Method for the determination of organophosphorous triesters and precursors and metabolites thereof in environmental media

Abstract

The application discloses a method for determining organic phosphoric acid triesters and precursors and metabolites thereof in an environmental medium, and comprises the following steps: determining one or more of the organic phosphoric acid triesters, the precursors and the metabolites thereof in the environmental medium by liquid chromatography-tandem mass spectrometry, in particular, ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. The application can realize rapid identification and accurate quantification of various organic phosphoric acid triesters, precursors and metabolites thereof in the environmental medium, such as dust, and makes up for the deficiency of the prior art in the field.

Description

Technical Field

[0001] This invention belongs to the technical field of methods for determining organophosphate triesters, and particularly relates to a method for determining organophosphate triesters based on liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Organophosphate trypsins (tri-OPEs) are a class of chemicals widely used as additives in flame retardants, plasticizers, stabilizers, defoamers, wetting agents, hydraulic fluids, and commercial lubricants. Studies have found that tri-OPEs have endocrine-disrupting effects, carcinogenicity, reproductive and neurotoxicity, and because they do not form chemical bonds with the additives, they are easily released into the environment, posing a threat to human health and the environment.

[0003] In addition, organic phosphate ester antioxidants (OPAs) are a class of antioxidants widely used in the production process of polyolefins. During production, use and waste disposal, they are easily oxidized to pentavalent triphosphates and released into the environment, making them one of the potential sources of organic phosphate triesters (tri-OPEs).

[0004] On the other hand, numerous in vivo and in vitro experiments have found that tri-OPEs can be further metabolized into organophosphate ester metabolites (mOPEs), including organophosphate diesters (di-OPEs) and hydroxylated organophosphate esters (OH-OPEs), which have also been shown to have adverse effects such as endocrine disruption, contact allergy, and cytotoxicity. Some mOPEs are even more toxic than their parent compounds.

[0005] Currently, traditional tri-OPEs, OPAs and their oxidized novel tri-OPEs, as well as mOPEs, have been detected in various environmental media such as soil, dust, water, and atmosphere. However, there is no standard detection method for OPAs, tri-OPEs, and mOPEs in the existing technology to achieve rapid and accurate assessment of the concentration level of OPAs and possible metabolites in environmental media. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to establish a method for simultaneously determining a variety of organophosphate triesters (tri-OPEs) and their precursors such as organophosphite antioxidants (OPAs) and metabolites such as organophosphate diesters (di-OPEs) in environmental media. This method is highly efficient and accurate, effectively making up for the deficiencies of the prior art.

[0007] The technical solution of the present invention is as follows:

[0008] A method for determining organophosphates and their precursors and metabolites in environmental media, comprising: using an internal standard method, determining one or more of organophosphates, their precursors, and their metabolites in environmental media by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0009] According to some preferred embodiments of the present invention, the liquid chromatography-tandem mass spectrometry is ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0010] According to some preferred embodiments of the present invention, the organophosphate triester is selected from one or more of the following: triethyl phosphate, tripropyl phosphate, tri-n-butyl phosphate, tri(butoxyethyl) phosphate, tri(2-ethylhexyl) phosphate, triphenyl phosphate, diphenyl 2-ethylhexyl phosphate, tri(4-methylphenyl) phosphate, tri(2-chloroethyl) phosphate, tri(2-chloropropyl) phosphate, tri(1,3-dichloroisopropyl) phosphate, methyl diphenyl phosphate, tris(2,4-di-tert-butyl)phenyl phosphate, bis(2,4-di-tert-butylphenyl)pentaerythritol diphosphate, and tris(4-nonylphenyl) phosphate.

[0011] According to some preferred embodiments of the present invention, the precursor is selected from one or more of tri(2-chloroethyl) phosphite, triphenyl phosphite, tris(2,4-di-tert-butyl)phenyl phosphite, bis(2,4-di-tert-butylphenyl)pentaerythritol diphosphite, tris(4-nonylphenyl) phosphite, and triisodecyl phosphite.

