Method for analyzing residual quantity of metalaxyl and metalaxyl acid metabolite thereof in fish body

Abstract

The invention discloses a method for analyzing residual quantity of metalaxyl and metalaxyl acid as a metalaxyl metalaxyl metalaxyl in a fish body, the metalaxyl and metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl metalaxyl According to the method, the enantiomers of the metalaxyl are subjected to refined detection, so that metabolism of the chiral medicine metalaxyl in aquatic animals, especially fish bodies, is better known, the residual elimination rule characteristics of the enantiomers of the chiral medicine metalaxyl in the aquatic animals are disclosed, an accurate and reasonable administration scheme is guided to be formulated, the use amount of the medicine is reduced, and the production cost is reduced. The method is helpful for more accurate risk assessment of metalaxyl, and is of great significance to improvement of fish food quality and protection of ecological environment.

Description

Technical Field

[0001] The invention relates to the technical field of environmental detection, and in particular to a method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish. Background Art

[0002] Metalaxyl, also known as D,LN-(2,6-dimethylphenyl)-N-(2'-methoxyacetyl)alanine methyl ester, is an organic compound belonging to the systemic amide fungicide class. It is mainly used to control downy mildew of cucumber, grape, Chinese cabbage, late blight of potato, and early and late blight of tomato. In aquaculture, it is mainly used to control Saprolegniasis, and fish is the main product of aquaculture. Metalaxyl is a chiral compound with an asymmetric carbon atom in its molecular structure, forming a pair of enantiomers (R-metalaxyl and S-metalaxyl). The basic substances of life, such as amino acids, proteins and carbohydrates, all have chiral characteristics. Therefore, chiral drugs will show different characteristics of metabolism, elimination, activity and toxicity when entering the large chiral environment of the organism. In recent years, metalaxyl has been widely used in aquaculture, which has promoted the development of aquaculture and improved the quality and safety of aquatic products. At present, there are few reports on the anti-Saprolegnia activity of metalaxyl enantiomers and their stereoselective metabolism, pharmacokinetics, tissue distribution and residual elimination differences in aquatic animals. The establishment of a residual analysis method for metalaxyl enantiomers in fish (plasma and tissues) and the study of its stereoselective metabolism and pharmacokinetics in aquatic animals are very meaningful. However, there are few studies on the analysis of the residual amount of metalaxyl enantiomers and its metabolite metalaxyl acid in fish. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides a method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish. The establishment of this method is of great significance for studying the metabolism, pharmacokinetics and residual elimination of the chiral drug metalaxyl in aquatic animals, especially the metabolism in fish. It can not only be used to reveal the regular characteristics of the residual elimination of metalaxyl enantiomers in aquatic animals, but also guide the formulation of accurate and reasonable dosing plans, reduce the amount of drugs used, help to conduct a more accurate risk assessment of metalaxyl, and help improve the quality of fish food and protect the ecological environment.

[0004] The technical solution of the present invention is:

[0005] A quantitative analysis method for the residues of metalaxyl and its metabolite metalaxyl acid in fish, wherein the metalaxyl and its metabolite metalaxyl acid include R-metalaxyl and S-metalaxyl, R-metalaxyl acid and S-metalaxyl acid, R-metalaxyl-D6 and S-metalaxyl-D6;

[0006] The steps include:

[0007] 1) Pretreatment of samples to be tested

[0008] Fish plasma sample pretreatment:

[0009] The obtained fish plasma was homogenized, internal standard solution was added, vortexed and then allowed to stand;

[0010] Add formic acid acetonitrile solution, vortex, centrifuge to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution;

[0011] Fish tissue sample pretreatment:

[0012] After adding the internal standard solution to the homogenized fish tissue sample, vortex and let it stand;

[0013] Add formic acid ethyl acetate solution, vortex, sonicate, and centrifuge to collect the supernatant; add n-hexane to the supernatant to remove fat, vortex, centrifuge, heat the supernatant in a water bath until it is almost dry, take the residue and add the composite solution to re-dissolve, vortex, centrifuge, take the supernatant and filter through a 0.22 μm filter membrane to obtain the test solution;

[0014] 2) Selection of chromatographic and mass spectrometric conditions

[0015] Selection of chromatographic conditions:

[0016] Chromatographic column Y1, column temperature 30-40°C; flow rate 0.2-0.5 mL / min; injection volume: 2-10 μL; mobile phase A is 0.1 vt% formic acid acetonitrile solution; mobile phase B is 0.1 vt% formic acid aqueous solution of 5 mM ammonium acetate, isocratic elution: mobile phase A: mobile phase B = 75:25 (v:v);

[0017] The conditions for mass spectrometry detection are shown in the table below:

[0018]

[0019] Note: * Quantitative ion.

