CN117310051A - Method for detecting benzoic acid in gastrointestinal tract contents of monogastric animals - Google Patents
Method for detecting benzoic acid in gastrointestinal tract contents of monogastric animals Download PDFInfo
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- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 title claims abstract description 301
- 239000005711 Benzoic acid Substances 0.000 title claims abstract description 154
- 235000010233 benzoic acid Nutrition 0.000 title claims abstract description 154
- 241001465754 Metazoa Species 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 36
- 210000001035 gastrointestinal tract Anatomy 0.000 title claims description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 117
- 238000001514 detection method Methods 0.000 claims abstract description 104
- 150000002500 ions Chemical class 0.000 claims abstract description 46
- 239000007864 aqueous solution Substances 0.000 claims abstract description 44
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 29
- 239000000243 solution Substances 0.000 claims abstract description 28
- 210000003736 gastrointestinal content Anatomy 0.000 claims abstract description 25
- 230000003544 deproteinization Effects 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 51
- 239000007788 liquid Substances 0.000 claims description 26
- 229960000583 acetic acid Drugs 0.000 claims description 18
- 239000012362 glacial acetic acid Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000010829 isocratic elution Methods 0.000 claims description 15
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 15
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 15
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 13
- 239000011701 zinc Substances 0.000 claims description 13
- 229910052725 zinc Inorganic materials 0.000 claims description 13
- 238000002137 ultrasound extraction Methods 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 239000004246 zinc acetate Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000013467 fragmentation Methods 0.000 claims description 5
- 238000006062 fragmentation reaction Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 5
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 abstract description 27
- 238000002414 normal-phase solid-phase extraction Methods 0.000 abstract description 15
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 abstract description 8
- 238000001195 ultra high performance liquid chromatography Methods 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000000746 purification Methods 0.000 abstract description 5
- 239000002904 solvent Substances 0.000 abstract description 5
- 241000287828 Gallus gallus Species 0.000 description 73
- 239000012071 phase Substances 0.000 description 37
- 239000000126 substance Substances 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000012086 standard solution Substances 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 230000002183 duodenal effect Effects 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 150000001559 benzoic acids Chemical class 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 210000001630 jejunum Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 210000004534 cecum Anatomy 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 238000012417 linear regression Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 210000001198 duodenum Anatomy 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000003405 ileum Anatomy 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- WXBLLCUINBKULX-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1.OC(=O)C1=CC=CC=C1 WXBLLCUINBKULX-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a method for detecting benzoic acid in gastrointestinal contents of monogastric animals, and belongs to the technical field of analysis and detection. The detection method provided by the invention does not need to carry out solid phase extraction column purification on the sample to be detected, only needs acetonitrile aqueous solution extraction and deproteinization, and is simple and convenient to operate; the conventional acetonitrile water solution is used as the solvent, so that the cost is low, and the method has important practical value; the benzoic acid in the gastrointestinal contents of monogastric animals is efficiently and accurately tested by utilizing the rapid analysis function of ultra-high performance liquid chromatography detection and the characteristic ion selection function of mass spectrum, and the method has the characteristics of high sensitivity, high selectivity, strong specificity and good reproducibility; the interference of complex matrixes can be effectively overcome by adopting tandem mass spectrometry detection, a detection result with high selectivity and high sensitivity is obtained, and the detection limit of residual benzoic acid in gastrointestinal contents of monogastric animals is further reduced.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting benzoic acid in gastrointestinal contents of monogastric animals.
Background
Benzoic acid (benzoic acid) has broad-spectrum antibacterial effect and strong antibacterial power, and is an effective antibiotic substitute. Benzoic acid is commonly used in the prior art as an acidity regulator and preservative in livestock and poultry diets (for example, as a feed additive for broiler chickens and laying hens). Therefore, the method for accurately and rapidly detecting the residual quantity of the benzoic acid in different parts of the gastrointestinal tract of the livestock and poultry has important significance for promoting the evaluation of the effectiveness and the safety of the benzoic acid and determining the proper adding quantity.
At present, the benzoic acid in livestock meat or viscera is detected by the following method: the sample is extracted by methanol, purified by a solid phase extraction column, eluted by methanol, dried by nitrogen, re-dissolved by water and detected by UPLC-MS. However, this detection method requires solid phase extraction column purification of the sample, and the pretreatment step is complicated.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting benzoic acid in gastrointestinal contents of monogastric animals, which does not need to purify a sample by a solid phase extraction column and has simple steps.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a method for detecting benzoic acid in gastrointestinal contents of monogastric animals, which comprises the following steps:
extracting a sample of gastrointestinal tract contents of a monogastric animal to be detected by adopting an acetonitrile aqueous solution to obtain an extracting solution;
deproteinizing the extracting solution to obtain a sample solution to be detected;
performing ultra-high performance liquid-tandem mass spectrometry detection on the sample liquid to be detected to obtain a detection result of benzoic acid;
the detection result comprises a qualitative detection result and/or a quantitative detection result;
the conditions for ultra performance liquid chromatography detection of the ultra performance liquid-tandem mass spectrometry detection include: the chromatographic column is a C18 chromatographic column; mobile phase A is water, mobile phase B is acetonitrile; the elution mode is isocratic elution, and the volume fraction of the mobile phase A in the isocratic elution process is 20-40%; the flow rate of the mobile phase is 0.2-1.0 mL/min; the column temperature is 25-45 ℃; the sample injection amount is 2-20 mu L;
the conditions for tandem mass spectrometry detection of the ultra-high performance liquid-tandem mass spectrometry detection include: the detection mode is negative ion multiple reaction monitoring; the scanning mode is multi-reaction ion scanning; the ion source is an electrospray ion source; the temperature of the ion source is 250-380 ℃; the capillary voltage is 3500-4500V; the voltage of the photomultiplier is 300-450V; the source internal fragmentation voltage is 60-100V; the collision energy is 5-15 eV; the qualitative ion pair was 121.0/77.0 and the quantitative ion pair was 77.0.
