A method for simultaneously detecting 27 bisphenol compounds in dairy products

Abstract

The application discloses a method for simultaneously detecting 27 bisphenol compounds in dairy products, which adopts an ultra-high performance liquid chromatograph for detection, an ACQUITY UPC 2 HSS Torus 2-Pic chromatographic column separation, EMR-Lipid technology for purification, can simultaneously detect 27 bisphenol compounds in dairy products, has the advantages of high flux, fast speed and good recovery, and has good application prospect.

Description

TECHNICAL FIELD

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

[0002] Bisphenols (BPs) are a class of substances with two hydroxyphenyl structures. This class of compounds has stable chemical properties, good ductility, and low production cost, and is an important raw material for the production of polycarbonate and other high molecular materials. It is widely used in the industrial production of various dairy product packaging materials such as milk powder bags, yogurt bottles, and milk bottles. However, this kind of compound has endocrine disrupting effects on the human body, affects reproductive and developmental functions, and has toxic effects such as teratogenicity, carcinogenicity, and mutagenicity. Moreover, it is easy to accumulate and difficult to degrade, which has potential harm to human health and the ecological environment.

[0003] At present, the main methods for detecting bisphenol compounds are liquid chromatography (LC), liquid chromatography-tandem mass spectrometry (LC-MS), and gas chromatography-tandem mass spectrometry (GC-MS). Most bisphenol compounds have high thermal stability and are not volatile, so they are not suitable for direct detection by GC or GC-MS. Derivatization treatment is usually required before analysis, which is time-consuming and easy to cause loss of target substances. LC-MS has high sensitivity, good selectivity, and strong anti-interference ability, and is the most commonly used method for detecting bisphenol compounds. However, due to the large number of bisphenol compounds and their similar structures, there are still many challenges in existing analysis methods, mainly including: (1) the sample pretreatment method is complex and has low extraction efficiency; (2) the separation effect of similar structures is poor, and high-throughput analysis of multiple bisphenol compounds cannot be achieved; and (3) the solvent consumption is large, which does not meet the green and environmental development requirements of the detection method. Therefore, it is necessary to establish a rapid, efficient, and green high-throughput analysis method for the detection of bisphenol compounds. SUMMARY

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

[0005] To solve the above technical problems, the 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 kinds of bisphenol compounds are prepared into standard solutions with different concentrations, then the standard solutions are gradient eluted by using an ultra-high performance liquid chromatograph, and chromatograms and standard curves of the standard solutions at different concentrations are obtained;

[0008] Among them, the 27 kinds of bisphenol compounds are bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), 4-cinnamyl phenol (4-CP), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), bisphenol A bisdiphenyl phosphate (BDP), bisphenol F glycidyl (2-chloro-1-propanol) ether (BFDGE-HCl), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE-H2O), bisphenol F di(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), bisphenol A di(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), bisphenol AF (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-methylethylene) 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: 0.5-2g of the sample to be detected is dissolved in 10mL of acetonitrile, then a water removal agent and a salting agent are added, and after vortex oscillation, centrifugation is performed, the supernatant is concentrated to near dryness under nitrogen blowing at 40℃, 1mL of methanol is used for redissolution, then polytetrafluoroethylene filter membrane is used for filtration, and the obtained filtrate is prepared for use;

[0010] Step 3: The filtrate obtained by the treatment in step 2 is gradient eluted by using an ultra-high performance liquid chromatograph, and the chromatogram of the sample solution to be detected is obtained, the retention time of each chromatographic peak in the chromatogram of the sample solution to be detected is compared with that of each chromatographic peak in the chromatogram of the standard solution, the bisphenol compounds in the sample to be detected are qualitatively analyzed, and the peak area of each chromatographic peak is brought into the standard curve obtained in step 1 to calculate the content of the bisphenol compounds contained in the sample.

