阅读说明：本技术 一种人体尿液中磷系阻燃剂及其降解产物的检测方法 (Method for detecting phosphorus flame retardant and degradation product thereof in human urine ) 是由 朱婷婷 邓臣 胡蓉 刘云浪 王金玲 陈建义 齐秀娟 谢林伸 于 2020-06-05 设计创作，主要内容包括：本发明公开一种人体尿液中磷系阻燃剂及其降解产物的检测方法,包括样本预处理步骤、固相萃取的步骤、定容的步骤、液相色谱的步骤和标准曲线的绘制步骤。本发明提供了一种人体尿液中磷系阻燃剂及其部分降解产物的检测方法,从而为人体尿液中磷系阻燃剂的调查、风险评估等提供参考。该发明方法操作简单,过程简便,能够在尽可能节约试剂的同时达到最佳萃取效果,同时保证了较高的回收率和数据的准确性。(The invention discloses a method for detecting phosphorus flame retardants and degradation products thereof in human urine, which comprises the steps of sample pretreatment, solid-phase extraction, volume determination, liquid chromatography and standard curve drawing. The invention provides a method for detecting a phosphorus flame retardant and a partial degradation product thereof in human urine, thereby providing references for investigation, risk assessment and the like of the phosphorus flame retardant in the human urine. The method disclosed by the invention is simple to operate, the process is simple and convenient, the optimal extraction effect can be achieved while the reagent is saved as much as possible, and meanwhile, the higher recovery rate and the data accuracy are ensured.)

1. A method for detecting phosphorus flame retardants and degradation products thereof in human urine is characterized by comprising the following steps: the method comprises the following steps:

step S1, sample preprocessing step; adding a human urine sample into a standard substitute, and uniformly mixing to be detected;

step S2, solid phase extraction; in the step, the pretreated human urine sample is subjected to solid phase extraction to obtain an eluent;

step S3, fixing the volume; in this step, the eluent is subjected to constant volume;

step S4, a step of liquid chromatography; in the step, a triple quadrupole rod LC-MS is adopted for detection;

and step S5, drawing a standard curve.

2. The method for detecting phosphorus flame retardants and their degradation products in human urine according to claim 1, wherein: in the sample pretreatment step, 0.5-1ml of human urine sample is taken.

3. The method for detecting phosphorus-based flame retardant and degradation products thereof in human urine according to claim 2, wherein the method comprises the following steps: the solid phase extraction step comprises:

step S201, activating a column; after the MCX extraction column is installed on a solid phase extraction device, adding methanol with the same amount as the human urine sample into the column, naturally dripping the solution, and adding the same amount of ultrapure water for the same operation when the solution is equal to the substances in the column;

step S202, sample loading and enrichment; passing the human urine sample added with the standard substitute through the activated small column, and discarding the effluent;

step S203, leaching; leaching the small column by using ultrapure water containing 8 per mill formic acid with the same dosage as the human urine sample;

step S204, drying and eluting; after washing, opening the regulating valve, and pumping for 10-30min by using a vacuum pump; ensuring the drying of the solid phase extraction column; and (3) eluting the dried small column with a methanol and acetonitrile mixture with the same dosage as the human urine sample, wherein the volume ratio of methanol to acetonitrile in the methanol and acetonitrile mixture is 3 to 7, soaking the column with the eluent for 1 to 5 minutes, naturally lowering the eluent, and collecting the eluent.

4. The method for detecting phosphorus flame retardants in human urine and their degradation products according to claim 3, wherein: in the step of loading and enriching, after the solid phase extraction column is activated, the human urine sample should pass through the column immediately, and the water of the column should form trickling at the lower end thereof in the process of passing through the column, so that a continuous liquid column cannot be formed.

5. The method for detecting phosphorus flame retardants in human urine and their degradation products according to claim 3, wherein: in the volume fixing step, the eluent is fixed to 0.5-1mL by ultrapure water.

