CN108375635B - Method for detecting enramycin residue in animal tissue - Google Patents

Abstract

The invention relates to a method for detecting enramycin residue in animal tissues, which comprises the following steps of 1) extracting a sample before detection by adopting an HP L C-MS/MS method, namely, homogenizing the sample, extracting the sample by using an extracting solution, removing organic matters in the solution by blowing nitrogen after extraction, then fully mixing the solution with a hydrochloric acid solution, centrifuging, and taking supernatant, and 2) purifying the sample, namely purifying the supernatant by using an ENV solid-phase extraction column to obtain a sample to be detected.

Description

Method for detecting enramycin residue in animal tissue

Technical Field

The invention relates to the field of detection of enramycin, in particular to a method for detecting residual enramycin in animal tissues.

Background

Enramycin (enramycin) is a polypeptide antibiotic obtained by fermenting actinomycetes (Streptomyces fungicidicus), and has main components of enramycin A and B, which are applied in the form of hydrochloride. White or slightly yellowish white powder (crude gray or grayish brown powder, with peculiar smell).

Enramycin is an organic base composed of 13 different types of 17 amino acid molecules and fatty acid molecules. Wherein the amino acid molecule forms a cyclic polypeptide structure, and the type of the terminal fatty acid is different. Can be divided into enramycin A (C)₁₀₇H₁₃₈Cl₂N₂₆O₃₁) And enramycin B (C)₁₀₈H₁₄₀Cl₂N₂₆O₃₁) It is a mixture of these two components. The hydrochloride of the enramycin is white crystalline powder, has the molecular weight of about 2500, the melting point of 238-245 ℃, is easily soluble in dimethyl sulfoxide and dilute hydrochloric acid, is soluble in methanol and aqueous ethanol, is insoluble in acetone and is insoluble in benzene and chloroform. Enramycin AThe hydrochloride salt of the element has excellent stability to heat, light and moisture. The enramycin has high stability in feed, is less degraded after being stored for a long time at room temperature, is very stable in the process of preparing granules, and has little reduced titer after being mixed with the feed after being stored for a long time at room temperature. The enramycin is not degraded in intestinal tracts and can keep the original antibacterial activity, and the structural formula of the enramycin is as follows:

enramycin has a powerful bactericidal effect on major gram-positive bacteria under both aerobic and anaerobic conditions. The enramycin not only has a bacteriolytic effect, but also has a bactericidal effect. The enramycin has wide gram-positive bacteria resistance, excellent effects of promoting growth and improving the utilization rate of feed, and can promote growth and improve feed reward by adding the enramycin into the piglet opening feed. But also can reduce the occurrence of diarrhea of piglets. The enramycin mixing material can play a role in promoting growth and improving feed reward for both broilers and reserve chickens, and enramycin mainly acts on intestinal flora to improve the bad condition of defecation. Enramycin can enhance the anticoccidial activity of anticoccidial drugs or reduce the incidence of coccidiosis.

In order to ensure the safety of animal food and public health, the enramycin residue in the animal food needs to be detected and supervised, and the highest residue limit of enramycin in the animal food is not established in China. The European Union, the United states and the International Committee for food code council (CAC) have not reported the maximum residual limit of enramycin. The dosage of the enramycin premix per ton of feed specified by the veterinary drug dictionary in China is as follows: 2.5-20 g of pig and 1-5 g of chicken. The rest period of the pigs and the chickens is 7 days.

Enramycin belongs to the difficulty of polypeptide antibiotic detection, and a method for detecting enramycin residue in animal tissues is not established in China. Related reports on the world are few, only meat detection methods are established, and other tissue detection methods are blank. Therefore, the establishment of the method for detecting enramycin residue in animal food is of great significance.

Disclosure of Invention

The invention aims to provide a method for detecting residual enramycin in animal tissues, which is mainly improved by carrying out the following pretreatment on a sample before detection by adopting an HP L C-MS/MS method:

1) sample extraction: homogenizing and homogenizing an animal tissue sample, extracting the animal tissue sample with an extracting solution, blowing nitrogen to remove organic matters in the solution after extraction is finished, then fully mixing the residual solution with a hydrochloric acid solution, and centrifuging to obtain a supernatant;

2) sample purification: purifying the supernatant by using an ENV solid phase extraction column to obtain a sample to be detected;

the preparation method of the extracting solution comprises the following steps:

A. mixing methanol and hydrochloric acid to prepare hydrochloric acid methanol mixed solution with the volume fraction of the hydrochloric acid being 1.8%;

B. and mixing the 1.8% hydrochloric acid methanol mixed solution with water to prepare a solution with the volume fraction of the hydrochloric acid methanol mixed solution of 54-56%.

According to the structural characteristics of enramycin, the method preferably selects a certain proportion of acidified methanol water as an extraction solvent, and effectively reduces matrix interference through purification by an ENV solid phase extraction cartridge. The method has the characteristics of simple operation, low detection limit, good reproducibility and low determination cost, and is suitable for quantitative analysis of enramycin residue in animal food. The detection method has wide application range, and the detection object can be tissues of various animals, such as muscle, liver, kidney, fat and the like.

