CN108693004B - A method for detecting whether the rapeseed meal is doped with antibiotic residues - Google Patents

Abstract

The invention discloses a method for detecting whether the rapeseed meal is doped with antibiotic filter residues. The method includes the following steps: detecting whether the rapeseed meal contains characteristic markers of antibiotic filter residues, and detecting the characteristic markers means that the rapeseed meal is doped with antibiotic filter residues. It specifically includes sample pretreatment, volatile component collection, gas chromatography separation, and ion mobility spectrometry detection methods to detect whether there are characteristic markers of antibiotic filter residues in the sample, so as to determine whether the rapeseed meal is doped with antibiotic filter residues. The invention has both the high separation ability of gas chromatography and the high sensitivity of ion mobility spectrum, and at the same time, it adopts the static headspace sampling method, which can accurately and non-destructively detect trace volatile components. The invention can detect streptomycin filter residue, cephalosporin Characteristic markers of bacterial residues and colistin sulfate residues to identify adulteration in rapeseed meal.

Description

A method for detecting whether the rapeseed meal is doped with antibiotic residues

technical field

The invention relates to a method for detecting whether rapeseed meal is doped with antibiotic filter residues, and belongs to the field of feed quality and safety detection.

Background technique

Rapeseed meal is a by-product of oil extraction from rapeseed as raw material. Its protein content is between 34% and 38%, and the proportion of methionine and lysine in the amino acid composition is relatively high.

Antibiotic filter residue is a high-concentration industrial waste containing a variety of refractory organic substances formed during the production of antibiotics by biological fermentation. high protein content. Since the filter residue contains antibiotic residues that have not been fully extracted and secondary metabolites that have not been evaluated for safety, the use of feed materials containing antibiotic filter residues in the breeding process will endanger the health of farmed animals, affect food safety, and induce bacterial resistance. potential risks. Therefore, there is a need for identification technology in the supervision of antibiotic residues in rapeseed meal by agricultural administrative departments.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for detecting whether the rapeseed meal is doped with antibiotic residues. The present invention has both the high separation ability of gas chromatography and the high sensitivity of ion mobility spectrum. For accurate and non-destructive detection of trace volatile components, the invention can detect the characteristic markers of streptomycin filter residue, cephalosporin filter residue and colistin sulfate filter residue, thereby identifying whether rapeseed meal is adulterated.

A method for detecting whether the rapeseed meal is doped with antibiotic residues provided by the present invention comprises the following steps: detecting whether the rapeseed meal contains characteristic markers of antibiotic residues, and detecting the characteristic markers is the rapeseed Seed meal mixed with antibiotic residues.

In the above method, the method specifically includes the following steps: 1) pretreatment of the sample: the rapeseed meal sample is pulverized;

2) Collection of volatile components: heat incubation of the crushed rapeseed meal sample in step 1), and then collect volatile components;

3) separation by gas chromatography: the volatile components are separated by gas chromatography;

4) ion mobility spectrometry detection: through step 3) the volatile components after separation are detected by ion mobility spectrometry, if the characteristic marker of the antibiotic filter residue is detected, then the rapeseed meal is doped with the antibiotic filter residue.

In the present invention, the pretreatment of the sample specifically includes the following steps: using a cyclone mill or other grinding device to pulverize the rapeseed meal sample, and the particle size of the pulverized sample reaches 18 meshes; weighing 0.5 g of the pulverized sample to a clean In the glass headspace bottle, the rubber stopper is sealed, and the aluminum cap is sealed with the gland to ensure that the headspace bottle is sealed and air-tight.

In the above method, the antibiotic filter residue is one or more of streptomycin filter residue, cephalosporin filter residue and colistin sulfate filter residue.

In the above method, when the antibiotic filter residue is the streptomycin filter residue, the characteristic markers are heptanol, ethyl hexanoate and ethyl propionate;

When the antibiotic filter residue is the cephalosporin filter residue, the characteristic markers are 3-methylbutanal, phenylacetaldehyde, methylthiopropionaldehyde and 3-methylbutanol;

When the antibiotic filter residue is the colistin sulfate filter residue, the characteristic markers are dimethyltrisulfide and 2,6-nonadienal.

In the above method, the particle size of the pulverization is greater than or equal to 18 meshes, specifically 18 meshes or 18-40 meshes.

