CN113075338A - Method for detecting phthalate esters in animal food - Google Patents
Method for detecting phthalate esters in animal food Download PDFInfo
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- CN113075338A CN113075338A CN202110423029.0A CN202110423029A CN113075338A CN 113075338 A CN113075338 A CN 113075338A CN 202110423029 A CN202110423029 A CN 202110423029A CN 113075338 A CN113075338 A CN 113075338A
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- G—PHYSICS
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- 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
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The application relates to the technical field of food chemical detection, and particularly discloses a method for detecting phthalate in animal food, which comprises the steps of mixing and oscillating an extraction solvent and a sample to be detected, centrifuging and layering, drying supernatant, adding a purification solvent, mixing uniformly, purifying by a solid phase extraction column, drying, adding a constant volume solvent for constant volume, and detecting by a gas chromatography-mass spectrometry method; the extraction solvent comprises n-hexane and ethyl acetate, wherein the n-hexane: the volume ratio of ethyl acetate is 1: (1-3). The method solves the problems of difficult qualitative and inaccurate quantitative detection of phthalate in animal food.
Description
Technical Field
The application relates to the technical field of food chemical detection, in particular to a method for detecting phthalate esters in animal food.
Background
Phthalate is a general name of ester substances formed by phthalic acid, and is mainly used in polyvinyl chloride materials to change polyvinyl chloride from hard plastic into elastic plastic and play a role of a plasticizer. Phthalate substances are widely applied to living products such as toys, food packaging bags, detergents, lubricating oil and the like, but the phthalate substances can interfere endocrine in human bodies and animal bodies, so that the number of sperms is reduced, the sperm motility is low, the sperm morphology is abnormal, and the male reproduction problem is easily caused.
Animal food, such as liquid milk, livestock and poultry meat and aquatic products, is often packaged in food packaging bags during transportation and sale, and because the phthalate ester substances are fat-soluble compounds and are easily dissolved in the fat of the animal food, the intake of human body is increased, which is not good for human health. Therefore, the phthalate ester substance needs to be detected for animal food to test whether the content of the phthalate ester substance exceeds the standard or not.
However, there are a great variety of phthalate esters, and there are 15 phthalate esters that are commonly detected, including: dimethyl phthalate (DMP), diisobutyl phthalate (DIBP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP), di (2-methoxy) ethyl phthalate (DMEP), di-n-octyl phthalate (DNOP), di (2-ethyl) hexyl phthalate (DEHP), dicyclohexyl phthalate (DCHP), di (2-butoxy) ethyl phthalate (DBEP), butylbenzyl phthalate (BBP), dihexyl phthalate (DHXP), dipentyl phthalate (DPP), di (2-ethoxy) ethyl phthalate (DEEP), diphenyl phthalate (DPHP), di (4-methyl-2-pentyl) phthalate (BMPP).
The related art, see application No. 201010217703.1, discloses a method for detecting phthalate plasticizers, comprising the following steps: 1. pretreatment: putting a sample to be detected into an organic solvent for dissolving and processing to obtain a detected solution dissolved with a plasticizer; 2. and (3) measuring the content of the plasticizer: and (3) determining the type and the content of the plasticizer in the detected solution by adopting gas chromatography and chemical ionization mass spectrometry, wherein the chemical ionization reagent adopted in the chemical ionization mass spectrometry is isobutane, methane or ammonia gas.
With respect to the related art among the above, the inventors consider that the following drawbacks exist: the phthalate detection method is mainly used for detecting polyvinyl chloride plastic samples, however, when animal food samples are detected, other macromolecules or macromolecular substances in the animal food are easily dissolved in a solvent when phthalate substances in the animal food samples are extracted by using the solvent, the detected substances are required to be heated and gasified when gas chromatography-mass spectrometry (GC-MS) is used for detection, the macromolecules or the macromolecular substances are easily decomposed, so that the detection result is easily influenced by the occurrence of miscellaneous peaks, particularly when 15 phthalate substances are detected, the ionic characteristic peaks are more, the difficulty in qualitative identification is higher, and the ionic characteristic peaks introduced by impurities easily cover the qualitative ionic characteristic peaks of the phthalate substances, so that the difficulty in qualitative identification is caused; meanwhile, in quantitative detection, the introduced miscellaneous peak is easily connected with a quantitative characteristic peak to form a cross-linked double peak or a triple peak, so that the integral of the quantitative characteristic peak is inaccurate, and the detection result is influenced.
