CN112345661A - Method for determining content of florfenicol in veterinary drug gardenia flaviviridae oral liquid - Google Patents

Method for determining content of florfenicol in veterinary drug gardenia flaviviridae oral liquid Download PDF

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CN112345661A
CN112345661A CN202011133777.7A CN202011133777A CN112345661A CN 112345661 A CN112345661 A CN 112345661A CN 202011133777 A CN202011133777 A CN 202011133777A CN 112345661 A CN112345661 A CN 112345661A
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florfenicol
supernatant
gardenia
residue
oral liquid
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CN112345661B (en
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张萌萌
李宗进
姬桂花
郭超晖
李震
杜志毅
王璐
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Beijing Jingjie Testing Technology Co ltd
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Beijing Jingjie Testing Technology Co ltd
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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Abstract

The application relates to the technical field of detection, and particularly discloses a method for determining the content of florfenicol in a veterinary medicine gardenia flavivia oral liquid. The method comprises the following steps: s1, adding water into the sample to obtain a sample solution A to be detected; s2, adding ethyl acetate into the sample liquid A to be detected, and centrifuging to obtain a supernatant A and a residue A; repeatedly extracting the residue A to obtain a supernatant B, mixing the supernatants A and B to obtain a supernatant C, and blowing nitrogen to the supernatant C to obtain a residue B; s3, adding a methanol aqueous solution into the residue B for redissolving, adding n-hexane for degreasing, fully and uniformly mixing, centrifuging, and removing the upper n-hexane layer; repeatedly removing fat to obtain a standby liquid; s4, passing the standby liquid through a solid phase extraction column, then leaching with water, eluting with methanol, collecting the eluent, and blowing and drying with nitrogen to obtain residue C; and S5, adding water into the residue C for redissolving, uniformly mixing by vortex, centrifuging, filtering by a membrane to obtain a supernatant D, and detecting the supernatant D by a liquid chromatography-tandem mass spectrometer. The method and the device have the effects of high detection efficiency and accurate detection result.

Description

Method for determining content of florfenicol in veterinary drug gardenia flaviviridae oral liquid
Technical Field
The application relates to the technical field of medicine detection, in particular to a method for determining the content of florfenicol in a veterinary medicine gardenia flavivia oral liquid.
Background
Florfenicol is a synthetic monofluoro derivative of thiamphenicol, is white or grey white crystalline powder, is odorless, is a novel broad-spectrum antibacterial agent of veterinary special chloramphenicol, is successfully developed in the late eighties, has broad antibacterial spectrum, good absorption, wide in-vivo distribution, safe and efficient, and has obvious treatment effect on bacterial diseases of livestock and poultry caused by sensitive bacteria. It can enter bacteria, and can prevent protein synthesis to achieve antibacterial effect, and can be widely used for preventing and treating bacterial diseases of livestock and poultry in domestic and foreign animal husbandry.
The veterinary gardenia oral liquid is a livestock product, and the main components of the veterinary gardenia oral liquid comprise Chinese pulsatilla root, coptis root, phellodendron bark, ash bark, oriental wormwood, gardenia, ligusticum chuanxiong hort, astragalus root, tripterygium wilfordii, poria cocos and rhizoma alismatis.
However, in the related technology, the detection limit of the method for detecting the content of florfenicol in the veterinary gardenia flavivia oral liquid is too high, and the problem of providing a detection method with a lower detection limit is urgently needed to be solved in the field.
