Simultaneous determination of six chlorinated disinfection by-products in serum by gas chromatography

Abstract

The application provides a gas phase detection method for simultaneously determining six chloro disinfectant by-products in serum, comprising the following steps: S1 liquid-liquid extraction-acidified methanol derivatization pretreatment; S2 instrument detection: S21 chromatographic conditions: using a 6890N gas chromatograph, a DB-1701 capillary column, a sample inlet temperature of 200-250 DEG C, and a detector temperature of 280-320 DEG C; S22 carrier gas and flow rate; S23 programmed temperature conditions; S3 qualitative identification and quantitative concentration calculation: the application also optimizes the detection conditions, improves the recovery rate of HAAs, and reduces the detection limit of the method, and the sensitivity of the application is appropriate, the sample usage is smaller, the operation is relatively simple, and the application can be widely used in the detection of HAAs in serum in clinical large-scale cohort.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a gas chromatography method for simultaneously determining six chlorine-based disinfectant byproducts in serum. Background Technology

[0002] Chlorination disinfection is one of the most important disinfection methods in drinking water treatment, ensuring water quality safety by effectively inhibiting pathogenic microorganisms in the water. However, during the disinfection process, chlorine-based disinfectants (including chlorine gas Cl2, hypochlorous acid HOCl, and hypochlorite OCl-) inevitably react with dissolved organic matter, bromides, and iodides, generating disinfection byproducts (DBPs). Currently, more than 800 disinfection byproducts have been identified, among which haloacetic acids (HAAs) account for 13% of the total organic halogens in water, making them the second largest category of disinfection byproducts after trihalomethanes.

[0003] Haloacetic acids (HAAs) have attracted considerable attention due to their widespread exposure in water bodies and their potential health risks. These compounds can accumulate in the human body through drinking water and are associated with various potential toxicities, including reproductive and developmental toxicity, cytotoxicity, and genotoxicity. Common haloacetic acids found in water include monochloroacetic acid (CAA), monobromoacetic acid (BAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), iodoacetic acid (IAA), and dibromoacetic acid (DBAA). As representative HAAs, DCAA and TCAA have been shown in studies to have significantly higher carcinogenic risks than trihalomethanes (THMs), being 50 times and 100 times higher, respectively, than THMs. Furthermore, HAAs account for over 91.9% of the carcinogenic risk of DBPs.

[0004] Previous epidemiological studies have often relied on the concentration of haloacetic acids in water supply systems to assess population exposure levels and health risks. However, this approach ignores the temporal and spatial variability of water in the piped network, as well as individual differences in water usage habits and physiological activities, easily leading to misclassification of exposure levels. Furthermore, traditional epidemiological studies exploring the distribution of haloacetic acids in the human body primarily focus on two biomarkers: dichloroacetic acid and trichloroacetic acid in urine, lacking data on blood samples. Serum samples, as biological samples that directly reflect the internal environment of the human body, can more accurately reveal an individual's actual internal exposure level to haloacetic acids. Therefore, the detection of haloacetic acids in serum is of great significance for comprehensively assessing an individual's exposure level and health risk.

[0005] Currently, internationally accepted methods for detecting haloacetic acids in drinking water mainly include EPA methods 552.1 and 552.2, and standard method 6251B. However, methods for detecting haloacetic acids in blood samples are very limited and face challenges in terms of serum volume, sensitivity, and resistance to interference from complex blood matrices. Therefore, it is necessary to develop a blood haloacetic acid (HAAs) detection method with appropriate sensitivity, small sample volume, simple operation, and wide applicability in large-scale cohort studies. Summary of the Invention

[0006] This invention aims to provide a liquid-liquid extraction-acidified methanol derivatization-gas chromatography method to establish a rapid qualitative and quantitative analysis method for six HAAs in serum. Through optimization, the recovery rate of HAAs is improved and the method detection limit is lowered, facilitating rapid detection of HAAs in serum and monitoring the risk of carcinogens in the human body. The technical solution adopted in this invention is as follows:

