A method for simultaneously determining 16 oxidative stress biomarkers in urine based on UPLC-MS / MS

Abstract

This invention relates to the field of chemical analysis and biomedical detection technology, and discloses a method for the simultaneous determination of 16 oxidative stress biomarkers in urine based on UPLC-MS / MS. The method includes: sample pretreatment: adding an isotope internal standard mixture and diluent to a urine sample, centrifuging at low temperature, and using the supernatant as the test solution; UPLC-MS / MS determination: liquid chromatography analysis conditions: a reverse-phase column, mobile phase A being a 0.01%-0.1% (v / v) formic acid aqueous solution, and mobile phase B being a methanol-acetonitrile mixture containing formic acid, where the formic acid volume fraction in mobile phase B is 0.01%-0.1%, and the methanol to acetonitrile volume ratio is 1:3 to 1:5; mass spectrometry analysis conditions: using an electrospray ionization source, performing segmented multiple reaction monitoring (MRM) scanning in positive and negative ion switching modes. This invention offers a simple and rapid pretreatment, low sample consumption, high detection throughput, and good sensitivity, enabling the simultaneous determination of 16 biomarkers with different physicochemical properties within 15 minutes.

Description

Technical Field

[0001] This invention relates to the fields of chemical analysis and biomedical detection technology, specifically to a method for simultaneously determining 16 oxidative stress biomarkers in urine based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Background Technology

[0002] Oxidative stress refers to an imbalance between oxidation and antioxidation in the body, leading to inflammatory infiltration of neutrophils, increased protease secretion, and the production of numerous oxidative stress biomarkers. Oxidative stress is a significant factor contributing to aging and various diseases (such as cancer, cardiovascular disease, diabetes, and neurodegenerative diseases). During oxidative stress, reactive oxygen species attack biological macromolecules (such as DNA, lipids, and proteins), producing a series of stable metabolites, namely oxidative stress biomarkers.

[0003] Currently, the detection of oxidative stress biomarkers mainly relies on enzyme-linked immunosorbent assay (ELISA) and chromatography-mass spectrometry (LC-MS). While ELISA is simple to operate, it has poor specificity and typically only detects a single indicator, making it difficult to comprehensively assess the body's oxidative stress status. Existing LC-MS methods, although highly sensitive, often suffer from the following problems: 1) Complex pretreatment, often requiring solid-phase extraction, which is time-consuming, labor-intensive, and consumes a large amount of solvent; 2) Low throughput, making it difficult to simultaneously detect different types of biomarkers with vastly different chemical properties (such as polarity) in a single injection; 3) Insufficient sensitivity in some methods, making it difficult to detect trace amounts of biomarkers in urine; 4) For biomarkers with very similar molecular weights, such as isomers, effective simultaneous separation and determination in a single injection is not possible. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and develop a high-throughput detection method that is simple to preprocess, highly sensitive, and can simultaneously cover nucleic acid oxidation, lipid peroxidation, protein oxidation and nitration damage products.

[0005] This invention provides a method for the simultaneous determination of 16 oxidative stress biomarkers in urine based on UPLC-MS / MS, the method comprising the following steps: (S1) Sample pretreatment: Take a urine sample, add an isotope internal standard mixture and diluent, let it stand at 2-8℃ for 5-20 minutes, centrifuge at 2-8℃ and take the supernatant as the test solution. The diluent is a mixed solution of methanol and water with a volume ratio of 1:1. (S2) UPLC-MS / MS determination: The liquid chromatography analysis conditions are as follows: the chromatographic column is a C18 reverse-phase column, mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%-0.1%, mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in mobile phase B is 0.01%-0.1%, and the volume ratio of methanol to acetonitrile is 1:3 to 1:5; The mass spectrometry analysis conditions are as follows: an electrospray ionization source is used, and segmented multiple reaction monitoring is performed in positive and negative ion switching modes; The 16 oxidative stress biomarkers are 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α ; The isotope internal standard mixture includes 8-isoprostaglandin F. 2α -d4, 4-hydroxy-2-nonenal mercaptouric acid -d3, 4-hydroxy-2-nonenal glutathione -d3, 3-bromotyrosine 13 C9, 15 N,L,L-dityrosine- 13 C 12 3-Nitro-L-Tyrosine 13 C6 and 8-hydroxy-2'-deoxyguanosine- 15 N5.

[0006] Preferably, in step (S1), the mixture is left to stand at 4°C for 10 minutes.

[0007] Preferably, step (S1) includes: adding an equal volume of the diluent and the isotope internal standard mixture to the urine sample to obtain a test solution before centrifugation, such that the concentration of the isotope internal standard in the test solution before centrifugation is 20 µg / L; letting it stand at 4°C for 10 minutes, and then centrifuging at 4°C and 1000 rpm for 10 minutes, and taking the supernatant as the test solution, wherein the solvent of the isotope internal standard mixture is methanol.

[0008] Preferably, in step (S2), the chromatographic column is a Poroshell 120 Aq-C18, the volume fraction of formic acid in mobile phase A is 0.01%, the volume fraction of formic acid in mobile phase B is 0.01%, and the volume ratio of methanol to acetonitrile in mobile phase B is 1:4.

[0009] Preferably, in step (S2), the column temperature for liquid chromatography separation is 35-45℃; the flow rate is 0.3 mL / min; the injection volume is 1 μL; and the elution program is gradient elution, wherein the gradient elution is as follows: 0-0.5 min, 5% (v / v) mobile phase B; 0.5-8 min, 5%-95% (v / v) mobile phase B; 8-12 min, 95% (v / v) mobile phase B; 12-12.5 min, 95%-5% (v / v) mobile phase B; 12.5-15 min, 5% (v / v) mobile phase B.

[0010] Preferably, in step (S2), the column temperature for liquid chromatography separation is 40°C.

[0011] Preferably, in step (S2), the switching between positive and negative ion modes includes: detecting 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, and 4-hydroxy-2-nonenal glutathione in positive ion mode; and detecting 4-hydroxy-2-nonenal mercaptouric acid and 11β-prostaglandin F in negative ion mode. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α .

[0012] Preferably, in step (S2), the mass spectrometry conditions are as follows: capillary voltage is 1 kV in positive ion mode and 3 kV in negative ion mode; ion source temperature is 150℃; desolvation gas temperature is 550℃; cone gas flow rate is 150 L / h; desolvation gas flow rate is 1000 L / h; cone voltage is 8-40 V; and collision energy is 10-40 eV.

[0013] More preferably, the internal standards, retention times, quantitative ion pairs and their corresponding cone voltages and collision energies for the 16 oxidative stress biomarkers are as follows: .

[0014] More preferably, the residence time and scanning window corresponding to the 16 oxidative stress biomarkers are as follows: .

