A high-sensitivity detection method for multi-residues of veterinary drugs in pig urine

Abstract

The application provides a high-sensitivity detection method for multi-residues of veterinary drugs in pig urine, and belongs to the technical field of detection of veterinary drug residues. The application first performs enzymatic treatment on the sample to be detected, thereby improving the detection sensitivity of salbutamol and ractopamine; then performs acid hydrolysis and derivatization treatment on the sample to be detected, thereby improving the reversed-phase chromatographic retention performance and mass spectrometry ionization efficiency of nitrofurans in the compound to be detected, improving the sensitivity, improving the polarity of the compound to be detected, thereby improving the chromatographic separation effect, effectively separating the interfering substances in the matrix, improving the anti-interference ability of the detected substance, and indirectly improving the detection sensitivity; clenbuterol, metronidazole, dimetridazole, olaquindox, chlorpromazine and chloramphenicol are mainly in free state, and are not affected by enzymatic and acid hydrolysis and derivatization during detection, can be detected together with the above-mentioned compounds to be detected, and the mass spectrometry derivatization and sensitivity detection of multi-residues of cross-class veterinary drugs are realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of veterinary drug residue detection, and particularly relates to a high-sensitivity detection method for veterinary drug multi-residues in pig urine. BACKGROUND

[0002] With the development of large-scale breeding industry, veterinary drugs are commonly used in animal breeding process, such as nitrofuran (furazolidone, furaltadone, furanitroline, furazolium), nitroimidazole (metronidazole, dimetridazole, lozidazole), beta-agonists (clenbuterol, salbutamol, ractopamine), chloramphenicol (chloramphenicol) and phenothiazine (chlorpromazine) and the like. The monitoring of veterinary drug residues can avoid the flow of unqualified products into the market, which not only ensures the safety of food, but also avoids the loss of breeding enterprises, and accordingly the detection technology of veterinary drug residues becomes an important field of research and development.

[0003] The existing veterinary drug residue detection technology is often classified and processed according to the properties of veterinary drug residues in order to detect metabolites, and thus mainly focuses on the detection of the same type of veterinary drugs or single veterinary drugs. For example, the prior art (Yang Junhua, Chen Haiyan, Ma Jing, et al. Method for determination of nitrofuran prohibited drugs in fish feed by HPLC [J] Ningxia Forestry Science and Technology, 2020, Vol. 61, No. 11) discloses a high performance liquid chromatography analysis method for four kinds of nitrofuran drugs (furazolidone, furanitroline, furazolidone and furaltadone) in feed. After the fish feed is extracted and purified, high performance liquid chromatography is used for detection and analysis. The prior art (Li Xiangbo, Wang Yahui. Establishment of detection method for nitroimidazole drug residues in chicken meat based on HPLC-MS / MS [J] Food Industry, 2021, Vol. 42, No. 2) discloses an analysis method for metronidazole, dimetridazole, levamisole and lozidazole in chicken meat. The sample is extracted with ethyl acetate, the extract is purified by MCX column, and then detected and analyzed by high performance liquid chromatography-tandem triple quadrupole mass spectrometry. The prior art (Shi Jingfei. Metabolism and tissue residue prediction of salbutamol and ractopamine in pigs and goats [D] Central China Agricultural University, June 2014) discloses a quantitative confirmation method for five kinds of beta-receptor agonists (salbutamol, zilpaterol, clenbuterol, ractopamine and phenylethanolamine A). The sample is hydrolyzed by beta-glucuronidase, ultrasonically extracted with alkalized ethyl acetate, degreased with n-hexane, purified by MCX solid phase extraction column, eluted with ammonia water-methanol, dried with nitrogen, redissolved with the initial mobile phase, and detected by HPLC-MS / MS. However, the technical solutions disclosed in the above prior arts are only for the detection of the same type of veterinary drugs or single veterinary drugs, and cannot realize the cross-class detection of veterinary drug multi-residues in pig urine. SUMMARY

[0004] Therefore, the present application aims to provide a high-sensitivity detection method for multiple residues of veterinary drugs in pig urine.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a high-sensitivity detection method for multiple residues of veterinary drugs in pig urine, characterized by comprising the following steps:

[0007] The pig urine sample, the mixed internal standard solution, the ammonium acetate buffer solution and the enzymatic reagent are first mixed for enzymolysis to obtain an enzymolysis mixture;

[0008] The enzymolysis mixture, the acidolysis reagent and o-nitrobenzaldehyde are secondly mixed for derivatization to obtain a derivatization mixture;

[0009] The derivatization mixture and ethyl acetate are thirdly mixed, and after extraction and centrifugation, an upper layer liquid and a lower layer liquid are obtained;

[0010] The lower layer liquid is subjected to cation exchange column separation with water, methanol and ammonia-methanol mixed solution as eluents in sequence, and the ammonia-methanol mixed solution eluate is collected; the ammonia-methanol mixed solution eluate and the upper layer liquid are combined, dried and then redissolved in acetonitrile water solution to obtain a sample liquid to be tested;

[0011] The sample liquid to be tested is detected by liquid chromatography tandem mass spectrometry to obtain a detection result of multiple residues of veterinary drugs;

[0012] The multiple residues of veterinary drugs include at least two of metabolites of nitrofuran compounds, nitroimidazole compounds, beta-agonist compounds, chloramphenicol compounds and phenothiazine compounds.

[0013] Preferably, the metabolites of nitrofuran compounds include one or more of furazolidone metabolites, nitrofurantoin metabolites, furaltadone metabolites and furazolium metabolites; the nitroimidazole compounds include one or more of metronidazole, dimetridazole and ronidazole; the beta-agonist compounds include one or more of clenbuterol, salbutamol and ractopamine; the chloramphenicol compounds include chloramphenicol; and the phenothiazine compounds include chlorpromazine.

[0014] Preferably, the enzymatic reagent comprises β-glucuronidase / arylsulfatase; the volume ratio of the swine urine sample to the enzymatic reagent is 2:0.02-0.04; the volume ratio of the swine urine sample to ammonium acetate is 2:4-5; the concentration of the ammonium acetate buffer solution is 0.19-0.21 mol / L;

[0015] The enzymolysis time is 2-3 h.

[0016] Preferably, the acidolysis reagent comprises hydrochloric acid and / or formic acid; the volume ratio of the swine urine sample to the acidolysis reagent is 2:1.4-1.6.

[0017] Preferably, the volume ratio of the swine urine sample to o-nitrobenzaldehyde is 2:0.09-0.11.

[0018] Preferably, the derivatization temperature is 36-38℃, and the time is 15-17 h.

[0019] Preferably, the volume ratio of the swine urine sample to ethyl acetate is 2:6-10.

[0020] Preferably, in the cation exchange column separation process, the amount of water used is 1-1.5 BV, the amount of methanol used is 1-1.5 BV, and the amount of ammonia-methanol mixed solution used is 1.5-2 BV.

[0021] Preferably, the liquid chromatography-mass spectrometry detection conditions of the liquid chromatography-mass spectrometry comprise: the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is 0.1 v / v% formic acid aqueous solution, the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.25-0.3 mL / min; the column temperature is 35-40℃; and the injection amount is 2-5 μL.

[0022] The elution mode is gradient elution; and the gradient elution program is as follows: at 0.00 min, the volume fraction of the mobile phase A is 98%; at 0.00-2.50 min, the volume fraction of the mobile phase A decreases from 98% to 95%; at 2.50-3.00 min, the volume fraction of the mobile phase A decreases from 95% to 85%; at 3.00-5.00 min, the volume fraction of the mobile phase A decreases from 85% to 65%; at 5.00-7.00 min, the volume fraction of the mobile phase A decreases from 65% to 45%; at 7.00-9.00 min, the volume fraction of the mobile phase A increases from 45% to 95%; at 9.00-11.00 min, the volume fraction of the mobile phase A is 95%; at 11.00-11.01 min, the volume fraction of the mobile phase A increases from 95% to 98%; and at 11.01-14.00 min, the volume fraction of the mobile phase A is 98%.

[0023] Preferably, the mass spectrometry detection conditions of the liquid chromatography tandem mass spectrometry comprise: the ion source is an electrospray ion source; the detection mode is multiple reaction monitoring; the scanning mode is positive and negative ion scanning; the positive mode scanning ion source parameters are: the spray voltage is 5500eV; the ion source temperature is 550 DEG C; the negative mode scanning ion source parameters are: the spray voltage is -4500eV; the ion source temperature is 550 DEG C.

