Analysis method for detecting oxidative stress biomarker in urine

Abstract

The invention relates to the field of environmental chemistry and biological sample analysis, in particular to an analysis method for detecting oxidative stress biomarkers in urine. The analysis method capable of simultaneously detecting dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid in human urine is provided on the basis of liquid chromatography-tandem mass spectrometry, effective purification of a urine sample is achieved through specific pretreatment, the matrix effect is reduced, and the detection sensitivity is improved. And specific liquid chromatogram parameter conditions are combined, so that simultaneous detection of the four oxidative stress biomarkers is realized. The method is good in selectivity, high in sensitivity and high in detection speed, the problem that polar and non-polar components cannot be measured at the same time at present is solved, the contained oxidative stress biomarkers are more comprehensive and more reasonable, and the oxidative stress damage condition of the organism can be better reflected.

Description

Technical Field

[0001] The present invention relates to the field of environmental chemistry and biological sample analysis, and in particular to an analytical method for detecting oxidative stress biomarkers in urine. Background Art

[0002] With the advancement of science and technology, humans have been exposed to more and more chemicals in their daily lives. Since these chemicals will cause harm to human health after entering the human body, they are also defined as new pollutants. At present, evaluating the potential toxic effects and health hazards of these new pollutants to humans has become a research hotspot in the fields of environmental science and public health at home and abroad.

[0003] Oxidative stress is an important biological response of the body to the exposure of chemicals (such as pollutants, drugs, heavy metals, etc.) in the external environment. It can not only cause cell damage in the body, but also stimulate the body's defense response. Therefore, evaluating the body's oxidative stress damage helps to better understand the impact of environmental factors on health and provide a theoretical basis for the prevention and treatment of related diseases. Determination of the concentration of oxidative stress biomarkers in urine can be used to evaluate oxidative stress damage caused by pollutant exposure, and these oxidative stress biomarkers can reflect the DNA, RNA, lipid and protein damage produced by the body. Liquid chromatography-tandem mass spectrometry has become the best choice for the determination of oxidative stress biomarkers because it can achieve high sensitivity and high specificity of multi-component simultaneous determination. In liquid chromatography-tandem mass spectrometry, sample pretreatment is an important part of LC-MS / MS analysis. By performing appropriate sample pretreatment, interference can be effectively removed, matrix effects can be reduced, sensitivity and selectivity can be improved, thereby ensuring the accuracy and reliability of the final analysis results.

[0004] At present, the commonly used sample pretreatment methods for determining oxidative stress biomarkers in urine are direct dilution method and solid phase extraction method. Among them, the direct dilution method is to dilute the urine 10 times and then directly measure it on the machine. Although this method is simple to operate, it has the following problems: First, it cannot effectively remove the inorganic salt components in the urine, and it is easy to precipitate during elution, causing blockage of the mass spectrometry pipeline; second, the interference in the urine cannot be effectively removed, so it has a strong matrix effect and is not suitable for large-scale sampling analysis; third, the dilution affects the analytical sensitivity, resulting in an increase in undetected samples. The solid phase extraction method generally only includes individual types of oxidative stress damage markers. For example, the treatment method disclosed in Chinese patent CN104391071B is only suitable for enriching and extracting three biomarkers used to reflect DNA, RNA and lipid oxidative stress damage, and does not include biomarkers reflecting protein oxidative damage. In addition, the biomarker of lipid damage involved in this method is 8-isoprostaglandin F2α, which is derived from the oxidation of arachidonic acid. Although it is a product of oxidation reaction, its production pathway is complex and more dependent on a specific biosynthetic pathway. 4-Hydroxynonenalthiouric acid (HNEMA) is a direct product of lipid peroxidation (especially during the oxidation of cell membrane phospholipids), and therefore directly reflects the degree of lipid oxidation. Compared with 8-isoprostaglandin F2α, HNEMA is a better biomarker reflecting the degree of lipid oxidation.

