Extraction solvent and detection method of bacitracin preparation in feed

Abstract

This invention discloses an extraction solvent for bacitracin preparations in feed. It belongs to the field of product testing and analysis technology. The extraction solvent of this invention is a 12-15% ammonia-methanol-acetonitrile (1:1:1) solution, which can be used for extraction and analysis of bacitracin zinc and methylene salicylate bacitracin in different feeds using the same detection method, improving analytical efficiency, simplifying and increasing efficiency, and saving production costs. This invention also provides a detection method for bacitracin preparations in feed, using 12-15% ammonia-methanol-acetonitrile (1:1:1) as the extraction reagent. This not only optimizes the extraction conditions but also the purification conditions, greatly improving the accuracy and precision of the detection method of this invention, and providing technical support for the standardized use of bacitracin preparations in feed.

Description

Technical Field

[0001] This invention relates to the field of product testing and analysis technology, and more specifically to an extraction solvent and detection method for bacitracin preparations in feed. Background Technology

[0002] Bacitracin, discovered in 1943, is a polypeptide antibiotic secreted by Bacillus. Its antibacterial spectrum is similar to penicillin, and it is primarily used to treat penicillin-resistant staphylococcal infections. Bacitracin is unstable and, when used as a feed additive, needs to form a stable complex with metal ions or disalicylic acid. Currently, bacitracin formulations mainly include bacitracin zinc and bacitracin methylenedisalicylate. Bacitracin zinc effectively prevents animal mortality and liver lesions caused by high doses of Clostridium perfringens in feed. Bacitracin methylenedisalicylate, because it dissolves in the slightly acidic environment of the small intestine, exerts its antibacterial and growth-promoting effects better than bacitracin zinc. Therefore, both bacitracin zinc and bacitracin methylenedisalicylate are widely used in animal husbandry and feed production both domestically and internationally.

[0003] However, further research by relevant scholars has found that the use of bacitracin preparations may cause nephrotoxicity and adverse reactions such as rashes and itching. The official release of Announcement No. 194 by the Ministry of Agriculture and Rural Affairs on July 9, 2019, marked the removal of bacitracin zinc and methylene salicylate bacitracin preparations from the list of feed additives. To better regulate the use of bacitracin preparations as feed additives, how to detect bacitracin zinc and methylene salicylate bacitracin in feed has become a hot topic of concern.

[0004] A review of domestic and international literature revealed only detection methods for single bacitracin zinc in feed, with no reports on methylene salicylic acid bacitracin. Furthermore, bacitracin zinc and methylene salicylic acid bacitracin have significantly different physicochemical properties.

[0005] Therefore, how to overcome the significant differences in physicochemical properties and achieve the simultaneous detection of bacitracin zinc and methylene salicylate bacitracin is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides an extraction solvent and a detection method for bacitracin preparations in feed.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An extraction solvent for bacitracin preparations in feed, wherein the extraction solvent is an ammonia-methanol-acetonitrile solution, wherein the mass concentration of ammonia is 12-15%, and the volume ratio of the three is 1:1:1.

[0009] Preferably, the bacitracin preparation is one or both of bacitracin zinc and methylene salicylate bacitracin.

[0010] Preferably, the feed is one or more of compound feed, concentrated feed, and premixed feed.

[0011] The beneficial effects of the above operation are as follows: The extraction solvent of the present invention can effectively extract bacitracin zinc and methylene salicylic acid bacitracin from any one of the compound feed, concentrated feed, and premixed feed, with an extraction rate of more than 80%, providing technical support for the detection of bacitracin preparations in feed.

[0012] Another object of the present invention is to provide a method for detecting bacitracin preparations in feed, comprising the following steps:

[0013] (1) Extraction of blank feed sample: Weigh the blank feed sample, add the above extraction solvent, and centrifuge to obtain the supernatant;

[0014] (2) Purification of blank feed sample: Take the supernatant from step (1), add EDTA to dilute, purify by C18 solid phase extraction column, after rinsing and elution, collect the eluent, blow dry with nitrogen, add bacitracin A standard solution of known concentration to reconstitute, mix well and filter to obtain blank feed sample standard solution.

[0015] (3) Standard curve of blank feed sample: The standard solution of blank feed sample was subjected to liquid chromatography-tandem mass spectrometry. The peak area of ​​bacitracin quantitative ion chromatography was used as the vertical axis and the mass concentration of the corresponding bacitracin standard solution was used as the horizontal axis to obtain the standard curve of blank feed sample.

