Method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant

Abstract

The invention discloses a method for simultaneously detecting bisphenol diglycidyl ether and a p-phenylenediamine antioxidant and a pretreatment method for simultaneously detecting the bisphenol diglycidyl ether and the p-phenylenediamine antioxidant, and the pretreatment method comprises the following steps: mixing a to-be-detected sample, an extraction agent and a phase separation inducer; extracting and separating, and taking supernate to obtain a pretreated sample; the extracting agent comprises at least one of ethyl acetate, acetonitrile, tert-butyl methyl ether or dichloromethane. According to the method, an extraction method is adopted for pretreatment, the phase separation inducer is combined with the specific extraction agent, rapid and simple pretreatment can be achieved, the detection efficiency is greatly improved, and then bisphenol diglycidyl ether and a p-phenylenediamine antioxidant are extracted and detected at the same time.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant. Background Art

[0002] Bisphenol diglycidyl ethers (BDGEs) are aromatic ether compounds, including bisphenol A-diglycidyl ether (BADGE) and bisphenol F-diglycidyl ether (BFDGE) and their derivatives. They are mainly used in canned food coatings and are a typical class of estrogen-like environmental endocrine disruptors (EEDs). p-Phenylenediamine antioxidants (PPDs) are aromatic amine compounds that are widely used in the tire rubber industry. During long-term use, tires produce tire wear particles that enter the environmental medium. Bisphenol diglycidyl ethers and p-Phenylenediamine antioxidants have similar structures and are widely detected in various environmental media and biological matrices. They can enter the body through oral administration, skin contact and inhalation. They have reproductive and developmental toxicity and are biomagnified through the food chain, posing a threat to human health. Human urine plays a vital role in excreting various environmental pollutants from the body to the outside of the body. It is relatively easy to collect and store, and urine samples have been widely used to assess the level of human exposure to environmental pollutants. At present, related research has problems such as cumbersome pretreatment and long time consumption. For example, the existing pretreatment method often uses enzymatic hydrolysis, which generally takes more than 12 hours; at the same time, the existing detection methods are mostly single detection, and there is a lack of research on the simultaneous detection methods of multiple pollutants. In addition, since the detection time required for each substance is relatively long, mostly more than 15 minutes, if multiple substances are detected, the detection time needs to be doubled. Since the human population is in a long-term low-dose mixed exposure to multiple pollutants, it is necessary to develop a stable and sensitive method for the simultaneous detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants. Summary of the invention

[0003] In order to overcome at least one of the problems existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a pretreatment method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant.

[0004] The second object of the present invention is to provide a kit for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant.

[0005] The third object of the present invention is to provide a method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a pretreatment method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, comprising the following steps: mixing a sample to be tested, an extractant and a phase separation inducer, extracting and separating, and taking a supernatant to obtain a pretreated sample; the extractant comprises at least one of ethyl acetate, acetonitrile, tert-butyl methyl ether or dichloromethane.

[0008] Preferably, the bisphenol diglycidyl ether includes at least one of bisphenol A diglycidyl ether (BADGE), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE-H2O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl-H2O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), bisphenol A bis (3-chloro-2-hydroxypropyl) ether (BADGE-2HCl), bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE-2H2O), bisphenol F diglycidyl ether (BFDGE), bisphenol F bis (2,3-dihydroxypropyl) ether (BFDGE-2H2O) or bisphenol F bis (3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl).

[0009] Preferably, the p-phenylenediamine antioxidant includes at least one of N-cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-(1,3-dimethyl)butyl-N'-phenyl-p-phenylenediamine (6PPD), N,N'-diphenyl-p-phenylenediamine (DPPD) or N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone (6PPD-Q).

[0010] Preferably, the extractant comprises at least one of ethyl acetate, acetonitrile or tert-butyl methyl ether; further preferably, the extractant is selected from ethyl acetate.

[0011] Preferably, the phase separation inducer includes at least one of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate or zinc sulfate; further preferably, the phase separation inducer includes at least one of sodium chloride, potassium chloride or calcium chloride; further preferably, the phase separation inducer is selected from sodium chloride.

[0012] Sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and zinc sulfate can be used as phase separation inducers to achieve better extraction and separation effects. In particular, since sodium chloride itself is hygroscopic, it can effectively separate and remove the water in the supernatant of the extractant during the extraction process; and in the extraction process, the salting-out effect of sodium chloride is also conducive to increasing the salt content of the water phase and increasing the density of the water phase, thereby making the target compound have a higher distribution coefficient between the salt-containing phase and the organic extraction phase, thereby achieving a more thorough separation effect.