In the above method, the temperature of the thermal incubation is 90°C, the time is 10min, and the vibration speed is 500rpm;

The volatile components are collected by a static headspace volatile component collecting device.

In the present invention, the thermal incubation is performed using a thermal incubator; the static headspace volatile component collection device includes a headspace bottle;

The sample-filled headspace vial is placed on a thermal incubator for a heated incubation to facilitate the release of volatiles from the sample into the headspace for collection.

In the above method, the separation conditions of the gas chromatography are as follows: the chromatographic column is an SE54 chromatographic column; the column temperature is 40°C; the carrier gas is nitrogen, and the inlet temperature is 80°C; 2~20min, the carrier gas flow rate is 100mL/min; 20~30min, the carrier gas flow rate is 150mL/min.

In the above-mentioned method, the bars detected by the ion mobility spectrum are as follows: a tritium source β-ray ionization source is used; the temperature of the migration tube is 45°C, the drift gas is nitrogen, and the flow rate is 150mL/min.

In the above method, the injection conditions for the gas chromatographic separation are as follows: the temperature of the injection needle is 95° C., the injection volume is 500 μL, and the injection speed is 100 μL/s.

In the present invention, the results of the gas chromatography separation and ion mobility spectrometry detection are judged by the spectrum. In the spectrum of gas chromatography-ion mobility spectrum, the ordinate is the gas retention time, the abscissa is the ion migration time, the color represents the concentration of the detected substance, the red indicates a higher concentration, and the darker the red, the higher the concentration. The detected substances were identified according to the gas phase retention time and ion migration time. If the characteristic markers of antibiotic filter residues were detected, it was determined that the rapeseed meal was adulterated with antibiotic filter residues.

The present invention has the following advantages:

1. The pretreatment of the present invention is simple, the detection time is short, and the discrimination is accurate, which can meet the detection purpose of identifying the adulteration of antibiotic filter residues in rapeseed meal.

2. The present invention has both the high separation ability of gas chromatography and the high sensitivity of ion mobility spectrum, and at the same time it adopts the static headspace sampling method, which can accurately and non-destructively detect trace volatile components.

3. The present invention provides a method for identifying adulteration of rapeseed meal based on gas chromatography-ion mobility spectrometry for detecting characteristic markers of streptomycin filter residue, cephalosporin filter residue and colistin sulfate filter residue.

Description of drawings

Figure 1 is the gas chromatography-ion mobility spectrum of the streptomycin filter residue sample.

Figure 2 is a gas chromatography-ion mobility spectrum of a sample of the filter residues spiked with streptomycin in rapeseed meal.

Figure 3 is a gas chromatography-ion mobility spectrogram of a rapeseed meal sample.

Figure 4 is a gas chromatography-ion mobility spectrogram of a cephalosporin filter residue sample.

Figure 5 is the gas chromatography-ion mobility spectrum of the rapeseed meal spiked with the cephalosporin filter residue sample.

Figure 6 is the gas chromatography-ion mobility spectrum of the colistin sulfate filter residue sample.

Figure 7 is a gas chromatography-ion mobility spectrum of a sample of the filter residues spiked with colistin sulfate in rapeseed meal.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1,

(1) Sample pretreatment

The rapeseed meal, the streptomycin filter residue, and the rapeseed meal mixed with the streptomycin filter residue (10%, m/m) were pulverized by a cyclone respectively, and the particle size after pulverization reached 18 meshes. Weigh 0.5 g of the crushed samples into clean glass headspace vials, seal them with rubber stoppers, and seal them with aluminum caps to ensure that the headspace vials are airtight.

(2) Static headspace volatile component collection

The headspace vial filled with the sample was heated and incubated on a thermal incubator to promote the release of volatile substances in the sample into the headspace. The thermal incubation temperature was 90 °C, the incubation time was 10 min, and the incubator vibration speed was 500 rpm. After the thermal incubation, the static headspace volatile components were aspirated with a syringe, the volume was 500 μL, and the injection speed was 100 μL/s.

(3) Gas chromatography separation

①chromatographic column: SE54 column (5% phenyl-95% dimethylpolysiloxane, non-polar stationary phase);

②Carrier gas: nitrogen;

③Column temperature: 40℃;

④Injector temperature: 80℃;

⑤Injection volume: 500μL;

⑥Injection speed: 100μL/s;

⑦ The gas phase separation conditions are: 0-2min, the carrier gas flow rate is 2mL/min, 2-20min, the carrier gas flow rate is 100mL/min, 20-30min, the carrier gas flow rate is 150mL/min.