[0015] Preferably, in step (S2), the following substances are used: 3-nitro-L-tyrosine, 3-bromotyrosine, 8-hydroxy-2′-deoxyguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α Using the isotope internal standard method, a linear regression analysis was performed with the peak area ratio of the target analyte to the internal standard as the ordinate and the target analyte mass concentration as the abscissa to construct the first set of standard curves. For 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 5-methylcytidine, 8-hydroxyguanosine, and 8-nitroguanosine, the external standard method was used, with the peak area of ​​the target analyte as the ordinate and the target analyte mass concentration as the abscissa to construct the second set of standard curves. The content of the oxidative stress biomarkers in the test solution was calculated based on the first and second set of standard curves.

[0016] More preferably, a series of standard working solutions containing the 16 oxidative stress biomarkers of different concentrations are prepared using a stepwise dilution method and artificial urine. The isotope internal standard mixture and the artificial urine are added to the standard working solutions to obtain a standard curve working solution, such that the concentration of the isotope internal standard in the standard curve working solution is the same as the concentration of the isotope internal standard in the test solution before centrifugation. The standard curve working solution is used to construct the first set and the second set of standard curves.

[0017] More preferably, a mixed standard intermediate solution containing the 16 oxidative stress biomarker standards is prepared using methanol, and the mixed standard intermediate solution is added to the artificial urine to prepare the standard working solution.

[0018] This invention also provides a kit for the simultaneous determination of 16 oxidative stress biomarkers in urine based on UPLC-MS / MS. The kit includes artificial urine, an isotope internal standard mixture, a diluent, a mixed standard solution of oxidative stress biomarkers, mobile phase A, and mobile phase B; wherein the isotope internal standard mixture includes 8-isoprostaglandin F. 2α -d4, 4-hydroxy-2-nonenal mercaptouric acid -d3, 4-hydroxy-2-nonenal glutathione -d3, 3-bromotyrosine 13 C9, 15 N,L,L-dityrosine- 13 C 12 3-Nitro-L-Tyrosine 13 C6 and 8-hydroxy-2'-deoxyguanosine- 15N5, the diluent is a 1:1 volume ratio of methanol to water, and the mixed standard solution of oxidative stress biomarkers includes 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α The mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%-0.1%, and the mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in the mobile phase B is 0.01%-0.1%, and the volume ratio of methanol to acetonitrile is 1:3 to 1:5.

[0019] The beneficial effects of this invention are as follows: The design logic of chromatographic separation systems differs depending on the chemical complexity and polarity range of the analytes. For analytes with significant molecular weight differences and narrow polarity ranges, differentiation is easily achieved in conventional chromatographic systems. However, for analytes with large polarity ranges, simultaneously retaining and separating compounds with such a wide polarity range in a single chromatographic system is extremely challenging and of great significance for comprehensively assessing oxidative stress levels and large-scale population screening. This invention, by optimizing sample pretreatment processes, chromatographic separation conditions, and mass spectrometry parameters, achieves high-sensitivity, high-throughput, and high-accuracy qualitative and quantitative analysis of 16 different oxidative stress biomarkers (including nucleosides, prostaglandins, and tyrosine derivatives) in urine within a 15-minute chromatographic detection cycle, significantly improving detection efficiency. Method validation results show that this method has an extremely low limit of detection (MDL as low as 0.05 μg / L) and good linearity (R² > 0.999). At different spiking levels, the recovery rate and precision of each target analyte meet the requirements for biological sample analysis. The matrix effect has little impact, and it can accurately determine trace biomarkers in complex urine matrices.

[0020] Furthermore, the technical solution of this invention has achieved several significant systemic advantages in actual clinical and epidemiological applications: Ultimate Pretreatment Efficiency and High Throughput: This invention simplifies pretreatment to "direct dilution + low-temperature centrifugation" through sophisticated chemical environment control, requiring only about 20 minutes in total. Combined with a single chromatographic run cycle of only 15 minutes, one instrument can process approximately 40 clinical samples per day, achieving an order-of-magnitude (approximately 50-fold) leap in detection efficiency. This provides irreplaceable technical support for large-scale epidemiological cohort studies. Breakthrough in meeting the need for ultra-small sample volumes: The synergistic combination of the pretreatment and high-sensitivity mass spectrometry methods in this invention reduces the amount of urine sample required for a single test to an extremely low 200 μL. This technological achievement has significant practical implications for biomonitoring of clinically challenging special populations (such as infants and children without the ability to urinate independently, and critically ill patients requiring multi-omics joint monitoring). Unprecedented Detection Dimensions and Coverage: Current monitoring methods in this field typically only perform fragmented detection of 1-2 oxidative stress pathways (e.g., detecting only 8-OHdG and 8-HdG). This invention, through a systematic reconstruction of the chromatography, mass spectrometry, and pretreatment systems, is the first in the world to achieve a single 15-minute injection while comprehensively covering 16 core biomarkers across five major oxidative damage pathways: DNA, RNA, proteins (including oxidative and nitration / halogenation damage), and lipids. This multi-dimensional, panoramic detection provides data value far exceeding the simple summation of existing single-indicator detection methods, offering a novel methodological platform for systematically assessing human oxidative stress status. Attached Figure Description

[0021] Figure 1 UPLC-MS / MS extraction ion chromatograms for 16 oxidative stress biomarker standards; Figure 2 This is a standard curve of oxidative stress biomarkers in Example 1; Figure 3 This represents the average level of oxidative stress biomarkers in the urine of healthy individuals in Example 1; Figure 4 Example 1: Evaluation of the matrix effect of artificial urine based on the slope ratio method; Figure 5 This is a bar chart showing the recovery rates of the method of the present invention at different spiking concentrations; Figure 6 Comparison of signal intensities of 16 oxidative stress biomarkers when the volume fraction of formic acid in the mobile phase is 0.01%, 0.05%, and 0.1%; Figure 7 To compare the signal-to-noise ratio of oxidative stress biomarkers in the segmented multiple response monitoring (MRM) scanning mode and the full scan mode in Example 1; Figure 8To compare the co-elution phenomenon of 8-HG and 8-HdG on the HSS T3 column in Comparative Example 2; Figure 9 For comparison, the analytical chromatogram based on the HILIC column in Example 3; Figure 10 For comparison example 4, the chromatographic resolution of prostaglandin isomers was compared using methanol-acetonitrile at different volume ratios. Figure 11 For comparison example 5, the chromatographic detection results of HNE-GSH were obtained when methanol:acetonitrile (1:1) was used as mobile phase B; Figure 12 To compare and evaluate the effectiveness of different packed SPE columns in the pretreatment of oxidative stress biomarkers in Example 6. Detailed Implementation

[0022] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. Unless otherwise specified, the methods used in the present invention are conventional methods; the reagents used, unless otherwise specified, are conventional commercially available products. The standards used in the present invention, their abbreviations, CAS numbers, and sources are shown in Table 1 below.

