Method for determining 91 pollutants in serum

Abstract

The invention provides a method for determining 91 pollutants in serum, and belongs to the technical field of analytical chemistry. According to the method, the serum sample is subjected to isotope labeling, standing for impurity removal, centrifugal constant volume and other treatments, and pollutants in the sample are efficiently extracted; then, the pollutants in the serum are detected by adopting an ultra-high performance liquid chromatography-mass spectrometry technology, so that rapid and accurate determination of 91 pollutants in the serum can be realized at the same time. The method disclosed by the invention is simple to operate and relatively good in separation effect, precision, accuracy and stability, and can be used for evaluating the exposure condition of pollutants in a large number of biological samples.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for determining 91 pollutants in serum. Background Art

[0002] Environmental pollutants refer to substances that are produced or enter the environment through human activities, change the normal composition and properties of the environment, and directly or indirectly cause harm to the human body. With the improvement of agricultural production technology and the development of modern industry, environmental pollutants have been integrated into people's lives along with many new chemical synthetic substances, and are widely exposed in the air, food and daily necessities. Environmental pollutants can enter the human body through various pathways such as diet, skin, oral and respiratory mucosa, and accumulate in the human body, thereby interfering with the synthesis, secretion, transportation and binding of natural hormones in the human body. They can also imitate the effects of natural hormones in the body to destroy the balance of human hormones, thereby causing various diseases in the human reproductive system, endocrine system, heart and nervous system, etc., posing a major threat to human health.

[0003] Bisphenol compounds are organic compounds containing two phenol groups. Common bisphenol compounds include bisphenol A, bisphenol F, bisphenol S, etc. Bisphenol compounds are widely used in plastic products, food packaging, personal care products, etc. Flame retardant compounds are a class of compounds widely used in various materials. Their function is to reduce the flame and smoke produced when the material burns to improve the flame retardant properties of the material. Common flame retardants include brominated flame retardants, nitrogen flame retardants, phosphorus flame retardants, etc. Phthalates are one of the main environmental pollutants in the world. Phthalates are often used in plastic products as plasticizers to improve the plasticity, durability, stability and other functional properties of the products. Perfluorinated and polyfluoroalkyl substances are a class of synthetic chemicals with fully or partially fluorinated alkyl chains and one or more polar functional groups. Due to their water and oil resistance, high temperature resistance and surface activity, perfluorinated and polyfluoroalkyl substances are widely used in non-stick kitchen utensils, food packaging, cleaning products and other products.

[0004] Environmental pollutant mass spectrometry detection technology is an analytical technology based on the principle of mass spectrometry. This technology can determine the type and content of pollutants in the sample by detecting and analyzing the mass spectrometry characteristics of the compounds in the sample. At present, different methods and instruments have been developed for pollutant mass spectrometry detection technology, mainly including gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. The development of pollutant mass spectrometry detection technology has made the detection and analysis of trace pollutants in the environment more accurate and sensitive. However, there are still some shortcomings in pollutant detection technology, such as high cost of detection equipment, complicated operation steps, and inability to directly analyze certain compounds. Therefore, it is necessary to continue to improve and perfect mass spectrometry analysis technology, increase analysis speed, reduce costs, and simplify operation procedures to meet the needs of pollutant detection in different scenarios. At present, the detection technology of environmental pollutants is not sensitive, the detection substances are single, and there is a lack of detection methods for comprehensive evaluation of environmental pollutant exposure.

[0005] At present, there is no unified standard for the detection of 91 pollutants in serum. Although there are patents for related detection of pollutants in China, there is no detection method for 91 pollutants in serum. Therefore, in order to fill the technical gap of the determination of 91 pollutants in serum, the present invention establishes a method for processing and detecting 91 pollutants in serum quickly, accurately and stably, which provides a basis and premise for further exploration of the relationship between pollutants and human health, and has broad medical and public health significance. Summary of the invention

[0006] In view of the above shortcomings of the prior art, the present invention provides a method for determining 91 pollutants in serum. The present invention efficiently extracts pollutants in the sample by performing isotope internal standardization, static impurity removal, and centrifugation to fix the volume of the serum sample; and then uses ultra-high performance liquid chromatography-mass spectrometry to detect the pollutants in the serum, thereby achieving rapid and accurate determination of the content of 91 pollutants in the serum.

