CN114755334A - Method for simultaneously determining bacitracin B2 and B3 contents in pig tissues - Google Patents
Method for simultaneously determining bacitracin B2 and B3 contents in pig tissues Download PDFInfo
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- CN114755334A CN114755334A CN202210380721.4A CN202210380721A CN114755334A CN 114755334 A CN114755334 A CN 114755334A CN 202210380721 A CN202210380721 A CN 202210380721A CN 114755334 A CN114755334 A CN 114755334A
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- 108010001478 Bacitracin Proteins 0.000 title claims abstract description 101
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- 238000000034 method Methods 0.000 title claims abstract description 38
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 14
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000019253 formic acid Nutrition 0.000 claims abstract description 7
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract 12
- 238000004458 analytical method Methods 0.000 claims description 25
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 20
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- 238000012417 linear regression Methods 0.000 description 8
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- 238000004445 quantitative analysis Methods 0.000 description 7
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- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 5
- 238000002203 pretreatment Methods 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 1
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- 241000283690 Bos taurus Species 0.000 description 1
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- 241000282887 Suidae Species 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention discloses a method for simultaneously determining bacitracin B2 and B3 contents in pig tissues, which is determined by a liquid chromatography tandem mass spectrometry method, wherein the chromatographic conditions are as follows: the mobile phase is chromatographic pure acetonitrile and 0.1 percent formic acid solution; the proportion of the mobile phase is as follows: the mixture is 20 percent of chromatographically pure acetonitrile at 0 min-0.2 min, the mixture is changed from 20 percent of chromatographically pure acetonitrile to 30 percent of chromatographically pure acetonitrile at 0.2 min-6.0 min, the mixture is changed from 30 percent of chromatographically pure acetonitrile to 80 percent of chromatographically pure acetonitrile at 6.0 min-6.1 min, the mixture is 80 percent of chromatographically pure acetonitrile at 6.1 min-7.0 min, the mixture is changed from 80 percent of chromatographically pure acetonitrile to 20 percent of chromatographically pure acetonitrile at 7.0 min-7.1 min, and the mixture is 20 percent of chromatographically pure acetonitrile at 7.1 min-9.0 min; flow rate of mobile phase: 0.300 mL/min; sample introduction amount: 10.0. mu.L.
Description
Technical Field
The invention relates to the technical field of bacitracin detection, in particular to a method capable of simultaneously determining bacitracin B2 and B3 contents in pig tissues.
Background
Bacitracin (bacitracin), an important polypeptide antibiotic, is mainly produced by bacillus licheniformis and bacillus subtilis, and the like, can interfere the synthesis of cell walls by inhibiting dephosphorylation of lipid carriers, is effective on staphylococcus, streptococcus, and the like, and is widely applied to the field of livestock breeding. Bacitracin is a multicomponent compound containing various components such as A, B, C, D, E. Currently, commercial bacitracin preparations have bacitracin a as the main active ingredient followed by bacitracin B, which contains B1, B2, B3, etc.
Aiming at the drug residue risk generated by the use of bacitracin, a series of Maximum Residual Limit (MRL) of bacitracin is established at home and abroad for monitoring, for example, national standard GB31650-2019 stipulates that the MRL of bacitracin in edible tissues of cattle, pigs and poultry is 500 microgram/kg, wherein the stipulated bacitracin residue markers are three components of bacitracin A, B, C, but currently, the existing national standard only measures bacitracin A, or partial literature measures bacitracin B, and currently, no residue analysis method respectively accurately quantifies bacitracin B2 and B3 in the bacitracin residue markers, and since bacitracin B2 and B3 have certain toxicological differences, the quantitative analysis of the bacitracin residue markers is more favorable for scientific evaluation of the bacitracin residue risk. Therefore, it is necessary to establish an analytical method capable of simultaneously and separately determining the residual content of bacitracin B2 and B3 in the porcine tissue.