Disclosure of Invention
Aiming at solving the problem that the content detection limit of florfenicol in the oral liquid of the veterinary drug gardenia flavivirida is too high. The application provides a method for determining florfenicol in gardenia flavivis oral liquid, which adopts the following technical scheme:
a method for determining the content of florfenicol in an oral liquid containing gardenia jasminoides comprises the following steps:
s1, adding water into the veterinary drug gardenia oral liquid to be detected to obtain a sample liquid A to be detected;
s2, adding ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating and centrifuging to obtain supernatant A and residue A; adding ethyl acetate into the residue A, repeatedly extracting to obtain a supernatant B, combining the supernatant B and the supernatant A to obtain a supernatant C, and blowing and drying the supernatant C by nitrogen to obtain a residue B;
s3, adding a methanol aqueous solution into the residue B for redissolution to obtain a redissolved solution A, adding n-hexane for degreasing, fully mixing uniformly, and centrifuging at the rotating speed of 3000-5000r/min for 2-8min to remove the n-hexane on the upper layer;
adding n-hexane, fully mixing, centrifuging at the rotating speed of 3000-5000r/min for 2-8min, removing the n-hexane at the upper layer, and repeatedly removing grease once to obtain a standby liquid;
s4, passing the standby liquid through a solid phase extraction column, then leaching with water, eluting with methanol, collecting the eluent, and blowing and drying with nitrogen to obtain residue C;
s5, adding water into the residue C to obtain a redissolved solution B, uniformly mixing the redissolved solution B in a vortex mode, centrifuging for 2-8min at the rotating speed of 9000-15000r/min, then passing through a microfiltration membrane to obtain a supernatant D, and filtering the supernatant D to perform liquid chromatography-tandem mass spectrometer detection.
By adopting the technical scheme, the sample liquid A to be detected is obtained by adding water into the veterinary drug gardenia flaviviridae oral liquid for dissolution.
In step S2, adding ethyl acetate into sample liquid a to be tested, mixing and oscillating, and then centrifuging, wherein in the process, ethyl acetate is used as an extractant to extract the target compound florfenicol, a supernatant a is obtained after extraction is completed, and a part of florfenicol remains in residue a after primary extraction; at the moment, ethyl acetate is added into the residual residue A again to repeatedly extract the florfenicol once to obtain a supernatant B, so that the extraction degree of the florfenicol reaches the maximum limit, the maximum extraction rate of the florfenicol is further reached, the florfenicol content in the sample liquid finally used for liquid chromatography detection is high, the detection limit is low, and the detection is more accurate.
In step S3, adding methanol aqueous solution into residue B for redissolving, wherein the florfenicol has high solubility in the methanol aqueous solution, and the florfenicol can be fully dissolved by adding the methanol aqueous solution; then adding n-hexane saturated by methanol aqueous solution, wherein the n-hexane can fully extract fat in the gardenia jasminoides oral liquid, so that the fat removing effect is realized, and the interference of the fat on the subsequent purification treatment and the liquid chromatography-tandem mass spectrometry detection is reduced; adding n-hexane, fully mixing uniformly, and then carrying out centrifugal treatment to remove the n-hexane on the upper layer, so that impurities are prevented from being mixed, the florfenicol is prevented from being interfered in the extraction, and the purpose of separating and removing fat is further realized; repeating the above operation, repeating the degreasing once, and removing the fat twice to remove the impurity fat in the oral liquid of the veterinary drug gardenia jasminoides to the maximum extent so as to improve the purity of the target florfenicol and further improve the accuracy of the experimental result.
In step S4, the solid phase extraction column is purified by water and methanol to remove impurities in the solid phase extraction column and create a certain solvent environment; meanwhile, the methanol has stronger penetrability in the solid phase extraction column, so that the standby liquid can flow through the solid phase extraction column at a higher speed to prepare for further concentration treatment; and (3) finishing the elution process of the standby solution, and further removing the interferents to the maximum extent, so that the target florfenicol is retained, and the target florfenicol with the highest purity is provided for the subsequent experimental steps.
In step S5, redissolving the residue B obtained in step S4 with water to obtain a redissolved solution B, then performing vortex mixing, centrifuging, and finally passing through a microfiltration membrane to further remove the remaining impurities in the redissolved solution, thereby obtaining florfenicol with higher purity, so that the detection result of the subsequent test is more accurate, and simultaneously, the damage of impurities to the liquid chromatography-tandem mass spectrometer can be reduced, and the service life of the experimental instrument can be prolonged.
In step S6, the redissolved solution B is centrifuged by using a centrifuge, so that solid insoluble substances with small particle sizes, which cannot be dissolved in the redissolved solution B, are separated, the influence of impurities on florfenicol during subsequent liquid chromatography tandem mass spectrometry is reduced, the impurities in the redissolved solution B are effectively removed, the occurrence of impurity peaks during subsequent liquid chromatography tandem mass spectrometry is reduced, and the influence of impurities on the florfenicol detection result is reduced.