[0007] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0008] A method for determining 91 pollutants in serum comprises the following steps: adding an internal standard solution containing bisphenol isotope compounds, flame retardant isotope compounds, phthalate isotope compounds, and perfluoro / polyfluoroalkyl isotope compounds to a serum sample, adding a precipitant, mixing and centrifuging, and collecting supernatant 1; standing supernatant 1 at 4°C and centrifuging to collect supernatant 2; drying supernatant 2, adding a reconstitution solution, mixing and centrifuging, and collecting supernatant 3 for ultra-high performance liquid chromatography-mass spectrometry detection;

[0009] The precipitant is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 3:7;

[0010] The reconstitution solution is acetonitrile and water, and the volume ratio of acetonitrile to water is 2:8;

[0011] The chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column, and the mobile phases used for elution include mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.01% acetic acid and 25% methanol, and the mobile phase B is an acetonitrile solution containing 0.01% acetic acid and 25% methanol.

[0012] The present invention uses a specific pretreatment method (using methanol and acetonitrile as precipitants, acetonitrile and water as resolvents, standing the serum sample for impurity removal, and centrifuging to fix the volume) to process the serum sample, and cooperates with specific elution conditions (using an aqueous solution containing 0.01% acetic acid and 25% methanol as mobile phase A, and an acetonitrile solution containing 0.01% acetic acid and 25% methanol as mobile phase B) to elute the pollutants, thereby achieving simultaneous qualitative and quantitative detection of at least 91 pollutants.

[0013] Furthermore, the pollutants include one or more of bisphenol compounds, flame retardant compounds, phthalate compounds, and perfluoro / polyfluoroalkyl compounds.

[0014] Furthermore, the bisphenol compound includes one or more of bisphenol A (BPA), bisphenol P (BPP), bisphenol S (BPS), bisphenol AP (BPAP), hexafluorobisphenol A (BPAF), bisphenol B (BPB), bisphenol F (BPF), bisphenol Z (BPZ), diisononyl cyclohexane-1,2-dicarboxylate (DINCH), diphenyl phosphate (DPHP), hexabromocyclododecane (HBCDD), tribromophenol (TBP), tetramethylbisphenol A (TMBPA), bisphenol C (BPC), bisphenol M (BPM), bisphenol BP (BPBP), and bisphenol fluorene (BPFL).

[0015] Furthermore, the flame retardant compounds include aluminum diethylphosphinate (ALPI), tetraphenyl resorcinol diphosphate (BPDPP), isooctyl diphenyl phosphate (EHDPP), tricresyl phosphate (TCP), tributoxyethyl phosphate (TBOEP), bisphenol A diphenyl phosphate (BPA-BDPP), diphenyl phosphate (DCP), 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO), trioctyl phosphate (TEHP), triethyl phosphate (TEP), phosphorus One or more of trimethyl phosphate (TMP), triphenyl phosphate (TPHP), triisobutyl phosphate (TIBP), tributyl phosphate (TNBP), tri(2-chloropropyl) phosphate (TCIPP), tri(1,3-dichloroisopropyl) phosphate (TDCIPP), tri(2-chloroethyl) phosphate (TCEP), potassium 3-phenylsulfonylbenzenesulfonate (KSS), decabromodiphenyl ether (DO), tribromophenol (TBP), 2,3,5,6-tetrabromo-p-xylene (TBBPA), and decabromodiphenyl ethane (DBDPE).

