CN112986446A - Synchronous detection method and application of organic phosphate and metabolite in poultry egg products - Google Patents

Abstract

The invention discloses a synchronous detection method and application of organic phosphate and metabolites in poultry egg products, wherein the method comprises the following steps: 1) constructing a risk assessment mathematical model, 2) determining the components to be detected and the samples to be detected, 3) preprocessing the samples, and 4) detecting the samples; 5) internal standard method to obtain detection data 6) to carry out health exposure risk assessment. The invention also discloses application of the detection data obtained by the detection method to the evaluation and analysis of the healthy exposure risk of the food ingested by the organophosphate and metabolite crowd, including daily exposure analysis, pollution source analysis, exposure sensitive crowd analysis, safety limit analysis of markers, risk analysis and classification. According to the invention, a risk assessment mathematical model is constructed, 19 specific substances in the OPEs and the mOPEs are selected as components to be detected, and the synchronous detection is carried out by adopting the ultra-performance liquid chromatography-tandem mass spectrometry, so that the content of the 19 specific substances can be simultaneously obtained, and the detection data and the risk analysis result can be rapidly and efficiently obtained.

Description

Synchronous detection method and application of organic phosphate and metabolite in poultry egg products

Technical Field

The invention relates to the technical field of organic phosphate detection and health risk assessment, in particular to a synchronous detection method and application of organic phosphate and metabolites in poultry and egg products.

Background

Organic phosphate ester parent compounds (OPEs) and metabolites thereof (mOPEs) are mainly used as flame retardants and plasticizers, are widely applied to electronic products, plastic products, textiles, building materials and the like, and are widely detected in various environmental media and organisms. OPEs, a class of endocrine disruptors, have been shown to be biologically toxic and potentially health-hazardous by dietary exposure into humans, and have attracted attention. However, recent studies have shown that OPEs are rapidly metabolised to mOPEs upon entry into the organism, and that some mOPEs are equally or even more toxic than OPEs. Furthermore, since OPEs are rapidly metabolically transformed in vivo, the content of mOPEs in biological samples is likely to be higher than OPEs. Thus, for food products of the biological sample type (e.g. meat, poultry eggs, etc.), merely detecting the levels of OPEs therein is likely to greatly underestimate the health risk of such environmental contaminants.

In the prior art, chinese patent application CN 201911416523.3 discloses a method for detecting organic phosphate, comprising the following steps: s1: collecting a sample to be detected and filtering; s2: detecting the sample by using online solid phase extraction-ultra high performance liquid chromatography tandem mass spectrometry to obtain a total ion flow diagram chromatogram of the sample; s3: drawing a working curve of the organic phosphate standard solution; s4: and (4) comparing the corresponding quantitative peak area of the organic phosphate in the chromatogram of the total ion flow diagram obtained in the step S2 with the working curve obtained in the step S3, and converting to obtain the content of the organic phosphate in the sample. However, the detection method of the organic phosphate disclosed by the invention can only detect the OPEs, and cannot synchronously detect the OPEs and the mOPEs, so that the operation time is longer, the number of detected components is small, and the data summarizing and analyzing speed is slow.

The applicant finds that OPEs and mOPEs exist in biological samples such as poultry eggs and the like at the same time through research, but the two substances have different components and characteristics, and the accurate detection result can be obtained only by respectively carrying out pretreatment and instrument analysis on the two pollutants in the conventional detection method. For example, Liu et al establish a set of analysis methods for respectively measuring OPEs and mOPEs in biological samples, wherein a purification method of freezing degreasing combined with solid-phase extraction of a dispersing agent is adopted as a pretreatment method, acetone, ethyl acetate, n-hexane and dichloromethane are selected as elution solvents, and a gas chromatography-mass spectrometry (GC-MS) instrument and a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) instrument are adopted to respectively detect the OPEs and the mOPEs. By adopting the method, the two types of pollutants are respectively pretreated and analyzed by an instrument, the time consumption is respectively 4 hours and 8 hours, and the total time is 12 hours; moreover, the results detected by respective instruments need to be recorded, counted and analyzed, and the defects of long analysis and detection time, overhigh consumption cost of manpower, machine time and consumables and the like exist.

Meanwhile, the exposure characteristics of organic phosphate and metabolites in poultry egg products in crowds are not sufficiently researched at present, a scientific and effective informatization analysis process, an analysis model and an analysis tool are lacked, the obtained data is detected and can only be manually researched and judged, a large amount of manpower is consumed, the researched and judged result is related to the professional level of an analyst, the repeatability of an analysis method and the process is poor, the researched and judged analysis result of each batch cannot be guaranteed to have objectivity and stability, and the objectivity and the accuracy of health risk assessment are influenced.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a combined detection method and application of organic phosphate and metabolites thereof in poultry egg products, overcome the defects of the existing detection method and the limitations of manual evaluation, and by constructing a risk evaluation mathematical model, selecting 19 specific substances in organic phosphate parent compounds (OPEs) and metabolites (mOPEs) thereof in the poultry egg products as components to be detected according to parameters related to a health exposure risk assessment mathematical model, and samples to be detected are extracted according to the requirements of health exposure risk assessment specifications, and are synchronously detected by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, the content of 19 specific substances can be obtained simultaneously, and the method is applied to the evaluation of the risk of the ingestion health exposure of the population so as to quickly and efficiently obtain detection data and a risk analysis result.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a synchronous detection method for organic phosphate and metabolites in poultry egg products is characterized by comprising the following steps:

1) constructing a risk assessment mathematical model: an organophosphate parent compound and metabolite thereof for the population to ingest a health exposure risk assessment model;

2) determining a component to be detected and a sample to be detected: selecting 19 specific substances in organic phosphate parent compounds (OPEs) and metabolites (mOPEs) of the organic phosphate parent compounds as components to be detected according to parameters related to a health exposure risk assessment mathematical model, and extracting samples to be detected according to health exposure risk assessment specification requirements;

3) sample pretreatment: pretreating a sample, extracting supernate from a unit sample and an internal standard substance by adopting a solid-liquid extraction method, and purifying the supernate by adopting a solid-phase extraction column to obtain a sample extracting solution to be detected;

4) sample detection: synchronously detecting the extracting solution by adopting ultra-high performance liquid chromatography-tandem mass spectrometry to synchronously obtain an integral chromatogram and a mass spectrogram of an organic phosphate parent compound and a metabolite thereof of a sample;

5) obtaining a quantitative detection result: calculating the content of each component to be detected, namely 19 specific substances in the organic phosphate parent compound and the metabolite thereof in the sample according to an internal standard method;

6) and (3) exposure risk assessment: and substituting the data obtained by the sample detection into the risk assessment mathematical model to carry out the evaluation of the exposure risk of the ingestion health of the crowd.

The method comprises the following steps of 1) constructing a risk assessment mathematical model, and specifically comprises the following steps:

calculating the total exposure amount per day after food intake according to the content of the organic phosphate parent compound and the metabolite thereof in the sample, and correspondingly evaluating the exposure risk level;

wherein the total daily intake (EDIs, ng/(kg. d)) of the organophosphate parent compound and its metabolites is calculated by the following formula:

in the formula: ADDp-daily average exposure of organophosphate parent compound via food intake, ng/(kg · d);

ADDm-daily average exposure of organophosphate metabolites via food intake, ng/(kg. d).

