Extraction and purification method of endogenous metabolites after exposure of zebrafish to patulin

Abstract

The application discloses a method for extracting and purifying endogenous metabolites after exposure of zebra fish to patulin, which comprises the following steps: weighing a sample to be detected and adding acetonitrile-water mixed extraction liquid, oscillating and centrifuging to obtain extraction liquid and residues, taking out the extraction liquid, repeatedly extracting the residues, combining the extraction liquid, adding ammonium acetate, oscillating and centrifuging to obtain upper acetonitrile phase and lower water phase, taking the water phase layer to pass through a diatomite column, balancing, using pure acetonitrile as an eluent to perform elution, obtaining eluent, adding the acetonitrile phase to neutral alumina powder, oscillating and then low-temperature standing, then low-temperature high-speed centrifuging to obtain supernatant, combining the obtained eluent and the obtained supernatant, nitrogen blowing to near dryness, using a methanol-water mixed solution to constant volume, then high-speed centrifuging, and finally being ready for analysis. The application has high detection efficiency, can determine important biomarkers generated after exposure of zebra fish to patulin, and can provide a detection basis for poisoning monitoring of the compounds.

Description

TECHNICAL FIELD

[0001] The application relates to a detection method of metabolites of patulin exposed zebra fish, in particular to a rapid extraction and purification method and a detection method of patulin and metabolites of patulin exposed zebra fish. BACKGROUND

[0002] Patulin is a secondary metabolite produced by fungi, which widely exists in moldy fruits and their products, and enters the body through food intake to produce toxic effects. Since patulin is more toxic than other mycotoxins and has a more direct mode of action, the European Union, China, the United States and other countries have made strict regulations on the maximum limit of patulin in food (10-50 mu g / L, 50 mu g / kg). The half lethal dose (LD50) of patulin in rodents is 20-100 mg / kg, and that in domestic chickens is 50-170 mg / kg. The International Agency for Research on Cancer also classifies it as a class 3 carcinogen. Since mycotoxin poisoning incidents occur frequently and have a high mortality rate, identifying the toxic substance and implementing targeted rescue measures are the main measures to reduce the mortality rate of poisoning. However, current research has found that mycotoxins rapidly undergo metabolic reactions under the action of cytochrome P450 enzymes (CYP450) and other enzymes, and are converted into water-soluble metabolites with strong polarity, resulting in almost no detection of the presence of the parent compound in the body fluid. Moreover, the metabolic pathways of these mycotoxins are complex, and the products differ greatly, making it difficult to detect the toxic substance in the biological samples of the mistaken eaters, increasing the difficulty of rescue. At present, there have been reports on the related metabolism of some mycotoxins in grains and other cereals, and some metabolites often show stronger toxicity than the parent compounds. However, it is not clear what metabolites patulin exists in the body. Therefore, establishing an extraction and purification method of endogenous metabolites after patulin exposure and determining important biomarkers produced after patulin exposure can provide detection basis for the poisoning monitoring of this kind of compound.

[0003] Patulin detection mainly involves two aspects of technology: pretreatment technology and instrument analysis. Common pretreatment technologies include liquid-liquid extraction, solid-phase extraction and matrix dispersed solid-phase extraction, etc. These pretreatment technologies have their own characteristics, but all have the disadvantage of selectivity, and are aimed at the extraction and purification analysis of exogenous added patulin in food matrix (feed, moldy fruits and their products, etc.), and cannot realize the synchronous extraction of patulin and strong polarity patulin metabolites in endogenous tissue samples. For example, Liu Chunlin et al. used solid-phase extraction technology to detect and analyze patulin in hawthorn and its oral liquid products, and acetonitrile and ethyl acetate were used as extraction solutions in the pretreatment, which had strong specificity MFC 228 multi-functional purification column for purification treatment, the strong polarity and the physicochemical properties of the different penicillin metabolites cannot be compatible at the same time; similarly, Sadok et al. detected and analyzed the patulin in fruit products by liquid chromatography-mass spectrometry technology, the sample was extracted by acetonitrile, and then purified by QuEChERS, but the recovery rate of the hydrophilic metabolites cannot be guaranteed. In addition, the emerging QuEChERS purification method has obvious advantages in removing impurities such as pigments, organic acids and metal ions, but there is still complex matrix interference for tissue samples with high protein and fat content. After the zebrafish is exposed to patulin, the metabolites thereof have not been separated and identified, and the establishment of a comprehensive and efficient pretreatment method is an important prerequisite for ensuring that unknown compounds are identified. Therefore, the establishment of a rapid extraction and purification method for endogenous metabolites of zebrafish exposed to patulin has important clinical significance for targeted treatment of patulin poisoning symptoms.

[0004] Zebrafish has been widely used in preliminary research of drug and toxicant metabolism as a mature in vivo metabolic model. So far, it has been used to simulate a variety of human diseases, including melanoma, leukemia, etc., and shows remarkable similarity to human diseases. Compared with rodents, zebrafish has some unique physiological advantages: small size, low price, large-scale breeding; high egg production, sufficient sample amount for experiments; transparent body, convenient for real-time observation in vivo; fast growth and development, which can effectively shorten the experimental period. Biologically, the zebrafish genome sequencing work has been completed, with a high homology of 87% to human genes, and the early embryonic development is very similar to that of humans. The phase I metabolic enzyme CYP450 and phase II metabolic enzyme uridine diphosphate glucuronyltransferase, sulfotransferase, etc. contained therein are a vertebrate model that can predict drug metabolites, and have been widely used in safety evaluation of in vivo drug efficacy, toxicity, etc. SUMMARY