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

[0008] S1 Liquid-Liquid Extraction-Acidified Methanol Derivatization Pretreatment: Take 0.1–1 mL of serum into a 10–100 mL headspace vial, add 0.2–5 mL of sulfuric acid to acidify, making the pH < 0.5. After mixing, quickly add 10–200 mg of anhydrous sodium sulfate to dissolve the solid completely. Then add 0.5–15 mL of methyl tert-butyl ether (MTBE) containing the internal standard, shake vigorously for 1–10 min, vortex, mix thoroughly, centrifuge for 5 minutes, and transfer 1–3 mL of the supernatant to a 10–20 mL glass centrifuge tube. Add 2–6 mL of [unclear text - possibly a specific ingredient or solution] to the solution. After vortexing and mixing a 10% sulfuric acid methanol solution, the derivatization reaction is carried out. An ice-water mixture is prepared in advance. After derivatization, the mixture is immediately cooled to room temperature in an ice-water bath. Sodium sulfate solution is added, the mixture is shaken, and the cap is quickly opened to release the gas to terminate the esterification reaction. After standing and separating the layers, 300-800 μL of the upper organic phase is transferred to a centrifuge tube. 0.5-5 mL of saturated sodium bicarbonate solution is added for neutralization. After mixing and standing, the upper ester layer is transferred, and anhydrous sodium sulfate is added to remove moisture. The tube is then sealed and stored in a refrigerator at -30 to -10°C.

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

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

[0011] Furthermore, the vortex is generated by oscillating on a vortex oscillator at 1500–2000 rpm for 2–20 minutes.

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

[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 hours.

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

[0016] S2 instrument testing:

[0017] S21 chromatographic conditions: 6890N gas chromatograph, DB-1701 capillary column, injection port temperature 200~250℃, detector temperature 280~320℃;

[0018] S22 Carrier Gas and Flow Rate: Both carrier gas and 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 program temperature rise conditions: initial temperature 40℃, hold for 2-10 min, rise at 5℃ / min to 80-100℃, hold for 2-10 min, rise at 20℃ / min to 200-220℃, hold for 0-1 min, total duration 18-40 min;

[0020] S3 Qualitative Identification:

[0021] Based on retention time, the retention times for each component, MCAA, MABA, DCAA, TCAA, IAA, and DBAA, are determined as follows:

[0022]

[0023] S4 quantitative detection:

[0024] S42 quantitative concentration calculation:

[0025] S41. Standard Curve Construction: Six single-standard haloacetic acids—monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, monoiodoacetic acid, and dibromoacetic acid—were prepared in methanol to form a mixed standard solution of haloacetic acids with a concentration of 180–220 mg / L. This solution was then further diluted to prepare standard dilutions of haloacetic acids (HAAs) at 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. Different concentrations of these haloacetic acid solutions were added to blank fetal bovine serum using a microsyringe to form a standard spiking gradient series of solutions with final concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. Blank fetal bovine serum was used as a blank control solution. The standard solutions were then injected into a gas chromatograph for detection, and the response values ​​at each concentration point were measured.

[0026] S42 Quantitative Analysis and Concentration Calculation: S421 Calculation using the internal standard method: After integrating the spectrum, the peak area of ​​the target compound is obtained as Starget, and the peak area of ​​the internal standard is Sinternal. The ratio of the two, i.e., the relative response value R = Starget. 标目 / S 标内 With R as the ordinate and the concentration of the HAA series standard dilution solution as the abscissa, the standard curve of the target substance was plotted by forcing it through the origin, and the slope α of the standard curve of several types of target substances was obtained.

[0027] S422 Quantitative Analysis: First, the sample was pretreated using liquid-liquid extraction-acidification with methanol derivatization. Then, the sample was mixed with an internal standard and analyzed by gas chromatography under the same conditions as the standard curve. After integrating the sample chromatogram, the peak area S of the target compound was obtained. 样目 Peak area S of internal standard 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, through formula C 样 =R 样 / a calculates the concentration of the target compound in the sample;

[0028] Beneficial effects

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

[0030] This invention provides a rapid qualitative and quantitative analysis method for six HAAs in serum simultaneously. By optimizing the detection conditions, the recovery rate of HAAs is improved and the detection limit of the method is reduced. In addition, this invention has appropriate sensitivity, requires a small sample volume, and is relatively simple to operate, and can be widely used in large-scale clinical cohorts for the detection of HAAs in serum. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0032] Figure 1 The blank chromatogram represents a mixture of six HAA standard solutions with a concentration of 100 μg / L. The peaks were identified as MCAA(1); 1,2-DBP(2); MBAA(3); DCAA(4); TCAA(5); IAA(6); DBAA(7);