[0016] Furthermore, the phthalate compounds include di(2-ethylhexyl) phthalate (DEHP), dioctyl phthalate (DOP), diisobutyl phthalate (DIBP), dicyclohexyl phthalate (DCHP), dimethoxyethyl phthalate (DMEP), dimethyl phthalate (DMP), dipentyl phthalate (DPP), diethyl phthalate (DEP), dinonyl phthalate (DNP), butyl benzyl phthalate (BBP), diisononyl phthalate (DINP), dibutyl phthalate (DBP), diisodecyl phthalate (DIDP), diisopropyl phthalate (DIPRP), dibenzyl phthalate (DBZP), dipropyl phthalate (DPRP), dibutyl phthalate (DBP), diisodecyl phthalate (DIDP), diisopropyl phthalate (DIPRP ... One or more of dibutyl phthalate (DNHP), diallyl phthalate (DAP), diisopentyl phthalate (DIPP), dibutoxyethyl phthalate (DBEP), monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), monoisobutyl phthalate (MIBP), monobenzyl phthalate (MBZP), monoethylhexyl phthalate (MEHP), diheptyl phthalate (DNHPP), diheptyl phthalate (DIHPP), diundecyl 1,2-benzenedicarboxylate (DUP), butylcyclohexyl phthalate (BCP), n-pentyl isopentyl phthalate (PIPP), monoisopropyl phthalate (MIPP), and monocyclohexyl phthalate (MCHP).

[0017] Furthermore, the perfluoro / polyfluoroalkyl compounds include heptafluorobutyric acid (PFBA), nonafluoropentanoic acid (PFPeA), undecafluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), nonadecafluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), perfluorooctadecanoic acid (PFOcDA), perfluorobutyl sulfonic acid (PFBS), heptadecafluorooctane sulfonic acid (PFOS), perfluorodecane sulfonic acid (PFDS), perfluorooctane sulfonamide (PFOSA), perfluorooctane sulfonyl fluoride (FX-8), and perfluorohexane sulfonic acid (PFHxS).

[0018] Furthermore, the internal standard solution is deuterated ethylhexyl phthalate (MEHP-D 4 ), perfluorooctanoic acid- 13 C 4 (PFOA- 13 C 4 ), triethyl phosphate-D 15 (TEP-D 15 )、Bisphenol A- 13 C 12 (BPA- 13 C 12 ) mixed solution.

[0019] Furthermore, the added amount of the bisphenol isotope compound, flame retardant isotope compound, phthalate isotope compound, and perfluoro / polyfluoroalkyl isotope compound is 1 ng per 100 μL of serum.

[0020] Furthermore, the volume ratio of the serum sample to the precipitant is 1:4.

[0021] Furthermore, the volume ratio of the serum sample to the reconstitution solution is 1:1.

[0022] Furthermore, the drying is carried out under a nitrogen flow.

[0023] Furthermore, the ultra-high performance liquid chromatography condition parameters are as follows:

[0024] C18 column specifications: 100 mm × 2.1 mm, 2.0 μm;

[0025] Column temperature: 40 °C;

[0026] Injection volume: 5 μL;

[0027] Flow rate of mobile phase: 0.3 mL / min;

[0028] Elution gradient: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 - 27 min, 98% B; 27 -28 min, 98% -5% B; 28 - 35 min, 5% B.

[0029] Furthermore, the condition parameters of the mass spectrometry detection are as follows:

[0030] Ionization source: electrospray ionization source;

[0031] Ionization mode: positive and negative switching mode;

[0032] Collision gas: ultra-high purity nitrogen;

[0033] Scan mode: Multiple reaction monitoring mode.

