CN109813820B - Method for identifying metabolite of phoxim in different tissues of freshwater fish - Google Patents
Method for identifying metabolite of phoxim in different tissues of freshwater fish Download PDFInfo
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Abstract
The invention discloses a method for identifying metabolic products of phoxim in different tissues of freshwater fish, which adopts a soaking method for drug administration, and qualitatively detects the metabolic products of phoxim in different tissues through ultra-high performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry (UPLC-QTOFMS), and comprises the following steps: (1) soaking freshwater fish in phoxim solution, and collecting different tissues; (2) extracting a sample by using acetonitrile and ethyl acetate in a volume ratio of 5:1, mixing the purificant with anhydrous magnesium sulfate and octadecylsilane chemically bonded silica in a mass ratio of 10: 1; (3) UPLC-QTOFMS qualitative analysis: with phoxim, O’,O’Comparing the retention time and the ion mass of tetraethyl phosphorodithioate and O, O-diethylthiophosphate, and judging the metabolic products of each tissue sample.
Description
Technical Field
The invention belongs to the technical field of aquaculture and detection analysis, and particularly relates to a method for qualitatively analyzing phoxim and metabolites thereof in freshwater fish tissues based on ultra-high performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry.
Background
Phoxim (Phoxim) is an organophosphorus insecticide currently used as a veterinary drug to control mites, lice and other ectoparasites in pigs and sheep. Phoxim is also approved in China for killing or expelling sinergasilus, lernaea, carpea, louse, third generation worm, dactylogyrus, nematode and other parasites which parasitize on freshwater fishes such as megalobrama amblycephala, tilapia, grass carp, herring, silver carp, bighead carp, crucian and the like. If the phoxim is improperly used, the phoxim can be caused to generate residues in aquatic animal tissues, and further harm the human health.
Major degradation steps of phoxim in rats, pigs and rabbits include hydrolysis and dealkylation of the phosphate ester linkages, cyanobenzaldoxime (further detoxified in rats and pigs to benzoylamino ester acids and degraded in rabbits to benzonitrile) and desethylphoxim. In rats and calves, oxidation of the cyano group is an important pathway of degradation, and in combination with the carboxylic acid phoxim, the oxidized analogue of phoxim is responsive to highly pesticidally active insecticides, found in fly extracts but not in mammals, although the possible transient intermediate of this compound was identified temporarily as desethylphoxim in rats. Metabolism of phoxim in pigs: radiolabeled phoxim was administered as gelatin capsules to pigs in a single dose of 5mg/kg bw. The highest radiation concentration occurred 24h after administration, the radioactivity in fat amounted to a maximum of 1320. mu.g/kg, in liver to 600. mu.g/kg, in kidney to 350. mu.g/kg and in muscle to 50. mu.g/kg, and the concentration in tissue at 72h was approximately half of the above-mentioned concentration, and only phoxim and cyanobenzoylcyanoxime in tissue could be identified and only in fat could be quantified. Phoxim is found in fat (90% radioactivity), loin and muscle, and cyanobenzaldoxime is not found in loin, muscle and liver. Seven pigs are administered through the skin with radiolabeled phoxim as a pour-on, the dose is 100mg/kg bw, and the skin bioavailability is 1.2% -2.9% (European Medicines Agency, 2005). Liurongfei et al (2013) use high performance liquid chromatography tandem ion trap mass spectrometer to study the metabolites and metabolic pathways of liver microsome in crucian body. The research adopts a method for preparing crucian liver microsomes, and phoxim is directly added into the liver microsomes for in vitro incubation. The method is different from the actual use condition of the phoxim in fish bodies, and the high concentration of the phoxim in vitro liver microsome, namely 100 mu mol/L (35.796mg/L), is not existed in the actual condition, so that the metabolite of the phoxim in the fish bodies may be different from the metabolite of the phoxim which is incubated by the liver microsome in vitro in high concentration. In order to disclose the metabolic products of phoxim in tissues in a fish body, the research researches the metabolic products of phoxim in tissues such as megalobrama amblycephala plasma, muscle, skin, liver, kidney, gill, intestine and the like according to the practical situation of the phoxim used in the fish, the research result is closer to the practical situation, and the research also provides scientific basis for establishing the maximum residual limit of the phoxim in aquatic products.
The ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) is composed of four parts of ultra-high performance liquid phase, an ion source, a mass analyzer and a detector, and adopts quadrupole ion focusing transmission, positive and negative double-pulse repulsion, vertical acceleration and reverseThe injection type time-of-flight mass spectrometer analyzes the molecular mass of the compound in a microchannel plate detection mode, and has the characteristics of high efficiency, high sensitivity, strong specificity, various detection modes, high analysis speed and abundant data information and data processing. The ultra-high performance liquid chromatography has extremely fine particle size, higher separation degree, analysis speed and sensitivity than the high performance liquid chromatography, has lower processing requirement on a complex sample biological sample, and is beneficial to saving manpower, material resources and time; and the method has good compatibility with mass spectrum, can improve the resolution of the chromatogram on the whole, reduce ion inhibition, reduce co-elution phenomenon, improve the sensitivity and reliability of the mass spectrum, can analyze low-content substances in a complex matrix, and can eliminate the interference of the matrix to the maximum extent. QTOFMS combines the advantages of quadrupole mass spectrometry and time-of-flight mass spectrometry, and in a full scan mode, UPLC effluent is ionized by an electrospray ion source and then directly passes through a quadrupole rod and a LINACTMThe linear acceleration collision cell is introduced into a time-of-flight mass analyzer for accurate mass analysis in reflection mode. And (3) using a powerful information correlation data acquisition mode (IDA) and a high-resolution and high-accuracy mass number primary scanning and secondary scanning mode to obtain a corresponding high-resolution and high-accuracy mass number primary mass spectrogram and a corresponding high-accuracy mass number secondary mass spectrogram. The resolution of the QTOFMS in a low nucleus ratio (m/z 100) is more than or equal to 25000(FWHM), the resolution of a high nucleus ratio (m/z 950) can reach 40000(FWHM), and meanwhile, the nucleus ratio (m/z) of a target object can be accurately measured to 4 bits after a decimal point, so that the certainty of analyzing the target object is greatly increased. Even though the tandem quadrupole mass spectrometry with high resolution (0.1u) has a resolution of 1000 at a low nucleus ratio (m/z 100), the nucleus ratio (m/z) of the target can be measured to 1 bit after the decimal point with a resolution of 10000(FWHM) at a high nucleus ratio (m/z 1000). Therefore, the UPLC-QTOFMS can well separate and identify the metabolites of phoxim in the tissues of freshwater fish.
At present, the identification of the metabolite of phoxim in tissues such as plasma, muscle, skin, liver, kidney, gill, intestine and the like in aquatic animals is not reported, and the accurate molecular weight identification of phoxim and the metabolite thereof in the tissues in freshwater fish body, which is administrated by soaking phoxim, by using ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry (UPLC-QTOFMS) adopted by the invention, belongs to the first time.
Disclosure of Invention
The invention aims to provide a method for identifying the metabolite of phoxim in different tissues of fishes by adopting ultra-high performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry (UPLC-QTOFMS), wherein the sample pretreatment method in the method is suitable for various different tissues (blood, muscle, skin, liver, kidney, gill, intestine and the like) of freshwater fish bodies, and the treatment method is simple and quick to operate; the method has the advantages that the method is based on the separation and identification of the phoxim and the metabolites thereof in the tissues of the freshwater fish by the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS), not only is the analysis speed high, but also the accurate mass number of the phoxim and the metabolites thereof can be obtained, and the accuracy of the identification result of the phoxim and the metabolites thereof is ensured.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for identifying a metabolite of phoxim in different tissues of freshwater fish is characterized by comprising the following steps:
(1) phoxim soaking administration
Soaking freshwater fish in 1-5 mg/L phoxim solution (prepared by dissolving phoxim missible oil with acetone and diluting with distilled water), respectively collecting blood plasma, liver, kidney, gill, intestine, skin and muscle tissues after soaking for 2h, 4h, 6h, 8h, 10h, 24h and 48h, and homogenizing;
(2) sample processing
Respectively weighing the samples of the tissues, sequentially adding an extracting agent A and a purifying agent B, performing vortex oscillation and centrifugation, collecting an extracting solution, repeatedly extracting sample residues by using the extracting agent A, combining the extracting solutions, drying by using nitrogen, performing constant volume by using methanol, filtering by using a filter membrane of 0.22 mu m, and detecting a filtrate by using UPLC-Q-TOFMS;
the extractant A is acetonitrile and ethyl acetate, and the volume ratio of the extractant A to the extractant B is 5:1, mixing;
the purifying agent is prepared by mixing anhydrous magnesium sulfate and octadecylsilane chemically bonded silica in a mass ratio of 10: 1;
(3) UPLC-QTOFMS qualitative analysis
Detecting each tissue sample by using an ultra-high performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry technology, and comparing the retention time and the ion mass of the detected compound with the retention time and the ion mass of phoxim, O, O, O ', O', -tetraethyl phosphorodithioate and O, O-diethylthiophosphoric acid to judge the metabolite of each tissue sample.
