CN113109474B - Method for detecting sedative drug metabolism residues in animal tissues - Google Patents

Abstract

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

Description

Method for detecting sedative drug metabolism residues in animal tissues

Technical Field

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

Background

Phenothiazines such as chlorpromazine, promethazine, and meothiazine have found particular utility in addition to clinical therapeutic sedation, such as improving meat quality (like "clenbuterol"), preserving water, and the like. In order to relieve stress reaction of pigs in the transportation and slaughtering processes, some slaughter houses use sedatives for the pigs to eliminate agitation. Some producers are driven by economic benefits, and the medicines are added in the feeding process to achieve the effects of weight increment fattening, column outlet time shortening, loss reduction caused by stress, collaborative water injection and the like. The medicines and metabolites thereof inevitably remain in pigs, and the medicine residues enter the human body through a food chain to reach a certain concentration, so that symptoms such as nausea, vomiting, dizziness, weakness, limb numbness, mouth and tongue numbness and the like can appear, and serious people have short-time mental disorder. The main metabolites of chlorpromazine include chlorpromazine sulfoxide, 2-chlorophenothiazine, and promethazine sulfoxide, and 2, 6-Dimethylaniline (DMA).

The research of chlorpromazine, promethazine and meothiazine at home and abroad mainly has the following defects:

1. the related research mainly focuses on the residual detection of the original medicine, and the quantitative detection of the metabolites is lack of attention. It is reported that chlorpromazine, promethazine and meothiazine have very high absorption, metabolism and decomposition rates in vivo, and only a small amount of chlorpromazine, promethazine and meothiazine is discharged from urine in a form of a protomer, so that few reports of detecting an original drug in livestock and poultry products exist. However, it has been reported that when animal derived food sedative drugs and their metabolite residues are subjected to general investigation, a part of pork may contain a trace amount of promethazine sulfoxide.

2. The residual hazard of chlorpromazine, promethazine and meothiazine metabolites is not known enough. It has been reported that chlorpromazine, promethazine metabolite moiety has crude drug activity, can cause leukopenia and granulocytopenia, thereby causing lesions, ocular complications and the like of human liver, kidney, and the main metabolite 2, 6-Dimethylaniline (DMA) of xylazine has genotoxicity and carcinogenesis. At present, no regulation on the metabolite limit of chlorpromazine, promethazine and meothiazine is available in China, and specific research reports on related metabolites are lacking.

3. Due to lack of detection technical means, exposure risk researches on chlorpromazine, promethazine and tolthiazine original drugs and metabolites are lacking. At present, research reports about chlorpromazine, promethazine, meothiazine and metabolite dietary exposure risks are not seen, but in view of the existing cases reports about acute poisoning events caused by chlorpromazine and meothiazine in meat, the residual risks of the drugs should be highly valued.

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

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for detecting sedative drug metabolism residues in animal tissues, which aims to solve the defects in the prior art.

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

preparing a sample, cleaning the unfrozen animal tissue to be tested, removing hair, skin, blood stasis, tendons and bones, cutting into pieces, mashing, subpackaging in a sample container, and preserving for later use;

extracting a sample, namely weighing 5g of the sample, putting the sample into 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 for 10min at 35-55 ℃, taking out the centrifuge tube, cooling the mixed liquid in the centrifuge tube to room temperature, centrifuging for 5min at 4500r/min, and transferring the obtained supernatant to a 100mL chicken heart bottle;

and (3) extracting the solids in the centrifuge tube after removing the supernatant twice to obtain a supernatant of the second time, combining the supernatants of the two times, adding 10mL of isopropanol, and performing rotary evaporation to near dryness at 45 ℃ to obtain residues.

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

sample detection, namely detecting the filtered supernatant by a machine, wherein detection conditions comprise:

chromatographic column: agilent Eclipse XDB-C18 chromatographic column; flow rate, 0.2mL/min; mobile phase: phase A is 0.1% ammonia water; the phase B is methanol, and the gradient elution is carried out; column temperature: 40 ℃; sample injection amount: 10. Mu.L; quantitative determination by an internal standard method;

the temperature of the ESI ion source is 100 ℃; the dry air flow is 10L/h; capillary voltage 4000V; the temperature of the air flow is 350 ℃; atomization gas pressure 40psi; the collision gas type is nitrogen; positive ions; the detection mode is mass spectrum multi-reaction monitoring.

Preferably, the adsorbent is 50mg C18,10mg PSA,50mg neutral alumina.

