Solid-phase extraction-vacuum centrifugal concentration-liquid chromatography-tandem mass spectrometry detection method for flavor nucleotide chlorinated product

Abstract

The invention discloses a solid-phase extraction-vacuum centrifugal concentration-liquid chromatography-tandem mass spectrometry detection method for flavor nucleotide chlorinated products, which comprises the following steps: enriching target substances in a water sample to be detected by using solid-phase extraction, and then concentrating the extracted sample by using a vacuum centrifugal concentrator, and finally, determining the concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine and 8-chloro-inosine by using a multiple reaction monitoring mode of liquid chromatography tandem mass spectrometry. According to the method, solid-phase extraction and vacuum centrifugal concentration are combined, and finally, a liquid chromatography-tandem mass spectrometry detection technology is combined for use, so that the trace flavor nucleotide chlorinated product is detected under a complex water body matrix condition; the method has the advantages of small sample required volume, low detection limit, high sensitivity, short detection time and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of disinfection by-product detection, and particularly relates to a solid-phase extraction-vacuum centrifugal concentration-liquid chromatography tandem mass spectrometry detection method for taste nucleotide chlorinated products. BACKGROUND

[0002] Drinking water disinfection is one of the greatest public health achievements of the 20th century. In order to inhibit the growth of bacteria and other microorganisms in drinking water distribution networks, the "Drinking Water Health Standards" (GB 5749-1985) stipulates that the chlorine and free chlorine preparation in the finished water of tap water should be within the range of 0.3-4 mg / L. At the same time, the chlorinated disinfectant reacts with natural or artificial organic matter, bromide, iodide in water to form disinfection by-products (DBPs). So far, more than 700 DBPs have been found in drinking water. Epidemiological studies have shown that long-term drinking of chlorinated disinfection water is associated with an increased risk of cancer, but the currently regulated DBPs cannot explain the related cancer risk. Epidemiological studies have shown that long-term drinking of chlorinated disinfection water is associated with an increased risk of cancer.

[0003] A cooking process, for example making soup, usually means mixing tap water, food materials, seasonings and food additives together, heating and continuing for several minutes to several hours. Food and food additives can react with residual disinfectants in tap water used for cooking food. Some compounds can be oxidized to form various products during cooking, which can serve as precursors for the formation of halogenated DBPs.

[0004] With the rapid development of the food industry and the increasing demand for food flavor, taste nucleotides (such as inosinic acid disodium and guanylic acid disodium) are a new type of food additive. As an important new type of nucleotide flavor enhancer, it is widely used in catering cooking. In the "National Food Safety Standard Food Additive Use Standard" (GB 2760-2024) of our country, the maximum use amount of taste nucleotide disodium is not clearly specified, but in the commonly used food added with taste nucleotide, its concentration is significantly higher than that of nucleic acid in the environment. In the cooking process, it reacts with chlorinated / chloraminated tap water to generate new halogenated nucleic acid disinfection by-products. This kind of by-product may have potential genotoxicity and carcinogenicity, which poses a serious threat to human health. At present, there is no research on taste nucleotides as precursors of disinfection by-products. Its generation mechanism, analysis method and toxicity evaluation have not been systematically studied, which brings new challenges to the field of food safety water and drinking water safety.

[0005] Due to the complexity of water matrix and large matrix interference, it is urgent to develop a method for detecting taste nucleotide chlorinated products in water with high sensitivity and low detection limit, to provide a theoretical basis for evaluating health risks. Summary of the Invention

[0006] The purpose of this invention is to address the potential health risks posed by chlorinated flavor nucleotides in existing technologies, which present technical challenges such as analytical difficulty, low concentration, and significant matrix interference. This invention provides a solid-phase extraction-vacuum centrifugation-liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the detection of chlorinated flavor nucleotides. This invention combines high sensitivity and low detection limit, and features small sample pretreatment volume and simple operation, enabling the detection of trace amounts of chlorinated flavor nucleotides under complex aquatic matrix conditions.

