CN116183773A - Method for rapidly determining flavomycin A in animal-derived food by utilizing liquid chromatography-mass spectrometry - Google Patents

Abstract

The invention belongs to the technical field of chromatographic detection, and discloses a method for rapidly determining flavomycin A in animal-derived foods by utilizing liquid chromatography-mass spectrometry. Mixing a sample to be detected and an extraction reagent, uniformly mixing by vortex, centrifuging to obtain clear liquid, and obtaining liquid to be purified; adding water saturated normal hexane into the liquid to be purified, mixing uniformly by vortex, centrifuging to obtain the purified liquid, taking purified liquid nitrogen gas for blow-drying, using acetonitrile to fix the volume, filtering the liquid to be detected, and detecting the purified liquid by using an ultra-high performance liquid chromatography-tandem mass spectrometer. The method has the advantages of no need of purifying by a purifying column, simple and convenient operation, good improvement of the problems of large matrix effect, low sensitivity and the like in the prior art for detecting animal-derived foods, especially foods with high fat and protein contents, ideal recovery rate and precision, 1.0 mug/kg of the minimum detection limit of the flavomycin A method, improvement of the detection accuracy, and great significance for detecting meat products, and effective guarantee of the repeatability and reproducibility of analysis results.

Description

Method for rapidly determining flavomycin A in animal-derived food by utilizing liquid chromatography-mass spectrometry

Technical Field

The invention belongs to the technical field of chromatographic detection, and particularly relates to a method for rapidly determining flavomycin A in animal-derived foods by utilizing liquid chromatography-mass spectrometry.

Background

Flavomycin (Flavomycin) also known as moenomycin, placard, yellow phospholipidol (flavosporipol) or yellow phospholipidol, fuleomycin, fulafos, and the world health organization is commonly named Huang Linzhi (FV). Flavomycin is a multicomponent phosphoglycolipid antibiotic produced by anaerobic fermentation of Streptomyces griseus (Streptomyces macitensis, streptomyces bambergiensis). The flavomycin is weak acid, belongs to a strong polar compound, is easily dissolved in water, methanol, dimethylformamide and other small molecular substances, and is not dissolved in benzene, chloroform and other organic solvents; no definite melting point exists, and decomposition is started at 200 ℃; stable in neutral water and methanol solutions, but is susceptible to failure under strong acid and base conditions.

As one of the earliest antibiotic feed additives in the market abroad, flavomycin was the only phosphorus-containing glycolipid antibiotic allowed to be used in the breeding industry abroad. The feed additive has small dosage and outstanding growth promoting effect on animals such as cattle, pigs, chickens and the like, and improves the utilization efficiency of the feed; can be used for preventing coccidiosis; the flavomycin mainly inhibits gram-positive bacteria such as staphylococcus aureus, streptococcus, diplococcus and the like, and partial gram-negative bacteria such as Pasteurella, brucella and the like are also sensitive to the flavomycin; has no incompatibility with other medicine additives such as vitamins, amino acids, microelements, etc. These remarkable advantages make the flavomycin widely used for livestock and poultry cultivation and aquaculture. In the united states, flavomycin is approved for cattle, pigs and poultry but should be used at a level of 0.5-20mg/kg, either alone or in combination with a monensin, rasagilin or salinomycin plasma carrier antibiotic to increase the rate of weight gain and feed efficiency.

The biological synthesis of peptidoglycan, which is a substance of cell wall, is interfered by the inhibition of the activity of transglycosylase to cause cell lysis and death, so that a very strong sterilization effect is achieved, the European Union has authorized the use of the flavomycin in feed within the range of 1-25mg/kg of pharmacological dose, but when the flavomycin is found to have a remarkable growth promoting effect, the conference of European agricultural ministry of the 2005 decides that the flavomycin is forbidden to be added in the feed from 1 st 2006, and the flavomycin is only allowed to be used in the feeding process of rabbits from 9 th and 30 th 2009. Other scholars have shown that flavomycin can cause an adaptive response in rumen bacteria, thereby significantly altering its antibiotic susceptibility. The antigenicity of the flavomycin itself and other common antibiotics have no cross antigenicity, and the case of the flavomycin itself and other common antibiotics has to be studied deeply. As non-metabolic medicines, the flavomycin is hardly absorbed after being orally taken, on one hand, the flavomycin possibly remains in animal products, on the other hand, animal feces are inevitably discharged into the environment, and the environment such as soil, surface water and the like is easily polluted, so that the threat to human safety is possibly brought. The animal-derived food with the residual flavomycin after long-term eating can destroy intestinal flora of human body; has effects on animal bone development, and massive intake can even produce hematopoietic dysfunction; the crude product of the flavomycin is contacted with human in the production process, and adverse allergic reactions such as cough, high fever, diarrhea and the like are easy to generate, and other harm to animals and human bodies is still in need of intensive study.

