Method for simultaneously detecting 27 bisphenol compounds in dairy products

Abstract

The invention discloses a method for simultaneously detecting 27 bisphenol compounds in a dairy product, an ultra performance convergence chromatograph is adopted for detection, ACQUITY UPC2HSS Torus 2-Pic chromatographic column is adopted for separation, an EMR-Lipid technology is adopted for purification, the 27 bisphenol compounds in the dairy product can be qualitatively and quantitatively detected at the same time, and the method has the advantages of being high in flux, high in speed and good in recovery and has a very good application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of food detection, and in particular to a method for simultaneously detecting 27 bisphenol compounds in dairy products. Background Art

[0002] Bisphenols (BPs) are a class of substances with two hydroxyphenyl structures. These compounds have stable chemical properties, high temperature resistance, good ductility, and low production costs. They are important raw materials for the production of various polymer materials such as polycarbonate and are widely used in the industrial production of various dairy packaging materials such as milk powder bags, yogurt bottles, and milk bottles. However, these compounds have endocrine disrupting effects on the human body, affecting reproductive and developmental functions, and have toxic effects such as teratogenicity, carcinogenicity, and mutagenicity. They are also easy to enrich and difficult to degrade, posing potential hazards to human health and the ecological environment.

[0003] At present, the main methods for detecting bisphenol compounds include liquid chromatography (LC), liquid chromatography-tandem mass spectrometry (LC-MS) and gas chromatography-tandem mass spectrometry (GC-MS). Since most bisphenol compounds have high thermal stability and are not easy to volatilize, they are not suitable for direct detection by GC or GC-MS. Derivatization treatment is usually required before analysis. Such methods are cumbersome and time-consuming, and are prone to loss of target substances. LC-MS has high sensitivity, good selectivity, and strong anti-interference ability. It is currently the most commonly used detection method for bisphenol substances. However, due to the wide variety of bisphenol compounds and their similar structures, the existing analytical methods still face many challenges, mainly including: (1) the sample pretreatment method is complicated and the extraction efficiency is low; (2) the separation effect of structurally similar substances is poor, and it is impossible to achieve high-throughput analysis of multiple bisphenol compounds; in addition, the detection time is long (usually more than 10 minutes), and the analysis cost is high; (3) the solvent consumption is large, which does not meet the development requirements of green and environmentally friendly detection methods. Therefore, it is very necessary to establish a fast, efficient, and green high-throughput analysis method for the detection of bisphenol compounds. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a method for simultaneously detecting 27 bisphenol compounds in dairy products, so as to solve the problems of large matrix interference, poor recovery rate and low sensitivity in the prior art, thereby improving the detection efficiency of bisphenol compounds in dairy products.

[0005] In order to solve the above technical problems, this application adopts the following technical solutions:

[0006] A method for simultaneously detecting 27 bisphenol compounds in dairy products, the specific steps are as follows:

[0007] Step 1: 27 bisphenol compounds are prepared into standard solutions of different concentrations, and then the standard solutions are gradient eluted using an ultra-high performance convergence chromatograph to obtain chromatograms and standard curves of the standard solutions at various concentrations;

[0008] Among them, the 27 bisphenol compounds are bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), 4-cinnamphenol (4-CP), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), bisphenol A diphenyl phosphate (BDP), bisphenol F glycidyl (2-chloro-1-propanol) ether (BFDGE-HCl), bisphenol A (2,3-dihydroxypropyl) glyceryl ether (BADGE-H2O), bisphenol F di(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), bisphenol A di(3-chloro-2-hydroxypropyl) ether (BADGE-2HCl), bisphenol A F (BPAF), tetrachlorobisphenol A (TCBPA), bisphenol C (BPC), bisphenol M (BPM), bisphenol TMC (BPTMC), bisphenol B (BPB), bisphenol A (BPA), bisphenol A (3-chloro-2-hydroxypropyl) (EPI-001), bisphenol E (BPE), tetrabromobisphenol A (TBBPA), bisphenol F (BPF), bisphenol PH (BPPH), bisphenol Z (BPZ), 4,4'-(1-methylethylidene) bis(2-methylphenol) (BPC2), bisphenol T (BPT), bisphenol S (BPS), bisphenol BP (BPBP), bisphenol A di(2,3-dihydroxypropyl) ether (BADGE-2H2O);