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

Preferably, the secondary extraction of the solids after removal of the supernatant in the centrifuge tube comprises: and (3) dispersing the solid in the centrifuge tube, adding 15mL of acetonitrile, shaking up and down by hands for 30s, performing ultrasonic extraction for 10min at the temperature of 35-55 ℃, taking out the centrifuge tube, cooling the mixed liquid in the centrifuge tube to room temperature, and centrifuging for 5min at 4500r/min to obtain a supernatant liquid of the second time.

Preferably, the filtering of the supernatant comprises: the supernatant was filtered through a 0.22 μm filter.

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

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

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

transferring 100 mu L of promazine, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline standard stock solution, chlorpromazine and a toluene thiazine standard product with the mass concentration of 1000 mu g/mL, and preparing a mixed intermediate solution with the mass concentration of 1000ng/mL by using methanol to fix the volume in a 10mL brown volumetric flask, and freezing and preserving in a dark place;

and transferring chlorpromazine-D6, promethazine-D6 and tolylthiazine-D6 standard stock solutions, and fixing the volume 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 performing light-proof freezing preservation.

Preferably, the preparation is a mixed intermediate solution with the mass concentration of 1000ng/mL, the temperature of the mixed intermediate solution is between-16 and 20 ℃ in a dark freezing preservation mode, and the preparation is a mixed internal standard use solution with the mass concentration of 1000ng/mL, and the temperature of the mixed intermediate solution is between-16 and 20 ℃ in a dark freezing preservation mode.

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

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

according to the method for detecting the sedative drug metabolism residue in the animal tissues, provided by the embodiment of the invention, the sedative drug in the animal tissues can be accurately detected, the detection result is accurate and reliable, reliable data is provided for the measurement of the sedative drug metabolism residue, and the method has remarkable economic benefits and wide market prospects. Has good reference value for guiding the measurement of drug residues.

Drawings

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

FIG. 2 is a graph showing the mass correlation of 2, 6-dimethylaniline and internal standard characteristic ions according to the present invention;

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

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

FIG. 5 is a graph of the mass correlation of promethazine sulfoxide and internal standard characteristic ions according to the present invention;

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

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

FIG. 8 is a standard graph of the present invention for xylazine;

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

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

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

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

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

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

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The instrument and the device of the invention:

6410B liquid chromatograph-tandem quadrupole mass spectrometer, agilent company, USA;

XS205 analytical balance, mettler company, switzerland;

TE212-L electronic balance, sartorius, germany;

an adjustable range pipette, VITLAB company, germany;

TDL-40B desk-top centrifuge, shanghai's pavilion scientific instrument factory.

The material of the invention is as follows:

TABLE 1 list of standard substances

Standard substance name	CAS number	Standard value	Manufacturing factories
				Chlorpromazine	50-53-3	1000μg/mL，1.2mL	Jar ink quality inspection 91437JA
Promethazine hydrochloride	58-33-3	250mg，≥99.5％	Jar ink quality test 71696-250mg
				Toluene thiazine	7361-61-7	1000μg/mL1.2mL	Jar ink quality inspection BW902738-1000-A
Chlorpromazine sulfoxide	969-99-3	25mg，≥96％	Canadian TRCC424755
				Promethazine sulfoxide	7640-51-9	10mg，≥95％	Jar ink quality inspection 709972-10mg
2, 6-dimethylaniline	87-62-7	100mg，≥99.8％	Jar ink quality control 70989-100mg
				2-chlorophenothiazine	92-39-7	10mg，≥98.0％	Jar ink quality inspection 712899-10mg
chlorpromazine-D6 hydrochloride	1228182-46-4	10mg，≥99.4％	Quality control 72640-120mg of ink in jar
				Promethazine hydrochloride-D6	1189947-02-1	50mg，≥99.7％	Witegatr018-50, germany
Toluene thiazine-D6	1228182-53-3	25mg，≥99.4	Witegatr010-25 in germany

Acetonitrile, methanol, isopropanol, n-hexane, all chromatographically pure, fisher, USA;

formic acid (LC/MS), fisher, USA;

ammonia (LC/MS), limited in Shanghai's science and technology;

ammonium acetate (chromatographic purity), merck, usa;

Bondesil-C18 bulk adsorbent (40 um), agilent, U.S.;

Bondesil-PSA bulk adsorbent (40 um), agilent, U.S.;

the test water meets the requirements of GB/T6682 primary water, and the rest reagents are all of domestic analytical purity.

