Analytical methods for detecting oxidative stress biomarkers in urine

Abstract

This invention relates to the fields of environmental chemistry and biological sample analysis, specifically to an analytical method for detecting oxidative stress biomarkers in urine. Based on liquid chromatography-tandem mass spectrometry (LC-MS / MS), this invention provides an analytical method for the simultaneous detection of dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid in human urine. This method achieves effective purification of urine samples through specific pretreatment, reducing matrix effects, and, combined with specific LC parameters, enables the simultaneous detection of four oxidative stress biomarkers. This method exhibits good selectivity, high sensitivity, and fast detection speed, solving the current problem of the inability to simultaneously determine polar and nonpolar components. It includes a more comprehensive and rational list of oxidative stress biomarkers, better reflecting the body's oxidative stress damage.

Description

Technical Field

[0001] This invention relates to the fields of environmental chemistry and biological sample analysis, specifically to an analytical method for detecting oxidative stress biomarkers in urine. Background Technology

[0002] With technological advancements, humans are increasingly exposed to a growing number of chemicals in their daily lives. Because these chemicals can harm human health after entering the body, they are defined as new pollutants. Currently, assessing the potential toxic effects and health hazards of these new pollutants has become a research hotspot in environmental science and public health both domestically and internationally.

[0003] Oxidative stress is an important biological response of the body to exposure to chemical substances (such as pollutants, drugs, and heavy metals) in the external environment. It can induce cellular damage and trigger the body's defense responses. Therefore, assessing oxidative stress damage helps to better understand the impact of environmental factors on health and provides a theoretical basis for the prevention and treatment of related diseases. Measuring the concentration of oxidative stress biomarkers in urine can be used to assess oxidative stress damage caused by pollutant exposure, and these biomarkers can reflect DNA, RNA, lipid, and protein damage produced by the body. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the optimal choice for measuring oxidative stress biomarkers due to its ability to achieve simultaneous determination of multiple components with high sensitivity and specificity. In LC-MS / MS, sample pretreatment is a crucial step in the analysis. Appropriate sample pretreatment can effectively remove interferences, reduce matrix effects, and improve sensitivity and selectivity, thereby ensuring the accuracy and reliability of the final analytical results.

[0004] Currently, the commonly used sample pretreatment methods for determining oxidative stress biomarkers in urine include direct dilution and solid-phase extraction. Direct dilution involves diluting urine 10-fold before direct analysis. While simple, this method has several drawbacks: first, it cannot effectively remove inorganic salt components from urine, which tend to precipitate during elution, causing blockage in the mass spectrometer tubing; second, it cannot effectively remove interfering substances in urine, resulting in a strong matrix effect and making it unsuitable for large-scale sample analysis; and third, dilution affects analytical sensitivity, leading to an increase in undetectable samples. Solid-phase extraction generally only includes a few types of oxidative stress damage biomarkers. For example, the processing method disclosed in Chinese patent CN104391071B is only suitable for enriching and extracting three biomarkers reflecting DNA, RNA, and lipid oxidative stress damage, and does not include biomarkers reflecting protein oxidative damage. Furthermore, the biomarker for lipid damage involved in this method is 8-isoprostaglandin F2α, which originates from the oxidation of arachidonic acid. Although it is an oxidation product, its formation pathway is complex and more dependent on specific biosynthetic pathways. In contrast, 4-hydroxynonenal thiouric acid (HNEMA) is a direct product of lipid peroxidation (particularly generated during the oxidation of cell membrane phospholipids), thus directly reflecting the degree of lipid oxidation. Compared to 8-isoprostaglandin F2α, HNEMA is a superior biomarker for reflecting the degree of lipid oxidation.

[0005] Given the shortcomings of existing analytical methods for oxidative stress biomarkers, developing an analytical method that can simultaneously reflect oxidative stress damage to proteins, DNA, RNA, and lipids has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide an analytical method for detecting oxidative stress biomarkers in urine. This method can simultaneously detect four oxidative stress biomarkers, thereby reflecting the oxidative stress damage of proteins, DNA, RNA and lipids in the body.

[0007] To achieve the above objectives, the present invention provides an analytical method for detecting oxidative stress biomarkers in urine. This method can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid, and includes the following steps:

[0008] (1) Prepare a mixed standard stock solution and a mixed isotope internal standard stock solution. The mixed standard stock solution includes four oxidative stress biomarkers: dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid. The mixed isotope internal standard stock solution includes dityrosine- 13 C 12 8-Hydroxyguanosine-13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Four internal standard compounds: N2 and 4-hydroxynonenal mercaptouric acid-d3;

[0009] (2) Sample pretreatment: Formic acid aqueous solution and mixed isotope internal standard solution are added to urine sample to obtain diluent. The mixed isotope internal standard solution is the mixed isotope internal standard stock solution diluted with water.

[0010] (3) Solid phase extraction: The diluted solution was transferred to an activated Water Oasis HLB solid phase extraction column. After passing through the column by gravity, a first eluent containing 1-3% methanol aqueous solution was used to obtain a first eluent including dityrosine. Then, a second eluent containing 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid was obtained by a second eluent using methanol. The first eluent and the second eluent were mixed and then nitrogen was blown to the injection volume to obtain the sample to be tested.