Therefore, how to provide a method for determining bacitracin B2 and B3 content in pig tissues is a problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of this, the invention provides a method capable of simultaneously determining bacitracin B2 and B3 content in pig tissue, which is beneficial to comprehensively evaluating bacitracin B2 and B3 content in pig tissue.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for simultaneously determining bacitracin B2 and B3 content in pig tissue comprises analyzing extracted bacitracin B2 and B3 by liquid chromatography tandem mass spectrometry, wherein the chromatographic conditions are as follows: the mobile phase is chromatographic pure acetonitrile and 0.1 percent formic acid solution; the proportion of the mobile phase is as follows: the chromatographic pure acetonitrile content is 20% at 0 min-0.2 min, is changed from 20% to 30% at 0.2 min-6.0 min, is changed from 30% to 80% at 6.0 min-6.1 min, is 80% at 6.1 min-7.0 min, is changed from 80% to 20% at 7.0 min-7.1 min, is 20% at 7.1 min-9.0 min; flow rate of mobile phase: 0.300 mL/min; sample injection amount: 10.0. mu.L.
The advantage of this elution condition is that bacitracin B2, B3 can be separated in a short analysis time with good separation, B2 and B3 are the same detection ion pair, so good separation is a prerequisite for accurate quantification thereof.
As a preferable embodiment of the above-mentioned means, the column is a C18 column, and the column temperature is 35 ℃.
As a preferable embodiment of the above-mentioned technical means, the mass spectrometry conditions in the analysis are: adopting an electrospray ion source positive source mode, wherein the voltage is 1.5KV, the desolventizing air flow rate is 800L/h, and the atomizing air temperature is 600 ℃; the airflow speed of the taper holes is 20L/h; the ion source temperature is 150 ℃; the ion scanning mode is a multiple reaction monitoring mode; the detected ion pairs are 705/199.3 and 705/110.2.
As a preferable technical scheme of the technical scheme, the method for extracting bacitracin B2 and B3 content in pig tissues comprises the following steps:
1) weighing a pig tissue sample, adding an extraction solvent, homogenizing at a high speed of 10000r/min, dispersing and extracting for 1min, centrifuging at 9500r/min for 2min, layering to obtain a supernatant A, repeatedly extracting the residual residues for 1 time according to the steps to obtain a supernatant B, and combining the two supernatants to obtain a solution C; the extraction solvent is 1% formic acid methanol, the function is to extract the residual bacitracin B2 and B3 in the sample, and the step has better extraction efficiency so as to ensure that the whole pretreatment method meets the requirement of residual analysis (namely the final recovery rate meets 70% -110%).
2) Adding n-hexane into the solution C obtained in the step 1), shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D; the n-hexane mainly has the functions of removing fat, removing lipid impurities in a sample and reducing the adverse effect of the n-hexane on subsequent detection. The step is primary purification, and removes a part of impurities to reduce impurity interference.
3) Uniformly mixing the solution D obtained in the step 2) with PSA and C18, shaking and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E; the step is a second step of purification, impurities with high acidity and polarity are removed, impurity interference is further reduced, matrix effect is reduced, and sensitivity is improved.
4) And (4) filtering the solution E obtained in the step 3) through a 0.22-micron filter membrane to obtain bacitracin B2 and B3 to be detected. This step can filter out the bigger particle impurity, prevents to get into detecting system, damages the instrument.
As a preferable technical scheme of the technical scheme, in the step 1), the mass-to-volume ratio of the pig tissue sample to the extraction solvent is 0.2-0.4 g/mL.
As a preferable technical scheme of the above technical scheme, in the step 2), the volume ratio of the solution C to n-hexane is 1: 1 to 2.
As a preferable embodiment of the above technical means, in the step 3), the volume-to-mass ratio of the solution D, PSA and C18 is 1ml to 50 mg.