[0005] The purpose of the present application is to provide an extraction and purification method for endogenous metabolites of zebrafish exposed to patulin, to solve the problems raised in the above background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solution: a rapid extraction and purification method for patulin and metabolites of zebrafish exposed to patulin, characterized by comprising the following steps:

[0007] (1) 1 g of the sample to be tested is added into 10 mL of acetonitrile-water mixed extraction solution, shaken for 5 min, and centrifuged at 4500 r / min for 5 min to obtain an extraction solution and a residue, and the extraction solution is taken out, and the residue is extracted once more;

[0008] (2) Combine the extraction liquid, add 2 g of ammonium acetate, shake for 5 min, and centrifuge at 4500 r / min for 5 min to obtain the upper acetonitrile phase and the lower water phase;

[0009] (3) Take the water phase layer to a diatomite column, balance for 15 min, and elute with 15 ml of pure acetonitrile as an eluent to obtain an eluate;

[0010] (4) Add 1 g of neutral alumina powder to the remaining acetonitrile phase, shake for 5 min, and stand at -20℃ for 1.0 h, then centrifuge at -4℃ at a high speed of 15000 r / min for 5 min to obtain a supernatant;

[0011] (5) Combine the eluate obtained in step (3) and the supernatant obtained in step (4), dry under nitrogen blowing, and dilute to 1.0 ml with a methanol-water mixture, then centrifuge at a high speed and analyze.

[0012] 2. The rapid extraction and purification method of patulin and metabolites after patulin exposure of zebrafish according to claim 1, characterized in that the sample to be tested in step (1) is a sample of fish meat after patulin exposure of zebrafish.

[0013] 3. The rapid extraction and purification method of patulin and metabolites after patulin exposure of zebrafish according to claim 1, characterized in that the volume ratio of acetonitrile to water in the acetonitrile-water mixed extraction liquid in step (1) is 1:4.

[0014] 4. The rapid extraction and purification method of patulin and metabolites after patulin exposure of zebrafish according to claim 1, characterized in that the volume ratio of methanol to water in the methanol-water mixture in step (5) is 1:4.

[0015] 5. A detection method of endogenous metabolites after patulin exposure of zebrafish, characterized by comprising the following steps:

[0016] (1) Sample pretreatment

[0017] Take 1 g of the sample from the zebrafish exposed to patulin and add 10 mL of acetonitrile-water mixed extraction solution, shake for 5 min, centrifuge at 4500 r / min for 5 min, obtain the extraction solution and residue, take out the extraction solution, repeat the extraction of the residue once; combine the extraction solutions, add 2 g of ammonium acetate, shake for 5 min, centrifuge at 4500 r / min for 5 min, obtain the upper acetonitrile phase and the lower water phase; take the water phase layer on the diatomite column, balance for 15 minutes, use 15 ml of pure acetonitrile as the eluent for elution, obtain the eluent; add 1 g of neutral alumina powder to the remaining acetonitrile phase, shake for 5 min, stand at -20℃ for 1.0 h, centrifuge at -4℃ at 15000 r / min for 5 min, obtain the supernatant; combine the eluent and the supernatant, dry under nitrogen, use methanol-water mixture to make up to 1.0 ml, high-speed centrifuge, and analyze after pretreatment, see the pretreatment flow chart Figure 1 ;

[0018] (2) Preparation of matrix standard curve

[0019] Prepare a patulin standard curve, the concentration range is: 0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL, 20 ng / mL, take the concentration as the abscissa and the standard peak area as the ordinate as the basis for the quantification of the target;

[0020] (3) Chromatography and mass spectrometry conditions

[0021] Liquid phase conditions: chromatographic column Syncronis C18 column, 2.1 mm x 150 mm, 1.7 μm, mobile phase A: 5% methanol in water, mobile phase B: 5% water in methanol solution, flow rate: 0.3 mL / min, sample size: 5.0 μL, gradient see Table 1 below:

[0022] Table 1 HPLC elution program in positive and negative ion modes

[0023]

[0024] Mass spectrometry conditions: mass spectrometry is carried out in positive / negative ion switching mode, mass range: m / z 50-700, resolution 120,000, automatic gain control target value 5e 5 ; negative ion mode 2700V, positive ion mode 3800V, ion transmission tube temperature 300℃, sheath gas pressure 35arb, auxiliary gas pressure 10arb, gasification chamber temperature 350℃; before sample operation, the instrument is corrected in positive and negative ion modes respectively; secondary uses automatic trigger mode, resolution 35,000, automatic gain control target value 2e 5 , collision energy is 15%, 30% and 50% respectively;

[0025] (4) Concentration calculation method

[0026] The content of the analyte in the sample is calculated according to the following formula (1):

[0027] X = C * V / m, wherein:

[0028] X is the content of the analyte in the sample, in μg / kg; the analyte is patulin and its metabolites

[0029] C is the concentration of the analyte in the sample treatment solution, calculated according to the matrix standard curve, in μg / L

[0030] V is the constant volume, in mL;

[0031] m is the mass of the sample, in g;

[0032] 6. The method according to claim 5, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed extraction solution is 1:4, and the volume ratio of methanol to water in the methanol-water mixed solution is 1:4.

[0033] Technical effects and advantages of the present application:

[0034] The solid phase assisted liquid / liquid extraction technology (SLLE) used in the present application is an important sample pretreatment technology, which uses macroporous diatomite as a carrier. The sample uses acetonitrile-water (1:4, v:v) as an extraction solvent, which completely meets the extraction requirements of patulin and strong polar metabolites. By adding salt substances, the solution will actively separate into two phases: organic phase (upper layer) and aqueous phase (lower layer). The water and polar compounds in the aqueous phase are retained and adsorbed by the diatomite column, and pure acetonitrile is used as an eluent to complete the step-by-step elution of different polar substances through dynamic distribution of the two phases, successfully extracting strong polar metabolites from the aqueous phase and achieving purification and concentration. Compared with traditional liquid-liquid extraction, SLLE does not need to use a separatory funnel and a large amount of organic solvent to extract the analyte multiple times, and has the advantages of simple operation steps, small solvent consumption, good reproducibility, etc. The upper organic phase is directly concentrated by nitrogen blowing, and through the combination of low temperature and neutral alumina powder, the oil interference is removed to the maximum extent.