[0034] Furthermore, the method for determining 91 pollutants in serum comprises the following steps:

[0035] (1) Serum pretreatment: 1 μL of isotope internal standard solution and 400 μL of precipitant (methanol:acetonitrile volume ratio of 3:7) were added to 100 μL of serum sample, and the mixture was thoroughly vortexed for 5 min, and then centrifuged at 12000 rpm for 10 min at 4 °C to collect supernatant 1; supernatant 1 was allowed to stand at 4 °C for 30 min, and then centrifuged at 12000 rpm for 10 min at 4 °C to collect supernatant 2; supernatant 2 was dried under nitrogen flow, and then redissolved in 100 μL of acetonitrile:water volume ratio of 2:8, and thoroughly vortexed for 5 min, and then centrifuged at 12000 rpm for 10 min at 4 °C to collect supernatant 3 for determination by the instrument, with an injection volume of 5 μL; the isotope internal standard solution was 1 μg / mL deuterated monoethylhexyl phthalate (MEHP-D 4 ), perfluorooctanoic acid- 13 C 4 (PFOA- 13 C 4 ), triethyl phosphate-D 15 (TEP-D 15 )、Bisphenol A- 13 C 12 (BPA- 13 C 12 ) mixed solution (i.e., in the mixed solution, MEHP-D 4 PFOA- 13 C 4 、TEP-D 15 、BPA- 13 C12 The concentration of each was 1 μg / mL).

[0036] (2) 91 pollutants were detected by ultra-high performance liquid chromatography-mass spectrometry: the ultra-high performance liquid chromatography method used a C18 chromatographic column with a size of 100 mm × 2.1 mm, 2.0 μm, a column temperature of 40 °C, and mobile phases A and B. Mobile phase A was an aqueous solution containing 0.01% acetic acid and 25% methanol, and mobile phase B was an acetonitrile solution containing 0.01% acetic acid and 25% methanol. The flow rate of the mobile phase was 0.3 mL / min. The mass spectrometry detection conditions were as follows: the ionization source was an electrospray ionization source, the ionization mode was a positive and negative switching mode, the collision gas was ultra-high purity nitrogen, and the scanning mode was a multiple reaction monitoring mode.

[0037] Furthermore, the method also includes quantitative analysis of pollutants, and the quantitative analysis includes the following steps: preparing standard solutions containing 91 pollutants at different concentrations; performing ultra-high performance liquid chromatography-mass spectrometry detection on the standard solutions containing 91 pollutants at different concentrations; drawing a standard curve based on the concentrations of the standard solutions of 91 pollutants and the corresponding peak areas of ultra-high performance liquid chromatography-mass spectrometry; based on the peak time and peak area of ​​the pollutants in the serum sample, comparing the peak time and peak area of ​​the standard, and adjusting the peak area according to the internal standard isotope content, quantitatively analyzing the 91 pollutants in the serum sample.

[0038] Compared with the prior art, the present invention is beneficial in that:

[0039] (1) The present invention performs isotope internal standardization, static impurity removal, centrifugal volume determination on serum samples, and then uses ultra-high performance liquid chromatography-mass spectrometry to detect pollutants in serum, thereby achieving rapid and accurate simultaneous determination of the contents of 91 pollutants in serum.

[0040] (2) The present invention can efficiently extract pollutants from serum samples through simple pretreatment and perform qualitative and quantitative detection thereof. The method of the present invention has the advantages of simplicity, high efficiency and low cost, and can be used to evaluate the exposure of pollutants in a large number of biological samples.

[0041] (3) The present invention uses an aqueous solution containing 0.01% acetic acid and 25% methanol and an acetonitrile solution containing 0.01% acetic acid and 25% methanol as mobile phases in ultra-high performance liquid chromatography-mass spectrometry detection, which can improve the life of the chromatographic column and the separation degree of the target substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a scatter plot of the retention time of pollutants eluted with different mobile phases. DETAILED DESCRIPTION

[0043] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0044] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The present invention provides a method for determining 91 pollutants in serum, comprising the following steps: adding an internal standard solution containing a bisphenol isotope compound, a flame retardant isotope compound, a phthalate isotope compound, and a perfluoro / polyfluoroalkyl isotope compound to a serum sample, adding a precipitant, mixing and centrifuging, and collecting a supernatant 1; standing the supernatant 1 at 4°C and centrifuging to collect a supernatant 2; drying the supernatant 2, adding a reconstitution solution, mixing and centrifuging, and collecting a supernatant 3 for ultra-high performance liquid chromatography-mass spectrometry detection;

[0046] The precipitant is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 3:7;

[0047] The reconstitution solution is acetonitrile and water, and the volume ratio of acetonitrile to water is 2:8;

[0048] The chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column, and the mobile phases used for elution include mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.01% acetic acid and 25% methanol, and the mobile phase B is an acetonitrile solution containing 0.01% acetic acid and 25% methanol.