[0009] Step 2: Dissolve 0.5-2 g of the sample to be tested in 10 mL of acetonitrile, add a dehydrating agent and a salting-out agent, vortex and centrifuge, concentrate the supernatant to near dryness under nitrogen blowing at 40°C, re-dissolve with 1 mL of methanol, and then filter with a polytetrafluoroethylene filter membrane. The filtrate is used for standby use;

[0010] Step 3: Use an ultra-high performance convergence chromatograph to perform gradient elution on the filtrate obtained in step 2 to obtain a chromatogram of the sample solution to be tested, compare the retention time of each chromatographic peak in the chromatogram of the sample solution to be tested with that of each chromatographic peak in the chromatogram of the standard solution, perform qualitative analysis on the bisphenol compounds in the sample to be tested, and substitute the peak area of ​​each chromatographic peak into the standard curve obtained in step 1 to calculate the content of bisphenol compounds in the sample.

[0011] Preferably, in step 1, 27 bisphenol compound standards are taken respectively, dissolved and fixed to volume with methanol, and prepared into a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, standard solutions of different concentrations are prepared with organic solvents, and the organic solvent is one or more of methanol, n-hexane, tetrahydrofuran and ethyl acetate. Wherein, the concentration range of the standard solution is 50-1000 μg / L, and such a concentration range ensures that the peak area is within a relatively reasonable range, thereby ensuring a better detection range.

[0012] Preferably, in step 2, the acetonitrile contains 0.1% formic acid calculated by mass percentage; the amount of water removal agent added is 4 g, and the amount of salting-out agent added is 1 g; the specific steps of centrifugal concentration after vortex oscillation are:

[0013] After adding the dehydrating agent and salting-out agent, vortex and oscillate for 2 minutes, transfer the supernatant to the EMR-Lipid purification tube, vortex and oscillate for 2 minutes, centrifuge at 5000r / min for 5 minutes, concentrate the supernatant to near dryness under nitrogen blowing, re-dissolve with methanol, and then filter with a membrane.

[0014] Preferably, the dehydrating agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride.

[0015] Preferably, the salting-out agent is one or more of sodium chloride, sodium sulfate, and sodium dihydrogen phosphate.

[0016] Preferably, the test conditions of the ultra-high performance convergence chromatograph are:

[0017] The chromatographic column is ACQUITY UPC, an achiral column. 2 HSS Torus 2-Pic (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS Torus DEA (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS Torus Diol (150mm×3mm, 1.7μm) and ACQUITY UPC 2 One of the HSS Torus 1-AA (150 mm × 3 mm, 1.7 μm);

[0018] Mobile phase A is supercritical CO2, and mobile phase B is an organic modifier;

[0019] Column temperature is 20-50°C;

[0020] Flow rate: 1.0-1.5 mL / min;

[0021] System back pressure is 1700-2100 psi;

[0022] The detection wavelength is 200-300nm;

[0023] The injection volume is 1 to 10 μL.

[0024] Preferably, the organic modifier is one or more of methanol, ethanol, isopropanol and acetonitrile.

[0025] Preferably, the elution gradient program is: from 0 to 1.5 minutes, the volume fraction of mobile phase B increases from 4% to 14%; then maintain for 2 minutes; from 3.5 to 4 minutes, the volume fraction of mobile phase B increases from 14% to 16%; from 4 to 5 minutes, the volume fraction of mobile phase B increases from 16% to 20% and then maintain for 0.5 minutes; from 5.5 to 6.5 minutes, the volume fraction of mobile phase B increases from 20% to 25%; finally, after 0.5 minutes, the volume fraction of mobile phase B decreases from 25% to 4%. Separation under this elution gradient program has better separation effect and higher sensitivity.

[0026] Preferably, the sample to be tested is a dairy product, which refers to milk, yogurt, milk powder, etc., and various foods and beverages made by processing cow's milk, sheep's milk, goat's milk or milk of other mammals.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] 1. The method described in this application is the first to use ACQUITY UPC 2 The Torus 2-Pic column is used for the separation and detection of bisphenol compounds. It can quickly separate 27 bisphenol compounds simultaneously within 6.5 minutes.

[0029] 2. The method described in the present application uses supercritical CO2 as the mobile phase and a small amount of organic solvent as a modifier, which expands the limitations of gas chromatography and makes it unaffected by the boiling point and volatility of the substance. It has a wider selection range for the target, a higher diffusion rate, and a faster analysis speed. Compared with conventional liquid chromatography, it greatly reduces the use of organic solvents, greatly reduces costs and toxicity, is economical and environmentally friendly, and has good market promotion prospects.