[0020] 3) injecting the test solution obtained in step 1) and performing detection according to the above-mentioned chromatographic mass spectrometry conditions to obtain a chromatogram of metalaxyl and its metabolite metalaxyl acid, and analyzing to obtain quantitative data.

[0021] Furthermore, in step 2), the column temperature is 30° C.; the flow rate is 0.25 mL / min; and the injection volume is 5 μL.

[0022] Furthermore, the concentration of the formic acid acetonitrile solution in steps 1) and 2) is 0.1 vt%.

[0023] Furthermore, the concentration of the ethyl formate solution in steps 1) and 2) is 0.1 vt%.

[0024] Furthermore, during the fish tissue sample pretreatment in step 1), the composite solution is prepared from 0.1 vt% formic acid acetonitrile and 0.1 vt% formic acid aqueous solution in a volume ratio of 3:7.

[0025] Furthermore, during the fish tissue sample pretreatment in step 1), the water bath heating temperature is 40-50°C.

[0026] Furthermore, in step 2), the mass spectrometer selects electrospray ionization positive ion mode (ESI + ), multiple reaction monitoring mode;

[0027] Furthermore, electrospray ion source, positive ion scanning; mass spectrometry scanning mode is multiple reaction monitoring (MRM); ion source temperature is 150°C; desolvation gas flow rate is 1000L / h; desolvation gas temperature is 500°C; cone gas flow rate is 150L / h; capillary voltage is 3.0kV.

[0028] Furthermore, the fish tissue sample in step 1) is at least one of muscle tissue, liver tissue, kidney tissue, heart tissue, spleen tissue, swim bladder tissue, and fish intestinal tissue.

[0029] The beneficial effects achieved by the technical solution of the present invention are:

[0030] The present invention utilizes LC-MS / MS to quantitatively detect metalaxyl and its metabolite metalaxyl acid in fish bodies (including fish plasma and fish tissues), and has the advantages of low limits of detection (LODs) and limits of quantification (LOQs), good reproducibility, high precision, etc.

[0031] And carry out detailed detection of metalaxyl enantiomers, so as to better understand the metabolism of the chiral drug metalaxyl in aquatic animals, especially fish, reveal the residual elimination characteristics of its enantiomers in aquatic animals, guide the formulation of precise and reasonable dosing plans, reduce the amount of drugs used, and contribute to a more accurate risk assessment of metalaxyl, which is of great significance to improving the quality of fish food and protecting the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Attached Figure 1 The chromatogram in the plasma sample of the embodiment.

[0033] Attached Figure 2 4 is a chromatogram of the swim bladder tissue sample in the embodiment.

[0034] Attached Figure 3 Graph showing the chromatogram in the heart tissue sample of the embodiment.

[0035] Attached Figure 4 Graph showing the chromatogram of the liver tissue sample in the embodiment.

[0036] Attached Figure 5 Graph showing the chromatogram in the kidney tissue sample of the embodiment.

[0037] Attached Figure 6 The chromatogram in the muscle tissue sample of the embodiment.

[0038] Attached Figure 7 Graph showing the chromatogram of the intestinal tissue sample of the embodiment.

[0039] Attached Figure 8 : is a chromatogram of a spleen tissue sample in the embodiment. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The instrument used in the following examples: Acquity UPLC I-Class / Xevo TQ-S ultra-high performance liquid chromatograph tandem triple quadrupole mass spectrometer (Waters, USA).

[0044] Reagents: Racemic metalaxyl standard (purity 99.3%) was purchased from Dr. Ehrenstorfer, Germany; racemic metalaxyl acid standard (purity 99.4%) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.; internal standard racemic metalaxyl-D6 (purity 98.9%) was purchased from Tianjin Alta Technology Co., Ltd.; acetonitrile and ethyl acetate (chromatographically pure, Thermo Fisher Scientific, USA), formic acid (mass spectrometry pure, TIC, Japan).

[0045] The fish used in the following experiment were purchased from Hunan Yi Ze Agricultural Products Co., Ltd. Several crucian carps weighing 300±50g were collected, and several blank plasma and tissue samples of crucian carps were collected. 10 fish were randomly selected and bathed in the medicine for 2 hours according to the recommended dose (9mg / L) of "Compound Metalaxyl Powder" in Announcement No. 2505 of the Ministry of Agriculture and Rural Affairs. One day after the administration, the plasma and tissue samples of the crucian carps were collected. Other feeding was normal.

