Method for simultaneously detecting eight haloacetic acid disinfection byproducts in human urine

Abstract

The invention provides a method for simultaneously detecting eight haloacetic acid disinfection byproducts in human urine. The method comprises the following specific steps: S1, preparing a mixed standard solution; s2, preparing an internal standard solution; s3, drawing a standard working curve; s4, sample pretreatment: putting urine into a 15mL centrifugal tube, sequentially adding concentrated sulfuric acid and anhydrous sodium sulfate, uniformly mixing, adding an extracting agent MTBE containing an internal standard, vigorously oscillating, centrifuging, putting an upper organic phase into the 15mL centrifugal tube, adding acidified methanol, uniformly mixing by vortex, and carrying out esterification reaction in a water bath at 50 DEG C for 2 hours; after derivation is finished, taking out the centrifugal tube, placing the centrifugal tube in a refrigerator for cooling, adding an anhydrous sodium sulfate solution with the concentration of 150g / L, uniformly shaking to terminate esterification reaction, standing for layering, taking an upper-layer organic phase, adding an isometric saturated sodium bicarbonate solution, uniformly mixing, standing, transferring the upper-layer organic phase into a small sample injection bottle, and standing for on-machine detection. The method has the advantages of being high in sensitivity, small in sample usage amount and high in practicability, and can be used for conventional biological monitoring of HAAs of people.

Description

Technical Field

[0001] The invention belongs to the technical field of disinfection by-product detection, and specifically relates to a method for simultaneously detecting 8 types of haloacetic acid disinfection by-products in human urine. Background Art

[0002] Drinking water disinfection is an effective way to remove pathogenic microorganisms in water and prevent the spread of waterborne infectious diseases such as typhoid, cholera, and dysentery. It is also an important measure to ensure water quality safety. However, the chlorine in disinfectants can react with natural organic matter, man-made pollutants, and halogen ions in water to generate a class of potentially toxic environmental pollutants, collectively referred to as disinfection by-products (DBPs). Currently, there are more than 800 confirmed DBPs. Haloacetic acids (HAAs), as a representative of non-volatile DBPs, are the second most common DBPs in chlorinated water, accounting for more than 25% of the total DBPs.

[0003] People are directly exposed to HAAs through drinking water. A large number of studies have shown that HAAs have reproductive and developmental toxicity, carcinogenicity, genotoxicity, cytotoxicity, neurotoxicity, and endocrine disruption effects. Dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in HAAs have been identified as animal carcinogenic compounds. Previous epidemiological studies usually assess the exposure level and health risks of the population based on the concentration of HAAs in the water supply system, but this method does not fully consider the spatiotemporal variability of pipe network water and the differences in individual water use habits and physiological activities, which can easily lead to misclassification of exposure levels. In contrast, internal exposure assessment can more accurately reflect the actual exposure of individuals to HAAs. After oral ingestion, HAAs are rapidly absorbed by the gastrointestinal tract and excreted in the urine. Therefore, the level of HAAs in urine is an important indicator for measuring human exposure load.

[0004] HAAs include chloroacetic acid (Cl-HAAs), bromoacetic acid (Br-HAAs) and iodoacetic acid (I-HAAs). Studies have shown that the toxicity of HAAs is I-HAAs>Br-HAAs>Cl-HAAs. At present, the detection methods of HAAs in urine are relatively limited internationally, and none of them covers iodinated haloacetic acids; there is also a poor balance between urine dosage, sensitivity and the production of unstable products. Therefore, it is necessary to develop a detection method for HAAs in urine that has appropriate sensitivity, small sample usage, and simple operation, and can be widely used in large-scale cohorts, covering iodinated, brominated and chlorinated HAAs.

[0005] The present invention establishes a method for simultaneously detecting eight types of haloacetic acid disinfection by-products in human urine by using liquid-liquid extraction-acidified methanol derivatization-gas chromatography, which has the advantages of small dosage (5 mL), high sensitivity (detection limit of 0.013-0.321 μg / L), and simple operation (pretreatment: 7-8 min / sample). Summary of the invention

[0006] The present invention provides a method for simultaneously detecting 8 kinds of haloacetic acid disinfection by-products in human urine. The method has a linear range of 0.1-100 μg / L, the recovery rates of the 8 kinds of HAAs are 84.9%-122.6%, the relative standard deviations are between 1.7% and 14.2%, the detection limits are 0.013-0.321 μg / L, and the quantitative limits are 0.043-1.070 μg / L. The method has the advantages of high sensitivity, small sample usage, and strong practicality, and can be used for routine biological monitoring of HAAs in the human population.

