I), 0.1% formic acid aqueous solution-methanol (II) and 0.05% formic acid (containing 2mmoL/L ammonium acetate) -ethylmethanol (III);
fig. 3: different nano-ZrO 2 /PSA/C 18 Me of 3 brassinolides under the scavenger combination;
fig. 4: me of 3 brassinolides at different MWCNTs dosages;
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Methodological verification
The 28-epibrassinolide standard used in the following embodiments of the application has a content of 90%, the 28-Gao Yuntai brassinolide standard has a content of 90%, and the 22,23, 24-epibrassinolide standard has a content of 92%, all of which are purchased from Beijing service Chengcheng also trusted technology development Co., ltd; the microbial agent is a biological pesticide microbial agent and a microbial fertilizer microbial agent;
1 materials and methods
1.1 preparation of Standard solution and matrix solution
Accurately weighing a certain amount of brassinolide standard substances (3 effective components, 28-epibrassinolide, 28-Gao Yuntai-epibrassinolide and 22,23, 24-epibrassinolide), respectively preparing standard mother solutions with the concentration of 500mg/L by acetonitrile, mixing and diluting to obtain 3 mixed standard solutions with the concentration of 10mg/L of the effective components, and diluting to 200, 100, 50, 25, 10, 5 and 2.5 mug/L by acetonitrile and a microbial agent matrix solution (the microbial agent matrix solution is obtained by the following 1.2 sample pretreatment step).
1.2QuEChERS sample pretreatment
Fully grinding a microbial agent sample in a frozen state by adopting a clean mortar, accurately weighing 2.5g of the ground sample in a 50mL centrifuge tube, adding 10mL of acetonitrile, shaking uniformly rapidly, adding 1g of sodium chloride and 4g of anhydrous magnesium sulfate, extracting for 10min by vortex oscillation at 2500rpm, centrifuging for 5min at 5000rpm, and purifying the supernatant.
1.5mL of the supernatant was placed in a 2mL centrifuge tube (containing nano-ZrO) 2 /PSA/C 18 MWCNTs=30/20/30/3 mg (which is the most preferred scavenger combination, see "QuEChERS optimization" section for specific screening results) and 200mg anhydrous magnesium sulfate) were vortexed at 2500rpm for 5min, centrifuged at 10000rpm for 3min, and the supernatant was subjected to 0.22 μm organic filter.
1.3 analytical method with instruments
And detecting by adopting a UPLC-MSMS (ultra-high performance liquid chromatography-tandem mass spectrometry) method.
The conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column is C 18 (2.6 μm x 2.1mm x 50 mm); the separation of the target compounds was performed by gradient elution using 0.1% formic acid in water (phase a) and methanol (phase B) as mobile phases (which are the most preferred mobile phases, see section "analytical method optimization" for specific screening results), and the gradient elution procedure was shown in table 1 (which is the most preferred gradient elution procedure, determined by section "analytical method optimization" screening). The sample volume was 2. Mu.L and the column temperature was 35 ℃.
TABLE 1 28-epibrassinolide, 28-Gao Yuntai lactone, 22,23, 24-epibrassinolide gradient elution procedure
The mass spectrum conditions are as follows: electrospray ionization (ESI); a positive ion scanning mode, wherein the scanning mode is multi-reaction monitoring (MRM), the temperature is 450 ℃, and the voltage is 5.5kV; the atomizing gas GS1 pressure is 40psi; the atomizing gas GS2 pressure was 40psi.
4. Matrix effect
The matrix effect (Me) refers to a phenomenon that the signal of a target compound is enhanced and weakened due to interference of co-eluting impurities, and in the analysis process of a complex sample, the ionization degree of ions is affected by the matrix effect, so that the quantitative result of the target compound is affected.
The research adopts an additive test method after extraction to calculate the effect of the extracted additive matrix, and adopts Me to evaluate the purifying effect, and the calculation formula is shown in the following formula.
Me＝Se 2 /Se 1
In the formula, se 1 Response intensity of pure standard solution, se 2 Adding the response intensity of the same amount of analyte to the sample matrix; se (Se) 1 And Se (Se) 2 The same amount of analyte must be added.
When Me is between 0.8 and 1.2, no obvious matrix effect is considered, namely, the purifying effect is good; when Me is less than 0.8, the matrix inhibition effect is stronger, and the purification effect is poorer when the numerical value is smaller; when Me is more than 1.2, the matrix enhancement effect is larger as the numerical value is larger, and the purifying effect is poorer.
2. Results
2.1 Analysis method optimization
A200. Mu.g/L mixed standard solution of 28-epibrassinolide, 28-Gao Yuntai-lactone and 22,23, 24-mixed epibrassinolide was injected into the mass spectrum by a syringe pump for parameter tuning and scanning in ESI positive and negative modes. The results show that the parent ions of the 3 target compounds are [ M+H ]] + A mode. And finally obtaining mass spectrum acquisition parameters (table 2) of 3 compounds in the MRM mode by tuning and optimizing parent ions and child ions of the target compound and the optimal taper hole voltage and collision energy, selecting two groups of ion pairs with optimal sensitivity for detection, wherein one group is used for quantification, and the other group is used for auxiliary qualitative.
