Gas phase detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum

Abstract

The invention provides a gas phase detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum. The gas phase detection method comprises the following steps: S1, liquid-liquid extraction-acidification methanol derivatization pretreatment; s2, instrument detection: S21, chromatographic conditions: a 6890N gas chromatograph and a DB-1701 capillary column are adopted, the temperature of a sample inlet is 200-250 DEG C, and the temperature of a detector is 280-320 DEG C; s22, carrying gas and flow velocity; s23, temperature programming conditions are set; according to the method, by optimizing detection conditions, the recovery rate of the HAAs is increased, and the detection limit of the method is reduced, and the method is proper in sensitivity, small in sample usage amount, simple and convenient to operate and capable of being widely applied to detection of the HAAs in clinical large-scale queues and serum.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a gas detection method for simultaneously determining 6 kinds of by-products of chlorine-based disinfectants in serum. Background Art

[0002] Chlorination disinfection is one of the most important disinfection methods in drinking water treatment, which ensures water quality safety by effectively inhibiting pathogenic microorganisms in water. However, during the disinfection process, chlorine-based disinfectants (including chlorine Cl 2 , hypochlorous acid HOCl and hypochlorite OCl-) will inevitably react with dissolved organic matter, bromide and iodide, thus generating disinfection by-products (DBPs). Currently, more than 800 kinds of disinfection by-products have been identified, among which haloacetic acids (HAAs) account for 13% of the total organic halogens in water and are the second largest category of disinfection by-products after trihalomethanes.

[0003] Haloacetic acids (HAAs) have attracted much attention due to their wide exposure in water bodies and potential health risks. These compounds can not only accumulate in the human body through drinking water, but also are related to a variety of potential toxicities, including reproductive and developmental toxicity, cytotoxicity and genotoxicity. The more common haloacetic acids in water bodies include chloroacetic acid (CAA), bromoacetic acid (BAA), dichloroacetic acid (DCAA), trichloroacetic acid, (TCAA), iodoacetic acid, (IAA), dibromoacetic acid (DBAA), dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) as representative substances of HAAs. Existing studies have shown that their carcinogenic risks are significantly higher than those of trihalomethanes (THMs), 50 times and 100 times that of THMs respectively. In addition, the carcinogenic risk of HAAs accounts for more than 91.9% of the carcinogenic risk of DBPs.

[0004] In previous epidemiological studies, evaluating the population exposure level and health risks of haloacetic acids often relied on the concentration of haloacetic acids in the water supply system. However, this method ignored the variability of pipe network water in time and space, as well as the differences in individual water use habits and physiological activities, which was prone to misclassification of exposure levels. In addition, when traditional epidemiological studies explored the distribution of haloacetic acids in the human body, they mainly focused on the two biomarkers of dichloroacetic acid and trichloroacetic acid in urine, lacking research data in blood. Serum samples, as biological samples directly reflecting the internal environment of the human body, can more accurately reveal the actual internal exposure level of individuals to haloacetic acids. Therefore, haloacetic acids in serum are of great significance for comprehensively evaluating the exposure level and health risks of individuals.

[0005] At present, the international methods for detecting haloacetic acids in drinking water mainly include EPA methods 552.1 and 552.2, and standard method 6251B. However, the detection methods for haloacetic acids in blood samples are very limited, and face challenges in terms of serum dosage, sensitivity, and resistance to interference from complex blood matrices. Therefore, it is necessary to develop a blood HAAs detection method with appropriate sensitivity, small sample usage, simple operation, and can be widely used in large-scale cohorts. Summary of the invention

[0006] The present invention aims to provide a liquid-liquid extraction-acidified methanol derivatization-gas chromatography method to establish a set of rapid qualitative and quantitative analysis methods for 6 types of HAAs in serum at the same time, and through optimization, improve the recovery rate of HAAs and reduce the detection limit of the method, so as to facilitate the rapid detection of HAAs in serum and monitor the risk of carcinogens in the human body. The present invention adopts the following technical solutions:

[0007] The present invention provides a qualitative and quantitative gas phase detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum, comprising the following steps:

