CN112881554A - Detection method for nicotine drug chloride and metabolite thereof in mutton - Google Patents
Detection method for nicotine drug chloride and metabolite thereof in mutton Download PDFInfo
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- CN112881554A CN112881554A CN202110057417.1A CN202110057417A CN112881554A CN 112881554 A CN112881554 A CN 112881554A CN 202110057417 A CN202110057417 A CN 202110057417A CN 112881554 A CN112881554 A CN 112881554A
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- mutton
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Abstract
The invention relates to a method for detecting nicotine chloride medicines and metabolites thereof in mutton, which comprises the steps of firstly obtaining an extracting solution by a QuEChERS pretreatment method, then measuring chromatographic peaks, full-scanning mass-to-charge ratios and secondary characteristic breaking fragments of 11 nicotine chloride medicines and metabolites thereof by ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrometry, simultaneously obtaining chromatographic peaks, full-scanning mass-to-charge ratios, secondary characteristic breaking fragments and breaking ways of corresponding standard substances, comparing the chromatographic peaks in normal distribution with the chromatographic peaks, then obtaining molecular formulas of substances corresponding to each chromatographic peak, multiplying the standard solution concentration of the corresponding substances by the ratio of the peak area of the chromatographic peak of each substance to be detected to the peak area of the chromatographic peak of the corresponding substance to obtain the concentration of each nicotine chloride medicine or metabolite thereof in the extracting solution, and finally obtaining the concentration of each nicotine chloride medicine or metabolite thereof in mutton, and completing the detection of nicotine chloride medicines and metabolites thereof in the mutton.
Description
Technical Field
The invention relates to the technical field of detection of medicines and metabolites thereof, in particular to a detection method of nicotine chloride medicines and metabolites thereof in mutton.
Background
With the increase of meat products in diet of people, the mutton has the characteristics of high protein, low fat, rich vitamins and trace elements, and is deeply loved by consumers in the market. The industrial chain of meat products has close relation among all the links, including a plurality of links of feed culture, sheep breeding, meat product processing and terminal sale, and pesticide residue pollution is an important factor influencing the quality of meat products. The nicotine medicine and the metabolite thereof both contain a nitromethylene heterocyclic structure, have the characteristics of remarkable insecticidal activity, strong selectivity and the like, and are widely used in culture and planting of animal feed, and the nicotine medicine and the metabolite thereof are finally transferred to mutton through food chain transfer and ecological enrichment.
The detection method for the nicotine chloride medicine in the animal derived food mainly comprises a Liquid Chromatography (LC) method, a liquid chromatography-tandem mass spectrometry (LC-MS) method, a gas chromatography-tandem mass spectrometry (GC-MS) method and the like. At present, the accurate quantification of the nicotine chloride medicine in the animal derived food can be realized by applying a liquid chromatography-tandem mass spectrometry technology, but the high-throughput qualitative and quantitative analysis of the metabolite of the nicotine chloride medicine cannot be realized.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a detection method of nicotine chloride medicines and metabolites thereof in mutton, which has the advantages of high selectivity, high-flux qualitative and quantitative analysis, high scanning speed, high detection sensitivity, simplicity and convenience, and provides a new method for detecting the nicotine chloride medicines and the metabolites thereof.
The specific technical scheme of the invention is as follows:
a method for detecting nicotine chloride medicines and metabolites thereof in mutton comprises the following steps:
step 1, homogenizing mutton, then carrying out vortex by using an acetic acid solution of acetonitrile to obtain a mixed system A, adding anhydrous magnesium sulfate and sodium acetate into the mixed system A, carrying out vortex, then carrying out oscillation extraction to obtain an extracting solution, and centrifuging the extracting solution to obtain a mixed system B;
step 2, adding ethylenediamine-N-propyl silica gel, octadecyltrichlorosilane and MgSO (MgSO) into the mixed system B4Obtaining a mixed system C, centrifuging the mixed system C after swirling, and extracting supernatant to obtain an extracting solution;
step 3, diluting the extracting solution with an ammonium acetate solution, filtering, and determining the filtered solution in an ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrum to obtain chromatographic peaks, full-scan mass-to-charge ratios and secondary characteristic broken fragments of 11 nicotine drugs and metabolites thereof in mutton, wherein the 11 nicotine drugs and the metabolites thereof are respectively imidacloprid, imidacloprid urea, pentahydroxy imidacloprid, nicotine imidacloprid, acetamiprid-metabolite-IM-1-4, acetamiprid-metabolite-IM-1-2, acetamiprid-metabolite-6-fluoronicotinic acid, acetamiprid-metabolite-IM-2-1, dinotefuran and dinotefuran-metabolite-UF;