Flavomycin as an antibacterial agent will also be subject to risk assessment without exception. The inspection and quarantine sector has now listed yellow mycin as a feed and animal-derived food residue monitoring program.

The prior researches show that five active ingredients A, A, C1, C3 and C4 of the flavomycin have similar chemical characteristics and antibacterial activity. The most predominant component of the flavomycin is flavomycin A, and the relative amount of the flavomycin A to the total active components is more than 50%, generally between 60 and 80%. The relative amount of the flavomycin A is specified to be more than 50% in China. Therefore, the content of the flavomycin in the sample can be calculated indirectly by detecting the content of the flavomycin A.

At present, the detection technology for the flavomycin A mainly comprises liquid chromatography, liquid chromatography-tandem mass spectrometry and ion pair high performance liquid chromatography, and the detection objects are mainly feed, and are pork and poultry tissues respectively.

In 2013, li Jinjiang and the like, normal hexane is added in pretreatment of a sample to remove fat, so that a detection method for determining the residual quantity of the flavomycin A in the livestock and poultry meat by using a liquid chromatography-tandem mass spectrometry method is established. The method mainly researches two matrixes of pork and chicken, the flavomycin A is well separated, the method can be used for measuring the residual quantity of the flavomycin A in livestock and poultry meat, the linear range is 10-1000 mug/kg, the detection limit of the method is 3 mug/kg, the quantitative limit of the method is 10 mug/kg, the recovery rate is 70-100% when the adding concentration is 10-50 mug/kg, and the variation coefficient is 4.6-8.8%. The sensitivity of the method is greatly improved, but HLB column purification is needed, which is not beneficial to the rapid detection of a large number of samples. According to the method, the flavomycin is used as a standard substance, the content of flavomycin A is calculated according to the measurement result through content conversion, the detection error is increased, the purification can be performed only after the process is performed before the purification, and the whole process is too complex, so that the method is not beneficial to large-scale rapid inspection and detection.

In 2014, xu Hui and the like, an ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting the residue of the flavomycin A in poultry tissues (muscle, fat, liver and kidney) is established, a sample is extracted by 10% ammoniated methanol, acetonitrile is settled and protein is purified, 0.3% formic acid-5% acetonitrile-water solution and 0.3% formic acid-5% water-acetonitrile are taken as mobile phases, and Agilent Poroshell SB-C18 chromatographic columns are used for ultra-efficient separation, multi-reaction detection MRM mode detection and external standard method quantification are carried out. The standard substance is the flavomycin A, and the correlation coefficient r is more than or equal to 0.995 within the linear range of 20-200 mug/L; the quantitative limit of the method is 10 mug/kg; the recovery rate is 66.5 to 89.4 percent; the Relative Standard Deviation (RSD) is 4.7% -10.2%. The method utilizes acetonitrile to remove impurities, is more convenient and rapid than solid phase extraction column purification, can be effectively used for rapid determination of the yellow mould A residue in poultry tissues, but uses 10% ammonia water as an extraction reagent, has high alkali solution concentration, pollutes the environment and has relatively high detection limit.

In order to provide technical support for food risk early warning and food safety supervision, it is imperative to establish a detection method of the flavomycin A in animal-derived food with high efficiency, rapidness, convenience and low cost.

Disclosure of Invention

In order to overcome the defects of small application range (limited to livestock and poultry meat), low sensitivity, complex operation, large pollution and the like of a detection method of the flavomycin A in the prior art, the invention aims to provide a method for rapidly detecting the flavomycin A in animal-derived foods by utilizing liquid chromatography-mass spectrometry.

The aim of the invention is achieved by the following technical scheme:

a method for rapidly determining flavomycin A in animal-derived food by utilizing liquid chromatography-mass spectrometry, comprising the following operations:

(1) Mixing 1.0-5.0 g of sample to be detected with an extraction reagent, uniformly vortex mixing, centrifuging and taking clear liquid to obtain liquid to be purified; wherein the extraction reagent is ammonia water and methanol mixed solution, the volume ratio of ammonia water to methanol is (0.5-5) (99.5-95.0), and the mass volume ratio of the sample to be detected to the extraction reagent is (1.0-5.0) g (2-10) mL;

(2) Adding 10mL of water-saturated n-hexane into the liquid to be purified prepared in the step (1), mixing uniformly by vortex, freezing and centrifuging to obtain a supernatant, and taking a lower layer liquid to obtain a purified liquid;

(3) Taking 1-5 mL of purified liquid, drying with nitrogen at 40-50 ℃, using 1mL of acetonitrile to fix the volume, performing ultrasonic treatment for 5min, uniformly mixing, and passing through an organic microporous filter membrane of 0.22 mu m to obtain liquid to be detected;

(4) And (3) detecting the liquid to be detected prepared in the step (3) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer, wherein the minimum detection limit of the flavomycin A is 1.0 mug/kg.