[0008] S1 Liquid-liquid extraction-acidification methanol derivatization pretreatment: Take 0.1-1 mL of serum to a 10-100 mL headspace bottle, add 0.2-5 mL of sulfuric acid to acidify to pH < 0.5, mix well and quickly add 10-200 mg of anhydrous sodium sulfate to dissolve all the solids, then add 0.5-15 mL of methyl tert-butyl ether (MTBE) containing internal standard and shake vigorously for 1-10 min, then vortex and shake, mix thoroughly and centrifuge for 5, aspirate 1-3 mL of supernatant and transfer to a 10-20 mL glass centrifuge tube, add 2-6 mL 10% sulfuric acid methanol solution, vortex mix and then derivatize, prepare ice-water mixture in advance, cool to room temperature in an ice-water bath immediately after derivatization, add sodium sulfate solution, shake and quickly open the bottle cap to release gas to terminate the esterification reaction, let stand and separate, draw 300-800 μL of the upper organic phase and transfer it to a centrifuge tube, add 0.5-5mL saturated sodium bicarbonate solution for neutralization, mix and stand, transfer the upper ester layer, add anhydrous sodium sulfate to remove moisture, and seal and store in a -30--10℃ refrigerator;

[0009] Further, the concentration of the sulfuric acid is 50 to 80 v / v% sulfuric acid;

[0010] Further, the internal standard is 1,2-dibromopropane (i.e., 1,2-DBP); further, the internal standard concentration is 2 to 100 μg / L,

[0011] Furthermore, the vortexing condition is oscillating on a vortex oscillator at 1500-2000 rpm for 2-20 min;

[0012] Furthermore, the conditions for centrifugation are centrifuging at 3000 - 6000 rpm for 2 - 8 min;

[0013] Furthermore, the concentration of the sulfuric acid - methanol solution is 8 - 12 wt%;

[0014] Furthermore, the derivatization temperature is 40 - 60 °C; furthermore, the derivatization time is 1 - 3 h;

[0015] Furthermore, the concentration of the sodium sulfate solution is 100 - 200 g / L; furthermore, the volume of sodium sulfate added is 5 - 10 m;

[0016] S2 Instrument detection:

[0017] S21 Chromatographic conditions: Using a 6890N gas chromatograph, a DB - 1701 capillary column, the inlet temperature is 200 - 250 °C, and the detector temperature is 280 - 320 °C;

[0018] S22 Carrier gas and flow rate: Both the carrier gas and the make - up gas are high - purity nitrogen, with a purity ≥99.99%, the make - up gas flow rate is 50 - 80 mL / min; the injection volume is 0.5 - 2 μL, the column flow rate is 1 - 2 mL / min, the total flow rate is 4 - 6 mL / min, the pressure is 6 - 8 psi, and the average flow rate is 25 cm / sec;

[0019] S23 Programmed temperature conditions: The initial temperature is 40 °C, held for 2 - 10 min, heated to 80 - 100 °C at a rate of 5 °C / min, held for 2 - 10 min, heated to 200 - 220 °C at a rate of 20 °C / min, held for 0 - 1 min, and the total duration is 18 - 40 min;

[0020] S3 Qualitative identification:

[0021] According to the retention time, each component is determined. The retention times of MCAA, MBAA, DCAA, TCAA, IAA, and DBAA are as follows:

[0022]

[0023] S4 Quantitative detection:

[0024] S42 Calculation of quantitative concentration:

[0025] S41 Drawing the standard curve: Take six single-standard haloacetic acid substances, namely chloroacetic acid, bromoacetic acid, dichloroacetic acid, trichloroacetic acid, iodoacetic acid, and dibromoacetic acid, add methanol to prepare a haloacetic acid mixed standard solution with a concentration of 180 - 220 mg / L. Subsequently, further dilute this solution into haloacetic acid HAAs standard dilution solutions with concentrations of 5 mg / L, 2.5 mg / L, 1 mg / L, 500 μg / L, 250 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, and 10 μg / L. In blank fetal bovine serum, use a microinjector to separately add the above-mentioned haloacetic acid solutions with different concentrations, thereby forming a standard spiked gradient series solution with final concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. Use blank fetal bovine serum as the blank control solution, and separately inject the above-mentioned standard solutions into the gas chromatograph for detection to obtain the response values at each concentration point;

[0026] S42 Quantitative analysis and concentration calculation: S421 Use the internal standard method for calculation: After integrating the spectrum, the peak area of the target compound is S_target and the peak area of the internal standard substance is S_internal. Take the ratio of the two, that is, the relative response value R = S 标目 / S 标内 , and use R as the ordinate and the concentration of the haloacetic acid HAAs series standard dilution solution as the abscissa, force it to pass through the origin, draw the standard curve of the target substance, and obtain the calibration curve slope a of several types of target substances;

