Kit and method for simultaneously detecting catecholamine compounds, metabolites thereof and creatinine in urine or plasma of human body based on derivatization method

Abstract

The invention belongs to the technical field of biomedical detection, and relates to a kit and a method for simultaneously detecting catecholamine compounds, metabolites thereof and creatinine in human urine or plasma based on a derivatization method. A method for simultaneously detecting catecholamine compounds, metabolites thereof and creatinine in human urine or plasma based on a derivatization method comprises the following steps: respectively adding an internal standard working solution into a series of calibration products, a series of quality control products, a plasma sample and a urine sample, and uniformly mixing; adding a precipitant, uniformly mixing and centrifuging; taking supernate, carrying out nitrogen blowing and drying, adding a sample adjusting solution, and uniformly mixing; a derivatization reagent is added for a derivatization reaction, and a derivatization reaction solution is taken for LC-MS / MS detection analysis. The method breaks through the bottleneck of synchronous detection, realizes synchronous detection of catecholamine, metabolites thereof and creatinine in plasma and random urine for the first time, does not need multiple operations, obtains a whole set of core indexes required by clinical diagnosis at one time, and greatly improves the detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection technology, and relates to a kit and method for simultaneously detecting catecholamine compounds and metabolites thereof and creatinine in human urine or plasma based on a derivatization method. BACKGROUND

[0002] Catecholamine and its metabolic abnormalities are closely related to the diagnosis and treatment monitoring of various neuroendocrine tumors. At present, the clinical detection value of catecholamine and its metabolites has been recognized in many authoritative guidelines. Among them, the Chinese "Guidelines for the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma (2020 Edition)" and the European Society of Endocrinology (ESE) clinical practice guidelines both point out that adrenaline (E), norepinephrine (NE), dopamine (DA) and their key end metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) are of great importance to the diagnosis and efficacy evaluation of pheochromocytoma, neuroblastoma and other diseases.

[0003] The sample types for clinical detection of catecholamines and metabolites are mainly plasma and urine, and each has its own focus: plasma detection focuses on adrenaline, norepinephrine and dopamine; urine detection focuses on the three compounds and their metabolic end products vanillylmandelic acid (VMA) and homovanillic acid (HVA). In the field of urine detection, the traditional method requires the collection of 24-hour urine samples, but this method has the problems of complicated collection process, poor patient compliance and insufficient convenience. In recent years, with the development of detection technology, studies have shown that the use of random urine samples combined with creatinine correction can effectively replace the traditional 24-hour urine collection method. This technological innovation not only ensures the accuracy of the detection results, but also significantly improves the feasibility and convenience of clinical detection, providing more efficient support for clinical diagnosis and treatment.

[0004] Before the widespread application of LC-MS / MS (liquid chromatography-tandem mass spectrometry) technology in the field of clinical detection, chemiluminescence and high-performance liquid chromatography (HPLC) were the mainstream technologies for detecting catecholamines and metabolites in clinical settings. Among them, chemiluminescence, with its advantages of simple operation and high automation, has been applied in clinical settings, but a large number of studies have shown that this method is easily interfered by various endogenous substances in blood and urine (such as hormone metabolites and drug metabolites), has poor specificity, and cannot meet the needs of precise diagnosis of diseases in clinical settings. Therefore, the Chinese "Guidelines for the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma (2020 Edition)" and the American "Clinical Practice Guidelines for Pheochromocytoma and Paraganglioma" both recommend LC-MS / MS technology as the detection method for catecholamines and metabolites.