[0033] Figure 2 The diagram shows the optimized pretreatment conditions. The recovery rates of HAAs methyl ester under different conditions are: (a) different sulfuric acid ratios; (b) amount of anhydrous sodium sulfate added; (c) volume of added extractant; (d) volume ratio of acidified methanol to the extracted organic phase (A / M); (e) derivatization time; (f) storage time; (g) volume of added sodium sulfate solution; and (h) volume of added saturated sodium bicarbonate solution. The concentration of HAAs was 100 μg / L (mean ± standard deviation, n = 3). Detailed Implementation

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

[0035] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, unless otherwise specified, the following embodiments and features in the embodiments can be combined with each other.

[0036] Example

[0037] 1. Standards and reagents

[0038] Halogenated acetic acid standards were purchased from Cato Research Chemicals, Inc. (Cato), USA, including monochloroacetic acid (CAA, >99%), monobromoacetic acid (BAA, >99%), dichloroacetic acid (DCAA, >99%), trichloroacetic acid (TCAA, >19%), iodoacetic acid (IAA, >97%), and dibromoacetic acid (DBAA, >99%). The internal standard 1,2-dibromopropane (1,2-DBP), with a purity exceeding 99.0%, was obtained from Sigma-Aldrich, USA. Fetal bovine serum was purchased from China Biotechnology Co., Ltd. Chromatographic grade MTBE was provided by Merck AG, Germany. Analytical grade anhydrous sodium sulfate and concentrated sulfuric acid were obtained from China National Pharmaceutical Group Chemical Reagent 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 pretreated using a liquid-liquid extraction-acidification-methanol derivatization method. 0.5 mL of serum was transferred to a 20 mL headspace vial, and 1 mL of 75% sulfuric acid was added for acidification (pH < 0.5). After mixing, 50 mg of anhydrous sodium sulfate was quickly added, and the mixture was shaken until the solid was completely dissolved. Then, 3 mL of methyl tert-butyl ether (MTBE) containing an internal standard (1,2-DBP) at a concentration of 20 μg / L was added. The mixture was vigorously shaken by hand for 2 min, and then vortexed at 1800 rpm for 10 min at room temperature. 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. The mixture was vortexed and derivatized at 50 °C for 2 hours. Prepare an ice-water mixture in advance. After derivatization, immediately cool the solution to room temperature in an ice-water bath. Add 7 mL of 150 g / L sodium sulfate solution, shake well, and quickly open the cap to release gas to terminate the esterification reaction. After allowing the layers to separate, transfer 600 μL of the upper organic phase to a 10 mL centrifuge tube. Add 1 mL of saturated sodium bicarbonate solution for neutralization, mix well, and let stand for 2 min. Transfer the upper ester layer to a brown autosampler vial, add 50 mg of anhydrous sodium sulfate to the vial to remove moisture, seal, and store at -20°C. Before GC-ECD detection, remove the vial from the freezer and vortex for 1 min before analysis.

[0041] 3. Chromatographic conditions

[0042] A 6890N gas chromatograph (Agilent, USA) with an ECD detector and a DB-1701 capillary column (Agilent, USA, 30m × 320μm, 0.25mm). Gas chromatographic conditions were as follows: injector temperature 230℃, detector temperature 300℃. Both carrier gas and make-up gas were high-purity nitrogen (purity ≥99.99%), with a make-up gas flow rate of 60 mL / min. The injection volume was 1 μL, using splitless injection, with a column flow rate of 1.4 mL / min, a total flow rate of 4.9 mL / min, a pressure of 6.3 psi, and an average flow rate of 25 cm / sec. Optimized temperature programming conditions were: initial temperature 40℃, hold for 2 min, increase to 80℃ at 5℃ / min, hold for 2 min, increase to 215℃ at 20℃ / min, hold for 0 min, for a total duration of 18.75 min.