The sample to be detected in the step (1) is animal-derived food of the to-be-detected flavomycin A.

The sample to be tested in the step (1) comprises dairy products, eggs, livestock and poultry meat and meat products thereof, and animal viscera or backfat with high fat content.

The vortex mixing in the step (1) is vortex oscillation for 5-10 min;

the centrifugation condition in the step (1) is 3000-6000 r/min for 3-5 min.

The vortex mixing in the step (2) is vortex oscillation for 10-30 s;

the refrigerated centrifugation in the step (2) is carried out at the temperature of-3 to 0 ℃ and the centrifugation time of 3000 to 6000r/min is 3 to 5min.

The detection conditions in the step (4) are as follows:

chromatographic conditions:

chromatographic column: 1.7 μm,2.1 mm. Times.150 mm BEH C18 column; column temperature: 35 ℃; sample injection amount: 5.0. Mu.L; flow rate: 0.3mL/min;

the mobile phase gradient elution procedure was: 0.0 to 1.0min,98 percent of 10mmol/L ammonium formate solution and 2 percent of acetonitrile; 1.0 to 6.0min,2 percent of 10mmol/L ammonium formate solution and 98 percent of acetonitrile; 6.0 to 7.0min,98 percent of 10mmol/L ammonium formate solution and 2 percent of acetonitrile;

mass spectrometry conditions:

an electrospray ion source; a negative ion mode; detecting multiple reactions; capillary voltage: 3.0KV; taper hole voltage: 30.0KV; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 1000L/Hr; taper hole nitrogen flow rate: 150L/Hr; nozzle pressure 7.0bar.

The detection conditions in the step (4) are as follows: qualitative ion pairs 790.3/576.2m/z, channel voltage 40V, collision energy 35V; ion pair 790.3/554.4m/z, channel voltage 40V, collision energy 43V.

Compared with the prior art, the invention has the following advantages and effects:

(1) The traditional extraction method mostly adopts high-concentration ammoniated methanol for extraction, but the invention adopts low-concentration ammonia methanol mixed solution as an extraction reagent, thereby not only greatly reducing environmental pollution, but also being beneficial to quick concentration, having better extraction effect, more convenient operation and small matrix effect.

(2) Compared with 2013 Li Jinjiang and other methods, the method does not need SPE small column purification, has lower cost, further reduces matrix effect by separating and concentrating, and has better purification effect.

(3) In the purification mode, firstly, normal hexane is saturated with water to remove fat, then acetonitrile is selected to remove protein to the maximum extent during redissolution, and the matrix effect is reduced by separating and taking, so that three action points together reach a low detection limit, the problem that the detection in the prior art is limited to livestock and poultry meat is well improved, the method is applicable to animal-derived foods, has ideal recovery rate and precision, the detection limit of the lowest method is 1.0 mug/kg, the quantitative limit is 3.0 mug/kg, and the repeatability and reproducibility of analysis results are effectively ensured.

(4) The mobile phase and chromatographic column adopted in the detection of the invention ensure that the peak time is fast, the good peak time is maintained, the detection time is saved, and the quantitative analysis is more accurate.

Drawings

FIG. 1 is a TIC diagram corresponding to column number 1.

FIG. 2 is a TIC diagram corresponding to column No. 2.

FIG. 3 is a TIC diagram corresponding to column number 3.

FIG. 4 is a TIC diagram of a10 mmol/L ammonium formate solution in mobile phase with acetonitrile.

Fig. 5 is a total ion flow diagram of a negative fish sample.

FIG. 6 is a graph showing the recovery rate analysis of flavomycin A after the fish meat sample is extracted by using the mixed solution of ammonia water and methanol with different concentrations.

FIG. 7 is a graph showing the recovery rate analysis of flavomycin A after the fish meat sample was extracted with a mixture of aqueous ammonia acetonitrile and aqueous ammonia methanol, respectively.