[0030] 3. The method described in this application adopts EMR-Lipid technology for purification, which has a strong adsorption effect on long-chain lipid substances in dairy products. This pretreatment method has the advantages of fast adsorption speed and good purification effect, and can significantly improve the recovery rate of the target substance.

[0031] 4. The method described in the present application can perform high-throughput separation and detection of 27 bisphenol compounds by precisely adjusting and optimizing the chromatographic column, mobile phase, system back pressure, and column temperature, while gradiently changing the ratio of carbon dioxide to organic solvent in the elution gradient, and has the advantages of high throughput, fast speed, and good recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the standard chromatogram for detecting 27 bisphenol compounds in Example 1.

[0033] Figure 2 The chromatograms obtained after separation with different chromatographic columns in Example 3: where a is ACQUITY UPC 2 HSSTorus Diol (150mm×3mm, 1.7μm); b is ACQUITY UPC 2 HSS Torus 2-Pic (150 mm × 3 mm, 1.7 μm); c is ACQUITY UPC 2 HSS Torus DEA (150 mm × 3 mm, 1.7 μm); d is ACQUITY UPC 2 HSS Torus1-AA (150mm×3mm, 1.7μm).

[0034] Figure 3 It is the result diagram of the recovery rate under different purification conditions of Example 4.

[0035] In the figure: 1 is bisphenol A diglycidyl ether (BADGE), 2 is bisphenol F diglycidyl ether (BFDGE), 3 is 4-cinnamphenol (4-CP), 4 is bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), 5 is bisphenol A diphenyl phosphate (BDP), 6 is bisphenol F glycidyl (2-chloro-1-propanol) ether (BFDGE-HCl), 7 is bisphenol A (2,3-dihydroxypropyl) glyceryl ether (BADGE-H2O), 8 is bisphenol F di(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), 9 is bisphenol A di(3-chloro-2-hydroxypropyl) ether (BADGE-2HCl), 10 is bisphenol AF (BPAF), and 11 is tetrachlorobisphenol A (TCBPA) , 12 is bisphenol C (BPC), 13 is bisphenol M (BPM), 14 is bisphenol TMC (BPTMC), 15 is bisphenol B (BPB), 16 is bisphenol A (BPA), 17 is bisphenol A (3-chloro-2-hydroxypropyl) (EPI-001), 18 is bisphenol E (BPE), 19 is tetrabromobisphenol A (TBBPA), 20 is bisphenol F (BPF), 21 is bisphenol PH (BPPH), 22 is bisphenol Z (BPZ), 23 is 4,4'-(1-methylethylidene) bis(2-methylphenol) (BPC2), 24 is bisphenol T (BPT), 25 is bisphenol S (BPS), 26 is bisphenol BP (BPBP), and 27 is bisphenol A di(2,3-dihydroxypropyl) ether (BADGE-2H2O). DETAILED DESCRIPTION

[0036] This application will combine the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on this application belong to the scope of protection of this application.

[0037] Unless otherwise indicated in specific cases in this application, the numerical ranges listed herein include the upper and lower limits, as well as all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0038] 1. A method for simultaneous detection of 27 bisphenol compounds in dairy products

[0039] Step 1: 27 bisphenol compounds are prepared into standard solutions of different concentrations, and then the standard solutions are gradient eluted using an ultra-high performance convergence chromatograph to obtain chromatograms and standard curves of the standard solutions at various concentrations;

[0040] Among them, the 27 bisphenol compounds are bisphenol A diglycidyl ether, bisphenol F diglycidyl ether, 4-cinnamic phenol, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A diphenyl phosphate, bisphenol F glycidyl (2-chloro-1-propanol) ether, bisphenol A (2,3-dihydroxypropyl) glyceryl ether, bisphenol F di(3-chloro-2-hydroxypropyl) ether, bisphenol A di(3- Bisphenol A (2-chloro-2-hydroxypropyl) ether, bisphenol AF, tetrachlorobisphenol A, bisphenol C, bisphenol M, bisphenol TMC, bisphenol B, bisphenol A, bisphenol A (3-chloro-2-hydroxypropyl), bisphenol E, tetrabromobisphenol A, bisphenol F, bisphenol PH, bisphenol Z, 4,4'-(1-methylethylidene) bis(2-methylphenol), bisphenol T, bisphenol S, bisphenol BP, bisphenol A di(2,3-dihydroxypropyl) ether;