diluting 11 standard solutions of nicotine-based medicaments and metabolites thereof with an ammonium acetate solution to obtain corresponding diluents, respectively measuring all the diluents in an ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap mass spectrum to obtain a chromatographic peak, a full-scan mass-to-charge ratio, secondary characteristic fragmentation fragments and a fragmentation path of corresponding substances, wherein the chromatographic peak, the full-scan mass-to-charge ratio and the secondary characteristic fragmentation fragments of all the substances form a standard substance database;
step 4, extracting the chromatographic peaks obtained in the step 3 one by one to obtain corresponding chromatographic peaks in normal distribution, comparing each chromatographic peak in normal distribution with chromatographic peaks in a standard substance database to obtain the category of substances corresponding to each chromatographic peak, comparing the retention time of the chromatographic peak, the full-scan mass-to-charge ratio, the retention time of the secondary characteristic fragmentation fragment and the chromatographic peaks in the standard substance database, the full-scan mass-to-charge ratio, the secondary characteristic fragmentation fragment and the fragmentation path obtained in the step 3 to obtain the molecular formula of the substances corresponding to each chromatographic peak, and completing the one-to-one correspondence of the chromatographic peaks in the step 3 with the corresponding nicotine chloride drugs and metabolites thereof;
step 5, multiplying the peak area of the chromatographic peak corresponding to each substance to be detected and the peak area ratio of the chromatographic peak of the corresponding substance by the concentration of the standard solution of the corresponding substance to obtain the concentration of each nicotine chloride medicine or metabolite thereof in the extracting solution, and finally obtaining the concentration of each nicotine chloride medicine or metabolite thereof in the mutton by using the following formula to finish the detection of the nicotine chloride medicine and metabolite thereof in the mutton;
ci＝(c0v)/m, wherein ciThe concentration of each chloronicotinyl drug or metabolite thereof in the mutton is measured in units of mu g/kg, c0Is the concentration of each nicotine drug or metabolite thereof in the extracting solution, and has the unit of microgram/L, v is the volume of the extracting solution in the step 3, and has the unit of L, and m is the mass of the mutton in the step 1, and has the unit of kg.
Preferably, the mass ratio of the mutton to the anhydrous magnesium sulfate to the sodium acetate in the step 1 is (5-10): 5-6): 2-3; in step 2, ethylenediamine-N-propyl silica gel, octadecyltrichlorosilane and MgSO4The weight ratio of mutton to mutton is (0.3-0.4): (0.2-0.4): (0.6-0.8): 5-10).
Preferably, the volume ratio of acetonitrile to acetic acid in the acetic acid solution of acetonitrile in the step 1 is 1%, and the ratio of the acetic acid solution of acetonitrile to mutton is (5-20) mL and (5-10) g.
Preferably, Accucore aQ is selected in the ultra-high performance liquid chromatography in the step 3, and the specific conditions are shown as follows, wherein the column temperature of the ultra-high performance liquid chromatography is 50-55 ℃, the spraying voltage is 4-5 kV, the ion source temperature is 60-65 ℃, and the capillary temperature is 325-375 ℃;
the quadrupole rod electrostatic field orbit ion trap mass spectrum adopts a two-stage scanning mode, the two-stage scanning mode is variable data independent acquisition, and the scanning time is 0-20 min; resolution was taken at 95000FWHM and divided into two quality scan segments: the m/z-150-.
Further, in the condition of the ultra-high performance liquid chromatography, the mobile phase A is a mixed solution composed of formic acid, ammonium formate and water, wherein the formic acid accounts for 0.1% of the total volume of the mixed solution, the concentration of the ammonium formate is 4mM, the mobile phase B is a mixed solution composed of formic acid, methanol and ammonium formate, wherein the formic acid accounts for 0.1% of the total volume of the mixed solution, and the concentration of the ammonium formate is 4 mM;
the gradient elution procedure is within 0-1 min, and the volume proportion of the mobile phase A is 80%; linearly reducing the volume proportion of the mobile phase A from 80% to 0 within 1-10 min; the volume ratio of the mobile phase A is 0 within 10-14 min; the volume proportion of the mobile phase A is linearly increased to 80% from 0 within 14-16 min; within 16-20 min, the volume proportion of the mobile phase A is 80%, and the flow rate is 0.3-0.5 mL/min.
Further, in the condition of the ultra-high performance liquid chromatography, the flow rate of sheath gas is 55-60 arb, the flow rate of auxiliary gas is 40-45 arb, the flow rate of gas curtain is 5-8 arb, and the heating temperature is 325-375 ℃.
Furthermore, in the mass spectrum of the quadrupole rod electrostatic field orbital ion trap, the collision energy is 20.0eV, 35.0eV and 50.0eV respectively.
Preferably, in step 3, the corresponding dilution is subjected to a secondary scan in a data-dependent scan mode, and other measurement conditions are the same as those of the extract.
Preferably, in step 3, when the secondary characteristic fragment corresponding to each of the 11 chloronicotinyls and metabolites thereof is obtained, the ratio of signal intensity between ion fragments is selected to be greater than 15% to distinguish the secondary characteristic fragment of the same drug.
Preferably, the secondary characteristic fragmentation fragments in the standard substance database in step 4 are the abundance ratio and the mass-to-charge ratio of the secondary characteristic fragmentation fragments.