[0011] (4) LC-MS / MS detection: The mixed standard stock solution was diluted to prepare mixed standard working solutions of different mass concentrations and LC-MS / MS detection was performed. The test sample was also detected by LC-MS / MS. The ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​the corresponding internal standard compound in the test sample was used as the ordinate, and the mass concentration of each oxidative stress biomarker in the mixed standard working solution was used as the abscissa. The standard curve was fitted by 1 / x for curve weighting and quantification.

[0012] The LC-MS / MS detection conditions included: using a Waters Acquity UPLC HSS T3 column; mobile phase A being 0.04-0.06% (v / v) formic acid aqueous solution and mobile phase B being methanol; total injection time being 12 min; and elution gradients being 0-0.5 min, 99% A, 0.5-3.5 min, 99% A-35% A, 3.5-4 min, 35% A-0% A, 4-8.5 min, holding at 0% A, 8.5-5.6 min, 0% A-99% A, 8.6-12 min, holding at 99% A.

[0013] This invention provides an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) for the simultaneous detection of dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid. This method achieves effective purification of urine samples through specific pretreatment, reducing matrix effects, and, combined with specific LC parameters, enables the simultaneous detection of four oxidative stress biomarkers. This method exhibits high selectivity, high sensitivity, and fast detection speed, solving the current problem of the inability to simultaneously determine polar and nonpolar components. It includes a more comprehensive and rational list of oxidative stress biomarkers, better reflecting the body's oxidative stress damage. Detailed Implementation

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] This invention provides an analytical method for detecting oxidative stress biomarkers in urine. This method can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid, and includes the following steps:

[0016] (1) Prepare a mixed standard stock solution and a mixed isotope internal standard stock solution. The mixed standard stock solution includes four oxidative stress biomarkers: dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid. The mixed isotope internal standard stock solution includes dityrosine- 13 C 12 8-Hydroxyguanosine- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Four internal standard compounds: N2 and 4-hydroxynonenal mercaptouric acid-d3;

[0017] (2) Sample pretreatment: Formic acid aqueous solution and mixed isotope internal standard solution are added to urine sample to obtain diluent. The mixed isotope internal standard solution is the mixed isotope internal standard stock solution diluted with water.

[0018] (3) Solid phase extraction: The diluted solution was transferred to an activated Water Oasis HLB solid phase extraction column. After passing through the column by gravity, a first eluent containing 1-3% methanol aqueous solution was used to obtain a first eluent including dityrosine. Then, a second eluent containing 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid was obtained by a second eluent using methanol. The first eluent and the second eluent were mixed and then nitrogen was blown to the injection volume to obtain the sample to be tested.

[0019] (4) LC-MS / MS detection: The mixed standard stock solution was diluted to prepare mixed standard working solutions of different mass concentrations and LC-MS / MS detection was performed. The test sample was also detected by LC-MS / MS. The ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​the corresponding internal standard compound in the test sample was used as the ordinate, and the mass concentration of each oxidative stress biomarker in the mixed standard working solution was used as the abscissa. The standard curve was fitted by 1 / x for curve weighting and quantification.

[0020] The LC-MS / MS detection conditions included: using a Waters Acquity UPLC HSS T3 column; mobile phase A being 0.04-0.06% (v / v) formic acid aqueous solution and mobile phase B being methanol; total injection time being 12 min; and elution gradients being 0-0.5 min, 99% A, 0.5-3.5 min, 99% A-35% A, 3.5-4 min, 35% A-0% A, 4-8.5 min, holding at 0% A, 8.5-5.6 min, 0% A-99% A, 8.6-12 min, holding at 99% A.

[0021] According to the present invention, before pretreatment and LC-MS / MS detection of urine samples, a mixed standard stock solution and a mixed isotope internal standard stock solution need to be prepared for use to facilitate subsequent steps and obtain better analytical results. The concentrations of the mixed standard stock solution and the mixed isotope internal standard stock solution can be selected within a wide range. Preferably, in step (1), the concentrations of each oxidative stress biomarker in the mixed standard stock solution are simultaneously 3-5 μg / mL, preferably 3.5-4.5 μg / mL, for example, values ​​such as 3.5 μg / mL, 4 μg / mL, 4.2 μg / mL, and 4.5 μg / mL, and any range thereof. Preferably, the concentrations of each internal standard compound in the mixed isotope internal standard stock solution are simultaneously 3-5 μg / mL, preferably 3.5-4.5 μg / mL, for example, values ​​such as 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL, and 4.3 μg / mL, and any range thereof. Preferably, the concentrations of each oxidative stress biomarker in the mixed standard stock solution are the same as the concentrations of each internal standard compound in the mixed isotope internal standard stock solution. The solvents for the mixed standard stock solution and the mixed isotope internal standard stock solution can be selected from a wide range; preferably, the solvent for both isotope internal standard stock solutions is methanol.