6. The method for detecting phosphorus flame retardants in human urine and their degradation products according to claim 3, wherein: the liquid chromatography step comprises:

chromatographic conditions are as follows: using Agilent 1260 series liquid chromatography for separating the target compound, wherein the chromatographic column is; the column temperature is 35 ℃; the sample amount is 10 mu L, the mobile phase is methanol (A) and 0.005mol/L ammonium acetate solution (B), and the flow rate of the mobile phase is 250 mu L/min;

mass spectrum conditions: electrospray ionization source (ESI); multiple reaction monitoring mode (MRM); ion source ionization mode: positive;

the capillary voltage is 3500V, the atomizer pressure is 45psi, the carrier gas is high-purity nitrogen, the carrier gas temperature is 330 ℃, the flow rate is 9.5L/min, the concentration of the analytes is 500 mug/L by detecting with a multi-reaction monitoring mode (MRM); determining the parent ion and the ion of each compound through the total ion flow diagram and the corresponding mass spectrogram result, and optimizing the Collision Energy (CE) and the cluster removal voltage (DP) according to the response condition.

7. The method for detecting phosphorus flame retardants in human urine and their degradation products according to claim 3, wherein: the standard curve is drawn:

the target samples used for the instrumental analysis in the experiment contained 14 phosphorus-based flame retardants and 3 metabolites, triethyl phosphate (TEP), diethyl phosphate (DEP), tripropyl phosphate (TPP), tributyl phosphate (TBP), tripentyl phosphate (TNP), trioctyl phosphate (TEHP), tri-m-tolyl phosphate (TMCP), diphenyl cresyl phosphate (CDP), tris (2-butoxyethyl) phosphate (TBOEP), triphenylphosphine oxide (TPPO) and 2-ethylhexyl diphenyl phosphate (EHDPP), triphenyl phosphate (TPHP), resorcinol tetraphenyl phosphate (RDP) and triisobutyl phosphate (TIBP)14 phosphorus-based flame retardants, bis (2-butoxyethyl) -2- (3-hydroxybutoxy) ethyl triester phosphate (BHEPT), bis (2-butoxyethyl) -2-hydroxyethyl triester phosphate (BHP), Bis (butoxyethyl) phosphate (BBP)3 phosphorus flame retardant degradation products of tris (2-butoxyethyl) phosphate (TBOEP) utilizing tri-n-butyl phosphate-d₂₇(TBP-d₂₇) Tris (2-butoxyethyl) phosphate-d₂₇(TBOEP-d₂₇) As a standard substitute, trimethyl phosphate-C₁₃（TMP- C₁₃) As an internal standard, standard curves of the target were prepared at concentrations of 2, 5, 10, 50, 100, 500. mu.g/L.

8. The method for detecting phosphorus-based flame retardants in human urine and degradation products thereof according to any one of claims 1 to 8, wherein: also comprises a method blank step; when the step is used for processing samples, a blank sample is inserted into each batch of experiment, ultrapure water is used for replacing the samples, the processing steps identical to those of the samples are carried out, and the samples are tested on a computer.

Technical Field

The invention relates to the field of detection methods of phosphorus flame retardants and degradation products thereof, in particular to a detection method of phosphorus flame retardants and degradation products thereof in human urine.

Background

Organic Phosphorus Flame Retardants (OPFRs) are widely used in electronic products, foams, plastics and textiles due to their low smoke, low toxicity and high heat resistance. The organic phosphorus flame retardant is mainly added into the material in an adding mode rather than a chemical bond mode, so that the organic phosphorus flame retardant is easily leached from the treated material in modes of product abrasion, volatilization and permeation and is released into various environmental media, thereby affecting human beings and various organisms. Researchers have detected different concentrations of OPFRs in vivo and in various environmental media. Research shows that the organic phosphorus flame retardant can produce various toxic effects such as biological toxicity, potential carcinogenicity and neurotoxicity, and reduce the immunity of the organism. For example, TPHP (triphenyl phosphate) inhibits the activity of monocyte carboxylesterase in human blood, which in turn has an adverse effect on the human immune system. TCP has potential biological toxicity and central nerve toxicity, and can be absorbed by human body and contact with skin to cause toxicity. TBOEP can have interfering effects on the endocrine system of the organism. Therefore, researches on the influence of OPFRs on human health gradually attract attention of students, and urine is a most widely applied human health monitoring material due to the characteristics of easy collection and large sample amount.