Disclosure of Invention
In order to solve the problems that the qualitative determination is difficult and the quantitative determination is inaccurate when the phthalate esters substances are detected in the animal food, the application provides a method for detecting the phthalate esters in the animal food.
The method for detecting the phthalate esters in the animal food adopts the following technical scheme: a method for detecting phthalate esters in animal food comprises mixing extraction solvent with sample to be detected, shaking, centrifuging, layering, oven drying the layered clear liquid, adding purification solvent, mixing, purifying with solid phase extraction column, oven drying, adding constant volume solvent, and detecting by gas chromatography-mass spectrometry; the extraction solvent comprises n-hexane and ethyl acetate, wherein the n-hexane: the volume ratio of ethyl acetate is 1: (1-3).
By adopting the technical scheme, the extraction solvent can extract phthalate substances in the animal food sample to obtain an extracting solution, the extracting solution is dried and then is added with a purifying solvent to be purified by a solid-phase extraction column, the purifying solvent can drive the phthalate substances to flow in the solid-phase extraction column, meanwhile, the solid-phase extraction column can adsorb macromolecules and macromolecular substances dissolved in the purifying solvent, the macromolecules and the macromolecular substances are separated from the phthalate substances, then the separated effluent is dried, and a constant volume solvent is added for constant volume for GC-MS detection; the method reduces the possibility that high molecules or macromolecular substances mixed in the sample are easily decomposed to generate impurities when the sample is heated and gasified in the gas chromatography, so that the qualitative and the quantitative accuracy of the detection result are difficult, and the accuracy of the detection of the phthalate substances in the animal food is improved.
Preferably, the extraction solvent is a mixture of n-hexane, ethyl acetate and a supramolecular solvent, the volume ratio of the n-hexane to the ethyl acetate to the supramolecular solvent is 1:1 (0.2-0.8), the supramolecular solvent is a mixture of tetrahydrofuran and long-chain alkyl alcohol, and the volume ratio of the tetrahydrofuran to the long-chain alkyl alcohol is (0.2-0.4): (0.1-0.2).
By adopting the technical scheme, the n-hexane, the ethyl acetate and the supramolecular solvent can have a good extraction effect on phthalate substances in the sample, and meanwhile, the supramolecular solvent can also filter the extracted macromolecules and macromolecular substances, so that the influence on GC-MS (gas chromatography-mass spectrometry) detection of the phthalate substances caused by the fact that the macromolecules or the macromolecular substances in the sample to be detected are dissolved in the extraction solvent is further reduced.
Preferably, the extraction solvent is a mixture of n-hexane, ethyl acetate and a supramolecular solvent, the volume ratio of the n-hexane to the ethyl acetate to the supramolecular solvent is 1:1 (0.35-0.5), the supramolecular solvent is a mixture of tetrahydrofuran and long-chain alkyl alcohol, and the volume ratio of the tetrahydrofuran to the long-chain alkyl alcohol is (0.2-0.4): (0.1-0.2).
By adopting the technical scheme, the accuracy of detecting the phthalate ester substances is further improved by the specific proportion of the extraction solvent.
Preferably, the preparation method of the supramolecular solvent comprises the following steps of mixing ultrapure water, tetrahydrofuran and long-chain alkyl alcohol according to a volume ratio of (1-2): (0.2-0.4): (0.1-0.2), uniformly mixing, shaking and centrifuging, and taking supernatant to obtain the supramolecular solvent.
Through adopting above-mentioned technical scheme, ultrapure water, tetrahydrofuran and long chain alkyl alcohol mix and vibrate the back, tetrahydrofuran and long chain alkyl alcohol can utilize hydrogen bond, dispersion power etc. to take place self-assembling in ultrapure water, form hydrophilic head in, the outside reverse micelle structure of hydrophobic afterbody, this kind of reverse micelle structure aperture is less, can hinder polymer and macromolecular substance to get into and draw the solvent in, has promoted the selectivity of drawing the solvent.
Preferably, the carbon chain of the long-chain alkyl alcohol is C8-C12.
