Method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic mushroom dregs

Abstract

The invention belongs to the technical field of solid waste detection, and discloses a method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C. The method comprises the following steps: (1) preparing an antibiotic mushroom dreg sample into a test solution by using an organic phase extracting agent and a water phase extracting agent in sequence; (2) detecting the test solution by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method to obtain a detection result of each component; wherein the chromatographic conditions are as follows: a chromatographic column is a C18 column; the mobile phase comprises a weak polar phase and a strong polar phase, the weak polar phase is selected from methanol or acetonitrile, and the strong polar phase is selected from a formic acid aqueous solution, an acetic acid aqueous solution, formic acid-ammonium formate or acetic acid-ammonium acetate solution. The method has the advantages of high accuracy, high sensitivity and strong specificity, can quickly and accurately detect the content of various antibiotics in the antibiotic mushroom dregs, and provides a detection method basis for disposal and utilization and safety risk evaluation of the antibiotic mushroom dregs.

Description

Technical Field

[0001] The invention belongs to the technical field of solid waste detection, and in particular relates to a method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residues. Background Art

[0002] my country is a major producer and user of antibiotics, among which β-lactam antibiotics and macrolide antibiotics are the most widely used. Such antibiotics produced by microbial fermentation will produce a large amount of antibiotic residues, which contain high concentrations of antibiotic residues. If improperly disposed of, it may lead to potential risks of environmental bacterial resistance and pose a potential threat to human public health. Therefore, establishing detection and analysis methods for such substances is crucial to assess their potential resistance risks to bacteria and other microorganisms in the ecological environment.

[0003] Regarding the detection of such antibiotics, common detection areas include water, aquatic products, soil, feed and food. The current national standards GB / T 22975-2008 "Determination of amoxicillin, ampicillin, piperacillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin and dicloxacillin residues in milk and milk powder by liquid chromatography-tandem mass spectrometry", GB 29684-2013 "Determination of erythromycin residues in aquatic products by liquid chromatography-tandem mass spectrometry", local standards DB65 / T4370-2021 "Determination of erythromycin and penicillin in water quality by liquid chromatography-triple quadrupole mass spectrometry", DB22 / T2695-2017 "Determination of lincomycin and erythromycin in feed by liquid chromatography-mass spectrometry / mass spectrometry", group standard T / PIAC00001-2021 "Detection method of penicillin in antibiotic bacterial residues and organic fertilizer base materials, crops and environmental media" Detection methods such as T / PIAC00002-2021 "Detection Method for Cephalosporins in Antibiotic Bacterial Residues and Organic Fertilizer Bases, Crops and Environmental Media", T / PIAC00003-2021 "Detection Method for Erythromycin in Antibiotic Bacterial Residues and Organic Fertilizer Bases, Crops and Environmental Media", and Announcement No. 316-5-2020 of the Ministry of Agriculture and Rural Affairs "Determination of 17 Cephalosporins in Feed by Liquid Chromatography-Tandem Mass Spectrometry" are related to the detection items (erythromycin, penicillin, cephalosporin) of the present invention.

[0004] However, the scope of application of the above national standards, local standards, inspection and quarantine standards, and methods announced by the Ministry of Agriculture and Rural Affairs is limited and does not include the field of solid waste, especially antibiotic residue matrix samples. Although the scope of application of the above group standards includes the field of solid waste, the detection method for antibiotic residue in the group standards is liquid chromatography, which has limited sensitivity and is easily interfered by the sample matrix. In addition, the sample detection time is long and the efficiency is low, which cannot meet the needs of current environmental risk assessment.

[0005] At present, there is no report on the simultaneous determination method of erythromycin A, penicillin G and cephalosporin C in microbial fermentation pharmaceutical residues, which will not be able to guarantee the impact on environmental safety during the disposal and utilization of antibiotic residues. Summary of the invention

[0006] In view of this, in order to solve the problems existing in the prior art, the present invention provides a rapid, accurate and highly sensitive detection method for simultaneously determining multiple antibiotics in antibiotic bacterial residue.

