A method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people

Abstract

The present invention proposes a method for preparing a quality control sample of phthalate plasticizer metabolites in urine of the Chinese population, which relates to the technical field of chemical exposure risk assessment and pollutant human monitoring. The urine from multiple healthy volunteers is screened and mixed, and the urine is purified by freezing and multi-stage filtration. Then, phthalate metabolites of corresponding components are added to the purified urine, and after sufficient mixing, a quality control sample of phthalate plasticizer metabolites in real urine that matches the exposure index and level of the general population in my country is obtained. The method proposed in the present invention has a simple preparation process, simple experimental conditions and operation, economical manufacturing cost, and a wide range of practical applications. At the same time, the quality control sample prepared by the present invention has good uniformity and stability, and can be used for quality control in the determination process of phthalate metabolites in urine of the general population in my country.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical exposure risk assessment and pollutant human biological monitoring, and in particular to a method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people. Background Art

[0002] Phthalates (PAEs) are the most common plasticizers, which are widely added to various plastic products such as packaging materials, electrical appliances, toys, etc. to increase the plasticity of these products. PAEs are generally added to plastic products in a non-chemically bound manner, and can be continuously released by leaching, leaching, evaporation, etc. during the production, processing, use and recycling of related products. Food, breathing, drinking water, medication and personal care products are all important ways for people to ingest PAEs. PAEs have been proven to be a type of endocrine disruptor with potential reproductive toxicity, developmental toxicity and neurotoxicity, which can cause health hazards to the human body and increase the risk of obesity, asthma and diabetes.

[0003] Human biomonitoring is the gold standard for chemical exposure assessment. The most commonly used method for PAEs exposure assessment is to measure the concentration of PAEs metabolites in human urine and then estimate the PAEs prototype. At present, this method is widely used in PAEs population monitoring projects carried out in many countries and regions, such as the US National Biomonitoring Project, the German Environmental Survey Project, the Canadian Health Measurement Survey Project, the EU Human Biomonitoring Initiative, and my country's National Human Biomonitoring Project. Accurately determining the concentration level of PAEs metabolites in urine is the key and basis for PAEs exposure assessment. The urine sample matrix is ​​complex, and the detection of PAEs metabolites at extremely low concentrations (trace levels) also increases the difficulty of laboratory analysis.

[0004] To ensure the accuracy and comparability of the analysis results, quality control can be strengthened by means of spike recovery, participation in proficiency verification or proficiency comparison projects of authoritative institutions, and determination of quality control samples or standard reference materials. Among these, the determination of quality control samples or standard reference materials as a quality control method is the most practical and widely recognized. At present, urine quality control samples containing PAEs metabolites are very limited. Only NIST 3672 (smokers) and NIST 3673 (non-smokers) produced by the National Institute of Standards and Technology of the United States are available for use. NIST 3672 and NIST 3673 have homologous components with the samples being tested, but there are still many problems in their use in my country, such as (1) standard reference materials rely on imports, which are expensive and have a long transportation cycle, resulting in high testing costs; (2) standard reference materials require storage at -80°C, and it is difficult to achieve the relevant cold chain during transportation, which may affect the quality of samples; (3) standard reference materials are prepared based on urine from foreign populations, and the PAEs metabolite indicators do not match the PAEs metabolite indicators and exposure levels of the Chinese population. For example, in NIST 3673, the concentrations of mono-n-butyl phthalate (mnBP) and mono-isobutyl phthalate (miBP) are 5.18 ng / mL and 11.2 ng / mL, respectively. However, studies have shown that the median values ​​(50% percentile concentrations) of the above two indicators in the Chinese population are 38.5-57.6 ng / mL and 47.1-77.6 ng / mL, respectively. Therefore, using NIST as a quality control sample for quality control will be limited by differences in detection indicators and concentrations. Most importantly, there is still a lack of quality control samples or standard reference materials that match the detection indicators and concentrations of PAEs metabolites in the general population of my country. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a quality control sample of phthalate plasticizer metabolites in urine of the Chinese population. The quality control sample prepared by the method has good uniformity and stability, and the detection indexes and concentrations involved in the quality control sample match well with the exposure level of phthalate metabolites in urine of the general population of my country. It is suitable for carrying out the quality control of the experimental process of determining phthalate metabolites in urine of the Chinese population or comparative analysis of results.