[0007] The terms involved in the present invention are:

[0008] In the present invention, CAA is chloroacetic acid, BAA is bromoacetic acid, DCAA is dichloroacetic acid, TCAA is trichloroacetic acid, IAA is iodoacetic acid, BCAA is chlorobromoacetic acid, BDCAA is monobromodichloroacetic acid, and DBAA is dibromoacetic acid.

[0009] In the present invention, the purity of CAA standard is 99.6%, the purity of BAA standard is 99+%, the purity of DCAA standard is 99+%, the purity of TCAA standard is 97.8%, the purity of IAA standard is 97+%, the purity of BCAA standard is 97+%, the purity of BDCAA standard is 97+%, and the purity of DBAA standard is 99.4%. The above standards are all purchased from CATO Research Chemicals (Guangzhou, China).

[0010] In the present invention, the internal standard 1,2-dibromopropane (1,2-DBP) has a purity of 97% and is purchased from Sigma-Aldrich (Madrid, Spain).

[0011] In the present invention, methanol is of chromatographic grade, ammonium bicarbonate is of analytical grade, and methyl tert-butyl ether MTBE is of chromatographic grade, all of which are purchased from Merck (Darmstadt, Germany); anhydrous sodium sulfate is of analytical grade, and concentrated sulfuric acid is of process ultrapure with a purity of 96.0+%, all of which are purchased from Sinopharm Chemical Reagent (Shanghai, China); artificial urine (containing creatinine, pH 4.7) is purchased from Phygene (Fuzhou, China).

[0012] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: a method for simultaneously detecting 8 types of haloacetic acid disinfection byproducts in human urine, the specific steps are as follows:

[0013] S1: Prepare mixed standard solution: dilute 8 haloacetic acid standards with methanol to prepare a single standard stock solution with a concentration of 20 mg / mL, add 1.5 mL of methanol solution to a 2 mL volumetric flask, transfer 20 μL of the single standard stock solution to the volumetric flask, mix to volume to form a mixed standard stock solution with a final concentration of 200 μg / mL, add 1.5 mL of methanol solution to a 2 mL volumetric flask, transfer 50 μL of the mixed standard stock solution to the volumetric flask, mix to volume to form a mixed standard working solution with a final concentration of 5 μg / mL, take a 1 mL volumetric flask and add 800 μL of methanol solution, accurately transfer 100 μL of the mixed standard working solution to the volumetric flask, mix to volume to form a mixed standard solution with a concentration of 500 μg / L, continue to dilute in the same way to obtain a mixed standard solution with a concentration of 50 μg / L;

[0014] S2: Prepare internal standard solution: add 9.5mL of MTBE solution to a 10mL volumetric flask, pipette 51.6μL of internal standard standard into the volumetric flask, mix to volume to form an internal standard stock solution with a final concentration of 10mg / mL, transfer to a brown glass bottle, cover and oscillate for 1min; take a 10mL volumetric flask and add 9.5mL of MTBE solution, accurately pipette 200μL of internal standard stock solution with a concentration of 10mg / mL, mix to volume to form an internal standard primary dilution with a final concentration of 200μg / mL; pipette 100μL of the internal standard primary dilution into a wide-mouth bottle containing 200mLMTBE, mix to form an internal standard solution with a concentration of 100μg / L;