[0013] Preferably, the sample to be tested is a urine sample. In some embodiments of the present invention, the urine sample is an animal urine sample, such as a human urine sample.

[0014] Preferably, the matrix concentration in the sample to be tested is ≥ 0.005 ng / mL; more preferably 0.005-0.1 ng / mL.

[0015] Preferably, the volume ratio of the sample to be tested to the extractant is 1:(2-10); more preferably 1:(4-8); and even more preferably (5-7).

[0016] Preferably, the dosage ratio of the sample to be tested to the phase separation inducer is 1 mL: (0.1-0.3) g; more preferably 1 mL: (0.13-0.27) g; and even more preferably 1 mL: (0.16-0.24) g;

[0017] Preferably, the extraction and separation are performed 1 to 5 times; more preferably 2 to 4 times; and even more preferably 3 times.

[0018] Preferably, the time for mixing the sample to be tested, the extractant and the phase separation inducing agent is 20 to 40 minutes; more preferably, it is 25 to 35 minutes.

[0019] Preferably, the method of mixing the sample to be tested, the extractant and the phase separation inducer includes vortexing, oscillation or a combination thereof; further preferably, the method of mixing the sample to be tested, the extractant and the phase separation inducer includes vortexing and oscillation; even further preferably, the method of mixing the sample to be tested, the extractant and the phase separation inducer is: vortexing for 0.1 to 1 min and oscillating for 25 to 35 min.

[0020] Preferably, the separation method is selected from centrifugation; the centrifugal rotation speed is preferably 6000-8000 rpm.

[0021] Preferably, the extraction and separation process further comprises the steps of drying, re-dissolving and filtering after taking the supernatant.

[0022] Preferably, the drying method is selected from nitrogen blowing drying.

[0023] Preferably, the solvent used for the re-dissolution includes methanol, ethanol or a combination thereof; more preferably methanol.

[0024] Preferably, the amount of the solvent used for the reconstitution is 0.05-0.1 mL.

[0025] Preferably, the filtration pore diameter is 0.2-0.25 μm.

[0026] The second aspect of the present invention provides a kit for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, wherein the kit is prepared by the pretreatment method described in the first aspect of the present invention.

[0027] The third aspect of the present invention provides a method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, comprising the following steps:

[0028] S1. Pretreatment: pretreating the sample to be tested by the pretreatment method described in the first aspect of the present invention, and using the obtained pretreated sample as the test sample; or using the kit described in the second aspect of the present invention as the test sample;

[0029] S2. Detection: Using ultra high performance liquid chromatography-tandem mass spectrometry to detect the test sample obtained in step S1; the mobile phase used in the ultra high performance liquid chromatography-tandem mass spectrometry includes mobile phase A and mobile phase B, mobile phase A is selected from formic acid aqueous solution, and mobile phase B is selected from formic acid methanol solution.

[0030] Preferably, in step S2, a standard curve of bisphenol diglycidyl ether and p-phenylenediamine antioxidant is established by the ultra-high performance liquid chromatography-tandem mass spectrometry, and the concentrations of bisphenol diglycidyl ether and p-phenylenediamine antioxidant are obtained by using the standard curve.

[0031] Preferably, in step S2, the detection result is also corrected by an internal standard method; further preferably, the internal standard used in the internal standard method includes hexadeuterated bisphenol A diglycidyl ether (BADGE-d6) and / or pentadeuterated N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone (6PPD-Q-d5).

[0032] Preferably, in step S2, the mass concentration of the formic acid aqueous solution in the mobile phase A of the ultra-high performance liquid chromatography-tandem mass spectrometry is 0.03-0.2%; more preferably 0.05-0.1%.

[0033] Preferably, in step S2, the mass concentration of the formic acid methanol solution in the mobile phase B of the ultra-high performance liquid chromatography-tandem mass spectrometry is 0.03-0.2%; more preferably 0.05-0.1%.

[0034] Preferably, in step S2, the elution method used in the ultra-high performance liquid chromatography-tandem mass spectrometry is gradient elution; the elution procedure of the gradient elution is as follows:

[0035] 0-2min: 90-95% mobile phase A, 5-10% mobile phase B;

[0036] 2-5 min: 85-90% mobile phase A, 10-15% mobile phase B;

[0037] 5-7min: 40-50% mobile phase A, 50-60% mobile phase B;

[0038] 7-10min: 20-30% mobile phase A, 70-80% mobile phase B;

[0039] 10-11 min: 0.1-1% mobile phase A, 99-99.9% mobile phase B;

[0040] 11-11.5min: 0.1-1% mobile phase A, 99-99.9% mobile phase B;

[0041] 11.5~14min: 95~99% mobile phase A, 1~5% mobile phase B.