According to the technical scheme, the total amount of bacitracin A or B is analyzed in the field of residual analysis according to published literature data, but B2 and B3 are not analyzed, the invention improves the problem, and the residual analysis literature does not realize good separation of bacitracin B2 and B3, particularly in short 9min instrument analysis time. Some foreign literatures realize separation of B2 and B3 by increasing the analysis time of an instrument (about 60 min) for analyzing the components of bacitracin, but the high efficiency is sacrificed, and large-batch sample detection is basically impossible for residual analysis, so that the method has the advantages of higher analysis efficiency, simplicity and high sensitivity on the whole, compared with the prior art, the method adopts UPLC-MSMS to simultaneously and respectively perform qualitative and quantitative analysis on bacitracin B2 and B3 in the pig tissues, and has short analysis time and high sensitivity; and the pretreatment process is simpler, the result is reliable, and the analysis efficiency is greatly improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 shows the chemical structures of bacitracin B2 (top) and B3 (bottom);
FIG. 2 is a quantitative ion chromatogram of bacitracin B2, B3;
FIG. 3 is a qualitative ion chromatogram of bacitracin B2, B3;
FIG. 4 is a graph showing the standard curve of bacitracin B2 in example 1;
FIG. 5 is a graph showing the standard curve of bacitracin B3 in example 1;
FIG. 6 is a qualitative ion chromatogram of bacitracin B2, B3 in comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The raw materials, equipment, etc. used in all examples are known products and can be obtained by purchasing commercially available products, mainly as follows:
UPLC-MS/MS (Waters Acquity UPLC-XEVO TQ-S Micro, Waters, USA); refiner (T25, IKA, germany); high speed centrifuge (3K15, Sigma, germany); vortex mixers (GENIUS3, IKA, germany); electronic balance (Quintix124, Mettler Toledo, switzerland); a one-tenth-ten-thousandth electronic balance (XPE205, Mettler Toledo, switzerland);
acetonitrile (chromatographically pure, Merk, germany); methanol (chromatographically pure, Merk, germany); formic acid (chromatographically pure, Sigma, germany); sodium chloride (analytical grade, chemical reagents of national drug group, ltd); N-Propylethylenediamine (PSA) and octadecylsilane bonded silica (C18) (tianjin bannajie technologies ltd); pure water (Wahaha group, Inc.); bacitracin B2 standard (92.80% pure, TOKU-E, Japan) and bacitracin B3 standard (99.72% pure, TOKU-E, Japan).
In order to measure the accuracy and precision of the analysis method of the present invention, an experiment was conducted by using an addition recovery method in which bacitracin B2 and B3 mixed standard solutions were added to blank samples at concentrations of 0.01mg/kg, 0.1mg/kg and 1mg/kg, respectively, and each concentration was repeated 5 times. The specific implementation is as follows:
example 1:
(1) pretreatment method
a. Accurately weighing 2.00g of homogenized pork sample, adding 10mL of extraction solvent (1% formic acid methanol), homogenizing at 10000r/min at high speed, dispersing and extracting for 1min, centrifuging at 9500r/min for 2min, layering to obtain supernatant A, and repeatedly extracting the residual residue for 1 time according to the steps to obtain supernatant B. Mixing the two supernatants to obtain solution C;
b. adding 10mL of n-hexane into the solution C obtained in the step a, shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D;
c. taking 2mL of the solution D obtained in the step b, 100mg of N-propyl ethylenediamine (PSA) and 100mg of octadecyl bonded silica gel (C)18) Uniformly mixing, vibrating and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E;
d. and (c) taking 1.0mL of the solution E obtained in the step (c), and filtering the solution E through a 0.22-micron filter membrane to obtain a sample to be detected.
(2) Standard solution preparation
a. Preparation of a standard stock solution: weighing 10.78mg bacitracin B2 standard substance with the purity of 92.80%, and fixing the volume to 10mL by using acetonitrile to obtain bacitracin B2 standard stock solution with the concentration of 1000 mg/L; additionally weighing 10.03mg of bacitracin B3 standard substance with the purity of 99.72%, and diluting to 10mL with acetonitrile to obtain a standard stock solution of bacitracin B3 with the concentration of 997 mg/L; 1mL of the bacitracin B2 and bacitracin B3 standard stock solutions were accurately pipetted, and the volume of each stock solution was adjusted to 10mL with acetonitrile to obtain a 100mg/L mixed standard stock solution of bacitracin B2 and bacitracin B3.
b. Preparing a matrix standard working solution: accurately transferring the bacitracin B2 and bacitracin B3 mixed standard stock solution, diluting with a pig tissue blank sample constant volume solution step by step to obtain a series of matrix standard working solutions with the concentrations of 0.0005mg/L, 0.001mg/L, 0.01mg/L, 0.1mg/L and 0.2mg/L, and waiting for UPLC-MSMS detection.
(3) Detection conditions
Liquid phase conditions: the column was Waters acquitylbeh C18 (2.1X 100mm,1.7 μm); the temperature of the chromatographic column is 35 ℃; the sample injection amount is 10.0 mu L; the mobile phase is chromatographic pure acetonitrile (A) and 0.1 percent formic acid solution (B), and gradient elution is adopted (shown in a table 1); the flow rate of the mobile phase was 0.30 mL/min.
