Method for detecting halogenated nucleoside based on ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting halogenated nucleoside based on ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: aiming at drinking water and extracellular free halogenated nucleoside substances, firstly enriching target substances by using solid-phase extraction, and then concentrating and enriching by using vacuum centrifugation; for nucleic acid molecules in cells, firstly, intracellular DNA and RNA are extracted by using a kit, then enzymolysis is performed step by step by using multiple enzymes, and vertical ultrafiltration centrifugal purification is performed; and finally, measuring the concentrations of the 20 halogenated nucleosides by using a multi-reaction monitoring mode of ultra-high performance liquid chromatography-tandem mass spectrometry. Aiming at samples of different matrixes, pretreatment methods such as solid-phase extraction, vacuum centrifugal concentration, composite enzymolysis and vertical ultrafiltration purification are respectively combined, and finally, the detection technology of ultra-high performance liquid chromatography-tandem mass spectrometry is applied, so that simultaneous qualitative and quantitative analysis of various trace halogenated nucleosides under the complex matrix condition is realized.

Description

Technical Field

[0001] This invention relates to the field of drinking water disinfection byproduct detection technology, and in particular to a method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Drinking water is a basic human need, and its safety is directly related to the health of the general public. Disinfection is an essential step in drinking water treatment, effectively controlling the occurrence of drinking water-related diseases and ensuring that it meets human health requirements. Meanwhile, to inhibit the growth of bacteria and other microorganisms in drinking water distribution networks, the "Standards for Drinking Water Quality" (GB 5749-2022) stipulates that the free chlorine concentration in tap water leaving the treatment plant in my country must be within the range of 0.3–2 mg / L. However, while chlorination disinfection kills pathogenic microorganisms, it also reacts with organic matter in the water to form harmful disinfection byproducts (DBPs). Epidemiological studies have shown a correlation between long-term consumption of chlorinated water and an increased risk of various cancers.

[0003] Liquid chlorine disinfection is currently the most mature and widely used disinfection technology. The main bactericidal and disinfecting agents are hypochlorous acid and its derivatives, such as chloramine, bromamine, chlorine dioxide, and hypobromic acid. These active halides can act not only on cell walls and viral capsids, but also, due to their small molecular size and lack of charge, can penetrate into bacteria (viruses) and react with biological macromolecules to kill pathogenic microorganisms. Nucleic acids are the basic genetic material of almost all organisms and can be released into water sources from various sources (such as agricultural runoff, livestock discharge, industrial wastewater, and urban sewage). Simultaneously, nucleic acids can naturally decompose into nucleotides, nucleosides, or nucleobases. Therefore, nucleic acids and their components are widely present in water sources and are likely to produce halogenated nucleic acid (containing nucleosides) disinfection byproducts. Existing studies have shown that some halogenated nucleoside products have both cytotoxic and genotoxic properties. Because their structure is similar to that of normal nucleosides, they can be misidentified and incorporated into nucleic acid chains, affecting normal gene replication and expression, and even leading to base mismatches and gene mutations. They have been shown to be closely related to the occurrence and development of human diseases such as atherosclerosis, diabetes, and various types of cancer.

[0004] Whether in their free state in drinking water or in their bound state incorporating nucleic acid chains, halogenated nucleosides exist in complex matrices, leading to significant detection interference. Therefore, in order to rapidly and accurately detect halogenated nucleosides in various complex matrices, it is urgent to develop an analytical method for halogenated nucleosides with high sensitivity and low detection limit. This would provide a theoretical basis for elucidating the occurrence characteristics and genotoxicity of halogenated nucleosides in the drinking water environment and assessing their health risks. Summary of the Invention

[0005] The purpose of this invention is to address the technical problems of low concentration, complex matrix, and significant detection interference of halogenated nucleoside products in existing technologies, and to provide a method for the detection of halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry. This invention combines the two core advantages of high sensitivity and low detection limit, and features small sample pretreatment volume and simple operation, enabling the detection and analysis of trace halogenated nucleoside substances under various complex matrix conditions.

[0006] The objective of this invention is achieved through the following technical solution: a method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0007] (1) For different matrices, corresponding pretreatment methods are used to pretreat them to obtain mixed solutions for detection by liquid chromatography-tandem mass spectrometry; wherein, the matrices include drinking water, extracellular fluid and intracellular nucleic acid molecules;

[0008] (2) A 0.1% formic acid / water 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, and BEH was used. The C18 liquid chromatography column was used to separate and quantify the concentrations of halogenated nucleosides using a multiple reaction monitoring method to determine the qualitative and quantitative concentrations of 8-bromoadenosine, 8-bromo-2'-deoxyadenosine, 5-bromocytidine, 5-bromo-2'-deoxycytidine, 8-bromoguanosine, 8-bromo-2'-deoxyguanosine, 5-bromouridine, 5-bromo-2'-deoxyuridine, 2-chloroadenosine, 2-chloro-2'-deoxyadenosine, 8-chloroadenosine, 8-chloro-2'-deoxyadenosine, 6-chloroguanosine, 6-chloro-2'-deoxyguanosine, 8-chloroguanosine, 8-chloroguanosine, 8-chloro-2'-deoxyguanosine, 5-chlorouridine, 5-chloro-2'-deoxyuridine, 5-chlorocytidine, and 5-chloro-2'-deoxycytidine in the corresponding matrix.

