Method for determining residual quantity of penicillin and tetracycline antibiotics in citrus products

Abstract

The invention discloses a method for determining the residual quantity of penicillin and tetracycline antibiotics in a citrus product. The method comprises the following steps: S1, preparing a sample; s2, respectively extracting penicillin and tetracyclines in the sample; s3, a liquid chromatography-tandem mass spectrometer is adopted for determination, the liquid chromatography-tandem mass spectrometer comprises chromatographic conditions and mass spectrometry conditions, and the chromatographic conditions are as follows: a mobile phase A is a 0.1% formic acid aqueous solution, a mobile phase B is a methanol-acetonitrile solution, and the methanol-acetonitrile solution is prepared by mixing methanol and acetonitrile according to the volume ratio of 2: 1. Compared with the prior art, the method has the advantages that a sample pretreatment method is changed, measurement conditions are adjusted, an antibiotic residue calculation formula is designed, and the recovery rate and precision of the penicillin and tetracycline antibiotics in the citrus product obtained through measurement are high.

Description

Technical Field

[0001] The invention relates to the technical field of fruit detection, and in particular to a method for determining the residual amounts of penicillin and tetracycline antibiotics in citrus products. Background Art

[0002] At present, there are more than 140 countries and regions in the world that produce citrus. However, quarantine diseases such as citrus Huanglongbing, ulcer disease, and anthrax seriously threaten the development of the world's citrus industry. Among them, citrus Huanglongbing is caused by infection with Bacillus asiatica and is widely spread by citrus psyllids. It is a disease that is extremely devastating to citrus and is called the "cancer" of citrus. In order to strictly control citrus Huanglongbing and reduce economic losses, in recent years, a method of preventing and controlling citrus Huanglongbing by injecting antibiotics such as penicillin, tetracycline and aminosugar antibiotics into the trunk has emerged in the main citrus producing areas. Among them, ampicillin, oxytetracycline, tetracycline and doxycycline have obvious prevention and control effects compared with other antibiotics, and are the preferred agents for trunk injection to treat Huanglongbing.

[0003] Although "trunk injection of antibiotics" can effectively prevent and treat citrus Huanglongbing, the antibiotics injected into the trunk remain in the fruit trees and citrus fruits in the form of original drugs or metabolites after being absorbed and metabolized by the fruit trees. Irrational, irregular, or even blind use of antibiotics will lead to serious excess of antibiotic residues in fruits, endangering the health of consumers. Therefore, in order to better supervise and manage the quality and safety of imported and exported citrus products to protect the physical and mental health of the people, a standard method for determining the residual amount of tetracycline antibiotics in citrus and its juice was developed.

[0004] However, antibiotic pesticides are highly active, and citrus products are a complex matrix. There is a serious matrix effect during the detection process, which makes it difficult to sensitively and accurately obtain the antibiotic content in citrus products. Overcoming the matrix effect and obtaining target compounds from citrus products quickly, with high quality and in large quantities are the detection difficulties in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products, aiming to overcome the matrix effect of citrus products and obtain a detection method with high sensitivity and good accuracy, which is used to detect the residual amount of penicillin and tetracycline antibiotics in citrus products on the market and citrus products to be exported, so as to achieve a sound antibiotic monitoring system in citrus products and protect the health of consumers.

[0006] To achieve the above object, the present invention provides a method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products, the method comprising:

[0007] S1, sample preparation;

[0008] S2, extracting penicillins and tetracyclines from the samples respectively;

[0009] S3, using liquid chromatography-tandem mass spectrometry for determination, the liquid chromatography-tandem mass spectrometry includes chromatographic conditions and mass spectrometry conditions, the chromatographic conditions are as follows:

[0010] Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol acetonitrile solution, wherein the methanol acetonitrile solution is prepared by mixing methanol and acetonitrile in a volume ratio of 2:1;

[0011] Gradient elution program:

[0012] At 0 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%;

[0013] At 1.0 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%;

[0014] At 4.5 min, the flow rate was 0.25 mL / min, mobile phase A was 10%, and mobile phase B was 90%;

[0015] At 6.5 min, the flow rate was 0.25 mL / min, mobile phase A was 10%, and mobile phase B was 90%;

[0016] At 6.6 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%;

[0017] At 8 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%.

