CN113406239B - A method for the determination of fluoxastrobin and its metabolites in water - Google Patents

Abstract

The invention belongs to the technical field of substance detection, and provides a method for determining fluoxastrobin and metabolites thereof in water. The determination method provided by the invention has the advantages of simple pretreatment and high detection sensitivity on fluoxastrobin and metabolites thereof in water. The invention adopts APCI ^‑ The linear range of the detection of the o-chlorophenol is 0.0005 to 0.05mg/L, the LOQ is 0.001mg/L, and the LOD is 1.00 multiplied by 10 ^‑2 ng, the response degree of ESI source detection is higher than that of the prior art; LODs of fluoxastrobin, fluoxastrobin metabolite M48-E, and fluoxastrobin M40 were 1.00X 10, respectively ^‑3 ng、1.00×10 ^‑3 ng and 2.5X 10 ^‑3 ng; the minimum detectable concentration (LOQ) of fluoxastrobin, fluoxastrobin metabolite M48-E, and fluoxastrobin M40 in water was 0.00200 mg.L ^‑1 。

Description

A method for the determination of fluoxastrobin and its metabolites in water

technical field

The invention relates to the technical field of substance detection, in particular to a method for determining fluoxastrobin and its metabolites in water.

Background technique

Fluoxastrobin is a wide-spectrum dihydro-dioxazines systemic fungicide variety for stem and leaf treatment developed by Bayer Company in Germany. The product has fast absorption, rain erosion resistance, long application period, It has the advantages of many applicable crops and excellent control. Fluoxastrobin is mainly used to control all fungal pathogens in potatoes, vegetables, cereals and coffee. It has the dual characteristics of rapid sterilization and long-lasting effect, and has good compatibility with crops. Under the conditions of recommended dosage, it is relatively safe for crops, groundwater and the environment. It has good activity against almost all fungal diseases (Basidiomycetes, Ascomycetes, Fungi and Oomycetes) such as rust, net spot, downy mildew and dozens of diseases.

Fluoxastrobin metabolites include o-chlorophenol, M48-E and M40. Fluoxastrobin metabolite o-chlorophenol is very harmful to the environment, especially to water and soil can cause serious pollution. If fluoxastrobin is not used properly, it will pollute the groundwater to a certain extent, and in turn do certain harm to the human body. At present, there are only reports on the synthesis of fluoxastrobin, residue detection in fruits and beverages, the influence of soil microorganisms and soil enzyme activities, residue detection on rice plants, and no detection methods for fluoxastrobin and its metabolites in water. Related reports.

Contents of the invention

In view of this, the object of the present invention is to provide a method for the determination of fluoxastrobin and its metabolites in water. The determination method provided by the invention can accurately detect fluoxastrobin and its metabolites in water.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for determining fluoxastrobin and its metabolites in water, wherein the fluoxastrobin metabolites include o-chlorophenol, M48-E and M40, comprising the following steps:

Mix the water sample to be tested with acetonitrile to obtain the fluoxastrobin-M48-E-M40 machine sample;

Filter the water sample to be tested to obtain the o-chlorophenol sample;

Perform the first ultra-high performance liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 sample on the machine, and obtain the chromatographic information of fluoxastrobin and M48-E;

The second ultra-high liquid chromatography tandem mass spectrometry detection is performed on the fluoxastrobin-M48-E-M40 sample on the machine to obtain M40 chromatographic information;

The third ultra-high liquid chromatography tandem mass spectrometry detection is carried out on the sample of the o-chlorophenol to obtain the chromatographic information of the o-chlorophenol;

Substitute the fluoxastrobin concentration-peak area standard curve, the M48-E concentration-peak area standard curve, the M40 concentration - peak area standard curve and o-chlorophenol concentration-peak area standard curve, obtain the content of fluoxastrobin and its metabolites in water;

The parameters detected by the first ultra-high performance liquid chromatography tandem mass spectrometry include the first mass spectrometry detection parameters and the first chromatographic detection parameters;

The first mass spectrometry detection parameters include: ion source: ESI ⁺ ; capillary voltage: 5.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi; spray gas: 50psi; Injection voltage: 10V; Collision chamber injection voltage: 16V;

The first chromatographic detection parameters include: chromatographic column: ACQUITY UPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, Waters Company, USA; chromatographic column temperature: 40°C; injection volume: 2.00μL; flow rate: 0.3mL min ⁻¹ ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure acetonitrile; gradient washing Off program: 0～0.5min, 65%A; 0.5～2.0min, 65%A～15%A; 2.0～4.0min, 15%A～2%A; 4.0～4.5min, 2%A; 4.5～4.6 min, 2%～65%A; 4.6～6.5min, 65%A;