[0009] Compared with existing technologies, the beneficial effects of this invention are as follows: For free halogenated nucleosides in drinking water and extracellular fluid, solid-phase extraction is used to enrich and reduce matrix interference; for halogenated nucleosides incorporated into nucleic acid chains, target analytes are extracted and purified through complex enzymatic digestion and vertical ultrafiltration; finally, combined with multiple reaction monitoring (MRM) methods of chromatography-tandem mass spectrometry, high-sensitivity, high-precision, and high-accuracy qualitative and quantitative analysis of trace halogenated nucleosides in different complex matrices can be achieved; this invention achieves the enrichment and concentration of halogenated nucleosides in drinking water through solid-phase extraction, followed by rinsing with pure water and a solution containing 2% formic acid. Methanol eluent improves elution efficiency and significantly reduces matrix interference. This invention employs chromatographic tandem mass spectrometry analysis in a mixed positive and negative ion mode, enabling the differentiation and identification of chloroadenosine isomers. By using typical deribose characteristic fragment ions of halogenated nucleosides as daughter ions in the MRM method for quantitative halogenated nucleosides, the invention achieves the detection and quantitative assessment of halogenated nucleoside disinfection byproducts in drinking water at levels as low as ng / L. This invention has advantages such as small sample volume requirement, low detection limit, high sensitivity, and short detection time, and is suitable for the detection and analysis of trace halogenated nucleosides under various complex matrix conditions. Attached Figure Description

[0010] Figure 1 This is a flowchart of the method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry of the present invention;

[0011] Figure 2 This study describes the detection signal intensity and chromatographic peaks of chloroadenosine isomers under different gradient elution modes in positive ion mode using liquid chromatography-tandem mass spectrometry. Figure 2 In the figure, (a) shows the detection signal intensity and chromatogram of the six chloroadenosine peaks in sample group a. Figure 2 (b) shows the detection signal intensity and chromatogram of the six chloroadenosine peaks in group b samples. Figure 2 In the diagram, (c) represents the detection signal intensity and chromatogram of the six chloroadenosine peaks in sample group c. Figure 2 (d) represents the detection signal intensity and chromatogram of the six chloroadenosine peaks in sample group d;

[0012] Figure 3 This is a standard curve plot showing the fitting relationship between the concentration of six halogenated nucleosides (2-chloro-2'-deoxyadenosine, 2-chloroadenosine, 5-bromo-2'-deoxycytidine, 5-bromocytidine, 5-bromo-2'-deoxyuridine, and 5-bromouridine) and the detection peak area in negative ion mass spectrometry mode.

[0013] Figure 4This is a standard curve plot showing the fitting relationship between the concentration of six halogenated nucleosides (5-chloro-2'-deoxycytidine, 5-chlorocytidine, 5-chloro-2'-deoxyuridine, 5-chlorouridine, 6-chloro-2'-deoxyguanosine, and 6-chloroguanosine) and the detection peak area in negative ion mass spectrometry mode.

[0014] Figure 5 This is a standard curve plot showing the fitting relationship between the concentration of six halogenated nucleosides (8-bromo-2'-deoxyadenosine, 8-bromoadenosine, 8-bromo-2'-deoxyguanosine, 8-bromoguanosine, 8-chloro-2'-deoxyguanosine, and 8-chloroguanosine) and the detection peak area in negative ion mass spectrometry mode.

[0015] Figure 6 This is a standard curve plot showing the fitting relationship between the concentration of two chloroadenosines (8-chloro-2'-deoxyadenosine and 8-chloroadenosine) as standard samples and the detection peak area in positive ion mass spectrometry mode.

[0016] Figure 7 The figure shows the effect of eluents containing different proportions of methanol on the detection signal intensity of five typical halogenated nucleosides.

[0017] Figure 8 Chromatograms comparing 8-chloroguanosine in raw water and disinfected drinking water; among which, Figure 8 (a) in the figure represents the chromatogram of 8-chloroguanosine in raw water. Figure 8 (b) in the figure represents the chromatogram of 8-chloroguanosine in drinking water;

[0018] Figure 9 Chromatograms comparing 8-chloro-2'-deoxyguanosine in raw water and disinfected drinking water; where, Figure 9 (a) The chromatogram of 8-chloro-2'-deoxyguanosine in raw water. Figure 9 (b) in the figure represents the chromatogram of 8-chloro-2'-deoxyguanosine in drinking water;

[0019] Figure 10 Chromatograms comparing 8-bromoguanosine in raw water and disinfected drinking water; among which, Figure 10 (a) in the figure represents the chromatogram of 8-bromoguanosine in raw water. Figure 10 (b) in the figure represents the chromatogram of 8-bromoguanosine in drinking water;

[0020] Figure 11 Chromatograms comparing 8-bromoadenosine in raw water and disinfected drinking water; among which, Figure 11 (a) in the figure represents the chromatogram of 8-bromoadenosine in the raw water. Figure 11 (b) in the figure represents the chromatogram of 8-bromoadenosine in drinking water;

[0021] Figure 12Chromatograms comparing 8-bromo-2'-deoxyguanosine in raw water and disinfected drinking water; where, Figure 12 (a) in the figure represents the chromatogram of 8-bromo-2'-deoxyguanosine in the raw water. Figure 12 (b) in the figure represents the chromatogram of 8-bromo-2'-deoxyguanosine in drinking water;