[0042] Preferably, in step S2, the ion source used in the ultra-high performance liquid chromatography-tandem mass spectrometry is selected from an electrospray ionization source ESI.

[0043] Preferably, in step S2, the detection mode adopted by the ultra-high performance liquid chromatography-tandem mass spectrometry is selected from the positive ion mode.

[0044] Preferably, in step S2, the ion source temperature used in the ultra-high performance liquid chromatography-tandem mass spectrometry is 450-550°C.

[0045] Preferably, in step S2, the ionization voltage used in the ultra-high performance liquid chromatography-tandem mass spectrometry is 4000-5000V.

[0046] Preferably, in step S2, the collection method used by the ultra-high performance liquid chromatography-tandem mass spectrometry is selected from a multiple reaction detection mode.

[0047] Preferably, in step S2, the chromatographic column used in the ultra-high performance liquid chromatography-tandem mass spectrometry is selected from an Agilent ZOBRAX SB-C18 chromatographic column; further preferably, the length of the chromatographic column is 80 to 120 mm, the inner diameter is 2 to 2.5 mm, and the filler particle diameter is 3 to 4 μm.

[0048] Preferably, in step S2, the column temperature used in the ultra-high performance liquid chromatography-tandem mass spectrometry is 20-30°C.

[0049] Preferably, in step S2, the injection volume used in the ultra-high performance liquid chromatography-tandem mass spectrometry is 1 to 3 μL.

[0050] Preferably, in step S2, the mobile phase flow rate used in the ultra-high performance liquid chromatography-tandem mass spectrometry is 0.2-0.3 mL / min.

[0051] The beneficial effects of the present invention are as follows: the present invention adopts an extraction method for pretreatment, and utilizes a phase separation inducer in combination with a specific extractant, so as to achieve rapid and simple pretreatment, and greatly improve the detection efficiency; wherein, the phase separation inducer can induce the stratification of the organic phase and the aqueous phase, so that the target compound has a higher distribution coefficient in the original phase and the extractant phase, thereby achieving better extraction and separation effects; the pretreated sample obtained by the present invention through a specific pretreatment step is tested, and bisphenol diglycidyl ether and p-phenylenediamine antioxidant can be detected simultaneously, so as to achieve simultaneous detection of the above two types of substances, and the detection efficiency is high.

[0052] Specifically, compared with the prior art, the present invention has the following advantages:

[0053] 1. The present invention realizes the simultaneous extraction and detection analysis of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine samples by improving sample pretreatment and liquid phase analysis methods. It is simple and convenient and solves the problems of complicated and time-consuming pretreatment steps.

[0054] 2. The present invention does not require enzymatic hydrolysis, but only needs to add a phase separation inducer and an extractant for pretreatment, which greatly simplifies the steps and effectively improves the experimental efficiency of pretreatment. The method of the present invention is used to detect 14 substances, and the total time is about 14 minutes, which greatly saves sample detection time and is more economical and effective. At the same time, the method of the present invention does not require solid phase extraction column enrichment and purification, which is more economical and environmentally friendly. The method provided by the present invention provides a basic condition for further exploring the relationship between mixed exposure to pollutants and human health outcomes, and expands the scope of application of health risk assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure is a schematic flow chart of the steps of the method for simultaneous extraction and detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants in urine in Example 1.

[0056] Figure 2 This is one of the extraction chromatograms of bisphenol diglycidyl ether in the target analyte of Example 1.

[0057] Figure 3 This is the second extraction chromatogram of bisphenol diglycidyl ether in the target analyte of Example 1.

[0058] Figure 4 This is the extraction chromatogram of the p-phenylenediamine antioxidant in the target analyte of Example 1.

[0059] Figure 5 It is the average recovery test result figure of Examples 1 to 3.

[0060] Figure 6 It is the average recovery test result diagram of Example 1 and Examples 4-5.

[0061] Figure 7 It is a graph showing the average recovery test results of Example 1 and Examples 6-7.

[0062] Figure 8 It is a graph showing the average recovery test results of Example 1 and Examples 8 to 10.

[0063] Fig. 9 This is a graph showing the average recovery test results of Example 1 and Example 11.

[0064] Fig.10 It is the average recovery test result diagram of Example 1 and Comparative Example 1.