By adopting the technical scheme, the supermolecular solvent formed by the long-chain alkyl alcohol with the carbon chain of C8-C12 has better selectivity on phthalate substances; the carbon chain is too short, the hydrogen bond effect is enhanced, the selectivity to the large polar substance is high, and the extraction of the phthalate ester substance is not facilitated; the carbon chain is too long, so that the dispersion force of the alkyl alcohol is reduced, the intermolecular action is weakened, and the extraction rate of the supermolecule solvent is reduced.
Preferably, the purifying solvent is one or a mixture of acetonitrile, acetone and ethyl acetate.
By adopting the technical scheme, the selectivity of the purifying solvents to the phthalate ester substances is better, the separation of the phthalate ester substances from other impurities can be further promoted, and the separation of the impurity peaks is facilitated.
Preferably, the constant volume solvent is n-hexane.
By adopting the technical scheme, the n-hexane has weaker polarity, the gas chromatographic column cannot be damaged, the n-hexane serving as a mobile phase cannot generate other impurities after gasification, and the n-hexane has better selectivity on phthalate substances and can drive the phthalate substances to flow in the gas chromatographic column.
Preferably, the matrix in the solid phase extraction column is fine-pore silica gel, the mesh number of the silica gel is 200-300 meshes, and the particle size is 45-75 μm.
Through adopting above-mentioned technical scheme, pore silica gel is compared in macroporous silica gel and adsorbs polymer and macromolecular substance more easily, and when the silica gel mesh was greater than 300 meshes, the silica gel granularity undersize led, leads to filter speed too slow, reduce efficiency, and the silica gel mesh is greater than 200 meshes, and the silica gel granularity is too big, and adsorption effect worsens, has further improved the degree of accuracy that phthalate ester class material detected through the matrix in the preferred solid phase extraction post.
In summary, the present application has the following beneficial effects:
1. according to the method, the phthalic acid ester substances in the animal food sample can be extracted by the extraction solvent to obtain an extracting solution, the extracting solution is dried and then is added with a purifying solvent to be purified by a solid-phase extraction column, the purifying solvent can drive the phthalic acid ester substances to flow in the solid-phase extraction column, meanwhile, the solid-phase extraction column can adsorb the high molecules and the high molecules dissolved in the purifying solvent, the high molecules and the high molecules are separated from the phthalic acid ester substances, the separated effluent is dried, and a constant volume solvent is added for constant volume for GC-MS detection; the method reduces the possibility that high molecules or macromolecular substances mixed in the sample are easily decomposed to generate impurities when the sample is heated and gasified in the gas chromatography, so that the qualitative and the quantitative accuracy of the detection result are difficult, and the accuracy of the detection of the phthalate substances in the animal food is improved.
2. The extraction solvent comprises n-hexane, ethyl acetate and a supramolecular solvent, and can extract phthalate substances in a sample; the supermolecule solvent is prepared by mixing tetrahydrofuran and long-chain alkyl alcohol, the tetrahydrofuran and the long-chain alkyl alcohol can be self-assembled in water by utilizing hydrogen bonds, dispersion force and the like to form a reverse micelle structure with a hydrophilic head part inside and a hydrophobic tail part outside, the pore diameter of the reverse micelle structure is small, high molecules and macromolecular substances can be prevented from entering an extraction phase, and the selectivity of the extraction solvent is improved.
Detailed Description
The reagents used in the present application are all commercial products, and the specification is chromatographic purity, the matrix in the solid phase extraction column (SPE) is microporous silica gel, the mesh number of the silica gel is 200-300 meshes, and the particle size is 45-75 μm.
Preparation example of supramolecular solvent
Preparation example 1
Preparation of a supramolecular solvent: mixing 10mL of ultrapure water, 2mL of tetrahydrofuran and 1mL of n-octanol, shaking up to obtain a mixed solution, centrifuging the mixed solution at 4000r/min for 2min, and taking supernatant to obtain the supramolecular solvent.
Preparation example 2
Preparation of a supramolecular solvent: mixing 15mL of ultrapure water, 3mL of tetrahydrofuran and 1.5mL of n-octanol, shaking up to obtain a mixed solution, centrifuging the mixed solution at 4000r/min for 2min, and taking supernatant to obtain the supramolecular solvent.
Preparation example 3
Preparation of a supramolecular solvent: mixing 20mL of ultrapure water, 4mL of tetrahydrofuran and 2mL of n-octanol, shaking up to obtain a mixed solution, centrifuging the mixed solution at 4000r/min for 2min, and taking supernatant to obtain the supramolecular solvent.