[0011] Preferably, in step 1, 27 bisphenol standard samples are respectively taken, dissolved and constant volume with methanol, and configured into a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, standard solutions with different concentrations are prepared by using an organic solvent, which is one or more of methanol, n-hexane, tetrahydrofuran and ethyl acetate. Among them, 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 reasonable range, thereby ensuring a better detection range.

[0012] Preferably, in step 2, the acetonitrile contains 0.1% formic acid 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 vortex oscillation and centrifugal concentration are as follows:

[0013] After adding the water removal agent and the salting-out agent, vortex oscillation is performed for 2 min, the supernatant is transferred to an EMR-Lipid purification tube, vortex oscillation is performed for 2 min, and centrifugal concentration is performed at 5000 r / min for 5 min; the supernatant is concentrated to near dryness under nitrogen blowing, redissolved with methanol, and then filter membrane filtration is performed.

[0014] Preferably, the water removal 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 liquid chromatograph are as follows:

[0017] The chromatographic column is one of non-chiral chromatographic columns ACQUITY UPC 2 HSS Torus 2-Pic (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus DEA (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus Diol (150 mm x 3 mm, 1.7 μm) and ACQUITY UPC 2 HSS Torus 1-AA (150 mm x 3 mm, 1.7 μm);

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

[0019] The column temperature is 20-50°C;

[0020] The flow rate is 1.0-1.5 mL / min;

[0021] The system back pressure is 1700-2100 psi;

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

[0023] The sample injection volume is 1-10 μL.

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

[0025] Preferably, the elution gradient program is as follows: 0-1.5 min, the volume fraction of mobile phase B is increased from 4% to 14%; then maintained for 2 min; 3.5-4 min, the volume fraction of mobile phase B is increased from 14% to 16%, 4-5 min, the volume fraction of mobile phase B is increased from 16% to 20%, then maintained for 0.5 min; 5.5-6.5 min, the volume fraction of mobile phase B is increased from 20% to 25%; finally, the volume fraction of mobile phase B is decreased from 25% to 4% in 0.5 min. Under this elution gradient program, the separation effect is better and the sensitivity is higher.

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

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

[0028] 1. The method described in the application first applies ACQUITY UPC 2 Torus 2-Pic chromatographic column to the separation and detection of bisphenol compounds, and 27 kinds of bisphenol compounds can be separated rapidly within 6.5 min.

[0029] 2. The method described in the application uses supercritical CO2 as the mobile phase and a small amount of organic solvent as the modifier, which expands the limitations of gas chromatography, makes it not affected by the boiling point and volatility of the substance, has a larger selection range of target substances, a higher diffusion rate and a faster analysis speed; compared with conventional liquid chromatography, the use of organic solvents is greatly reduced, the cost and toxicity are greatly reduced, it is economic and environmentally friendly, and has a good market prospect.

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

[0031] 4. The method described in this application enables 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 simultaneously varying the ratio of carbon dioxide to organic solvent in the elution gradient. It has the advantages of high throughput, fast speed, and good recovery. Attached Figure Description

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

[0033] Figure 2 Here are the chromatograms obtained after separation using 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 (150mm×3mm, 1.7μm); c stands for ACQUITY UPC 2 HSS Torus DEA (150mm×3mm, 1.7μm); d represents ACQUITY UPC 2 HSS Torus1-AA (150mm×3mm, 1.7μm).

[0034] Figure 3 The graph shows the recovery rate results under different purification conditions in Example 4.

[0035] Figure: 1 is bisphenol A diglycidyl ether (BADGE), 2 is bisphenol F diglycidyl ether (BFDGE), 3 is 4-cinnamyl phenol (4-CP), 4 is bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), 5 is bisphenol A bisdiphenyl phosphate (BDP), 6 is bisphenol F glycidyl (2-chloro-1-propanol) ether (BFDGE-HCl), 7 is bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE-H2O), 8 is bisphenol F bis(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), 9 is bisphenol A bis(3-chloro-2-hydroxypropyl) ether (BADGE-2HCl), 10 is bisphenol AF (BPAF), 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), 27 is bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE-2H2O). DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be described clearly and completely in the present application combined with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. All other embodiments obtained by a person of ordinary skill in the art based on the present application belong to the scope of protection of the present application.