(4) Ion mobility spectrometry detection

①Ionization source: tritium source (beta ray);

②Migration tube temperature: 45℃;

③Drift gas: nitrogen;

④Flow rate: 150mL/min.

(5) Result judgment:

In the gas chromatography-ion mobility spectrum, by comparing the gas retention time and the ion migration time, it is determined whether the characteristic markers of streptomycin filter residues are detected, so as to determine whether streptomycin filter residues are added to the sample. Antibiotic filter residue characteristics Marker name, gas phase retention time and ion migration time are shown in Table 1.

Table 1 Name, gas phase retention time and ion migration time of characteristic markers of antibiotic filter residues

The results showed that heptanol, ethyl hexanoate and ethyl propionate were detected in the streptomycin filter residue, as shown in Figure 1; ester, ethyl propionate, as shown in Figure 2; no heptanol, ethyl caproate, and ethyl propionate were detected in pure rapeseed meal, as shown in Figure 3. It can be seen from the above results that this method can accurately determine whether streptomycin filter residue is added to rapeseed meal.

Embodiment 2,

(1) Sample pretreatment

The rapeseed meal, the cephalosporin filter residue, and the rapeseed meal mixed with the cephalosporin filter residue (10%, m/m) were pulverized by a cyclone mill respectively, and the particle size after pulverization reached 18 meshes. Weigh 0.5 g of the crushed samples into clean glass headspace vials, seal them with rubber stoppers, and seal them with aluminum caps to ensure that the headspace vials are airtight.

(2) Static headspace volatile component collection

The headspace vial filled with the sample was heated and incubated on a thermal incubator to promote the release of volatile substances in the sample into the headspace. The thermal incubation temperature was 90 °C, the incubation time was 10 min, and the incubator vibration speed was 500 rpm. After the thermal incubation, the static headspace volatile components were aspirated with a syringe, the volume was 500 μL, and the injection speed was 100 μL/s.

(3) Gas chromatography separation

①chromatographic column: SE54 column (5% phenyl-95% dimethylpolysiloxane, non-polar stationary phase);

②Carrier gas: nitrogen;

③Column temperature: 40℃;

④Injector temperature: 80℃;

⑤Injection volume: 500μL;

⑥Injection speed: 100μL/s;

⑦ The gas phase separation conditions are: 0-2min, the carrier gas flow rate is 2ml/min, 2-20min, the carrier gas flow rate is 100mL/min, 20-30min, the carrier gas flow rate is 150mL/min.

(4) Ion mobility spectrometry detection

①Ionization source: tritium source (beta ray);

②Migration tube temperature: 45℃;

③Drift gas: nitrogen;

④Flow rate: 150mL/min.

(5) Result judgment:

In the gas chromatography-ion mobility spectrum, by comparing the gas retention time and the ion migration time, it is determined whether the characteristic markers of the cephalosporin filter residue are detected, so as to determine whether the cephalosporin filter residue is added to the sample. The names of the characteristic markers, gas phase retention time and ion migration time of antibiotic filter residues are shown in Table 1.

The results showed that 3-methylbutyraldehyde, phenylacetaldehyde, methylthiopropanal and 3-methylbutanol were detected in the cephalosporin filter residue, as shown in Figure 4; 3-methylbutyraldehyde, phenylacetaldehyde, methylthiopropanal, and 3-methylbutanol were detected in the seed meal, as shown in Figure 5; 3-methylbutyraldehyde was not detected in pure rapeseed meal , phenylacetaldehyde, methylthiopropionaldehyde, 3-methylbutanol, as shown in Figure 3. It can be seen from the above results that this method can accurately determine whether the rapeseed meal is mixed with cephalosporin filter residue.

Embodiment 3,

(1) Sample pretreatment

The rapeseed meal, the colistin sulfate filter residue, and the rapeseed meal mixed with the colistin sulfate filter residue (10%, m/m) were pulverized by a cyclone respectively, and the particle size after pulverization reached 18 meshes. Weigh 0.5 g of the crushed samples into clean glass headspace vials, seal them with rubber stoppers, and seal them with aluminum caps to ensure that the headspace vials are airtight.