The parameters detected by the second ultra-high performance liquid chromatography tandem mass spectrometry include a second mass spectrometry detection parameter and a second chromatographic detection parameter;

The second mass spectrometry detection parameters include: ion source: ESI ^- ; capillary voltage: -4.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi; spray gas: 50psi ;Injection voltage: -10V, collision chamber injection voltage: -16V;

The second chromatographic detection parameters include: chromatographic column: ACQUITY UPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, Waters Company, USA; chromatographic column temperature: 40°C; injection volume: 5.00μL; flow rate: 0.3mL min ⁻¹ ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure acetonitrile; gradient washing Off program: 0～0.5min, 55%A; 0.5～3.5min, 55%A～5%A; 3.5～4.0min, 5%A; 4.0～4.1min, 5%～55%A; 4.1～6.0min , 55%A;

The parameters detected by the third ultra-high performance liquid chromatography tandem mass spectrometry include the third mass spectrometry detection parameters and the third chromatographic detection parameters;

The third mass spectrometry detection parameters include: ion source: APCI ^- ; needle current: -3.5mA; ion source temperature: 350°C; curtain gas: 25psi; impingement gas: 8psi; spray gas: 50psi; injection voltage: - 10V; Collision chamber injection voltage: -14V;

Omega Polar C₁₈色谱柱， 3μm，2.1mm×50mm，美国Phenomenex公司；流速：0.40mL/min；柱温： 40℃；进样体积：20.0μL；流动相A为2mmol/L乙酸铵水溶液；流动相B 为色谱纯乙腈；梯度洗脱程序：0～0.5min，80％A；0.5～2.0min，80％A～40％A； 2.0～3.0min，40％A～20％A；3.0～3.5min，20％A；3.5～3.6min，20％A～80％A； 3.6～5.5min，80％A。The third chromatographic detection parameters include: chromatographic column:

Omega Polar C ₁₈ chromatographic column, 3μm, 2.1mm×50mm, American Phenomenex Company; flow rate: 0.40mL/min; column temperature: 40℃; injection volume: 20.0μL; mobile phase A is 2mmol/L ammonium acetate aqueous solution; Phase B is chromatographically pure acetonitrile; gradient elution program: 0~0.5min, 80%A; 0.5~2.0min, 80%A~40%A; 2.0~3.0min, 40%A~20%A; 3.0~3.5 min, 20%A; 3.5～3.6min, 20%A～80%A; 3.6～5.5min, 80%A.

Preferably, in the process of preparing fluoxastrobin-M48-E-M40 on-machine samples, the volume ratio of the water sample to be tested and acetonitrile is 1:1.

Preferably, after the water sample to be tested is mixed with acetonitrile, the resulting mixture is filtered, and the pore size of the filter membrane is 0.22 μm.

Preferably, the pore size of the filter membrane for filtering the water sample to be tested is 0.22 μm.

The invention provides a method for determining fluoxastrobin and its metabolites in water. The determination method of the invention has a simple pretreatment method, and has high detection accuracy and sensitivity for fluoxastrobin and its metabolites in water. The present invention adopts APCI-source to detect o-chlorophenol in the linear range of 0.0005～0.05 mg/L, LOQ is 0.001mg/L, and LOD is 1.00× ^{10-2 ng} , which is higher than the responsivity of ESI source detection in the prior art ( The linear range is 0.05～10mg/L, and the LOQ is 0.03～1.5mg/kg); the LOD of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 are 1.00×10 ^-3 ng, 1.00×10 ^-3 ng and 2.5×10 ^-3 ng; the lowest detection concentration (LOQ) of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 in water is 0.00200mg·L ^{- 1} .

Description of drawings

Fig. 1 is the regression equation of fluoxastrobin standard curve;

Fig. 2 is the standard curve regression equation of fluoxastrobin metabolite M48-E;

Fig. 3 is the standard curve regression equation of fluoxastrobin metabolite M40;

Fig. 4 is the regression equation of the standard curve of fluoxastrobin metabolite o-chlorophenol;

Figure 5 is a diagram of the peak area response results for different ion source temperatures.

Detailed ways

The invention provides a method for determining fluoxastrobin and its metabolites in water, wherein the fluoxastrobin metabolites include o-chlorophenol, M48-E and M40, comprising the following steps:

Mix the water sample to be tested with acetonitrile to obtain the fluoxastrobin-M48-E-M40 machine sample;

Filter the water sample to be tested to obtain the o-chlorophenol sample;

Perform the first ultra-high performance liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 sample on the machine, and obtain the chromatographic information of fluoxastrobin and M48-E;

The second ultra-high liquid chromatography tandem mass spectrometry detection is performed on the fluoxastrobin-M48-E-M40 sample on the machine to obtain M40 chromatographic information;

The third ultra-high liquid chromatography tandem mass spectrometry detection is carried out on the sample of the o-chlorophenol to obtain the chromatographic information of the o-chlorophenol;

Substitute the fluoxastrobin concentration-peak area standard curve, the M48-E concentration-peak area standard curve, the M40 concentration - peak area standard curve and o-chlorophenol concentration-peak area standard curve to obtain the content of fluoxastrobin and its metabolites in water.