Compared with the prior art, the invention has the following advantages:
the invention relates to a detection method of nicotine chloride medicine and metabolite thereof in mutton, which comprises the steps of firstly utilizing a QuEChERS pretreatment method to extract a solid sample in mutton homogenate by acetonitrile, removing most of interferents existing in the acetonitrile by dispersion matrix extraction, directly carrying out mass spectrometry on extract liquor, then utilizing ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap mass spectrometry to collect chromatographic peaks, full-scanning mass-to-charge ratios and secondary characteristic fracture fragments of 11 nicotine chloride medicine and metabolite standard substance compounds thereof, establishing a standard database of the 11 nicotine chloride medicine and the metabolite thereof, then utilizing the chromatographic peaks, the full-scanning mass-to-charge ratios and the secondary characteristic fracture fragments to complete one-to-one correspondence of the chromatographic peaks, the corresponding nicotine chloride medicine and the metabolite thereof, and multiplying the ratio of the peak area of the chromatographic peak corresponding to each substance to be detected to the peak area of the chromatographic peak of the corresponding substance to the peak area of the corresponding substance by the peak area of the corresponding substance to be detected to the peak area of the corresponding substance The concentration of each nicotine drug or metabolite thereof in the extracting solution is obtained by standard solution concentration, the concentration of each nicotine drug or metabolite thereof in the mutton is finally obtained, the detection of the nicotine drugs and the metabolites thereof in the mutton is completed, the non-directional screening method of the nicotine drugs and the metabolites thereof in the mutton is constructed, the accurate qualitative and accurate quantification of the nicotine drugs and the metabolites thereof in the mutton can be realized, the method has the characteristics of large peak capacity, high accuracy and precision and accurate quantification, the loss of the drugs in the analytes is reduced, the high drug recovery rate can be obtained, and the method has important application value. The method can be used for carrying out non-directional drug screening on mutton, and can identify and screen out unknown drugs in the mutton, so that accurate screening and high-throughput analysis of a large amount of data are realized, and the method is suitable for monitoring nicotine chloride drugs and metabolites thereof in the mutton at present.
Drawings
FIG. 1 is a mass spectrogram of ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap mass spectrometry for determining acetamiprid, a nicotine drug of the present invention;
FIG. 2 is a diagram of the breakage mechanism of acetamiprid according to the present invention.
Detailed Description
The principles and advantages of the present invention are explained and illustrated below with reference to specific embodiments so that those skilled in the art may better understand the present invention. The following description is exemplary only, and is not intended to limit the scope of the present disclosure.
The invention relates to a method for detecting nicotine chloride medicine in mutton and metabolites thereof, which comprises the following steps of preparing instruments, reagents and solutions,
1. instrument for measuring the position of a moving object
Vortex mixer model 2T vortex mixer (Scientific Industries, usa); an Avnti J-26 XPI model high speed refrigerated centrifuge (Beckman Coulter, USA); ultra performance liquid chromatography-quadrupole electrostatic field orbitals ion trap mass spectrometry equipped with an ion source (Thermo Fisher Scientific, usa); thermo Hypersil GoldAccucore aQ chromatographic column (Thermo Fisher Scientific, USA); a speed-regulating multipurpose oscillator; analytical balance (Mettler Toledo, switzerland); vortex mixer (IKA).
2. Reagent
Mutton, LC-MS acetonitrile, ammonium formate, formic acid, ammonium acetate, glacial acetic acid (Sigma, USA), a 0.22 mu m microporous filter membrane (Pall, USA), high-grade pure anhydrous magnesium sulfate, sodium acetate (Supelco, USA), nicotine chloride and related chemical standard substances of metabolites thereof (Sigma-Aldrich, Germany and Dr.
3. Preparation of the solution
11 kinds of nicotine chloride medicine and its metabolite specifically refer to imidacloprid and imidacloprid urea, pentahydroxy imidacloprid, nicotine imidacloprid; acetamiprid, acetamiprid-metabolite-IM-1-4, acetamiprid-metabolite-IM-1-2, acetamiprid-metabolite-6-fluoronicotinic acid, and acetamiprid-metabolite-IM-2-1; dinotefuran and dinotefuran-metabolite-UF.
Respectively weighing 11 standard substances of nicotine chloride medicines and metabolites thereof, respectively weighing 1.0mg (accurate to 0.01mg) of each standard substance, placing the standard substances in a 10mL volumetric flask, selecting corresponding solvents to dissolve and fix the volume to 10mL according to the solubility of the standard substances in different solvents (normal hexane, acetone and acetonitrile) and the measurement requirements, preparing 100mg/L single compound standard stock solution, and storing the standard stock solution in the dark at-15 ℃. And (3) transferring 0.5mL of each single compound standard stock solution into a 100mL volumetric flask, diluting with an acetonitrile-water solution (1:1, v/v) and fixing the volume to a scale, and preparing a standard solution with a corresponding concentration. The standard solution was stored in a brown closed bottle at-15 ℃ in the dark. All standard solutions used in the invention are prepared as before.