In the formula: cpi-concentration of organophosphate precursor compound in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

In the formula: cmi-concentration of organophosphate metabolites in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

The steps for assessing the exposure level are:

a) setting a two-dimensional matrix mathematical model of the health exposure risk level: setting the total concentration of OPEs and mOPEs in the target food as five content grades of 0-5, 5-10, 10-20, 20-40 and 40-80 as row elements; setting the daily average intake of the target food as four intake grades of 0-10, 10-20, 20-40 and 40-80 as row elements; the two form a 4 x 5 two-dimensional matrix risk grade mathematical model together;

b) substituting the data of sample detection into formula (4), and calculating content grade and intake grade respectively, wherein the daily intake ratio coefficient f of fowl egg is assigned to 0.415, and the value of RfD is assigned to 1.5 × 10³；

In the formula: r-maximum health risk coefficient;

EDIs-daily average exposure of the compound of interest, ng/(kg. d);

f-the proportion of a certain food product to the total daily dietary intake,%;

RfD-reference dose, known minimum in the detected target.

c) Filling the calculated content grade and intake grade elements into a two-dimensional matrix risk grade mathematical model respectively to obtain corresponding health exposure risk grades; the grade is one of low risk, medium risk, high risk.

The components to be detected in the step 2), specifically TBP, TCEP, TCPP, TDCPP, TPHP, EHDPP, TBOEP, TEHP, TMPP in organic phosphate parent compounds (OPEs), and metabolites thereof (mOPEs): BBOEHEP, OH-TBOEP, DBP, BCEP, BCPP, BDCPP, DPHP, BBOEP, BEHP, BMPP.

The step 3) of pretreating the sample specifically comprises the following steps:

3-1) crushing a single poultry egg product sample, adding Acetonitrile (ACN) and toluene (Tol) together with an internal standard substance, carrying out ultrasonic extraction, taking supernate, and filtering to obtain an extract; adding 20ng of mixed internal standard substances of d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP for every two grams of biological samples;

3-2) blowing and concentrating the extracted liquid nitrogen obtained in the step 3-1) to obtain a crude extract;

3-3) purifying the crude extract obtained in the step 3-2) by adopting a CNWBOND Si silica gel column, and collecting eluent containing a target substance;

3-4) carrying out nitrogen blow concentration on the eluent containing the target substance in the step 3-3), and carrying out grease removal by adopting Z-Sep/C18 to obtain a solution containing the target substance;

3-5) further purifying the solution containing the target object in the step 3-4) by adopting a Strata X-AW column to obtain a sample extracting solution to be detected.

The ultrasonic extraction in the step 3-1) is specifically as follows: extracting for 2 times; each time of extraction, adding 4.5mL acetonitrile and 0.5mL toluene into each gram of poultry egg product sample, performing ultrasonic treatment for 30min, and filtering supernate to obtain extract liquor; for every two grams of sample supernatant nitrogen was blown near dry and re-dissolved with 1mL of n-hexane to obtain crude extract.

The step 3-3) further comprises: the crude extract obtained was purified using CNWBOND Si silica gel column and 500mg of neutral silica gel filler was added per two g of sample.

The step 3-4) further comprises the following steps: the eluate containing the target was dried by blowing nitrogen, redissolved with 1.5mL of acetonitrile, degreased with Z-Sep/C18, and added with 60mg of Z-Sep and 30mL of C18 for every two g of samples.

The step 3-5) further comprises the following steps: the degreased solution containing the target was further purified using a Strata X-AW column, and 500mg of filler was added for every two grams of sample.

The working conditions of the ultra-high performance liquid chromatography in the step 4) are set as follows:

a chromatographic column: poroshell 120 EC-C18 column, length 50mm, internal diameter 4.6mm, filler particle size 2.7 μm; mobile phase: phase A is 0.1% acetic acid in methanol-water (5:95, V/V); phase B was 0.1% acetic acid in methanol-water (95:5, V/V); gradient elution procedure: 0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A; flow rate: 600. mu.L/min^-1(ii) a Column temperature: 45 ℃; sample introduction amount: 5 μ L.

The working conditions of the mass spectrometer in the step 4) are set as follows:

an ionization mode: electrospray ion source, positive and negative ion mode (ESI +); detecting a triple quadrupole mass spectrum; spraying voltage: +/-4000V; mass spectrum scanning mode: multiple reactive ion monitoring (MRM); inlet voltage: -10V; collision cell exit voltage: -15V.

And 5) calibrating the contents of the organic phosphate parent compound and the metabolite thereof by adopting a standard curve without matrix matching.

The synchronous detection method for the organic phosphate and the metabolites in the poultry egg products is characterized in that the obtained detection data are applied to the health exposure risk assessment analysis of the ingestion of the organic phosphate and the metabolites by people, and the analysis comprises daily exposure analysis, pollution source analysis, exposure sensitive people analysis, safety limit analysis of markers, risk analysis and classification.

The method for synchronously detecting the organic phosphate and the metabolite in the poultry egg product is characterized by comprising the following steps:

A) setting a computer-based organic phosphate and metabolite human ingestion health exposure risk assessment and analysis system, which comprises a detection instrument, a main control computer and a built-in analysis program; the analysis program comprises the following components which are connected in sequence and are in interactive communication: the system comprises a control and operation module, an input and output management module, a detection sample management module, a detection component management module, a detection instrument management module, a detection data management module, a daily exposure calculation module, a pollution source analysis module, an exposure sensitive population analysis module, a safety limit management module of a marker and a risk analysis and grading management module;

B) the main control computer is electrified to work, and mathematical models and parameters in the sensitive biomarker management module, the detection sample management module, the detection component management module, the detection instrument management module, the detection data management module, the daily exposure calculation module, the pollution source analysis module, the exposure sensitive population analysis module, the safety limit management module of the marker and the risk analysis and grading management module are respectively set through the input and output management module;

C) the detection instrument outputs the detection data of the sample to a main control computer through a data interface, and a control and operation module of an analysis program, a daily exposure calculation module, an exposure sensitive population analysis module, a safety limit management module of a marker, a risk analysis and grading management module are matched with each other, respectively operate according to preset mathematical models and parameters of the modules, and output corresponding operation results.

The exposure sensitive population analysis module is used for screening and judging sensitive populations exposed to the OPEs and the mOPEs according to sample detection data.

The sensitive biomarker management module is used for screening and judging the management of the markers and the safety limit values thereof according to the sample detection data.

And the pollution source analysis module is used for analyzing and judging the pollution source according to the sample detection data.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a synchronous detection method and application of organic phosphate and metabolites in poultry egg products, wherein a risk assessment mathematical model is constructed, 19 specific substances in organic phosphate parent compounds (OPEs) and metabolites (mOPEs) in the poultry egg products are selected as components to be detected according to parameters related to a crowd ingestion health exposure risk assessment mathematical model, samples to be detected are extracted according to health exposure risk assessment specification requirements, and synchronous detection is carried out by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, so that the content of the 19 specific substances can be simultaneously obtained, the content of the 19 specific substances is applied to crowd ingestion health exposure risk assessment, detection data and risk analysis results are rapidly and efficiently obtained, the defects of the existing detection method that the types of the detected substances are few, the time is long, synchronous detection cannot be carried out, and the like, the manual assessment speed is slow, the rate of the detection is slow, and the content of the mOPEs and the risk analysis results are rapidly and efficiently obtained, Unstable result and the like.