The application provides a scheme of florfenicol in animal remedy gardenia flavivia oral liquid is drawed completely to high efficiency, each step in the scheme is simple and high-efficient, gradually realize the high purity extraction to the florfenicol in animal remedy gardenia flavivia oral liquid, provide certain basis for follow-up liquid chromatogram-tandem mass spectrometry detection florfenicol content's survey, accurate quantitative determination to florfenicol content that must accomplish, simultaneously, have that the testing result detects the limit low, the accuracy is high and the high advantage of sensitivity.
Preferably, in step S1, the concentration of the veterinary drug gardenia yellow oral liquid in the sample liquid a to be tested is 0.8-1.2 g/mL.
By adopting the technical scheme, the veterinary gardenia yellow oral liquid is dissolved by adopting water, the dosage of the veterinary gardenia yellow oral liquid and the dosage of the veterinary gardenia yellow oral liquid are limited in a certain range, so that the water-soluble components in the veterinary gardenia yellow oral liquid are distributed into the water phase, and the florfenicol in the veterinary gardenia yellow oral liquid is convenient to extract.
Preferably, in step S2, the volume ratio of the added amount of ethyl acetate to the added water in step S1 is 1: (3-5).
By adopting the technical scheme, ethyl acetate is added into the sample liquid A to be detected twice, and the florfenicol is repeatedly extracted twice, so that the amount of the extracted florfenicol is higher, and the extraction rate of the florfenicol reaches the maximum value.
Preferably, in step S2, the oscillation time is not less than 10 min.
By adopting the technical scheme, the oscillation with sufficient time is adopted to ensure that different components in the sample liquid to be detected are fully contacted with the ethyl acetate, so that the different components are distributed to the components to which the different components belong, and the effect of separating the different components in the sample to be detected is achieved.
Preferably, in step S2, centrifugation is performed at 3000-5000r/min for 5-15min after oscillation.
By adopting the technical scheme, the centrifugal treatment is carried out at a certain rotating speed, so that the layered components are distributed more stably, and the supernatant A is obtained.
Preferably, in step S3, the n-hexane is a solution of n-hexane saturated with a 5% volume fraction of aqueous methanol.
By adopting the technical scheme, the redissolved solution A is degreased by adding the organic solvent n-hexane, impurity fat contained in the redissolved solution A is removed, the influence of the fat on the subsequent detection and determination of the florfenicol content by liquid chromatography-tandem mass spectrometry is reduced, and the effect of reducing the mixed impurities can be achieved.
Preferably, in step S3, the aqueous methanol solution added to the residue B is a 5% aqueous methanol solution, and the volume ratio of the aqueous methanol solution to the added n-hexane is 1: 1, and the volume ratio of the water to the water added in the step S1 is (1.5-2.5): 1.
by adopting the technical scheme, the volume of the added methanol aqueous solution and the volume of the n-hexane have a certain ratio, so that the n-hexane can fully extract fat, and the interference of the fat on subsequent purification treatment and liquid chromatography-tandem mass spectrometry detection is reduced.
Preferably, in step S5, the membrane pore size of the microfiltration membrane is 0.22 μm.
By adopting the technical scheme, due to the adoption of the microfiltration membrane, impurity particles with the particle size of more than 0.22 mu m in the redissolution B can be removed, the subsequent detection of the florfenicol is facilitated, and the aim of more accurate detection result is fulfilled.
In summary, the present application has the following beneficial effects:
1. according to the detection method, in the pretreatment process before detection, the sample liquid to be detected is extracted twice by using ethyl acetate, so that the amount of the extracted florfenicol is more, the extraction rate of the sample liquid to be detected is higher, a good preparation basis is provided for the subsequent liquid chromatography tandem mass spectrometry detection, the quantitative detection of the florfenicol is realized, the sensitivity is high, the detection limit is low, and the detection result precision is high.
2. According to the detection method, n-hexane is added in the pretreatment process before detection, so that the effect of removing fat is effectively achieved, and meanwhile, impurities except for a target can be reduced and mixed into florfenicol, so that the purpose of separating and removing fat is achieved; meanwhile, the repeated degreasing is carried out once, so that the purity of the target florfenicol is higher, and the interference of impurities on subsequent purification treatment and liquid chromatography-tandem mass spectrometry detection is reduced.