[0022] According to the present invention, in step (1), the mixed standard stock solution and the mixed isotope internal standard stock solution can be obtained by conventional preparation methods in the art, for example, by the following methods:

[0023] Mixed standard stock solution: Weigh each oxidative stress biomarker into a single volumetric flask, dilute to volume with methanol, and repeat this process to obtain single-standard stock solutions for the four oxidative stress biomarkers. Transfer equal volumes of the single-standard stock solutions for each of the four oxidative stress biomarkers into volumetric flasks, dilute to volume with methanol, and obtain the mixed standard stock solution.

[0024] Mixed isotope internal standard stock solution: Weigh each internal standard compound into a single volumetric flask, dilute to volume with methanol, and repeat this process to obtain single standard stock solutions for each of the four internal standard compounds. Transfer equal volumes of the single standard stock solutions for each of the four internal standard compounds into volumetric flasks, dilute to volume with methanol, and obtain the mixed isotope internal standard stock solution.

[0025] According to the present invention, in step (2), the specific pretreatment of the urine sample can make the obtained sample have more suitable properties, so as to obtain better separation effect in subsequent solid phase extraction and to facilitate qualitative and quantitative analysis in LC-MS / MS detection.

[0026] The inventors of this invention discovered that diluting the sample can significantly improve the adsorption of dityrosine in urine by the solid-phase extraction column, while reducing the matrix effect of urine and increasing the detection rate of dityrosine. Furthermore, dityrosine is a highly polar substance and is not easily retained on a reversed-phase solid-phase extraction column. However, by adding formic acid aqueous solution to adjust the sample pH, both dilution and alteration of the ionization state of dityrosine can be achieved, thereby changing its polarity and significantly increasing the retention ratio of dityrosine on the solid-phase extraction column. As the volume fraction of formic acid in the formic acid aqueous solution increases, the retention ratio of dityrosine on the solid-phase extraction column shows a trend of first increasing and then decreasing. To obtain better separation and analysis results, preferably, in step (2), the volume fraction of formic acid in the formic acid aqueous solution is 0.2-0.8%, preferably 0.4-0.6%, for example, it can be 0.4%, 0.45%, 0.5%, and 0.6%, or any value between these values. Preferably, the amount of formic acid aqueous solution used relative to 1 mL of the urine sample is 3-5 mL, more preferably 3.5-4.5 mL, for example, it can be 3.5 mL, 4 mL, 4.2 mL and 4.5 mL and any range between these values.

[0027] The inventors of this invention also discovered that the proportion of methanol in the sample also affects the retention ratio of dityrosine on the solid-phase extraction column. The lower the methanol proportion, the higher the retention ratio of dityrosine on the solid-phase extraction column. Therefore, in order to obtain a better separation effect, the mixed isotope internal standard stock solution needs to be diluted with water to form the mixed isotope internal standard working solution before being added to the urine sample to improve the retention ratio of dityrosine on the solid-phase extraction column. In addition, adding the mixed isotope internal standard working solution to the urine sample introduces internal standard compounds corresponding to the four oxidative stress biomarkers, which can make the qualitative and quantitative results in the subsequent LC-MS / MS detection more accurate. In order to achieve a better separation and analysis effect, preferably, in step (2), the concentration of each internal standard compound in the mixed isotope internal standard working solution is simultaneously 0.8-1.2 μg / mL, preferably 0.9-1.1 μg / mL, for example, it can be 0.9 μg / mL, 1 μg / mL, 1.05 μg / mL and 1.1 μg / mL and any range between these values. Preferably, the volume of the mixed isotope internal standard solution used relative to 1 mL of the urine sample is 0.04-0.06 mL, more preferably 0.045-0.055 mL, for example, it can be 0.045 μg / mL, 0.05 μg / mL, 0.053 μg / mL and 0.055 μg / mL and any range between these values.

[0028] According to the present invention, in step (3), the Water Oasis HLB solid-phase extraction column can effectively adsorb and elute dityrosine. Compared with the Water Oasis HLB solid-phase extraction column, other existing solid-phase extraction columns are less effective. For example, dityrosine cannot be effectively retained on the Water Oasis MAX solid-phase extraction column; while on the Envi-carb solid-phase extraction column, the adsorption capacity is too strong, making effective elution of dityrosine impossible.

[0029] According to the present invention, in step (3), in order to obtain better separation effect, preferably, the Water Oasis HLB solid-phase extraction column is activated with at least 1 mL of methanol and at least 1 mL of water. More preferably, the Water Oasis HLB solid-phase extraction column is activated with 1-4 mL of methanol (e.g., values ​​of 1 mL, 2 mL, 3 mL, and 4 mL) and 1-4 mL of water (e.g., values ​​of 1 mL, 2 mL, 3 mL, and 4 mL).