FIG. 8 is a graph showing the effect of different purification modes on recovery.

Fig. 9 is a total ion flow diagram of a negative fish sample after standard addition.

FIG. 10 is an extract ion flow chromatogram of flavomycin A in a negative fish sample following standard addition.

FIG. 11 is a graph showing the recovery rate analysis of flavomycin A from beef samples extracted with aqueous ammonia methanol mixtures of different concentrations.

FIG. 12 is a graph showing the recovery rate analysis of flavomycin A after the egg sample is extracted by using a mixed solution of ammonia water and methanol with different concentrations.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The experimental apparatus and reagents involved in the following examples are shown below:

XEVO TQ ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Waters, USA); VX-iii vortex oscillator (beijing pedal science and technology limited); 5810 type R centrifuge (Eppendorf, germany); XPE type 205 analytical balance (sensitivity 0.01 mg) (Mettler-Toledo, switzerland); milli-Q Advantage A10 ultra-pure water System (Dalmschtatamer group, germany) and the like.

Flavomycin a was purchased from the company Tianjin allta technologies limited; ammonium formate, acetonitrile, methanol (all mass spectroscopically pure) were purchased from us Fisher Scientific company; ammonia (analytically pure) content 25-28%, purchased from the company Miou, tianjin, and other reagents are all made analytically pure.

Example 1

In this example, the best chromatographic conditions and mass spectrometry conditions were searched and determined with the yellow mycin A standard, and the specific experimental method is as follows:

(1) Preparing a standard stock solution and a series of standard working solutions of the flavomycin A:

(1) preparation of standard stock solution: precisely weighing a proper amount of yellow mould A standard substance, dissolving with methanol, and fixing volume to obtain yellow mould A standard stock solution with concentration of 100ng/mL, and storing at 4deg.C in dark place for three months.

(2) Preparing a series of standard working solutions: the standard stock solutions of the yellow mould A are respectively sucked precisely, and are diluted by methanol to form serial standard working solutions with the concentration of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL and 50.0ng/mL for use.

(2) The detection is carried out on the machine by adopting a XEVO TQ ultra-high performance liquid chromatography-tandem mass spectrometer, and the specific liquid chromatography conditions are as follows:

chromatographic column: ACQUITY

Peptides BEH C18 column (2.1 mm. Times.150 mm,1.7 μm Watertian technologies (Shanghai); column temperature: 35 ℃; sample injection amount: 5.0. Mu.L.

Mobile phase A was 10mmol/L ammonium formate solution and B was acetonitrile, and the mobile phase gradient elution procedure is shown in Table 1.

TABLE 1 gradient elution procedure for mobile phases

Mass spectrometry conditions: an electrospray ion source; a negative ion mode; multiple reaction monitoring (multiple reaction monitoring, MRM); capillary voltage: 3.0KV; taper hole voltage: 30.0KV; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 1000L/Hr; taper hole nitrogen flow rate: 150L/Hr; nozzle pressure 7.0bar.

Other mass spectral parameters are shown in table 2.

Table 2 mass spectrometry parameters

* And quantifying ions.

Analysis of results:

(1) Selection of chromatographic columns

The test is to select No. 1 chromatographic column ACQUITY

HILIC Core-shell (2.1 mm. Times.50 mm,1.7 μm), column No. 2 ACQUITY +.>

Peptides BEH C18 (2.1 mm. Times.150 mm,1.7 μm), column 3 ACQUITY +.>

The analysis and detection of the standard working solution of the same concentration of the flavomycin by three chromatographic columns of Pepeptide BEH C18 (2.1 mm multiplied by 50mm,1.7 μm) are shown in the figures 1-3. As can be seen from the figure analysis, the hydrophilicity of the No. 1 chromatographic column makes it difficult to effectively retain and separate the flavomycin A, the length of the No. 3 chromatographic column is insufficient to effectively separate the flavomycin A, the target peak cannot be detected, and the No. 3 chromatographic column can separate the target object relatively better, the peak type is relatively better, so that the test selects ACQUITY->

Peptides BEH C18 (2.1 mm. Times.150 mm,1.7 μm) column.

(2) Selection of mobile phase

ACQUITY for test

Peptides BEH C18 (2.1 mm×150mm,1.7 μm) chromatographic column, acetonitrile as the organic phase in the mobile phase, 10mmol/L ammonium formate solution and 10mmol/L ammonium acetate solution as the aqueous phase were respectively selected to analyze and detect the same concentration of standard working solution of flavomycin, and the result shows that 10mmol/L ammonium formateThe better response of the flavomycin A peak when the solution was in mobile phase with acetonitrile is higher, see FIGS. 2 and 4. Therefore, the test selects 10mmol/L ammonium formate solution and acetonitrile as mobile phase, and simultaneously, the proportion of the two solutions is further optimized, so that the peak is faster to shorten the detection time.