[0046] Embodiment 1:

[0047] Reagent preparation

[0048] The preparation of standard stock solution (standard solution) is as follows:

[0049] Weigh 10 mg of each of the metalaxyl standard and metalaxyl acid standard into a 10 mL volumetric flask, add chromatographic acetonitrile and mix by ultrasonication, then dilute to the mark to prepare a 1 mg / mL standard solution, and store at -20°C;

[0050] The preparation of internal standard stock solution (internal standard solution) is as follows:

[0051] Weigh 1 mg of internal standard metalaxyl-D6 into a 10 mL volumetric flask, add chromatographic acetonitrile and mix by ultrasonication, then dilute to the mark to prepare a 100 μg / mL internal standard solution, and store at -20°C;

[0052] The preparation of the mixed standard working solution is as follows:

[0053] Take an appropriate volume of standard solution, place it in a 10 mL volumetric flask, dilute with acetonitrile to make a 10 μg / mL mixed standard working solution, and store it at 4 °C;

[0054] Sample pretreatment

[0055] Fish plasma sample pretreatment:

[0056] Take 0.2mL of plasma sample in a 2mL centrifuge tube, add 100μL, 100ng / mL internal standard solution, vortex for 30s, and let stand for 20min. Add 0.7mL of 0.1vt% formic acid acetonitrile solution for extraction, vortex for 30s, and centrifuge at 10000×g at 4℃ for 5min. Take 0.5mL of supernatant in a 2mL centrifuge tube, add 0.5mL of ultrapure water, vortex for 30s, and centrifuge at 15000×g at 4℃ for 10min. Pass the supernatant through a 0.22μm filter membrane into a sample injection bottle for use;

[0057] Fish tissue sample pretreatment:

[0058] Take 2g of homogenized muscle, liver, and intestinal tissue samples, 0.5g of kidney, heart, spleen, and swim bladder (swim bladder and intestine cut into pieces), respectively, and put them in 50mL centrifuge tubes, add 100μL, 100μg / mL internal standard solution, vortex for 30s, and let stand for 20min. Add 10mL 0.1vt% formic acid ethyl acetate solution to extract, vortex for 30s, ultrasonic for 10min, 10000×g, 4℃ centrifuge for 10min. Take the supernatant in a 25mL glass test tube, dilute to 15mL with 0.1% formic acid ethyl acetate, and vortex for 30s. Then take 7.5mL in a 15mL centrifuge tube, add 5mL n-hexane to remove fat, vortex for 30s, and centrifuge at 10000×g at 4℃ for 10min. Collect the fat-free extract in a 10mL glass tube and spin it in a 45℃ water bath until it is almost dry. The residue was re-dissolved with 1 mL of 0.1 vt% formic acid acetonitrile: 0.1 vt% formic acid aqueous solution (3:7, v / v), vortexed and ultrasonicated for 10 min, centrifuged at 15000×g, 4°C for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane into a sampling bottle for later use.

[0059] Instrumental detection method

[0060] Liquid phase conditions: chromatographic column Y1 (150 mm × 2.1 mm id, 5 μm); column temperature: 30 °C; flow rate: 0.25 mL / min; injection volume: 5 μL; mobile phase: A, 0.1% formic acid acetonitrile; B, 5 mM ammonium acetate containing 0.1% formic acid water, isocratic elution (A: B = 75: 25 (v: v))

[0061] Mass spectrometry conditions: electrospray ionization positive ion mode (ESI + ), multiple reaction monitoring mode;

[0062] Electrospray ion source, positive ion scanning; mass spectrometry scanning mode is multiple reaction monitoring (MRM); ion source temperature is 150℃; desolvation gas flow rate is 1000L / h; desolvation gas temperature is 500℃; cone gas flow rate is 150L / h; capillary voltage is 3.0kV. The mass spectrometry parameters of metalaxyl, metalaxyl acid and metalaxyl-D6 are shown in Table 1.

[0063] Table 1 Main mass spectrometry parameters of target analytes

[0064]

[0065] Note: * Quantitative ion.

[0066] The test solution obtained in step 1) was sampled and detected according to the above-mentioned chromatographic mass spectrometry conditions to obtain a chromatogram of metalaxyl and its metabolite metalaxyl acid. The quantitative data were analyzed and the results are shown in Table 2.

[0067] Table 2: Drug content in each tissue (n=6, ±SD)

[0068]

[0069] Each sample from a different source of matrix was repeated 20 times to evaluate the selectivity of the method of the present invention. The results showed that the method had good selectivity and no interfering peaks within 2.5% of the relative retention time of the target analyte. Typical MRM chromatograms of blank fish plasma and tissue extracts spiked with a concentration of 50 ng / mL (20 ng / g) are shown in the attached figure.

[0070] In the concentration range of 1-200 ng / mL (internal standard concentration was 10 ng / mL), the internal standard solution was prepared by adding exo-metalaxyl-D6 to acetonitrile. Metalaxyl and metalaxyl acid showed good linearity in crucian carp plasma, muscle, kidney, liver, heart, spleen, swim bladder and intestinal tissues, with r greater than 0.99, as shown in Table 3.