The specific detection method comprises the following steps:
1, processing a mutton sample by adopting a QuEChERS pretreatment method;
the method comprises the following specific steps:
weighing 5-10 g (accurate to 0.01g) of mutton sample, chopping, and grinding into homogenate.
Placing the homogenate into a 50mL centrifuge tube, adding 15-20 mL of 1% acetonitrile acetic acid solution (the volume ratio of acetonitrile to acetic acid is 1%), performing vortex mixing for 1-3 min, adding 5-6 g of anhydrous magnesium sulfate and 2-3 g of sodium acetate, performing vortex mixing for 1-3 min, performing oscillation extraction for 2-5 min, and performing centrifugation for 5-10 min (0-5 ℃) at 2000-4000 rpm.
Taking 2-5 mL of supernatant fluid into a 15mL polypropylene centrifugal tube, adding 300-400 mg of solid dispersion purifying agent PSA (namely ethylenediamine-N-propyl silica gel for adsorbing medium-polarity and polar impurities), 200-400 mg of solid dispersion purifying agent C18 (namely octadecyl trichlorosilane for adsorbing reversed-phase solid adsorbent for adsorbing lipid and other non-polar impurities) and 600-800 mg of MgSO4Centrifuging at 2000-4000 rpm for 10-20 min after swirling for 1-3 min, and transferring supernatant to obtain an extracting solution;
the mechanism of the QuEChERS pretreatment method is that solid samples in the meat product homogenate are extracted by acetonitrile, then most of interferents existing in the acetonitrile are removed by extraction of a dispersion matrix, and the extract can be directly subjected to mass spectrometry, so that the method is a method for effectively separating trace pesticide residues.
In the implementation of the present invention, the optimal data is adopted in the above data, and since the range of the data is small, other data is not provided, which is specifically as follows.
Mutton sample 5g (accurate to 0.01g) was weighed, minced, and ground to homogenate.
The homogenate was placed in a 50mL centrifuge tube, 20mL of a 1% acetonitrile acetic acid solution (acetonitrile to acetic acid volume ratio of 1%) was added, vortexed for 1min, 6g of anhydrous magnesium sulfate and 2g of sodium acetate were added, vortexed for 1min, extracted with shaking for 2min, and centrifuged at 3000rpm for 5min (3 ℃).
Taking 2mL of supernatant, putting into a 15mL polypropylene centrifuge tube, adding 300mg of solid dispersion purifying agent PSA, 200mg of solid dispersion purifying agent C18 and 800mg of MgSO4Vortex for 1min, centrifuging at 3000rpm for 15min, and collecting supernatant to obtain extractive solution.
2, measuring the chlorinated nicotine medicine and metabolites thereof by adopting a high performance liquid chromatography-quadrupole electrostatic field orbit ion trap high resolution mass spectrometer;
mu.L of the extract was mixed with 500. mu.L of 0.2M ammonium acetate solution, thus mixing 1: diluting the extracting solution by 1(v/v) to strengthen ionization, weighing 154g of ammonium acetate, dissolving the ammonium acetate in 1L of purified water to obtain 0.2mol/L of ammonium acetate solution, filtering the ammonium acetate solution by a 0.22 mu m organic microporous membrane to obtain a sample to be detected, introducing the sample to be detected into a sample introduction small disc for sample introduction, and collecting chromatographic peaks, full-scanning mass-to-charge ratios and secondary characteristic fragmentation fragments of 11 nicotine chloride medicines and metabolites thereof by using ultra-high performance liquid chromatography-quadrupole electrostatic field rail ion trap mass spectrometry.
The chromatographic measurement conditions were as follows:
the chromatographic column was Accucore aQ, a UHPLC guard column (10 mm. times.2.1 mm, 2.6 μm, Thermo Fisher Scientific Co., USA). The column temperature is 50-55 ℃. The mobile phase A is a mixed solution composed of formic acid, ammonium formate and water, wherein the formic acid accounts for 0.1% of the total volume of the mixed solution, the concentration of the ammonium formate is 4mM, the mobile phase B is a mixed solution composed of formic acid, methanol and ammonium formate, wherein the formic acid accounts for 0.1% of the total volume of the mixed solution, and the concentration of the ammonium formate is 4 mM. The gradient elution procedure is within 0-1 min, and the volume proportion of the mobile phase A is 80%; linearly reducing the volume proportion of the mobile phase A from 80% to 0 within 1-10 min; the volume ratio of the mobile phase A is 0 within 10-14 min, and the volume ratio of the mobile phase A is linearly increased from 0 to 80% within 14-16 min; within 16-20 min, the volume proportion of the mobile phase A is 80%, and the flow rate is 0.3-0.5 mL/min. The collision gas is high-purity nitrogen (the purity is 99.99%), the ion source is an electrospray ion source, the compound is measured by adopting a positive mode, the flow rate of the sheath gas is 55-60 arb, the flow rate of the auxiliary gas is 40-45 arb, the flow rate of the gas curtain is 5-8 arb, the spraying voltage is 4-5 kV, the temperature of the ion source is 60-65 ℃, the temperature of the capillary tube is 325-375 ℃, the heating temperature is 325-375 ℃, and the sample injection amount is 10 mu L.
