阅读说明：本技术 一种检测烟用香精或水基胶中邻苯二甲酸酯的方法 (Method for detecting phthalic acid ester in tobacco essence or water-based adhesive ) 是由 朱静 陆冰琳 许蔼飞 杨飞 宋凌勇 农李政 刘珊珊 唐桂芳 林莉 潘玉灵 于 2019-12-19 设计创作，主要内容包括：本发明提供了一种检测烟用香精或水基胶中的邻苯二甲酸酯的方法,首先将烟用香精或水基胶称取后加有机溶剂振荡提取,取部分萃取液氮吹,用水复溶,磁性石墨烯固相萃取净化、洗脱后,以气相色谱-串联质谱测定烟用香精或水基胶中的邻苯二甲酸酯。本方法能够快速、准确检测邻苯二甲酸酯残留量,具有方法环保、结果准确、干扰少、灵敏度高及重复性好的优点。特别适用于检测烟用香精或水基胶中残留的邻苯二甲酸酯。(The invention provides a method for detecting phthalic acid ester in tobacco essence or water-based adhesive, which comprises the steps of weighing the tobacco essence or water-based adhesive, adding an organic solvent for oscillation extraction, blowing part of extracted liquid nitrogen, redissolving with water, carrying out solid-phase extraction, purification and elution by magnetic graphene, and measuring the phthalic acid ester in the tobacco essence or water-based adhesive by gas chromatography-tandem mass spectrometry. The method can be used for rapidly and accurately detecting the residual quantity of the phthalic acid ester, and has the advantages of environmental protection, accurate result, less interference, high sensitivity and good repeatability. Is particularly suitable for detecting the residual phthalic acid ester in the tobacco essence or the water-based adhesive.)

1. A method for detecting phthalic acid ester in tobacco essence or water-based adhesive comprises the steps of weighing a tobacco sample, adding an organic solvent, carrying out vortex oscillation, carrying out centrifugal filtration, drying 2.0mL of filtrate by nitrogen, redissolving by 2.0mL of ultrapure water, adding 20mg of magnetic graphene particles, carrying out vortex oscillation at 2000r/min for 3min, and separating a solid phase from a liquid phase by a magnet. Discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and determining phthalate in the essence by gas chromatography-tandem mass spectrometry, wherein the method specifically comprises the following steps:

a. weighing 0.2g of sample, and placing the sample in a 15mL triangular flask with a plug;

b. and (3) extracting a sample: accurately adding 10mL of organic solvent, and performing vortex oscillation extraction for 5 min;

c. sample purification: centrifuging at 8000rpm for 3min, sucking 2mL of filtrate, drying with nitrogen, re-dissolving with 2.0mL of ultrapure water, adding 20mg of magnetic graphene particles into a centrifuge tube, performing vortex oscillation at 2000r/min for 3min, and separating solid and liquid phases with a magnet; discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and determining phthalate in the sample by gas chromatography-tandem mass spectrometry;

d. preparing an internal standard solution: weighing 10mgd₄Dissolving the DBP internal standard substance in a 10mL volumetric flask by using methanol, and fixing the volume to a scale to prepare a standard stock solution of the internal standard substance. Transferring 1mL of internal standard stock solution into a 100mL volumetric flask, and performing constant volume to a scale by using methanol to obtain an internal standard working solution;

e. preparation of mixed standard working solutions: weighing 13 phthalate standard substances, and preparing a mixed standard working solution with a concentration gradient by using methanol;

f. gas chromatography-tandem mass spectrometry: sucking prepared mixed standard working solution with different concentrations and sample solution to be detected, and respectively injecting the mixed standard working solution and the sample solution to be detected into a gas chromatography-tandem mass spectrometer;

the gas chromatography conditions used were: a chromatographic column: DB-5MS, 30m (length) × 0.25mm (inside diameter) × 0.25 μm (film thickness); temperature rising procedure: the initial temperature is 60 ℃, the temperature is kept for 1min, the speed is 20 ℃/min to 220 ℃, the temperature is kept for 1min, and then the speed is 5 ℃/min to 280 ℃, and the temperature is kept for 15 min; carrier gas: helium (purity is more than or equal to 99.999%) in a constant-current mode; flow rate: 1.0 mL/min; sample inlet temperature: 280 ℃; sample introduction amount: 1 μ L, split-flow sample injection, the split-flow ratio is: 10: 1; .