In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

The invention mixes the water sample to be tested with acetonitrile to obtain the fluoxastrobin-M48-E-M40 machine sample.

In the present invention, the volume ratio of the water sample to be tested and acetonitrile is preferably 1:1. In the present invention, after the water sample to be tested is mixed with acetonitrile, it is preferred to vortex and filter the resulting mixed system, and the vortex time is preferably 30s; the pore size of the filter membrane is preferably 0.22 μm. In the present invention, the concentration range of each substance in the fluoxastrobin-M48-E-M40 upper machine sample is preferably within the concentration range when the standard curve is established, when the fluoxastrobin-M48-E-M40 upper machine When the concentration of each substance in the sample is higher than the concentration range when the standard curve is established, it preferably also includes: after diluting the fluoxastrobin-M48-E-M40 upper machine sample and then filtering; the diluted reagent is preferably A mixed solvent of deionized water and acetonitrile with a volume ratio of 1:1.

In the invention, the water sample to be tested is filtered to obtain the machine sample of o-chlorophenol. In the present invention, the pore size of the filter membrane for filtering the water sample to be tested is preferably 0.22 μm. In the present invention, the concentration range of o-chlorophenol in the sample on the ortho-chlorophenol is preferably in the concentration range when establishing a standard curve, when the concentration of ortho-chlorophenol in the sample on the ortho-chlorophenol is higher than that when the standard curve is established When the concentration range, preferably also include: filter again after diluting described ortho-chlorophenol upper machine sample; The reagent of described dilution is preferably drinking pure water.

After obtaining the fluoxastrobin-M48-E-M40 on-machine sample, the present invention performs the first ultra-high performance liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 on-machine sample to obtain the fluoxastrobin-M48-E-M40 on-machine sample Chromatographic information for esters and M48-E.

In the present invention, the parameters detected by the first ultra-high performance liquid chromatography tandem mass spectrometry include the first mass spectrometry detection parameters and the first chromatographic detection parameters.

In the present invention, the first mass spectrometry detection parameters include: ion source: ESI ⁺ ; capillary voltage: 5.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi; Spray gas: 50psi; injection voltage: 10V; collision chamber injection voltage: 16V.

In the present invention, the first chromatographic detection parameters include: chromatographic column: ACQUITYUPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, American Waters Company; chromatographic column temperature: 40°C; injection volume: 2.00μL; Flow rate: 0.3mL·min ^-1 ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure Acetonitrile; Gradient elution program: 0～0.5min, 65%A; 0.5～2.0min, 65%A～15%A; 2.0～4.0min, 15%A～2%A; 4.0～4.5min, 2%A ;4.5～4.6min, 2%～65%A; 4.6～6.5min, 65%A.

After obtaining the fluoxastrobin-M48-E-M40 on-board sample, the present invention performs second ultra-high liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 on-board sample to obtain M40 chromatographic information .

In the present invention, the parameters detected by the second ultra-high performance liquid chromatography tandem mass spectrometry include the second mass spectrometry detection parameters and the second chromatographic detection parameters.

In the present invention, the second mass spectrometry detection parameters include: ion source: ESI ⁻ ; capillary voltage: -4.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi ; Spray gas: 50psi; Injection voltage: -10V, Collision chamber injection voltage: -16V.

In the present invention, the second chromatographic detection parameters include: chromatographic column: ACQUITYUPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, Waters Company, USA; chromatographic column temperature: 40°C; injection volume: 5.00μL; Flow rate: 0.3mL·min ^-1 ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure Acetonitrile; Gradient elution program: 0~0.5min, 55%A; 0.5~3.5min, 55%A~5%A; 3.5~4.0min, 5%A; 4.0~4.1min, 5%~55%A; 4.1～6.0min, 55%A.

The on-machine sample of o-chlorophenol is obtained, and the present invention performs third ultra-high liquid chromatography tandem mass spectrometry detection on the on-machine sample of o-chlorophenol to obtain chromatographic information of o-chlorophenol.

In the present invention, the parameters detected by the third ultra-high performance liquid chromatography tandem mass spectrometry include the third mass spectrometry detection parameters and the third chromatography detection parameters.

In the present invention, the third mass spectrometry detection parameters include: ion source: APCI ^- ; needle current: -3.5mA; ion source temperature: 350°C; curtain gas: 25psi; impingement gas: 8psi; spray gas: 50 psi ; Injection voltage: -10V; Collision chamber injection voltage: -14V.