[0006] To achieve the above content, the technical solution of the present invention is as follows.

[0007] 1. A method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people, characterized in that it comprises the following steps:

[0008] (1) Prepare standard working solutions of monoethyl phthalate (mEP), mono-n-butyl phthalate (mnBP), mono-isobutyl phthalate (miBP), mono-(2-ethyl-5-oxyhexyl) phthalate (mEOHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (mEHHP), and mono-(2-ethylhexyl) phthalate (mEHP) respectively for standby use;

[0009] (2) Preparation of low-concentration urine samples, including urine collection, low-concentration urine screening, low-concentration urine mixing and natural precipitation;

[0010] (3) Filtration of low-concentration urine samples, including coarse filtration and fine filtration;

[0011] (4) Urine sample determination and spiking: To determine the concentration of phthalate metabolites in urine, add the standard working solution to the filtered urine;

[0012] (5) Mixing;

[0013] (6) Repackaging.

[0014] The urine quality control sample prepared by the above method has an intra-bottle precision of less than 7.6% and an inter-bottle precision of less than 5.2%. When stored at below -20°C, it can be stably stored for 9 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a flow chart of the quality control sample preparation method of the present invention;

[0016] Figure 2 The difference between the effects of natural precipitation at 4°C and precipitation at room temperature in the quality control sample preparation method of the present invention;

[0017] Figure 3 Mix at 4°C in the quality control sample preparation method of the present invention;

[0018] Figure 4 Mixing in the quality control sample preparation method of the present invention;

[0019] Figure 5 The stability of substance 1 (mEP) in the quality control sample of the present invention;

[0020] Figure 6 The stability of substance 2 (miBP) in the quality control sample of the present invention;

[0021] Figure 7 The stability of substance 3 (mnBP) in the quality control sample of the present invention;

[0022] Figure 8 The stability of substance 4 (mEOHP) in the quality control sample of the present invention;

[0023] Fig. 9 The stability of substance 5 (mEHHP) in the quality control sample of the present invention;

[0024] Fig.10 This is the stability of substance 6 (mEHP) in the quality control sample of the present invention. DETAILED DESCRIPTION

[0025] To better illustrate the objectives, technical solutions and advantages of the embodiments of the present invention, this section clearly and completely describes the technical solutions in the embodiments of the present invention.

[0026] The present invention provides a method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people. The phthalate metabolites involved mainly include: monoethyl phthalate (mEP), mono-n-butyl phthalate (mnBP), monoisobutyl phthalate (miBP), mono(2-ethyl-5-oxyhexyl) phthalate (mEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (mEHHP), and mono(2-ethylhexyl) phthalate (mEHP).

[0027] The quality control sample prepared by the method of the present invention has good uniformity and stability.

[0028] The present invention adopts the quality control sample prepared by the real urine matrix of the general population, and can eliminate the influence of the matrix effect on the determination during the determination process, and can be used for quality control in the determination process and analyze the analytical deviation caused by various reasons.

[0029] Example:

[0030] This embodiment provides a method for preparing a quality control sample of phthalate plasticizer metabolites in urine of Chinese people. Since the content introduced in this section belongs to the embodiments of the present invention, it should not be regarded as limiting the scope of each phthalate metabolite in the quality control sample.

