CN116338029A - Method for measuring ethyl carbamate content in white spirit - Google Patents

Method for measuring ethyl carbamate content in white spirit Download PDF

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Publication number
CN116338029A
CN116338029A CN202310088387.XA CN202310088387A CN116338029A CN 116338029 A CN116338029 A CN 116338029A CN 202310088387 A CN202310088387 A CN 202310088387A CN 116338029 A CN116338029 A CN 116338029A
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content
ethyl carbamate
mass
sample
white spirit
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靳晓婷
郝建国
任文吉
杨晓婧
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Luliang Vocational And Technical College
Shanxi Xinghuacun Fenjiu Group Wine Industry Development Zone Co Ltd
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Luliang Vocational And Technical College
Shanxi Xinghuacun Fenjiu Group Wine Industry Development Zone Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Abstract

The application discloses a method for measuring the content of ethyl carbamate in white spirit, which relates to the technical field of testing the content of ethyl carbamate in white spirit, and is characterized in that a wine sample is filtered, an internal standard substance is added, a certain two types of butyl carbamate serving as the internal standard substance is added into the filtered wine sample, and the liquid chromatography-mass spectrometer analysis is carried out, so that the content of ethyl carbamate in the sample is detected. The method omits the process of sample elution and concentration, avoids potential safety hazards to environment and experimenters possibly generated in the process, simultaneously reduces the loss of measured content possibly caused in the pretreatment process, does not need complex pretreatment, adopts a direct sample injection mode to be simple and quick, does not use an organic solvent, can make up for potential safety hazards caused by using a large amount of dangerous chemicals such as diethyl ether and the like in the traditional method, and adopts an internal standard method by taking butyl amino acetate as an internal standard substance to avoid detection result deviation caused by different matrixes.