[0030] According to the present invention, in step (3), in order to better separate the components, avoid component loss, and improve the extraction efficiency of each component, it is necessary to use different elution solutions based on the polarity differences of each component. The inventors of the present invention have discovered that a methanol aqueous solution with a volume fraction of 1-3% can effectively elute dityrosine. After the dityrosine is eluted, methanol can effectively elute the remaining components. To achieve better elution results, preferably, the amount of methanol aqueous solution used in the first elution is at least 1 mL, preferably 1-3 mL, for example, 1 mL, 2 mL, 2.5 mL, and 3 mL, or any value between these values. Preferably, the amount of methanol used in the second elution is at least 1 mL, preferably 1-3 mL, for example, 1 mL, 2 mL, 2.5 mL, and 3 mL, or any value between these values. To facilitate subsequent LC-MS / MS detection, the injection volume of the sample to be tested can be adjusted. Preferably, the injection volume is 0.5-2 mL, more preferably 0.8-1.2 mL, for example, it can be 0.8 mL, 1 mL, 1.1 mL and 1.2 mL and any range between these values.

[0031] According to the present invention, in step (4), after LC-MS / MS detection, four oxidative stress biomarkers in the sample can be qualitatively analyzed, and quantitative analysis of the four oxidative stress biomarkers in the sample can be achieved by establishing a standard curve using the isotope internal standard method. To obtain a more reasonable standard curve, preferably, the mass concentrations of the mixed standard solution are 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.

[0032] According to the present invention, in step (4), in order to obtain a faster detection speed and improve the stability and accuracy of the detection, it is necessary to further adjust the LC-MS / MS detection conditions. Preferably, the LC-MS / MS detection conditions further include: a mobile phase flow rate of 0.1-0.3 mL / min, preferably 0.2-0.3 mL / min (for example, values ​​such as 0.2 mL / min, 0.25 mL / min, 0.28 mL / min, and 0.3 mL / min, and any range thereof); an injection volume of 6-12 μL, preferably 8-10 μL (for example, values ​​such as 8 μL, 9 μL, and 10 μL, and any range thereof); a column oven temperature of 30-50℃, preferably 35-45℃ (for example, values ​​such as 35℃, 40℃, 42℃, and 45℃, and any range thereof); and a sample room temperature. The temperature is 4-12℃, preferably 6-10℃ (e.g., values ​​such as 6℃, 7℃, 8℃, and 10℃, or any range thereof); the ion source is an electrospray ionization source; the ionization mode is positive ion mode and negative ion mode; the scanning mode is multi-ion reaction detection mode; the capillary voltage is 2.3-2.6kV, preferably 2.4-2.5kV (e.g., values ​​such as 2.4kV, 2.45kV, 2.48kV, and 2.5kV, or any range thereof); the ion source temperature is 120-180℃, preferably 140-160℃ (e.g., values ​​such as 140℃, 145℃, 150℃, and 160℃, or any range thereof); the desolventizing temperature is 400-600℃, preferably 450-550℃ (e.g., values ​​such as 450℃, 480℃, 500℃, and 530℃, or any range thereof).

[0033] According to the present invention, among the four oxidative stress biomarkers, dityrosine is abbreviated as diY, 8-hydroxyguanosine as 8-OHG, 8-hydroxydeoxyguanosine as 8-OHdG, and 4-hydroxynonenal thiouric acid as HNEMA. Among the four internal standard compounds, dityrosine- 13 C 12 The abbreviation is diY- 13 C12 8-Hydroxyguanosine- 13 C- 15 N2 is abbreviated as 8-OHG- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 N2 is abbreviated as 8-OHdG- 13 C- 15 The abbreviation for N2,4-hydroxynonenal mercaptouric acid-d3 is HNEMA-d3.

[0034] This invention provides an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) for the simultaneous detection of dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid. This method achieves effective purification of urine samples through specific pretreatment, reducing matrix effects, and, combined with specific LC parameters, enables the simultaneous detection of four oxidative stress biomarkers. This method exhibits high selectivity, high sensitivity, and fast detection speed, solving the current problem of the inability to simultaneously determine polar and nonpolar components. It includes a more comprehensive and rational list of oxidative stress biomarkers, better reflecting the body's oxidative stress damage.

[0035] The present invention will be described in detail below through embodiments.

[0036] Unless otherwise specified, the apparatus used in the following examples are all standard experimental apparatuses in the field, the experimental procedures are all standard procedures in the field, and the raw materials and reagents used are all commercially available. The instrument used for LC-MS / MS detection is an I-Class Plus ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (TQ-XS, Waters Corporation, USA).

[0037] Example 1

[0038] This embodiment is used to evaluate the spiked recovery rate and relative standard deviation of the analytical method of the present invention.

[0039] (1) Prepare mixed standard stock solution and mixed isotope internal standard stock solution.

[0040] Weigh 1 mg of dityrosine into a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL dityrosine single-standard stock solution. Following the same method, obtain single-standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid, each with a concentration of 100 μg / mL. Transfer 1 mL of each of the aforementioned four oxidative stress biomarkers' single-standard stock solutions into a 25 mL volumetric flask and dilute to volume with methanol to obtain a 4 μg / mL mixed standard stock solution (the concentration of each of the four oxidative stress biomarkers is 4 μg / mL).

