CN112461823A - Rapid detection method of florfenicol - Google Patents
Rapid detection method of florfenicol Download PDFInfo
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- CN112461823A CN112461823A CN202011246196.4A CN202011246196A CN112461823A CN 112461823 A CN112461823 A CN 112461823A CN 202011246196 A CN202011246196 A CN 202011246196A CN 112461823 A CN112461823 A CN 112461823A
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
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Abstract
The invention discloses a method for quickly detecting florfenicol, which comprises the following steps: the method comprises the following steps: pretreatment, namely cleaning a sample and extracting an organic solvent; step two: adding 5ml of acetonitrile ethyl acetate into the extracting solution of the organic solvent, fully oscillating for about 30 seconds, oscillating through a vortex oscillator, adding 10 ml of ethyl acetate, uniformly mixing in a vortex manner for 2 minutes, and centrifuging at 5000r/min for 5 minutes. According to the method for rapidly detecting the florfenicol, a large amount of fat and other endogenous interferents are removed by washing a hydrolysate with ethyl acetate in the sample pretreatment process; the anti-interference capability of the detection method on other substances in the edible tissue of the pig is obviously improved, the detection cost is reduced, the using amount of organic solvents is reduced, the quantitative analysis result is reliable, and the method is suitable for basic detection units to perform daily monitoring on the florfenicol total residues in the edible tissue of the pig and adopts an internal standard method for quantification.
Description
Technical Field
The invention relates to the technical field of florfenicol detection, in particular to a rapid detection method of florfenicol.
Background
Florfenicol, also known as florfenicol, is a fluorinated derivative of chloramphenicol, has no nitro group which is potentially caused by aplastic anemia in a molecular structure, and has wide antibacterial spectrum and easy absorption. Florfenicol has therefore become a major replacement for chloramphenicol and is widely used in the treatment of gram-positive, gram-negative and chloramphenicol resistant bacteria in animals. However, the result of the reproductive toxicity test shows that florfenicol has the effects of blood toxicity, embryo toxicity and immunosuppression, and adverse reactions such as anorexia, diarrhea and the like can also occur after the florfenicol is taken, and the human health can be greatly harmed by eating a large amount of pork with overproof florfenicol. Currently, due to the clinical misuse of veterinary drugs, the resistance of bacteria to florfenicol is also increasing and multiple resistances are present over time. For a long time, the drug resistance of bacteria can be greatly improved, and common drugs have no effective inhibition effect on the bacteria. Not only does this result in economic waste, but it also increases the difficulty of veterinary clinical treatment, and even threatens human life safety.
Drug residue monitoring typically detects urine or blood samples from live animals, or tissue samples, such as muscle, liver, etc., from slaughtered animals. Due to the time lag of veterinary drug residue detection, and the fact that veterinary drugs are affected by biological macromolecules such as enzymes in biological tissues, the veterinary drugs are usually easy to metabolize and have high clearance rate, so that sometimes, it is difficult to trace some abnormal or illegal veterinary drugs. Establishing a rapid and sensitive detection method and searching a biopsy sample which can be sampled in vivo are two important directions for veterinary drug residue analysis and research. In the past decade, the detection of illicit drugs such as stimulants in human hair by forensic medicine has been inspired, and the feasibility of animal hair as a test material has been discussed and studied. Because animal hair is easy to collect, transport and store, and more importantly, the characteristic of safety evaluation can be made before slaughter, the hair lacks blood circulation, lacks various active substances for degrading drugs and has a quick excretion way, so that the drug metabolism is slow, and the retention time in the hair is far longer than that of other tissues. Compared with general tissue samples (muscle, liver, kidney, fat, lung, etc.), hair can be obtained without slaughtering the animal, and is simple and convenient. In addition, due to the structural and component characteristics of the hair and lower metabolic activity, the long retention time of the medicine in the hair after entering the hair is determined, the medicine can be used as a long-term detection sample, which is incomparable with urine or blood, especially for the medicine with short half-life period, such as florfenicol and the like, after a certain period of medicine holiday, the medicine residue can not be detected from edible tissues or blood and urine to prove whether the forbidden medicine is used, and the hair can still be used as the detection sample for a long time due to slow metabolism of the medicine in the hair, so that the proof basis for the forbidden medicine is provided. The method for detecting the florfenicol drug residue in the pig hair has important significance for effectively monitoring the use of the drugs and ensuring the safety of animal food in China.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme:
the invention relates to a method for rapidly detecting florfenicol, which comprises the following steps:
the method comprises the following steps: pretreatment, namely cleaning a sample and extracting an organic solvent;
step two: adding 5ml of acetonitrile ethyl acetate into the extracting solution of the organic solvent, fully oscillating for about 30 seconds, oscillating through a vortex oscillator, adding 10 ml of ethyl acetate, uniformly mixing in a vortex manner for 2 minutes, and centrifuging at 5000r/min for 5 minutes;
step three: blowing the clear liquid at the temperature of 60 ℃ by using nitrogen, dissolving the residue by adding 1 ml of water, swirling for 30 seconds, and filtering by using a filter membrane to obtain a liquid to be detected;
step four: and (4) extracting 100 mu L of the solution to be detected on the upper layer of the solution to be detected, inserting a detection strip, and reading a detection result.