[0037] Unless otherwise specified in specific cases, the numerical ranges listed in the present application include the upper limit and the lower limit, and all integers and fractions within the range, and are not limited to the specific values listed in the defined range.

[0038] I. A method for simultaneously detecting 27 bisphenol compounds in dairy products

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

[0040] The 27 kinds of bisphenol compounds are bisphenol A diglycidyl ether, bisphenol F diglycidyl ether, 4-cinnamyl phenol, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A bis-diphenyl phosphate, bisphenol F glycidyl (2-chloro-1-propanol) ether, bisphenol A (2,3-dihydroxypropyl) glycidyl ether, bisphenol F di (3-chloro-2-hydroxypropyl) ether, bisphenol A di (3-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-methylethylene) bis (2-methylphenol), bisphenol T, bisphenol S, bisphenol BP, bisphenol A di (2,3-dihydroxypropyl) ether.

[0041] Step 2: 0.5-2 g of the sample to be tested is dissolved in 10 mL of acetonitrile, and then a water removal agent and a salting agent are added. After vortex oscillation, centrifugation is performed, and the supernatant is concentrated to near dryness under nitrogen blowing at 40℃. Then, 1 mL of methanol is used for redissolution, followed by filtration with a polytetrafluoroethylene filter membrane to obtain a filtrate for standby use.

[0042] Step 3: A blank sample solution is used to prepare a matrix series standard solution of the 27 kinds of bisphenol compounds. Then, the matrix series standard solution is subjected to gradient elution by using an ultra-high performance liquid chromatograph to obtain a chromatogram and a standard curve of the matrix series standard solution at each concentration. The blank sample solution is obtained by treating a sample containing no target substance according to step 2.

[0043] Step 4: The filtrate obtained by step 2 is subjected to gradient elution by using an ultra-high performance liquid chromatograph to obtain a chromatogram of the sample solution to be tested. The retention time of each chromatographic peak in the chromatogram of the sample solution to be tested is compared with that in the chromatogram of the matrix series standard solution. The bisphenol compounds in the sample to be tested are qualitatively analyzed, and the peak area of each chromatographic peak is brought into the standard curve obtained by step 3 to calculate the content of the bisphenol compounds contained in the sample.

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

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

[0046] In some embodiments of the present application, the treatment method of the sample to be tested is adjusted, and it is found that other purification methods cannot simultaneously achieve efficient extraction of 27 bisphenols. Some purification methods only have certain extraction effects on a few bisphenols, but the extraction effect on other types of bisphenols is not good, as shown in Figure 3 Therefore, in step 2 of the method described in the present application, 0.1% formic acid is contained in acetonitrile according to 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:

[0047] After adding the water removal agent and the salting-out agent, vortex oscillation for 2 min, transfer the supernatant to the EMR-Lipid purification tube, vortex oscillation for 2 min, centrifugal concentration at 5000 r / min for 5 min, and then concentrate the supernatant to near dryness under nitrogen blowing at 40°C, and then redissolve with 1 mL of methanol, followed by filter membrane filtration. Among them, the water removal 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, it is found that due to the large number of bisphenol compounds and the very similar structure and polarity between them, it is difficult to separate them. After using four different chromatographic columns for separation, completely different separation effects are obtained, and the chromatograms obtained also have very significant differences. Therefore, in the present application, the test conditions of the ultra-high performance liquid chromatograph are as follows:

