Method for simultaneous detection of 11-dehydrothromboxane b2 and creatinine and use thereof

Abstract

The application provides a method for simultaneously detecting 11-dehydro-thromboxane B2 and creatinine and application thereof, and relates to the technical field of analytical chemistry, and the method comprises the following steps: detecting pretreated samples by using liquid chromatography tandem mass spectrometry, bringing the detection value into a standard curve, and calculating the content of 11-dehydro-thromboxane B2 and creatinine in the samples; wherein the pretreatment comprises extracting 11-dehydro-thromboxane B2 and creatinine in the samples by using an organic phase. The method alleviates the problem that 11-dehydro-thromboxane B2 and creatinine cannot be simultaneously detected in the prior art.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for the simultaneous detection of 11-dehydrothromboxane B2 and creatinine, and its application. Background Technology

[0002] 11-Dehydrothromboxane B2 (11-dh-TXB2) is a metabolite of thromboxane A2 (TXA2) and is mainly excreted by the kidneys. TXA2 is an important product of arachidonic acid metabolism, primarily released by activated platelets. TXA2 has two main activities: firstly, it has a strong platelet aggregation effect, promoting platelet aggregation and thrombus formation; secondly, it can cause vasoconstriction, bronchial and smooth muscle contraction, and regulate vascular tone. Current methods for detecting 11-dehydrothromboxane B2 mainly include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), gas chromatography-tandem mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0003] Creatinine (Cre) is a metabolic byproduct of muscle metabolism in the human body, primarily excreted through glomerular filtration, and its levels are related to kidney function. The 24-hour clearance rate of creatinine can also be used to determine sample integrity or to correct urine sample concentration using creatinine ratios. Existing methods for creatinine detection mainly include the Jaffe method, enzymatic spectrophotometry, HPLC, capillary electrophoresis, capillary zone electrophoresis, gas chromatography-tandem mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0004] Currently, there is no method for simultaneously detecting 11-dehydro-thromboxane B2 and creatinine. In scenarios where it is necessary to obtain 11-dehydro-thromboxane B2 and creatinine from a sample simultaneously, the two analytes need to be detected separately, which increases the workload and the amount of sample required. Therefore, how to combine 11-dehydro-thromboxane B2 and creatinine into a single detection procedure is a problem that needs to be solved.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for simultaneously detecting 11-dehydrothromboxane B2 and creatinine, thereby alleviating the problem that 11-dehydrothromboxane B2 and creatinine cannot be detected simultaneously in the prior art.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a method for simultaneously detecting 11-dehydrothromboxane B2 and creatinine is provided. The method includes detecting a pretreated sample using liquid chromatography-tandem mass spectrometry, substituting the detected values ​​into a standard curve, and calculating the contents of 11-dehydrothromboxane B2 and creatinine in the sample. The pretreatment includes extracting 11-dehydrothromboxane B2 and creatinine from the sample using an organic phase.

[0009] In an optional embodiment, the organic phase comprises acetonitrile.

[0010] In an optional embodiment, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution containing ammonium formate, and mobile phase B is a methanol solution containing ammonium formate.

[0011] In an optional embodiment, the chromatographic column used for liquid chromatography detection is a pentafluorophenyl liquid chromatography column.

[0012] In an optional embodiment, the column temperature is 20–50°C, preferably 40°C.

[0013] In an optional implementation, the elution procedure for liquid chromatography is as follows:

[0014] 0 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0015] 1.5 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0016] 1.6 min, mobile phase A 1-10% v / v, mobile phase B 90-99% v / v;

[0017] 3.7 min, mobile phase A1 ~ 10% v / v, mobile phase B 90 ~ 99% v / v;

[0018] 3.8 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0019] 5.0 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v.

[0020] In optional implementations, the mass spectrometry conditions include the following (a) to (c):

[0021] (a) Scanning mode: Electrospray ionization, positive and negative switching mode;

[0022] (b) Acquisition mode: Multiple reaction monitoring;

[0023] (c) Ion source parameters:

[0024] Positive ion mode: Electrospray voltage: 5200~5800V; curtain gas: 23~27psi; atomizing gas: 47~53psi; auxiliary gas: 61~69; collision gas: 8~10; ion source temperature: 475~525℃;

[0025] And, negative ion mode: electrospray voltage: -3300~3700V; air curtain gas: 23~27psi; atomizing gas: 47~53psi; auxiliary gas: 61~69; collision gas: 8~10; ion source temperature: 475~525℃.

