Method for simultaneous determination of multiple nucleotides in drinking water by solid phase extraction-liquid chromatography-mass spectrometry

Abstract

The application discloses a kind of solid phase extraction-liquid quality connection simultaneously detecting multiple nucleotides in drinking water in the technical field of water pollution monitoring, first using solid phase extraction enrichment target material, nitrogen blow concentration, finally using the concentration of guanosine acid, adenosine acid, cytidine acid, uridine acid, deoxyguanosine acid, deoxyadenosine acid, deoxycytidine acid and deoxythymidine acid is determined using the multiple reaction monitoring mode of liquid chromatography tandem mass spectrometry, the present application combines solid phase extraction pretreatment technology with the detection technology of liquid chromatography tandem mass spectrometry, realizes the detection of trace nucleotide under complex matrix condition, the present application can realize the simultaneous determination of multiple nucleotides by one sample injection, with the advantages of high recovery rate, short detection time, many detection types and the like.

Description

Technical Field

[0001] This invention relates to the field of water pollutant monitoring technology, and in particular to a method for detecting multiple nucleotides in drinking water. Background Technology

[0002] To inhibit the growth of bacteria and other microorganisms in drinking water distribution networks, the "Standards for Drinking Water Quality" (GB5749-1985) stipulates that the chlorine and free chlorine concentrations in tap water leaving treatment plants in my country must be within the range of 0.3–4 mg / L. However, while chlorination disinfection kills pathogenic microorganisms, the disinfectant can also react chemically with halogen ions and natural organic matter to generate disinfection byproducts (DBPs). Epidemiological studies have shown a correlation between long-term consumption of chlorinated water and an increased risk of cancer.

[0003] Halogenated nucleic acids (HNOAs) are a typical class of mutagens that can induce DNA mutations, leading to chronic inflammation, cancer, and other complications. They can explain the increased carcinogenic risk of drinking water and are a class of DBPs with mutagenic potential. Studies have found that biomolecules such as nucleic acids and proteins react with chlorine at high reactivity and rates. Their precursor is the biomolecule nucleic acid, a common genetic material in all organisms. Nucleic acids have an ordered structure and high potential concentration, and are commonly found in plants, animals, and microorganisms. Nucleic acids enter the environment through dander, saliva, gametes, feces, and remains. Environmental nucleic acids in water bodies account for approximately 1.3–9.6% of the total organic nitrogen in natural water and urban wastewater. However, free single nucleotide molecules in the aquatic environment exist at trace levels, and a comprehensive and accurate understanding of their concentration and type is lacking.

[0004] Due to the complexity of drinking water matrices and the significant matrix interference, there is an urgent need to develop an efficient, rapid, and comprehensive detection method to quickly and accurately detect the presence characteristics of nucleotides in drinking water and provide a theoretical basis for assessing health risks. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry (LC-MS). This method features high recovery rate, short sample processing time, detection of multiple nucleotides, and simple operation, enabling efficient and rapid analysis and detection of various trace nucleotides under complex matrix conditions.

[0006] The objective of this invention is achieved as follows: a method for the simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry, comprising the following steps:

[0007] Step 1) After collecting the drinking water sample, add a dechlorinating agent of equal mass to the residual chlorine in the sample to quench the residual chlorine, and then refrigerate the sample in a refrigerator.

[0008] Step 2) Filter the sample to be tested obtained in Step 1) through filter paper to obtain a filtered sample;

[0009] Step 3) Activate the WAX ​​column used for solid-phase extraction with 6-12 mL of methanol and 6-18 mL of pure water respectively;

[0010] Step 4) The filtered sample obtained in Step 2) is enriched by passing it through a WAX column at a rate of 3-6 mL per minute. The WAX ​​column is then dried under vacuum until the bottom turns white.

[0011] Step 5) Elute the target substance retained on the WAX ​​column with methanol containing a set proportion of ammonia under standard atmospheric pressure to obtain a test sample containing the target substance.

[0012] Step 6) The sample containing the target substance obtained in step 5) is blown with nitrogen to A mL under a nitrogen flow, and then 1-A mL of ultrapure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0013] Step 7) Using ultrapure water containing ammonium acetate and diethylamine as the aqueous phase and acetonitrile solution containing ammonium acetate and diethylamine as the organic phase, liquid chromatography-tandem mass spectrometry analysis was performed. The elution gradient was set at a specific time. The concentrations of eight nucleotides in drinking water were separated and quantitatively analyzed using a liquid chromatography column and multiple reaction monitoring method.

[0014] Furthermore, the WAX ​​cartridges described in steps 3)-5) are 6 cc, 200 mg.

[0015] Furthermore, in step 7), the liquid chromatography column used is a Thermo Hypercarb column with parameters of 2.1 × 100 mm and 5 μm.