In the implementation of the present invention, the optimal data is adopted in the above data, and since the range of the data is small, other data is not provided, which is specifically as follows.
The column temperature was 50 ℃. The flow rate of the sheath gas was 55arb, the flow rate of the auxiliary gas was 40arb, the flow rate of the gas curtain was 5arb, the spray voltage was 4kV, the ion source temperature was 65 ℃, the capillary temperature was 375 ℃, and the heating temperature was 375 ℃.
The mass spectrometry conditions were as follows:
the first-level scanning mode: in the full scan mode, the resolution is 95,000FWHM, and the target value of the automatic gain control is set to 8.0 × 106。
The secondary scanning mode is Variable Data Independent Acquisition, the English name is Variable Data Independent Acquisition, the abbreviated vDIA, the scanning time is 0-20 min, the resolution adopts 95,000FWHM, the maximum injection time is 100ms, the target value of automatic gain control is set to be 3.0 multiplied by 106The list of information (i.e., m/z) can be divided into: 150.00000, 200.00000, 250.00000, 300.00000, 350.00000, 400.00000, 450.00000, 500.00000, 550.00000, 600.00000, 700.00000, 800.00000, 900.00000, wherein m/z-150.00000-550.00000 is set as a first vDIA scan, an isolation window range is set as 54.0Da, a cycle count is set as 9, m/z-600.00000-900.00000 is set as a second vDIA scan, the isolation window range is set as 104.0Da, and the cycle count is set as 4. The collision energy was 20.0eV, 35.0eV, and 50.0eV, respectively.
Establishment of standard substance database of 3, 11 kinds of nicotine chloride medicines and metabolites thereof
The standard solution of 11 kinds of nicotine-based drug chloride and metabolite thereof is diluted to prepare solution with the concentration of 500 mug/L, 500 mug/L solution and 500 mug/L0.2M ammonium acetate solution are respectively taken, and the ratio of 1: 1(v/v) diluting the extracting solution for collecting information of a standard substance database, wherein the standard substance database of the nicotine drug and the metabolite thereof comprises a chromatographic peak, a full-scanning mass-to-charge ratio, a secondary characteristic fragmentation fragment and a molecular formula; the secondary characteristic fragmentation fragment covers the abundance ratio and the mass-to-charge ratio of the secondary characteristic fragmentation fragment, the chromatographic peak covers retention time, peak area and peak width, the sample is parallelly injected and analyzed for 6 times, and the measuring condition of the chromatographic-mass spectrum is only different from the diluted extracting solution in a secondary scanning mode, so that the accuracy and the reproducibility of the standard substance database of the nicotine-based chloride medicine and the metabolite thereof are realized.
Firstly, collecting the full-scan chromatogram, the full-scan mass spectrum, the secondary fracture chromatogram and the secondary fracture mass spectrum of each standard substance. After 1 second and 3 times of scanning in a two-stage scanning mode with data dependent on a scanning mode, collision and fragmentation are carried out through three different collision energies of low (20eV), medium (35eV) and high (50eV), and a standard substance collision and fragmentation ion spectrogram is obtained through a normalization result of mass spectrum information of different collision energy levels. The second-order broken fragments of the similar medicines are distinguished by selecting the signal intensity ratio between the ion fragments of the chloronicotinyl medicines and the metabolites thereof to be more than 15 percent as quantitative information.
Establishing a standard substance database of nicotine chloride drugs and metabolites thereof based on the analysis result of the actually measured fracture information, wherein the standard substance database comprises 4 adduct forms ([ M-H ] in positive ion mode and negative ion mode]-、[M+HCOO]-、[M+H]+、[M+Na]+And (c) spectrum information will be described below using fig. 1 as a representative, and will be described in detail below.
Specifically, a secondary characteristic fragmentation is obtained through the research on fragmentation paths and mechanisms, and the secondary characteristic fragmentation is applied to screening of nicotine chloride medicines and metabolites thereof in mutton, so that a non-directional screening method is established.
(1) Cleavage pathway and mechanism study
The invention analyzes the mechanism of the fragment breaking of 11 kinds of nicotine chloride medicines and metabolites thereof, and obtains the reaction paths of all the fragment breaking. Taking acetamiprid as an example, a high resolution mass spectrum of acetamiprid by HPLC-quadrupole electrostatic field orbited ion trap is shown in FIG. 1.