[0026] In an optional implementation, the sample may include a urine sample.

[0027] In an optional implementation, the internal standard method is used to construct the standard curve.

[0028] Secondly, the method for simultaneous detection of 11-dehydrothromboxane B2 and creatinine in the first aspect is also provided for application in the preparation of 11-dehydrothromboxane B2 and creatinine monitoring products.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The method provided by this invention enables the simultaneous detection of 11-dehydrothromboxane B2 and creatinine in a sample. After sample pretreatment, the sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), which offers the advantages of convenience and speed. Furthermore, the method provided by this invention has a simple pretreatment step, requiring only organic phase extraction of the analytes from the sample, eliminating the need for complex operations such as solid-phase extraction, further simplifying the detection method.

[0031] The method provided by this invention features high sensitivity, good repeatability, high accuracy, and good specificity. In the preferred embodiment, the linear range for 11-dehydrothromboxane B2 is 0.1–50 ng / mL, and the linear range for creatinine is 10–5000 ng / mL. Within these linear ranges, the correlation coefficients (r) are all ≥0.990. The accuracy of low, medium, and high-value quality control samples is between 85.83% and 113.21%, the RSD% of the quality control samples is <9.71%, and the spiked recovery rate is 85%–110%. The simultaneous detection of 11-dehydrothromboxane B2 and creatinine provided by this invention meets the relevant regulatory requirements for linearity, repeatability, batch-to-batch variation, and accuracy of 11-dehydrothromboxane B2 and creatinine, and plays an important role in the study of the pathological mechanisms of related diseases and the development of related drugs. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 A chromatogram for detecting creatinine in a substitute matrix sample using the method established in Example 1;

[0034] Figure 2 The chromatogram for detecting 11dH-TXB2 in the substitute matrix sample using the method established in Example 1;

[0035] Figure 3 A chromatogram for detecting creatinine in a standard solution sample using the method established in Example 1;

[0036] Figure 4 The chromatogram for detecting 11dH-TXB2 in a standard solution sample using the method established in Example 1;

[0037] Figure 5 A chromatogram for detecting creatinine in human urine samples using the method established in Example 1;

[0038] Figure 6 The chromatogram for detecting 11dH-TXB2 in human urine samples using the method established in Example 1;

[0039] Figure 7 The chromatogram for detecting creatinine in a blank solution sample using the method established in Example 1;

[0040] Figure 8 The chromatogram for detecting 11dH-TXB2 in a blank solution sample using the method established in Example 1;

[0041] Figure 9 The chromatogram of 11dH-TXB2 is shown when the salt solution in the mobile phase is ammonium formate.

[0042] Figure 10 The chromatogram of creatinine is shown when the salt solution in the mobile phase is ammonium formate.

[0043] Figure 11 The chromatogram of 11dH-TXB2 is shown when the salt solution in the mobile phase is ammonium fluoride.

[0044] Figure 12 This is a chromatogram of creatinine when the salt solution in the mobile phase is ammonium fluoride. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] In a first aspect, a method for simultaneously detecting 11-dehydrothromboxane B2 and creatinine is provided. The method includes detecting a pretreated sample using liquid chromatography-tandem mass spectrometry, substituting the detected values ​​into a standard curve, and calculating the contents of 11-dehydrothromboxane B2 and creatinine in the sample. The pretreatment includes extracting 11-dehydrothromboxane B2 and creatinine from the sample using an organic phase.

[0047] In an optional embodiment, the organic phase used for extraction in the pretreatment step includes acetonitrile.

[0048] In an optional embodiment, the volume ratio of the sample to the organic phase in the extraction step is 1:(0.5-2), for example, but not limited to 1:0.5, 1:1, 1:1.5 or 1:2, preferably 1:1.

[0049] In an optional embodiment, during the extraction step, 50 μL of sample and 50 μL of mixture are taken to extract 11-dehydrothromboxane B2 and creatinine from the sample.