[0015] S3: Draw a standard working curve: Take 11 15mL centrifuge tubes and number them 1-11, add 5mL of blank artificial urine to each tube, add 100μL, 50μL, 20μL and 10μL of 5μg / mL mixed standard solution to bottles 1-4, add 50μL, 20μL and 10μL of 500μg / L mixed standard solution to bottles 5-7, add 50μL, 20μL and 10μL of 50μg / L mixed standard solution to bottles 8-10, and use bottle 11 as a blank control to control the interference generated during the experiment; add 1.25mL of concentrated sulfuric acid and 4g of anhydrous sodium sulfate to 11 centrifuge tubes, mix well, and add 3mL of internal standard extract Add MTBE, shake vigorously for 2 minutes, centrifuge at 4000r for 5 minutes, take 2mL of the upper organic phase in a 15mL centrifuge tube, add 5mL of acidified methanol, vortex mix for 1min, and carry out esterification reaction in a 50℃ water bath for 2h; after the derivatization, take out the centrifuge tube and cool it in a refrigerator for 10min, add 6mL of anhydrous sodium sulfate solution with a concentration of 150g / L, shake well to terminate the esterification reaction, let it stand for stratification, take 200μL of the upper organic phase, add an equal volume of saturated sodium bicarbonate solution, mix well and let it stand for 2min, transfer the upper organic phase to the injection vial, and leave it for detection on the machine; draw a working curve with the concentration of each concentration point as the horizontal axis and the instrument response intensity as the vertical axis, and the linear relationship strength is quantified by the correlation coefficient R;

[0016] S4: Sample pretreatment: Take 5mL of urine in a 15mL centrifuge tube, add 1.25mL of concentrated sulfuric acid and 4g of anhydrous sodium sulfate in turn, mix well, add 3mL of extractant MTBE containing internal standard, shake vigorously for 2min, centrifuge at 4000r for 5min, take 2mL of the upper organic phase in a 15mL centrifuge tube, add 5mL of acidified methanol, vortex mix for 1min, and carry out esterification reaction in a 50℃ water bath for 2h; after the derivatization, take out the centrifuge tube and cool in the refrigerator for 10min, add 6mL of 150g / L anhydrous sodium sulfate solution, shake well to terminate the esterification reaction, stand for stratification, take 200μL of the upper organic phase, add an equal volume of saturated sodium bicarbonate solution, mix well and stand for 2min, transfer the upper organic phase to the injection vial, and leave it for machine detection;

[0017] S5: Detection and result analysis: Use gas chromatography to detect and analyze the content of haloacetic acid disinfection by-products in urine.

[0018] Preferably, the haloacetic acid disinfection by-products include iodohaloacetic acid disinfection by-products, bromohaloacetic acid disinfection by-products and chlorohaloacetic acid disinfection by-products.

[0019] Preferably, the haloacetic acid disinfection byproducts include CAA, BAA, DCAA, TCAA, IAA, BCAA, BDCAA and DBAA.

[0020] Preferably, the concentration of the internal standard 1,2-DBP in S2 is 1.937 g / mL.

[0021] Preferably, the concentration of the internal standard 1,2-DBP in the extractant MTBE containing the internal standard in S4 is 100 μg / L.

[0022] Preferably, the acidified methanol in S4 contains 10% concentrated sulfuric acid.

[0023] Preferably, in S5, the gas chromatograph is a 6890N gas chromatograph equipped with an ECD detector, and the capillary column is an Agilent capillary column, and the specification of the capillary column is 30m×320μm, 0.25mm.

[0024] Preferably, the working conditions of the gas chromatograph are:

[0025] The injection port temperature was 230°C, the detector temperature was 300°C, the carrier gas and tail gas were both high-purity nitrogen, the tail gas flow rate was 60mL / min, the injection volume was 1μL, the injection method was splitless injection, the column flow rate was 1.4mL / min, the total flow rate was 4.9mL / min, the pressure was 6.3psi, and the average flow rate was 25cm / sec; the programmed temperature conditions were: initial temperature 40°C, maintained for 2min, increased to 80°C at 5°C / min, maintained for 2min, increased to 215°C at 20°C / min, maintained for 0min, and the total time was 18.75min.

[0026] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0027] The invention establishes a method for simultaneously detecting eight kinds of haloacetic acid disinfection byproducts in human urine by adopting liquid-liquid extraction-acidified methanol derivatization-gas chromatography, and tests the method from the aspects of linearity, accuracy, precision, detection limit, etc. The linear range of the method is 0.1-100 μg / L, the recovery rate of the eight kinds of HAAs is in the range of 84.9%-122.6%, the relative standard deviation is between 1.7%-14.2%, the detection limit of the method is 0.013-0.321 μg / L, and the quantitative limit is 0.043-1.070 μg / L. The method has the advantages of small sample dosage (5 mL), high sensitivity (detection limit is 0.013-0.321 μg / L) and simple operation (pretreatment: 7-8 min / sample), and can be used for routine biological monitoring of HAAs in the human population. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying creative work.