(2) The synchronous detection method provided by the invention adopts rapid sample pretreatment and high performance liquid chromatography-tandem mass spectrometry, can be suitable for detecting pollutants of various poultry egg products such as eggs, duck eggs, goose eggs, quail eggs and the like, synchronously detects the content of organophosphates and metabolites thereof in the poultry egg products, has the advantages of large detectable component quantity, high accuracy, high efficiency, convenience and high sensitivity, has the detection limit of 0.0004-0.33 ng/g, the lower limit of quantification of 0.12-0.55 mug/kg, the standard recovery rate of an actual sample of 60.6-91.1 percent, consumes about 3 hours in the whole detection process, greatly shortens the detection time by more than 50 percent compared with the existing detection method (needing to respectively detect OPEs and mOPEs by adopting different instruments), and can meet the synchronous quantitative detection and risk evaluation analysis of the organophosphates and the metabolites thereof in the poultry egg products.

(3) The synchronous detection method provided by the invention adopts a quick and effective sample pretreatment step, the extracted sample extracting solution to be detected can be extracted while ensuring that the organic phosphate and the metabolite thereof are effective, and the method can be applied to an ultra-high liquid chromatography tandem mass spectrometer to carry out quick and sensitive synchronous detection on the organic phosphate and the metabolite thereof, and quantitatively detect the contents of 19 organic phosphate and the metabolite thereof, thereby solving the problems that the existing organic phosphate and the metabolite thereof pretreatment consumes too long time, the solvent consumption is large, and the detection cannot be finished by adopting a small instrument and the like. The usage amount of the organic solvent in the pretreatment process is greatly reduced, and the number of used instruments is also reduced, so that the detection cost is reduced.

(4) The synchronous detection method and the application provided by the invention have the advantages that a set of crowd ingestion risk assessment mathematical model and a detection sample assessment data system are established in advance, a quick and effective pretreatment and instrument synchronous detection method matched with the crowd ingestion risk assessment mathematical model is provided, and a computer system is combined for data processing and analysis, so that the content of 19 substances, namely organic phosphate and metabolites thereof, in poultry egg products is synchronously determined, the analysis operation process is greatly simplified, the detection and analysis time is shortened, and a stable and reliable analysis result is obtained.

(5) According to the synchronous detection method and the application, the computer system and the detection instrument are adopted to acquire data online without manually processing and analyzing the data, the main control computer and the built-in analysis program automatically complete the daily exposure calculation of the ingestion of people, the pollution source analysis, the exposure sensitive people analysis, the safety limit management of the marker, the risk analysis, the grading management and the like according to the detection data, and output the analysis result, so that the synchronous detection method has the advantages of comprehensive functions, high efficiency and good stability and objectivity of the analysis result.

Drawings

FIG. 1 is a schematic flow chart of a synchronous detection method and application according to an embodiment of the present invention;

FIG. 2 is a total ion current chromatogram of target OPEs and mOPEs in a 200 ng/mL standard solution according to example of the present invention;

FIG. 3 is a schematic diagram of a two-dimensional matrix structure for assessing the level of risk of human ingestion health exposure in accordance with an embodiment of the present invention;

FIG. 4 is a schematic diagram of the composition structure of a human feeding health exposure assessment analysis system according to an embodiment of the present invention;

fig. 5 is a schematic diagram of a module composition of the analysis program in fig. 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example (b):

referring to fig. 1 to 5, the method for synchronously detecting organophosphates and metabolites in poultry egg products provided by the present invention comprises the following steps:

1) constructing a risk assessment mathematical model: an organophosphate parent compound and metabolite thereof for a population ingestion health exposure risk assessment model;

2) determining a component to be detected and a sample to be detected: selecting 19 specific substances in organic phosphate parent compounds (OPEs) and metabolites (mOPEs) of the organic phosphate parent compounds as components to be detected according to parameters related to a health exposure risk assessment mathematical model, and extracting samples to be detected according to health exposure risk assessment specification requirements;

3) sample pretreatment: pretreating a sample, extracting supernate from a unit sample and an internal standard substance by adopting a solid-liquid extraction method, and purifying the supernate by adopting a solid-phase extraction column to obtain a sample extracting solution to be detected;

4) sample detection: synchronously detecting the extracting solution by adopting ultra-high performance liquid chromatography-tandem mass spectrometry to synchronously obtain an integral chromatogram and a mass spectrogram of an organic phosphate parent compound and a metabolite thereof of a sample;

5) obtaining a quantitative detection result: calculating the content of each component to be detected, namely 19 specific substances in the organic phosphate parent compound and the metabolite thereof in the sample according to an internal standard method;

6) and (3) exposure risk assessment: and substituting the data obtained by the sample detection into the risk assessment mathematical model to carry out the evaluation of the exposure risk of the ingestion health of the crowd.

The method comprises the following steps of 1) constructing a risk assessment mathematical model, and specifically comprises the following steps:

calculating the total exposure amount per day after food intake according to the content of the organic phosphate parent compound and the metabolite thereof in the sample, and correspondingly evaluating the exposure risk level;

wherein the total daily intake (EDIs, in ng/(kg. d)) of the organophosphate parent compound and its metabolites is calculated by the following formula:

in the formula: ADDp-daily average exposure of organophosphate parent compound via food intake, ng/(kg · d);

ADDm-daily average exposure of organophosphate metabolites via food intake, ng/(kg. d).

In the formula: cpi-concentration of organophosphate precursor compound in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

In the formula: cmi-concentration of organophosphate metabolites in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

The steps for assessing the exposure level are:

a) setting a two-dimensional matrix mathematical model of the health exposure risk level: setting the total concentration of OPEs and mOPEs in the target food as five content grades of 0-5, 5-10, 10-20, 20-40 and 40-80 as row elements; setting the daily average intake of the target food as four intake grades of 0-10, 10-20, 20-40 and 40-80 as row elements; the two form a 4 x 5 two-dimensional matrix risk grade mathematical model together;

b) substituting the data of sample detection into formula (4), and calculating content grade and intake grade respectively, wherein the daily intake ratio coefficient f of fowl egg is assigned to 0.415, and the value of RfD is assigned to 1.5 × 10³；

In the formula: r-maximum health risk coefficient;

EDIs-daily average exposure of the compound of interest, ng/(kg. d);

f-the proportion of a certain food product to the total daily dietary intake,%;

RfD-reference dose, known minimum in the detected target.

c) Filling the calculated content grade and intake grade elements into a two-dimensional matrix risk grade mathematical model respectively to obtain corresponding health exposure risk grades; the grade is one of low risk, medium risk, high risk.

The components to be detected in the step 2), specifically TBP, TCEP, TCPP, TDCPP, TPHP, EHDPP, TBOEP, TEHP, TMPP in organic phosphate parent compounds (OPEs), and metabolites thereof (mOPEs): BBOEHEP, OH-TBOEP, DBP, BCEP, BCPP, BDCPP, DPHP, BBOEP, BEHP, BMPP.

