CN113109474A

CN113109474A - Method for detecting metabolic residues of sedative drugs in animal tissues

Info

Publication number: CN113109474A
Application number: CN202110400252.3A
Authority: CN
Inventors: 张惠峰; 魏春雁; 范宏; 刘笑笑; 孟繁磊; 宋志峰; 张慧; 郑妍婕
Original assignee: Jilin Academy of Agricultural Sciences
Current assignee: Jilin Academy of Agricultural Sciences
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-07-13
Anticipated expiration: 2041-04-14
Also published as: CN113109474B

Abstract

The invention discloses a method for detecting metabolic residues of sedative drugs in animal tissues, which adopts a dispersion solid-phase extraction-high performance liquid chromatography triple quadrupole mass spectrometry to simultaneously detect chlorpromazine, promethazine, xylazine and metabolites thereof, such as chlorpromazine sulfoxide, 2-chlorophenothiazine, promethazine sulfoxide and 2, 6-dimethylaniline, in pork, pork liver, pork kidney, pig heart and pig lung, optimizes detection conditions, can accurately detect the sedative drugs in the animal tissues, has accurate and reliable detection results, provides reliable data for the measurement of the metabolic residues of the sedative drugs, has obvious economic benefits and wide market prospect. Has good reference value for guiding the measurement of the drug residue.

Description

Method for detecting metabolic residues of sedative drugs in animal tissues

Technical Field

The invention relates to the technical field of detection, in particular to a method for detecting metabolic residues of sedative drugs in animal tissues.

Background

Phenothiazines such as chlorpromazine, promethazine, xylazine and the like are found to have special effects except for clinical treatment and sedation, such as improvement of meat quality (like "clenbuterol"), water retention and the like. In order to reduce the stress reaction of pigs in the transportation and slaughtering processes, some slaughterhouses use sedatives for pigs to eliminate restlessness. Some producers are driven by economic benefits, and the medicines are added in the feeding process to increase weight and promote fertility, shorten slaughter time, reduce loss caused by stress, cooperate with water injection and the like. The drugs and their metabolites will inevitably remain in the pig body, and the drug residues enter the human body through the food chain to reach a certain concentration, which will cause symptoms such as nausea, vomiting, dizziness, weakness, numbness of limbs, mouth and tongue, etc., and serious patients will have short-time mental disorder. The main metabolites of chlorpromazine include chlorpromazine sulfoxide, 2-chlorophenothiazine, promethazine sulfoxide, the main metabolite of promethazine, and 2, 6-Dimethylaniline (DMA), the main metabolite of xylazine.

The researches on chlorpromazine, promethazine and xylazine at home and abroad mainly have the following defects:

first, related research mainly focuses on residual detection of raw drugs, and the quantitative detection of metabolites thereof is of insufficient concern. Chlorpromazine, promethazine and xylazine are reported to be fast in absorption, metabolism and decomposition speed in vivo, and only a small amount of chlorpromazine, promethazine and xylazine is discharged from urine in the form of protomer, so that few reports exist on the detection of original drugs in livestock and poultry products. However, when the residual quantity of animal-derived food sedative drugs and metabolites thereof is generally checked, a part of pork may contain trace promethazine sulfoxide.

Secondly, the understanding of the residual harm of chlorpromazine, promethazine and xylazine metabolites is insufficient. The chlorpromazine and promethazine metabolites have the original drug activity and can cause leucopenia and agranulocytosis, so that pathological changes of the liver and the kidney, eye complications and the like of a human body are caused, and the main metabolite 2, 6-Dimethylaniline (DMA) of xylazine has the genotoxicity and carcinogenic effect. At present, the limitation of chlorpromazine, promethazine and xylazine metabolites is not specified in China, and specific research reports of related metabolites are lacked.