[0029] Figure 1 The chromatograms of 8 HAAs, where 1 is CAA, 2 is 1,2-DBP, 3 is BAA, 4 is DCAA, 5 is TCAA, 6 is IAA, 7 is BCAA, 8 is BDCAA, and 9 is DBAA;

[0030] Figure 2 The optimization results analysis diagram of the sample pretreatment procedure, where (A) is the effect of the amount of sulfuric acid added on the HAAs extraction efficiency, (B) is the effect of the amount of anhydrous sodium sulfate added on the extraction efficiency, (C) is the effect of the volume ratio of acidified methanol to the extracted organic phase on the derivatization efficiency, (D) is the effect of the water bath time on the derivatization efficiency, (E) is the effect of the volume of sodium sulfate solution on the response value, and (F) is the effect of the A / M ratio on the recovery volume of MTBE after secondary extraction. DETAILED DESCRIPTION

[0031] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0032] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0033] Example 1 Gas Chromatography Conditions

[0034] The gas chromatograph used in the present invention is a US 6890N gas chromatograph, equipped with an ECD detector, and a capillary column is an Agilent capillary column, wherein the capillary column specifications are 30m×320μm, 0.25mm. The working conditions of the gas chromatograph are: the injection port temperature is 230°C, the detector temperature is 300°C, the carrier gas and the tail gas are both high-purity nitrogen, the tail gas flow rate is 60mL / min, the injection volume is 1μL, the injection mode is non-divided flow injection, the column flow rate is 1.4mL / min, the total flow rate is 4.9mL / min, the pressure is 6.3psi, and the average flow rate is 25cm / sec; the program heating conditions are: the initial temperature is 40°C, maintained for 2min, increased to 80°C at 5°C / min, maintained for 2min, increased to 215°C at 20°C / min, maintained for 0min, and the total time is 18.75min. Eight HAAs standard solutions with a concentration of 20 μg / L were added to blank artificial urine (the standard solutions were CAA, BAA, DCAA, TCAA, IAA, BCAA, BDCAA and DBAA standard solutions). Under this condition, the chromatograms of the eight HAAs were as follows: Figure 1 shown.

[0035] Example 2 Optimization of sample pretreatment procedures

[0036] 1. Determination of the amount of concentrated sulfuric acid added

[0037] Haloacetic acid HAAs are almost completely ionized in the aqueous phase (degree of ionization > 99%), and ionic HAAs are difficult to be extracted by organic extractants. In order to improve the extraction efficiency, acidic substances (such as concentrated sulfuric acid) can be added before extraction to lower the pH value of the solution, so that HAAs are converted from ionic state to molecular state, making them easier to be extracted by organic solvents. According to the provisions of the USEPA 552.3 method, the pH value of the water sample needs to be adjusted to below 0.5. However, there is currently a lack of relevant research on urine as a biological matrix for reference. The present invention uses blank urine matrix spiked samples with a concentration of 20 μg / L as the research object, and explores the effect of concentrated sulfuric acid addition in the range of 0 to 1.5 mL on the extraction efficiency of HAAs. The results are shown in Figure 2. Figure 2 A (the peak area when the amount of concentrated sulfuric acid added was 1.25 mL was taken as 100%).

[0038] Figure 2A shows that when the amount of concentrated sulfuric acid added is between 0 and 1.25 mL, the response intensity of most HAAs increases with the increase in the volume of concentrated sulfuric acid added, especially IAA, DCBAA, TCAA and DBAA, the peak area increases by more than 50%, and when the volume of concentrated sulfuric acid continues to increase, except for BCAA, the response values ​​of the remaining HAAs no longer increase significantly, and even show a downward trend. In addition, by measuring the pH value of samples with different volumes of concentrated sulfuric acid added, it was found that when 1 mL of concentrated sulfuric acid was added, the pH value of the solution had dropped to 0.41, which meets the requirements of the USEPA 552.3 method. Based on the results of this study and the relatively complex situation of the real urine matrix, this study added 1.25 mL of concentrated sulfuric acid before extraction.