(3) Standard curve equation and correlation coefficient

And respectively precisely absorbing a proper amount of the standard stock solution of the flavomycin A, diluting the standard stock solution with methanol to obtain a series of standard working solutions with the concentration of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL and 50.0ng/mL, and measuring according to the liquid chromatography-mass spectrometry condition, wherein the linear equation of the flavomycin A is Y= 11.3373X-0.0813643, X is the mass concentration of the flavomycin A, Y is the peak area of a flavomycin A quantitative ion pair, and the correlation coefficient (r) is 0.999596.

Example 2 measurement of fish meat sample

In this example, the optimal chromatographic conditions and mass spectrometric conditions determined in example 1 were used to determine the fish sample to be tested, and whether it contained flavomycin A; the method comprises the steps of adding a yellow mould A standard substance into the determined negative fish meat, further determining the optimal extraction reagent and purification reagent, and determining the recovery rate, precision and minimum detection limit of the method, wherein the specific experimental method is as follows:

(1) Sample preparation: randomly extracting 500g of edible parts of weever, dividing the edible parts into small blocks, and completely crushing the small blocks by using a meat grinder;

(2) And (3) preparing a marked sample: accurately weighing three (5.00+/-0.01) g of crushed fish samples into a polypropylene centrifuge tube, respectively adding 15.0 mu L, 30.0 mu L and 150.0 mu L of the standard stock solution of the flavomycin A prepared in the example 1, uniformly mixing, standing overnight in an opening, obtaining a negative fish sample after the solvent in the working solution is completely volatilized, and then extracting and purifying according to the sample pretreatment step in the step (3);

(3) Sample pretreatment:

(1) adding 10mL of extraction reagent into the sample prepared in the step (1) or the negative fish labeling sample prepared in the step (2), and performing vortex oscillation for 10min and centrifugation for 6000r/min for 5min to obtain liquid to be purified (supernatant);

(2) adding 5mL of water-saturated normal hexane into the liquid to be purified prepared in the step (1), uniformly mixing by vortex for 30s, centrifuging at 0 ℃ for 5min at 6000r/min, and taking the lower layer liquid to obtain a purified liquid;

(3) taking 2.5mL of purified liquid, drying with nitrogen at 40 ℃, fixing the volume with 1mL of acetonitrile, carrying out ultrasonic treatment for 5min, uniformly mixing, and passing through an organic microporous filter membrane with the thickness of 0.22 mu m to obtain liquid to be detected;

(4) Detecting the liquid to be detected prepared in the steps (3) and (3) on a machine by using a XEVO TQ ultra-high performance liquid chromatography-tandem mass spectrometer, wherein the specific method is the same as that in the step (2) of the embodiment 1, and quantitatively analyzing the flavomycin A in the liquid to be detected according to the standard curve obtained in the embodiment 1;

analysis of results:

(1) Detection of fish sample to be detected

The crushed fish meat is pretreated to prepare a purified liquid, and the purified liquid is detected by a machine to determine that the fish meat does not contain the flavomycin A (shown in figure 5).

(2) Sample extraction

In this embodiment, when the negative fish meat is extracted by adding the standard sample, the extraction effects of different extraction reagents are compared, wherein the extraction reagents are respectively: acetonitrile, methanol, ammoniated acetonitrile (volume ratio of ammonia water to acetonitrile: 0.1:99.9, 0.2:99.8, 0.5:99.5, 1.0:99.0, 2.0:98.0, 5.0:95.0, 10.0:90.0) and ammoniated methanol (volume ratio of ammonia water to methanol: 0.1:99.9, 0.2:99.8, 0.5:99.5, 1.0:99.0, 2.0:98.0, 5.0:95.0, 10.0:90.0), and the recovery rates thereof were analyzed, and the results are shown in fig. 6 and 7. As can be seen from fig. 6: with the increase of the volume fraction of the ammonia water, the recovery rate of the flavomycin A gradually increases, and when the concentration of the ammonia water is more than 1.0%, the recovery rate is slowly increased, and the ammoniated methanol with the concentration of the ammonia water of 1.0% is selected in an experiment to be suitable in consideration of environmental protection. As can be seen from fig. 7, the extraction efficiency of acetonitrile and ammoniated acetonitrile is significantly lower than that of methanol and ammoniated methanol, which may be due to the strong polarity of methanol, so that the flavomycin a in the sample can be better extracted, and the purpose of better removing impurities such as protein can be achieved. Therefore, the experiment selects ammoniated methanol, namely the mixed solution of ammonia water and methanol as an extraction reagent, and the ammoniated methanol with the concentration of 1.0% of ammonia water is the optimal extraction reagent for fish samples.