[0065] Fig.11 This is a graph showing the average recovery test results of the method in Example 1 at different spiked concentrations. DETAILED DESCRIPTION

[0066] The content of the present invention is further described in detail below through specific examples. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the principles set forth in the present invention all belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific data exemplified below. The raw materials, reagents or devices used in the following examples and comparative examples, unless otherwise specified, can all be obtained from conventional commercial sources, or can be obtained by existing known methods.

[0067] Example 1

[0068] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, the flow chart of the steps in this example is as follows Figure 1 As shown, the specific steps are as follows:

[0069] S1. Sample pretreatment:

[0070] S1-1. Take 0.5 mL of the urine sample to be tested and thaw it at 4°C, place it in a 15 mL centrifuge tube, and then add 25 μL of a mixed standard solution of the target analyte and the internal standard with a concentration of 100 ng / mL (i.e., a spike concentration of 5 ng / mL);

[0071] S1-2, add 3 mL of ethyl acetate as an extractant, add 0.1 g of sodium chloride, vortex for 30 seconds and shake for 0.5 hours, and then centrifuge at 8000 rpm for 5 minutes to extract the supernatant;

[0072] S1-3, repeat the above step S1-2 twice, that is, use the extraction times of step S1-2 three times in total; collect and combine the supernatant in another spare 15mL centrifuge tube, blow nitrogen to dryness, and then re-dissolve with 0.1mL methanol. After sufficient vortexing, pass through a 0.22μm organic phase nylon filter membrane, and transfer the filtrate to a 9mm threaded brown glass injection vial equipped with a glass liner tube to obtain the sample to be tested, and store it in a refrigerator at -20℃;

[0073] S2. Sample testing:

[0074] Ultra high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to detect and analyze the test samples obtained in step S3, and a standard curve of each substance was established using the 10-point method. The average recoveries, intra-day and inter-day relative standard deviations of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine were calculated. When the recovery range was 70% to 120% and the relative standard deviation was less than 20%, the accuracy of the method was considered to be good.

[0075] The above-mentioned method for simultaneous extraction and detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants in urine, the ultra-high performance liquid chromatography conditions are shown in Table 3.

[0076] Table 3 Positive ion mode detection conditions

[0077]

[0078]

[0079] The above-mentioned method for simultaneous extraction and detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants in urine, the mass spectrometry detection conditions are as follows: the mass spectrometry detection conditions are: positive ion mode (ESI + ), ion source temperature 500℃; ion spray voltage 5000V; column temperature 25℃; GSⅠ40psi; GSⅡ40psi; CUR 35psi.

[0080] In the above method for simultaneous extraction and detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants in urine, the mass spectrometry parameters are shown in Table 4.

[0081] Table 4 Mass spectrometry parameters in positive ion mode

[0082]

[0083]

[0084] In the above method for simultaneous extraction and detection of bisphenol diglycidyl ether and p-phenylenediamine antioxidants in urine, the basic information of the target analytes is shown in Table 5.

[0085] Table 5 Basic characteristics of target analytes

[0086]

[0087] In this example, the target analytes in the urine sample to be tested include: bisphenol A diglycidyl ether (BADGE), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE-H2O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl-H2O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), bisphenol A bis (3-chloro-2-hydroxypropyl) ether (BADGE-2HCl), bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE-2H2O), bisphenol F diglycidyl ether (BADGE), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE-H2O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE-HCl), bisphenol A (2,3-dihydroxypropyl) ether (BADGE-2H2O), bisphenol F diglycidyl ether (BADGE-HCl ... A (3-chloro-2-hydroxypropyl) glycidyl Glycerol ether (BFDGE), bisphenol F bis(2,3-dihydroxypropyl) ether (BFDGE-2H2O), bisphenol F bis(3-chloro-2-hydroxypropyl) ether (BFDGE-2HCl), N-cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-(1,3-dimethyl)butyl-N'-phenyl-p-phenylenediamine (6PPD), N,N'-diphenyl-p-phenylenediamine (DPPD) and N(-1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone (6PPD-Q).

[0088] The extraction chromatograms of the above target analytes are shown in Figures 2 to 4 shown.

[0089] Specifically, Figure 2 This is one of the extraction chromatograms of bisphenol diglycidyl ether in the target analyte of Example 1, wherein a is BADGE, b is BADGE-H2O, c is BADGE-HCl-H2O, d is BFDGE, and e is BADGE-HCl; Figure 3 The second extraction chromatogram of bisphenol diglycidyl ether in the target analyte of Example 1, wherein a is BADGE-2H2O, b is BFDGE-2H2O, c is BADGE-2HCl, and d is BFDGE-2HCl; Figure 4This is the extraction chromatogram of the p-phenylenediamine antioxidant in the target analyte of Example 1, wherein a is CPPD, b is IPPD, c is DPPD, d is 6PPD, and e is 6PPD-Q.