[0022] Figure 13 Chromatograms comparing 8-bromo-2'-deoxyadenosine in raw water and disinfected drinking water; where, Figure 13 (a) in the figure represents the chromatogram of 8-bromo-2'-deoxyadenosine in the raw water. Figure 13 (b) in the figure represents the chromatogram of 8-bromo-2'-deoxyadenosine in drinking water;

[0023] Figure 14 Chromatograms comparing 5-chlorouridine in raw water and disinfected drinking water; among which, Figure 14 (a) in the figure represents the chromatogram of 5-chlorouridine in the raw water. Figure 14 (b) in the figure represents the chromatogram of 5-chlorouridine in drinking water;

[0024] Figure 15 Chromatograms comparing 5-chlorocytidine in raw water and disinfected drinking water; among which, Figure 15 (a) in the figure represents the chromatogram of 5-chlorocytidine in the raw water. Figure 15 (b) in the figure represents the chromatogram of 5-chlorocytidine in drinking water;

[0025] Figure 16 Chromatograms comparing 5-chloro-2'-deoxycytidine in raw water and disinfected drinking water; where, Figure 16 (a) in the figure represents the chromatogram of 5-chloro-2'-deoxycytidine in the raw water. Figure 16 (b) in the figure represents the chromatogram of 5-chloro-2'-deoxycytidine in drinking water;

[0026] Figure 17 Chromatograms comparing 5-bromouridine in raw water and disinfected drinking water; among which, Figure 17 (a) in the figure represents the chromatogram of 5-bromouridine in the raw water. Figure 17 (b) in the figure represents the chromatogram of 5-bromouridine in drinking water;

[0027] Figure 18 Chromatograms comparing 5-bromocytidine in raw water and disinfected drinking water; among which, Figure 18 (a) in the figure represents the chromatogram of 5-bromocytidine in the raw water. Figure 18 (b) in the figure represents the chromatogram of 5-bromocytidine in drinking water;

[0028] Figure 19Chromatograms comparing 5-bromo-2'-deoxyuridine in raw water and disinfected drinking water; where, Figure 19 (a) in the figure represents the chromatogram of 5-bromo-2'-deoxyuridine in the raw water. Figure 19 (b) in the figure represents the chromatogram of 5-bromo-2'-deoxyuridine in drinking water;

[0029] Figure 20 Chromatograms comparing 5-bromo-2'-deoxycytidine in raw water and disinfected drinking water; where, Figure 20 (a) in the figure represents the chromatogram of 5-bromo-2'-deoxycytidine in the raw water. Figure 20 (b) in the figure represents the chromatogram of 5-bromo-2'-deoxycytidine in drinking water;

[0030] Figure 21 Chromatograms comparing 2-chloroadenosine in raw water and disinfected drinking water; where, Figure 21 (a) in the figure represents the chromatogram of 5-bromodeoxyuridine in the raw water. Figure 21 (b) in the figure represents the chromatogram of 5-bromodeoxyuridine in drinking water;

[0031] Figure 22 The chromatograms show a comparison of 2-chloro-2'-deoxyadenosine in raw water and disinfected drinking water; among which, Figure 22 (a) in the figure represents the chromatogram of 5-bromodeoxyuridine in the raw water. Figure 22 (b) in the figure represents the chromatogram of 5-bromodeoxyuridine in drinking water. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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."

[0035] 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.

[0036] The present invention provides a method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry, which can simultaneously detect multiple halogenated nucleosides and is applicable to the detection of halogenated nucleosides in different matrices such as drinking water, extracellular fluid, and intracellular nucleic acid molecules. Figure 1 As shown, the specific steps include the following:

[0037] (1) For different matrices, corresponding pretreatment methods are used to pretreat them to obtain mixed solutions for detection by liquid chromatography-tandem mass spectrometry. Among them, the matrices include drinking water, extracellular fluid and intracellular nucleic acid molecules, etc.

[0038] Furthermore, in step (1), the pretreatment method for drinking water specifically includes the following sub-steps:

[0039] (a1.1) After collecting drinking water, sodium sulfite of the same amount as the residual chlorine in the drinking water is added to quench the residual chlorine, and the sample to be tested is obtained and stored at 4°C.

[0040] (a1.2) The sample to be tested obtained in step (a1.1) is filtered through filter paper with a pore size of 0.45 μm to obtain the filtered sample.

[0041] (a1.3) The MAX column required for solid-phase extraction was activated sequentially with 12 mL of methanol and 12 mL of pure water. The MAX column specifications were 6 cc and 150 mg.

[0042] (a1.4) The filtered sample obtained in step (a1.2) is enriched by passing it through a MAX column and the MAX column is dried under vacuum until the bottom turns white.

[0043] (a1.5) Under standard atmospheric pressure, use 3 mL of methanol solution containing 2% formic acid to elute the target substance retained on the MAX column to obtain a test sample containing the target substance.

[0044] (a1.6) The sample containing the target substance obtained in step (a1.5) is concentrated and dried in a vacuum centrifuge, then reconstituted with 1 mL of pure water, and vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0045] Furthermore, in step (1), the pretreatment method for the extracellular fluid specifically includes the following sub-steps:

[0046] (b1.1) After collecting the extracellular fluid sample, the sample was filtered through an aqueous filter membrane with a pore size of 0.22 μm to obtain the filtered sample.

[0047] (b1.2) The required MAX column for solid-phase extraction was activated sequentially with 6 mL of methanol and 6 mL of pure water. The specifications of the MAX column were 3 cc and 60 mg.