Omega Polar C₁₈色谱柱，3μm，2.1mm×50mm，美国Phenomenex公司；流速：0.40mL/min；柱温：40℃；进样体积：20.0μL；流动相A为2mmol/L乙酸铵水溶液；流动相B为色谱纯乙腈；梯度洗脱程序：0～0.5min，80％A；0.5～2.0min， 80％A～40％A；2.0～3.0min，40％A～20％A；3.0～3.5min，20％A；3.5～3.6min， 20％A～80％A；3.6～5.5min，80％A。In the present invention, the third chromatographic detection parameters include: chromatographic column:

Omega Polar C ₁₈ chromatographic column, 3μm, 2.1mm×50mm, American Phenomenex Company; flow rate: 0.40mL/min; column temperature: 40℃; injection volume: 20.0μL; mobile phase A is 2mmol/L ammonium acetate aqueous solution; Phase B is chromatographically pure acetonitrile; gradient elution program: 0-0.5min, 80%A; 0.5-2.0min, 80%A-40%A; 2.0-3.0min, 40%A-20%A; 3.0-3.5 min, 20%A; 3.5-3.6min, 20%A-80%A; 3.6-5.5min, 80%A.

In the present invention, the qualitative and quantitative modes of fluoxastrobin and its metabolites are all MRM multiple reaction monitoring modes, and Table 1 is the quantitative and qualitative parameters of fluoxastrobin and its metabolites in the MRM multiple reaction monitoring mode .

Table 1 Quantitative and qualitative parameters of fluoxastrobin and its metabolites in MRM multiple reaction monitoring mode

*Qualitative ion

After obtaining the fluoxastrobin chromatographic information, M48-E chromatographic information, M40 chromatographic information and o-chlorophenol chromatographic information, the present invention combines the fluoxastrobin chromatographic information, M48-E chromatographic information, M40 chromatographic information and o-chlorophenol The phenol chromatographic information was substituted into the standard curve of fluoxastrobin concentration-peak area, M48-E concentration-peak area standard curve, M40 concentration-peak area standard curve and o-chlorophenol concentration-peak area standard curve to obtain fluoxastrobin and the content of its metabolites.

In the present invention, the acquisition method of the fluoxastrobin concentration-peak area standard curve, M48-E concentration-peak area standard curve and M40 concentration-peak area standard curve preferably includes the following steps:

Take by weighing 0.0120g of fluoxastrobin standard substance, and set the volume to 10.00mL with acetonitrile to obtain a concentration of 1.19×10 ³ mg/L fluoxastrobin standard stock solution; the purity of the fluoxastrobin standard substance is preferably 99.33%;

Weigh 0.0183g of fluoxastrobin metabolite M48-E, and dilute to 10.00mL with acetonitrile to obtain a standard stock solution of fluoxastrobin metabolite M48-E with a concentration of 1.73× ¹⁰³ mg/L; The purity of the strytostrobin metabolite M48-E is preferably 94.6%;

Weigh 0.0120 g of fluoxastrobin metabolite M40, and dilute to 10.00mL with acetonitrile to obtain a standard stock solution of fluoxastrobin metabolite M40 with a concentration of 1.11× ¹⁰³ mg/L; the fluoxastrobin metabolite The purity of the compound M40 is preferably 92.45%;

Dilute the fluoxastrobin standard stock solution, fluoxastrobin metabolite M48-E standard stock solution and fluoxastrobin metabolite M40 standard stock solution with acetonitrile to obtain fluoxastrobin and fluoxastrobin metabolite M48 -E and fluoxastrobin metabolite M40 concentration are both 10.0mg L ^-1 mixed standard working solution;

Use acetonitrile and deionized water with a volume ratio of 1:1 to dilute the 10.0 mg·L ^-1 mixed standard working solution step by step to obtain concentrations of 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500 mg· L ^-1 mixed standard working solution;

The series of mixed standard working solutions were detected by the first ultra-high performance liquid chromatography tandem mass spectrometry, and the chromatographic information of fluoxastrobin and fluoxastrobin metabolite M48-E were obtained; the chromatographic information of fluoxastrobin and fluoxastrobin The concentration was fitted to obtain the fluoxastrobin concentration-peak area standard curve; the chromatographic information of the fluoxastrobin metabolite M48-E and the concentration of the fluoxastrobin metabolite M48-E were fitted to obtain, M48-E Concentration-peak area standard curve.

In the present invention, the parameters detected by the first ultra-high liquid chromatography tandem mass spectrometry are preferably consistent with the above technical solution and will not be repeated here.