[0024] The application provides a detection method for multi-residues of veterinary drugs in pig urine. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is an IDA TOF MS / MS scan accurate mass spectrum diagram of a test sample liquid of Example 1 Test 1.

[0026] Figure 2 Figure 2 is an IDA TOF MS / MS scan clenbuterol accurate mass spectrum diagram of a test sample liquid of Example 1 Test 1.

[0027] Figure 3 Figure 3 is an IDA TOF MS / MS scan mass-to-charge ratio spectrum (410.0-414.0) of a test sample liquid of Example 1 Test 1.

[0028] Figure 4 Figure 4 is an IDA TOF MS / MS scan accurate mass spectrum diagram of a test sample liquid of Example 1 Test 2.

[0029] Figure 5 Figure 5 is an IDA TOF MS / MS scan clenbuterol accurate mass spectrum diagram of a test sample liquid of Example 1 Test 2.

[0030] Figure 6 Figure 6 is an IDA TOF MS / MS scan mass-to-charge ratio spectrum (410.0-414.0) of a test sample liquid of Example 1 Test 2.

[0031] Figure 7 Clenbuterol exact mass plot for Example 1 Test 3 sample liquid under test IDA TOF MS / MS scan;

[0032] Figure 8 Clenbuterol exact mass plot for Example 1 Test 3 sample liquid under test IDA TOF MS / MS scan;

[0033] Figure 9 Exact mass plot (410.0-414.0) for Example 1 Test 3 sample liquid under test IDA TOF MS / MS scan;

[0034] Figure 10 Chlorpromazine chromatogram for Example 1 Test 1 sample liquid under test;

[0035] Figure 11 Chlorpromazine chromatogram for Example 1 Test 2 sample liquid under test;

[0036] Figure 12 Chlorpromazine chromatogram for Example 1 Test 3 sample liquid under test;

[0037] Figure 13 Comparison of test compound response for Example 1 Tests 1-3 sample liquids under test;

[0038] Figure 14 Comparison of test compound response for acid hydrolysis and enzymatic hydrolysis of pig urine sample (left) and standard sample (right);

[0039] Figure 15 Bar graph of effect of acetic acid buffer solution concentration and hydrochloric acid addition on solution pH;

[0040] Figure 16 Comparison of test compound response for different extraction methods for pig urine sample (left) and standard sample (right);

[0041] Figure 17 Comparison of test compound response for methanol eluent and ammonia-methanol mixture eluent for pig urine sample (left) and standard sample (right);

[0042] Figure 18 Comparison of test compound response for different amounts of ammonia-methanol mixture for pig urine sample (left) and standard sample (right);

[0043] Figure 19 Characteristic ion reference chromatograms for 2-NP-AMOZ (left) and 2-NP-AOZ (right);

[0044] Figure 20Reference chromatograms for characteristic ions for 2-NP-AHD (left) and 2-NP-SEM (right);

[0045] Figure 21 Reference chromatograms for characteristic ions for clenbuterol (left) and salbutamol (right);

[0046] Figure 22 Reference chromatograms for characteristic ions for ractopamine (left) and metronidazole (right);

[0047] Figure 23 Reference chromatograms for characteristic ions for dimetridazole (left) and roxidazole (right);

[0048] Figure 24 Reference chromatograms for characteristic ions for chloφromazine (left) and chloramphenicol (right);

[0049] Figure 25 Clenbuterol-spiked sample chromatogram for Example 6;

[0050] Figure 26 Ractopamine-spiked sample chromatogram for Example 6;

[0051] Figure 27 Salbutamol-spiked sample chromatogram for Example 6;

[0052] Figure 28 Furazolidone metabolite-spiked sample chromatogram for Example 6;

[0053] Figure 29 Furagilin metabolite-spiked sample chromatogram for Example 6;

[0054] Figure 30 Furantoin metabolite-spiked sample chromatogram for Example 6;

[0055] Figure 31 Furaltadone metabolite-spiked sample chromatogram for Example 6;

[0056] Figure 32 Metronidazole-spiked sample chromatogram for Example 6;

[0057] Figure 33 Dimetridazole-spiked sample chromatogram for Example 6;

[0058] Figure 34 Roxidazole-spiked sample chromatogram for Example 6;

[0059] Figure 35 Chloφromazine-spiked sample chromatogram for Example 6;

[0060] Figure 36 Chloramphenicol-spiked sample chromatogram for Example 6;

[0061] Figure 37 Clenbuterol concentration and peak area standard curve for Example 7;

[0062] Figure 38 Ractopamine concentration and peak area standard curve for Example 7;

[0063] Figure 39 Salbutamol concentration and peak area standard curve for Example 7;

[0064] Figure 40 Furazolidone metabolite concentration and peak area standard curve for Example 7;

[0065] Figure 41 Furazolidone metabolite concentration and peak area standard curve for Example 7;

[0066] Figure 42 Furantoin metabolite concentration and peak area standard curve for Example 7;

[0067] Figure 43 Furantoin metabolite concentration and peak area standard curve for Example 7;

[0068] Figure 44 Metronidazole concentration and peak area standard curve for Example 7;

[0069] Figure 45 Dimetridazole concentration and peak area standard curve for Example 7;

[0070] Figure 46 Ronidazole concentration and peak area standard curve for Example 7;

[0071] Figure 47 Chlorpromazine concentration and peak area standard curve for Example 7;

[0072] Figure 48 Chloramphenicol concentration and peak area standard curve for Example 7. DETAILED DESCRIPTION

[0073] The present application provides a high-sensitivity detection method for veterinary drug multi-residues in pig urine, characterized in that it comprises the following steps:

[0074] The pig urine sample, mixed internal standard solution, ammonium acetate buffer solution and enzyme digestion reagent are first mixed for enzyme digestion to obtain an enzyme digestion mixture;

[0075] The enzyme digestion mixture, acid hydrolysis reagent and o-nitrobenzaldehyde are secondly mixed for derivation to obtain a derivatization mixture;

[0076] The derivatization mixture and ethyl acetate are thirdly mixed, and after extraction and centrifugation, an upper layer liquid and a lower layer liquid are obtained;

[0077] The lower liquid is separated by a cation exchange column with water, methanol and ammonia-methanol mixed solution as eluents in sequence, the ammonia-methanol mixed solution eluent is collected, the ammonia-methanol mixed solution eluent and the upper liquid are combined, and then are redissolved in acetonitrile water solution after drying to obtain a sample liquid to be detected;

[0078] The sample liquid to be detected is detected by liquid chromatography tandem mass spectrometry to obtain the detection result of the veterinary drug multi-residues;

[0079] The veterinary drug multi-residues comprise at least two of metabolites of nitrofurans, nitroimidazoles, beta-agonists, chloramphenicols and phenothiazines.

[0080] In the present application, the reagents used are all commercially available products known to those skilled in the art, unless otherwise specified.

[0081] The pig urine sample, mixed internal standard liquid, ammonium acetate and enzymatic reagent are first mixed for enzymolysis to obtain an enzymolysis mixed liquid. In the present application, the types of internal standards in the mixed internal standard liquid are preferably the same as those of the veterinary drug multi-residues. In the present application, the enzymatic reagent preferably comprises beta-glucuronidase / arylsulfatase; the volume ratio of the pig urine sample to the enzymatic reagent is preferably 2:0.02-0.04, more preferably 2:0.024-0.036, and most preferably 2:0.028-0.032; and the volume ratio of the pig urine sample to ammonium acetate is preferably 2:4-5, more preferably 2:4.2-4.8, and most preferably 2:4.4-4.6. In the present application, the first mixing is preferably vortex mixing; the vortex mixing time is preferably 10-30 s, more preferably 15-25 s, and most preferably 18-20 s; and the vortex mixing temperature is preferably room temperature. In the present application, the enzymolysis temperature is preferably 36-38℃, more preferably 36.5-37.5℃, and most preferably 36.8-37℃; and the enzymolysis time is preferably 2-3 h, more preferably 2.2-2.8 h, and most preferably 2.4-2.5 h. The pig urine sample is first enzymolyzed in the present application, and the conjugation rate of salbutamol and ractopamine aniline type beta-receptor agonists with glucuronic acid in tissues is low, so enzymolysis is helpful for the sufficient extraction of beta-receptor agonists, and improves the accuracy and sensitivity of detection.