[0039] 2. Determination of the amount of anhydrous sodium sulfate added

[0040] Adding anhydrous sodium sulfate can increase the ionic strength of the solution, help weaken the interaction between HAAs with strong polarity and water molecules, promote the transfer of HAAs from the aqueous phase to the organic phase, and thus improve the extraction efficiency. In addition, it can reduce the solubility of organic solvents in the aqueous phase, help separate the aqueous layer and the organic layer after liquid-liquid extraction, and improve the recovery rate and purity of the organic solvent. The effect of the amount of anhydrous sodium sulfate added on the extraction efficiency can be seen in Figure 2 B (the peak area is 100% when the addition amount is 4 g). The results show that when the addition amount of anhydrous sodium sulfate is 4 g, the extraction efficiency of most target HAAs reaches the optimal state.

[0041] 3. Determination of the amount of acidified methanol added

[0042] In the present invention, we have explored the volume ratio (A / M) of acidified methanol to the extracted organic phase during the derivatization process. In current research, A / M is generally not more than 1:1, such as 1:1 in USEPA 552.3 and 1:3 in USEPA 552.2. The present invention sets 6 A / M values, namely 1:1, 1.5:1, 2:1, 2.5:1, 3:1 and 3.5:1, to conduct experimental analysis and explore the effect of A / M on the derivatization efficiency. The results are shown in Table 1. Figure 2 C (peak area is 100% when A / M=1). Experimental data show that, except for CAA and BAA, the response values ​​of other HAAs show a significant growth trend with the increase of A / M, especially for DCBAA, TCAA, IAA, and DBAA with higher molecular weight, the area can increase by more than 2 times, indicating that HAAs with higher molecular weight need to add more acidified methanol to achieve better derivatization effect.

[0043] However, the increase of A / M ratio will affect the recovery volume of MTBE (methyl tert-butyl ether) after secondary extraction. Figure 2As shown in F, when the volume of anhydrous sodium sulfate is fixed, the higher the A / M, the smaller the volume of MTBE recovered, and the MTBE volume of at least 400 μL can meet the needs of subsequent experiments.

[0044] 4. Determination of water bath temperature and time

[0045] Since the boiling point of the extractant MTBE is 60°C, the water bath temperature is set to 50°C to prevent the esterification product generated by excessive temperature from volatilizing with MTBE. Figure 2 D (the peak area when the water bath time is 120 min is 100%). In the range of 0 to 120 min, the peak area of ​​each HAAs ester increases with the increase of water bath time. After continuing to increase to 150 min, the peak area does not change significantly. Therefore, water bath heating for 120 min is the optimal condition.

[0046] 5. Determination of the amount of anhydrous sodium sulfate solution added

[0047] The purpose of adding anhydrous sodium sulfate solution with a concentration of 150g / L is to achieve secondary extraction, promote the transfer of methyl haloacetate from the aqueous phase to the organic phase, and improve the extraction efficiency. Figure 2 As shown in F. When A / M is less than 2.5, the volume of MTBE recovered is large. When A / M is 3.0 or 3.5, more anhydrous sodium sulfate solution (>8mL) needs to be added to separate MTBE, which is not conducive to experimental operation. Therefore, A / M of 2.5 is a suitable condition. Keep A / M at 2.5 to study the effect of the volume of anhydrous sodium sulfate solution on the response value. The results are shown in 2E (peak area is 100% when the volume of anhydrous sodium sulfate solution is 5mL). The response intensity decreases with the increase of the volume of anhydrous sodium sulfate solution. When the amount of anhydrous sodium sulfate solution added is 6mL, the volume of MTBE recovered is about 450μL, which can meet the needs of subsequent experiments.