[0016] (4) Take the sample to be tested, extract it according to the method in step (1), purify it according to the method in step (2), blow it dry with nitrogen, add methanol aqueous solution to redissolve it, mix well and filter to obtain the sample solution to be tested.

[0017] (5) Take the sample solution obtained in step (4) and perform liquid chromatography-tandem mass spectrometry to determine the chromatographic peak area of ​​the sample solution. Calculate the content of bacitracin A in the sample based on the feed blank sample standard curve.

[0018] The beneficial effects of the above operation are as follows: by extracting the sample to be tested with the specific extraction solvent of the present invention and purifying it with a specific C18 solid phase extraction column, the qualitative and quantitative detection of bacitracin zinc and methylene salicylic acid bacitracin with large differences in physicochemical properties is realized, which improves the accuracy and precision of the detection method of the present invention, increases the detection efficiency, simplifies the detection steps, and saves costs.

[0019] Preferably, the mass-to-volume ratio of the sample extracted to the extraction solvent in step (1) is 0.2-0.4 g / mL.

[0020] Preferably, the extraction method in step (1) is vortex extraction, with a vortex time of 0.5-1 min, an oscillation speed of 900-1000 r / min, and an oscillation extraction time of 15-20 min.

[0021] Preferably, the centrifugation step in step (1) includes a first centrifugation and a second centrifugation, wherein the centrifugation speed is 9000-10000 r / min and the centrifugation time is 3-5 min.

[0022] Preferably, in step (2), the mass-to-volume ratio of the supernatant to the EDTA solution is 0.25-0.5 ml; before passing the blank sample solution through the column, the extraction column is pre-activated with 5 ml of methanol and water in sequence; the eluent is an acetonitrile aqueous solution with a mass concentration of 15-20% and a volume of 3-5 ml; the elution buffer is methanol and a volume of 3-5 ml.

[0023] Preferably, in step (2), the amount of bacitracin A standard solution added during reconstitution is 1 ml, with concentrations of 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L and 1.0 mg / L, respectively; after reconstitution, vortex for 30 s to mix, and filter through a 0.22 μm microporous membrane.

[0024] Preferably, in step (4), the mass concentration of the methanol aqueous solution is 20%, and the amount used is 1 ml.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The bacitracin preparation extraction solvent of the present invention solves the problem that bacitracin zinc and methylene salicylic acid bacitracin have large differences in physicochemical properties and cannot be extracted at the same time; at the same time, the bacitracin extraction solvent of the present invention also solves the influence of different feed types on extraction efficiency, so that the extraction efficiency of the bacitracin preparation in different feed types is as high as 80% or more.

[0027] (2) The extraction solvent of this invention is used to perform qualitative and quantitative analysis on the feed to be tested, and a specific C18 solid phase extraction column is used for purification, which improves the accuracy and precision of the detection method and provides technical support for the standardized use of bacitracin preparations in feed.

[0028] (3) Full scanning of the bacitracin zinc and methylene salicylate bacitracin standard solutions within the range of 500-2000 revealed a major ion mass-to-charge ratio of 712, which is the mass-to-charge ratio of bacitracin A (molecular weight 1422.7) carrying two charges. No ions formed by directly charged bacitracin zinc (molecular weight 1486) and methylene salicylate bacitracin (molecular weight 1985) molecules were observed. This indicates that bacitracin zinc and methylene salicylate bacitracin in feed were analyzed using liquid chromatography-tandem mass spectrometry, and the analyte was determined to be bacitracin A. Based on this, bacitracin A was selected to reconstitute blank feed to prepare a blank feed sample standard solution. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The attached image shows a full-scan mass spectrum of bacitracin zinc.

[0031] Figure 2 The attached image shows a full-scan mass spectrum of methylene salicylic acid bacitracin.

[0032] Figure 3 The attached figure shows the chromatogram of bacitracin daughter ions;

[0033] Figure 4 The attached figure is a comparison chart of the recovery effects of different ammoniation extracts of the present invention (left: premixed feed, middle: concentrated feed, right: compound feed);

[0034] Figure 5 The attached figure is a comparison chart of the recovery effects of ammonia water concentrations in different extracts of the present invention (top: premixed feed, middle: concentrated feed, bottom: compound feed). Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] The standard solutions of bacitracin zinc and methylene salicylate bacitracin were subjected to a full scan in the range of 500-2000. The full scan mass spectrum is shown below. Figure 1 and Figure 2 The attached figure shows the chromatogram of bacitracin's daughter ions. Figure 3 As shown in the attached figure, the mass-to-charge ratio of the main ion formed is 712, which is the mass-to-charge ratio when bacitracin A (molecular weight 1422.7) carries 2 charges. No ions formed by directly carrying charges from bacitracin zinc (molecular weight 1486) and methylene salicylate bacitracin (molecular weight 1985) molecules were observed.