The step 3) of pretreating the sample specifically comprises the following steps:

3-1) crushing a single poultry egg product sample, adding Acetonitrile (ACN) and toluene (Tol) together with an internal standard substance, carrying out ultrasonic extraction, taking supernate, and filtering to obtain an extract; adding 20ng of mixed internal standard substances of d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP for every two grams of biological samples;

3-2) blowing and concentrating the extracted liquid nitrogen obtained in the step 3-1) to obtain a crude extract;

3-3) purifying the crude extract obtained in the step 3-2) by adopting a CNWBOND Si silica gel column, and collecting eluent containing a target substance;

3-4) carrying out nitrogen blow concentration on the eluent containing the target substance in the step 3-3), and carrying out grease removal by adopting Z-Sep/C18 to obtain a solution containing the target substance;

3-5) further purifying the solution containing the target object in the step 3-4) by adopting a Strata X-AW column to obtain a sample extracting solution to be detected.

The ultrasonic extraction in the step 3-1) is specifically as follows: extracting for 2 times; adding 4.5mL of acetonitrile and 0.5mL of toluene into each gram of poultry egg product sample, performing ultrasonic treatment for 30min, and filtering supernate to obtain extract liquor; for every two grams of sample supernatant nitrogen was blown near dry and re-dissolved with 1mL of n-hexane to obtain crude extract.

The step 3-3) further comprises: the crude extract obtained was purified using CNWBOND Si silica gel column and 500mg of neutral silica gel filler was added per two g of sample.

The step 3-4) further comprises the following steps: the eluate containing the target was dried by blowing nitrogen, redissolved with 1.5mL of acetonitrile, degreased with Z-Sep/C18, and added with 60mg of Z-Sep and 30mL of C18 for every two g of samples.

The step 3-5) further comprises the following steps: the degreased solution containing the target was further purified using a Strata X-AW column, and 500mg of filler was added for every two grams of sample.

The working conditions of the ultra-high performance liquid chromatography in the step 4) are set as follows:

a chromatographic column: poroshell 120 EC-C18 column, length 50mm, internal diameter 4.6mm, filler particle size 2.7 μm; mobile phase: phase A is 0.1% acetic acid in methanol-water (5:95, V/V); phase B was 0.1% acetic acid in methanol-water (95:5, V/V); gradient elution procedure: 0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A; flow rate: 600. mu.L/min^-1(ii) a Column temperature: 45 ℃; sample introduction amount: 5 μ L.

The working conditions of the mass spectrometer in the step 4) are set as follows:

an ionization mode: electrospray ion source, positive and negative ion mode (ESI +); detecting a triple quadrupole mass spectrum; spraying voltage: +/-4000V; mass spectrum scanning mode: multiple reactive ion monitoring (MRM); inlet voltage: -10V; collision cell exit voltage: -15V.

The conditions of detected ions adopted by the internal standard method of the mass spectrometer are as follows:

table 1: detected ion conditions adopted by internal standard method of mass spectrometer

And 5) calibrating the contents of the organic phosphate parent compound and the metabolite thereof by adopting a standard curve without matrix matching.

According to the synchronous detection method of the organic phosphate and the metabolites in the poultry egg products, the obtained detection data is applied to the evaluation and analysis of the exposure risks of the crowd ingesting the organic phosphate and the metabolites, including daily exposure analysis, pollution source analysis, exposure sensitive crowd analysis, safety limit analysis of markers, risk analysis and classification; the method specifically comprises the following steps:

A) setting a computer-based organic phosphate and metabolite human ingestion health exposure risk assessment and analysis system, which comprises a detection instrument, a main control computer and a built-in analysis program; the analysis program comprises the following components which are connected in sequence and are in interactive communication: the system comprises a control and operation module, an input and output management module, a detection sample management module, a detection component management module, a detection instrument management module, a detection data management module, a daily exposure calculation module, a pollution source analysis module, an exposure sensitive population analysis module, a safety limit management module of a marker and a risk analysis and grading management module;

B) the main control computer is electrified to work, and mathematical models and parameters in the sensitive biomarker management module, the detection sample management module, the detection component management module, the detection instrument management module, the detection data management module, the daily exposure calculation module, the pollution source analysis module, the exposure sensitive population analysis module, the safety limit management module of the marker and the risk analysis and grading management module are respectively set through the input and output management module;

C) the detection instrument outputs the detection data of the sample to a main control computer through a data interface, and a control and operation module of an analysis program, a daily exposure calculation module, an exposure sensitive population analysis module, a safety limit management module of a marker, a risk analysis and grading management module are matched with each other, respectively operate according to preset mathematical models and parameters of the modules, and output corresponding operation results.

The exposure sensitive population analysis module is used for screening and judging sensitive populations exposed to the OPEs and the mOPEs according to sample detection data.

The sensitive biomarker management module is used for screening and judging the management of the markers and the safety limit values thereof according to the sample detection data.

And the pollution source analysis module is used for analyzing and judging the pollution source according to the sample detection data.

Specific example 1:

the synchronous detection method of the organic phosphate and the metabolite in the poultry egg product provided by the invention is basically the same as the previous embodiment, and the difference is that the synchronous detection of the organic phosphate and the metabolite thereof specifically comprises the following steps:

1) sample pretreatment: extracting supernate from a single poultry egg product sample and an internal standard substance by adopting a solid-liquid extraction method, and purifying the supernate by adopting a solid-phase extraction column to obtain a sample extracting solution to be detected;

1-1) crushing a single poultry egg product sample, adding acetonitrile and toluene together with an internal standard substance, carrying out ultrasonic extraction, taking supernatant, and separating to obtain an extract; adding 20 mu L of mixed internal standard substances of d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP for every two grams of biological samples; in order to improve the extraction effect, the ultrasonic extraction step is repeated for 2 times; extracting every time, adding 4.5mL acetonitrile and 0.5mL toluene into every two grams of poultry egg product samples, performing ultrasonic treatment for 30min, and taking supernate to obtain extract liquor;

1-2) blowing and concentrating the extracted liquid nitrogen obtained in the step (1-1) to be nearly dry, and redissolving by using normal hexane to obtain a crude extract; redissolving every two grams of poultry egg product samples by using 1mL of normal hexane;

1-3) adopting CNWBOND Si silica gel solid phase extraction column to purify the sample from the crude extract obtained in the step 1-2), and collecting the eluent containing the target substance; adding 500mg of neutral silica gel filler to every two grams of samples;

1-4) blowing nitrogen into the solution containing the target object in the step 1-3) to be nearly dry, redissolving the solution by using acetonitrile, and removing lipid in the poultry egg product by using a Z-Sep/C18 column to obtain a solvent containing the target object after the lipid is removed; blowing nitrogen to the utmost extent for every two grams of poultry egg product samples, redissolving with 1.5mL of acetonitrile, carrying out oscillation for 30min and centrifugation for 10min through a 60mg Z-Sep and 30mL C18 packed column, and removing lipid in the poultry egg products to obtain a solvent containing a target object after the lipid removal;

1-5) further purifying the solvent containing the target object after the fat removal in the step 1-4) by using a Strata X-AW column, eluting by using triethylamine and acetonitrile, and collecting eluent to obtain a refined extract; purifying each two grams of poultry egg product sample by a Strata X-AW column, activating a solid phase extraction small column by 3mL of ultrapure water and 3mL of acetonitrile, flushing the extraction column by 2mL of ultrapure water, vacuumizing for 2min, eluting by 5mL of triethylamine and acetonitrile (5:95, V: V), and collecting eluent to obtain a refined extract;

1-6) drying the refined extract obtained in the step (1-5) by nitrogen, and then fixing the volume by using acetonitrile and water; for every two grams of poultry egg product samples, 100 mul of acetonitrile and water (5:95, V: V) are used for constant volume.