[0049] The chromatographic column is a non-chiral chromatographic column ACQUITY UPC 2 HSS Torus 2-Pic (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus DEA (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus Diol (150 mm x 3 mm, 1.7 μm) and ACQUITY UPC 2one of HSS Torus 1-AA (150mm x 3mm, 1.7μm); further preferably ACQUITY UPC 2 HSS Torus 2-Pic (150mm x 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] The column temperature is 20-50℃;

[0052] The flow rate is 1.0-1.5mL / min;

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

[0054] In some embodiments of the present application, the procedure of the elution gradient is as follows: from 0 to 1.5min, the volume fraction of the mobile phase B is increased from 4% to 14%; then maintained for 2min; from 3.5 to 4min, the volume fraction of the mobile phase B is increased from 14% to 16%, from 4 to 5min, the volume fraction of the mobile phase B is increased from 16% to 20%, then maintained for 0.5min; from 5.5 to 6.5min, the volume fraction of the mobile phase B is increased from 20% to 25%; and finally, the volume fraction of the mobile phase B is decreased from 25% to 4% in 0.5min. Under the condition of the elution gradient described in the present application, the separation effect is better and the sensitivity is higher.

[0055] II. Examples and Comparative Examples

[0056] Example 1: Establishment of high-throughput method

[0057] Step 1: 25.0mg of each of 27 bisphenol compounds was accurately weighed into a 25mL volumetric flask, dissolved with methanol, and the methanol was made up to volume to prepare a standard stock solution with a concentration of 1000mg / L; based on the standard stock solution, a series of standard solutions with different concentrations were prepared by using organic solvents;

[0058] Step 2: The standard solution prepared in step 1 was gradient eluted by ultra-high performance liquid chromatography, wherein the chromatographic column was ACQUITY UPC 2Torus 2-Pic chromatographic column; mobile phase A is supercritical CO2, mobile phase B is methanol; column temperature is 35℃; flow rate is 1.5 mL / min; system back pressure is 1800 psi; detection wavelength is 220 nm; injection volume is 1 μL; the procedure of elution gradient is as follows: 0-1.5 min, the volume fraction of mobile phase B is increased from 4% to 14%; then maintained for 2 min; 3.5-4 min, the volume fraction of mobile phase B is increased from 14% to 16%, 4-5 min, the volume fraction of mobile phase B is increased from 16% to 20%, then maintained for 0.5 min; 5.5-6.5 min, the volume fraction of mobile phase B is increased from 20% to 25%; finally, the volume fraction of mobile phase B is decreased from 25% to 4% in 0.5 min. The chromatogram of the standard solution obtained by the above gradient elution is shown in FIG. 1. Figure 1 As shown in FIG. 1, the method of the present application can realize the rapid detection of 27 bisphenol compounds within 6.5 min, and compared with the detection speed of conventional gas phase and liquid phase methods, the flux is higher, the speed is faster, and the analysis efficiency is greatly improved. Figure 1

[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), add 4 g of water removal agent and 1 g of salting-out agent, vortex for 2 min, transfer the supernatant to an EMR-Lipid purification tube, vortex for 2 min, centrifuge at 5000 r / min for 5 min, take the supernatant and concentrate to near dryness under nitrogen blowing at 40℃, redissolve with 1 mL of methanol, filter with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, the filtrate is a blank sample solution for UPC-C testing;

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

[0062] Step 3: gradient elute the matrix series standard solutions prepared in step 2 by using ultra-high performance liquid chromatography to obtain the chromatograms of the matrix series standard solutions at different concentrations, plot the peak area as the vertical coordinate y and the concentration as the horizontal coordinate x to obtain a matrix series standard curve. The test conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column is an ACQUITY UPC 2 ​Torus2-Pic column; mobile phase A was supercritical CO2, mobile phase B was methanol; column temperature was 35℃; flow rate was 1.5 mL / min; system back pressure was 1800 psi; detection wavelength was 220 nm; injection volume was 1 μL.