Preferably, the specific operation of the step 2) is that the supernatant obtained in the step 1) passes through the ENV solid-phase extraction column, then the ENV solid-phase extraction column is leached once by using a mixed solution of methanol and a 5% ammonia water solution, then the ENV solid-phase extraction column is leached once by using a mixed solution of formic acid and ethyl acetate, finally the ENV solid-phase extraction column is eluted by using a mixed solution of methanol and 0.1M hydrochloric acid, and the obtained eluent passes through an organic filter membrane to obtain a sample to be detected;

preferably, the volume ratio of the methanol to the 5% ammonia water solution is 20:80, the volume ratio of the formic acid to the ethyl acetate is 1:99, and the volume ratio of the methanol to the 0.1M hydrochloric acid is 80: 20.

Preferably, the specific operation of the extraction in the step 1) is to mix the sample and the extracting solution according to the mass-volume ratio of 1: 4.5-5.5, sufficiently shake and mix the mixture, centrifuge the mixture, take the supernatant, repeat the operation for 3 times, and combine the supernatants;

and mixing the solution subjected to nitrogen blowing and organic matter removal with 0.1M hydrochloric acid solution according to the volume ratio of 5: 0.8-1.2, fully shaking and uniformly mixing, performing ultrasonic treatment, and finally centrifuging to obtain a supernatant.

Preferably, the conditions of the liquid chromatography detection are as follows: and (3) adopting a C18 chromatographic column, wherein the column temperature is 28-32 ℃, and selecting aqueous solution of formic acid with the volume fraction of 0.2% and methanol as eluent for gradient elution.

Preferably, the specific operation of gradient elution is as follows: 0-1.5 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is from 90:10 to 50: 50; 1.5-3.3 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is 50:50 is adjusted to 30:70 for 3.3-3.5 min, and the volume ratio of 0.2% formic acid water solution to methanol is adjusted from 30:70 to 5: 95; 3.5-6 min, and maintaining the volume ratio of 0.2% formic acid water solution to methanol at 5:95, 6 to 6.1min, wherein the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is adjusted from 5:95 to 90:10 and 6.1 to 9min, and the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is maintained at 90: 10. (see Table 1 in the examples for details)

Preferably, during the mass spectrometric detection, enramycin A has qualitative ion pairs of 786.1 > 1089.6, 786.1 > 178.9 and 786.1 > 95.3, corresponding to collision energies of 25eV, 25eV and 30eV, quantitative ion pairs of 786.1 > 1089.6, corresponding to collision energies of 25 eV;

the qualitative ion pair of enramycin B is 790.9 > 1089, 790.9 > 193.2 and 790.9 > 95, corresponding to collision energies of 25eV, 25eV and 30eV, the quantitative ion pair is 790.9 > 1089, corresponding to a collision energy of 25 eV.

Preferably, the mass spectrometry detection conditions comprise that an electrospray ion source is selected, the temperature of a dry gas is 300 ℃, the flow rate of the dry gas is 7L/min, the pressure of the atomization gas is 35Psi, the temperature of a sheath gas is 300 ℃, the flow rate of the sheath gas is 11L/min, the voltage of a capillary is 3500V, the voltage of a nozzle is 1500V, and the corona current is 0.16 uA.

Preferably, the method of the present invention comprises the steps of:

1) pretreatment of samples

Homogenizing and homogenizing an animal tissue sample, mixing the animal tissue sample with an extracting solution according to the mass-volume ratio of 1: 4.5-5.5, fully shaking and uniformly mixing, centrifuging, taking supernate, repeating the operation for 3 times, and combining the supernate;

mixing the solution after removing the organic matters by nitrogen blowing with 0.1M hydrochloric acid solution according to the volume ratio of 5: 0.8-1.2, fully shaking and uniformly mixing, then carrying out ultrasonic treatment, and finally centrifuging to obtain supernatant;

the extracting solution is prepared by the following method:

A. mixing methanol and hydrochloric acid to prepare hydrochloric acid methanol mixed solution with the volume fraction of the hydrochloric acid being 1.8%;

B. mixing the 1.8% hydrochloric acid methanol mixed solution with water to prepare a solution with the volume fraction of the hydrochloric acid methanol mixed solution of 556%, namely the extracting solution;

2) purification of the supernatant

Enabling the supernatant obtained in the step 1) to pass through an ENV solid-phase extraction column, leaching the ENV solid-phase extraction column once with a mixed solution of methanol and a 5% ammonia water solution, leaching the ENV solid-phase extraction column once with a mixed solution of formic acid and ethyl acetate, eluting the ENV solid-phase extraction column once with a mixed solution of methanol and 0.1M hydrochloric acid, and filtering the obtained eluent with an organic filter membrane to obtain a sample to be detected;

the volume ratio of the methanol to a 5% ammonia water solution is 20:80, the volume ratio of the formic acid to the ethyl acetate is 1:99, and the volume ratio of the methanol to the 0.1M hydrochloric acid is 80: 20;

