1. A method for measuring fluoxastrobin and metabolites thereof in water, the fluoxastrobin metabolites comprise o-chlorophenol, M48-E and M40, and the method comprises the following steps:

mixing a water sample to be detected with acetonitrile to obtain a fluoxastrobin-M48-E-M40 computer sample;

filtering a water sample to be detected to obtain an o-chlorophenol on-machine sample;

performing first ultra performance liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain the chromatographic information of fluoxastrobin and M48-E;

performing second ultrahigh liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain M40 chromatographic information;

performing third ultrahigh liquid chromatography tandem mass spectrometry detection on the o-chlorophenol on-machine sample to obtain o-chlorophenol chromatographic information;

substituting the fluoxastrobin chromatographic information, the chromatographic information of M48-E, the chromatographic information of M40 and the o-chlorophenol chromatographic information into a fluoxastrobin concentration-peak area standard curve, an M48-E concentration-peak area standard curve, an M40 concentration-peak area standard curve and an o-chlorophenol concentration-peak area standard curve to obtain the content of fluoxastrobin and metabolites thereof in the water;

the parameters of the first ultra performance liquid chromatography tandem mass spectrometry detection comprise a first mass spectrometry detection parameter and a first chromatographic detection parameter;

the first mass spectrometry detection parameter comprises: an ion source: ESI⁺(ii) a Capillary voltage: 5.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: 10V; collision cell emission voltage: 16V;

the first chromatographic detection parameters include: a chromatographic column: ACQUITY UPLC^TMBEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 2.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 65% A; 0.5-2.0 min, 65-15% A; 2.0-4.0 min, 15-2% A; 4.0-4.5 min, 2% A; 4.5-4.6 min, 2% -65% A; 4.6-6.5 min, 65% A;

the parameters of the second ultra performance liquid chromatography tandem mass spectrometry detection comprise a second mass spectrometry detection parameter and a second chromatographic detection parameter;

the second mass spectrometry detection parameters include: an ion source: ESI^-(ii) a Capillary voltage: -4.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: -10V, collision cell ejection voltage: -16V;

the second color spectrumThe detection parameters include: a chromatographic column: ACQUITY UPLC^TMBEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 5.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 55% A; 0.5-3.5 min, 55-5% A; 3.5-4.0 min, 5% A; 4.0-4.1 min, 5% -55% A; 4.1-6.0 min, 55% A;

the third ultra performance liquid chromatography tandem mass spectrometry detection parameters comprise a third mass spectrometry detection parameter and a third chromatography detection parameter;

the third mass spectrometric detection parameter comprises: an ion source: APCI^-(ii) a Needle current: -3.5 mA; ion source temperature: 350 ℃; air curtain air: 25 psi; and (3) collision gas spraying: 8 psi; spraying mist: 50 psi; injection voltage: -10V; collision cell emission voltage: -14V;

the third spectral detection parameters include: a chromatographic column:Omega Polar C₁₈column, 3 μm, 2.1mm × 50mm, Phenomenex, USA; flow rate: 0.40 mL/min; column temperature: 40 ℃; sample introduction volume: 20.0 mu L; the mobile phase A is 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 80% A; 0.5-2.0 min, 80-40% A; 2.0-3.0 min, 40-20% A; 3.0-3.5 min, 20% A; 3.5-3.6 min, 20-80% A; 3.6-5.5 min, 80% A.

2. The determination method according to claim 1, wherein in the process of preparing the fluoxastrobin-M48-E-M40 computer sample, the volume ratio of the water sample to be determined to acetonitrile is 1: 1.

3. the method according to claim 1, wherein the mixture of the sample of water to be tested and acetonitrile is filtered, and the pore size of the filter membrane is 0.22 μm.

4. The method according to claim 1, wherein the pore size of the filter for filtering the sample of water to be tested is 0.22 μm.