Table 2 Mass Spectrometry information for 28-Meta brassinolide, 28-Gao Yuntai lactone, 22,23, 24-Meta brassinolide
Note that: "x" is the quantitative ion pair
Due to ESI of formic acid and ammonium acetate + Enhancement of target Compounds [ M+H ] in mode] + And [ M+NH ] 4 ] + The ionization degree of the common reagent and the two can effectively improve the peak type at the same time, so that the peak type is more sharp and symmetrical, thus the influence of a mobile phase system of 0.05 percent formic acid aqueous solution-methanol (I), 0.1 percent formic acid aqueous solution-methanol (II) and 0.05 percent formic acid (containing 2mmoL/L ammonium acetate) aqueous solution-ethylmethanol (III) on the sensitivity and the peak type of 28-epibrassinolide, 28-Gao Yuntai lactone, 22,23, 24-mixed-epibrassinolide is respectively examined when the gradient elution of a target compound is carried out.
The results of the study showed that the sensitivity of the target compound was low when the mobile phase was III, and that the peak pattern of the 3 compounds was optimal and the sensitivity was highest when the mobile phase was II (fig. 2).
In summary, 0.1% formic acid-methanol is finally determined as a mobile phase system, and all compounds can be effectively separated within 7min by continuously optimizing a gradient elution program, and the total ion flow chromatogram of the target compound is shown in fig. 1. The elution procedure optimally determined is shown in table 1 above.
2.2QuEChERS optimization
At an addition level of 100. Mu.g/kg and a fixed MWCNTs dose of 4mg, nano-ZrO was examined 2 /PSA/C 18 The purifying effect of the purifying agent combination on 28-epibrassinolide, 28-Gao Yuntai-brassinolide and 22,23, 24-mixed-epibrassinolide in the microbial agent matrix (table 3).
As a result of the study, when nano-ZrO 2 /PSA/C 18 When the amount of (C) is 30/20/30mg, me of 3 compounds is between 0.81 and 0.96 (FIG. 3), and the purification effect is best.
Thus, the study finally selects nano-ZrO 2 /PSA/C 18 The next purification optimization of the microbial agent matrix was performed =30/20/30 mg.
TABLE 3 amount of detergent combinations in microbial agent matrices
The MWCNTs can effectively adsorb impurities such as pigment, heavy metal, inorganic nonmetallic ions, aromatic hydrocarbon, antibiotics and the like, and are high-efficiency adsorbents, however, the MWCNTs can adsorb target compounds while adsorbing the impurities, so that the dosage of the MWCNTs needs to be optimized. Based on the above-mentioned results, nano-ZrO was immobilized 2 /PSA/C 18 When the MWCNTs are respectively 3, 5, 8 and 12mg, me of 28-epibrassinolide, 28-Gao Yuntai lactone and 22,23, 24-mixed-epibrassinolide in the microbial agent matrix is examined.
The results show that when the MWCNTs are used in an amount of 3-8mg, me of 3 compounds in the microbial agent matrix is 0.77-1.11, and the purifying effect is good (figure 4). On the basis of ensuring good purifying effect, the principle of cost saving is considered, and the dosage of the MWCNTs is finally determined to be 3mg.
In conclusion, by optimizing the combination of the purifying agents, the optimal combination dosage of the purifying agents for analyzing trace 28-epi-homobrassinolide, 28-Gao Yuntai-lactone and 22,23, 24-mixed-epi-brassinolide in the microbial agent matrix is finally determined to be nano-ZrO 2 /PSA/C 18 /MWCNTs＝30/20/30/3mg。
3 method verification
3.1 detection limit, quantitative limit, standard Curve and R 2
The analysis is carried out by adopting the method obtained by the screening, the mass concentration is taken as an X axis, the corresponding peak area is taken as a Y axis, and a solvent standard curve and a substrate standard curve of the microbial agent substrate are drawn. The results show that 28-surface high brassinolide, 28-Gao Yuntai lactone and 22,23, 24-mixed surface brassinolide in the microbial agent matrix have good linear relationship in the concentration range of 5-100 mug/L, R 2 Between 0.9995 and 0.9999; the LODs of 3 compounds in the microbial agent obtained by using the signal to noise ratio (S/N) of 3 times as LOD and the lowest addition level as LOQ were 0.1-0.3 μg/kg, and the LOQs were 10 μg/kg (Table 4).
Table 4 3 kindsDetection limit, quantitative limit, standard curve and R of brassinolide in solvent and microbial agent matrix 2
3.2 accuracy and precision
The residual limit of brassinolide is not established in China, and with reference to the minimum value of GB 2763-2021MRLs, 4 addition levels of 10, 100, 1000 and 5000 mug/kg are set in a blank microbial agent for 5 parallel tests. The results showed that the average recovery of 3 compounds in the microbial agent matrix was between 79.8% and 103.2% with Relative Standard Deviations (RSDs) of 2.6% to 9.4% at addition levels of 10 to 5000. Mu.g/kg (Table 5). In conclusion, all the results meet the pesticide residue detection standard requirements.