[0082] After obtaining the enzymatic hydrolysis mixture, the enzymatic hydrolysis mixture, acidolysis reagent and o-nitrobenzaldehyde are secondly mixed to perform derivatization to obtain a derivatized mixture. In the application, the acidolysis reagent preferably comprises hydrochloric acid and formic acid; the volume ratio of the pig urine sample to the acidolysis reagent is preferably 2:1.4-1.6, more preferably 2:1.45-1.55, and most preferably 2:1.48-1.5; and the volume ratio of the pig urine sample to the mass of o-nitrobenzaldehyde is preferably 2:0.09-0.11, more preferably 2:0.095-0.105, and most preferably 2:0.098-0.1. In the application, the second mixing is preferably vortex mixing; the time of the vortex mixing is preferably 20-30 s, more preferably 25-28 s, and most preferably 26-27 s. In the application, the temperature of the derivatization is preferably 36-38℃, more preferably 36.5-37.5℃, and most preferably 36.8-37℃; and the time of the derivatization is preferably 15-17 h, more preferably 15.5-16.5 h, and most preferably 15.8-16.2 h.

[0083] In the application, the enzymatic hydrolysis mixture is subjected to acidolysis derivatization, and the derivatization mechanism is shown in formula I: the carbonyl nucleophilic addition reaction is utilized, i.e. the derivative is subjected to derivatization with o-nitrobenzaldehyde, the nitrogen atom with a lone pair of electrons in the primary amine group attacks the carbon atom with a positive charge on the carbonyl group of o-nitrobenzaldehyde to complete the nucleophilic addition reaction, and an intermediate α-hydroxy amine compound is formed, and then further dehydration is performed to form a Schiff base derivative product. From the analysis of the aldehyde amine nucleophilic addition reaction mechanism, the tertiary amine and quaternary amine compounds do not undergo the nucleophilic addition reaction with the aldehyde group, and the steric hindrance also affects the activity of the nucleophilic addition reaction (such as clenbuterol, salbutamol, ractopamine, levamisole and chloramphenicol). The chemical structures of the 12 kinds of veterinary drug residues to be detected all have amino structures, but metronidazole, dimenidazole and chlorpromazine only have tertiary amine structures, and thus do not undergo the derivatization reaction with o-nitrobenzaldehyde. The chemical structures of the 4 kinds of nitrofurans metabolites (furacillin metabolite, furantoin metabolite, furaltadone metabolite and furazolidone metabolite) meet the structural characteristics of the derivatization reaction with o-nitrobenzaldehyde.

[0084]

[0085] The application improves the ionization efficiency of the mass spectrum of the to-be-tested compounds by adding acidolysis reagents and o-nitrobenzaldehyde to derive 4 nitrofuran metabolites to form derivative products, thereby improving sensitivity; another derivative group can improve the polarity of the to-be-tested compounds, thereby improving the chromatographic separation effect, effectively separating the interfering substances in the matrix, and improving the anti-interference ability of the to-be-tested substances, thereby indirectly improving the detection sensitivity. Moreover, the derivative conditions provided by the application are specific reactions, which can precisely control the compounds that occur in the derivative reaction. The to-be-tested compounds (including clenbuterol, metronidazole, dimetridazole, ronidazole, chlorpromazine and chloramphenicol) in free state have no difference in response value after enzyme hydrolysis and acidolysis derivation, and can be detected together with other to-be-tested compounds, thereby realizing high-sensitivity detection of multi-residues of cross-class veterinary drugs.

[0086] After obtaining the derivative mixed solution, the derivative mixed solution and ethyl acetate are mixed in the application, and after extraction and centrifugation, the upper liquid and the lower liquid are obtained. In the application, the purity of the ethyl acetate is preferably chromatographically pure; the volume ratio of the pig urine sample to ethyl acetate is preferably 2:6-10, more preferably 2:7-9, and most preferably 2:7.5-8.5. In the application, the extraction is preferably vortex extraction; the extraction time is preferably 5-10 s, more preferably 6-9 s, and most preferably 7-8 s; and the vortex extraction temperature is preferably room temperature. In the application, the centrifugal speed is preferably 4500-5500 rpm, more preferably 4800-5200 rpm, and most preferably 4900-5100 rpm; and the centrifugal time is preferably ≥5 min, more preferably 6-9 min, and most preferably 7-8 min. In the application, the upper liquid is preferably an organic phase; and the lower liquid is preferably an acidic aqueous phase.

[0087] After the upper layer liquid and the lower layer liquid are obtained, the lower layer liquid is separated by a cation exchange column with water, methanol and ammonia-methanol mixed solution as eluent in sequence, the ammonia-methanol mixed solution eluent is collected, the ammonia-methanol mixed solution eluent and the upper layer liquid are combined, and after drying, the sample liquid to be detected is obtained by re-dissolving in acetonitrile aqueous solution. In the present application, the volume percentage content of ammonia in the ammonia-methanol mixed solution is preferably 5%, and the concentration of ammonia in the ammonia-methanol mixed solution is preferably 28 v / v%. In the present application, the amount of water used in the cation exchange column separation process is preferably 1-1.5 BV, more preferably 1.1-1.4 BV, and most preferably 1.2-1.3 BV; the amount of methanol used is; the amount of methanol used is preferably 1-1.5 BV, more preferably 1.1-1.4 BV, and most preferably 1.2-1.3 BV; the amount of ammonia-methanol mixed solution used is preferably 1.5-2 BV, more preferably 1.6-1.9 BV, and most preferably 1.7-1.8 BV. In the present application, the cation exchange column is preferably a mixed type cation exchange column. In the present application, after the lower layer liquid is separated by a cation exchange column with water and methanol as eluent in sequence, it is preferably further comprising discarding the water and methanol eluent, and drying the cation exchange column by air blowing. The present application does not have special limitations on the time and air flow rate of the air drying, and the cation exchange column can be dried by air blowing. In the present application, the concentration of the acetonitrile aqueous solution is preferably 5-10 v / v%, more preferably 5-9 v / v%, and most preferably 5-6 v / v%; the volume ratio of the swine urine sample to the acetonitrile aqueous solution is preferably 2:0.25-1, more preferably 2:0.5-1, and most preferably 2:0.9-1. In the present application, after re-dissolving, it is preferably further comprising filtering the re-dissolved solution to obtain the sample liquid to be detected; the filter membrane for filtering is preferably an organic filter membrane with a pore size of 0.22 μm.

[0088] After obtaining the sample liquid to be detected, the liquid chromatography tandem mass spectrometry is used to detect the sample liquid to be detected, and the detection result of the veterinary drug multi-residues is obtained. The veterinary drug multi-residues include at least two of metabolites of nitrofuran compounds, nitroimidazole compounds, β-agonist compounds, chloramphenicol compounds and phenothiazine compounds. In the present application, the metabolites of nitrofuran compounds preferably include one or more of furazolidone metabolites, furantoin metabolites, furaltadone metabolites and furazolide metabolites; the β-agonist compounds preferably include one or more of clenbuterol, salbutamol and ractopamine; the nitroimidazole compounds preferably include one or more of metronidazole, dimetridazole and lozidazole; the phenothiazine compounds preferably include chlorpromazine; and the chloramphenicol compounds preferably include chloramphenicol. In the present application, the veterinary drug multi-residues preferably include furazolidone metabolites, furantoin metabolites, furaltadone metabolites, furazolide metabolites, clenbuterol, salbutamol, ractopamine, metronidazole, dimetridazole, lozidazole, chlorpromazine and chloramphenicol.

[0089] In the present application, the conditions of liquid chromatography detection of the liquid chromatography tandem mass spectrometry preferably include that the chromatographic column is a C 18 The chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is 0.1v / v% formic acid aqueous solution, the mobile phase B is acetonitrile, the flow rate of the mobile phase is 0.3mL / min; the column temperature is 40℃; the injection volume is 5μL; the elution mode is gradient elution; the program of the gradient elution is as follows: 0.00min, the volume fraction of the mobile phase A is 98%; 0.00-2.50min, the volume fraction of the mobile phase A decreases from 98% to 95%; 2.50-3.00min, the volume fraction of the mobile phase A decreases from 95% to 85%; 3.00-5.00min, the volume fraction of the mobile phase A decreases from 85% to 65%; 5.00-7.00min, the volume fraction of the mobile phase A decreases from 65% to 45%; 7.00-9.00min, the volume fraction of the mobile phase A increases from 45% to 95%; 9.00-11.00min, the volume fraction of the mobile phase A is 95%; 11.00-11.01min, the volume fraction of the mobile phase A increases from 95% to 98%; 11.01-14.00min, the volume fraction of the mobile phase A is 98%.