There are 14 phosphorus flame retardants and 3 metabolites in human urine, which are triethyl phosphate (TEP), diethyl phosphate (DEP), tripropyl phosphate (TPP), tributyl phosphate (TBP), tripentyl phosphate (TNP), trioctyl phosphate (TEHP), tri-m-tolyl phosphate (TMCP), diphenyl cresyl phosphate (CDP), tris (2-butoxyethyl) phosphate (TBOEP), triphenylphosphine oxide (TPPO) and 2-ethylhexyl di-cresyl phosphate (2-ethylhexyl-di-phenyl-phosphate (2-butoxyethyl-ethyl-phenyl-phosphate (TBOEP), respectivelyPhenyl Phosphate (EHDPP), triphenyl phosphate (TPHP), resorcinol tetraphenyl phosphate (RDP) and triisobutyl phosphate (TIBP)14 phosphorus flame retardants, bis (2-butoxyethyl) -2- (3-hydroxybutoxy) ethyl phosphotriester (BHEPT), bis (2-butoxyethyl) -2-hydroxyethyl phosphotriester (BHP), bis (butoxyethyl) phosphate (BBP)3 tris (2-butoxyethyl) phosphate (TBOEP) phosphorus flame retardant degradation products, in the analysis of domestic literature on the phosphorus flame retardant content in human urine, use tri-n-butyl phosphate-d₂₇(TBP-d₂₇) Tris (2-butoxyethyl) phosphate-d₂₇(TBOEP-d₂₇) As an indicator within standard substitutes.

At present, a few scholars have studied the OPFRs in urine, but the existing detection method for the OPFRs in urine has few targets, and the detection method for studying the OPFRs and metabolites thereof in urine has fewer methods. Chinese patent publication No. CN105699527A discloses a method for detecting the content of bromine-based flame retardant and phosphorus-based flame retardant in serum, which is incorporated herein by reference, and in this patent application, the method for detecting the content of phosphorus-based flame retardant in serum comprises the following steps:

(1b) mixing the serum to be detected with diatomite, adding an internal standard indicator, then adding a first organic solvent to extract a brominated flame retardant in the serum, and fully extracting to obtain an extracting solution;

(2b) concentrating the extracting solution obtained in the step (1b), transferring the extracting solution to a solid phase extraction column for purification, eluting the phosphorus flame retardant from the solid phase extraction column by using a second organic solvent, collecting the eluent, and concentrating; transferring the concentrated eluent to a composite alumina-silica gel column for further purification, eluting again by using a second organic solvent, and fixing the volume of the eluted eluent again;

(3b) preparing a phosphorus flame retardant standard solution containing the internal standard indicator in the step (1b), measuring the solution with the constant volume in the step (2b) and the phosphorus flame retardant standard solution containing the internal standard indicator by using a gas chromatography-mass spectrometer, and performing data processing and quantitative analysis based on an internal standard method to obtain the content of the phosphorus flame retardant in serum.

The detection method can well detect the phosphorus flame retardant in the serum, and provides an effective method for the human exposure risk assessment of novel pollutants.

However, no method for detecting phosphorus flame retardants and degradation products thereof in human urine exists at present, and after all, human urine is more easily obtained than human serum.

Therefore, a set of complete detection method for the phosphorus flame retardant and the degradation products thereof in human urine is established, and the method has important guiding significance for guaranteeing the health of residents and the safety of regional ecological environment, and making pollution control strategies and elimination measures of the organic phosphorus flame retardant according with national conditions and places.

Disclosure of Invention

The invention aims to provide a method for detecting phosphorus flame retardants and degradation products thereof in human urine, aiming at the problem that no good method for detecting the phosphorus flame retardants and the degradation products thereof in human urine exists at present and the pollution control strategy and elimination measure of organic phosphorus flame retardants according with the national conditions and places need to be formulated.

The technical scheme adopted by the invention for realizing the purpose is as follows: a method for detecting phosphorus flame retardants and degradation products thereof in human urine comprises the following steps:

step S1, sample preprocessing step; adding a human urine sample into a standard substitute, and uniformly mixing to be detected;

step S2, solid phase extraction; in the step, the pretreated human urine sample is subjected to solid phase extraction to obtain an eluent;

step S3, fixing the volume; in this step, the eluent is subjected to constant volume;

step S4, a step of liquid chromatography; in the step, a triple quadrupole rod LC-MS is adopted for detection;

and step S5, drawing a standard curve.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: in the sample pretreatment step, 0.5-1ml of human urine sample is taken.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: the solid phase extraction step comprises:

step S201, activating a column; after the MCX extraction column is installed on a solid phase extraction device, adding methanol with the same amount as the human urine sample into the column, naturally dripping the solution, and adding the same amount of ultrapure water for the same operation when the solution is equal to the substances in the column;

step S202, sample loading and enrichment; passing the human urine sample added with the standard substitute through the activated small column, and discarding the effluent;