[0005] In view of the shortcomings of existing analytical methods for oxidative stress biomarkers, how to develop an analytical method that can simultaneously reflect oxidative stress damage to proteins, DNA, RNA and lipids has become a problem that needs to be solved urgently. Summary of the invention

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

[0007] In order to achieve the above object, the present invention provides an analytical method for detecting oxidative stress biomarkers in urine, which can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercapturic acid, comprising the following steps:

[0008] (1) preparing a mixed standard stock solution and a mixed isotope internal standard stock solution, wherein the mixed standard stock solution includes four oxidative stress biomarkers: bistyrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the mixed isotope internal standard stock solution includes bistyrosine- 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: adding a formic acid aqueous solution and a mixed isotope internal standard working solution to a urine sample to obtain a diluted solution, wherein the mixed isotope internal standard working solution is the mixed isotope internal standard stock solution diluted with water;

[0010] (3) Solid phase extraction treatment: the diluted solution is transferred to an activated Water Oasis HLB solid phase extraction column, and after passing through the column by natural gravity, a first elution is performed with a methanol aqueous solution having a volume fraction of 1-3% to obtain a first eluent including dityrosine, and then a second elution is performed with methanol to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the first eluent and the second eluent are mixed and blown with nitrogen to the injection volume to obtain a sample to be tested;

[0011] (4) LC-MS / MS detection: diluting the mixed standard stock solution into mixed standard working solutions of different mass concentrations and performing LC-MS / MS detection, and performing LC-MS / MS detection on the sample to be tested; using the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​each corresponding internal standard compound in the sample to be tested as the ordinate, and using the mass concentration of each oxidative stress biomarker in the mixed standard working solution as the abscissa, a standard curve of four oxidative stress biomarkers is established, and the standard curve is fitted by using 1 / x for curve weighting and then quantification;

[0012] The conditions for the LC-MS / MS detection include: using a Waters Acquity UPLC HSS T3 chromatographic column; using a formic acid aqueous solution with a volume fraction of 0.04-0.06% as mobile phase A, and using methanol as mobile phase B; a full injection time of 12 minutes; an elution gradient of 0-0.5 minutes, 99% A, 0.5-3.5 minutes, 99% A-35% A, 3.5-4 minutes, 35% A-0% A, 4-8.5 minutes, maintaining 0% A, 8.5-5.6 minutes, 0% A-99% A, 8.6-12 minutes, maintaining 99% A.

[0013] The present invention provides an analytical method for simultaneously detecting dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid based on liquid chromatography tandem mass spectrometry. The method realizes effective purification of urine samples through specific pretreatment, reduces matrix effect, and realizes simultaneous detection of four oxidative stress biomarkers in combination with specific liquid chromatography parameter conditions. The method has good selectivity, high sensitivity, and fast detection speed, and solves the problem that polar and non-polar components cannot be measured simultaneously at present. The oxidative stress biomarkers contained are more comprehensive and more reasonable, and can better reflect the oxidative stress damage of the body. DETAILED DESCRIPTION

[0014] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0015] The present invention provides an analytical method for detecting oxidative stress biomarkers in urine, which can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercapturic acid, and comprises the following steps:

[0016] (1) preparing a mixed standard stock solution and a mixed isotope internal standard stock solution, wherein the mixed standard stock solution includes four oxidative stress biomarkers: bistyrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the mixed isotope internal standard stock solution includes bistyrosine- 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: adding a formic acid aqueous solution and a mixed isotope internal standard working solution to a urine sample to obtain a diluted solution, wherein the mixed isotope internal standard working solution is the mixed isotope internal standard stock solution diluted with water;

[0018] (3) Solid phase extraction treatment: the diluted solution is transferred to an activated Water Oasis HLB solid phase extraction column, and after passing through the column by natural gravity, a first elution is performed with a methanol aqueous solution having a volume fraction of 1-3% to obtain a first eluent including dityrosine, and then a second elution is performed with methanol to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the first eluent and the second eluent are mixed and blown with nitrogen to the injection volume to obtain a sample to be tested;

[0019] (4) LC-MS / MS detection: diluting the mixed standard stock solution into mixed standard working solutions of different mass concentrations and performing LC-MS / MS detection, and performing LC-MS / MS detection on the sample to be tested; using the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​each corresponding internal standard compound in the sample to be tested as the ordinate, and using the mass concentration of each oxidative stress biomarker in the mixed standard working solution as the abscissa, a standard curve of four oxidative stress biomarkers is established, and the standard curve is fitted by using 1 / x for curve weighting and then quantification;

[0020] The conditions for the LC-MS / MS detection include: using a Waters Acquity UPLC HSS T3 chromatographic column; using a formic acid aqueous solution with a volume fraction of 0.04-0.06% as mobile phase A, and using methanol as mobile phase B; a full injection time of 12 minutes; an elution gradient of 0-0.5 minutes, 99% A, 0.5-3.5 minutes, 99% A-35% A, 3.5-4 minutes, 35% A-0% A, 4-8.5 minutes, maintaining 0% A, 8.5-5.6 minutes, 0% A-99% A, 8.6-12 minutes, maintaining 99% A.