3) liquid chromatography detection

Performing liquid chromatography detection on the sample to be detected prepared in the step 2), wherein the detection conditions are as follows: adopting a C18 chromatographic column, controlling the column temperature at 30 ℃, and selecting aqueous solution of formic acid with volume fraction of 0.2% and methanol as eluent to carry out gradient elution;

the specific operation of the gradient elution is as follows: 0-1.5 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is from 90:10 to 50: 50; 1.5-3.3 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is 50:50 is adjusted to 30:70 for 3.3-3.5 min, and the volume ratio of 0.2% formic acid water solution to methanol is adjusted from 30:70 to 5: 95; 3.5-6 min, and maintaining the volume ratio of 0.2% formic acid water solution to methanol at 5:95, 6 to 6.1min, wherein the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is adjusted from 5:95 to 90:10, 6.1 to 9min, and the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is maintained at 90: 10;

4) mass spectrometric detection

Detecting the sample after the liquid chromatography detection by a secondary mass spectrum, wherein the mass spectrum detection conditions are as follows:

selecting an electrospray ion source, wherein the temperature of dry gas is 300 ℃, the flow of the dry gas is 7L/min, the pressure of the atomization gas is 35Psi, the temperature of sheath gas is 300 ℃, the flow of the sheath gas is 11L/min, the voltage of a capillary tube is 3500V, the voltage of a nozzle is 1500V, and the corona current is 0.16 uA;

in the process of mass spectrometry detection, qualitative ion pairs of enramycin A are 786.1 & gt 1089.6, 786.1 & gt 178.9 and 786.1 & gt 95.3, the corresponding collision energy is 25eV, 25eV and 30eV respectively, quantitative ion pairs are 786.1 & gt 1089.6, and the corresponding collision energy is 25 eV;

the qualitative ion pair of enramycin B is 790.9 > 1089, 790.9 > 193.2 and 790.9 > 95, corresponding to collision energies of 25eV, 25eV and 30eV, the quantitative ion pair is 790.9 > 1089, corresponding to a collision energy of 25 eV.

The method of the invention has the following beneficial effects:

1) according to the method, a certain proportion of acidified methanol water is preferably selected as an extraction solvent according to the structural characteristics of enramycin, and purification is performed by an ENV solid phase extraction cartridge, so that matrix interference is effectively reduced, and enramycin can be effectively extracted.

2) The method further optimizes a liquid chromatography detection method and a mass spectrometry detection method, can accurately detect the enramycin in the animal tissue, has the characteristics of simple operation, low detection limit, good reproducibility and low detection cost, and is suitable for quantitative analysis of enramycin residue in animal food.

3) The method of the invention has wide application range and can be applied to any kind of animal tissues.

Drawings

FIG. 1 shows the recovery rates of enramycin A in different proportions;

FIG. 2 shows the recovery rates of enramycin B in different proportions;

FIG. 3 shows the recovery of 0.1MHC L for different ratios of enramycin A elution;

FIG. 4 shows the recovery of 0.1MHC L for different ratios of enramycin B elution;

FIG. 5 blank muscle enramycin AMRM chromatogram;

FIG. 6 blank muscle enramycin B MRM chromatogram;

FIG. 7 blank Mycosphaerella sarmentosa (50. mu.g/kg) MRM chromatogram;

FIG. 8 blank Mycosphaerella gemmifera B (50. mu.g/kg) MRM chromatogram;

FIG. 9 is a blank enramycin AMRM chromatogram;

FIG. 10 is a blank fatty enramycin B MRM chromatogram;

FIG. 11 is a MRM chromatogram of blank enramycin A (50. mu.g/kg);

FIG. 12 is a MRM chromatogram of blank enramycin B (50. mu.g/kg);

FIG. 13 blank liver enramycin AMRM chromatogram;

FIG. 14 blank liver enramycin B MRM chromatogram;

FIG. 15 blank liver adding enramycin A (50. mu.g/kg) MRM chromatogram;

FIG. 16 blank liver adding enramycin B (50. mu.g/kg) MRM chromatogram;

FIG. 17 blank renal enramycin AMRM chromatogram;

FIG. 18 blank renal enramycin B MRM chromatogram;

FIG. 19 is a MRM chromatogram of blank kidney supplemented with enramycin A (50. mu.g/kg);

FIG. 20 is a MRM chromatogram of a blank kidney supplemented with enramycin B (50. mu.g/kg);

FIG. 21 MRM chromatogram (25. mu.g/kg) of enramycin A standard solution;

FIG. 22 MRM chromatogram (25. mu.g/kg) of enramycin B standard solution;

FIG. 23a is a standard curve of stroma-added enramycin A in porcine muscle tissue;

FIG. 23b is a standard curve of stroma addition enramycin A for porcine kidney;

FIG. 23c Standard Curve for stroma addition of enramycin A in porcine liver;