[0041] Step 2: Dissolve 0.5-2 g of the sample to be tested in 10 mL of acetonitrile, add a dehydrating agent and a salting-out agent, vortex and centrifuge, concentrate the supernatant to near dryness under nitrogen blowing at 40°C, re-dissolve with 1 mL of methanol, and then filter with a polytetrafluoroethylene filter membrane. The filtrate is used for standby use;

[0042] Step 3: Prepare a matrix series standard solution of 27 bisphenol compounds using a blank sample solution; then, use an ultra-high performance convergence chromatograph to perform gradient elution on the matrix series standard solution to obtain a chromatogram and a standard curve of the matrix series standard solution at each concentration; wherein the blank sample solution is a sample without the target substance that is processed in step 2.

[0043] Step 4: Use an ultra-high performance convergence chromatograph to perform gradient elution on the filtrate obtained in step 2 to obtain a chromatogram of the sample solution to be tested, compare the retention time of each chromatographic peak in the chromatogram of the sample solution to be tested with that of each chromatographic peak in the chromatogram of the matrix series standard solution, perform qualitative analysis on the bisphenol compounds in the sample to be tested, and substitute the peak area of ​​each chromatographic peak into the standard curve obtained in step 3 to calculate the content of bisphenol compounds in the sample.

[0044] In some embodiments of the present application, the sample to be tested is a dairy product, which refers to milk, yogurt, milk powder, etc., specifically various foods and beverages made by processing cow's milk, sheep's milk, goat's milk or milk of other mammals.

[0045] In some embodiments of the present application, in step 1, 27 bisphenol compound standards are taken respectively, dissolved and fixed to volume with methanol, and prepared into a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, standard solutions of different concentrations are prepared with organic solvents, and the organic solvent is one or more of methanol, n-hexane, tetrahydrofuran and ethyl acetate. Among them, the concentration range of the standard solution is 50 to 1000 μg / L, and such a concentration range ensures that the peak area is within a relatively reasonable range, thereby ensuring a better detection range.

[0046] In some embodiments of the present application, the processing method of the sample to be tested is adjusted, and it is found that other purification methods cannot achieve efficient extraction of 27 bisphenol compounds at the same time. Some purification methods only have a certain extraction effect on several bisphenol compounds, but the extraction effect on other types of bisphenol compounds is not good, such as Figure 3 Therefore, in step 2 of the method described in the present application, the acetonitrile contains 0.1% formic acid according to the mass percentage; the amount of dehydrating agent added is 4g, and the amount of salting-out agent added is 1g; the specific steps of centrifugal concentration after vortex oscillation are:

[0047] After adding the dehydrating agent and the salting-out agent, vortex and oscillate for 2 minutes, take the supernatant and transfer it to the EMR-Lipid purification tube, vortex and oscillate for 2 minutes, centrifuge at 5000r / min for 5 minutes, concentrate the supernatant to near dryness under nitrogen blowing at 40°C, re-dissolve with 1mL of methanol, and then filter through a membrane. The dehydrating agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride. The salting-out agent is one or more of sodium chloride, sodium sulfate and sodium dihydrogen phosphate.

[0048] In some embodiments of the present application, the present application found that since there are many types of bisphenol compounds, and their structures and polarities are very similar, there is a problem of difficulty in separation. After using four different chromatographic columns to separate them, completely different separation effects appear, and the obtained chromatograms also have very significant differences. Therefore, in the present application, the test conditions of the ultra-high performance convergence chromatograph are:

[0049] The chromatographic column is ACQUITY UPC, an achiral column. 2 HSS Torus 2-Pic (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS Torus DEA (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS Torus Diol (150mm×3mm, 1.7μm) and ACQUITY UPC 2HSS Torus 1-AA (150 mm × 3 mm, 1.7 μm); more preferably ACQUITY UPC 2 HSS Torus 2-Pic (150mm×3mm, 1.7μm);

[0050] The mobile phase A is supercritical CO2, and the mobile phase B is an organic modifier; wherein the organic modifier is one or more of methanol, ethanol, isopropanol and acetonitrile;

[0051] Column temperature is 20-50°C;

[0052] Flow rate: 1.0-1.5 mL / min;

[0053] The system back pressure is 1700-2100 psi; the detection wavelength is 200-300 nm; and the injection volume is 1-10 μL.