[0023] Table 1

[0024] The internal standards used in this invention, their abbreviations, and sources are shown in Table 2 below: Table 2

[0025] The UPLC-MS / MS used in the embodiments of the present invention is an ultra-high performance liquid chromatography (PREMIER, WATERS Corporation, USA)-triple quadrupole (TQ Absolute, WATERS Corporation, USA) column and a Poroshell 120 Aq-C18 column (2.1×150 mm, 1.9 µm, Agilent Technologies, USA).

[0026] This invention provides a method for the simultaneous determination of 16 oxidative stress biomarkers in urine based on UPLC-MS / MS, wherein the 16 oxidative stress biomarkers are 8-HG, 8-NG, CY, NY, m 5 C, BY, 8-OHdG, 8-HdG, 8-NdG, diy, HNE-GSH, HNE-MA, 11β-PGF 2α 5,6-trans-PGF 2α 8-PGF2α and 15-PGF 2α The aforementioned 16 oxidative stress biomarkers span five major oxidative damage pathways, with polarities ranging across multiple orders of magnitude (from highly polar nucleic acid bases like 8-HG to moderately polar prostaglandins like 15-PGF). 2α Simultaneously retaining and separating compounds with such a wide polarity range in a single chromatographic system is an extremely challenging task. This invention achieves highly sensitive and accurate qualitative and quantitative detection of the aforementioned 16 oxidative stress biomarkers by systematically reconstructing sample pretreatment, chromatography, and mass spectrometry through a single 15-minute injection.

[0027] The sample pretreatment steps of this invention include: taking a urine sample, adding an isotope internal standard mixture and a diluent, allowing it to stand at low temperature and centrifuging, and then taking the supernatant as the test solution. The diluent is a mixture of methanol and water in a volume ratio of 1:1. The isotope internal standard mixture includes 8-PGF. 2α -d4、HNE-MA-d3、HNE-GSH-d3、BY- 13 C9、diy- 13 C 12 NY- 13 C6 and 8-OHdG- 15 N5.

[0028] The temperature and time for settling and centrifuging urine samples after adding the isotope internal standard mixture and diluent can be set according to the temperature and time windows conventionally used for urine sample processing in the art, without particular limitations. For example, settling at 2-8°C for 5-20 minutes, followed by centrifugation at 2-8°C, more preferably settling at 4°C for 10 minutes, and then centrifuging at 4°C. 2-8°C can effectively inhibit the enzymatic degradation and non-enzymatic isomerization of the target substance, while avoiding salt precipitation, and 5-20 minutes of settling is sufficient to ensure that the isotope internal standard and the matrix are fully equilibrated.

[0029] In a preferred embodiment of the present invention, the specific operation of the sample pretreatment is as follows: an equal volume of the diluent and the isotope internal standard mixture is added to the urine sample to obtain the test solution before centrifugation. The concentration of the isotope internal standard in the test solution before centrifugation can be determined by those skilled in the art based on common knowledge, as long as the concentration of the isotope internal standard in the test solution before centrifugation is on the same order of magnitude as the concentration of the target sample; there are no other special limitations. Preferably, the concentration of the isotope internal standard in the test solution before centrifugation is 5µg / L-100µg / L, more preferably 20µg / L. At the above concentration, the accuracy of the method of the present invention is higher. The test solution before centrifugation is allowed to stand at 4°C for 10 minutes, and then centrifuged at 4°C and 1000rpm for 10 minutes. The supernatant is taken as the test solution, wherein the solvent of the isotope internal standard mixture is methanol.

[0030] The 16 oxidative stress biomarkers of this invention include base modifications such as 8-HG and PGF. 2α Isomer (8-PGF) 2α 11β-PGF 2α 5,6-trans-PGF 2α 15-PGF 2α Prolonged incubation poses a high risk of degradation or isomerization. To ensure the accuracy of the detection results, this invention adopts a pretreatment strategy of "dilution-addition of internal standard-low temperature centrifugation," shortening the single-sample processing time to less than 30 minutes, significantly reducing sample loss compared to traditional solid-phase extraction (SPE) or liquid-liquid extraction (LLE). This invention eliminates complex extraction or precipitation steps, employing a combination strategy of "dilution of urine sample + micro-injection" to ensure 100% retention of all biomarkers while perfectly controlling the matrix effect (ME) within a safe range of -19.45% to 4.52% (|ME|<20%). Furthermore, this invention uses methanol:water (1 / 1, v / v) as a diluent, a "compatibility with both solubility and chromatography" system specifically designed for 16 broadly polar oxidative stress biomarkers. For the 16 target compounds with logarithmic partition coefficients (logP) ranging from -1.1 to 2.6, pure water or pure organic solvents would cause a decrease in the solubility of some compounds. Methanol:water (1 / 1, v / v) not only provides a compromise in terms of solubility, but more importantly, it ensures that the volume percentage of organic solvent in the pretreated analyte is approximately 26%. Combined with subsequent micro-injection (1 µL), methanol:water (1 / 1, v / v) achieves perfect compatibility with the initial mobile phase (95% (v / v) mobile phase A) in subsequent chromatographic detection, avoiding peak broadening caused by solvent effects.

[0031] The UPLC-MS / MS determination of the present invention includes: The test solution was injected into a UPLC-MS / MS instrument, and the eluent was obtained by liquid chromatography separation. The chromatographic analysis conditions were as follows: a C18 reversed-phase column, preferably a Poroshell 120 Aq-C18 column, for analysis of 8-HG and m... 5 Biomarkers with high polarity, such as C, exhibit weaker retention (earlier retention time) on reversed-phase chromatography. Using a water-resistant Poroshell 120 Aq-C18 column can achieve better peak shape and reproducibility for these biomarkers under high aqueous phase conditions.

[0032] Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%-0.1%, wherein the volume fraction of formic acid is preferably 0.01%; mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in mobile phase B is 0.01%-0.1%, preferably 0.01%, and the volume ratio of methanol to acetonitrile is 1:3 to 1:5, preferably 1:4.

[0033] In a preferred embodiment of the present invention, the column temperature for liquid chromatography separation is 35-45℃, more preferably 40℃; the flow rate is 0.3 mL / min; the injection volume is 1 μL; and the elution program employs gradient elution, wherein the gradient elution is as follows: 0-0.5 min, 5% (v / v) mobile phase B; 0.5-8 min, 5%-95% (v / v) mobile phase B; 8-12 min, 95% (v / v) mobile phase B; 12-12.5 min, 95%-5% (v / v) mobile phase B; 12.5-15 min, 5% (v / v) mobile phase B. Through specific chromatographic gradient optimization, the present invention can achieve the simultaneous detection of structurally highly similar isoprostaglandin isomers (8-PGF) while ensuring the simultaneous detection of other markers with different polarities. 2α 15-PGF 2α Rapid baseline separation (e.g.) avoids quantitative inaccuracies caused by isomer interference.