(3) Sample purification

In the embodiment, ammoniated methanol with the ammonia water concentration of 1.0% is used as an extraction reagent, and when purifying liquid to be purified, the purifying effects of different purifying modes are compared. After purification by different purification modes, the recovery rate of the control sample without any purification mode is lower, the water saturated n-hexane has a certain purification effect, and the recovery rate after purification is relatively improved. And after the HLB and the C18 purifying column are adopted for purifying, the loss of the flavomycin A is larger, and as can be seen from the figure 8, the degreasing effect is optimal by selecting water saturated n-hexane.

(3) Selection of the multiple solutions

Taking a purified liquid of a negative fish meat added with a standard sample, and re-dissolving the purified liquid by using 1mL of water, methanol and acetonitrile respectively after the purified liquid is dried by nitrogen. The test finds that: after re-dissolving with water, the liquid to be detected is turbid and cannot pass through an organic microporous filter membrane with the thickness of 0.22 mu m, so that the detection standard of the machine can not be met; the precipitate was clearly seen by redissolving with methanol and acetonitrile, and was clear and transparent after filtration through a 0.22 μm organic microporous filter. And (3) detecting the two liquids to be detected on the machine, and calculating the recovery rate, wherein the recovery rate of the sample after acetonitrile redissolution is 72.17%, and the recovery rate of the sample after methanol redissolution is 59.05%, so that acetonitrile is selected for redissolution in the test.

(4) Recovery rate and precision

On the basis of the optimal extraction reagent (ammonia water and methanol mixed solution, wherein the volume ratio of ammonia water to methanol is 1.0:99.0) and water saturated normal hexane for purifying and degreasing, a repeatability test is carried out on negative fish meat added with a yellow mould A standard substance. The total ion flow chart of the negative fish sample added with the standard substance is shown in figure 9, and the ion flow chart of the extract of the flavomycin A in the negative fish sample added with the standard substance is shown in figure 10. 8 replicates were prepared for each concentration, and recovery and precision were calculated from the measurements, the results of which are shown in Table 3.

Table 3 labeled recovery and precision of flavomycin a in fish samples (n=8)

(5) Minimum method detection limit

On the basis of the optimal extraction reagent (ammonia water and methanol mixed solution, wherein the volume ratio of ammonia water to methanol is 1.0:99.0) and water saturated n-hexane purification and degreasing, the detection limit of the method is calculated according to the ratio of an instrument response value to noise of 3, and the detection limit of the method of the fulvin A in fish meat is 1.0 mug/kg.

Example 3 assay of beef samples

In this example, the optimal chromatographic conditions and mass spectrometric conditions determined in example 1 were used to determine the beef sample to be tested, and whether it contained flavomycin A; the method comprises the steps of adding a yellow mould A standard substance into negative beef, and determining the recovery rate, precision and minimum detection limit of the method. The specific experimental method is as follows:

(1) Sample preparation: randomly extracting 500g of edible beef, dividing into small blocks, and thoroughly crushing by using a meat grinder;

(2) And (3) preparing a marked sample: accurately weighing three parts of uniformly mixed negative beef in a polypropylene centrifuge tube, respectively adding 15.0 mu L, 30.0 mu L and 150.0 mu L of the standard stock solution of the flavomycin A prepared in the example 1 into the polypropylene centrifuge tube, uniformly mixing, standing overnight at an opening, obtaining a negative beef standard sample after the solvent in the working solution is completely volatilized, and then extracting and purifying according to the sample pretreatment step in the step (3);

(3) Sample pretreatment:

(1) adding 10mL of extraction reagent into the sample prepared in the step (1) or the negative beef labeled sample prepared in the step (2), and carrying out vortex oscillation for 10min and centrifugation for 4000r/min for 3min to obtain liquid to be purified (supernatant);

(2) adding 10mL of water-saturated normal hexane into the liquid to be purified prepared in the step (1), uniformly mixing for 15s by vortex, and centrifuging at 0 ℃ for 5min at 5000r/min to obtain a purified liquid;