And thirdly, due to the lack of detection technical means, the research on the exposure risk of chlorpromazine, promethazine and xylazine raw medicines and metabolites is lacked. At present, no research report about the dietary exposure risk of chlorpromazine, promethazine, xylazine and metabolites exists, but the residual risk of the drugs is highly regarded in view of the report about the cases related to acute poisoning events caused by chlorpromazine and xylazine in meat.

In conclusion, the establishment of the efficient and reliable drug detection method has very important significance for finding potential risks of chlorpromazine, promethazine and xylazine original drugs and metabolites and developing related researches.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for detecting metabolic residues of sedative drugs in animal tissues so as to overcome the defects in the prior art.

In order to achieve the above object, the present invention provides a method for detecting metabolic residues of sedative drugs in animal tissues, comprising:

preparing a sample, cleaning thawed animal tissues to be detected, removing hair, skin, blood stasis, tendons and bones, cutting into pieces, mashing, subpackaging in sample containers, and storing for later use;

sample extraction, namely weighing 5g of sample, placing the sample in a 50mL centrifuge tube, adding 50 mu L of mixed internal standard working solution, carrying out vortex mixing for 30s, standing for 10min, adding 15mL of acetonitrile, carrying out vortex mixing for 1min, adding 15g of anhydrous sodium sulfate, carrying out vortex mixing for 1min, carrying out ultrasonic extraction at 35-55 ℃ for 10min, taking out the centrifuge tube, cooling the mixed liquid in the centrifuge tube to room temperature, centrifuging at 4500r/min for 5min, and transferring the obtained first supernatant to a 100mL heart-shaped bottle;

and (3) carrying out secondary extraction on the solid in the centrifugal tube after the supernatant is removed to obtain a secondary supernatant, combining the two supernatants, adding 10mL of isopropanol, carrying out rotary evaporation at 45 ℃ until the supernatant is nearly dry to obtain a residue.

Purifying a sample, adding 100 mu L of methanol and a small amount of water into the residue, dissolving the residue, transferring the residue into a 2mL centrifuge tube, adding 1mL of n-hexane into the residue after the volume is determined to be 1.0mL by water, adding an adsorbent, carrying out vortex oscillation for 1min, centrifuging for 5min at 12000r/min, and filtering the lower layer clear liquid for later use;

and (3) sample detection, namely performing machine detection on the filtered lower layer clear liquid, wherein the detection conditions comprise:

a chromatographic column: an Agilent Eclipse XDB-C18 chromatography column; flow rate, 0.2 mL/min; mobile phase: phase A is 0.1% ammonia water; phase B is methanol, and gradient elution is carried out; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; quantifying by an internal and external standard method;

mass spectrum ion source ESI ion source temperature is 100 ℃; the flow rate of the drying gas is 10L/h; the capillary voltage is 4000V; the temperature of the airflow is 350 ℃; atomizing gas pressure 40 psi; collision gas type of nitrogen; the scanning mode is positive ion; the detection mode is mass spectrum multiple reaction monitoring.

Preferably, the adsorbent is 50mgC18,10mg psa,50mg neutral alumina.

Preferably, the gradient elution condition is that the flow rate is 0.2ml/min and the flow rate is 95% of the mobile phase A and 5% of the mobile phase B in 0-0.5 min in terms of volume fraction; in 0.5-3 min, the mobile phase A is 95% -60%, and the mobile phase B is 5% -40%; in 3-4 min, the mobile phase A is 60-20%, and the mobile phase B is 40-80%; in 4-5 min, the mobile phase A is 20-5%, and the mobile phase B is 80-95%; 5-17 min later, 5% of a mobile phase A and 95% of a mobile phase B; in 17-17.1 min, the mobile phase A is 5-95%, and the mobile phase B is 95-5%; and in 17.1-18 min, the mobile phase A is 95% and the mobile phase B is 5%.

Preferably, the supernatant-removed solid in the centrifuge tube is subjected to secondary extraction, which includes: shaking the solid in the centrifugal tube, adding 15mL of acetonitrile, shaking up and down by hand shaking for 30s, performing ultrasonic extraction at 35-55 ℃ for 10min, taking out the centrifugal tube, cooling the mixed liquid in the centrifugal tube to room temperature, and centrifuging at 4500r/min for 5min to obtain a second supernatant.

