CN110658279B - Method for detecting ribavirin and metabolites thereof in livestock and poultry hair - Google Patents

Method for detecting ribavirin and metabolites thereof in livestock and poultry hair Download PDF

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CN110658279B
CN110658279B CN201910988310.1A CN201910988310A CN110658279B CN 110658279 B CN110658279 B CN 110658279B CN 201910988310 A CN201910988310 A CN 201910988310A CN 110658279 B CN110658279 B CN 110658279B
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ribavirin
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formic acid
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徐俊
谢敏
周瑶敏
闵佳玲
邬磊
万伟杰
胡丽芳
许晓红
肖勇
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Institute Of Agricultural Products Quality Safety And Standard Jiangxi Academy Of Agricultural Sciences
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention belongs to the technical field of drug residue detection, and particularly relates to a method for detecting ribavirin and metabolites thereof in livestock and poultry hairs. The method comprises the steps of carrying out homogeneous extraction on residual ribavirin and metabolites thereof in a sample by using a methanol solution of formic acid, carrying out dispersion purification on an extracting solution by using ethylenediamine-N-Propylsilane (PSA) and octadecylsilane chemically bonded (C18) matrixes, carrying out ultra performance liquid chromatography tandem mass spectrometry detection and internal standard method quantification, wherein a mobile phase A is 0.1% (v/v) ammonium formate acetate aqueous solution, a mobile phase B is methanol, the flow rate is 0.4mL/min, and a chromatographic column adopts a SB-Aq polar column with the size of 1.8 mu m and the size of 3mm multiplied by 100 mm. The method overcomes the defects of difficult sampling, need of live slaughter and the like, has strong specificity, high sensitivity, short time consumption and low requirement on sample purification by optimizing the sample processing step, and provides reliable technical support for supervision and control on the quality safety of livestock and poultry products.

Description

Method for detecting ribavirin and metabolites thereof in livestock and poultry hair
Technical Field
The invention belongs to the technical field of drug residue detection, and particularly relates to a method for detecting ribavirin and metabolites thereof in livestock and poultry hair.
Background
Ribavirin is a human-used broad-spectrum antiviral drug, and is clinically used for treating lassa fever, infant respiratory syncytial virus pneumonia, influenza A, hepatitis C and the like (Khatami, 2014).
Exposure to the "fast-growing chicken" event in 2012 indicated that the "human veterinary" phenomenon of ribavirin was extremely common during animal farming. Although 560 bulletin issued as early as 2005 in China clearly forbids the use of antiviral drugs for veterinary use by ribavirin and the like, effective antiviral drugs for veterinary use are lacked at present, the choice of farmers is limited, and ribavirin has good drug effect and low price, and farmers pursue benefit maximization for reducing breeding risk, and can still add ribavirin in feed and drinking water for treatment and prevention of viral diseases, so that the control of livestock and poultry epidemic diseases and the quality safety of livestock and poultry products are influenced, and the generation of bacterial drug resistance is increased, and further the health of human beings is threatened.
Research shows that ribavirin has two main metabolic pathways in animals: the method comprises the steps of firstly, carrying out reversible phosphorylation reaction under the action of nucleated cell adenosine kinase to form ribavirin monophosphate, ribavirin diphosphate and ribavirin triphosphate; secondly, degradation reactions, including enucleation saccharification and amide hydrolysis, occur to form the metabolite TCONH2And RTCOOH. At present, the national standards for ribavirin detection include a local standard of Jiangsu province, namely determination of total amount of ribavirin and metabolite residues in chicken liver-liquid chromatography tandem mass spectrometry and a commercial inspection industry recommended standard, namely determination of residual amount of ribavirin in export animal-derived food-liquid chromatography/mass spectrometry, which establish a ribavirin detection method by hydrolyzing a ribavirin phosphorylation product into a ribavirin raw drug by phosphatase, and relate to ribavirin and main metabolite TCONH thereof2The detection method of (2) has not been promulgated at present. Research shows that the main metabolite of ribavirin in livestock and poultry is TCONH2Meanwhile, the detection substrates of ribavirin and metabolites thereof in the current related livestock and poultry products are mainly muscle, liver and blood, the muscle and liver tissues need to obtain samples by animal slaughter and other modes, the blood collection also needs professional personnel, and low-temperature preservation treatment is needed after the sampling, so that the sampling and storage are inconvenient.