[0034] The effluent was analyzed by mass spectrometry to collect signals of the 16 oxidative stress biomarkers. The mass spectrometry analysis conditions were as follows: electrospray ionization source was used, and segmented multiple reaction monitoring (ScheduledMRM) was performed in positive and negative ion switching mode. Scheduled MRM sets the monitoring window based on the retention time of each target and collects the corresponding ion pairs only within the expected elution period, which effectively prolongs the residence time and improves the signal-to-noise ratio.

[0035] In a preferred embodiment of the present invention, the switching between positive and negative ion modes includes: detecting 8-HG, 8-NG, CY, NY, and m in positive ion mode. 5C, BY, 8-OHdG, 8-HdG, 8-NdG, diy, and HNE-GSH; HNE-MA and 11β-PGF were detected in negative ion mode. 2α 5,6-trans-PGF 2α 8-PGF 2α and 15-PGF 2α .

[0036] In a preferred embodiment of the present invention, the mass spectrometry conditions are as follows: capillary voltage is 1 kV in positive ion mode and 3 kV in negative ion mode; ion source temperature is 150°C; desolvation gas temperature is 550°C; cone gas flow rate is 150 L / h; desolvation gas flow rate is 1000 L / h; the cone voltage is 8-40 V; and the collision energy is 10-40 eV. Preferably, the internal standards, retention times, quantitative ion pairs and their corresponding cone voltages and collision energies for 16 oxidative stress biomarkers, and the qualitative ion pairs and their corresponding cone voltages and collision energies are shown in Table 3 below: Table 3 .

[0037] More preferably, the residence time and scanning window corresponding to the 16 oxidative stress biomarkers are shown in Table 4 below: Table 4

[0038] The method of the present invention further includes using isotope internal standard method and / or external standard method to calculate the content of each oxidative stress biomarker in the urine sample to be tested according to a standard curve. In a preferred embodiment of the present invention, for NY, BY, 8-OHdG, diy, HNE-GSH, HNE-MA, 11β-PGF 2α 5,6-trans-PGF 2α 8-PGF 2α and 15-PGF 2α It has corresponding isotope internal standards, including four PGFs. 2α Isomers share 8-PGF 2α -d4 was used as an analogue to replace the internal standard. Using the isotopic internal standard method, linear regression analysis was performed with the peak area ratio of the target analyte to the internal standard as the ordinate and the target analyte mass concentration as the abscissa to construct the first set of standard curves. For 8-HG, 8-NG, CY, and m... 5C, 8-HdG, and 8-NdG are analyzed using the external standard method, with the peak area of ​​the target analyte as the ordinate and the mass concentration of the target analyte as the abscissa, to construct a second set of standard curves. The content of the oxidative stress biomarkers in the test solution is calculated based on the first and second set of standard curves. Preferably, a series of standard working solutions containing the 16 oxidative stress biomarkers of different concentrations are prepared using a stepwise dilution method with artificial urine. The isotope internal standard mixture and the artificial urine are added to the standard working solutions to obtain a standard curve working solution, such that the concentration of the isotope internal standard in the standard curve working solution is the same as the concentration of the isotope internal standard in the test solution before centrifugation. This standard curve working solution is used to construct the first and second sets of standard curves. Specifically, a mixed standard intermediate solution containing all 16 oxidative stress biomarker standards is prepared using methanol, and the mixed standard intermediate solution is added to the artificial urine to prepare the standard working solution. Preferably, when constructing the standard curve, the mass concentration gradient of the HNE-GSH standard is 10, 15, 20, 25, 30 and 50 µg / L; the mass concentration gradient of the remaining 15 oxidative stress biomarker standards is 0.5, 1, 2.5, 5, 10, 15, 20, 25, 30 and 50 µg / L.

[0039] Baseline separation of isomers is a core technical challenge in this field, and the four PGFs in this invention... 2α Isomers (prostaglandin isomers) have the same molecular weight (354) and identical MRM quantitative ion pairs (353.0 > 193.0), making them indistinguishable by mass spectrometry alone. Baseline separation requires a highly selective chromatographic system. This invention innovatively introduces a methanol:acetonitrile mixed organic phase in a specific ratio as mobile phase B, where the volume ratio of methanol to acetonitrile is 1:3 to 1:5, preferably 1:4. Utilizing the special selectivity of acetonitrile for structurally similar isomers, baseline separation is successfully achieved while simultaneously meeting the peak requirements of lipid peroxidation markers such as HNE-GSH. Compared to existing technologies that typically require time-consuming separate analytical methods for prostaglandin substances, this method can complete high-throughput analysis of 16 markers, including the aforementioned isomers, within 15 minutes, significantly improving detection efficiency and avoiding quantitative inaccuracies caused by isomer interference. If the acetonitrile concentration in the organic phase is too high, highly polar markers such as nucleic acid bases like 8-HG will be largely lost due to co-precipitation with protein flocculents. Directly injecting an organic phase containing a high concentration of acetonitrile into a reversed-phase C18 chromatography system will result in the complete loss of polar compounds. Furthermore, both mobile phase A and mobile phase B in the liquid chromatography separation process contain formic acid. This invention has determined the optimal formic acid concentration through system optimization, thereby improving the signal response of each target analyte.

[0040] The method of this invention involves up to 24 MRM channels (16 target analytes + internal standard) during mass spectrometry detection. Using a conventional full-scan mode would severely compress the dwell time of each channel, leading to a sharp decrease in sensitivity. This invention employs scheduled MRM based on a retention time window, avoiding the aforementioned problems. This is not a simple parameter selection but a further optimized solution tailored to the vast compound library of this invention, resulting in a significant increase in the signal-to-noise ratio of most target analytes, ranging from 1.06 to 5.63 times.

[0041] Furthermore, the method of this invention employs internal standard correction for analytes with corresponding isotopic internal standards, but not for isomers without corresponding isotopic internal standards (such as 11β-PGF). 2α (etc.), using structurally similar 8-PGF 2α -d4 was used as an alternative internal standard for quantification. Other target analytes were quantified using an external standard method based on an artificial urine matrix, with quantification calculated based on chromatographic peak area. For analytes without a matching isotopic internal standard, this invention employs an external standard quantification strategy based on an artificial urine matrix. By constructing a standard curve in artificial urine with physicochemical properties highly similar to real urine, the matrix inhibition effect that early elution components may experience is effectively simulated and offset, thus achieving low-cost, high-accuracy quantitative analysis without relying on expensive isotopic internal standards. For DIY, which also has weak retention, an isotopic internal standard (DIY-) is introduced. 13 C 12 This effectively corrects for matrix effects near the dead time, ensuring quantitative accuracy. Qualitative identification is achieved by comparing multiple reaction monitoring (MRM) ion channels and retention times.

