Neuroblastoma and catecholamine-related tumor diagnostic kit and application thereof

Abstract

The present application relates to the field of clinical laboratory medicine technology, in particular to a neuroblastoma and catecholamine-related tumor diagnosis kit and its application. The kit contains key components for detecting high vanillylmandelic acid and vanillylmandelic acid in urine, including isotope internal standard, specific chromatographic column, optimized mobile phase and sample pretreatment material. The detection method using the kit optimizes the collection, preservation and pretreatment process of instant urine samples, and combines the liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis conditions designed for low concentration and complex matrix samples to realize the rapid and accurate quantification of trace HVA and VMA in urine. It overcomes the shortcomings of traditional 24-hour urine detection method such as large trauma, long cycle and poor accuracy, and provides an efficient and reliable solution for non-invasive, early diagnosis and postoperative efficacy monitoring of neuroblastoma and other catecholamine-related tumors, which has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of clinical laboratory medicine technology, specifically to diagnostic kits for neuroblastoma and catecholamine-related tumors and their applications. Background Technology

[0002] Neuroblastoma (NB) is the most common extracranial malignant tumor in children, primarily affecting those under 5 years old. Early diagnosis and postoperative recurrence monitoring rely heavily on accurate detection of tumor markers. Homovanillic acid (HVA), a terminal metabolite of dopamine, and vanillylmandelic acid (VMA), a terminal metabolite of adrenaline and noradrenaline, are both involved in the production and release of catecholamines and their metabolites by tumor cells in patients with catecholamine-secreting tumors such as neuroblastoma. This results in a significant increase in the excretion of HVA and VMA in the urine. Therefore, quantitative detection of urinary HVA and VMA has become a core biochemical marker for the clinical diagnosis, risk stratification, efficacy evaluation, and recurrence monitoring of neuroblastoma. Changes in their levels directly reflect tumor activity and are crucial for clinical diagnosis and treatment.

[0003] Currently, the standard clinical method for detecting urinary HVA and VMA is to collect 24-hour urine samples for testing. However, this method has several insurmountable drawbacks: 1) The collection process is complicated and invasive: all urine needs to be collected continuously for 24 hours. Young children (the main population of neuroblastoma patients) have very low cooperation. Parents often need to supervise the whole process (including getting up frequently at night to collect urine). This not only disturbs the sleep of the children and parents, but also easily causes children to fear medical procedures, creating a double burden on both the physiological and psychological levels. 2) Poor controllability of sample quality: 24-hour urine collection is prone to omissions (such as when children urinate unconsciously and are not collected), resulting in low test results; preservatives such as glacial acetic acid need to be added, and long-term storage (such as more than 24 hours) is prone to HVA / VMA degradation, which further affects the accuracy of the results; 3) Long testing cycle and low efficiency: It takes 2 to 3 days from the start of urine collection to the completion of testing, which cannot meet the needs of rapid clinical diagnosis or emergency follow-up.

[0004] In terms of detection technology, existing HVA / VMA detection methods have limitations: Traditional methods (electrochemical methods, ELISA methods): sensitivity is only at the ng / mL level, specificity is insufficient, and they are easily interfered with by uric acid, vitamin C and dietary impurities (such as vanillin in chocolate and bananas). The false positive / false negative rate is high, making it difficult to meet the detection needs of low-concentration samples (such as early tumors or minimal residual lesions). Emerging LC-MS / MS methods: Although they possess high sensitivity (pg / mL level) and high specificity, existing technical solutions are all designed for 24-hour urine samples and are not adapted to immediate urine samples (single random urine). While immediate urine samples have the advantages of convenient collection and no retention time limit, the lower HVA / VMA concentration (approximately 1 / 10 to 1 / 20 of that in 24-hour concentrated urine samples) and more complex matrix interference (greater fluctuations in metabolic impurities in a single urine sample) make it difficult for existing LC-MS / MS methods to achieve accurate quantification and thus prevent their application in clinical diagnosis and treatment.