[0050] In an optional embodiment, the pretreatment step includes vortex mixing the sample and the organic phase, separating the supernatant, and then performing liquid chromatography-tandem mass spectrometry detection.

[0051] In an optional embodiment, separating the supernatant includes centrifuging at 24,000 rpm for 10 minutes and then taking the supernatant.

[0052] In an optional implementation, an internal standard is added during the pretreatment step, including mixing the sample, the internal standard, and the organic phase.

[0053] In an optional embodiment, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution containing ammonium formate, and mobile phase B is a methanol solution containing ammonium formate.

[0054] In an optional embodiment, the concentration of ammonium formate in mobile phase A is 0.1 to 1 mM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mM, preferably 0.2 mM.

[0055] In an optional embodiment, the concentration of ammonium formate in mobile phase B is 0.1 to 1 mM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mM, preferably 0.2 mM.

[0056] In an optional embodiment, the chromatographic column used for liquid chromatography detection is a pentafluorophenyl liquid chromatography column.

[0057] In an optional implementation, the chromatographic column used for liquid chromatography detection is a Phenomenex Kinetex F5 column with a specification of 2.6 μm and a diameter of 3 mm × 100 mm.

[0058] In an optional embodiment, the column temperature is 20 to 50°C, for example, but not limited to 20, 25, 30, 35, 40, 45 or 55°C, preferably 40°C.

[0059] In an optional implementation, the elution procedure for liquid chromatography is as follows:

[0060] 0 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0061] 1.5 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0062] 1.6 min, mobile phase A 1-10% v / v, mobile phase B 90-99% v / v;

[0063] 3.7 min, mobile phase A 1-10% v / v, mobile phase B 90-99% v / v;

[0064] 3.8 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v;

[0065] 5.0 min, mobile phase A 65-75% v / v, mobile phase B 25-35% v / v.

[0066] In a preferred embodiment, the elution procedure for liquid chromatography is as follows:

[0067] 0 min, mobile phase A 70% v / v, mobile phase B 30% v / v;

[0068] 1.5 min, mobile phase A 70% v / v, mobile phase B 30% v / v;

[0069] 1.6 min, mobile phase A 5% v / v, mobile phase B 95% v / v;

[0070] 3.7 min, mobile phase A 5% v / v, mobile phase B 95% v / v;

[0071] 3.8 min, mobile phase A 70% v / v, mobile phase B 30% v / v;

[0072] 5.0 min, mobile phase A 70% v / v, mobile phase B 30% v / v.

[0073] In an optional implementation, the mass spectrometry conditions in the method include the following (a) to (c):

[0074] (a) Scanning mode: Electrospray ionization, positive and negative switching mode;

[0075] (b) Acquisition mode: Multiple reaction monitoring;

[0076] (c) Ion source parameters:

[0077] Positive ion mode: Electrospray voltage: 5200~5800V; curtain gas: 23~27psi; atomizing gas: 47~53psi; auxiliary gas: 61~69; collision gas: 8~10; ion source temperature: 475~525℃;

[0078] In negative ion mode, the electrospray voltage is -3700 to -3300V; the curtain gas is 23 to 27 psi; the atomizing gas is 47 to 53 psi; the auxiliary gas is 61 to 69; the collision gas is 8 to 10; and the ion source temperature is 475 to 525℃.

[0079] In an optional implementation, the positive ion mode is as follows: electrospray voltage: 5500V; curtain gas: 25psi; atomizing gas: 50psi; auxiliary gas: 65; collision gas: 9; ion source temperature: 500℃.

[0080] In an optional implementation, the negative ion mode is as follows: electrospray voltage: -3500V; curtain gas: 25psi; atomizing gas: 50psi; auxiliary gas: 65; collision gas: 9; ion source temperature: 500℃.

[0081] In an optional implementation, the mass spectrometry parameters are as follows:

[0082]

[0083]

[0084] In an optional implementation, an internal standard method is used to construct the standard curve. Using the internal standard method can reduce matrix interference, pretreatment step interference, and the influence of instrument factors.

[0085] In an optional embodiment, the internal standard includes isotopically labeled 11-dehydrothromboxane B2 and isotopically labeled creatinine.

