A method for detecting a uranium chelating compound TAM-2LI-MAM2 in blood plasma

Abstract

This invention discloses a method for detecting the uranium excretion-promoting compound TAM-2LI-MAM2 in plasma, belonging to the field of bioanalytical technology. The method includes: adding buspirone solution to an isolated plasma sample, mixing, centrifuging to precipitate proteins, and using the supernatant as the test sample; sequentially performing liquid chromatography (LC) separation and tandem mass spectrometry (TMS) detection on the test sample; wherein the mobile phase used in the LC separation includes phase A and phase B, where phase A is a 2 mM ammonium acetate aqueous solution containing 0.4 wt% ammonia, and phase B is an acetonitrile solution containing 0.2 wt% ammonia. This detection method has advantages such as good reproducibility, high sensitivity, and fast analysis speed, and can be reliably used to determine the content of uranium excretion-promoting compounds in plasma.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a method for detecting the uranium excretion-promoting compound TAM-2LI-MAM2 in plasma. Background Technology

[0002] With the continuous development of technology, uranium is an important raw material for nuclear energy and is used extensively in the nuclear industry. In the event of a nuclear leak, uranium may diffuse into the environment and enter the body through the respiratory tract, digestive tract, or skin and mucous membranes, circulating throughout the body via the bloodstream, ultimately primarily as uranyl ions (UO2). 2+ Uranium exists in the form of uranium and is deposited in bones and kidneys. Due to the heavy metal toxicity and long-term radioactivity of uranium, uranium ester deposits in the body can cause kidney damage and increase the risk of diseases such as osteosarcoma. Chelation therapy, which uses excretion-inducing agents to effectively bind with uranium esters in the body and excrete them through the metabolic system, can effectively reduce the harm of uranium esters to the human body and is considered the most feasible means to solve this problem.

[0003] The prior art (“In Vivo Uranium Decorporation by a Tailor-Made Hexadentate Ligand”, Bin Chen, et al., Journal of the American Chemical Society, 2022, 144, 11054-11058) discloses a uranium expulsion-promoting compound (TAM-2LI-MAM2), the structure of which is as follows: Mouse ovulation induction experiments confirmed that this compound has excellent in vivo ovulation-inducing effects. After a single intraperitoneal administration, the renal ovulation rate reached an ideal 95%, and the skeletal ovulation rate reached 81%. Compared with previously reported bidentate and tetradentate ligands and clinical ovulation-inducing drugs under the same experimental conditions, the ovulation rate was significantly improved, setting a new record for actinide skeletal ovulation efficiency. Even with delayed administration, this ligand still exhibited a high ovulation rate (renal: 77%; skeletal: 54%).

[0004] Therapeutic drug monitoring and generic drug clinical studies require the measurement of blood drug concentrations in subjects or patients to study their pharmacokinetic behavior. To accelerate the clinical application of this compound, a simple, accurate, rapid, and highly sensitive bioanalytical method is needed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for detecting the uranium excretion-promoting compound TAM-2LI-MAM2 in plasma, which solves the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: A method for detecting the uranium excretion-promoting compound TAM-2LI-MAM2 in plasma for non-diagnostic purposes, comprising the following steps: Add buspirone solution to the isolated plasma sample, mix well, centrifuge to precipitate the protein, and take the supernatant as the sample to be tested; The sample to be tested was sequentially separated by liquid chromatography and detected by tandem mass spectrometry; wherein the mobile phase used in the liquid chromatography separation includes phase A and phase B, wherein phase A is a 2 mM ammonium acetate aqueous solution containing 0.4 vt% ammonia, and phase B is an acetonitrile solution containing 0.2 vt% ammonia.

[0007] Furthermore, the tandem mass spectrometry detection employs multiple reaction monitoring in positive ion mode, the quantitative analysis ion pair of the uranium expulsion compound is m / z 559.3→361.0, and the quantitative analysis ion pair of the buspirone solution is m / z 386.2→122.2.

[0008] Furthermore, the concentration of the buspirone solution is 5 ng / mL.