[0063] Step 4: Precision and recovery test: add high (500.0 μg / kg), medium (75.0 μg / kg), and low (25 μg / kg) concentrations of 27 bisphenol compound mixed standard solutions into the blank sample solution respectively, and perform the standard addition recovery experiment, and repeat the test 6 times at each concentration level.

[0064] The test results (Table 1) show that, within the respective linear range, the linear correlation coefficients of the 27 bisphenol compounds are all higher than 0.9980, the detection limits of the target substances are 3-5 μg / kg, the quantification limits are 10-25 μg / kg, the standard addition recoveries are 85.5%-114.8%, and the precision is 0.7-6.0%, indicating that the method described in the application can meet the detection requirements of actual samples.

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

[0066]

[0067]

[0068] Example 3: Influence of chromatographic column type on separation effect

[0069] Step 1: accurately weigh 25.0 mg of each of 27 bisphenol compound standard samples into a 25 mL volumetric flask, dissolve with methanol, and dilute with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, prepare a series of standard solutions with different concentrations by using organic solvents;

[0070] Step 2: weigh 2 g (accurate to 0.0001 g) of the sample into a 25 mL centrifuge tube, add 10 mL of acetonitrile (containing 0.1% formic acid), add 4 g of water removal agent and 1 g of salting-out agent, vortex for 2 min, transfer the supernatant to an 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℃, redissolve with 1 mL of methanol, then filter with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, and the filtrate is used for UPC-C test;

[0071] Step 3: perform gradient elution on the sample solution prepared in step 2 by using ultra-high performance liquid chromatography, and separate by using different types of chromatographic columns, the chromatographic column types are ACQUITY UPC 2HSS Torus 2-Pic (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus DEA (150 mm x 3 mm, 1.7 μm), ACQUITY UPC 2 HSS Torus Diol (150 mm x 3 mm, 1.7 μm) and ACQUITY UPC 2 HSS Torus 1-AA (150 mm x 3 mm, 1.7 μm); mobile phase A was supercritical CO2, mobile phase B was methanol; column temperature was 35 °C; flow rate was 1.5 mL / min; system back pressure was 1800 psi; detection wavelength was 220 nm; injection volume was 1 μL; elution gradient program was as follows: 0-1.5 min, volume fraction of mobile phase B was increased from 4% to 14%; then maintained for 2 min; 3.5-4 min, volume fraction of mobile phase B was increased from 14% to 16%, 4-5 min, volume fraction of mobile phase B was increased from 16% to 20%, then maintained for 0.5 min; 5.5-6.5 min, volume fraction of mobile phase B was increased from 20% to 25%; finally, volume fraction of mobile phase B was decreased from 25% to 4% in 0.5 min. The chromatograms of different chromatographic columns under the above conditions were shown in FIGS. 1-4. Figure 2

[0072] Since the bisphenol compounds are various in types, similar in structure and polarity, and difficult to separate, it can be known from the above that the 27 bisphenol compounds cannot be completely separated on the HSS Torus 1-AA, HSS Torus DEA, HSS Torus Diol and ACQUITY UPC Figure 2 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 cannot be completely separated, and phenomena such as stacking, tailing and broadening of chromatographic peaks occurred, but the ACQUITY UPC 2 HSS Torus 2-Pic chromatographic column, the overall chromatographic peak shape was sharp and symmetrical, and the 27 target substances could be completely separated within 6.5 min. The experiment proved that, through the optimization experiment of the chromatographic column, the method established in the application is a rapid high-throughput method, which is suitable for qualitative and quantitative analysis of the 27 bisphenol substances in dairy products.

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

[0074] ​​Step 1: 25.0 mg of each of 27 bisphenol compounds was accurately weighed into a 25 mL volumetric flask, dissolved with methanol, and diluted with methanol to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, a blank sample solution in step 1 of Example 2 was used to prepare a matrix series standard solution with different concentrations;

[0075] Step 2: Gradient elution was performed on the matrix series standard solution prepared in step 1 by using ultra-high performance liquid chromatography to obtain chromatograms of the matrix series standard solution at different concentrations, and a matrix series standard curve was obtained by plotting the peak area as the ordinate y and the concentration as the abscissa x.