[0012] According to some preferred embodiments of the present invention, the metabolite is selected from one or more of the following: triethyl phosphate, dibutyl phosphate, di(2-butoxyethyl) phosphate, di(2-ethylhexyl) phosphate, di(2-chloroethyl) phosphate, di(2-chloropropyl) phosphate, di(1,3-dichloro)isopropyl phosphate, diphenyl phosphate, di(2-methylphenyl) hydrogen phosphate, bis(2,4-di-tert-butylphenyl) phosphate, 2-(tert-butyl)phenylphenyl hydrogen phosphate, dibenzyl phosphate, (3-hydroxybutyl)dibutyl phosphate, di(2-butoxyethyl)2-(3-hydroxybutoxy)ethyl phosphate, di(2-butoxyethyl)2-hydroxyethyl phosphate, (2-ethyl-5-hydroxyhexyl)diphenyl phosphate, and di(1-chloro-2-propyl)1-hydroxy-2-propyl phosphate.

[0013] According to some preferred embodiments of the present invention, the internal standard compound used in the internal standard method includes one or more of the following: triethyl deuterated phosphate, tripropyl deuterated phosphate, tributyl deuterated phosphate, tri(2-butoxyethyl) deuterated phosphate, triphenyl deuterated phosphate, tri(2-chloroethyl) deuterated phosphate, tri(1,3-dichloro-2-propyl) deuterated phosphate, dibutyl deuterated phosphate, di(2-chloroethyl) deuterated phosphate, diphenyl deuterated phosphate, bis(1-chloro-2-propyl) phosphate, hydrogen deuterated di(2-methylphenyl) phosphate, di(2-butoxyethyl) deuterated phosphate, and di(1,3-dichloro) isopropyl deuterated phosphate.

[0014] According to some preferred embodiments of the present invention, the determination method specifically includes:

[0015] The preparation of standard solutions includes: dissolving the standard of the target compound in methanol under light-protected conditions to obtain a stock solution of each target compound with a concentration of 1.00 mg / mL; mixing the stock solutions of each target compound and diluting them with methanol to obtain a standard solution in which the concentration of each target compound is 1000 ng / mL.

[0016] The preparation of internal standard solutions includes: dissolving the standards of the internal standard compounds in methanol under light-protected conditions to obtain stock solutions of each internal standard compound with a concentration of 1.00 mg / mL; mixing the stock solutions of each internal standard compound and diluting them with methanol to obtain internal standard solutions in which the concentration of each internal standard compound is 1000 ng / mL.

[0017] The preparation of the standard curve sample solution includes: diluting the standard solution to different degrees with methanol to obtain standard solutions with concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.25 ng / mL, i.e., gradient standard solutions; adding the internal standard solution and methanol to each gradient standard solution to obtain a standard curve sample solution in which the concentration of the internal standard compound is 40 ng / mL.

[0018] A standard curve for the target compound is obtained based on the standard curve sample solution.

[0019] The environmental medium to be tested is pretreated to obtain the pretreated test sample;

[0020] The content of the pretreated sample was determined by liquid chromatography-tandem mass spectrometry.

[0021] The target compound is selected from one or more of the following: organophosphate triesters, their precursors organophosphites, their metabolites organophosphate diesters, and their metabolites hydroxylated organophosphate esters; the internal standard compound is selected from deuterated organophosphate triesters and / or deuterated organophosphate diesters.

[0022] According to some preferred embodiments of the present invention, the pretreatment includes: dividing the freeze-dried environmental medium with a particle size of less than 2 mm into two parts; adding the internal standard solution to the first part and then adding acetonitrile solution for multiple extractions and separations, and combining the supernatants to obtain a first extract; adding the internal standard solution to the second part and then adding ammonia in methanol solution for multiple extractions and separations, and combining the supernatants to obtain a second extract; mixing and concentrating the first and second extracts to obtain a pretreated test sample; wherein the volume percentage of ammonia in the ammonia in methanol solution is 0.1%.