[0007] The objective of this invention is achieved through the following technical solution: a method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides, comprising the following steps: (1) After collecting water samples, sodium sulfite of the same amount as the residual chlorine in the water samples is added to quench the residual chlorine, and the sample to be tested is obtained and stored at 4°C. (2) The sample to be tested obtained in step (1) is filtered through a filter membrane with a pore size of 0.45 µm to obtain a filtered sample; (3) Activate the MAX column required for solid-phase extraction with 6-12 mL of methanol and 6-18 mL of pure water respectively; (4) The filtered sample was enriched by passing it through a MAX column and then rinsed with 10 mL of pure water. (5) The target substance retained on the MAX column was eluted with 3 mL of methanol containing 2% formic acid under standard atmospheric pressure to obtain a test sample containing the target substance. (6) The sample containing the target substance is concentrated to 0.1 mL by vacuum centrifugation, and then 0.4 mL of pure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry. (7) A 0.1% formic acid aqueous solution and a 0.1% formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in water were separated and quantified using a BEH C18 liquid chromatography column via multiple reaction monitoring (MRM).

[0008] Furthermore, the MAX column has a specification of 6cc and 150mg.

[0009] Furthermore, the vacuum centrifugal concentrator operates at a temperature of 4°C and a rotation speed of 1000 r / min.

[0010] Furthermore, in the liquid chromatography-tandem mass spectrometry, the mass spectrometry ion source temperature is 650℃.

[0011] Furthermore, the temperature of the liquid chromatography column is 35°C.

[0012] Furthermore, the specific conditions for the gradient elution are as follows: Within 0-8 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 98:2 to 92:8; Within 8-12 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 92:8 to 80:20; Within 12-12.1 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution increased from 80:20 to 98:2; Within 12.1-15 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution was 98:2.

[0013] Furthermore, the specific conditions for the multiple reaction monitoring method are shown in the table below: Compared with existing technologies, the beneficial effects of this invention are as follows: This invention reduces matrix interference through solid-phase extraction enrichment and accurately quantifies chlorinated products of flavor nucleotides in water using a multiple reaction monitoring (MRM) method; This invention enables enrichment and concentration through solid-phase extraction under complex water matrix conditions, and improves elution efficiency by using an eluent containing formic acid, thereby reducing the method detection limit and matrix interference, and significantly lowering the method detection limit; This invention develops an MRM method for quantitative chlorinated products using characteristic fragment ions, and establishes a method for detecting trace chlorinated products of flavor nucleotides, achieving detection in water at levels as low as ng / L; This invention has the advantages of small sample volume requirement, low detection limit, high sensitivity, and short detection time, and is suitable for the detection of trace chlorinated products of flavor nucleotides under water matrix conditions. Attached Figure Description

[0014] Figure 1 This is a flowchart of the solid-phase extraction-vacuum centrifugation-liquid chromatography-tandem mass spectrometry method for detecting chlorinated flavor nucleotides of the present invention. Figure 2 The graph shows the effect of different ion source temperatures on the detection signal intensity of six flavor nucleotide chlorinated products. Figure 3 The graph shows the effect of different water-to-methanol ratios in the solvent on the detection signal intensity of six flavor nucleotide chlorinated products. Figure 4 Figure 1 shows the effect of different solid-phase extraction columns on the retention and recovery efficiency of six flavor nucleotide chlorinated products. Figure 5The effect of the same eluent volume on the recovery rate of chlorinated products of six flavor nucleotides is shown in the figure. Figure 6 Figure showing the effect of different enrichment methods on the recovery rate of chlorinated products of six flavor nucleotides; Figure 7 The MRM chromatogram and standard curve of 2-chloro-hypoxanthine are shown; among them, Figure 7 (a) in the figure is the MRM chromatogram of 2-chloro-hypoxanthine; Figure 7 (b) in the figure is the standard curve of 2-chloro-hypoxanthine; Figure 8 The MRM chromatogram and standard curve of 8-chloro-hypoxanthine are shown; among them, Figure 8 (a) is the MRM chromatogram of 8-chloro-hypoxanthine; Figure 8 (b) in the figure is the standard curve of 8-chloro-hypoxanthine; Figure 9 The MRM chromatogram and standard curve of 6-chloropurine are shown; among them, Figure 9 (a) is the MRM chromatogram of 6-chloropurine; Figure 9 (b) in the figure is the standard curve of 6-chloropurine; Figure 10 The MRM chromatogram and standard curve of 8-chloro-guanine are shown; among them, Figure 10 (a) is the MRM chromatogram of 8-chloro-guanine; Figure 10 (b) in the figure is the standard curve of 8-chloro-guanine; Figure 11 The MRM chromatogram and standard curve of 2-chloro-inosine are shown; among them, Figure 11 (a) in the figure is the MRM chromatogram of 2-chloro-inosine; Figure 11 (b) in the figure is the standard curve of 2-chloro-inosine; Figure 12 The MRM chromatogram and standard curve of 8-chloro-inosine are shown; among them, Figure 12 (a) is the MRM chromatogram of 8-chloro-inosine; Figure 12 (b) in the figure is the standard curve of 8-chloro-inosine; Figure 13 The following are MRM chromatograms of six chlorinated nucleotide products in different water bodies; among them, Figure 13 (a) shows the MRM chromatogram of the chlorinated products of six flavor nucleotides in the standard solution; Figure 13 (b) in the figure is the MRM chromatogram of the chlorinated products of the six flavor nucleotides in the raw water; Figure 13 (c) is the MRM chromatogram of six chlorinated nucleotides in drinking water. Detailed Implementation