[0035] The difference between the high efficiency liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technology adopted by the present application and the conventional triple quadrupole mass spectrometry is that the HPLC-Q-Orbitrap-HRMS technology does not need characteristic ion fragments of a known to-be-detected compound, but directly collects high-precision mass numbers (resolution > 70000, m / z 200) to perform directional and non-directional full-scan detection on the to-be-detected sample, and when a target compound needs to be added, the existing full-scan data can be analyzed again without repeating sampling, and the HPLC-Q-Orbitrap-HRMS technology is particularly suitable for qualitative analysis of unknown metabolites. The Q Exactive mass spectrometer is also equipped with a full-automatic screening software Compound Discoverer TM , the Compound Discoverer is a data processing auxiliary software based on high-resolution chromatography mass spectrometry, and is used for simultaneous screening of multiple components and qualitative and quantitative analysis of completely unknown substances. In addition to the accurate mass number, the Compound Discoverer also provides multiple methods such as retention time, isotope distribution, main secondary fragment confirmation and secondary mass spectrum graph similarity comparison, comprehensive judgment, to obtain accurate qualitative results. In the case that the resolution reaches 70,000, the obtained data reduces the interference of similar mass numbers co-eluted, and greatly avoids the generation of false positive results.

[0036] In summary, the present application provides a rapid extraction and purification method and a detection method for patulin and metabolites of patulin exposed zebrafish, combines solid-phase assisted liquid / liquid extraction and matrix dispersion purification technology, establishes an extraction and purification method for endogenous metabolites of patulin exposed zebrafish, realizes the extraction and purification of patulin and metabolites with great differences in physical and chemical properties through one pretreatment, and evaluates the extraction and purification effect of the method by using HPLC-Q-Orbitrap-HRMS, determines important biomarkers generated after patulin exposure, including Hydroascladiol, E-ascladiol and Z-ascladiol, and due to the similarity of the parent nucleus structure, relative semi-quantitative analysis can be performed according to the patulin standard curve. The results show that the patulin presents a good linear relationship in the concentration range of 1-20 ng / mL, and the correlation coefficient r 2 is greater than 0.999, which meets the requirements of quantitative analysis. According to the content value of each compound corresponding to the signal-to-noise ratio S / N≥3, the detection limit (LQD) of the compound is obtained in the range of 0.01-0.03 mg / kg. The method has strong compatibility for endogenous metabolites of patulin with different physical and chemical properties, has high detection efficiency, determines important biomarkers generated after patulin exposure, and can provide detection basis for poisoning monitoring of the compounds. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 This is a flowchart of the pretreatment extraction and purification process.

[0038] Figure 2 The images show the extracted ion chromatogram and secondary mass spectrometry of patulin.

[0039] Figure 3 The images show the extracted ion chromatograms of the patulin exposure group and the blank control group, as well as the secondary mass spectrometry mirror image of compound 1. The black line represents the patulin exposure group, and the red line represents the blank control group. The upper right corner shows the secondary mass spectrometry mirror image of the patulin standard and compound 1.

[0040] Figure 4 Secondary mass spectra of two metabolites of patulin in zebrafish: compounds 2 and 3 (A), and compound 4 (B).

[0041] Figure 5 The effects of extraction solvent ratio (A) and volume (B) on the extraction efficiency of patulin and its metabolites were investigated.

[0042] Figure 6 The effect of eluent volume on the extraction efficiency of patulin and its metabolites (A), and the effect of neutral alumina powder dosage and low-temperature freezing time on the oil removal rate (B).

[0043] Figure 7 The chromatographic separation of patulin and its three metabolites, Hydroascladiol, E-ascladiol, and Z-ascladiol, under three different chromatographic columns are shown in Figure 1. A: Syncronis C18 (2.1 mm × 150 mm, 1.7 μm); B: Hypersile Gold C8 (150 mm × 2.1 mm, 3 μm); C: Atlantis T3 (150 mm × 2.1 mm, 3 μm). Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] This invention provides, for example Figures 1-7 The method shown is for the extraction and purification of endogenous metabolites in zebrafish exposed to patulin: Specific Implementation

[0047] 1. Experimental instruments and reagents

[0048] Q-Exactive quadrupole electrostatic field orbitrap high resolution mass spectrometer (Thermo Fisher Scientific) equipped with H-ESI II source. UltiMate 3000 high pressure liquid chromatography with autosampler. Chromatographic column: Syncronis C18 column (2.1 mm x 150 mm, 1.7 μm). Milli-Q high purity water generator (Millipore, USA). Freeze dryer (Christ, Germany). Refrigerated centrifuge (SIGMA, Germany). Vortex shaker (Heldolph, Germany). Macroporous silica gel column (Chromabond XTR, 15 mL).

[0049] Ammonium acetate (chromatographically pure, Sigma-Aldrich, USA). Other reagents (chromatographically pure, Merck, Germany). Experimental water is Milli-Q ultrapure water (18.2 ΩM).

[0050] Patulin standard was purchased from Sigma, purity ≥ 99%, dissolved in acetonitrile, prepared into a standard solution of 1.0 mg / mL, and stored at -20°C in the dark.