The mass spectrometry conditions used were: the scanning mode is as follows: scanning positive ions; electron impact ionization source (EI); ionization energy: 70 eV; ion source temperature: 230 ℃; temperature of the quadrupole rods: 150 ℃; solvent retardation: 6min, multiple reactive ion detection mode scan (MRM), MRM parameters are shown in Table 1:

TABLE 1 Mass spectrometric parameters

g. Calculation of phthalate measurement results

And (3) carrying out quantitative analysis by an internal standard method, namely carrying out regression analysis on the corresponding concentration of the phthalic acid ester by comparing the chromatographic peak area of each phthalic acid ester with the peak area of an internal standard to obtain a standard curve, wherein the correlation coefficient is more than or equal to 0.99, measuring the extracted sample, measuring the ratio of the chromatographic peak area of the detected phthalic acid ester to the peak area of the internal standard, substituting the ratio into the standard curve, and obtaining the residual quantity of each phthalic acid ester in the sample.

2. The method of claim 1, wherein: the mixed standard working solution was prepared as follows: respectively and accurately weighing 10mg of 13 phthalic acid esters in the same 100mL volumetric flask, accurately measuring the quantity to 0.1mg, dissolving the phthalic acid esters by using normal hexane, and fixing the volume to prepare a mixed standard stock solution with the concentration of 100 mu g/mL; transferring 1000 mu L of the mixed standard stock solution into a 10mL volumetric flask, and using normal hexane for constant volume, wherein the concentration of the mixed standard solution is 10.0 mu g/mL; transferring 100, 200, 500, 1000, 2000 and 5000 mu L of mixed standard solution into a 10mL volumetric flask, adding 100 mu L of internal standard working solution, diluting with n-hexane to constant volume, and preparing the mixed standard working solution with different concentrations. The concentrations of the phthalic acid esters in the series of mixed standard working solutions are respectively as follows: 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, and 5000 ng/mL.

3. The method of claim 1, wherein: in step f, the MRM parameters are shown in table 1:

TABLE 1 Mass spectrometric parameters

4. The method of claim 1, wherein: when the sample weighed in the step a is the water-based adhesive for cigarettes, the organic solvent in the step b is n-hexane; and c, when the sample weighed in the step a is the tobacco essence, the organic solvent in the step b is ethanol.

Technical Field

The invention belongs to the technical field of physicochemical inspection of harmful substance residues in tobacco essence or water-based adhesive, and mainly relates to a method for measuring 13 phthalic acid esters in tobacco essence or water-based adhesive, in particular to a method for directly measuring by gas chromatography-tandem mass spectrometry after extraction by using an organic solvent and solid-phase extraction and purification by using magnetic graphene.

Background

Phthalates (PAEs) are a class of materials commonly referred to as plasticizers used in industrial products to increase the clarity, durability, elasticity, and longevity of plastic articles, including personal care products, pharmaceuticals, food packaging, nutritional supplements, and the like. It has been found that phthalates are biologically toxic, interfere with the normal regulation of the endocrine system, immunity and nerves and have mutagenic, teratogenic and carcinogenic properties. Essence is involved in Taiwan beverage plasticizer events which are developed in 2011, so that the plasticizer problem of essence is of social concern. The essence is formed by mixing a plurality of spices, the internal components of the essence are complex when the essence is often added into foods such as beverages, jellies, biscuits and the like, and part of the essence consists of oil-soluble components, so that the result has a false positive risk.

The prior literature reports the determination of phthalic acid ester in samples such as milk powder [ military soldiers and the like, chromatography, 2018, 36(8) ], textiles [ Liezarmy and the like, physicochemical inspection-chemical division, 2016, 52(7) ]. The phthalate ester is determined mainly by gas chromatography-mass spectrometry. The matrix of the essence sample is more complex than textiles, dairy products and the like, most of the essence sample is natural extract, the single quadrupole mass spectrum selectivity is poor, the quantitative limit is high, and the detection requirement of phthalate in the essence is difficult to meet. The method adopts a magnetic solid-phase extraction mode to pretreat the essence sample, and uses gas chromatography-triple quadrupole tandem mass spectrometry to determine the essence sample, thereby eliminating matrix interference to the maximum extent, improving the specificity of the method, reducing the risk of false positive, improving the detection sensitivity, and providing reference for realizing the rapid and accurate analysis of 13 phthalic acid esters in the essence.