As a preferred technical solution of the present invention, the pretreatment in the first step includes the following steps:
s1: cleaning the surface of the sample, sucking the surface moisture by using filter paper after cleaning, and drying the sample at the temperature of 50 ℃ for later use;
s2: shearing the dried sample, adding 2 g of the sheared sample into a centrifuge tube, adding 50 mu L of florfenicol-D3 standard working solution and 5mL of 0.5mol/L sodium hydroxide solution, uniformly mixing, keeping the temperature in a water bath at 80 ℃ for 1 hour, taking out, and cooling to room temperature.
In the second step, acetonitrile and ethyl acetate are mixed according to the volume ratio of 1: 1.
In the third step, after vortexing for 30 seconds, the supernatant is taken and filtered through a 0.22 μm organic filter membrane.
As a preferred technical solution of the present invention, in the fourth step, the result is interpreted:
negative (-): the color rendering ratio of the T line to the C line is strong, which indicates that the carbendazim in the sample is lower than the detection limit;
weakly positive (±): the line T is consistent with the line C, which indicates that the carbendazim in the sample is positioned near the detection limit;
positive (+): the color development of the T line is weaker than that of the C line, which indicates that the carbendazim in the sample is higher than the detection limit.
In the fourth step, the detection strip comprises absorbent paper, an NC film, a gold label pad and a sample pad, the gold label pad is formed by burning, marking, spraying and drying 1% chloroauric acid and 1% trisodium citrate, and then is cut into strips, and the sample pad is cut, soaked and dried.
As a preferred technical scheme, the detection box comprises a box cover and a box body, wherein a clamping sleeve seat is installed at the edge of the surface of the box body, a plurality of detection strips are arranged in the box body, two supporting top plates are arranged at the bottoms of the detection strips, the two ends of each supporting top plate are respectively arranged on the two sides of the box body in a sliding mode, elastic pieces are arranged at the bottoms of the supporting top plates, an outlet is formed in one side, close to the supporting top plates, of the box body, limiting grooves are respectively formed in the inner walls of the two sides of the box body, and the two ends of each supporting top plate are movably clamped in the limiting grooves.
As a preferable technical scheme of the invention, two positioning rollers are installed in the box cover, two ends of each positioning roller are respectively and rotatably connected to the inner walls of two sides of the box cover, a paper twisting roller is sleeved on the surface of each positioning roller, the positioning rollers are in transmission connection through a belt, and one end of each positioning roller movably penetrates through the box cover and is fixedly connected with a rotating block.
As a preferable technical scheme of the invention, the florfenicol-D3 standard working solution is added into the S2, and the concentration is 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L, 40.5 mug/L and 1-3 ml/bottle.