[0009] Furthermore, the liquid chromatography separation uses a C18 column.

[0010] Furthermore, the flow rate of the mobile phase is 0.5 mL / min to 1.0 mL / min.

[0011] Furthermore, the injection volume for liquid chromatography separation is 1 μL to 10 μL.

[0012] Furthermore, the liquid chromatography separation employs gradient elution, and the elution program is set according to time and mobile phase volume percentage as follows: From 0 to 0.4 min, phase A was 85% and phase B was 15%. Within 0.4–1.2 minutes, phase A decreased to 5%, while phase B increased to 95%. 1.2–1.8 min, phase A maintained at 5%, phase B maintained at 95%; Within 1.8–1.9 minutes, phase A rises to 20%, while phase B decreases to 80%. 1.9–2.5 min, phase A maintained at 20%, phase B maintained at 80%; Within 2.5–2.6 minutes, phase A decreased to 2%, while phase B increased to 98%. 2.6–4.5 min, phase A remains at 2%, phase B remains at 98%; 4.5–4.6 min, phase A rises to 50%, phase B decreases to 50%; 4.6–6.0 min, phase A maintained at 50%, phase B maintained at 50%; 6.0–6.1 min, phase A rises to 85%, phase B drops to 15%; 6.1–8.0 min, phase A is maintained at 85%, and phase B is maintained at 15%.

[0013] The above-mentioned detection method is used in the pharmacokinetic evaluation of uranium excretion-inducing compounds, the application of which includes: quantitatively determining the concentration of TAM-2LI-MAM2 in isolated plasma samples at multiple dosing time points using the detection method according to any one of claims 1 to 7.

[0014] Furthermore, the pharmacokinetic assessment includes constructing plasma drug concentration-time curves based on concentration data obtained from quantitative measurements at various time points to obtain pharmacokinetic parameters.

[0015] A detection kit for determining the concentration of the uranium excretion-inducing compound TAM-2LI-MAM2 in plasma, the kit comprising: Internal standard reagents, including buspirone solution; The chromatographic elution reagent includes separately packaged mobile phase A and mobile phase B, wherein mobile phase A is a 2 mM ammonium acetate aqueous solution containing 0.4 wt% ammonia, and mobile phase B is an acetonitrile solution containing 0.2 wt% ammonia.

[0016] The beneficial effects of this invention are: 1. This invention employs a slightly alkaline mobile phase system containing a specific ratio of ammonia (0.4% ammonia in ammonium acetate aqueous solution and 0.2% ammonia in acetonitrile solution), combined with specific mass spectrometry multiple reaction monitoring ion pairs in positive ion mode (m / z 559.3→361.0), and uses buspirone, whose physicochemical properties are similar to its chromatographic behavior, as an internal standard. The slightly alkaline mobile phase improves the ionization state of the TAM-2LI-MAM2 macromolecule with a multidentate ligand structure in mass spectrometry, thereby enhancing the signal response intensity. The buspirone internal standard effectively corrects the matrix effect bias in plasma sample mass spectrometry analysis. The combination of these two methods results in a limit of quantitation of 10 ng / mL, and the precision and accuracy meet the bioanalytical acceptance criteria.

[0017] 2. This invention employs a specific gradient elution program in the liquid chromatography stage, rapidly stepping the mobile phase B from 15% to 95% and maintaining it in segments. This gradient elution design facilitates the sequential elution of the target compound (retention time approximately 1.18 min) and the internal standard (retention time approximately 3.44 min) within a short time, achieving good baseline separation. Furthermore, the analysis cycle for a single sample is controlled within 8.0 minutes, avoiding prolonged column rinsing and equilibration.

[0018] 3. In the plasma pretreatment step, this invention employs a one-step protein precipitation and extraction process. This involves adding an organic reagent containing an internal standard to a mere 25 μL of plasma sample, vortexing to mix, and then directly centrifuging at high speed (13000 rpm) to separate the supernatant. This pretreatment method eliminates the need for complex solid-phase extraction or liquid-liquid extraction steps, making it simple and easy to operate, and effectively removing most protein interference. Furthermore, only 25 μL of plasma is needed to meet the injection requirements of LC-MS / MS, significantly reducing the difficulty of blood collection from experimental animals and making it possible to obtain complete pharmacokinetic curves at multiple time points in a single mouse.