However, not only does hair eliminate slaughter, but hair is also removed from the animal as compared to normal tissue samplingThe sampling is convenient and simple, the residual time of the medicine in the hair is long, the metabolic degradation is slow, and the medicine can still be used as a sample for detection and analysis in a long period of time. Therefore, research on ribavirin and TCONH (human transport protein) as a main metabolite in livestock and poultry hair2The residue detection method has very important significance for effectively monitoring the forbidden use of the medicines, and ensuring the quality safety of livestock and poultry products and the health of consumers. For example, chinese patent document CN104155398A discloses a method for detecting residual amount of antiviral drugs in animal hair, but the sample needs a solid phase extraction column for purification, which takes a long time and has a low recovery rate. Therefore, a method for detecting ribavirin and metabolites thereof in livestock and poultry hair with simplified steps, easy operation and high sample recovery rate needs to be developed, and related ribavirin and metabolites TCONH thereof in livestock and poultry hair are not found at present2The report on the residual detection method and the detection standard of (1) are provided.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to overcome ribavirin and TCONH in the prior art2The problems of inconvenient sample sampling, difficult storage, complex operation, long time consumption, low recovery rate and the like of a detection sample are solved, so that the method for detecting the residual quantity of ribavirin and metabolites thereof in the hair of livestock and poultry is provided. The method has the advantages of simple pretreatment, high recovery rate, low detection limit, high sensitivity and wide application prospect.
Therefore, the invention provides the following technical scheme:
the invention provides a method for detecting ribavirin and metabolites thereof in livestock and poultry hair, which comprises the following steps:
preparation of test solution
Cleaning hair of livestock and fowl to be tested, cutting into pieces, grinding with liquid nitrogen into powder, adding13C5Oscillating and extracting a ribavirin internal standard working solution and a formic acid-methanol mixed solution to obtain an extracting solution, dispersing and purifying the extracting solution by using ethylenediamine-N-propyl silane and octadecyl silane, and dissolving the extracting solution by using a formic acid aqueous solution to obtain a test solution;
ultra-high performance liquid chromatography-tandem mass spectrometry detection
Liquid chromatography conditions: a chromatographic column: ZORBAX SB-Aq, 3.0mm X100 mm, 1.8 μm; the mobile phase A is ammonium formate acetate aqueous solution with the volume concentration of 0.1 percent, the mobile phase B is methanol, and the flow rate is as follows: 0.4 mL/min; column temperature: 35 ℃; sample introduction amount: 5.0 mu L; gradient elution procedure: maintaining for 0-2.5min for 0% B; 2.5-3.5min, the 0% B is changed to 98% B linearly; maintaining 98% B for 3.5-6 min; after 6-7min, the linear change of 98% B is reduced to 0% B;
mass spectrum conditions: electrospray ionization Positive ion mode, ESI+(ii) a The detection mode is as follows: multiple reaction monitoring, MRM; capillary voltage: 3000V; ion source temperature TEM: 150 ℃; temperature of drying gas: 150 ℃; flow rate of drying gas: 15L/min; atomizing gas pressure: 35 psi; temperature of sheath gas: 300 ℃; sheath airflow: 11L/min.
Further, the ethylenediamine-N-Propylsilane (PSA) and the octadecylsilane (C18) were used in amounts of 10 to 30mg/mL, respectively.
Further, the dispersion purification step is: and adding ethylenediamine-N-propylsilane and octadecylsilane into the extracting solution, carrying out vortex centrifugation, carrying out nitrogen blow-drying at the temperature of 40-50 ℃, and adding formic acid aqueous solution to dissolve residues to obtain a test solution.