The ion formed by the compound losing one electron is an odd electron ion which comprises two activation centers, and the fracture mechanism of the compound is mainly a rearrangement reaction (namely rHRAnd rHC) Pi electron cloud migration reaction, charge-induced i-fragmentation reaction. The mass-to-charge ratio of acetamiprid is 223.07450, and the parent ion C6H5ClN+The major cleavage pathways are shown in FIG. 2:
C6H5ClN+cleavage pathway with m/z of 126.01050: the first cleavage route is i cleavage, the acetamiprid forms an odd-electron ion by adding a proton, the positive charge center is located on an unsaturated N atom connected with a double bond, the N atom is an N atom on an imino group, and a pair of electrons on a bond of a saturated N atom connected with a 6-chloropyridine structure are attracted by the positive charge center to cause charge transfer and cleavage of a corresponding single bond. The second cleavage route is the remote rearrangement reaction (rH)R) Due to the migration of pi electron cloud, N ═ C bonds No. 1 and 6, C ═ C bonds No. 2 and 3, and C ═ C bonds No. 4 and 5 in the 6-chloropyridine structure are broken and charge centers are formed on the carbon No. 4, which is extremely unstable, and in the radical-induced hydrogen rearrangement reaction, the hydrogen is not exchanged with the electron position in the 6-chloropyridine structure in pairs, and therefore, the bond of the saturated N atom to the 6-chloropyridine structure is broken.
(2) Second order characteristic fragmentation
The molecular formula of the acetamiprid is C10H11ClN4Belongs to the first generation of neonicotinoid medicine-chloropyridine medicine. The chloronicotinyl drug acts on acetylcholine receptors, acts on the central nervous system of insects, and has no cross resistance with traditional pesticides. Through the analysis of the mechanism and the route of the fragment of the chloronicotinyl medicament and the metabolite thereof with the 6-chloropyridine-3-methyl heterocyclic group structure, the chloronicotinyl medicament and the metabolite thereof with the chloropyridine structure are found out: acetamiprid-metabolite-IM-1-2, acetamiprid-metabolite-IM-1-4, acetamiprid-metabolite-6-fluoronicotinic acid, acetamiprid-metabolite-IM-2-1, acetamiprid, imidacloprid, acetamiprid, acetamipri,The pentahydroxyimidacloprid, nicotine imidacloprid, imidacloprid urea, dinotefuran and dinotefuran-metabolite-UF all have C6H5ClN+The functional properties of the fragments, i.e., the drugs, are consistent with the functional properties of the fragmented fragments, and the mass spectrum fragmentation patterns and molecular structures share commonalities.
The non-directional screening method comprises the following specific steps:
the secondary characteristic fragment is combined with a standard nicotine chloride medicine and a metabolite database thereof to be used for screening the nicotine chloride medicine and the metabolite thereof in the mutton.
Performing peak extraction by using secondary characteristic broken fragments and full-scanning mass-to-charge ratio of the nicotine drug and metabolite thereof in a standard database, and analyzing a chromatographic peak in normal distribution; comparing with chromatographic peaks in a standard nicotine drug and a metabolite database thereof, firstly judging the category of the substance, and then comparing the retention time of the chromatographic peaks, the full-scanning mass-to-charge ratio, the retention time of the secondary characteristic fracture fragments and the chromatographic peaks in the standard substance database, the full-scanning mass-to-charge ratio, the secondary characteristic fracture fragments and the fracture paths to conjecture the structural formula and the molecular formula of the compound so as to finish one-to-one correspondence of the chromatographic peaks with the corresponding nicotine drug and the metabolite thereof. And finally, quantitative and qualitative analysis is carried out by using the chromatographic and mass spectrum information of the standard analyte. The analyte concentration is calculated by multiplying the standard analyte concentration by the peak area ratio of the nicotine hydrochloride drug and the metabolite thereof in the mutton to the standard analyte. Obtaining the concentration of each nicotine chloride medicine or metabolite thereof in the mutton by the following formula, and completing the detection of the nicotine chloride medicine and the metabolite thereof in the mutton;
ci＝(c0v)/m, wherein ciThe concentration of each chloronicotinyl drug or metabolite thereof in the mutton is measured in units of mu g/kg, c0Is the concentration of each nicotine drug or metabolite thereof in the extracting solution, and the unit is mug/L, v is the volume of the extracting solution, and the unit is L, m is the mass of mutton, and the unit is kg. For example, in screening for chloronicotinyl and its metabolites in mutton, characteristic fragmentation m/z 126 is used01050 chromatographic peaks are extracted that are normally distributed and not present in the standard database.
The molecular formula of the analyte is presumed to be CxHyClNzComparing the broken fragments with high mass-to-charge ratio with other analyte breaking ways of a standard nicotine drug and metabolite database thereof, finding that the unknown analyte is similar to the breakage mechanism of acetamiprid, and comparing and analyzing the standard substance and a spectrogram to judge that the analyte is acetamiprid-metabolite-IM-1-4. The acetamiprid-metabolite-IM-1-4 concentration is calculated by multiplying the peak area ratio of the acetamiprid-metabolite-IM-1-4 to the standard analyte in the mutton with the standard analyte concentration. The final quantitative concentration was 0.29. mu.g/kg. Thereby realizing the high-flux qualitative and quantitative analysis of the nicotine medicament and the metabolite thereof.