Preparation example 4
Preparation 4 was compared with preparation 1 except that n-octanol was replaced with an equal volume of n-nonanol.
Preparation example 5
Preparation example 5 was compared with preparation example 1 except that n-octanol was replaced with an equal volume of n-decanol.
Preparation example 6
Preparation example 6 was compared with preparation example 1, except that n-octanol was replaced with an equal volume of n-undecanol.
Preparation example 7
Preparation example 7 was compared with preparation example 1, except that n-octanol was replaced with an equal volume of n-dodecanol.
Preparation example 8
Preparation example 8 was compared with preparation example 1, except that n-octanol was replaced with an equal volume of n-heptanol.
Preparation example 9
Production example 9 is different from production example 1 in that n-octanol is replaced with n-tridecanol of the same volume.
Examples
In examples 1 to 37, the accuracy of the method for detecting the phthalate ester substances in the animal food is verified by adopting standard recovery.
Example 1
The method for detecting the phthalate esters in the animal food comprises the following steps: all glassware used in the detection step is pretreated, soaked in n-hexane for 2 hours, and then vacuum-dried for 1 hour at the drying temperature of 160 ℃.
Randomly selecting 3 complete and independent packaging samples, wherein the packaging bag is made of polyethylene plastic, the samples are liquid milk, and uniformly mixing the 3 samples to obtain initial samples; taking 10g of an initial sample as a sample 1, taking 10g of the initial sample as a sample 2, adding 1mg of dimethyl phthalate (DMP) into the sample 2, and uniformly mixing to obtain a labeled sample 2.
Adding 2g of sample 1 into an extraction solvent, mixing for 2min at 2000r/min in a vortex manner, centrifuging for 2min at 4000r/min, transferring supernatant obtained after twice centrifugation to obtain an extracting solution, repeatedly extracting the sample 1 twice, combining the extracting solutions, and mixing the extracting solvent with 4mL of n-hexane and 4mL of ethyl acetate;
drying the extracting solution at 50 ℃ by nitrogen blowing, adding 1mL of acetonitrile, shaking up and fixing the volume to obtain a solution to be purified;
adding 5mL of acetonitrile into the SPE small column for activating and rinsing to obtain an activated SPE small column;
adding the solution to be purified into an activated SPE small column with the flow rate of 1mL/min, and collecting the effluent liquid; washing the glass tube containing the solution to be purified by using 2mL of acetonitrile to obtain a washing solution, adding the washing solution into the activated SPE small column, collecting the effluent again, and combining the effluent obtained in the two steps and shaking uniformly;
and (3) blowing and drying the effluent at 50 ℃ by nitrogen, adding 2mL of n-hexane for dissolving and fixing the volume to obtain a solution to be detected, and detecting by GC-MS.
The spiked sample 2 was also subjected to the same processing steps as sample 1 and sent to GC-MS detection.
Example 2
Example 2 compares to example 1 with the difference that the extraction solvent was formed from 2.67mL of n-hexane mixed with 5.33mL of ethyl acetate.
Example 3
Example 3 compares to example 1 with the difference that the extraction solvent is formed by mixing 2mL of n-hexane and 6mL of ethyl acetate.
Example 4
Example 4 compares to example 1 with the difference that the extraction solvent is formed by mixing 3.4mL of n-hexane, 3.4mL of ethyl acetate and 1.2mL of the supramolecular solvent of preparation 1.
Example 5
Example 5 compares to example 1 with the difference that the extraction solvent was formed from a mixture of 3.3mL of n-hexane, 3.3mL of ethyl acetate and 1.4mL of the supramolecular solvent of preparation 1.
Example 6
Example 6 compares to example 1 with the difference that the extraction solvent is formed by mixing 3.2mL of n-hexane, 3.2mL of ethyl acetate and 1.6mL of the supramolecular solvent of preparation 1.
Example 7
Example 7 was compared to example 1 except that the extraction solvent was formed from a mixture of 3.6mL of n-hexane, 3.6mL of ethyl acetate and 0.8mL of the supramolecular solvent of preparation 1.