[0018] Preferably, in the above technical solution, the extracting tetracycline antibiotics from the sample specifically comprises:

[0019] First, a preset amount of tetracycline extractant is added to the sample, vortexed and shaken for extraction, and centrifuged to obtain a supernatant;

[0020] Reactivate the PSA column, take the supernatant and pass it through the PSA column, rinse the PSA column with water and discard the effluent, elute the PSA column with 1% ammonia water and take the eluent, add the diluent to the eluent, mix well and filter with a filter membrane to obtain an extract containing tetracycline antibiotics.

[0021] Preferably, in the above technical solution, the purification filler in the PSA column is a PSA adsorbent.

[0022] According to the above technical scheme, the present application adopts a PSA column to fill the PSA adsorbent (powder) on the column. The PSA adsorbent selectively adsorbs penicillin and tetracycline targets through the ion exchange function. The impurities flow out with the water elution, and then the target compound is eluted by the eluent to achieve the purpose of purification.

[0023] Preferably, in the above technical solution, the preset amount of extractant is 0.1 mL 1% EDTA-2Na and 20 mL 0.5% formic acid acetonitrile.

[0024] Preferably, in the above technical solution, the diluent is 1% formic acid in a 1:1 ratio of acetonitrile to water.

[0025] Preferably, in the above technical solution, the filtering device is an organic phase filter membrane.

[0026] Preferably, in the above technical solution, the extracting penicillin from the sample specifically comprises:

[0027] Add acetonitrile to the sample, vortex mix, shake and extract, add buffer solution and shake and extract, centrifuge to obtain supernatant, take the supernatant, add water to mix, and filter with a filter device.

[0028] Preferably, in the above technical solution, the buffer solution is 0.2% acetic acid aqueous solution.

[0029] Preferably, in the above technical solution, the filtering device is a double-layer 0.22 μm hydrophobic PTFE needle filter.

[0030] Preferably, in the above technical solution, the compound parameters in the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometer are as follows:

[0031]

[0032]

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The test object is the antibiotic pesticide residues in citrus products, which are highly active and more difficult to detect than traditional pesticides;

[0035] (2) Since the target substance is lost during the pretreatment process and the matrix interference during the injection process may cause the content to be too high or too low, the present invention adds a standard solution of known concentration to the blank sample as a control. Because the interference of the standard reference substance during the pretreatment process and the injection process is consistent with that of the sample to be tested, the obtained results are more reliable than the traditional detection using the solvent as the standard solution for control. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a recovery rate diagram of Example 1 of the present invention and Comparative Examples 1-3;

[0037] Figure 2 is a graph of elution efficiency of different eluents in Comparative Example 4 of the present invention;

[0038] Figure 3 It is a graph of the elution efficiency of different eluents in Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0039] 1. Preparation of standard stock solution:

[0040] (1) 200 mg / L tetracycline, doxycycline (doxycycline), and oxytetracycline standard stock solutions: Accurately weigh 10 mg of each tetracycline, doxycycline, and oxytetracycline standard solution into a 50 mL volumetric flask, dissolve it in methanol and make up to the mark, transfer it to a brown reagent bottle, and store it at -18°C away from light. The shelf life is 12 months.

[0041] (2) 200 mg / L ampicillin and benzylpenicillin standard stock solutions: accurately weigh 10 mg of ampicillin and benzylpenicillin standard substances respectively into a 50 mL volumetric flask, dissolve with 20 mL of water, add acetonitrile to the mark, and dispense into brown vials (5-10 mL per vial). Immediately store at -18°C away from light. The shelf life is 3 months and repeated freezing and thawing is prohibited.

[0042] 2. Mixed standard working solution:

[0043] Accurately measure 0.5mL of each standard stock solution into a 10mL volumetric flask, add 3:2 acetonitrile and water to dilute to the mark to prepare a 10mg / L standard mixed working solution, dispense into test tubes or reagent bottles, and store at -18℃ away from light. The shelf life is 1 month and it must not be repeatedly frozen and thawed.

[0044] 3. Instruments used: 1290 Infinity II liquid chromatograph, Eclipse Plus C18 chromatographic column (2.1×100 mm, 1.8 μm) (Agilent, USA); Qtrap 4500 mass spectrometer (AB SCIEX, USA); Feige DL-5-B centrifuge (Shanghai Anting Scientific Instrument Factory); VOTEX GENIE2 vortex mixer (Scientific Industries, USA); GM200 knife-type mixer-grinder (Retsch, Germany); PSA column (self-made), wherein the preparation method of PSA column is as follows:

[0045] (1) Recovering the used commercial solid phase extraction column, removing all the original fillers, taking out the gasket, cleaning the gasket and solid phase extraction column and drying them;

[0046] (2) Weigh 200 mg of PSA adsorbent, load it into a small column and compact it to obtain a PSA small column.