Background

The fluoxastrobin is a broad-spectrum dihydrooxazine (dihydro-dioxazines) systemic stem and leaf treatment bactericide variety developed by Bayer in Germany, and the product has the advantages of quick absorption, rain wash resistance, long application adaptation period, more applicable crops and excellent prevention and treatment. The fluoxastrobin is mainly used for preventing and controlling all fungi germs in potatoes, vegetables, cereals and coffee, has the dual characteristics of quick sterilization and long lasting time, and has good compatibility to crops. Is relatively safe to crops, underground water and environment under the condition of recommended dosage. Has good activity on almost all diseases of fungi (Basidiomycetes, ascomycetes, fungi known as fungi and oomycetes) such as rust disease, net blotch, downy mildew and other dozens of diseases.

The fluoxastrobin metabolites include o-chlorophenol, M48-E and M40. The fluoxastrobin metabolite o-chlorophenol has great harm to the environment, and can cause serious pollution to water and soil in particular. If the fluoxastrobin is improperly applied, the fluoxastrobin causes certain pollution to underground water and further has certain harm to human bodies. At present, only synthesis, residual detection in fruits and beverages, influence of soil microorganisms and soil enzyme activity and residual detection on rice plants are reported on fluoxastrobin, and no related report is found on an aquatic detection method of fluoxastrobin and metabolites thereof.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for measuring fluoxastrobin and metabolites thereof in water. The determination method provided by the invention can accurately detect fluoxastrobin and metabolites thereof in water.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for measuring fluoxastrobin and metabolites thereof in water, wherein the fluoxastrobin metabolites comprise o-chlorophenol, M48-E and M40, and the method comprises the following steps:

mixing a water sample to be detected with acetonitrile to obtain a fluoxastrobin-M48-E-M40 computer sample;

filtering a water sample to be detected to obtain an o-chlorophenol on-machine sample;

performing first ultra performance liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain the chromatographic information of fluoxastrobin and M48-E;

performing second ultrahigh liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain M40 chromatographic information;

performing third ultrahigh liquid chromatography tandem mass spectrometry detection on the o-chlorophenol on-machine sample to obtain o-chlorophenol chromatographic information;

substituting the fluoxastrobin chromatographic information, the chromatographic information of M48-E, the chromatographic information of M40 and the o-chlorophenol chromatographic information into a fluoxastrobin concentration-peak area standard curve, an M48-E concentration-peak area standard curve, an M40 concentration-peak area standard curve and an o-chlorophenol concentration-peak area standard curve to obtain the content of fluoxastrobin and metabolites thereof in the water;

the parameters of the first ultra performance liquid chromatography tandem mass spectrometry detection comprise a first mass spectrometry detection parameter and a first chromatographic detection parameter;

the first mass spectrometry detection parameter comprises:an ion source: ESI⁺(ii) a Capillary voltage: 5.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: 10V; collision cell emission voltage: 16V;

the first chromatographic detection parameters include: a chromatographic column: ACQUITY UPLC^TM BEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 2.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 65% A; 0.5-2.0 min, 65-15% A; 2.0-4.0 min, 15-2% A; 4.0-4.5 min, 2% A; 4.5-4.6 min, 2% -65% A; 4.6-6.5 min, 65% A;

the parameters of the second ultra performance liquid chromatography tandem mass spectrometry detection comprise a second mass spectrometry detection parameter and a second chromatographic detection parameter;

the second mass spectrometry detection parameters include: an ion source: ESI^-(ii) a Capillary voltage: -4.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: -10V, collision cell ejection voltage: -16V;

the second chromatographic detection parameters include: a chromatographic column: ACQUITY UPLC^TMBEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 5.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 55% A; 0.5-3.5 min, 55-5% A; 3.5-4.0 min, 5% A; 4.0-4.1 min, 5% -55% A; 4.1-6.0 min, 55% A;

the third ultra performance liquid chromatography tandem mass spectrometry detection parameters comprise a third mass spectrometry detection parameter and a third chromatography detection parameter;

the third mass spectrometric detection parameter comprises: an ion source: APCI^-(ii) a Needle current: -3.5 mA; ion source temperature: 350 ℃; air curtain air: 25 psi; and (3) collision gas spraying: 8 psi; spraying mist: 50 psi; injection voltage: -10V; collision cell emission voltage: -14V;

the third spectral detection parameters include: a chromatographic column:Omega Polar C₁₈column, 3 μm, 2.1mm × 50mm, Phenomenex, USA; flow rate: 0.40 mL/min; column temperature: 40 ℃; sample introduction volume: 20.0 mu L; the mobile phase A is 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 80% A; 0.5-2.0 min, 80-40% A; 2.0-3.0 min, 40-20% A; 3.0-3.5 min, 20% A; 3.5-3.6 min, 20-80% A; 3.6-5.5 min, 80% A.