TABLE 1 liquid phase gradient elution conditions
Mass spectrum conditions: the ion source is an electrospray ion source positive source mode (ESI)+) (ii) a The capillary voltage is 1.5 KV; the desolventizing air flow rate is 800 ℃; the temperature of atomizing gas is 600 ℃; the air flow rate of the taper hole is 20L/h; the ion source temperature is 150 ℃; the ion scan mode is a multiple reaction monitoring mode (MRM) as shown in table 2.
TABLE 2 multiple reaction monitoring conditions
(4) Qualitative and quantitative analysis
The results of sample injection detection of the series of matrix standard working solutions show that the linear relation between the sample injection concentration and the peak area is good, and the method can be used for accurate quantification of bacitracin B2 and B3. The linear regression equation and the correlation coefficient r are shown in table 3.
When the addition concentration range of bacitracin B2 and B3 in pork is 0.01-1 mg/kg, the result shows that (table 4), the average recovery rate of bacitracin B2 and B3 is 81.9-93.9%, and the accuracy is good; the relative standard deviation is 1.8-4.1%, which indicates that the method has good precision; the method meets the requirement of residue analysis.
TABLE 3 Linear relationship between bacitracin B2 and B3 standard solutions of pork matrix
| Analysis of Components | Linear regression equation | Coefficient of correlation r |
| Bacitracin B2 | y＝3027897.0x+1422.2 | 0.9999 |
| Bacitracin B3 | y＝2202175.1x+1211.5 | 0.9999 |
Table 4 accuracy and precision of the pork add recovery test
Example 2:
(1) pretreatment method
a. Accurately weighing 2.00g of homogenized pork liver sample, adding 10mL of extraction solvent (1% formic acid methanol), homogenizing at 10000r/min at high speed, dispersing and extracting for 1min, centrifuging at 9500r/min for 2min, layering to obtain supernatant A, and repeatedly extracting the residual residue for 1 time according to the steps to obtain supernatant B. Mixing the two supernatants to obtain solution C;
b. adding 10mL of n-hexane into the solution C obtained in the step a, shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D;
c. taking 2mL of the solution D obtained in the step b, 100mg of N-Propylethylenediamine (PSA) and 100mg of octadecyl silica gel (C)18) Uniformly mixing, vibrating and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E;
d. and (c) taking 1.0mL of the solution E obtained in the step (c), and filtering the solution E through a 0.22-micron filter membrane to obtain a sample to be detected.
(2) Standard solution preparation
The same as in example 1.
(3) Detection conditions
The same as in example 1.
(4) Qualitative and quantitative analysis
The results of sample injection detection of the series of matrix standard working solutions show that the linear relation between the sample injection concentration and the peak area is good, and the method can be used for accurate quantification of bacitracin B2 and B3. The linear regression equation and the correlation coefficient r are shown in table 5.
When the adding concentration range of bacitracin B2 and B3 in the pig liver is 0.01-1 mg/kg, the result shows that (table 6), the average recovery rate of bacitracin B2 and B3 is 78.9-92.7%, and the accuracy is good; the relative standard deviation is 1.9-4.2%, which shows that the method has good precision; the method meets the requirement of residue analysis.
TABLE 5 Linear relationship between bacitracin B2 and B3 porcine liver stroma standard solutions
| Analytical Components | Linear regression equation | Coefficient of correlation r |
| Bacitracin B2 | y＝3344757.0x+2287.9 | 0.9999 |
| Bacitracin B3 | y＝2160775.6x+611.7 | 1.0000 |
TABLE 6 accuracy and precision of the additive recovery test in pork liver
Example 3:
(1) pretreatment method
a. Accurately weighing 2.00g of homogenized pig kidney sample, adding 10mL of extraction solvent (1% formic acid methanol), homogenizing at 10000r/min at high speed, dispersing and extracting for 1min, centrifuging at 9500r/min for 2min, layering to obtain supernatant A, and repeatedly extracting the residual residue for 1 time according to the steps to obtain supernatant B. Mixing the two supernatants to obtain solution C;
b. adding 10mL of n-hexane into the solution C obtained in the step a, shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D;
c. 2mL of the solution D obtained in step b were taken and mixed with 100mg of N-Propylethylenediamine (PSA) and 100mg of octadecyl silica gel (C)18) Uniformly mixing, vibrating and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E;
d. and (c) taking 1.0mL of the solution E obtained in the step (c), and filtering the solution E through a 0.22-micron filter membrane to obtain a sample to be detected.