2) An instrumental analysis step: obtaining an organic phosphate ester and metabolite correlation chromatogram and mass spectrogram of the sample to be detected by using the sample extracting solution to be detected obtained in the step (1) through ultra-high performance liquid chromatography-tandem mass spectrometry;

the working conditions of the ultra-high performance liquid chromatography of the organic phosphate and the metabolite thereof are set as follows:

a chromatographic column: poroshell 120 EC-C18 column, 4.6mm x 50mm, 2.7 μm, column specification: the length is 50mm, the inner diameter is 4.6mm, and the grain diameter of the filler is 2.7 mu m;

mobile phase: phase A: 0.1% acetic acid in methanol-water (5:95, V/V); phase B: 0.1% acetic acid in methanol-water (95:5, V/V)

Elution procedure: see table 2 below;

flow rate: 600. mu.L/min^-1

Column temperature: at a temperature of 45 c,

the sample volume is 5 mu L

0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A, as shown in the following table:

table 2: liquid chromatography gradient elution procedure for organophosphates and their metabolites

The working conditions of the mass spectrometer in the step 2) are set as follows:

an ionization mode: electrospray ion source, positive and negative ion mode (ESI +); detecting a triple quadrupole mass spectrum;

spraying voltage: +/-4000V;

mass spectrum scanning mode: multiple reactive ion monitoring (MRM);

inlet voltage: -10V;

collision cell exit voltage: -15V

3) And calculating the contents of the organic phosphate and the metabolites thereof in the sample according to an internal standard method.

And step 3) calibrating the contents of the organic phosphate and the metabolites thereof by adopting a standard curve without matrix matching.

The mass spectrum working conditions optimized for OPEs and mOPEs in the embodiment of the invention are as follows in Table 3:

table 3: mass spectrometry operating conditions optimized for OPEs and mOPEs

4) And (3) exposure risk assessment: calculating and evaluating the total exposure to food intake based on the data of the contents of the organophosphate parent compound and its metabolites in the sample measured in step 2).

Wherein the total daily intake (EDIs, in ng/(kg. d)) of organophosphates and their metabolites is calculated by the following formula:

in the formula: ADDp-daily average exposure of organophosphate parent compound via food intake, ng/(kg · d);

ADDm-daily average exposure of organophosphate metabolites via food intake, ng/(kg. d).

In the formula: cpi-concentration of organophosphate precursor compound in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

In the formula: cmi-concentration of organophosphate metabolites in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

Specific example 2:

the method for synchronously detecting organic phosphate and metabolites in the poultry egg products provided by the embodiment is basically the same as the previous embodiment, and is different in that the sample to be detected is suspected low-concentration exposed eggs, and the method specifically comprises the following steps:

1) sample pretreatment: extracting supernate from a single poultry egg product sample and an internal standard substance by adopting a solid-liquid extraction method, and purifying the supernate by adopting a solid-phase extraction column to obtain a sample extracting solution to be detected;

step 1) sampling a unit sample and an internal standard substance by a solid-liquid extraction method and a solid-phase extraction method to obtain a detection sample extracting solution;

1-1) accurately weighing 2g of low-concentration exposed egg sample, crushing the egg sample into a 15mL glass centrifuge tube, mixing the egg sample with internal standard substances of 20ng, d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP, extracting for each time, adding 4.5mL of acetonitrile and 0.5mL of toluene into each gram of egg product sample, performing ultrasonic extraction for 30min, repeatedly extracting for 2 times, and combining supernate to obtain extract liquor;

1-2) blowing and concentrating the extraction liquid nitrogen obtained in the step 1-1) to be dry, and redissolving by using normal hexane to obtain a crude extract; redissolving every two grams of poultry egg product samples by using 1mL of normal hexane;

1-3) adopting CNWBOND Si silica gel solid phase extraction column to purify the sample from the crude extract obtained in the step 1-2), and collecting the eluent containing the target substance; for every two grams of sample, 500mg of neutral silica gel filler was added.

1-4) blowing nitrogen into the solution containing the target object in the step 1-3) to be nearly dry, redissolving the solution by using acetonitrile, and removing lipid in the poultry egg product by using a Z-Sep/C18 column to obtain a solvent containing the target object after the lipid is removed; redissolving every two grams of poultry egg product samples by using 1mL of acetonitrile, oscillating for 30min through a Z-Sep/C18 column, centrifuging for 10min, removing lipid in the poultry egg products, and obtaining the solvent containing the target object after the lipid is removed.

1-5) further purifying the solvent containing the target object after the fat removal in the step 1-4) by using a Strata X-AW column, eluting by using triethylamine and acetonitrile, and collecting eluent to obtain a refined extract; purifying each two grams of poultry egg product sample by a Strata X-AW column, activating a solid phase extraction small column by 3mL of ultrapure water and 3mL of acetonitrile, flushing the extraction column by 2mL of ultrapure water, vacuumizing for 2min, eluting by 5mL of triethylamine and acetonitrile (5:95, V: V), and collecting eluent to obtain a refined extract;

1-6) carrying out nitrogen blow drying on the refined extract obtained in the step 1-5), and then fixing the volume by using acetonitrile and water; for every two grams of poultry egg product samples, 100 mul of acetonitrile and water (5:95, V: V) are used for constant volume.

2) Acquiring mass spectrum data of related peaks of the organic phosphate and metabolites thereof of the sample to be detected by adopting the ultra-high performance liquid chromatography-tandem mass spectrometry on the sample extracting solution to be detected obtained in the step 1);

the working conditions of the ultra-high performance liquid chromatography for detecting the organic phosphate and the metabolites thereof are as follows:

a chromatographic column: poroshell 120 EC-C18 column, 4.6mm x 50mm, 2.7 μm, column specification: length 50mm, inner diameter 4.6mm, filler particle size 2.7 μm.

Mobile phase: phase A: 0.1% acetic acid in methanol-water (5:95, V/V); phase B: 0.1% acetic acid in methanol-water (95:5, V/V)

Elution procedure: see Table 4 below

Flow rate: 600. mu.L/min^-1

Column temperature: 45 deg.C

The sample volume is 5 mu L

0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A, as shown in Table 4 below:

table 4: liquid chromatography gradient elution procedure for organophosphate metabolites

The working conditions of the mass spectrometer for detecting the organic phosphate and the metabolites thereof are as follows:

an ionization mode: electrospray ion source, positive and negative ion mode (ESI +); detecting a triple quadrupole mass spectrum;

spraying voltage: +/-4000V;

mass spectrum scanning mode: multiple reactive ion monitoring (MRM);

inlet voltage: -10V;

collision cell exit voltage: -15V.

(3) And calculating the contents of the organic phosphate and the metabolites thereof in the sample according to an internal standard method.

In step (3), the contents of organophosphates and their metabolites are preferably calibrated using a standard curve without matrix matching.