[0086] In an optional implementation, the standard curve is the correspondence between the content of the analyte in the standard and the detection value of the standard; the detection value of the standard is the peak value ratio of the standard and the internal standard, and the peak value includes peak height or peak area.

[0087] In an optional embodiment, the standard is composed of several standards with a series of concentrations. Among the several standards with a series of concentrations, the concentration of 11-dehydrothromboxane B2 is distributed from 0.1 to 50 ng / mL, and the concentration points are preferably set to 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL; the concentration of creatinine is distributed from 10 to 5000 ng / mL, and the concentration points are preferably set to 10 ng / mL, 20 ng / mL, 50 ng / mL, 200 ng / mL, 1000 ng / mL, 2500 ng / mL and 5000 ng / mL.

[0088] In an optional implementation, the method further includes testing quality control samples to evaluate the test results.

[0089] This invention provides a method for the simultaneous detection of 11-dehydrothromboxane B2 and creatinine. The sample source can be, for example, but not limited to, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, or humans. It should be noted that the method for the simultaneous detection of 11-dehydrothromboxane B2 and creatinine provided by this invention is not for diagnostic or therapeutic purposes. Specifically, samples from the aforementioned mammals are experimental animal samples. The measurement method of this invention is used to provide experimental results, which can then be further used in fields such as detecting the physiological state of subjects, studying drug mechanisms of action, or conducting pharmacokinetic studies. Furthermore, knowing the content of 11-dehydrothromboxane B2 and creatinine in the sample does not mean that the result directly points to a specific disease diagnosis. Therefore, the method for the simultaneous detection of 11-dehydrothromboxane B2 and creatinine provided by this invention is not for diagnostic or therapeutic purposes. The method provided by this invention does not limit the sample type; conventional sample types in the art can be selected, such as, for example, but not limited to, serum, plasma, tissue or cell extracts, and body fluids. In an optional embodiment, the sample is urine. When the sample is urine, the matrix for standards and / or controls includes PBS.

[0090] Secondly, the method for simultaneously detecting 11-dehydrothromboxane B2 and creatinine, as described in the first aspect, is also provided for use in the preparation of 11-dehydrothromboxane B2 and creatinine monitoring products. This method can be used as an evaluation standard to assess the detection effectiveness of other 11-dehydrothromboxane B2 and / or creatinine detection methods, or it can be pre-installed in the detection product as a running module, with the reagents used in this method serving as complementary reagents in the assay product.

[0091] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0092] The mass spectrometry system used in the following examples is Citrine. TM Triple Quad TM System, Sciex; chromatographic column: Phenomenex Kinetex F5 (2.6 μm, 3 mm × 100 mm);

[0093] The main experimental reagents and materials used in the following examples include:

[0094] Dehydrothromboxane B2 standard, Cayman, Lot#:0473293-129; Creatinine standard, Merck, C4255;

[0095] Dehydrothromboxane B2-d4 isotope labeled standard, Cayman, Lot#:0663023-3; Creatinine-D3 isotope standard, Tianjin Alta Technology Co., Ltd., LOT#:S118501;

[0096] Formic acid: MREDA, USA, chromatographic grade, batch number LOT095224;

[0097] Acetonitrile: Fisher Scientific, USA, mass spectrometry grade;

[0098] Methanol: Fisher Scientific, USA, mass spectrometry grade;

[0099] Ultrapure water: Watsons triple-distilled water;

[0100] HyClone phosphate-buffered saline (PBS), Cytiva, LOT: SH30256.02.

[0101] Example 1

[0102] This embodiment provides a method for simultaneously detecting dehydrothromboxane B2 and creatinine, comprising the following steps:

[0103] (I) Reagent preparation:

[0104] Dehydrothromboxane B2 (11dH-TXB2) standard dilution solution: pure water: methanol: acetonitrile = 8:1:1;

[0105] Alternative matrix: HyClone phosphate-buffered saline (PBS).

[0106] The 11dH-TXB2 stock solution was prepared by dissolving 10 mg of powder in 10 mL of pure water:methanol:acetonitrile = 8:1:1 to obtain 1 mg / mL.