[0005] In summary, existing technologies lack a solution that adapts to the characteristics of immediate urine samples to achieve non-invasive, highly accurate, and efficient detection of HVA and VMA in urine. This limits the efficiency of early diagnosis of neuroblastoma and the convenience of postoperative follow-up, and targeted improvements are urgently needed. Summary of the Invention

[0006] The purpose of this invention is to provide a diagnostic kit for neuroblastoma and catecholamine-related tumors and its application, which solves the problem that existing technologies are difficult to adapt to the characteristics of immediate urine samples, and achieves non-invasive, highly accurate and efficient detection of HVA and VMA in urine.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a diagnostic kit for neuroblastoma and catecholamine-associated tumors, comprising the following core components: Isotope internal standard solutions, containing HVA-d5 and VMA-d3, are used to correct for sample pretreatment losses and differences in mass spectrometry ionization efficiency; For chromatographic columns, Poroshell 120 EC-C18 (50 mm × 2.1 mm, 2.7 μm) or ACQUITY UPLCHSS PFP (2.1 × 100 mm, 1.8 μm) are preferred to effectively separate HVA and VMA. The mobile phase system consists of mobile phase A, which is an ammonium acetate solution containing 0.1% formic acid; and mobile phase B, which contains... A 0.1% formic acid solution in acetonitrile is beneficial for the ionization of target analytes and improves the chromatographic peak shape; For solid-phase extraction columns used in sample pretreatment, Waters Oasis MAX columns are preferred; for batch detection, WCX (4.0 mg) + MAX (3.0 mg) tandem columns are optional; used for enriching low concentrations of target analytes and removing matrix interference. Glacial acetic acid solution used to prevent sample degradation.

[0008] The method of using the neuroblastoma and catecholamine-associated tumor diagnostic kit includes the following steps: S1. Sample collection and preprocessing; Collect a single, immediate urine sample of 5–10 mL and place it in a sterile polyethylene urine cup. Immediately after collection, add 0.1% glacial acetic acid (e.g., 5 μL of glacial acetic acid for 5 mL of urine sample) to the urine cup and gently shake to avoid HVA / VMA degradation.

[0009] Take a portion of the urine sample, dilute it, add ultrapure water (LC-MS grade), and vortex mix for 1 min. (3000 r / min), reduce the matrix concentration; Centrifuge at 10,000 rpm for 5 minutes (at room temperature) to remove cell debris and protein precipitate; Microfiltration: The supernatant is filtered through a 0.22μm aqueous membrane (polyethersulfone), and the filtrate is collected. Prepare for use in order to obtain the test solution.

[0010] For samples with extremely low target analyte concentrations or exceptionally complex matrices, selective addition of solids can be considered. Phase extraction steps are performed to further enrich and purify the product.

[0011] S2, liquid chromatography separation; The test solution was separated by gradient elution using a chromatographic column and mobile phase; the processed test solution was then injected... Integrate into the LC-MS system. Use the specified column and mobile phase, and execute the optimized gradient elution program: 0–1 min, 2% mobile phase; In 1–2 minutes, the mobile phase content increased from 2% to 80%. 2–3.5 min, maintaining 80% mobile phase; 3.5–4 min, 80% of the mobile phase decreases to 2% of the mobile phase; 4–4.5 min, maintain 2% mobile phase; Baseline separation of HVA and VMA was achieved at a flow rate of 0.4 mL / min.

[0012] The chromatographic column temperature is 30–35 °C; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an ammonium acetate solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

[0013] S3, Mass spectrometry detection; The separated components are analyzed using either electrospray ionization in negative ion mode or multiple reaction monitoring mode. Mass spectrometry analysis was used to detect characteristic ion pairs of HVA and VMA. Among them, the characteristic ion pairs of HVA are parent ion 181.0 → daughter ion 137.0 (collision energy 8-35 eV) and parent ion 181.0 → daughter ion 122.0 (collision energy 14-35 eV). The characteristic ion pairs of VMA are parent ion 197.1 → daughter ion 138.1 (collision energy 11-35 eV) and parent ion 197.1 → daughter ion 137.1 (collision energy 20 eV). The characteristic ion pair of the internal standard HVA-d5 is 186.1 for the parent ion and 142.1 for the daughter ion, while the characteristic ion pair of the internal standard VMA-d3 is 200.0 for the parent ion and 138.0 for the daughter ion.

[0014] The capillary voltage is 3.0–3.2 kV; the source temperature is 150–300 °C; the desolvation gas temperature is 500–600 °C; and the desolvation gas flow rate is 900–1100 L / h.

[0015] S4. Quantitative analysis; Using the isotope internal standard method, a series of concentration standard curves are first prepared using the standards in the kit. By comparing the peak area ratio of the target analyte to the internal standard in the test sample and substituting it into the standard curve equation, the accurate concentrations of HVA and VMA in urine can be calculated.