[0038] The results showed that bacitracin zinc and methylene salicylate bacitracin in feed were analyzed by liquid chromatography-tandem mass spectrometry, and the analyte was identified as bacitracin A.

[0039] Using the detection method of the bacitracin preparation of the present invention, Examples 2-4 were set up for different types of feed (compound feed, concentrated feed and premixed feed), and experiments were conducted by adding and recovering methods to measure the accuracy and precision of the detection method of the present invention.

[0040] Example 2

[0041] (1) Preparation of blank feed sample standard solution

[0042] Weigh 2.00 g of blank feed sample and place it in a 50 mL plastic centrifuge tube. Accurately add 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1). Vortex for 30 s, then extract by shaking at 900 r / min for 15 min. Centrifuge at 9500 r / min for 3 min. Transfer the supernatant to another centrifuge tube. Add another 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) to the sample residue and repeat the extraction once. Combine the supernatants, accurately transfer 5 mL of the supernatant, add 10 mL of EDTA solution, and mix well for later use.

[0043] The solid-phase extraction column was pre-activated sequentially with 5 mL of methanol and water. The prepared solution was passed through the column, washed with 3 mL of 15% acetonitrile solution, and then eluted with 3 mL of methanol. The eluent was collected in a 10 mL centrifuge tube and dried under nitrogen at 40°C to obtain the residue. 1 mL of a known concentration of bacitracin A standard solution was accurately transferred to redissolve the residue. After redissolving, the solution was vortexed for 30 s, mixed thoroughly, and then filtered through a 0.22 μm microporous membrane to obtain a blank feed sample standard solution.

[0044] (2) Standard curve of blank feed sample

[0045] Six concentration gradients were set for bacitracin A standard solution: 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L and 1.0 mg / L. Based on these six concentration gradients, corresponding standard curves were established for quantitative addition recovery.

[0046] (3) Preparation of the sample solution to be tested

[0047] Four comparative experiments were set up for chicken compound feed, with a total feed amount of 2.00g in each group. Each group had 5 parallel experiments, and the comparative experiments are as follows:

[0048] Blank sample + bacitracin A standard solution with a concentration of 0.05 mg / kg added;

[0049] Blank sample + bacitracin A standard solution with a concentration of 0.2 mg / kg added;

[0050] Blank sample + bacitracin zinc standard solution with a concentration of 200 mg / kg;

[0051] Blank sample + bacitracin methylene salicylate standard solution with a concentration of 200 mg / kg.

[0052] The four groups of chicken feeds containing different amounts of bacitracin were placed in 50 mL plastic centrifuge tubes. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was accurately added. After vortexing for 30 s, the mixture was extracted by shaking at 900 r / min for 15 min, followed by centrifugation at 9500 r / min for 3 min. The supernatant was transferred to another centrifuge tube. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was added to the sample residue, and the extraction was repeated once. The supernatants were combined, and 5 mL of the supernatant was accurately transferred. 10 mL of EDTA solution was added, and the mixture was stirred well for later use.

[0053] The solid-phase extraction column was pre-activated with 5 mL of methanol and water in sequence. The prepared solution was passed through the column, washed with 3 mL of 15% acetonitrile solution, and eluted with 3 mL of methanol. The eluent was collected in a 10 mL centrifuge tube, dried under nitrogen at 40 °C, and reconstituted with 1 mL of 20% water-methanol solution. After mixing thoroughly, the solution was filtered through a 0.22 μm microporous membrane to obtain the sample solution to be tested.

[0054] (4) Take the sample solution obtained in step (3) and perform liquid chromatography-tandem mass spectrometry to determine the chromatographic peak area of ​​the sample solution. Calculate the content of bacitracin A in the sample based on the standard curve of the blank feed sample.