[0051] 2. Establishment of patulin exposed zebrafish model

[0052] Select 60 adult zebrafish (mass (1.0 ± 0.2) g, provided by Shanghai Feixi Biotechnology Co., Ltd.), randomly divided into experimental group and blank control group, 10 zebrafish in each group, 3 parallel treatments. Ultraclean water was used for breeding, and the volume of the breeding water in the independent water tank was 500 mL. The breeding adaptation period before toxicant exposure was 24 h. Experimental group: add appropriate amount of patulin standard to the solution for culturing zebrafish to make its concentration reach 10 mg / L, and culture for 2.0 h (constant temperature, illumination); blank control group: do not give drug treatment to the solution for culturing zebrafish, and culture for 2.0 h (constant temperature, illumination).

[0053] After the experiment, the surface of the zebrafish was washed with PBS buffer for 3 times, and after euthanasia, freeze-drying treatment was carried out, and the parameters were set as follows: 1) pre-freezing: gradient cooling to -50°C within 0.5 h, and keeping for 1.5 h; 2) main drying: vacuum extraction to 0.1 mbar within 0.5 h, slowly increasing the temperature to -20°C within 2.0 h, and keeping for 10 h; 3) secondary main drying: increasing the temperature to -5°C within 0.5 h, and keeping for 10 h; 4) final drying: reducing the vacuum degree to 0.01 mbar within 0.25 h, increasing the temperature to 25°C within 0.5 h, and keeping for 5 h. After drying, the sample was ground into powder with liquid nitrogen, and stored at -20°C in the dark.

[0054] 3. Pretreatment steps

[0055] Due to the large polarity difference of patulin and its metabolites, the extraction solvent is 10 mL water-acetonitrile (4:1, v:v) according to the pKa value and polarity range of the extraction solvent, and the steps are as follows:

[0056] (1) Take 1.00 g of sample, add 10 mL of acetonitrile-water (1:4, v:v), shake for 5 min, ultrasonic extraction for 10 min, centrifuge at 4500 r / min for 5 min, obtain the extract and residue, take out the extract, and repeat the extraction once for the residue;

[0057] (2) Combine the extract, add 2.0 g of ammonium acetate, shake for 5 min, centrifuge at 4500 r / min for 5 min, obtain the acetonitrile phase (upper) and the water phase (lower);

[0058] (3) Take the water phase layer on the diatomite column, equilibrate for 15 minutes, and elute with 15 ml of pure acetonitrile as eluent to obtain the eluent;

[0059] (4) Add 1.0 g of neutral alumina powder to the remaining organic phase layer, shake for 5 min, and stand at -20℃ for 1.0 h, then centrifuge at 15000 r / min at low temperature for 5 min (-4℃) to obtain the supernatant;

[0060] (5) Combine the collected liquids of (3) and (4), dry under nitrogen, and dilute to 1.0 ml with methanol-water (1:1, v:v), then centrifuge and analyze.

[0061] 4. Chromatography and mass spectrometry conditions

[0062] Liquid phase conditions: Syncronis C18 column (2.1 mm x 150 mm, 1.7 μm), mobile phase A: water containing 5% methanol, mobile phase B: methanol containing 5% water, flow rate: 0.3 mL / min, injection volume: 5.0 μL, gradient as shown in Table 1 below:

[0063] Table 1 HPLC elution program in positive and negative ion modes

[0064]

[0065] Mass spectrometry conditions: mass spectrometry is performed in full scan mode under positive / negative ion conversion mode, mass range: m / z 50-700, resolution 120,000, automatic gain control (AGC) target value 5e 5; negative ion mode 2700 V, positive ion mode 3800 V, ion transfer tube temperature 300 °C, sheath gas pressure (N2) 35 arb, auxiliary gas pressure (N2) 10 arb, vaporizer temperature 350 °C; the instrument was calibrated for positive and negative ions respectively before sample running; the second stage was in automatic trigger mode, resolution 35,000, automatic gain control (AGC) target value 2e 5 , and the collision energies were 15%, 30% and 50% respectively.

[0066] 5. Qualitative and quantitative analysis

[0067] Qualitative part: the mass error was required to be less than 5*10 -6 ; Compound Discoverer simultaneously compared the retention time, isotope distribution, main secondary fragments and secondary mass spectrum similarity to comprehensively judge to obtain accurate qualitative results and avoid false positive results.

[0068] Quantitative part: the cerulenin standard curve was prepared, the concentration range was 0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL and 20 ng / mL, and the concentration was taken as the horizontal coordinate and the standard peak area was taken as the vertical coordinate as the basis for the target quantitative analysis; other detected cerulenin metabolites (not commercially available or not purchased due to low purity) were semi-quantitatively analyzed by the above standard curve due to the structural similarity.

[0069] Table 2. Results of tandem mass spectrometric analysis of E / Z ascladiol and hydroascladiol compared with Ricelli et al. (2007)

[0070]

[0071] II. Analysis of experimental results

[0072] 1. Chromatographic and mass spectrometric analysis of cerulenin and its metabolites