Preferably, the subnatant filtration comprises: the lower layer was filtered through a 0.22 μm filter.

Preferably, the sedative drug comprises: chlorpromazine, promethazine hydrochloride, xylazine, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline, 2-chlorophenothiazine, chlorpromazine-D6 hydrochloride, promethazine hydrochloride-D6 and xylazine-D6.

Preferably, the detection method of the present invention further comprises:

preparation of standard stock solution: respectively preparing standard stock solutions with the mass concentration of 1000 mug/mL of promethazine hydrochloride, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline, 2-chlorophenothiazine, chlorpromazine-D6 hydrochloride, promethazine hydrochloride-D6 and xylazine-D6;

transferring 100 mu L of promethazine, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline standard stock solution, chlorpromazine and xylazine standard substance with the mass concentration of 1000 mu g/mL, dissolving the mixture in a 10mL brown volumetric flask by using methanol to prepare mixed intermediate solution with the mass concentration of 1000ng/mL, and freezing and storing the mixed intermediate solution in a dark place;

and (3) transferring chlorpromazine-D6, promethazine-D6 and xylazine-D6 standard stock solutions, dissolving the stock solutions in a 10mL brown volumetric flask by using methanol to prepare a mixed internal standard use solution with the mass concentration of 1000ng/mL, and freezing and storing the mixed internal standard use solution in a dark place.

Preferably, the preparation is a mixed intermediate solution with the mass concentration of 1000ng/mL, the temperature of the mixed intermediate solution subjected to light-shielding freezing storage is-16-20 ℃, the preparation is a mixed internal standard use solution with the mass concentration of 1000ng/mL, and the temperature of the mixed internal standard use solution subjected to light-shielding freezing storage is-16-20 ℃.

Preferably, the animal tissue to be tested is pork, pork liver, pork kidney, pork heart or pork lung.

The detection method provided by the embodiment of the invention at least has the following beneficial effects:

according to the method for detecting the metabolic residues of the sedative drugs in the animal tissues, the sedative drugs in the animal tissues can be accurately detected, the detection result is accurate and reliable, reliable data are provided for measuring the metabolic residues of the sedative drugs, and the method has remarkable economic benefit and wide market prospect. Has good reference value for guiding the measurement of the drug residue.

Drawings

FIG. 1 is a mass correlation spectrum of xylazine and internal standard characteristic ions of the present invention;

FIG. 2 is a mass correlation spectrum of 2, 6-dimethylaniline and an internal standard characteristic ion of the invention;

FIG. 3 is a mass correlation spectrum of chlorpromazine sulfoxide and internal standard characteristic ions of the invention;

FIG. 4 is a mass correlation spectrum of chlorpromazine and internal standard characteristic ions of the invention;

FIG. 5 is a mass correlation spectrum of promethazine sulfoxide and internal standard characteristic ions of the invention;

FIG. 6 is a mass correlation spectrum of promethazine and internal standard characteristic ions of the present invention;

FIG. 7 is a mass-correlation spectrum of 2-chlorophenothiazine and an internal standard characteristic ion of the invention;

FIG. 8 is a standard curve of xylazine according to the invention;

FIG. 9 is a graph of a standard curve for 2, 6-dimethylaniline according to the present invention;

FIG. 10 is a graph of chlorpromazine sulfoxide standards according to the invention;

FIG. 11 is a standard graph of chlorpromazine according to the invention;

FIG. 12 is a graph of a promethazine sulfoxide standard of the present invention;

FIG. 13 is a promethazine standard curve of the present invention;

FIG. 14 is a standard graph of 2-chlorophenothiazine according to the invention.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The apparatus and equipment of the invention:

6410B liquid chromatography-tandem quadrupole mass spectrometer, Agilent, USA;

XS205 analytical balance, Mettler, switzerland;

TE212-L electronic balance Sartorius, Germany;

range-adjustable pipettors, VITLAB, germany;

TDL-40B desk centrifuge, Shanghai' an pavilion scientific instrument factory.