Methodology investigation
a standard curve, linear range and correlation coefficient of method
Preparing nicotine drug and metabolite matrix matching standard substance solutions thereof, wherein the concentrations are respectively 2 mug/kg, 5 mug/kg, 8 mug/kg, 11 mug/kg, 15 mug/kg, 30 mug/kg, 50 mug/kg, 80 mug/kg, 200 mug/kg and 500 mug/kg. When a standard analyte calibration curve is plotted, the ordinate y represents the peak area of the chromatographic peak of the nicotine drug and the metabolite thereof, and x represents the standard analyte concentration (μ g/kg). The results show that the nicotine-based drug chloride and the metabolite thereof have correlation coefficients larger than 0.99 in the respective corresponding concentration ranges and are in good linear relation.
b detection limit, quantitative lower limit, relative standard deviation and recovery rate of the invention
The lower limit of quantitation and the limit of detection are considered by the detection capacity (i.e., CC β) and the determination limit (i.e., CC α). The detection capacity and the determination limit were 0.08. mu.g/kg to 0.63. mu.g/kg and 0.03. mu.g/kg to 0.32. mu.g/kg, respectively, as shown in Table 1. The accuracy of the invention is evaluated through the average recovery rate and the relative standard deviation of 12 times of parallel experimental results, mixed standard substance solutions of nicotine chloride medicines and metabolites thereof at three concentration levels of CC beta, 2-time CC beta and 4-time CC beta are added into mutton samples, and the relative standard deviation and the average recovery rate of the nicotine chloride medicines and the metabolites thereof are calculated by using ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap high-resolution mass spectrometry detection. The final highest relative standard deviation and average recovery results are respectively 5% -7.6% and 83% -111% as shown in table 2, and the accuracy and precision of the method can meet the requirement of non-directional screening of nicotine chloride medicines and metabolites thereof in mutton.
TABLE 1 Linear Range, correlation coefficient and detection Capacity, determination Limit of the method
TABLE 2 relative standard deviation and mean recovery of three concentration levels of CC beta, 2-fold CC beta and 4-fold CC beta for the method
6) Actual sample detection
By adopting the ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap high-resolution mass spectrometry rapid screening method established by the invention, 121 batches of nicotine chloride medicines and metabolites thereof in mutton are screened. The result shows that the method can be used for rapidly screening the nicotine chloride medicines and the metabolites thereof in the mutton.
The results show that all potential target compounds are in the respective corresponding concentration ranges, the correlation coefficient r2Are all larger than 0.99, and have good linear relation. The method determines that the limit and the detection capacity are respectively 0.005 mu g/kg-768 mu g/kg and 0.008 mu g/kg-650 mu g/kg, and the average recovery rate and the relative standard deviation result are respectively 89-106 percent and 1.2-6.5 percent. The method has the advantages of high analysis speed, simple process, high precision and accuracy, and can be used for well analyzing nicotine chloride medicine in mutton and its metabolismAnd (5) measuring a product.
Claims (10)
1. A method for detecting nicotine chloride medicines and metabolites thereof in mutton is characterized by comprising the following steps:
step 1, homogenizing mutton, then carrying out vortex by using an acetic acid solution of acetonitrile to obtain a mixed system A, adding anhydrous magnesium sulfate and sodium acetate into the mixed system A, carrying out vortex, then carrying out oscillation extraction to obtain an extracting solution, and centrifuging the extracting solution to obtain a mixed system B;
step 2, adding ethylenediamine-N-propyl silica gel, octadecyltrichlorosilane and MgSO (MgSO) into the mixed system B4Obtaining a mixed system C, centrifuging the mixed system C after swirling, and extracting supernatant to obtain an extracting solution;
step 3, diluting the extracting solution with an ammonium acetate solution, filtering, and determining the filtered solution in an ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrum to obtain chromatographic peaks, full-scan mass-to-charge ratios and secondary characteristic broken fragments of 11 nicotine drugs and metabolites thereof in mutton, wherein the 11 nicotine drugs and the metabolites thereof are respectively imidacloprid, imidacloprid urea, pentahydroxy imidacloprid, nicotine imidacloprid, acetamiprid-metabolite-IM-1-4, acetamiprid-metabolite-IM-1-2, acetamiprid-metabolite-6-fluoronicotinic acid, acetamiprid-metabolite-IM-2-1, dinotefuran and dinotefuran-metabolite-UF;
diluting 11 standard solutions of nicotine-based medicaments and metabolites thereof with an ammonium acetate solution to obtain corresponding diluents, respectively measuring all the diluents in an ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap mass spectrum to obtain a chromatographic peak, a full-scan mass-to-charge ratio, secondary characteristic fragmentation fragments and a fragmentation path of corresponding substances, wherein the chromatographic peak, the full-scan mass-to-charge ratio and the secondary characteristic fragmentation fragments of all the substances form a standard substance database;
step 4, extracting the chromatographic peaks obtained in the step 3 one by one to obtain corresponding chromatographic peaks in normal distribution, comparing each chromatographic peak in normal distribution with chromatographic peaks in a standard substance database to obtain the category of substances corresponding to each chromatographic peak, comparing the retention time of the chromatographic peak, the full-scan mass-to-charge ratio, the retention time of the secondary characteristic fragmentation fragment and the chromatographic peaks in the standard substance database, the full-scan mass-to-charge ratio, the secondary characteristic fragmentation fragment and the fragmentation path obtained in the step 3 to obtain the molecular formula of the substances corresponding to each chromatographic peak, and completing the one-to-one correspondence of the chromatographic peaks in the step 3 with the corresponding nicotine chloride drugs and metabolites thereof;
step 5, multiplying the peak area of the chromatographic peak corresponding to each substance to be detected and the peak area ratio of the chromatographic peak of the corresponding substance by the concentration of the standard solution of the corresponding substance to obtain the concentration of each nicotine chloride medicine or metabolite thereof in the extracting solution, and finally obtaining the concentration of each nicotine chloride medicine or metabolite thereof in the mutton by using the following formula to finish the detection of the nicotine chloride medicine and metabolite thereof in the mutton;
ci＝(c0v)/m, wherein ciThe concentration of each chloronicotinyl drug or metabolite thereof in the mutton is measured in units of mu g/kg, c0Is the concentration of each nicotine drug or metabolite thereof in the extracting solution, and has the unit of microgram/L, v is the volume of the extracting solution in the step 3, and has the unit of L, and m is the mass of the mutton in the step 1, and has the unit of kg.