[0027] S422 Quantitative analysis: First, use liquid-liquid extraction - acidified methanol derivatization to pretreat the sample. Then, after mixing the sample with the internal standard, perform gas chromatographic analysis under the same conditions as the standard curve; After integrating the sample spectrum, obtain the peak area S 样目 of the target compound and the peak area S 样内 of the internal standard substance, calculate the relative response value R 样 = S 样目 / S 样内 , and use the slope a of the standard curve to calculate the concentration of the target compound in the sample through the formula C 样 = R 样 / a;

[0028] Advantageous effects

[0029] Compared with the prior art, the advantageous effects of the present invention at least include:

[0030] The present invention provides a method for rapid qualitative and quantitative analysis of 6 kinds of HAAs in serum simultaneously. By optimizing the detection conditions, the recovery rate of HAAs is improved and the method detection limit is reduced. In addition, the present invention has appropriate sensitivity, a small sample usage, and relatively simple operation, and can be widely used for the detection of HAAs in large-scale clinical cohorts and serum. Brief description of the drawings

[0031] To more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings required for the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the accompanying drawings.

[0032] Figure 1 It represents the blank chromatogram of a mixture of six HAAs standard solutions with an incorporated concentration of 100 μg / L. Among them, the peaks are identified as MCAA (1); 1,2-DBP (2); MBAA (3); DCAA (4); TCAA (5); IAA (6); DBAA (7);

[0033] Figure 2 It represents the optimized diagram of conditional pretreatment. The recovery rates of HAAs methyl esters under different conditions: (a) different sulfuric acid ratios; (b) the amount of anhydrous sodium sulfate added; (c) the volume of the extraction agent added; (d) the volume ratio of acidified methanol to the extraction organic phase (A / M); (e) derivatization time; (f) storage time; (g) the volume of the sodium sulfate solution added; (h) the volume of the saturated sodium bicarbonate solution added. The concentration of HAAs is 100 μg / L (mean ± standard deviation, n = 3). Specific Embodiments

[0034] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0035] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content 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 manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0036] Embodiment

[0037] 1. Standards and Reagents

[0038] Haloacetic acid standards were purchased from Cato Research Chemicals, Inc. (Cato), USA, including monochloroacetic acid (CAA, concentration>99%), monobromoacetic acid (BAA, concentration>99%), dichloroacetic acid (DCAA, concentration>99%), trichloroacetic acid (TCAA, concentration>19%), iodoacetic acid (IAA, concentration>97%), and dibromoacetic acid (DBAA, concentration>99%). The internal standard 1,2-dibromopropane (1,2-DBP) had a purity of more than 99.0% and was from Sigma-Aldrich, USA. Fetal bovine serum was purchased from China Biotechnology Co., Ltd. Chromatographic grade MTBE was provided by Merck, Germany. Analytical grade anhydrous sodium sulfate and concentrated sulfuric acid were both from Chemical Reagent Co., Ltd., China National Pharmaceutical Group Co., Ltd. Ultrapure water (LC-MS grade), methanol (chromatographic grade), and sodium bicarbonate (analytical grade) were all purchased from Sigma-Aldrich, USA.

[0039] 2. Sample Pretreatment

[0040] Haloacetic acids (HAAs) were pre-treated using liquid-liquid extraction-acidified methanol derivatization. 0.5 mL of serum was placed in a 20 mL headspace bottle, and 1 ml of 75% sulfuric acid was added to acidify (pH < 0.5). After mixing, 50 mg of anhydrous sodium sulfate was quickly added, and the solid was completely dissolved by shaking. Then 3 mL of methyl tert-butyl ether (MTBE) containing the internal standard (1,2-DBP) was added, and the internal standard concentration was 20 μg / L. After vigorous hand shaking for 2 min, the mixture was shaken on a vortex oscillator at 1800 rpm at room temperature for 10 min. After thorough mixing, the mixture was centrifuged at 5000 rpm for 5 min. 2 ml of the supernatant was transferred to a 15 mL glass centrifuge tube, and 4 ml of 10% sulfuric acid methanol solution was added. After vortex mixing, the mixture was derivatized at 50 ° C for 2 hours. Prepare the ice-water mixture in advance. After the derivatization is completed, cool it to room temperature in an ice-water bath immediately. Add 7mL of 150g / L sodium sulfate solution, shake well and quickly open the bottle cap to release gas to terminate the esterification reaction. After standing and stratifying, take 600μL of the upper organic phase and transfer it to a 10mL centrifuge tube. Add 1mL of saturated sodium bicarbonate solution for neutralization reaction, mix well and let stand for 2min. Transfer the upper ester layer to a brown automatic injection bottle, add 50mg of anhydrous sodium sulfate to the injection bottle to remove moisture, and seal it in a -20℃ refrigerator. Take it out of the refrigerator and vortex for 1min before GC-ECD detection, and then detect it on the machine.