[0107] The creatinine stock solution was prepared by dissolving 10 mg of powder in 10 mL of pure water to obtain a concentration of 1 mg / mL.

[0108] The standard working solutions were diluted with PBS (a substitute matrix) to the following concentrations: 11dH-TXB2: 50 ng / mL, 25 ng / mL, 10 ng / mL, 2 ng / mL, 0.5 ng / mL, 0.2 ng / mL, 0.1 ng / mL; creatinine: 5000 ng / mL, 2500 ng / mL, 1000 ng / mL, 200 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL. Linear working solutions S1–S7 were obtained as shown in Table 1.

[0109] Table 1. Concentrations of 11dH-TXB2 and creatinine in the working solutions of each standard solution.

[0110]

[0111] The 11dH-TXB2 and creatinine mixed internal standard were diluted with PBS from the stock solution to 50 ng / mL and 1 μg / mL, respectively.

[0112] (II) Sample Pretreatment:

[0113] (1) Urine sample: Add 50 μL of patient urine sample to a clean 1.5 mL EP tube, add 10 μL of isotope internal standard, and then add 50 μL of pure acetonitrile solution. Vortex evenly in a shaker, centrifuge at 24000 rpm for 10 minutes, and then inject the supernatant.

[0114] (2) Standard curve and quality control (QC) samples: Add 50 μL of linear working solution and quality control working solution samples to a clean 1.5 mL EP tube, add 10 μL of isotope internal standard, and then add 50 μL of pure acetonitrile solution. Vortex evenly in a shaker, centrifuge at 24000 rpm for 10 minutes, and then inject the supernatant.

[0115] (III) Liquid Chromatography Conditions:

[0116] Column: Phenomenex Kinetex F5 (2.6μm, 3*100mm);

[0117] The gradient elution conditions for the chromatographic column are shown in Table 2:

[0118] Table 2 Gradient elution conditions for the column

[0119] Time (min) A (% v / v) B (% v / v) 0 70 30 1.5 70 30 1.6 5 95 3.7 5 95 3.8 70 30 5.0 70 30

[0120] Mobile phase A: pure water containing 0.2 mM ammonium formate;

[0121] Mobile phase B: pure methanol containing 0.2 mM ammonium formate;

[0122] Flow rate: 300 μL / min;

[0123] Column temperature: 40℃;

[0124] Injection volume: 10 μL.

[0125] (iv) Mass spectrometry conditions:

[0126] Scanning mode: Electrospray ionization, positive and negative switching mode;

[0127] Acquisition mode: Multiple response monitoring (MRM);

[0128] Ionization mode: Electrospray voltage: -3500 / 5500V; Curtain gas: 25; Atomizing gas (gas 1): 50; Auxiliary gas (gas 2): 65; Ion source temperature (TEM): 500℃.

[0129] Mass spectrometry parameters are shown in Table 3:

[0130] Table 3 Mass Spectrometry Parameters

[0131]

[0132]

[0133] In Table 3, ID represents the ion channel, ID-1 and -2 represent the quantitative and qualitative ion pairs of the corresponding analytes on the mass spectrometer detector, respectively, and IS represents the internal standard.

[0134] (V) Data Processing:

[0135] Data was collected and processed using MultiQuant software, and the results are represented in Mean, SD, and RSD formats.

[0136] Example 2 Methodological Validation

[0137] The sample pretreatment, liquid chromatography conditions, and mass spectrometry conditions in this embodiment were performed according to the method in Example 1.

[0138] (I) Intra-batch and inter-batch precision and accuracy assessment

[0139] Multiple human urine samples with low 11dH-TXB2 and creatinine background values ​​were selected and mixed to serve as a blank matrix for method validation. Appropriate concentrations of 11dH-TXB2 and creatinine standard solutions were added to the urine blank matrix to prepare three quality control samples (low, medium, and high concentrations), following the procedures outlined in "Sample Pretreatment." Within the same batch, six samples were prepared and measured for each concentration to examine intra-batch variability. Three different batches of urine samples with low, medium, and high concentrations were prepared using the same method and analyzed using a standard curve prepared on the same day to examine inter-batch variability. The accuracy of the method was examined based on the closeness of the measured concentration to the theoretical true concentration. The results are shown in Table 4. The experimental results show that the accuracy of all quality control samples was within the error range, and all samples were included in the statistics. The accuracy of all three different concentrations of quality control samples ranged from 85.83% to 113.21%, and the RSD% of all quality control samples was <9.71%. This indicates that the quantitative method is stable, reliable, and meets the requirements for testing human urine samples.

