Method for simultaneously detecting 17 organophosphate substances in urine

Abstract

The invention relates to a method for simultaneously detecting 17 organophosphate substances in urine, and belongs to the technical field of detection. The invention relates to a method for simultaneously detecting 17 organophosphorus ester substances in urine, and the 17 organophosphorus ester substances are diethyl phosphate, di (2-chloroethyl) phosphate, bis (1-chloro-2-propyl) phosphate, diphenyl phosphate, dibutyl phosphate, xylene phosphate, di-p-tolyl phosphate, triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, 2, 3, 4-trimethyl-1, 3-pentanediol monoisobutyrate, 2, 3, 4-trimethyl-1, 3-pentanediol monoisobutyrate, 2, 3, 4-trimethyl-1, 3-pentanediol monoisobutyrate, 2, 3, 4-trimethyl-1, 3-pentanediol monoisobutyrate and 2, 3, 4-trimethyl-1, 3-pentanediol monoisobutyrate. The invention relates to a flame-retardant and flame-retardant composite material which is prepared from the following components: 1, 2-dichloromethyl-trimethylene-bis [bis (2-chloroethyl) phosphate], bis (2-ethylhexyl) phosphate, tris (1, 3-dichloro-2-propyl) phosphate, triisobutyl phosphate, tributyl phosphate, tris (2-n-butoxyethyl) phosphate, resorcinol tetraphenyl diphosphate and bisphenol A bis diphenyl phosphate. The method has the characteristics of simplicity, convenience, high efficiency, accuracy, sensitivity, economy and the like.

Description

Technical Field

[0001] The invention relates to a method for simultaneously detecting 17 kinds of organophosphate substances in urine, belonging to the technical field of detection. Background Art

[0002] With the gradual banning of brominated flame retardants, organophosphate esters (OPEs) have been used as substitutes, and their production and use have gradually increased in recent years. Organophosphate esters are widely used in various industries as flame retardants, plasticizers and anti-foaming agents, such as building materials, textiles, chemicals, electronics and petroleum. Organophosphate esters are added to products by physical methods. Since there is no chemical bond, they can be slowly released into the environment through wear and volatilization. Studies have shown that OPEs have become a global environmental pollutant and are widely distributed in air, water, dust, soil, sediments and organisms. After entering the human body through dietary intake, respiratory inhalation and other routes, organophosphate esters are easily absorbed by the human body. Toxicological studies have confirmed that OPEs have neurotoxic, carcinogenic, teratogenic, endocrine disrupting effects, etc., and have potential serious hazards to human health. Therefore, it is urgent to conduct research on the internal exposure level of OPEs in the human body to provide basic scientific data for the health risk assessment of OPEs.

[0003] Animal experiments and in vitro cell experiments have shown that OPEs are mainly metabolites in vivo after the side chains are removed to form phosphodiester compounds. After entering the human body, OPEs flow through the blood, liver and kidneys, and urine is one of the main excretion routes. Since urine is a non-invasive biological sample, it has attracted much attention and is widely used in biomarker research. Therefore, determining the level of OPEs diester metabolites in human urine can be used as the main method to evaluate the exposure level of OPEs in the human body.

[0004] However, there is currently no standard method for the detection of 17 organophosphate substances in human urine at home and abroad. Therefore, it is of great practical significance to establish a method for the simultaneous determination of 17 organophosphate substances in human urine by liquid chromatography-tandem mass spectrometry. It can provide reliable technical support for the monitoring and analysis of organophosphate substances in human urine, accurately reflect the exposure level of organophosphate substances in the human body, and scientifically evaluate the safety of organophosphate substances.

[0005] At present, the detection methods of organophosphates reported in the literature mainly include gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, enzyme-linked immunosorbent assay, biosensor method, etc. However, since OPEs contain phosphate groups, they are of medium polarity and have a high boiling point, which makes them suitable for liquid chromatography analysis. However, liquid chromatography has low sensitivity and relies only on retention time for qualitative analysis, which is prone to false positives or false negatives. Analysis using gas chromatography-mass spectrometry requires derivatization operations, which is time-consuming and laborious. Liquid chromatography-tandem mass spectrometry has significant advantages such as high sensitivity, good selectivity, and strong anti-interference ability. It has been applied to the qualitative and quantitative determination of organophosphates in food and food packaging materials.

[0006] Li Pei, Zeng Xiangying, Cui Juntao, et al. Determination of 7 organophosphate metabolites in urine by liquid chromatography-tandem mass spectrometry [J]. Analytical Chemistry, 2017, 45(11): 1648-1654. A method for detecting 7 organophosphate metabolites in human urine was established by liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). For organophosphate diesters with different physicochemical properties, solid phase extraction technology was used for enrichment and purification, and an efficient solid phase extraction column was screened and its elution conditions were optimized; at the same time, the mobile phase and mass spectrometry parameters were optimized to obtain characteristic ion pairs for qualitative and quantitative analysis of each metabolite. The results showed that the pretreatment was enriched by OasisWAX solid phase extraction column, and the target compounds were eluted with 2 mL of methanol containing 5% ammonia and 2 mL of methanol. Except for diethyl phosphate (17.8%-36.2%), the recoveries of the other target compounds were between 60.5% and 104.0%. Under the optimized HPLC conditions, the seven compounds could achieve complete baseline separation, and the detection limits and quantification limits of the seven organophosphate diesters were between (0.005-0.2) μg / L and (0.02-0.5) μg / L, respectively. The intra-day and inter-day precision results (RSD≤15.4%) showed that the method had good stability and reproducibility. The method was used for the detection of organophosphate diesters in urine of the general population. All seven compounds were detected in urine, and the total concentration was between (0.5-6.7) μg / L.