Preferably, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-90%.
Preferably, the volume fraction of mobile phase a during the isocratic elution is 30%.
Preferably, the solid-to-liquid ratio of the gastrointestinal tract content sample of the monogastric animal to be tested and the acetonitrile aqueous solution is 1g: 2.5-10 mL.
Preferably, the extraction is ultrasonic extraction; the ultrasonic extraction frequency is 30-60 Hz, the power is 75-250W, and the time is 15-45 min.
Preferably, the deproteinization is performed by using a protein precipitant, which includes an aqueous solution of potassium ferrocyanide and an aqueous solution of zinc acetate-glacial acetic acid mixture.
Preferably, the concentration of the potassium ferrocyanide aqueous solution is 50-150 g/L.
Preferably, the solid-to-liquid ratio of the gastrointestinal tract content sample of the monogastric animal to be tested and the potassium ferrocyanide aqueous solution is 1g:0.1 to 0.4mL.
Preferably, the concentration of zinc acetate in the zinc acetate-glacial acetic acid mixed aqueous solution is 150-250 g/L, and the concentration of glacial acetic acid is 90-150 mL/L.
Preferably, the solid-to-liquid ratio of the sample of the gastrointestinal tract content of the monogastric animal to be tested and the zinc acetate-glacial acetic acid mixed aqueous solution is 1g:0.1 to 0.4mL.
The invention provides a method for detecting benzoic acid in gastrointestinal tract contents of monogastric animals, which comprises the steps of firstly extracting a sample of the gastrointestinal tract contents of monogastric animals to be detected by adopting acetonitrile aqueous solution to obtain an extracting solution; then deproteinizing the obtained extract to obtain a sample solution to be tested; and carrying out ultra-high performance liquid-tandem mass spectrometry detection on the sample liquid to be detected to obtain qualitative detection and/or quantitative detection results of the benzoic acid. The detection method provided by the invention does not need to carry out solid-phase extraction column purification on the sample to be detected, solves the problems of complex operation and special instrument and equipment requirement caused by adopting the solid-phase extraction column purification, only needs acetonitrile aqueous solution to extract and remove protein, and has the advantages of simple and convenient operation, good adaptability and high detection accuracy. In addition, the invention adopts the conventional acetonitrile aqueous solution as the solvent to extract the gastrointestinal tract content sample of the monogastric animal to be detected, has low cost and important practical value. The method utilizes the rapid analysis function of ultra-high performance liquid chromatography detection and the characteristic ion selection function of mass spectrum to test the benzoic acid in the gastrointestinal contents of monogastric animals efficiently and accurately, and has the characteristics of high sensitivity, high selectivity, strong specificity and good reproducibility; the interference of complex matrixes can be effectively overcome by adopting tandem mass spectrometry detection, a detection result with high selectivity and high sensitivity is obtained, and the detection limit of residual benzoic acid in gastrointestinal contents of monogastric animals is further reduced. In addition, under the ultra-high performance liquid chromatography detection condition adopted by the invention, the obtained benzoic acid chromatographic peak has good peak shape, no obvious impurity peak interference measurement, can effectively avoid false positive results, and realizes accurate detection of residual benzoic acid in gastrointestinal contents of monogastric animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram of a benzoic acid standard;
FIG. 2 is a standard quality spectrum of benzoic acid;
fig. 3 shows a benzoic acid chromatogram-mobile phase ratio 80 in the jejunal content of broilers: 20, a step of;
fig. 4 shows a benzoic acid chromatogram-mobile phase ratio 70 in the jejunal content of broilers: 30;
fig. 5 is a benzoic acid chromatogram-mobile phase ratio 60 in the jejunal content of broilers: 40, a step of performing a;
fig. 6 is a benzoic acid chromatogram-mobile phase ratio 50 in the jejunal content of broilers: 50;
fig. 7 is a benzoic acid chromatogram-mobile phase ratio 40 in the jejunal content of broilers: 60;
fig. 8 is a benzoic acid chromatogram-mobile phase ratio 30 in the jejunal content of broilers: 70;
fig. 9 is a benzoic acid chromatogram-mobile phase ratio 20 in the jejunal content of broilers: 80;
FIG. 10 is a chromatogram of benzoic acid in the stomach contents of broiler chickens;
FIG. 11 is a mass spectrum of benzoic acid in the stomach contents of broiler chickens;
FIG. 12 is a chromatogram of benzoic acid in the duodenal contents of broiler chickens;
FIG. 13 is a mass spectrum of benzoic acid in the contents of the duodenum of broilers;
FIG. 14 is a chromatogram of benzoic acid in the jejunal content of broilers;
FIG. 15 is a mass spectrum of benzoic acid in the jejunal content of broilers;
FIG. 16 is a chromatogram of benzoic acid in the ileal content of broiler chickens;
FIG. 17 is a mass spectrum of benzoic acid in the ileal content of broiler chickens;
FIG. 18 is a chromatogram of benzoic acid in the cecal content of broilers;
FIG. 19 is a mass spectrum of benzoic acid in the cecum content of broilers;
FIG. 20 is a chromatogram of benzoic acid in the rectal content of broiler chickens;
fig. 21 is a mass spectrum of benzoic acid in the rectal content of broiler chickens.