The material of the invention is:

TABLE 1 list of standard substances

Name of Standard substance	CAS number	Standard value	Manufacturer of the product
				Chlorpromazine	50-53-3	1000μg/mL，1.2mL	Ink quality inspection 91437JA
Promethazine hydrochloride	58-33-3	250mg，≥99.5％	Ink quality test 71696 of jar 250mg
				Xylazine	7361-61-7	1000μg/mL1.2mL	Ink quality detection BW902738-1000-A
Chlorpromazine sulfoxide	969-99-3	25mg，≥96％	Canadian TRCC424755
				Promethazine sulfoxide	7640-51-9	10mg，≥95％	Ink quality inspection of jar 709972-10mg
2, 6-dimethylaniline	87-62-7	100mg，≥99.8％	70989 ink quality test 100mg
				2-chlorophenothiazines	92-39-7	10mg，≥98.0％	Ink quality inspection of jar 712899-10mg
chlorpromazine-D6 hydrochloride	1228182-46-4	10mg，≥99.4％	Ink quality detection 72640-
				Promethazine hydrochloride-D6	1189947-02-1	50mg，≥99.7％	Germany WitegaTR018-50
xylazine-D6	1228182-53-3	25mg，≥99.4	German WitegaTR010-25

Acetonitrile, methanol, isopropanol, n-hexane, all chromatographically pure, Fisher, usa;

formic acid (LC/MS), Fisher corporation, USA;

ammonia water (LC/MS), Shanghai' an spectral experiment science and technology with limited shares;

ammonium acetate (chromatographically pure), Merck, usa;

Bondersil-C18 bulk adsorbent (40um), Agilent, USA;

Bondersil-PSA bulk adsorbent (40um), Agilent, USA;

the test water meets the requirements of GB/T6682 first-grade water, and the other reagents are domestic analytical purifiers.

The selection and detection process of parameters and technical indicators in the present invention will be described with reference to the following embodiments.

Preparation of Standard stock solution

Respectively weighing appropriate amounts of promethazine hydrochloride, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline, 2-chlorophenothiazine, chlorpromazine-D6 hydrochloride, promethazine hydrochloride-D6 and xylazine-D6, and diluting the solution to a 10mL brown volumetric flask with methanol to obtain a standard stock solution with the mass concentration of 1000 mug/mL.

And (3) transferring 100 mu L of promethazine standard stock solution, chlorpromazine sulfoxide, promethazine sulfoxide and 2, 6-dimethylaniline standard stock solution, and chlorpromazine and xylazine standard product with the mass concentration of 1000 mu g/mL sold in the market, dissolving the mixture in a 10mL brown volumetric flask by using methanol to prepare a mixed intermediate solution with the mass concentration of 1000ng/mL, and freezing and storing the mixed intermediate solution at the temperature of minus 16-20 ℃ in the dark. When in use, the mixture is gradually diluted to the concentration of 100ng/mL and 10ng/mL by methanol and is prepared as before use.

And (3) transferring chlorpromazine-D6, promethazine-D6 and xylazine-D6 standard stock solutions, dissolving the standard stock solutions in a 10mL brown volumetric flask by using methanol to prepare a mixed internal standard use solution with the mass concentration of 1000ng/mL, and freezing and storing the mixed internal standard use solution at the temperature of minus 16-20 ℃ in a dark place.

50 muL, 100 muL, 0.5mL and 1mL of 10ng/mL mixed standard solution and 50 muL, 100 muL and 200 muL of 1000ng/mL mixed standard solution are respectively added into a blank matrix, the mixture is processed according to the same extraction and purification steps with the sample, 7 standard solutions (0.5, 1, 5, 10, 50, 100 and 200ng/mL) with different mass concentrations are prepared, and a working curve is drawn by an internal standard method.