[0016] Preparation of standard curve: Stock solution: Prepare a 2 mg / mL stock solution of HVA / VMA standard with methanol and store at -80°C protected from light. A series of standard working solutions: diluted with pretreated mixed immediate urine from healthy individuals to 20, 50, 100, 500, 2000, 10000, 20000 ng / mL (HVA) and 0.5, 1.0, 5.0, 10.0, 50.0, 100.0 ng / mL (VMA), covering the concentration range of immediate urine; Linear regression: Plot a curve with "standard concentration (x-axis)" vs "standard peak area / internal standard peak area (y-axis)" and a weight factor of 1 / x², requiring r² ≥ 0.99.

[0017] Method validation metrics: Limits of detection (LOD): HVA 0.25–10 ng / mL, VMA 0.10–5 ng / mL; Limits of quantitation (LOQ): HVA 1.00–156.25 ng / mL, VMA 0.40–78.13 ng / mL; Precision: Intra-batch CV 2.0%–10.1%, inter-batch CV 2.1%–12.5% ​​(both < 15%); Recovery rate: 88%–104% (spiking concentration 20–15000 ng / mL); Matrix effect: 85.25%~109.88%.

[0018] Preferably, for samples with extremely low target analyte concentrations or exceptionally complex matrices, a solid-phase extraction step is added: For solid phase extraction column selection, Waters Oasis MAX column is preferred; for batch detection, WCX (4.0mg) + MAX (3.0mg) tandem column is an option. Activation and equilibration: The solid-phase extraction column was activated with 600 μL of methanol and then equilibrated with 600 μL of ultrapure water (flow rate 0.3 mL / min). For sample loading and rinsing, take 200 μL of the filtered filtrate, add 20 μL of isotope internal standards (HVA-d5, VMA-d3, final concentration 10 ng / mL), mix well and load the sample; rinse with 600 μL of ultrapure water and 600 μL of methanol to remove impurities. After elution and resolubilization, elute with 200 μL of 2% formic acid-methanol solution, dry under nitrogen at 30°C, and then resolubilize with 100 μL of ultrapure water for instrumental analysis.

[0019] On the other hand, the present invention also provides the application of the aforementioned neuroblastoma and catecholamine-associated tumor diagnostic kit and its method of use in the preparation of diagnostic products for the auxiliary diagnosis or detection of neuroblastoma and catecholamine-associated tumors.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. The diagnostic kit for neuroblastoma and catecholamine-associated tumors involved in this invention and its application are non-invasive, which facilitates a better patient experience. Using immediate urine instead of 24-hour urine, sample collection takes only a few minutes, greatly reducing the pain and fear of children (especially young children). Patient cooperation rate has increased from approximately 30% to over 90%, while also relieving families of the heavy burden of waiting day and night to collect urine, reflecting humanistic care.

[0021] 2. The diagnostic kit for neuroblastoma and catecholamine-associated tumors involved in this invention and its application significantly improve the efficiency of detecting HVA and VMA in urine, facilitating a shorter diagnosis and treatment cycle. The entire process from sample collection to obtaining test results can be completed within hours, much faster than the 2-3 days required by traditional methods. This meets the needs of rapid clinical diagnosis and urgent condition assessment, and helps to shorten the diagnosis and treatment cycle.

[0022] 3. The diagnostic kit for neuroblastoma and catecholamine-related tumors involved in this invention and its application result in high accuracy in detecting HVA and VMA in urine, reducing the rate of misdiagnosis / missed diagnosis. The optimized LC-MS method can achieve detection limits for HVA and VMA in real-time urine as low as ng / mL or even pg / mL, effectively detecting low concentrations of biomarkers released by early tumors or minimal residual disease, reducing the risk of missed diagnosis; it has high specificity, and through MRM mode and isotope internal standard correction, combined with chromatographic retention time locking, it can effectively eliminate interference from various complex matrices in urine such as uric acid and vitamin C, with false positive and false negative rates of less than 5%; it has the advantage of good stability, and the optimized sample preservation and storage scheme (e.g., storage at -80℃ for 30 days) ensures the stability of samples and target analytes, supporting remote sample transport and centralized testing.

[0023] 4. The diagnostic kit for neuroblastoma and catecholamine-related tumors involved in this invention is flexible and highly scalable. It includes a basic pretreatment process and an optional SPE enrichment process, adaptable to laboratory conditions and testing needs at different levels, from primary hospitals to top-tier medical centers. Besides neuroblastoma, it can also be widely used for the diagnosis and monitoring of other tumors causing abnormal catecholamine metabolism, possessing broad clinical application prospects and market value. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the entire process of using the neuroblastoma and catecholamine-related tumor diagnostic kit of the present invention. Detailed Implementation

[0025] 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.