[0043] 4. Results and Discussion

[0044] 4.1 Standard curve and gas chromatogram of haloacetic acid

[0045] In this experiment, six haloacetic acid single standards were first prepared into a 200 mg / L haloacetic acid mixed standard solution using methanol. This solution was then further diluted to 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. 10 μL of different concentrations of haloacetic acid solution were added to 0.5 mL of blank fetal bovine serum using a microsyringe, thus forming a standard spiking 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 any added standards was used as a process blank control. The column temperature program was optimized, and this method significantly shortened the programmed temperature time, as shown in Table 1 and... Figure 1 It can be seen that the minimum retention time difference of the six methyl haloacetic acids is greater than 0.6 min, indicating good separation, which meets the requirements for qualitative analysis on the chromatographic column used in this experiment. Meanwhile, the linear correlation of the calibration curves for the six haloacetic acids is all higher than 0.996, meeting the requirements for quantitative analysis.

[0046] a. Plotting the standard curve: Six haloacetic acid single standards (including monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, monoiodoacetic acid, and dibromoacetic acid) were prepared into a 200 mg / L haloacetic acid mixed standard solution using methanol. This solution was then further diluted to prepare mixed standard dilutions of haloacetic acids (HAAs) at 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. 10 μL of each concentration of haloacetic acid solution was added to 0.5 mL of blank fetal bovine serum using a microsyringe, thus forming a standard spiking 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 any added standard substances was used as a process blank control; the above standard solutions were introduced into a gas chromatograph and detected by an electron capture detector to obtain the response values ​​at each concentration point;

[0047] b. Concentration Calculation: The internal standard method is used for calculation. After integrating the spectrum, the peak area of ​​the target compound (Starget) and the peak area of ​​the internal standard (Sinternal) are obtained. The ratio of the two, i.e., the relative response value R = Starget / Sinternal, is plotted with R as the ordinate and the haloacetic acid (HAAs) series standard dilutions as the abscissa, forced to pass through the origin, to obtain the standard curve slope 'a' for several types of target substances. After completing the plotting of the haloacetic acid standard curve and concentration calculation, quantitative analysis is performed. First, the actual sample is pretreated. After mixing the pretreated sample with the internal standard, gas chromatography analysis is performed under the same conditions as the standard curve. After integrating the sample spectrum, the peak area of ​​the target compound (Ssample) and the peak area of ​​the internal standard (Sinternal) are obtained, and the relative response value R of the sample is calculated. 样 =S 样目 / S 样 The concentration of the target compound in the sample is calculated using the formula Csample = Rsample / a, based on the slope α of the standard curve.

[0048] Table 1. Linear regression equations, regression coefficients (R²), and retention times for six HAA standards.

[0049]

[0050] Figure 1 The blank chromatogram represents a mixture of six HAA standard solutions with a concentration of 100 μg / L. The peaks were identified as MCAA (1); 1,2-DBP (2); MBAA (3); DCAA (4); TCAA (5); IAA (6); and DBAA (7).

[0051] 4.2 Optimization of Preprocessing Methods

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

[0053] HAAs (haloacetic acids) have an ionization degree exceeding 99% in water and exist primarily in ionic form. Compared to molecular HAAs, ionic HAAs are more difficult to extract effectively from the aqueous phase using extractants.

[19] Therefore, acidic substances are usually added to the water sample to lower the pH value before the extraction process. However, there is currently a lack of relevant research on blood as a biological matrix for reference. According to US Environmental Protection Agency (EPA) methods 552.2 and 552.3, the pH value of the water sample needs to be adjusted to below 0.5 to ensure efficient extraction of HAAs. However, studies have shown that acidic samples can shorten the lifespan of chromatographic columns, and excessive sulfuric acid in water can increase the solubility of ethers and water.

[16] Therefore, this paper ensures that the pH value is less than 0.5. [1] The effect of different sulfuric acid ratios on the extraction efficiency of HAAs (100 μg / L, spiked in blank fetal bovine serum) was investigated. Three replicates were set up for each group, and the results are shown in Fig. 2a.

[0054] Experimental results showed that the recoveries of all six haloacetic acids increased with the increase of the sulfuric acid ratio from 25% to 50%, especially TCAA (trichloroacetic acid) and DBAA (dibromoacetic acid). When the sulfuric acid ratio was further increased to 75%, no significant change in recovery was observed. Given the impact of acidic samples on column lifespan and considering the complexity of real blood sample matrices, 1 mL of 50% sulfuric acid solution was added before extraction in this study.