Preferably, in the process of preparing the fluoxastrobin-M48-E-M40 computer sample, the volume ratio of the water sample to be detected to the acetonitrile is 1: 1.

preferably, after the water sample to be detected and acetonitrile are mixed, filtering the obtained mixed solution, wherein the aperture of the filtered filter membrane is 0.22 μm.

Preferably, the aperture of the filter membrane for filtering the water sample to be detected is 0.22 μm.

The invention provides a method for measuring fluoxastrobin and metabolites thereof in water, and the method has the advantages of simple pretreatment method, high detection accuracy and high sensitivity for fluoxastrobin and metabolites thereof in water. The linear range of the detection of the o-chlorophenol by adopting an APCI-source is 0.0005-0.05 mg/L, the LOQ is 0.001mg/L, and the LOD is 1.00 multiplied by 10^{- 2}ng is higher than the responsiveness (the linear range is 0.05-10 mg/L, and the LOQ is 0.03-1.5 mg/kg) of ESI source detection in the prior art; LOD of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 were 1.00X 10 respectively^-3ng、1.00×10^{- 3}ng and 2.5X 10^-3ng; the minimum detection concentration (LOQ) of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 in water was 0.00200Mg·L^-1。

Drawings

FIG. 1 is a regression equation of the fluoxastrobin standard curve;

FIG. 2 is a regression equation of the standard curve for fluoxastrobin metabolite M48-E;

FIG. 3 is a regression equation of the standard curve for fluoxastrobin metabolite M40;

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

FIG. 5 is a graph of peak area response results for different ion source temperatures.

Detailed Description

The invention provides a method for measuring fluoxastrobin and metabolites thereof in water, wherein the fluoxastrobin metabolites comprise o-chlorophenol, M48-E and M40, and the method comprises the following steps:

mixing a water sample to be detected with acetonitrile to obtain a fluoxastrobin-M48-E-M40 computer sample;

filtering a water sample to be detected to obtain an o-chlorophenol on-machine sample;

performing first ultra performance liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain the chromatographic information of fluoxastrobin and M48-E;

performing second ultrahigh liquid chromatography tandem mass spectrometry on the fluoxastrobin-M48-E-M40 computer sample to obtain M40 chromatographic information;

performing third ultrahigh liquid chromatography tandem mass spectrometry detection on the o-chlorophenol on-machine sample to obtain o-chlorophenol chromatographic information;

substituting the fluoxastrobin chromatographic information, the chromatographic information of M48-E, the chromatographic information of M40 and the o-chlorophenol chromatographic information into a fluoxastrobin concentration-peak area standard curve, an M48-E concentration-peak area standard curve, an M40 concentration-peak area standard curve and an o-chlorophenol concentration-peak area standard curve to obtain the content of fluoxastrobin and metabolites thereof in the water.

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

According to the invention, a water sample to be detected and acetonitrile are mixed to obtain the fluoxastrobin-M48-E-M40 computer sample.

In the invention, the volume ratio of the water sample to be detected to the acetonitrile is preferably 1: 1. in the invention, after the water sample to be detected is mixed with acetonitrile, preferably, the obtained mixed system is subjected to vortex and filtration, wherein the vortex time is preferably 30 s; the pore size of the filtration membrane is preferably 0.22 μm. In the present invention, the concentration range of each substance in the fluoxastrobin-M48-E-M40 computer sample is preferably in the concentration range when a standard curve is established, and when the concentration of each substance in the fluoxastrobin-M48-E-M40 computer sample is higher than the concentration range when the standard curve is established, the method preferably further comprises: diluting the fluoxastrobin-M48-E-M40 computer sample and then filtering; the diluted reagent is preferably deionized water and acetonitrile in a volume ratio of 1: 1.