[0007] Luo Xianru, Zhang Jinzhou, Huang Haiyan, et al. Determination of 8 organophosphate metabolites in human urine by liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Health Laboratory Technology, 2022, 32(10):1170-1174. A simple, rapid and accurate liquid chromatography-tandem mass spectrometry (LC-MS / MS) analytical method for the determination of 8 organophosphate metabolites in human urine was established. Methods CNW weak anion exchange (SPE) column was used to extract and enrich the 8 organophosphate metabolites in human urine. 18(2.1 mm × 150 mm, 5 μm) as the chromatographic column, methanol as the organic phase, water (10 mmol ammonium acetate) as the inorganic phase, gradient elution, tandem mass spectrometry electrospray negative ion mode (ESI - ) was used for determination, and the isotope internal standard method was used for quantification. The results showed that the average spike recovery of the target compounds was 83.2%-115.7%, the intra-day precision was 2.8%-11.5%, and the inter-day precision was 3.2%-12.9%. The eight target compounds had a good linear relationship in the linear range of 0.01 μg / L-100.0 μg / L, with a correlation coefficient (r) of more than 0.99 and a limit of quantification of 0.010 μg / L-0.10 μg / L. This method is simple, accurate, has good precision and stability, and is suitable for the determination of the contents of eight organophosphate metabolites in urine.

[0008] However, these existing methods of detecting organophosphates in urine by liquid chromatography-tandem mass spectrometry are applicable to a small number of organophosphate types, have low detection efficiency and high cost, and no detection methods have been reported for some organophosphates, making it impossible to accurately detect the contents of 17 organophosphates at the same time. Summary of the invention

[0009] The purpose of the present invention is to provide a new method for simultaneously detecting 17 kinds of organophosphate substances in urine.

[0010] To achieve the above purpose, the 17 kinds of organophosphate substances are diethyl phosphate, di(2-chloroethyl) phosphate, bis(1-chloro-2-propyl) phosphate, diphenyl phosphate, dibutyl phosphate, ditolyl phosphate, di-p-tolyl phosphate, triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate], di(2-ethylhexyl) phosphate, tri(1,3-dichloro-2-propyl) phosphate, triisobutyl phosphate, tributyl phosphate, tri(2-n-butoxyethyl) phosphate, resorcinol tetraphenyl diphosphate and bisphenol A bisdiphenyl phosphate; The method comprises: A. Sample treatment: adjust the pH value of the urine to be tested to 2 with hydrochloric acid solution, add water to the fixed volume, mix well, centrifuge, take the supernatant and pass it through a pre-activated WAX column, elute with 2% formic acid solution, 30% methanol solution and 50% methanol solution in sequence, discard the eluent, add 5% ammoniated methanol for elution, collect the eluent, add formic acid to the eluent, mix well and pass through a 0.22 μm organic phase filter membrane to obtain the solution to be tested; the volume ratio of urine, volume after adding water to the fixed volume, supernatant passed through the pre-activated WAX column, 2% formic acid solution, 30% methanol solution, 50% methanol solution, 5% ammoniated methanol, and formic acid added to the eluent is: 5:10:2:2:2:2:0.95:0.05; B. preparing a standard solution, respectively detecting the test solution and the standard solution by liquid chromatography-tandem mass spectrometry, and comparing the test results of the test solution with those of the standard solution to obtain a test result; The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent Eclipse XDB-C 18 , 3.0 mm×100 mm, particle size 1.8 µm; mobile phase: methanol and 1 mmol / L ammonium acetate solution; flow rate: 0.20 mL / min~0.32 mL / min; column temperature: 40 ℃; injection volume: 1 µL; The conditions of the mass spectrometry are: Ion source: electrospray ion source; scanning mode: positive ion mode and negative ion mode; detection mode: multiple reaction monitoring; drying gas N2 temperature: 260℃; drying gas N2 flow rate: 5.0 L / min; nebulizer gas pressure: 35 psi (241 kPa); capillary voltage: 4000 V; sheath gas N2 temperature: 350℃; sheath gas N2 flow rate: 12 L / min; nozzle voltage: 0 V.

[0011] In a specific embodiment, the centrifugation in step A is performed at 4000 r / min for 5 min.

[0012] The concentration of the hydrochloric acid solution may be 50% (V / V).

[0013] In a specific embodiment, the test solution in step A is stored away from light.

[0014] In a specific embodiment, the pre-activation method of the WAX ​​column comprises sequentially activating with 3 mL of 5% ammoniacal methanol, 3 mL of methanol, and 3 mL of 2% formic acid solution.

[0015] In a specific embodiment, the mobile phase gradient elution conditions of the liquid chromatography are shown in Table 1: Table 1 Mobile phase gradient elution program

[0016] In a specific embodiment, the liquid chromatography condition is: a flow rate of 0.30 mL / min.

[0017] In a specific embodiment, the retention times and MRM parameters of the 17 organophosphate substances are shown in Table 2: Table 2 Retention times and mass spectrometry parameters of 17 organophosphates

[0018] In a specific embodiment, the method further comprises step C. subtracting the test result of a blank experiment from the test result of step B to obtain a final test result; urine is not added to the blank experiment, and the rest is the same as steps A and B.

[0019] In a specific embodiment, the linear range, detection limit and quantification limit of the 17 organophosphate substances in urine are shown in Table 3: Table 3 Linear range, detection limit and quantification limit of 17 organophosphates in urine

[0020] In a specific embodiment, the recovery rate of the 17 organophosphate substances in the urine ranges from 70.91% to 103.64%; The relative standard deviations of the 17 organophosphate substances in the urine ranged from 0.31% to 13.01%.