[0048] (b1.3) The filtered sample obtained in step (b1.1) is enriched by passing it through MAX columns, with a loading volume of 2 mL for each MAX column.

[0049] (b1.4) Under standard atmospheric pressure, after rinsing with 2 mL of pure water, the MAX column is dried under vacuum until the bottom turns white.

[0050] (b1.5) Under standard atmospheric pressure, use 2 mL of methanol solution containing 2% formic acid to elute the target substance retained on the MAX column to obtain a test sample containing the target substance.

[0051] (b1.6) The sample containing the target substance obtained in step (b1.5) is concentrated and dried in a vacuum centrifuge, then reconstituted with 1 mL of pure water, and vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0052] Furthermore, in step (1), the pretreatment method for intracellular nucleic acid molecules specifically includes the following sub-steps:

[0053] (c1.1) DNA and RNA were extracted from cells using a commercially available kit. The purity and concentration of the extracted DNA and RNA samples were qualitatively and quantitatively assessed using a NanoDrop micro-spectrophotometer. The commercially available kit was an RNA / DNA co-extraction kit (Beyotime).

[0054] (c1.2) The DNA and RNA solutions extracted in step (c1.1) were concentrated and dried using a vacuum centrifuge, and stored at -20℃ and -80℃, respectively.

[0055] (c1.3) Dissolve and mix the nucleic acid sample saved in step (c1.2) with sterile double-distilled water according to the nucleic acid content determined in step (c1.1) to prepare a nucleic acid solution with a concentration of 1 μg / μL, and mix well.

[0056] (c1.4) Take 16 μL of the nucleic acid solution obtained in step (c1.3), add 2 μL of nuclease P1 and 2 μL of (1×) nuclease P1 reaction buffer, vortex to mix, briefly centrifuge, and then place in a 37°C water bath for 2 h. The concentration of nuclease P1 is 100,000 units / mL; the nuclease P1 reaction buffer is a 50 mmol / L sodium acetate solution with a pH of 5.5.

[0057] (c1.5) Add 50 μL of alkaline phosphatase solution to the solution obtained from enzymatic hydrolysis in step (c1.4), mix well, and briefly centrifuge. Then place in a 37°C water bath for 1 hour. The alkaline phosphatase solution is prepared by dissolving lyophilized powder (≥10 DEA units / mg) derived from bovine intestinal mucosa in alkaline phosphatase buffer, i.e., a 500 mmol / L Tris-HCl buffer at pH=8. Each 1 mg of lyophilized alkaline phosphatase powder is dissolved in 50 μL of alkaline phosphatase buffer.

[0058] (c1.6) Add 90 μL of sterile double-distilled water to the solution after enzymatic hydrolysis in step (c1.5), mix well, transfer to a vertical ultrafiltration centrifuge tube, and centrifuge at 4°C and 12000g for 15 minutes. Collect the filtrate as a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0059] Furthermore, the vacuum centrifugal concentrator operates at a temperature of 4°C and a rotation speed of 2000 r / min.

[0060] (2) A 0.1% formic acid / water 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, and BEH was used. The C18 liquid chromatography column was used to separate and quantify the concentrations of halogenated nucleosides using a multiple reaction monitoring method to determine the qualitative and quantitative concentrations of 8-bromoadenosine, 8-bromo-2'-deoxyadenosine, 5-bromocytidine, 5-bromo-2'-deoxycytidine, 8-bromoguanosine, 8-bromo-2'-deoxyguanosine, 5-bromouridine, 5-bromo-2'-deoxyuridine, 2-chloroadenosine, 2-chloro-2'-deoxyadenosine, 8-chloroadenosine, 8-chloro-2'-deoxyadenosine, 6-chloroguanosine, 6-chloro-2'-deoxyguanosine, 8-chloroguanosine, 8-chloroguanosine, 8-chloro-2'-deoxyguanosine, 5-chlorouridine, 5-chloro-2'-deoxyuridine, 5-chlorocytidine, and 5-chloro-2'-deoxycytidine in the corresponding matrix.

[0061] Furthermore, the specific conditions for gradient elution include:

[0062] ① When analyzing in negative ion mode of mass spectrometry, the corresponding gradient elution conditions are as follows:

[0063] Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 50:50.

[0064] Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 50:50 to 2:98;

[0065] Within 12 to 12.1 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 2:98 to 98:2;

[0066] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0067] ② When analyzing in positive ion mode of mass spectrometry, the specific gradient elution conditions are as follows:

[0068] Within 0–10 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:20; within 10–12 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 50:50; within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 50:50 to 98:2; within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0069] Furthermore, the mass ion source temperature for liquid chromatography-tandem mass spectrometry is 600℃.

[0070] Furthermore, the specific conditions for the multiple reaction monitoring method are shown in Table 1:

[0071] Table 1: Conditions for Multiple Reaction Monitoring Methods

[0072]

[0073]

[0074] Compounds marked with an asterisk (*) are analyzed in positive ion mode of mass spectrometry, while other compounds are analyzed in negative ion mode by default.

[0075] The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry of the present invention will be described in detail below with reference to the embodiments, and the purpose and effect of the present invention will become more apparent.

[0076] Example 1

[0077] This embodiment investigated the effects of different elution gradients on the detection signal intensity and chromatographic peak resolution of two groups of chloroadenosine isomers in positive ion mode of mass spectrometry, specifically including the following steps:

[0078] (1) Prepare mixed standard solutions containing the same concentration of six chloroadenosines (2-chloro-2'-deoxyadenosine, 8-chloro-2'-deoxyadenosine, 6-chloro-2'-deoxyguanosine, 2-chloroadenosine, 8-chloroadenosine, and 6-chloroguanosine) using pure water, and divide them into four equal parts: a, b, c, and d.