step 203, leaching; leaching the small column by using ultrapure water containing 8 per mill formic acid with the same dosage as the human urine sample;

step S204, drying and eluting; after washing, opening the regulating valve, and pumping for 10-30min by using a vacuum pump; ensuring the drying of the solid phase extraction column; and (3) eluting the dried small column with a methanol and acetonitrile mixture with the same dosage as the human urine sample, wherein the volume ratio of methanol to acetonitrile in the methanol and acetonitrile mixture is 3 to 7, soaking the column with the eluent for 1 to 5 minutes, naturally lowering the eluent, and collecting the eluent.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: in the step of loading and enriching, after the solid phase extraction column is activated, the human urine sample should pass through the column immediately, and the water of the column should form trickling at the lower end thereof in the process of passing through the column, so that a continuous liquid column cannot be formed.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: in the volume fixing step, the eluent is fixed to 0.5-1mL by ultrapure water.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: the liquid chromatography step comprises:

chromatographic conditions are as follows: using Agilent 1260 series liquid chromatography for separating the target compound, wherein the chromatographic column is; the column temperature is 35 ℃; the sample amount is 10 mu L, the mobile phase is methanol (A) and 0.005mol/L ammonium acetate solution (B), and the flow rate of the mobile phase is 250 mu L/min;

mass spectrum conditions: electrospray ionization source (ESI); multiple reaction monitoring mode (MRM); ion source ionization mode: positive;

the capillary voltage is 3500V, the atomizer pressure is 45psi, the carrier gas is high-purity nitrogen, the carrier gas temperature is 330 ℃, the flow rate is 9.5L/min, the concentration of the analytes is 500 mug/L by detecting with a multi-reaction monitoring mode (MRM); determining the parent ion and the ion of each compound through the total ion flow diagram and the corresponding mass spectrogram result, and optimizing the Collision Energy (CE) and the cluster removal voltage (DP) according to the response condition.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: the standard curve is drawn:

the target samples used for the instrumental analysis in the experiment contained 14 phosphorus-based flame retardants and 3 metabolites, triethyl phosphate (TEP), diethyl phosphate (DEP), tripropyl phosphate (TPP), tributyl phosphate (TBP), tripentyl phosphate (TNP), trioctyl phosphate (TEHP), tri-m-tolyl phosphate (TMCP), diphenyl cresyl phosphate (CDP), tris (2-butoxyethyl) phosphate (TBOEP), triphenylphosphine oxide (TPPO) and 2-ethylhexyl diphenyl phosphate (EHDPP), triphenyl phosphate (TPHP), resorcinol tetraphenyl phosphate (RDP) and triisobutyl phosphate (TIBP)14 phosphorus-based flame retardants, bis (2-butoxyethyl) -2- (3-hydroxybutoxy) ethyl triester phosphate (BHEPT), bis (2-butoxyethyl) -2-hydroxyethyl triester phosphate (BHP), Bis (butoxyethyl) phosphate (BBP)3 phosphorus flame retardant degradation products of tris (2-butoxyethyl) phosphate (TBOEP) utilizing tri-n-butyl phosphate-d₂₇(TBP-d₂₇) Tris (2-butoxyethyl) phosphate-d₂₇(TBOEP-d₂₇) As a standard substitute, trimethyl phosphate-C₁₃(TMP-C₁₃) As an internal standard, standard curves of the target were prepared at concentrations of 2, 5, 10, 50, 100, 500. mu.g/L.

Further, in the method for detecting phosphorus flame retardants and degradation products thereof in human urine, the following steps are performed: also comprises a method blank step; when the step is used for processing samples, a blank sample is inserted into each batch of experiment, ultrapure water is used for replacing the samples, the processing steps identical to those of the samples are carried out, and the samples are tested on a computer.

The invention provides a method for detecting phosphorus flame retardants and degradation products thereof in human urine, which makes an environmental pollution control strategy and elimination measures by quantitatively detecting the phosphorus flame retardants and the degradation products thereof in the human urine.

The invention provides a method for detecting a phosphorus flame retardant and a partial degradation product thereof in human urine, thereby providing references for investigation, risk assessment and the like of the phosphorus flame retardant in the human urine. The method disclosed by the invention is simple to operate, the process is simple and convenient, the optimal extraction effect can be achieved while the reagent is saved as much as possible, and meanwhile, the higher recovery rate and the data accuracy are ensured.

The present invention will be described in more detail with reference to the following examples.

Detailed Description