[0016] Furthermore, the specific conditions for the elution gradient described in step 7) are as follows:

[0017] Within 0-10 min, the volume ratio of the aqueous phase to the organic phase decreased from 98:2 to 80:20;

[0018] Within 10-10.01 min, the volume ratio of the aqueous phase to the organic phase increased from 80:20 to 98:2;

[0019] Within 10.01-14 min, the volume ratio of the aqueous phase to the organic phase remained at 98:2.

[0020] Furthermore, the specific parameters of the multiple reaction monitoring method described in step 7) are shown in the table below:

[0021]

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention improves the recovery rate of nucleotides and enhances the detection sensitivity of nucleotides by optimizing the type of solid phase extraction column; it reduces the sample analysis time of liquid chromatography-mass spectrometry (LC-MS) methods; and it enables the simultaneous determination of more types of nucleotides by constructing a multiple reaction monitoring mode.

[0024] The method of the present invention achieves enrichment and concentration of multiple nucleotides under complex matrix conditions by optimizing solid phase extraction, thereby improving the detection sensitivity of the method and reducing the detection limit and matrix interference.

[0025] This invention develops a quantitative MRM method for multiple nucleotides using characteristic fragment ions, establishes a method for detecting trace nucleotides in drinking water, and realizes the detection of nucleotides in drinking water at levels as low as ng / L.

[0026] This invention has the advantages of low detection limit, high sensitivity and short detection time, and is suitable for the simultaneous detection of trace and multiple nucleotides under complex matrix conditions;

[0027] This invention can be widely used to detect the presence characteristics of various nucleotides in natural water bodies and drinking water. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 This is a flowchart of the present invention.

[0030] Figure 2 This is a graph showing the nucleotide recovery rate of the WAX ​​solid-phase extraction column in this invention.

[0031] Figure 3 This is a standard curve diagram of the eight nucleotides of this invention.

[0032] Figure 4 This is a liquid chromatogram of the mixed standard of eight nucleotides of the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] like Figure 1 The following is a flowchart of a method for the simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry:

[0035] Step 1) After collecting the drinking water sample, add a dechlorinating agent of equal mass to the residual chlorine in the sample to quench the residual chlorine, and then store the sample at 4°C.

[0036] Step 2) Filter the sample to be tested obtained in Step 1) through filter paper with a pore size of 0.45 µm to obtain a filtered sample;

[0037] Step 3) Activate the WAX ​​column used for solid-phase extraction with 10 mL of methanol and 10 mL of pure water respectively. The WAX ​​column specifications are 6 cc, 200 mg.

[0038] Step 4) The filtered sample obtained in Step 2) is enriched by passing it through a WAX column at a rate of about 4 mL per minute, and the WAX ​​column is dried under vacuum until the bottom turns white.

[0039] Step 5) Elute the target substance retained on the WAX ​​column with 1 mL of methanol containing 2% ammonia under standard atmospheric pressure to obtain the test sample of the target substance.

[0040] Step 6) The sample containing the target substance obtained in step (5) is blown down to 0.05 mL under a nitrogen flow, and then 0.95 mL of ultrapure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry.

[0041] Step 7) Using ultrapure water containing 2 mM ammonium acetate and 0.06% diethylamine as the aqueous phase, and acetonitrile solution containing 2 mM ammonium acetate and 0.06% diethylamine as the organic phase, liquid chromatography-tandem mass spectrometry analysis was performed. The elution gradient was 14 minutes. A Thermo Hypercarb column (2.1 × 100 mm, 5 μm) was used as the liquid chromatography column to separate and quantify the concentration of eight nucleotides in drinking water using multiple reaction monitoring. The parameters of the liquid chromatography column were 2.1 × 100 mm and 5 μm.

[0042] Specifically, the conditions for gradient elution in step 7) are as follows:

[0043] Within 0-10 min, the volume ratio of the aqueous phase to the organic phase decreased from 98:2 to 80:20;

[0044] Within 10-10.01 min, the volume ratio of the aqueous phase to the organic phase increased from 80:20 to 98:2;

[0045] Within 10.01-14 min, the volume ratio of the aqueous phase to the organic phase remained at 98:2.

[0046] Specifically, the parameters for the multiple reaction monitoring method in step 7) are shown in the table below:

[0047]

[0048] Step 8) After the instrument is set up, first use a single standard to determine the retention time of the 8 nucleotides, and then use a mixed standard to plot a standard curve; the 8 nucleotides are guanosine monophosphate, adenosine monophosphate, cytidine monophosphate, uridine monophosphate, deoxyguanosine monophosphate, deoxyadenosine monophosphate, deoxycytidine monophosphate and deoxythymidine monophosphate.

[0049] Specifically, the standard curves were plotted as follows: Eight nucleotides were prepared into mixed standard solutions, which were then diluted with ultrapure water to obtain a series of standard solutions with concentrations of 1, 10, 20, 50, 100, and 200 μg / L. After analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS), standard curves for the eight nucleotides were plotted with concentration on the x-axis and peak area on the y-axis. The LC chromatograms and standard curves for the eight nucleotides are shown below. Figure 2 and Figure 3 As shown. The standard curves for the 8 nucleotides exhibit good linearity, R0 2 Between 0.998 and 0.999.