The series of mixed standard working solutions were detected by the second ultra-high performance liquid chromatography tandem mass spectrometry to obtain the chromatographic information of the metabolite M40 of fluoxastrobin, and the chromatographic information of the metabolite M40 of fluoxastrobin and the The concentrations were fitted to obtain the concentration-peak area standard curve of fluoxastrobin metabolite M40.

In the present invention, the parameters detected by the second ultra-high performance liquid chromatography tandem mass spectrometry are preferably consistent with the above technical solution, and will not be repeated here.

In the present invention, the acquisition method of the o-chlorophenol concentration-peak area standard curve preferably comprises the following steps:

Weigh 0.0116g of o-chlorophenol standard substance, and dilute to 10.00mL with acetonitrile to obtain o-chlorophenol standard stock solution with a concentration of 1.16×10 ³ mg/L; the purity of the o-chlorophenol standard substance is preferably 99.8%;

Dilute the o-chlorophenol standard stock solution with drinking purified water to obtain a concentration of o-chlorophenol standard working solution that is 10.0, 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500mg/L;

Perform the third ultra-high liquid chromatography tandem mass spectrometry detection on a series of o-chlorophenol standard working solutions to obtain the chromatographic information of o-chlorophenol, and fit the chromatographic information of o-chlorophenol to the concentration of o-chlorophenol to obtain the concentration of o-chlorophenol - Peak area standard curve.

In the present invention, the parameters detected by the third ultra-high liquid chromatography tandem mass spectrometer are preferably consistent with the above technical solution and will not be repeated here.

The determination methods of fluoxastrobin and its metabolites in water provided by the present invention will be described in detail below in conjunction with the examples, but they cannot be interpreted as limiting the protection scope of the present invention.

Example 1

1.1 Instruments and reagents

Instruments and equipment: ABSciex5500 liquid chromatography tandem mass spectrometry (ABSciex, USA), ACQUITYUPLC ^TM BEH C ₁₈ chromatographic column (1.7μm, 2.1mm×100mm, Waters, USA), BSA224S electronic balance (sensitivity 0.0001g, Germany Sartorius Group), MX-S adjustable mixer (Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.), 50mL PPTE centrifuge tube, 0.22μm organic syringe filter membrane.

Reagents: fluoxastrobin (purity: 99.33%, Dr. Ehrenstorfer GmbH), o-chlorophenol (purity: 99.8%), fluoxastrobin metabolite M48-E (purity: 94.6%), fluoxastrobin metabolite M40 (purity: 92.45%), three metabolites are all provided by Beijing Kefa Weiye Pesticide Technology Center, acetonitrile (chromatographically pure, Merck company), formic acid and ammonium acetate (chromatographically pure, Anaqua Chemicals Supply company), drinking purified water ( Guangzhou Watsons Food & Beverage Co., Ltd).

1.2 Preparation of standard solution

Weigh 0.0120 g of fluoxastrobin standard substance (purity is 99.33%), and dilute to 10.00 mL with acetonitrile to obtain a standard stock solution with a concentration of 1.19×10 ³ mg/L; weigh fluoxastrobin metabolite M48- E 0.0183 g (purity 94.6%), dilute to 10.00mL with acetonitrile to obtain a standard stock solution with a concentration of 1.73× ¹⁰³ mg/L; weigh 0.0120 g of fluoxastrobin metabolite M400.0120 g (purity ), and dilute to 10.00mL with acetonitrile to obtain a standard stock solution with a concentration of 1.11× ¹⁰³ mg/L; weigh 0.0116g of o-chlorophenol standard substance (purity is 99.8%), and dilute to 10.00mL with acetonitrile to obtain The standard stock solution with a concentration of 1.16×10 ³ mg/L should be refrigerated at 0°C to 5°C for future use.

Dilute the standard stock solution of fluoxastrobin and its metabolites (M48-E and M40) with acetonitrile to obtain a mixed standard working solution with a concentration of 10.0 mg L ^-1 ; use V _acetonitrile : V _water = 1:1 for stepwise dilution Mixed standard working solution of 10.0mg L ^-1 to obtain mixed standard working solution with concentrations of 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500mg L ^-1 ; dilute o-chlorophenol standard stock solution with drinking pure water , to obtain a concentration of 10.0, 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500mg/L o-chlorophenol standard working solution.

1.3 Sample pretreatment

Use acetonitrile to dilute 5.000mL of the water sample to 10.00mL, vortex for 30s; pass through a 0.22 μm filter membrane or dilute with a mixed solvent of V _acetonitrile : V _water = 1:1 and pass through a 0.22 μm filter membrane to obtain fluorine Azoxystrobin-M48-E-M40 on-board samples.

O-chlorophenol: pass the water sample to be tested through a 0.22 μm filter membrane, or pass through a 0.22 μm filter membrane after dilution with drinking purified water, to obtain the o-chlorophenol sample on the machine.