3. The detection method adopted by the application has strong repeatability, when the content of florfenicol in the veterinary drug gardenia flavivirida oral liquid is lower (5.0 mug/kg), the recovery rate of the detection method is 81.2%, the national standard (60-120%) is met, and the standard deviation of the detection method meets the national standard and is as low as 3.18%; when the content of florfenicol in the veterinary drug gardenia flaviviridae oral liquid is slightly high (10.0 mug/kg), the recovery rate of the detection method is 91%, the national standard (60-120%) is met, and the standard deviation of the detection method meets the national standard and is as low as 3.87%; when the content of florfenicol in the veterinary drug gardenia flavivirida oral liquid is higher (50.0 mug/kg), the recovery rate of the detection method is 82.9 percent, the national standard (60-120 percent) is met, and the standard deviation of the detection method meets the national standard and is as low as 2.22 percent.
Drawings
FIG. 1 is a LC-MS/MS Multiple Reaction Monitoring (MRM) chromatogram of a blank gardenia oral liquid of the present application;
FIG. 2 is an LC-MS/MS Multiple Reaction Monitoring (MRM) chromatogram of a blank gardenia jasminoides oral solution to which florfenicol was added at 5.0. mu.g/kg (detection limit concentration) in the present example.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
The concentration of the veterinary drug gardenia oral liquid in the sample liquid A to be detected is 0.8-1.2g/mL, namely 0.8-1.2g of the veterinary drug gardenia oral liquid is added into 1mL of water.
Examples
Example 1
A method for detecting the content of florfenicol in a veterinary drug gardenia flaviviridae oral liquid comprises the following steps:
s1, taking a homogeneous sample of 2.5g of veterinary drug gardenia oral liquid and 2.5mL of water, and dissolving 2.5g of veterinary drug gardenia oral liquid in 2.5mL of water to obtain sample liquid A to be detected;
s2, adding 10mL of ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating for 10min, then carrying out centrifugal treatment on a centrifugal machine at the rotation speed of 4000r/min for 10min to obtain supernatant A, and transferring the supernatant A into a 50mL centrifugal tube;
adding 10mL of ethyl acetate into the residue A again for repeated extraction once to obtain a supernatant B, combining the supernatant A and the supernatant B into the same 50mL centrifuge tube to obtain a supernatant C, drying the supernatant C under a nitrogen blowing instrument at 45 ℃, removing the ethyl acetate in the supernatant C, and obtaining a residue B;
s3, adding 4mL of 5% methanol aqueous solution into the residue B obtained in the step to obtain a redissolved solution A, adding 4mL of n-hexane saturated with 5% methanol aqueous solution into the redissolved solution A to remove fat, fully mixing uniformly, and centrifuging on a centrifuge at the rotation speed of 4000r/min for 5min so as to remove the n-hexane in the redissolved solution A and reduce the influence of impurities on the detection result;
adding n-hexane, fully mixing, centrifuging at the rotating speed of 3000-5000r/min for 2-8min, removing the n-hexane at the upper layer, and repeatedly removing grease once to obtain a standby liquid;
s4, extracting the standby liquid by using a C18 solid-phase extraction column, activating the solid-phase extraction column (with the specification of 200mg and 6mL) by using 3mL of methanol and 3mL of water in sequence to finish the process of rinsing the solid-phase extraction column, and allowing all the standby liquid to pass through the column to finish the purification process; after the standby liquid passes through the column, leaching with 3mL of water, activating with 3mL of water, collecting eluent, and drying the eluent under a nitrogen blowing instrument at 45 ℃ to obtain residue C;
s5, adding 1mL of water into the residue C for redissolving to obtain a redissolution B, uniformly mixing by vortex, then completely transferring the redissolution B into a 1.5mL centrifuge tube for centrifugation at 12000r/min for 6min, and finally passing through a 0.22-micron microfiltration membrane to obtain a supernatant D; and filtering the supernatant D, and detecting by using a liquid chromatography-tandem mass spectrometer. In the liquid phase detection, the selected chromatographic column is a C18 column, 2.1 x 50mm and 1.7 μm (namely, the inner diameter of the C18 column is 2.1mm, the length of the C18 column is 50mm, and the pore diameter of the filler in the C18 column is 1.7 μm), wherein the mobile phase A used in the liquid phase chromatography is acetonitrile, and the mobile phase B is water. The mass spectrum conditions are as follows: by adopting negative ion ionization, the ion pair is 356.15 & gt 185.2, the voltage of a cone hole is 30V, the energy of collision gas is 22V, the ion pair is 356.15 & gt 336.15, the voltage of the cone hole is 30V, and the energy of the collision gas is 16V.