[0073] Cerulenin is a lactone compound formed by connecting a pyran ring with a hydroxyl group and a furan ring with a ketone group. The extracted ion chromatogram and secondary mass spectrum distribution of cerulenin are shown in Figure 2 . The accurate determination of the molecular ion peak is a prerequisite for the qualitative analysis of this type of compound. The base peak with high response intensity and complete structural information is usually selected as the molecular ion peak of the target, and it is used as the target for secondary high-resolution mass spectrometric analysis. By comparing the response intensities of each target in ESI+ and ESI- modes, the best ionization mode and the existence form of the molecular ion peak of each substance are determined. From the primary mass spectrum of cerulenin, Figure 2), the stable molecular ion peak of the compound is *a[M-H]-m / z 153.0186, which is formed after the loss of H atom of the hydroxyl group on the pyran ring. The secondary mass spectrum of [M-H]- is scanned, and the graphs showing obvious differences in the secondary mass spectrum are superimposed as the collision energy increases. The main fragmentation fragments presented include: m / z 135.0080, which is analyzed by element simulation (error value less than 5×10 -6 ), and the corresponding elemental composition of the fragmentation fragment is C7H3O3-, which is speculated to be *a'[M-H-18Da]- formed after the hydroxyl bond on the pyran ring is broken and a molecule of water is removed based on m / z 153.0186. Similarly, the elemental composition of the m / z 109.0286 fragmentation fragment is C6H5O2-, which is speculated to be *b[M-H-CO2]- formed after the oxygen atom on the furan ring and the adjacent carbonyl C=O are broken and the pyran ring is negatively charged. This fragmentation mode forms the most abundant fragmentation fragments; the elemental composition of the m / z 125.0235 fragmentation fragment is C6H5O3-, which is speculated to be *c[M-H-CO]- formed after the C-O branch on the pyran ring is broken. The above three fragmentation fragments are all formed after the molecular ion peak m / z 153.0186 [M-H]- is broken, which is consistent with the reported secondary spectrum of expansin [28, 29]. In addition, the elemental composition of the m / z 81.0336 fragmentation fragment is C5H5O-, and the elemental composition of the m / z 97.0286 fragmentation fragment is C5H5O2-, which are speculated to be new fragmentation ions *b'[*b-28Da]- and *c'[*c-28Da]- formed after the pyran ring and furan ring branch lose C=O (28Da) and C-O (28Da) based on *b and *c. Studies have shown that although mycotoxins are extensively metabolized by cytochrome P450 enzymes and other enzymes after being ingested by the body, the newly synthesized metabolites often chemically modify the branches of the parent drug compounds, and the backbone structure remains basically unchanged, thereby presenting similar mass spectrometry fragmentation pathways to the parent drug compounds. Based on this, the secondary mass spectrometry fragmentation rule of expansin established above can be used to rapidly identify its metabolites.

[0074] By comparing and analyzing the total ion chromatograms of the expansin exposure group and the blank control group, the extraction ion chromatograms of the endogenous differential metabolites are obtained Figure 3). Compound 1 : retention time 4.82 min, molecular ion peak [M-H]"at m / z 153.0186, secondary mass spectrum scan of [M-H]"showed obvious difference in the mass spectrum with the increase of collision energy, the superposition of the mass spectrum showing obvious difference was superimposed, the main fragmentation fragments included: m / z 135.0080, m / z 125.0235, m / z 109.0286, m / z 97.0286 and m / z 81.0336, etc. By comparing with the retention time, isotope distribution, main secondary fragments and secondary mass spectrum of the standard substance of patulin (Figure 1, upper right), it can be determined that compound 1 is the original drug compound-patulin remaining in the body of zebrafish after patulin exposure. Figure 1 Figure 3 , upper right), it can be determined that compound 1 is the original drug compound-patulin remaining in the body of zebrafish after patulin exposure.

[0075] Compound 2 ( Figure 3 ) : retention time 4.16 min, the stable molecular ion peak of the compound is [M-H]"at m / z 155.0344, the corresponding elemental composition C7H7O4"is obtained by element simulation (error value less than 5x10"6), secondary mass spectrum scan of [M-H]"( Figure 4 ​A), with the increase of collision energy, the main fragmentation fragments include: m / z 137.0237 fragmentation fragment corresponding to the elemental composition of C7H5O3-, is the formation of *a'[M-H-H2O]- after the mother ion loses a molecule of water (18 Da); In addition, m / z 111.0446 corresponds to the elemental composition of C6H7O2-, which is the formation of *b[M-H-CO2]- after the mother ion loses CO2 (44 Da), compared with the corresponding fragmentation fragments (m / z 109.0286 and 135.0080) in the above patulin, the molecular weight difference is 2 Da, which can be initially speculated that compound 2 is the metabolic product after the hydrogenation reaction of the pyran ring of patulin. In addition, m / z 125.0235 fragmentation fragment corresponds to the elemental composition of C6H5O3-, which is the formation of fragment ion *c[M-H-CH2O]- after the branch on the pyran ring is broken, which is consistent with the corresponding fragment *c(m / z 125.0235, C6H5O3-) of patulin, further verifying the above speculation that the hydrogenation reaction occurs on the pyran ring, and the furan ring structure remains consistent with patulin. According to the literature reports, the above three fragmentation fragments of compound 2 are consistent with the secondary spectrum of Ascladiol reported in the literature (Table 2), so it can be known that: compound 2 is the structure with two hydroxyl groups formed after the ring-opening reaction of the pyran ring of patulin. Further analysis, compound 2 also exists 2 groups of fragmentation ions with a difference of 28 Da under high collision energy, m / z 83.0492 and 111.0446, m / z 97.0286 and 125.0235, which are the new fragment ions *b'[*b-28Da]- and *c'[*c-28Da]- produced after the loss of C=O (28 Da) and C-O (28 Da) in the pyran ring and furan ring branch based on *b and *c, which further confirms the above conclusion. Compound 3 Figure 3 ): retention time 3.99 min, the stable molecular ion peak formed by the compound is [M-H]- m / z 155.0344, the secondary mass spectrum scan of [M-H]- shows a similar secondary mass spectrum to compound 2. Studies have shown that Ascladiol exists in two configurations, of which E-ascladiol is the main metabolite of patulin, and the other is its isomer Z-ascladiol, both of which have the same ion fragments and are difficult to distinguish by mass spectrometry, only showing the difference in retention time, in which E-ascladiol elution time is slightly earlier than Z-ascladiol.