[0049] In some examples, the pollutants include one or more of bisphenol compounds, flame retardant compounds, phthalate compounds, and perfluoro / polyfluoroalkyl compounds. In the following specific examples, information on the 91 pollutants involved is shown in Table 1.

[0050] In the following specific examples, the isotope internal standard solution is deuterated phthalic acid monoethylhexyl ester (MEHP-D 4 ), perfluorooctanoic acid- 13 C 4 (PFOA- 13 C 4 ), triethyl phosphate-D15 (TEP-D 15 )、Bisphenol A- 13 C 12 (BPA- 13 C 12 ), the information of the above four isotopic internal standard compounds is shown in Table 1.

[0051] Table 1: Information on 91 pollutants and 4 isotope-labeled compounds

[0052]

[0053] Continued

[0054] Continued

[0055] In the following specific examples, the ultra-high performance liquid chromatography condition parameters are as follows:

[0056] Analytical instrument: LC-30AD system;

[0057] C18 column: Shimadzu Shim-pack GIST C18 column, 100 mm × 2.1 mm, 2.0 μm;

[0058] Column temperature: 40 °C;

[0059] Injection volume: 5 μL;

[0060] Flow rate of mobile phase: 0.3 mL / min;

[0061] Elution gradient: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 - 27 min, 98% B; 27 -28 min, 98% -5% B; 28 - 35 min, 5% B.

[0062] In the following specific examples, the mass spectrometry parameters are as follows:

[0063] Analytical instrument: triple quadrupole Shimadzu 8045 mass spectrometer;

[0064] Ionization source: electrospray ionization source;

[0065] Ionization mode: positive and negative switching mode;

[0066] Collision gas: ultra-high purity nitrogen;

[0067] Scan mode: Multiple reaction monitoring mode.

[0068] In the following specific examples, various raw materials, reagents, instruments and equipment used can be purchased from the market or prepared by existing methods.

[0069] Example 1

[0070] In this example, serum samples of 76 people, totaling 38 pairs of mothers and infants, were collected for pollutant determination in the following steps:

[0071] The serum samples were taken out of the -80 ℃ freezer, thawed on ice, and the precipitant (methanol: acetonitrile = 3:7); the reconstitution solution (acetonitrile: water = 2:8); 1 μg / mL MEHP-D 4 PFOA- 13 C 4 、TEP-D 15 、BPA- 13 C 12 Mixed solution (in the mixed solution, MEHP-D 4 PFOA- 13 C 4 、TEP-D 15 、BPA- 13 C 12 The concentration of each was 1 μg / mL).

[0072] Take 100 μL of serum sample in a centrifuge tube, add 1 μL of isotope internal standard solution and 400 μL of precipitant, vortex for 5 min, and centrifuge at 12000 rpm for 10 min; take supernatant 1 and add it to a new centrifuge tube, put it in a 4 ℃ refrigerator for 30 min, and centrifuge it at 12000 rpm for 10 min; take supernatant 2 and add it to a new centrifuge tube, blow dry with a nitrogen blower, add 100 μL of reconstitution solution, and centrifuge it at 12000 rpm for 10 min; take supernatant 3 and transfer it to the injection bottle, and wait for injection at 4 ℃.