[0048] The sample pretreatment procedure of the present invention is determined by the above conclusions as follows: take 5mL of urine sample in a 15mL glass centrifuge tube, add 1.25mL of concentrated sulfuric acid, 4g of anhydrous sodium sulfate in turn, mix well, add 3mL of MTBE extractant containing internal standard (internal standard 1,2-DBP concentration 100μg / L), shake vigorously by hand for 2min, and centrifuge at 4000r for 5min. Take 2mL of the upper organic phase in a 15mL glass centrifuge tube, add 5mL of acidified methanol (10% sulfuric acid), vortex mix for 1min, and carry out esterification reaction in a 50℃ water bath for 2h. After the derivatization is completed, take out the centrifuge tube and place it in a refrigerator to cool for 10min, add 6mL of 150g / L anhydrous sodium sulfate solution, shake well to terminate the esterification reaction, take 200μL of the upper organic phase after standing and stratification, add an equal volume of saturated sodium bicarbonate solution, mix well and stand for 2min, transfer the upper organic phase to the injection vial with an inner liner, and leave it for machine detection.

[0049] Example 3 Method Verification

[0050] Method validation includes linearity, precision, accuracy, method detection limit (MDL), method quantify limit (MQL) and quality control

[0051] 1. Linearity, precision and accuracy

[0052] In this study, linearity was evaluated using a 10-point calibration curve, with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. The strength of the linear relationship was quantified using the correlation coefficient (R).

[0053] The internal standard method was used to quantify the target HAAs. The accuracy and precision of the method were evaluated by calculating the matrix spike recoveries and relative standard deviations (RSDs) of six parallel samples at low (0.5 μg / L), medium (5 μg / L), and high (20 μg / L) (5 μg / L, 20 μg / L, and 50 μg / L for DCBAA).

[0054] 2. Limit of detection and limit of quantification

[0055] Six process blanks were prepared using blank artificial urine instead of real urine samples, and the blank samples were analyzed. For HAAs that could be detected in the process blanks, LODs and LOQs were defined as 3 and 10 times the standard deviation of the blank sample detection value, respectively. The final urine sample detection concentration was calculated by deducting the average blank concentration from the initial concentration. For HAAs that were not detected in the blanks, LODs and LOQs were defined as the blank matrix spike concentrations corresponding to the signal-to-noise ratio of 3 or 10, respectively.

[0056] 3. Quality Control

[0057] The glassware used in the experiment was soaked in an alkali cylinder overnight, then washed 5 times with tap water and ultrapure water, and used after drying to prevent possible contamination. The working curve was redrawn before each sample analysis, and calibrated with a 5μg / L standard to prevent the retention time of the target from changing during the analysis. During the instrument analysis process, 5μg / L standard was used for calibration after every 10 samples were measured to test the stability of the instrument. If the deviation exceeded 15%, the sample was re-injected.

[0058] According to the above description, the linearity, accuracy, precision and detection limit of the method were evaluated and verified using the optimized conditions, and the results are shown in Table 1. The eight target HAAs showed good linear relationships in the range of 0.1-100 μg / L. Except for DCBAA, the linear coefficients R of the remaining HAAs were all greater than 0.999. Six parallel experiments were conducted at low, medium and high concentration levels, and the results showed that the spiked recoveries of the eight HAAs ranged from 84.9% to 122.6%, with RSDs of 1.7% to 14.2%. All HAAs were not detected in blank urine, and the detection limits were calculated according to the concentrations corresponding to the signal-to-noise ratios of 3 and 10 for the matrix spiked samples. The detection limits ranged from 0.013 to 0.321 μg / L, and the quantification limits were 0.043 to 1.070 μg / L.

[0059] Table 1. Linear range, linear coefficient and methodological parameters of working curves of 8 haloacetic acids

[0060]

[0061] Example 4 Method Characteristics Performance Comparison

[0062] Due to the long half-life of TCAA in the human body (1-6 days), the high detection rate (>75%) and the high detection level in human urine, it has been confirmed to be the best DBPs biomarker for drinking chlorinated tap water and has been used in many population epidemiological studies. Nevertheless, TCAA levels alone cannot fully represent the cumulative health effects of all HAAs.