[0047] Example 1

[0048] A method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products, the method steps are as follows:

[0049] S1, sample preparation: according to the "Sampling Method for Fresh Fruits and Vegetables" (GB / T 8855-2008), citrus fruit samples were collected, the samples were shredded, chopped, mixed, put into a knife-type mixer grinder to crush, and prepared into samples to be tested, which were stored in a -18.0°C refrigerator for later use;

[0050] S2, comprising extracting tetracyclines and penicillins from the sample

[0051] S21, the steps of extracting tetracyclines from the sample are as follows:

[0052] Accurately weigh 5 g of sample and place it in a polypropylene centrifuge tube. Add 0.1 mL of 1% EDTA-2Na (w / v) aqueous solution, add 20 mL of 0.5% formic acid acetonitrile, vortex mix for 1 min, shake and extract for 20 min, centrifuge at 4000 r / min for 5 min, and take the supernatant.

[0053] Take 1 mL of acetonitrile to activate the PSA column, take 2 mL of the supernatant to pass through the PSA column, rinse the PSA column with 2 mL of water and discard the effluent, rinse the PSA column with 2 mL of 1% ammonia water and take the eluent, add 2 mL of diluent (1% formic acid in 1:1 acetonitrile water) to the eluent, mix well and filter the organic phase with a membrane filter to obtain a sample solution containing tetracyclines;

[0054] Prepare the standard sample solution, the preparation method is as follows:

[0055] Weigh 7 portions of 5.00 g of blank samples respectively into 50 mL plastic centrifuge tubes with stoppers, add appropriate amounts of mixed standard solution respectively, so that the sample spike contents are 0, 0.005, 0.010, 0.020, 0.050, 0.100, and 0.200 mg / kg, and then proceed as in step S21;

[0056] S22, the steps of extracting penicillin in the sample are as follows:

[0057] Accurately weigh 5 g of sample, add 15 mL of acetonitrile, vortex mix for 1 min, shake and extract for 25 min, add 5 mL of buffer (0.2% acetic acid aqueous solution), shake and extract for 5 min, centrifuge at 4000 r / min for 5 min, take the supernatant, take 1 mL of the supernatant, add 1 mL of water and mix well, filter with a filtration device (double-layer 0.22 μm hydrophobic PTFE needle filter) and bottle to obtain a sample solution containing penicillin;

[0058] Prepare the standard sample solution, the preparation method is as follows:

[0059] Weigh 7 portions of 5.00 g of blank samples respectively into 50 mL plastic centrifuge tubes with stoppers, add appropriate amounts of mixed standard solution respectively, so that the sample spike contents are 0, 0.005, 0.010, 0.020, 0.050, 0.100, and 0.200 mg / kg, and then proceed as in step S22;

[0060] S3, using liquid chromatography-tandem mass spectrometry for determination, the liquid chromatography-tandem mass spectrometry includes chromatographic conditions and mass spectrometry conditions, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry include chromatographic column, column temperature, mobile phase, injection volume and gradient elution program, and the specific settings are as follows:

[0061] Chromatographic column: ZORBAX Eclipse Plus C 18 , 2.1×100mm, 1.8μm;

[0062] Column temperature: 40°C;

[0063] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol acetonitrile solution, wherein the methanol acetonitrile solution is prepared by mixing methanol and acetonitrile in a volume ratio of 2:1;

[0064] Injection volume: 5 μL;

[0065] The gradient elution program is described in Table 1 below.

[0066] Table 1 Liquid chromatography gradient elution program

[0067]

[0068]

[0069] The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometer include ion source, scanning mode, detection mode, mass spectrometry conditions and mass spectrometry parameters of 12 target compounds, which are as follows:

[0070] Ion source: electrospray ionization (ESI)

[0071] Scanning mode: positive ion scanning

[0072] Detection method: Multiple reaction monitoring (MRM)

[0073] Mass spectrometry conditions, Curtain Gas (CUR): 30; Collision Gas (CAD): medium; IonsprayVoltage (IS): 4500V; Temperature (TEM): 500°C; Ion Soerce Gas1: 55°C; Ion SoerceGas2: 55°C.

[0074] The mass spectrometry parameters of the 12 target compounds are shown in Table 2.