[0090] In the present application, the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry preferably include: the ion source is an electrospray ion source; the detection mode is multiple reaction monitoring; the scanning mode is positive and negative ion scanning; the positive mode scanning ion source parameters are: the spray voltage is 5500 eV; the ion source temperature is 550℃; the atomization gas flow is 40 psi; the dry gas flow is 40 psi; the curtain gas flow is 40 psi; the collision gas flow is 9 psi; the negative mode scanning ion source parameters are: the spray voltage is -4500 eV; the ion source temperature is 550℃; the atomization gas flow is 40 psi; the dry gas flow is 40 psi; the curtain gas flow is 40 psi; the collision gas flow is 9 psi. The qualitative ion pairs, declustering voltage (DP) and fragmentation voltage (CE) of the compounds to be tested are shown in Table 1.

[0091] Table 1 Mass spectrometry parameters of compounds to be tested

[0092]

[0093] In the embodiments of the present application, the specific instruments, reagents and specifications of the samples to be tested are as follows:

[0094] Furantoin metabolites, furazolidone metabolites, furantoin metabolites, furacillin metabolites, clenbuterol, salbutamol, ractopamine, metronidazole, dimetridazole, lornidazole, chlorpromazine, chloramphenicol and corresponding isotopic internal standards, all with a purity of ≥98% (Dr. Ehrenstorfer Company, Germany); API 5500 type ultra-high performance liquid chromatography-tandem mass spectrometry / mass spectrometer with an electrospray ion source (AB SCIEX Company, USA); centrifuge (Shanghai Anting Company); G560E type vortex mixer (Scientific Industries Company, USA); hydrochloric acid, ammonia (analytical pure, Shanghai National Pharmaceutical Group); o-nitrobenzaldehyde (chromatographically pure, Shanghai National Pharmaceutical Group); β-glucuronidase / arylsulfatase (enzyme activity greater than 30 U / mL, Shanghai Anpu Company); acetonitrile, methanol, ethyl acetate are chromatographically pure (Shanghai Anpu Company); mixed cation exchange column (60 mg, 3 mL, waters company, USA).

[0095] The liquid chromatography detection conditions include: the chromatographic column is a C 18The chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is 0.1v / v% formic acid aqueous solution, the mobile phase B is acetonitrile, the flow rate of the mobile phase is 0.3mL / min; the column temperature is 40℃; the injection volume is 5μL; the elution mode is gradient elution; the procedure of the gradient elution is as follows: 0.00min, the volume fraction of the mobile phase A is 98%; 0.00-2.50min, the volume fraction of the mobile phase A decreases from 98% to 95%; 2.50-3.00min, the volume fraction of the mobile phase A decreases from 95% to 85%; 3.00-5.00min, the volume fraction of the mobile phase A decreases from 85% to 65%; 5.00-7.00min, the volume fraction of the mobile phase A decreases from 65% to 45%; 7.00-9.00min, the volume fraction of the mobile phase A increases from 45% to 95%; 9.00-11.00min, the volume fraction of the mobile phase A is 95%; 11.00-11.01min, the volume fraction of the mobile phase A increases from 95% to 98%; 11.01-14.00min, the volume fraction of the mobile phase A is 98%.

[0096] The conditions of mass spectrometric detection include: the ion source is electrospray ion source; the detection mode is multiple reaction monitoring; the scanning mode is positive and negative ion scanning; the positive mode scanning ion source parameters: the spray voltage is 5500eV; the ion source temperature is 550℃; the atomization gas flow is 40psi; the dry gas flow is 40psi; the curtain gas flow is 40psi; the collision gas flow is 9psi; the negative mode scanning ion source parameters: the spray voltage is -4500eV; the ion source temperature is 550℃; the atomization gas flow is 40psi; the dry gas flow is 40psi; the curtain gas flow is 40psi; the collision gas flow is 9psi. The qualitative ion pairs, deprotonation voltages (DP) and fragmentation voltages (CE) of the compounds to be tested are shown in Table 1.

[0097] Example 1

[0098] 12 compound derivation reaction influence test: in order to investigate whether the nucleophilic addition reaction of o-nitrobenzaldehyde as a derivative reagent affects other test substances, accurately weigh 10mg of furan itaconate metabolite, furazolidone metabolite, furantoin metabolite, furacilin metabolite, clenbuterol, salbutamol, ractopamine, metronidazole, dimetridazole, ornidazole, chlorpromazine, chloramphenicol standard substance, dissolve in methanol and dilute to 10mL volumetric flask, prepare a standard stock solution with a concentration of 1.0mg / mL, and dilute with methanol before use to prepare a mixed standard solution with an appropriate concentration. The corresponding isotopic internal standard of each compound is prepared in the same way.

[0099] The comparative experiments of mixed standard solution + water (Test 1), mixed standard solution + acid (Test 2), and mixed standard solution + acid + o-nitrobenzaldehyde (Test 3) were respectively used to determine whether there was a significant difference in the response values of each test sample. The test samples are shown in Table 2 (the concentration of the mixed standard is 1 μg / mL).

[0100] Table 2 Test samples of mixed standard solution + water, mixed standard solution + acid, and mixed standard solution + acid + o-nitrobenzaldehyde

[0101]

[0102] The high-resolution mass spectrometer was used to determine the accurate molecular weight of the Test 1, Test 2, and Test 3 test sample liquids. The scanning mode was IDA TOF MS / MS (time-of-flight high-resolution mass spectrometry trigger type one-two mass spectrometry simultaneous scanning). The possible products of the nucleophilic addition reaction of the test sample with o-nitrobenzaldehyde were observed and located. The test results are shown in Table 3, wherein, Figures 1-9 Figure 1 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 1 test sample liquid; Figure 2 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 1 test sample liquid; Figure 3 is the IDA TOF MS / MS scanning mass-to-charge ratio spectrum (410.0-414.0) of the Test 1 test sample liquid; Figure 4 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 2 test sample liquid; Figure 5 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 2 test sample liquid; Figure 6 is the IDA TOF MS / MS scanning mass-to-charge ratio spectrum (410.0-414.0) of the Test 2 test sample liquid; Figure 7 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 3 test sample liquid; Figure 8 is the IDA TOF MS / MS scanning accurate mass-to-charge ratio spectrum of the Test 3 test sample liquid; Figure 9 is the IDA TOF MS / MS scanning mass-to-charge ratio spectrum (410.0-414.0) of the Test 3 test sample liquid. The molecular ion mass-to-charge ratio of the test sample was extracted and observed. The corresponding molecular ion mass-to-charge ratio could be observed in the Test 1, Test 2, and Test 3 test sample liquids. The possible products of the o-nitrobenzaldehyde derivative reaction were observed to be dehydrated products after two molecules were combined. No corresponding products with accurate mass-to-charge ratio and natural isotopes were observed.

[0103] ​Take clenbuterol as an example: the accurate mass-to-charge ratio of clenbuterol is 277.08690, and the accurate mass-to-charge ratios of 277.0878 / 4.43, 277.0879 / 4.38 and 277.0879 / 4.44 are monitored in the Test1, Test2 and Test3 sample solutions respectively, and the corresponding retention times are also consistent. The observed isotopic accurate mass-to-charge ratios are 279.0849 / 4.40, 279.0848 / 4.39 and 279.0855 / 4.45, and the retention times of the isotopes are also consistent. The product of the possible derivative reaction of clenbuterol with o-nitrobenzaldehyde is calculated to have an accurate mass-to-charge ratio of 411.11110, and the mass spectrum of 411.11110 within a range of 3 Da is observed in the analysis data of Test1, Test2 and Test3. The peaks observed in Test1, Test2 and Test3 are 411.1868 / 4.23, 411.2980 / 4.61 and 411.0408 / 5.83, respectively, and the mass number deviation from the theoretical number is 184 ppm, 454 ppm and 171 ppm, respectively, which obviously exceeds the requirement of high-resolution mass spectrometry that the mass number deviation of the same compound should be within 5 ppm. In addition, the peak of the isotopes corresponding to the mass-to-charge ratio is not observed at the same retention time, which obviously does not match the characteristic peak of the chlorine element in clenbuterol. The response intensity of the chromatographic peak of the ion of clenbuterol extracted from Test1, Test2 and Test3 is also consistent. From the above experimental data analysis, it can be seen that under the above conditions, clenbuterol does not react with o-nitrobenzaldehyde.