[0140] Table 4. Intra- and inter-batch precision and accuracy data for 11dH-TXB2 and creatinine.

[0141]

[0142] (II) Selective Examination:

[0143] The chromatographic resolution of 11dH-TXB2 and creatinine from interfering peaks in the matrix was tested. The results showed a resolution greater than 1.5, and interference around the elution times of 11dH-TXB2 and creatinine did not affect quantitative analysis. Chromatograms of 11dH-TXB2 and creatinine in alternative matrices, standard solutions, human urine, and blank solutions were compared. The results are shown in [Figure number missing]. Figures 1-8 .

[0144] (III) Linear Range:

[0145] Five samples were prepared using standard solutions of 11dH-TXB2 and creatinine at seven relative concentrations, with PBS as the substitute matrix, and analyzed in the same run. The allowable deviation for linearity was ±15% of the true value. Regression equations were obtained by plotting the concentrations of 11dH-TXB2 and creatinine on the x-axis and the ratio of peak area to internal standard peak area on the y-axis, as shown in Table 5.

[0146] Table 5. Linear range data for 11dH-TXB2 and creatinine

[0147]

[0148] (iv) Recovery rate and matrix effect

[0149] According to the CLSI guidelines, the recovery rate and matrix effect are calculated using the following quality control materials, with working concentrations of the quality control materials shown in Table 6.

[0150] Table 6. Concentration of working solution for quality control products

[0151]

[0152] Prepare three mixed quality control working solutions of different concentrations as shown in Table 6, and add them to urine samples to obtain quality control samples in triplicate. Group A contains pure aqueous solution, while groups B and C contain low-value urine.

[0153] The working solution was incorporated into groups A and C. Samples were prepared for groups B and C. After extracting the supernatant, group B was reconstituted using the working solution.

[0154] The relative recovery rate of the analytes was obtained by calculating the ratio of the area ratio of group C analytes to that of group B at the corresponding concentration. The matrix effect of the analytes was obtained by calculating the ratio of the area ratio of group B analytes to that of group A at the corresponding concentration. The results are shown in Table 7.

[0155] Table 7. Relative recovery rates and matrix effects of 11dH-TXB2 and creatinine.

[0156]

[0157]

[0158] (V) Carryover effect

[0159] Residue was assessed by injecting samples containing 11dH-TXB2 and creatinine at their highest linear concentration points, as well as blank samples. Furthermore, during validation, samples were injected in the CLSI-recommended concentration sequence (i.e., medium, high, low, medium, medium, low, low, high, high, medium). Residue was required to be less than 20% of the analyte LLOQ and 5% of the isotopic internal standard. Results showed that this method was free from carryover interference.

[0160] (vi) Stability

[0161] Three quality control solutions of concentrations L, M, and H were selected from Table 6 and added to urine samples with low background values ​​to prepare urine quality control samples. The stability of the urine quality control samples was examined after being placed at room temperature for 6 hours and the stability of the pretreated samples after being refrigerated in an autosampler for 6 hours. The accuracy results are shown in Table 8, and the values ​​in parentheses are relative standard deviations.

[0162] Table 8. Stability data of 11dH-TXB2 and creatinine

[0163]

[0164] (vii) Limit of Quantification

[0165] Urine quality control samples were prepared by adding 11dH-TXB2 and creatinine standard solutions at the lowest limit of quantitation (LOQ) concentration to a urine blank matrix, following the procedure outlined in Example 1 under "Sample Pretreatment". Six samples were prepared and measured at each LOQ concentration to examine the fluctuation of the LOQ. The accuracy results are shown in Table 9, with values ​​in parentheses representing relative standard deviations.