[0015] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0016] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0017] It should be understood that although the terms first, second, third, etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first information may also be referred to as second information without departing from the scope of this invention, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0018] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0019] like Figure 1 As shown, the solid-phase extraction-vacuum centrifugation-liquid chromatography-tandem mass spectrometry detection method for chlorinated flavor nucleotides of the present invention specifically includes the following steps: (1) After collecting water samples, sodium sulfite of equal mass to the residual chlorine in the water samples is added to quench the residual chlorine, and the sample to be tested is obtained and stored at 4°C. The water includes drinking water, piped water and cooking water.

[0020] (2) The sample to be tested obtained in step (1) is filtered through a filter membrane with a pore size of 0.45 µm to obtain a filtered sample.

[0021] (3) The MAX column required for solid-phase extraction was activated using 6-12 mL of methanol and 6-18 mL of pure water, respectively. The specifications of the MAX column are 6cc and 150mg.

[0022] (4) The filtered sample obtained in step (2) is enriched by passing it through a MAX column and then rinsed with 10 mL of pure water.

[0023] (5) The target substance retained on the MAX column was eluted with 3 mL of methanol containing 2% formic acid under standard atmospheric pressure to obtain a test sample containing the target substance.

[0024] (6) The sample containing the target substance obtained in step (5) is concentrated to 0.1 mL by vacuum centrifugation, and then 0.4 mL of pure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry (LC-MA / MS).

[0025] Furthermore, the vacuum centrifugal concentrator operates at a temperature of 4°C and a rotation speed of 1000 r / min.

[0026] (7) A 0.1% formic acid aqueous solution and a 0.1% formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. The concentrations of six chloroformed nucleotides in water, namely 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine, were separated and quantified using a BEH C18 liquid chromatography column and multiple reaction monitoring method.

[0027] Furthermore, in liquid chromatography-tandem mass spectrometry, the mass spectrometry ion source temperature is 650℃.

[0028] Furthermore, the temperature of the liquid chromatography column was 35°C.

[0029] Furthermore, the specific conditions for gradient elution are as follows: Within 0-8 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 98:2 to 92:8; Within 8-12 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 92:8 to 80:20; Within 12-12.1 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution increased from 80:20 to 98:2; Within 12.1-15 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution was 98:2.

[0030] Furthermore, the specific conditions for the multiple reaction monitoring method are shown in Table 1.

[0031] Table 1: Setup conditions for multiple reaction monitoring methods The following describes in detail the solid-phase extraction-vacuum centrifugation-liquid chromatography-tandem mass spectrometry detection method for the chlorinated products of flavor nucleotides of the present invention with reference to the embodiments. The purpose and effect of the present invention will become more apparent.

[0032] Example 1 This embodiment investigated the effect of different ion source temperatures on the detection signal intensity of six flavor nucleotide chlorinated products, specifically including the following steps: (1) Prepare aqueous solutions containing the same concentration of the chlorinated products of the six flavor nucleotides.

[0033] (2) A 0.1% (v / v) formic acid aqueous solution and a 0.1% (v / v) formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in the aqueous solution were determined by separating and quantifying the analytes using a BEH C18 LC column via multiple reaction monitoring. The mass spectrometry ion source temperatures were set to room temperature, 200℃, 250℃, 300℃, 350℃, 400℃, 450℃, 500℃, 550℃, 600℃, 650℃, and 700℃, respectively.

[0034] The specific conditions for gradient elution are as follows: Within 0-8 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 98:2 to 92:8; Within 8-12 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 92:8 to 80:20; Within 12-12.1 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution increased from 80:20 to 98:2; Within 12.1-15 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution was 98:2.

[0035] The setup conditions for the multiple reaction monitoring method are shown in Table 1.