[0104] The derivatives and non-derivatives of Test 1, Test2 and Test3 are tested by liquid chromatography tandem triple quadrupole mass spectrometry using the aforementioned instrument conditions, and the influence of the derivative is determined by quantitative analysis. Take chlorpromazine as an example to illustrate that the mass spectrometry detection method used in the present application can detect each compound in the mixed standard solution by single channel detection, and then obtain the detection result of a single test compound. Figure 10 The chromatogram of chlorpromazine in the Test1 sample solution is shown in Figure 1; Figure 11 The chromatogram of chlorpromazine in the Test2 sample solution is shown in Figure 2; Figure 12 The chromatogram of chlorpromazine in the Test3 sample solution is shown in Figure 3. Figures 10-12It can be seen that compared with the aqueous solution environment, the addition of formic acid has no effect on the stability of chlorpromazine, but under acidic conditions, the aldehyde amine nucleophilic addition reaction occurs to the compound containing primary amine group. The furanocin metabolite, nitrofurantoin metabolite, furaltadone metabolite and furazolidone metabolite are monitored to be derivatized with o-nitrobenzaldehyde under the test conditions of Test3, and the derivatives of the four metabolites are not monitored in the tests of Test1 and Test2, and the standard addition tests of the other eight compounds in Test1, Test2 and Test3 all monitor the original compounds, which indicates that the o-nitrobenzaldehyde derivatization reaction under acidic conditions can realize the simultaneous determination of the above-mentioned 12 kinds of compounds, and the experimental results verify the feasibility of the technical principle. Figure 13 The figure is a comparison of the response values of the test compounds in the Test1-3 sample solutions, and according to Figure 13 It can be seen that after the addition of formic acid and o-nitrobenzaldehyde in Test3, the four nitrofuran metabolites form derivative products, and the response values of the other eight test substances with aqueous solution and acid addition without derivative reagent are basically the same, which indicates that the other eight test substances do not have derivatization reaction with o-nitrobenzaldehyde, and the experimental results prove that the eight compounds can be treated under acidic and o-nitrobenzaldehyde derivatization conditions, and the mass spectrometry is determined by measuring the original molecular ion. The above qualitative and quantitative analysis shows that the derivatization conditions determined in the application are specific reactions, which can be accurately controlled.

[0105] Example 2

[0106] Selection of nitrofuran metabolite enzymatic derivatization and acid hydrolysis derivatization: enzyme hydrolysis derivatization experiment and acid hydrolysis derivatization experiment are set to compare the derivatization efficiency of nitrofuran metabolites, and the specific experimental steps are as follows:

[0107] Enzymatic hydrolysis experiment: 2mL of pig urine sample was taken, 8mL of 0.2mol / L ammonium acetate was added, 40μL of β-glucuronidase / arylsulfatase was added, 100μL of o-nitrobenzaldehyde was added, and it was placed in a 37℃ water bath for vibration derivatization for 16h, and then 1mol / L sodium hydroxide solution was used to adjust the pH to 7.0, 7.5mL of acetonitrile and 7.5mL of ethyl acetate were added, vortex extraction was performed for 30s, 6g of sodium chloride was added, vortex mixing was performed for 15s, centrifugation was performed at a speed of 4500rpm for 5min, the supernatant was taken and nitrogen was blown to dryness, 1mL of 5v / v% acetonitrile aqueous solution was added, vortex ultrasonic dissolution was performed, and then 0.22μm organic membrane was used for determination. At the same time, the standard sample solution was prepared according to the above test operation.

[0108] Acidolysis experiment: 2 mL of pig urine sample was taken, 5 mL of 0.2 mol / L hydrochloric acid was added, 100 μL of o-nitrobenzaldehyde was added, and it was placed in a 37°C water bath for 16 h of derivatization. Then, 1 mol / L sodium hydroxide solution was used to adjust the pH to 7.0. Then, 7.5 mL of acetonitrile and 7.5 mL of ethyl acetate were added, vortex extraction was performed for 30 s, 6 g of sodium chloride was added, vortex mixing was performed for 15 s, centrifugation was performed at a speed of 4500 rpm for 5 min, the supernatant was taken, nitrogen blowing was performed until dryness, 1 mL of 5 v / v% acetonitrile aqueous solution was added, vortex ultrasonic dissolution was performed, and then 0.22 μm organic membrane was used for determination. At the same time, the standard sample solution was prepared according to the above experimental operation.

[0109] Figure 14 The acidolysis and enzymolysis of pig urine samples (left) and standard samples (right) were compared. It can be seen from Figure 14 that for the nitrofuran metabolites that need to be derivatized, the effect of acidolysis on the response values of the test compounds in the acidolysis and enzymolysis samples is more obvious (except for SEM, the other three test compounds). Acidolysis is better than enzymolysis, which may be because the strength of the acid affects the formation of a positively charged carbon atom on the carbonyl group of o-nitrobenzaldehyde. The response values of the β-receptor agonists in the standard solution treated by acidolysis and enzymolysis are basically the same, but in the pig urine matrix, the response value of clenbuterol treated by enzymolysis is higher than that treated by acidolysis, and the response value of ractopamine and salbutamol treated by acidolysis is higher than that treated by enzymolysis. This may be due to the influence of the matrix effect. The acidolysis and enzymolysis environment is more reliable based on the experimental results of the standard solution. Under the conditions of acidolysis and enzymolysis, the response values of lonomide treated by the two methods are relatively close, the response value of metronidazole treated by enzymolysis is higher than that treated by acidolysis, and the trend in the standard solution and pig urine matrix is consistent. Under the conditions of acidolysis and enzymolysis, the response values of chlorpromazine in the standard solution are basically the same, and the response value of acidolysis is slightly higher than that of enzymolysis. In the pig urine matrix, the response value of acidolysis is much higher than that of enzymolysis. Under the conditions of acidolysis and enzymolysis, the results of chloramphenicol in the standard solution are basically the same, and the response value of enzymolysis is higher than that of acidolysis. From the results of the two treatment methods, except for nitrofuran metabolites, enzymolysis and acidolysis are basically the same for the determination of other test compounds.

[0110] Example 3

[0111] Selection of acidolysis and enzymolysis and determination of enzymolysis time for β-receptor agonist drugs: positive pig urine samples (pig urine samples containing clenbuterol, ractopamine, and salbutamol) were treated by acidolysis and enzymolysis, respectively. Acidolysis was performed at 38°C for 16 h and 2 h, respectively, to obtain acidolysis sample A and acidolysis sample B. Enzymolysis was performed at 38°C for 16 h and 2 h, respectively, to obtain enzymolysis sample A and enzymolysis sample B. GB / T 22286-2008 was used for extraction and purification, and the residual amounts of the above three test compounds were determined. The experimental results are shown in Table 3.

[0112] Table 3 Comparison of extraction efficiency of β-receptor agonist drugs by acidolysis and enzymolysis (unit: μg / kg)

[0113]

[0114]

[0115] From Table 3, it can be seen that the residues of ractopamine and salbutamol in the enzymolysis sample are higher than those in the trichloroacetic acid acidolysis sample, and there is no significant difference in the residues of clenbuterol in the two samples, which further confirms that the conjugation rate of aniline type β-receptor agonists with glucuronic acid in the tissue is low, and the effect of acidolysis and time is not obvious. The enzymolysis effect is more thorough, and the effect of enzymolysis time is not significant. Only ractopamine has a difference, but the enzymolysis effect of 2h also reaches 67.9%~91.3% enzymolysis efficiency.

[0116] From the above enzymolysis and acidolysis experimental results, it is concluded that enzymolysis helps to fully extract β-receptor agonist drugs, and acidolysis after derivation can improve the derivation efficiency of nitrofuran metabolites. The enzymolysis efficiency of β-receptor agonist drugs is sufficient after 2h of enzymolysis. Based on this, the sample is treated by enzymolysis and acidolysis derivation in stages, i.e., enzymolysis for 2h, and then adding hydrochloric acid and o-nitrobenzaldehyde derivative reagent for derivation.

[0117] Example 4

[0118] Determination of acidolysis and derivation of acid addition: β-receptor agonist drugs are enzymolyzed using 0.2mol / L acetate buffer solution (pH 5.2), which can maintain a stable pH environment. However, the above experiments have proved that the derivation reaction of nitrofuran metabolites and o-nitrobenzaldehyde requires an acidic condition, and the acidic condition has an impact on sample acidolysis and derivation. This experiment compares the amount of hydrochloric acid added to control the pH of the acetate buffer solution to meet the requirements of the derivation reaction of nitrofuran metabolites and o-nitrobenzaldehyde.