[0055] Liquid chromatography conditions: The column was a Waters Peptide BEH C18 (2.1×100mm, 1.7μm); the column temperature was 35℃; the injection volume was 10.0μL; the mobile phase was 0.1% formic acid aqueous solution (A) and chromatographic grade acetonitrile (B), with gradient elution (as shown in Table 1); the mobile phase flow rate was 0.30mL / min.

[0056] Table 1 Liquid phase gradient elution conditions

[0057]

[0058] Mass spectrometry conditions: Ion source was electrospray ionization positive source mode (ESI+); capillary voltage was 3.0 kV; desolvation temperature was 600 °C; desolvation gas was nitrogen 1000 L / Hr; cone gas flow rate was 50 L / h; ion source temperature was 150 °C; ion scanning mode was multiple reaction monitoring (MRM) mode as shown in Table 2.

[0059] Table 2 Multiple reaction monitoring conditions

[0060]

[0061] Qualitative and quantitative analysis:

[0062] The above-mentioned series of matrix standard working solutions were injected and analyzed. The results showed a good linear relationship between the injection concentration and the peak area, which can be used for the accurate quantification of bacitracin. The linear regression equation was y = 1097.4x + 8457.8 and the correlation coefficient r was 0.9996.

[0063] When the concentration of bacitracin A in chicken feed ranged from 0.05 to 0.2 mg / kg, and the concentrations of bacitracin zinc and methylene salicylic acid bacteria were 200 mg / kg, the recovery rates of the bacitracin preparations were shown in Table 3. The average recovery rate of bacitracin was 84.5% to 87.7%, indicating good accuracy; its relative standard deviation was 3.9% to 9.2%, indicating good method precision; the method met the requirements for residue analysis.

[0064] Table 3. Accuracy and precision of the recovery test in chicken compound feed.

[0065]

[0066] Example 3

[0067] (1) Preparation of blank feed sample standard solution

[0068] Same as Example 2

[0069] (2) Standard curve of blank feed sample

[0070] Same as Example 2

[0071] (3) Preparation of the sample solution to be tested

[0072] Four comparative experiments were set up for chicken concentrate feed, with a total feed amount of 2.00g in each group and 5 parallel experiments in each group. The comparative experiments are as follows:

[0073] Blank sample + bacitracin A standard solution with a concentration of 0.05 mg / kg added;

[0074] Blank sample + bacitracin A standard solution with a concentration of 0.2 mg / kg added;

[0075] Blank sample + bacitracin zinc standard solution with a concentration of 200 mg / kg;

[0076] Blank sample + bacitracin methylene salicylate standard solution with a concentration of 200 mg / kg.

[0077] The four groups of chicken concentrates with different amounts of bacitracin were placed in 50 mL plastic centrifuge tubes. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was accurately added. After vortexing for 30 s, the mixture was extracted by shaking at 900 r / min for 15 min, followed by centrifugation at 9500 r / min for 3 min. The supernatant was transferred to another centrifuge tube. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was added to the sample residue, and the extraction was repeated once. The supernatants were combined, and 5 mL of the supernatant was accurately transferred. 10 mL of EDTA solution was added, and the mixture was stirred well for later use.

[0078] The solid-phase extraction column was pre-activated with 5 mL of methanol and water in sequence. The prepared solution was passed through the column, washed with 3 mL of 15% acetonitrile solution, and eluted with 3 mL of methanol. The eluent was collected in a 10 mL centrifuge tube, dried under nitrogen at 40 °C, and reconstituted with 1 mL of 20% water-methanol solution. After mixing thoroughly, the solution was filtered through a 0.22 μm microporous membrane to obtain the sample solution to be tested.

[0079] (4) Same as Example 2.

[0080] Chromatographic conditions: Same as in Example 2.

[0081] Mass spectrometry conditions: Same as in Example 2.

[0082] Qualitative and quantitative analysis:

[0083] The above-mentioned series of matrix standard working solutions were injected and analyzed. The results showed a good linear relationship between the injection concentration and the peak area, which can be used for the accurate quantification of bacitracin. The linear regression equation was y = 938.1x + 7229.9, and the correlation coefficient r was 0.9996.

[0084] When the concentration of bacitracin A in chicken concentrate ranged from 0.05 to 0.2 mg / kg, and the concentrations of bacitracin zinc and methylene salicylic acid bacteria were 200 mg / kg, the recovery rates of the bacitracin preparations were shown in Table 4. The average recovery rate of bacitracin was 89.7% to 90.7%, indicating good accuracy; its relative standard deviation was 3.2% to 8.0%, indicating good method precision; the method met the requirements for residue analysis.