Further, the UHPLC analysis conditions are specifically: c18 chromatography column, 100mm × 2.1mm × 1.8 μm; column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient of the methanol containing 10% of organic phase and the 0.1% of formic acid aqueous solution containing 90% of aqueous phase is maintained for 1min, then the organic phase linearly rises to the methanol containing 90% of volume concentration within 5min, the aqueous phase linearly drops to the 0.1% of formic acid aqueous solution containing 10% of aqueous phase, the maintenance is carried out for 4min, the organic phase linearly drops to the methanol containing 10% of volume within 0.1min, the aqueous phase linearly rises to 90% of volume, and finally the maintenance is carried out for 1.9 min; the injection volume was 5.0. mu.L.
Further, the QTOFMS analysis conditions are specifically: an electrospray ion source is in a positive ion mode, the scanning range is M/Z100-1000 Da, and the accumulation time is 0.199957 secs; the mass range of product ions scanned by the time-of-flight mass spectrometry is 50-1000 Da, and the cluster removing voltage is as follows: 45V, the collision energy is 35eV, and the expansion collision energy is 15.0 eV; the ion release delay is 67, the ion release width is 25, the spray gas is 50Psi, the auxiliary heating gas is 55Psi, the air window gas is 25Psi, the ionization temperature is 400 ℃, and the positive ion spray voltage is 5500V.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. in order to reveal the metabolic products of phoxim in fish tissues, the method comprises the steps of soaking freshwater fish in a water body containing phoxim, absorbing and enriching phoxim into the body for metabolism by the fish body, and then collecting different tissues for qualitative analysis, so that the analysis result is closer to the actual situation.
2. The ultra-high performance liquid chromatography (UPLC) is characterized in that the particle size is extremely fine on the basis of High Performance Liquid Chromatography (HPLC) (the UPLC adopts a sub-two-micron chromatographic column with the diameter of 1.7 mu m and is shorter in column length, the separation speed is respectively improved by 9 times and 3 times compared with the traditional chromatographic columns with the diameter of 5 mu m and 3 mu m of HPLC), the UPLC has higher resolution than the HPLC, the peak capacity is enlarged by 2-3 times, and the sensitivity is improved by 2-3 times compared with the HPLC. The UPLC has lower processing requirements on complex biological samples, and is beneficial to saving manpower, material resources and time; and the method has good compatibility with mass spectrum, can improve the resolution of the chromatogram on the whole, reduce ion inhibition, reduce co-elution phenomenon, improve the sensitivity and reliability of the mass spectrum, can analyze low-content substances in a complex matrix, and can eliminate the interference of the matrix to the maximum extent.
3. The quadrupole tandem time-of-flight mass spectrometry (Q-TOFMS) has strong qualitative capability (the qualitative capability is better than that of a triple quadrupole mass spectrometry QQQ/MS), and the analysis is carried out in a primary mass spectrometry MS1 mode and a secondary mass spectrometry MS2 mode, so that the accurate mass of parent ions and fragment ions is provided, the accurate mass of compound molecules can be accurately measured to 4 positions after a decimal point, the elementary composition and the multilevel fragment ions are provided, the qualitative result is more accurate, and the QQQ/MS can only measure the compound molecules to 1 position after the decimal point, and compared with the QQQ/MS, the Q-TOF/MS has higher resolution, more efficient mass identification and higher selectivity. For in vivo metabolite identification, HPLC-QQQ/MS is adopted, and due to the fact that the tandem mass spectrum is not high in accuracy, low in sensitivity, weak in resolution and limited in mass detection width, accurate molecular mass of metabolites cannot be given, isomers with short retention time are difficult to distinguish, the qualitative effect is not accurate, and the number of the compounds to be determined is small. The UPLC-QTOFMS is adopted to qualitatively determine the metabolites in the body, and the problems can be solved one by one.
4. The invention optimizes the extraction method of phoxim and the metabolite thereof, the extraction rate of the phoxim and the metabolite thereof in the tissues of the freshwater fish is the highest by the extractant prepared from acetonitrile and ethyl acetate according to the volume ratio of 5:1, and MgSO 1 is adopted4+ C18 the mixed scavenger decontaminated sample at a mass ratio of 10:1 produced the lowest matrix effect.
Drawings
FIG. 1: the effect of different extractants is compared.
FIG. 2: the effects of different purifiers are compared.
FIG. 3: chromatogram and mass spectrum of megalobrama amblycephala plasma sample and blank plasma sample.