[0031] like Figure 1-4 As shown, a method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people is provided, and the preparation process is as follows:

[0032] (1) Standard configuration

[0033] The 6 PAEs metabolite standards involved in this embodiment were purchased from AccuStandard, USA, and the isotope internal standards were purchased from Cambridge Isotope Laboratories, USA. The concentrations of the 6 PAEs metabolite standards and internal standards are all 100 μg / mL. The 6 standards were diluted to 25.0 mL with methanol to prepare a 4 μg / mL single standard solution. Take 1 mL of each of the 6 internal standard stock solutions and mix them, then dilute to 25.0 mL with methanol to prepare a 4.0 μg / mL mixed internal standard solution of 6 PAEs metabolites.

[0034] (2) Low-concentration urine mixing and natural precipitation

[0035] a. Urine collection

[0036] (2.1) Recruit 20 volunteers, including male, female, smokers and non-smokers, to collect urine. All volunteers are in good health and have not taken any medication within 7 days. The urine cup used is made of polypropylene.

[0037] (2.2) Each volunteer collects approximately 100 mL of urine (morning urine / random urine). The collected urine can be stored at 4°C for one week.

[0038] b. Low concentration urine screening

[0039] (2.3) Take 2 mL of each urine sample and transfer it to 20 5 mL centrifuge tubes. Add 0.5 mL of ammonium acetate (1 mol / L), 20 μL of β-glucuronidase, and 40 ng of isotope internal standard to the urine samples, mix well, and then enzymolyze at 37°C for 2 h.

[0040] (2.4) Activate the MAX solid phase extraction column with 3 mL of methanol and 3 mL of water, and pass the enzymatically hydrolyzed urine sample through the column. After passing through the column, rinse with 3 mL of water and 3 mL of methanol in sequence; then elute with 2 mL of methanol containing 2% formic acid. Dilute the eluate 5 times with 5% acetonitrile water and measure it using a liquid chromatography-tandem triple quadrupole mass spectrometer.

[0041] (2.5) The chromatographic column was Waters ACQUITY UPLC BEH Phenyl, with physical property parameters: 100 mm × 2.1 mm, 1.7 μm. Mobile phase A was 0.1% acetic acid in water (v / v, volume ratio), and mobile phase B was 0.1% acetic acid in acetonitrile (v / v). Gradient elution: 0-14.5 min, 95% A-50% A; 14.5-18.0 min, 50% A-10% A; 18.0-19.0 min, 10% A; 19.0-19.5 min, 10% A-95% A; 19.5-23.0 min, 95% A. The flow rate was 0.3 mL / min, the column oven temperature was room temperature, and the injection volume was 10 μL.

[0042] (2.6) Ionization mode: ESI ion source; Scan mode: negative ionization; Detection mode: multiple ion reaction monitoring (MRM); Collision gas (CAD): medium; Curtain gas (CUR): 30 psi; Nebulizer gas (GS1): 30 psi; Heating gas (GS2): 50 psi; Spray voltage (IS): -4500 V; Desolvation temperature (TEM): 450 °C; Entrance voltage (EP): -10 V; Scan time: 30 ms. The retention times, ion pairs, collision voltages and declustering voltages of the 12 substances are shown in Table 1.

[0043] Table 1 Ion pair information, collision voltage and declustering voltage of 6 substances

[0044]

[0045] * is the quantitative ion

[0046] The concentrations of six PAEs metabolites in the urine of 20 volunteers are shown in Table 2.

[0047] Table 2 Urine test results of 20 volunteers (μg / L)