Detailed Description
The invention provides a method for detecting benzoic acid in gastrointestinal contents of monogastric animals, which comprises the following steps:
extracting a sample of gastrointestinal tract contents of a monogastric animal to be detected by adopting an acetonitrile aqueous solution to obtain an extracting solution;
deproteinizing the extracting solution to obtain a sample solution to be detected;
performing ultra-high performance liquid-tandem mass spectrometry detection on the sample liquid to be detected to obtain a detection result of benzoic acid;
the detection result comprises a qualitative detection result and/or a quantitative detection result;
the conditions for ultra performance liquid chromatography detection of the ultra performance liquid-tandem mass spectrometry detection include: the chromatographic column is a C18 chromatographic column; mobile phase A is water, mobile phase B is acetonitrile; the elution mode is isocratic elution, and the volume fraction of the mobile phase A in the isocratic elution process is 20-40%; the flow rate of the mobile phase is 0.2-1.0 mL/min; the column temperature is 25-45 ℃; the sample injection amount is 2-20 mu L;
the conditions for tandem mass spectrometry detection of the ultra-high performance liquid-tandem mass spectrometry detection include: the detection mode is negative ion multiple reaction monitoring; the scanning mode is multi-reaction ion scanning; the ion source is an electrospray ion source; the temperature of the ion source is 250-380 ℃; the capillary voltage is 3500-4500V; the voltage of the photomultiplier is 300-450V; the source internal fragmentation voltage is 60-100V; the collision energy is 5-15 eV; the qualitative ion pair was 121.0/77.0 and the quantitative ion pair was 77.0.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
The invention adopts acetonitrile aqueous solution to extract the gastrointestinal tract content sample of monogastric animal to be detected to obtain extract. In the invention, the monogastric animal in the gastrointestinal tract content sample of the monogastric animal to be detected is preferably chicken and/or pig, more preferably broiler chicken or laying hen. In the invention, the gastrointestinal tract content of the monogastric animal to be detected in the gastrointestinal tract content sample of the monogastric animal preferably comprises one or more of myogastric content, duodenal content, jejunal content, ileal content, cecal content and rectal content. In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50 to 90%, more preferably 60 to 80%, and most preferably 70 to 75%. In the invention, the solid-to-liquid ratio of the gastrointestinal tract content sample of the monogastric animal to be tested and the acetonitrile aqueous solution is preferably 1g:2.5 to 10mL, more preferably 1g: 4-8 mL, most preferably 1g: 5-6 mL. In the present invention, the extraction is preferably ultrasonic extraction; the frequency of ultrasonic extraction is preferably 30-60 Hz, more preferably 35-55 Hz, and most preferably 40-50 Hz; the power of ultrasonic extraction is preferably 75-250W, more preferably 100-200W, and most preferably 120-150W; the ultrasonic extraction time is preferably 15-45 min, more preferably 25-40 min, and most preferably 30-35 min. The invention adopts the conventional acetonitrile aqueous solution as the solvent to extract the gastrointestinal tract content sample of the monogastric animal to be detected, and has low cost.
After the extracting solution is obtained, the extracting solution is deproteinized to obtain the sample solution to be detected. In the present invention, the deproteinization is preferably performed using a protein precipitation agent; the protein precipitant preferably comprises an aqueous solution of potassium ferrocyanide and an aqueous solution of zinc acetate-glacial acetic acid mixture. In the invention, the concentration of the potassium ferrocyanide aqueous solution is preferably 50-150 g/L, more preferably 80-120 g/L, and most preferably 90-100 g/L; the solid-to-liquid ratio of the gastrointestinal tract content sample of the monogastric animal to be tested and the potassium ferrocyanide aqueous solution is preferably 1g:0.1 to 0.4mL, more preferably 1g:0.15 to 0.3mL, most preferably 1g:0.18 to 0.2mL. In the invention, the concentration of zinc acetate in the zinc acetate-glacial acetic acid mixed aqueous solution is preferably 150-250 g/L, more preferably 180-220 g/L, and most preferably 190-200 g/L; the concentration of glacial acetic acid is preferably 90-150 mL/L, more preferably 110-130 mL/L, and most preferably 115-120 mL/L; the solid-to-liquid ratio of the sample of the gastrointestinal tract content of the monogastric animal to be tested and the zinc acetate-glacial acetic acid mixed aqueous solution is preferably 1g:0.1 to 0.4mL, more preferably 1g:0.15 to 0.3mL, most preferably 1g:0.18 to 0.2mL.