(II) sample preparation

Cleaning the cold fresh pig muscle product, removing hair, skin, blood stasis, tendons and bones, and preparing. Thawing frozen pork at room temperature, slightly thawing and softening the sample, removing hair, skin, tendons and bones when the sample is just thawed and frozen water does not flow out, and preparing after the heart, liver, kidney, lung and other byproducts of the pig are cleaned by using experimental water. Cutting the sample into 1cmX1cm small pieces, mixing, dividing to 500g by quartering method, mashing with a mashing machine, mixing, and packaging into a plurality of sample containers for later use. Dicing or cutting into small pieces after pretreating livestock and poultry viscera, mashing with a pounder, mixing well, and subpackaging into sample containers.

(III) sample preservation

After the sample is prepared, if the sample cannot be detected in time, the sample is immediately frozen and stored, and the sample cannot be melted and deteriorated before detection. The sample detected on the day can be temporarily stored in a refrigerated mode for no more than 8 hours. The frozen sample should be thawed at 45 deg.C below for 15min or 2-5 deg.C for 18h before being tested.

(IV) extraction

Weighing 5g of sample, accurately measuring the sample to 0.01g, adding 50 mu L of mixed internal standard working solution into a 50mL centrifuge tube, carrying out vortex mixing for 30s, standing for 10min, adding 15mL of acetonitrile, carrying out vortex mixing for 1min, adding 15g of anhydrous sodium sulfate, carrying out vortex mixing for 1min, carrying out ultrasonic extraction at 35-55 ℃ for 10min, taking out, cooling to room temperature, centrifuging at 4500r/min for 5min, and transferring the supernatant into a 100mL heart-shaped flask.

Shaking off the solid in the centrifugal tube, adding 15mL of acetonitrile, shaking up and down by hand shaking for 30s, repeatedly extracting for 1 time, and combining the supernatants; 10mL of isopropanol was added, 45 ℃ and rotary evaporated to near dryness.

(V) extraction

Adding 100 μ L methanol, small amount of water, dissolving the residue, transferring to 2mL centrifuge tube, adding water to constant volume of 1.0mL, adding 1mL n-hexane, adding adsorbent (50mgC18,10mg PSA,50mg neutral alumina), vortex shaking for 1min, centrifuging at 12000r/min for 5min, filtering the lower layer clear solution with 0.22 μm filter membrane, and supplying to liquid.

(VI) detection

The detection conditions of a 6410B liquid chromatography-tandem quadrupole mass spectrometer, Agilent company, USA:

a chromatographic column: agilent Eclipse XDB-C18(3 mm. times.100 mm, 1.8 μm); flow rate: 0.2 mL/min; mobile phase: phase A is 0.1% ammonia water; phase B is methanol, gradient elution is carried out, and specific set conditions are shown in Table 2; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; quantification by internal and external standard method.

TABLE 2 mobile phase gradient elution conditions

Mass spectrometry ion source: ESI ion source temperature: 100 ℃; flow rate of drying gas: 10L/h; capillary voltage: 4000V; temperature of gas flow: 350 ℃; atomizing gas pressure: 40 psi; collision gas type: nitrogen gas; the scanning mode is positive ion; the detection mode is as follows: mass spectrometry multiple reactions monitor MRM. Wherein, the conditions of the mass spectrum acquisition parameters are detailed in Table 3.

TABLE 3 Mass Spectrometry Collection parameter conditions

(VII) results and analysis

In the test process, in order to obtain the best experimental result, a great amount of tests and comparisons are carried out on the selection of an extracting agent, the extraction mode, the material-liquid ratio extraction time and the like, and the result is analyzed. The analysis of the results is described in detail below:

1. spectrogram analysis

And (4) analyzing according to chromatographic conditions, completely separating the target compound (the sedative drug to be detected) from the interfering substance, and having good peak shape. See table 4 and figures 1 to 7 for details.