[0048] mE miBP mxD mEOHP mxDV mEH Volunteer 1 18.3 19.8 108 12.1 22.2 4.44 Volunteer 2 8.4 68.4 116 12.6 21.0 1.58 Volunteer 3 4.08 10.4 36.9 4.89 7.86 1.51 Volunteer 4 1.06 5.48 8.55 2.33 3.57 0.15 Volunteer 5 2.81 25.1 128 4.79 8.73 0.71 Volunteer 6 1.57 35.4 37.8 5.23 8.21 1.26 Volunteer 7 4.00 19.0 40.4 3.45 6.00 2.08 Volunteer 8 13.2 20.3 45.5 3.58 5.10 1.77 Volunteer 9 6.91 18.2 35.3 4.68 6.57 1.99 Volunteer 10 3.99 21.1 38.4 2.59 3.49 1.20 Volunteer 11 17.0 32.9 117 7.74 12.4 3.68 Volunteer 12 8.69 16.8 51.0 3.81 5.25 1.31 Volunteer 13 11.3 22.3 134 11.1 18.7 2.35 Volunteer 14 4.00 20.6 42.0 8.47 13.7 0.73 Volunteer 15 8.50 24.1 49.4 6.51 12.9 2.85 Volunteer 16 3.77 14.1 32.1 6.10 6.82 3.24 Volunteer 17 22.4 29.4 136 5.46 9.73 2.80 Volunteer 18 8.76 31.2 33.1 4.62 7.20 2.84 Volunteer 19 7.41 6.80 37.1 1.70 2.09 0.91 Volunteer 20 0.88 24.6 36.2 4.87 9.13 2.19

[0049] Urine samples that simultaneously meet the requirements of mEP less than 15 μg / L, miBP less than 40 μg / L, mnBP less than 50 μg / L, mEOHP less than 8 μg / L, mEHHP less than 10 μg / L, and mEHP less than 3 μg / L are selected. Based on the screening conditions.

[0050] In this example, 13 cases of low-concentration urine that met the screening criteria were selected.

[0051] c. Low-concentration urine samples were prepared by mixing low-concentration urine and natural precipitation

[0052] Take a 2L glass beaker (wash the beaker with clean water and then rinse it with ultrapure water three times, then place it in an oven at 120 degrees Celsius for 24 hours). Pour the 13 screened urine samples into the glass beaker, and place the beaker in a 4°C refrigerator for 24 hours to complete the natural precipitation process. The precipitation effect of 100mL urine is as follows Figure 2 As shown in the right figure, it can be seen that the precipitation effect of natural precipitation at 4°C is better than that of the left figure (natural precipitation at room temperature).

[0053] (3) Filtration of low concentration urine samples

[0054] a. Coarse filtration

[0055] (3.1) Transfer the clear urine layer after precipitation to another glass beaker, and do not take the lower sediment in glass beaker 1.

[0056] (3.2) Take 2 quantitative filter papers and install them on the glass sand core filter device. Then slowly pour the urine into the glass sand core measuring cylinder and turn on the pump. At this time, the urine begins to undergo the first coarse filtration. Replace the filter paper every time 80mL of urine is filtered.

[0057] (3.3) After the first coarse filtration of urine is completed, the above filtration steps are repeated once to complete the second coarse filtration of urine.

[0058] b. Fine filtration

[0059] Install the 0.45μm water filter membrane on the glass sand core filter device. Pour 50mL of primary filtered urine into the glass sand core measuring cylinder and turn on the pump. At this time, the urine begins to be finely filtered. Replace the filter membrane every time about 50mL of urine is filtered.

[0060] Table 3 shows the time required to filter 50 mL of sample and 1 L of sample by different filtering methods. It can be seen from the data that the filtering method provided by this method can save time while taking into account the filtering effect.

[0061] Table 3 Comparison of filtering effects of different filtering methods

[0062]

[0063] c. After the urine has been finely filtered, transfer the urine to the glass beaker 3.

[0064] (4) Sample determination and spike addition

[0065] The concentration of pollutants in fine filtered urine was determined by liquid chromatography-tandem triple quadrupole mass spectrometry, using the same method as that shown in the urine screening process. The measured urine concentrations are shown in Table 4.

[0066] Table 4 Concentration of mixed urine in the embodiment (μg / L)

[0067]

[0068] In order to prepare quality control samples that match the exposure level of the general Chinese population, this method is based on several publicly published PAEs exposure assessment studies conducted on the Chinese population. The average of the median concentrations reported in each study is first calculated, and then the approximate value of the mean is selected as the target concentration value of the quality control sample. The median, mean and target concentrations of the six PAEs metabolites are shown in Table 5.