FIG. 23d Standard Curve for adding enramycin A to swine fat base;

FIG. 24a is a standard curve of stroma-additive enramycin B in porcine muscle tissue;

FIG. 24B is a standard curve of stroma addition enramycin B on porcine kidney;

FIG. 24c Standard Curve for stroma addition of enramycin B in porcine liver;

FIG. 24d Standard Curve for adding enramycin B to ground substance of pig fat.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

This example relates to the detection of enramycin in the muscle tissue of pigs:

1.1 instruments and reagents used in the experiment:

ultra-high performance liquid chromatography-tandem mass spectrometry: 6470, Agilent, USA;

a high-speed refrigerated centrifuge: SIGMR 3K15, eastern scientific and technological development, Beijing Wuzhou;

vortex mixer: VX-III, Beijing Pedal science and technology Co., Ltd;

an ultrasonic cleaner: HS3120, tianjin henkouzhi technical development ltd;

nitrogen blowing instrument: DC-12, Shanghai' an Spectroscopy scientific instruments, Inc.;

solid phase extraction column ENV (200mg/3m L), Agilent, USA;

enramycin a standard: the content is more than or equal to 95 percent, BOC SCIENCES company;

enramycin B standard: the content is more than or equal to 85 percent, BOC SCIENCES company;

the chromatographic purity of methanol, ethyl acetate and formic acid is high;

hydrochloric acid, ammonia water, analytically pure.

Water, secondary water according to GB/T6682.

1.2 preparation of the Main test solutions

The enramycin standard stock solution (1mg/m L) is prepared by accurately weighing 5.0mg of enramycin standard, placing in a 5m L volumetric flask, dissolving with methanol, metering to a certain volume, and shaking to obtain the enramycin standard stock solution which is stored in a refrigerator at-20 ℃.

The preparation of the enramycin standard working solution comprises accurately sucking 1M L of enramycin stock solution into a 10M L volumetric flask, dissolving and diluting the enramycin stock solution to scale with methanol and 0.1M hydrochloric acid (8:2), diluting the solution to 100 mu g/M L, and sequentially diluting the standard working solutions with concentrations of 0.05, 0.1, 0.5, 1 and 2 mu g/M L, and storing the solution at-20 ℃ for 6 months of validity period.

1.8% hydrochloric acid methanol solution 491m L methanol and 9m L hydrochloric acid were transferred to a 500m L reagent bottle and mixed by ultrasound for use.

55 percent (1.8 percent hydrochloric acid) methanol water solution, namely taking 1.8 percent hydrochloric acid methanol 220m L and 180m L water in a reagent bottle of 500m L, and ultrasonically mixing the mixture for standby.

0.1M hydrochloric acid, namely transferring 1M L hydrochloric acid into a 120M L volumetric flask, adding water to the volumetric flask to reach a constant volume to a scale, and ultrasonically mixing the solution uniformly for later use.

1% ethyl formate-ethyl acetate 495m L and 5m L were pipetted into a 500m L vial and mixed by sonication for further use.

methanol-0.1M hydrochloric acid (80: 20): mixing methanol and 0.1M hydrochloric acid at a ratio of 8:2 by ultrasonic wave.

And (3) transferring 5m of L ammonia water and 95m of L water into a 100m L reagent bottle, and ultrasonically mixing for later use.

Methanol-5% ammonia solution (20:80) 80m of L5% ammonia solution and 20m of L% methanol are transferred into a 100m L reagent bottle and mixed evenly by ultrasound for standby.

1.3 sample pretreatment

1.3.1 sample preparation

Taking out 30-50 g of frozen sample, thawing, cutting, homogenizing at high speed, and homogenizing for later use.

1.3.2 sample extraction

Accurately weighing (2.0 +/-0.05) g of a homogeneous tissue sample in a 50M L centrifuge tube, adding 10M L55% (1.8% hydrochloric acid) methanol aqueous solution, uniformly mixing in a vortex manner, carrying out vortex oscillation for 10min, centrifuging at 1000r/min at low temperature for 10000r/min, centrifuging for 10min, transferring a supernatant into a 50M L centrifuge tube, adding 10M L55% (1.8% hydrochloric acid) methanol aqueous solution into residues, repeatedly extracting for two times, combining three extracting solutions, blowing nitrogen to 12.5M L, adding 2.5M L0.1.1M hydrochloric acid, uniformly mixing in a vortex manner, carrying out ultrasound for 5min, centrifuging at low temperature for 10000r/min, centrifuging for 10min, and taking the supernatant to be purified by an upper column.

1.3.3 sample clarification

Activating an ENV (200mg, 3M L) solid phase extraction column by using 5M L methanol and 5M L water in sequence, passing all standby liquid through the column, leaching the standby liquid by using 6M L methanol-5% ammonia water solution (20:80) after all sample liquid flows out, draining for 1min, leaching by using 6M L1% ethyl formate once, draining for 2min, finally eluting by using 8M L methanol-0.1M hydrochloric acid (80:20), draining for 1min, vortexing, uniformly mixing, and passing through a microporous organic filter membrane to be detected.