[0119] Figure 15 The column chart of the influence of the concentration of acetate buffer solution and the amount of hydrochloric acid added on the pH value of the solution is as follows: Figure 15It can be seen that the pH of 0.2 mol / L acetate buffer solution and 2.0 mol / L acetate buffer solution after adding 0.5 mL hydrochloric acid is between 4.4 and 4.6, and the stable balance system of the buffer solution is basically not broken, and the higher the concentration of acetate, the smaller the influence of the added acid on the pH. To reach the level of the pH of 0.5 mL hydrochloric acid added in water, 1.5 mL hydrochloric acid is added in 0.2 mol / L acetate buffer solution, so the enzyme hydrolysis buffer solution is determined as 0.2 mol / L ammonium acetate buffer solution (pH 4.8), 1.5 mL hydrochloric acid is added after 2 h of enzyme hydrolysis, 100 μL of 0.1 mol / L o-nitrobenzaldehyde is added, and acid hydrolysis and derivation are carried out at 37°C for 16 h according to the requirements of GB / T 21311-2007 "Determination of nitrofurans metabolite residues in animal-derived food by high performance liquid chromatography / tandem mass spectrometry".

[0120] Example 5

[0121] Determination of extraction and purification steps: The current standard extraction and purification steps of the mass spectrometry method for detecting β-receptor agonist residues mainly include: after enzyme hydrolysis, the sample is subjected to acidic protein precipitation, pH adjustment to alkaline, liquid-liquid extraction, solvent replacement, and cation exchange solid phase extraction column purification; the extraction and purification steps of the mass spectrometry method for detecting nitrofurans metabolite residues are basically the same, after acid hydrolysis and derivation, the sample is subjected to pH adjustment to neutral, liquid-liquid extraction, solution replacement, and liquid-liquid extraction purification or HLB solid phase extraction column purification; the extraction steps of the mass spectrometry method for detecting nitroimidazole residues are also relatively consistent, the sample is extracted with an organic solvent, concentrated after solvent replacement, and purified by GPC, and then purified by C18 solid phase extraction column after solvent replacement; the determination of chlorpromazine residues adopts acid organic solvent extraction, HLB column purification after solvent replacement and concentration; the detection of chloramphenicol residues adopts organic solvent extraction, liquid-liquid extraction purification or silica gel column purification. The above extraction and purification pretreatment methods combine the chemical properties of the detected substances and remove the interfering substances in the sample matrix as much as possible. According to the chemical properties of the detected substances and the above-mentioned enzyme hydrolysis and acid hydrolysis derivation operation requirements, the extraction and purification technical route is designed as follows: after enzyme hydrolysis and acid hydrolysis and derivation, the sample solution is extracted with an organic solvent, the non-ionic detected substances are extracted in an acidic environment, the pH is adjusted, and then extracted or the lower solution is enriched and purified by a cation exchange solid phase extraction column, the two extraction solutions are combined, and the nitrogen is blown to dryness and then determined on the instrument. Different extraction methods, types and amounts of eluent are designed for comparison experiments, and the specific experimental steps and results are as follows:

[0122] Experiment 1: 3 groups of experiments, respectively, 3 2 mL pig urine samples were taken, named sample a, sample b and sample c, 4.5 mL of 0.2 mol / L ammonium acetate was added to sample b, 40 μL of β-glucuronidase / arylsulfatase was added to sample c, and after 2 h of 37℃ water bath oscillation, 100 μL of o-nitrobenzaldehyde and 1 mol / L hydrochloric acid 1.5 mL were added, and after 16 h of 37℃ water bath oscillation, sample a was adjusted to pH 7.5, then 8 mL of acetonitrile and 6 g of sodium chloride were added, sample b was adjusted to pH 7.5, then 8 mL of ethyl acetate was added, sample c was not adjusted to pH, and 8 mL of ethyl acetate was added, and vortex extraction was carried out for 30 s, and the upper liquid was taken out at a speed of 5000 r / min. The lower solution of sample c was passed through the MCX column (60 mg / 3 mL), the column was activated with 6 mL of methanol and 6 mL of water in turn, and then the sample was added, and then 6 mL of water, 6 mL of methanol and 6 mL of 5v / v% ammonia-methanol mixed solution were eluted in turn, and the 5v / v% ammonia-methanol mixed solution eluent was collected and combined with the upper liquid. The three groups of extraction solutions were dried by nitrogen blowing at 45℃ water bath, 1 mL of 5v / v% acetonitrile aqueous solution was added for constant volume, and the machine test was carried out. At the same time, the standard sample solution test was carried out for comparison.

[0123] Figure 16 The response value comparison chart of the compounds to be tested in different extraction ways of pig urine samples (left) and standard samples (right) is shown in the following figure: Figure 16 It can be seen that there is a certain difference among the three extraction methods. Sample a is adjusted to pH 7.5 and extracted with acetonitrile, and the extraction efficiency of the standard solution is acceptable, but the response value of the pig urine sample after adding standard is worse than that of the other two methods, which may be caused by the extraction of polar interfering substances leading to the enhancement of matrix effect; sample b is adjusted to pH 7.5 and extracted with ethyl acetate, and the response value of the standard solution is worse than that of the other methods, and the β-receptor agonist is more obvious, in which the response value of salbutamol is 2 orders of magnitude, and the extraction efficiency of ethyl acetate for polar target compounds is poor; sample c adopts the combination of ethyl acetate extraction and MCX column solid phase extraction, and the weakly polar compound is effectively extracted by ethyl acetate, and the cation exchange solid phase extraction column has a sufficient extraction for polar and basic target compounds. This combined extraction method has good extraction efficiency for all target compounds, so the enzyme digestion, acid hydrolysis, ethyl acetate extraction and solid phase extraction are combined for pretreatment.

[0124] Experiment two: 2 mL of pig urine sample was taken, 4.5 mL of 0.2 mol / L ammonium acetate was added, then 40 μL of β-glucuronidase / arylsulfatase was added, after 2 h of oscillation in 37 °C water bath, 100 μL of o-nitrobenzaldehyde and 1 mol / L hydrochloric acid 1.5 mL were added, after 16 h of oscillation in 37 °C water bath, 8 mL of ethyl acetate was extracted, then the solution after derivatization was passed through MCX column (60 mg / 3 mL), the column was activated with 6 mL of methanol and 6 mL of water in turn, then the sample was loaded, and then 6 mL of water, 6 mL of methanol were used for elution, 6 mL of 5 v / v% ammonia water-methanol mixed solution was used for elution, and the methanol eluent and 5 v / v% ammonia water-methanol mixed solution eluent were collected respectively, and then they were dried in 45 °C water bath under nitrogen blowing, 1 mL of 5 v / v% acetonitrile aqueous solution was added for constant volume, and then they were determined by machine. At the same time, a standard sample solution control experiment was performed.

[0125] Figure 17 The response value comparison chart of the test compounds in the methanol eluent and the ammonia water-methanol mixed solution eluent of the pig urine sample (left) and the standard sample (right) is shown in the following figure. Figure 17 It can be seen that after ethyl acetate extraction and MCX solid phase extraction enrichment, part of the test compounds will be eluted in the methanol elution step. Except that the amount of metronidazole eluted from the standard sample solution and the amount of tinidazole eluted from the pig urine sample are slightly high (accounting for 11.5% and 16.3% of the amount of ammonia water-methanol mixed solution elution respectively), the amounts of other dimetridazole, furacilin metabolites account for less than 5% of the amount of ammonia water-methanol mixed solution elution. Therefore, the solution after derivatization is first extracted with ethyl acetate, and most of the molecules in the solution system have been extracted, so the MCX column purification step still uses methanol elution to reduce the matrix effect as much as possible.

[0126] Experiment three: 2 mL of pig urine sample was taken, 4.5 mL of 0.2 mol / L ammonium acetate was added, then 40 μL of β-glucuronidase / arylsulfatase was added, after 2 h of oscillation in 37 °C water bath, 100 μL of o-nitrobenzaldehyde and 1 mol / L hydrochloric acid 1.5 mL were added, after 16 h of oscillation in 37 °C water bath, 8 mL of ethyl acetate was extracted, then the solution after derivatization was passed through MCX column (60 mg / 3 mL), the column was activated with 6 mL of methanol and 6 mL of water in turn, then the sample was loaded, and then 6 mL of water, 6 mL of methanol were used for elution, 6 mL of 5 v / v% ammonia water-methanol mixed solution was used for elution, and then 3 mL of 5 v / v% ammonia water-methanol mixed solution was used for elution, the total 5% ammonia water-methanol mixed solution eluent was collected, dried in 45 °C water bath under nitrogen blowing, 1 mL of 5 v / v% acetonitrile aqueous solution was added for constant volume, and then it was determined by machine. Compared with 6 mL of 5 v / v% ammonia water-methanol mixed solution elution in experiment two, a standard sample solution control experiment was also performed.