Further, the reagent for cleaning the livestock and poultry hair is SDS with the mass concentration of 1% or Tween 80 with the mass concentration of 5%, and the cleaning time is 30-60 min.
Further, the mass of the livestock and poultry hair to be measured is measured, and the mass is measured13C5The usage amount of the ribavirin internal standard working solution is 100-250 mu L/g, and the concentration is 0.2-1.0 mg/L.
Further, in the formic acid-methanol mixed solution, the volume fraction of formic acid is 1-4%;
the using amount of the formic acid-methanol mixed solution is 50-100mL/g based on the mass of the livestock and poultry hair.
Further, the shaking extraction is extraction at 45-90 ℃ for 1-12 h;
preferably, the shake extraction is performed at 60-70 deg.C for 2-4 h.
Further, after the oscillation extraction, the method also comprises the steps of ultrasonic treatment and centrifugal treatment, wherein the ultrasonic time is 10-30 min.
Further, the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%.
Further, the quantitative ion of the ribavirin under the mass spectrum condition is m/z 245.1>113, qualitative ion is m/z 245.1>96；TCONH2The quantitative ion of (b) is m/z 113>96 and the qualitative ion is m/z 113>69 and13C5quantification of ribavirin ion of m/z 250>113。
The livestock and poultry hair in the invention includes but is not limited to: common animal hair such as pig hair, cow hair, wool, chicken hair, duck hair, goose hair, etc.
The technical scheme of the invention has the following advantages:
the invention provides a method for detecting ribavirin and metabolites thereof in livestock and poultry hairs, which comprises the following steps: cleaning hair of livestock and fowl to be tested, cutting into pieces, grinding with liquid nitrogen into powder, adding13C5Oscillating and extracting a ribavirin internal standard working solution and a formic acid-methanol mixed solution to obtain an extracting solution, dispersing and purifying the extracting solution by using ethylenediamine-N-propylsilane and octadecylsilyl, and dissolving the extracting solution by using a formic acid aqueous solution to obtain a test sample solution; liquid chromatography conditions: and (3) chromatographic column: ZORBAX SB-Aq, 3.0mm X100 mm, 1.8 μm; the mobile phase A is ammonium formate acetate aqueous solution with the volume concentration of 0.1 percent, the mobile phase B is methanol, and the flow rate is as follows: 0.4 mL/min; column temperature: 35 ℃; sample introduction amount: 5.0 mu L; gradient elution procedure: maintaining for 0-2.5min for 0% B; 2.5-3.5min, the 0% B is changed to 98% B linearly; maintaining 98% B for 3.5-6 min; after 6-7min, the linear change of 98% B is reduced to 0% B; mass spectrum conditions: electrospray ionization positive ion mode, ESI+(ii) a The detection mode is as follows: multiple reaction monitoring, MRM; capillary voltage: 3000V; ion source temperature TEM: 150 ℃; temperature of drying gas: 150 ℃; flow rate of drying gas: 15L/min; atomizing gas pressure: 35 psi; temperature of sheath gas: 300 ℃; the flow rate of the sheath gas: 11L/min. According to the detection method provided by the invention, the hair of the livestock and poultry is taken as a detection object, the livestock and poultry such as pigs, cattle, sheep, chickens, ducks and the like do not need to be killed, the sample is convenient to obtain, and the living body detection of the livestock and poultry can be realized; meanwhile, because the hair growth period is long, the medicine is easy to accumulate in the hair and is slowly degraded, the hair growth period is long, so that the medicine is easy to degrade, and the hair growth period is shortThe determination of the selected hair can more accurately implement risk early warning and safety supervision on the use of the forbidden medicine ribavirin in the production process of livestock and poultry. The preparation steps of the test solution provided by the invention have the advantages of clean treatment on the hair of livestock and poultry, low matrix effect, good extraction effect, less interference on the detection process, high accuracy of the detection result, great avoidance of the occurrence of results such as false positive, false negative and the like, ribavirin and TCONH2The recovery rate of the method is high, and particularly, the recovery rate of a sample can be greatly improved in the liquid nitrogen grinding step; the method has the advantages that the hairs are simultaneously digested and extracted by using formic acid-methanol, and the extract is dispersed and purified by using ethylenediamine-N-propyl silane and octadecylsilane, so that the use of a solid phase extraction column is avoided, the sample pretreatment time is greatly shortened, the operation is simple, and the ribavirin and TCONH in the hairs are efficiently separated and extracted2Low detection limit and high sensitivity.