[0076] Compound 4 Figure 3) with a stable molecular ion peak at a retention time of 0.92 min, and the elemental composition of C7H9O4- was obtained by elemental simulation (error value less than 5 x 10-6). The secondary mass spectrum of [M-H]- was scanned Figure 4 B) with the increase of collision energy, the main fragmentation fragments included: m / z 139.0391 corresponding to the elemental composition of C7H7O3-, which was a' [M-H-H2O]- formed by the loss of one molecule of water (18 Da) from the parent ion; in addition, m / z 113.0609 corresponding to the elemental composition of C6H9O2-, which was b [M-H-CO2]- formed by the loss of CO2(44 Da) from the parent ion; compared with the corresponding fragmentation fragments (m / z 111.0446 and 137.0237) in Ascladiol, the molecular weight difference was 2 Da, which could be preliminarily speculated that compound 4 was a metabolite of Ascladiol based on further hydrogenation of the pyran ring. Further analysis, m / z 127.0401 corresponding to the elemental composition of C6H7O3-, which was c [M-H-CH2O]- formed by the loss of the branch on the pyran ring, compared with the corresponding fragment composition c (m / z 125.0235, C6H5O3-) in Ascladiol, the molecular weight difference was 2 Da, which could be speculated that the hydrogenation reaction occurred on the double bond of the pyran ring. Combined with the literature reports, the above three fragmentation fragments of compound 4 were consistent with the secondary spectrum of Hydroascladiol reported in the literature (Table 2), which indicated that compound 4 was a new metabolite of the patulin on the basis of Ascladiol, which was formed by hydrogenation of the double bond on the pyran ring. Further analysis, compound 4 also had two sets of fragmentation ion groups with a difference of 28 Da under high collision energy, m / z 85.0659 and 113.0609, m / z 99.0451 and 127.0401, which were new fragmentation ions b' [*b-28Da]- and c' [*c-28Da]- produced by the loss of C=O (28 Da) and C-O (28 Da) in the pyran ring and furan ring branch on the basis of b and c, which further confirmed the above conclusion by comparing with the corresponding fragmentation fragments (m / z 83.0492 and 97.0286) in ascladiol.

[0077] In summary, by comparing and analyzing the secondary spectrum of the target compound in the literature, it was found that zebrafish mainly produced three metabolites during the exposure of patulin: one was E-ascladiol, the main metabolite of patulin, the other was its isomer Z-ascladiol, and a new product HydroascladiolFigure 3 ) This is consistent with Wei et al. using Lactobacillus plantarum in vitro degradation of patulin to form metabolites, it is speculated that the zebrafish intestinal microbial flora may play a major role in the degradation of patulin, the following focuses on the extraction efficiency of the above three patulin and its metabolites are optimized.

[0078] 2, the optimization of the ratio and volume of the extraction solvent

[0079] The following focuses on the extraction efficiency of the above-mentioned patulin and its three metabolites are optimized.

[0080] The extraction solvent used in the application is water-acetonitrile system, the polarity range of the two solvents completely meets the extraction demand of patulin and its metabolites. Among them, high proportion of water solution can well guarantee the extraction efficiency of strong polar metabolites, acetonitrile as the auxiliary extraction agent of water, its addition amount has great influence on the extraction rate of medium polar patulin, acetonitrile has good solubility and strong penetration, which has become a commonly used solvent for endogenous compound extraction. According to the above pretreatment steps, the proportion of water in the mixed extraction solvent is optimized, 20mL of different ratio of extraction solution is added respectively, including: 0%, 50%, 60%, 80%, 90% and 100%, the influence of water proportion on the extraction effect of patulin and its metabolites (calculated by peak area) is investigated. The results show that Figure 5 A): patulin metabolites, including Hydroascladiol, E-ascladiol and Z-ascladiol are greatly affected by water proportion, when the water proportion is ≤50%, the extraction efficiency is obviously low, and it is significantly improved with the increase of water proportion ratio (60%-90%), when the water proportion is 100%, the extraction rate begins to decrease, which is also the biggest disadvantage of pure water solution in the extraction process of endogenous polar compounds; the extraction rate of patulin is the highest and stable when the water proportion is 50%-80%, it may be due to the high proportion of acetonitrile, which can cause the excessive extraction of fat components in the tissue sample, resulting in strong matrix inhibition effect, considering comprehensively, the extraction ratio is finally determined as 80% water-acetonitrile (4:1, v:v).

[0081] On this basis, by adding different volumes of water-acetonitrile (4:1, v:v) solution, the influence of 10mL, 10mL×2 and 10mL×3 extraction volumes on the extraction rate of the above substances is further investigated. The results show that Figure 5 B), when the extraction liquid volume is 10mL, the extraction efficiency is low, when the extraction liquid volume is 10mL×2 and 10mL×3, the extraction efficiency tends to be stable. From the perspective of saving the time and cost of nitrogen blowing concentration, the elution volume of water-acetonitrile (4:1, v:v) as eluent is determined as 10mL×2.

[0082] 3. Selection of purification conditions

[0083] After 2 times extraction by 10 mL water-acetonitrile (4:1, v:v) solution, ammonium acetate oscillation distribution obtained two phase after layering: water phase (lower layer) and acetonitrile phase (upper layer), the experiment respectively investigated the influence of 0.5 g, 1.0 g, 2.0 g, 3.0 g and 4.0 g ammonium acetate on the effect of mixed extraction liquid layering. The results showed that when the amount of ammonium acetate was 0.5 g and 1.0 g, the acetonitrile phase and water phase were not completely separated. When the amount was 2.0 g, the water phase reached saturation state, the layering interface was clear, and the acetonitrile layer was clear and transparent. When the amount was 3.0 g and 4.0 g, the lower water solution reached supersaturation state, and part of the salt precipitated at the bottom. In order to avoid the pollution of the instrument by the salt solution as much as possible, the amount of ammonium acetate was finally determined as 2.0 g.