[0127] Figure 18The response value of the test compound was compared with the different amounts of ammonia-methanol mixed solution for pig urine sample (left) and standard sample (right). The results are shown in Figure 1. Figure 18 It can be seen that after elution with 6 mL of 5 v / v% ammonia-methanol mixed solution, and then elution with 3 mL of 5 v / v% ammonia-methanol mixed solution, less than 0.7% of the test β-receptor agonist was eluted, and other test substances were completely eluted. Therefore, the amount of ammonia-methanol mixed solution eluent in the present application is determined to be 6 mL.

[0128] Example 6

[0129] Determination of detection limit and quantification limit: The detection limit and quantification limit were determined by calculating the signal-to-noise ratio using sample spiking determination. 2 mL of pig urine sample was taken, 4.5 mL of 0.2 mol / L ammonium acetate was added, 40 μL of β-glucuronidase / arylsulfatase was added, and after 2 h of shaking in a 37°C water bath, 100 μL of o-nitrobenzaldehyde and 1.5 mL of 1 mol / L hydrochloric acid were added, and after 16 h of derivatization in a 37°C water bath, 8 mL of ethyl acetate was added, and the mixture was vortexed for 30 s, and the upper layer was transferred to a centrifuge tube, and the lower layer was transferred to a MCX column activated with 6 mL of water and 6 mL of methanol, and then eluted with 6 mL of water and 6 mL of methanol, respectively, and the column was air-dried, and then eluted with 6 mL of 5% ammonia-methanol mixed solution, and the eluent was collected in the same centrifuge tube as the ethyl acetate extract, and nitrogen blowing was performed at 45°C water bath until dryness, and 1 mL of 5 v / v% acetonitrile aqueous solution was added for ultrasonic redissolution, and at the same time, a spiked sample control experiment was performed (spiked concentration: chloramphenicol 0.1 μg / L, and the remaining 11 test compounds 0.5 μg / L).

[0130] The pig urine sample and the spiked sample were detected by liquid chromatography tandem mass spectrometry, and the qualitative ion pairs, deprotonation voltage (DP) and fragmentation voltage (CE) of the test compounds are shown in Table 1, and the characteristic ion reference chromatograms of the test compounds are shown in Figure 2, wherein, Figures 19-24 Figure 19 is the characteristic ion reference chromatogram of 2-NP-AMOZ (left) and 2-NP-AOZ (right); Figure 20 is the characteristic ion reference chromatogram of 2-NP-AHD (left) and 2-NP-SEM (right); Figure 21 is the characteristic ion reference chromatogram of clenbuterol (left) and salbutamol (right); Figure 22 is the characteristic ion reference chromatogram of ractopamine (left) and metronidazole (right); Figure 23 is the characteristic ion reference chromatogram of dimetridazole (left) and roxidazole (right); Figure 24 is the characteristic ion reference chromatogram of chlorpromazine (left) and chloramphenicol (right).

[0131] The determination results are shown in Table 2, wherein, Figures 25-36 Figure 25 ​​Chloramphenicol spiked sample chromatogram; Figure 26 Ractopamine spiked sample chromatogram; Figure 27 Salbutamol spiked sample chromatogram; Figure 28 Furazolidone metabolite spiked sample chromatogram; Figure 29 Furagilin metabolite spiked sample chromatogram; Figure 30 Alloxazine metabolite spiked sample chromatogram; Figure 31 Furaltadone metabolite spiked sample chromatogram; Figure 32 Metronidazole spiked sample chromatogram; Figure 33 Dimetridazole spiked sample chromatogram; Figure 34 Ronidazole spiked sample chromatogram; Figure 35 Chlorpromazine spiked sample chromatogram; Figure 36 Chloramphenicol spiked sample chromatogram. By Figures 25-36 The signal-to-noise ratio of each test compound in the pig urine sample can be obtained, as shown in Table 4.

[0132] Table 4 Signal-to-noise ratio of test compounds in pig urine sample

[0133] Test compound Signal to noise ratio Test compound Signal to noise ratio Clenbuterol 66.5 Furazolidone metabolite 43.8 Ractopamine 63.3 Metronidazole 15.0 Albuterol 66.0 Dimetridazole 11.1 Furazocin metabolite 18.4 Ronidazole 20.0 Furazolidone metabolite 30.7 Chlorpromazine 9.0 Allopurinol metabolite 9.0 Chloramphenicol 24.1

[0134] As can be seen from Table 4, the response intensity of chloramphenicol at an addition level of 0.1 μg / L can meet the quantitative requirements of the method, and the signal-to-noise ratio of the other 11 test compounds at an addition level of 0.5 μg / L can meet the quantitative requirements, so it is determined that in the present application, when the sample sampling amount is 2 mL, the detection limit of chloramphenicol is 0.05 μg / L, the quantification limit is 0.1 μg / L, and the detection limit of the other 11 compounds is 0.25 μg / L, and the quantification limit is 0.5 μg / L.

[0135] Example 7

[0136] Test compound standard curve, linear equation and correlation coefficient: mixed series intermediate solution and mixed series standard solution are configured:

[0137] Mixed standard intermediate solution: 20 μL of chloramphenicol standard stock solution with a concentration of 1.0 mg / mL is taken, and 100 μL to 10 mL of other concentrations of 1.0 mg / mL standard stock solution is taken into a volumetric flask, which is diluted to constant volume with methanol to prepare a mixed standard intermediate solution with a concentration of 2 μg / mL of chloramphenicol and 10 μg / mL of other compounds. Store below -20℃, and the effective period is 1 month.

[0138] Mixed internal standard intermediate solution: 20 μL of chloramphenicol-D5 standard stock solution with a concentration of 1.0 mg / mL and 100 μL of other internal standard stock solution with a concentration of 1.0 mg / mL were taken into a 10 mL volumetric flask, which was diluted to the mark with methanol to prepare a mixed internal standard intermediate solution with a concentration of 2 μg / mL of chloramphenicol-D5 and 10 μg / mL of other internal standard compounds. The solution was stored below -20 ℃ and was valid for 1 month.

[0139] Mixed standard working solution: 10 μL of the mixed standard intermediate solution was taken and 990 μL of methanol was added, which was mixed uniformly to prepare a mixed standard intermediate solution with a concentration of 20 ng / mL of chloramphenicol and 100 ng / mL of other compounds. The solution was prepared immediately before use.

[0140] Mixed internal standard working solution: 10 μL of the mixed internal standard intermediate solution was taken and 990 μL of methanol was added, which was mixed uniformly to prepare a mixed internal standard intermediate solution with a concentration of 20 ng / mL of chloramphenicol-D5 and 100 ng / mL of other internal standard compounds. The solution was prepared immediately before use.

[0141] Six 2 mL of water were taken respectively, 5 μL, 10 μL, 20 μL, 50 μL, 100 μL and 200 μL of the mixed standard working solution were added respectively, and then 50 μL of the mixed internal standard working solution was added. The series of mixed standard working solutions with a concentration of 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 1.0 ng / mL, 2.0 ng / mL and 4.0 ng / mL of chloramphenicol and 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL and 20.0 ng / mL of other 11 kinds of compounds to be tested were prepared by using the pig urine sample pretreatment method provided by the application. The series of mixed standard working solutions were detected by using liquid chromatography tandem mass spectrometry. The concentration and peak area standard curve of the compound to be tested is shown in Figures 37-48 Figure 37 is the concentration and peak area standard curve of clenbuterol; Figure 38 is the concentration and peak area standard curve of ractopamine; Figure 39 is the concentration and peak area standard curve of salbutamol; Figure 40 is the concentration and peak area standard curve of furanciline metabolite; Figure 41 is the concentration and peak area standard curve of furazolidone metabolite; Figure 42 is the concentration and peak area standard curve of furantoin metabolite; Figure 43 is the concentration and peak area standard curve of furaltadone metabolite; Figure 44 is the concentration and peak area standard curve of metronidazole; Figure 45 is the concentration and peak area standard curve of dimetridazole; Figure 46 is the concentration and peak area standard curve of lornidazole.​Figure 47 Chlorpromazine concentration and peak area standard curve; Figure 48 Chloramphenicol concentration and peak area standard curve. Linear equation and correlation coefficient are shown in Table 5.