[0023] According to some preferred embodiments of the present invention, the environmental medium is soil in the environment.

[0024] According to some preferred embodiments of the present invention, the target compound includes triethyl phosphate, tripropyl phosphate, tri-n-butyl phosphate, tri(butoxyethyl) phosphate, tri(2-ethylhexyl) phosphate, triphenyl phosphate, diphenyl 2-ethylhexyl phosphate, tri(4-methylphenyl) phosphate, tri(2-chloroethyl) phosphate, tri(2-chloropropyl) phosphate, tri(1,3-dichloroisopropyl) phosphate, methyl diphenyl phosphate, tris(2,4-di-tert-butyl) phosphate, and bis(2,4-di-tert-butyl) phosphate. Pentaerythritol diphosphate (Phenyl)phosphite, tris(4-nonylphenyl) phosphate, tris(2-chloroethyl) phosphite, triphenyl phosphite, tris(2,4-di-tert-butyl)phosphite, bis(2,4-di-tert-butylphenyl)pentaerythritol diphosphate, tris(4-nonylphenyl) phosphite, triisodecyl phosphite, triethyl phosphate, dibutyl phosphate, di(2-butoxyethyl) phosphate, di(2-ethylhexyl) phosphate, di(2-chloroethyl) phosphate, di(2-chloropropyl) phosphate, di(1,3-di ... The internal standard compounds include: di(2-methylphenyl)hydrogen phosphate, di(2,4-di-tert-butylphenyl)phosphate, 2-(tert-butyl)phenylphenylhydrogen phosphate, dibenzyl phosphate, (3-hydroxybutyl)dibutyl phosphate, di(2-butoxyethyl)2-(3-hydroxybutoxy)ethyl phosphate, di(2-butoxyethyl)2-hydroxyethyl phosphate, (2-ethyl-5-hydroxyhexyl)diphenyl phosphate, and di(1-chloro-2-propyl)1-hydroxy-2-propyl phosphate; the internal standard compounds include: deuterated... Triethyl phosphate, tripropyl deuterated phosphate, tributyl deuterated phosphate, tri(2-butoxyethyl) deuterated phosphate, triphenyl deuterated phosphate, tri(2-chloroethyl) deuterated phosphate, tri(1,3-dichloro-2-propyl) deuterated phosphate, dibutyl deuterated phosphate, di(2-chloroethyl) deuterated phosphate, diphenyl deuterated phosphate, bis(1-chloro-2-propyl) phosphate, hydrogen deuterated di(2-methylphenyl) phosphate, di(2-butoxyethyl) deuterated phosphate, and di(1,3-dichloro)isopropyl deuterated phosphate.

[0025] According to some preferred embodiments of the present invention, the liquid chromatography-tandem mass spectrometry is an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, wherein the chromatographic column used is a C18 column with a size of 100 mm × 2.1 mm and a packing particle size of 1.9 μm; the mass spectrometer used is in positive ion mode, with an ion injection voltage of 5500 V, a temperature of 450 °C, a collision gas voltage of 8 psi, a declustering voltage and pressure of 10 psi, and a curtain gas pressure of 35 psi.

[0026] According to some preferred embodiments of the present invention, in the determination by liquid chromatography-tandem mass spectrometry, the mobile phase A is methanol and acetonitrile in a volume ratio of 1:1, and the mobile phase B is an aqueous solution of acetic acid with a volume percentage of 0.2%. The elution process is set as follows:

[0027] 0 0.5 95 5 0.5 0.5 95 5 4 0.5 0 100 11 0.5 0 100 11.1 0.5 95 5 14 0.5 95 5

[0028] The determination method of the present invention can simultaneously and accurately determine a variety of OPAs, tri-OPEs and mOPEs compounds and their contents. In particular, when using high performance liquid chromatography tandem triple quadrupole mass spectrometry, it can simultaneously and rapidly and accurately determine a variety of OPAs, their precursors and metabolites, significantly improving the analysis rate, shortening the analysis time, effectively reducing background interference, and lowering the method detection limit.