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

Preparation of (one) Standard stock solution

And respectively weighing a proper amount of promethazine hydrochloride, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline, 2-chlorophenothiazine, chlorpromazine-D6 hydrochloride, promethazine-D6 hydrochloride and tolylthiazine-D6 which are 10mg, and preparing the standard stock solution with the mass concentration of 1000 mug/mL by using methanol to a brown volumetric flask with the volume of 10 mL.

And (3) transferring 100 mu L of promazine, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline standard stock solution, commercially available chlorpromazine and a toluene thiazine standard product with the mass concentration of 1000 mu g/mL, and fixing the volume 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 between-16 and 20 ℃ in a dark place. When in use, the mixture is gradually diluted to the concentration of 100ng/mL and 10ng/mL by methanol, and the mixture is prepared on site.

And transferring chlorpromazine-D6, promethazine-D6 and tolylthiazine-D6 standard stock solutions, and fixing the volume 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 preserving the mixed internal standard use solution at the temperature of minus 16-20 ℃ in a dark place.

To the blank substrate, 50. Mu.L, 100. Mu.L, 0.5mL and 1mL of a mixed standard solution of 10ng/mL were added, and 50. Mu.L, 100. Mu.L and 200. Mu.L of a mixed standard solution of 1000ng/mL were added, and the mixture was treated in the same steps as the extraction and purification of the sample to prepare 7 standard solutions (0.5, 1, 5, 10, 50, 100 and 200 ng/mL) of different mass concentrations, and an internal standard method was used to draw a working curve.

(II) sample preparation

After the cold fresh pig muscle products are cleaned, the hair, the skin, the blood stasis, the tendons and the bones are removed, and the pig muscle products are prepared. Thawing frozen pork at room temperature, slightly thawing the sample, softening, removing hair, skin, tendons and bones when the interior is just thawed and the frozen water is not flowing out, preparing, and cleaning the byproducts such as pig heart, liver, kidney and lung with experimental water. The sample is cut into small pieces of 1cmX cm, uniformly mixed, reduced to 500g by a quartering method, smashed by a smashing machine, uniformly mixed and packaged into a plurality of sample containers for standby. Pretreating viscera of livestock and fowl, dicing or cutting, mashing with a masher, mixing, and packaging into sample containers.

(III) sample preservation

If the sample cannot be detected in time after preparation, the sample should be frozen and preserved immediately to ensure that the sample cannot be melted and deteriorated before detection. The test sample tested on the same day can be stored in a temporary refrigeration way for no more than 8 hours. The frozen sample should be subjected to test after thawing at 45 ℃ or below for no more than 15min, or 2-5 ℃ for no more than 18 h.

(IV) extraction

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

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

(V) extraction

100. Mu.L of methanol was added, a small amount of water was added, the residue was dissolved, transferred to a 2mL centrifuge tube, 1mL of n-hexane was added to a volume of 1.0mL of water, an adsorbent (50 mg C18,10mg PSA,50mg neutral alumina) was added, and the mixture was centrifuged by vortexing for 1min,12000r/min for 5min, and the supernatant was filtered through a 0.22 μm filter membrane for liquid use.

(sixth) detection

Detection conditions were measured using a 6410B liquid chromatograph-tandem quadrupole mass spectrometer, agilent, usa:

chromatographic column: agilent Eclipse XDB-C18 (3 mm. Times.100 mm,1.8 μm); flow rate: 0.2mL/min; mobile phase: phase A is 0.1% ammonia water; the phase B is methanol, the gradient elution is carried out, and the specific setting conditions are shown in Table 2; column temperature: 40 ℃; sample injection amount: 10. Mu.L; and (5) quantifying by an internal standard method.

TABLE 2 gradient elution conditions for mobile phases

Mass spectrometry ion source: ESI ion source temperature: 100 ℃; dry air flow: 10L/h; capillary voltage: 4000V; temperature of air flow: 350 ℃; atomization gas pressure: 40psi; type of collision gas: nitrogen gas; positive ions; the detection mode is as follows: mass spectrometry multiple reaction monitoring MRM. Wherein, the mass spectrum acquisition parameter conditions are shown in Table 3.