TABLE 4 target Compound Retention time and relative abundance of the second-to-strong fragment ion

2. Selection of the extractant

The extraction effects of acetone, methanol, acetonitrile and acidified acetonitrile (0.1% formic acid) on 7 target compounds are respectively considered, the addition standard recovery rate of the 4 different extracting agents on the target compounds is not obviously influenced by comparison, but after the acetonitrile extraction, the peak area response value of the characteristic ion fragment of each target compound in pork, pork liver, pork heart, pork lung and pork kidney is obviously higher than that of other extracting agents, the analysis recovery rate is not obviously influenced mainly because the loss of the extracting process is offset by an internal standard method, and the acetonitrile is preferably used as the extracting agent.

3. Selection of extraction method, time and temperature

The complexity of each sample of pork, pork liver, pork heart, pork lung and pork kidney is different, and the tissue body fluid components and the proportion contained in the samples are different, so the quality of the required anhydrous sodium sulfate is influenced, after 5g, 10g, 15g and 20g of anhydrous sodium sulfate with different qualities are added for comparison, the pork liver and the pork lung need more anhydrous sodium sulfate, 15-20 g of anhydrous sodium sulfate can well remove water in the samples, and the 15g of anhydrous sodium sulfate is preferably selected for dewatering through comprehensive consideration.

Pork, pork liver, pig heart, pig lung, pig kidney are in the extraction process, caking and wall built-up adhesion degree are different, through modes such as hand-operated oscillation, vortex mixing, convolution oscillation, ultrasonic extraction and the like of a large number of tests, it is found that the extraction mode of vortex mixing and ultrasonic combination can fully extract a target compound, the sample is easy to cake and wall built-up after being extracted and centrifuged for the first time, vortex and ultrasonic can not effectively disperse the sample after an extracting agent is directly added, so that the extraction efficiency is influenced, but the sample can be dispersed by adopting a simple mechanical oscillation mode before the agent is added, and the condition of wall built-up of the pig lung, pig liver and pig kidney sample can be effectively improved by adopting an up-down hand-operated mode after the agent is added. And (3) comprehensively considering the extraction effect and the working efficiency, and selecting an extraction mode of vortex for 1min and ultrasonic for 10 min. In addition, the proper temperature can effectively accelerate the deterioration of the protein and improve the purification efficiency, but the high temperature is easy to cause the decomposition of the target compound, and after a large number of experiments, the ultrasonic temperature of 35-55 ℃ is selected.

4. Selection of the concentration mode

Two sample concentration modes of nitrogen blowing and rotary evaporation are considered, and rotary evaporation is preferred due to more extraction solvents. In the test process, the fact that due to the fact that matrixes of pork, pork liver, pork heart, pork lung and pork kidney are different, pressure and time required by rotary steaming are greatly different, the matrixes need to be adjusted at any time according to different matrixes, and much labor is consumed is found. 10mL of isopropanol was tested as suitable for use in the present process.

5. Selection of purification conditions

The purifying effects of the adsorbing and purifying agents such as C18, PSA, neutral alumina, diatomite filter aids and the like on different matrix samples such as pork, pork liver, pork heart, pork lung, pork kidney and the like are examined, the adding standard recovery rate of each adsorbent is not obviously influenced by using an internal standard method for quantifying, but the adding standard recovery rate of each adsorbent has important influence on the signal response value and characteristic ion interference of a target compound, particularly 2, 6-dimethylaniline and 2-chlorophenothiazine, the purifying effects on the samples are obvious by comparing C18, PSA and neutral alumina, but the target compound is adsorbed to different degrees when the using amount is large, after the experiment, 50mgC18,10mgPSA and 50mg neutral alumina are preferably used for purification, and 1mL of n-hexane is added for degreasing.