According to the method for detecting ribavirin and the metabolites thereof in the livestock and poultry hair, provided by the invention, the recovery rate and the detection sensitivity of the sample can be further improved and the detection limit is reduced by optimizing and selecting the conditions in each step.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows ribavirin, TCONH2And13C5-a TIC chart of ribavirin standards.
FIG. 2 is a TIC diagram of a blank chicken feather in example 1 of the present invention;
FIG. 3 shows the addition of ribavirin and TCONH to the blank chicken feather in example 1 of the present invention2And13C5-a ribavirin standard TIC profile.
FIG. 4 is a graph of a positive chicken feather sample and an internal standard TIC in example 2 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
Condition investigation experiment:
1. preparation of standard solution:
standard stock solutions: accurately weighing appropriate amount of ribavirin and TCONH2And13C5-ribavirin standard, prepared into 1.0mg/mL standard stock solutions with methanol respectively, and stored at-20 ℃ in the dark.
Standard intermediate solution: and transferring an appropriate amount of standard stock solution into a 10mL volumetric flask, diluting the stock solution with methanol to obtain standard intermediate solution with the mass concentration of 10.0mg/L respectively, and storing the intermediate solution at the temperature of minus 20 ℃ in a dark place.
Standard working solution: and (3) transferring a proper amount of standard intermediate solution into a 10mL volumetric flask, diluting the intermediate solution into standard working solution with the mass concentration of 1.0mg/L by using methanol respectively, and storing the working solution at the temperature of minus 20 ℃ in a dark place.
2. Optimization of sample processing method conditions
(1) Selection of hydrolysis solvent
The structure of hair is dense, resulting in the drug being firmly retained in the biological structure of hair, and thus a hydrolysis method is required to break the structure of hair, so that the drug is released from the keratinized tissue. Common hair hydrolysis methods include acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and organic agent hydrolysis. The hydrolysis effects of 0.1mol/L HCl, 0.1mol/L NaOH, 2% formic acid-methanol, and 2% formic acid-acetonitrile were compared in this experiment. The specific results are shown in the following table:
TABLE 1 different extraction methods ribavirin and TCONH2Recovery of (N ═ 3)
Object of interest 2% formic acid-methanol 2% formic acid-acetonitrile 0.1mol/L HCL 0.1mol/L NaOH
Ribavirin (ribavirin) 103.4±3.6 90.9±3.0 77.1±3.2 43.5±2.8
TCONH2 99.2±4.5 87.4±4.3 68.9±6.0 39.1±1.4
The results show that: the extraction efficiency of 0.1mol/L NaOH in the four extraction solvents is the worst, the recovery rate of ribavirin is only 43.5 percent, and TCONH2The recovery was only 39.1%, probably due to the base with ribavirin and TCONH2The reaction, which causes a decrease in protonation efficiency, affects the formation of the excimer ion peak. MiningRibavirin and TCONH by extraction with 2% formic acid-methanol solvent2The recovery rate is highest and reaches 103.4 percent and 99.2 percent respectively, and is also obviously higher than 2 percent formic acid-acetonitrile and 0.1mol/L HCL. Combination of ribavirin and TCONH2Results of recovery of ribavirin and its metabolite TCONH after 2h of hydrolysis of the hair with 2% formic acid-methanol2The highest recovery rate is obtained.