(2) Standard solution preparation
The same as in example 1.
(3) Detection conditions
The same as in example 1.
(4) Qualitative and quantitative analysis
The results of sample injection detection of the series of matrix standard working solutions show that the linear relation between the sample injection concentration and the peak area is good, and the method can be used for accurate quantification of bacitracin B2 and B3. The linear regression equation and the correlation coefficient r are shown in table 7.
When the adding concentration range of bacitracin B2 and B3 in the pig tissue is 0.01-1 mg/kg, the result shows (Table 8) that the average recovery rate of bacitracin B2 and B3 is 84.0% -94.3%, which shows that the accuracy is good; the relative standard deviation is 2.2-6.5%, which shows that the method has good precision; the method meets the requirement of residue analysis.
TABLE 7 Linear relationship of bacitracin B2, B3 porcine kidney matrix standard solutions
| Analysis of Components | Linear regression equation | Coefficient of correlation r |
| Bacitracin B2 | y＝3193026.1x+970.5 | 0.9999 |
| Bacitracin B3 | y＝2133412.7x+101.2 | 1.0000 |
TABLE 8 accuracy and precision of the addition recovery test in porcine kidney
The chemical structural formulas of bacitracin B2 and B3 extracted in example 1 are shown in FIG. 1, the quantitative and qualitative ion pairs thereof are shown in FIG. 2 and FIG. 3, respectively, the standard curve of bacitracin B2 is shown in FIG. 4, and the standard curve of bacitracin B3 in example is shown in FIG. 5.
Comparative example 1 (existing elution conditions)
(1) Standard solution preparation
a. Preparation of a standard stock solution: weighing 10.78mg bacitracin B2 standard substance with purity of 92.80%, and diluting to 10mL with acetonitrile to obtain bacitracin B2 standard stock solution with concentration of 1000 mg/L; weighing 10.03mg bacitracin B3 standard substance with purity of 99.72%, and diluting to 10mL with acetonitrile to obtain standard stock solution of bacitracin B3 with concentration of 997 mg/L; 1mL of the bacitracin B2 and bacitracin B3 standard stock solutions were accurately pipetted, and the volume of each stock solution was adjusted to 10mL with acetonitrile to obtain a 100mg/L mixed standard stock solution of bacitracin B2 and bacitracin B3.
b. Preparing a matrix standard working solution: accurately transferring the mixed standard stock solution of bacitracin B2 and bacitracin B3, diluting with a pig tissue blank sample constant volume solution step by step to obtain a series of matrix standard working solutions with the concentrations of 0.0005mg/L, 0.001mg/L, 0.01mg/L, 0.1mg/L and 0.2mg/L, and waiting for UPLC-MSMS detection.
(2) Detection conditions
Liquid phase conditions: the column was Waters Acquity BEH C18 (2.1X 100mm,1.7 μm); the temperature of the chromatographic column is 35 ℃; the sample injection amount is 10.0 mu L; the mobile phase was chromatographically pure acetonitrile (a) and 0.1% formic acid solution (B), with gradient elution (as shown in table 9); the flow rate of the mobile phase was 0.30 mL/min.
TABLE 9 liquid phase gradient elution conditions
| Time/min | A proportion/%) | Proportion of B/%) |
| 0.00 | 20.0 | 80.0 |
| 0.50 | 20.0 | 80.0 |
| 1.00 | 80.0 | 20.0 |
| 6.00 | 80.0 | 20.0 |
| 6.50 | 20.0 | 80.0 |
| 9.00 | 20.0 | 80.0 |
Mass spectrum conditions: the ion source is an electrospray ion source positive source mode (ESI)+) (ii) a The capillary voltage is 1.5 KV; the desolventizing airflow rate is 800 ℃; the temperature of atomizing gas is 600 ℃; the air flow rate of the taper hole is 20L/h; the ion source temperature is 150 ℃; the ion scan mode is a multiple reaction monitoring mode (MRM) as shown in table 10.