[0036] The final result is as follows Figure 2 As shown, the horizontal axis represents different mass spectrometry ion source temperatures, and the vertical axis represents the ratio of the detection signal intensity to the highest signal intensity of the six odor nucleotide chlorinated products at different ion source temperatures. Figure 2 The experimental results show that as the ion source temperature increases, the detection signal intensity of the six flavor nucleotide chlorinated products also increases. However, since 700℃ is the upper limit of the mass spectrometer temperature, the mass spectrometer ion source temperature is set to 650℃ in order to ensure the long-term use of the mass spectrometer.

[0037] Example 2 This embodiment investigated the effect of the ratio of water to methanol in the solvent on the detection signal intensity of six odor nucleotide chlorinated products, specifically including the following steps: (1) Prepare water / methanol solutions containing the same concentration of the chlorinated products of six flavor nucleotides, with water to methanol ratios of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0, respectively.

[0038] (2) A 0.1% (v / v) formic acid aqueous solution and a 0.1% (v / v) formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in the water were determined by separating and quantifying the analytes using a BEH C18 LC column via multiple reaction monitoring (MRM). The mass spectrometry ion source temperature was set to 650 °C; the gradient elution conditions were the same as in Example 1; and the MRM settings are shown in Table 1.

[0039] The final result is as follows Figure 3 As shown, the horizontal axis represents the different ratios of water and methanol in the solvent, and the vertical axis represents the ratio of the detection signal intensity of the six odor-causing nucleotide chlorinated products to the highest signal intensity under different ratios. Figure 3 The experimental results show that the overall detection signal intensity is best when the ratio of water to methanol is 100:0. Therefore, a ratio of water to methanol of 100:0, i.e., pure water, is more suitable as a detection solvent.

[0040] Example 3 This embodiment investigated the effect of different solid-phase extraction columns on the recovery rate of chlorinated products of six flavor nucleotides, specifically including the following steps: (1) Prepare aqueous solutions containing the same concentration of the chlorinated products of the six flavor nucleotides.

[0041] (2) Activate the MAX, WAX and HLB columns required for solid phase extraction with 6-12 mL methanol and 6-18 mL pure water respectively.

[0042] (3) The sample prepared in step (1) was enriched by passing it through MAX, WAX and HLB columns respectively, and then rinsed with 10 mL of pure water.

[0043] (4) Under standard atmospheric pressure, the target substance retained on the MAX column was eluted with 3 mL of methanol solution containing 2% formic acid, the target substance retained on the WAX ​​column was eluted with 3 mL of methanol solution containing 5% ammonia, and the target substance retained on the HLB column was eluted with 3 mL of methanol solution containing 2% formic acid (denoted as HLB a) and methanol solution containing 5% ammonia (denoted as HLB b) respectively, to obtain the test sample containing the target substance.

[0044] (5) The sample containing the target substance obtained in step (4) is concentrated to 0.1 mL using a vacuum centrifuge, and then 0.4 mL of pure water is added. The mixture is then vortexed to obtain a mixed solution for subsequent liquid chromatography-tandem mass spectrometry detection. The vacuum centrifuge operates at a temperature of 4℃ and a rotation speed of 1000 r / min.

[0045] (6) A 0.1% (v / v) formic acid aqueous solution and a 0.1% (v / v) formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in the aqueous solution were determined by separating and quantifying the analytes using a BEH C18 LC column via multiple reaction monitoring (MRM). The mass spectrometry ion source temperature was set to 650 °C; the gradient elution conditions were the same as in Example 1; and the MRM settings were shown in Table 1.

[0046] The final result is as follows Figure 4 As shown, the horizontal axis represents the chlorinated products of six flavor nucleotides, and the vertical axis represents the recovery rates of the halogenated products of flavor nucleotides using four eluents from three different solid-phase extraction columns. Figure 4 The experimental results shown indicate that the overall recovery rate is high when using the Oasis MAX solid-phase extraction column.

[0047] Example 4 This example investigated the effect of different eluent volumes on the recovery rates of six chloroformed nucleotides, specifically including the following steps: (1) Prepare aqueous solutions containing the same concentration of the chlorinated products of the six flavor nucleotides.

[0048] (2) 6-12 mL of methanol and 6-18 mL of pure water were used to activate the MAX column required for solid phase extraction.

[0049] (3) The sample prepared in step (1) is enriched by passing it through a MAX column and then rinsed with 10 mL of pure water.

[0050] (4) Under standard atmospheric pressure, the target substance retained on the MAX column was eluted with 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL of methanol solution containing 2% formic acid, respectively, to obtain the test sample containing the target substance.