(3) taking 2.0mL of purified liquid, drying with nitrogen at 50 ℃, fixing the volume with 1mL of acetonitrile, carrying out ultrasonic treatment for 5min, uniformly mixing, and passing through an organic microporous filter membrane with the thickness of 0.22 mu m to obtain liquid to be detected;

(4) Detecting the purifying liquid prepared in the step (3) and the step (2) on the machine by using a XEVO TQ ultra-high performance liquid chromatography-tandem mass spectrometer, and quantitatively analyzing the flavomycin A in the liquid to be detected according to the standard curve obtained in the embodiment 1 by adopting the specific method as in the embodiment 1 and the step (2);

analysis of results:

(1) Detection of beef to be detected

And (3) preprocessing the uniformly mixed beef to be tested to prepare a purified liquid, and detecting on a machine, wherein the result shows that the beef to be tested does not contain the flavomycin A.

(2) Sample extraction

In this example, when the negative beef is extracted with the standard sample, the extraction effects of ammoniated methanol with different volume ratios are compared in combination with example 1, the volume ratio of ammonia water to methanol is 0.2:99.8, 0.5:99.5, 1.0:99.0, 2.0:98.0, 5.0:95.0, and 10.0:90.0, respectively, and the recovery rate is calculated, and the result is shown in fig. 11. As can be seen from fig. 11: as the volume fraction of the ammonia water increases, the recovery rate of the flavomycin A gradually increases, and when the concentration of the ammonia water is more than 2.0%, the recovery rate starts to decrease, so that an ammoniated methanol solution with the volume ratio of the ammonia water to the methanol of 2.0:98.0 is selected as the optimal extraction reagent for beef samples in an experiment.

(3) Recovery rate and precision

Repeatability tests were performed on negative beef to which the yellow mycin A standard was added. 8 replicates were prepared for each concentration, and recovery and precision were calculated from the measurements, the results of which are shown in Table 4.

Table 4 labeled recovery and precision of flavomycin a in beef sample (n=8)

(4) Minimum detection limit

The detection limit of the method is calculated according to the ratio of the instrument response value to the noise of 3, and then the detection limit of the method of the flavomycin A in the beef is 1.0 mug/kg.

Example 4 determination of egg samples

In the embodiment, the best chromatographic conditions and mass spectrum conditions determined in the embodiment 1 are used for measuring the egg sample to be tested, and determining whether the egg sample contains the flavomycin A or not; the method comprises the steps of adding a yellow mould A standard substance into a negative egg sample, and determining the recovery rate, the precision and the minimum detection limit of the method. The specific experimental method is as follows:

(1) Sample preparation: randomly extracting 500g of eggs, removing shells, homogenizing egg liquid by using a homogenizer for later use;

(2) And (3) preparing a marked sample: accurately weighing three evenly mixed egg samples of 2.00+/-0.01 g into a polypropylene centrifuge tube, respectively adding 15.0 mu L, 30.0 mu L and 150.0 mu L of the standard stock solution of the flavomycin A prepared in the example 1, evenly mixing, standing overnight at an opening, and extracting and purifying according to the sample pretreatment step of the step (3) after the solvent in the working solution is completely volatilized;

(3) Sample pretreatment:

(1) adding 5mL of extraction reagent into the sample prepared in the step (1) or the negative egg prepared in the step (2), and performing vortex oscillation for 10min and centrifugation for 6000r/min for 5min to obtain liquid to be purified (supernatant);

(2) adding 10mL of water-saturated normal hexane into the liquid to be purified prepared in the step (1), uniformly mixing for 15s by vortex, centrifuging at 0 ℃ for 5min at 6000r/min, and taking the lower layer liquid to obtain a purified liquid;

(3) taking 2.0mL of purified liquid, drying with nitrogen at 45 ℃, fixing the volume with 1mL of acetonitrile, carrying out ultrasonic treatment for 5min, uniformly mixing, and passing through an organic microporous filter membrane with the thickness of 0.22 mu m to obtain liquid to be detected;

(4) Detecting the purifying liquid prepared in the step (3) and the step (2) on the machine by using a XEVO TQ ultra-high performance liquid chromatography-tandem mass spectrometer, and quantitatively analyzing the flavomycin A in the liquid to be detected according to the standard curve obtained in the embodiment 1 by adopting the specific method as in the embodiment 1 and the step (2);

analysis of results:

(1) Detection of egg sample to be detected

And (3) preprocessing the uniformly mixed egg sample to be tested to prepare a purified liquid, and detecting on a machine, wherein the result shows that the egg sample to be tested does not contain the flavomycin A.