[0054] In some embodiments of the present application, the elution gradient program is: from 0 to 1.5 minutes, the volume fraction of mobile phase B increases from 4% to 14%; then maintain for 2 minutes; from 3.5 to 4 minutes, the volume fraction of mobile phase B increases from 14% to 16%; from 4 to 5 minutes, the volume fraction of mobile phase B increases from 16% to 20% and then maintain for 0.5 minutes; from 5.5 to 6.5 minutes, the volume fraction of mobile phase B increases from 20% to 25%; finally, after 0.5 minutes, the volume fraction of mobile phase B decreases from 25% to 4%. Separation under the conditions of the elution gradient described in the present application can obtain better separation effect and higher sensitivity.

[0055] 2. Examples and Comparative Examples

[0056] Example 1: High-throughput method establishment

[0057] Step 1: Accurately weigh 25.0 mg of each of the 27 bisphenol compound standards into a 25 mL volumetric flask, dissolve them in methanol, and make up to volume with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, use organic solvents to prepare a series of standard solutions of different concentrations;

[0058] Step 2: Use ultra-high performance convergence chromatography to perform gradient elution on the standard solution prepared in step 1, where the chromatographic column is ACQUITY UPC 2Torus 2-Pic chromatographic column; mobile phase A is supercritical CO2, mobile phase B is methanol; column temperature is 35°C; flow rate is 1.5mL / min; system back pressure is 1800psi; detection wavelength is 220nm; injection volume is 1μL; elution gradient program is: from 0 to 1.5min, the volume fraction of mobile phase B increases from 4% to 14%; then maintain for 2min; from 3.5 to 4min, the volume fraction of mobile phase B increases from 14% to 16%; from 4 to 5min, the volume fraction of mobile phase B increases from 16% to 20%, and then maintain for 0.5min; from 5.5 to 6.5min, the volume fraction of mobile phase B increases from 20% to 25%; finally, after 0.5min, the volume fraction of mobile phase B decreases from 25% to 4%. The chromatogram of the standard solution obtained after the above gradient elution is as follows Figure 1 As shown. Figure 1 It can be seen that the method of the present application can achieve rapid detection of 27 bisphenol compounds within 6.5 minutes. Compared with the detection speed of conventional gas phase and liquid phase methods, it has higher throughput and faster speed, which greatly improves the analysis efficiency.

[0059] Example 2: Method Evaluation

[0060] Step 1: Weigh 2 g (accurate to 0.0001 g) of negative milk sample into a 25 mL centrifuge tube, add 10 mL of acetonitrile (containing 0.1% formic acid), 4 g of dehydrating agent, 1 g of salting-out agent, vortex for 2 min, transfer the supernatant to the EMR-Lipid purification tube, vortex for 2 min, centrifuge at 5000 r / min for 5 min, concentrate the supernatant to near dryness under nitrogen blowing at 40 ° C, re-dissolve with 1 mL of methanol, filter with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, and the filtrate is the blank sample solution for UPCC test;

[0061] Step 2: Accurately weigh 25.0 mg of each of the 27 bisphenol compound standards into a 25 mL volumetric flask, dissolve them in methanol, and make up to volume with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, dilute with the blank sample solution in step 1 and prepare a series of matrix standard solutions of different concentrations;

[0062] Step 3: Use ultra-high performance convergence chromatography to perform gradient elution on the matrix series standard solution prepared in step 2 to obtain the chromatogram of the matrix series standard solution at various concentrations, and plot the peak area as the ordinate y and the concentration as the abscissa x to obtain the matrix series standard curve. The test conditions of the ultra-high performance convergence chromatography are: the chromatographic column is ACQUITY UPC 2Torus2-Pic chromatographic column; mobile phase A is supercritical CO2, mobile phase B is methanol; column temperature is 35°C; flow rate is 1.5mL / min; system back pressure is 1800psi; detection wavelength is 220nm; injection volume is 1μL.

[0063] Step 4: Precision and recovery test: Add three concentrations of 27 bisphenol compounds, high (500.0 μg / kg), medium (75.0 μg / kg), and low (25 μg / kg), into the blank sample solution to perform spike recovery experiments. Repeat the test six times at each concentration level.

[0064] The test results (Table 1) show that within their respective linear ranges, the linear correlation coefficients of the 27 bisphenol compounds are all higher than 0.9980, the detection limit of each target substance is 3-5 μg / kg, the quantification limit is 10-25 μg / kg, the spiked recovery rate is 85.5%-114.8%, and the precision is 0.7-6.0%, indicating that the method described in this application can meet the detection requirements of actual samples.