[0041] Weigh 1 mg of dityrosine - 13 C 12 Dilute to 10 mL in a volumetric flask and bring to a final volume with methanol to obtain a 100 μg / mL concentration of bistyrosine- 13 C 12 Single-standard stock solutions were prepared using the same method to obtain 100 μg / mL concentrations of 8-hydroxyguanosine- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Single-standard stock solutions of N2 and 4-hydroxynonenal thiouric acid-d3. Transfer 1 mL of each of the aforementioned four internal standard compounds into a 25 mL volumetric flask, and dilute to volume with methanol to obtain a mixed isotope internal standard stock solution of 4 μg / mL (the concentration of each of the four internal standard compounds is 4 μg / mL).

[0042] (2) Sample spikes

[0043] The same urine sample was divided into four portions. Three of these portions were adjusted to spiked concentrations of 5 ng / mL (the concentrations of the four oxidative stress biomarkers in the urine sample were all 5 ng / mL), 10 ng / mL, and 50 ng / mL, respectively, by adding appropriate amounts of mixed standard stock solution.

[0044] All four urine samples were subsequently processed and analyzed using the same method, with each urine sample being tested in six parallel experiments.

[0045] (3) Sample pretreatment

[0046] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (formic acid volume fraction is 0.05%) and 10 μL of mixed isotope internal standard working solution (mixed isotope internal standard stock solution diluted with water to 1 μg / mL mixed isotope internal standard working solution) to obtain the diluent.

[0047] (4) Solid-phase extraction treatment

[0048] The Water Oasis HLB solid-phase extraction column was activated with 3 mL of methanol and 3 mL of water. The diluted solution was then transferred into the Water Oasis HLB solid-phase extraction column. After passing through the column by gravity, the column was dried under air for 2 min. The first eluent, consisting of dityrosine, was obtained by elution with 1 mL of 2% methanol-water solution. The column was dried under air for 1 min, and then eluted with 1 mL of methanol to obtain a second eluent consisting of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid. The first and second eluents were mixed and then purged with nitrogen to 1 mL to obtain the sample to be tested.

[0049] (5) LC-MS / MS detection

[0050] The mixed standard stock solution was diluted with methanol to prepare mixed standard working solutions with mass concentrations of 0.01, 0.1, 1, 5, 10, 20, 50 and 100 ng / mL, respectively, and detected by LC-MS / MS. The test samples were also detected by LC-MS / MS. A standard curve for the four oxidative stress biomarkers was established with the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​the corresponding internal standard compound in the test sample as the ordinate and the mass concentration of each oxidative stress biomarker in the mixed standard working solution as the abscissa. The standard curves were fitted using 1 / x weighting before quantification.

[0051] The LC-MS / MS detection conditions included: a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); mobile phase A of 0.05% formic acid aqueous solution and mobile phase B of methanol; total injection time of 12 min; and an elution gradient of 0–0.5 min, 99% A, 0.5–3.5 min, 99% A–35% A, 3.5–4 min, 35% A–0% A, 4–8.5 min, and a hold of 0% A for 8.5–5.6 min. 0% A-99% A, 8.6-12 min, maintain 99% A; mobile phase flow rate: 0.25 mL / min; injection volume: 10 μL; column oven temperature: 40℃; sample chamber temperature: 8℃; ion source: electrospray ionization source; ionization modes: positive ion mode and negative ion mode; scanning mode: multi-ion reaction detection mode; capillary voltage: 2.46 kV; ion source temperature: 150℃; desolvation temperature: 500℃.

[0052] (6) Evaluation of spiked recovery rate and relative standard deviation

[0053] Spiked recovery rate = (measured value of spiked sample - measured value of unspiked sample) ÷ spiked amount × 100%;

[0054] Relative standard deviation (RSD) = Deviation of spiked recovery rate ÷ Average of spiked recovery rate × 100%.

[0055] The recoveries and relative standard deviations at each spike concentration are shown in Table 1.

[0056] Table 1

[0057]

[0058] As shown in Table 2, the recoveries of the four oxidative stress biomarkers involved in this invention are all at high levels at various spiking concentrations, and the relative standard deviations are also very low. This demonstrates that the results obtained using the analytical method of this invention have excellent accuracy and reliability.

[0059] Example 2

[0060] This embodiment is used to evaluate the matrix effect of the analytical method of the present invention.

[0061] (1) Prepare mixed standard stock solution and mixed isotope internal standard stock solution.

[0062] Weigh 1 mg of dityrosine into a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL dityrosine single-standard stock solution. Following the same method, obtain single-standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid, each with a concentration of 100 μg / mL. Transfer 1 mL of each of the aforementioned four oxidative stress biomarkers' single-standard stock solutions into a 25 mL volumetric flask and dilute to volume with methanol to obtain a 4 μg / mL mixed standard stock solution (the concentration of each of the four oxidative stress biomarkers is 4 μg / mL).

[0063] Weigh 1 mg of dityrosine - 13 C 12 Dilute to 10 mL in a volumetric flask and bring to a final volume with methanol to obtain a 100 μg / mL concentration of bistyrosine- 13 C 12 Single-standard stock solutions were prepared using the same method to obtain 100 μg / mL concentrations of 8-hydroxyguanosine- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Single-standard stock solutions of N2 and 4-hydroxynonenal thiouric acid-d3. Transfer 1 mL of each of the aforementioned four internal standard compounds into a 25 mL volumetric flask, and dilute to volume with methanol to obtain a mixed isotope internal standard stock solution of 4 μg / mL (the concentration of each of the four internal standard compounds is 4 μg / mL).