The invention content is as follows:

the invention aims to provide a method for detecting 13 phthalic acid esters in tobacco essence or water-based adhesive by gas chromatography-tandem mass spectrometry based on the existing conditions. The method overcomes the defects of the prior art, can quickly and accurately detect the residual quantity of phthalic acid ester in tobacco essence or water-based adhesive, has accurate determination result and less interference, can recycle the magnetic solid phase extraction material, reduces the use of organic solvent, and is economic and efficient.

In view of the existing analysis method, the content of phthalate in the tobacco essence or water-based adhesive is low, the essence solvent is generally an organic solvent, the matrix is complex, and the interference is large, so that the magnetic graphene particles are adopted for solid-phase extraction and purification, and then the tandem mass spectrometry with high sensitivity and strong anti-interference capability is adopted for analysis.

The purpose of the invention is realized by the following technical scheme:

a method for detecting phthalic acid ester in tobacco essence or water-based adhesive by combining magnetic solid-phase extraction with gas chromatography tandem mass spectrometry comprises the steps of weighing the tobacco essence, adding an organic solvent, carrying out vortex oscillation, centrifuging, taking 2.0mL of filtrate, drying with nitrogen, redissolving with 2.0mL of ultrapure water, adding 20mg of magnetic graphene particles, carrying out vortex oscillation at 2000r/min for 3min, and separating a solid phase from a liquid phase by using a magnet. Discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and determining phthalate in the essence by gas chromatography-tandem mass spectrometry. The method specifically comprises the following steps:

a. weighing 0.2g of sample (accurate to 0.01g) and placing the sample in a 15mL triangular flask with a plug;

b. and (3) extracting a sample: accurately adding 10mL of ethanol, and performing vortex oscillation extraction for 5 min;

c. sample purification: centrifuging at 8000rpm for 3min, sucking 2mL of filtrate, blowing to dry with nitrogen, redissolving with 2.0mL of ultrapure water, adding 20mg of C18 functionalized magnetic particles into the centrifuge tube, performing vortex oscillation at 2000r/min for 3min, and separating solid and liquid phases with magnet. Discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and measuring phthalate in the essence by gas chromatography-tandem mass spectrometry;

d. preparing an internal standard solution: weighing 10mg (accurate to 0.1mg) of d4-DBP internal standard substance into a 10mL volumetric flask, dissolving with methanol and fixing the volume to the scale to prepare the standard stock solution of the internal standard substance. Transferring 1mL of internal standard stock solution into a 100mL volumetric flask, and performing constant volume to a scale by using methanol to obtain an internal standard working solution;

e. preparation of mixed standard working solutions: weighing 13 phthalate standard substances, and preparing a mixed standard working solution with a concentration gradient by using methanol;

f. gas chromatography-tandem mass spectrometry: sucking prepared mixed standard working solution with different concentrations and sample solution to be detected, and respectively injecting the mixed standard working solution and the sample solution to be detected into a gas chromatography-tandem mass spectrometer;

g. calculation of phthalate measurement results

And (3) carrying out quantitative analysis by an internal standard method, namely carrying out regression analysis on the corresponding concentration of each phthalate according to the chromatographic peak area of each phthalate to obtain a standard curve, wherein the correlation coefficient is more than or equal to 0.99, measuring the extracted sample to obtain the chromatographic peak area of the detected phthalate, substituting the chromatographic peak area into the standard curve to obtain the residual quantity of each phthalate in the sample.