1.4 sample determination

1.4.1 apparatus conditions

1.4.1.1 chromatographic working conditions

Chromatographic column Agilent EC-C18 chromatographic column (Dim: 2.1 × 100mm, particle size: 2.7 μm);

mobile phase: gradient elution with 0.2% formic acid and methanol, gradient as shown in table 1;

column temperature: 30 ℃;

the injection volume was 10. mu. L.

TABLE 1 gradient elution conditions

1.4.1.2 Mass Spectrometry conditions

An ion source: electrospray ion source AJS ESI (+);

drying Gas temperature (Gas Temp): 300 ℃;

dry Gas Flow (Gas Flow) 7L/min;

atomization gas pressure (Nebulier): 35 Psi;

sheath Gas temperature (Sheath Gas Temp): 300 ℃;

sheath Gas Flow (Sheath Gas Flow) 11L/min;

capillary voltage (Capillary): 3500V;

nozzle Voltage (Nozzle Voltage): 1500V;

corona Current (Corona Current): 0.16 uA.

The qualitative and quantitative ion pairs, the voltage of the taper hole and the collision energy are shown in Table 2

TABLE 2 qualitative and quantitative determination of ion pairs

1.5 assay method

Single-point calibration:

or matrix matching standard curve calibration: from As aCs + b, a and b are determined

Calculating the residual quantity of enramycin according to the following formula:

in the formula:

x is the residual quantity (mu g/kg) of enramycin in a test sample;

cs-the concentration of the corresponding enramycin drug in the matrix-matched solution (ng/m L);

c-corresponding enramycin drug concentration (ng/m L) in the test material solution;

as is the area of the peak of the corresponding enramycin medicament in the matrix matching solution;

a is the peak area of the corresponding enramycin medicament in the test material solution;

v-volume of residue (m L);

m-test sample mass (g).

Note: the blank value is subtracted from the calculation result, the measurement result is represented by the arithmetic mean value of the parallel measurement, and three significant figures are reserved.

Detection shows that after 5 days of medicine feeding, the residual quantity of the enramycin A in the pig muscle is 0, and the residual quantity of the enramycin B in the pig muscle is 17.34 mu g/kg.

Example 2

The difference compared to example 1 is that the sample tested was porcine liver. Detection shows that after 5 days of medicine feeding, the residual quantity of enramycin A and enramycin B in the pig liver is 0.

Example 3

The difference compared to example 1 is that the sample tested was porcine kidney. Detection shows that after 5 days of medicine feeding, the residual quantity of enramycin A in the pig kidney is 19.12 mu g/kg, and the residual quantity of enramycin B in the pig kidney is 14.92 mu g/kg.

Example 4

The difference compared to example 1 is that the sample tested was pig fat. The detection proves that no enramycin A or enramycin B remains in the pig fat.

Comparative example 1

The comparative example compares the influence of different extracting solutions on the extraction effect, and the selected extracting solutions are respectively as follows:

A. 2% Formic Acid (FA) in methanol, wherein the formic acid is 2% by volume;

B. acetonitrile;

C. 2% trichloroacetic acid-EDTA phosphate buffer solution: methanol (40:60, v/v),

D. EDTA phosphate buffer solution: methanol (40:60, v/v),

E. 1% trifluoroacetic acid (TFA) in methanol: 1% trifluoroacetic acid (TFA) in water (5: 3, v/v)),

F. 0.1M HC L solution methanol (20:80, v/v),

G. 1% HC L methanol

Enramycin was extracted in the same manner as in example 1. The experiment shows that: (1) under the same operation steps, the extraction solvent A, F, G can obtain better adding recovery rate of the enramycin; (2) the acidified organic solvent has stronger protein precipitation capacity, and the extracting solution contains a plurality of endogenous interferents, particularly for processing liver and kidney samples; (3) because enramycin is easily dissolved in dilute hydrochloric acid, although the recovery rate of F, G is better, the ideal recovery rate is not achieved.

Further, samples were extracted using methanol containing 1% HC L in different ratios of 50% methanol (2% HC L) water, 55% methanol (1.8% HC L) water, 60% methanol (1.6% HC L) water, and 70% methanol (1.4% HC L) water, respectively, and the results were compared and recovered by repeating three days (see FIGS. 1 and 2) (the above solutions were prepared in the same manner as the extracts mentioned in the description)

From the figure, it can be seen that the recovery rate of 55% methanol (1.8% HC L) water is relatively stable after repeating three days, so 55% methanol (1.8% HC L) water is selected as the extract.

Comparative example 2

This comparative example relates to a comparison of the effect of different solid phase extraction columns comparing the purification capacity of the C18, H L B and WCX, PCX and MCX columns, different purification step operations (table 3).

TABLE 3 purification conditions of different extraction columns

According to the analysis, the molecular weight of enramycin is larger, the inner diameter of the column compared in the experiment is smaller, and enramycin is not retained on an E column, so that a column ENV (200mg/3m L) with large-aperture filler is selected.