[0079] It should be understood that since this is a mixed standard with a fixed concentration, there is no need for steps such as activation and enrichment of the water sample. The concentration can be directly detected using different elution gradients.

[0080] (2) A 0.1% formic acid / water solution (as mobile phase A) and a 0.1% formic acid / methanol solution (as mobile phase B) were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. The signal intensities of 2-chloro-2'-deoxyadenosine, 8-chloro-2'-deoxyadenosine, 6-chloro-2'-deoxyguanosine, 2-chloroadenosine, 8-chloroadenosine, and 6-chloroguanosine in group a sample were separated and detected by multiple reaction monitoring.

[0081] The specific conditions for gradient elution are as follows:

[0082] Within 0–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 2:90;

[0083] Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 2:90 to 98:2;

[0084] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0085] The specific conditions for the multiple reaction monitoring method are shown in Table 2:

[0086] Table 2: Setup conditions for multiple reaction monitoring methods in positive ion mode

[0087]

[0088]

[0089] (3) A 0.1% formic acid / water 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 signal intensities of 2-chloro-2'-deoxyadenosine, 8-chloro-2'-deoxyadenosine, 6-chloro-2'-deoxyguanosine, 2-chloroadenosine, 8-chloroadenosine, and 6-chloroguanosine in the samples of group b were separated and detected by multiple reaction monitoring.

[0090] The specific conditions for gradient elution are as follows:

[0091] Within 0 to 2 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:20;

[0092] Within 2 to 12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 50:50.

[0093] Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 50:50 to 98:2;

[0094] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0095] The specific conditions for the multiple reaction monitoring method are shown in Table 2.

[0096] (4) A 0.1% formic acid / water 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 signal intensities of 2-chloro-2'-deoxyadenosine, 8-chloro-2'-deoxyadenosine, 6-chloro-2'-deoxyguanosine, 2-chloroadenosine, 8-chloroadenosine, and 6-chloroguanosine in the C group samples were separated and detected by multiple reaction monitoring.

[0097] The specific conditions for gradient elution are as follows:

[0098] Within 0 to 6 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:20;

[0099] Within 2 to 12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 2:90;

[0100] Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 2:90 to 98:2;

[0101] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0102] The specific conditions for the multiple reaction monitoring method are shown in Table 2.

[0103] (5) A 0.1% formic acid / water 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 signal intensities of 2-chloro-2'-deoxyadenosine, 8-chloro-2'-deoxyadenosine, 6-chloro-2'-deoxyguanosine, 2-chloroadenosine, 8-chloroadenosine, and 6-chloroguanosine in the group d samples were separated and detected by multiple reaction monitoring.

[0104] The specific conditions for gradient elution are as follows:

[0105] Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:20.

[0106] Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 50:50.

[0107] Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 50:50 to 98:2;

[0108] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0109] The specific conditions for the multiple reaction monitoring method are shown in Table 2.

[0110] The final result is as follows Figure 2 As shown, the chromatogram results of group a are as follows: Figure 2 As shown in (a) above, the chromatogram results of group b are as follows: Figure 2 As shown in (b) above, the chromatogram results of group c are as follows: Figure 2 As shown in (c), the chromatogram results of group d samples are as follows: Figure 2 As shown in (d) in the figure, the horizontal axis represents the injection time, and the vertical axis represents the signal intensity. The effects of four gradient elution programs with a total duration of 15 min on the detection signal intensity and resolution of the six chloroadenosine chromatographic peaks were tested. The experimental results showed that although the signal intensities of the six chloroadenosine chromatographic peaks were all within 10 min using the three gradient elution programs in steps (2) to (4), the results showed that the three gradient elution programs in steps (2) to (4) all had a signal intensity of less than 10 min. 6 While the chromatographic peaks were relatively even, there was overlap, resulting in poor separation. However, the gradient elution procedure in step (5) not only ensured that the peak signal intensity remained within 10... 6 The peak shapes and separation of the six chloroadenosines were good, and the gradient elution procedure in step (5) was used for the differentiation and quantitative analysis of chloroadenosine isomers.

[0111] Example 2

[0112] This embodiment examines the linear fitting results between the concentration of standard samples of 20 halogenated nucleosides and the detection peak area, specifically including the following steps:

[0113] (1) Prepare a mixed aqueous solution of 20 halogenated nucleosides, wherein the concentrations of the 20 halogenated nucleosides in the mixed aqueous solution are the same, namely 0, 1, 2.5, 5, 10, 25, 50 and 100 μg / L.

[0114] (2) A 0.1% formic acid / water 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, and 20 halogenated nucleosides in the sample were separated and quantified by multiple reaction monitoring.

[0115] Specifically, the gradient elution conditions for analysis in negative ion mass spectrometry mode are as follows:

[0116] Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 50:50.

[0117] Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 50:50 to 2:98;

[0118] Within 12 to 12.1 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 2:98 to 98:2;

[0119] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0120] In the positive ion mode of mass spectrometry, the specific gradient elution conditions are as follows:

[0121] Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:20.

[0122] Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 50:50.

[0123] Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 50:50 to 98:2;

[0124] Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2.