[0055] 4.2.2 Effect of Anhydrous Sodium Sulfate Addition Amount on Recovery Rate

[0056] Anhydrous sodium sulfate, a commonly used salting-out agent, primarily functions to reduce the solubility of HAAs in water, thereby promoting their transfer from the aqueous phase to the organic phase and improving extraction efficiency. Furthermore, the dehydrating effect of anhydrous sodium sulfate is crucial for esterification reactions, as the presence of water can inhibit the reaction towards ester formation, affecting reaction equilibrium.

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

[0057] Experimental results showed that the recoveries of MCAA, MBAA, and IAA exhibited a non-linear relationship of first increasing and then decreasing with increasing anhydrous sodium sulfate dosage, reaching peak recoveries at 100 mg of anhydrous sodium sulfate. Conversely, the recoveries of trihaloacetic acid (TCAA) and dibromoacetic acid (DBAA) continuously increased with increasing anhydrous sodium sulfate dosage, possibly due to their easier separation from the aqueous phase at higher salt concentrations. However, the recovery of dichloroacetic acid (DCAA) decreased with increasing anhydrous sodium sulfate dosage; this anomalous trend may be related to the solubility behavior of DCAA at high salt concentrations or other unknown mechanisms. Considering all six HAAs, this study ultimately selected 100 mg of anhydrous sodium sulfate as the optimal condition during extraction.

[0058] 4.2.3 Effect of extractant dosage on 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 (MTBE) was used as the extractant. To verify the effect of MTBE addition on the recovery rate of HAAs, 3 mL, 4 mL, and 5 mL of MTBE were added to the blank spiked sample, and three parallel samples were set up. The results are shown in Fig. 2c.

[0060] The experimental results showed that the levels of all six HAAs decreased to varying degrees as the amount of extractant added increased. Considering that adding 3 mL of MTBE was sufficient to obtain the required 2 mL of organic phase for the esterification step, this experiment opted to add 3 mL of MTBE to all samples.

[0061] 4.2.4 Effect of Acidified Methanol Addition Amount on Derivatization Efficiency

[0062] Haloacetic acid derivatization is a chemical reaction that occurs under acid-catalyzed conditions, primarily through a bimolecular nucleophilic substitution mechanism (Sn). 2 The reaction involves the reaction of HAAs with methanol to generate the corresponding methyl ester derivatives. This process, also known as Fischer-Speier esterification, involves an acid-catalyzed reaction with dimethyl sulfate as the reaction mediator and is a standard derivatization method adopted in US EPA 552.2 and 552.3. Most current studies use a 1:1 volume ratio (A / M) of acidified methanol to the extracted organic phase during derivatization. However, some studies have shown that increasing the amount of acidic methanol in the reaction can improve methylation efficiency.

[16] Therefore, this study investigated the volume ratio (A / M) of acidified methanol to extracted organic phase during the derivatization process, setting up three groups with A / M ratios of 1:1, 1:2, and 1:3, with three parallel samples in each group. The results are shown in Fig. 2d. The results showed that with the increase of the A / M ratio, the recovery rate of DCAA (dichloroacetic acid) showed a slight decreasing trend, while the recovery rates of the other five HAAs increased significantly, especially IAA and DBAA. However, the experiment found that although increasing the A / M ratio could improve the recovery rate of methyl haloacetate, it affected the recovery volume of MTBE after the secondary extraction. This is because the increased amount of acidified methanol leads to increased solubility of MTBE in the lower methanol and sodium sulfate mixed solution, thereby reducing the recovery volume of the MTBE organic phase. When the A / M ratio reached 1:3, it was difficult to obtain a sufficient organic phase recovery volume for subsequent experiments.

[0063] 4.2.5 Effect of Derivation Time on Recovery Rate

[0064] In existing studies on HAA derivatization, the water bath temperature is generally set at 50℃, a choice based on the boiling point of the extractant MTBE (55.2℃). Maintaining the temperature at 50℃ aims to minimize the loss of esters generated during derivatization due to MTBE volatilization. Following the aforementioned experimental procedure, this study established three methyl esterification reaction times (1 h, 2 h, and 3 h) to evaluate the effect of derivatization time on esterification efficiency. The results are shown in Fig. 2e.