[0090] Example 2

[0091] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-3 of this example, step S1-2 is not repeated, that is, the extraction times of step S1-2 are used once; the other steps are the same as Example 1.

[0092] Example 3

[0093] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-3 of this example, step S1-2 is repeated once, that is, the extraction times of step S1-2 are used twice in total; the other steps are the same as Example 1.

[0094] Example 4

[0095] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, the amount of ethyl acetate used as the extractant is 1 mL each time; the other steps are the same as Example 1.

[0096] Example 5

[0097] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, the amount of ethyl acetate used as the extractant is 2 mL each time; the other steps are the same as Example 1.

[0098] Example 6

[0099] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, the amount of sodium chloride used is 0.05 g; the other steps are the same as Example 1.

[0100] Example 7

[0101] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, the amount of sodium chloride used is 2.0 g; the other steps are the same as Example 1.

[0102] Example 8

[0103] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that in step S1-2 of this example, an equal amount of acetonitrile is used as an extractant instead of ethyl acetate in Example 1; the other steps are the same as Example 1.

[0104] Example 9

[0105] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, an equal amount of dichloromethane is used instead of ethyl acetate in Example 1 as an extractant; the other steps are the same as Example 1.

[0106] Example 10

[0107] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that: in step S1-2 of this example, an equal amount of tert-butyl methyl ether is used instead of ethyl acetate in Example 1 as an extractant; the other steps are the same as Example 1.

[0108] Embodiment 11

[0109] A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant in urine, which differs from Example 1 only in that in step S2 of this example, an equal amount of methanol is used instead of 0.1% formic acid methanol in Example 1 as mobile phase B; the other steps are the same as Example 1.

[0110] Comparative Example 1

[0111] A method for detecting bisphenol diglycidyl ether and paraphenylenediamine antioxidants in urine, which differs from Example 1 only in that: in step S1-1 of this example, 50 μL of a mixed enzyme solution of β-glucuronidase and arylsulfatase is added to the urine sample to be tested for enzymatic hydrolysis for 12 hours; the other steps are the same as Example 1.

[0112] Effect detection

[0113] (1) The average recovery rates of the methods of the embodiment and the comparative example are compared, and the calculation formula of the average recovery rate is shown in the following formula ①:

[0114] Average recovery rate (%) = (C ss -C s ) / C st ×100%①;

[0115] Among them, C ss is the concentration of the spiked sample, i.e. the measured value of the spiked sample; C s is the concentration of the unspiked sample, that is, the measured value of the unspiked sample, also known as the matrix blank; Cst is the spike concentration.

[0116] The average recovery test results of Examples 1 to 3 are as follows Figure 5 As shown, it can be seen that a good extraction effect can be obtained by using the specific number of extractions in the embodiments of the present invention; further, the average recovery rates of the methods of embodiments 1 to 3 are compared. The average recovery rate of each substance in urine in embodiment 1 is 71% to 122%, while when the number of extractions is 1 time (embodiment 2) or 2 times (embodiment 3), the average recovery rates of substances in bisphenol diglycidyl ether and p-phenylenediamine antioxidant are low, among which the average recovery rates of BADGE, BFDGE, BADGE-HCL, BFDGE-2H2O, BFDGE-2HCL of bisphenol diglycidyl ether and CPPD, IPPD, DPPD, 6PPD, and 6PPD-Q of p-phenylenediamine antioxidant are all lower than 71%. It can be seen that, relative to embodiments 2 to 3, the three extraction methods of embodiment 1 can obtain better extraction effects.

[0117] The average recovery test results of Example 1 and Example 4-5 are as follows Figure 6 As shown, it can be seen that a good extraction effect can be obtained by using the specific amount of the extractant in the embodiment of the present invention; further, compared with Example 1, when the amount of ethyl acetate extraction each time in Example 4 is 1 mL and the amount of ethyl acetate extraction each time in Example 5 is 2 mL, the average recovery rate of the substances in bisphenol diglycidyl ether and p-phenylenediamine antioxidant is reduced. It can be seen that, relative to Examples 4 to 5, the method of Example 1 can achieve better synchronous extraction and detection effects.