[0021] Beneficial effects: The present invention establishes for the first time a method for the simultaneous and accurate determination of the contents of 17 organophosphate substances in human urine, filling the gap of the lack of a method for the simultaneous and accurate determination of the contents of more than 15 organophosphate substances in human urine. It can provide reliable technical support for relevant units to carry out monitoring and analysis of the contents of organophosphate substances in human urine, accurately reflect the exposure level of organophosphate substances in the human body, and scientifically evaluate the safety of organophosphate substances, and has broad economic and social benefits.

[0022] The present invention does not require the use of any isotope internal standard and the nitrogen blowing concentration and dilution. The method of the present invention has a good linear relationship, the correlation coefficient r is greater than 0.999, the recovery rate ranges from 70.91% to 103.64%, and the relative standard deviation ranges from 0.31% to 13.01%.

[0023] The method of the invention has good repeatability and is simple, efficient, accurate, sensitive and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the linear equation and standard curve of diethyl phosphate.

[0025] Figure 2 17 organophosphates were analyzed in Agilent Eclipse XDB-C 18 Total ion current (TIC) of the (3.0 mm × 100 mm, 1.8 μm) column. DETAILED DESCRIPTION

[0026] To achieve the above purpose, the 17 kinds of organophosphate substances are diethyl phosphate, di(2-chloroethyl) phosphate, bis(1-chloro-2-propyl) phosphate, diphenyl phosphate, dibutyl phosphate, ditolyl phosphate, di-p-tolyl phosphate, triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate], di(2-ethylhexyl) phosphate, tri(1,3-dichloro-2-propyl) phosphate, triisobutyl phosphate, tributyl phosphate, tri(2-n-butoxyethyl) phosphate, resorcinol tetraphenyl diphosphate and bisphenol A bisdiphenyl phosphate; The method comprises: A. Sample treatment: adjust the pH value of the urine to be tested to 2 with hydrochloric acid solution, add water to the fixed volume, mix well, centrifuge, take the supernatant and pass it through a pre-activated WAX column, elute with 2% formic acid solution, 30% methanol solution and 50% methanol solution in sequence, discard the eluent, add 5% ammoniated methanol for elution, collect the eluent, add formic acid to the eluent, mix well and pass through a 0.22 μm organic phase filter membrane to obtain the solution to be tested; the volume ratio of urine, volume after adding water to the fixed volume, supernatant passed through the pre-activated WAX column, 2% formic acid solution, 30% methanol solution, 50% methanol solution, 5% ammoniated methanol, and formic acid added to the eluent is: 5:10:2:2:2:2:0.95:0.05; B. preparing a standard solution, respectively detecting the test solution and the standard solution by liquid chromatography-tandem mass spectrometry, and comparing the test results of the test solution with those of the standard solution to obtain a test result; The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent Eclipse XDB-C 18 , 3.0 mm×100 mm, particle size 1.8 µm; mobile phase: methanol and 1 mmol / L ammonium acetate solution; flow rate: 0.20 mL / min~0.32 mL / min; column temperature: 40 ℃; injection volume: 1 µL; The conditions of the mass spectrometry are: Ion source: electrospray ion source; scanning mode: positive ion mode and negative ion mode; detection mode: multiple reaction monitoring; drying gas N2 temperature: 260℃; drying gas N2 flow rate: 5.0 L / min; nebulizer gas pressure: 35 psi (241 kPa); capillary voltage: 4000 V; sheath gas N2 temperature: 350℃; sheath gas N2 flow rate: 12 L / min; nozzle voltage: 0 V.

[0027] In a specific embodiment, the centrifugation in step A is performed at 4000 r / min for 5 min.

[0028] Because human urine contains a certain amount of sodium and potassium salts, and organophosphate substances are metabolites of exogenous pollutants and are at trace or even ultra-trace levels in urine, it is necessary to use the sample processing method of the present invention to extract, enrich and purify the urine to be tested. The pretreatment method of the present invention does not affect the determination of the content of organophosphate substances in the sample.

[0029] In addition to inorganic salts, urine also contains some organic matter. The method of the present invention can remove other matrix interfering components to the greatest extent while retaining organic phosphate substances.

[0030] The experiment showed that the recovery rates of 17 organophosphate esters were good when purified by WAX solid phase extraction cartridge (60 mg / 3 mL). However, when purified by other cartridges, such as HLB cartridge (60 mg / 3 mL), MAX cartridge (60 mg / 3 mL), and MCX cartridge (60 mg / 3 mL), the recovery rates of several organophosphate esters were poor. When using WAX cartridge, the recovery rates of the target diethyl phosphate and di(2-chloroethyl) phosphate were significantly better than those of HLB cartridge, MAX cartridge, and MCX cartridge.

[0031] Experiments show that the pH value of urine affects the enrichment and purification effect of organophosphate substances. Under pH 2.0 conditions, the recovery rate of the target di(2-chloroethyl) phosphate is significantly better than that under other conditions, while for phosphate diester substances under pH 2.0 and pH 3.0 conditions, the recovery rate of the target diethyl phosphate is significantly better than that under other conditions. The optimal urine pH value is 2.0.

[0032] Experiments show that when the methanol concentration of the eluent reaches 70% (v / v), some weakly polar compounds are eluted, and the recovery rate of 50% of the organic phosphate substances is significantly reduced. When the eluent is a 50% methanol solution, the recovery rate of the target diethyl phosphate and di(2-chloroethyl) phosphate is significantly higher than that of the eluent of 30% methanol solution, and after eluting with a 50% methanol solution, the impurity peaks next to the diethyl phosphate chromatographic peak in urine are significantly reduced. In order to ensure that the impurities are eluted more completely and reduce the interference of urine matrix components, a 50% (v / v) methanol solution should be selected as the WAX ​​column eluent. Experiments show that nitrogen blowing concentration will affect the recovery rate of some targets, especially resorcinol tetraphenyl diphosphate and bisphenol A diphenyl phosphate. The recovery rate loss of nitrogen blowing under alkaline conditions is relatively large, and the recovery rates of triisobutyl phosphate, tributyl phosphate and tri-(2-chloroisopropyl) phosphate are also affected to a certain extent after drying. The present invention does not require nitrogen blowing concentration and dilution.