[0021] According to the present invention, before the urine sample is pre-treated and LC-MS / MS detected, it is necessary to prepare a mixed standard stock solution and a mixed isotope internal standard stock solution for standby use, so as to facilitate the subsequent steps and obtain better analysis results. The concentrations of the mixed standard stock solution and the mixed isotope internal standard stock solution can be selected in a wide range. Preferably, in step (1), the concentration of each oxidative stress biomarker in the mixed standard stock solution is 3-5 μg / mL at the same time, preferably 3.5-4.5 μg / mL, for example, it can be 3.5 μg / mL, 4 μg / mL, 4.2 μg / mL and 4.5 μg / mL and the range between any values ​​thereof. Preferably, the concentration of each internal standard compound in the mixed isotope internal standard stock solution is 3-5 μg / mL at the same time, preferably 3.5-4.5 μg / mL, for example, it can be 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL and 4.3 μg / mL and the range between any values ​​thereof. Preferably, the concentration of each oxidative stress biomarker in the mixed standard stock solution is the same as the concentration of each internal standard compound in the mixed isotope internal standard stock solution. The solvent of the mixed standard stock solution and the mixed isotope internal standard stock solution can be selected in a wide range. Preferably, the solvent of the mixed standard stock solution and the mixed isotope internal standard stock solution 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 method:

[0023] Mixed standard stock solution: Weigh a single oxidative stress biomarker into a single volumetric flask, dilute to volume with methanol, and obtain the single standard stock solutions of four oxidative stress biomarkers in this way. Transfer the same volume of the single standard stock solutions of the four oxidative stress biomarkers to a volumetric flask, dilute to volume with methanol, and obtain the mixed standard stock solution.

[0024] Mixed isotope internal standard stock solution: Weigh a single internal standard compound into a single volumetric flask, dilute to volume with methanol, and obtain the single standard stock solutions of four internal standard compounds in this way. Transfer the same volume of the single standard stock solutions of the four internal standard compounds to a volumetric flask, dilute to volume with methanol, and obtain the mixed isotope internal standard stock solution.

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

[0026] The inventors of the present invention have found that diluting the sample can significantly improve the adsorption of bistyrosine in urine by the solid phase extraction column, while reducing the matrix effect of urine and improving the detection rate of bistyrosine. In addition, bistyrosine is a highly polar substance and is not easy to be retained on the reverse solid phase extraction column. By adding a formic acid aqueous solution to adjust the sample pH, the dilution effect can be achieved, and the ionization state of bistyrosine can be changed, thereby changing its polarity, and significantly improving the retention ratio of bistyrosine on the solid phase extraction column. As the volume fraction of formic acid in the formic acid aqueous solution increases, the retention ratio of bistyrosine on the solid phase extraction column shows a trend of first rising and then falling. In order to obtain a better separation and analysis effect, 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% and the range between any values ​​thereof. Preferably, relative to 1 mL of the urine sample, the amount of the formic acid aqueous solution is 3-5 mL, 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 therebetween.

[0027] The inventors of the present invention also found that the methanol ratio in the sample will also affect the retention ratio of dityrosine on the solid phase extraction column. The lower the methanol ratio, 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 the mixed isotope internal standard solution before adding it to the urine sample to increase the retention ratio of dityrosine on the solid phase extraction column. In addition, adding the mixed isotope internal standard solution to the urine sample and introducing the internal standard compounds corresponding to the four oxidative stress biomarkers 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 solution is 0.8-1.2 μg / mL at the same time, 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 the range between any values ​​thereof. Preferably, relative to 1 mL of the urine sample, the amount of the mixed isotope internal standard solution is 0.04-0.06 mL, 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 value in between.

[0028] According to the present invention, in step (3), the Water Oasis HLB solid phase extraction column can adsorb and elute dityrosine well. Compared with the Water Oasis HLB solid phase extraction column, other existing solid phase extraction columns have poor effects. For example, dityrosine cannot be effectively retained on the Water Oasis MAX solid phase extraction column; and on the Envi-carb solid phase extraction column, due to the strong adsorption capacity, effective elution of dityrosine cannot be achieved.