Table 3 conditions for mass spectrum acquisition parameters

(seventh) 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 the extractant, the extraction mode, the extraction time of the feed liquid ratio and the like, and the result is analyzed. The analysis procedure of the results is specifically described below:

1. spectrogram analysis

The target compound (sedative drug to be detected) is completely separated from the interfering substances by analysis according to chromatographic conditions, and the peak shape is good. See table 4 and figures 1 to 7 for details.

TABLE 4 target compound retention time and relative abundance of next strongest fragment ions

2. Selection of extractant

The extraction effects of acetone, methanol, acetonitrile and acidified acetonitrile (0.1% formic acid) on 7 target compounds are examined respectively, and compared with the extraction results, the 4 different extractants have no obvious influence on the standard recovery rate of the target compounds, but after acetonitrile extraction, the peak area response value of characteristic ion fragments of each target compound in pork, pork liver, pig heart, pig lung and pig kidney is obviously higher than that of other extractants, and the analysis recovery rate has no obvious influence on the main reason that the internal standard method counteracts the loss of species in the extraction process, so that the acetonitrile is preferably used as the extractant.

3. Selection of extraction mode, time and temperature

The pork, pork liver, pork heart, pork lung and pork kidney have different complex degree, and the tissue and body fluid components and the proportion are different, so that the quality of the required anhydrous sodium sulfate is affected, 5g, 10g, 15g and 20g of anhydrous sodium sulfate with different quality are added, more anhydrous sodium sulfate is needed by the pork liver and the pork lung, 15-20 g of anhydrous sodium sulfate can better remove the moisture in the sample, and 15g of anhydrous sodium sulfate is preferably used for removing water by comprehensive consideration.

Pork, pork liver, pork heart, pork lung and pork kidney are different in caking and wall attachment degree in the extraction process, modes such as shaking by a large number of tests, vortex mixing, rotary shaking, ultrasonic extraction and the like are compared, the extraction mode of vortex mixing and ultrasonic combination is found to be capable of fully extracting target compounds, the samples are easy to agglomerate and wall attachment after centrifugation for the first extraction, the samples cannot be effectively dispersed by vortex and ultrasonic after the extractant is directly added, so that the extraction efficiency is affected, the samples can be dispersed by adopting a simple mechanical shaking mode before the reagent is added, and the conditions of the pork lung, the pork liver and the pork kidney sample wall attachment can be effectively improved by an up-down shaking mode after the reagent is added. Comprehensively considering the extraction effect and the working efficiency, selecting an extraction mode of vortex for 1min and ultrasonic for 10 min. In addition, the proper temperature can effectively accelerate the protein deterioration and improve the purification efficiency, but the temperature is too high to easily cause the decomposition of target compounds, and the ultrasonic temperature is selected to be 35-55 ℃ after a large number of experiments.

4. Selection of concentration mode

The two sample concentration modes of nitrogen blowing and rotary evaporation are examined, and rotary evaporation is preferred because of more extraction solvent. In the test process, the pressure and time required by rotary evaporation are found to be very different due to different substrates of pork, pork liver, pork heart, pork lung and pork kidney samples, the required pressure and time are required to be adjusted at any time according to different substrates, more manpower is consumed, the problem is improved by adding a certain amount of isopropanol according to the actual requirement of mass sample test, and the samples are effectively evaporated to near dryness. 10mL of isopropanol was tested to be suitable for use in the present process.

5. Selection of the purification conditions

The purification effect of the adsorption purifying agents such as C18, PSA, neutral alumina and diatomite filter aid on pork, pork liver, pork heart, pork lung, pork kidney and other different matrix samples is examined, the quantitative determination of each adsorbent by using an internal standard method has no obvious influence on the sample standard adding recovery rate, but has important influence on the signal response value of a target compound and the interference of characteristic ions, particularly the purification effect of 2, 6-dimethylaniline and 2-chlorophenothiazine on the samples is obvious by comparing C18, PSA and neutral alumina, but the adsorption of different degrees on the target compound can be carried out when the using amount is more, and 50mg of C18,10mg of PSA and 50mg of neutral alumina are preferable for purification after the experiment, and 1mL of n-hexane is added for degreasing.