The method filters a water sample to be detected to obtain an on-machine sample of o-chlorophenol. In the invention, the pore diameter of the filter membrane for filtering the water sample to be detected is preferably 0.22 μm. In the present invention, the concentration range of the o-chlorophenol in the o-chlorophenol on-machine sample is preferably within the concentration range when the standard curve is established, and when the concentration of the o-chlorophenol in the o-chlorophenol on-machine sample is higher than the concentration range when the standard curve is established, the method further preferably includes: diluting the o-chlorophenol sample on a machine and then filtering; the diluted reagent is preferably purified drinking water.

After the fluoxastrobin-M48-E-M40 computer sample is obtained, the invention carries out first ultra performance liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 computer sample to obtain the chromatographic information of fluoxastrobin and M48-E.

In the invention, the parameters of the first ultra performance liquid chromatography tandem mass spectrometry detection comprise a first mass spectrometry detection parameter and a first chromatographic detection parameter.

In the present invention, the first mass spectrometric detection parameters comprise: an ion source: ESI⁺(ii) a Capillary voltage: 5.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: 10V; collision cell emission voltage: 16V.

In the present invention, the firstThe chromatographic detection parameters comprise: a chromatographic column: ACQUITYUPLC^TM BEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 2.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 65% A; 0.5-2.0 min, 65-15% A; 2.0-4.0 min, 15-2% A; 4.0-4.5 min, 2% A; 4.5-4.6 min, 2% -65% A; 4.6-6.5 min, 65% A.

After the fluoxastrobin-M48-E-M40 computer sample is obtained, the invention carries out second ultrahigh liquid chromatography tandem mass spectrometry detection on the fluoxastrobin-M48-E-M40 computer sample to obtain M40 chromatographic information.

In the invention, the second parameter for the ultra performance liquid chromatography tandem mass spectrometry detection comprises a second mass spectrometry detection parameter and a second chromatography detection parameter.

In the present invention, the second mass spectrometry detection parameters include: an ion source: ESI^-(ii) a Capillary voltage: -4.5 kV; ion source temperature: 550 ℃; air curtain air: 35 psi; and (3) collision gas spraying: 7 psi; auxiliary heating gas: 50 psi; spraying mist: 50 psi; injection voltage: -10V, collision cell ejection voltage: -16V.

In the present invention, the second spectrum detection parameters include: a chromatographic column: ACQUITYUPLC^TM BEH C₁₈Column, 1.7 μm, 2.1mm × 100mm, Waters corporation, USA; temperature of the column: 40 ℃; sample introduction amount: 5.00 mu L; flow rate: 0.3 mL/min^-1(ii) a The mobile phase A is a 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 2 mmol/L; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 55% A; 0.5-3.5 min, 55-5% A; 3.5-4.0 min, 5% A; 4.0-4.1 min, 5% -55% A; 4.1-6.0 min, 55% A.

And (3) obtaining an on-machine sample of the o-chlorophenol, and carrying out third ultra-high liquid chromatography tandem mass spectrometry detection on the on-machine sample of the o-chlorophenol to obtain the chromatographic information of the o-chlorophenol.

In the invention, the third parameter for the hplc-tandem mass spectrometry detection includes a third mass spectrometry detection parameter and a third chromatography detection parameter.

In the present invention, the third mass spectrometric detection parameters comprise: an ion source: APCI^-(ii) a Needle current: -3.5 mA; ion source temperature: 350 ℃; air curtain air: 25 psi; and (3) collision gas spraying: 8 psi; spraying mist: 50 psi; injection voltage: -10V; collision cell emission voltage: -14V.

In the present invention, the third spectral detection parameters include: a chromatographic column:Omega Polar C₁₈column, 3 μm, 2.1mm × 50mm, Phenomenex, USA; flow rate: 0.40 mL/min; column temperature: 40 ℃; sample introduction volume: 20.0 mu L; the mobile phase A is 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 80% A; 0.5-2.0 min, 80-40% A; 2.0-3.0 min, 40-20% A; 3.0-3.5 min, 20% A; 3.5-3.6 min, 20-80% A; 3.6-5.5 min, 80% A.