[0029] In some more specific embodiments, under the preferred chromatographic conditions and tandem mass spectrometry parameters of the present invention, it is possible to simultaneously determine 15 OPEs, 6 OPAs, and 17 metabolites from three major categories (chloroalkyl, non-chloroalkyl, and aromatic), and the detection has excellent selectivity and sensitivity, enabling rapid identification and accurate quantification of OPEs and their precursors and metabolites in soil media, thus making up for the shortcomings of current technologies in this field. Attached Figure Description

[0030] Figure 1 Chromatograms of tri-OPEs and their deuterated internal standards;

[0031] Figure 2 Chromatograms of di-OPEs and their deuterated internal standards;

[0032] Figure 3 The chromatogram of OH-OPEs;

[0033] Figure 4 The chromatogram of OPAs;

[0034] Figure 5 Chromatograms of BCEP, BCIPP, BDCIPP, DBP, DPHP, and their corresponding deuterated internal standard compounds. Detailed Implementation

[0035] The present invention will now be described in detail with reference to embodiments and accompanying drawings. However, it should be understood that the embodiments and drawings are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention. All reasonable modifications and combinations included within the inventive spirit of the present invention fall within the scope of protection of the present invention.

[0036] Example 1

[0037] The following process was used to determine OPEs, OPAs, and their degradation product mOPEs in soil media:

[0038] (1) Obtaining the target compound and internal standard compound

[0039] The following 40 target compounds were used in this embodiment;

[0040] Fifteen tri-OPEs compounds: triethyl phosphate (TEP), tripropyl phosphate (TPP), tri-n-butyl phosphate (TNBP), tri(butoxyethyl) phosphate (TBOEP), tri(2-ethylhexyl) phosphate (TEHP), triphenyl phosphate (TPHP), diphenyl 2-ethylhexyl phosphate (EHDPP), tri(4-methylphenyl) phosphate (TCrP), tri(2-chloroethyl) phosphate (TCEP), tri(2-chloropropyl) phosphate (TCIPP), tri(1,3-dichloroisopropyl) phosphate (TDCIPP), methyl diphenyl phosphate (MDPP), tris(2,4-di-tert-butyl) phosphate (AO168=O), pentaerythritol diphosphate (AO626=O2), and tris(4-nonylphenyl) phosphate (TNPP);

[0041] Six OPA compounds: tri(2-chloroethyl) phosphite (TCEPi), triphenyl phosphite (TPHPi), tris(2,4-di-tert-butyl)phosphite (AO168), bis(2,4-di-tert-butylphenyl)pentaerythritol diphosphite (AO626), tris(4-nonylphenyl)phosphite (TNPPi), and triisodecyl phosphite (TiDePi);

[0042] Eleven di-OPEs compounds: triethyl phosphate (DEP), dibutyl phosphate (DNBP), di(2-butoxyethyl) phosphate (BBOEP), di(2-ethylhexyl) phosphate (BEHP), di(2-chloroethyl) phosphate (BCEP), di(2-chloropropyl) phosphate (BCIPP), di(1,3-dichloro)isopropyl phosphate (BDCIPP), diphenyl phosphate (DPHP), di(2-methylphenyl) phosphate (DoCP), bis(2,4-di-tert-butylphenyl) phosphate (B2,4DtBPP), 2-(tert-butyl)phenylphenyl phosphate (tb-PPP), and dibenzyl phosphate (DTP);

[0043] Five OH-OPE compounds: (3-hydroxybutyl)dibutyl phosphate (3OH-TNBP), di(2-butoxyethyl)2-(3-hydroxybutoxy)ethyl phosphate (3OH-TBOEP), di(2-butoxyethyl)2-hydroxyethyl phosphate (BBOEHEP), di(2-ethyl-5-hydroxyhexyl)diphenyl phosphate (5-OH-EHDPHP), and di(1-chloro-2-propyl)1-hydroxy-2-propyl phosphate (BCIPHIPP).