[0084] After the lower water phase was loaded, the water solution was dispersed on the surface of the chemically inert matrix particles of the diatomite column to form a thin layer covering the surface. The three ascladiol metabolites in the water phase, including Hydroascladiol, E-ascladiol and Z-ascladiol, were adsorbed in the thin layer of the water phase on the surface of the carrier. Pure acetonitrile was used as an eluent for elution, and a high specific surface area interface was formed between the two phases. Through the gradual infiltration of acetonitrile, the compounds were eluted in turn according to the size of the polarity. The whole process kept the water solution on the diatomite column, and the obtained eluent (acetonitrile) could be directly concentrated by nitrogen. In order to save the nitrogen blowing concentration time and the amount of acetonitrile, the experiment further optimized different elution volumes, including 10 mL, 15 mL, 20 mL and 25 mL acetonitrile, to investigate the influence on the elution effect of the three ascladiol metabolites (calculated by peak area). The results showed that when the volume of acetonitrile was 15 mL, 20 mL and 25 mL, the response values of the three target compounds tended to be stable. From the perspective of cost saving, the final volume of the eluent was determined as 15 mL. Figure 6 A).

[0085] In addition, the acetonitrile in the upper layer as an auxiliary extraction solvent can well ensure the extraction efficiency of the medium-polar ascladiol, but inevitably, a large amount of fat components in the zebrafish tissue will be extracted and produce strong matrix inhibition effect. The pretreatment of the present application uses neutral alumina powder and low-temperature freezing to eliminate the interference of oil. The amount of neutral alumina powder and the low-temperature freezing time are associated with the oil removal rate. Under the premise of sufficient purification, the waste of cost should also be avoided. In the experiment, the oil removal rates of adding 0.1 g, 0.2 g, 0.5 g, 1.0 g and 2.0 g of alumina powder, and low-temperature standing for 0 h, 0.5 h, 1.0 h, 1.5 h and 2.0 h at-20℃ were compared. From the perspective of cost saving, the final amount of alumina powder was determined as 0.5 g. Figure 6BIt can be seen that when the addition amount is 0.1 g, 0.2 g and 0.5 g, the amount of alumina powder is obviously insufficient, and is greatly affected by the freezing time: the oil removal rate of 2.0 h freezing time has obvious advantages, and the oil removal rate ranges from 43.9% to 98.1%; when the amount of alumina powder is ≥1.0 g, the oil removal rate of 1.0 h is obviously higher than that of 0.5 h, and the oil removal rates of 1.0 h, 1.5 h and 2.0 h are very small, and the oil removal rates are all ≥99.2% or more, that is, when the amount of alumina powder is sufficient, the freezing time is dominant, the longer the time, the better the oil removal rate, and finally remains stable. From the perspective of saving time and cost, the purification conditions are finally determined as follows: the addition amount of alumina powder is 1.0 g, and the freezing time is 1.0 h.

[0086] 4. Optimization of instrument conditions

[0087] The experiment adopts full scan and positive and negative ion switching mode for mass spectrometry, and the accurate mass number of the first mass spectrum is extracted for qualitative and quantitative analysis to obtain the best ionization mode and the existence form of the molecular ion peak of each compound. The results show that in the ESI - mode, the patulin and its metabolites show the best mass spectrometry response. At the same time, the secondary mode is triggered to further improve the accuracy of the qualitative analysis, and the ultra-high resolution helps to analyze complex samples and ensures the maximum detection and identification of metabolites in one chromatographic run.

[0088] The experiment compares three different specifications of chromatographic columns covering three types of fillers, including: 1) Syncronis C18 (2.1 mm x 150 mm, 1.7 μm) silica gel bonded medium chain C18 functional group chromatographic column; 2) Hypersile Gold C8 column (2.1 mm x 150 mm, 3.0 μm) silica gel bonded short chain C8 functional group chromatographic column; and 3) Atlantis T3 (150 mm x 2.1 mm, 3 μm) C18 functional group chromatographic column based on ultra-pure silica gel matrix. The chromatographic separation effect of the above chromatographic columns on two E-ascladiol and Z-ascladiol isomers is discussed. The results show that by optimizing the mobile phase properties and gradient, Syncronis C18 (2.1 mm x 150 mm, 1.7 μm) can obtain the best chromatographic peak shape and separation effect Figure 7 A). The silica gel bonded short chain C8 functional group chromatographic column and the Atlantis T3 chromatographic column (150 mm x 2.1 mm, 3 μm) have low retention for the target compounds, and through the trial of different mobile phases and gradients, the effective separation of the target compounds cannot be achieved Figure 7 B and 7C).

[0089] Therefore, the experiment selects a Syncronis C18 chromatographic column (2.1mm*150mm, 1.7um) for chromatographic separation. In addition, two different specifications of Syncronis C18 chromatographic columns (100mm*2.1mm, 1.7um and 100mm*2.1mm, 3um) are compared, and the results also show only a small difference in retention time under the same pressure, and both can meet the needs of mass spectrometric detection in terms of resolution. Since the small-particle chromatographic column has a lower flow rate, it can save organic solvents well, and therefore the Syncronis C18 chromatographic column (2.1mm*150mm, 1.7um) is finally selected for analysis and detection.