[0075] Table 2 Compound parameters of 12 antibiotic target compounds

[0076]

[0077]

[0078] Note: EP, EntrancePotential; DP, DeclusteringPotential; CE, CollisionEnergy; CXP: CollisionCellExitPotential

[0079] The peak areas of the standard sample solutions are measured by liquid chromatography-tandem mass spectrometry, and the penicillin standard curve and the tetracycline antibiotic standard curve are drawn respectively with the concentration as the horizontal coordinate and the peak area as the vertical coordinate. The peak areas of the sample solution containing penicillin are measured by liquid chromatography-tandem mass spectrometry, and the penicillin standard curve is substituted to obtain the penicillin concentration in the sample solution; the peak areas of the sample solution containing tetracycline antibiotics are measured by liquid chromatography-tandem mass spectrometry, and the tetracycline antibiotic standard curve is substituted to obtain the tetracycline concentration in the sample solution.

[0080] Optimization of mass spectrometry conditions

[0081] In order to obtain the best mass spectrometry parameters for 4 tetracyclines and 8 penicillins, single standard solutions of 12 compounds with a concentration of 1.0 μg / mL were prepared and injected into the mass spectrometer in sequence using the needle pump mode for analysis optimization.

[0082] First, search for parent ions in Q1 mode. The results show that all seven compounds have [M+H] +The peaks are all the optimal parent ion peaks. Then, the daughter ion analysis is performed in the product ion (MS2) mode. The parent ion is broken by gradually increasing the collision energy, and two fragment ions with stronger responses are selected from the fragments as the qualitative ions, and the qualitative ion with the highest response is selected as the quantitative ion. Finally, the declustering voltage (DP), collision energy (CE) and exit voltage (CXP) are optimized one by one in the MRM mode to obtain the optimal compound ion pair parameters.

[0083] Matrix Effects

[0084] Matrix effect (ME) refers to the phenomenon that the co-eluted components in the sample matrix affect the ionization of the target compound during the instrument detection process, resulting in a weakening or enhancement of the detection signal of the target compound. In this study, 6 negative samples were weighed for extraction and purification. Before bottling, penicillin and tetracycline antibiotic standard solutions were added to make the final concentrations of 0.5, 1.0, 2.0, 5.0, 10, and 20 μg / L, respectively. Then, a series of standard solutions of the same concentration were prepared with the constant volume solution. After injection, the matrix curve and solvent curve were fitted with the concentration as the x-axis and the peak area as the y-axis. The linear correlation coefficient R of the standard curve of each compound was required to be ≥0.9950, and then the matrix effect ME = (BA) / A×100% (A is the slope of the linear equation of the solvent standard curve, and B is the slope of the linear equation of the matrix standard curve). The results are shown in Table 3.

[0085] Table 3 Matrix effect

[0086]

[0087] The results are shown in Table 3. The matrix effect of tetracycline antibiotics extracted from citrus by the above method ranges from 71% to 80%. This indicates that the above method is used to extract four tetracycline antibiotics, which produces a more obvious matrix enhancement effect in citrus samples. This means that the results obtained by using solvent curve quantification of tetracycline antibiotics will be too high. It is not suitable to use solvent curve for quantification of tetracycline antibiotics in citrus products. In order to ensure that the accuracy of the detection meets the requirements of verification of the coherent standard method, it is necessary to use sample spike curve for quantification.

[0088] The results are shown in Table 3. The matrix effect of penicillins extracted from citrus by the above method ranges from -8.3% to -70.7%. This indicates that the above method is used to extract 8 penicillins, which produces a more obvious matrix inhibition effect in citrus samples. This means that the results obtained by using solvent curve quantification for penicillins will be low. It is not suitable to use solvent curve for quantification of penicillins in citrus products. In order to ensure that the accuracy of the detection meets the requirements of verification of the coherent standard method, it is necessary to use sample spike curve for quantification.

[0089] Linear range, recovery and precision

[0090] In this study, 0.010, 0.020, and 0.100 mg / kg mixed standard solutions were added to negative citrus samples, respectively. Six parallel runs were set for each added concentration to conduct spike recovery experiments. At the same time, standard samples were prepared according to the preparation of standard sample solutions and used for quantification. The results are shown in Table 4.

[0091] Table 4 Linear equation, recovery and precision of spike test

[0092]

[0093]

[0094] The results are shown in Table 4. Within the linear range of 0.005-0.200 mg / kg, the linear correlation of each item was good, and the correlation coefficient R was greater than or equal to 0.9984; the spiked recoveries at low, medium and high concentration levels ranged from 91.2% to 120%, and the relative standard deviation was 1.7% to 11.0%.