[0041] 3. Chromatographic conditions

[0042] The 6890N gas chromatograph (Agilent, USA) is equipped with an ECD detector and a DB-1701 capillary column (Agilent, USA, 30 m × 320 μm, 0.25 mm). The gas chromatographic conditions are as follows: the inlet temperature is 230 °C, and the detector temperature is 300 °C. The carrier gas and make-up gas are both high-purity nitrogen (purity ≥ 99.99%), and the make-up gas flow rate is 60 mL / min. The injection volume is 1 μL, the injection mode is splitless injection, the column flow rate is 1.4 mL / min, the total flow rate is 4.9 mL / min, the pressure is 6.3 psi, and the average flow velocity is 25 cm / sec. The optimized temperature programming conditions: the initial temperature is 40 °C, held for 2 min, increased to 80 °C at 5 °C / min, held for 2 min, increased to 215 °C at 20 °C / min, held for 0 min, and the total duration is 18.75 min.

[0043] 4. Results and Discussion

[0044] 4.1 Standard Curves and Gas Chromatograms of Haloacetic Acids

[0045] In this experiment, six single-standard haloacetic acid substances were first used to prepare a 200 mg / L mixed standard solution of haloacetic acids with methanol. Subsequently, this solution was further diluted to mixed standard dilutions of haloacetic acids (HAAs) at 5 mg / L, 2.5 mg / L, 1 mg / L, 500 μg / L, 250 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, and 10 μg / L. In 0.5 ml of blank fetal bovine serum, 10 μL of haloacetic acid solutions at different concentrations were respectively added using a microsyringe to form a standard spike gradient series with final concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. In the experiment, blank fetal bovine serum without adding any standard substances was used as a process blank control, and the temperature programming of the chromatographic column was optimized. This method significantly shortened the time of temperature programming. As can be seen from Table 1 and Figure 1 it can be seen that the minimum difference in the retention times of the six methyl haloacetates is more than 0.6 min, and the resolution is good, meeting the requirements for qualitative analysis on the chromatographic column used in this experiment. At the same time, the linear correlation degrees of the calibration curves of the six haloacetic acids are all higher than 0.996, meeting the requirements for quantitative analysis.

[0046] a. Drawing the standard curve: Six single-standard haloacetic acid substances (including monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, monoiodoacetic acid, and dibromoacetic acid) were used to prepare a mixed standard solution of haloacetic acid with a concentration of 200 mg / L using methanol. Subsequently, this solution was further diluted into mixed standard dilution solutions of haloacetic acids (HAAs) with concentrations of 5 mg / L, 2.5 mg / L, 1 mg / L, 500 μg / L, 250 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, and 10 μg / L. In 0.5 ml of blank fetal bovine serum, 10 μL of haloacetic acid solutions with different concentrations were respectively added using a microinjector to form a series of standard spike gradients with final concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. In the experiment, blank fetal bovine serum without adding any standard substances was used as the process blank control; the above standard solutions were injected into a gas chromatograph and detected with an electron capture detector to obtain the response values at each concentration point;

[0047] b. Concentration calculation: The internal standard method was used for calculation; after integrating the chromatogram, the peak area of the target compound (S_target) and the peak area of the internal standard substance (S_internal) were obtained. The ratio of the two, that is, the relative response value R = S_target / S_internal, was used. With R as the ordinate and the series of standard dilution solutions of haloacetic acids HAAs as the abscissa, forcing it through the origin, the standard curve of the target substance was drawn to obtain the calibration curve slope a of several types of target substances; after completing the drawing of the haloacetic acid standard curve and concentration calculation, quantitative analysis was continued. First, the actual sample was pretreated. After the treated sample was mixed with the internal standard, gas chromatographic analysis was carried out under the same conditions as the standard curve. After integrating the sample chromatogram, the peak area of the target compound (S_sample_target) and the peak area of the internal standard substance (S_sample_internal) were obtained, and the relative response value R of the sample was calculated 样 =S 样目 / S 样 ), and using the slope a of the standard curve, the concentration of the target compound in the sample was calculated through the formula C_sample = R_sample / a.