In the present invention, deproteinizing the extract preferably comprises: and mixing the extracting solution with a potassium ferrocyanide aqueous solution and a zinc acetate-glacial acetic acid mixed aqueous solution, and centrifuging to obtain a sample solution to be detected. In the invention, the rotation speed of the centrifugation is preferably 6000-9000 rpm, more preferably 6500-8500 rpm, and most preferably 7000-8000 rpm; the centrifugation time is preferably 8-15 min, more preferably 9-14 min, and most preferably 10-12 min. In the invention, the supernatant obtained by centrifugation is collected as a sample liquid to be tested.
After the sample liquid to be detected is obtained, the sample liquid to be detected is subjected to ultra-high performance liquid-tandem mass spectrometry detection to obtain a detection result of benzoic acid; the detection result comprises a qualitative detection result and/or a quantitative detection result.
In the invention, the sample liquid to be detected is preferably diluted and filtered in sequence before the ultra-high performance liquid-tandem mass spectrometry detection. In the present invention, the diluting solvent is preferably an aqueous acetonitrile solution; the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50-90%, more preferably 60-80%, and most preferably 70-75%. In the present invention, the filtration membrane is preferably a 0.22 μm microporous membrane. In the present invention, the type of the gastrointestinal contents of the monogastric animal is preferably the same as that described above, and will not be described in detail herein.
In the present invention, the conditions for ultra performance liquid-tandem mass spectrometry preferably include conditions for ultra performance liquid chromatography and conditions for tandem mass spectrometry.
In the present invention, the conditions for the ultra performance liquid chromatography detection include: the chromatographic column is C 18 Chromatographic column, preferably Zorbax SB-C 18 A chromatographic column; the C is 18 The particle size of the stationary phase of the chromatographic column is less than 2 mu m; mobile phase A is water, mobile phase B is acetonitrile; the elution mode is isocratic elution, wherein the volume fraction of the mobile phase A in the isocratic elution process is 20-40%, preferably 25-35%, more preferably 25-30%, and most preferably 30%; the flow rate of the mobile phase is 0.2-1.0 mL/min, preferably 0.3-0.9 mL/min, more preferably 0.4-0.8 mL/min; the column temperature is 25-45 ℃, preferably 30-40 ℃, more preferably 35-38 ℃; the sample amount is 2 to 20. Mu.L, preferably 5 to 15. Mu.L, more preferably 8 to 12. Mu.L. The ultra-high performance liquid chromatography detection adopts small particles and high performance particles C 18 The stationary phase further improves the separation efficiency, the analysis speed and the sensitivity.
In the present invention, the conditions for tandem mass spectrometry detection include: the detection mode is negative ion multiple reaction monitoring; the scanning mode is multi-reaction ion scanning; the ion source is an electrospray ion source; the temperature of the ion source is 250-380 ℃, preferably 280-350 ℃, more preferably 300-320 ℃; the capillary voltage is 3500-4500V, preferably 3700-4200V, more preferably 3900-4000V; the photomultiplier voltage is 300-450V, preferably 350-420V, more preferably 380-400V; the source internal fragmentation voltage is 60-100V, preferably 65-90V, more preferably 70-80V; the collision energy is 5 to 15eV, preferably 6 to 12eV, more preferably 8 to 9eV; the collision gas is nitrogen, and the air flow of the collision gas is 3-8L/min, preferably 4-7L/min, and more preferably 5-6L/min; the sprayer pressure is 10-20 psi, preferably 12-18 psi, more preferably 15-17 psi; the qualitative ion pair was 121.0/77.0 and the quantitative ion pair was 77.0.
According to the ultra-high performance liquid chromatography-tandem mass spectrometry detection method provided by the invention, the running time of the ultra-high performance liquid chromatography detection is within 3-5 min, so that the rapid and efficient detection of the benzoic acid content is realized. The animal gastrointestinal tract content contains various nutritional components such as protein, fat, carbohydrate, vitamins, minerals and the like, and the benzoic acid is easy to produce false positive results by the detection of the benzoic acid by a common liquid chromatography method. The invention uses the rapid analysis function of the ultra-high performance liquid chromatography and the characteristic ion selection function of the mass spectrum through the ultra-high performance liquid-tandem mass spectrum detection, can rapidly, efficiently and accurately test the benzoic acid in the gastrointestinal contents of animals, and has the characteristics of high sensitivity, high selectivity and strong specificity. Under the chromatographic conditions used in the invention, the benzoic acid peak shape is good, no obvious impurity peak interference measurement is carried out, and a method is provided for the rapid detection of the benzoic acid in the gastrointestinal contents of animals.
In the present invention, the step of obtaining the qualitative detection result preferably includes: comparing chromatographic peak data of the sample to be detected obtained by the ultra-high performance liquid-tandem mass spectrometry detection with chromatographic peak data of a standard substance obtained by the ultra-high performance liquid-tandem mass spectrometry detection of a series of benzoic acid standard substance solutions to obtain a qualitative detection result. When a chromatographic peak (the variation range is within +/-2.5%) consistent with the retention time of benzoic acid with a certain concentration in a serial benzoic acid standard solution is detected in the sample liquid to be detected, and the deviation between the relative abundance ratio of the selected monitoring ion pair in the chromatogram of the sample liquid to be detected and the ion relative abundance ratio (k) of the benzoic acid standard solution with a corresponding concentration is not more than the range specified in the table 1, determining that the corresponding compound is detected in the sample to be detected. In the invention, the detection conditions of the ultra-high performance liquid-tandem mass spectrum of the serial benzoic acid standard solution are the same as those of the ultra-high performance liquid-tandem mass spectrum detection of the sample liquid to be detected, and the description is omitted here.