[0166] Table 9. Limits of Quantification (LOQ) for 11dH-TXB2 and creatinine

[0167] Analyte Limit of quantification (CV) 11 dH-TXB2 106.09(3.11) JG 91.38(3.80)

[0168] Example 3: Biological Sample Analysis Data

[0169] The detection method established in Example 1 was applied to the urine of 8 individuals to detect the concentrations of 11dH-TXB2 and creatinine. The concentrations of 11dH-TXB2 and creatinine in the urine are shown in Table 10.

[0170] Table 10. Concentrations of 11dH-TXB2 and JG in the urine of 8 human cases.

[0171] Sample name 11 -DH-Thromboxane (ng / mL) Creatinine (pg / mL) Unine-1 6.45 390.27 Unine-2 2.53 396.31 Unine-3 2.74 471.78 Unine-4 3.44 641.25 Unine-5 4.55 953.66 Unine-6 3.41 269.02 Unine-7 28.19 517.05 Unine-8 6.77 451.48

[0172] Example 4

[0173] Three pretreatment methods were compared: methanol precipitation of protein, acetonitrile precipitation of protein, and solid phase extraction. The methanol precipitation of protein pretreatment method was the same as in Example 1, except that acetonitrile was replaced with an equal amount of methanol. Except for the pretreatment step, the other steps were the same as in Example 1.

[0174] The recovery rates of 11dH-TXB2 and creatinine, as well as the complexity of the pretreatment procedure, were investigated. The results showed that methanol precipitation of proteins provided good separation of 11dH-TXB2 from interferences in urine, with a recovery rate of 93%. Furthermore, the pretreatment procedure was simpler than solid-phase extraction, which is beneficial for clinical testing. Therefore, acetonitrile precipitation of proteins was chosen as the pretreatment condition.

[0175] Example 5

[0176] Comparison of different scanning modes: In view of the problem that high concentrations of creatinine in urine can easily lead to oversaturation of creatinine channels, positive and negative switching modes and negative ion mode alone were investigated to detect 11dH-TXB2 and creatinine. Except for the difference in scanning mode, the other operation steps were the same as in Example 1.

[0177] The results showed that creatinine had an excessively high response in negative ion mode. If positive ion mode was used to scan creatinine, the signal saturation problem at the upper limit of clinical reference JG could be guaranteed. At the same time, the sensitivity of 11dH-TXB2 in negative ion mode could meet the requirements of the lower limit of clinical reference. Therefore, the positive and negative ion switching scanning mode was selected.

[0178] Example 6

[0179] Comparing different chromatographic conditions:

[0180] The effects of different salt solutions (ammonium formate and ammonium fluoride) and the ratios of ammonium formate buffer (0.1 mM, 0.2 mM, and 1 mM) in the mobile phase on the chromatographic separation were compared. The mobile phases for each experimental group are shown in Table 11, and the remaining steps and parameters are the same as in Example 1.

[0181] Table 11 Mobile phase composition of each experimental group

[0182]

[0183]

[0184] Experimental results show that the response is low when the ammonium formate buffer solution in the mobile phase is 0.1 mM and 1.0 mM, while the chromatographic separation effect is better when the ammonium formate buffer solution in the mobile phase is 0.2 mM, with a stable baseline, symmetrical peak shape, and good resolution. Furthermore, the addition of ammonium formate can significantly increase the stability of the chromatographic peak retention time. Therefore, the preferred mobile phase is: phase A (aqueous solution containing 0.2 mM ammonium formate) - phase B (methanol solution containing 0.2 mM ammonium formate).

[0185] After adjusting the elution ratio of the mobile phase at different times, the retention times of each chromatographic peak were moderate, and the baseline was stable and not prone to drift. This improved the resolution of the chromatogram and effectively avoided chromatogram tailing, which is beneficial for the detection and analysis of 11dH-TXB2 and creatinine. Figures 9-12 As shown.

[0186] Example 7

[0187] Comparing different chromatographic columns:

[0188] The effects of three chromatographic columns—Phenomenex Kinetex F5 (2.6 μm, 3 mm × 100 mm), Phenomenex Kinetex C18 (2.6 μm, 3 mm × 100 mm), and Phenomenex Kinetex EVO C18 (2.6 μm, 3 mm × 100 mm)—on the detection results of 11dH-TXB2 and creatinine were compared. Except for the chromatographic columns, all other operating procedures and parameters were the same as in Example 1.