[0065] Table 1 Standard curve equations and related parameters of 27 bisphenol compound standard solutions

[0066]

[0067]

[0068] Example 3: Effect of chromatographic column type on separation performance

[0069] Step 1: Accurately weigh 25.0 mg of each of the 27 bisphenol compound standards into a 25 mL volumetric flask, dissolve them in methanol, and make up to volume with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, use organic solvents to prepare a series of standard solutions of different concentrations;

[0070] Step 2: Weigh 2 g (accurate to 0.0001 g) of sample into a 25 mL centrifuge tube, add 10 mL of acetonitrile (containing 0.1% formic acid), add 4 g of dehydrating agent, 1 g of salting-out agent, vortex oscillate for 2 min, transfer the supernatant to the EMR-Lipid purification tube, vortex oscillate for 2 min, centrifuge at 5000 r / min for 5 min, concentrate the supernatant to near dryness under nitrogen blowing at 40 ° C, re-dissolve with 1 mL of methanol, and then filter with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane. The filtrate is used for UPCC testing;

[0071] Step 3: Use ultra-high performance convergence chromatography to perform gradient elution on the sample solution prepared in step 2, and use different types of chromatographic columns for separation. The chromatographic column models are ACQUITY UPC 2HSS Torus 2-Pic (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS Torus DEA (150mm×3mm, 1.7μm), ACQUITY UPC 2 HSS TorusDiol (150mm×3mm, 1.7μm) and ACQUITY UPC 2 HSS Torus 1-AA (150mm×3mm, 1.7μm); mobile phase A is supercritical CO2, mobile phase B is methanol; column temperature is 35℃; flow rate is 1.5mL / min; system back pressure is 1800psi; detection wavelength is 220nm; injection volume is 1μL; elution gradient program is: from 0 to 1.5min, the volume fraction of mobile phase B increases from 4% to 14%; then maintain for 2min; from 3.5 to 4min, the volume fraction of mobile phase B increases from 14% to 16%; from 4 to 5min, the volume fraction of mobile phase B increases from 16% to 20%, and then maintain for 0.5min; from 5.5 to 6.5min, the volume fraction of mobile phase B increases from 20% to 25%; finally, after 0.5min, the volume fraction of mobile phase B decreases from 25% to 4%. The chromatograms obtained by separation of different chromatographic columns under the above conditions are as follows Figure 2 shown.

[0072] Since bisphenol compounds are of various types, similar in structure and polarity, they are difficult to separate. Figure 2 It can be seen that in ACQUITY UPC 2 HSS Torus DEA, ACQUITY UPC 2 HSS Torus Diol and ACQUITY UPC 2 On the HSS Torus 1-AA column, the 27 bisphenol compounds could not be completely separated by baseline, and the chromatographic peaks showed stacking, tailing, and broadening. However, the ACQUITY UPC 2 When the HSS Torus 2-Pic column was used for separation, the overall peak shape of the chromatographic peak was sharp and symmetrical, and the 27 target substances could be completely separated within 6.5 minutes. The experiment proved that after the optimization experiment of the chromatographic column, the method of this application established a rapid high-throughput method suitable for the qualitative and quantitative analysis of 27 bisphenols in dairy products.

[0073] Example 4: Effect of purification conditions on recovery rate

[0074] Step 1: Accurately weigh 25.0 mg of each of 27 bisphenol compound standards into a 25 mL volumetric flask, dissolve them in methanol, and make up to volume with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, use the blank sample solution in step 1 of Example 2 to prepare a series of matrix standard solutions of different concentrations;

[0075] Step 2: Use ultra-high performance convergence chromatography to perform gradient elution on the matrix series standard solution prepared in step 1 to obtain the chromatogram of the matrix series standard solution at various concentrations, and plot with the peak area as the ordinate y and the concentration as the abscissa x to obtain the matrix series standard curve.

[0076] Step 3: Preparation of sample solution: Weigh 2 g (accurate to 0.0001 g) of milk sample into a 25 mL centrifuge tube, add 10 mL of acetonitrile (containing 0.1% formic acid), 4 g of dehydrating agent, 1 g of salting-out agent, vortex for 2 min, and transfer the supernatant to EMR-Lipid, Osia-HLB, Al2O3, C 18 In the purification tube, each purified solution was vortexed for 2 min and centrifuged at 5000 r / min for 5 min. The supernatant was concentrated to near dryness under nitrogen blowing at 40 °C, re-dissolved with 1 mL of methanol, and then filtered with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane. The filtrate was used for UPCC testing.