In the present invention, the qualitative and quantitative modes of fluoxastrobin and its metabolites are both MRM multi-reaction monitoring modes, and table 1 is the quantitative and qualitative parameters of fluoxastrobin and its metabolites under the MRM multi-reaction monitoring mode.

TABLE 1 Fluoroazoxystrobin and its metabolites quantitative and qualitative parameters in MRM multiple reaction monitoring mode

Quantitative ion (quaternary ion)

After fluoxastrobin chromatographic information, M48-E chromatographic information, M40 chromatographic information and o-chlorophenol chromatographic information are obtained, the fluoxastrobin chromatographic information, the chromatographic information of M48-E, the chromatographic information of M40 and the o-chlorophenol chromatographic information are substituted into a fluoxastrobin concentration-peak area standard curve, an M48-E concentration-peak area standard curve, an M40 concentration-peak area standard curve and an o-chlorophenol concentration-peak area standard curve, so that the content of fluoxastrobin and metabolites thereof in water is obtained.

In the present invention, the obtaining manner of the fluoxastrobin concentration-peak area standard curve, the M48-E concentration-peak area standard curve and the M40 concentration-peak area standard curve preferably comprises the following steps:

weighing 0.0120g of fluoxastrobin standard substance, and fixing the volume to 10.00mL by acetonitrile to obtain the concentration of 1.19 multiplied by 10³A standard stock solution of fluoxastrobin in mg/L; the purity of the fluoxastrobin standard product is preferably 99.33%;

weighing 0.0183g of fluoxastrobin metabolite M48-E, and diluting to 10.00mL with acetonitrile to obtain the concentration of 1.73 × 10³A standard stock solution of fluoxastrobin metabolite M48-E in mg/L; the purity of the fluoxastrobin metabolite M48-E is preferably 94.6%;

weighing 400.0120 g of fluoxastrobin metabolite M, and diluting to 10.00mL with acetonitrile to obtain the concentration of 1.11 × 10³A standard stock solution of fluoxastrobin metabolite M40 in mg/L; the purity of the fluoxastrobin metabolite M40 is preferably 92.45%;

diluting the fluoxastrobin standard stock solution, the fluoxastrobin metabolite M48-E standard stock solution and the fluoxastrobin metabolite M40 standard stock solution with acetonitrile to obtain the fluoxastrobin, the fluoxastrobin metabolite M48-E and the fluoxastrobin metabolite M40, wherein the concentrations of the fluoxastrobin, the fluoxastrobin metabolite M48-E and the fluoxastrobin metabolite M40 are all 10.0 mg.L^-1Mixed standard working solution of (1);

the volume ratio of acetonitrile to deionized water is 1:1, diluting by 10.0 mg.L step by step^-1The standard working solutions of (1) to (0.00), 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500 mg. L^-1Mixed standard working solution of (1);

performing first ultra performance liquid chromatography tandem mass spectrometry detection on the series of mixed standard working solutions to obtain chromatographic information of fluoxastrobin and fluoxastrobin metabolite M48-E; fitting the fluoxastrobin chromatographic information and the fluoxastrobin concentration to obtain a fluoxastrobin concentration-peak area standard curve; fitting the chromatographic information of the fluoxastrobin metabolite M48-E and the concentration of the fluoxastrobin metabolite M48-E to obtain an M48-E concentration-peak area standard curve.

In the present invention, the parameters of the first hplc-ms detection are preferably consistent with the above technical solutions and are not described herein again.

And performing second ultra performance liquid chromatography tandem mass spectrometry on the series of mixed standard working solutions to obtain chromatographic information of the fluoxastrobin metabolite M40, and fitting the chromatographic information of the fluoxastrobin metabolite M40 and the concentration of the fluoxastrobin metabolite M40 to obtain a fluoxastrobin metabolite M40 concentration-peak area standard curve.

In the present invention, the parameters of the second hplc-tandem mass spectrometry are preferably consistent with the above technical solution, and are not described herein again.