[0063] To our knowledge, only three methods have been developed to detect multiple HAAs in urine, but none of them include IAA. [1]100 μL of sample was pretreated by liquid-liquid extraction-acidified methanol derivatization-headspace solid phase microextraction, and HAAs in urine were determined by gas chromatography-electron capture detector (GC-ECD). The linear range of the method was 5-20000 μg / L, and the detection limit was 0.6 μg / L-7.2 μg / L, which was not sensitive enough to determine the level of HAAs in human urine. Ji et al. [2] HAAs are derivatized with pentafluorobenzyl bromide (PFB-Br) and detected by gas chromatography-tandem mass spectrometry (GC-TMS) with a detection limit of 0.025-1 μg / L. However, this method will initially form unstable PFB-ester derivatives for trihalogenated HAAs and quickly decarboxylate to PFB-ethane. In addition, the high sensitivity obtained is related to the high response of the PFB derivative detection instrument. The present invention establishes a detection method for HAAs in human urine, and a large number of verification experiments are carried out on the pretreatment steps to obtain the best experimental conditions, thereby increasing the sensitivity of the target HAAs in the GC-ECD instrument. This method uses less urine, and the sensitivity meets the level of HAAs in human urine, and can be used for routine biological monitoring of HAAs in the population.

[0064] References:

[0065] [1]Wang,Y.-X.,et al.,Profiles,variability and predictors ofconcentrations of blood trihalomethanesand urinary haloacetic acids alongpregnancy among 1760Chinese women.EnvironmentalResearch,2019.172:p.665-674.

[0066] [2]Jia,M.,et al.,Simultaneous Determination of Trace Levels of NineHaloacetic Acids inBiological Samples as Their Pentafluorobenzyl Derivativesby GasChromatography / Tandem Mass Spectrometry in Electron Capture NegativeIon ChemicalIonization Mode.Analytical Chemistry,2003.75(16):p.4065-4080.

[0067] Example 5 Determination of actual samples

[0068] This study was approved by the Medical Ethics Committee of the School of Public Health, Fudan University (IRB#2021-TYSQ-03-116). Urine was collected from 155 community residents in two communities in Shanghai. All participating volunteers read and signed the informed consent form before sampling. The collected urine was morning urine. After packaging on the day of sampling, it was transported back to the laboratory through the cold chain. All urine samples obtained were frozen at -80°C before processing. The results of HAAs detection in urine of community residents are shown in Table 2.

[0069] Table 2. HAAs concentrations in urine samples from community residents in Shanghai (μg / L)

[0070]

[0071]