2. The method for detecting chloronicotinyl drugs and metabolites thereof in mutton according to claim 1, wherein the mass ratio of the mutton, the anhydrous magnesium sulfate and the sodium acetate in the step 1 is (5-10): (5-6): (2-3); in step 2, ethylenediamine-N-propyl silica gel, octadecyltrichlorosilane and MgSO4The weight ratio of mutton to mutton is (0.3-0.4): (0.2-0.4): (0.6-0.8): 5-10).
3. The method for detecting nicotine-containing drugs and metabolites thereof in mutton according to claim 1, wherein the volume ratio of acetonitrile to acetic acid in the acetic acid solution of acetonitrile in step 1 is 1%, and the ratio of the acetic acid solution of acetonitrile to mutton is (5-20) mL (5-10) g.
4. The method for detecting nicotine-containing chloride drugs and metabolites thereof in mutton according to claim 1, wherein Accucore aQ is selected in the ultra-high performance liquid chromatography in the step 3, and the specific conditions are as follows, wherein the column temperature of the ultra-high performance liquid chromatography is 50-55 ℃, the spray voltage is 4-5 kV, the ion source temperature is 60-65 ℃, and the capillary temperature is 325-375 ℃;
the quadrupole rod electrostatic field orbit ion trap mass spectrum adopts a two-stage scanning mode, the two-stage scanning mode is variable data independent acquisition, and the scanning time is 0-20 min; resolution was taken at 95000FWHM and divided into two quality scan segments: the m/z-150-.
5. The method for detecting nicotine-containing drug chloride and metabolites thereof in mutton according to claim 4, wherein the mobile phase A is a mixed solution of formic acid, ammonium formate and water, wherein formic acid accounts for 0.1% of the total volume of the mixed solution, the concentration of ammonium formate is 4mM, and the mobile phase B is a mixed solution of formic acid, methanol and ammonium formate, wherein formic acid accounts for 0.1% of the total volume of the mixed solution, and the concentration of ammonium formate is 4 mM;
the gradient elution procedure is within 0-1 min, and the volume proportion of the mobile phase A is 80%; linearly reducing the volume proportion of the mobile phase A from 80% to 0 within 1-10 min; the volume ratio of the mobile phase A is 0 within 10-14 min; the volume proportion of the mobile phase A is linearly increased to 80% from 0 within 14-16 min; within 16-20 min, the volume proportion of the mobile phase A is 80%, and the flow rate is 0.3-0.5 mL/min.
6. The method for detecting nicotine-containing drugs and metabolites thereof in mutton according to claim 4, wherein the flow rate of the sheath gas is 55-60 arb, the auxiliary gas flow rate is 40-45 arb, the air curtain gas flow rate is 5-8 arb, and the heating temperature is 325-375 ℃ under the condition of the ultra-high performance liquid chromatography.
7. The method for detecting nicotine-containing drugs and metabolites thereof in mutton according to claim 4, wherein the collision energy in the quadrupole electrostatic field orbitron ion trap mass spectrum is 20.0eV, 35.0eV and 50.0eV, respectively.
8. The method for detecting chloronicotinyl agents and metabolites thereof in mutton according to claim 1, wherein the corresponding dilutions are subjected to secondary scanning in a data-dependent scanning mode in step 3, and other measurement conditions are the same as those of the extract.
9. The method for detecting chloronicotinyl agents and metabolites thereof according to claim 1, wherein in step 3, when the secondary characteristic fragments corresponding to 11 kinds of chloronicotinyl agents and metabolites thereof are obtained, the secondary characteristic fragments of the same type of agents are distinguished by selecting the signal intensity ratio of more than 15% between ion fragments.
10. The method for detecting chloronicotinyl agents and metabolites thereof in mutton according to claim 1, wherein the secondary characteristic fragmentation in the standard substance database of step 4 is the abundance ratio and the mass-to-charge ratio of the secondary characteristic fragmentation.
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