Example 2
The method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the embodiment 1, and the difference between the embodiment and the embodiment 1 is that the setting parameters of each operation in the embodiment are different, and the method specifically comprises the following steps:
s1, taking a homogeneous sample of 2g of veterinary gardenia jasminoides oral liquid and 2.5mL of water which are the same as those in the embodiment 1, and dissolving the veterinary gardenia jasminoides oral liquid in the water to obtain a sample liquid A to be detected;
s2, adding 5mL of ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating for 10min, then carrying out centrifugal treatment, wherein the selected rotating speed is 3000r/min, the centrifugal time is 15min, and transferring the obtained supernatant A into a 50mL centrifugal tube after the centrifugal treatment is finished;
adding 5mL of ethyl acetate into the residue A for repeated extraction to obtain a supernatant B, combining the supernatant A and the supernatant B into the same 50mL centrifuge tube to obtain a supernatant C, and finally drying the supernatant C under a 45 ℃ nitrogen blowing instrument to obtain a residue B;
s3, adding 3.75mL of 5% methanol aqueous solution into the residue B obtained in the step to obtain a redissolved solution A, adding 3.75mL of n-hexane saturated with 5% methanol aqueous solution into the redissolved solution A, fully mixing uniformly, and centrifuging on a centrifuge at the rotating speed of 3000r/min for 8min so as to remove the n-hexane in the redissolved solution A and reduce the influence of impurities on the detection result; repeatedly removing fat once to obtain a standby liquid;
s4, adopting a C18 solid phase extraction column to carry out an extraction process, taking a fixed extraction column (the specification is 200mg, 6mL) to activate with 3mL of methanol and 3mL of water in sequence, completing the process of rinsing the solid phase extraction column, taking all standby liquid to pass through the column, and completing the purification process of the standby liquid; after the standby liquid passes through the column, leaching with 3mL of water, activating with 3mL of water, collecting eluent, and drying the eluent under a nitrogen blowing instrument at 45 ℃ to obtain residue C;
s5, adding 1mL of water into the residue C for redissolving to obtain a redissolution B, uniformly mixing by vortex, then completely transferring the redissolution B into a 1.5mL centrifuge tube for centrifugation, wherein the selected centrifugation speed is 9000r/min, the time is 8min, and finally passing through a 0.22 mu m microfiltration membrane to obtain a supernatant D; and filtering the supernatant D, and detecting by using a liquid chromatography-tandem mass spectrometer.
The other operations were the same as in example 1.
Example 3
The method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the embodiment 1, and the difference between the embodiment and the embodiment 1 is that the setting parameters of each operation in the embodiment are different, and the method specifically comprises the following steps:
s1, taking 3g of veterinary gardenia yellow oral liquid and 2.5mL of water which are the same as the homogeneous sample in the embodiment 1, and dissolving 2.5g of veterinary gardenia yellow oral liquid in 2.5mL of water to obtain sample liquid A to be detected;
s2, adding 15mL of ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating for 20min, then carrying out centrifugal treatment, wherein the selected rotating speed is 5000r/min, the centrifugal time is 5min, and transferring the obtained supernatant A into a 50mL centrifugal tube after the centrifugal treatment is finished;
adding 15mL of ethyl acetate into the residue A for repeated extraction to obtain a supernatant B, combining the supernatant A and the supernatant B into the same centrifugal tube to obtain a supernatant C, and finally blowing the supernatant C to dry under a 45 ℃ nitrogen blowing instrument to obtain a residue B;
s3, adding 6.25mL of 5% methanol aqueous solution into the residue B obtained in the step to obtain a redissolved solution A, adding 6.25mL of n-hexane saturated with 5% methanol aqueous solution into the redissolved solution A, fully mixing uniformly, and centrifuging on a centrifuge at the rotation speed of 5000r/min for 2min so as to remove the n-hexane in the redissolved solution A and reduce the influence of impurities on a detection result; repeatedly removing fat once to obtain a standby liquid;
s4, adopting a C18 solid phase extraction column to carry out an extraction process, taking the solid phase extraction column (the specification is 200mg, 6mL) to activate with 3mL of methanol and 3mL of water in sequence, completing the process of rinsing the solid phase extraction column, taking all standby liquid to pass through the column, and completing the purification process; after the standby liquid passes through the column, leaching with 3mL of water, activating with 3mL of water, collecting eluent, and drying the eluent under a nitrogen blowing instrument at 45 ℃ to obtain residue C;
s5, adding 1mL of water into the residue C for redissolving to obtain a redissolution B, uniformly mixing by vortex, then completely transferring the redissolution B into a 1.5mL centrifuge tube, centrifuging at the selected centrifugal speed of 15000r/min for 5min, and finally passing through a 0.22-micron microfiltration membrane to obtain a supernatant D; and filtering the supernatant D, and detecting by using a liquid chromatography-tandem mass spectrometer. The other operations were the same as in example 1.