(2) Selection of hydrolysis time and temperature
Hair hydrolysis time and temperature are controlled for ribavirin and TCONH2The extraction effect of (2) has a more obvious influence. The experiment compares the hydrolysis time of 1h, 2h and 12h at 45 ℃, 65 ℃, 90 ℃ with that of 1h, 2h and 12h on ribavirin and TCONH respectively2The result shows that when the hair is hydrolyzed at 65 ℃ for 2 hours, the ribavirin and TCONH2The recovery rate was highest, and the specific results are shown in Table 2.
TABLE 2 different hydrolysis times and temperatures for ribavirin and TCONH2Influence of recovery
Figure BDA0002237416750000071
(3) Effect of liquid nitrogen milling
Comparing the influence of whether liquid nitrogen milling is carried out on the recovery rate in the test of chicken feather, pig hair and cattle hair, and the specific results are shown in the following table 3:
TABLE 3 liquid nitrogen milling to hair with ribavirin and TCONH2Influence of recovery (N ═ 3)
Figure BDA0002237416750000081
From the data in the table above, it can be seen that the sample milled with liquid nitrogen can significantly improve the recovery of the target.
(4) Optimization of mass spectrometry conditions
Respectively adding 1.0mg/L of ribavirin and TCONH2And13C5-ribavirin standard solution, directly entering ion source without chromatography column, under positive and negative ion mode, respectively performing full sweepTo select the appropriate ionization regime and parent ions. The results show that ribavirin and TCONH2At ESI+Ionization in the mode can obtain higher abundance of parent ions. The fragmentation voltage was then optimized to maximize the parent ion strength. In the determination of ribavirin and TCONH2And performing secondary mass spectrometry by adopting a daughter ion mode, selecting 1 pair of fragment ions with relatively strongest abundance as quantitative ions, selecting 1 pair or 2 pairs of fragment ions with second strongest abundance as qualitative ions, and optimizing the collision energy of the daughter ions respectively. The optimized mass spectrum conditions are shown in table 4.
TABLE 4 ribavirin and TCONH in MRM monitoring mode2Mass spectrum optimization conditions of
Figure BDA0002237416750000082
2. Linear regression equation and detection limit
Formulation of ribavirin and TCONH2Mixing the standard solutions with mass concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100.0 μ g/L, and adding into the mixed solution13C5-ribavirin standard working solution 20.0 μ g/L was measured to quantify the ion peak area (y) versus concentration (x, μ g/L) for a standard curve. The results show that ribavirin and TCONH2Has a good linear relationship (r) within a range of 1.0 to 100.0. mu.g/L2>0.99). Ribavirin and TCONH2Respectively, Y is 0.078572X +0.060047 (R)20.9987) and Y0.100857X-0.028160 (R)2＝0.9997)。
Ribavirin and TCONH were added to different concentrations in blank chicken feather samples2And (3) detecting the standard solution according to the optimized experimental conditions from high to low mass concentration until the signal-to-noise ratio is equal to 3 (S/N-3) and the signal-to-noise ratio is equal to 10 (S/N-10), and determining ribavirin and TCONH in the chicken feather sample2The detection Limits (LOD) and the quantification Limits (LOQ) were 0.2. mu.g/kg and 0.5. mu.g/kg, respectively.
3. Recovery and precision
Taking a blank chicken feather sample, adding ribavirin and TCONH2Standard solutionThe solutions were 0.5, 1.0, 5.0. mu.g/kg, respectively, and then sample pretreatment and measurement were carried out according to the method, with 6 measurements for each addition level. The ribavirin and TCONH are measured by experiment2Recovery and relative standard deviation.