The invention has the beneficial effects that: according to the method for rapidly detecting the florfenicol, a large amount of fat and other endogenous interferents are removed by washing a hydrolysate with ethyl acetate in the sample pretreatment process; the anti-interference capability of the detection method on other substances in the edible tissue of the pig is obviously improved, the detection cost is reduced, the using amount of an organic solvent is reduced, the quantitative analysis result is reliable, the method is suitable for a basic detection unit to carry out daily monitoring on the florfenicol total residue in the edible tissue of the pig by adopting an internal standard method for quantification, the detection Limit (LOD) of the florfenicol is 0.5 mu g/kg, the quantification Limit (LOQ) is 1.5 mu g/kg, the method has higher recovery rate and lower detection limit and quantification limit, the detection cost is reduced, the using amount of the organic solvent is reduced, the quantitative analysis result is reliable, and the method is suitable for the basic detection unit to carry out daily monitoring on the florfenicol total residue in the edible tissue of the pig.
Drawings
FIG. 1 is a flow chart of a method for rapid detection of florfenicol of the present invention;
FIG. 2 is a flow chart of the pretreatment of a method for the rapid detection of florfenicol of the present invention;
FIG. 3 is a processing flow chart of a detection strip of the rapid detection method of florfenicol of the present invention;
FIG. 4 is an exploded view of a detection box of the rapid detection method of florfenicol of the present invention.
In the figure: 1. a box cover; 2. rotating the block; 3. a positioning roller; 4. twisting paper rolls; 5. a belt; 6. a test strip; 7. a clamping sleeve seat; 8. a limiting groove; 9. supporting a top plate; 10. a spring plate; 11. an outlet; 12. and (5) a box body.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example (b): as shown in figures 1-4, the method for rapidly detecting florfenicol comprises the following steps:
the method comprises the following steps: pretreatment, namely cleaning a sample and extracting an organic solvent;
step two: adding 5ml of acetonitrile ethyl acetate into the extracting solution of the organic solvent, fully oscillating for about 30 seconds, oscillating through a vortex oscillator, adding 10 ml of ethyl acetate, uniformly mixing in a vortex manner for 2 minutes, and centrifuging at 5000r/min for 5 minutes;
step three: blowing the clear liquid at the temperature of 60 ℃ by using nitrogen, dissolving the residue by adding 1 ml of water, swirling for 30 seconds, and filtering by using a filter membrane to obtain a liquid to be detected;
step four: and (4) extracting 100 mu L of the solution to be detected on the upper layer of the solution to be detected, inserting a detection strip, and reading a detection result.
Wherein, the pretreatment in the first step comprises the following steps:
s1: cleaning the surface of the sample, sucking the surface moisture by using filter paper after cleaning, and drying the sample at the temperature of 50 ℃ for later use;
s2: shearing the dried sample, adding 2 g of the sheared sample into a centrifuge tube, adding 50 mu L of florfenicol-D3 standard working solution and 5mL of 0.5mol/L sodium hydroxide solution, uniformly mixing, keeping the temperature in a water bath at 80 ℃ for 1 hour, taking out, and cooling to room temperature.
Wherein in the second step, acetonitrile and ethyl acetate in the acetonitrile ethyl acetate are mixed according to the volume ratio of 1: 1.
Wherein, in the third step, after vortexing for 30 seconds, the supernatant was taken and filtered through a 0.22 μm organic filter membrane.
And in the fourth step, the result is interpreted:
negative (-): the color rendering ratio of the T line to the C line is strong, which indicates that the carbendazim in the sample is lower than the detection limit;
weakly positive (±): the line T is consistent with the line C, which indicates that the carbendazim in the sample is positioned near the detection limit;
positive (+): the color development of the T line is weaker than that of the C line, which indicates that the carbendazim in the sample is higher than the detection limit.
In the fourth step, the detection strip comprises absorbent paper, an NC membrane, a gold label pad and a sample pad, wherein the gold label pad is cut into strips after gold burning, marking, gold spraying and drying are carried out on 1% of chloroauric acid and 1% of trisodium citrate, and the sample pad is cut, soaked and dried.
Wherein, the detection box includes lid 1 and box body 12, the surperficial edge of box body 12 installs the card sleeve seat 7, and be equipped with a plurality of test strips 6 in the box body 12, the bottom of test strip 6 is equipped with two roof supports 9, and the both ends of roof support 9 slide respectively and set up the both sides at box body 12, roof support 9 bottom is equipped with shell fragment 10, and be close to one side of roof support 9 on the box body 12 and seted up export 11, spacing groove 8 has been seted up on the both sides inner wall of box body 12 respectively, and the both ends activity card of roof support 9 establishes in spacing groove 8.