The standard working solution in the present invention is formulated as follows: respectively and accurately weighing 10mg of 13 phthalic acid esters in the same 100mL volumetric flask, accurately measuring the quantity to 0.1mg, dissolving the phthalic acid esters by using methanol, fixing the volume and preparing a mixed standard stock solution with the concentration of 100 mu g/mL; transferring 1000 mu L of mixed standard stock solution into a 10mL volumetric flask, and performing constant volume by using methanol, wherein the concentration of the mixed standard solution is 10.0 mu g/mL; transferring 100, 200, 500, 1000, 2000 and 5000 mu L of mixed standard solution into a 10mL volumetric flask, adding 100 mu L of internal standard working solution, diluting with methanol to constant volume, and preparing the mixed standard working solution with different concentrations. The concentrations of the phthalic acid esters in the series of mixed standard working solutions are respectively as follows: 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL and 5000 ng/mL;

the gas chromatography conditions used were: a chromatographic column: DB-5MS, 30m (length) × 0.25mm (inside diameter) × 0.25 μm (film thickness); temperature rising procedure: the initial temperature is 60 ℃, the temperature is kept for 1min, the speed is 20 ℃/min to 220 ℃, the temperature is kept for 1min, and then the speed is 5 ℃/min to 280 ℃, and the temperature is kept for 15 min; carrier gas: helium (purity is more than or equal to 99.999%) in a constant-current mode; flow rate: 1.0 mL/min; sample inlet temperature: 280 ℃; sample introduction amount: 1 μ L, split-flow injection (split ratio: 10: 1).

The mass spectrometry conditions used were: the scanning mode is as follows: scanning positive ions; electron impact ionization source (EI); ionization energy: 70 eV; ion source temperature: 230 ℃; temperature of the quadrupole rods: 150 ℃; solvent retardation: 6 min; multiple reactive ion detection mode scanning (MRM); the qualitative ion and quantitative ion pairs for each phthalate are shown in table 1.

TABLE 1 Mass spectrometric parameters

When the sample weighed in the step a is the water-based adhesive for cigarettes, the organic solvent in the step b is n-hexane; and c, when the sample weighed in the step a is the tobacco essence, the organic solvent in the step b is ethanol.

The method overcomes the defects of the sample treatment method in the prior art, and optimizes the sample pretreatment method and the instrument detection conditions for the tobacco essence or water-based adhesive sample. Compared with the prior art, the method has the following excellent effects:

(1) the method utilizes GC-MS/MS to determine the content of phthalic acid ester in the tobacco essence or water-based adhesive, has strong matrix interference resistance and simple pretreatment method.

(2) The extraction solvent used in the invention is ethanol, which has no pollution to the environment and is green and environment-friendly.

(3) According to the invention, magnetic graphene is adopted for purification, so that impurities such as pigment in a sample matrix can be effectively removed, and the sample can be effectively purified. And compared with solid phase extraction, the magnetic solid phase extraction has simple and convenient operation and can be recycled.

(4) The method has the advantages of accurate operation, high sensitivity and good repeatability.

① detection limit of the method of the invention:

mixed standard working solutions of different concentrations were injected into GC-MS/MS and the limit of detection (LOD) was calculated as a 3-fold signal-to-noise ratio (S/N-3).

② repeatability and recovery from spiking of the process of the invention:

a standard solution of phthalate was added to a blank sample, and then pretreatment and GC-MS/MS analysis were performed, respectively, and the recovery thereof was calculated according to the addition amount of the scalar and the measured value.

The linear range, recovery, relative standard deviation and detection limit of the phthalate are shown in tables 2 and 3.

TABLE 2 Linear coefficient, recovery, relative standard deviation, detection limit (essence for tobacco)

TABLE 3 Linear coefficient, recovery, relative standard deviation, detection limit for phthalate (in water-based glue for cigarettes)

Drawings

FIG. 1 is a flow chart of the measurement method of the present invention.

FIG. 2 is a standard solution selective ion chromatogram.

Detailed Description

The invention is further described below with reference to examples, but without limiting the invention.

FIG. 1 is a flow chart of the measurement method of the present invention.

FIG. 2 is a standard solution selective ion chromatogram.

Example 1:

1. instruments and reagents:

the 13 kinds of phthalate and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.

Agilent gas chromatography-tandem mass spectrometer; mettler AE 163 Switzerland electronic balance (sensory: 0.0001 g).