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

[0077] Step 4: Sample detection: Gradient elution was performed on the sample solution treated with different purification materials prepared in step 3 by using ultra-high performance liquid chromatography to obtain chromatograms of the sample solution. The retention time of each chromatographic peak in the chromatogram of the sample solution was compared with that of the standard solution, and the bisphenol compounds in the sample were qualitatively analyzed. The peak area of each chromatographic peak was brought into the standard curve obtained in step 3 to calculate the content of bisphenol compounds in the sample. Separation was performed by using an ACQUITY UPC-C 2 HSS Torus 2-Pic (150 mm x 3 mm, 1.7 μm) column; mobile phase A was supercritical CO2, mobile phase B was methanol; column temperature was 35°C; flow rate was 1.5 mL / min; system back pressure was 1800 psi; detection wavelength was 220 nm; injection volume was 1 μL; elution gradient program was as follows: 0-1.5 min, volume fraction of mobile phase B increased from 4% to 14%; then maintained for 2 min; 3.5-4 min, volume fraction of mobile phase B increased from 14% to 16%, 4-5 min, volume fraction of mobile phase B increased from 16% to 20%, then maintained for 0.5 min; 5.5-6.5 min, volume fraction of mobile phase B increased from 20% to 25%; finally, volume fraction of mobile phase B decreased from 25% to 4% in 0.5 min.

[0078] The recovery rates under different purification conditions obtained by the above conditions are shown in Table 2. Figure 3 The experimental results show that Osia-HLB, Al2O3 and C 18 None of the three purification methods can simultaneously achieve efficient extraction of 27 bisphenol compounds. The recovery rate of 27 bisphenol compounds after purification by the EMR-Lipid purification technology can reach more than 90%. The experiments prove that the method has the advantages of simple operation, good purification effect and high recovery rate.

[0079] Example 5: Actual sample detection

[0080] 30 dairy products (including 10 portions of milk, 10 portions of yogurt and 10 portions of milk powder) were randomly purchased from a local supermarket, and the bisphenol compound residue was detected according to the above optimal conditions. The experimental steps are as follows:

[0081] Step 1: 25.0 mg of each of 27 bisphenol compounds was accurately weighed into a 25 mL volumetric flask, dissolved with methanol, and the methanol was diluted to the mark to prepare a standard stock solution with a concentration of 1000 mg / L; based on the standard stock solution, a series of standard solutions with different concentrations were prepared by using organic solvents;

[0082] Step 2: 2 g (accurate to 0.0001 g) of sample was weighed into a 25 mL centrifuge tube, 10 mL of acetonitrile (containing 0.1% formic acid) was added, 4 g of water removal agent and 1 g of salting-out agent were added, vortexed for 2 min, the supernatant was transferred to an EMR-Lipid purification tube, vortexed for 2 min, centrifuged at 5000 r / min for 5 min, the supernatant was concentrated to near dryness under nitrogen blowing at 40°C, 1 mL of methanol was used for redissolution, and then filtered with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane. The filtrate was used for UPCC test;

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

[0084] Step 4: Sample detection: the sample solution prepared in step 2 was gradient eluted by using ultra-high performance liquid chromatography to obtain the chromatogram of the sample solution, the retention time of each chromatographic peak in the chromatogram of the sample solution was compared with that of the standard solution, the bisphenol compounds in the sample were qualitatively analyzed, and the peak area of each chromatographic peak was brought into the standard curve obtained in step 3 to calculate the content of bisphenol compounds in the sample.

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

[0086] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the technical solutions. Those of ordinary skill in the art should understand that modifications or equivalent replacements of the technical solutions of the present application without departing from the purpose and scope of the technical solutions should be covered in the scope of the claims of the present application.