[0051] (5) The sample containing the target substance obtained in step (4) is concentrated to 0.1 mL using a vacuum centrifuge, and then 0.4 mL of pure water is added. The mixture is then vortexed to obtain a mixed solution for subsequent liquid chromatography-tandem mass spectrometry detection. The vacuum centrifuge operates at a temperature of 4℃ and a rotation speed of 1000 r / min.

[0052] (6) A 0.1% (v / v) formic acid aqueous solution and a 0.1% (v / v) formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in the water were determined by separating and quantifying the analytes using a BEH C18 LC column via multiple reaction monitoring (MRM). The mass spectrometry ion source temperature was set to 650°C; the gradient elution conditions were the same as in Example 1; and the MRM settings were shown in Table 1.

[0053] The final result is as follows Figure 5 As shown, the horizontal axis represents the chlorinated products of the six flavor nucleotides, and the vertical axis represents the recovery rate of the halogenated products of the flavor nucleotides with different elution buffer volumes. Figure 5 The experimental results show that when the volume of the eluent increased from 1 mL to 3 mL, the recovery rate of most chloroformed flavor nucleotides increased; however, when the volume of the eluent was greater than 3 mL, the recovery rate of all six chloroformed flavor nucleotides decreased to varying degrees with increasing eluent volume. Therefore, the eluent volume was set to 3 mL.

[0054] Example 5 This embodiment evaluated the effect of different enrichment methods on the recovery rate of six chloroforms of flavor nucleotides, specifically including the following steps: (1) Prepare aqueous solutions containing the same concentration of the chlorinated products of the six flavor nucleotides.

[0055] (2) Activate the Oasis MAX column required for solid phase extraction with 6-12 mL of methanol and 6-18 mL of pure water respectively, and then rinse with 10 mL of pure water.

[0056] (3) The sample prepared in step (1) is enriched by passing it through a MAX column.

[0057] (4) The target substance retained on the MAX column was eluted with 3 mL of methanol solution containing 2% formic acid under standard atmospheric pressure to obtain a test sample containing the target substance.

[0058] (5) The test samples containing the target substance obtained in step (4) were concentrated using two methods: nitrogen blowing (i.e., SPE+N2 method) and vacuum centrifugation (i.e., SPE+VCC method). Both were concentrated to 0.1 mL, and then 0.4 mL of pure water was added. The mixture was then vortexed to obtain a mixed solution for subsequent liquid chromatography-tandem mass spectrometry detection. The working temperature of the vacuum centrifuge was 4℃, and the rotation speed was 1000 r / min.

[0059] (6) A 0.1% (v / v) formic acid aqueous solution and a 0.1% (v / v) formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in the water were determined by separating and quantifying the analytes using a BEH C18 LC column via multiple reaction monitoring (MRM). The ion source temperature was set to 650°C; the gradient elution conditions were the same as in Example 1; and the MRM settings were shown in Table 1.

[0060] The final result is as follows Figure 6 As shown, the horizontal axis represents the chlorinated products of the six flavor nucleotides, and the vertical axis represents the recovery rate of the chlorinated products of the flavor nucleotides by different concentration methods. Figure 6 The experimental results show that the two concentration methods have little difference in their effect on the recovery rate of chlorinated nucleotides, but overall, vacuum centrifugation concentration yields a better recovery rate. Therefore, vacuum centrifugation concentration was chosen as the concentration method for the sample.