[0118] The average recovery test results of Example 1 and Example 6-7 are as follows Figure 7 As shown, it can be seen that a good extraction effect can be obtained by using the specific amount of sodium chloride in the embodiment of the present invention; further, compared with Example 1, when the amount of sodium chloride in Example 6 is 0.05g and the amount of sodium chloride in Example 7 is 0.2g, the average recoveries of BADGE-HCl-H2O, BADGE-HCl, BADGE-2H2O, BFDGE-2H2O, BADGE-2HCl, CPPD, IPPD, DPPD and 6PPD in Example 6 are all lower than 71%, and the average recoveries of Example 7 and Example 1 are both good. Considering the principle of economy, relative to Examples 6 to 7, the method of Example 1 can achieve better synchronous extraction and detection effects.

[0119] The average recovery test results of Example 1 and Examples 8 to 10 are as follows Figure 8As shown, it can be seen that good extraction effect can be obtained by using the specific extractant in the embodiment of the present invention; further, compared with Example 1, when acetonitrile is used as the solvent in Example 8, dichloromethane is used as the solvent in Example 9, and tert-butyl methyl ether is used as the solvent in Example 10, the average recovery rate of the target analytes is low, among which the average recovery rates of BADGE, BADGE-H2O, BFDGE, BADGE-HCl, BFDGE-2H2O, BFDGE-2HCl, CPPD, IPPD, DPPD and 6PPD are all lower than 71%. It can be seen that, relative to Examples 8 to 10, the method of Example 1 can achieve better synchronous extraction and detection effect. In the method of the embodiment of the present invention, using acetonitrile, dichloromethane or tert-butyl methyl ether as a single extractant can only extract part of the substances, and it is impossible to fully realize the simultaneous extraction and detection of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants; while using ethyl acetate as an extractant, it is possible to realize the simultaneous extraction and detection of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants, and the extraction and detection efficiency is high.

[0120] The average recovery test results of Example 1 and Example 11 are as follows Fig. 9 As shown, it can be seen that good extraction effect can be obtained by using the specific mobile phase in the embodiment of the present invention; further, compared with Example 1, when methanol is used as the mobile phase B in Example 11, the average recovery rate of the target analyte is relatively unstable, the recovery rate of BADGE-2H2O is higher than 140%, and the recovery rate of BADGE, BADGE-HCl, BADGE-2HCl, CPPD, IPPD, 6PPD and 6PPD-Q is decreased. Therefore, relative to Example 11, the method of Example 1 can achieve better synchronous extraction and detection effect.

[0121] The recovery test results of Example 1 and Comparative Example 1 are as follows: Fig.10 As shown, compared with Example 1, 50 μL of a mixed enzyme solution of β-glucuronidase and arylsulfatase was added to Comparative Example 1 for enzymatic hydrolysis for 12 hours and then extracted. The recovery rate of the unhydrolyzed substance in Example 1 was between 71% and 122%, and the recovery rate of the substance extracted after enzymatic hydrolysis in Comparative Example 9 was between 76% and 116%. The effects were comparable and relatively stable. Considering the experimental time consumption and economic principles, the method of Example 1 can meet the analysis requirements and achieve better synchronous extraction and detection effects, and Example 1 can greatly save pretreatment time and effectively improve detection efficiency.

[0122] (2) The average recovery rate of the methods of the embodiment and the comparative example at different spiked concentrations was tested to evaluate the accuracy of the method. The specific test steps are as follows: 12 human urine samples were taken, 0.5 mL of each was placed in a 15 mL centrifuge tube, and three concentration levels of target analyte and internal standard mixed standard solution (2, 5 and 10 ng / mL) were added respectively. Four parallels were set for each concentration. At the same time, a urine sample with 0.5 mL of ultrapure water added and a urine sample with only the corresponding concentration of internal standard solution added were set as the program blank and matrix blank respectively. The sample spiked recovery rate was corrected by the isotope internal standard method. The internal standard corresponding to the target analyte is shown in Table 4. Within one day, urine samples with three concentration levels (2, 5 and 10 ng / mL, n=4) of the original were added for intra-day evaluation; for three consecutive days, urine samples with three concentration levels (2, 5 and 10 ng / mL, n=12) of the original were added for inter-day evaluation. The relative standard deviations (RSDs) of the average recovery of each substance were calculated to evaluate the intra-day and inter-day precision, repeatability and reproducibility of the method of the present invention.

[0123] The average recovery test results of the method of Example 1 at different spiked concentrations are shown in Table 6, and the test result bar graph is shown in Fig.11 As shown, the test correlation coefficient (r 2 ), limit of detection (LOD) and limit of quantitation (LOQ) are shown in Table 7.