TABLE 10 multiple reaction monitoring conditions
Comparative example 2:
(1) pretreatment method
a. Accurately weighing 2.00g of homogenized pork sample, adding 2mL of extraction solvent (1% formic acid methanol), homogenizing at a high speed of 5000r/min for dispersion and extraction for 1min, centrifuging at 9500r/min for 2min, layering to obtain supernatant A, and repeatedly extracting the residual residue for 1 time according to the steps to obtain supernatant B. Mixing the two supernatants to obtain solution C;
b. adding 10mL of n-hexane into the solution C obtained in the step a, shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D;
c. taking 1mL of the solution D obtained in the step b, 100mg of N-propyl ethylenediamine (PSA) and 100mg of octadecyl bonded silica gel (C)18) Uniformly mixing, vibrating and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E;
d. and (c) taking 1.0mL of the solution E obtained in the step (c), and filtering the solution E through a 0.22-micron filter membrane to obtain a sample to be detected.
(2) Standard solution preparation
As in comparative example 1.
(3) Detection conditions
Liquid phase conditions: the column was Waters acquitylbeh C18 (2.1X 100mm,1.7 μm); the column temperature of the chromatographic column is 35 ℃; the sample injection amount is 10.0 mu L; the mobile phase was chromatographically pure acetonitrile (a) and 0.1% formic acid solution (B), with gradient elution (as shown in table 11); the flow rate of the mobile phase was 0.30 mL/min.
TABLE 11 liquid phase gradient elution conditions
| Time/min | A proportion/%) | Proportion of B/%) |
| 0.00 | 20.0 | 80.0 |
| 0.20 | 20.0 | 80.0 |
| 6.00 | 30.0 | 70.0 |
| 6.10 | 80.0 | 20.0 |
| 7.00 | 80.0 | 20.0 |
| 7.10 | 20.0 | 80.0 |
| 9.00 | 20.0 | 80.0 |
Mass spectrum conditions: as in comparative example 1.
(4) Qualitative and quantitative analysis
The sample injection detection of the series of matrix standard working solutions shows that the linear relation between the sample injection concentration and the peak area is good within the concentration range of 0.001-0.2 mg/L. The linear regression equation and the correlation coefficient r are shown in table 12.
Bacitracin B2, B3 mixed standard solutions were added to the raw pork for additive recovery tests, which were repeated 2 times per concentration. When the addition concentration is in the range of 0.01-1 mg/kg, the results show (Table 13) that the average recovery rates of bacitracin B2 and B3 are 36.8-57.5%, and the relative standard deviation is 1.4-12.1%.
TABLE 12 bacitracin B2, B3 pork matrix standard solution linearity relationship
| Analytical Components | Linear regression equation | Coefficient of correlation r |
| Bacitracin B2 | y＝1,522,760.4x+2,423.1 | 0.9990 |
| Bacitracin B3 | y＝1,033,330.5x+3,037.3 | 0.9963 |
TABLE 13 accuracy and precision of pork add recovery test
Comparative example 1 a qualitative and quantitative ion chromatogram is shown in fig. 6.
The results of the experiment were analyzed as follows:
(1) as can be seen from comparative example 1 and FIG. 6, bacitracin B2 and B3 could not be separated effectively under the general instrumental detection conditions, and thus could not be analyzed qualitatively and quantitatively accurately.
(2) As can be seen from comparative example 2, unlike the conditions of the method of the present invention, the lowest point of the linear range of bacitracin B2, B3 became high due to the more significant matrix effect, etc., indicating that the detection sensitivity became poor and the recovery rate of addition did not satisfy the requirement of the residual analysis.
(3) As can be seen from FIGS. 2 and 3, the background of the chromatogram is clean, the interference of impurities is less, and bacitracin B2 and B3 can be well separated under the chromatographic conditions of the method, so that the qualitative and quantitative analysis of the bacitracin B2 and the bacitracin B3 are more accurate. While the sensitivity is relatively high.
(4) Compared with the prior relevant published documents, the pretreatment process of the method is greatly simplified, the SPE purification process is reduced, the impurity interference degree is small, and the time cost is saved by more than 60 percent on the whole.
(5) As can be seen from tables 3, 5 and 7 in combination with FIGS. 3 and 4, the linear relationship between the injection concentration and the response value of different porcine tissue matrix standard solutions in the range of 0.0005-0.2 mg/L is good, and the correlation coefficients r are all above 0.999, which indicates that the method can be used for accurate quantification.