[0019] 4. This invention uses a C18 column for high-performance liquid chromatography (HPLC) for separation, and a mass spectrometer (MS / MS) with two-stage mass analyzers in series for directional fragmentation monitoring. The multiple reaction monitoring (MRM) mode of the mass spectrometer is equivalent to performing two mass "filters" on the target molecules. Combined with the retention time difference brought about by the C18 column, it effectively eliminates the co-eluting interference of complex endogenous substances in the plasma matrix. No obvious interference peaks were observed at the retention times of the target analyte and the internal standard in the blank plasma, ensuring the reliability of the results of the determination of trace drug concentrations in vivo. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Mass spectrometry scan of fragment ions for TAM-2LI-MAM2; Figure 2 Ion chromatograms of TAM-2LI-MAM2 and internal standard buspirone in blank plasma of CD-1 mice (bottom: TAM-2LI-MAM2, top: buspirone).

[0022] Figure 3 Ion chromatograms of TAM-2LI-MAM2 and internal standard buspirone after adding 100.0 ng / mL standard working solution to CD-1 mouse plasma (bottom: TAM-2LI-MAM2, top: buspirone).

[0023] Figure 4 Mean plasma concentration versus time curves of CD-1 mice after intravenous administration of 5 mg / kg TAM-2LI-MAM2. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] A method for detecting the uranium excretion-promoting compound TAM-2LI-MAM2 in plasma, comprising the following steps: S1. Plasma sample pretreatment: Add internal standard solution to plasma sample, mix well, centrifuge to precipitate protein, and take supernatant to obtain the sample to be tested. S2. The sample obtained in S1 was analyzed by HPLC-MS / MS. The mass spectrometer was supplied with high-purity nitrogen gas throughout the process. The mass spectrometry detection was performed using an electrospray ionization (ESI) source. The scanning mode was positive ion mode with multiple reaction monitoring (MRM) scan and the scan time was 100ms. The detection results were analyzed using Analyst 1.7.2. The uranium excretion-inducing compound is (TAM-2LI-MAM2).

[0026] When using the internal standard method, the selection of the internal standard is a crucial task. An ideal internal standard should be able to be added to the sample in an accurate and known amount, and should have essentially the same or as consistent as possible with the physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be able to completely separate from the components in the sample.

[0027] In some embodiments, the internal standard solution described in this invention is a buspirone solution.

[0028] In chromatography, the selection of the chromatographic column is crucial, requiring high column efficiency, good selectivity, and fast analysis speed. In some embodiments, the HPLC (High Performance Liquid Chromatography) column described in this invention is an Xbridge C18 column or a column with equivalent performance.

[0029] In some embodiments, the chromatographic column has dimensions of 100 mm × 4.6 mm.

[0030] In some embodiments, the packing material of the chromatographic column has a particle size of 3.5 μm.

[0031] In some embodiments, the mobile phase of the HPLC includes phase A and phase B, wherein phase A is a 2 mM ammonium acetate aqueous solution containing 0.4% ammonia and phase B is an acetonitrile solution containing 0.2% ammonia.

[0032] In some embodiments, the HPLC employs gradient elution.

[0033] In some implementations, the gradient elution procedure is shown in Table 1 below: Table 1 Gradient elution program In some embodiments, the flow rate of the mobile phase is 0.5-1.0 mL / min.

[0034] In some embodiments, the flow rate of the mobile phase is 0.6 mL / min.

[0035] In some embodiments, the column temperature of the HPLC is 25°C to 35°C.

[0036] In some embodiments, the column temperature of the HPLC is room temperature.

[0037] In some embodiments, the injection volume of the HPLC is 1-10 μL.

[0038] In some embodiments, the injection volume of the HPLC is 1 μL.