[0085] Table 4. Accuracy and precision of the recovery test in chicken concentrate.

[0086]

[0087] Example 4

[0088] (1) Preparation of blank feed sample standard solution

[0089] Same as Example 2

[0090] (3) Standard curve of blank feed sample

[0091] Same as Example 2

[0092] (3) Preparation of the sample solution to be tested

[0093] Four comparative experiments were set up for chicken premixed feed, with a total feed amount of 2.00g in each group and 5 parallel experiments in each group. The comparative experiments are as follows:

[0094] Blank sample + bacitracin A standard solution with a concentration of 0.05 mg / kg added;

[0095] Blank sample + bacitracin A standard solution with a concentration of 0.2 mg / kg added;

[0096] Blank sample + bacitracin zinc standard solution with a concentration of 200 mg / kg;

[0097] Blank sample + bacitracin methylene salicylate standard solution with a concentration of 200 mg / kg.

[0098] The four groups of chicken premixed feeds with different amounts of bacitracin were placed in 50 mL plastic centrifuge tubes. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was accurately added. After vortexing for 30 s, the mixture was extracted by shaking at 900 r / min for 15 min, followed by centrifugation at 9500 r / min for 3 min. The supernatant was transferred to another centrifuge tube. 10 mL of 12% ammonia-acetonitrile-methanol solution (1+1+1) was added to the sample residue, and the extraction was repeated once. The supernatants were combined, and 5 mL of the supernatant was accurately transferred. 10 mL of EDTA solution was added, and the mixture was stirred well for later use.

[0099] The solid-phase extraction column was pre-activated with 5 mL of methanol and water in sequence. The prepared solution was passed through the column, washed with 3 mL of 15% acetonitrile solution, and eluted with 3 mL of methanol. The eluent was collected in a 10 mL centrifuge tube, dried under nitrogen at 40 °C, and reconstituted with 1 mL of 20% water-methanol solution. After mixing thoroughly, the solution was filtered through a 0.22 μm microporous membrane to obtain the sample solution to be tested.

[0100] (4) Same as Example 2.

[0101] Chromatographic conditions: Same as in Example 2.

[0102] Mass spectrometry conditions: Same as in Example 2.

[0103] Qualitative and quantitative analysis:

[0104] The above-mentioned series of matrix standard working solutions were injected and analyzed. The results showed a good linear relationship between the injected concentration and the peak area, which can be used for the accurate quantification of bacitracin A. The linear regression equation was y = 1286.8x + 9917.4, and the correlation coefficient r was 0.9996.

[0105] When the concentration of bacitracin A in chicken premixed feed ranged from 0.05 to 0.2 mg / kg, and the concentrations of bacitracin zinc and methylene salicylic acid bacteria were 200 mg / kg, the recovery rates of the bacitracin preparations were shown in Table 5. The average recovery rate of bacitracin was 84.8% to 92.5%, indicating good accuracy; its relative standard deviation was 6.9% to 9.6%, indicating good method precision; the method met the requirements for residue analysis.

[0106] Table 5. Accuracy and precision of the recovery test in chicken premix feed.

[0107]

[0108] Example 5

[0109] Optimization of different extraction solvents

[0110] a: Bacitracin is readily soluble in organic solvents such as water and methanol, but bacitracin zinc and methylene salicylate bacitracin are both bacitracin complexes, and their solubility in solvents will change.

[0111] This experiment first explored the extraction efficiency of formic acid water + acetonitrile (1+1), formic acid acetonitrile, ammoniated acetonitrile, ammoniated methanol, and formic acid methanol on different feeds (chicken compound feed, chicken concentrated feed, and chicken compound premixed feed). Bacitracin standard solution (BTC), bacitracin zinc standard solution (BZ), and methylene salicylate bacitracin technical solution (BMD) were added and recovered separately to ensure that the actual bacitracin concentration was 1.0 mg / kg.