TABLE 1 maximum allowable deviation of relative ion abundance for characterization
In the present invention, the concentration of benzoic acid standard in the series of benzoic acid standard solutions is preferably 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 25. Mu.g/mL, and 50. Mu.g/mL, respectively, and the solvent in the series of benzoic acid standard solutions is preferably acetonitrile aqueous solution; the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50-90%, more preferably 60-80%, and most preferably 70-75%.
In the invention, the detection result is preferably automatically optimized by using an automatic optimization software Optimizer, data Acquisition is used for Data measurement, and Qualitative Analysis 10.0.0 software is used for Data analysis.
In the present invention, the obtaining of the quantitative detection result preferably includes the steps of: comparing chromatographic peak data of a sample to be detected obtained by ultra-high performance liquid-tandem mass spectrometry detection with a preset standard curve to obtain the quantitative detection result, wherein the standard curve is a linear relation curve between the concentration of a benzoic acid standard in a benzoic acid standard solution and the chromatographic peak area of the benzoic acid standard.
In the present invention, the method for producing the standard curve preferably includes the steps of:
and performing ultra-high performance liquid-tandem mass spectrometry detection on the serial benzoic acid standard solution to obtain the chromatographic peak area of the serial benzoic acid standard, and drawing a standard curve by taking the concentration of the benzoic acid standard in the serial benzoic acid standard solution as an abscissa and the chromatographic peak area of the benzoic acid standard as an ordinate to obtain a linear regression equation. In the invention, the detection conditions of the ultra-high performance liquid-tandem mass spectrum of the serial benzoic acid standard solution are the same as those of the ultra-high performance liquid-tandem mass spectrum detection of the sample liquid to be detected, and the description is omitted here.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In an embodiment of the present invention, the specifications of the specific instrument, reagent and sample to be tested are as follows:
the instrument is specifically: ultra performance liquid chromatography-mass spectrometry (Agilent company 1290 information II-6470B); ultrasonic breaker (ShenNa SN-PS 250); a high-speed cryocentrifuge (Eppendorf, germany); analytical balance (Shimadzu Corp., japan).
The reagent specifically comprises: acetonitrile was chromatographically pure and purchased from Sigma-Aldrich company; potassium ferrocyanide, zinc acetate and glacial acetic acid are all excellent purity and purchased from Sigma-Aldrich company; the dilution water is drohent water; benzoic acid standards were purchased from the national institute of metrology.
The conditions of the ultra-high performance liquid chromatography detection of the ultra-high performance liquid-tandem mass spectrometry detection are as follows: the chromatographic column is Agilent Zorbax SB-C 18 Chromatographic column (2.1X105 mm,1.8 μm); mobile phase A is water, mobile phase B is acetonitrile; the elution mode is isocratic elution, wherein the volume fraction of the mobile phase A is 30% and the volume fraction of the mobile phase B is 70% in the isocratic elution process, and the isocratic elution time is 5min; the flow rate of the mobile phase is 0.2mL/min; the column temperature is 35 ℃; the sample loading was 5. Mu.L.
The conditions for tandem mass spectrometry detection of the ultra-high performance liquid-tandem mass spectrometry detection are as follows: the detection mode is negative ion multiple reaction monitoring; the scanning mode is multi-reaction ion scanning; the ion source is an electrospray ion source; the ion source temperature is preferably 300 ℃; the capillary voltage is 4000V; the photomultiplier voltage is 400V; the in-source fragmentation voltage was 72V; collision energy is 8eV; the collision gas is nitrogen, and the air flow of the collision gas is 7L/min; sprayer pressure was 15psi; the qualitative ion pair is 121.0/77.0, and the quantitative ion pair is 77.0; the benzoic acid retention time was 1.127min.
In all embodiments of the invention, the broiler gastrointestinal content is myogastric content, duodenal content, jejunal content, ileal content, cecal content, or rectal content.
Example 1
Determination of Linear regression equation, correlation coefficient, linear Range, detection limit and quantitative limit of benzoic acid
Preparing a series of benzoic acid standard substance solutions: 1mg/mL of standard benzoic acid solution is diluted into 5000 mug/mL of benzoic acid standard stock solution by using the chen water, the benzoic acid standard stock solution is diluted into microporous filter membranes with the concentration of 0.25 mug/mL, 0.5 mug/mL, 1 mug/mL, 2.5 mug/mL, 5 mug/mL, 25 mug/mL and 50 mug/mL respectively by using the 70v/v% acetonitrile water solution, and then the microporous filter membranes are filtered, so that a series of benzoic acid standard solution is obtained. And (3) taking a series of benzoic acid standard substance solutions for ultra-high performance liquid-tandem mass spectrometry detection to obtain a benzoic acid standard substance chromatogram and a benzoic acid standard substance chromatogram. Fig. 1 is a chromatogram of a benzoic acid standard substance, fig. 2 is a chromatogram of a benzoic acid standard substance, as can be seen from fig. 1, water and acetonitrile are selected as mobile phases, isocratic elution is used, the concentration of the benzoic acid standard substance solution is 1 μg/mL, and the proportion of the mobile phases is water: acetonitrile (v/v) =30: at 70, the benzoic acid peak type is good; as can be seen from fig. 2, since benzoic acid is an acidic compound, protons are liable to lose one unit of negative charge, and thus negative ion mode detection is employed. When the concentration of the benzoic acid standard substance solution is 1 mug/mL, the excimer ion signal obtained by ESI negative ion mode is strong, the interference is less, and the mass spectrum is automatically optimized to obtain benzoic acid: m/z 77.0 as quantitative ion of benzoic acid.