Example 8
Example 8 compares to example 1 with the difference that the extraction solvent was formed from 2.9mL of n-hexane, 2.9mL of ethyl acetate and 2.2mL of the supramolecular solvent of preparation 1 mixed together.
Example 9
Example 9 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation 2.
Example 10
Example 10 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation example 3.
Example 11
Example 11 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation 4.
Example 12
Example 12 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation example 5.
Example 13
Example 13 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation example 6.
Example 14
Example 14 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation example 7.
Example 15
Example 15 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation 8.
Example 16
Example 16 compares to example 1 with the difference that the supramolecular solvent in the extraction solvent is provided by preparation 9.
Example 17
Example 17 was compared to example 1 except that acetonitrile was replaced with an equal volume of acetone.
Example 18
Example 18 was compared to example 1 except that acetonitrile was replaced with an equal volume of ethyl acetate.
Example 19
Example 19 was compared with example 1 except that acetonitrile was replaced with an equal volume of a mixed solvent composed of ethyl acetate and acetone in a volume ratio of 1: 1.
Example 20
Example 20 was compared to example 1 except that acetonitrile was replaced with an equal volume of n-hexane.
Example 21
Example 21 was compared to example 1, except that the n-hexane in the constant volume process was replaced with dichloromethane.
Example 22
Example 22 was compared to example 1, except that n-hexane in the volumetric process was replaced with acetonitrile.
Example 23
Example 23 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of diisobutyl phthalate (DIBP).
Example 24
Example 24 is compared with example 1 except that dimethyl phthalate (DMP) in the spiked sample 2 was replaced with equal mass of di-n-butyl phthalate (DBP).
Example 25
Example 25 is compared to example 1 except that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of diethyl phthalate (DEP).
Example 26
Example 26 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of di (2-methoxy) ethyl phthalate (DMEP).
Example 27
Example 27 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of di-n-octyl phthalate (DNOP).
Example 28
Example 28 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of di (2-ethyl) hexyl phthalate (DEHP).
Example 29
Example 29 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of dicyclohexyl phthalate (DCHP).
Example 30
Example 30 is compared to example 1 except that the dimethyl phthalate (DMP) in spiked sample 2 was replaced with an equal mass of di (2-butoxy) ethyl phthalate (DBEP).
Example 31
Example 31 is compared to example 1 except that the dimethyl phthalate (DMP) in spiked sample 2 was replaced with an equal mass of di (2-butoxy) ethyl phthalate (DBEP).
Example 32
Example 32 is compared to example 1 except that the dimethyl phthalate (DMP) in spiked sample 2 was replaced with an equal mass of Butyl Benzyl Phthalate (BBP).
Example 33
Example 33 is compared to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of dihexyl phthalate (DHXP).
Example 34
Example 34 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of dipentyl phthalate (DPP).
Example 35
Example 35 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of di (2-ethoxy) ethyl phthalate (DEEP).
Example 36
Example 36 compares to example 1 with the difference that the dimethyl phthalate (DMP) in the spiked sample 2 was replaced with an equal mass of diphenyl phthalate (DPHP).
Example 37
Example 37 compares to example 1 with the difference that the dimethyl phthalate (DMP) in spiked sample 2 was replaced with an equal mass of di (4-methyl-2-pentyl) phthalate (BMPP).
Comparative example
Comparative example 1
Compared with the embodiment 1, the difference of the comparative example 1 is that 2mL of normal hexane is added for constant volume for GC-MS detection after the extracted extracting solution of the sample is directly dried by nitrogen blowing.
Comparative example 2
Comparative example 2 is compared to example 1 except that the extraction solvent was 8mL of n-hexane.
Performance test
Detecting 15 phthalate esters by adopting GC-MS, wherein the types of gas chromatographic columns are as follows: HP-5MS quartz capillary column [30m × 0.25mm (internal diameter) × 0.25 μm (film thickness) ]; the temperature of a sample inlet is 250 ℃; a temperature raising program, wherein the initial column temperature is 80 ℃, the temperature is kept for 1min, the temperature is raised to 220 ℃ at the speed of 20 ℃/min, the temperature is kept for 1min, and then the temperature is raised to 280 ℃ at the speed of 5 ℃/min, and the temperature is kept for 1 min; the carrier gas is helium, the purity is more than or equal to 99.999 percent, and the flow rate is 1.0 mL/min; the sample introduction mode is not divided; the sample injection amount is 1.0 mu L; the temperature of the color transmission line is 280 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; ionization mode, electron bombardment source (EI); a monitoring mode, selecting an ion monitoring mode (SIM); the ionization energy is 70 eV. The characteristic ions of the phthalate esters are shown in Table 1.