[0050] Step 9) Determination of Recovery Rate: After preparing a mixed standard solution of 8 nucleotides, dilute it with ultrapure water to a concentration of 0.1 μg / L in 1L of water, which is the pre-spiked sample. The control samples consist of two 1L ultrapure water aliquots. After steps 2) to 7) above, add the mixed standard solution of nucleotides to one of the control samples to a concentration of 100 μg / L, which is called the post-spiked sample. Measure the concentration of the 8 nucleotides in each of the three samples. The recovery rate is calculated as: Recovery Rate = (Pre-spiked - Control) / (Post-spiked - Control) * 100%. The recovery rates of the 8 nucleotides are shown below. Figure 4 As shown, the recovery rates of this method were 88.6%-105.0%, with an average recovery rate of 95.7%.

[0051] This invention compared the recoveries of two solid-phase extraction columns, MAX and WAX. MAX columns showed recoveries ranging from 0-54.4%, exhibited no enrichment or recovery performance for GMP, and had an average recovery of 34.5%. WAX columns, on the other hand, showed recoveries ranging from 88.6-105.0%, with an average recovery of 95.7%, significantly higher than the MAX column. Optimizing the solid-phase extraction column type improved nucleotide recovery and enhanced nucleotide detection sensitivity; it also reduced the sample processing time for liquid chromatography-mass spectrometry (LC-MS) and enabled the simultaneous determination of more nucleotide types by constructing a multiple reaction monitoring mode.

[0052] The method of this invention achieves enrichment and concentration of multiple nucleotides under complex matrix conditions by optimizing solid-phase extraction, thereby improving the detection sensitivity of the method and reducing the detection limit and matrix interference.

[0053] This invention develops a quantitative MRM method for various nucleotides using characteristic fragment ions, establishes a method for detecting trace nucleotides in drinking water, and realizes the detection of nucleotides in drinking water at levels as low as ng / L.

[0054] This invention has the advantages of low detection limit, high sensitivity and short detection time, and is suitable for the simultaneous detection of trace and multiple nucleotides under complex matrix conditions.

[0055] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: Step 1) After collecting the drinking water sample, add a dechlorinating agent of equal mass to the residual chlorine in the sample to quench the residual chlorine, and then refrigerate the sample in a refrigerator. Step 2) Filter the sample to be tested obtained in Step 1) through filter paper to obtain a filtered sample; Step 3) Activate the WAX ​​column used for solid-phase extraction with 6-12 mL of methanol and 6-18 mL of pure water respectively; Step 4) The filtered sample obtained in Step 2) is enriched by passing it through a WAX column at a rate of 3-6 mL per minute. The WAX ​​column is then dried under vacuum until the bottom turns white. Step 5) Elute the target substance retained on the WAX ​​column with methanol containing a set proportion of ammonia under standard atmospheric pressure to obtain a test sample containing the target substance. Step 6) The sample containing the target substance obtained in step 5) is blown with nitrogen to A mL under a nitrogen flow, and then 1-A mL of ultrapure water is added. The mixture is vortexed to obtain a mixed solution for detection by liquid chromatography-tandem mass spectrometry. Step 7) Using ultrapure water containing ammonium acetate and diethylamine as the aqueous phase and an acetonitrile solution containing ammonium acetate and diethylamine as the organic phase, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was performed. The elution gradient was set at a specific time. The concentrations of eight nucleotides in drinking water were separated and quantitatively analyzed using a Thermo Hypercarb column. The eight nucleotides were: guanosine monophosphate, adenosine monophosphate, cytidine monophosphate, uridine monophosphate, deoxyguanosine monophosphate, deoxyadenosine monophosphate, deoxycytidine monophosphate, and deoxythymidine monophosphate. The specific elution gradient conditions were as follows: Within 0-10 min, the volume ratio of the aqueous phase to the organic phase decreased from 98:2 to 80:20; Within 10-10.01 min, the volume ratio of the aqueous phase to the organic phase increased from 80:20 to 98:2; Within 10.01-14 min, the volume ratio of the aqueous phase to the organic phase remained at 98:

2.

2. The method for simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry according to claim 1, characterized in that, The WAX ​​cartridges described in steps 3)-5) are 6 cc, 200 mg.

3. The method for simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry according to claim 1 or 2, characterized in that, In step 7), the liquid chromatography column used is a Thermo Hypercarb column with parameters of 2.1 × 100 mm and 5 μm.

4. The method for simultaneous detection of multiple nucleotides in drinking water using solid-phase extraction-liquid chromatography-mass spectrometry according to claim 1 or 2, characterized in that, The specific parameters of the multiple reaction monitoring method described in step 7) are shown in the table below: 。

Images