[0090] 5. Actual sample analysis

[0091] Using the above established pretreatment method, the endogenous metabolites of patulin exposed zebrafish are extracted, purified, and quantitatively analyzed by HPLC-Q-Orbitrap-HRMS for patulin and its three metabolites, including: Hydroascladiol, E-ascladiol and Z-ascladiol. The results show that patulin shows a good linear relationship in the concentration range of 1-20ng / mL, and the correlation coefficient r 2 is greater than 0.999, which meets the requirements of quantitative analysis. According to the content value of each compound corresponding to the signal-to-noise ratio S / N≥3, the detection limit (LOD) of the compound is obtained. As shown in Table 3,

[0092] Table 3 Content value, detection limit and linear equation of endogenous patulin and its metabolites in patulin exposed zebrafish

[0093]

[0094] The method has strong applicability to protein powder food matrix, and can solve the extraction and purification process of both fat-soluble and water-soluble hormones in one step.

[0095] In summary, the present application uses water-acetonitrile (4:1, v:v) extraction, and based on the salting-out effect, two phases are obtained after layering: the water phase (upper layer) and the organic phase (lower layer). Using the unique retention of water molecules by diatomite column, the extraction and purification of patulin and its metabolites, including: Hydroascladiol, E-ascladiol and Z-ascladiol, which have very different physicochemical properties, are realized by one pretreatment. Based on the similarity of the parent nucleus structure, the obtained patulin metabolites are relatively semi-quantitatively analyzed, and the detection limit range of the metabolites is obtained.

[0096] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting and purifying endogenous metabolites of zebrafish after exposure to patulin, characterized in that, Includes the following steps: (1) Weigh the sample to be tested and add it to the acetonitrile-water mixed extraction solution. After shaking and centrifuging, the extract and residue are obtained. Take out the extract and extract the residue repeatedly. (2) Combine the extracts, add ammonium acetate, shake and centrifuge to obtain the upper acetonitrile phase and the lower aqueous phase; (3) Take the diatomaceous earth column on the aqueous phase layer, after equilibrium, use pure acetonitrile as the eluent to elute and obtain the eluent; (4) Add the acetonitrile phase to the neutral alumina powder, shake and let it stand at low temperature, then centrifuge at low temperature and high speed to obtain the supernatant; (5) Combine the eluent obtained in step (3) and the supernatant obtained in step (4), blow them to near dryness with nitrogen, make up to volume with methanol-water mixture, centrifuge at high speed, and then analyze; the sample to be tested in step (1) is the fish meat sample to be tested after being exposed to patulin; the volume ratio of acetonitrile to water in the acetonitrile-water mixed extract in step (1) is 1:4; Metabolites include Hydroascladiol, E-ascladiol, and Z-ascladiol.

2. The method for extracting and purifying endogenous metabolites of zebrafish after patulin exposure according to claim 1, characterized in that, In step (5), the volume ratio of methanol to water in the methanol-water mixture is 1:

4.

3. A method for detecting endogenous metabolites in zebrafish after exposure to patulin, characterized in that, Includes the following steps: (1) Sample pretreatment: Weigh the zebrafish meat sample to be tested after exposure to patulin and add it to the acetonitrile-water mixture for extraction. After shaking and centrifugation, the extract and residue are obtained. The extract is removed and the residue is extracted again. Combine the extracts, add ammonium acetate, shake and centrifuge to obtain the upper acetonitrile phase and the lower aqueous phase; Take a diatomaceous earth column from the aqueous phase layer, and after equilibrium, elute with pure acetonitrile to obtain the eluent; Then, the acetonitrile phase was added to neutral alumina powder, shaken, and allowed to stand at low temperature. Finally, it was centrifuged at low temperature and high speed to obtain the supernatant. Combine the eluent and supernatant, blow them to near dryness with nitrogen, bring the volume up with a methanol-water mixture, centrifuge at high speed, and then analyze. (2) Preparation of matrix standard curve: Prepare a standard curve for patulin with a concentration range of 0-20 ng / mL. Plot the concentration on the x-axis and the peak area of ​​the standard on the y-axis to serve as the basis for quantification of the target analyte. (3) Chromatographic and mass spectrometric conditions Liquid chromatography conditions: Syncronis C18 column, 2.1 mm × 150 mm, 1.7 μm; mobile phase A: aqueous solution containing 5% methanol; mobile phase B: methanol solution containing 5% water; flow rate: 0.3 mL / min; injection volume: 5.0 μL. Mass spectrometry conditions: Full scan mass spectrometry was performed in positive / negative ion conversion mode, mass range: m / z 50–700, resolution 120,000, automatic gain control target value 5e. 5 ; Negative ion mode 2700V, positive ion mode 3800V, ion transmission tube temperature 300℃, sheath gas pressure 35 arb, auxiliary gas pressure 10 arb, vaporization chamber temperature 350℃. Before running the sample, the instrument was calibrated for both positive and negative ions. The second stage uses an automatic trigger mode, with a resolution of 35,000 and an automatic gain control target value of 2e. 5 The collision energies were 15%, 30%, and 50%, respectively. (4) Concentration calculation method: The content of the analyte in the sample is obtained according to the following formula (1): In the formula: X – The concentration of the analyte in the sample, in μg / kg; the analyte is patulin and its metabolites. C – The concentration of the analyte in the sample treatment solution, calculated based on the matrix standard curve, in μg / L. V — Constant volume, in mL; m – Sample mass, in g; In step (1), the volume ratio of acetonitrile to water in the acetonitrile-water mixed extract is 1:

4. Metabolites include Hydroascladiol, E-ascladiol, and Z-ascladiol.

4. The method for detecting endogenous metabolites in zebrafish after patulin exposure according to claim 3, characterized in that: The volume ratio of methanol to water in the methanol-water mixture is 1:4.

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