According to the signal to noise ratio S/N=10 of the chromatographic peak of the quantitative ion as the quantitative limit of the method, the detection limit of the benzoic acid is 0.53 mug/mL, and the quantitative limit is 1.81 mug/mL.
Benzene is prepared by taking the concentration of benzoic acid standard substances in a series of benzoic acid standard substance solutions as the abscissaAnd (3) drawing a standard curve by taking the chromatographic peak area of the formic acid standard product as an ordinate to obtain a linear regression equation. The linear regression equation is y=47328x+607.6, the correlation coefficient r 2 =1.0000, the linear range is 0.25 to 50 μg/mL.
Comparative example 1
Selection of mobile phase ratio
Benzoic acid standard substances with the concentration of 5 mug/g are added into the jejunum contents of the broiler chickens, the detection of the ultra-high performance liquid-tandem mass spectrum is respectively carried out, the conditions of the detection of the ultra-high performance liquid-tandem mass spectrum are the same as those of the embodiment, and the difference from the embodiment is that in the isocratic elution process, the ratio of mobile phases is water: acetonitrile (v/v) =80: 20. 70: 30. 60: 40. 50: 50. 40: 60. 30:70 and 20:80. fig. 3 shows a benzoic acid chromatogram-mobile phase ratio 80 in the jejunal content of broilers: 20, a step of; fig. 4 shows a benzoic acid chromatogram-mobile phase ratio 70 in the jejunal content of broilers: 30; fig. 5 is a benzoic acid chromatogram-mobile phase ratio 60 in the jejunal content of broilers: 40, a step of performing a; fig. 6 is a benzoic acid chromatogram-mobile phase ratio 50 in the jejunal content of broilers: 50; fig. 7 is a benzoic acid chromatogram-mobile phase ratio 40 in the jejunal content of broilers: 60; fig. 8 is a benzoic acid chromatogram-mobile phase ratio 30 in the jejunal content of broilers: 70; fig. 9 is a benzoic acid chromatogram-mobile phase ratio 20 in the jejunal content of broilers: 80. from fig. 3 to 9, from the peak shape and the peak-out time, the mobile phase ratio is water: acetonitrile (v/v) =30: and a better detection effect can be achieved at 70.
Example 2
Method recovery rate, RSD and precision
About 0.200g of jejunum content of broiler chickens is weighed and placed in a 1.5mL centrifuge tube, benzoic acid standard substances with different concentration levels and 1.0mL acetonitrile water solution (the volume fraction of acetonitrile is 70%) are precisely added, and ultrasonic extraction is carried out for 30min (the frequency of ultrasonic is 40 Hz) after shaking and mixing. Then 40 mu L of 100g/L potassium ferrocyanide aqueous solution and 40 mu L of 200g/L zinc acetate-glacial acetic acid mixed aqueous solution are added to remove protein, and after uniform mixing, the mixture is centrifuged at 8000rpm for 10min to precipitate protein. Transferring all the supernatant to a volumetric flask, fixing the volume to 10mL by using acetonitrile water solution (the volume fraction of acetonitrile is 70%), shaking uniformly, filtering by using a microporous filter membrane with the size of 0.22 mu m, and respectively performing ultra-high performance liquid-tandem mass spectrometry detection. Wherein, the concentration levels of benzoic acid standard addition in the jejunum content of the broiler chickens are respectively 0.5 mug/g, 5 mug/g and 50 mug/g. Table 2 shows the recovery of benzoic acid in the jejunal content of broilers and the relative standard deviation (n=6, i.e. 6 determinations per addition level).
Table 2 recovery of benzoic acid in jejunal contents of broiler chickens and relative standard deviation
As can be seen from Table 2, the average recovery of benzoic acid was between 91.37 and 98.04% with a Relative Standard Deviation (RSD) of 1.24 to 2.46% at 3 different concentration levels of 0.5. Mu.g/g, 5. Mu.g/g and 50. Mu.g/g.
The precision of the method was evaluated by repeated measurements on the labeled samples, and table 3 shows the results of the benzoic acid precision measurement (n=6) in the jejunal contents of broilers.
Table 3 determination of benzoic acid precision in jejunal contents of broiler chickens
As can be seen from Table 3, the method provided by the invention has good repeatability, accords with the quantitative methodology regulation, and is suitable for quantitative determination of benzoic acid in the gastrointestinal tract of broiler chickens.