[0071] Table 3: Linear range, linear equation and correlation coefficient of metalaxyl and metalaxyl acid in crucian carp plasma and tissue extracts

[0072]

[0073] Blank crucian carp plasma and tissue samples were spiked with 5, 20 and 100 ng / mL (or ng / g) concentrations, the internal standard solution concentration was 10 ng / mL (internal standard solution was metalaxyl-D6 added to acetonitrile to make up to volume), 5 replicates per level, 3 batches were separated by one day or several days, and the recovery and precision of the method were evaluated. As shown in Tables 4 and 5, the average recovery of the analyte in plasma and tissue matrix was between 75% and 102%, and the intra-day and inter-day precisions were less than 15%.

[0074] Table 4: Recoveries of target analytes in fish plasma and tissues

[0075]

[0076]

[0077] The unit of drug concentration in tissue is ng / g.

[0078] Table 5: Precision of target analytes in porcine plasma and tissue

[0079]

[0080] The unit of drug concentration in tissue is ng / g.

[0081] The sample addition concentration corresponding to the signal-to-noise ratio (S / N) ≥ 3 was the detection limit, and the sample addition concentration corresponding to the S / N ≥ 10 was the quantification limit. The detection limit and quantification limit were 1 ng / mL and 2 ng / mL in plasma samples, and 0.5 ng / g and 1 ng / g in tissue samples, respectively. The established method is simple, sensitive and reliable, meeting the requirements of veterinary drug residue analysis and detection, as well as pharmacokinetics and residue elimination studies.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish, characterized in that: The metalaxyl and its metabolite metalaxyl include R-metalaxyl and S-metalaxyl, R-metalaxyl acid and S-metalaxyl acid, R-metalaxyl-D6 and S-metalaxyl-D6; The steps include: 1) Sample pretreatment Fish plasma sample pretreatment: The obtained fish plasma was homogenized, internal standard solution was added, vortexed and then allowed to stand; Add formic acid acetonitrile solution, vortex, centrifuge to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution; Fish tissue sample pretreatment: After adding the internal standard solution to the homogenized fish tissue sample, vortex and let it stand; Add formic acid ethyl acetate solution, vortex, sonicate, and centrifuge to collect the supernatant; add n-hexane to the supernatant to remove fat, vortex, centrifuge, heat the supernatant in a water bath until it is almost dry, take the residue and add the composite solution to re-dissolve, vortex, centrifuge, take the supernatant and filter through a 0.22 μm filter membrane to obtain the test solution; 2) Selection of chromatographic and mass spectrometric conditions Selection of chromatographic conditions: Chromatographic column Y1, column temperature 30-40°C; flow rate 0.2-0.5 mL / min; injection volume: 2-10 μL; mobile phase A is formic acid acetonitrile solution; mobile phase B is ammonium acetate formic acid aqueous solution, isocratic elution: mobile phase A: mobile phase B = 75:25 (v:v); The conditions for mass spectrometry detection are shown in the table below: Note: * Quantitative ion. 3) injecting the test solution obtained in step 1) and performing detection according to the above-mentioned chromatographic mass spectrometry conditions to obtain a chromatogram of metalaxyl and its metabolite metalaxyl acid, and analyzing to obtain quantitative data.

2. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: Step 2) Column temperature: 30°C; flow rate: 0.25 mL / min; injection volume: 5 μL.

3. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: The concentration of the ethyl formate solution in step 1) is 0.1 vt%.

4. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: The concentration of the formic acid acetonitrile solution in step 1) is 0.1 vt%.

5. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: During the fish tissue sample pretreatment process of step 1), the composite solution is prepared from 0.1 vt% formic acid acetonitrile and 0.1 vt% formic acid aqueous solution in a volume ratio of 3:

7.

6. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: During the fish tissue sample pretreatment process of step 1), the water bath heating temperature is 40-50°C.

7. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: In step 2), the mass spectrometer was set to electrospray ionization positive ion mode (ESI + ), multiple reaction monitoring mode.

8. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 7, characterized in that: In step 2), the ion source temperature is 150°C; the desolvation gas flow rate is 1000 L / h; the desolvation gas temperature is 500°C; the cone gas flow rate is 150 L / h; and the capillary voltage is 3.0 kV.

9. The method for analyzing the residual amount of metalaxyl and its metabolite metalaxyl acid in fish according to claim 1, characterized in that: The fish tissue sample in step 2) is at least one of muscle tissue, liver tissue, kidney tissue, heart tissue, spleen tissue, swim bladder tissue, fish intestinal tissue, and fish brain.

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