[0039] In some embodiments, the injection volume of the HPLC is 5 μL.

[0040] In some embodiments, the ion source of the mass spectrometer is an electrospray ionization source.

[0041] In some embodiments, the mass spectrometer is used in positive ion mode for multiple reaction monitoring (MRM) scanning, with a scan time of 100 ms.

[0042] In some embodiments, the analytical ion pair for the uranium expulsion compound TAM-2LI-MAM2 in the mass spectrometer is 559.3→361.0; the analytical ion pair for the internal standard is 386.2→122.2.

[0043] In some embodiments, the concentration of the internal standard solution is 5 ng / mL.

[0044] In some embodiments, plasma sample pretreatment includes: taking 25 μL of plasma sample and adding 100 μL of internal standard solution (buspirone solution, concentration of 5 ng / mL) using a continuous pipette, mixing with a multi-tube vortex mixer, centrifuging at 13000 rpm for 15 min in a refrigerated centrifuge to precipitate proteins, and taking 5 μL or 1 μL of the supernatant for LC-MS / MS analysis.

[0045] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0046] In the following examples, unless otherwise specified, all temperatures are in Celsius, and unless otherwise specified, all starting materials and reagents are commercially available or synthesized according to known methods.

[0047] Unless otherwise specified in the examples, the solutions used refer to aqueous solutions.

[0048] Unless otherwise specified in the examples, the room temperature is 20℃~30℃.

[0049] Example 1 1. Materials and Instruments The chromatography-mass spectrometry system consists of a Shimadzu LC-40D liquid chromatography system from Japan and an ABSciex TripleQuad5500+ triple quadrupole mass spectrometer from the United States.

[0050] The uranium excretion-promoting compound TAM-2LI-MAM2 was obtained from the State Key Laboratory of Radiation Medicine and Radiation Protection at Soochow University.

[0051] 2. Liquid chromatography-mass spectrometry (LC-MS) conditions Chromatographic conditions: The internal standard was buspirone; mobile phase A consisted of 2 mM ammonium acetate aqueous solution (0.4% ammonia), and mobile phase B consisted of acetonitrile (0.2% ammonia); the chromatographic column was an Xbridge C18 column with dimensions of 100 mm × 4.6 mm and a particle size of 3.5 μm; the column temperature was set to room temperature, the mobile phase flow rate was 0.6 mL / min, the injection volume was 5 μL, the run time was 8.0 min, and gradient elution was used. The mobile phase gradient is shown in Table 2 below.

[0052] The mass spectrometer used high-purity nitrogen as the gas supply throughout the entire process. Mass spectrometry detection employed electrospray ionization (ESI) with multiple reaction monitoring (MRM) in positive ion mode for a scan time of 100 ms. Ion information for the uranium-induced expulsion compound TAM-2LI-MAM2 and the internal standard (IS) buspirone is shown in Table 2.

[0053] Table 2. Mobile phase gradient and reactant ion information for the detection of TAM-2LI-MAM2 and buspirone (internal standard). Note: In Table 2, the mass spectrometry voltage parameters are represented as follows: DP represents declustering voltage, CE represents collision energy, EP represents inlet voltage, and CXP represents collision chamber outlet voltage.

[0054] 3. Methods and Steps 3.1 Preparation of standard reference solution Accurately weigh an appropriate amount of TAM-2LI-MAM2 reference standard, dissolve it in DMSO and dilute to volume to prepare a stock solution with a TAM-2LI-MAM2 concentration of 1.00 mg / mL.

[0055] 3.2 Preparation of working solutions for the standard curve Accurately pipette appropriate amounts of each stock solution and dilute them stepwise with human methanol to obtain a series of standard samples. The concentration range of TAM-2LI-MAM2 is 10.00–4000 ng / mL.

[0056] 3.3 Preparation of internal standard solution Accurately weigh buspirone reference standard, dissolve it in methanol and dilute to volume to prepare an internal standard stock solution with a concentration of approximately 1.00 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution and dilute it with acetonitrile:water (50:50, v / v) to obtain an internal standard solution with a buspirone concentration of 5 ng / mL.