1.4 Mass Spectrometry Conditions

Fluoxastrobin, fluoxastrobin metabolite M48-E: ion source: ESI ⁺ ; capillary voltage 5.5kV, ion source temperature 550 ℃, air curtain gas 35psi, spray gas 7psi, auxiliary heating gas 50psi, spray gas 50psi, The injection voltage is 10V, and the collision chamber injection voltage is 16V;

Fluoxastrobin metabolite M40: ion source: ESI ^- ; capillary voltage -4.5kV, ion source temperature 550°C, air curtain gas 35psi, spray gas 7psi, auxiliary heating gas 50psi, spray gas 50psi, injection voltage -10V, Collision chamber injection voltage -16V;

O-chlorophenol: ion source: APCI ^- ; needle current -3.5mA, ion source temperature 350°C, curtain gas 25psi, impingement gas 8psi, spray gas 50psi, injection voltage -10V, collision chamber injection voltage -14V.

The qualitative and quantitative modes of fluoxastrobin and its metabolites are MRM multiple reaction monitoring mode, and the qualitative parameters are shown in Table 1.

1.5 Chromatographic conditions

Fluoxastrobin, fluoxastrobin metabolite M48-E: Chromatographic column: ACQUITYUPLC ^TM BEH C ₁₈ column (1.7μm, 2.1mm×100mm, American Waters Company); chromatographic column temperature is 40°C; injection volume is 2.00 μL; flow rate is 0.3mL·min ^-1 ; mobile phase A is 0.1% formic acid + 2mmol/L ammonium acetate aqueous solution; mobile phase B is chromatographically pure acetonitrile; gradient elution program: 0～0.5min, 65%A; 0.5～ 2.0min, 65%A～15%A; 2.0～4.0min, 15%A～2%A; 4.0～4.5min, 2%A; 4.5～4.6min, 2%～65%A; 4.6～6.5min, 65% A.

Fluoxastrobin metabolite M40: Chromatographic column: ACQUITYUPLC ^TM BEH C ₁₈ column (1.7μm, 2.1mm×100mm, Waters, USA); column temperature is 40°C; injection volume is 5.00μL; flow rate is 0.3mL. min ^-1 ; mobile phase A is 0.1% formic acid + 2mmol/L ammonium acetate aqueous solution; mobile phase B is chromatographically pure acetonitrile; gradient elution program: 0～0.5min, 55%A; 0.5～3.5min, 55%A～ 5%A; 3.5~4.0min, 5%A; 4.0~4.1min, 5%~55%A; 4.1~6.0min, 55%A.

OmegaPolar C₁₈色谱柱(3μm，2.1mm×50mm，美国Phenomenex公司)；流速为0.40mL/min；柱温为40℃；进样体积为20.0 μL；流动相A为2mmol/L乙酸铵水溶液；流动相B为色谱纯乙腈；梯度洗脱程序：0～0.5min，80％A；0.5～2.0min，80％A～40％A；2.0～3.0min， 40％A～20％A；3.0～3.5min，20％A；3.5～3.6min，20％A～80％A；3.6～5.5min， 80％A。o-Chlorophenol: Column:

OmegaPolar C ₁₈ chromatographic column (3μm, 2.1mm×50mm, American Phenomenex Company); flow rate is 0.40mL/min; column temperature is 40°C; injection volume is 20.0 μL; mobile phase A is 2mmol/L ammonium acetate aqueous solution; Phase B is chromatographically pure acetonitrile; gradient elution program: 0-0.5min, 80%A; 0.5-2.0min, 80%A-40%A; 2.0-3.0min, 40%A-20%A; 3.0-3.5 min, 20%A; 3.5-3.6min, 20%A-80%A; 3.6-5.5min, 80%A.

2 Results and Analysis

2.1 Regression equation

The mixed standard working solution with the concentration of 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500mg ^L The phenol standard working solution was detected and analyzed according to the above-mentioned mass spectrometry conditions-chromatographic conditions, and a standard curve was constructed based on concentration-peak area; the results are shown in Figures 1 to 4, specifically:

Fluoxastrobin: y=415730151x+82490, R ² =0.9999;

Fluoxastrobin metabolite M48-E: y=65518514x-5619, R ² =1.0000;

Fluoxastrobin metabolite M40: y=1964557x-381, R ² =0.9996;

Fluoxastrobin metabolite o-chlorophenol: y=9337889x+5757, R ² =0.9995.