[0064] (2) Sample selection

[0065] Take six urine samples from different sources, and then divide each sample into six parallel samples for further processing.

[0066] All 36 urine samples underwent the same pretreatment and solid-phase extraction process.

[0067] (3) Sample pretreatment

[0068] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (formic acid volume fraction is 0.05%) and 10 μL of mixed isotope internal standard working solution (mixed isotope internal standard stock solution diluted with water to 1 μg / mL mixed isotope internal standard working solution) to obtain the diluent.

[0069] (4) Solid-phase extraction treatment

[0070] The Water Oasis HLB solid-phase extraction column was activated with 3 mL of methanol and 3 mL of water. The diluted solution was then transferred to the Water Oasis HLB solid-phase extraction column and allowed to pass through the column under gravity. After air drying for 2 min, a first eluent containing dityrosine was obtained by eluting with 1 mL of 2% methanol-water solution. After air drying for 1 min, a second eluent containing 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid was obtained by eluting with 1 mL of methanol. The first and second eluents were mixed and then blown with nitrogen to 1 mL to obtain one urine matrix sample.

[0071] (5) Mixed samples

[0072] Six urine matrix samples from the same source were mixed to obtain six urine matrix samples from different sources.

[0073] (6) LC-MS / MS detection

[0074] Following the method in step (5) of Example 1, urine matrix standard curves were established for urine matrix samples from six different sources, and solution matrix standard curves were established using the initial mobile phase. The difference is that the abscissa represents six concentration points: 1, 5, 10, 20, 50, and 100 ng / mL.

[0075] The LC-MS / MS detection conditions included: a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); mobile phase A of 0.04-0.06% (v / v) formic acid aqueous solution and mobile phase B of methanol; total injection time of 12 min; and an elution gradient of 0-0.5 min, 99% A, 0.5-3.5 min, 99% A-35% A, 3.5-4 min, 35% A-0% A, 4-8.5 min, and a hold of 0% A, 8.5-5.6 min. The incubation time was 8.6-12 min, with a mobile phase flow rate of 0.25 mL / min; the injection volume was 10 μL; the column oven temperature was 40 °C; the sample chamber temperature was 8 °C; the ion source was an electrospray ionization source; the ionization modes were positive ion mode and negative ion mode; the scanning mode was multi-ion reaction detection mode; the capillary voltage was 2.46 kV; the ion source temperature was 150 °C; and the desolvation temperature was 500 °C. The ion-pair mass spectrometry information for the four oxidative stress biomarkers is shown in Table 2.

[0076] (7) Matrix effect assessment

[0077] The urine matrix standard curve and the solution matrix standard curve were obtained through step (6). For each oxidative stress biomarker, the matrix effect was evaluated by the ratio of the slope of the standard curve obtained by fitting the six urine matrix standard curves with 1 / x to the slope of the solution matrix standard curve. The matrix effects of the four oxidative stress biomarkers were all weak matrix effects (0.94-1.08), as shown in Table 3.

[0078] Table 2

[0079]

[0080]

[0081] Table 3

[0082] markers Urine sample 1 Urine sample 2 Urine sample 3 Urine sample 4 5 urine samples Urine sample 6 8-OHdG 1.02 0.94 0.98 1.05 1.04 0.99 8-OHG 1.05 0.98 1.02 1.03 1.01 0.96 HNEMA 0.99 1.05 0.94 0.95 0.94 1.03 diY 0.96 0.98 1.03 1.05 1.08 0.99

[0083] Example 3

[0084] This embodiment is used to measure actual samples.

[0085] (1) Prepare mixed standard stock solution and mixed isotope internal standard stock solution.

[0086] Weigh 1 mg of dityrosine into a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL dityrosine single-standard stock solution. Following the same method, obtain single-standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid, each with a concentration of 100 μg / mL. Transfer 1 mL of each of the aforementioned four oxidative stress biomarkers' single-standard stock solutions into a 25 mL volumetric flask and dilute to volume with methanol to obtain a 4 μg / mL mixed standard stock solution (the concentration of each of the four oxidative stress biomarkers is 4 μg / mL).

[0087] Weigh 1 mg of dityrosine - 13 C 12 Dilute to 10 mL in a volumetric flask and bring to a final volume with methanol to obtain a 100 μg / mL concentration of bistyrosine- 13 C 12 Single-standard stock solutions were prepared using the same method to obtain 100 μg / mL concentrations of 8-hydroxyguanosine- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Single-standard stock solutions of N2 and 4-hydroxynonenal thiouric acid-d3. Transfer 1 mL of each of the aforementioned four internal standard compounds into a 25 mL volumetric flask, and dilute to volume with methanol to obtain a mixed isotope internal standard stock solution of 4 μg / mL (the concentration of each of the four internal standard compounds is 4 μg / mL).

[0088] (3) Sample pretreatment

[0089] Forty urine samples from different sources were selected and processed separately.