6. Selection of detection conditions

(1) Conditions for liquid phase separation

A plurality of mobile phase proportions such as methanol and acetonitrile are considered as the organic phase, water phase is water, 0.1% formic acid, 5mmol ammonium acetate (0.1% formic acid water) solution, 0.01% ammonia water-0.1% ammonia water and the like, a large number of experiments show that the separation effect of each target compound is better than acetonitrile when the organic phase is methanol, and when the water phase is water, 0.1% formic acid and 5mmol ammonium acetate (0.1% formic acid water) solution, the 2-chlorophenothiazine signal response is extremely low and only has response when the concentration is higher. When the water phase is ammonia water, the response of the 2-chlorophenothiazine is good, the response value of 7 target compounds is greatly enhanced, the peak type is good, the interference influence is avoided, and the signal response value is continuously increased along with the increase of the concentration of the ammonia water. Considering the tolerance degree and signal response of the pH value of the chromatographic column, preferably 0.1% ammonia water is used as a water phase flow item, 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 requirement is met.

(2) Mass spectrometry conditions

Through a large number of experiments, various mass spectrum parameters are tuned, and the response degree of each fragment ion is comprehensively compared, so that the mass spectrum ion source conditions, the fragmentation voltage, the collision voltage and qualitative and quantitative ions are determined, and the specific reference is made to the table 2.

7. Selection of internal standard

The synchronous response degree of 4 substances such as SKF-525A, tolylthiazide-D6, chlorpromazine-D6, promethazine-D6 and the like to 7 target compounds is examined, and compared, the 4 substances have better synchronous response to 7 compounds in the same substrate, when a pork sample is used as a substrate to draw a working curve, the synchronous response is greatly influenced by 5 different substrates such as pork, pork liver, pig heart, pig lung and pig kidney, wherein SKF-525A is easily influenced by substrate interference due to higher polarity, and is not suitable for being used as an internal standard due to the fact that the SKF-525A is easily influenced by substrate interference due to the fact that the 7 target compounds have certain synchronous correspondence in the pork sample. The tolylthiazide-D6, the chlorpromazine-D6 and the promethazine-D6 correspond well with the tolylthiazide, the chlorpromazine and the promethazine, and the tolylthiazide-D6 and the chlorpromazine sulfoxide, the promethazine sulfoxide, the 2, 6-dimethylaniline and the 2-chlorophenothiazine synchronously respond due to similar polarities and similar peak-out times, so the tolylthiazide-D6 is selected as a reference internal standard.

8. Verification of methods

(1) Linear range, detection limit and quantification limit

To the blank substrate, 50. Mu.L, 100. Mu.L, 0.5mL and 1mL of a mixed standard solution of 10ng/mL were added, 50. Mu.L, 100. Mu.L and 200. Mu.L of a mixed standard solution of 1000ng/mL were added, and the mixture was treated according to the above steps of sample extraction and purification to prepare 7 standard solutions (0.5, 1, 5, 10, 50, 100 and 200 ng/mL) of different mass concentrations, and an internal standard method was used to draw a working curve. Standard and standard samples were measured, the standard deviation of the measurement results of 7 target compounds was calculated by measuring 7 times the sample added at the lowest acceptable concentration of 0.1 μg/kg, the limit of detection was calculated as lod=0+3s, and the limit of quantification was calculated as loq=0+10s. The detection limit and the quantitative limit of the linear regression equation and the method are shown in Table 5.

Table 5 7 Linear regression equation of target compounds and method detection limit and quantitative limit

(2) Method precision and recovery

7 parts of positive samples with the concentration level of 10.0 mug/kg are respectively added into different pig edible tissues such as pig hearts, pig lungs, pig kidneys, pork and pig livers, and the measurement results are shown in table 4. The recovery rate of the tolylthiazide 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 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%, and the detailed results are shown in tables 6-10.

TABLE 6 pig heart sample recovery and precision test results

TABLE 7 pig heart sample recovery and precision test results

Table 8 results of the pig kidney sample recovery and precision test

TABLE 9 results of pork sample recovery and precision test

Table 10 results of pig liver sample recovery and precision test

(3) Stability of

The same standard concentration level (10.0 mug/kg) sample is measured by different operators respectively at intervals of 0 day, 3 day and 10 day, the average measuring result RSD of 3 times is calculated, the repeatability of 7 target compounds in 5 different matrixes is 2.33% -11.8%, the repeatability is 15%, the results show that the method is reliable and stable, and the detailed table 11-17 is shown.

Table 11 results of pig heart sample recovery and precision test

Table 12 results of pig lung sample recovery and precision test

TABLE 13 results of pig kidney sample recovery and precision test

Table 14 results of pork sample recovery and precision test

Table 15 results of pig liver sample recovery and precision test

While the foregoing has been disclosed in the specification and drawings, it will be apparent to those skilled in the art that various substitutions and modifications may be made without departing from the spirit of the invention, and it is intended that the scope of the invention be limited not by the specific embodiments disclosed, but by the appended claims.