[0112] The results showed that the extraction efficiencies of 1% formic acid acetonitrile and 5% ammoniated acetonitrile were both less than 10% in different feeds. When 2% formic acid water + acetonitrile (1+1) was used as the extraction solution, the extraction efficiencies of bacitracin and bacitracin zinc were in the range of 69.2%-102%, and the extraction efficiencies of methylene salicylate bacitracin were in the range of 36.8%-54.1%, indicating that the presence of a certain proportion of aqueous solution in the extraction solution could improve the extraction efficiency. When 5% ammoniated methanol and 1% formic acid methanol were used as the extraction solution, it was found that methylene salicylate bacitracin could not be effectively extracted in 1% formic acid methanol. When 5% ammoniated methanol was used as the extraction solvent, the extraction recoveries of bacitracin, bacitracin zinc, and methylene salicylate bacitracin were between 66.6%-103.3%, while the extraction recoveries of bacitracin standard solution were between 66.6%-79.6%, lower than the extraction recoveries of bacitracin zinc and methylene salicylate bacitracin. Furthermore, the extraction efficiency of premixed feed was lower than that of compound feed and concentrated feed. Therefore, further optimization of the ammoniated extraction solution is needed to improve extraction efficiency.

[0113] b. Compare the extraction efficiencies of ammonia water, ammoniated methanol, ammonia water + acetonitrile (1+1), ammonia water + methanol (1+1), and ammonia water + acetonitrile + methanol (1+1+1) on different feeds (chicken compound feed, chicken concentrated feed, and chicken compound premixed feed). Bacitracin standard solution (BTC), bacitracin zinc standard solution (BZ), and methylene salicylate bacitracin technical solution (BMD) were added for recovery, ensuring an actual bacitracin concentration of 1.0 mg / kg. The recovery effects of different ammoniated extracts are compared in the following figure. Figure 4 As shown in the attached figure.

[0114] The results showed that 5% ammonia water had poor extraction efficiency for concentrated and premixed feeds. For both premixed and compound feeds, the extraction efficiency of 5% ammoniated methanol and 10% ammonia water + methanol (1+1) extracts was lower than the other two extracts when bacitracin was added. The extraction efficiency of concentrated feed indicated that 15% ammonia water + methanol + acetonitrile (1+1+1) as the extractant yielded the best extraction efficiency for all different standard solutions. Under these conditions, the extraction efficiency of both compound and premixed feeds was higher than 80%.

[0115] c. The extraction efficiency of ammonia concentration in the extract was investigated for different feeds (chicken compound feed, chicken concentrated feed, and chicken compound premixed feed). Bacitracin standard solution (BTC), bacitracin zinc standard solution (BZ), and methylene salicylate bacitracin technical solution (BMD) were added for recovery, ensuring an actual bacitracin concentration of 1.0 mg / kg. The recovery efficiency of different ammonia concentrations in the extract is compared in the following figure. Figure 5 As shown in the attached figure.

[0116] The results showed that the concentration of ammonia water had a significant impact on the extraction and recovery efficiency of bacitracin and bacitracin preparations, especially bacitracin. When the ammonia water concentration increased to 12%, the extraction and recovery efficiency of the three standard solutions was greater than 80%, which was not significantly different from the recovery efficiency of the 15% ammonia water + methanol + acetonitrile (1+1+1) extract. Therefore, 12%-15% ammonia water + methanol + acetonitrile (1+1+1) was selected as the extraction solvent.

[0117] Example 6

[0118] Optimization of sample purification conditions

[0119] Because the extract contains a high amount of organic solvent, it cannot be directly purified using a solid-phase extraction column. Therefore, this invention uses 5 mL of the extract, diluted with 10 mL of EDTA solution, before loading. Since bacitracin zinc and the premixed feed contain metal ions, these ions are excessively retained in the solid-phase extraction column and are difficult to elute. Methylene salicylic acid bacitracin easily forms flocculent precipitates in acidic solutions; therefore, the dilution solution before column loading was determined to be EDTA solution (pH 7.0).

[0120] To obtain a cleaner sample solution, this invention employs QuEChERS and solid-phase extraction (SPE) columns for purification. The purification effect and recovery were tested using QuEChERS (PSA adsorbent specification 200 mg) and solid-phase extraction methods (Prime HLB, WCX, C18, and Agilent EMR columns).

[0121] The results showed that bacitracin was partially adsorbed in both EMR and Prime HLB purification columns, but flowed directly through the WCX column without retention, while retention was too strong and elution was difficult in the HLB column. The PSA adsorbent purification method was not suitable for subsequent nitrogen drying due to the 33% water content in the extract. However, the addition of bacitracin and methylene salicylate bacitracin standard solutions to the C18 column met the purification requirements.

[0122] Note:

[0123] All raw materials and equipment used in the experimental examples and embodiments are known products and can be obtained by purchasing commercially available products.