[0033] The concentration of the hydrochloric acid solution may be 50% (V / V).

[0034] Experiments show that Agilent Eclipse XDB-C 18 The (3.0 mm×100 mm, 1.8 μm) chromatographic column is currently the only chromatographic column that meets the requirements of the method of the present invention.

[0035] The mobile phase composition of the present invention is a 1 mmol / L ammonium acetate-methanol system. The response of other mobile phases, such as a water-methanol system, is the worst, and the target di(2-chloroethyl) phosphate is severely tailed, and the peak shapes of dibutyl phosphate, ditolyl phosphate, and di-p-tolyl phosphate are also poor. Compared with a 0.02% acetic acid system, the addition of acid in the 1 mmol / L ammonium acetate system significantly reduces the response of most of its targets. Compared with a 1 mmol / L ammonium acetate-acetonitrile system, most of the targets in the 1 mmol / L ammonium acetate-methanol system respond better. The experiment further investigates the influence of different concentrations of ammonium acetate systems and different types of salt systems on the response of the target. The concentration of ammonium acetate salt ranges from 0.5 mmol / L to 5 mmol / L, and the difference in the response of the target is not large. The response of most of the targets in the ammonium acetate system is significantly higher than that in the ammonium formate system. After comprehensive consideration, the mobile phase composition of 1 mmol / L ammonium acetate-methanol system is the best.

[0036] The experiment showed that as the column temperature increased, the elution time of organophosphates became slightly faster, but it did not affect the separation of organophosphates. In order to maximize the analysis efficiency and reduce the column pressure, the column temperature was selected to be 40 °C after comprehensive consideration.

[0037] The experiment shows that when the temperature of drying gas N2 is 260 ℃, the response of each target peak is better than other conditions; when the flow rate of drying gas N2 is 5 L / min, the response of each target peak is better than other conditions; when the pressure of nebulizing gas N2 is 35 psi (241 kPa), the response of each target peak is better than other conditions; when the capillary voltage is 4000 V, the response of each target peak is better than other conditions; when the temperature of sheath gas N2 is 350 ℃, the response of each target peak is better than other conditions; when the flow rate of sheath gas N2 is 12 L, the response of each target peak is better than other conditions; when the nozzle voltage is 0 V, the response of each target peak is better than other conditions. Therefore, the mass spectrometry ion source conditions are selected as drying gas N2 temperature 260 ℃, drying gas N2 flow rate 5 L / min, nebulizing gas N2 pressure 35 psi (241 kPa), capillary voltage 4000 V, sheath gas N2 temperature 350 ℃, sheath gas N2 flow rate 12 L, and nozzle voltage 0 V.

[0038] In a specific embodiment, the test solution in step A is stored away from light.

[0039] In a specific embodiment, the pre-activation method of the WAX ​​column comprises sequentially activating with 3 mL of 5% ammoniacal methanol, 3 mL of methanol and 3 mL of 2% formic acid solution.

[0040] In a specific embodiment, the mobile phase gradient elution conditions of the liquid chromatography are shown in Table 1 above.

[0041] In a specific embodiment, the chromatographic conditions of the liquid chromatography are: a flow rate of 0.30 mL / min.

[0042] Experiments show that when the chromatographic flow rate of liquid chromatography is in the range of 0.20 mL / min to 0.32 mL / min, the separation degree of each target does not change. When the flow rate gradually decreases, the response of each analyte is improved to a certain extent. However, as the flow rate continues to decrease, the retention time of each analyte is also delayed, and the chromatographic peak becomes wider. 0.30 mL / min is the best choice.

[0043] In a specific embodiment, the retention times and MRM parameters of the 17 organophosphate substances are shown in Table 2 above.

[0044] In a specific embodiment, the method further comprises step C. The test result of step B is deducted from the test result of the blank test to obtain the final test result; the blank test does not add urine, and the rest is the same as steps A and B. Organic phosphates are widely present in various environmental media. During the experiment, it was found that even without injection, high levels of triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, triisobutyl phosphate and tri(2-n-butoxyethyl) phosphate were detected. This may be due to the presence of trace amounts of the target substance in the mobile phase or instrument pipeline material, which interferes with its determination. Therefore, a shorter C is installed after the liquid phase pump. 18 The chromatographic column (e.g., 3.0 mm×50 mm, particle size 1.8 µm) can cause the chromatographic peaks of triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, triisobutyl phosphate, and tris(2-n-butoxyethyl) phosphate remaining in the system to shift backward, thereby effectively distinguishing them from the chromatographic peaks of related target substances in the sample, thereby effectively eliminating the interference of the related target substances remaining in the system itself on the sample detection results.

[0045] In a specific embodiment, the linear range, detection limit and quantification limit of the 17 organophosphate substances in urine are shown in Table 3 above.

[0046] In a specific embodiment, the recovery rate of the 17 organophosphate substances in the urine ranges from 70.91% to 103.64%; The relative standard deviations of the 17 organophosphate substances in the urine ranged from 0.31% to 13.01%.