[0026] Example 1: The diagnostic kit for neuroblastoma and catecholamine-associated tumors of the present invention comprises the following components: Isotope internal standard solutions, containing HVA-d5 and VMA-d3, are used to correct for sample pretreatment losses and differences in mass spectrometry ionization efficiency; For chromatographic columns, Poroshell 120 EC-C18 (50 mm × 2.1 mm, 2.7 μm) or ACQUITY UPLCHSS PFP (2.1 × 100 mm, 1.8 μm) are preferred to effectively separate HVA and VMA. The mobile phase system consists of mobile phase A, which is an ammonium acetate solution containing 0.1% formic acid; and mobile phase B, which contains... A 0.1% formic acid solution in acetonitrile is beneficial for the ionization of target analytes and improves the chromatographic peak shape; For solid-phase extraction columns used in sample pretreatment, Waters Oasis MAX columns are preferred; for batch detection, WCX (4.0 mg) + MAX (3.0 mg) tandem columns are optional; used for enriching low concentrations of target analytes and removing matrix interference. Glacial acetic acid solution used to prevent sample degradation.

[0027] Example 2: Specific steps for using the neuroblastoma and catecholamine-associated tumor diagnostic kit: Taking the immediate urine sample from a child undergoing follow-up after neuroblastoma surgery as an example: S1. Sample Collection and Preservation: Instruct the child to collect approximately 10 mL of morning midstream urine in a sterile polyethylene urine cup. Immediately add 10 μL of glacial acetic acid using a micropipette and gently mix. Record the information and place in a 4°C freezer for testing within 24 hours. For long-term storage, freeze at -80°C (good stability within 30 days; based on previous experimental data: HVA / VMA concentration variation coefficient < 10% after 30 days of storage at -80°C).

[0028] S2. Sample preprocessing (basic workflow): Take 1 mL of the mixed urine sample into a centrifuge tube, add 2 mL of ultrapure water, and vortex for 1 minute (3000 r / min) to reduce the matrix concentration.

[0029] Centrifuge at 10,000 rpm for 5 minutes at room temperature to remove cell debris and protein precipitate.

[0030] Carefully aspirate the supernatant and filter it through a 0.22μm aqueous syringe filter to collect the filtrate.

[0031] Take 50 μL of filtrate, add 450 μL of initial mobile phase (98% A + 2% B) and 5 μL of isotope internal standard working solution, vortex to mix, and use as the test solution.

[0032] S3. Sample pretreatment (SPE enrichment process, suitable for low concentration samples): SPE column selection: Waters Oasis MAX column is preferred, and WCX (4.0mg) + MAX (3.0mg) tandem column can be selected for batch detection.

[0033] The SPE column was activated with 600 μL of methanol and then equilibrated with 600 μL of ultrapure water (flow rate 0.3 mL / min).

[0034] Take 200 μL of the filtrate after filtration in step S2, add 20 μL of isotope internal standard working solution (HVA-d5, VMA-d3, final concentration 10 ng / mL), mix well and then load the sample.

[0035] Load the sample mixture onto the container and rinse it sequentially with 600 μL of ultrapure water and 600 μL of methanol.

[0036] The target analyte was eluted with 200 μL of 2% formic acid-methanol solution, and the eluent was collected.

[0037] The eluent was dried with gentle nitrogen gas in a 30°C water bath.

[0038] The residue was redissolved in 100 μL of ultrapure water and vortexed to mix, thus becoming the test solution.

[0039] S4, LC-MS / MS analysis: The test solution was separated by gradient elution using a chromatographic column and mobile phase; the processed test solution was then injected... Integrate into the LC-MS system. Use the specified column and mobile phase, and execute the optimized gradient elution program: 0–1 min, 2% mobile phase; In 1–2 minutes, the mobile phase content increased from 2% to 80%. 2–3.5 min, maintaining 80% mobile phase; 3.5–4 min, 80% of the mobile phase decreases to 2% of the mobile phase; 4–4.5 min, maintain 2% mobile phase; Baseline separation of HVA and VMA was achieved at a flow rate of 0.4 mL / min.

[0040] The column temperature was 35℃; the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A contained an ammonium acetate solution of 0.1% formic acid, and mobile phase B contained an acetonitrile solution of 0.1% formic acid.