[0065] Experimental data showed that the recovery rate of MBAA did not change significantly within a derivatization time of 1 to 3 hours. For DCAA and TCAA, the recovery rate showed a slight increase with increasing derivatization time. For MCAA, IAA, and DBAA, the recovery rate showed an increasing trend within 1 to 2 hours, but a slight decreasing trend was observed when the derivatization time was further increased to 3 hours. This change may be related to the decomposition of esterification products due to excessively long derivatization time. Therefore, this study selected 2 hours as the optimal derivatization time.

[0066] 4.2.6 Effect of storage time on the degradation of methyl haloacetate

[0067] Studies have shown that the derived samples degrade over time.

[20] After derivatization, the samples were left at room temperature (24℃) for different periods of time for further processing. The results are shown in Fig. 2f. The experiment showed that the determination results of the six haloacetic acids all degraded to varying degrees with prolonged storage time. This phenomenon may be because the esterification reaction is a reversible process, and hydrolysis at room temperature causes the degradation of methyl haloacetates, thus reducing the recovery rate. Therefore, to reduce the impact of hydrolysis on the recovery rate, the derivatized samples should be processed immediately to maintain the stability of the derivatized products and the accuracy of the analytical results. 4.2.7 Amount of sodium sulfate aqueous solution added.

[0068] Following 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 increases the ionic strength in the aqueous phase, which reduces the interfacial tension between the organic and aqueous phases, optimizing the phase separation. Furthermore, the addition of sodium sulfate may enhance the salting-out effect, further promoting the transfer of methyl HAAs from the aqueous phase to the organic phase, thus improving extraction efficiency and methyl ester recovery. This experiment used an A / M ratio of 1:2 as the baseline condition. After the water bath was completed and the mixture cooled to room temperature, 6 mL, 7 mL, and 8 mL of sodium sulfate solution were added to three groups of samples, with three parallel samples per group. The experimental results are shown in Fig. 2.

[0069] The experimental results showed that increasing the sodium sulfate solution concentration significantly affected the recovery rates of MBAA, DCAA, IAA, and DBAA. In particular, a significant increase in the recovery rates of these substances was observed when the sodium sulfate solution concentration was increased to 7 mL. However, when the sodium sulfate solution concentration was further increased to 8 mL, the improvement in recovery rates was not significant. In contrast, the recovery rates of MCAA and TCAA were not sensitive to the increase in sodium sulfate solution concentration, and the changes were not significant. Therefore, this study selected the addition of 7 mL of sodium sulfate solution to the samples.

[0070] 4.2.8 Amount of saturated sodium bicarbonate aqueous solution added

[0071] In the derivatization of haloacetic acids (HAAs), acidic conditions are typically used to promote the reaction of HAAs with methanol to generate the corresponding methyl esters. This acidic environment is crucial for improving the efficiency of the derivatization reaction. However, acidic samples can not only shorten the lifespan of the chromatographic column, but excess sulfuric acid in water can also increase the solubility of ethers 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 sample. To this end, this study added 0.5 mL–1.5 mL of saturated sodium bicarbonate solution to three groups of blank spiked samples to investigate the effect of the amount of saturated sodium bicarbonate solution added on the extraction efficiency of HAAs. Three parallel samples were set up for each group. The results are shown in Fig. 2h.

[0072] The results showed that the recoveries of MCAA, DCAA, TCAA, and IAA increased with the increase of the amount of saturated sodium bicarbonate solution added. However, for MBAA and DBAA, the recoveries showed a trend of first increasing and then decreasing. This trend may be related to the hydrolysis or return of some HAA methyl esters to the aqueous phase. Considering that 1 mL of saturated sodium bicarbonate solution was sufficient for the experimental requirements, this study chose to use 1 mL of saturated sodium bicarbonate solution as the standard addition amount in subsequent experiments.

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

[0074] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A qualitative and quantitative gas chromatography method for simultaneously determining six chlorine-based disinfectant byproducts in serum, characterized in that, Includes the following steps: S1 Liquid-Liquid Extraction-Acidification Methanol Derivatization Pretreatment: Take 0.1-1 mL of serum into a 10-100 mL headspace vial, add 0.2-5 mL of sulfuric acid (50-80 v / v%) to acidify, making the pH < 0.