A phoxim extraction ion flow chromatogram in a 3-a megalobrama amblycephala plasma sample, a phoxim primary mass chromatogram in a 3-b megalobrama amblycephala plasma sample, a phoxim secondary mass chromatogram in a 3-c megalobrama amblycephala plasma sample, a 3-d megalobrama amblycephala plasma blank sample extraction ion flow chromatogram, a 3-e megalobrama amblycephala plasma blank sample primary mass chromatogram, an O, O ', O', tetraethyl phosphorodithioate extraction ion flow chromatogram in a 3-f megalobrama amblycephala plasma sample, a 3-g blank plasma extraction ion flow chromatogram, a 3-h megalobrama plasma O, O, O ', O', -tetraethyl diphosphothioate primary mass chromatogram, a 3-i megalobrama amblycephala plasma primary mass chromatogram, an O, O, O, O ', O', -tetraethyl diphosphothioate secondary mass chromatogram in a 3-j megalobrama plasma, an extraction ion flow chromatogram of O, O-diethyl thiophosphoric acid in the plasma of the 3-k megalobrama amblycephala, a blank extraction ion flow chromatogram of the plasma of the 3-l megalobrama amblycephala, a primary mass spectrogram of O, O-diethyl thiophosphoric acid in the plasma of the 3-m megalobrama amblycephala, a blank primary mass spectrogram of the plasma of the 3-n megalobrama amblycephala and a secondary mass spectrogram of O, O-diethyl thiophosphoric acid in the plasma sample of the 3-O megalobrama amblycephala.
FIG. 4: chromatogram and mass spectrum of megalobrama amblycephala muscle sample and blank muscle sample.
Extracting an ion flow chromatogram from phoxim in a 4-a megalobrama amblycephala muscle sample, extracting a first-order mass spectrogram from phoxim in a 4-b megalobrama amblycephala muscle sample, extracting a second-order mass spectrogram from phoxim in a 4-c megalobrama amblycephala muscle sample, extracting an ion flow chromatogram from a 4-d megalobrama amblycephala muscle blank sample, extracting a first-order mass chromatogram from a 4-e megalobrama amblycephala muscle sample, extracting an ion flow chromatogram from O, O, O ', O', -tetraethyl disulfide phosphorrama in a 4-f megalobrama amblycephala muscle sample, extracting an ion flow chromatogram from a 4-i megalobrama amblycephala blank muscle sample, extracting an ion flow chromatogram from a 4-g megalobrama muscle sample, extracting O, O in a 4-j megalobrama muscle sample, o ', O', -tetraethyl diphosphorus sibiricus secondary mass spectrogram, O-diethyl thiophosphoric acid extraction ion flow chromatogram in a 4-k megalobrama amblycephala muscle sample, 4-l megalobrama amblycephala blank muscle sample extraction ion flow chromatogram, O-diethyl thiophosphoric acid primary mass spectrogram in a 4-m megalobrama amblycephala muscle sample, 4-n megalobrama blank muscle sample primary mass spectrogram, and O, O-diethyl thiophosphoric acid secondary mass spectrogram in a 4-O megalobrama amblycephala muscle sample.
FIG. 5: chromatogram and mass spectrum of megalobrama amblycephala skin sample and blank skin sample.
Extracting an ion current chromatogram from phoxim in a skin sample of the megalobrama amblycephala 5-a, extracting a primary mass chromatogram from phoxim in a skin sample of the megalobrama amblycephala 5-b, extracting a secondary mass chromatogram from phoxim in a skin sample of the megalobrama amblycephala 5-d, extracting an ion current chromatogram from a blank sample of the megalobrama 5-e, extracting an ion current chromatogram from O, O, O ', O', -tetraethyl disulfide monophosphate in a skin tissue of the megalobrama amblycephala 5-f, extracting an ion current chromatogram from O, O, O ', O', -tetraethyl disulfide monophosphate diphosphate primary mass chromatogram in a skin tissue of the megalobrama amblycephala 5-h, extracting an ion current chromatogram from a skin tissue of the megalobrama amblycephala 5-i, 5-j megalobrama amblycephala skin blank tissue primary mass spectrogram.
FIG. 6: chromatogram and mass spectrum of megalobrama amblycephala liver sample and blank liver sample.