[0047] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0048] Example 1 1 Experimental materials and instruments 1.1 Reagents and Materials Diethyl phosphate standard: purity ≥95%; molecular formula: C4H 11 O4P; CAS number: 598-02-7. Di(2-chloroethyl) phosphate standard: purity ≥ 99.0%; molecular formula: C4H9O4PC l2 ; CAS number: 3040-56-0. Bis(1-chloro-2-propyl) phosphate standard: purity ≥98%; molecular formula: C6H 13 C l2 O4P; CAS number: 789440-10-4. Diphenyl phosphate standard: purity ≥ 99.0%; molecular formula: C 12 H 11O4P; CAS number: 838-85-7. Dibutyl phosphate standard: purity ≥ 97.0%; molecular formula: C8H 19 O4P; CAS number: 107-66-4. Ditoluene phosphate standard: purity ≥ 99%; molecular formula: C 14 H 15 O4P; CAS No.: 1623-08-1. Di-p-tolyl phosphate standard: purity ≥ 98%; molecular formula: C 14 H 14 O4P; CAS number: 843-24-3. Triphenylphosphine oxide standard: purity ≥ 98%; molecular formula: C 18 H 15 OP; CAS No.: 791-28-6. Tris-(2-chloroisopropyl) phosphate standard: purity ≥ 98.2%; molecular formula: C9H 18 C l3 O4P; CAS number: 13674-84-5. 2,2-Bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate] standard: purity ≥99%; molecular formula: C 13 H 24 C l6 O8P2; CAS number: 38051-10-4. Di(2-ethylhexyl) phosphate standard: purity ≥ 96.9%; molecular formula: C 16 H 35 O4P; CAS number: 298-07-7. Tris(1,3-dichloro-2-propyl) phosphate standard: purity ≥ 95%; molecular formula: C9H 15 C l6 O4P; CAS number: 13674-87-8. Triisobutyl phosphate standard: purity ≥ 99.7%; molecular formula: C 12 H 27 O4P; CAS number: 126-71-6. Tributyl phosphate standard: purity ≥ 99.5%; molecular formula: C 12 H 27 O4P; CAS number: 126-73-8. Tris (2-n-butoxyethyl) phosphate standard: purity ≥ 95%; molecular formula: C 18 H 39 O7P; CAS number: 78-51-3. Resorcinol tetraphenyl diphosphate standard: purity ≥ 98%; molecular formula: C 30 H 24 O8P2; CAS number: 57583-54-7. Bisphenol A bis diphenyl phosphate standard: purity ≥ 98%; molecular formula: C 39 H 34 O8P2; CAS number: 5945-33-5.

[0049] WAX solid phase extraction cartridge, specification: 60 mg / 3mL; ammonia NH3·H2O, high grade; methanol CH3OH, chromatographic grade; formic acid CH2O2, chromatographic grade; hydrochloric acid HCl, high grade; ammonium acetate CH3COOH, high grade; 0.22 μm nylon filter membrane; 0.45 μm aqueous filter membrane; the experimental water is first-grade water, for specific requirements, refer to GB / T 6682.

[0050] 1.2 Solution preparation 5% ammonia methanol solution: Pipette 5 mL of ammonia water, dilute to 100 mL with methanol, and shake well. 30% methanol solution: Pipette 30 mL of methanol, dilute to 100 mL with water, and shake well. 50% methanol solution: Pipette 50 mL of methanol, dilute to 100 mL with water, and shake well. 2% formic acid solution: Pipette 2 mL of formic acid, dilute to 100 mL with water, and shake well. Hydrochloric acid (1+1) solution: Pipette 50 mL of water, slowly add 50 mL of hydrochloric acid, and shake well. 1 mmol / L ammonium acetate solution: Weigh 0.77 g of ammonium acetate, dissolve in water to 1L, mix well, filter the aqueous phase with a membrane filter, and degas by ultrasonication.

[0051] Single standard stock solution: Take about 10 mg (accurate to 0.1 mg) of each organophosphate standard substance, dissolve them in methanol and make up to 10 mL, prepare a single standard stock solution with a mass concentration of 1000 mg / L, store at -18 ℃, and the validity period is half a year.

[0052] Mixed standard stock solution: Pipette an appropriate amount of each organophosphate standard stock solution into the same 100 mL volumetric flask, dilute to volume with methanol, shake well, and prepare a mixed standard stock solution with a mass concentration of 20 mg / L for di(2-chloroethyl) phosphate and a mass concentration of 10 mg / L for the remaining organophosphates. Store at -18 ℃ with a shelf life of one month.

[0053] Mixed standard intermediate solution: Pipette 1.0 mL of the mixed standard stock solution into a 100 mL volumetric flask, dilute with methanol to make up the volume, and prepare a mixed standard intermediate solution with a mass concentration of di(2-chloroethyl) phosphate of 200 ng / mL and a mass concentration of 100 ng / mL for the other organophosphate substances. Prepare and use immediately.

[0054] A series of standard working solutions: Accurately pipette appropriate amount of the mixed standard intermediate solution, dilute to volume with 50% methanol solution, shake well to prepare a series of standard working solutions with mass concentrations of 2-chloroethyl phosphate being 0.4 ng / mL, 1.0 ng / mL, 2 ng / mL, 4ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and mass concentrations of other organophosphate substances being 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively. Prepare and use immediately.

[0055] 1.3 Instruments and Equipment Liquid chromatography-tandem mass spectrometry: equipped with electrospray ionization source (ESI source). Analytical balance: sensitivity 0.1 g, 0.01 mg. Acidity meter: accuracy 0.01 pH unit, pH value measurement range 0-14. Vortex mixer. Electric pipette: 0 mL-50 mL.

[0056] 2 Experimental methods Human midstream urine is collected in a glass or plastic centrifuge tube that does not contain organophosphates and stored in an ultra-low temperature refrigerator for later use. Before use, thaw at room temperature, mix, centrifuge, and wait for pre-treatment.