Comparative example 3

This comparative example compares the effect of different eluents and eluents during the solid phase extraction.

The mixed solution of 5% ammonia water and methanol with different proportions is used as eluent, the mixed solution of 6m L5% ammonia water with the volume ratio of 50%, 60%, 70% and 80% is used for eluting the ENV small column respectively, the detection shows that the methanol solution with the volume ratio of 70% and 80% of ammonia water has no target object, and in order to avoid excessive organic phase, the methanol solution with the volume fraction of 80% of ammonia water is selected as eluent 1.

According to the solubility of enramycin, a mixed solution of methanol and 0.1M hydrochloric acid is selected as an eluent, the proportions of 0.1MHC L and methanol are compared, 9M L0.1 MHC L: methanol (70:30v/v), 0.1MHC L: methanol (50:50v/v), 0.1MHC L: methanol (40:60v/v), 0.1MHC L: methanol (30:70v/v) and 0.1MHC L: methanol (20:80v/v) are respectively used as the eluent, the recovery rate of 0.1MHC L: methanol (20:80v/v) in an on-machine test can reach more than 80% (as shown in figures 3 and 4), and 0.1MHC L: methanol solution (20:80v/v) is selected as the eluent.

Comparative example 4

This comparative example compares the effect of different flows on the results of the liquid chromatography analysis. Acetonitrile, methanol and formic acid solutions are respectively selected as mobile phases, the influence of the mobile phases on the enramycin peak appearance is examined, and the result shows that: methanol is superior to acetonitrile as a mobile phase; if the mobile phase contains 0.2% formic acid aqueous solution, the ion response degree of enramycin and internal standard thereof can be obviously enhanced; therefore, methanol is selected: 0.2% formic acid water as mobile phase.

Examples of the experiments

1. Specificity testing

Blank samples and blank labeled samples, 2g +/-0.02 g, are accurately weighed, operated according to the sample processing method in the embodiment 1, and subjected to UP L C-MS/MS analysis, and the result is shown in figures 5-22 (the abscissa in the figures is the acquisition time).

2. Method of Linear test

Extracting and purifying muscle, liver, kidney and fat matrixes of the blank pigs by adopting the sample pretreatment method in example 1 to prepare blank matrixes, precisely measuring 100 mu l of an enramycin standard solution working solution respectively, adding the working solution into 900 mu l of matrixes to prepare 2, 10, 12.5, 25, 50 and 100 mu g/L series matrix addition concentrations, performing liquid chromatography-tandem mass spectrometry according to the detection conditions in example 1, repeatedly measuring each concentration for 3 times, drawing a standard curve by using the measured peak area and the corresponding concentration, fitting and solving a regression equation and a correlation coefficient.

The results show that the linear relation is good in the concentration range of 2-100.0 mug/L, the correlation coefficients are all above 0.999, and the specific data are shown in tables 4 and 5 and figures 23-24.

TABLE 4 matrix matching standard curve of enramycin A in edible porcine tissue

Tissue of	Regression equation	Linear range (μ g/L)	Correlation coefficient
				Pork	y＝706.83x-700.68	2～100	0.9997
Pig kidney	y＝829.52x-746.96	2～100	0.9990
				Pork liver	y＝614.26-347.36	2～100	0.9985
Pig fat	y＝1920.85x-1404.06	2～100	0.9997

TABLE 5 matrix matching standard curve of enramycin B in porcine edible tissue

Tissue of	Regression equation	Linear range (μ g/L)	Correlation coefficient
				Pork	y＝319.27x-91.33	2～100	0.9993
Pig kidney	y＝257.95x+89.66	2～100	0.9989
				Pork liver	y＝206.21x+424.30	2～100	0.9995
Pig fat	y＝1035.20x-742.5	2～100	0.9992

3. Sensitivity of the probe

Preparing enramycin A and enramycin B standard working solutions with a series of concentrations, adding the prepared enramycin A and enramycin B standard working solutions into blank samples (pig muscles, fat, kidneys and livers) to obtain corresponding sample addition concentrations (10-100 mu g/kg), performing UP L C-MS/MS analysis according to the sample pretreatment method operation of example 1, determining qualitative ion baseline noise values (S/N) at the retention time of the drugs, repeating the steps for 5 times, and calculating an average value, wherein the concentration when the signal-to-noise ratio (S/N) is more than or equal to 3 is a detection limit, the concentration when the signal-to-noise ratio (S/N) is more than or equal to 10 and the RSD is less than or equal to 20% is a quantitative limit, and determining the detection limits of the pig muscles, fat, livers and kidneys are 5 mu g/kg, the quantitative limits are 10 mu g/kg.4, and the accuracy of the method are

The method recovery was determined using standard addition methods. Accurately weighing a plurality of samples of the muscle, fat, liver and kidney of the blank pig, and respectively adding standard working solution to prepare a muscle test material with the drug content of 10, 50 and 100 mu g/kg; the content of the drug in the fat sample is 10, 50 and 100 mug/kg; the content of the medicine in the liver sample is 10, 50 and 100 mug/kg; the drug content in the kidney sample was 10, 50, 100. mu.g/kg, and 5 of each drug was added in parallel, and the samples were continuously examined for 3 days. The recovery rate was calibrated by single point or standard curve and the recovery rate and the intra-day and inter-day variation coefficients are shown in tables 6-13. As can be seen from the table, under the low (or quantitative limit), medium and high concentrations, the recovery rate of the method is 60.10-106.77%, and the daily variation coefficient is less than or equal to 11.24%; the coefficient of variation between days is less than or equal to 10.92%.