(6) As can be seen from tables 4, 6 and 8, when the bacitracin B2 and B3 addition concentration in different pig tissues is in the range of 0.01-1 mg/kg, the average addition recovery rate is 78.9-94.3%, and the RSD is 1.8-6.5%, which indicates that the analysis method has good accuracy and precision and meets the related requirements of residual analysis.
In conclusion, the method adopts UPLC-MSMS to detect and simultaneously and respectively determine bacitracin B2 and bacitracin B3 residues in the pig tissues, the pretreatment process is safe and efficient, the detection sensitivity of the instrument is high, the accuracy, precision and the like of the analysis method also meet the requirements of residue analysis, and a certain technical support is provided for scientific evaluation of bacitracin residues in the pig tissues.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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 (7)
1. A method for simultaneously determining bacitracin B2 and B3 contents in pig tissues is characterized in that extracted bacitracin B2 and B3 are analyzed by a liquid chromatography tandem mass spectrometry method, and during analysis, the chromatographic conditions are as follows: the mobile phase is chromatographic pure acetonitrile and 0.1 percent formic acid solution; the proportion of the mobile phase is as follows: the mixture is 20 percent of chromatographically pure acetonitrile at 0 min-0.2 min, the mixture is changed from 20 percent of chromatographically pure acetonitrile to 30 percent of chromatographically pure acetonitrile at 0.2 min-6.0 min, the mixture is changed from 30 percent of chromatographically pure acetonitrile to 80 percent of chromatographically pure acetonitrile at 6.0 min-6.1 min, the mixture is 80 percent of chromatographically pure acetonitrile at 6.1 min-7.0 min, the mixture is changed from 80 percent of chromatographically pure acetonitrile to 20 percent of chromatographically pure acetonitrile at 7.0 min-7.1 min, and the mixture is 20 percent of chromatographically pure acetonitrile at 7.1 min-9.0 min; flow rate of mobile phase: 0.300 mL/min; sample introduction amount: 10.0. mu.L.
2. The method for simultaneously determining bacitracin B2 and B3 in pig tissue as claimed in claim 1, wherein the chromatographic column is C18 column and the column temperature is 35 ℃.
3. The method for simultaneously determining bacitracin B2 and B3 content in pig tissue as claimed in claim 1, wherein the mass spectrometric conditions are: adopting an electrospray ion source positive source mode, wherein the voltage is 1.5KV, the desolventizing air flow rate is 800L/h, and the atomizing air temperature is 600 ℃; the air flow rate of the taper hole is 20L/h; the ion source temperature is 150 ℃; the ion scanning mode is a multiple reaction monitoring mode; the detected ion pairs are 705/199.3 and 705/110.2.
4. The method for simultaneously determining the bacitracin B2 and B3 content in pig tissue as claimed in claim 1, wherein the method for extracting the bacitracin B2 and B3 content in pig tissue comprises the following steps:
1) weighing a pig tissue sample, adding an extraction solvent, homogenizing at a high speed of 10000r/min, dispersing and extracting for 1min, centrifuging at 9500r/min for 2min, layering to obtain a supernatant A, repeatedly extracting the residual residues for 1 time according to the steps to obtain a supernatant B, and combining the two supernatants to obtain a solution C;
2) adding n-hexane into the solution C obtained in the step 1), shaking for 1min, centrifuging at 9500r/min for 2min, layering, and removing the upper layer to obtain a solution D;
3) uniformly mixing the solution D obtained in the step 2) with PSA and C18, shaking and purifying for 1min, centrifuging at 9500r/min for 2min, and layering to obtain a solution E;
4) taking the solution E obtained in the step 3), and filtering the solution E through a 0.22 mu m filter membrane to obtain bacitracin B2 and B3 to be detected.
5. The method for simultaneously determining the bacitracin B2 and B3 content in the pig tissue as claimed in claim 4, wherein in step 1), the mass-to-volume ratio of the pig tissue sample and the extraction solvent is 0.2-0.4 g/mL.
6. The method for simultaneously determining the bacitracin B2 and B3 contents in pig tissues as claimed in claim 4, wherein in the step 2), the volume ratio of the solution C to n-hexane is 1: 1 to 2.
7. The method for simultaneously determining the bacitracin B2 and B3 content in pig tissues as claimed in claim 4, wherein in step 3), the volume-to-mass ratio of the solution D, PSA and C18 is 1ml to 50 mg.
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