[0057] 2.1 Sample pretreatment Accurately transfer 5 mL of the sample into a 10 mL glass centrifuge tube, adjust the pH value to 2 with hydrochloric acid (1+1), add water to make up to 10 mL, mix well and centrifuge at 4000 r / min for 5 min, take 2.0 mL of the supernatant and pass it through a pre-activated WAX column (activated with 3 mL 5% ammoniated methanol, 3 mL methanol, and 3 mL 2% formic acid solution in sequence), elute with 2 mL 2% formic acid solution, 2 mL30% methanol solution and 2 mL50% methanol solution in sequence, discard the eluent, accurately add 950 μL ammoniated methanol for elution, collect the eluate, add 50 μL formic acid, vortex mix well, filter through a 0.22 μm nylon filter membrane and store in a brown injection vial for testing.

[0058] Blank experiment: Except for not adding urine, the other steps are the same as above.

[0059] 2.2 Chromatographic reference conditions Column: Agilent Eslipse XDB-C 18 Column, 3.0 mm × 100 mm, particle size 1.8 µm; mobile phase: methanol and 1 mmol / L ammonium acetate solution, gradient elution program see Table 1 above; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 1 μL.

[0060] 2.3 Mass spectrometry reference conditions Ion source: electrospray ion source (ESI source); scanning mode: positive ion mode and negative ion mode; detection mode: multiple reaction monitoring (MRM); drying gas N2 temperature: 260 ℃; drying gas N2 flow rate: 5.0 L / min; nebulizer gas N2 pressure: 35 psi (241 kPa); capillary voltage: 4000 V; sheath gas N2 temperature: 350 ℃; sheath gas N2 flow rate: 12 L / min; nozzle voltage: 0 V; qualitative ion pairs, quantitative ion pairs, collision energy, and in-source fragmentation voltage refer to Table 2 above.

[0061] 2.4 Qualitative determination Under the same experimental conditions, the retention time of the chromatographic peak of the analyte in the sample solution is the same as that of the standard working solution (within the range of ±2.5%), and the selected ions all appear in the spectrum of the sample solution after background subtraction, and the deviation of the relative abundance of each qualifier ion and the relative abundance ratio of the standard ion does not exceed the range specified in Table 4, then it can be determined that the corresponding analyte exists in the sample.

[0062] Table 4 Maximum allowable deviation of relative ion abundance for qualitative confirmation 2.5 Quantitative determination Under the same experimental conditions, a series of mixed standard working solutions are injected and analyzed in sequence from low to high concentrations. The standard working curve is plotted with the peak area of ​​the analyte as the ordinate and the mass concentration of the analyte solution as the abscissa. The standard working curve is used to quantify the samples. The mass concentration of each analyte in the sample solution should be within the mass concentration range of the standard working curve. Samples exceeding the upper limit of the mass concentration of the standard working curve should be appropriately diluted according to the measured concentration before analysis.

[0063] 2.6 Results Analysis The content of the target substance in the sample is calculated according to formula (1).

[0064] Where: w ——The content of the target substance in the sample, in nanograms per milliliter (ng / mL); ρ ——The mass concentration of the target substance in the sample obtained from the standard working curve, in nanograms per milliliter (ng / mL); V ——The fixed volume of the sample, in milliliters (mL); V 样 ——The sample volume, in milliliters (mL). The blank value should be deducted from the calculation result.

[0065] 3 In-laboratory validation of the method 3.1 Verification of the linear range, detection limit and quantification limit of the method Table 5 Method linearity and sensitivity results Under the optimal working conditions of the instrument, a series of mixed standard working solutions for determination were injected into the liquid chromatography-tandem mass spectrometer from low to high concentrations, and a standard working curve was drawn with the peak area of ​​the target as the ordinate and the concentration of the standard working solution as the abscissa, as shown in Table 5 and Figure 1 As shown in the table. It can be seen from the table that the linear relationship of each target substance is good within the range of the limit of quantification ~ 20 ng / mL (the limit of quantification of di(2-chloroethyl) phosphate is ~ 40 ng / mL), and the correlation coefficient r is all > 0.999. In order to investigate the sensitivity of this method, the signal-to-noise ratio method was used to calculate the limit of detection (LOD) and limit of quantification (LOQ) of this method. The concentration with a signal-to-noise ratio S / N ≥ 3 was determined as the limit of detection concentration of this method, and the concentration with a signal-to-noise ratio S / N ≥ 10 was determined as the limit of quantification concentration of this method. The specific results are shown in Table 5. It can be seen from the table that this method has good sensitivity for the determination of organophosphate substances, and can well meet the needs of trace or even ultra-trace determination of 17 organophosphate substances in urine.

[0066] 3.2 Verification of method accuracy 11 human urine samples were taken, 2 of which were used to prepare the test solution in full accordance with the sample pretreatment method specified in 2.1, and the other 9 were added with appropriate amounts of mixed standard intermediate solution to make the final test solution concentrations constitute three different concentration levels of spiked samples: low concentration, medium concentration, and high concentration. The test solution was then prepared according to the sample pretreatment method specified in 2.1 to examine the spiked recovery of this method. The specific results are shown in Tables 6 to 22. As can be seen from the table, the recovery range of 17 organophosphate substances in human urine determined by this method is 70.91% to 103.64%, and the relative standard deviation range is 0.31% to 13.01%, which meets the requirements of GB / T 32465-2015 "Requirements for Validation and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for Validation and Verification of Chemical Analysis Methods for Conformity Assessment" and other relevant standards for method recovery and precision, indicating that this method can meet the needs of accurate determination of the content of 17 organophosphate substances in human urine.