[0042] This invention also provides a kit for the method of this invention, the kit comprising artificial urine, an isotope internal standard mixture, a diluent, a mixed standard solution of oxidative stress biomarkers, mobile phase A, and mobile phase B; wherein the isotope internal standard mixture comprises 8-PGF 2α -d4、HNE-MA-d3、HNE-GSH-d3、BY- 13 C9、diy- 13 C 12 NY- 13 C6 and 8-OHdG- 15 N5, the diluent is a 1:1 volume ratio of methanol to water, and the mixed standard solution of oxidative stress biomarkers includes 8-HG, 8-NG, CY, NY, and m. 5C, BY, 8-OHdG, 8-HdG, 8-NdG, diy, HNE-GSH, HNE-MA, 11β-PGF 2α 5,6-trans-PGF 2α 8-PGF 2α and 15-PGF 2α The mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%-0.1%, and the mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in the mobile phase B is 0.01%-0.1%, and the volume ratio of methanol to acetonitrile is 1:3 to 1:5.

[0043] In a preferred embodiment of the present invention, the solvent for the isotope internal standard mixture and the working solution for the oxidative stress biomarker standard curve is methanol. In a preferred embodiment of the present invention, mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%, and mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in mobile phase B is 0.01%, and the volume ratio of methanol to acetonitrile is 1:4. Example 1

[0044] Construction of standard curve Sixteen oxidative stress biomarkers were prepared: 8-OHdG, NY, HNE-MA, 8-NG, BY, 8-HG, CY, and 11β-PGF. 2α , 8-NdG, 15-PGF 2α m 5 C, DIY, 8-PGF 2α A mixed standard intermediate solution of 8-HdG, 5-trans-PGF2α and HNE-GSH, and 8-PGF2α internal standard of 7 isotopes. 2α -d4、HNE-MA-d3、HNE-GSH-d3、BY- 13 C9、diy- 13 C 12 NY- 13 C6 and 8-OHdG- 15The internal standard mixture for N5 was prepared using methanol as the solvent. The concentrations of the individual standard stock solutions ranged from 0.1 to 5 mg / mL, while the concentrations of the mixed standard intermediate solution and the mixed internal standard intermediate solution were 5 µg / mL and 2 µg / mL, respectively. Specific preparation methods are shown in Tables 5 and 6 below. Specifically, the individual standard stock solutions of 16 standards were added to a 10 mL volumetric flask according to the sampling volumes in Table 5, and the volume was adjusted to obtain a 5 µg / mL mixed standard intermediate solution. The individual standard stock solutions of 7 internal standards were added to a container according to the sampling volumes in Table 6, followed by the addition of 2338 μL of methanol to obtain a 2 µg / mL mixed internal standard intermediate solution. This mixed internal standard intermediate solution was then further diluted with methanol to obtain an 800 µg / L internal standard mixture.

[0045] Table 5

[0046] Table 6

[0047] A series of standard working solutions of varying concentrations were prepared by serially diluting and mixing intermediate standard solutions with artificial urine. Then, 200 µL of each of these standard working solutions was taken and mixed thoroughly with 190 µL of artificial urine and 10 µL of internal standard solution (800 µg / L) to obtain a series of standard curve working solutions. When plotting the standard curves, the final concentration gradients of the 15 target compounds (excluding HNE-GSH) were 0.5, 1, 2.5, 5, 10, 15, 20, 25, 30, and 50 µg / L. For HNE-GSH, the final concentration gradients were 10, 15, 20, 25, 30, and 50 µg / L (0.5 µg / L, 1 µg / L, 2.5 µg / L, and 5 µg / L were below the limit of quantitation for HNE-GSH and were excluded from the standard curve calculation). The final concentration of the internal standard was constant at 20 µg / L.

[0048] The following “ UPLC-MS / MS detection using ultra-high performance liquid chromatography-tandem mass spectrometry The described method was used to analyze a series of standard curve working solutions at different concentrations. The UPLC-MS / MS extracted ion chromatograms of 16 oxidative stress biomarker standards are shown below. Figure 1 As shown. By Figure 1It is evident that this invention can simultaneously separate 16 target analytes in a single 15-minute chromatographic run, achieving high throughput and simultaneous measurement of multiple target analytes with a single injection. A linear regression analysis was performed with the peak area ratio of the target analyte to the internal standard as the ordinate (Y) and the target analyte mass concentration as the abscissa (X) to construct an internal standard curve. For target analytes without corresponding internal standards, a linear regression analysis was performed with the peak area of ​​the target analyte as the ordinate (Y) and the target analyte mass concentration as the abscissa (X) to construct an external standard curve. The standard curves are as follows: Figure 2 As shown, it can be seen that each target compound exhibits good linearity within the linear range (10-50 µg / L for HNE-GSH, and 0-50 µg / L for the other 15 compounds), with a correlation coefficient (R0). 2 All values ​​are greater than 0.999, as shown in Table 7 below: Table 7 .

[0049] Pretreatment of test samples Accurately transfer 200 μL of thawed urine samples from healthy individuals (the urine samples were collected from 10 healthy volunteers in the Jinan City, Shandong Province, morning midstream urine after informed consent, and stored at -80℃ for later use) into a vial, add 190 μL of diluent (methanol:water = 1:1, v / v) and 10 μL of isotope internal standard mixture (800 µg / L, final system concentration 20 µg / L), and vortex to mix. Incubate the mixture at 4℃ for 10 minutes, then centrifuge at 1000 rpm for 10 minutes at the same temperature. Accurately transfer 200 μL of the supernatant to an inner liner tube as the analyte for UPLC-MS / MS analysis.

[0050] UPLC-MS / MS detection using ultra-high performance liquid chromatography-tandem mass spectrometry The test solution was separated by liquid chromatography to obtain the eluent. The chromatographic separation conditions were as follows: the chromatographic column was a Poroshell 120 Aq-C18 (2.1×150 mm, 1.9 µm); the mobile phase consisted of an aqueous formic acid solution (mobile phase A) and a methanol-acetonitrile mixture containing formic acid (methanol:acetonitrile = 1:4, V / V) (mobile phase B), with the volume fraction of formic acid in both phases being 0.01%; the elution gradient was: 0–0.5 min, 5% (v / v) mobile phase B; 0.5–8 min, 5%–95% (v / v) mobile phase B; 8–12 min, 95% (v / v) mobile phase B; 12–12.5 min, 95%–5% (v / v) mobile phase B; 12.5–15 min, 5% (v / v) mobile phase B; the mobile phase flow rate was 0.3 mL / min; the column temperature was 40℃; and the injection volume was 1 mL / min. μL.

[0051] The effluent was detected by tandem mass spectrometry in segmented multiple reaction monitoring (MRM) mode. The mass spectrometry detection conditions were as follows: electrospray ionization (ESI) source with rapid positive / negative ion switching; capillary voltage of 1 kV in positive ion mode and 3 kV in negative ion mode; ion source temperature of 150 °C; desolvation gas temperature of 550 °C; cone gas flow rate of 150 L / Hr; and desolvation gas flow rate of 1000 L / Hr. Table 8 shows the internal standard, detection mode, retention time, quantitative ion pair and its corresponding cone voltage (V) and collision energy (EV), qualitative ion pair and its corresponding cone voltage (V) and collision energy (EV), residence time, and scanning window for each oxidative stress biomarker.