[0072] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting eight types of haloacetic acid disinfection byproducts in human urine, characterized in that: The specific steps are as follows: S1: Prepare mixed standard solution: dilute 8 haloacetic acid standards with methanol to prepare a single standard stock solution with a concentration of 20 mg / mL, add 1.5 mL of methanol solution to a 2 mL volumetric flask, transfer 20 μL of the single standard stock solution to the volumetric flask, mix to volume to form a mixed standard stock solution with a final concentration of 200 μg / mL, add 1.5 mL of methanol solution to a 2 mL volumetric flask, transfer 50 μL of the mixed standard stock solution to the volumetric flask, mix to volume to form a mixed standard working solution with a final concentration of 5 μg / mL, take a 1 mL volumetric flask and add 800 μL of methanol solution, accurately transfer 100 μL of the mixed standard working solution to the volumetric flask, mix to volume to form a mixed standard solution with a concentration of 500 μg / L, continue to dilute in the same way to obtain a mixed standard solution with a concentration of 50 μg / L; S2: Prepare internal standard solution: add 9.5mL of MTBE solution to a 10mL volumetric flask, pipette 51.6μL of internal standard standard into the volumetric flask, mix to volume to form an internal standard stock solution with a final concentration of 10mg / mL, transfer to a brown glass bottle, cover and oscillate for 1min; take a 10mL volumetric flask and add 9.5mL of MTBE solution, accurately pipette 200μL of internal standard stock solution with a concentration of 10mg / mL, mix to volume to form an internal standard primary dilution with a final concentration of 200μg / mL; pipette 100μL of the internal standard primary dilution into a wide-mouth bottle containing 200mLMTBE, mix to form an internal standard solution with a concentration of 100μg / L; S3: Draw a standard working curve: Take 11 15mL centrifuge tubes and number them 1-11, add 5mL of blank artificial urine to each tube, add 100μL, 50μL, 20μL and 10μL of 5μg / mL mixed standard solution to bottles 1-4, add 50μL, 20μL and 10μL of 500μg / L mixed standard solution to bottles 5-7, add 50μL, 20μL and 10μL of 50μg / L mixed standard solution to bottles 8-10, and use bottle 11 as a blank control to control the interference generated during the experiment; add 1.25mL of concentrated sulfuric acid and 4g of anhydrous sodium sulfate to 11 centrifuge tubes, mix well, and add 3mL of internal standard extract Add MTBE, shake vigorously for 2 minutes, centrifuge at 4000r for 5 minutes, take 2mL of the upper organic phase in a 15mL centrifuge tube, add 5mL of acidified methanol, vortex mix for 1min, and carry out esterification reaction in a 50℃ water bath for 2h; after the derivatization, take out the centrifuge tube and cool it in a refrigerator for 10min, add 6mL of anhydrous sodium sulfate solution with a concentration of 150g / L, shake well to terminate the esterification reaction, let it stand for stratification, take 200μL of the upper organic phase, add an equal volume of saturated sodium bicarbonate solution, mix well and let it stand for 2min, transfer the upper organic phase to the injection vial, and leave it for detection on the machine; draw a working curve with the concentration of each concentration point as the horizontal axis and the instrument response intensity as the vertical axis, and the linear relationship strength is quantified by the correlation coefficient R; S4: Sample pretreatment: Take 5mL of urine in a 15mL centrifuge tube, add 1.25mL of concentrated sulfuric acid and 4g of anhydrous sodium sulfate in turn, mix well, add 3mL of extractant MTBE containing internal standard, shake vigorously for 2min, centrifuge at 4000r for 5min, take 2mL of the upper organic phase in a 15mL centrifuge tube, add 5mL of acidified methanol, vortex mix for 1min, and carry out esterification reaction in a 50℃ water bath for 2h; after the derivatization, take out the centrifuge tube and cool in the refrigerator for 10min, add 6mL of 150g / L anhydrous sodium sulfate solution, shake well to terminate the esterification reaction, stand for stratification, take 200μL of the upper organic phase, add an equal volume of saturated sodium bicarbonate solution, mix well and stand for 2min, transfer the upper organic phase to the injection vial, and leave it for machine detection; S5: Detection and result analysis: Use gas chromatography to detect and analyze the content of haloacetic acid disinfection by-products in urine.

2. The method according to claim 1, characterized in that The haloacetic acid disinfection by-products include iodohaloacetic acid disinfection by-products, bromohaloacetic acid disinfection by-products and chlorohaloacetic acid disinfection by-products.

3. The method according to claim 2, characterized in that The haloacetic acid disinfection byproducts include CAA, BAA, DCAA, TCAA, IAA, BCAA, BDCAA and DBAA.

4. The method according to claim 1, characterized in that The concentration of the internal standard 1,2-DBP in the S2 is 1.937 g / mL.

5. The method according to claim 1, characterized in that The concentration of the internal standard 1,2-DBP in the extractant MTBE containing the internal standard in the S4 is 100 μg / L.

6. The method according to claim 1, characterized in that The acidified methanol in S4 contains 10% concentrated sulfuric acid.

7. The method according to claim 1, characterized in that In the S5, the gas chromatograph is a 6890N gas chromatograph equipped with an ECD detector, and the capillary column is an Agilent capillary column, and the specification of the capillary column is 30m×320μm, 0.25mm.

8. The method according to claim 7, characterized in that The working conditions of the gas chromatograph are: The injection port temperature was 230°C, the detector temperature was 300°C, the carrier gas and tail gas were both high-purity nitrogen, the tail gas flow rate was 60mL / min, the injection volume was 1μL, the injection method was splitless injection, the column flow rate was 1.4mL / min, the total flow rate was 4.9mL / min, the pressure was 6.3psi, and the average flow rate was 25cm / sec; the programmed temperature conditions were: initial temperature 40°C, maintained for 2min, increased to 80°C at 5°C / min, maintained for 2min, increased to 215°C at 20°C / min, maintained for 0min, and the total time was 18.75min.

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