Claims

1. A method for simultaneous detection of 27 bisphenolic compounds in dairy products, characterized by, The specific steps are as follows: Step 1: 27 kinds of bisphenol compounds are prepared into standard solutions with different concentrations, then the standard solutions are gradient eluted by using an ultra-high performance liquid chromatograph, and chromatograms and standard curves of the standard solutions at different concentrations are obtained; The 27 kinds of bisphenol compounds are bisphenol A diglycidyl ether, bisphenol F diglycidyl ether, 4-cinnamyl phenol, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A bis-diphenyl phosphate, bisphenol F glycidyl (2-chloro-1-propanol) ether, bisphenol A (2,3-dihydroxypropyl) glycerol ether, bisphenol F di (3-chloro-2-hydroxypropyl) ether, bisphenol A di (3-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-methylethylene) bis (2-methylphenol), bisphenol T, bisphenol S, bisphenol BP, bisphenol A di (2,3-dihydroxypropyl) ether; Step 2: 0.5-2 g of the sample to be measured is dissolved in 10 mL of acetonitrile, and according to the mass percentage, 0.1% of formic acid is contained in the acetonitrile; then a water removal agent and a salting-out agent are added, the water removal agent is added in an amount of 4 g, the salting-out agent is added in an amount of 1 g, vortex oscillation is performed for 2 min, the supernatant is transferred to an EMR-Lipid purification tube, vortex oscillation is performed for 2 min, centrifugation is performed at 5000 r / min for 5 min, the supernatant is concentrated to near dryness under nitrogen blowing, and then redissolved with methanol, followed by filtration with a polytetrafluoroethylene filter membrane to obtain a filtrate for standby; Step 3: The filtrate obtained in step 2 is gradient eluted by using an ultra-high performance liquid chromatograph, and a chromatogram of the sample solution to be measured is obtained; the retention time of each chromatographic peak in the chromatogram of the sample solution to be measured is compared with that of each chromatographic peak in the chromatogram of the standard solution, qualitative analysis of the bisphenol compounds in the sample to be measured is performed, and the peak area of each chromatographic peak is brought into the standard curve obtained in step 1 to calculate the content of the bisphenol compounds contained in the sample; The test conditions of the ultra-high performance liquid chromatograph are as follows: The chromatographic column is an achiral chromatographic column ACQUITY UPC 2 HSS Torus 2-Pic; The mobile phase A is supercritical CO2, and the mobile phase B is methanol; The column temperature is 20-50℃; The flow rate is 1.0-1.5 mL / min; The system back pressure is 1700-2100 psi; the detection wavelength is 200-300 nm; and the injection amount is 1-10 μL; The program of the elution gradient is as follows: from 0 to 1.5 min, the volume fraction of the mobile phase B is increased from 4% to 14%; then maintained for 2 min; from 3.5 to 4 min, the volume fraction of the mobile phase B is increased from 14% to 16%, from 4 to 5 min, the volume fraction of the mobile phase B is increased from 16% to 20%, and then maintained for 0.5 min; from 5.5 to 6.5 min, the volume fraction of the mobile phase B is increased from 20% to 25%; and finally, the volume fraction of the mobile phase B is decreased from 25% to 4% in 0.5 min.

2. The method of claim 1, wherein, In step 1, 27 bisphenol standard samples are respectively taken, dissolved and constant volume with methanol to configure standard stock solution with concentration of 1000 mg / L; based on the standard stock solution, standard solutions with different concentrations are prepared by using organic solvents, which are one or more of methanol, n-hexane, tetrahydrofuran and ethyl acetate.

3. The method of claim 1, wherein, The water removing agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride.

4. The method of claim 1, wherein, The salting agent is one or more of sodium chloride, sodium sulfate and sodium dihydrogen phosphate.

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