[0007] In order to achieve the above object, the object of the present invention is to provide a method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residues, using the following technical scheme:

[0008] A method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residues comprises the following steps:

[0009] (1) using an organic phase extractant and an aqueous phase extractant in sequence to prepare a test solution from an antibiotic bacterial residue sample for later use;

[0010] (2) using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the test solution and obtain qualitative or quantitative detection results of each component;

[0011] Wherein, the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase includes a weak polar phase and a strong polar phase, the weak polar phase is selected from methanol or acetonitrile, and the strong polar phase is selected from formic acid aqueous solution, acetic acid aqueous solution, formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content is 0.02% to 0.2% by volume, and the ammonium salt concentration is 0.01M to 0.05M.

[0012] It is worth noting that erythromycin A belongs to the macrolide antibiotics, penicillin G and cephalosporin C belong to the β-lactam antibiotics, and the properties of the two classes of compounds are significantly different. In the existing detection methods, whether it is the commonly used liquid chromatography or liquid chromatography-triple quadrupole tandem mass spectrometry, different sample pretreatment methods are generally used for classification and detection, and the detection efficiency is low. The matrix of antibiotic residue samples is complex. After only extraction with an extractant, it is directly tested in a liquid chromatograph (ultraviolet detector). The chromatogram obtained has a high baseline, serious impurity interference, and is prone to false positive results. Solid phase extraction columns are required for purification, which not only increases the sample pretreatment cost, but also affects the recovery rate of the target to a certain extent. In view of the different solubilities of three antibiotics, the present invention first uses an organic phase extractant to extract erythromycin A, and then adds an aqueous phase extractant to extract penicillin G and cephalosporin C. Through two-step extraction, the pretreatment methods of the three antibiotic residue samples are unified, and liquid chromatography-triple quadrupole tandem mass spectrometry is used for detection, so that the three antibiotics are simultaneously determined. The detection result has strong specificity, high accuracy and high sensitivity, and the detection method is simple and efficient.

[0013] Furthermore, the antibiotic residue is the culture medium residue remaining after microbial fermentation production, specifically one or more of the residues of erythromycin, penicillin G, and cephalosporin.

[0014] Furthermore, the step (1) specifically includes:

[0015] (1.1) Weigh 0.5-1.0 g of sample, add 5-10 mL of organic phase extractant A, vortex mix, and perform ultrasonic extraction for 10 min. Then, add aqueous phase extractant B and make up to 25-50 mL. Vortex mix, and perform ultrasonic extraction for another 10 min.

[0016] (1.2) Centrifuge the extract at 4000 rpm for 10 min, take the supernatant and filter it through a microporous membrane to obtain the extract.

[0017] Furthermore, in the step (1.1), the organic phase extractant A is methanol or acetonitrile, and the aqueous phase extractant B is water or 0.1M ammonium acetate solution; in the step (1.2), the microporous filter membrane is 0.22 μm or 0.45 μm.

[0018] Furthermore, in step (2), the particle size of the chromatographic filler is 1.7 μm; the mobile phase uses methanol as a weak polar phase and 0.1% formic acid aqueous solution as a strong polar phase.

[0019] Furthermore, the chromatographic conditions also include a gradient elution program of:

[0020] The duration of the initial stage is 1.0 to 5.0 min, and the volume ratio of methanol is 5% to 20%;

[0021] The duration of the intermediate stage is 3.0 to 10.0 min, and the volume ratio of methanol is 60% to 90%;

[0022] The duration of the final stage is 2.0 to 5.0 minutes, and the volume ratio of methanol is 5% to 20%; and,

[0023] The flow rate is 0.2-0.4 mL / min, the column temperature is 25-40 °C, and the injection volume is 1-15 μL.

[0024] Furthermore, the gradient elution procedure is:

[0025] 0.00-3.00min, 10% methanol, 3.01-7.00min, 90% methanol, 7.01-10.00min, 10% methanol; and, the flow rate was 0.25mL / min, the column temperature was 35°C, and the injection volume was 2μL.