[0044] And the following 14 internal standard compounds:

[0045] The internal standard compounds for seven deuterated tri-OPEs are: triethyl deuterated phosphate (TEP-D15), tripropyl deuterated phosphate (TPP-D21), tri-n-butyl deuterated phosphate (TNBP-D27), tri(2-butoxyethyl) deuterated phosphate (TBOEP-D27), triphenyl deuterated phosphate (TPHP-D15), tri(2-chloroethyl) deuterated phosphate (TCEP-D12), and tri(1,3-dichloro-2-propyl) deuterated phosphate (TDCIPP-D15).

[0046] The internal standard compounds for the seven deuterated di-OPEs are: di-n-butyl deuterated phosphate (DNBP-D18), di(2-chloroethyl) deuterated phosphate (BCEP-D8), diphenyl deuterated phosphate (DPHP-D10), bis(1-chloro-2-propyl) phosphate (BCIPP-D12), hydrogen di(2-methylphenyl) phosphate (DocP-D14), di(2-butoxyethyl) deuterated phosphate (BBOEP-D8), and di(1,3-dichloro)isopropyl deuterated phosphate (BDCIPP-D10).

[0047] The information of the target compounds is shown in Table 1 below:

[0048] Table 1 Information on target compounds

[0049]

[0050]

[0051]

[0052]

[0053] (2) Prepare standard and internal standard solutions according to the target compound and internal standard compound:

[0054] Weigh 10 mg of each of the above 40 target compounds of OPAs, tri-OPEs, di-OPEs and OH-OPEs, place them in 40 15 mL brown glass bottles, add 10 mL of methanol to each bottle to dissolve them, vortex and mix well to prepare a single standard stock solution of the target compound with a concentration of 1.00 mg / mL.

[0055] Take 10 μL of each of the 40 single standard stock solutions with a concentration of 1.00 mg / mL into a 15 mL brown glass reagent bottle, then add 9.6 mL of methanol solution, and after vortex mixing, obtain mixed working solution 1. The concentration of the target compounds OPAs, tri-OPEs, di-OPEs and OH-OPEs in mixed working solution 1 is 1000 ng / mL.

[0056] Weigh 10 mg of each of the 14 deuterated tri-OPEs and di-OPEs internal standard compounds and place them in 14 15 mL brown glass bottles. Add 10 mL of methanol to each bottle to dissolve them. Vortex the mixtures to prepare individual standard stock solutions of the internal standard compounds with a concentration of 1.00 mg / mL.

[0057] Take 10 μL of each of the 14 internal standard compounds into a 15 mL brown glass reagent bottle, then add 9.86 mL of methanol solution, and after vortex mixing, obtain mixed working solution 2. The concentration of deuterated tri-OPEs and di-OPEs internal standard compounds in mixed working solution 2 is 1000 ng / mL.

[0058] (3) Prepare the standard curve samples:

[0059] Take 400 μL of mixed working solution 1 and add it to a 1.5 mL liquid chromatography vial. Add 600 μL of methanol solution and vortex to mix, obtaining a first mixed solution with a concentration of 400 ng / mL. Then take 500 μL of the first mixed solution and add it to a 1.5 mL liquid chromatography vial. Add 500 μL of methanol solution and vortex to dilute, obtaining a second mixed solution with a concentration of 200 ng / mL. Based on the process of obtaining the first and second mixed solutions, mix working solution 1 is diluted stepwise to finally obtain 8 sets of standard sample solutions with concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.25 ng / mL. Then, 40 μL of mixed working solution 2 and 460 μL of methanol were added to the 8 standard sample solutions. The 8 mixed solutions were diluted to the same extent to obtain 8 standard curve sample solutions containing internal standard compounds. The concentration of the deuterated internal standard compound in the 8 standard curve sample solutions was 40 ng / ml, and the concentration of the target compound was gradient-distributed in the range of 1.56-200 ng / ml.