The mass spectrum operating conditions for this example optimized for OPEs and mOPEs detection are as follows in Table 5:

TABLE 5 Mass Spectrometry working conditions optimized for OPEs and mOPEs detection

Specific example 3:

the method for synchronously detecting organic phosphate and metabolites in the poultry egg products provided by the embodiment is basically the same as the previous embodiment, and is different in that the sample to be detected is suspected high-concentration exposed eggs, and the method specifically comprises the following steps:

1) sample pretreatment: extracting a supernatant from a single-unit duck egg product sample and an internal standard substance by a solid-liquid extraction method, and purifying the supernatant by a solid-phase extraction column to obtain a sample extracting solution to be detected;

step 1) sampling a unit sample and an internal standard substance by a solid-liquid extraction method and a solid-phase extraction method to obtain a detection sample extracting solution;

1-1) accurately weighing 2g of high-concentration exposed egg sample, crushing the egg sample into a 15mL glass centrifuge tube, mixing the egg sample with internal standard substances of 20ng, d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP, extracting for each time, adding 4.5mL of acetonitrile and 0.5mL of toluene into each gram of egg product sample, performing ultrasonic extraction for 30min, repeating the extraction for 2 times, and combining the supernatants to obtain an extract.

1-2) blowing and concentrating the extracted liquid nitrogen obtained in the step 1-1) to be nearly dry, and redissolving by using normal hexane to obtain a crude extract; redissolving every two grams of poultry egg product samples by using 1mL of normal hexane;

1-3) adopting CNWBOND Si silica gel solid phase extraction column to purify the sample from the crude extract obtained in the step (1-2), and collecting the eluent containing the target substance; for every two grams of sample, 500mg of neutral silica gel filler was added.

1-4) blowing nitrogen into the solution containing the target substance in the step (1-3) to be nearly dry, redissolving the solution with acetonitrile, and removing lipid in poultry egg products through a Z-Sep/C18 column to obtain a solvent containing the target substance after lipid removal; redissolving every two grams of poultry egg product samples by using 1.5mL of acetonitrile, oscillating for 30min through a Z-Sep/C18 column, centrifuging for 1min, removing lipid in the poultry egg products, and obtaining the solvent containing the target object after the lipid is removed.

1-5) further purifying the solvent containing the target object after the fat removal in the step 1-4) by using a Strata X-AW column, eluting by using triethylamine and acetonitrile, and collecting eluent to obtain a refined extract; and (2) further purifying every two grams of poultry egg product samples by using a Strata X-AW column, firstly activating a solid phase extraction small column by using 3mL of ultrapure water and 3mL of acetonitrile, flushing the extraction column by using 2mL of ultrapure water, vacuumizing for 2min, then eluting by using 5mL of triethylamine and acetonitrile (5:95, V: V), and collecting eluent to obtain a refined extract.

1-6) carrying out nitrogen blow drying on the refined extract obtained in the step 1-5), and then fixing the volume by using acetonitrile and water; for every two grams of poultry egg product samples, 100 mul of acetonitrile and water (5:95, V: V) are used for constant volume.

2) Obtaining an organic phosphate ester and metabolite correlation chromatogram and mass spectrogram of the sample to be detected by using the sample extracting solution to be detected obtained in the step (1) through ultra-high performance liquid chromatography-tandem mass spectrometry;

the ultra-high performance liquid chromatography conditions of the organic phosphate and the metabolite thereof are as follows:

a chromatographic column: poroshell 120 EC-C18 column, 4.6mm x 50mm, 2.7 μm, column specification: length 50mm, inner diameter 4.6mm, filler particle size 2.7 μm.

Mobile phase: phase A: 0.1% acetic acid in methanol-water (5:95, V/V); phase B: 0.1% acetic acid in methanol-water (95:5, V/V)

Elution procedure: see Table 6 below

Flow rate: 600. mu.L/min^-1

Column temperature: 45 deg.C

The sample volume is 5 mu L

0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A, as shown in the following table:

table 6: liquid chromatography gradient elution procedure for organophosphate metabolites

The working conditions of the mass spectrometer for detecting the organophosphate and the metabolite thereof in the embodiment are as follows:

an ionization mode: electrospray ion source, positive and negative ion mode (ESI +); detecting a triple quadrupole mass spectrum;

spraying voltage: +/-4000V;

mass spectrum scanning mode: multiple reactive ion monitoring (MRM);

inlet voltage: -10V;

collision cell exit voltage: -15V

3) And calculating the contents of the organic phosphate and the metabolites thereof in the sample according to an internal standard method.

And 3) calibrating the contents of the organic phosphate and the metabolite thereof by adopting a standard curve without matrix matching.

The working conditions for the mass spectra optimized for OPEs and mOPEs of this example are as follows:

TABLE 7 OPEs and mOPEs optimized Mass Spectroscopy working conditions

The data of the sample detection results of the specific examples 2 and 3 are shown in the following table:

TABLE 8 detection results (ng/g) of OPEs and mOPEs in different blank spiked samples

The detection result of the embodiment shows that the concentration range of the OPEs in the detected low-concentration (2.5 ng/g) blank standard-adding sample is 2.01-2.69 ng/g, and the content range of the mOPEs is 1.76-2.77 ng/g; the concentration range of the OPEs in the high-concentration (20 ng/g) blank standard-added sample is 16.90-20.48 ng/g, and the concentration range of the mOPEs is 16.48-20.17 ng/g. The accuracy of the detection result is high.

Specific example 4

The method for synchronously detecting organic phosphate and metabolites in poultry egg products provided by the embodiment is basically the same as the previous embodiments, and is different in that two egg blank samples are selected and used in the embodiment, and the effects of detecting a linear range, a detection limit and a recovery rate are observed, and the method specifically comprises the following steps:

under optimized experimental conditions, the standard solvents are used for preparing 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200 and 500 ng.mL^-1A series of standard solutions. The mass concentration of the target substance (X, ng. mL)^-1) The ratio (Y) of the peak area of the response to the peak area of the corresponding internal standard is the abscissa, and the ordinate is the response piece matrix standard curve. The results show that the concentration of the surfactant is 0.2 to 500 ng/mL^-1Good range linear dependence (r)> 0.997）。

In the embodiment, two egg blank samples are selected, and the OPEs and the mOPEs are subjected to a standard addition recovery test, wherein the addition levels are 2.5 ng/g and 20ng/g, each addition level is repeatedly measured for 1 time, and the internal standard method is used for quantification. The result shows that the recovery rate of the OPEs and the mOPEs is 55.23-91.01%.

The recovery rates of different scalar addition amounts and the detection limit detection results of the method are shown in the following table.

TABLE 9 recovery rates for different spiked amounts and detection results of detection limits of the methods

Specific example 5

The method for synchronously detecting organic phosphate and metabolites in poultry egg products provided by the embodiment is basically the same as the previous embodiments, and is different in that the method for synchronously detecting organic phosphate and metabolites in poultry egg products observes the influence of ultra-high performance liquid chromatography conditions and specifically comprises the following steps:

the Poroshell 120 EC-C18 (4.6 mm multiplied by 50mm, 2.7 μm) chromatographic column is selected, the methanol-water and acetic acid solution can obtain good separation effect, and the target peak response value is lower when the methanol-water is selected as the mobile phase than when the methanol-water and acetic acid are selected. Therefore, methanol-water plus acetic acid was selected as the mobile phase in this example. The total ion current chromatogram of target OPEs and mOPEs in 200 ng/mL standard solution is shown in figure 2.