Comparative example 2
Selection of pretreatment method
Weighing about 0.200g of jejunum content of broiler chickens, placing into a 1.5mL centrifuge tube, precisely adding benzoic acid standard substances with different concentration levels and 1.0mL acetonitrile water solution (the volume fraction of acetonitrile is 70%), carrying out ultrasonic extraction for 30min (the ultrasonic frequency is 40 Hz) after shaking and mixing uniformly, centrifuging for 5min at a rotating speed of 8000rpm, extracting supernatant obtained by centrifugation by a PRiME HLB solid phase extraction column (the solid phase extraction column is pre-activated by methanol and ultrapure water), eluting by anhydrous methanol, collecting eluent, drying by a gentle nitrogen flow, adding acetonitrile water solution with the volume fraction of 70% to dissolve residues, carrying out vortex re-dissolution, and respectively carrying out ultra-high performance liquid phase-tandem mass spectrum detection by a 0.22 mu m filter membrane. Wherein, the concentration levels of benzoic acid standard addition in the jejunum content of the broiler chickens are respectively 0.5 mug/g, 5 mug/g and 50 mug/g. Table 4 shows the recovery rate of benzoic acid in the jejunal content of broilers and the relative standard deviation (n=6, solid phase extraction treatment).
Table 4 recovery of benzoic acid in jejunal content of broiler chickens and relative standard deviation (n=6, solid phase extraction treatment)
As shown in Table 4, under the condition of 3 different standard concentration levels of 0.5 mug/g, 5 mug/g and 50 mug/g, the average recovery rate of benzoic acid in a sample to be detected obtained through solid phase extraction column purification is 92.03-98.82%, the Relative Standard Deviation (RSD) is 0.64-1.30%, the average recovery rate of benzoic acid is equivalent to that of the detection method provided by the invention, and the relative standard deviation of benzoic acid in the detection method provided by the invention is also similar to that of the solid phase extraction method, so that the detection method provided by the invention has the advantages that the detection accuracy is not reduced, and the cost, time and steps of pretreatment of the sample to be detected are saved.
The precision of the method was evaluated by repeated measurement of the labeled sample, and table 5 shows the results of the benzoic acid precision measurement (n=6, solid phase extraction treatment) in the jejunal contents of broilers.
Table 5 determination of benzoic acid precision in jejunal contents of broiler chickens (n=6, solid phase extraction treatment)
As can be seen from table 3 and table 5, the detection method provided by the invention has good repeatability when compared with the detection method using the solid phase extraction method as the pretreatment method, meets the quantitative methodology rule, is suitable for quantitative determination of benzoic acid in the gastrointestinal tract of broiler chickens, but the sample pretreatment method has simpler steps and lower cost.
Example 3
Detection of absorption and distribution of different types of benzoic acid in gastrointestinal tract of broiler chickens
The chickens at 40 days are randomly divided into 2 groups for treatment, 8 times are repeated for each group, 4 chickens are repeated for each time, and pure benzoic acid and benzoic acid treated by a coating process are respectively fed, wherein the feeding dosage is 60mg. At 0.5h, 1h, 2h and 4h after feeding, samples of the gastrointestinal tract of broiler chickens (myogastric, duodenal, jejunal, ileal, cecal and rectal contents) were taken at each time point and the absorption and distribution of benzoic acid in each segment of the gastrointestinal tract of broiler chickens was examined.
Taking 5.00g of potassium ferrocyanide, adding water to a volume of 50mL after dissolving a proper amount of water, and obtaining a potassium ferrocyanide aqueous solution with the concentration of 100 g/L.
10.00g of zinc acetate is taken, after a small amount of water is dissolved, 6mL of glacial acetic acid is added, water is added to fix the volume to 50mL, and the zinc acetate-glacial acetic acid mixed aqueous solution with the zinc acetate concentration of 200g/L is obtained.
About 0.200g of each sample of the gastrointestinal tract contents (myostomach, duodenum, jejunum, ileum, cecum and rectum contents) of the broiler chickens are weighed respectively, placed in a 1.5mL centrifuge tube, 1.0mL acetonitrile water solution (the volume fraction of acetonitrile is 70%) is precisely added, and after shaking and mixing, ultrasonic extraction is carried out for 30min (the ultrasonic frequency is 40 Hz). Then 40 mu L of 100g/L potassium ferrocyanide aqueous solution and 40 mu L of 200g/L zinc acetate-glacial acetic acid mixed aqueous solution are added to remove protein, and after uniform mixing, the mixture is centrifuged at 8000rpm for 10min to precipitate protein. Transferring all the supernatant into a volumetric flask, fixing the volume to 10mL by using acetonitrile water solution (the volume fraction of acetonitrile is 70%), shaking uniformly, and filtering by using a microporous filter membrane with the size of 0.22 mu m to obtain the to-be-detected sample liquid of the gastrointestinal tract contents (myogastric, duodenal, jejunal, ileum, cecum and rectal contents) of the broiler after feeding pure benzoic acid and feeding the benzoic acid-coated broiler respectively.