[0067] Table 6 Recovery rate of diethyl phosphate Table 7 Recovery rate of di(2-chloroethyl) phosphate Table 8 Recovery rate of added bis(1-chloro-2-propyl) phosphate Table 9 Recovery rate of diphenyl phosphate addition Table 10 Recovery rate of dibutyl phosphate Table 11 Recovery rate of ditolyl phosphate Table 12 Recovery rate of di-p-tolyl phosphate Table 13 Recovery rate of triphenylphosphine oxide Table 14 Recovery rate of tri-(2-chloroisopropyl) phosphate Table 15 Addition recovery of 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl)phosphate] Table 16 Recovery rate of di(2-ethylhexyl) phosphate Table 17 Recovery rate of tris(1,3-dichloro-2-propyl) phosphate Table 18 Recovery rate of triisobutyl phosphate Table 19 Recovery rate of tributyl phosphate Table 20 Recovery rate of tri(2-n-butoxyethyl) phosphate Table 21 Addition recovery rate of resorcinol tetraphenyl diphosphate Table 22 Recovery rate of bisphenol A diphenyl phosphate 3.3 Verification of method precision A 10 ng / mL mixed standard solution (2-chloroethyl phosphate concentration is 20 ng / mL) was prepared, and 6 injections were made continuously. The injection precision of the method was calculated. The specific results are shown in Table 23. Six urine samples were taken, 100 µL of a 100 ng / mL mixed standard solution (2-chloroethyl phosphate concentration is 200 ng / mL) was added, and the test solution was prepared according to the sample pretreatment method of this embodiment. One injection was made for each sample, and the repeatability of each spiked solution was calculated. The specific results are shown in Tables 23 to 25. Under the same experimental conditions, person B took 6 urine samples, added 100 µL of a 100 ng / mL mixed standard solution (2-chloroethyl phosphate concentration is 200 ng / mL), and the test solution was prepared according to the sample pretreatment method of this embodiment. One injection was made for each sample. The intermediate precision of each spiked solution was calculated by comparing with the repeatability data. The specific results are shown in Table 25. As can be seen from the table, the precision of this method for determining 17 kinds of organophosphates in human urine fully meets the requirements of GB / T 32465-2015 "Validation and Internal Quality Control Requirements for Chemical Analysis Methods", GB / T 27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment" and other relevant standards for method repeatability and precision, indicating that this method has good precision in determining the content of 17 kinds of organophosphate substances in urine. The 17 organophosphates in the following table are represented by abbreviations, ester A is diethyl phosphate; ester B is di(2-chloroethyl) phosphate; ester C is bis(1-chloro-2-propyl) phosphate; ester D is diphenyl phosphate; ester E is dibutyl phosphate; ester F is ditolyl phosphate; ester G is di-p-tolyl phosphate; ester H is triphenylphosphine oxide; ester I is tri-(2-chloroisopropyl) phosphate; ester J is 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate]; ester K is di(2-ethylhexyl) phosphate; ester L is tri(1,3-dichloro-2-propyl) phosphate; ester M is triisobutyl phosphate; ester N is tributyl phosphate; ester O is tri(2-n-butoxyethyl) phosphate; ester P is resorcinol tetraphenyl diphosphate; ester Q is bisphenol A bisdiphenyl phosphate.

[0068] Table 23 Injection precision results of 17 organophosphate esters Table 24 Repeatability results of 17 organophosphates Table 25 Precision results of 17 kinds of organophosphates 3.4 Verification of method stability 1 μL of the mixed standard working solution and the urine spiked test solution were respectively taken and injected into the liquid chromatography-tandem mass spectrometer for analysis at 0 h, 2 h, 4 h, 8 h, 16 h and 24 h according to the optimal instrument and equipment conditions optimized by this method to investigate the stability of each test solution. The specific results are shown in Table 26 and Table 27. As can be seen from the table, there was no obvious regular decrease or increase in the response results of each solution within 24 hours. According to the requirements of GB / T 32465-2015 "Requirements for Validation and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment" and other relevant standards, the RSD of the test results of each test solution met the requirements, indicating that the test solutions prepared by this method were all stable within 24 hours.

[0069] Table 26 Stability test results of mixed standard working solution Table 27 Stability test results of spiked solution in urine 4. Inter-laboratory validation of the method The key parameters such as detection limit, quantification limit, linearity and range, accuracy and precision were verified in the laboratory. The verification samples were entrusted to three universities and testing institutions in Sichuan, Tianjin and Guizhou respectively. The method of this embodiment was used for inter-laboratory verification. The average recovery rate was 71.96% to 99.70%, and the RSD was between 0.43% and 9.86%. The feedback results showed that this method had good accuracy and precision in determining the contents of 17 organophosphate substances in human urine, which fully met the relevant standard requirements.

[0070] Example 2 and Comparative Examples 1-5 Example 2 Using Agilent Eclipse XDB-C 18 (3.0 mm × 100 mm, 1.8 μm), Comparative Examples 1-5 were made using Agilent Extend C 18 (2.1 mm × 100 mm, 1.8 μm), Agilent RRHD SB-C 18(2.1 mm×100 mm, 1.8 μm), HSS T3 (2.1 mm×100 mm, 1.8 μm), ACE Excel 3 AQ (2.1 mm×100 mm, 1.8 μm), Agilent SB-AQ (2.1 mm×100 mm, 1.8 μm), numbered 1 to 6, the separation and response effects of columns 1 to 6 on organophosphate substances are shown in Table 28, Agilent Eclipse XDB-C 18 The total ion current (TIC) of the chromatographic column (3.0 mm × 100 mm, 1.8 μm) is shown in Figure 2 .Depend on Figure 2 As shown in Table 28, when Agilent SB-AQ (column 6) was used as the chromatographic column, the peaks of most organophosphates were asymmetric and had tailing. When HSS T3 (column 4) was used as the chromatographic column, the isomers of triisobutyl phosphate and tributyl phosphate could not be separated. 18 When (column 2) was used as the chromatographic column, the peak shapes of target compounds such as di(2-chloroethyl) phosphate, dibutyl phosphate, ditolyl phosphate, and di-p-tolyl phosphate were slightly poor and asymmetrical; when using Agilent RRHD SB-C 18 When (column 3) was used as the chromatographic column, the peak of di(2-chloroethyl) phosphate was slightly fatter, and the response of di-p-cresyl phosphate was reduced to 0.38% of that of column 1. When ACE Excel 3 AQ (column 5) was used as the chromatographic column, the responses of diphenyl phosphate, di-p-cresyl phosphate, and triphenylphosphine oxide were significantly reduced.