Specific example 6

The method for synchronously detecting organic phosphate and metabolites in poultry egg products provided by the embodiment is basically the same as the previous embodiments, and is different in that the method for synchronously detecting organic phosphate and metabolites in poultry egg products observes the influence of tandem mass spectrometry conditions and specifically comprises the following steps:

by adopting a needle pump sample injection mode, 200 mu g/L of each single-standard solution of OPEs and mOPEs is continuously injected into ESI at the flow rate of 5 mu L/min, Q1 and Q3 are respectively scanned in a positive ion detection mode and a negative ion detection mode to determine a parent ion and a daughter ion pair, then mass spectrum parameters such as cluster removal voltage and collision energy of each compound are optimized, and the optimized results are shown in Table 10.

TABLE 10 OPEs and mOPEs optimized Mass Spectrometry conditions

In this embodiment, under the optimized experimental condition, the ops and the mpopes in the actual egg sample are synchronously detected, and the detection result is as follows:

TABLE 11 content of OPEs and mOPEs in eggs (ng/g ww)

The results show that the concentration range of the OPEs in the egg samples tested in the example is nd-1.79 ng/g ww, and the concentration range of the mOPEs is nd-1.44 ng/g ww. Based on the measured data, the daily average exposure dose of OPEs and mOPEs ingested by eating the eggs was further evaluated.

The Intake (IR) of eggs and products thereof by children and adults in China is respectively 12.5 g/d and 25.9 g/d; exposure Frequency (EF) 365 d/a; duration of Exposure (ED) 1 a; average exposure time (AT) 365 d; the average Body Weight (BW) of residents of children and adults in China is 21.58 kg and 61.52 kg respectively. Substituting the values of the parameters into formula (1) to calculate EDIs, formula (2) to calculate ADDp, and formula (3) to calculate ADDm.

Through calculation, the daily exposure total dose EDIs of the OPEs and the mOPEs ingested by children and adults in China through eating eggs are 3.06 ng/(kg.d) and 2.22 ng/(kg.d).

The total daily exposure dose for children was 38% higher than for adults, and thus children were determined to be sensitive to exposure to OPEs and mOPEs. According to the calculation result, sensitive markers of the children exposed OPEs and mOPEs are selected and determined to be TCPP, DPHP, BEHP, TBP and TPHP, and the exposure amount of 5 markers accounts for 90% of the total exposure dose.

Safety limit management of markers: further calculations indicate that a low risk is achieved when the daily average exposure dose EDIs for children is < 6.2 ng/(kg. d), i.e. the safety limits (recommended values) for the levels of OPEs and mOPEs in eggs are 10.7 ng/g ww.

For the analysis of the contamination source: the chicken food mainly comprises feeds such as corn, grains and the like, and the OPEs and the mOPEs in the eggs can be derived from polluted feeds. In addition, drinking water, soil, air, etc. may also be a source of contamination of OPEs and mOPEs in eggs. When the risk level is higher, each possible pollution source needs to be checked, and the poultry can be prevented from contacting polluted feed, water, soil, air and the like.

And importing the data into data obtained by detecting the sample, inputting the data into a computer-based organic phosphate and metabolite health exposure risk assessment analysis system, calculating to obtain calculated content grade and intake grade elements, respectively filling the calculated content grade and intake grade elements into a two-dimensional matrix risk grade mathematical model, substituting the two-dimensional matrix risk grade mathematical model into a risk assessment mathematical model, and performing health exposure risk assessment, wherein in the embodiment, the children expose 5 sensitive markers to combine with the content grade, and the health exposure risk grade is judged to be low risk.

Specific example 7

The method for synchronously detecting organic phosphate and metabolites in poultry egg products provided by the embodiment is basically the same as the previous embodiments, and is different in that the sample to be detected is duck eggs.

In this embodiment, under the optimized experimental condition, the ops and the mpopes in the actual duck egg sample are synchronously detected, and the detection result is as follows:

TABLE 12 content of OPEs and mOPEs in Duck eggs (ng/g ww)

Substituting the values of the parameters into the formulas (1), (2) and (3) to respectively calculate EDIs, ADDp and ADDm.

The daily exposure total dose of the OPEs and the mOPEs ingested by children and adults in China through eating duck eggs is calculated to be 5.71 ng/(kg.d) and 4.15 ng/(kg.d).

The total daily exposure dose for children was 38% higher than for adults, and thus children were determined to be sensitive to exposure to OPEs and mOPEs. According to the calculation result, sensitive markers of the children exposed OPEs and mOPEs are selected and determined to be DPHP, BEHP, DBP, BDCPP, BBOEP and TCPP, and the exposure amount of 6 markers accounts for 83% of the total exposure dose.

The daily exposure dose EDIs of children is less than 6.2 ng/(kg. d), and the risk level is low.

According to the description of the above embodiments of the present invention, the samples to be detected can be eggs, eggs of other fowl and egg products except for duck eggs, such as goose eggs, quail eggs, etc., and can be applied to achieve the technical effects described in the present invention, and therefore, the samples are not listed one by one.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of this invention and is not intended to limit the invention, and that any modification, equivalent replacement or improvement made within the spirit and principle of the invention is included in the scope of the invention.

Claims (17)

1. A synchronous detection method for organic phosphate and metabolites in poultry egg products is characterized by comprising the following steps:

1) constructing a risk assessment mathematical model: an organophosphate parent compound and metabolite thereof for a population to ingest a health exposure risk assessment model;

2) determining a component to be detected and a sample to be detected: selecting 19 specific substances in organic phosphate parent compounds OPEs and metabolites mOPEs of the organic phosphate parent compounds OPEs in the poultry egg products as components to be detected according to parameters related to a health exposure risk assessment mathematical model, and extracting samples to be detected according to health exposure risk assessment specification requirements;

3) sample pretreatment: pretreating a sample, extracting supernate from a unit sample and an internal standard substance by adopting a solid-liquid extraction method, and purifying the supernate by adopting a solid-phase extraction column to obtain a sample extracting solution to be detected;

4) sample detection: synchronously detecting the extracting solution by adopting ultra-high performance liquid chromatography-tandem mass spectrometry to synchronously obtain an integral chromatogram and a mass spectrogram of an organic phosphate parent compound and a metabolite thereof of a sample;

5) obtaining a quantitative detection result: calculating the content of each component to be detected, namely 19 specific substances in the organic phosphate parent compound and the metabolite thereof in the sample according to an internal standard method;

6) and (3) exposure risk assessment: and substituting the data obtained by the sample detection into the risk assessment mathematical model to carry out health exposure risk assessment.

2. The method for synchronously detecting organic phosphate and metabolites in the poultry egg products according to claim 1, wherein the step 1) of constructing a risk assessment mathematical model specifically comprises the following steps:

calculating the total exposure amount per day after food intake according to the content of the organic phosphate parent compound and the metabolite thereof in the sample, and correspondingly evaluating the exposure risk level;

wherein the total daily intake of the organophosphate parent compound and its metabolites EDIs, in ng/(kg. d), is calculated by the following formula:

in the formula: ADDp-daily average exposure of organophosphate parent compound via food intake, ng/(kg · d);

ADDm — daily exposure of organophosphate metabolites via food intake, ng/(kg · d);

in the formula: cpi-concentration of organophosphate precursor compound in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d;

in the test: cmi-concentration of organophosphate metabolites in food, ng/g wet weight;

IR-food intake, g/d;

EF-frequency of exposure, d/a;

ED-years of exposure, a;

BW-weight of the population, kg;

AT-mean exposure time, d.