[0125] The quantitative results of the concentrations of 20 halogenated nucleosides are as follows: Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, the horizontal axis represents the concentration of halogenated nucleosides, and the vertical axis represents the integrated peak area of ​​halogenated nucleosides. Experimental results show that the R² values ​​of the standard curves for 20 halogenated nucleosides... 2 All values ​​reached above 0.99, indicating a good fitting effect.

[0126] Example 3

[0127] This embodiment evaluated the recovery, limit of detection, and limit of quantitation of 20 halogenated nucleosides using an Oasis MAX solid-phase extraction column, specifically including the following steps:

[0128] (1) A mixed standard solution of 20 halogenated nucleosides was prepared using pure water. Three concentration levels (low, medium, and high) were selected within the concentration range of the standard curve, and the concentrations of the 20 halogenated nucleosides in the mixed aqueous solution were kept the same.

[0129] (2) The sample to be tested obtained in step (1) is filtered through filter paper with a pore size of 0.45 μm to obtain the filtered sample.

[0130] (3) The MAX column required for solid-phase extraction was activated by 12 mL of methanol and 12 mL of pure water, respectively.

[0131] (4) The filtered sample obtained in step (2) is enriched by passing it through a MAX column and the MAX column is dried under vacuum until the bottom turns white.

[0132] (5) Under standard atmospheric pressure, the target substance retained on the MAX column was eluted with 3 mL of methanol containing 2% formic acid to obtain the test sample containing the target substance.

[0133] (6) The sample to be tested obtained in step (5) is concentrated and dried in a vacuum centrifuge, then 1 mL of pure water is added to redissolve it, and after vortexing, a mixed solution is obtained for detection by liquid chromatography-tandem mass spectrometry.

[0134] (7) 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. Gradient elution was performed for 15 minutes, and 20 halogenated nucleosides in the sample were separated and quantified by multiple reaction monitoring. The gradient elution conditions were the same as those in Example 2.

[0135] (8) The specific calculation formulas for the recovery rate, detection limit, and quantitation limit of the 20 halogenated nucleosides are as follows:

[0136] The mixed standard solution at low concentration levels was subjected to the same sample pretreatment and the measurement was repeated 7 times to calculate the standard deviation (SD).

[0137] The method detection limit = 3.14 × SD (t = 3.14 when the degrees of freedom are 6);

[0138] Method limit of quantitation = 10 × SD.

[0139] The recoveries, limits of detection, and limits of quantitation for 20 halogenated nucleosides were calculated based on the above formulas, and the results are shown in Table 3.

[0140] Table 3: Recovery rates, limits of detection, and limits of quantitation for 20 halogenated nucleosides

[0141]

[0142]

[0143] As shown in Table 3, the method of the present invention exhibits low limits of detection and quantitation and high recovery rates for a variety of halogenated nucleosides, and the error range of matrix effect is within ±15%, which meets the analytical requirements.

[0144] Example 4

[0145] This embodiment investigated the effect of different eluent types on the signal intensity of halonucleotide measurements, specifically including the following steps:

[0146] (1) Five halogenated nucleosides were randomly selected, and aqueous solutions containing the same concentrations of 2-chloroadenosine, 6-chloroguanosine, 5-bromouridine, 5-bromocytidine, and 8-bromoguanosine were prepared. The solutions were then filtered through filter paper with a pore size of 0.45 μm to obtain filtered samples.

[0147] (2) The MAX column required for solid-phase extraction was activated by using 6 mL of methanol and 6 mL of pure water respectively.

[0148] (3) The filtered samples obtained in step (1) are enriched by MAX columns.

[0149] (4) Under standard atmospheric pressure, the column was rinsed with aqueous solutions containing different proportions of methanol (0, 0.5%, 1%, 2%, 3%, 4%) and then dried under vacuum until the bottom of the MAX column turned white.

[0150] (5) The halogenated nucleosides retained on the MAX column were eluted with 2 mL of methanol containing 2% formic acid under standard atmospheric pressure to obtain a sample containing five halogenated nucleosides.

[0151] (6) After the sample to be tested obtained in step (5) is concentrated, enriched and dried in a vacuum centrifuge, 1 mL of pure water is added to redissolve it, and the mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0152] (7) A solution containing 0.1% formic acid / water and a solution containing 0.1% formic acid / methanol were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. A BEH C18 liquid chromatography column was used, and the five halogenated nucleosides were analyzed by multiple reaction monitoring.

[0153] The final result is as follows Figure 7 As shown, the horizontal axis represents different types of eluents, and the vertical axis represents the signal intensity of the mass spectrometry response. Although the instructions for use of the extraction column and the physicochemical properties of halogenated nucleosides suggest that an aqueous solution containing methanol should be used for elution, the experimental results show that pure water provides the best elution effect.

[0154] Example 5

[0155] This embodiment evaluated the potential for the generation of chloronucleotides in the intracellular nucleic acid chains of Chinese hamster ovary cells under different effective chlorine concentrations of exposure, specifically including the following steps:

[0156] (1) Sodium hypochlorite mother liquor was diluted with HBSS buffer to obtain exposure solutions of different concentrations, with available chlorine contents of 1.5, 3, 5 and 7.5 mg / L respectively.

[0157] (2) Select Chinese hamster ovary cells in the logarithmic growth phase, count them after trypan blue staining, and inoculate 106 cells per dish (60 mm in diameter) and incubate overnight.

[0158] (3) After the cells adhere to the wall, discard the original culture medium, gently rinse twice with phosphate buffer, and then replace with different concentrations of exposure solution. Incubate in a biochemical incubator at 37°C for 30 min.