The data show that the method is stable and reliable and meets the requirements of accuracy and precision.

Table 6 enramycin a addition recovery and intra-and inter-day precision in porcine muscle (n ═ 5, day ═ 3)

Table 7 enramycin a recovery and intra-and inter-day precision (n ═ 5, day ═ 3) in porcine kidney

Table 8 enramycin a addition recovery and intra-and inter-day precision in porcine liver (n ═ 5, day ═ 3)

TABLE 9 recovery rate of enramycin A addition in pig fat and daily and diurnal precisions (n. 5, day3)

Table 10 enramycin B addition recovery in porcine muscle and intra-and inter-day precision (n ═ 5, day ═ 3)

Table 11 enramycin B addition recovery and intra-and inter-day precision in porcine kidney (n ═ 5, day ═ 3)

TABLE 12 enramycin B addition recovery and intra-and inter-day precision in porcine liver (n-5, day-3)

TABLE 13 recovery rate of enramycin B addition in pig fat and daily and diurnal precisions (n. 5, day3)

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. A method for detecting enramycin residue in animal tissues is characterized in that before the detection is carried out by adopting an HP L C-MS/MS method, the following pretreatment is carried out on a sample:

1) sample extraction: homogenizing and homogenizing an animal tissue sample, extracting the animal tissue sample with an extracting solution, blowing nitrogen to remove organic matters in the solution after extraction is finished, then fully mixing the residual solution with a hydrochloric acid solution, and centrifuging to obtain a supernatant;

2) sample purification: purifying the supernatant by using an ENV solid phase extraction column to obtain a sample to be detected;

the extracting solution is prepared by the following method:

A. mixing methanol and hydrochloric acid to prepare hydrochloric acid methanol mixed solution with the volume fraction of the hydrochloric acid being 1.8%;

B. mixing the 1.8% hydrochloric acid methanol mixed solution with water to prepare a solution with 54-56% volume fraction of the hydrochloric acid methanol mixed solution, namely the extracting solution;

the specific operation of the step 2) is that the supernatant obtained in the step 1) passes through the ENV solid phase extraction column, then the ENV solid phase extraction column is leached once by using a mixed solution of methanol and 5% ammonia water solution, then the ENV solid phase extraction column is leached once by using a mixed solution of formic acid and ethyl acetate, finally the ENV solid phase extraction column is eluted by using a mixed solution of methanol and 0.1M hydrochloric acid, and the obtained eluent passes through an organic filter membrane to obtain a sample to be detected; the volume ratio of the methanol to the 5% ammonia water solution is 20:80, the volume ratio of the formic acid to the ethyl acetate is 1:99, and the volume ratio of the methanol to the 0.1M hydrochloric acid is 80: 20.

2. The method according to claim 1, wherein the specific operation of the extraction in the step 1) is that the animal tissue sample is homogenized and homogenized, then the animal tissue sample is mixed with the extracting solution according to the mass-to-volume ratio of 1: 4.5-5.5, the mixture is fully shaken and uniformly mixed, the supernatant is obtained after centrifugation, the operation is repeated for 3 times, and the supernatant is combined;

and mixing the solution subjected to nitrogen blowing and organic matter removal with 0.1M hydrochloric acid solution according to the volume ratio of 5: 0.8-1.2, fully shaking and uniformly mixing, performing ultrasonic treatment, and finally centrifuging to obtain a supernatant.

3. The method according to claim 1 or 2, characterized in that the conditions of the liquid chromatography detection are: and (3) adopting a C18 chromatographic column, controlling the column temperature to be 28-32 ℃, and selecting aqueous solution of formic acid with the volume fraction of 0.2% and methanol as eluent to carry out gradient elution.

4. The method according to claim 3, characterized in that the specific operation of the gradient elution is: 0-1.5 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is from 90:10 to 50: 50; 1.5-3.3 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is 50:50 is adjusted to 30:70 for 3.3-3.5 min, and the volume ratio of 0.2% formic acid water solution to methanol is adjusted from 30:70 to 5: 95; 3.5-6 min, and maintaining the volume ratio of 0.2% formic acid water solution to methanol at 5:95, 6 to 6.1min, wherein the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is adjusted from 5:95 to 90:10 and 6.1 to 9min, and the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is maintained at 90: 10.