[0073] The LC-MS / MS system for detection consisted of an LC-30AD ultra-high performance liquid chromatograph and a Shimadzu 8045 mass spectrometer. The chromatographic column used for ultra-high performance liquid chromatography was a Shimadzu Shim-pack GIST C18 column (100 mm×2.1 mm, 2.0 μm). The pollutants were separated at a column temperature of 40 ℃ and a flow rate of 0.3 mL / min, and the injection volume was 5 μL. The mobile phases used for elution consisted of an aqueous solution containing 0.01% acetic acid and 25% methanol (mobile phase A) and an acetonitrile solution containing 0.01% acetic acid and 25% methanol (mobile phase B). The separation gradient was as follows: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 - 27 min, 98% B; 27 - 28 min, 98% -5% B; 28 - 35 min, 5% B.

[0074] The exposure concentrations of pollutants detected in maternal serum and cord blood serum samples are shown in Table 2 .

[0075] Table 2: Distribution of contaminants in maternal and cord blood serum (ng / mL)

[0076]

[0077] As shown in Table 2, Example 1 detected 38 pollutants in serum samples, including 5 bisphenol compounds, 15 organophosphorus flame retardants, 4 phthalates and 14 perfluoro / polyfluoroalkyl substances. The present invention defines the content corresponding to a signal-to-noise ratio of 3 as the limit of detection (LOD), and the content corresponding to a signal-to-noise ratio of 10 as the limit of quantification (LOQ). The LOD range of the 38 pollutants in Example 1 is 0.001 ng / mL to 0.007 ng / mL; the LOQ range is 0.003 ng / mL to 0.025 ng / mL.

[0078] The inventors also used different re-dissolving solutions (such as: the volume ratio of acetonitrile to water is 1:9; or, the volume ratio of acetonitrile to water is 5:5) to re-dissolve the sample. The experimental results show that the use of other re-dissolving solutions to re-dissolve the sample failed to achieve the effect of the present invention.

[0079] The inventors prepared 91 pollutants in Table 1 into 100 ng / mL standard solutions, the solvent of which was acetonitrile: water (v:v) = 2: 8, and took 0.1 mL of each solution to form a mixed solution. 1 μg / mL of each isotope internal standard solution (MEHP-D 4 PFOA- 13C 4 、TEP-D 15 、BPA- 13 C 12 ), the mixed solution was used for ultra-high performance liquid chromatography-mass spectrometry detection. The LC-MS / MS system for detection consisted of an LC-30AD ultra-high performance liquid chromatograph and a Shimadzu 8045 mass spectrometer; the chromatographic column used for ultra-high performance liquid chromatography was a Shimadzu Shim-pack GIST C18 chromatographic column (100 mm×2.1 mm, 2.0 μm), and the mixed solution was separated under the conditions of a column temperature of 40 ℃ and a flow rate of 0.3 mL / min, and the injection volume was 5 μL; the mobile phase used for elution consisted of an aqueous solution containing 0.01% acetic acid and 25% methanol (mobile phase A) and an acetonitrile solution containing 0.01% acetic acid and 25% methanol (mobile phase B), and the separation gradient was as follows: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 -27 min, 98% B; 27 - 28 min, 98% -5% B; 28 - 35 min, 5% B. The test results are shown in Table 3.

[0080] Table 3: Detection results of 91 pollutants and isotope-labeled compounds in standard solutions

[0081]

[0082] Continued

[0083] Continued

[0084] The test results in Table 3 show that the method of the present invention can detect 91 pollutants simultaneously and has a good separation effect.

[0085] The inventors used spiked recovery to evaluate the recovery and matrix effect of 91 pollutants, that is, after adding standard substances to the serum, the serum was treated by the pretreatment method described in Example 1, and then the pollutants were detected by the detection method described in Example 1. The recovery of the detected pollutants ranged from 69.3 ± 1.3% to 143.1 ± 6.7%, which can meet the requirements of sample analysis; the range of matrix effect was 64.4 ± 5.1% to 132.3 ± 2.6%, indicating that the enhancement and inhibition effects of the matrix on the mass spectrometry signal can be ignored.