[0124] UPLC-MS / MS (Waters Acquity UPLC-XEVO TQ-S Micro, Waters, USA); Homogenizer (T25, IKA, Germany); High-speed centrifuge (3K15, Sigma, Germany); Vortex mixer (GENIUS3, IKA, Germany); Electronic balance (Quintix124, Mettler Toledo, Switzerland); 0.0001 g electronic balance (XPE205, Mettler Toledo, Switzerland);

[0125] Acetonitrile, methanol (chromatographic grade, Merk, Germany); N-propylethylenediamine (PSA) (Tianjin Bona Aier Technology Co., Ltd.); disodium ethylenediaminetetraacetate, ammonia (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); purified water (Wahaha Group Co., Ltd.); bacitracin A (C 66 H 103 N 17 O 16 S (CAS: 22601-59-8) was purchased from TOKU-E, bacitracin zinc was purchased from TRC, and methylene salicylate bacitracin was purchased from Greencon Biochemical Co., Ltd.

[0126] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0127] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An extraction solvent for bacitracin preparation in a feed, characterized by, The extraction solvent is an ammonia water-methanol-acetonitrile solution, wherein the mass concentration of ammonia water is 12-15%, and the volume ratio of the three is 1:1:1; The bacitracin preparation is one or both of bacitracin zinc and methylene salicylic acid bacitracin. The feed is one or more of compound feed, concentrated feed and premixed feed.

2. A method for detecting a bacitracin preparation in a feed, characterized in that, It comprises the following steps: (1) blank feed sample extraction: weigh the blank feed sample, use the extraction solvent of claim 1, centrifuge for extraction, and obtain the supernatant; (2) blank feed sample purification: take the supernatant in step (1), dilute with EDTA, purify with a C18 solid phase extraction column, after elution and washing, collect the eluate, dry with nitrogen, add a known concentration of bacitracin A standard solution, mix well, filter, and obtain the blank feed sample standard solution; (3) blank feed sample standard curve: take the blank feed sample standard solution for liquid chromatography-tandem mass spectrometry determination, take the bacitracin quantitative ion chromatographic peak area as the ordinate, and the corresponding bacitracin standard solution mass concentration as the abscissa, and obtain the blank feed sample standard curve; (4) take the sample to be tested, extract according to the method of step (1), purify according to the method of step (2), dry with nitrogen, redissolve with methanol water solution, mix well, filter, and obtain the sample solution to be tested; (5) take the sample solution to be tested obtained in step (4) for liquid chromatography-tandem mass spectrometry determination, obtain the chromatographic peak area of the sample solution to be tested, and calculate the content of bacitracin A in the sample to be tested according to the blank feed sample standard curve.

3. A method for detecting a bacitracin preparation in a feed according to claim 2, characterized in that, The mass-volume ratio of the extracted sample to the extraction solvent in step (1) is 0.2-0.4 g / mL.

4. The method for detecting a bacitracin preparation in a feed according to claim 2, characterized by, The extraction method in step (1) is vortex shaking extraction, the vortex time is 0.5-1 min, the shaking speed is 900-1000 r / min, and the shaking extraction time is 15-20 min.

5. The method for detecting a bacitracin preparation in a feed according to claim 2, characterized by, The centrifugation step in step (1) includes primary centrifugation and secondary centrifugation, and the centrifugal speed of the centrifugation is 9000-10000 r / min, and the centrifugation time is 3-5 min.

6. The method for detecting a bacitracin preparation in a feed according to claim 2, characterized by, In step (2), the mass-volume ratio of the supernatant to the EDTA solution is 0.25-0.5 ml; before the blank sample solution passes through the column, the extraction column is pre-activated with 5 ml of methanol and water in turn; the elution liquid is an acetonitrile water solution with a mass concentration of 15-20%, and the volume is 3-5 ml; the elution liquid is methanol, and the volume is 3-5 ml.

7. The method of claim 2, wherein the bacitracin preparation is a feed-grade bacitracin preparation. In step (2), the addition amount of bacitracin A standard solution during redissolution is 1 ml, and the concentrations are 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L and 1.0 mg / L respectively; after redissolution, vortex for 30 s, mix well, and filter through a 0.22 μm microporous filter membrane.

8. The method of claim 2, wherein the bacitracin preparation is a feed-grade bacitracin preparation. In step (4), the mass concentration of the methanol water solution is 20%, and the amount used is 1 ml.

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