[0029] According to the present invention, in step (3), in order to obtain a 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 (for example, values ​​such as 1 mL, 2 mL, 3 mL and 4 mL) and 1-4 mL of water (for example, values ​​such as 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 eluents using the polarity differences of the components. The inventors of the present invention have found through research that a methanol aqueous solution with a volume fraction of 1-3% can elute dityrosine well. After the elution of dityrosine is completed, methanol can be used to elute the remaining components well. In order to achieve a better elution effect, preferably, the amount of methanol aqueous solution in the first elution is at least 1mL, preferably 1-3mL, for example, it can be 1mL, 2mL, 2.5mL and 3mL and the range between any values ​​thereof. Preferably, the amount of methanol in the second elution is at least 1mL, preferably 1-3mL, for example, it can be 1mL, 2mL, 2.5mL and 3mL and the range between any values ​​thereof. In order 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, 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 the values.

[0031] According to the present invention, in step (4), after LC-MS / MS detection, the four oxidative stress biomarkers in the sample can be qualitatively analyzed, and the four oxidative stress biomarkers in the sample can be quantitatively analyzed by establishing a standard curve using the isotope internal standard method. In order to obtain a more reasonable standard curve, preferably, the mass concentrations of the mixed standard solution are 0.01ng / mL, 0.1ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL and 100ng / 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 conditions of the LC-MS / MS detection. Preferably, the conditions of the LC-MS / MS detection also include: a mobile phase flow rate of 0.1-0.3 mL / min, preferably 0.2-0.3 mL / min (for example, it can be 0.2 mL / min, 0.25 mL / min, 0.28 mL / min and 0.3 mL / min and the range between any values ​​thereof); an injection volume of 6-12 μL, preferably 8-10 μL (for example, it can be 8 μL, 9 μL and 10 μL and the range between any values ​​thereof); a column oven temperature of 30-50°C, preferably 35-45°C (for example, it can be 35°C, 40°C, 42°C and 45°C and the range between any values ​​thereof); and a sample room temperature of 100 °C. The temperature of the ion source is 4-12°C, preferably 6-10°C (for example, it can be 6°C, 7°C, 8°C and 10°C and the range between any values ​​thereof); the ion source is an electrospray ionization source; the ionization modes are positive ion mode and negative ion mode; the scanning mode is a multiple ion reaction detection mode; the capillary voltage is 2.3-2.6 kV, preferably 2.4-2.5 kV (for example, it can be 2.4 kV, 2.45 kV, 2.48 kV and 2.5 kV and the range between any values ​​thereof); the ion source temperature is 120-180°C, preferably 140-160°C (for example, it can be 140°C, 145°C, 150°C and 160°C and the range between any values ​​thereof); the desolvation temperature is 400-600°C, preferably 450-550°C (for example, it can be 450°C, 480°C, 500°C and 530°C and the range between any values ​​thereof).

[0033] According to the present invention, among the four oxidative stress biomarkers, dityrosine is abbreviated as diY, 8-hydroxyguanosine is abbreviated as 8-OHG, 8-hydroxydeoxyguanosine is abbreviated as 8-OHdG, and 4-hydroxynonenalthiouric acid is abbreviated as HNEMA. 13 C 12 The abbreviation is diY- 13 C12 ,8-hydroxyguanosine- 13 C- 15 The abbreviation of N2 is 8-OHG- 13 C- 15 N2,8-hydroxydeoxyguanosine- 13 C- 15 The abbreviation of N2 is 8-OHdG- 13 C- 15 The abbreviation of N2,4-hydroxynonenalmercaptouric acid-d3 is HNEMA-d3.

[0034] The present invention provides an analytical method for simultaneously detecting dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid based on liquid chromatography tandem mass spectrometry. The method realizes effective purification of urine samples through specific pretreatment, reduces matrix effect, and realizes simultaneous detection of four oxidative stress biomarkers in combination with specific liquid chromatography parameter conditions. The method has good selectivity, high sensitivity, and fast detection speed, and solves the problem that polar and non-polar components cannot be measured simultaneously at present. The oxidative stress biomarkers contained are more comprehensive and more reasonable, and can better reflect the oxidative stress damage of the body.