(2) Sample extraction

In this example, when the negative egg is extracted by adding the standard sample, the extraction effect of ammoniated methanol with different volume ratios is compared by combining with example 1, the volume ratio of ammonia water to methanol is 1.0:99.0, 2.0:98.0, 5.0:95.0, 10.0:90.0 and 15.0:85.0 respectively, and the recovery rate is calculated, and the result is shown in fig. 12. As can be seen from fig. 12: with the increase of the volume fraction of the ammonia water, the recovery rate of the flavomycin A is rapidly increased, and when the concentration of the ammonia water is more than 5.0%, the recovery rate is reduced, so that an ammoniated methanol solution with the volume ratio of the ammonia water to the methanol of 5.0:95.0 is selected as an optimal extraction reagent for egg samples in an experiment.

(3) Recovery rate and precision

Repeatability tests were performed on negative egg samples with the addition of the yellow mycin A standard. 8 replicates were prepared for each concentration, and recovery and precision were calculated from the measurements, the results of which are shown in Table 5.

Table 5 labeled recovery and precision of flavomycin a in egg sample (n=8)

(4) Minimum detection limit

The detection limit of the method is calculated according to the ratio of the instrument response value to the noise of 3, and then the detection limit of the method of the flavomycin A in the egg sample is 1.0 mug/kg.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (7)

1. A method for rapidly determining flavomycin A in animal-derived food by utilizing liquid chromatography-mass spectrometry, which is characterized by comprising the following steps:

(1) Mixing 1.0-5.0 g of sample to be detected with an extraction reagent, uniformly vortex mixing, centrifuging and taking clear liquid to obtain liquid to be purified; wherein the extraction reagent is ammonia water and methanol mixed solution, the volume ratio of ammonia water to methanol is (0.5-5) (99.5-95.0), and the mass volume ratio of the sample to be detected to the extraction reagent is (1.0-5.0) g (2-10) mL;

(2) Adding 10mL of water-saturated n-hexane into the liquid to be purified prepared in the step (1), mixing uniformly by vortex, freezing and centrifuging to obtain a supernatant, and taking a lower layer liquid to obtain a purified liquid;

(3) Taking 1-5 mL of purified liquid, drying with nitrogen at 40-50 ℃, using 1mL of acetonitrile to fix the volume, performing ultrasonic treatment for 5min, uniformly mixing, and passing through an organic microporous filter membrane of 0.22 mu m to obtain liquid to be detected;

(4) And (3) detecting the liquid to be detected prepared in the step (3) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer, wherein the minimum detection limit of the flavomycin A is 1.0 mug/kg.

2. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the sample to be detected in the step (1) is animal-derived food of the to-be-detected flavomycin A.

3. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the sample to be tested in the step (1) comprises dairy products, eggs, livestock and poultry meat and meat products thereof, and animal viscera or backfat with high fat content.

4. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the vortex mixing in the step (1) is vortex oscillation for 5-10 min;

the centrifugation condition in the step (1) is 3000-6000 r/min for 3-5 min.

5. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the vortex mixing in the step (2) is vortex oscillation for 10-30 s;

the refrigerated centrifugation in the step (2) is carried out at the temperature of-3 to 0 ℃ and the centrifugation time of 3000 to 6000r/min is 3 to 5min.

6. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the detection conditions in the step (4) are as follows:

chromatographic conditions:

chromatographic column: 1.7 μm,2.1 mm. Times.150 mm BEH C18 column; column temperature: 35 ℃; sample injection amount: 5.0. Mu.L; flow rate: 0.3mL/min;

the mobile phase gradient elution procedure was: 0.0 to 1.0min,98 percent of 10mmol/L ammonium formate solution and 2 percent of acetonitrile; 1.0 to 6.0min,2 percent of 10mmol/L ammonium formate solution and 98 percent of acetonitrile; 6.0 to 7.0min,98 percent of 10mmol/L ammonium formate solution and 2 percent of acetonitrile;

mass spectrometry conditions:

an electrospray ion source; a negative ion mode; detecting multiple reactions; capillary voltage: 3.0KV; taper hole voltage: 30.0KV; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 1000L/Hr; taper hole nitrogen flow rate: 150L/Hr; nozzle pressure 7.0bar.

7. The method for rapidly determining the flavomycin A in the animal-derived food by utilizing the liquid chromatography-mass spectrometry according to claim 1, wherein the method comprises the following steps of:

the detection conditions in the step (4) are as follows: qualitative ion pairs 790.3/576.2m/z, channel voltage 40V, collision energy 35V; ion pair 790.3/554.4m/z, channel voltage 40V, collision energy 43V.

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