Extracting an ion flow chromatogram from phoxim in a 6-a megalobrama amblycephala liver sample, extracting a first-grade mass chromatogram from phoxim in a 6-b megalobrama amblycephala liver sample, extracting a second-grade mass chromatogram from phoxim in a 6-c megalobrama amblycephala liver sample, extracting an ion flow chromatogram from a 6-d megalobrama amblycephala blank liver sample, extracting an ion flow chromatogram from O, O, O ', O', -tetraethyl disulfide monophosphate in a 6-f megalobrama amblycephala liver tissue, extracting an ion flow chromatogram from O, O, O ', O', -tetraethyl disulfide monophosphate in a 6-g megalobrama liver tissue, extracting an ion flow chromatogram from an 6-i megalobrama amblycephala liver tissue, o, O, O ', O', -tetraethyl diphosphonate disulfide primary mass spectrogram in the liver tissue of the 6-j megalobrama amblycephala, an O, O-diethyl thiophosphoric acid extraction ion flow chromatogram in the liver tissue of the 6-k megalobrama amblycephala, an O, O-diethyl thiophosphoric acid primary mass spectrogram in the liver tissue of the 6-l megalobrama amblycephala, an O, O-diethyl thiophosphoric acid secondary mass spectrogram in the liver tissue of the 6-m megalobrama amblycephala, an O-diethyl thiophosphoric acid extraction ion flow chromatogram in the blank liver tissue of the 6-n megalobrama amblycephala and a primary mass spectrogram in the blank liver tissue of the 6-O megalobrama amblycephala.
FIG. 7 is the chromatogram and mass spectrum of megalobrama amblycephala kidney sample and blank kidney sample.
Extracting a phoxim from a 7-a megalobrama amblycephala kidney sample to obtain an ion current chromatogram, extracting a phoxim primary mass spectrum from a 7-b megalobrama amblycephala kidney sample, extracting a phoxim secondary mass spectrum from a 7-c megalobrama amblycephala kidney sample, extracting an ion current chromatogram from a 7-d megalobrama amblycephala blank kidney sample, extracting a primary mass spectrum from a 7-e megalobrama amblycephala blank kidney sample, extracting an ion current chromatogram from a 7-f megalobrama amblycephala kidney tissue by O and O-diethyl thiophosphoric acid, extracting an ion current chromatogram from a 7-g megalobrama kidney tissue by O and O-diethyl thiophosphoric acid, extracting an ion current chromatogram from a 7-i megalobrama amblycephala kidney tissue, and extracting a primary mass spectrum from a 7-j megalobrama kidney tissue.
FIG. 8 is the chromatogram and mass spectrogram of the Megalobrama amblycephala branchia sample and the blank branchia sample.
An extraction ion flow chromatogram of phoxim in an 8-a megalobrama amblycephala branchia sample, a primary mass chromatogram of phoxim in an 8-b megalobrama amblycephala branchia sample, a secondary mass chromatogram of phoxim in an 8-c megalobrama amblycephala branchia sample, an extraction ion flow chromatogram of an 8-d megalobrama amblycephala branchia sample, a primary mass chromatogram of an 8-e megalobrama branchia blank sample, an extraction ion flow chromatogram of O, O-diethyl thiophosphoric acid in an 8-f megalobrama amblycephala branchia tissue, an extraction ion flow chromatogram of O, O-diethyl thiophosphoric acid in an 8-g megalobrama amblycephala branchia tissue, an extraction ion flow chromatogram of an O, O-diethyl thiophosphoric acid secondary mass chromatogram of an 8-i megalobrama branchia tissue, and a primary mass chromatogram of the 8-j megalobrama branchia blank tissue.
FIG. 9 is the chromatogram and mass spectrum of Megalobrama amblycephala in intestinal and blank intestinal samples.
Extracting a chromatographic chart of an ion flow from phoxim in a 9-a megalobrama amblycephala intestinal sample, extracting a chromatographic chart of a first-order phoxim in a 9-b megalobrama amblycephala intestinal sample, extracting a chromatographic chart of an ion flow from a 9-d megalobrama amblycephala intestinal blank sample, extracting a chromatographic chart of a first-order phoxim in a 9-e megalobrama intestinal blank sample, and extracting a second-order phorama amblycephala intestinal blank sample from a 9-f megalobrama amblycephala intestinal blank sample.
FIG. 10 shows the structural formula of phoxim and its metabolites
10-a phoxim, 10-b O, O, O ', O', -tetraethyl phosphorodithioate, 10c-O, O-diethylthiophosphoric acid.