3. The method for simultaneously detecting organophosphates and metabolites in avian egg products according to claim 2, wherein the step of assessing the exposure level comprises:

a) setting a two-dimensional matrix mathematical model of the health exposure risk level: setting the total concentration of OPEs and mOPEs in the target food as five content grades of 0-5, 5-10, 10-20, 20-40 and 40-80 as row elements; setting the daily average intake of the target food as four intake grades of 0-10, 10-20, 20-40 and 40-80 as row elements; the two form a 4 x 5 two-dimensional matrix risk grade mathematical model together;

b) substituting the data of sample detection into formula (4), and calculating content grade and intake grade respectively, wherein the daily intake ratio coefficient f of fowl egg is assigned to 0.415, and the value of RfD is assigned to 1.5 × 10³；

In the formula: r-maximum health risk coefficient;

EDIs-daily average exposure of the compound of interest, ng/(kg. d);

f-the proportion of a certain food product to the total daily dietary intake,%;

RfD-reference dose, known minimum in the detected target;

c) filling the calculated content grade and intake grade elements into a two-dimensional matrix risk grade mathematical model respectively to obtain corresponding health exposure risk grades; the grade is one of low risk, medium risk, high risk.

4. The method for simultaneously detecting organophosphates and metabolites in poultry egg products according to claim 1, wherein the components to be detected in step 2), specifically TBP, TCEP, TCPP, TDCPP, TPHP, EHDPP, TBOEP, TEHP, TMPP, and the metabolites mpopes thereof, are selected from the group consisting of the organophosphate parent compounds ops: BBOEHEP, OH-TBOEP, DBP, BCEP, BCPP, BDCPP, DPHP, BBOEP, BEHP, BMPP.

5. The method for synchronously detecting organic phosphate and metabolites in the poultry egg products according to claim 1, wherein the step 3) of pretreating the samples comprises the following steps:

3-1) crushing a single poultry egg product sample, adding acetonitrile ACN and toluene Tol together with an internal standard substance, carrying out ultrasonic extraction, taking supernate, and filtering to obtain an extract liquid; adding 20ng of mixed internal standard substances of d27-TBOEP, d15-TPHP, d27-TBP, d18-TCPP, d12-TCEP, d8-BBOEP, d4-BBOEHEP, d10-DPHP, d8-BCEP and d10-BDCPP for every two grams of biological samples;

3-2) blowing and concentrating the extracted liquid nitrogen obtained in the step 3-1) to obtain a crude extract;

3-3) purifying the crude extract obtained in the step 3-2) by adopting a CNWBOND Si silica gel column, and collecting eluent containing a target substance;

3-4) carrying out nitrogen blow concentration and degreasing on the eluent containing the target object in the step 3-3) to obtain a solution containing the target object;

3-5) further purifying the solution containing the target object in the step 3-4) to obtain a sample extracting solution to be detected.

6. The method for synchronously detecting organic phosphate and metabolites in the poultry egg product according to claim 5, wherein the ultrasonic extraction in the step 3-1) is specifically as follows: extracting for 2 times; each time of extraction, adding 4.5mL acetonitrile and 0.5mL toluene into each gram of poultry egg product sample, performing ultrasonic treatment for 30min, and filtering supernate to obtain extract liquor; for every two grams of sample supernatant nitrogen was blown near dry and re-dissolved with 1mL of n-hexane to obtain crude extract.

7. The method for synchronously detecting organic phosphate and metabolites in the poultry egg product according to claim 5, wherein the step 3-3) further comprises: the crude extract obtained was purified using CNWBOND Si silica gel column and 500mg of neutral silica gel filler was added per two g of sample.

8. The method for synchronously detecting organic phosphate and metabolites in the poultry egg product according to claim 5, wherein the steps 3-4) further comprise: the eluate containing the target was dried by blowing nitrogen, redissolved with 1.5mL of acetonitrile, degreased with Z-Sep/C18, and added with 60mg of Z-Sep and 30mL of C18 for every two g of samples.

9. The method for synchronously detecting organic phosphate and metabolites in the poultry egg product according to claim 5, wherein the steps 3-5) further comprise: the degreased solution containing the target was further purified using a Strata X-AW column, and 500mg of filler was added for every two grams of sample.

10. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 1, wherein the working conditions of the ultra high performance liquid chromatography in the step 4) are set as follows:

a chromatographic column: poroshell 120 EC-C18 column, length 50mm, internal diameter 4.6mm, filler particle size 2.7 μm; mobile phase: phase A is 0.1% acetic acid in methanol-water (5:95, V/V); phase B was 0.1% acetic acid in methanol-water (95:5, V/V); gradient elution procedure: 0-6 min, 95-5% A; 6-11 min, 5% A; 11-15.5 min, 5-95% A; 15.5-18 min, 95% A; flow rate: 600. mu.L/min^-1(ii) a Column temperature: 45 ℃; sample introduction amount: 5 μ L.

11. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 1, wherein the working conditions of the mass spectrometer in the step 4) are set as follows:

an ionization mode: an electrospray ion source with positive and negative ion modes ESI +/-II; detecting a triple quadrupole mass spectrum; spraying voltage: +/-4000V; mass spectrum scanning mode: multiple reactive ion monitoring, MRM; inlet voltage: -10V; collision cell exit voltage: -15V.

12. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 1, wherein in the step 5), the contents of the parent compounds of organic phosphate and the metabolites thereof are calibrated by using a standard curve without matrix matching.

13. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to any one of claims 1 to 12, wherein the obtained detection data are applied to the analysis of the risk assessment of the exposure to the health of the ingestion of organic phosphate and metabolites by people, including daily exposure analysis, pollution source analysis, exposure-sensitive people analysis, safety limit analysis of markers, risk analysis and classification.

14. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 13, comprising the steps of:

A) setting a computer-based organic phosphate and metabolite health exposure risk assessment and analysis system, which comprises a detection instrument, a main control computer and a built-in analysis program; the analysis program comprises the following components which are connected in sequence and are in interactive communication: the system comprises a control and operation module, an input and output management module, a detection sample management module, a detection component management module, a detection instrument management module, a detection data management module, a daily exposure calculation module, a pollution source analysis module, an exposure sensitive population analysis module, a safety limit management module of a marker and a risk analysis and grading management module;

B) the main control computer is electrified to work, and mathematical models and parameters in the sensitive biomarker management module, the detection sample management module, the detection component management module, the detection instrument management module, the detection data management module, the daily exposure calculation module, the pollution source analysis module, the exposure sensitive population analysis module, the safety limit management module of the marker and the risk analysis and grading management module are respectively set through the input and output management module;

C) the detection instrument outputs the detection data of the sample to a main control computer through a data interface, and a control and operation module of an analysis program, a daily exposure calculation module, an exposure sensitive population analysis module, a safety limit management module of a marker, a risk analysis and grading management module are matched with each other, respectively operate according to preset mathematical models and parameters of the modules, and output corresponding operation results.

15. The method for synchronously detecting organic phosphate and metabolites in avian egg products according to claim 14, wherein the exposure sensitive population analysis module is used for screening and judging sensitive populations exposed to OPEs and mOPEs according to sample detection data.

16. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 14, wherein the sensitive biomarker management module is used for screening and judging the management of markers and their safety limits according to sample detection data.

17. The method for synchronously detecting organic phosphate and metabolites in poultry egg products according to claim 14, wherein the pollution source analysis module is used for analyzing and judging pollution sources according to sample detection data.

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