Example 4
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, except that the addition amount of ethyl acetate in the step S2 is 5mL, and the operation is the same as that in the example 1.
Example 5
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, except that the addition amount of ethyl acetate in the step S2 is 15mL, and the operation is the same as that in the example 1.
Example 6
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, except that in the step S3, the addition amount of a 5% methanol aqueous solution is 3.75mL, and the operation is the same as that in the example 1.
Example 7
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, except that in the step S3, the addition amount of a 5% methanol aqueous solution is 6.25mL, and the operation is the same as that in the example 1.
Example 8
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, and the difference is that in the step S3, the addition amount of 5% methanol aqueous solution is 4mL, and the addition amount of n-hexane saturated by 5% methanol aqueous solution is 6mL, and the operation is the same as that in the example 1.
Example 9
A method for detecting the content of florfenicol in a veterinary drug gardenia flavivia oral liquid is carried out according to the method in the example 1, and the difference is that in the step S3, the addition amount of 5% methanol aqueous solution is 4mL, and the addition amount of n-hexane saturated by 5% methanol aqueous solution is 3mL, and the operation is the same as that in the example 1.
Comparative example
Comparative example 1
A method for detecting the content of florfenicol in a veterinary drug gardenia flaviviridae oral liquid is carried out according to the method in the embodiment 1, and the difference between the comparative example and the embodiment 1 is that: in step S2, the ammonia water-ethyl acetate mixed solution is used to extract the target florfenicol, and the other operations are the same as those in example 1.
Comparative example 2
A method for detecting the content of florfenicol in a veterinary drug gardenia flaviviridae oral liquid is carried out according to the method in the embodiment 1, and the difference between the comparative example and the embodiment 1 is as follows: in step S2, the ethyl acetate is used to extract the florfenicol as the target compound for 1 time, specifically: s2, adding 10mL of ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating for 10min, then carrying out centrifugal treatment on a centrifugal machine at the rotation speed of 4000r/min for 10min to obtain supernatant A, transferring the supernatant A into a 50mL centrifugal tube, drying the supernatant A under a nitrogen blowing instrument at 45 ℃, removing the ethyl acetate, and obtaining residue B; the other operations were the same as in example 1.
Comparative example 3
A method for detecting the content of florfenicol in a veterinary drug gardenia flaviviridae oral liquid is carried out according to the method in the embodiment 1, and the difference between the comparative example and the embodiment 1 is as follows: in the step S3, n-hexane is adopted to carry out degreasing on the redissolved solution for 1 time, specifically: s3, adding 4mL of 5% methanol aqueous solution into the residue B obtained in the step to obtain a redissolved solution A, adding 4mL of n-hexane saturated with 5% methanol aqueous solution into the redissolved solution A, fully mixing uniformly, removing fat, and centrifuging on a centrifuge at the rotation speed of 4000r/min for 5min so as to remove the n-hexane in the redissolved solution A and reduce the influence of impurities on a detection result; the other operations were the same as in example 1.