2.2 Recovery and precision

Fluoxastrobin and its metabolites (M48-E and M40) at three spiked levels (0.002mg·L ^-1 , 0.100mg·L ^-1 and 10.0mg·L ^-1 ), the average of 5 replicates The recoveries and relative standard deviations are shown in Table 2. O-chlorophenol was spiked at three levels (0.001, 0.100 and 10.0 mg·L ^-1 ), and five parallel samples were set for each concentration. The average recoveries and relative standard deviations are shown in Table 2.

The recovery and relative standard deviation of fluoxastrobin and its metabolites in table 2 water

It can be seen from Table 2 that the average recoveries of fluoxastrobin and its metabolites (M48-E and M40) were 81.8% to 109% at the addition levels of 0.00200, 0.100 and 10.0 mg·L ^-1 of the samples, compared with the standard Deviation RSD (n=5) is 1.72%～7.38%; o-Chlorophenol at 0.00100, 92.8mg·L ^-1 addition level, the average recovery rate is 89.6% and 102%, relative standard deviation RSD (n=3) is 6.06% and 8.84%.

2.3 Detection limit and quantification limit

The minimum detection amount (LOD) of the instrument was multiplied by the lowest level concentration of the standard curve by the injection volume. The LODs of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 were 1.00×10 ^-3 ng, 1.00×10 ^-3 ng and 2.5×10 ^{- 3} ng; the lowest detectable concentration (LOQ) of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 in water was 0.00200 mg·L ^-1 ; the minimum detectable amount (LOD) of o-chlorophenol was 1.00×10 ^-2 ng, and the minimum detectable concentration (LOQ) of o-chlorophenol in water was 0.00100 mg·L ^-1 .

2.4 Optimization of pre-processing method

Fluoxastrobin and its metabolites (M48-E and M40): Two organic solvents (ethyl acetate and acetonitrile) extraction and acetonitrile dilution three pretreatment methods were selected. The results showed that ethyl acetate extracted fluoxastrobin and The recoveries of its metabolites (M48-E and M40) (n=3) were 84.5%～94.4%, 39.8%～46.9%, 39.0%～39.7%, and the recoveries of M48-E and M40 could not meet the requirement The recoveries of acetonitrile to extract fluoxastrobin and its metabolites (M48-E and M40) (n=3) were respectively 98.2%～103%, 89.3%～97.2%, 104%～112%, and the recoveries were all Can meet the requirements; the recoveries of fluoxastrobin and its metabolites (M48-E and M40) (n=3) diluted with acetonitrile are respectively 99.3%～118%, 99.5%～104%, 100%～107%, The recovery rate can meet the requirements; since the standard curve of the standard working solution prepared by acetonitrile is not linear, in order to eliminate the solvent effect, the method of acetonitrile dilution is selected as the optimal pretreatment method

2.5 Selection of o-chlorophenol mass spectrometry conditions

O-chlorophenol: Compared with ESI ^- source and APCI ^- source, the response value of APCI ^- source is higher for samples with the same concentration, so APCI ^- source is selected for detection.

Optimize the ion source parameters: compare the response values of the peak area when the curtain gas is 25psi, 30psi and 35psi respectively, the results are: when the curtain gas is 25psi, the peak area is 253347, when the curtain gas is 30psi, the peak area is 145218, when the curtain gas is 35psi, The peak area is 170770. It can be seen that the response value of air curtain air is the highest at 25psi, and the air curtain air is 25psi.

Ion source temperature: compare the peak area response values at ion source temperatures of 400°C, 350°C, 300°C and 250°C, and the results are shown in Figure 5. It can be seen from Figure 5 that when the ion source temperature is 350°C, the response value of the peak area is the highest. For o-chlorophenol, the sensitivity is greatly improved by optimizing the mass spectrometry parameters.

2.6 Optimization of chromatographic conditions

Using 0.1% formic acid aqueous solution-acetonitrile, 2mmol/L ammonium acetate aqueous solution-acetonitrile, 0.1% formic acid+2mmol/L ammonium acetate aqueous solution-acetonitrile as the elution solvent, compare the response and peak shape of the target object Esters and their metabolites (M48-E and M40) have high sensitivity, moderate retention time and good peak shape under the elution conditions of 0.1% formic acid + 2mmol/L ammonium acetate aqueous solution and acetonitrile; Under the elution conditions of ammonium acetate aqueous solution and acetonitrile, the response value is higher and the peak shape is better.