[0095] Comparative Example 1

[0096] The process is the same as steps S1 and S3 of Example 1, except that the purification filler of the PSA column in S21 is different.

[0097] Accurately weigh 5 g of sample and place it in a polypropylene centrifuge tube. Add 20 mL of 0.5% formic acid acetonitrile, vortex mix for 1 min, shake and extract for 20 min, centrifuge at 4000 r / min for 5 min, and take the supernatant.

[0098] Take 1mL acetonitrile to activate the PSA column (the purification filler is C 18 ), take 2mL of the supernatant and pass it through a PSA column, rinse the PSA column with 2mL of water and discard the effluent, elute the PSA column with 2mL of 1% ammonia water and take the eluent, add 2mL of 1% formic acid acetonitrile to the eluent, mix well and filter the organic phase with a membrane filter to obtain an extract containing tetracyclines.

[0099] Comparative Example 2

[0100] The process is the same as steps S1 and S3 of Example 1, except that the purification filler of the PSA column in S21 is different.

[0101] Accurately weigh 5 g of sample and place it in a polypropylene centrifuge tube. Add 20 mL of 0.5% formic acid acetonitrile, vortex mix for 1 min, shake and extract for 20 min, centrifuge at 4000 r / min for 5 min, and take the supernatant.

[0102] Take 1 mL of acetonitrile to activate the PSA column (the purification filler is GCB), take 2 mL of the supernatant to pass through the PSA column, rinse the PSA column with 2 mL of water and discard the effluent, elute the PSA column with 2 mL of 1% ammonia water and take the eluent, add 2 mL of 1% formic acid acetonitrile to the eluent, mix well and filter the organic phase with a membrane filter to obtain an extract containing tetracyclines.

[0103] Comparative Example 3

[0104] The process is the same as steps S1 and S3 of Example 1, except that the purification filler of the PSA column in S21 is different.

[0105] Accurately weigh 5 g of sample and place it in a polypropylene centrifuge tube. Add 20 mL of 0.5% formic acid acetonitrile, vortex mix for 1 min, shake and extract for 20 min, centrifuge at 4000 r / min for 5 min, and take the supernatant.

[0106] Take 1 mL of acetonitrile to activate the PSA column (the purification filler is Al2O3), take 2 mL of the supernatant to pass through the PSA column, rinse the PSA column with 2 mL of water and discard the effluent, elute the PSA column with 2 mL of 1% ammonia water and take the eluent, add 2 mL of 1% formic acid acetonitrile to the eluent, mix well and filter the organic phase with a membrane filter to obtain an extract containing tetracyclines.

[0107] The recovery rates of tetracycline antibiotics in Example 1 and Comparative Examples 1-3 are shown in Tables 5 and Figure 1 The standard deviation is shown in Table 6.

[0108] Table 5 Recovery rates of tetracycline antibiotics in Example 1 and Comparative Examples 1-3

[0109] filler Doxycycline tetracycline Oxytetracycline Chlortetracycline Comparison 100 100 100 100 <![CDATA[C 18 ]]> 107.9 102.5 99.4 105.4 GCB 26.8 49.9 25.0 26.1 <![CDATA[Al2O3]]> 20.2 17.3 17.5 20.6 PSA 1.6 1.2 1.4 2.5

[0110] The results are shown in Table 5 and Figure 1 As shown, C 18The filler has no adsorption effect on tetracycline compounds in acetonitrile, while GCB, Al2O3 and PSA all have different degrees of adsorption, among which PSA has the highest adsorption rate (100-recovery rate), 98.4% of doxycycline, 98.8% of tetracycline, 98.6% of oxytetracycline and 97.5% of chlortetracycline are adsorbed by PSA filler. Therefore, PSA filler can be used as an adsorbent for tetracycline compounds.

[0111] Comparative Example 4

[0112] Tests for screening eluate components:

[0113] Elution method 1: Use 2 mL of 5% ammonia and acetonitrile to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 2 mL of 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0114] Elution method 2: Use 2 mL of 5% ammonia methanol to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 2 mL of 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0115] Elution method 3: Use 2 mL of 5% ammonia water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 2 mL of 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0116] The elution effects of the above elution methods 1-3 are as follows: Figure 2 As shown, from Figure 2 It can be seen that the elution effect of 5% ammonia acetonitrile is the worst, followed by 5% ammonia methanol, and 5% ammonia has the best elution effect on tetracycline antibiotics, with elution efficiency exceeding 70%. Therefore, using ammonia alone is the best way to extract tetracycline antibiotics from samples.