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

[0036] In the following examples, unless otherwise specified, the devices used are all conventional experimental devices in the art, the experimental operations adopted are all conventional operations in the art, and the raw materials, reagents, etc. used can be obtained commercially. The instrument model for LC-MS / MS detection is an I-Class Plus ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (TQ-XS, Waters, USA).

[0037] Example 1

[0038] This example is used to evaluate the spiked recovery 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 bistyrosine into a 10 mL volumetric flask, dilute to volume with methanol, and obtain a single standard stock solution of bistyrosine with a concentration of 100 μg / mL. In the same way, obtain single standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenalthiouric acid with a concentration of 100 μg / mL. Transfer 1 mL of each single standard stock solution of the above four oxidative stress biomarkers to a 25 mL volumetric flask, dilute to volume with methanol, and obtain a mixed standard stock solution of 4 μg / mL (the concentration of the four oxidative stress biomarkers is 4 μg / mL).

[0041] Weigh 1 mg of bis-tyrosine 13 C 12 Add to a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL bis-tyrosine 13 C 12 Single-label stock solutions were prepared using the same method to obtain 8-hydroxyguanosine- 13 C- 15 N2, 8-hydroxydeoxyguanosine- 13 C- 15 Single standard stock solutions of N2 and 4-hydroxynonenal mercaptouric acid-d3. Pipette 1 mL of each of the four internal standard compounds into a 25 mL volumetric flask, dilute to volume with methanol, and obtain a 4 μg / mL mixed isotope internal standard stock solution (the concentration of the four internal standard compounds is 4 μg / mL).

[0042] (2) Sample spike

[0043] The same urine was divided into four parts, three of which were added with appropriate amounts of mixed standard stock solution to adjust the spiked concentrations to 5 ng / mL (the concentrations of the four oxidative stress biomarkers in the urine samples were all 5 ng / mL), 10 ng / mL, and 50 ng / mL, respectively.

[0044] The four urine samples were subsequently processed and analyzed using the same method, and each urine sample was subjected to six parallel experiments.

[0045] (3) Sample pretreatment

[0046] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (the volume fraction of formic acid 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 a diluted solution.

[0047] (4) Solid phase extraction

[0048] The Water Oasis HLB solid phase extraction column was activated with 3 mL of methanol and 3 mL of water, and then the dilution was transferred to the Water Oasis HLB solid phase extraction column. After passing through the column by natural gravity, the column was air-dried for 2 minutes. 1 mL of a 2% methanol aqueous solution was used for the first elution to obtain a first eluent including dityrosine. The column was air-dried for 1 minute, and then 1 mL of methanol was used for the second elution to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid. The first eluent and the second eluent were mixed and blown to 1 mL with nitrogen to obtain a sample to be tested.

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

[0050] The mixed standard stock solution was diluted with methanol to mixed standard working solutions with mass concentrations of 0.01, 0.1, 1, 5, 10, 20, 50 and 100 ng / mL, respectively, and LC-MS / MS detection was performed, and the samples to be tested were also tested by LC-MS / MS detection; the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​each corresponding internal standard compound in the sample to be tested 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 to establish standard curves of the four oxidative stress biomarkers, and the standard curves were fitted using 1 / x for curve weighting and then quantification.

[0051] The conditions of the LC-MS / MS detection include: using a Waters Acquity UPLC HSS T3 chromatographic column (100 mm×2.1 mm, 1.8 μm); using a 0.05% volume fraction of formic acid aqueous solution as mobile phase A, and using methanol as mobile phase B; the full injection time is 12 min; the elution gradient is 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 holding 0% A, 8.5-5.6 min , 0% A-99% A, 8.6-12min, maintain 99% A; mobile phase flow rate is 0.25mL / min; injection volume is 10μL; column oven temperature is 40℃; sample chamber temperature is 8℃; ion source is electrospray ionization source; ionization mode is positive ion mode and negative ion mode; scanning mode is multiple ion reaction detection mode; capillary voltage is 2.46kV; ion source temperature is 150℃; desolvation temperature is 500℃.

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

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

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

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

[0056] Table 1

[0057]

[0058] It can be seen from Table 2 that the spiked recoveries of the four oxidative stress biomarkers involved in the present invention at various spiked concentrations are at a relatively high level, and the relative standard deviations are also at a very low level. It can be seen that the results obtained by the analytical method of the present invention have excellent accuracy and reliability.