[0077] Step 4: Sample detection: Use ultra-high performance convergence chromatography to perform gradient elution on the sample solution treated with different purification materials prepared in step 3 to obtain a chromatogram of the sample solution. Compare the retention time of each chromatographic peak in the chromatogram of the sample solution with that of the chromatographic peak of the standard solution, perform qualitative analysis on the bisphenol compounds in the sample, and bring the peak area of ​​each chromatographic peak into the standard curve obtained in step 3 to calculate the content of bisphenol compounds in the sample. 2 HSS Torus 2-Pic (150mm×3mm, 1.7μm) column was used for separation; mobile phase A was supercritical CO2, and mobile phase B was methanol; column temperature was 35°C; flow rate was 1.5mL / min; system back pressure was 1800psi; detection wavelength was 220nm; injection volume was 1μL; elution gradient program was as follows: from 0 to 1.5min, the volume fraction of mobile phase B increased from 4% to 14%; then maintained for 2min; from 3.5 to 4min, the volume fraction of mobile phase B increased from 14% to 16%; from 4 to 5min, the volume fraction of mobile phase B increased from 16% to 20%, and then maintained for 0.5min; from 5.5 to 6.5min, the volume fraction of mobile phase B increased from 20% to 25%; finally, after 0.5min, the volume fraction of mobile phase B decreased from 25% to 4%.

[0078] The recovery rates under different purification conditions were obtained under the above conditions. Figure 3 As shown in the experimental results, Osia-HLB, Al2O3 and C 18 The three purification methods cannot achieve efficient extraction of 27 bisphenol compounds at the same time. The recovery rate of 27 bisphenol compounds after purification by EMR-Lipid purification technology can reach more than 90%. Experiments have proved that the method of the application has the significant advantages of simple operation, good purification effect and high recovery rate.

[0079] Example 5: Actual sample testing

[0080] 30 dairy products (including 10 each of milk, yogurt and milk powder) were randomly purchased from a local supermarket and tested for bisphenol residues under the above optimal conditions. The experimental steps are as follows:

[0081] Step 1: Accurately weigh 25.0 mg of each of the 27 bisphenol compound standards into a 25 mL volumetric flask, dissolve them in methanol, and make up to volume with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, use organic solvents to prepare a series of standard solutions of different concentrations;

[0082] Step 2: Weigh 2 g (accurate to 0.0001 g) of sample into a 25 mL centrifuge tube, add 10 mL of acetonitrile (containing 0.1% formic acid), add 4 g of dehydrating agent, 1 g of salting-out agent, vortex oscillate for 2 min, transfer the supernatant to the EMR-Lipid purification tube, vortex oscillate for 2 min, centrifuge at 5000 r / min for 5 min, concentrate the supernatant to near dryness under nitrogen blowing at 40 ° C, re-dissolve with 1 mL of methanol, and then filter with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane. The filtrate is used for UPCC testing;

[0083] Step 3: Based on the standard stock solution prepared in step 1, the blank sample solution in step 1 of Example 2 is diluted and prepared into a series of matrix standard solutions of different concentrations;

[0084] Step 4: Sample detection: Use ultra-high performance convergence chromatography to perform gradient elution on the sample solution prepared in step 2 to obtain a chromatogram of the sample solution, compare the retention time of each chromatographic peak in the chromatogram of the sample solution with that of the chromatographic peak of the standard solution, perform qualitative analysis on the bisphenol compounds in the sample, and substitute the peak area of ​​each chromatographic peak into the standard curve obtained in step 3 to calculate the content of bisphenol compounds in the sample.