[0052] Table 8

[0053] Data acquisition and processing were performed using MassLynx software. The retention times of each target analyte were determined by injection analysis of standard solutions (e.g., ...). Figure 1 As shown in the figure, qualitative identification is achieved by comparing segmented multi-reaction monitoring ion channels and retention times, while quantitative calculation is based on chromatographic peak area. For analytes with corresponding isotope internal standards, the internal standard method is used for correction, while for analytes without corresponding isotope internal standards, the external standard method is used for correction. Finally, 13 of the 16 target oxidative stress biomarkers were successfully detected and quantified in actual healthy human urine samples.

[0054] For PGF 2α Isomers, HNE-MA, and DIY, etc., are based on isotopic internal standards (such as 8-PGF). 2α -d4, HNE-MA-d3, diy- 13 C 12 Concentration is calculated from the peak area after correction (etc.); for m 5 For substances such as C that lack a matched internal standard, absolute quantification was performed using a standard curve established by the external standard method. The results showed that the concentration levels of each biomarker in the tested samples were (mean ± standard deviation): m 5 The highest concentration was C, at 92.43±35.83 μg / L; the concentration of DIY was 22.64±13.77 μg / L; the concentration of HNE-MA was 18.38±27.82 μg / L; the concentration of 8-NG was 15.79±23.50 μg / L; the concentrations of 8-OHdG, 8-NdG, and 8-HdG were 6.12±4.50 μg / L, 6.03±3.37 μg / L, and 5.32±2.75 μg / L, respectively; the concentrations of 8-HG, CY, and BY were 3.18±4.56 μg / L, 2.73±0.98 μg / L, and 2.47±11.63 μg / L, respectively; PGF2α The average concentrations of isomers ranged from 0.15 μg / L to 0.74 μg / L; see details below. Figure 3 .

[0055] Method detection limit Following the US Environmental Protection Agency (EPA) Detection Limit Determination Procedure (EPA 821-R-16-006), a spiked recovery experiment was performed using an artificial urine matrix. 200 µL of a mixed standard solution of a specific concentration and 10 µL of internal standard solution (final system concentration 20 µg / L) were added to 190 µL of artificial urine matrix. After mixing, the mixture was allowed to stand at 4°C for 10 minutes, centrifuged at 1000 rpm for 10 minutes, and 200 µL of the supernatant was collected. UPLC-MS / MS analysis was performed according to the above method, with 7 parallel determinations (n=7). The method detection limit (MDL) was determined by three times the standard deviation (SD) of the results, and the quantitation limit (MQL) was determined by ten times the standard deviation. For HNE-GSH, the method detection limit was determined by adding a 10 μg / L mixed standard solution containing 16 oxidative stress biomarkers to the matrix. For the other 15 oxidative stress biomarkers besides HNE-GSH, the method detection limit was determined by adding a 3.5 μg / L mixed standard solution containing 16 oxidative stress biomarkers to the matrix. The MDL, MQL, and spiked recoveries are shown in Table 9 below.

[0056] Table 9

[0057] Evaluation of the matrix effect of artificial urine This study uses the standard curve slope comparison method: based on the above... Construction of standard curve The standard curve obtained in the above method was used as the matrix standard curve. The solvent standard curve was obtained as follows: a single-standard stock solution was prepared with methanol, then a mixed standard intermediate solution was prepared using water as the solvent, and the solution was gradually diluted with water to obtain a series of working standard solutions containing 16 oxidative stress biomarkers. The remaining components were the same as those described above. Construction of standard curve "Same. The matrix effect is calculated using the formula ME(%) = (slope of matrix standard curve / slope of solvent standard curve - 1) × 100%. The evaluation results are as follows..." Figure 4 As shown, the method of the present invention can ensure 100% retention of all markers while perfectly controlling the matrix effect (ME) within a safe range of -19.45% to 4.52% (|ME|<20%).

[0058] Real urine sample spiked recovery rate To further investigate the applicability of the method in real complex matrices, this study collected urine samples from 10 healthy individuals from different sources. These samples were mixed in equal volumes to prepare a urine mixing tank. 200 µL of mixed standard solutions at concentrations of 3.5 μg / L (low concentration), 20 μg / L (medium concentration), or 50 µg / L (high concentration) and 10 µL of internal standard solution were added to each 190 µL urine mixing tank (final system concentration 20 µg / L). For the low concentration level, the recoveries of the 15 target analytes (excluding HNE-GSH) were achieved using a 3.5 μg / L mixed standard concentration containing 16 oxidative stress biomarkers. Due to method sensitivity limitations, the low concentration level for HNE-GSH was achieved using a 10 µg / L mixed standard solution containing 16 oxidative stress biomarkers and 10 µL of internal standard solution (final system concentration 20 µg / L). After mixing the above solutions, let them stand at 4°C for 10 minutes, centrifuge at 1000 rpm for 10 minutes, and take 200 µL of the supernatant for UPLC-MS / MS analysis according to the above method. Spike recovery experiments were performed. Considering that endogenous oxidative stress markers are commonly present in actual urine, the background response values ​​of each sample were subtracted in the calculations. The spike recoveries of oxidative stress markers at low, medium, and high concentration levels are shown in Table 10. Figure 5 As shown.

[0059] Table 10 .

[0060] The results are shown in Table 10 and Figure 5 As shown, using the method of the present invention, the spiked recovery rate in real urine samples is between 80% and 120%, indicating that the detection method of the present invention is stable and the results are reliable. Example 2

[0061] Except for changing the volume fractions of formic acid in mobile phases A and B during liquid chromatography separation to 0.05% and 0.1% respectively, oxidative stress biomarkers in a mixed standard solution of 16 oxidative stress biomarkers with a mass concentration of 10 µg / L (methanol as solvent) were detected under the same conditions as in Example 1. The signal intensities of the 16 oxidative stress biomarkers at the three formic acid concentrations were compared using a normalization method.

[0062] The results are as follows Figure 6 As shown, the simultaneous detection of 16 oxidative stress biomarkers can be achieved when the volume fraction of formic acid in mobile phase A and mobile phase B is 0.01%, 0.05%, and 0.1%, respectively. However, the signal response of each target analyte varies with different volume fractions of formic acid. When the volume fraction of formic acid is 0.01%, the signal intensity of the 16 oxidative stress biomarkers is the strongest overall.

[0063] Comparative Example 1 Except for using full-scan multiple reaction monitoring (MRM) during mass spectrometry detection, a mixed standard solution of 16 oxidative stress biomarkers (methanol as solvent) at a mass concentration of 10 µg / L was detected under the same conditions as in Example 1. The signal-to-noise ratio of the oxidative stress biomarkers in Example 1 and Comparative Example 1 was compared.