[0059] Example 2

[0060] This example 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 bistyrosine into a 10 mL volumetric flask, dilute to volume with methanol, and obtain a single standard stock solution of bistyrosine with a concentration of 100 μg / mL. In the same way, obtain single standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenalthiouric acid with a concentration of 100 μg / mL. Transfer 1 mL of each single standard stock solution of the above four oxidative stress biomarkers to a 25 mL volumetric flask, dilute to volume with methanol, and obtain a mixed standard stock solution of 4 μg / mL (the concentration of the four oxidative stress biomarkers is 4 μg / mL).

[0063] Weigh 1 mg of bis-tyrosine 13 C 12 Add to a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL bis-tyrosine 13 C 12 Single-label stock solutions were prepared using the same method to obtain 8-hydroxyguanosine- 13 C- 15 N2, 8-hydroxydeoxyguanosine- 13 C- 15 Single standard stock solutions of N2 and 4-hydroxynonenal mercaptouric acid-d3. Pipette 1 mL of each of the four internal standard compounds into a 25 mL volumetric flask, dilute to volume with methanol, and obtain a 4 μg / mL mixed isotope internal standard stock solution (the concentration of the four internal standard compounds is 4 μg / mL).

[0064] (2) Sample selection

[0065] Six urine samples from different sources were collected and each sample was divided into six parallel samples.

[0066] Thirty-six urine samples were subsequently subjected to the same pretreatment and solid phase extraction methods.

[0067] (3) Sample pretreatment

[0068] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (the volume fraction of formic acid 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 a diluted solution.

[0069] (4) Solid phase extraction

[0070] The Water Oasis HLB solid phase extraction column was activated with 3 mL of methanol and 3 mL of water, and then the dilution was transferred to the Water Oasis HLB solid phase extraction column. After passing through the column by natural gravity, the column was air-dried for 2 minutes. 1 mL of a 2% methanol aqueous solution was used for the first elution to obtain a first eluent including dityrosine. The column was air-dried for 1 minute, and then 1 mL of methanol was used for the second elution to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid. The first eluent and the second eluent were mixed and nitrogen blown to 1 mL to obtain a 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 urine sources.

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

[0074] Referring to the method of step (5) of Example 1, urine matrix standard curves of urine matrix samples from six different sources were established, and a solution matrix standard curve was established with the initial mobile phase. The difference was that the horizontal axis was six concentration points of 1, 5, 10, 20, 50 and 100 ng / mL.

[0075] The LC-MS / MS detection conditions include: using a Waters Acquity UPLC HSS T3 chromatographic column (100 mm×2.1 mm, 1.8 μm); using a 0.04-0.06% volume fraction of formic acid aqueous solution as mobile phase A, and using methanol as mobile phase B; the full injection time is 12 min; the elution gradient is 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, keep 0% A, 8.5-5.6 min, 0% A-99% A, 8.6-12min, keep 99% A; mobile phase flow rate is 0.25mL / min; injection volume is 10μL; column oven temperature is 40℃; sample chamber temperature is 8℃; ion source is electrospray ionization source; ionization mode is positive ion mode and negative ion mode; scanning mode is multiple ion reaction detection mode; capillary voltage is 2.46kV; ion source temperature is 150℃; desolvation temperature is 500℃. The mass spectral information of ion pairs of 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 by step (6). For each oxidative stress biomarker, the ratio of the slope of the standard curve after 1 / x fitting of the six urine matrix standard curves to the slope of one solution matrix standard curve was used as the matrix effect for evaluation. The matrix effects of the four oxidative stress biomarkers all showed 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 Urine sample 5 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 Y 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 bistyrosine into a 10 mL volumetric flask, dilute to volume with methanol, and obtain a single standard stock solution of bistyrosine with a concentration of 100 μg / mL. In the same way, obtain single standard stock solutions of 8-hydroxyguanosine, 8-hydroxydeoxyguanosine, and 4-hydroxynonenalthiouric acid with a concentration of 100 μg / mL. Transfer 1 mL of each single standard stock solution of the above four oxidative stress biomarkers to a 25 mL volumetric flask, dilute to volume with methanol, and obtain a mixed standard stock solution of 4 μg / mL (the concentration of the four oxidative stress biomarkers is 4 μg / mL).