Detailed Description
The following detailed description of the present invention will be provided in conjunction with the accompanying drawings
Example 1:
test materials and methods
1.1 test drugs, test fish and culture conditions
Phoxim emulsifiable concentrate (content: 98% or more, available from pesticide Co., Ltd., from Shichentai), acetone (super grade pure, national pharmaceutical group chemical Co., Ltd.), Phoxim control (content: 98% or more, Dr. Ehrenstontorfer, Germany, CAS number: 14816-18-3), O, O, O ', O', -tetraethyl phosphorodithioate (content: 94.3% or more, American Advanced ChemTech, CAS number: 3689-24-5), O, O-diethylthiophosphoric acid (content: 98.0% or more, American Advanced ChemTech, CAS number: 5871017-0), and phosphorus oxyoctanoate (content: 96.7% or more, Germany Drfer. Ehrenstontorr, CAS number: 14816-17-2).
The healthy megalobrama amblycephala is purchased from the Baishazhou big market in Wuhan, the megalobrama amblycephala is 340 +/-19 g, and is placed in an aquarium (160cm multiplied by 60cm multiplied by 80cm) sterilized by potassium permanganate for temporary culture for 7 days, the experimental water is tap water which is fully aerated and dechlorinated, an oxygen filling pump is adopted for continuous oxygenation in the culture process, the water temperature is 24 ℃, and the healthy megalobrama amblycephala is selected for experiments after the temporary culture is finished.
1.2 Experimental methods
1.2.1 modes of administration
Soaking for administration, wherein the concentration of phoxim aqueous solution for soaking is 1 mg/L.
1.2.2 preparation and administration of Phoxim solution
3g of phoxim missible oil is dissolved by acetone, the volume is determined to be 100mL, and phoxim acetone solution with the concentration of 30mg/mL is prepared. Adding the prepared phoxim solution into aerated and dechlorinated tap water to prepare a water solution with the phoxim concentration of 1mg/L, respectively injecting the prepared 1mg/L water solution into a glass fish tank with the thickness of 60cm multiplied by 35cm multiplied by 40cm, respectively putting 5 megalobrama amblycephala in each glass for soaking, wherein 40L of the phoxim water solution is in each glass tank. Taking out the megalobrama amblycephala from the glass jar after soaking for 2, 4, 6, 8, 10, 24 and 48 hours, washing the body surface with distilled water for 3 times, and sucking the water on the body surface of the megalobrama amblycephala with filter paper. The fish blood, muscle, skin, liver, kidney, gill, intestine, etc. tissues are collected separately (per time period). The blood is injected into a syringe which is pre-rinsed with heparin sodium, the blood is put into a centrifuge tube rinsed with heparin sodium, the centrifuge tube is centrifuged for 5min at 4000r/min, and the upper plasma is taken and stored at-80 ℃. Taking out the intestine, washing the intestinal contents with a syringe filled with distilled water, cutting with scissors, homogenizing the intestine with a homogenizer, and freezing at-80 deg.C for storage. Shearing muscle, skin and gill with scissors, homogenizing with homogenizer, and freezing at-80 deg.C. Homogenizing the liver and kidney tissues by a homogenizer, and freezing and storing at-80 ℃ to be tested.
1.2.3 Ultra Performance Liquid Chromatography (UPLC) analysis conditions
Agilent ZORBAX Eclipse Plus C18 column (100 mm. times.2.1 mm,1.8 μm), column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient was 10% methanol by volume and 90% aqueous 0.1% formic acid for 1min, followed by a linear ramp up to 90% methanol by volume in 5min for 4min, followed by a linear ramp down to 10% methanol by volume in 0.1min for 1.9 min. The injection volume was 5.0. mu.L.
1.2.4 sample pretreatment
1) Pretreatment of samples such as plasma, liver, kidney, gill, skin and intestine: weighing 1g of liver, kidney, gill, skin and intestine and 1mL of blood, adding 5mL of extractant A, performing vortex for 30s and ultrasonic treatment for 3min, adding 0.5g of purifying agent B, performing vortex for 30s and centrifugation for 5min at 7000r/min, transferring supernatant into a 10mL centrifuge tube, adding 5mL of extractant A, repeatedly extracting once, combining two extracting solutions, blowing nitrogen at 50 ℃ to dry, performing constant volume with 1mL of methanol, filtering through a 0.22-micron filter head, and performing upper UPLC-Q-TOFMS analysis.
2) Pre-treating a muscle sample, namely putting 5.0g of muscle into a 50mL centrifuge tube, adding 20mL of extractant A, performing vortex for 30s, performing ultrasonic treatment for 3min, adding 3.0g of purifying agent B, performing vortex for 30s, centrifuging for 5min at 7000r/min, transferring supernatant into a 150mL chicken heart bottle, adding 15mL of extractant A, repeatedly extracting once, combining two extracting solutions, performing rotary evaporation at 45 ℃ to about 3mL, transferring 3mL liquid into a 10mL centrifuge tube, washing the chicken heart bottle twice by using 5mL of extractant A, combining washing solutions, blowing nitrogen at 50 ℃ to dryness, fixing volume by 1mL methanol, filtering by using a 0.22 mu m filter head, and performing UPLC-Q-TOFMS analysis.