[0064] The results are as follows Figure 7 As shown, compared with the full-scan mode of multi-response monitoring (MRM), the segmented MRM based on the retention time window can significantly improve the signal-to-noise ratio of most targets by 1.06 to 5.63 times, and the method has higher reliability and accuracy.

[0065] Comparative Example 2 Except that the chromatographic column used in the liquid chromatography separation was an ACQUITY HSS T3 column, the oxidative stress biomarkers in the mixed standard solution of 16 oxidative stress biomarkers with a mass concentration of 10 µg / L (solvent was methanol) were detected under the same conditions as in Example 1.

[0066] Test results as follows Figure 8 As shown, the results indicate that the ACQUITY HSS T3 column cannot effectively separate the key compounds 8-HdG and 8-HG, as they undergo severe co-elution, failing to meet the prerequisites for quantitative analysis.

[0067] Comparative Example 3 Except that the chromatographic column used in the liquid chromatography separation process was a conventional HILIC column, the oxidative stress biomarkers in a mixed standard solution of 16 oxidative stress biomarkers with a mass concentration of 10 µg / L (solvent was methanol) were detected under the same conditions as in Example 1.

[0068] Test results as follows Figure 9 As shown, four prostaglandin F 2α Isomer (8-PGF) 2α 11β-PGF 2α 5,6-trans-PGF 2α With 15-PGF 2α The DIY target analyte could not be separated, the BY peak showed severe tailing, and the DIY target analyte was completely undetectable.

[0069] Comparative Example 4 Except that methanol:acetonitrile (8:2, V / V) was used as mobile phase B in the liquid chromatography separation process, the oxidative stress biomarkers in the mixed standard solution of 16 oxidative stress biomarkers with a mass concentration of 50 µg / L (solvent is methanol) were detected under the same conditions as in Example 1.

[0070] The results are as follows Figure 10 As shown, mobile phase B of methanol:acetonitrile (8:2) cannot provide sufficient selectivity to separate the four PGFs. 2α Isomers. Compared to mobile phase B of methanol:acetonitrile (8:2), the method of this invention produces 5,6-trans-PGF. 2α With 15-PGF 2α 8-PGF 2α With 11β-PGF 2α The separation rates were improved by 21.4% and 11.1%, respectively, while also meeting the peak requirements of lipid peroxidation markers such as HNE-GSH.

[0071] Comparative Example 5 Except that methanol:acetonitrile (1:1) was used as mobile phase B in the liquid chromatography separation process, the oxidative stress biomarkers in the mixed standard solution of 16 oxidative stress biomarkers with a mass concentration of 50 µg / L (solvent is methanol) were detected under the same conditions as in Example 1.

[0072] The results are as follows Figure 11 The chromatographic detection results of HNE-GSH, a key marker of the lipid peroxidation pathway, showed that only baseline noise signal was present at the target retention time, with no obvious effective chromatographic peak, and the substance could not be effectively detected.

[0073] Comparative Example 6 Solid-phase extraction (SPE) (using ABS Elute Nexus and Oasis Premier HLB columns) was employed to pretreat a mixed standard solution of 16 oxidative stress biomarkers (20 µg / L) in methanol. The solution was then processed using the method described in Example 1. UPLC-MS / MS detection using ultra-high performance liquid chromatography-tandem mass spectrometry "Conduct testing and compare the effectiveness of solid phase extraction pretreatment."

[0074] The solid-phase extraction pretreatment step includes: Solid-phase extraction (SPE) on an ABS Elute Nexus column: 190 μL of water was added to 200 μL of a 20 µg / L mixed standard solution (methanol) and 10 μL of an internal standard mixture (800 µg / L). The ABS Elute Nexus column was activated with 2 mL of methanol and 2 mL of ultrapure water. The mixed standard solution sample (after adding water and internal standard) was transferred to the ABS Elute Nexus column, washed with 2 mL of 95:5 (v / v) water:methanol, dried under vacuum, and eluted with 1 mL of methanol and 1 mL of ethyl acetate. The eluent was concentrated at low temperature and evaporated to dryness, then reconstituted with 0.2 mL of 8:2 (v / v) water:methanol. UPLC-MS / MS detection was performed using the same method as in Example 1. The relative loss rate of each oxidation marker was determined based on the peak area after the pretreatment method of this invention: Loss rate (%) = (1 - peak area after SPE treatment / peak area after the pretreatment method of this invention) × 100%. Figure 12 As shown, the ABS EluteNexus column exhibited varying degrees of relative loss for all types of biomarkers. Specifically, the relative loss rate for DNA oxidative damage markers ranged from 68.70% to 91.01%, such as 8-OHdG and 8-HG; the loss for RNA oxidative damage markers was more significant, ranging from 91.21% to 100.00%, such as 8-HdG and m... 5 C, among which 8-HdG showed complete loss. The relative loss rates of protein oxidative damage and nitration damage markers were generally high and similarly distributed, concentrated in the ranges of 83.98%–96.12% and 81.12%–97.25%, respectively. Protein oxidative damage markers included diy, BY, and CY, while nitration damage markers included 8-NG, NY, and 8-NdG. Notably, lipid oxidative damage markers showed significant variability within their respective groups (40.99%–100.00%), for example, 15-PGF. 2α 8-PGF 2α 11β-PGF 2α 5, 6-trans-PGF 2α HNE-MA and HNE-GSH, PGF in this group 2α The loss of isomers was relatively controlled, but HNE-MA and HNE-GSH showed extreme losses as high as 100.00%. This result indicates that the ABS Elute Nexus column is difficult to effectively enrich the various biomarkers with large polarity differences involved in this study.

[0075] Oasis Premier HLB column solid-phase extraction: 190 μL of water was added to 200 μL of a 20 µg / L mixed standard solution (methanol) and 10 μL of an internal standard mixture (800 µg / L). The sample was directly loaded and washed with 10% methanol-water solution, eluted twice with 200 μL of pure methanol, concentrated at low temperature for 90 min, and then redissolved in 200 μL of methanol:acetonitrile (2:8, v / v) containing 0.01% formic acid. UPLC-MS / MS analysis was performed using the same method as in Example 1. The analyte loss during the SPE process was quantitatively assessed using the peak area (100%) after the pretreatment method of this invention. Results are as follows... Figure 12 As shown, except for 8-HG (loss of 2.00%), the relative loss rates of the other markers all exceeded 40%, with 13 exceeding 60%. RNA damage markers showed the most severe loss (m 5 C reached 92.70%, 8-HdG reached 82.10%, the loss rate of protein damage markers was 64.30% to 84.80%, the nitration damage markers varied greatly (8-NG reached 85.30%, 8-NdG was 41.60%), and the loss of lipid peroxidation markers was concentrated in the range of 58.10% to 78.50%.