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

weighing 0.0116g of o-chlorophenol standard substance, and fixing the volume to 10.00mL by acetonitrile to obtain the concentration of 1.16 multiplied by 10³mg/L of o-chlorophenol standard stock solution; the purity of the o-chlorophenol standard is preferably 99.8%;

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

and performing third ultrahigh liquid chromatography-tandem mass spectrometry detection on the series of o-chlorophenol standard working solutions to obtain the chromatographic information of the o-chlorophenol, and fitting the chromatographic information of the o-chlorophenol and the concentration of the o-chlorophenol to obtain an o-chlorophenol concentration-peak area standard curve.

In the present invention, the parameters of the third hplc-tandem mass spectrometry detection are preferably consistent with the above technical solutions and are not described herein again.

The method for measuring fluoxastrobin and its metabolites in water according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

1.1 instruments and reagents

Instruments and devices: ABSciex5500 liquid chromatography tandem mass spectrometer (ABSciex, USA), ACQUITY UPLC^TM BEH C₁₈Chromatographic column (1.7 μm, 2.1 mm. times.100 mm, Waters corporation, USA), BSA224S electronic balance (sensitivity 0.0001g, Sidorist group, Germany), MX-S adjustable mixer (Peking DarongXingIng laboratory instruments, Ltd.), 50mL PPTE centrifuge tube, 0.22 μm organic syringe filter membrane.

Reagent: 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%), all three metabolites supplied by Beijing Kefa Weinwei pesticide technology center, acetonitrile (chromatografic, Merck), formic acid and ammonium acetate (chromatografic, Anaqua Chemicals Supply), and purified drinking water (Guangzhou Drift food and beverage Co., Ltd.).

1.2 preparation of Standard solution

0.0120g of fluoxastrobin standard substance (with the purity of 99.33%) is weighed, and the volume is determined to be 10.00mL by acetonitrile, so that the concentration is 1.19 multiplied by 10³mg/L of standard stock solution; 0.0183g of fluoxastrobin metabolite M48-E (purity 94.6%) is weighed, and the volume is adjusted to 10.00mL by acetonitrile to obtain the concentration of 1.73X 10³mg/L of standard stock solution; weighing 400.0120 g of fluoxastrobin metabolite M (purity is 92.45%), and diluting to 10.00mL with acetonitrile to obtain concentration of 1.11 × 10³mg/L of standard stock solution; 0.0116g of o-chlorophenol standard substance (purity is 99.8%) is weighed, and acetonitrile is used for fixing the volume to 10.00mL to obtain the concentration of 1.16 multiplied by 10³And (4) refrigerating and storing the mg/L standard stock solution at 0-5 ℃ for later use.

Diluting fluoxastrobin and its metabolite (M48-E and M40) standard stock solution with acetonitrile to obtain concentration of 10.0 mg.L^-1Mixed standard working solution of (1); by V_Acetonitrile:V_{Water (W)}1:1 dilution step by step of 10.0 mg.L^-1Of (2) a mixed standardWorking solutions were obtained at concentrations of 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500 mg. L^-1Mixed standard working solution of (1); diluting the o-chlorophenol standard stock solution with purified drinking water to obtain the o-chlorophenol standard working solution with the concentrations of 10.0, 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500 mg/L.

1.3 sample pretreatment

Fixing the volume of a 5.000mL sample to 10.00mL by acetonitrile in a water sample to be detected, and performing vortex for 30 s; passing through 0.22 μm filter or V_Acetonitrile：V_{Water (W)}After diluting with the mixed solvent at a ratio of 1:1, the mixed solvent is filtered through a 0.22 mu M filter membrane, so that a machine sample of fluoxastrobin-M48-E-M40 is obtained.

O-chlorophenol: and (3) filtering the water sample to be detected through a 0.22-micron filter membrane, or diluting the water sample with drinking purified water and then filtering the water sample through the 0.22-micron filter membrane to obtain the o-chlorophenol sample on the computer.

1.4 Mass Spectrometry conditions

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

fluoxastrobin metabolite M40: an ion source: ESI^-(ii) a Capillary voltage is-4.5 kV, ion source temperature is 550 ℃, gas curtain gas is 35psi, jet collision gas is 7psi, auxiliary heating gas is 50psi, spray gas is 50psi, injection voltage is-10V, and injection voltage of a collision chamber is-16V;

o-chlorophenol: an ion source: APCI^-(ii) a The needle current is minus 3.5mA, the ion source temperature is 350 ℃, the air curtain gas is 25psi, the spraying collision gas is 8psi, the spraying mist gas is 50psi, the injection voltage is minus 10V, and the injection voltage of the collision chamber is minus 14V.