[0060] (4) Perform sample pretreatment:

[0061] Soil sample collection and sieving: Soil was collected using a clean iron shovel rinsed with methanol to remove gravel, insect shells, roots and other materials. The soil was then placed in an aluminum foil bag rinsed with methanol and freeze-dried in a vacuum freeze dryer. The freeze-dried soil sample was then homogenized by passing it through a 2mm sieve to obtain the soil sample.

[0062] Ultrasonic extraction of soil samples: Weigh two 0.5g soil samples into 15mL glass centrifuge tubes 1 and 2, add 20μL of mixed working solution 2 to each, then add 3mL of acetonitrile solution to centrifuge tube 1 and vortex for 10min. After ultrasonic extraction for 30min, centrifuge at 4000rpm / min and transfer the supernatant to a clean 15mL glass centrifuge tube 3. Repeat the extraction process once, transferring the obtained supernatant to centrifuge tube 3. Add the volume percentage of the solution to centrifuge tube 2. A methanol solution containing 0.1% ammonia was vortexed for 10 min, followed by ultrasonic extraction for 30 min. After high-speed centrifugation at 4000 rpm / min, the supernatant was transferred to a glass centrifuge tube 3. This extraction process was repeated once, and the resulting supernatant was also transferred to centrifuge tube 3. The entire extract in centrifuge tube 3 was then concentrated to near dryness by nitrogen blowing, and methanol was added to bring the volume to 500 μL. After high-speed centrifugation, the supernatant was collected and placed in a sample vial to obtain the pretreated test sample. This sample was then placed in a -20℃ refrigerator for testing.

[0063] In the above process, the acetonitrile solution added to centrifuge tube 1 can be used for the extraction of OPAs, and the methanol solution of ammonia added to centrifuge tube 2 can be used for the extraction of tri-OPEs, di-OPEs and OH-OPEs.

[0064] (5) The pretreated samples were analyzed by LC-MS / MS:

[0065] The chromatographic and mass spectrometric conditions for the LC-MS / MS method are as follows:

[0066] Ultra-high performance liquid chromatography-electrospray ionization triple and quadrupole mass spectrometry (AB SCIEX 6500+ system; ABSCIEX, Santa Clara, CA) was used for separation with a Hypersil GOLD aQ column (C18, 100 mm × 2.1 mm, 1.9 μm, Thermo Fisher Scientific, Bellefonte, PA). ESI 6500+ mass spectrometry was used with positive ions, an ion injection voltage of 5500 V, a temperature of 450 °C, a collision gas voltage of 8 psi, a declustering voltage and pressure of 10 psi, a curtain gas pressure of 35 psi, and multiple reaction monitoring (MRM) mode. The qualitative and quantitative ion pairs and mass spectrometry parameters of the target compounds are shown in Table 2 below.

[0067] Table 2 Instrument detection parameters for the target compound

[0068]

[0069]

[0070]

[0071] The mobile phase A used in the detection was a 1:1 volume ratio of methanol and acetonitrile, and the mobile phase B was an aqueous solution of 0.2% acetic acid by volume. The initial mobile phase volume ratio was mobile phase A: mobile phase B = 95:5, the flow rate was 0.5 mL / min, and the injection volume was 5 μL. The total running time of the mobile phase was 14 min.

[0072] With the initial mobile phase set to 95% A and held for 0.5 min, phase A linearly decreased to 0% and was held for 7 min. Then, within 0.1 min, it linearly recovered to the initial mobile phase conditions of 95% A and 5% B and was held for 2.9 min.

[0073] Table 3 Mobile phase conditions

[0074] 0 0.5 95 5 0.5 0.5 95 5 4 0.5 0 100 11 0.5 0 100 11.1 0.5 95 5 14 0.5 95 5

[0075] The concentrations of OPAs, tri-OPEs, di-OPEs, and OH-OPEs in the dust sample were quantitatively determined by LC-MS / MS, based on the peak area ratio of the analyte to the internal standard, and a standard curve was obtained by normalization.