Wherein, install two registration rollers 3 in the lid 1, and the both ends of registration roller 3 rotate respectively and connect on the both sides inner wall of lid 1, and the surface cover of registration roller 3 is equipped with twists with fingers paper roll 4, and connects through belt 5 transmission between the registration roller 3, and lid 1 is passed in the activity of one end of registration roller 3 to fixedly connected with changes piece 2.
Wherein, in S2, florfenicol-D3 standard working solution with the concentration of 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L, 40.5 mug/L and 1-3 ml/bottle is added.
The working principle is as follows: determination of the ratio of acetonitrile: the ethyl acetate is extracted according to the volume ratio of 1:1, the detection limit is low, the sensitivity is high, the recovery is stable, a reliable analysis method is established for the determination of the florfenicol amine residual quantity in the animal-derived matrix, and a large amount of fat and other endogenous interferents are removed by washing hydrolysate with ethyl acetate in the sample pretreatment process; the invention obviously improves the anti-interference capability of the detection method to other substances in the edible tissue of the pig, simultaneously reduces the detection cost, reduces the dosage of organic solvent, has reliable quantitative analysis result, is suitable for the basal layer detection unit to carry out daily monitoring on the florfenicol total residue in the edible tissue of the pig by adopting an internal standard method for quantification, has the detection Limit (LOD) of 0.5 mu g/kg and the quantification Limit (LOQ) of 1.5 mu g/kg, has higher recovery rate and lower detection limit and quantification limit, simultaneously reduces the detection cost, reduces the dosage of organic solvent, has reliable quantitative analysis result, is suitable for the basal layer detection unit to carry out daily monitoring on the florfenicol total residue in the edible tissue of the pig, can carry out easy extraction after detection, obtain the florfenicol, avoids the waste, and the rotary block 2 drives the positioning roller 3 to rotate, therefore, the paper twisting roller 4 moves on the surface of the detection strip 6, the detection strip 6 is exposed out of the box body 12 through the outlet 11, and the bottom of the detection strip 6 is supported through the two supporting top plates 9, so that the paper twisting roller 4 is always attached to the surface of the detection strip 6.
Finally, it should be noted that: in the description of the present invention, it should be noted that the terms "vertical", "upper", "lower", "horizontal", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for rapidly detecting florfenicol is characterized by comprising the following steps:
the method comprises the following steps: pretreatment, namely cleaning a sample and extracting an organic solvent;
step two: adding 5ml of acetonitrile ethyl acetate into the extracting solution of the organic solvent, fully oscillating for about 30 seconds, oscillating through a vortex oscillator, adding 10 ml of ethyl acetate, uniformly mixing in a vortex manner for 2 minutes, and centrifuging at 5000r/min for 5 minutes;
step three: blowing the clear liquid at the temperature of 60 ℃ by using nitrogen, dissolving the residue by adding 1 ml of water, swirling for 30 seconds, and filtering by using a filter membrane to obtain a liquid to be detected;
step four: and (4) extracting 100 mu L of the solution to be detected on the upper layer of the solution to be detected, inserting a detection strip, and reading a detection result.
2. The method for rapidly detecting florfenicol as claimed in claim 1, wherein the pretreatment in the first step comprises the following steps:
s1: cleaning the surface of the sample, sucking the surface moisture by using filter paper after cleaning, and drying the sample at the temperature of 50 ℃ for later use;
s2: shearing the dried sample, adding 2 g of the sheared sample into a centrifuge tube, adding 50 mu L of florfenicol-D3 standard working solution and 5mL of 0.5mol/L sodium hydroxide solution, uniformly mixing, keeping the temperature in a water bath at 80 ℃ for 1 hour, taking out, and cooling to room temperature.
3. The method for rapidly detecting florfenicol as claimed in claim 1, wherein in the second step, acetonitrile and ethyl acetate in the acetonitrile ethyl acetate are mixed according to a volume ratio of 1: 1.