[0086] The inventors prepared a series of mixed solutions of pollutant standards with different contents, and added equal amounts of isotopic internal standards (MEHP-D4, PFOA- 13 C4 、TEP-D 15 、BPA- 13 C 12 1 μg / mL), and the standard curve was constructed by plotting the relationship between the peak area ratio (pollutant standard / isotope) and the content of the pollutant standard. The results showed that the linear correlation coefficient of the standard curves of all pollutants was R 2 All of them are greater than 0.99, which indicates that the analytical method of the present invention has good linearity. In order to evaluate the accuracy and precision of the method of the present invention, the inventors selected three concentrations of low (1 ng / mL), medium (10 ng / mL), and high (100 ng / mL) from the linear range of the standard curve for intra-day and inter-day precision experiments. The results showed that the relative error (RE) of accuracy was less than 20.3%, and the relative standard deviation (RSD) within and between days was less than 9.0% and 17.6%, respectively, which indicates that the method of the present invention has good accuracy and precision.

[0087] In addition, the inventors prepared 91 pollutants in Table 1 into 100 ng / mL standard solutions, the solvent of the standard solution was acetonitrile: water (v:v) = 2: 8, 0.1 mL of each solution was mixed to form a mixed solution, and 1 μg / mL of isotope internal standard solution (MEHP-D 4 PFOA- 13 C 4 、TEP-D 15 、BPA- 13 C 12 ) After that, the mixed solution was used for ultra-high performance liquid chromatography-mass spectrometry detection. The LC-MS / MS system for detection consisted of an LC-30AD ultra-high performance liquid chromatograph and a Shimadzu 8045 mass spectrometer; the chromatographic column used for ultra-high performance liquid chromatography was a Shimadzu Shim-pack GIST C18 chromatographic column (100 mm×2.1 mm, 2.0 μm), and the mixed solution was separated under the conditions of a column temperature of 40 ℃ and a flow rate of 0.3 mL / min, and the injection volume was 5 μL; the mobile phase used for this elution was an aqueous solution containing 0.01% acetic acid and 25% methanol as mobile phase A, and an acetonitrile solution containing 0.01% acetic acid as mobile phase B, and the separation gradient was as follows: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 - 27min, 98% B; 27 - 28 min, 98% -5% B; 28 - 35 min, 5% B. A scatter plot is drawn with the pollutant number as the horizontal axis and the retention time as the vertical axis to represent the separation effect. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the separation effect of pollutants is better when the mobile phase of the present invention is used to elute the pollutants, indicating that the method of the present invention can achieve effective separation of 91 types of pollutants.

[0088] In summary, the method of the present invention is simple, direct, low-cost, and can quickly and accurately detect and quantitatively analyze 91 pollutants in serum. In addition, the method of the present invention is simple and rapid and can be used to assess the exposure of pollutants in a large number of biological samples.

[0089] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for determining 91 pollutants in serum, characterized in that: The method comprises the following steps: adding an internal standard solution containing a bisphenol isotope compound, a flame retardant isotope compound, a phthalate isotope compound, and a perfluoro / polyfluoroalkyl isotope compound to a serum sample, adding a precipitant, mixing and centrifuging, and collecting supernatant 1; allowing supernatant 1 to stand and then centrifuging to collect supernatant 2; drying supernatant 2, adding a reconstitution solution, mixing and then centrifuging, and collecting supernatant 3 for ultra-high performance liquid chromatography-mass spectrometry detection; The precipitant is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 3:7; The reconstitution solution is acetonitrile and water, and the volume ratio of acetonitrile to water is 2:8; The chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column, and the mobile phases used for elution include mobile phase A and mobile phase B; wherein the mobile phase A is an aqueous solution containing 0.01% acetic acid and 25% methanol, and the mobile phase B is an acetonitrile solution containing 0.01% acetic acid and 25% methanol.

2. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The pollutants include one or more of bisphenol compounds, flame retardant compounds, phthalate compounds, and perfluoro / polyfluoroalkyl compounds.

3. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The internal standard solution is MEHP-D4, PFOA- 13 C4, TEP-D 15 、BPA- 13 C 12 of mixed solution.

4. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The added amount of the bisphenol isotope compound, the flame retardant isotope compound, the phthalate isotope compound, and the perfluoro / polyfluoroalkyl isotope compound is 1 ng per 100 μL of serum.

5. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The volume ratio of the serum sample to the precipitant is 1:

4.

6. The method for determining 91 pollutants in serum according to claim 1, characterized in that: The volume ratio of the serum sample to the reconstitution solution is 1:

1.

7. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The condition parameters of the ultra-high performance liquid chromatography are as follows: C18 column specifications: 100 mm × 2.1 mm, 2.0 μm; Column temperature: 40 °C; Injection volume: 5 μL Flow rate of mobile phase: 0.3 mL / min; Elution gradient: 0 - 3 min, 5% B; 3 - 20 min, 5 - 98% B; 20 - 27 min, 98% B; 27 - 28min, 98% -5% B; 28 - 35 min, 5% B.

8. A method for determining 91 pollutants in serum according to claim 1, characterized in that: The mass spectrometry detection conditions are as follows: Ionization source: electrospray ionization source; Ionization mode: positive and negative switching mode; Collision gas: ultra-high purity nitrogen; Scan mode: Multiple reaction monitoring mode.

9. The method for determining 91 pollutants in serum according to claim 1, characterized in that: The method for determining 91 pollutants in serum comprises the following steps: (1) Serum pretreatment: 1 μL of isotope internal standard solution and 400 μL of precipitant (methanol:acetonitrile volume ratio of 3:7) were added to 100 μL of serum sample, and the mixture was thoroughly vortexed for 5 min, then centrifuged at 12000 rpm for 10 min at 4 °C, and supernatant 1 was collected; supernatant 1 was allowed to stand at 4 °C for 30 min, then centrifuged at 12000 rpm for 10 min at 4 °C, and supernatant 2 was collected; supernatant 2 was dried under nitrogen flow, then redissolved in 100 μL of acetonitrile:water volume ratio of 2:8, and thoroughly vortexed for 5 min, then centrifuged at 12000 rpm for 10 min at 4 °C, and supernatant 3 was collected for determination on the instrument, with an injection volume of 5 μL; the isotope internal standard solution was 1 μg / mL MEHP-D4 and PFOA- 13 C4, TEP-D 15 、BPA- 13 C 12 A mixed solution; (2) 91 pollutants were detected by ultra-high performance liquid chromatography-mass spectrometry: the ultra-high performance liquid chromatography method used a C18 chromatographic column with a specification of 100 mm × 2.1 mm, 2.0 μm, a column temperature of 40 °C, and mobile phases A and B. Mobile phase A was an aqueous solution containing 0.01% acetic acid and 25% methanol, and mobile phase B was an acetonitrile solution containing 0.01% acetic acid and 25% methanol. The flow rate of the mobile phase was 0.3 mL / min. The mass spectrometry detection conditions were as follows: the ionization source was an electrospray ionization source, the ionization mode was a positive and negative switching mode, the collision gas was ultra-high purity nitrogen, and the scanning mode was a multiple reaction monitoring mode.

10. The method for determining 91 pollutants in serum according to claim 1, characterized in that: The method also includes quantitative analysis of pollutants, and the quantitative analysis includes the following steps: preparing standard solutions containing 91 pollutants of different concentrations; performing ultra-high performance liquid chromatography-mass spectrometry detection on the standard solutions containing the 91 pollutants of different concentrations; drawing a standard curve according to the concentrations of the standard solutions of the 91 pollutants and the corresponding peak areas of the ultra-high performance liquid chromatography-mass spectrometry; and performing quantitative analysis on the pollutants in the serum sample according to the peak time and peak area of ​​the pollutants in the serum sample, compared with the peak time and peak area of ​​the standard, and adjusting the peak area according to the internal standard isotope content.

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