[0090] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (formic acid volume fraction is 0.05%) and 10 μL of mixed isotope internal standard working solution (mixed isotope internal standard stock solution diluted with water to 1 μg / mL mixed isotope internal standard working solution) to obtain the diluent.

[0091] (4) Solid-phase extraction treatment

[0092] The Water Oasis HLB solid-phase extraction column was activated with 3 mL of methanol and 3 mL of water. The diluted solution was then transferred into the Water Oasis HLB solid-phase extraction column. After passing through the column by gravity, the column was dried under air for 2 min. The first eluent, consisting of dityrosine, was obtained by elution with 1 mL of 2% methanol-water solution. The column was dried under air for 1 min, and then eluted with 1 mL of methanol to obtain a second eluent consisting of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid. The first and second eluents were mixed and then purged with nitrogen to 1 mL to obtain the sample to be tested.

[0093] (5) LC-MS / MS detection

[0094] The mixed standard stock solution was diluted with methanol to prepare mixed standard working solutions with mass concentrations of 0.01, 0.1, 1, 5, 10, 20, 50 and 100 ng / mL, respectively, and detected by LC-MS / MS. The test samples were also detected by LC-MS / MS. A standard curve for the four oxidative stress biomarkers was established with the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​the corresponding internal standard compound in the test sample as the ordinate and the mass concentration of each oxidative stress biomarker in the mixed standard working solution as the abscissa. The standard curves were fitted using 1 / x weighting before quantification.

[0095] The LC-MS / MS detection conditions included: a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); mobile phase A of 0.04-0.06% (v / v) formic acid aqueous solution and mobile phase B of methanol; total injection time of 12 min; and an elution gradient of 0-0.5 min, 99% A, 0.5-3.5 min, 99% A-35% A, 3.5-4 min, 35% A-0% A, 4-8.5 min, and a hold of 0% A, 8.5-5.6 min. The incubation time was 8.6-12 min, with a mobile phase flow rate of 0.25 mL / min and an injection volume of 10 μL. The column oven temperature was 40 °C, the sample chamber temperature was 8 °C, the ion source was an electrospray ionization source, the ionization modes were positive ion mode and negative ion mode, the scanning mode was multi-ion reaction detection mode, the capillary voltage was 2.46 kV, the ion source temperature was 150 °C, and the desolventizing temperature was 500 °C.

[0096] (6) Measurement results

[0097] Four oxidative stress biomarkers—dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid—were all detected with a detection rate of 100%. The detection concentration ranges were 8.9–82.3 μg / L, 2.7–38.2 μg / L, 0.52–14.4 μg / L, and 1.7–575.3 μg / L, respectively, with median detection concentrations of 37.8 μg / L, 12.4 μg / L, 2.89 μg / L, and 96.9 μg / L, respectively. The results are shown in Table 4.

[0098] Table 4

[0099]

[0100] Comparative Example 1

[0101] According to the method of Example 1, except that in step (3), the formic acid aqueous solution was replaced with pure water, and the final spike recovery rate and relative standard deviation at each spike concentration are shown in Table 5.

[0102] Table 5

[0103]

[0104] As shown in Table 5, the recovery rate of dityrosine decreased significantly at various spiking concentrations, indicating that the accuracy and reliability of this analytical method for dityrosine are not high.

[0105] Comparative Example 2

[0106] According to the method of Example 1, the difference is that in step (3), the mixed isotope internal standard solution is replaced by a mixed isotope internal standard stock solution diluted with methanol to a mixed isotope internal standard methanol dilution solution of 1 μg / mL. The final spike recovery rate and relative standard deviation at each spike concentration are shown in Table 6.

[0107] Table 6

[0108]

[0109] As shown in Table 5, the recovery rate of dityrosine decreased significantly at various spiking concentrations, indicating that the accuracy and reliability of this analytical method for dityrosine are not high.

[0110] Comparative Example 3

[0111] According to the method of Example 1, except that in step (4), the 2% methanol aqueous solution was replaced with a 4% methanol aqueous solution, and the final spike recovery rate and relative standard deviation at each spike concentration are shown in Table 7.

[0112] Table 7

[0113]

[0114]

[0115] As shown in Table 5, the recovery rate of dityrosine was low and the relative standard deviation was high when using a 4% methanol aqueous solution for the first elution, indicating that the accuracy and reliability of the analytical method deteriorated.