Claims (5)

1. A method for detecting sedative drug metabolic residues in animal tissues, comprising:

preparing a sample, cleaning the unfrozen animal tissue to be tested, removing hair, skin, blood stasis, tendons and bones, cutting into pieces, mashing, and subpackaging in a sample container, and preserving for later use, wherein the animal tissue to be tested comprises pork, pork liver, pork kidney, pork heart or pork lung;

extracting a sample, namely weighing 5g of the sample, putting the sample into 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 for 10min at 35-55 ℃, taking out the centrifuge tube, cooling the mixed liquid in the centrifuge tube to room temperature, centrifuging for 5min at 4500r/min, and transferring the obtained supernatant to a 100mL chicken heart bottle;

extracting the solids in the centrifuge tube after removing the supernatant twice to obtain a supernatant of the second time, combining the supernatants of the second time, adding 10mL of isopropanol, and performing rotary evaporation to near dryness at 45 ℃ to obtain residues;

sample purification, namely adding 100 mu L of methanol into the residue, dissolving the residue with a small amount of water, transferring the residue into a 2mL centrifuge tube, adding 1mL of n-hexane into the centrifuge tube with water to a volume of 1.0mL, adding 50mg of C18,10mg of PSA and 50mg of neutral alumina, carrying out vortex oscillation for 1min, centrifuging for 5min with 12000r/min, and filtering the supernatant for later use;

sample detection, namely detecting the filtered supernatant by a machine, wherein detection conditions comprise:

chromatographic column: an Agilenteclipse XDB-C18 column, 3mm 100mm,1.8 μm; flow rate, 0.2mL/min; mobile phase: phase A is 0.1% ammonia water; the phase B is methanol, and the gradient elution is carried out; column temperature: 40 ℃; sample injection amount: 10. Mu.L; quantitative determination by an internal standard method;

the temperature of the ESI ion source is 100 ℃; the dry air flow is 10L/h; capillary voltage 4000V; the temperature of the air flow is 350 ℃; atomization gas pressure 40psi; the collision gas type is nitrogen; positive ions; the detection mode is mass spectrum multi-reaction monitoring;

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

mobile phase a is 5% and mobile phase B is 95%; the phase A of the mobile phase is 5 to 95 percent and the phase B of the mobile phase is 95 to 5 percent within 17 to 17.1 minutes; at 17.1-18 min, the mobile phase A phase is 95% and the mobile phase B phase is 5%;

wherein the sedative drug comprises: chlorpromazine, promethazine hydrochloride, tolylthiazide, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline and 2-chlorophenothiazine.

2. The method of claim 1, wherein the secondary extraction of the supernatant-removed solids in the centrifuge tube comprises: and (3) dispersing the solid in the centrifuge tube, adding 15mL of acetonitrile, shaking up and down by hands for 30s, performing ultrasonic extraction for 10min at the temperature of 35-55 ℃, taking out the centrifuge tube, cooling the mixed liquid in the centrifuge tube to room temperature, and centrifuging for 5min at 4500r/min to obtain a supernatant liquid of the second time.

3. The method of claim 1, wherein the filtering of the supernatant comprises: the supernatant was filtered through a 0.22 μm filter.

4. The method of detecting according to claim 1, further comprising:

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

transferring 100 mu L of promethazine hydrochloride, chlorpromazine sulfoxide, promethazine sulfoxide, 2, 6-dimethylaniline standard stock solution, chlorpromazine and a toluene thiazine standard product with the mass concentration of 1000 mu g/mL, and preparing a mixed intermediate solution with the mass concentration of 1000ng/mL by using methanol to fix the volume in a 10mL brown volumetric flask, and freezing and preserving in a dark place;

and transferring chlorpromazine-D6, promethazine-D6 hydrochloride and tolylthiazine-D6 standard stock solutions, and fixing the volume 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 performing light-proof freezing preservation.

5. The detection method according to claim 4, wherein the preparation is a mixed intermediate solution with a mass concentration of 1000ng/mL, the temperature of the mixed intermediate solution is between-16 and 20 ℃ in a dark freezing preservation mode, the preparation is a mixed internal standard use solution with a mass concentration of 1000ng/mL, and the temperature of the mixed intermediate solution is between-16 and 20 ℃ in a dark freezing preservation mode.