[0026] Furthermore, in step (2), the mass spectrometry conditions include:

[0027] The ionization mode is electrospray positive ion mode; the ion source temperature is 150-200°C; the desolvation gas temperature is 300-600°C; the desolvation gas flow rate is 400-1000L / Hr; the spray voltage is 2.8-4.0kv; the cone voltage is 10-40V; the collision voltage is 5-40V; and the scanning mode is multiple reaction monitoring mode.

[0028] Furthermore, the mass spectrometry conditions are: ion source temperature is 150°C; desolvation gas temperature is 450°C; desolvation gas flow rate is 900L / Hr; spray voltage is 4.0kv, and the multiple reaction monitoring conditions are shown in Table 1.

[0029] Table 1 Multiple reaction detection conditions for three antibiotics

[0030]

[0031] Furthermore, in step (2), the target component content is calculated using a standard curve method.

[0032] Compared with the prior art, the present invention aims at the different solubility of the three antibiotics, firstly extracts erythromycin A with an organic phase extractant, then adds an aqueous phase extractant to extract penicillin G and cephalosporin C, and unifies the pretreatment methods of the three antibiotic slag samples through two-step extraction, and successfully separates the three antibiotics in the antibiotic slag through the optimization of chromatographic conditions, and realizes the simultaneous determination of the three antibiotics in the antibiotic slag. In addition, the present invention adopts liquid chromatography-triple quadrupole mass spectrometry for detection, which can effectively improve the sensitivity of detection. In this method, the detection limits of erythromycin A, penicillin G and cephalosporin C can reach 0.02mg / kg, 0.07mg / kg and 0.07mg / kg respectively. When the spiked concentration of the three antibiotics is 1.0mg / kg, the recovery rate is between 70% and 120%. The method established by the present invention has high accuracy, high sensitivity and strong specificity, and can quickly and effectively detect the content of the three antibiotic substances in the antibiotic slag, providing a detection method basis for the disposal and utilization of antibiotic slag and safety risk assessment. In particular, in the preferred embodiment of the present invention, the pretreatment method of antibiotic bacterial residue only requires the addition of an extractant, ultrasonic extraction, centrifugation and filtration, without the need for solid phase extraction and repeated solvent extraction. The operation is simple, fast, low-cost and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1 This is the total ion current diagram of the mixed standard solution of three antibiotics in Example 1 of the present invention, wherein GK represents the penicillin G component; EA represents the erythromycin A component, and CPC represents the cephalosporin C component. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0037] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0038] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0039] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0040] The invention discloses a method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residues, and belongs to the technical field of solid waste detection. The invention adopts liquid chromatography-triple quadrupole mass spectrometry, and comprises the following steps: (1) using an organic phase extractant and an aqueous phase extractant in sequence to prepare a test solution from an antibiotic bacterial residue sample; (2) using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the test solution to obtain the detection results of each component; wherein the chromatographic conditions are: the chromatographic column is a C18 column; the mobile phase comprises a weak polar phase and a strong polar phase, the weak polar phase is selected from methanol or acetonitrile, and the strong polar phase is selected from formic acid aqueous solution, acetic acid aqueous solution, formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content of the strong polar phase is 0.02% to 0.2% by volume, and the ammonium salt concentration is 0.01M to 0.05M. The invention has high accuracy, high sensitivity and strong specificity, can quickly and accurately detect the content of multiple antibiotics in antibiotic bacterial residues, provides a detection method basis for the disposal and utilization of antibiotic bacterial residues and safety risk assessment, and has good application prospects.

[0041] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0042] In the following examples and experimental examples, the reagents and materials used are all commercially available.

[0043] Example 1

[0044] (1) Preparation of test solution:

[0045] Weigh 0.5 g of sample, add 5 mL of methanol, vortex mix, and ultrasonically extract for 10 min. Then, dilute to 25 mL with water, vortex mix, and ultrasonically extract for 10 min. Centrifuge the extract at 4000 rpm for 10 min, take the supernatant, and filter the supernatant through a 0.22 μm microporous filter membrane to obtain the test solution.