[0069] Table 5 Median exposure levels of PAEs metabolites in urine of the general population in China, median means, and target concentrations of quality control samples (μg / L)

[0070] mE miBP mxD mEOHP mxDV mEH Median value 1 18.7 38.5 47.1 17.6 11.8 5.9 Median 2 18.4 51.7 59.1 7.3 10.5 2.2 Median value 3 13.4 47.8 77.6 8.4 13.1 4.6 Median value 4 18.7 57.6 63.1 6.8 10.5 1.9 Median value 5 31.7 55.6 71.7 9.32 14.2 2.84 Median Mean 20.18 50.24 63.72 9.88 12.02 3.56 Target concentration value 20 50 64 10 12 4

[0071] Add the prepared 4 μg / mL single-labeled solution to the mixed urine. The amount added is the single-labeled solution added per 1000 mL of filtered urine (mL) and is calculated according to the following formula: mEP = (20-X mEP ) / 4mL, miBP=(50-X miBP ) / 4mL; mnBP = (64-X mnBP ) / 4mL; mEOHP = (10-X mEOHP ) / 4mL; mEHHP=(12-X mEHHP ) / 4mL; mEHP = (4-X mEHP ) / 4mL. X is the concentration of each phthalate metabolite in the fine filtered urine measured in Table 4. In this embodiment, the added amounts of mEP, miBP, mnBP, mEOHP, mEHHP, and mEHP are 3.6mL, 6.9mL, 5.9mL, 1.3mL, 1.1mL, and 0.5mL, respectively.

[0072] (5) Mixing

[0073] After the addition of the spike, the sample was sealed with aluminum foil and stirred on a magnetic stirrer at 4°C for 12 h.

[0074] (6) Urine packaging

[0075] After stirring and mixing, keep the urine in a stirring state and use a glass syringe to divide the urine. And evaluate the stability and uniformity of the divided quality control samples.

[0076] (7) Uniformity evaluation

[0077] According to the Technical Specifications for Primary Reference Materials (JJF 1006-1994) and the General Principles and Statistical Principles for the Determination of Reference Materials (JJF 1343-2012), the homogeneity of the quality control samples was tested. Eleven samples were randomly selected from the packaged quality control samples, and three subsamples were selected from each sample for inter-bottle and intra-bottle homogeneity tests. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to determine the PAEs metabolites in the quality control samples, and the determination method was the same as that shown in the urine screening process.

[0078] The data of the uniformity test is statistically processed using the F test. This method is to determine whether there is a systematic error between the measurement values ​​of each group by comparing the variability between groups and the variability within groups. The specific calculation method is as follows:

[0079] 11 samples were taken and 11 groups of measurement data with equal precision were measured under the same conditions.

[0080] x 11 , x 12 , x 13 ,average value

[0081] x 21 , x 22 , x 23 ,average value

[0082] ……………………………………;

[0083] x 111 , x 112 , x 113 ,average value

[0084] set up

[0085] Then, the sum of squares between groups is:

[0086] Within-group sum of squares:

[0087] Take v1=11-1=10, v2=11×(3-1)=22

[0088] Do statistics Then the statistic is an F-distributed variable with degrees of freedom (11, 22). Based on the degrees of freedom (11, 22) and the given significance level (0.05 in this test), the critical value F can be obtained from the F value table. crit =3.32. If the ratio of the two (F value) is less than the critical value F of the statistical test crit , the sample is considered to be homogeneous.

[0089] The results of the uniformity test of the six PAEs metabolites are shown in Tables 6 to 11.