TABLE 1
Determining the characteristic peak positions of different phthalate ester substances in a total ion flow spectrum by the charge-to-mass ratio of qualitative ions in 15 phthalate ester substances in the table 1 so as to determine the substances; and performing integral calculation on the characteristic peak area where the charge-to-mass ratio of the quantitative ions is located, so as to perform quantitative analysis on the substances.
Preparation of a standard solution: measured in equal amounts, dimethyl phthalate (DMP), diisobutyl phthalate (DIBP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP), di-2-methoxy ethyl phthalate (DMEP), di-n-octyl phthalate (DNOP), di-2-ethyl hexyl phthalate (DEHP), dicyclohexyl phthalate (DCHP), di-2-butoxy ethyl phthalate (DBEP), butylbenzyl phthalate (BBP), dihexyl phthalate (DHXP), dipentyl phthalate (DPP), di-2-ethoxy ethyl phthalate (DEEP), diphenyl phthalate (DPHP), di-4-methyl-2-pentyl phthalate (BMPP) pure samples were mixed well and formulated into 10.0mg/L stock solutions using n-hexane, the stock was then diluted stepwise to 0.40. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL, 4.0. mu.g/mL, 10. mu.g/mL so that the concentration covered 2 orders of magnitude.
The standard solutions with different concentrations were tested by GC-MS and the instrument automatically plotted as a standard curve. Repeating the blank for 20 times, calculating the standard deviation of the noise, and calculating the detection limit of the instrument. Multiplying the detection limit of the instrument by the dilution times, dividing by the sample weighing, and determining the detection limit and the quantitative limit by combining a matrix interference determination method. Table 2 shows the standard curve equation, correlation coefficient, detection limit and quantitative limit of the phthalate ester compounds in 15.
TABLE 2
According to the standard curve of each substance in Table 2, the quantitative characteristic peak area of the substance measured by GC-MS can be substituted into the y value in the standard curve to obtain the concentration x value, so as to measure the content of the substance. The detection limit represents the lowest content of the substance to be detected in the sample which can be detected by the detection method within a given reliability degree; the limit of quantitation is the lowest amount of the analyte in the sample that can be quantitatively determined.
Generally, the accuracy of the detection method is represented by the recovery rate, and the precision of the detection method is judged by the relative standard deviation. The phthalate concentrations in the spiked sample 2 and the sample 1 are calculated according to a standard curve, and then the recovery rate is calculated according to a recovery rate calculation formula:
the relative standard deviation (RSD%) is calculated according to the recovery value, and the calculation formula of the relative standard deviation is as follows:
xithe recovery rate value measured at each time is indicated,
the average value of the recovery rate is shown. Table 3 shows the normalized average recovery, the measured value of the relative standard deviation, and the degree of influence of the characteristic peak, which were calculated by repeating 5 experiments in examples and comparative examples.
TABLE 3
As can be seen by combining examples 1-6 and table 3, the average recovery rate of examples 1-3 is lower than that of examples 4-6, which indicates that the accuracy of the detection method can be effectively improved after the supramolecular solvent is introduced into the extraction solvent, and the RSD% values of the examples are all less than 3, which indicates that the error of the detection method is small and the detection value is reliable; in combination with examples 4-8, it can be seen that the volume ratio of n-hexane, ethyl acetate and supramolecular solvent in the extraction solvent is 1: (0.2-0.8), the recovery rate is increased and then decreased, when the volume ratio of n-hexane, ethyl acetate and supramolecular solvent is 1: (0.35-0.5), the recovery rate is relatively good, which indicates that the ratio of n-hexane, ethyl acetate and supramolecular solvent is 1: (0.35-0.5) the extractant can improve the accuracy of the test method.