[0057] 3.4 Plasma Sample Pretreatment After taking 25 μL of plasma sample, add 100 μL of internal standard solution from the same batch as the standard curve using a continuous pipette. Mix well using a multi-tube vortex mixer and centrifuge at 13000 rpm for 15 min in a refrigerated centrifuge to precipitate the protein. Take the supernatant and inject it for analysis (injection volume is 1 µL) for LC-MS / MS analysis of the sample.

[0058] In this embodiment, the specific steps for LC-MS / MS analysis of the sample are as follows: the mass spectrometer uses high-purity nitrogen as the gas supply throughout the process, the mass spectrometry detection adopts the form of electrospray ionization (ESI), the scanning mode is positive ion mode and multiple reaction monitoring (MRM) is performed, the scanning time is 100ms, and the detection results are analyzed using Analyst 1.7.2.

[0059] Example 2 In this embodiment, the detection method of Example 1 was validated according to the guidelines of Chinese Pharmacopoeia 9012, including selectivity, linearity, accuracy, and precision.

[0060] 1) Selectivity: Blank plasma and separately prepared samples for the lower limit of quantitation (LOQ) were processed and then injected for analysis. The peak area of ​​the chromatographically eluted interfering substances must be less than 20% of the peak area of ​​the analyte at the LOQ and less than 5% of the peak area of ​​the internal standard.

[0061] 2) Standard curve: The linear regression equation (weighting factor W=1 / x) is calculated by using the theoretical concentration of the analyte as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis. 2 The method was validated by performing two-sample analysis on each analytical batch of the standard curve samples.

[0062] 3) Precision and accuracy: Six samples from each of the three concentration quality control samples were measured. For each concentration level, the intra-batch and inter-batch precision of each component in the QC samples must be less than 15% to be acceptable, and the accuracy must be between -15% and 15% to be acceptable.

[0063] 4) Pharmacokinetic studies The concentration of the uranium excretion-inducing compound TAM-2LI-MAM2 in plasma samples was analyzed using the method established above, and the pharmacokinetic study of TAM-2LI-MAM2 in mice was conducted. Three ICR mice were intravenously administered 5 mg / kg of TAM-2LI-MAM2 at a volume of 5 mL / kg, with Saline (pH adjusted to 7-8 with NaOH) as the solvent. At different time points before administration (0 h) and within 24 h after administration, 30 μL of venous blood was collected and placed in K2EDTA anticoagulant centrifuge tubes. After centrifugation (4600 g, 4 °C) for 5 min, the plasma was separated and stored at -60 °C or below.

[0064] Results and Discussion: like Figure 1 As shown, the quantitative analysis of the uranium expulsion-inducing compound TAM-2LI-MAM2 confirmed the ion pair: m / z 559.3→361.0.

[0065] like Figures 2-3 As shown, the detection results were analyzed using Analyst 1.7.2. The retention times of uranium excretion-promoting compounds TAM-2LI-MAM2 and buspirone were approximately 1.18 min and 3.44 min, respectively. The similar retention times indicate that buspirone is a better internal standard choice. Moreover, the blank plasma sample showed no co-elution interference peaks at the corresponding retention times, indicating that the method has good selectivity for uranium excretion-promoting compounds TAM-2LI-MAM2 and buspirone.

[0066] The linear range of TAM-2LI-MAM2 in plasma samples from the clinical study was 10-4000 ng / mL. The typical linear regression equations for the standard curve of the analyte were: TAM-2LI-MAM2: y=0.0000367x-0.000161 (r=0.9925); The limit of quantitation (LOQ) in the sample had a TAM-2LI-MAM2 concentration of 10 ng / mL and a signal-to-noise ratio greater than 20.

[0067] The precision and accuracy results both meet the acceptance criteria, and the specific results are shown in Table 3.