[0041] The separated components are analyzed using either electrospray ionization in negative ion mode or multiple reaction monitoring mode. Mass spectrometry analysis was used to detect characteristic ion pairs of HVA and VMA. Among them, the characteristic ion pairs of HVA are parent ion 181.0 → daughter ion 137.0 (collision energy 8-35 eV) and parent ion 181.0 → daughter ion 122.0 (collision energy 14-35 eV). The characteristic ion pairs of VMA are parent ion 197.1 → daughter ion 138.1 (collision energy 11-35 eV) and parent ion 197.1 → daughter ion 137.1 (collision energy 20 eV). The characteristic ion pair of the internal standard HVA-d5 is 186.1 for the parent ion and 142.1 for the daughter ion, while the characteristic ion pair of the internal standard VMA-d3 is 200.0 for the parent ion and 138.0 for the daughter ion.

[0042] The capillary voltage is 3.0–3.2 kV; the source temperature is 150–300 °C; the desolvation gas temperature is 500–600 °C; and the desolvation gas flow rate is 900–1100 L / h.

[0043] S5. Quantitative Analysis: Using a mixed matrix of healthy human urine, a series of standard working solutions were prepared with HVA concentrations of 20, 50, 100, 500, 2000, 10000, and 20000 ng / mL, and VMA concentrations of 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 ng / mL. These solutions were then processed and analyzed using the same method.

[0044] Plot a standard curve using a linear regression with the concentration of the standard as the x-axis and the ratio of the peak area of ​​the standard to the corresponding peak area of ​​the internal standard as the y-axis, weighted by 1 / x². The correlation coefficient r² should be ≥ 0.99.

[0045] The peak area ratios of the samples to be tested are substituted into their respective standard curve equations to calculate the concentrations of HVA and VMA in urine.

[0046] Example 3: Validation of the usage method and results of the diagnostic kit for neuroblastoma and catecholamine-related tumors. To verify the reliability of this method, a comprehensive methodological validation was conducted: Linearity range: HVA showed good linearity in the range of 20–20000 ng / mL and VMA in the range of 0.5–100 ng / mL, with r² greater than 0.995 for both.

[0047] Sensitivity: The limit of quantitation (LOQ) for HVA is 1.0 ng / mL, and the LOQ for VMA is 0.4 ng / mL.

[0048] Precision: For quality control samples at three concentration levels (low, medium, and high), the intra-batch precision (CV) is 2.0%–8.5%, and the inter-batch precision (CV) is 2.1%–10.8%.

[0049] Accuracy (recovery): The average recoveries of HVA and VMA were between 92% and 105% in the range of spiked concentrations of 20 to 15,000 ng / mL.

[0050] Matrix effect: Five different sources of immediate urine matrix were examined. The matrix effect of HVA and VMA ranged from 88% to 112%, with a coefficient of variation of <15%, indicating that matrix interference was effectively controlled.

[0051] Stability: After immediate urine samples were stored at 4°C for 24 hours or at -80°C for 30 days, the concentration changes of HVA and VMA were less than ±10%.

[0052] The above embodiments demonstrate that the kit and method provided by the present invention can accurately, stably, and efficiently detect HVA and VMA in real-time urine, fully meeting the needs of clinical diagnosis and follow-up monitoring of neuroblastoma.

[0053] Example 4: Application of a diagnostic kit for neuroblastoma and catecholamine-associated tumors and its usage in preoperative diagnosis. Timing of testing: When a child suspected of having neuroblastoma (with symptoms such as abdominal pain, abdominal mass, or unexplained fever) first seeks medical attention; Diagnostic criteria: Refer to the age-specific normal range for children (e.g., for children aged 3-5 years, the upper limit of normal for immediate urine HVA is ≤50 ng / mL, and the upper limit of normal for VMA is ≤40 ng / mL). If the concentration of HVA and / or VMA in the sample exceeds twice the upper limit of normal, combined with imaging examinations (e.g., abdominal CT / MRI) and pathological biopsy results, neuroblastoma is diagnosed.

[0054] Example 5: Application of a diagnostic kit for neuroblastoma and catecholamine-associated tumors and its usage in postoperative follow-up. Testing frequency: 1 month, 3 months, 6 months and 12 months after surgery, and then once a year thereafter (for 3 years, the interval can be extended if there is no recurrence). Judgment criteria: ① If the HVA / VMA concentration drops to the normal range in the first postoperative test and remains normal in subsequent follow-ups, it indicates that the surgical resection was thorough and there was no recurrence; ② If the HVA / VMA concentration drops to normal after surgery and then rises again (e.g., from 30 ng / mL to 80 ng / mL), or remains above the normal range, it indicates a risk of tumor recurrence and requires immediate whole-body imaging examinations (e.g., whole-body bone scan, bone marrow aspiration) to confirm.