Respectively taking the sample liquid to be tested, and injecting the sample liquid into an ultra-high performance liquid chromatography-tandem mass spectrometer to obtain a chromatogram and a mass spectrum of benzoic acid in gastrointestinal tract contents (myogastric, duodenal, jejunal, ileum, cecum and rectal contents) of broiler chickens, as shown in fig. 10-21, wherein fig. 10-17 are chromatograms and mass spectra of benzoic acid in gastrointestinal tract contents of fed-batch benzoic acid broiler chickens: FIG. 10 is a chromatogram of benzoic acid in the stomach contents of broiler chickens; FIG. 11 is a mass spectrum of benzoic acid in the stomach contents of broiler chickens; FIG. 12 is a chromatogram of benzoic acid in the duodenal contents of broiler chickens; FIG. 13 is a mass spectrum of benzoic acid in the contents of the duodenum of broilers; FIG. 14 is a chromatogram of benzoic acid in the jejunal content of broilers; FIG. 15 is a mass spectrum of benzoic acid in the jejunal content of broilers; FIG. 16 is a chromatogram of benzoic acid in the ileal content of broiler chickens; fig. 17 is a mass spectrum of benzoic acid in the ileal content of broiler chickens. Fig. 18-21 are chromatograms and mass spectra of benzoic acid in gastrointestinal contents of pure benzoic acid fed chickens: FIG. 18 is a chromatogram of benzoic acid in the cecal content of broilers; FIG. 19 is a mass spectrum of benzoic acid in the cecum content of broilers; FIG. 20 is a chromatogram of benzoic acid in the rectal content of broiler chickens; fig. 21 is a mass spectrum of benzoic acid in the rectal content of broiler chickens. As shown in figures 10-21, benzoic acid in the gastrointestinal tract content of broiler chickens can be effectively detected after the pure benzoic acid is fed and the benzoic acid is coated.
The benzoic acid content in the samples of the gastrointestinal tract contents (myogastric, duodenal, jejunal, ileal, cecal, rectal contents) was calculated according to the above-described detection results and standard curves, and the results are shown in table 6 and table 7, wherein table 6 is the benzoic acid content in the gastrointestinal tract contents of the broiler chickens after the benzoic acid-coated pure product was fed, and table 7 is the benzoic acid content in the gastrointestinal tract contents of the broiler chickens after the benzoic acid-coated product was fed.
TABLE 6 benzoic acid content in the gastrointestinal contents of broiler chickens after being fed with pure benzoic acid (unit: μg/g)
TABLE 7 benzoic acid content in the gastrointestinal contents of broiler chickens fed with benzoic acid coating (unit: μg/g)
As can be seen from tables 6 to 7, the gastric-passing rate of the benzoic acid treated by the coating process is higher than that of the pure benzoic acid, and the benzoic acid can be slowly released in each section of the intestinal tract and enter the rear section of the small intestine to play a role in bacteriostasis.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A method for detecting benzoic acid in gastrointestinal contents of monogastric animals, comprising the steps of:
extracting a sample of gastrointestinal tract contents of a monogastric animal to be detected by adopting an acetonitrile aqueous solution to obtain an extracting solution;
deproteinizing the extracting solution to obtain a sample solution to be detected;
performing ultra-high performance liquid-tandem mass spectrometry detection on the sample liquid to be detected to obtain a detection result of benzoic acid;
the detection result comprises a qualitative detection result and/or a quantitative detection result;
the conditions for ultra performance liquid chromatography detection of the ultra performance liquid-tandem mass spectrometry detection include: the chromatographic column is C 18 A chromatographic column; mobile phase A is water, mobile phase B is acetonitrile; the elution mode is isocratic elution, and the volume fraction of the mobile phase A in the isocratic elution process is 20-40%; the flow rate of the mobile phase is 0.2-1.0 mL/min; the column temperature is 25-45 ℃; the sample injection amount is 2-20 mu L;
the conditions for tandem mass spectrometry detection of the ultra-high performance liquid-tandem mass spectrometry detection include: the detection mode is negative ion multiple reaction monitoring; the scanning mode is multi-reaction ion scanning; the ion source is an electrospray ion source; the temperature of the ion source is 250-380 ℃; the capillary voltage is 3500-4500V; the voltage of the photomultiplier is 300-450V; the source internal fragmentation voltage is 60-100V; the collision energy is 5-15 eV; the qualitative ion pair was 121.0/77.0 and the quantitative ion pair was 77.0.
2. The method according to claim 1, wherein the volume fraction of mobile phase a during the isocratic elution is 30%.
3. The detection method according to claim 1, wherein the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-90%.
4. The method according to claim 3, wherein the solid-to-liquid ratio of the sample of gastrointestinal contents of monogastric animal to be tested and acetonitrile aqueous solution is 1g: 2.5-10 mL.
5. The detection method according to any one of claims 1 to 3, wherein the extraction is ultrasonic extraction; the ultrasonic extraction frequency is 30-60 Hz, the power is 75-250W, and the time is 15-45 min.
6. The method according to claim 1, wherein the deproteinization is performed by using a protein precipitant comprising an aqueous solution of potassium ferrocyanide and an aqueous solution of zinc acetate-glacial acetic acid mixture.
7. The detection method according to claim 6, wherein the concentration of the potassium ferrocyanide aqueous solution is 50-150 g/L.
8. The method according to claim 6 or 7, wherein the solid-to-liquid ratio of the sample of gastrointestinal contents of the monogastric animal to be tested and the aqueous solution of potassium ferrocyanide is 1g:0.1 to 0.4mL.
9. The detection method according to claim 6, wherein the concentration of zinc acetate in the zinc acetate-glacial acetic acid mixed aqueous solution is 150-250 g/L and the concentration of glacial acetic acid is 90-150 mL/L.
10. The method according to claim 6 or 9, wherein the solid-to-liquid ratio of the sample of gastrointestinal contents of the monogastric animal to be tested and the zinc acetate-glacial acetic acid mixed aqueous solution is 1g:0.1 to 0.4mL.
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