Description

Method for measuring ethyl carbamate content in white spirit
Technical Field
The application relates to the technical field of testing of ethyl carbamate content in white spirit, in particular to a method for measuring ethyl carbamate content in white spirit.
Background
The white spirit is an alcoholic beverage which is prepared by taking cereal grains as raw materials, taking Daqu, xiaoqu or bran koji as saccharification starter, adopting solid state fermentation, distilling, storing and blending, and contains various trace substances. Urethane is a substance with genetic carcinogenesis, which can lead to the formation of tumors, lymphomas, etc. The ethyl carbamate consciouss the accompanying products of the white spirit in the fermentation process. The content of the white spirit in the white spirit has important significance for paying attention to food safety and public health.
At present, the most widely used method for detecting the content of ethyl carbamate in white spirit is a gas chromatography method, and the method needs complex pretreatment of samples, has complicated steps and low automation, and cannot meet the requirement of rapid mass detection. And consume a large amount of organic reagent, and have adverse effects on the environment and the health of experimental staff.
At present, the detection of the ethyl carbamate in the domestic general white spirit adopts GB5009.223-2014 'determination of the ethyl carbamate in the food safety national standard food'. The method is based on the measurement principle: and (3) adding an internal standard of the D5-carbamate into the sample, purifying and eluting the sample by an alkaline diatomite solid phase extraction column, concentrating eluent, and measuring by using a gas chromatograph-mass spectrometer, and quantifying by an internal standard method.
This method has the following problems:
(1) In the method, the alkaline diatomite solid phase extraction column is used for purification and elution, and the eluent is 5% ethyl acetate-diethyl ether solution. Wherein the diethyl ether solution is a dangerous chemical, and the use safety is required to be paid attention to in the process of purchase and use.
Concentrating after elution is finished, concentrating the eluent by using a nitrogen blowing instrument, so that organic reagents such as n-hexane, ethyl acetate, diethyl ether and the like can enter the air, and potential safety hazards can be brought to the environment and experimenters.
Disclosure of Invention
In order to solve the problem of the potential safety hazard, the invention provides a method for measuring the content of ethyl carbamate in white spirit.
The invention provides a method for measuring the content of ethyl carbamate in white spirit, which adopts the following technical scheme: a method for measuring the content of ethyl carbamate in white spirit comprises the following steps:
s101: filtering the wine sample, sequentially introducing each component of the sample after liquid chromatography separation into a mass spectrum detector, and ionizing each component in an ion source to generate ions with certain charges and different mass numbers;
s102: adding an internal standard substance, adding a certain two butyl carbamate serving as the internal standard substance into the filtered wine sample for analysis by a liquid chromatography-mass spectrometer, wherein the motion behaviors of different ions in an electromagnetic field are different, and separating the ions according to different mass-to-charge ratios (m/z) by a mass analyzer to obtain mass spectrograms arranged according to the sequence of the mass-to-charge ratios;
s103: and detecting the content of the ethyl carbamate in the sample, and obtaining qualitative and quantitative results of the sample through analysis and treatment of a mass spectrogram.
As a preferable scheme of the method for measuring the content of the ethyl carbamate in the white spirit, the invention comprises the following steps: and (3) matching an Agilent1290 liquid chromatograph with the analysis instrument in the S102, which is an Agilent 6470 triple quadrupole mass spectrometer.
As a preferable scheme of the method for measuring the content of the ethyl carbamate in the white spirit, the invention comprises the following steps: the reagent in the S102 is acetonitrile.
As a preferable scheme of the method for measuring the content of the ethyl carbamate in the white spirit, the invention comprises the following steps: the chromatographic conditions in S101 and S102 are:
chromatographic column: ZORBAX RRHD Eclipse Plus C18 2.1X105 mm,1.8 μm;
mobile phase a:0.1% (V/V) aqueous formic acid;
mobile phase B: acetonitrile;
flow rate: 0.25ml/min.
As a preferable scheme of the method for measuring the content of the ethyl carbamate in the white spirit, the invention comprises the following steps: the mass spectrometry conditions in S101 and S102 are:
ion source: electrospray ion source (EIC);
scanning mode: scanning positive ions;
the detection mode is as follows: MRM mode.
In summary, the present invention includes at least one of the following beneficial effects:
1. the method omits the process of eluting and concentrating the sample, thereby avoiding potential safety hazards to the environment and experimental staff possibly generated in the process, and simultaneously reducing the loss of the measured content possibly caused in the pretreatment process;
2. the method does not need complex pretreatment, adopts a direct sample injection mode, is simple and quick, does not use an organic solvent, can make up for the potential safety hazard caused by using a large amount of dangerous chemicals such as diethyl ether and the like in the traditional method, and can avoid the deviation of detection results caused by different matrixes by adopting an internal standard method with butyl amino acetate as an internal standard.
Drawings
FIG. 1 is an overview flow chart of the present invention;
FIG. 2 is a specific flow chart of the present invention;
FIG. 3 is an extracted ion flow chromatogram of an EC standard of the present invention;
FIG. 4 is an extracted ion flow chromatogram of a BC standard in accordance with the present invention;
FIG. 5 is a linear plot of the ratio of peak areas of characteristic fragment ions of EC and BC versus the mass concentration ratio of EC and BC of the present invention;
fig. 6 is an extracted ion flow chromatogram of EC and BC standards of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-4 and fig. 6, a method for determining the content of ethyl carbamate in white spirit comprises the following steps:
s101: filtering the wine sample, sequentially introducing each component of the sample after liquid chromatography separation into a mass spectrum detector, and ionizing each component in an ion source to generate ions with certain charges and different mass numbers;
s102: adding an internal standard substance, adding a certain two butyl carbamate serving as the internal standard substance into the filtered wine sample for analysis by a liquid chromatography-mass spectrometer, wherein the motion behaviors of different ions in an electromagnetic field are different, and separating the ions according to different mass-to-charge ratios (m/z) by a mass analyzer to obtain mass spectrograms arranged according to the sequence of the mass-to-charge ratios;
s103: and detecting the content of the ethyl carbamate in the sample, and obtaining qualitative and quantitative results of the sample through analysis and treatment of a mass spectrogram.
The analysis instrument in S102 is an Agilent 6470 triple quadrupole mass spectrometer and is matched with an Agilent1290 liquid chromatograph.
Acetonitrile for reagent in S102.
The chromatographic conditions in S101 and S102 are:
chromatographic column: ZORBAX RRHD Eclipse Plus C18 2.1X105 mm,1.8 μm;
mobile phase a:0.1% (V/V) aqueous formic acid;
mobile phase B: acetonitrile;
flow rate: 0.25ml/min.
Gradient elution procedure:
Time(min) A(％) B(％)
0.00 95.0 5.0
1.00 90.0 10.0
2.50 15.0 85.0
3.00 15.0 85.0
3.10 90.0 10.0
12.00 95.0 5.0
the mass spectrometry conditions in S101 and S102 are:
ion source: electrospray ion source (EIC);
scanning mode: scanning positive ions;
the detection mode is as follows: MRM mode.
The mass spectrometry conditions for Ethyl Carbamate (EC) and Butyl Carbamate (BC) are shown in the figure:
Figure SMS_1
working principle: the components of the sample after liquid chromatography separation sequentially enter a mass spectrum detector, the components are ionized in an ion source to generate ions with certain charges and different mass numbers, the movement behaviors of the different ions in an electromagnetic field are different, the ions are separated according to different mass-to-charge ratios (m/z) by a mass analyzer, mass spectrograms arranged according to the mass-to-charge ratios are obtained, and qualitative and quantitative results of the sample can be obtained through analysis and treatment of the mass spectrograms.
Example 1
Referring to FIG. 5, 0.01g of EC and BC standard are accurately weighed into a 10ml volumetric flask, and methanol is used for metering to scale marks to prepare standard stock solutions of 1000mg/L respectively. The EC standard solution was serially diluted with methanol-water (1:9, V/V) solution to prepare a standard curve with concentration gradients of 10.0, 20.0, 30.0, 50.0, 100.0, 200.0. Mu.g/L, and BC of 2. Mu.g/L was added thereto as an internal standard, and the standard curve was plotted. And (3) carrying out linear regression on the mass concentration ratio of the EC to the BC according to the ratio of the peak areas of the characteristic fragment ions of the EC to the BC to obtain a regression equation, a correlation coefficient and a linear range, wherein the table is as follows:
target object Carbamic acid ethyl ester
Linear equation y＝0.121781*x-0.001062
Linear range μg/L 10-200
Correlation coefficient 0.998
The unknown sample is subjected to 6 times of repeated tests by adopting the method and is compared with the standard method, and the results are shown in the following table:
Figure SMS_2
labeling the unknown sample, sucking 0.1ml of 10000 mug/L standard substance, fixing the volume to 10ml, and measuring the labeled sample, wherein the result is shown in the following table:
Figure SMS_3
Figure SMS_4
to sum up: the method utilizes an ultra-high performance liquid chromatography-mass spectrometer to complete the detection of the content of the ethyl carbamate in the white spirit by adopting an internal standard method. By adopting the method and a standard method, the same unknown sample is continuously measured, and the measurement deviation of the two methods is 1.2% -4.9%. The relative standard deviation of the same sample measured continuously for 6 times is 0.89% -2.43%, and is less than 5%, so that the method completely meets the requirements of the method. The unknown sample is subjected to labeling, the labeling recovery rate is calculated to be in the range of 90.6% -100.9%, the detection instrument is an ultra-high performance liquid chromatography-mass spectrometry combined instrument, the detection method is an internal standard method, and the sample can be directly subjected to on-machine analysis without pretreatment. Through verification, the precision, accuracy and repeatability of the method meet the detection requirements.
The above embodiments are not intended to limit the scope of the present invention, so: all equivalent changes in structure, shape and principle of the invention should be covered in the scope of protection of the invention.