1.2.5QTOFMS analysis conditions
Electrospray ionization (ESI), positive ion mode, mother ion TOFMS Masses scanning range is M/Z100 ~ 1000Da, accumulation time 0.199957 secs. Scanning range of product ion TOFMS Masses is M/Z50-1000 Da, and declustering voltage (DP): 45V, impact energy (CE) 35eV, extended impact energy (CES) 15.0 eV; the Ion Release Delay (IRD) was 67, the Ion Release Width (IRW) was 25, the spray gas (GS1) was 50Psi, the auxiliary heating gas (GS2) was 55Psi, the air window gas (CUR) was 25Psi, the ionization temperature was 400 ℃ and the positive ion spray voltage was 5500V.
1.2.6 screening of the extractant
Adding phoxim, O, O, O ', O', -tetraethyl diphosphonite, O, O-diethylthiophosphoric acid and octoxyphosphate standard mixed solution into megalobrama amblycephala muscle tissue to make the final concentration be 5mg/kg (n is 3). Methanol, acetonitrile, 0.1% methanol formate, 0.1% acetonitrile formate, dichloromethane, acetone, ethyl acetate, acetonitrile + ethyl acetate (v: v,5:1) are respectively used as extracting agents, the extracting agents are screened by taking the recovery rate as an evaluation standard, sample pretreatment and 1.2.5QTOFMS analysis conditions are carried out according to a 1.2.4 sample pretreatment method, the detection result is shown in figure 1, and the extraction recovery rates of the phoxim and the metabolites thereof are respectively more than 85.0% and better than the extraction recovery rates of the phoxim metabolites (50% -85.2%) of the extracting agents such as methanol, acetonitrile, 0.1% methanol formate, 0.1% acetonitrile formate, dichloromethane, acetone, ethyl acetate and the like from figure 1. Thus, acetonitrile + ethyl acetate (v: v,5:1) is preferred as the extractant in the present process.
1.2.7 screening of the purifying agent
The matrix effect (ME,%) refers to the components of the sample other than the analyte, as the matrix is oftenOften there is significant interference with the analyte analysis process and affects the accuracy of the analysis results, these effects and interferences are called matrix effects. The matrix effect has an ion suppression effect or an ion enhancement effect, and the response value of the matrix to the analysis target substance has a suppression effect when the ME value is a negative value according to the calculation formula, and a positive value indicates that the matrix has an enhancement effect on the response value of the analysis target substance. Adding phoxim, O, O, O ', O', -tetraethyl diphosphonite, O, O-diethylthiophosphoric acid and octoxyphosphate standard mixed solution into megalobrama amblycephala muscle tissue to make the final concentration be 5mg/kg (n is 3). Respectively adopting Graphitized Carbon Black (GCB), N-propyl ethylenediamine (PSA), octadecylsilane chemically bonded silica (C18) and anhydrous magnesium sulfate (MgSO)4) Anhydrous sodium sulfate (Na)2SO4) Multiwalled carbon nanotubes (MWNTs) and anhydrous magnesium sulfate (MgSO)4) + octadecylsilane chemically bonded silica (C18) (m: m,10:1) purified the extract of the sample, and the purification effect was evaluated by the matrix effect (ME,%), and the purifying agent was selected, the smaller the absolute value of the matrix effect, the better the purification effect. The results are shown in FIG. 2, with an optimized scavenger of anhydrous magnesium sulfate (MgSO)4) The purification effect of the octadecylsilane chemically bonded silica (C18) (m: m,10:1) on each tissue of the megalobrama amblycephala is superior to that of other purifiers, and MgSO (MgSO) is adopted4+ C18(m: m,10:1) as cleaning agent for matrix effect of megalobrama amblycephala tissue after cleaning on phoxim and its metabolite<12% | and matrix effect > 14% | after sample purification with the other purifiers described above), therefore, MgSO is preferred for this method4+ C18(m: m,10:1) as scavenger.
The matrix effect (ME,%) is calculated as follows:
1.2.8 analysis of metabolite of phoxim in megalobrama amblycephala tissue
Use of PeakView from AB SCIEXTMThe MaserView option in the software menu bar takes the total ion flow graph of UPLC-QTOFMS as the comparison, and the total ion flow graph of the actual sample subtracts the total ion flow graph of the blank comparison sampleAnd (3) comparing differences of the flow diagrams to obtain the accurate mass number of the different target object, and predicting the metabolic products of the phoxim in the plasma, muscle, skin, liver, kidney, branchia and intestinal tissues of the megalobrama amblycephala according to the structural formula and the position where metabolism is possible.