[0124] Table 6 Average recovery, intra-day and inter-day precision of target analytes in Example 1

[0125]

[0126]

[0127] Table 7 Test correlation coefficient r of Example 1 2 , LOD and LOQ

[0128]

[0129] As shown in Table 6, when the spike concentration of each substance was 2 ng / mL, the average recovery of the method ranged from 27% to 59%, the intra-day RSD was 9% to 38% (n=4), and the inter-day RSD was 22% to 39% (n=12); when the spike concentration of each substance was 5 ng / mL, the average recovery of the method ranged from 71% to 122%, the intra-day RSD was 1% to 18% (n=4), and the inter-day RSD was 5% to 37%.

[0130] (n=12); when the spiked concentration of each substance was 10 ng / mL, the average recovery of the method ranged from 83% to 118%, the intra-day RSD was 3% to 27% (n=4), and the inter-day RSD was 15% to 38% (n=12). Therefore, considering the principle of economy, when the spiked concentration of the substance was 5 ng / mL, the detection method of the present invention had good accuracy, repeatability and reproducibility, and met the analytical requirements for the simultaneous extraction and detection of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine.

[0131] (3) Practical application test, the specific implementation steps are as follows: 16 urine samples from normal people are selected, and the sample processing method is synchronously extracted according to the method in Example 1, and the obtained samples are detected in positive ion mode in turn. In the process of detecting real samples, two spiked quality control samples, a 10ng / mL retest sample in the standard curve, an instrument blank sample (methanol) and a program blank sample are set to perform quality control and assurance. The test results are shown in Table 8. Among them, the detection rate is calculated according to LOQ as the quantitative limit.

[0132] Table 8 Test results of real samples (values ​​below LOQ are defined as LOQ / 2)

[0133]

[0134] As shown in Table 8, the detection rates of nine bisphenol diglycidyl ethers and five p-phenylenediamine antioxidants in urine ranged from 6% to 100%, with median concentrations ranging from 0.001 to 3.725 ng / mL and geometric means ranging from 0.002 to 1.632 ng / mL.

[0135] In summary, the method for simultaneous extraction and detection of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine provided by the present invention overcomes many shortcomings of existing detection technologies, especially the problems of cumbersome steps, long time consumption and single detection of existing detection methods. By improving the instrumental analysis method, the simultaneous extraction and detection of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine is achieved, which is simple, fast, economical and environmentally friendly. At the same time, the average recovery rate of this method ranges from 71% to 122%, and the intra-day and inter-day relative standard deviations are within 18% and 20%, respectively; in addition, the standard curve of this method has good current performance, r 2 Both were greater than or equal to 0.99; the method had high sensitivity, with LOD and LOQ being 0.003-0.012 ng / mL and 0.01-0.04 ng / mL, respectively.

[0136] In the embodiment of the present invention, by improving the sample pretreatment and liquid phase analysis method, the synchronous extraction and detection analysis of 9 bisphenol diglycidyl ethers and 5 p-phenylenediamine antioxidants in urine samples are achieved, which is simple and convenient, and solves the problem of cumbersome and time-consuming pretreatment steps. In addition, the method in the embodiment of the present invention does not require enzymatic hydrolysis, and only needs to add phase separation inducers and extractants for pretreatment, which greatly simplifies the steps and effectively improves the experimental efficiency of pretreatment. The method of the present invention is used to detect 14 substances, and the total time is about 14 minutes, which greatly saves sample detection time and is more economical and effective; at the same time, the method of the present invention does not require solid phase extraction column enrichment and purification, which is more economical and environmentally friendly. The method provided by the present invention provides a basic condition for further exploring the relationship between mixed exposure to pollutants and human health outcomes, and expands the scope of application of health risk assessment.

[0137] In summary, the present invention adopts an extraction method for pretreatment, and utilizes a phase separation inducer in combination with a specific extractant, so as to achieve fast and simple pretreatment, and greatly improve the detection efficiency; wherein, the phase separation inducer can induce the stratification of the organic phase and the aqueous phase, so that the target compound has a higher distribution coefficient in the original phase and the extractant phase, thereby achieving better extraction and separation effects; after a specific pretreatment step, the obtained pretreated sample is detected by ultra-high performance liquid chromatography-tandem mass spectrometry with a specific mobile phase, and bisphenol diglycidyl ether and p-phenylenediamine antioxidant can be detected simultaneously, thereby achieving simultaneous detection of the above two substances, and the detection efficiency is high.