(2) Static headspace volatile component collection

The headspace vial filled with the sample was heated and incubated on a thermal incubator to promote the release of volatile substances in the sample into the headspace. The thermal incubation temperature was 90 °C, the incubation time was 10 min, and the incubator vibration speed was 500 rpm. After the thermal incubation, the static headspace volatile components were aspirated with a syringe, the volume was 500 μL, and the injection speed was 100 μL/s.

(3) Gas chromatography separation

①chromatographic column: SE54 column (5% phenyl-95% dimethylpolysiloxane, non-polar stationary phase);

②Carrier gas: nitrogen;

③Column temperature: 40℃;

④Injector temperature: 80℃;

⑤Injection volume: 500μL;

⑥Injection speed: 100μl/s;

⑦ The gas phase separation conditions are: 0-2min, the carrier gas flow rate is 2mL/min, 2-20min, the carrier gas flow rate is 100mL/min, 20-30min, the carrier gas flow rate is 150mL/min.

(4) Ion mobility spectrometry detection

①Ionization source: tritium source (beta ray);

②Migration tube temperature: 45℃;

③Drift gas: nitrogen;

④Flow rate: 150mL/min.

(5) Result judgment:

In the gas chromatography-ion mobility spectrum, by comparing the gas retention time and the ion migration time, it is determined whether the characteristic markers of colistin sulfate filter residues are detected, so as to determine whether the samples are spiked with colistin sulfate filter residues. Table 1 shows the name, gas phase retention time and ion migration time of antibiotic filter residue characteristic markers.

The results showed that dimethyl trisulfide and 2,6-nonadienal were detected in the colistin sulfate filter residue, as shown in Figure 6; dimethyl trisulfide was detected in the rapeseed meal mixed with the colistin sulfate filter residue Sulfur and 2,6-nonadienal, as shown in Figure 7; dimethyltrisulfide and 2,6-nonadienal were not detected in pure rapeseed meal, as shown in Figure 3. It can be seen from the above results that this method can accurately determine whether the rapeseed meal is mixed with colistin sulfate filter residue.

Claims (6)

1. a method for detecting whether the rapeseed meal is doped with antibiotic residues, comprising the steps of: 1) pretreatment of the sample: the rapeseed meal sample is pulverized; 2) Volatile component collection: thermally incubate the crushed rapeseed meal sample in step 1), and then collect volatile components; 3) separation by gas chromatography: the volatile components are separated by gas chromatography; 4) ion mobility spectrometry detection: the volatile components separated in step 3) are detected by ion mobility spectrometry, and if the characteristic marker of the antibiotic filter residue is detected, the rapeseed meal is doped with antibiotic residue; The antibiotic filter residue is one or more of streptomycin filter residue, cephalosporin filter residue and colistin sulfate filter residue; When the antibiotic filter residue is the streptomycin filter residue, the characteristic markers are heptanol, ethyl hexanoate and ethyl propionate; When the antibiotic filter residue is the cephalosporin filter residue, the characteristic markers are 3-methylbutyraldehyde, phenylacetaldehyde, methylthiopropionaldehyde, and 3-methylbutanol; When the antibiotic filter residue is the colistin sulfate filter residue, the characteristic markers are dimethyltrisulfide and 2,6-nonadienal. 2 . The method according to claim 1 , wherein the particle size of the pulverization is greater than or equal to 18 meshes. 3 . 3. The method according to claim 1 or 2, wherein the temperature of the thermal incubation is 90°C, the time is 10min, and the vibration speed is 500rpm; The volatile components are collected by a static headspace volatile component collecting device. 4. The method according to claim 1 or 2, wherein the separation conditions of the gas chromatography are as follows: the chromatographic column is an SE54 chromatographic column; the column temperature is 40°C; the carrier gas is nitrogen, and the inlet temperature is 80°C ; The gas phase separation conditions are: 0～2min, the carrier gas flow rate is 2mL/min; 2～20min, the carrier gas flow rate is 100mL/min; 20～30min, the carrier gas flow rate is 150mL/min. 5. The method according to claim 1 or 2, characterized in that: the bars detected by the ion mobility spectrum are as follows: a tritium source beta-ray ionization source is used; the temperature of the migration tube is 45°C, the drift gas is nitrogen, and the flow rate is 150mL/min. 6 . The method according to claim 1 or 2 , wherein the sampling conditions for the gas chromatographic separation are as follows: the temperature of the injection needle is 95° C., the injection volume is 500 μL, and the injection speed is 100 μL/s. 7 .

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