4. The method for rapidly detecting florfenicol as claimed in claim 1, wherein in step three, after vortexing for 30 seconds, supernatant is taken and filtered by 0.22 μm organic filter membrane.
5. The method for rapidly detecting florfenicol as claimed in claim 1, wherein in step four, the results are interpreted:
negative (-): the color rendering ratio of the T line to the C line is strong, which indicates that the carbendazim in the sample is lower than the detection limit;
weakly positive (±): the line T is consistent with the line C, which indicates that the carbendazim in the sample is positioned near the detection limit;
positive (+): the color development of the T line is weaker than that of the C line, which indicates that the carbendazim in the sample is higher than the detection limit.
6. The method for rapidly detecting florfenicol as claimed in claim 1, wherein in the fourth step, the detection strip comprises absorbent paper, NC membrane, gold label pad and sample pad, the gold label pad is cut into strips after being processed by gold burning, marking, gold spraying and drying by 1% chloroauric acid and 1% trisodium citrate, the sample pad is processed by cutting, soaking and drying, and the detection strip is arranged in the detection box.
7. The florfenicol rapid detection method as claimed in claim 6, wherein the detection box comprises a box cover (1) and a box body (12), a card sleeve seat (7) is installed at the edge of the surface of the box body (12), a plurality of detection strips (6) are arranged in the box body (12), two supporting top plates (9) are arranged at the bottom of the detection strips (6), two ends of the supporting top plates (9) are respectively arranged at two sides of the box body (12) in a sliding manner, an elastic sheet (10) is arranged at the bottom of the supporting top plates (9), an outlet (11) is arranged at one side of the box body (12) close to the supporting top plates (9), limiting grooves (8) are respectively arranged on the inner walls of two sides of the box body (12), and two ends of the supporting top plates (9) are movably clamped in the limiting grooves (8).
8. The florfenicol rapid detection method as claimed in claim 6, wherein two positioning rollers (3) are installed in the box cover (1), two ends of each positioning roller (3) are respectively rotatably connected to inner walls of two sides of the box cover (1), a twisting roller (4) is sleeved on the surface of each positioning roller (3), the positioning rollers (3) are in transmission connection through a belt (5), and one end of each positioning roller (3) movably penetrates through the box cover (1) and is fixedly connected with a rotating block (2).
9. The method for rapidly detecting florfenicol as claimed in claim 2, wherein the florfenicol-D3 standard working solution is added to S2 at concentrations of 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L and 1-3 ml/bottle.
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| CN202011246196.4A CN112461823A (en) | 2020-11-10 | 2020-11-10 | Rapid detection method of florfenicol |
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| CN202011246196.4A CN112461823A (en) | 2020-11-10 | 2020-11-10 | Rapid detection method of florfenicol |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005010168A2 (en) * | 2003-07-24 | 2005-02-03 | Charm Sciences, Inc. | Method and system for detecting chloramphenicol |
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| CN211122545U (en) * | 2019-10-25 | 2020-07-28 | 广州合尔拓生物科技有限公司 | Portable test paper |
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2020
- 2020-11-10 CN CN202011246196.4A patent/CN112461823A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005010168A2 (en) * | 2003-07-24 | 2005-02-03 | Charm Sciences, Inc. | Method and system for detecting chloramphenicol |
| CN106645477A (en) * | 2016-12-21 | 2017-05-10 | 青岛海润农大检测有限公司 | Method for detecting florfenicol amine residue and application |
| CN208488462U (en) * | 2018-08-13 | 2019-02-12 | 深圳市绿诗源生物技术有限公司 | A kind of reusable silaenafil detection card |
| CN111141907A (en) * | 2018-11-06 | 2020-05-12 | 武汉华美生物工程有限公司 | Florfenicol rapid detection kit and detection method |
| CN211122545U (en) * | 2019-10-25 | 2020-07-28 | 广州合尔拓生物科技有限公司 | Portable test paper |
Non-Patent Citations (1)
| Title |
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| 韩姣姣等: "氟苯尼考胶体金免疫层析定量检测方法的建立", 《分析化学》 * |
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