[0061] Figures 7-12 MRM chromatograms and standard curves are provided for six chloroforms of flavor nucleotides. The MRM chromatogram for 2-chloro-hypoxanthine is shown below. Figure 7 As shown in (a) above, the standard curve for 2-chloro-hypoxanthine is as follows: Figure 7 As shown in (b); the MRM chromatogram of 8-chloro-hypoxanthine is shown in Figure (b). Figure 8 The standard curve of (a), 8-chloro-hypoxanthine is as follows: Figure 8 As shown in (b); the MRM chromatogram of 6-chloropurine is shown in Figure (b). Figure 9 As shown in (a) above, the standard curve for 6-chloropurine is as follows: Figure 9 As shown in (b); the MRM chromatogram of 8-chloro-guanine is as follows. Figure 10 The standard curve of (a), 8-chloro-guanine is as follows: Figure 10As shown in (b); the MRM chromatogram of 2-chloro-inosine is shown in Figure (b). Figure 11 As shown in (a) above, the standard curve of 2-chloro-inosine is as follows: Figure 11 As shown in (b); the MRM chromatogram of 8-chloro-inosine is shown in Figure (b). Figure 12 As shown in (a) above, the standard curve of 8-chloro-inosine is as follows: Figure 12 As shown in (b) of the figure. In the MRM chromatogram, the horizontal axis represents retention time, and the vertical axis represents the peak signal intensity of the target substance. In the standard curve, the horizontal axis represents the concentration of the target substance in micrograms per liter, and the vertical axis represents the peak area of ​​the target substance. Figure 13 The following are MRM chromatograms of six chlorinated flavor nucleotides in different water bodies; among them, the MRM chromatograms of the six chlorinated flavor nucleotides in standard solutions are shown below. Figure 13 As shown in (a), the MRM chromatograms of the six chloroforms of flavor nucleotides in raw water are as follows. Figure 13 As shown in (b), the MRM chromatograms of the six chlorinated nucleotides in drinking water are as follows. Figure 13 As shown in (c); Figure 13 In this system, 1 represents 2-chloro-hypoxanthine, 2 represents 8-chloro-guanine, 3 represents 8-chloro-hypoxanthine, 4 represents 6-chloro-purine, 5 represents 2-chloro-inosine, and 6 represents 8-chloro-inosine. Figure 13 It can be seen that the six chlorinated nucleotides can only be detected in drinking water and not in raw water, thus indicating that these six chlorinated products are all disinfection byproducts.

[0062] The method described in this invention was applied to the testing of drinking water in 10 cities across China. The results are shown in Table 2. "Not detected" indicates that the substance was not detected, and "detected but not quantified" indicates that the concentration of the target substance reached the detection limit but was below the quantification limit. Table 2 shows that 6-chloropurine was detected in drinking water samples from all 10 cities, with a detection rate of 100%. The detection rates of 8-chloro-hypoxanthine and 8-chloro-guanine were both 90%, and the detection rates of 2-chloro-hypoxanthine and 2-chloro-inosine were both 80%. This demonstrates that these novel but uncontrolled disinfection byproducts warrant attention.

[0063] Table 2: Detection results of chlorinated products of six flavor nucleotides in drinking water The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for solid-phase extraction-vacuum centrifugation-liquid chromatography-tandem mass spectrometry detection of chlorinated flavor nucleotides, characterized in that, Includes the following steps: (1) After collecting water samples, sodium sulfite of the same amount as the residual chlorine in the water samples is added to quench the residual chlorine, and the sample to be tested is obtained and stored at 4°C. (2) The sample to be tested obtained in step (1) is filtered through a filter membrane with a pore size of 0.45 µm to obtain a filtered sample; (3) Activate the MAX column required for solid-phase extraction with 6-12 mL of methanol and 6-18 mL of pure water respectively; (4) The filtered sample was enriched by passing it through a MAX column and then rinsed with 10 mL of pure water. (5) The target substance retained on the MAX column was eluted with 3 mL of methanol containing 2% formic acid under standard atmospheric pressure to obtain a test sample containing the target substance. (6) The sample containing the target substance is concentrated to 0.1 mL by vacuum centrifugation, and then 0.4 mL of pure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry. (7) A 0.1% formic acid aqueous solution and a 0.1% formic acid methanol solution were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. The concentrations of 2-chloro-hypoxanthine, 8-chloro-hypoxanthine, 6-chloro-purine, 8-chloro-guanine, 2-chloro-inosine, and 8-chloro-inosine in water were separated and quantified using a BEH C18 liquid chromatography column and multiple reaction monitoring method.

2. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, The MAX cartridge has a specification of 6cc and 150mg.

3. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, The vacuum centrifuge concentrator operates at a temperature of 4°C and a rotation speed of 1000 r / min.

4. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, In the liquid chromatography-tandem mass spectrometry, the mass spectrometry ion source temperature is 650℃.

5. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, The temperature of the liquid chromatography column is 35°C.

6. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, The specific conditions for gradient elution are as follows: Within 0-8 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 98:2 to 92:8; Within 8-12 minutes, the volume ratio of formic acid aqueous solution to formic acid methanol solution decreased from 92:8 to 80:20; Within 12-12.1 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution increased from 80:20 to 98:2; Within 12.1-15 min, the volume ratio of formic acid aqueous solution to formic acid methanol solution was 98:

2.

7. The method for solid-phase extraction-vacuum centrifugation concentration-liquid chromatography-tandem mass spectrometry detection of chloroforms of flavor nucleotides according to claim 1, characterized in that, The specific conditions for the multiple reaction monitoring method are shown in the table below: 。

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