[0068] Table 3. Precision and accuracy of TAM-2LI-MAM2 determination in mouse plasma. The validated method was used to analyze TAM-2LI-MAM2 in plasma to evaluate its pharmacokinetic characteristics. Three ICR mice were intravenously administered 5 mg / kg of TAM-2LI-MAM2; the mean plasma drug concentration-time curves are shown below. Figure 4 It can be seen that the detection method can fully depict the pharmacokinetic characteristics of TAM-2LI-MAM2, and the linear range is close to the concentration level of the actual sample. The accuracy of the measurement is high, and the average plasma drug concentration-time curve can observe the complete elimination process in mice. Based on this elimination process, it can be inferred that TAM-2LI-MAM2 is eliminated in mice in phase II, that is, the elimination rate constant will be prolonged as the concentration in the body decreases.

[0069] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma, characterized in that, Includes the following steps: Add buspirone solution to the isolated plasma sample, mix well, centrifuge to precipitate the protein, and take the supernatant as the sample to be tested; The sample to be tested was sequentially separated by liquid chromatography and detected by tandem mass spectrometry; wherein the mobile phase used in the liquid chromatography separation includes phase A and phase B, wherein phase A is a 2 mM ammonium acetate aqueous solution containing 0.4 vt% ammonia, and phase B is an acetonitrile solution containing 0.2 vt% ammonia.

2. The method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The tandem mass spectrometry detection was performed using multiple reaction monitoring in positive ion mode. The quantitative analysis ion pair of the uranium expulsion compound was m / z 559.3→361.0, and the quantitative analysis ion pair of the buspirone solution was m / z 386.2→122.

2.

3. The method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The concentration of the buspirone solution was 5 ng / mL.

4. The method for detecting uranium excretion-inducing compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The liquid chromatography separation was performed using a C18 column.

5. The method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The flow rate of the mobile phase is 0.5 mL / min to 1.0 mL / min.

6. The method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The injection volume for liquid chromatography separation is 1 μL to 10 μL.

7. The method for detecting uranium excretion-promoting compound TAM-2LI-MAM2 in non-diagnostic plasma according to claim 1, characterized in that, The liquid chromatography separation employs gradient elution, and the elution program is set as follows based on time and mobile phase volume percentage: From 0 to 0.4 min, phase A was 85% and phase B was 15%. Within 0.4–1.2 minutes, phase A decreased to 5%, while phase B increased to 95%. 1.2–1.8 min, phase A maintained at 5%, phase B maintained at 95%; Within 1.8–1.9 minutes, phase A rises to 20%, while phase B decreases to 80%. 1.9–2.5 min, phase A maintained at 20%, phase B maintained at 80%; Within 2.5–2.6 minutes, phase A decreased to 2%, while phase B increased to 98%. 2.6–4.5 min, phase A remains at 2%, phase B remains at 98%; 4.5–4.6 min, phase A rises to 50%, phase B decreases to 50%; 4.6–6.0 min, phase A maintained at 50%, phase B maintained at 50%; 6.0–6.1 min, phase A rises to 85%, phase B drops to 15%; 6.1–8.0 min, phase A is maintained at 85%, and phase B is maintained at 15%.

8. The application of the detection method according to any one of claims 1-7 in the pharmacokinetics evaluation of uranium excretion-inducing compounds, characterized in that, The application includes: quantitatively determining the concentration of TAM-2LI-MAM2 in isolated plasma samples at multiple drug administration time points using the detection method according to any one of claims 1 to 7.

9. The application according to claim 8, characterized in that, The pharmacokinetic assessment includes constructing plasma drug concentration-time curves based on concentration data obtained from quantitative measurements at various time points to obtain pharmacokinetic parameters.

10. A detection kit for determining the concentration of the uranium excretion-inducing compound TAM-2LI-MAM2 in plasma, characterized in that, The kit contains: Internal standard reagents, including buspirone solution; The chromatographic elution reagent includes separately packaged mobile phase A and mobile phase B, wherein mobile phase A is a 2 mM ammonium acetate aqueous solution containing 0.4 wt% ammonia, and mobile phase B is an acetonitrile solution containing 0.2 wt% ammonia.

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