[0071] Table 28 Response values ​​of each compound on different chromatographic columns Note: Targets 1-17 are: 1. diethyl phosphate; 2. bis(2-chloroethyl) phosphate; 3. bis(1-chloro-2-propyl) phosphate; 4. diphenyl phosphate; 5. dibutyl phosphate; 6. ditolyl phosphate; 7. di-p-tolyl phosphate; 8. triphenylphosphine oxide; 9. tris-(2-chloroisopropyl) phosphate; 10. 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate]; 11. di(2-ethylhexyl) phosphate; 12. tris(1,3-dichloro-2-propyl) phosphate; 13. triisobutyl phosphate; 14. tributyl phosphate; 15. tris(2-n-butoxyethyl) phosphate; 16. tetraphenyl diphosphate of resorcinol; 17. bisphenol A diphenyl phosphate.

Claims

1. A method for simultaneously detecting 17 organophosphate substances in urine, characterized in that: The 17 kinds of organophosphate substances are diethyl phosphate, di(2-chloroethyl) phosphate, bis(1-chloro-2-propyl) phosphate, diphenyl phosphate, dibutyl phosphate, ditolyl phosphate, di-p-tolyl phosphate, triphenylphosphine oxide, tri-(2-chloroisopropyl) phosphate, 2,2-bischloromethyl-trimethylene-bis[bis(2-chloroethyl) phosphate], di(2-ethylhexyl) phosphate, tri(1,3-dichloro-2-propyl) phosphate, triisobutyl phosphate, tributyl phosphate, tri(2-n-butoxyethyl) phosphate, resorcinol tetraphenyl diphosphate and bisphenol A bisdiphenyl phosphate; The method comprises: A. Sample treatment: adjust the pH value of the urine to be tested to 2 with hydrochloric acid solution, add water to the fixed volume, mix well, centrifuge, take the supernatant and pass it through a pre-activated WAX column, elute with 2% formic acid solution, 30% methanol solution and 50% methanol solution in sequence, discard the eluent, add 5% ammoniated methanol for elution, collect the eluent, add formic acid to the eluent, mix well and pass through a 0.22 μm organic phase filter membrane to obtain the solution to be tested; the volume ratio of urine, volume after adding water to the fixed volume, supernatant passed through the pre-activated WAX column, 2% formic acid solution, 30% methanol solution, 50% methanol solution, 5% ammoniated methanol, and formic acid added to the eluent is: 5:10:2:2:2:2:0.95:0.05; B. preparing a standard solution, respectively detecting the test solution and the standard solution by liquid chromatography-tandem mass spectrometry, and comparing the test results of the test solution with those of the standard solution to obtain a test result; The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent Eclipse XDB-C 18 , 3.0 mm×100 mm, particle size 1.8 µm; mobile phase: methanol and 1 mmol / L ammonium acetate solution; flow rate: 0.20 mL / min~0.32 mL / min; column temperature: 40 ℃; injection volume: 1 µL; The mass spectrometry conditions are as follows: ion source: electrospray ion source; scanning mode: positive ion mode and negative ion mode; detection mode: multiple reaction monitoring; drying gas N2 temperature: 260°C; drying gas N2 flow rate: 5.0 L / min; nebulizing gas N2 pressure: 35 psi; capillary voltage: 4000 V; sheath gas N2 temperature: 350°C; sheath gas N2 flow rate: 12 L / min; nozzle voltage: 0 V.

2. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1, characterized in that: The centrifugation in step A is performed at 4000 r / min for 5 min.

3. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1, characterized in that: The test solution described in step A is stored away from light.

4. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The pre-activation method of the WAX ​​column includes sequentially activating with 3 mL of 5% ammoniacal methanol, 3 mL of methanol, and 3 mL of 2% formic acid solution.

5. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The mobile phase gradient elution conditions of the liquid chromatography are shown in Table 1: Table 1 Mobile phase gradient elution program 。 6. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The conditions of the liquid chromatography are: flow rate is 0.30 mL / min.

7. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The retention times and MRM parameters of the 17 organophosphate substances are shown in Table 2: Table 2 Retention times and mass spectrometry parameters of 17 organophosphates 。 8. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The method further comprises step C: subtracting the test result of the blank test from the test result of step B to obtain a final test result; the blank test does not add urine, and the rest is the same as steps A and B.

9. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The linear range, detection limit and quantification limit of the 17 organophosphate substances in urine are shown in Table 3: Table 3 Linear range, detection limit and quantification limit of 17 organophosphate substances in urine 。 10. The method for simultaneously detecting 17 kinds of organophosphate substances in urine according to claim 1 or 2, characterized in that: The recovery rates of the 17 organophosphate substances in urine ranged from 70.91% to 103.64%; The relative standard deviations of the 17 organophosphate substances in the urine ranged from 0.31% to 13.01%.

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