[0076] Example 2

[0077] Soil samples from agricultural land at different depths were collected using the collection method described in Example 1. Sample processing and instrumental analysis were performed as in Example 1. The linear regression coefficients of the target compounds were all greater than 0.99, indicating good linearity and allowing for accurate quantification. The limits of detection (LOD) and quantitation (LOQ) of the target compounds were calculated using signal-to-noise ratios of 3 and 10, respectively, and the results are shown in Table 4. Compared with existing reports, the analytical method established in this invention can achieve lower LOQs for most OPEs, especially novel tri-OPEs.

[0078] Table 4. Instrumental limits of quantitation and limits of detection for the target compounds.

[0079]

[0080]

[0081] Furthermore, the established LC-MS / MS method was applied to the detection of OPAs, tri-OPEs, di-OPEs, and OH-OPEs in actual soil samples. Based on retention times and characteristic ion pairs, the chromatographic peaks of OPAs, tri-OPEs, di-OPEs, OH-OPEs, and 14 isotopic internal standards were determined, as shown in the attached figure. Figure 1 As shown in Table 5, the sample concentration was calculated using the internal standard method based on the peak area ratio of the target compound and the corresponding internal standard.

[0082] Table 5. Concentration levels of the target compounds in agricultural soil samples (ng / g dry weight)

[0083]

[0084]

[0085] Example 3

[0086] Ultrapure water containing 0.2 mmol ammonium formate was used instead of the 0.2% (v / v) aqueous acetic acid solution in Example 1 as mobile phase B for LC-MS / MS detection. The tests revealed poor peak elution in negative ion mode. Chromatograms of BCEP, BCIPP, BDCIPP, DBP, DPHP, and their corresponding deuterated internal standard compounds with extremely poor peak elution are shown below. Figure 5 As shown.

[0087] and Figure 2 The comparison of peak elution results shows that selecting 0.2% (volume ratio) acetic acid aqueous solution as the precipitation phase B results in better detection.