[0048] Table 1 Linear regression equations, regression coefficients (R2), and retention times of six HAA standard products

[0049]

[0050] Figure 1 Represents the blank chromatogram of a mixture of six HAAs standard solutions with a spiked concentration of 100 μg / L. Among them, the peaks are identified as MCAA (1); 1,2-DBP (2); MBAA (3); DCAA (4); TCAA (5); IAA (6); DBAA (7).

[0051] 4.2 Optimization of the pretreatment method

[0052] 4.2.1 Influence of sulfuric acid ratio on recovery rate

[0053] The ionization degree of HAAs (haloacetic acids) in water exceeds 99%, and they mainly exist in ionic form. Compared with molecular HAAs, ionic HAAs are difficult to be effectively extracted from the aqueous phase by the extractant.

[19] . Therefore, before the extraction process, acidic substances are usually added to the water sample to lower the pH value. However, there is currently a lack of relevant research on this biological matrix of blood for reference. According to the regulations of the US EPA methods 552.2 and 552.3, to ensure the efficient extraction of HAAs, the pH value of the water sample needs to be adjusted to below 0.5. However, some studies have shown that acidic samples will shorten the service life of the chromatographic column, and excessive sulfuric acid in water will increase the solubility of ether and water.

[16] . Therefore, in this study, while ensuring that the pH value is less than 0.5 [1] , the influence of different sulfuric acid ratios on the extraction efficiency of HAAs (100 μg / L, spiked into blank fetal bovine serum) was investigated. In the experiment, three parallel samples were set in each group, and the results are shown in Fig. 2a.

[0054] The experimental results show that as the sulfuric acid ratio increases from 25% to 50%, the recovery rates of the six haloacetic acids all show an upward trend, especially for TCAA (trichloroacetic acid) and DBAA (dibromoacetic acid). When the sulfuric acid ratio is further increased to 75%, the recovery rate does not show a significant change. Considering the influence of acidic samples on the service life of the chromatographic column and the complexity of the real blood sample matrix, 1 mL of 50% sulfuric acid solution was added before extraction in this study.

[0055] 4.2.2 Influence of the amount of anhydrous sodium sulfate added on recovery rate

[0056] As a commonly used salting-out agent, the main function of anhydrous sodium sulfate is to reduce the solubility of HAAs in water, thereby promoting the transfer of HAAs from the aqueous phase to the organic phase and improving the extraction efficiency. In addition, the dehydration effect of anhydrous sodium sulfate is crucial for the esterification reaction because the presence of water may inhibit the reaction from proceeding in the direction of forming esters and affect the reaction equilibrium.

[17] . Therefore, in this study, 50 - 150 mg of anhydrous sodium sulfate was added to the blank spiked samples (100 μg / L) respectively to investigate the influence of the amount of anhydrous sodium sulfate added on the extraction efficiency of HAAs. Three parallel samples were set in each group, and the results are shown in Fig. 2b.

[0057] The experimental results show that the recoveries of MCAA, MBAA, and IAA exhibit a non-linear relationship of first increasing and then decreasing with the increase in the amount of anhydrous sodium sulfate added. Moreover, when the amount of anhydrous sodium sulfate added is 100 mg, the recoveries of these three HAAs reach their peaks. On the contrary, the recoveries of trihaloacetic acid (TCAA) and dibromoacetic acid (DBAA) continuously increase with the increase in the amount of anhydrous sodium sulfate added, which may be related to the characteristics of these two compounds being more easily separated from the aqueous phase at higher salt concentrations. For dichloroacetic acid (DCAA), its recovery decreases with the increase in the amount of anhydrous sodium sulfate added. This abnormal trend may be related to the dissolution behavior of DCAA at high salt concentrations or other unknown mechanisms. Considering the six HAAs comprehensively, in this study, 100 mg of anhydrous sodium sulfate was finally selected as the optimal condition during the extraction process.