5. After mixing, quickly add 10-200 mg of anhydrous sodium sulfate to completely dissolve the solid. Then add 0.5-15 mL of methyl tert-butyl ether containing an internal standard (1,2-dibromopropane) at a concentration of 2-100 mg / mL. μ g / L, shake vigorously by hand for 1-10 minutes, then vortex to mix thoroughly and centrifuge at 3000-6000 rpm for 2-8 minutes. Transfer 1-3 ml of the supernatant to a 10-20 mL glass centrifuge tube, add 2-6 ml of 10 wt% sulfuric acid methanol solution, vortex to mix, and then proceed with the derivatization reaction at 40-60℃ for 1-3 hours. Prepare an ice-water mixture in advance. After derivatization, immediately cool to room temperature in an ice-water bath, add sodium sulfate solution, shake well, and quickly open the cap to release gas to terminate the esterification reaction. After standing and separating the layers, transfer 300-800 μL of the upper organic phase to a centrifuge tube, add 0.5-5 mL of saturated sodium bicarbonate solution for neutralization, mix well, and let stand. Transfer the upper ester layer, add anhydrous sodium sulfate to remove moisture, and seal and store in a refrigerator at -30 to -10℃. S2 instrument testing: S21 chromatographic conditions: 6890N gas chromatograph, DB-1701 capillary column, injection port temperature 200~250℃, detector temperature 280~320℃; S22 Carrier Gas and Flow Rate: Both the carrier gas and the make-up gas are high-purity nitrogen, with a purity ≥99.99%, and the make-up gas flow rate is 50~80 mL / min; the injection volume is 0.5~2 mL / min. μ L, column flow rate 1~2 mL / min, total flow rate 4~6 mL / min, pressure 6~8 psi, average flow rate 25 cm / sec; S23 temperature program conditions: initial temperature 40 ℃, hold for 2~10 min, increase to 80~100℃ at 5 ℃ / min, hold for 2~10 min, increase to 200~220℃ at 20 ℃ / min, hold for 0~1 min, total duration 18~40 min; S3 Qualitative Identification: Based on retention time, the retention times of each component, MCAA, MBAA, DCAA, TCAA, IAA, and DBAA, are determined as follows: ； S4 quantitative detection: S42 quantitative concentration calculation: S41. Plotting a standard curve: Six haloacetic acid standards—monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, monoiodoacetic acid, and dibromoacetic acid—were prepared in methanol to form a mixed standard solution of haloacetic acids at concentrations of 180–220 mg / L. This solution was then further diluted to concentrations of 5 mg / L, 2.5 mg / L, 1 mg / L, and 500 mg / L. μ g / L, 250 μ g / L, 100 μ g / L, 50 μ g / L, 25 μ g / L, 10 μ A mixed standard dilution of haloacetic acids (HAAs) of g / L was prepared by adding different concentrations of the above haloacetic acid solution to blank fetal bovine serum using a microsyringe, resulting in final concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 g / L. μ A series of standard spiked solutions of g / L were used, with blank fetal bovine serum as a blank control solution. The above standard solutions were injected into a gas chromatograph for detection, and the response values ​​at each concentration point were measured. S42 Quantitative Analysis and Concentration Calculation: S421 Calculation using the internal standard method: After integrating the spectrum, the peak area of ​​the target compound is obtained as Starget, and the peak area of ​​the internal standard substance is Sinternal. The ratio of the two, i.e., the relative response value R = Starget / Sinternal, is plotted with R as the ordinate and the standard concentration C as the abscissa, forced to pass through the origin, to obtain the standard curve of the target substance and the slope a of the standard curves for several types of target substances. S422 Quantitative Analysis: First, the sample was pretreated by liquid-liquid extraction-acidification methanol derivatization. Then, the sample was 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 of ​​the target compound, Ssample_target, and the peak area of ​​the internal standard, Ssample_internal, were obtained. The relative response value of the sample, Rsample = Ssample_target / Ssample_internal, was calculated. The concentration of the target compound in the sample was calculated using the slope a of the standard curve, Csample = Rsample / a.

2. The qualitative and quantitative gas chromatography detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that, The vortex is oscillated on a 1500~2000 rpm vortex oscillator for 2~20 min.

3. The qualitative and quantitative gas chromatography detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum according to claim 1, characterized in that, The concentration of the sodium sulfate solution is 100~200g / L.

4. The qualitative and quantitative gas chromatography detection method for simultaneously determining six chlorine-based disinfectant byproducts in serum according to claim 3, characterized in that, The volume of sodium sulfate added is 5-10 m³.

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