Performance test
Detection limit of detection method
The detection limit of the method for detecting the content of florfenicol in the oral liquid of the gardenia flavivirida of the veterinary drugs in the examples 1 to 3, 4 to 9 and the comparative examples 1 to 3 is determined, and the detection method is as follows: 2.5g of the veterinary drug gardenia jasminoides oral liquid is taken, and added with a florfenicol standard substance to prepare the florfenicol-veterinary drug gardenia jasminoides oral liquid with a certain concentration (C, mu g/kg, which means the content of florfenicol in the veterinary drug gardenia oral liquid per unit), and the content of the florfenicol is determined by adopting the method in the embodiment 1.
When florfenicol under a certain florfenicol concentration is detected, calculating the signal-to-noise ratio S/N of the florfenicol, and when the S/N is more than or equal to 3, marking the concentration as C1(. mu.g/kg), followed by a formulation below C1The florfenicol-veterinary gardenia yellow oral liquid with other concentrations is detected, and C is obtained from all concentration results that florfenicol with certain concentration can be detected and S/N is more than or equal to 31、C2、C3、……、CnThe minimum value obtained in the screening is the detection limit of the method of this example, i.e., the detection limit is Min (C)1，C2，C3，……，Cn)。
Determining the detection limit of the method for detecting the content of florfenicol in the veterinary drug gardenia flavivirida oral liquid in example 1 according to the determination method, and determining a blank gardenia flavivirida oral liquid LC-MS/MS multiple reaction detection (MRM) chromatogram by adopting the detection method in example 1 as shown in figure 1;
as shown in fig. 2, the LC-MS/MS multiple reaction detection (MRM) chromatogram obtained by adding 5.0 μ g/kg (detection limit concentration) of florfenicol to a blank gardenia jasminoides ellis oral solution shows that fig. 2 shows that the detection limit of example 1 of the present application is 5.0 μ g/kg, and the signal-to-noise ratio S/N corresponding to the detection limit is 3.45.
Similarly, the detection limit of the method for detecting the content of florfenicol in the veterinary drug gardenia yellow oral liquid in the examples 2 to 9 and the comparative example is determined according to the method, and the determination results are shown in the following table 1.
TABLE 1 detection limits of florfenicol content detection method in veterinary drug gardenia flavivirida oral liquid
Figure BDA0002736005130000091
As can be seen from the above table 1, the detection method adopted by the application for detecting the oral liquid of the veterinary drug gardenia jasminoides has the detected detection limit value of 5.0 mug/kg and the lowest detection limit.
Calibration curve of (II) detection method
The method for correcting the calibration curve of the detection method comprises the following specific operations:
1. preparation of florfenicol standard stock solutions: accurately weighing a proper amount of florfenicol standard substance, dissolving with methanol, fixing the volume and preparing into 100mg/L florfenicol standard stock solution.
2. Preparation of matrix standard working solution: the blank sample extract is used to prepare a series of matrix standard working solutions with the concentration of 0, 12.5, 25.0, 50.0, 100.0 and 200.0 mu g/L.
3. And (3) filtering the solution through a 0.22-micron filter membrane, taking the filtrate, placing the filtrate in a sample injection bottle, and detecting under the experimental detection conditions in the example 1.
The specific results of the calibration curve based on the above detection method are shown in table 2.
Table 2 detection results of the detection method of the present application on florfenicol standard substance
c(μg/L) Peak area
0 0
25 2383.805
50 4934.464
100 9983.107
250 20167.068
500 38647.324
According to the data in table 1, when the liquid chromatography-tandem mass spectrometer is used for detection, the peak area is recorded as y, the concentration c (μ g/L) of florfenicol in the sample solution obtained by the correction curve is recorded as x, and then the linear regression equation between the peak area y and the concentration x of florfenicol is as follows: 196.389x +3.01177, R2＝0.999471。
(III) determination of reproducibility test
Calculating the detection result based on the standard curve of the detection method, wherein the calculation formula of the content X (mu g/kg) of the florfenicol in the sample is as follows: x is c × V/m × f × 1000/1000
Wherein, the meaning of the parameter indicated by each letter is as follows: c-the concentration of florfenicol (μ g/L) in the sample solution obtained in the standard working curve; m-the quality (g) of the veterinary drug gardenia jasminoides oral liquid; v-volume of reconstituted solution (mL) obtained after final reconstitution of the sample; f-sample dilution factor (that is, when the supernatant D finally obtained in step S5 is detected, if the content of florfenicol in the supernatant C is large and the measurement data cannot fall within the standard curve, the supernatant C needs to be diluted, and the dilution factor is denoted as f, where f is equal to or greater than 1).