[0046] (2) Drawing of standard curve:

[0047] Erythromycin A standard substance was dissolved with a small amount of methanol and then diluted with water to prepare a standard stock solution with a concentration of 1000 mg / L; penicillin G and cephalosporin C standard substances were prepared with water to prepare a standard stock solution with a concentration of 1000 mg / L; the above stock solutions were then mixed and diluted step by step with water to prepare mixed standard working solutions with concentrations of 1.0 μg / L, 5.0 μg / L, 25.0 μg / L, 100.0 μg / L, 200.0 μg / L and 500.0 μg / L, and detected and analyzed by liquid chromatography-triple quadrupole mass spectrometry, and a standard curve was drawn by measuring the peak area and the corresponding standard working solution concentration, and the regression equation and correlation coefficient were calculated.

[0048] (3) Detection and Analysis: The mixed standard working solution and the test sample were injected into a liquid chromatograph and detected and analyzed by liquid chromatography-triple quadrupole mass spectrometry, followed by quantitative analysis according to the external standard method.

[0049] In the above steps (2) and (3), the instrument for the liquid chromatography-triple quadrupole method is an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer.

[0050] The chromatographic conditions were as follows: the chromatographic column was a Waters-C18 column (2.1×100mm, 1.7μm); the mobile phase was methanol and 0.1% formic acid aqueous solution; gradient elution, 0.00-3.00min, 10% methanol, 3.01-7.00min, 90% methanol, 7.01-10.00min, 10% methanol; the flow rate was 0.25mL / min; the column temperature was 35°C; and the injection volume was 2μL.

[0051] Mass spectrometry conditions: electrospray positive ion mode; ion source temperature of 150°C; desolvation temperature of 450°C; desolvation flow of 900L / Hr; spray voltage of 4.0kv; specific parameters of the detected compounds are shown in Table 1.

[0052] The total ion current diagram of the three antibiotic mixed standard solutions obtained in this example is as follows Figure 1 As shown, the peak at the retention time of 1.15 min is cephalosporin C, the peak at the retention time of 4.73 min is erythromycin A, and the peak at the retention time of 4.88 min is penicillin G.

[0053] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the properties and application performance of the simultaneous detection method of erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residue disclosed in the present invention are further explained through the following methodological verification, but it should not be understood as a limitation of the present invention. The properties of the method obtained by other determination experiments conducted by technicians in this field according to the above invention content and the application based on the above properties are also regarded as falling within the protection scope of the present invention.

[0054] Experimental Example 1 Linear Investigation

[0055] The standard curve equations and linear correlation coefficients of erythromycin A, penicillin G and cephalosporin C calculated according to the method of step (2) in Example 1 are shown in Table 2:

[0056] Table 2 Standard curve equations and linear correlation coefficients of three antibiotics

[0057] Compound Name Regression equation Correlation coefficient r Erythromycin A Y=431.744X-657.709 0.9996 Penicillin G Y=141.07X+73.3673 0.9999 Cephalosporin C Y=47.1178X-237.639 0.9999

[0058] From the data in Table 2, it can be seen that the linearity of the three antibiotics is good.

[0059] Experimental Example 2 Detection limit, spike recovery and precision

[0060] In the manner that the target substance was not detected in the blank test, at least 7 parallel determinations were performed according to all the steps of sample analysis for samples with concentration values ​​or contents 3 to 5 times the detection limit of the estimated method, and the detection limit was calculated. The spike recovery rate when the sample spike concentration was 1.0 mg / kg was investigated, and 6 parallel determinations were performed, and the results are shown in Table 3.

[0061] Table 3 Detection limits, spiked recoveries and precision of three antibiotics

[0062]

[0063] As can be seen from Table 3, the sensitivity of the three antibiotic detection methods can reach below 0.1 mg / kg, and the spiked recoveries are all between 85% and 110.0%. The accuracy and sensitivity of the detection methods can meet the requirements for the determination of antibiotics in fungal residues.