[0087] Weigh 1 mg of bis-tyrosine 13 C 12 Add to a 10 mL volumetric flask and dilute to volume with methanol to obtain a 100 μg / mL bis-tyrosine 13 C 12 Single-label stock solutions were prepared using the same method to obtain 8-hydroxyguanosine- 13 C- 15 N2, 8-hydroxydeoxyguanosine- 13 C- 15 Single standard stock solutions of N2 and 4-hydroxynonenal mercaptouric acid-d3. Pipette 1 mL of each of the four internal standard compounds into a 25 mL volumetric flask, dilute to volume with methanol, and obtain a 4 μg / mL mixed isotope internal standard stock solution (the concentration of the four internal standard compounds is 4 μg / mL).

[0088] (3) Sample pretreatment

[0089] 40 urine samples from different sources were selected for subsequent processing.

[0090] Take 200 μL of urine sample, add 800 μL of formic acid aqueous solution (the volume fraction of formic acid 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 a diluted solution.

[0091] (4) Solid phase extraction

[0092] The Water Oasis HLB solid phase extraction column was activated with 3 mL of methanol and 3 mL of water, and then the dilution was transferred to the Water Oasis HLB solid phase extraction column. After passing through the column by natural gravity, the column was air-dried for 2 minutes. 1 mL of a 2% methanol aqueous solution was used for the first elution to obtain a first eluent including dityrosine. The column was air-dried for 1 minute, and then 1 mL of methanol was used for the second elution to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid. The first eluent and the second eluent were mixed and blown to 1 mL with nitrogen to obtain a sample to be tested.

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

[0094] The mixed standard stock solution was diluted with methanol to mixed standard working solutions with mass concentrations of 0.01, 0.1, 1, 5, 10, 20, 50 and 100 ng / mL, respectively, and LC-MS / MS detection was performed, and the samples to be tested were also tested by LC-MS / MS detection; the ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard working solution to the peak area of ​​each corresponding internal standard compound in the sample to be tested 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 to establish standard curves of the four oxidative stress biomarkers, and the standard curves were fitted using 1 / x for curve weighting and then quantification.

[0095] The LC-MS / MS detection conditions include: using a Waters Acquity UPLC HSS T3 chromatographic column (100 mm×2.1 mm, 1.8 μm); using a 0.04-0.06% volume fraction of formic acid aqueous solution as mobile phase A, and using methanol as mobile phase B; the full injection time is 12 min; the elution gradient is 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, keep 0% A, 8.5-5.6 min, 0% A-99% A, 8.6-12min, maintain 99% A; mobile phase flow rate is 0.25mL / min; injection volume is 10μL; column oven temperature is 40℃; sample chamber temperature is 8℃; ion source is electrospray ionization source; ionization mode is positive ion mode and negative ion mode; scanning mode is multiple ion reaction detection mode; capillary voltage is 2.46kV; ion source temperature is 150℃; desolvation temperature is 500℃.

[0096] (6) Measurement results

[0097] Four oxidative stress biomarkers, namely dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, were all detectable with a detection rate of 100%. The detection concentrations ranged from 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. The median values ​​of the detection concentrations were 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] The method of Example 1 is different from that in step (3), the formic acid aqueous solution is replaced by pure water. The final spiked recovery rates and relative standard deviations at various spiked concentrations are shown in Table 5.

[0102] Table 5

[0103]

[0104] It can be seen from Table 5 that the spiked recovery of bistyrosine at each spiked concentration is significantly reduced, indicating that the accuracy and reliability of this analytical method for bistyrosine are not high.

[0105] Comparative Example 2

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

[0107] Table 6

[0108]

[0109] It can be seen from Table 5 that the spiked recovery of bistyrosine at each spiked concentration is significantly reduced, indicating that the accuracy and reliability of this analytical method for bistyrosine are not high.

[0110] Comparative Example 3

[0111] The method of Example 1 is different in that in step (4), the methanol aqueous solution with a volume fraction of 2% is replaced by the methanol aqueous solution with a volume fraction of 4%. The final spiked recovery rates and relative standard deviations at various spiked concentrations are shown in Table 7.

[0112] Table 7

[0113]

[0114]

[0115] It can be seen from Table 5 that when a 4% methanol aqueous solution is used for the first elution, the recovery rate of bistyrosine added is low and the relative standard deviation is also high, indicating that the accuracy and reliability of the analytical method have deteriorated.