[0117] Comparative Example 5

[0118] To further optimize the concentration of ammonia water in the eluent, the following eluent concentration screening test was performed:

[0119] Elution method 1: Use 2 mL of pure water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 4 mL of 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0120] Elution method 2: Use 2 mL of 1% ammonia water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 4 mL of 1% formic acid in a 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0121] Elution method 3: Use 2 mL of 2% ammonia water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 4 mL of 2% formic acid in a 1:1 ratio of acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0122] Elution method 4: Use 2 mL of 5% ammonia water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 4 mL of 5% formic acid in a 1:1 acetonitrile water solution to the eluent, mix well, bottle and measure on the machine.

[0123] Elution method 5: Use 2 mL of 10% ammonia water to elute the PSA column adsorbed with an equal amount of the target substance to obtain an eluent, add 4 mL of 10% formic acid in a 1:1 acetonitrile water to the eluent, mix well, bottle and measure on the machine.

[0124] The elution effects of the above elution methods 1-5 are as follows: Figure 3 As shown, from Figure 3 It can be seen that the elution effect with pure water is poor, and there is no significant difference in the elution effect of doxycycline and oxytetracycline with 1% ammonia, 2% ammonia, 5% ammonia and 10% ammonia, but the elution recovery of tetracycline and chlortetracycline gradually decreases with the increase of ammonia content. Therefore, 1% ammonia is used as an eluent to obtain the best elution effect. At the same time, in order to maintain the stability of the target in the solvent, the present invention is rapidly added with 1%-10% formic acid 1: 1 acetonitrile water for rapid neutralization after eluting the target with a low-content ammonia solution, thereby ensuring that the target compound has a higher and more stable recovery rate.

[0125] The present invention can be implemented in various ways and is not limited to the embodiments described. A person skilled in the art can understand that the present invention can be implemented in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary and are not intended to limit the present invention.

Claims

1. A method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products, characterized in that: The method comprises: S1, sample preparation; S2, extracting penicillins and tetracyclines from the samples respectively; S3, using liquid chromatography-tandem mass spectrometry for determination, the liquid chromatography-tandem mass spectrometry includes chromatographic conditions and mass spectrometry conditions, the chromatographic conditions are as follows: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol acetonitrile solution, wherein the methanol acetonitrile solution is prepared by mixing methanol and acetonitrile in a volume ratio of 2:1; Gradient elution program: At 0 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%; At 1.0 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%; At 4.5 min, the flow rate was 0.25 mL / min, mobile phase A was 10%, and mobile phase B was 90%; At 6.5 min, the flow rate was 0.25 mL / min, mobile phase A was 10%, and mobile phase B was 90%; At 6.6 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%; At 8 min, the flow rate was 0.25 mL / min, mobile phase A was 90%, and mobile phase B was 10%.

2. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 1, characterized in that: The extracting of tetracycline antibiotics from the sample specifically comprises: First, a preset amount of tetracycline extractant is added to the sample, vortexed and shaken for extraction, and centrifuged to obtain a supernatant; Reactivate the PSA column, take the supernatant and pass it through the PSA column, rinse the PSA column with water and discard the effluent, elute the PSA column with 1% ammonia water and take the eluent, add the diluent to the eluent, mix well and filter with a filter membrane to obtain an extract containing tetracycline antibiotics.

3. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 2, characterized in that: The purification filler in the PSA column is a PSA adsorbent.

4. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 2, characterized in that: The preset amount of extractant is 0.1 mL 1% EDTA-2Na and 20 mL 0.5% formic acid acetonitrile.

5. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 2, characterized in that: The diluent is 1% formic acid in 1:1 acetonitrile water.

6. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 2, characterized in that: The filter membrane is an organic phase filter membrane.

7. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 1, characterized in that: The extracting of penicillin from the sample specifically comprises: Add acetonitrile to the sample, vortex mix, shake and extract, add buffer solution and shake and extract, centrifuge to obtain supernatant, take the supernatant, add water to mix, and filter with a filter device.

8. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 7, characterized in that: The buffer solution is 0.2% acetic acid aqueous solution.

9. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 7, characterized in that: The filtering device is a double-layer 0.22 μm hydrophobic PTFE needle filter.

10. The method for determining the residual amount of penicillin and tetracycline antibiotics in citrus products according to claim 1, characterized in that: The compound parameters in the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometer are as follows:

Images