[0058] 4.2.3 Effect of the amount of extractant added on the recovery rate

[0059] The role of the extractant is to effectively transfer HAAs from the aqueous phase to the organic phase for derivatization. In this experiment, methyl tert-butyl ether was used as the extractant. To verify the effect of the amount of MTBE added on the recovery rate of HAAs, in this study, 3 mL, 4 mL, and 5 mL of MTBE were respectively added to the blank spiked samples, and three groups of parallel samples were set up. The results are shown in Fig. 2c.

[0060] The experimental results show that with the increase in the amount of extractant added, all six HAAs decreased to varying degrees. Considering that adding 3 mL of MTBE can already meet the requirement of 2 mL of extraction organic phase for the esterification step, therefore, in this experiment, 3 mL of MTBE was selected to be added to all samples.

[0061] 4.2.4 Effect of the amount of acidified methanol added on the derivatization efficiency

[0062] The derivatization of haloacetic acid is a chemical reaction carried out under acid-catalyzed conditions, mainly through the bimolecular nucleophilic substitution mechanism (Sn 2 reaction) to react HAAs with methanol to form the corresponding methyl ester derivatives. This process is also known as Fischer-Speier esterification, which involves an acid-catalyzed reaction with dimethyl sulfate as the reaction mediator and is the standard derivatization treatment method adopted in US EPA 552.2 and 552.3. Currently, most studies use the method of a volume ratio of acidified methanol to extraction organic phase (A / M) of 1:1 during the derivatization process. However, some studies have shown that the methylation efficiency can be improved by increasing the amount of acidic methanol in the reaction.

[16] Therefore, in this study, the volume ratio of acidified methanol to extraction organic phase (A / M) during the derivatization process was explored. Three groups with A / M ratios of 1:1, 1:2, and 1:3 were set up, with 3 parallel samples in each group. The results are shown in Fig. 2d. The results showed that as the A / M ratio increased, the recovery rate of DCAA (dichloroacetic acid) showed a slight downward trend, while the recovery rates of the other five HAAs increased significantly, especially the recovery rates of IAA and DBAA. However, the experiment found that although increasing the A / M ratio could improve the recovery rate of methyl halide acetate, it would affect the recovered volume of MTBE after secondary extraction. This is because the increased amount of acidified methanol led to an increase in the solubility of MTBE in the lower-layer mixed solution of methanol and sodium sulfate, thereby reducing the recovered volume of the MTBE organic phase. When the A / M ratio reached 1:3, it was difficult to obtain enough recovered volume of the organic phase for subsequent experiments.

[0063] 4.2.5 Effect of Derivatization Time on Recovery Rate

[0064] In the existing studies on the derivatization treatment of HAAs, the water bath temperature is generally set at 50 °C. This choice is based on the boiling point of the extractant MTBE being 55.2 °C. The purpose of controlling the temperature at 50 °C is to minimize the loss caused by the volatilization of the esterified products generated during the derivatization process with MTBE. According to the aforementioned experimental procedures, this study set three methyl esterification reaction times (1 h, 2 h, 3 h) to evaluate the effect of derivatization time on the esterification efficiency. The results are shown in Fig. 2e.

[0065] The experimental data showed that there was no obvious change in MBAA within the derivatization time of 1 to 3 hours. For DCAA and TCAA, as the derivatization time increased, the recovery rate showed a slight upward trend. For MCAA, IAA, and DBAA, the recovery rate showed an upward trend within 1 to 2 hours, but when the derivatization time was further increased to 3 hours, there was a slight downward trend. This change may be related to the decomposition of the esterification products caused by too long derivatization time. Therefore, this study selected 2 hours as the optimal derivatization time.

[0066] 4.2.6 Effect of Standing Time on the Degradation of Methyl Halide Acetate

[0067] Some studies have shown that the derivatized samples will degrade as the standing time extends.