The qualitative and quantitative modes of fluoxastrobin and metabolites thereof are MRM multi-reaction monitoring modes, and qualitative parameters are shown in Table 1.

1.5 chromatographic conditions

Fluoxastrobin, fluoxastrobin metabolite M48-E: a chromatographic column: ACQUITYUPLC^TM BEH C₁₈Columns (1.7 μm, 2.1 mm. times.100 mm, Waters Corp.); the temperature of the chromatographic column is 40 ℃; the sample injection amount is 2.00 mu L; the flow rate was 0.3 mL/min^-1(ii) a The mobile phase A is 0.1 percent formic acid and 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 65% A; 0.5-2.0 min, 65-15% A; 2.0-4.0 min, 15-2% A; 4.0-4.5 min, 2% A; 4.5-4.6 min, 2% -65% A; 4.6-6.5 min, 65% A.

Fluoxastrobin metabolite M40: a chromatographic column: ACQUITYUPLC^TMBEH C₁₈Columns (1.7 μm, 2.1 mm. times.100 mm, Waters Corp.); the temperature of the chromatographic column is 40 ℃; the sample injection amount is 5.00 mu L; the flow rate was 0.3 mL/min^-1(ii) a The mobile phase A is 0.1 percent formic acid and 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 55% A; 0.5-3.5 min, 55-5% A; 3.5-4.0 min, 5% A; 4.0-4.1 min, 5% -55% A; 4.1-6.0 min, 55% A.

O-chlorophenol: a chromatographic column:OmegaPolar C₁₈chromatography columns (3 μm, 2.1 mm. times.50 mm, Phenomenex, USA); the flow rate is 0.40 mL/min; the column temperature was 40 ℃; the injection volume is 20.0 mu L; the mobile phase A is 2mmol/L ammonium acetate aqueous solution; the mobile phase B is chromatographic pure acetonitrile; gradient elution procedure: 0-0.5 min, 80% A; 0.5-2.0 min, 80-40% A; 2.0-3.0 min, 40-20% A; 3.0-3.5 min, 20% A; 3.5-3.6 min, 20-80% A; 3.6-5.5 min, 80% A.

2 results and analysis

2.1 regression equation

Mixing the mixture at concentrations of 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500 mg. L^-1The mixed standard working solution and the o-chlorophenol standard working solution with the concentration of 10.0, 1.00, 0.100, 0.0500, 0.0100, 0.00500, 0.00100 and 0.000500mg/L are detected and analyzed according to the mass spectrum condition-chromatographic condition, and a standard curve is constructed by the concentration-peak area; the results are shown in fig. 1 to 4, specifically:

fluoxastrobin: 415730151x +82490, R²＝0.9999；

Fluoxastrobin metabolite M48-E: 6551 for y8514x-5619，R²＝1.0000；

Fluoxastrobin metabolite M40: 1964557x-381, R²＝0.9996；

Fluoxastrobin metabolite o-chlorophenol: y 9337889x +5757, R²＝0.9995。

2.2 recovery and precision

Fluoxastrobin and its metabolites (M48-E and M40) were at 3 spiked levels (0.002 mg. L)^-1、0.100mg·L^-1And 10.0 mg. L^-1) The average recovery and relative standard deviation at 5 replicates are shown in table 2. O-chlorophenol at 3 spiking levels (0.001, 0.100, and 10.0 mg. multidot.L)^-1) The average recovery and relative standard deviation are shown in table 2, with 5 replicates per concentration.

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

As can be seen from table 2: samples were at 0.00200, 0.100 and 10.0 mg.L^-1At the addition level, the average recovery rate of fluoxastrobin and metabolites (M48-E and M40) is 81.8-109%, and the relative standard deviation RSD (n ═ 5) is 1.72-7.38%; . 0.00100 and 92.8 mg.L of o-chlorophenol^-1At the addition level, the average recovery was 89.6% and 102%, and the relative standard deviation RSD (n-3) was 6.06% and 8.84%.