TABLE 5 addition of ribavirin and TCONH to blank chicken feathers2Recovery and relative standard deviation of
Figure BDA0002237416750000091
The invention provides a method for detecting ribavirin and TCONH in livestock and poultry hair2A method for residual amount, which calculates ribavirin and TCONH by peak area according to an internal standard method2The amount of residue in the hair is calculated by the formula:
Figure BDA0002237416750000101
x- -ribavirin or TCONH in the sample2Residual amounts (. mu.g/kg);
Cs- - -ribavirin or TCONH2Concentration of standard working solution (. mu.g/L);
Csi- - -ribavirin or TCONH2Internal standard substance in standard working solution13C5-concentration of ribavirin (μ g/L);
Ciinternal standard substance in test solution13C5-concentration of ribavirin (μ g/L);
As- - -ribavirin or TCONH2Peak area of standard working solution;
a- -ribavirin or TCONH in the sample2Peak area of (a);
Asi- - -ribavirin or TCONH2Internal standard substance ribavirin or TCONH in standard working solution2Peak area of (a);
Ai- -in the sample13C5-peak area of ribavirin;
v- -sample volume (mL);
m- -sample weight (g).
If the calculation result requires subtraction of a blank value and the result is less than 0.2. mu.g/kg, it is judged as undetected.
Example 2
The embodiment provides a method for detecting ribavirin and metabolites thereof in chicken feathers, which comprises the following specific steps:
1. the samples were prepared from 5 positive and 5 blank chicken feather samples obtained by feeding oral ribavirin. The specific administration feeding mode is that 5 experimental group broilers are fed with a ribavirin raw powder aqueous solution according to a standard of 10mg/kg body weight and are continuously fed for 3 days, 5 blank group broilers replace the ribavirin raw powder aqueous solution with the same amount of physiological saline, and after the experiment is finished, hair samples are respectively collected to be tested.
2. Preparation of test solution
(1) Hair cleaning: the hair was washed with 1% SDS for 30min, then rinsed 3 times with distilled water until clean, the sample was dried in an oven at 45 deg.C, then cut into 1-2mm pieces, then ground into powder with liquid nitrogen, and placed in a desiccator.
(2) Hydrolysis and purification: 200mg of the treated hair sample was accurately weighed out, 50. mu.L of 0.2mg/L internal standard13C5-ribavirin, adding 10mL of 2% formic acid-methanol solution, placing at 65 ℃ for 2h, taking out, vortexing for 1min, performing ultrasound for 15min, centrifuging at 10000rpm for 5min, taking supernatant, putting the supernatant into a 15mL centrifuge tube in which 150mg of PSA and 150mg of C18 adsorbent are added in advance, vortexing for 30s, then centrifuging at 4500rpm for 5min, blowing the hydrolysate to be dry at 45 ℃ by nitrogen, dissolving residues with 1mL of 0.1% formic acid aqueous solution, and filtering with a 0.22 mu m filter membrane to obtain a test solution.
3. Ultra-high performance liquid chromatography-tandem mass spectrometry detection
Liquid chromatography conditions: a chromatographic column: ZORBAX SB-Aq, 3.0mm X100 mm, 1.8 μm; mobile phase a was an aqueous solution containing 0.1% (v/v) ammonium formate acetate (ammonium acetate concentration: 2mMol/L), mobile phase B was methanol, flow rate: 0.4 mL/min; column temperature: 35 ℃; sample injection amount: 5.0 mu L; gradient elution procedure: maintaining for 0-2.5min for 0% B; 2.5-3.5min, the 0% B is changed to 98% B linearly; maintaining 98% B for 3.5-6 min; after 6-7min, the linear change of 98% B is reduced to 0% B;
mass spectrum conditions: electrospray ionization Positive ion mode, ESI+(ii) a The detection mode comprises the following steps: multiple reaction monitoring, MRM; capillary voltage: 3000V; ion source temperature TEM: 150 ℃; temperature of the drying gas: 150 ℃; flow rate of drying gas: 15L/min; atomizing gas pressure: 35 psi; temperature of sheath gas: 300 ℃; sheath airflow: 11L/min. The quantitative ion of ribavirin is m/z 245.1>113, qualitative ion is m/z 245.1>96、TCONH2The quantitative ion of (b) is m/z 113>96 and the qualitative ion is m/z 113>69 and13C5quantification of ribavirin ion m/z 250>113。