Claims

1. A pretreatment method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, characterized in that: The following steps are involved: The sample to be tested, the extractant and the phase separation inducing agent are mixed, and the supernatant is taken after extraction and separation to obtain a pretreated sample; the extractant includes at least one of ethyl acetate, acetonitrile, tert-butyl methyl ether or dichloromethane.

2. The pretreatment method according to claim 1, characterized in that The bisphenol diglycidyl ether includes at least one of bisphenol A diglycidyl ether, bisphenol A (2,3-dihydroxypropyl) glycidyl ether, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A bis (3-chloro-2-hydroxypropyl) ether, bisphenol A bis (2,3-dihydroxypropyl) ether, bisphenol F diglycidyl ether, bisphenol F bis (2,3-dihydroxypropyl) ether or bisphenol F bis (3-chloro-2-hydroxypropyl) ether; And / or, the p-phenylenediamine antioxidant includes at least one of N-cyclohexyl-N'-phenyl-p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine, N-(1,3-dimethyl)butyl-N'-phenyl-p-phenylenediamine, N,N'-diphenyl-p-phenylenediamine or N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone.

3. The pretreatment method according to claim 1, characterized in that: The phase separation inducer includes at least one of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate or zinc sulfate; And / or, the sample to be tested is a urine sample.

4. The pretreatment method according to claim 1, characterized in that: The volume ratio of the sample to be tested to the extractant is 1:(2-10); And / or, the dosage ratio of the sample to be tested to the phase separation inducer is 1 mL: (0.1-0.3) g; And / or, the extraction and separation are performed 1 to 5 times.

5. A kit for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, characterized in that: The kit is prepared by the pretreatment method according to any one of claims 1 to 4.

6. A method for simultaneously detecting bisphenol diglycidyl ether and p-phenylenediamine antioxidant, characterized in that: The following steps are involved: S1. Pretreatment: pretreating the sample to be tested by the pretreatment method according to any one of claims 1 to 4, and using the obtained pretreated sample as the test sample; Or using the kit described in claim 5 as a test sample; S2. Detection: Using ultra high performance liquid chromatography-tandem mass spectrometry to detect the test sample obtained in step S1; the mobile phase used in the ultra high performance liquid chromatography-tandem mass spectrometry includes mobile phase A and mobile phase B, mobile phase A is selected from formic acid aqueous solution, and mobile phase B is selected from formic acid methanol solution.

7. The method according to claim 6, characterized in that In step S2, a standard curve of bisphenol diglycidyl ether and p-phenylenediamine antioxidant is established by the ultra-high performance liquid chromatography-tandem mass spectrometry, and the concentrations of bisphenol diglycidyl ether and p-phenylenediamine antioxidant are obtained by using the standard curve.

8. The method according to claim 6, characterized in that In step S2, the mass concentration of the formic acid aqueous solution in the mobile phase A of the ultra-high performance liquid chromatography-tandem mass spectrometry is 0.03-0.2%; And / or, in the mobile phase B of the ultra-high performance liquid chromatography-tandem mass spectrometry, the mass concentration of the formic acid methanol solution is 0.03-0.2%.

9. The method according to claim 6, characterized in that In step S2, the elution method used in the ultra-high performance liquid chromatography-tandem mass spectrometry is gradient elution; the elution procedure of the gradient elution is as follows: 0-2min: 90-95% mobile phase A, 5-10% mobile phase B; 2-5 min: 85-90% mobile phase A, 10-15% mobile phase B; 5-7min: 40-50% mobile phase A, 50-60% mobile phase B; 7-10min: 20-30% mobile phase A, 70-80% mobile phase B; 10-11 min: 0.1-1% mobile phase A, 99-99.9% mobile phase B; 11-11.5min: 0.1-1% mobile phase A, 99-99.9% mobile phase B; 11.5~14min: 95~99% mobile phase A, 1~5% mobile phase B.

10. The method according to claim 6, characterized in that In step S2, the detection conditions of the ultra-high performance liquid chromatography-tandem mass spectrometry method meet at least one of the following: 1) The ion source is selected from an electrospray ion source ESI; 2) the detection mode is selected from the positive ion mode; 3) The ion source temperature is 450-550°C; 4) The ionization voltage is 4000-5000V; 5) the collection method is selected from the multiple reaction detection mode; 6) The chromatographic column is selected from Agilent ZOBRAX SB-C18 chromatographic column; 7) The length of the chromatographic column is 80-120 mm, the inner diameter is 2-2.5 mm, and the diameter of the filler particles is 3-4 μm; 8) Column temperature is 20-30°C; 9) The injection volume is 1 to 3 μL; 10) The flow rate of the mobile phase is 0.2-0.3 mL / min.

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