[0090] Table 6 Homogeneity results of substance 1 (mEP) in quality control samples (μg / L)

[0091]

[0092] Table 7 Homogeneity results of substance 2 (miBP) in quality control samples (μg / L)

[0093]

[0094] Table 8 Homogeneity results of substance 3 (mnBP) in quality control samples (μg / L)

[0095]

[0096] Table 9 Homogeneity results of substance 4 (mEOHP) in quality control samples (μg / L)

[0097]

[0098] Table 10 Homogeneity results of substance 5 (mEHHP) in quality control samples (μg / L)

[0099]

[0100] Table 11 Homogeneity results of substance 6 (mEHP) in quality control samples (μg / L)

[0101]

[0102] Through the above statistical tests, see Figures 5 to 10 , mEP, miBP, mnBP, mEOHP, mEHHP, and mEHP all meet F <F crit (11,22) The data showed that there was no significant difference in the six target substances in urine between bottles and within bottles, indicating that mEP, miBP, mnBP, mEOHP, mEHHP, and mEHP were evenly distributed in the samples.

[0103] (8) Stability test

[0104] The samples were stored at -20°C for a 9-month stability study. The stability study was conducted by measuring one batch per month, with 3 packages measured each time. According to JJG 1343-2012, trend analysis was used to calculate the stability data. A linear model was selected as the basic model for stability evaluation, which can be expressed as Y = β0 - β1X, and the stability trend analysis results were calculated according to the model. If |β1| < t·s(β1) for a certain type of index, it indicates that the index is stable during the study period. The stability study results of this example are shown in Tables 12 to 17.

[0105] Table 12 Stability test results of Substance 1 (mEP) in quality control samples (μg / L)

[0106]

[0107] Table 13 Stability test results of Substance 2 (miBP) in quality control samples (μg / L)

[0108]

[0109] Table 14 Stability test results of Substance 3 (mnBP) in quality control samples (μg / L)

[0110]

[0111] Table 15 Stability test results of Substance 4 (mEOHP) in quality control samples (μg / L)

[0112]

[0113] Table 16 Stability test results of Substance 5 (mEHHP) in quality control samples (μg / L)

[0114]

[0115] Table 17 Stability test results of Substance 6 (mEHP) in quality control samples (μg / L)

[0116]

[0117] Through the above statistical tests, mEP, miBP, mnBP, mEOHP, mEHHP, and mEHP all satisfy |β1| < t·s(β1), indicating that these 6 target substances in urine are stable when stored at -20°C.

[0118] In summary, the present invention provides a method for preparing a control sample of phthalate plasticizer metabolites in urine of the Chinese population. The indexes and concentrations of the quality control samples prepared by this method match the actual exposure levels of phthalates in urine of the general population in my country. At the same time, it has good uniformity and stability. It is suitable for carrying out quality control of the experimental process of determining phthalate metabolites in urine of the Chinese population or comparative analysis of results. At the same time, the method has the characteristics of easy availability of materials, economical experimental cost, simple preparation process, simple experimental conditions and wide application range.

Claims

1. A method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people, characterized in that: The following steps are included: (1) Prepare standard working solutions of mEP, mnBP, miBP, mEOHP, mEHHP, and mEHP respectively for use; In step (1), the standard working solution concentration of mEP, mnBP, miBP, mEOHP, mEHHP, and mEHP is 4 μg / mL; (2) Preparation of low-concentration urine samples, including urine collection, low-concentration urine screening, low-concentration urine mixing and natural precipitation; The low-concentration urine must meet the following standards at the same time: mEP less than 15μg / L, miBP less than 40μg / L, mnBP less than 50μg / L, mEOHP less than 8μg / L, mEHHP less than 10μg / L, and mEHP less than 3μg / L; (3) Filtration of low-concentration urine samples, including coarse filtration and fine filtration; (4) Urine sample determination and spiking: To determine the concentration of phthalate metabolites in urine, add the standard working solution to the filtered urine; The amount of standard working solution added to each 1000 mL of filtered urine in step (4) is calculated according to the following formula: mEP = (20-X mEP ) / 4mL; miBP = (50-X miBP ) / 4mL; mnBP = (64-X mnBP ) / 4mL; mEOHP = (10-X mEOHP ) / 4mL; mEHHP=(12-X mEHHP ) / 4mL; mEHP = (4-X mEHP ) / 4mL; X is the concentration of each phthalate metabolite in filtered urine; (5) Mixing; (6) Repackaging.