Claims (5)

1. The method for measuring the content of the ethyl carbamate in the white spirit is characterized by comprising the following steps of:
s101: filtering the wine sample, sequentially introducing each component of the sample after liquid chromatography separation into a mass spectrum detector, and ionizing each component in an ion source to generate ions with certain charges and different mass numbers;
s102: adding an internal standard substance, adding a certain two butyl carbamate serving as the internal standard substance into the filtered wine sample for analysis by a liquid chromatography-mass spectrometer, wherein the motion behaviors of different ions in an electromagnetic field are different, and separating the ions according to different mass-to-charge ratios (m/z) by a mass analyzer to obtain mass spectrograms arranged according to the sequence of the mass-to-charge ratios;
s103: and detecting the content of the ethyl carbamate in the sample, and obtaining qualitative and quantitative results of the sample through analysis and treatment of a mass spectrogram.
2. The method for determining the content of ethyl carbamate in white spirit according to claim 1, wherein the method comprises the following steps: and (3) matching an Agilent1290 liquid chromatograph with the analysis instrument in the S102, which is an Agilent 6470 triple quadrupole mass spectrometer.
3. The method for determining the content of ethyl carbamate in white spirit according to claim 1, wherein the method comprises the following steps: the reagent in the S102 is acetonitrile.
4. The method for determining the content of ethyl carbamate in white spirit according to claim 1, wherein the chromatographic conditions in S101 and S102 are as follows:
chromatographic column: ZORBAX RRHD Eclipse Plus C18 2.1X105 mm,1.8 μm;
mobile phase a:0.1% (V/V) aqueous formic acid;
mobile phase B: acetonitrile;
flow rate: 0.25ml/min.
5. The method for determining the content of ethyl carbamate in white spirit according to claim 1, wherein mass spectrometry conditions in S101 and S102 are as follows:
ion source: electrospray ion source (EIC);
scanning mode: scanning positive ions;
the detection mode is as follows: MRM mode.
CN202310088387.XA 2023-02-09 2023-02-09 Method for measuring ethyl carbamate content in white spirit Pending CN116338029A (en)

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