[0189] The results showed that 11dH-TXB2 exhibited moderate retention on the Phenomenex Kinetex F5 column (2.6 μm, 3 mm × 100 mm), with symmetrical peak shapes and good separation from interfering substances in urine. However, its excessive retention on Phenomenex Kinetex C18 (2.6 μm, 3 mm × 100 mm) and Phenomenex Kinetex EVO C18 (2.6 μm, 3 mm × 100 mm) columns led to prolonged analysis time and reduced throughput. Therefore, the Phenomenex Kinetex F5 (2.6 μm, 3 mm × 100 mm) column was selected as the preferred column.

[0190] Example 8

[0191] Comparing different column temperatures:

[0192] The effects of different column temperatures (20℃, 30℃, 40℃, and 50℃) on the detection results of 11dH-TXB2 and creatinine were investigated. The results showed that at a column temperature of 40℃, the chromatographic peak retention time was suitable, the baseline was stable, the resolution of each chromatographic peak was good, and the peak shape was symmetrical. Therefore, a column temperature of 40℃ was selected.

[0193] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously detecting 11-dehydrothromboxane B2 and creatinine, characterized in that, The method includes detecting the pretreated sample using liquid chromatography-tandem mass spectrometry, substituting the detected values ​​into a standard curve, and calculating the contents of 11-dehydrothromboxane B2 and creatinine in the sample. The sample was a urine sample; The pretreatment step includes vortex mixing of the sample and organic phase, separating the supernatant, and then performing liquid chromatography-tandem mass spectrometry detection. The volume ratio of the sample to the organic phase is 1:(0.5~2); The organic phase is acetonitrile; The chromatographic column used for liquid chromatography detection was a Phenomenex Kinetex F5 column, 2.6 μm, 3 mm × 100 mm. The column temperature is 40℃; The mobile phases for liquid chromatography detection include mobile phase A and mobile phase B; mobile phase A is an aqueous solution containing ammonium formate, and mobile phase B is a methanol solution containing ammonium formate. The concentration of ammonium formate in mobile phase A is 0.2 mM; The concentration of ammonium formate in mobile phase B is 0.2 mM; The elution procedure for liquid chromatography is as follows: 0 min, mobile phase A 70% v / v, mobile phase B 30% v / v; 1.5 min, mobile phase A 70% v / v, mobile phase B 30% v / v; 1.6 min, mobile phase A 5% v / v, mobile phase B 95% v / v; 3.7 min, mobile phase A 5% v / v, mobile phase B 95% v / v; 3.8 min, mobile phase A 70% v / v, mobile phase B 30% v / v; 5.0 min, mobile phase A 70% v / v, mobile phase B 30% v / v; Mass spectrometry conditions include: (a) Scanning mode: electrospray ionization, positive and negative switching modes, and internal standard method to construct standard curve; Mass spectrometry conditions also include the following (b) to (c): (b) Acquisition mode: Multiple reaction monitoring; (c) Ion source parameters: Positive ion mode: Electrospray voltage: 5500V; curtain gas: 25psi; atomizing gas: 50psi; auxiliary gas: 65; collision gas: 9; ion source temperature: 500℃; And, negative ion mode: electrospray voltage: -3500V; air curtain gas: 25psi; atomizing gas: 50psi; auxiliary gas: 65; collision gas: 9; ion source temperature: 500℃; The internal standards include d4 isotope-labeled 11-dehydrothromboxane B2 and D3 isotope-labeled creatinine. The mass spectrometry parameters are as follows:

2. The method according to claim 1, characterized in that, The standard curve is the correspondence between the content of the analyte in the standard and the detection value of the standard; the detection value of the standard is the ratio of the peak value of the standard to the internal standard, and the peak value is the peak height or peak area.

3. The method according to claim 2, characterized in that, The standard consists of several standards with a series of concentrations, in which the concentration of 11-dehydrothromboxane B2 is distributed from 0.1 to 50 ng / mL and the concentration of creatinine is distributed from 10 to 5000 ng / mL.

4. The method according to claim 2, characterized in that, The matrix of the standard includes PBS.

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