2. Sample treatment:

0.2g of a sample of tobacco flavor (to the nearest 0.01g) was weighed out and placed in a 15mL triangular flask with a stopper. Accurately adding 10mL of ethanol, and performing vortex oscillation extraction for 5 min; centrifuging at 8000rpm for 3min, sucking 2mL of filtrate, drying with nitrogen, re-dissolving with 2.0mL of ultrapure water, adding 20mg of magnetic graphene particles into a centrifuge tube, performing vortex oscillation at 2000r/min for 3min, and separating solid and liquid phases with a magnet. Discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and measuring phthalate in the essence by gas chromatography-tandem mass spectrometry;

3. preparation of standard working solutions: the preparation method is shown in the summary part of the invention and is not repeated here.

4. GC-MS/MS measurement.

5. And (3) calculation of measurement results: and (3) carrying out quantitative analysis by an internal standard method, namely carrying out regression analysis on the corresponding concentration of each phthalate chromatographic peak area by comparing the peak area of each phthalate with the peak area of an internal standard to obtain a standard curve, wherein the correlation coefficient is more than or equal to 0.99, measuring the extracted sample to obtain the peak area ratio of the detected phthalate chromatographic peak area to the peak area of the internal standard, substituting the peak area ratio into the standard curve to obtain the residual quantity of the diisobutyl phthalate (DIBP) in the sample, namely 20.85 mg/kg.

For judging the accuracy of the method, a standard solution of DIBP is added into the sample, so that the theoretical content of the DIBP in the sample is 50.0mg/kg, the sample pretreatment is carried out, the peak area of a selective ion of the DIBP is measured by GC-MS/MS and is substituted into a standard curve, and the content of the DIBP in the sample at the moment is 48.82mg/kg, namely the standard recovery rate of the DIBP is 95.6 percent, which indicates that the method is accurate.

Example 2:

another sample of tobacco flavor was selected as described in example 1, and the 13 phthalates described above were not detected.

Example 3:

1. instruments and reagents:

the 13 kinds of phthalate and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.

Agilent gas chromatography-tandem mass spectrometer; mettler AE 163 Switzerland electronic balance (sensory: 0.0001 g).

2. Sample treatment:

a0.2 g sample of the water-based adhesive for cigarettes (to the nearest 0.01g) was weighed out and placed in a 15mL triangular flask with a stopper. Accurately adding 10mL of ethanol, and performing vortex oscillation extraction for 5 min; centrifuging at 8000rpm for 3min, sucking 2mL of filtrate, drying with nitrogen, re-dissolving with 2.0mL of ultrapure water, adding 20mg of magnetic graphene particles into a centrifuge tube, performing vortex oscillation at 2000r/min for 3min, and separating solid and liquid phases with a magnet. Discarding the water layer, adding 1mL of methanol, performing ultrasonic treatment for 2min, performing magnetic separation, transferring the supernatant into a chromatographic bottle, repeatedly eluting for 2 times, combining and collecting the eluates, and measuring phthalate in the water-based adhesive by gas chromatography-tandem mass spectrometry;

3. preparation of standard working solutions: the preparation method is shown in the summary part of the invention and is not repeated here.

4. GC-MS/MS measurement.

5. And (3) calculation of measurement results: and (3) carrying out quantitative analysis by an internal standard method, namely carrying out regression analysis on the corresponding concentration of the phthalate by comparing the chromatographic peak area of each phthalate with the peak area of an internal standard to obtain a standard curve, wherein the correlation coefficient is more than or equal to 0.99, measuring the extracted sample, measuring the ratio of the chromatographic peak area of the detected phthalate to the peak area of the internal standard, substituting the ratio into the standard curve, and obtaining that the residual quantity of the di (2-ethyl) hexyl phthalate (DEHP) in the sample is 10.24 mg/kg.

For judging the accuracy of the method, a DEHP standard solution is added into the sample, so that the theoretical content of the DEHP in the sample is 20.0mg/kg, the sample pretreatment is carried out, the peak area of a selective ion of the DEHP is measured by GC-MS/MS and is substituted into a standard curve, and the content of the DEHP in the sample at the moment is 19.44mg/kg, namely the standard recovery rate of the DEHP is 94.3%, which indicates that the method is accurate.

Example 4:

another sample of water-based glue for cigarettes was selected as described in example 1, and the above 13 phthalates were not detected.