The detection result shows that the ribavirin and TCONH in the sample are analyzed2The residual quantity of the drug is respectively between 54 mu g/kg-215 mu g/kg and 85 mu g/kg-119 mu g/kg, and the ribavirin and TCONH are not detected in the chicken feather sample which is not fed with ribavirin2And residual shows that the detection method is feasible.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (11)

1. A method for detecting ribavirin and metabolites thereof in livestock and poultry hairs is characterized by comprising the following steps:
preparation of test solution
Cleaning hair of livestock and fowl to be tested, cutting, grinding with liquid nitrogen into powder, adding13C5Oscillating and extracting a ribavirin internal standard working solution and a formic acid-methanol mixed solution to obtain an extracting solution, dispersing and purifying the extracting solution by PSA and C18 dispersing agent, and dissolving the extracting solution by formic acid aqueous solution to obtain a test sample solution;
ultra-high performance liquid chromatography-tandem mass spectrometry detection
Ultra-high performance liquid chromatography conditions: and (3) chromatographic column: ZORBAX SB-Aq, 3.0mm X100 mm, 1.8 μm; mobile phase a was an aqueous ammonium acetate solution containing 0.1% formic acid by volume, mobile phase B was methanol, flow rate: 0.4 mL/min; column temperature: 35 ℃; sample injection amount: 5.0 mu L; gradient elution procedure: maintaining for 0-2.5min for 0% B; 2.5-3.5min, the 0% B is changed to 98% B linearly; maintaining 98% B for 3.5-6 min; after 6-7min, the linear change of 98% B is reduced to 0% B;
mass spectrum conditions: electrospray ionization Positive ion mode, ESI+(ii) a The detection mode is as follows: multiple reaction monitoring, MRM; capillary voltage: 3000V; ion source temperature TEM: 150 ℃; temperature of the drying gas: 150 ℃; flow rate of drying gas: 15L/min; atomizing gas pressure: 35 psi; temperature of sheath gas: 300 ℃; sheath airflow: 11L/min.
2. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, wherein the dosage of PSA and C18 dispersant is 10-30mg/mL respectively.
3. The method for detecting ribavirin and metabolites thereof in the livestock and poultry hair according to claim 1, wherein the dispersion purification step comprises the following steps: adding PSA and C18 dispersant into the extract, vortex centrifuging, blow-drying with nitrogen at 40-50 deg.C, adding formic acid water solution to dissolve residue to obtain test solution.
4. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, wherein the reagent for cleaning livestock and poultry hair is SDS with a mass concentration of 1% or Tween 80 with a mass concentration of 5%, and the cleaning time is 30-60 min.
5. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, wherein the mass of the livestock and poultry hair is measured13C5The usage amount of the ribavirin internal standard working solution is 100-250 mu L/g, and the concentration is 0.2-1.0 mg/L.
6. The method for detecting ribavirin and metabolites thereof in the livestock and poultry hair according to claim 1, wherein the formic acid volume fraction of the formic acid-methanol mixture is 1-4%;
the using amount of the formic acid-methanol mixed solution is 50-100mL/g based on the mass of the livestock and poultry hair.
7. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, wherein the concussion extraction is extraction at 45-90 ℃ for 1-12 h.
8. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, characterized in that the method further comprises the steps of ultrasonic treatment and centrifugal treatment after the concussion extraction, wherein the ultrasonic treatment time is 10-30 min.
9. The method for detecting ribavirin and metabolites thereof in the livestock and poultry hair according to claim 1, wherein the formic acid in the aqueous formic acid solution has a volume fraction of 0.05-0.2%.
10. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 1, wherein the mass spectrometry condition is that the quantitative ion pair of ribavirin is m/z 245.1>113, qualitative ion pair of m/z 245.1>96；TCONH2The quantitative ion pair of (2) is m/z 113>96 and the qualitative ion pair is m/z 113>69 and13C5quantitative ion pair of ribavirin of m/z 250>113。
11. The method for detecting ribavirin and metabolites thereof in livestock and poultry hair according to claim 7, wherein the concussion extraction is performed at 60-70 ℃ for 2-4 h.
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