[0116] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An analytical method for detecting oxidative stress biomarkers in urine, characterized in that, This method can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal mercaptouric acid, and includes the following steps: (1) Prepare a mixed standard stock solution and a mixed isotope internal standard stock solution. The mixed standard stock solution includes four oxidative stress biomarkers: dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenal thiouric acid. The mixed isotope internal standard stock solution includes dityrosine- 13 C 12 8-Hydroxyguanosine- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 Four internal standard compounds: N2 and 4-hydroxynonenal mercaptouric acid-d3; (2) Sample pretreatment: Formic acid aqueous solution and mixed isotope internal standard solution were added to the urine sample to obtain a diluent. The mixed isotope internal standard solution was the mixed isotope internal standard stock solution diluted with water. The volume fraction of formic acid in the formic acid aqueous solution was 0.2-0.8%. (3) Solid phase extraction: The diluted solution is transferred to an activated Water Oasis HLB solid phase extraction column. After passing through the column by gravity, a first eluent containing 1-3% methanol aqueous solution is used to obtain a first eluent including dityrosine. Then, a second eluent containing 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid is obtained by a second eluent using methanol. The first eluent and the second eluent are mixed and then blown with nitrogen to the injection volume to obtain the sample to be tested. (4) LC-MS / MS detection: The mixed standard stock solution was diluted to prepare mixed standard working solutions of different mass concentrations and detected by LC-MS / MS, and the test sample was detected by LC-MS / MS. Using the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard solution to the peak area of ​​the corresponding internal standard compound in the test sample as the ordinate, and the mass concentration of each oxidative stress biomarker in the mixed standard solution as the abscissa, a standard curve for four oxidative stress biomarkers is established. When fitting the standard curve, 1 / x is used for curve weighting and quantification. The LC-MS / MS detection conditions included the use of a Waters Acquity UPLC HSS T3 column; The mobile phase A was a 0.04-0.06% (v / v) formic acid aqueous solution, and the mobile phase B was methanol; the total injection time was 12 min; the elution gradient was 0-0.5 min, 99% A, 0.5-3.5 min, 99% A-35% A, 3.5-4 min, 35% A-0% A, 4-8.5 min, holding at 0% A, 8.5-8.6 min, 0% A-99% A, 8.6-12 min, holding at 0% A. The system is designed to maintain 99% A concentration; the mobile phase flow rate is 0.1-0.3 mL / min; the injection volume is 6-12 μL; the column oven temperature is 30-50℃; the sample chamber temperature is 4-12℃; the ion source is an electrospray ionization source; the ionization modes are positive ion mode and negative ion mode; the scanning mode is multi-ion reaction detection mode; the capillary voltage is 2.3-2.6 kV; the ion source temperature is 120-180℃; and the desolvation temperature is 400-600℃.

2. The method according to claim 1, wherein, In step (1), the concentration of each oxidative stress biomarker in the mixed standard stock solution is simultaneously 3-5 μg / mL; And / or, the concentration of each internal standard compound in the mixed isotope internal standard stock solution is simultaneously 3-5 μg / mL; And / or, the solvent for the mixed standard stock solution and the mixed isotope internal standard stock solution is methanol.

3. The method according to claim 2, wherein, In step (1), the concentration of each oxidative stress biomarker in the mixed standard stock solution is simultaneously 3.5-4.5 μg / mL; And / or, the concentration of each internal standard compound in the mixed isotope internal standard stock solution is simultaneously 3.5-4.5 μg / mL.

4. The method according to claim 1, wherein, In step (2), the volume fraction of formic acid in the formic acid aqueous solution is 0.4-0.6%.

5. The method according to claim 1, wherein, In step (2), the amount of formic acid aqueous solution used is 3-5 mL relative to 1 mL of the urine sample.

6. The method according to claim 5, wherein, In step (2), the amount of formic acid aqueous solution used is 3.5-4.5 mL relative to 1 mL of the urine sample.

7. The method according to claim 1, wherein, In step (2), the concentration of each internal standard compound in the mixed isotope internal standard solution is simultaneously 0.8-1.2 μg / mL.

8. The method according to claim 7, wherein, In step (2), the concentration of each internal standard compound in the mixed isotope internal standard solution is simultaneously 0.9-1.1 μg / mL.

9. The method according to claim 1, wherein, In step (2), the amount of the mixed isotope internal standard solution used is 0.04-0.06 mL relative to 1 mL of the urine sample.

10. The method according to claim 9, wherein, In step (2), the amount of the mixed isotope internal standard solution used is 0.045-0.055 mL relative to 1 mL of the urine sample.

11. The method according to claim 1, wherein, In step (3), the Water Oasis HLB solid phase extraction column is activated with at least 1 mL of methanol and at least 1 mL of water.

12. The method according to claim 11, wherein, In step (3), the Water Oasis HLB solid phase extraction column is activated with 1-4 mL of methanol and 1-4 mL of water.

13. The method according to claim 1, wherein, In step (3), the amount of methanol aqueous solution used in the first elution is at least 1 mL; And / or, the amount of methanol used in the second elution is at least 1 mL; And / or, the injection volume is 0.5-2 mL.

14. The method according to claim 13, wherein, In step (3), the amount of methanol aqueous solution used in the first elution is 1-3 mL; And / or, the amount of methanol used in the second elution is 1-3 mL; And / or, the injection volume is 0.8-1.2 mL.

15. The method according to claim 1, wherein, In step (4), the mass concentrations of the mixed standard solutions are 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively.

16. The method according to claim 1, wherein, In step (4), the LC-MS / MS detection conditions also include: mobile phase flow rate of 0.2-0.3 mL / min; injection volume of 8-10 μL; column oven temperature of 35-45℃; sample chamber temperature of 6-10℃; capillary voltage of 2.4-2.5 kV; ion source temperature of 140-160℃; and desolvation temperature of 450-550℃.

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