The examiner performed the actual operation test, and the examples were all performed by the detection method of example 1. The practical operation test is carried out by adding and recovering the blank gardenia oral liquid, the adding concentrations are respectively shown in the following table, each concentration is 6 parallel concentrations, and the obtained recovery rate and the relative standard deviation are shown in the table 1.
Table 3 repeatability test of the test method of example 1 of the present application
Figure BDA0002736005130000101
According to the relevant regulations in appendix 4 of GB/T27404-: 60-120%, relative standard deviation range: 21-30 percent; when the concentration of florfenicol added is 10.0. mu.g/kg, the recovery rate range of the detection result is required to be: 60-120%, relative standard deviation range: 15 to 21 percent; when the concentration of florfenicol added is 50 mug/kg, the recovery rate of the detection result is required to be as follows: the range is as follows: 60-120%, relative standard deviation range: 15 to 21 percent. Therefore, the recovery rate of the detection result of the inspector meets the requirement in GB/T27404-2008, and the relative standard deviation of the recovery rate is better than the result in GB/T27404-2008.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims (8)

1. A method for measuring the content of florfenicol in a veterinary drug gardenia flaviviridae oral liquid is characterized by comprising the following steps:
s1, adding water into the veterinary drug gardenia oral liquid to be detected to obtain a sample liquid A to be detected;
s2, adding ethyl acetate into the sample liquid A to be detected, fully and uniformly mixing, oscillating and centrifuging to obtain supernatant A and residue A;
adding ethyl acetate into the residue A, repeatedly extracting to obtain a supernatant B, combining the supernatant B and the supernatant A to obtain a supernatant C, and blowing and drying the supernatant C by nitrogen to obtain a residue B;
s3, adding a methanol aqueous solution into the residue B for redissolution to obtain a redissolved solution A, adding n-hexane for degreasing, fully mixing uniformly, and centrifuging at the rotating speed of 3000-5000r/min for 2-8min to remove the n-hexane on the upper layer;
adding n-hexane, fully mixing, centrifuging at the rotating speed of 3000-5000r/min for 2-8min, removing the n-hexane at the upper layer, and repeatedly removing grease once to obtain a standby liquid;
s4, passing the standby liquid through a solid phase extraction column, then leaching with water, eluting with methanol, collecting the eluent, and blowing and drying with nitrogen to obtain residue C;
s5, adding water into the residue C to obtain a redissolved solution B, uniformly mixing the redissolved solution B in a vortex mode, centrifuging for 2-8min at the rotating speed of 9000-15000r/min, then passing through a microfiltration membrane to obtain a supernatant D, and filtering the supernatant D to perform liquid chromatography-tandem mass spectrometer detection.
2. The method for determining the content of florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S1, the concentration of the veterinary drug gardenia yellow oral liquid in the sample liquid A to be detected is 0.8-1.2 g/mL.
3. The method for determining the content of florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S2, the volume ratio of the added amount of ethyl acetate to the water added in step S1 is 1: (3-5).
4. The method for determining florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S2, the oscillation time is not less than 10 min.
5. The method for determining florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S2, after the oscillation, the mixture is centrifuged for 5-15min at the rotating speed of 3000-5000 r/min.
6. The method for determining florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S3, the n-hexane is saturated with an aqueous methanol solution having a volume fraction of 5%.
7. The method for determining florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S3, the aqueous methanol solution added to the residue B was a 5% aqueous methanol solution, and the volume ratio of the aqueous methanol solution to the added n-hexane was 1: 1, and the volume ratio of the water to the water added in the step S1 is (1.5-2.5): 1.
8. the method for determining florfenicol in a veterinary drug gardenia flavivis oral liquid according to claim 1, which is characterized in that: in step S5, the membrane pore size of the microfiltration membrane is 0.22 μm.
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