6. Selection of detection conditions

(1) Conditions of liquid phase separation

A large number of tests prove that the separation effect of each target compound is better than that of acetonitrile when the organic phase is methanol, and the signal response of 2-chlorophenothiazine is extremely low and the organic phase is only responsive at a higher concentration when water, 0.1% formic acid and 5mmol ammonium acetate (0.1% formic acid) solution are selected. When the aqueous phase is ammonia water, the 2-chlorophenothiazine has good response, the response values of the 7 target compounds are greatly enhanced, the peak pattern is good, no interference is caused, and the signal response value is continuously increased along with the increase of the concentration of the ammonia water. The pH tolerance degree and signal response of the chromatographic column are comprehensively considered, 0.1% ammonia water is preferably used as an aqueous phase flow term, a gradient elution program is determined through a large number of experiments, 7 target compounds in 5 matrixes are effectively separated in the shortest time, and the test requirements are met.

(2) Conditions of Mass Spectrometry

Through a large number of experiments, various mass spectrum parameters are tuned, the response degrees of fragment ions are comprehensively compared, and the ion source conditions, the fragmentation voltage, the collision voltage and qualitative and quantitative ions of the mass spectrum are determined, which is specifically referred to the table 2.

7. Selection of internal standards

The synchronous response degree of 4 substances such as SKF-525A, xylazine-D6, chlorpromazine-D6 and promethazine-D6 to 7 target compounds is examined, through comparison, the 4 substances have better synchronous response to the 7 compounds in the same matrix, when a working curve is drawn by taking a pork sample as a matrix, the synchronous response is greatly influenced by 5 different matrixes such as pork, pork liver, pork heart, pork lung and pork kidney, wherein the SKF-525A has higher polarity and earlier peak emergence and is easily influenced by matrix interference, and only the 7 target compounds in the pork sample have certain synchronous correspondence and are not suitable to be used as internal standards. The xylazine-D6, chlorpromazine-D6 and promethazine-D6 have good correspondence with xylazine, chlorpromazine and promethazine, and because the polarity is similar and the peak emergence time is similar, xylazine-D6 synchronously responds with chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline and 2-chlorophenothiazine, so that the xylazine is selected as a reference internal standard.

8. Verification of a method

(1) Linear range, detection limit and quantitation limit

50 muL, 100 muL, 0.5mL and 1mL of 10ng/mL mixed standard solution and 50 muL, 100 muL and 200 muL of 1000ng/mL mixed standard solution are respectively added into a blank substrate, and the mixture is processed according to the steps of sample extraction and purification to prepare 7 standard solutions (0.5, 1, 5, 10, 50, 100 and 200ng/mL) with different mass concentrations, and a working curve is drawn by an internal standard method. The standard substance and the spiked sample were measured, and the standard deviation of the measurement results of 7 kinds of target compounds was calculated by measuring the spiked sample 7 times at the lowest acceptable concentration of 0.1. mu.g/kg, and the detection limit of the method was calculated as LOD of 0+3S, and the quantification limit of the method was calculated as LOQ of 0+ 10S. The linear regression equation and method found limits, quantified limits, as shown in table 5.

TABLE 57 target compound linear regression equation and method detection limit, quantitative limit

(2) Method precision and recovery

The results of testing 7 portions of positive samples with concentration level of 10.0 mug/kg added to different edible tissues of pigs such as pig heart, pig lung, pig kidney, pork, pig liver and the like are shown in Table 4. The recovery rate of the xylazine in different tissues is 93.4% -101%, and the corresponding average relative standard deviation is 0.89-6.55%; the recovery rate of the 2, 6-dimethylaniline in different tissues is 96.0-101%, and the corresponding average relative standard deviation is 5.70-10.8%; the recovery rate of the chlorpromazine sulfoxide in different tissues is 78.5-94.5%, and the corresponding average relative standard deviation is 2.08-8.59%; the recovery rate of chlorpromazine in different tissues is 87.0-105%, and the corresponding average relative standard deviation is 3.12-5.39%; the recovery rate of promethazine sulfoxide in different tissues is 99.5-109%, and the corresponding average relative standard deviation is 5.23-9.98%; the recovery rate of promethazine in different tissues is 92.3% -107%, and the corresponding average relative standard deviation is 1.37-9.75%; the recovery rate of the 2-chlorophenothiazine in different tissues is 87.9-101%, and the corresponding average relative standard deviation is 5.53-8.91%, which is detailed in tables 6-10.