Example 2

Actual sample testing

In February 2021, screening of actual samples collected from 5 locations in Hangzhou, namely West Lake Water, Qiantang River Water, Canal Water, Jianqiao Hengtang River Water, and river water near the laboratory, found that the detection concentration of fluoxastrobin at these 5 points was Between 0.00000717 and 0.0000226 mg·L ^-1 , all were lower than the minimum detection concentration, and none of the three metabolites of fluoxastrobin were detected.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (4)

1. an assay method for fluoxastrobin and its metabolites in water, the fluoxastrobin metabolites comprise o-chlorophenol, M48-E and M40, comprising the following steps: Mix the water sample to be tested with acetonitrile to obtain the fluoxastrobin-M48-E-M40 machine sample; Filter the water sample to be tested to obtain the o-chlorophenol sample; Perform the first ultra-high performance liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 sample on the machine, and obtain the chromatographic information of fluoxastrobin and M48-E; Carry out the second ultra-high performance liquid chromatography tandem mass spectrometry detection to the fluoxastrobin-M48-E-M40 upper machine sample, obtain M40 chromatographic information; Carry out the third ultra-high performance liquid chromatography tandem mass spectrometry detection to described o-chlorophenol upper machine sample, obtain o-chlorophenol chromatographic information; Substitute the fluoxastrobin concentration-peak area standard curve, the M48-E concentration-peak area standard curve, the M40 concentration - peak area standard curve and o-chlorophenol concentration-peak area standard curve, obtain the content of fluoxastrobin and its metabolites in water; The parameters detected by the first ultra-high performance liquid chromatography tandem mass spectrometry include the first mass spectrometry detection parameters and the first chromatographic detection parameters; The first mass spectrometry detection parameters include: ion source: ESI ⁺ ; capillary voltage: 5.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi; spray gas: 50psi; Injection voltage: 10V; Collision chamber injection voltage: 16V; The first chromatographic detection parameters include: chromatographic column: ACQUITY UPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, Waters Company, USA; chromatographic column temperature: 40°C; injection volume: 2.00μL; flow rate: 0.3mL min ⁻¹ ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure acetonitrile; gradient washing Off program: 0～0.5min, 65%A; 0.5～2.0min, 65%A～15%A; 2.0～4.0min, 15%A～2%A; 4.0～4.5min, 2%A; 4.5～4.6 min, 2%～65%A; 4.6～6.5min, 65%A; The parameters detected by the second ultra-high performance liquid chromatography tandem mass spectrometry include a second mass spectrometry detection parameter and a second chromatographic detection parameter; The second mass spectrometry detection parameters include: ion source: ESI ^- ; capillary voltage: -4.5kV; ion source temperature: 550°C; curtain gas: 35psi; impingement gas: 7psi; auxiliary heating gas: 50psi; spray gas: 50psi ;Injection voltage: -10V, collision chamber injection voltage: -16V; The second chromatographic detection parameters include: chromatographic column: ACQUITY UPLC ^TM BEH C ₁₈ column, 1.7μm, 2.1mm×100mm, Waters Company, USA; chromatographic column temperature: 40°C; injection volume: 5.00μL; flow rate: 0.3mL min ⁻¹ ; mobile phase A is formic acid-ammonium acetate aqueous solution, the volume concentration of formic acid in the formic acid-ammonium acetate aqueous solution is 0.1%, and the concentration of ammonium acetate is 2mmol/L; mobile phase B is chromatographically pure acetonitrile; gradient washing Off program: 0～0.5min, 55%A; 0.5～3.5min, 55%A～5%A; 3.5～4.0min, 5%A; 4.0～4.1min, 5%～55%A; 4.1～6.0min , 55%A; The parameters detected by the third ultra-high performance liquid chromatography tandem mass spectrometry include the third mass spectrometry detection parameters and the third chromatographic detection parameters; The third mass spectrometry detection parameters include: ion source: APCI ^- ; needle current: -3.5mA; ion source temperature: 350°C; curtain gas: 25psi; impingement gas: 8psi; spray gas: 50psi; injection voltage: - 10V; Collision chamber injection voltage: -14V; The third chromatographic detection parameters include: chromatographic column:

Omega Polar C ₁₈ chromatographic column, 3μm, 2.1mm×50mm, American Phenomenex Company; flow rate: 0.40mL/min; column temperature: 40℃; injection volume: 20.0μL; mobile phase A is 2mmol/L ammonium acetate aqueous solution; Phase B is chromatographically pure acetonitrile; gradient elution program: 0-0.5min, 80%A; 0.5-2.0min, 80%A-40%A; 2.0-3.0min, 40%A-20%A; 3.0-3.5 min, 20%A; 3.5~3.6min, 20%A~80%A; 3.6~5.5min, 80%A. 2. The assay method according to claim 1, characterized in that in the process of preparing fluoxastrobin-M48-E-M40 on-board samples, the volume ratio of the water sample to be tested and acetonitrile is 1:1. 3. The assay method according to claim 1, characterized in that, after the water sample to be tested is mixed with acetonitrile, the resulting mixture is filtered, and the filter membrane pore size of the filter is 0.22 μm. 4. The assay method according to claim 1, characterized in that the pore size of the filter membrane for filtering the water sample to be tested is 0.22 μm.

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