Table 5 average recovery of 3 brassinolide and RSDs% at 4 addition levels in microbial inoculant matrix
In conclusion, the invention uses nano-ZrO 2 、C 18 PSA and MWCNTs are used as purifying agents, and a method for analyzing trace 28-surface high brassinolide, 28-Gao Yuntai lactone and 22,23, 24-mixed surface brassinolide in the microbial agent is established based on the QuEChERS-UPLC-MSMS technology, and the method has the advantages of high sensitivity, strong universality, high accuracy, good stability, simplicity and convenience in operation and the like, and provides technical support for detection of botanical pesticides of the microbial agent.
2. Examples
The content of brassinolide in the microbial agent obtained from the market is analyzed by the preferred analysis method determined by the present study, and the content of brassinolide in the commercial microbial agent is calculated by the microbial agent matrix standard curve. The analysis results are shown in Table 6.
TABLE 6 results of detection of the content of brassinolide in different microbial inoculants on the market
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (8)
1. A method for detecting brassinolide in a microbial agent based on nanomaterial QuEChERS-UPLC-MSMS, the method comprising the steps of:
(1) Sample collection
Collecting microbial agent products to be detected, marking, and sealing and storing in a refrigerator at-18 ℃ for later use;
(2) QuEChERS sample pretreatment
Sufficiently grinding a frozen microbial agent sample by adopting a clean mortar, accurately weighing 2-4g of the ground sample into a 50mL centrifuge tube, adding 8-15mL of acetonitrile, rapidly shaking uniformly, adding 0.5-2g of sodium chloride and 3-6g of anhydrous magnesium sulfate, carrying out vortex oscillation extraction at 2500rpm for 7-14min, centrifuging at 5000rpm for 3-6min, and discarding the precipitate to obtain supernatant;
taking 1.5mL of the supernatant in a 2mL centrifuge tube, wherein the centrifuge tube is pre-filled with a purifying agent combination and anhydrous magnesium sulfate, vortex oscillating for 3-8min at 2500rpm, centrifuging for 2-4min at 10000rpm, and passing the supernatant through a 0.22 mu m organic filter membrane for detection;
(3) UPLC-MSMS analysis and detection
And detecting the treated sample to be detected by adopting an UPLC-MSMS (ultra performance liquid chromatography-tandem mass spectrometry) method, and detecting brassinolide and the content thereof in the sample.
2. The method of claim 1, wherein the brassinolide comprises one or more of 28-epi-homobrassinolide, 28-Gao Yuntai-epi-brassinolide, 22,23, 24-epi-brassinolide.
3. The method according to claim 1, characterized in that the step "(2) sample pretreatment" is preferably:
fully grinding a microbial agent sample in a frozen state by adopting a clean mortar, accurately weighing 2.5g of the ground sample in a 50mL centrifuge tube, adding 10mL of acetonitrile, shaking uniformly rapidly, adding 1g of sodium chloride and 4g of anhydrous magnesium sulfate, performing vortex oscillation extraction for 10min at 2500rpm, centrifuging at 5000rpm for 5min, and discarding the precipitate to obtain a supernatant;
taking 1.5mL of the supernatant into a 2mL centrifuge tube, pre-loading a purifying agent combination and anhydrous magnesium sulfate into the centrifuge tube, carrying out vortex oscillation at 2500rpm for 5min, centrifuging at 10000rpm for 3min, and passing the supernatant through a 0.22 mu m organic filter membrane for detection.
4. A method according to claim 3, wherein the scavenger combination is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 、3-8mg MWCNTs。
5. The method of claim 4, wherein the scavenger combination is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 、3mg MWCNTs。
6. The method according to claim 1, characterized in that in said step "(3) UPLC-MSMS analytical test":
the ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is C 18 (2.6 μm x 2.1mm x 50 mm); separating the target compound by adopting a gradient elution mode by taking a formic acid aqueous solution (A phase) containing 0.1% and methanol (B phase) as mobile phases, wherein the gradient elution program is as follows: 0min 90%A+10%B,4.0min 5%A+95%B,5.5min5%A+95%B,5.6min 90%A+10%B,7.0min 90%A+10%B; flow rate: 0.40mL/min; the sample injection volume is 2 mu L, and the column temperature is 35 ℃;
the mass spectrum conditions are as follows: electrospray ionization (ESI); a positive ion scanning mode, wherein the scanning mode is multi-reaction monitoring (MRM), the temperature is 450 ℃, and the voltage is 5.5kV; the atomizing gas GS1 pressure is 40psi; the atomizing gas GS2 pressure was 40psi.
7. Use of a detergent composition for purifying a microbial inoculant sample when detecting brassinolide content in the microbial inoculant by a QuEChERS-UPLC-MSMS method, characterized in that the detergent composition is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 、3-8mg MWCNTs。
8. The use according to claim 7, wherein the detergent combination is: 30mg nano-ZrO 2 、20mg PSA、30mg C 18 、3mg MWCNTs。
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