5. The method of claim 1, wherein during mass spectrometric detection, enramycin a has qualitative ion pairs of 786.1 > 1089.6, 786.1 > 178.9, and 786.1 > 95.3, corresponding to collision energies of 25eV, and 30eV, respectively, quantitative ion pairs of 786.1 > 1089.6, corresponding to collision energies of 25 eV;

the qualitative ion pair of enramycin B is 790.9 > 1089, 790.9 > 193.2 and 790.9 > 95, corresponding to collision energies of 25eV, 25eV and 30eV, the quantitative ion pair is 790.9 > 1089, corresponding to a collision energy of 25 eV.

6. The method of claim 5, wherein the mass spectrometric detection is performed under conditions selected from the group consisting of electrospray ion source, dry gas temperature of 300 ℃, dry gas flow of 7L/min, atomization gas pressure of 35Psi, sheath gas temperature of 300 ℃, sheath gas flow of 11L/min, capillary voltage of 3500V, nozzle voltage of 1500V, and corona current of 0.16 uA.

7. A method according to any one of claims 1 to 6, comprising the steps of:

1) pretreatment of samples

Homogenizing and homogenizing an animal tissue sample, mixing the animal tissue sample with an extracting solution according to the mass-volume ratio of 1: 4.5-5.5, fully shaking and uniformly mixing, centrifuging, taking supernate, repeating the operation for 3 times, and combining the supernate;

mixing the solution after removing the organic matters by nitrogen blowing with 0.1M hydrochloric acid solution according to the volume ratio of 5: 0.8-1.2, fully shaking and uniformly mixing, then carrying out ultrasonic treatment, and finally centrifuging to obtain supernatant;

the extracting solution is prepared by the following method:

A. mixing methanol and hydrochloric acid to prepare hydrochloric acid methanol mixed solution with the volume fraction of the hydrochloric acid being 1.8%;

B. mixing the 1.8% hydrochloric acid methanol mixed solution with water to prepare a solution with 54-56% volume fraction of the hydrochloric acid methanol mixed solution, namely the extracting solution;

2) purification of the supernatant

Enabling the supernatant obtained in the step 1) to pass through an ENV solid-phase extraction column, leaching the ENV solid-phase extraction column once with a mixed solution of methanol and a 5% ammonia water solution, leaching the ENV solid-phase extraction column once with a mixed solution of formic acid and ethyl acetate, eluting the ENV solid-phase extraction column once with a mixed solution of methanol and 0.1M hydrochloric acid, and filtering the obtained eluent with an organic filter membrane to obtain a sample to be detected;

the volume ratio of the methanol to a 5% ammonia water solution is 20:80, the volume ratio of the formic acid to the ethyl acetate is 1:99, and the volume ratio of the methanol to the 0.1M hydrochloric acid is 80: 20;

3) liquid chromatography detection

Performing liquid chromatography detection on the sample to be detected prepared in the step 2), wherein the detection conditions are as follows: adopting a C18 chromatographic column, controlling the column temperature at 30 ℃, and selecting aqueous solution of formic acid with volume fraction of 0.2% and methanol as eluent to carry out gradient elution;

the specific operation of the gradient elution is as follows: 0-1.5 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is from 90:10 to 50: 50; 1.5-3.3 min, wherein the volume ratio of 0.2% formic acid water solution to methanol is 50:50 is adjusted to 30:70 for 3.3-3.5 min, and the volume ratio of 0.2% formic acid water solution to methanol is adjusted from 30:70 to 5: 95; 3.5-6 min, and maintaining the volume ratio of 0.2% formic acid water solution to methanol at 5:95, 6 to 6.1min, wherein the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is adjusted from 5:95 to 90:10, 6.1 to 9min, and the volume ratio of the 0.2 percent formic acid aqueous solution to the methanol is maintained at 90: 10;

4) mass spectrometric detection

Detecting the sample after the liquid chromatography detection by a secondary mass spectrum, wherein the mass spectrum detection conditions are as follows:

selecting an electrospray ion source, wherein the temperature of dry gas is 300 ℃, the flow of the dry gas is 7L/min, the pressure of the atomization gas is 35Psi, the temperature of sheath gas is 300 ℃, the flow of the sheath gas is 11L/min, the voltage of a capillary tube is 3500V, the voltage of a nozzle is 1500V, and the corona current is 0.16 uA;

in the process of mass spectrometry detection, qualitative ion pairs of enramycin A are 786.1 & gt 1089.6, 786.1 & gt 178.9 and 786.1 & gt 95.3, the corresponding collision energy is 25eV, 25eV and 30eV respectively, quantitative ion pairs are 786.1 & gt 1089.6, and the corresponding collision energy is 25 eV;

the qualitative ion pair of enramycin B is 790.9 > 1089, 790.9 > 193.2 and 790.9 > 95, corresponding to collision energies of 25eV, 25eV and 30eV, the quantitative ion pair is 790.9 > 1089, corresponding to a collision energy of 25 eV.

Images