[20] After derivatizing the samples, they were placed at room temperature of 24 °C for different times before being processed. The results are shown in Fig. 2f. The experiment shows that the measured results of the six haloacetic acids all degraded to varying degrees as the placement time extended. This phenomenon may be because the esterification reaction is a reversible process, and the hydrolysis reaction at room temperature causes the degradation of methyl haloacetate, thus reducing the recovery rate. Therefore, to reduce the influence of the hydrolysis reaction on the recovery rate, the derivatized samples should be processed immediately to maintain the stability of the derivatized products and the accuracy of the analysis results. 4.2.7 Amount of sodium sulfate aqueous solution

[0068] After derivatization, a 150 g / L sodium sulfate solution was added to the reaction system. Its main function is to promote the effective separation of the organic phase (MTBE) containing methyl HAAs from the acidified methanol aqueous phase. The addition of sodium sulfate increased the ionic strength in the aqueous phase. This change reduced the interfacial tension between the organic phase and the aqueous phase, optimizing the layering effect of the two phases. In addition, the addition of sodium sulfate may also enhance the salting-out effect, further promoting the transfer of methyl HAAs from the aqueous phase to the organic phase, improving the extraction efficiency and the recovery rate of methyl esters. In this experiment, with an A / M ratio of 1:2 as the basic condition, after the water bath ended and cooled to room temperature, 6 mL, 7 mL, and 8 mL of sodium sulfate solution were added to three groups of samples respectively, and three parallel samples were set for each group. The experimental results are shown in Fig. 2g.

[0069] The experimental results show that the increase in the sodium sulfate solution has a significant impact on the recovery rates of MBAA, DCAA, IAA, and DBAA. Especially when the sodium sulfate solution increased to 7 mL, the recovery rates of these substances increased significantly. However, when the sodium sulfate solution was further increased to 8 mL, the increase in the recovery rate was not obvious. In contrast, the recovery rates of MCAA and TCAA were not sensitive to the increase in the sodium sulfate solution, and the changes were not obvious. Therefore, in this study, 7 mL of sodium sulfate solution was selected to be added to the samples.

[0070] 4.2.8 Amount of saturated sodium bicarbonate aqueous solution

[0071] During the derivatization process of haloacetic acids (HAAs), acidic conditions are usually adopted to promote the reaction of HAAs with methanol to form the corresponding methyl esters. This acidic environment is crucial for improving the efficiency of the derivatization reaction. However, acidic samples may not only shorten the service life of the chromatographic column, but also the excessive sulfuric acid in water will increase the solubility of ether and water, thus affecting the chromatographic analysis results. Therefore, to neutralize the acidic conditions during the derivatization process, a saturated sodium bicarbonate solution needs to be added to the samples. For this purpose, 0.5 mL - 1.5 mL of saturated sodium bicarbonate solution was added to three groups of blank spiked samples respectively in this study to explore the influence of the amount of saturated sodium bicarbonate solution added on the extraction efficiency of HAAs. Three parallel samples were set for each group, and the results are shown in Fig. 2h.

[0072] The results showed that with the increase in the addition amount of saturated sodium bicarbonate solution, the recovery rates of MCAA, DCAA, TCAA, and IAA showed an upward trend. However, for MBAA and DBAA, the recovery rates first increased and then decreased. This trend may be related to the hydrolysis of some HAAs methyl esters or their return to the aqueous phase. Considering that using 1 mL of saturated sodium bicarbonate solution can already meet the requirements for analysis, in this study, 1 mL of saturated sodium bicarbonate solution was selected as the standard addition amount in subsequent experiments.

[0073] Figure 2 Represents the optimized diagram of condition pretreatment. Recovery rates of HAAs methyl esters under different conditions: (a) different sulfuric acid ratios; (b) addition amount of anhydrous sodium sulfate; (c) volume of extractant added; (d) volume ratio of acidified methanol to extraction organic phase (A / M); (e) derivatization time; (f) storage time; (g) volume of sodium sulfate solution added; (h) volume of saturated sodium bicarbonate solution added. The concentration of HAAs was 100 μg / L (mean ± standard deviation, n = 3).

[0074] The above embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those of ordinary skill in the art to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the essence of the content of the present invention should be covered within the protection scope of the present invention.