TABLE 6 porcine heart sample recovery and precision test results

TABLE 7 porcine heart sample recovery and precision test results

TABLE 8 porcine kidney sample recovery and precision test results

TABLE 9 pork sample recovery and precision test results

TABLE 10 porcine liver sample recovery and precision test results

(3) Stability of

The same standard concentration level (10.0 mug/kg) sample is respectively measured by different operators at intervals of 0 day, 3 days and 10 days, the method reproducibility of 7 target compounds in 5 different matrixes is 2.33-11.8 percent and is less than 15 percent by calculating the result RSD of 3 average measurements, and the results show that the method is reliable and can be stably reproduced, and the details are shown in tables 11-17.

TABLE 11 porcine heart sample recovery and precision test results

TABLE 12 porcine lung sample recovery and precision test results

TABLE 13 porcine kidney sample recovery and precision test results

TABLE 14 pork sample recovery and precision test results

TABLE 15 porcine liver sample recovery and precision test results

Therefore, the scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the scope of the present invention, which is defined by the claims of the present patent application.

Claims

1. A method for detecting metabolic residues of a sedative drug in animal tissue, comprising:

performing secondary extraction on the solid in the centrifugal tube after the supernatant is removed to obtain a secondary supernatant, combining the two supernatants, adding 10mL of isopropanol, performing rotary evaporation at 45 ℃ until the supernatant is nearly dried to obtain a residue;

2. The detection method according to claim 1, wherein the adsorbent is 50mgC18,10mg psa,50mg neutral alumina.

3. The detection method according to claim 1, wherein the gradient elution conditions are that the flow rate is 0.2ml/min and the flow rate is 95% for the mobile phase A and 5% for the mobile phase B in 0-0.5 min in volume fraction; in 0.5-3 min, the mobile phase A is 95% -60%, and the mobile phase B is 5% -40%; in 3-4 min, the mobile phase A is 60-20%, and the mobile phase B is 40-80%; in 4-5 min, the mobile phase A is 20-5%, and the mobile phase B is 80-95%; 5-17 min later, 5% of a mobile phase A and 95% of a mobile phase B; in 17-17.1 min, the mobile phase A is 5-95%, and the mobile phase B is 95-5%; and in 17.1-18 min, the mobile phase A is 95% and the mobile phase B is 5%.

4. The detection method according to claim 1, wherein the secondary extraction of the supernatant-removed solid in the centrifuge tube comprises: shaking the solid in the centrifugal tube, adding 15mL of acetonitrile, shaking up and down by hand shaking for 30s, performing ultrasonic extraction at 35-55 ℃ for 10min, taking out the centrifugal tube, cooling the mixed liquid in the centrifugal tube to room temperature, and centrifuging at 4500r/min for 5min to obtain a second supernatant.

5. The detection method according to claim 1, wherein the subnatant filtration comprises: the lower layer was filtered through a 0.22 μm filter.

6. The method for testing according to claim 1, wherein said sedative drug comprises: chlorpromazine, promethazine hydrochloride, xylazine, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline, 2-chlorophenothiazine, chlorpromazine-D6 hydrochloride, promethazine hydrochloride-D6 and xylazine-D6.

7. The detection method according to claim 6, further comprising:

8. The detection method according to claim 7, wherein the preparation is a mixed intermediate solution with a mass concentration of 1000ng/mL, the temperature for cryopreservation in the dark is-16-20 ℃, the preparation is a mixed internal standard use solution with a mass concentration of 1000ng/mL, and the temperature for cryopreservation in the dark is-16-20 ℃.

9. The detection method according to any one of claims 1 to 8, wherein the animal tissue to be detected is pork, pork liver, pork kidney, pork heart or pork lung.