[0064] Therefore, the present invention adopts a unique pretreatment method and liquid chromatography-triple quadrupole mass spectrometry to achieve the simultaneous determination of three antibiotics in antibiotic bacterial residues with high accuracy, high sensitivity and strong specificity. It can quickly and effectively detect the total amount of three antibiotic substances in antibiotic bacterial residues, and provide a detection method basis for the disposal and utilization of antibiotic bacterial residues and safety risk assessment, and has a good application prospect.

[0065] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting erythromycin A, penicillin G and cephalosporin C in antibiotic bacterial residues, characterized in that: The steps include: (1) using an organic phase extractant and an aqueous phase extractant in sequence to prepare a test solution from an antibiotic bacterial residue sample for later use; (2) using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the test solution and obtain qualitative or quantitative detection results of each component; Wherein, the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase includes a weak polar phase and a strong polar phase, the weak polar phase is selected from methanol or acetonitrile, and the strong polar phase is selected from formic acid aqueous solution, acetic acid aqueous solution, formic acid-ammonium formate or acetic acid-ammonium acetate solution, wherein the acid content is 0.02% to 0.2% by volume, and the ammonium salt concentration is 0.01M to 0.05M.

2. The detection method according to claim 1, characterized in that: The antibiotic bacterial residue is the culture medium residue remaining after microbial fermentation production.

3. The detection method according to claim 1, characterized in that: The step (1) specifically comprises: (1.1) Weigh 0.5-1.0 g of sample, add 5-10 mL of organic phase extractant A, vortex mix, and perform ultrasonic extraction for 10 min. Then, add aqueous phase extractant B and make up to 25-50 mL. Vortex mix, and perform ultrasonic extraction for another 10 min. (1.2) Centrifuge the extract at 4000 rpm for 10 min, take the supernatant and filter it through a microporous filter membrane to obtain the extract.

4. The detection method according to claim 3, characterized in that: In the step (1.1), the organic phase extractant A is methanol or acetonitrile, and the aqueous phase extractant B is water or 0.1M ammonium acetate solution; in the step (1.2), the microporous filter membrane is 0.22 μm or 0.45 μm.

5. The detection method according to claim 1, characterized in that: In the step (2), the particle size of the chromatographic filler is 1.7 μm; the mobile phase is methanol as a weak polar phase and 0.1% formic acid aqueous solution as a strong polar phase.

6. The detection method according to claim 5, characterized in that: The chromatographic conditions also include a gradient elution program of: The duration of the initial stage is 1.0 to 5.0 min, and the volume ratio of methanol is 5% to 20%; The duration of the intermediate stage is 3.0 to 10.0 min, and the volume ratio of methanol is 60% to 90%; The duration of the final stage is 2.0 to 5.0 minutes, and the volume ratio of methanol is 5% to 20%; and, The flow rate is 0.2-0.4 mL / min, the column temperature is 25-40 °C, and the injection volume is 1-15 μL.

7. The detection method according to claim 6, characterized in that: The gradient elution procedure is: 0.00-3.00min, 10% methanol, 3.01-7.00min, 90% methanol, 7.01-10.00min, 10% methanol; and, the flow rate was 0.25mL / min, the column temperature was 35°C, and the injection volume was 2μL.

8. The detection method according to claim 1, characterized in that: In the step (2), the mass spectrometry conditions include: The ionization mode is electrospray positive ion mode; the ion source temperature is 150-200°C; the desolvation gas temperature is 300-600°C; the desolvation gas flow rate is 400-1000L / Hr; the spray voltage is 2.8-4.0kv; the cone voltage is 10-40V; the collision voltage is 5-40V; and the scanning mode is multiple reaction monitoring mode.

9. The detection method according to claim 8, characterized in that: The mass spectrometry conditions are: ion source temperature of 150°C; desolvation gas temperature of 450°C; desolvation gas flow rate of 900L / Hr; spray voltage of 4.0kv. The multiple reaction monitoring conditions are shown in Table 1. Table 1 Multiple reaction detection conditions for three antibiotics 10. The detection method according to claim 1, characterized in that: In the step (2), the target component content is calculated using a standard curve method.

Images