Combining example 1 and examples 9-16, it can be seen that the chain length of the long-chain alkyl alcohol in the supramolecular solvent is less than C8 or greater than C12, which reduces the accuracy of the detection method, and when the chain length is less than C8, a hetero peak is generated to affect the characteristic peak of dimethyl phthalate (DMP), mainly because the carbon chain is too short, the hydrogen bonding effect is enhanced, the selectivity to the high-polar substance is high, and at this time, the extracted impurities are excessive phthalate, which results in increased impurities and generation of a hetero peak; the carbon chain is too long, so that the dispersion force of the alkyl alcohol is reduced, the intermolecular action is weakened, the extraction rate of the supramolecular solvent is reduced, and the recovery rate is reduced.
Combining the embodiment 1 and the embodiments 17-20, it can be seen that when the polar solvent is one or more of acetonitrile, acetone and ethyl acetate, the detection method has higher accuracy, and when the polar solvent is changed to n-hexane with weaker polarity, the mobile phase drives the phthalate substance to have poor separation effect on the SPE small column, the accuracy is lower, and meanwhile, a miscellaneous peak is generated; combining example 1 and examples 21-22, it can be seen that when entering GC-MS for detection, the recovery rate of n-hexane as a mobile phase is higher than that of other constant volume solvents, and higher accuracy is shown.
It can be seen from the combination of example 1 and examples 23-37 that examples 23-27 all have higher accuracy, which indicates that the detection method is suitable for detecting the 15 phthalate esters, and has good selectivity for the 15 phthalate esters.
By combining the example 1 and the comparative example 1 and combining the table 3, the sample to be detected does not undergo SPE (solid phase extraction) small column purification, so that the accuracy of the method is greatly influenced, and meanwhile, a characteristic peak is influenced by a hybrid peak; in combination with example 1 and comparative example 2, it can be seen that in comparative example 2, phthalate-based substances in the sample cannot be extracted by using n-hexane as an extraction solvent, and thus cannot be detected.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (8)
1. A method for detecting phthalate esters in animal food is characterized in that an extraction solvent is adopted to be mixed with a sample to be detected and vibrated, centrifugal layering is carried out, layered clear liquid is dried, a purification solvent is added to be mixed uniformly, the mixture is dried after being purified by a solid phase extraction column, a constant volume solvent is added to the mixture to achieve constant volume, and gas chromatography-mass spectrometry is used for detection; the extraction solvent comprises n-hexane and ethyl acetate, wherein the n-hexane: the volume ratio of ethyl acetate is 1: (1-3).
2. The method for detecting phthalates in animal food according to claim 1, wherein: the extraction solvent is a mixture of n-hexane, ethyl acetate and a supramolecular solvent, the volume ratio of the n-hexane to the ethyl acetate to the supramolecular solvent is 1:1 (0.2-0.8), the supramolecular solvent is a mixture of tetrahydrofuran and long-chain alkyl alcohol, and the volume ratio of the tetrahydrofuran to the long-chain alkyl alcohol is (0.2-0.4): (0.1-0.2).
3. The method for detecting phthalates in animal food according to claim 2, wherein: the extraction solvent is a mixture of n-hexane, ethyl acetate and a supramolecular solvent, the volume ratio of the n-hexane to the ethyl acetate to the supramolecular solvent is 1:1 (0.35-0.5), the supramolecular solvent is a mixture of tetrahydrofuran and long-chain alkyl alcohol, and the volume ratio of the tetrahydrofuran to the long-chain alkyl alcohol is (0.2-0.4): (0.1-0.2).
4. The method for detecting phthalates in animal food according to claim 3, wherein: the preparation method of the supramolecular solvent comprises the following steps of mixing ultrapure water, tetrahydrofuran and long-chain alkyl alcohol according to the volume ratio of (1-2): (0.2-0.4): (0.1-0.2), uniformly mixing, shaking and centrifuging, and taking supernatant to obtain the supramolecular solvent.
5. The method for detecting phthalates in animal food according to claim 4, wherein: the carbon chain of the long-chain alkyl alcohol is C8-C12.
6. The method for detecting phthalates in animal food according to claim 1 or 2, wherein: the purifying solvent is one or a mixture of acetonitrile, acetone and ethyl acetate.
7. The method for detecting phthalates in animal food according to claim 1 or 2, wherein: the constant volume solvent is n-hexane.
8. The method for detecting phthalates in animal food according to claim 1 or 2, wherein: the matrix in the solid phase extraction column is fine-pored silica gel, the mesh number of the silica gel is 200-300 meshes, and the particle size is 45-75 mu m.
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