[0076] It is evident that the traditional selective adsorption / elution principle inevitably leads to severe "selective loss" (with some RNA and lipid damage markers experiencing losses as high as 80%-100%) when faced with these 16 compounds with vastly different physicochemical properties.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for simultaneous determination of 16 oxidative stress biomarkers in urine based on UPLC-MS / MS, characterized in that, The method includes the following steps: (S1) Sample pretreatment: Take a urine sample, add an isotope internal standard mixture and diluent, let it stand at 2-8℃ for 5-20 minutes, centrifuge at 2-8℃ and take the supernatant as the test solution. The diluent is a mixed solution of methanol and water with a volume ratio of 1:

1. (S2) UPLC-MS / MS determination: The liquid chromatography analysis conditions are as follows: the chromatographic column is a C18 reversed-phase column, mobile phase A is a 0.01% (v / v) aqueous solution of formic acid, mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in mobile phase B is 0.01%, and the volume ratio of methanol to acetonitrile is 1:4; The column temperature for liquid chromatography separation was 35-45℃; the flow rate was 0.3 mL / min; the injection volume was 1 μL; and the elution program was gradient elution, which was as follows: 0-0.5 min, 5% mobile phase B; 0.5-8 min, 5%-95% mobile phase B; 8-12 min, 95% mobile phase B; 12-12.5 min, 95%-5% mobile phase B; 12.5-15 min, 5% mobile phase B. The mass spectrometry analysis conditions are as follows: an electrospray ionization source is used, and segmented multiple reaction monitoring is performed in positive and negative ion switching modes; The 16 oxidative stress biomarkers are 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α ; The isotope internal standard mixture includes 8-isoprostaglandin F. 2α -d4, 4-hydroxy-2-nonenal mercaptouric acid -d3, 4-hydroxy-2-nonenal glutathione -d3, 3-bromotyrosine 13 C9, 15 N,L,L-dityrosine- 13 C 12 3-Nitro-L-Tyrosine 13 C6 and 8-hydroxy-2'-deoxyguanosine- 15 N5.

2. The method according to claim 1, characterized in that, In step (S1), the mixture is left to stand at 4°C for 10 minutes.

3. The method according to claim 2, characterized in that, Step (S1) includes: adding an equal volume of the diluent and the isotope internal standard mixture to the urine sample to obtain a test solution before centrifugation, such that the concentration of the isotope internal standard in the test solution before centrifugation is 20 µg / L; letting it stand at 4°C for 10 minutes, and then centrifuging at 4°C and 1000 rpm for 10 minutes, and taking the supernatant as the test solution, wherein the solvent of the isotope internal standard mixture is methanol.

4. The method according to claim 1, characterized in that, In step (S2), the chromatographic column is a Poroshell 120 Aq-C18.

5. The method according to claim 1, characterized in that, In step (S2), the column temperature for liquid chromatography separation is 40°C.

6. The method according to claim 1, characterized in that, In step (S2), the switching between positive and negative ion modes includes: detecting 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, and 4-hydroxy-2-nonenal glutathione in positive ion mode; and detecting 4-hydroxy-2-nonenal mercaptouric acid and 11β-prostaglandin F in negative ion mode. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α .

7. The method according to claim 1, characterized in that, In step (S2), the mass spectrometry conditions are as follows: capillary voltage is 1 kV in positive ion mode and 3 kV in negative ion mode; ion source temperature is 150℃; desolvation gas temperature is 550℃; cone gas flow rate is 150 L / h; desolvation gas flow rate is 1000 L / h; cone voltage is 8-40 V; collision energy is 10-40 eV.

8. The method according to claim 7, characterized in that, The internal standards, retention times, quantitative ion pairs and their corresponding cone voltages and collision energies for the 16 oxidative stress biomarkers are as follows: 。 9. The method according to claim 8, characterized in that, The residence times and scanning windows corresponding to the 16 oxidative stress biomarkers are as follows: 。 10. The method according to any one of claims 1-9, characterized in that, In step (S2), the following substances are used: 3-nitro-L-tyrosine, 3-bromotyrosine, 8-hydroxy-2′-deoxyguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α Using the isotope internal standard method, a linear regression analysis was performed with the peak area ratio of the target analyte to the internal standard as the ordinate and the target analyte mass concentration as the abscissa to construct the first set of standard curves. For 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 5-methylcytidine, 8-hydroxyguanosine, and 8-nitroguanosine, the external standard method was used, with the peak area of ​​the target analyte as the ordinate and the target analyte mass concentration as the abscissa to construct the second set of standard curves. The content of the oxidative stress biomarkers in the test solution was calculated based on the first and second set of standard curves.

11. The method according to claim 10, characterized in that, A series of standard working solutions containing the 16 oxidative stress biomarkers were prepared using a stepwise dilution method and artificial urine at different concentrations. The isotope internal standard mixture and artificial urine were added to the standard working solutions to obtain a standard curve working solution, such that the concentration of the isotope internal standard in the standard curve working solution was the same as the concentration of the isotope internal standard in the test solution before centrifugation. The standard curve working solution was used to construct the first set and the second set of standard curves.

12. The method according to claim 11, characterized in that, A mixed standard intermediate solution containing the 16 oxidative stress biomarkers was prepared using methanol, and the standard working solution was prepared by adding the mixed standard intermediate solution to the artificial urine.

13. A kit for use in the method according to claim 1, characterized in that, The kit includes artificial urine, an isotope internal standard mixture, a diluent, a mixed standard solution of oxidative stress biomarkers, mobile phase A, and mobile phase B; wherein the isotope internal standard mixture includes 8-isoprostaglandin F. 2α -d4, 4-hydroxy-2-nonenal mercaptouric acid -d3, 4-hydroxy-2-nonenal glutathione -d3, 3-bromotyrosine 13 C9, 15 N,L,L-dityrosine- 13 C 12 3-Nitro-L-Tyrosine 13 C6 and 8-hydroxy-2'-deoxyguanosine- 15 N5, the diluent is a 1:1 volume ratio of methanol to water, and the mixed standard solution of oxidative stress biomarkers includes 8-hydroxyguanine, 8-nitroguanine, 3-chloro-L-tyrosine, 3-nitro-L-tyrosine, 5-methylcytidine, 3-bromotyrosine, 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, 8-nitroguanosine, L,L-dityrosine, 4-hydroxy-2-nonenal glutathione, 4-hydroxy-2-nonenal mercaptouric acid, and 11β-prostaglandin F. 2α 5,6-trans-prostaglandin F 2α 8-Isoprostaglandin F 2α and 15-prostaglandin F 2α The mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.01%, and the mobile phase B is a methanol-acetonitrile mixed solution containing formic acid, wherein the volume fraction of formic acid in the mobile phase B is 0.01%, and the volume ratio of methanol to acetonitrile is 1:4.

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