[0116] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An analytical method for detecting oxidative stress biomarkers in urine, characterized in that: The method can simultaneously detect dityrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenal mercaptouric acid, and comprises the following steps: (1) preparing a mixed standard stock solution and a mixed isotope internal standard stock solution, wherein the mixed standard stock solution includes four oxidative stress biomarkers: bistyrosine, 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the mixed isotope internal standard stock solution includes bistyrosine- 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: adding a formic acid aqueous solution and a mixed isotope internal standard working solution to a urine sample to obtain a diluted solution, wherein the mixed isotope internal standard working solution is the mixed isotope internal standard stock solution diluted with water; (3) Solid phase extraction treatment: the diluted solution is transferred to an activated Water Oasis HLB solid phase extraction column, and after passing through the column by natural gravity, a first elution is performed with a methanol aqueous solution having a volume fraction of 1-3% to obtain a first eluent including dityrosine, and then a second elution is performed with methanol to obtain a second eluent including 8-hydroxyguanosine, 8-hydroxydeoxyguanosine and 4-hydroxynonenalthiouric acid, and the first eluent and the second eluent are mixed and blown with nitrogen to the injection volume to obtain a sample to be tested; (4) LC-MS / MS detection: diluting the mixed standard stock solution into mixed standard working solutions of different mass concentrations and performing LC-MS / MS detection, and performing LC-MS / MS detection on the sample to be tested; The ratio of the peak area of ​​each oxidative stress biomarker in the mixed standard solution to the peak area of ​​each corresponding internal standard compound in the sample to be tested is used as the ordinate, and the mass concentration of each oxidative stress biomarker in the mixed standard solution is used as the abscissa to establish a standard curve of four oxidative stress biomarkers, and the standard curve is fitted by using 1 / x for curve weighting and then quantification; The conditions of the LC-MS / MS detection include: using a Waters Acquity UPLC HSS T3 chromatographic column; The mobile phase A was a formic acid aqueous solution with a volume fraction of 0.04-0.06%, and the mobile phase B was methanol; the full 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, maintaining 0% A, 8.5-5.6 min, 0% A-99% A, 8.6-12 min, maintaining 99% A.

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, preferably 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 μg / mL, preferably 3.5-4.5 μg / mL; And / or, the solvent of the mixed standard stock solution and the mixed isotope internal standard stock solution is methanol.

3. The method according to claim 1 or 2, wherein: 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%.

4. The method according to claim 3, wherein: Relative to 1 mL of the urine sample, the amount of the formic acid aqueous solution is 3-5 mL, preferably 3.5-4.5 mL.

5. The method according to any one of claims 1 to 4, 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, preferably 0.9-1.1 μg / mL.

6. The method according to claim 5, wherein: Relative to 1 mL of the urine sample, the amount of the mixed isotope internal standard solution is 0.04-0.06 mL, preferably 0.045-0.055 mL.

7. The method according to any one of claims 1 to 6, 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; Preferably, the Water Oasis HLB solid phase extraction column is activated with 1-4 mL of methanol and 1-4 mL of water.

8. The method according to any one of claims 1 to 7, wherein: In step (3), the amount of methanol aqueous solution used in the first elution is at least 1 mL, preferably 1-3 mL; and / or, the amount of methanol used in the second elution is at least 1 mL, preferably 1-3 mL; And / or, the injection volume is 0.5-2 mL, preferably 0.8-1.2 mL.

9. The method according to any one of claims 1 to 8, wherein: In step (4), 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.

10. The method according to any one of claims 1 to 9, wherein: In step (4), the conditions for LC-MS / MS detection also include: a mobile phase flow rate of 0.1-0.3 mL / min, preferably 0.2-0.3 mL / min; an injection volume of 6-12 μL, preferably 8-10 μL; a column oven temperature of 30-50°C, preferably 35-45°C; a sample chamber temperature of 4-12°C, preferably 6-10°C; an ion source of electrospray ionization; ionization modes of positive ion mode and negative ion mode; a scanning mode of multiple ion reaction detection mode; a capillary voltage of 2.3-2.6 kV, preferably 2.4-2.5 kV; an ion source temperature of 120-180°C, preferably 140-160°C; and a desolvation temperature of 400-600°C, preferably 450-550°C.

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