[0142] Table 5 Linear method and correlation coefficient of standard curve of compound to be tested

[0143] Test compound Linear equation and correlation coefficient Clenbuterol [y = 0.01832x - 0.00229 (r = 0.99815, r 2 = 0.99631)] Ractopamine [y = 0.02568x - 0.00497 (r = 0.99619, r 2 = 0.99239)] Albuterol y = 0.01682x - 0.00254 (r = 0.99729, r 2 = 0.99458) Furazocin metabolite [y = 0.02508x - 0.00351 (r = 0.99797, r 2 = 0.99594)] Furazolidone metabolite [y = 0.02651x - 0.00307 (r = 0.99789, r 2 = 0.99578)] Allopurinol metabolite [y = 0.02593x - 0.00499 (r = 0.99836, r 2 = 0.99672)] Furazolidone metabolite y = 0.07957x - 0.02212 (r = 0.99700, r 2 = 0.99401) Metronidazole y = 2.97391 x + 0.08512 (r = 0.99893, r 2 = 0.99785) Dimetridazole [y = 0.20281x - 0.03414 (r = 0.99813, r 2 = 0.99627)] Ronidazole [y = 0.02303x + 0.00323 (r = 0.99841, r 2 = 0.99681)] Chlorpromazine y = 0.20905x - 0.10134 (r = 0.99766, r 2 = 0.99533) Chloramphenicol y = 0.19058x - 0.00442 (r = 0.99667, r 2 = 0.99335)

[0144] From Figures 37-48 As can be seen from Table 5, the linear equation of the present application has good correlation at a level of more than one order of magnitude in the linear range, and the correlation coefficient is more than 0.99, meeting the requirements of quantitative methodology.

[0145] Example 8

[0146] Determination of sample spiking recovery: 1-fold, 2-fold and 10-fold spiking amounts were added respectively at the method limit of quantification level, 6 parallel samples were prepared at each concentration level, 2 mL of sample was accurately weighed and placed in a polypropylene centrifuge tube 1, 50 μL of mixed internal standard intermediate solution was added, 4.5 mL of 0.2 mol / L ammonium acetate solution and 40 μL of β-glucuronidase / arylsulfatase were added, vortexed to mix, and enzymatic hydrolysis was carried out at 37°C water bath oscillation for 2 h, 1.5 mL of 1.0 mol / L hydrochloric acid solution and 100 μL of 0.1 mol / L o-nitrobenzaldehyde solution were added, vortexed to mix, and derivatization was carried out at 37°C water bath oscillation for 16 h, then 8 mL of ethyl acetate was added, vortexed for 10 s, centrifuged at 5000 r / min for 5 min, the upper liquid was taken to centrifuge tube 2, and the lower liquid was transferred to a pre-activated MCX column, 6 mL of water and 6 mL of methanol were used for washing, the column was air-dried, 6 mL of 5% ammonia-methanol mixed solution was used for elution, the ammonia-methanol mixed solution eluate was collected in centrifuge tube 2, and nitrogen blowing was carried out at 45°C water bath until dry, 1 mL of constant volume liquid was added for ultrasonic redissolution, 0.22 μm organic filter membrane was used for filtration, and then the sample was determined.

[0147] Preparation of standard curve: 6 portions of 2 mL of water were taken, mixed standard working solution was added, mixed internal standard working solution was added, and sample pretreatment was carried out, thereby a series of mixed standard working solutions were prepared, in which the concentration of chloramphenicol was 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 1.0 ng / mL, 2.0 ng / mL and 4.0 ng / mL, and the concentration of other 11 compounds to be tested was 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL and 20.0 ng / mL. The accuracy and precision of spiking recovery are shown in Table 6.

[0148] Table 6 Spiking recovery and RSD value of sample to be tested

[0149]

[0150]

[0151] From Table 6, the spiked recoveries of the present technology are between 81.0-118.7%, and the precisions are between 2.20-12.34%, which meet the methodological requirements of the quantitative method, and the quantitative method is accurate and reliable.

[0152] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the embodiments of the present application without creative labor, which belong to the protection scope of the present application.

Claims

1. A highly sensitive method for detecting multiple veterinary drug residues in pig urine, characterized in that, Includes the following steps: The pig urine sample, mixed internal standard solution, ammonium acetate buffer solution and enzymatic hydrolysis reagent were first mixed and then enzymatically hydrolyzed to obtain an enzymatic hydrolysis mixture; the enzymatic hydrolysis reagent included β-glucuronidase / arylsulfatase. The enzymatic hydrolysis mixture, acid hydrolysis reagent, and o-nitrobenzaldehyde are mixed and derivatized to obtain a derivatized mixture. The derivatized mixture was mixed with ethyl acetate for the third time, extracted, and then centrifuged to obtain the upper and lower layers. The lower layer was sequentially separated by cation exchange column chromatography using water, methanol, and an ammonia-methanol mixture as eluents. The ammonia-methanol mixture eluent was collected, and the upper layer was combined with the upper layer after drying and redissolving in acetonitrile aqueous solution to obtain the sample solution to be tested. The test sample solution was analyzed by liquid chromatography-tandem mass spectrometry to obtain the detection results of multiple veterinary drug residues; The veterinary drug residues include metabolites of nitrofurans, nitroimidazoles, β-agonists, chloramphenicol, and phenothiazines.

2. The detection method according to claim 1, characterized in that, The metabolites of the nitrofuran compounds include one or more of the following: furacilin metabolites, nitrofurantoin metabolites, furazolidone metabolites, and furazolidone metabolites; the nitroimidazole compounds include one or more of the following: metronidazole, dimetridazole, and lonidazole; the β-agonist compounds include one or more of the following: clenbuterol, salbutamol, and ractopamine; the chloramphenicol compounds include chloramphenicol; and the phenothiazine compounds include chlorpromazine.

3. The detection method according to claim 1 or 2, characterized in that, The volume ratio of the pig urine sample to the enzymatic hydrolysis reagent is 2:0.02~0.04; the volume ratio of the pig urine sample to the ammonium acetate buffer solution is 2:4~5; and the concentration of the ammonium acetate buffer solution is 0.19~0.21 mol / L. The enzymatic hydrolysis time is 2-3 hours.

4. The detection method according to claim 1, characterized in that, The acid hydrolysis reagent includes hydrochloric acid and / or formic acid; the volume ratio of the pig urine sample to the acid hydrolysis reagent is 2:1.4~1.

6.

5. The detection method according to claim 1, characterized in that, The volume ratio of the pig urine sample to the volume of o-nitrobenzaldehyde was 2:0.09~0.

11.

6. The detection method according to claim 1, 4, or 5, characterized in that, The temperature for the derivation is 36~38℃, and the time is 15~17h.

7. The detection method according to claim 1, characterized in that, The volume ratio of the pig urine sample to ethyl acetate was 2:6~10.

8. The detection method according to claim 1, characterized in that, During the cation exchange column separation process, the amount of water used is 1~1.5 BV, the amount of methanol used is 1~1.5 BV, and the amount of ammonia-methanol mixed solution used is 1.5~2 BV.

9. The detection method according to claim 1, characterized in that, The conditions for liquid chromatography-tandem mass spectrometry detection include: a C10 column. 18 The chromatographic column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.1 v / v% formic acid aqueous solution, and mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.25~0.3 mL / min; the column temperature is 35~40℃; the injection volume is 2~5 μL; The elution method is gradient elution; the gradient elution program is as follows: 0.00 min, the volume fraction of mobile phase A is 98%; 0.00~2.50 min, the volume fraction of mobile phase A decreases from 98% to 95%; 2.50~3.00 min, the volume fraction of mobile phase A decreases from 95% to 85%; 3.00~5.00 min, the volume fraction of mobile phase A decreases from 85% to 65%; 5.00~7.00 min, the volume fraction of mobile phase A decreases from 65% to 45%; 7.00~9.00 min, the volume fraction of mobile phase A increases from 45% to 95%; 9.00~11.00 min, the volume fraction of mobile phase A is 95%; 11.00~11.01 min, the volume fraction of mobile phase A increases from 95% to 98%; 11.01~14.00 min, the volume fraction of mobile phase A is 98%.

10. The detection method according to claim 1, characterized in that, The mass spectrometry detection conditions for the liquid chromatography-tandem mass spectrometry include: the ion source is an electrospray ion source; the detection mode is multiple reaction monitoring; the scanning mode is positive and negative ion scanning; the ion source parameters for positive mode scanning are: spray voltage of 5500 eV and ion source temperature of 550 °C; the ion source parameters for negative mode scanning are: spray voltage of -4500 eV and ion source temperature of 550 °C.

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