[0088] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining organophosphates and their precursors and metabolites in environmental media, characterized in that, It includes: The preparation of standard solutions includes: dissolving the standard of the target compound in methanol under light-protected conditions to obtain a stock solution of each target compound with a concentration of 1.00 mg / mL; mixing the stock solutions of each target compound and diluting them with methanol to obtain a standard solution in which the concentration of each target compound is 1000 ng / mL. The preparation of internal standard solutions includes: dissolving the standards of the internal standard compounds in methanol under light-protected conditions to obtain stock solutions of each internal standard compound with a concentration of 1.00 mg / mL; mixing the stock solutions of each internal standard compound and diluting them with methanol to obtain internal standard solutions in which the concentration of each internal standard compound is 1000 ng / mL. The preparation of the standard curve sample solution includes: diluting the standard solution to different degrees with methanol to obtain standard solutions with concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.25 ng / mL, i.e., gradient standard solutions; adding the internal standard solution and methanol to each gradient standard solution to obtain a standard curve sample solution in which the concentration of the internal standard compound is 40 ng / mL. A standard curve for the target compound is obtained based on the standard curve sample solution. The environmental medium to be tested is pretreated to obtain a pretreated test sample. The pretreatment includes: dividing the freeze-dried environmental medium with a particle size of less than 2 mm into two parts; adding the internal standard solution to the first part and then adding acetonitrile solution for multiple extractions and separations, and combining the supernatants to obtain a first extract; adding the internal standard solution to the second part and then adding ammonia in methanol solution for multiple extractions and separations, and combining the supernatants to obtain a second extract; mixing and concentrating the first and second extracts to obtain the pretreated test sample; the volume percentage of ammonia in the ammonia in methanol solution is 0.1%; the environmental medium is soil in the environment. The content of the pretreated sample was determined by liquid chromatography-tandem mass spectrometry. The target compounds include: triethyl phosphate, tripropyl phosphate, tri-n-butyl phosphate, tri(butoxyethyl) phosphate, tri(2-ethylhexyl) phosphate, triphenyl phosphate, diphenyl 2-ethylhexyl phosphate, tri(4-methylphenyl) phosphate, tri(2-chloroethyl) phosphate, tri(2-chloropropyl) phosphate, tri(1,3-dichloroisopropyl) phosphate, methyl diphenyl phosphate, tris(2,4-di-tert-butyl) phosphate, bis(2,4-di-tert-butylphenyl) pentaerythritol diphosphate, tris(4-nonylphenyl) phosphate, tri(2-chloroethyl) phosphite, triphenyl phosphite, tris(2,4-di-tert-butyl) phosphite, bis(2,4-di-tert-butylphenyl) pentaerythritol diphosphate, tris(4-nonylphenyl) phosphite, and triisophosphite. Decyl phosphate, triethyl phosphate, dibutyl phosphate, di(2-butoxyethyl) phosphate, di(2-ethylhexyl) phosphate, di(2-chloroethyl) phosphate, di(2-chloropropyl) phosphate, di(1,3-dichloro)isopropyl phosphate, diphenyl phosphate, di(2-methylphenyl) hydrogen phosphate, bis(2,4-di-tert-butylphenyl) phosphate, 2-(tert-butyl)phenylphenyl hydrogen phosphate, dibenzyl phosphate, (3-hydroxybutyl)dibutyl phosphate, di(2-butoxyethyl)2-(3-hydroxybutoxy)ethyl phosphate, di(2-butoxyethyl)2-hydroxyethyl phosphate, (2-ethyl-5-hydroxyhexyl)diphenyl phosphate, and di(1-chloro-2-propyl)1-hydroxy-2-propyl phosphate; the internal standard compound is selected from deuterated organophosphate triesters and / or deuterated organophosphate diesters; The liquid chromatography-tandem mass spectrometry (LC-MS / MS) used is an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer. The chromatographic column used is a Hypersil GOLD aQ column with dimensions of 100 mm × 2.1 mm and a packing particle size of 1.9 μm. The mass spectrometer is used in positive ion mode with an ion injection voltage of 5500 V, a temperature of 450 °C, a collision gas voltage of 8 psi, a declustering voltage and pressure of 10 psi, and a curtain gas pressure of 35 psi. In the determination performed by the liquid chromatography-tandem mass spectrometry, the mobile phase A was methanol and acetonitrile in a 1:1 volume ratio, and the mobile phase B was an aqueous solution of acetic acid at a volume percentage of 0.2%. The elution process was set as follows:

2. The determination method according to claim 1, characterized in that, The internal standard compounds include one or more of the following: triethyl deuterated phosphate, tripropyl deuterated phosphate, tributyl deuterated phosphate, tri(2-butoxyethyl) deuterated phosphate, triphenyl deuterated phosphate, tri(2-chloroethyl) deuterated phosphate, tri(1,3-dichloro-2-propyl) deuterated phosphate, dibutyl deuterated phosphate, di(2-chloroethyl) deuterated phosphate, diphenyl deuterated phosphate, bis(1-chloro-2-propyl) phosphate, hydrogen deuterated di(2-methylphenyl) phosphate, di(2-butoxyethyl) deuterated phosphate, and di(1,3-dichloro) isopropyl deuterated phosphate.

3. The determination method according to claim 1, characterized in that, The internal standard compounds include triethyl deuterated phosphate, tripropyl deuterated phosphate, tributyl deuterated phosphate, tri(2-butoxyethyl) deuterated phosphate, triphenyl deuterated phosphate, tri(2-chloroethyl) deuterated phosphate, tri(1,3-dichloro-2-propyl) deuterated phosphate, dibutyl deuterated phosphate, di(2-chloroethyl) deuterated phosphate, diphenyl deuterated phosphate, bis(1-chloro-2-propyl) phosphate, hydrogen deuterated di(2-methylphenyl) phosphate, di(2-butoxyethyl) deuterated phosphate, and di(1,3-dichloro)isopropyl deuterated phosphate.

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