[0159] (4) A blank control group (i.e., HBSS buffer without sodium hypochlorite) was set up at the same time. Three replicates were set up for each experimental group and blank control group.

[0160] (5) After the exposure is complete, discard the exposure solution, rinse gently twice with phosphate buffer, and extract DNA and RNA from the cells according to the instructions of the commercial kit. The purity and concentration of the extracted DNA and RNA samples are qualitatively and quantitatively evaluated using a NanoDrop micro spectrophotometer.

[0161] (6) The extracted DNA and RNA solutions were concentrated and dried using a vacuum centrifuge. The nucleic acid content determined in step (5) was dissolved and mixed with sterile double-distilled water to prepare a 1 μg / μL nucleic acid solution.

[0162] (7) For every 16 μL of nucleic acid solution obtained in step (3), add 2 μL of nuclease P1 and 2 μL of (1×) nuclease P1 reaction buffer, vortex to mix, and after a short centrifugation, place in a 37°C water bath for 2 h.

[0163] (8) Add 50 μL of alkaline phosphatase solution to the solution after enzymatic hydrolysis in step 7, mix well and centrifuge briefly, then incubate in a water bath at 37°C for 1 hour.

[0164] (9) Add 90 μL of sterile double-distilled water to the solution after enzymatic hydrolysis in step (5), mix well, transfer to a vertical ultrafiltration centrifuge tube, and centrifuge at 4℃ and 12000g for 15 minutes. Collect the filtrate for detection by liquid chromatography-tandem mass spectrometry.

[0165] (10) A solution containing 0.1% formic acid / water and a solution containing 0.1% formic acid / methanol were used as the mobile phases for liquid chromatography-tandem mass spectrometry. Gradient elution was performed for 15 minutes. A BEH C18 liquid chromatography column was used. Chlorinated nucleosides were separated and quantified by multiple reaction monitoring to assess the generation potential of chlorinated nucleosides under cell-simulated chlorination conditions.

[0166] The experimental results are shown in Table 4. A total of 7 chloronucleotides were detected, among which 5-chloro-2'-deoxycytidine had the highest detection concentration and the greatest potential for generation under exposure to an effective chlorine concentration of 7.5 mg / L.

[0167] Table 4: Characteristics of chlorinated nucleoside production in Chinese hamster ovary cells under different effective chlorine concentrations.

[0168]

[0169] Figures 8-22 Chromatographic tandem mass spectra of 15 halogenated nucleosides in raw water and drinking water are presented respectively. The horizontal axis represents retention time, and the vertical axis represents peak signal intensity of the target substance. These correspond to 8-chloroguanosine, 8-chloro-2'-deoxyguanosine, 8-bromoguanosine, 8-bromoadenosine, 8-bromo-2'-deoxyguanosine, 8-bromo-2'-deoxyadenosine, 5-chlorouridine, 5-chlorocytidine, 5-chloro-2'-deoxycytidine, 5-bromouridine, 5-bromocytidine, 5-bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, 2-chloroadenosine, and 2-chloro-2'-deoxyadenosine. The chromatographic tandem mass spectra show that these 15 halogenated nucleosides can only be detected in drinking water, but not in raw water, indicating that these 15 halogenated nucleosides are all disinfection byproducts.

[0170] The method described in this invention was applied to the detection of 31 groups of drinking water samples. The results are shown in Table 5. "Not detected" indicates no detection, and "*" indicates detection but inability to be accurately quantified, meaning the target substance concentration reached the detection limit but was below the quantification limit. The results showed that 15 halogenated nucleoside disinfection byproducts were undetectable in the raw water of all 31 samples but detectable in the drinking water, with detection rates ranging from 87% to 100%. Among them, four disinfection byproducts (5-bromo-2'-deoxyuridine, 8-bromo-2'-deoxyadenosine, 2-chloro-2'-deoxyadenosine, and 2-chloroadenosine) achieved a 100% detection rate. The highest total cumulative concentration of halogenated nucleoside DBPs detected in a single sample reached 141.2 ng / L. Therefore, these novel but uncontrolled disinfection byproducts deserve our attention.

[0171] Table 5: Detection of 15 halogenated nucleosides in drinking water and raw water

[0172]

[0173]

[0174]

[0175]

[0176] 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 detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: (1) For different matrices, corresponding pretreatment methods are used to pretreat them to obtain mixed solutions for detection by liquid chromatography-tandem mass spectrometry; wherein, the matrices include drinking water, extracellular fluid and intracellular nucleic acid molecules; (2) A 0.1% formic acid / water 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, and BEH was used. The C18 liquid chromatography column was used to separate and quantify the concentrations of halogenated nucleosides using a multiple reaction monitoring method to determine the qualitative and quantitative concentrations of 8-bromoadenosine, 8-bromo-2'-deoxyadenosine, 5-bromocytidine, 5-bromo-2'-deoxycytidine, 8-bromoguanosine, 8-bromo-2'-deoxyguanosine, 5-bromouridine, 5-bromo-2'-deoxyuridine, 2-chloroadenosine, 2-chloro-2'-deoxyadenosine, 8-chloroadenosine, 8-chloro-2'-deoxyadenosine, 6-chloroguanosine, 6-chloro-2'-deoxyguanosine, 8-chloroguanosine, 8-chloroguanosine, 8-chloro-2'-deoxyguanosine, 5-chlorouridine, 5-chloro-2'-deoxyuridine, 5-chlorocytidine, and 5-chloro-2'-deoxycytidine in the corresponding matrix.

2. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (1), the pretreatment method for drinking water specifically includes the following sub-steps: (a1.1) After collecting drinking water, sodium sulfite of the same amount as the residual chlorine in the drinking water is added to quench the residual chlorine, and the sample to be tested is obtained and stored at 4°C. (a1.2) The sample to be tested obtained in step (a1.1) is filtered through filter paper with a pore size of 0.45 μm to obtain a filtered sample; (a1.3) The MAX column required for solid-phase extraction was activated sequentially with 12 mL of methanol and 12 mL of pure water; wherein, the specification of the MAX column is 6 cc, 150 mg; (a1.4) The filtered sample obtained in step (a1.2) is enriched by passing it through a MAX column and the MAX column is dried under vacuum until the bottom turns white. (a1.5) Under standard atmospheric pressure, use 3 mL of methanol solution containing 2% formic acid to elute the target substance retained on the MAX column to obtain a test sample containing the target substance. (a1.6) The sample containing the target substance obtained in step (a1.5) is concentrated and dried in a vacuum centrifuge, then reconstituted with 1 mL of pure water and vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

3. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (1), the pretreatment method for extracellular fluid specifically includes the following sub-steps: (b1.1) After collecting the extracellular fluid sample, it was filtered through an aqueous filter membrane with a pore size of 0.22 μm to obtain the filtered sample; (b1.2) The MAX column required for solid-phase extraction was activated sequentially with 6 mL of methanol and 6 mL of pure water; wherein, the specification of the MAX column is 3cc, 60mg; (b1.3) The filtered sample obtained in step (b1.1) is enriched by passing it through MAX columns, with a loading volume of 2 mL for each MAX column; (b1.4) Under standard atmospheric pressure, after rinsing with 2 mL of pure water, the MAX column was dried under vacuum until the bottom turned white; (b1.5) Under standard atmospheric pressure, use 2 mL of methanol solution containing 2% formic acid to elute the target substance retained on the MAX column and obtain a test sample containing the target substance. (b1.6) The sample containing the target substance obtained in step (b1.5) is concentrated and dried in a vacuum centrifuge, then reconstituted with 1 mL of pure water, and vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

4. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (1), the pretreatment method for intracellular nucleic acid molecules specifically includes the following sub-steps: (c1.1) DNA and RNA were extracted from cells using a kit, and the purity and concentration of the extracted DNA and RNA samples were qualitatively and quantitatively evaluated using a NanoDrop micro spectrophotometer. (c1.2) The DNA and RNA solutions extracted in step (c1.1) were concentrated and dried using a vacuum centrifuge, and stored in freezers at -20℃ and -80℃, respectively. (c1.3) Dissolve and mix the nucleic acid sample saved in step (c1.2) with sterile double-distilled water according to the nucleic acid content determined in step (c1.1) to prepare a nucleic acid solution with a concentration of 1 μg / μL, and mix well; (c1.4) Take 16 μL of the nucleic acid solution obtained in step (c1.3), add 2 μL of nuclease P1 and 2 μL of (1×) nuclease P1 reaction buffer, vortex to mix, and then briefly centrifuge and place in a 37°C water bath for 2 h. (c1.5) Add 50 μL of alkaline phosphatase solution to the solution after enzymatic hydrolysis in step (c1.4), mix well and centrifuge briefly, then place in a 37°C water bath for 1 h. (c1.6) Add 90 μL of sterile double-distilled water to the solution after enzymatic hydrolysis in step (c1.5), mix well, transfer to a vertical ultrafiltration centrifuge tube, and centrifuge at 4°C and 12000g for 15 minutes. Collect the filtrate as a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

5. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The concentration of nuclease P1 is 100,000 units / mL; the reaction buffer for nuclease P1 is a sodium acetate solution with a pH of 5.5 and a concentration of 50 mmol / L.

6. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The alkaline phosphatase solution is prepared by dissolving lyophilized powder derived from bovine intestinal mucosa in alkaline phosphatase buffer, i.e., Tris-HCl buffer with a concentration of 500 mmol / L and pH=8, wherein each 1 mg of alkaline phosphatase lyophilized powder is dissolved in 50 μL of alkaline phosphatase buffer.

7. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 2-4, characterized in that, The vacuum centrifuge concentrator operates at a temperature of 4°C and a rotation speed of 2000 r / min.

8. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The specific conditions for gradient elution include: ① When analyzing in negative ion mode of mass spectrometry, the corresponding gradient elution conditions are as follows: Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 50:

50. Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 50:50 to 2:98; Within 12 to 12.1 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 2:98 to 98:2; Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:2; ② When analyzing in positive ion mode of mass spectrometry, the specific gradient elution conditions are as follows: Within 0 to 10 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 98:2 to 80:

20. Within 10–12 minutes, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution decreased from 80:20 to 50:

50. Within 12–12.1 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution increased from 50:50 to 98:2; Within 12.1–15 min, the volume ratio of formic acid / aqueous solution and formic acid / methanol solution was 98:

2.

9. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The mass ion source temperature for the liquid chromatography-tandem mass spectrometry is 600℃.

10. The method for detecting halogenated nucleosides based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The specific conditions for the multiple reaction monitoring method are shown in the table below: Compounds marked with an asterisk (*) are analyzed in positive ion mode of mass spectrometry, while other compounds are analyzed in negative ion mode by default.

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