2. The method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 1, characterized in that: The low-concentration urine screening in step (2) includes: (2.3) Take 2 mL of urine and transfer it to multiple 5 mL centrifuge tubes; add 0.5 mL of 1 mol / L ammonium acetate, 20 μL of β-glucuronidase, and 40 ng of isotope internal standard respectively, mix well and perform enzymatic hydrolysis at 37 degrees Celsius for 2 hours; (2.4) Activate the MAX solid phase extraction column with 3 mL of methanol and 3 mL of water, and pass the enzymatically hydrolyzed urine through the column; after the column is completed, rinse with 3 mL of water and 3 mL of methanol in sequence; then elute with 2 mL of methanol containing 2% formic acid; dilute the eluate 5 times with 5% acetonitrile water, and measure it using a liquid chromatography-tandem triple quadrupole mass spectrometer; (2.5) The chromatographic column was Waters ACQUITY UPLC BEH Phenyl, with the following physical properties: 100 mm × 2.1 mm, 1.7 μm; mobile phase A was 0.1% acetic acid in water by volume, and mobile phase B was 0.1% acetic acid in acetonitrile by volume; gradient elution: 0-14.5 min, 95% A-50% A; 14.5-18.0 min, 50% A-10% A; 18.0-19.0 min, 10% A; 19.0-19.5 min, 10% A-95% A; 19.5-23.0 min, 95% A; flow rate was 0.3 mL / min, the column oven temperature was room temperature, and the injection volume was 10 μL; (2.6) Ionization mode: ESI ion source; Scan mode: negative ionization; Detection mode: multiple ion reaction monitoring; Collision gas: medium; Curtain gas: 30 psi; Nebulizer gas: 30 psi; Heating gas: 50 psi; Spray voltage: -4500 V; Desolvation temperature: 450 °C; Inlet voltage: -10 V; Scan time: 30 ms.

3. A method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 1, characterized in that: The urine collection in step (2) comprises: (2.1) Recruiting volunteers, including male and female smokers and non-smokers; all volunteers are in good health and have not taken any medication within 7 days; (2.2) Collect 100 mL of urine from each volunteer.

4. The method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 1, characterized in that: The ambient temperature for the natural precipitation of low-concentration urine in the low-concentration urine mixing and natural precipitation in step (2) is 4°C.

5. The method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 1, characterized in that: The coarse filtration in step (3) comprises: (3.1) Transfer the clear urine layer after precipitation to another glass beaker; (3.2) Take 2 quantitative filter papers and install them on the glass sand core filter device; then slowly pour the low-concentration urine sample into the glass sand core measuring cylinder and turn on the pump; at this time, the low-concentration urine sample begins to undergo the first coarse filtration; the filter paper is replaced every time 80 mL of the low-concentration urine sample is filtered; (3.3) After the low-concentration urine sample is filtered through the first coarse filtration, the above filtration steps are repeated once to complete the second coarse filtration.

6. A method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 5, characterized in that: The fine filtration in step (3) includes installing a 0.45 μm water filter membrane on a glass sand core filtration device; pouring 50 mL of the coarsely filtered low-concentration urine sample into a glass sand core measuring cylinder, turning on the pump, and starting fine filtration; replacing the filter membrane every time 50 mL is filtered.

7. The method for preparing a controlled sample of phthalate plasticizer metabolites in urine of Chinese people as claimed in claim 1, characterized in that: The mixing in step (5) includes stirring on a magnetic stirrer at 4° C. for 12 hours.

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