Identification of phoxim and metabolite thereof in internal tissue of megalobrama amblycephala by UPLC-QTOFMS
The major metabolites of phoxim in plasma, muscle and liver are O, O, O ', O', -tetraethyl phosphorodithioate and O, O-diethylthiophosphate, the metabolites in gill and kidney are mainly O, O-diethylthiophosphate, and the major metabolites in skin are O, O, O ', O', -tetraethyl phosphorodithioate. Phoxim exists in the intestine primarily as the prototypical drug, phoxim. QTOFMS measured retention time, molecular composition and accurate molecular weight M of phoxim and metabolite thereof in megalobrama amblycephala tissuesATheoretical molecular weight MTAnd mass accuracy, daughter ion (see table 1). Ion chromatogram and primary and secondary mass spectrograms of phoxim and its metabolite extracted from blood plasma, muscle, skin, liver, kidney, branchia and intestinal tissues of megalobrama amblycephala are shown in FIGS. 3-9. The structural formula of phoxim and its metabolite is shown in figure 10.
TABLE 1 molecular composition, exact molecular weight M of phoxim and its metabolitesATheoretical molecular weight MTAnd accuracy of mass
Based on the identification method, the applicant also identifies the metabolites of the phoxim in different tissues of freshwater fishes such as tilapia, channel catfish and the like, and the results show that the types of the metabolites of the phoxim in different tissues of the freshwater fishes (including megalobrama amblycephala, tilapia and channel catfish) are the same, but the contents of the metabolites are different. Therefore, the method provides a detection target for detecting the residues of the phoxim in the tissues of the freshwater fish, and also provides a technical basis and a scientific basis for the maximum residue limit of the phoxim in the tissues of the freshwater fish.
Claims (1)
1. A method for identifying a metabolite of phoxim in different tissues of freshwater fish is characterized by comprising the following steps:
(1) sample processing
Respectively weighing plasma and samples of liver, kidney, gill, intestine, skin and muscle tissues, sequentially adding an extracting agent A and a purifying agent B, centrifuging after vortex oscillation, collecting an extracting solution, repeatedly extracting sample residues by using the extracting agent A, combining the extracting solutions, drying by blowing nitrogen, fixing the volume by using methanol, filtering by using a filter membrane of 0.22 mu m, and detecting filtrate by using UPLC-Q-TOFMS;
the extractant A is acetonitrile and ethyl acetate, and the volume ratio of the extractant A to the extractant B is 5:1, mixing;
the purifying agent is prepared by mixing anhydrous magnesium sulfate and octadecylsilane chemically bonded silica in a mass ratio of 10: 1;
(2) UPLC-QTOFMS qualitative analysis
Detecting each tissue sample by using ultra-high performance liquid chromatography-quadrupole time of flight high-resolution mass spectrometry technology to obtain retention time and ion mass of the compound, and phoxim, O’,O’Comparing the retention time and the ion mass of tetraethyl phosphorodithioate and O, O-diethylthiophosphoric acid, and judging the metabolic products of each tissue sample;
the UHPLC analysis conditions are specifically as follows: c18 chromatographic column, 100mm × 2.1mm × 1.8 μm; column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient of the methanol containing 10% of organic phase and the 0.1% of formic acid aqueous solution containing 90% of aqueous phase is maintained for 1min, then the organic phase linearly rises to the methanol containing 90% of volume concentration within 5min, the aqueous phase linearly drops to the 0.1% of formic acid aqueous solution containing 10% of aqueous phase, the maintenance is carried out for 4min, the organic phase linearly drops to the methanol containing 10% of volume within 0.1min, the aqueous phase linearly rises to 90% of volume, and finally the maintenance is carried out for 1.9 min; the sample introduction volume is 5.0 muL;
the QTOFMS analysis conditions are as follows: an electrospray ion source is in a positive ion mode, the scanning range is M/Z100-1000 Da, and the accumulation time is 0.199957 secs; the mass range of product ions scanned by the time-of-flight mass spectrometry is 50-1000 Da, and the cluster removing voltage is as follows: 45V, the collision energy is 35eV, and the expansion collision energy is 15.0 eV; ion release delay was 67, ion release width was 25, ion source: the double-spray ion source has the spray gas of 50Psi, the auxiliary heating gas of 55Psi, the air window gas of 25Psi, the ionization temperature of 400 ℃ and the positive ion spray voltage of 5500V.
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