2.3 detection and quantitation limits

The lowest first-order concentration of the standard curve is multiplied by the sample injection amount to be taken as the minimum detection amount (LOD) of the instrument, and the LODs of fluoxastrobin, fluoxastrobin metabolite M48-E and fluoxastrobin M40 are respectively 1.00 multiplied by 10^-3ng、1.00×10^-3ng and 2.5X 10^{- 3}ng; minimum detected concentrations of fluoxastrobin, fluoxastrobin metabolite M48-E, and fluoxastrobin M40 in water (LOQ)) Is 0.00200 mg.L^-1(ii) a The minimum detection amount (LOD) of the instrument to the o-chlorophenol is 1.00 multiplied by 10 respectively^-2ng, the lowest detected concentration (LOQ) of o-chlorophenol in water is 0.00100 mg.L^-1。

2.4 optimization of the pretreatment method

Fluoxastrobin and its metabolites (M48-E and M40): the results of three pretreatment methods of extracting by two organic solvents (ethyl acetate and acetonitrile) and diluting by acetonitrile show that the recovery rates of fluoxastrobin extracted by ethyl acetate and metabolites thereof (M48-E and M40) (n ═ 3) are respectively 84.5-94.4%, 39.8-46.9%, 39.0-39.7%, and the recovery rates of M48-E and M40 do not meet the requirements; the recovery rates of fluoxastrobin and its metabolites (M48-E and M40) (n is 3) extracted from acetonitrile are respectively 98.2-103%, 89.3-97.2% and 104-112%, and the recovery rates can meet the requirements; the recovery rates of the fluoxastrobin diluted by acetonitrile and metabolites (M48-E and M40) (n is 3) thereof are respectively 99.3-118%, 99.5-104% and 100-107%, and the recovery rates can meet the requirements; since the standard curve of the standard working solution for preparing M40 from acetonitrile is not linear, in order to eliminate the solvent effect, the method of diluting acetonitrile is selected as the optimal pretreatment method

2.5 selection of Mass Spectrometry conditions for ortho-chlorophenol

O-chlorophenol: comparative ESI^-Source and APCI^-Source, same concentration sample, APCI^-The source response value is high, so APCI is selected^-And (4) detecting a source.

Optimizing ion source parameters: the peak area response values for the comparative air curtain at 25psi, 30psi and 35psi, respectively, are: the peak area is 253347 when the air curtain gas is 25psi, 145218 when the air curtain gas is 30psi, and 170770 when the air curtain gas is 35 psi. It can be seen that: the air curtain air response value is the highest at 25psi, and the air curtain air is selected to be 25 psi.

Ion source temperature: the results of comparing the peak area response values at 400 deg.C, 350 deg.C, 300 deg.C and 250 deg.C of the ion source are shown in FIG. 5. As can be seen from fig. 5: when the ion source temperature is 350 ℃, the peak area response value is the highest, and for o-chlorophenol, the sensitivity is greatly improved by optimizing mass spectrum parameters.

2.6 chromatographic Condition optimization

The three conditions of 0.1% formic acid aqueous solution-acetonitrile, 2mmol/L ammonium acetate aqueous solution-acetonitrile and 0.1% formic acid +2mmol/L ammonium acetate aqueous solution-acetonitrile are respectively used as elution solvents, compared with the response and peak shape of a target object, the fluoxastrobin and metabolites thereof (M48-E and M40) have higher sensitivity, moderate retention time and better peak shape under the elution conditions of 0.1% formic acid +2mmol/L ammonium acetate aqueous solution and acetonitrile; the o-chlorophenol has higher response value and better peak shape under the elution condition of 2mmol/L ammonium acetate water solution and acetonitrile.

Example 2

Actual sample detection

Actual samples collected from 5 places of Hangzhou west lake water, Qiantangjiang river water, Yun river water, Portulaca transverse pond river water and river water near a laboratory in 2021 month 2 are screened, and the detected concentration of fluoxastrobin at the 5 points is 0.00000717-0.0000226 mg.L^-1In between, all are less than the lowest detected concentration, and no 3 metabolites of fluoxastrobin were detected.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.