Claims

1. A qualitative and quantitative gas phase detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum, characterized in that: The following steps are involved: S1 Liquid-liquid extraction-acidification methanol derivatization pretreatment: Take 0.1-1 mL of serum to a 10-100 mL headspace bottle, add 0.2-5 mL of sulfuric acid to acidify to pH < 0.5, mix well and quickly add 10-200 mg of anhydrous sodium sulfate to dissolve all the solids, then add 0.5-15 mL of methyl tert-butyl ether (MTBE) containing internal standard and shake vigorously for 1-10 min, then vortex and shake, mix thoroughly and centrifuge for 5, aspirate 1-3 mL of supernatant and transfer to a 10-20 mL glass centrifuge tube, add 2-6 mL 10% sulfuric acid methanol solution, vortex mix and then derivatize, prepare ice-water mixture in advance, cool to room temperature in an ice-water bath immediately after derivatization, add sodium sulfate solution, shake and quickly open the bottle cap to release gas to terminate the esterification reaction, let stand and separate, draw 300-800 μL of the upper organic phase and transfer it to a centrifuge tube, add 0.5-5mL saturated sodium bicarbonate solution for neutralization, mix and stand, transfer the upper ester layer, add anhydrous sodium sulfate to remove moisture, and seal and store in a -30--10℃ refrigerator; S2 instrument detection: S21 Chromatographic conditions: 6890N gas chromatograph, DB-1701 capillary column, injection port temperature 200-250°C, detector temperature 280-320°C; S22 carrier gas and flow rate: both carrier gas and tail gas are high-purity nitrogen, purity ≥99.99%, tail gas flow rate 50-80mL / min; injection volume 0.5-2μL, column flow rate 1-2mL / min, total flow rate 4-6mL / min, pressure 6-8psi, average flow rate 25cm / sec; S23 program heating conditions: initial temperature 40°C, hold for 2-10 min, increase to 80-100°C at 5°C / min, hold for 2-10 min, increase to 200-220°C at 20°C / min, hold for 0-1 min, total time 18-40 min; S3 Qualitative identification: According to the retention time, the retention times of each component are determined as follows: S4 quantitative detection: S42 quantitative concentration calculation: S41 draws a standard curve: take six haloacetic acid single standard substances, monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, monoiodoacetic acid, and dibromoacetic acid, add methanol to prepare a haloacetic acid mixed standard solution of 180-220 mg / L, then further dilute the solution into 5 mg / L, 2.5 mg / L, 1 mg / L, 500 μg / L, 250 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, and 10 μg / L haloacetic acid HAAs mixed standard dilutions, and use a microinjector to add the above haloacetic acid solutions of different concentrations into blank fetal bovine serum, thereby forming a standard spiked gradient series solution with a final concentration of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L, and use blank fetal bovine serum as a blank control solution, respectively enter the above standard solutions into a gas chromatograph for detection, and measure the response value of each concentration point; S42 quantitative analysis and concentration calculation: S421 uses the internal standard method for calculation: after spectrum integration, the peak area of ​​the target compound is Sstandard, and the peak area of ​​the internal standard substance is Sstandard. The ratio of the two, i.e. the relative response value R=S 标目 / S 标内 With R as the ordinate and the standard sample concentration C as the abscissa, the standard curve of the target substance is drawn through the origin, and the slope a of the standard line of several types of target substances is obtained; S422 quantitative analysis: First, the sample is pre-treated by liquid-liquid extraction-acidified methanol derivatization. Then, the sample is mixed with the internal standard and analyzed by gas chromatography under the same conditions as the standard curve. After integrating the sample spectrum, the peak area S of the target compound is obtained. 样目 and the peak area S of the internal standard substance 样内 , calculate the relative response value R of the sample 样 =S 样目 / S 样内 , and using the slope a of the standard curve, the formula C 样 =R 样 / aCalculate the concentration of target compounds in samples.

2. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The concentration of the sulfuric acid is 50 to 80 v / v% sulfuric acid.

3. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The internal standard is 1,2-dibromopropane (ie, 1,2-DBP); further, the internal standard concentration is 2 to 100 μg / L.

4. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The vortexing condition is to oscillate on a vortex oscillator at 1500-2000 rpm for 2-20 minutes.

5. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The centrifugal conditions are 3000-6000 rpm and centrifugation for 2-8 min.

6. The method for simultaneously determining six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The concentration of the sulfuric acid methanol solution is 8-12 wt %.

7. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The derivatization temperature is 40-60° C.; further, the derivatization time is 1-3 hours.

8. The method for simultaneously determining the qualitative and quantitative gas phase detection of six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that: The concentration of the sodium sulfate solution is 100-200 g / L; further, the volume of the sodium sulfate added is 5-10 m3.

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