[0085] The experimental results show that among the 30 dairy samples, bisphenol compound residues were found in 2 samples, including 1 milk (BPA 159.0μg / kg) and 1 yogurt (BPF 50.8μg / kg), indicating that there is a risk of residual bisphenol compounds in actual dairy products. The method of this application can effectively extract and analyze bisphenol compounds in actual samples.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the technical solution. Those skilled in the art should understand that those modifications or equivalent substitutions of the technical solution of the present invention that do not depart from the purpose and scope of the technical solution should be included in the scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting 27 bisphenol compounds in dairy products, characterized in that: The specific steps are as follows: Step 1: 27 bisphenol compounds are prepared into standard solutions of different concentrations, and then the standard solutions are gradient eluted using an ultra-high performance convergence chromatograph to obtain chromatograms and standard curves of the standard solutions at various concentrations; Among them, the 27 bisphenol compounds are bisphenol A diglycidyl ether, bisphenol F diglycidyl ether, 4-cinnamic phenol, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A diphenyl phosphate, bisphenol F glycidyl (2-chloro-1-propanol) ether, bisphenol A (2,3-dihydroxypropyl) glyceryl ether, bisphenol F di(3-chloro-2-hydroxypropyl) ether, bisphenol A di(3- Bisphenol A (2-chloro-2-hydroxypropyl) ether, bisphenol AF, tetrachlorobisphenol A, bisphenol C, bisphenol M, bisphenol TMC, bisphenol B, bisphenol A, bisphenol A (3-chloro-2-hydroxypropyl), bisphenol E, tetrabromobisphenol A, bisphenol F, bisphenol PH, bisphenol Z, 4,4'-(1-methylethylidene) bis(2-methylphenol), bisphenol T, bisphenol S, bisphenol BP, bisphenol A di(2,3-dihydroxypropyl) ether; Step 2: Dissolve 0.5-2 g of the sample to be tested in 10 mL of acetonitrile, add a dehydrating agent and a salting-out agent, vortex and centrifuge, concentrate the supernatant to near dryness under nitrogen blowing, re-dissolve with methanol, and then filter with a polytetrafluoroethylene filter membrane to obtain a filtrate for use; Step 3: Use an ultra-high performance convergence chromatograph to perform gradient elution on the filtrate obtained in step 2 to obtain a chromatogram of the sample solution to be tested, compare the retention time of each chromatographic peak in the chromatogram of the sample solution to be tested with that of each chromatographic peak in the chromatogram of the standard solution, perform qualitative analysis on the bisphenol compounds in the sample to be tested, and substitute the peak area of ​​each chromatographic peak into the standard curve obtained in step 1 to calculate the content of bisphenol compounds in the sample.

2. The method according to claim 1, characterized in that: In step 1, 27 bisphenol compound standards are taken respectively, dissolved and fixed to volume with methanol, and prepared into a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, standard solutions of different concentrations are prepared with an organic solvent, and the organic solvent is one or more of methanol, n-hexane, tetrahydrofuran and ethyl acetate.

3. The method according to claim 1, characterized in that: In step 2, the acetonitrile contains 0.1% formic acid according to the mass percentage; the amount of water removal agent added is 4 g, and the amount of salting-out agent added is 1 g; the specific steps of centrifugal concentration after vortex oscillation are as follows: After adding the dehydrating agent and salting-out agent, vortex and oscillate for 2 minutes, transfer the supernatant to the EMR-Lipid purification tube, vortex and oscillate for 2 minutes, centrifuge at 5000r / min for 5 minutes, concentrate the supernatant to near dryness under nitrogen blowing, re-dissolve with methanol, and then filter with a membrane.

4. The method according to claim 3, characterized in that: The dehydrating agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride.

5. The method according to claim 3, characterized in that: The salting-out agent is one or more of sodium chloride, sodium sulfate, and sodium dihydrogen phosphate.

6. The method according to claim 1, characterized in that: The test conditions of the ultra-high performance convergence chromatograph are: The chromatographic column is ACQUITY UPC, an achiral column. 2 HSS Torus 2-Pic, ACQUITY UPC 2 HSS TorusDEA, ACQUITY UPC 2 HSS Torus Diol and ACQUITY UPC 2 One of the HSS Torus 1-AA; Mobile phase A is supercritical CO2, and mobile phase B is an organic modifier; Column temperature is 20-50°C; Flow rate: 1.0-1.5 mL / min; The system back pressure is 1700-2100 psi; the detection wavelength is 200-300 nm; and the injection volume is 1-10 μL.

7. The method according to claim 6, characterized in that: The organic modifier is one or more of methanol, ethanol, isopropanol and acetonitrile.

8. The method according to claim 1, characterized in that: The elution gradient program is as follows: at 0-1.5 minutes, the volume fraction of mobile phase B increases from 4% to 14%; then it is maintained for 2 minutes; at 3.5-4 minutes, the volume fraction of mobile phase B increases from 14% to 16%; at 4-5 minutes, the volume fraction of mobile phase B increases from 16% to 20% and then it is maintained for 0.5 minutes; at 5.5-6.5 minutes, the volume fraction of mobile phase B increases from 20% to 25%; finally, after 0.5 minutes, the volume fraction of mobile phase B decreases from 25% to 4%.

9. The method according to claim 1, characterized in that: The sample to be tested is a dairy product.

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