[0055] It should be noted that in this article, relational terms such as first and second are only used to refer to... Distinguishing one entity or operation from another does not necessarily require or imply any such actual relationship or order between those entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A diagnostic kit for neuroblastoma and catecholamine-associated tumors, characterized in that, include: Isotope internal standard solution containing HVA-d5 and VMA-d3; The chromatographic column was selected from Poroshell 120 EC-C18 column or ACQUITY UPLC HSS PFP column; Mobile phase A is an ammonium acetate solution containing 0.1% formic acid; Mobile phase B is an acetonitrile solution containing 0.1% formic acid; Solid-phase extraction columns for sample pretreatment; as well as Glacial acetic acid solution used to prevent sample degradation.

2. The diagnostic kit for neuroblastoma and catecholamine-associated tumors according to claim 1, characterized in that: The solid-phase extraction column is selected from Waters Oasis MAX column or WCX and MAX tandem column.

3. The diagnostic kit for neuroblastoma and catecholamine-associated tumors according to claim 2, characterized in that: It also includes HVA and VMA standard stock solutions for preparing standard curves.

4. A method of using the diagnostic kit for neuroblastoma and catecholamine-associated tumors as described in claim 1, characterized in that, Includes the following steps: S1. Sample collection and preprocessing; Collect immediate urine samples, add glacial acetic acid as a preservative, dilute, centrifuge, and filter to obtain the filtrate; S2, liquid chromatography separation; The test solution was separated by gradient elution using a chromatographic column and mobile phase. S3, Mass spectrometry detection; The separated components are analyzed using either electrospray ionization in negative ion mode or multiple reaction monitoring mode. Mass spectrometry analysis was used to detect characteristic ion pairs of HVA and VMA. S4. Quantitative analysis; Using the isotope internal standard method, the concentrations of HVA and VMA in urine are calculated based on the mass spectrometry signal intensity obtained in step S4.

5. The method of using the diagnostic kit for neuroblastoma and catecholamine-associated tumors according to claim 4, characterized in that: In step S1, the immediate urine sample is a single, randomly collected urine sample with a volume of 5–10 mL; the volume of glacial acetic acid added is 0.1% of the urine sample volume.

6. The method of using the neuroblastoma and catecholamine-associated tumor diagnostic kit according to claim 4, characterized in that, The gradient elution procedure in step S2 is as follows: 0–1 min, 2% mobile phase; In 1–2 minutes, the mobile phase content increased from 2% to 80%. 2–3.5 min, maintaining 80% mobile phase; 3.5–4 min, 80% of the mobile phase decreases to 2% of the mobile phase; 4–4.5 min, maintaining a 2% mobile phase; flow rate 0.4 mL / min; The chromatographic column temperature is 30–35 °C; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an ammonium acetate solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

7. The method of using the neuroblastoma and catecholamine-associated tumor diagnostic kit according to claim 4, characterized in that: In step S3, the characteristic ion pairs of HVA are mother ion 181.0 → daughter ion 137.0 and mother ion 181.0 → daughter ion 122.0; The characteristic ion pairs of VMA are parent ion 197.1 → daughter ion 138.1 and parent ion 197.1 → daughter ion 137.1; The characteristic ion pair of the internal standard HVA-d5 is 186.1 for the parent ion and 142.1 for the daughter ion, while the characteristic ion pair of the internal standard VMA-d3 is 200.0 for the parent ion and 138.0 for the daughter ion.

8. The method of using the neuroblastoma and catecholamine-associated tumor diagnostic kit according to claim 4, characterized in that: In step S1, when the concentration of HVA and / or VMA in the sample to be tested is below the limit of quantitation or the matrix interference is severe, a solid phase extraction step is used for enrichment and purification. The solid phase extraction step includes activation equilibration, sample loading, rinsing and elution redissolution.

9. The method of using the neuroblastoma and catecholamine-associated tumor diagnostic kit according to claim 1 or the neuroblastoma and catecholamine-associated tumor diagnostic kit according to claim 4 in the preparation of diagnostic products for the auxiliary diagnosis or detection of neuroblastoma.

10. The method of using the diagnostic kit for neuroblastoma and catecholamine-associated tumors according to claim 1 or the diagnostic kit for neuroblastoma and catecholamine-associated tumors according to claim 4 in the preparation of diagnostic products for the auxiliary diagnosis or detection of catecholamine-associated tumors.

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