[0005] However, the prior art still has the following defects: (1) catecholamine detection has the difficulties of low throughput and high cost: the concentration of catecholamine in plasma is extremely low, only at the pg (picogram) level. When detected by LC-MS / MS, the plasma sample usually needs to go through a complex pretreatment process, and a high-sensitivity mass spectrometer needs to be matched to achieve accurate detection. The current clinical commonly used technical solution is: through the weak cation exchange solid phase extraction column (WCX-SPE) or weak cation exchange magnetic beads (WCX-MB) to enrich and purify the sample, and then combine with Waters TQ-S and other high-sensitivity mass spectrometers to complete the detection. Although LC-MS / MS has greatly improved the detection accuracy compared with chemiluminescence, its clinical application still has obvious limitations: on the one hand, the sample pretreatment operation is complex, and the technical level of the experiment personnel is more stringent; on the other hand, the cost of consumables required for sample processing is high, and the performance and maintenance requirements of the instrument platform are also more stringent. These factors limit its popular application in some medical institutions to some extent. (2) Cannot simultaneously detect catecholamine prototype and metabolites: when using LC-MS / MS (liquid chromatography-tandem mass spectrometry) technology to simultaneously detect catecholamine and its neutral / acid metabolites, there are significant technical bottlenecks: catecholamine prototype (such as adrenaline, norepinephrine, dopamine) is an alkaline compound, while its key metabolites (such as vanillylmandelic acid, homovanillic acid) are acidic compounds. Due to the great difference in the acid-base properties of the two types of substances, the commonly used cation exchange type solid phase extraction method (such as the WCX-SPE mentioned earlier) can only enrich the alkaline catecholamine prototype, and cannot simultaneously capture the acid metabolites, resulting in that the two cannot be simultaneously detected by the same pretreatment process and detection method. In actual operation, independent sample pretreatment schemes and detection processes need to be designed for catecholamine prototype and acid metabolites, respectively, and detection is completed in two times. This technical limitation not only increases the sample usage, prolongs the detection period, but also increases the operation complexity and reagent consumable cost. (3) Urine detection lacks a method of creatinine correction: In clinical detection, urine creatinine is often used to calibrate the detection results of urine markers to eliminate the influence of urine dilution or concentration on the detection value and improve data accuracy. At present, when detecting vanillylmandelic acid and homovanillic acid, two catecholamine metabolites, 24-hour urine is often used as the sample to be detected. This kind of sample not only has a long collection period, but also needs to accurately measure the total urine volume to calculate the final concentration in the operation process, and the process is relatively cumbersome. If urine creatinine is used for calibration, the sample collection process can be greatly simplified: there is no need to strictly control the 24-hour collection time, and random urine can meet the detection requirements, greatly reducing the operation difficulty and time cost of sample collection, and improving the cooperation degree of patients. However, this optimization idea currently faces a key technical shortcoming: there is still a lack of an integrated detection method that can simultaneously detect catecholamine metabolites (vanillylmandelic acid and homovanillic acid) and urine creatinine in urine.In order to realize the creatinine calibration, the concentrations of vanillylmandelic acid, homovanillic acid and urine creatinine are obtained through two independent detection processes, and subsequent calibration calculation is carried out. This not only increases the operation steps and detection time, but also may affect the consistency of the calibration results due to the differences between the two detection samples, instrument errors and other factors, and also increases the use cost of reagent consumables, which restricts the clinical detection efficiency and economy to a certain extent. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a clinical detection method based on LC-MS / MS (liquid chromatography-tandem mass spectrometry) technology, which can simultaneously and accurately detect adrenaline, norepinephrine, dopamine (three types of catecholamine prototypes), homovanillic acid, vanillylmandelic acid (two types of catecholamine metabolites) and creatinine (calibration reference) in plasma and random urine samples, and has the core advantages of low cost and high throughput, which can better adapt to the actual needs of clinical detection scenes.

[0007] The core of the present application is to use benzoyl chloride to derive adrenaline, norepinephrine, dopamine (three types of catecholamine prototypes), homovanillic acid, vanillylmandelic acid (two types of catecholamine metabolites) and creatinine. All target compounds require benzoyl chloride to efficiently undergo derivatization reaction, and the derivatization reaction can significantly improve the mass spectrometry response signal of each product, laying a foundation for subsequent accurate detection. At the same time, the derivatization reagent also has the characteristics of practical operation: the derivatization reaction process is simple and easy to operate, without complex pretreatment steps; and the reaction specificity is strong, the rate of side reaction is low, and the interference of impurities can be minimized. Through the application of this core reagent, the simultaneous and accurate quantification of catecholamines, catecholamine metabolites and creatinine in the same detection system is finally realized (the derivatization reaction process is shown in Figure 1 ).

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] The present application discloses a kit for simultaneously detecting catecholamine compounds and their metabolites and creatinine in human urine or plasma based on derivatization method,

[0010] The catecholamine compounds are adrenaline, norepinephrine and dopamine;

[0011] The catecholamine compound metabolites are vanillylmandelic acid and homovanillic acid;

[0012] The kit comprises the following reagents:

[0013] (1) a series of calibration samples:

[0014] The series of calibrators are a series of calibrator working solutions with 6 different concentration levels, which are calibrator working solutions with 6 different concentration levels diluted by a first solvent from a matrix working solution with a known concentration;

[0015] The 6 different concentration levels of the calibrator working solution are:

[0016] The adrenaline concentration in the calibrator C1 working solution is 20 pg / mL, the norepinephrine concentration is 100 pg / mL, the dopamine concentration is 20 pg / mL, the homovanillyl acid concentration is 2000 pg / mL, the vanillyl mandelic acid concentration is 2000 pg / mL, and the creatinine concentration is 20000 pg / mL;

[0017] The adrenaline concentration in the calibrator C2 working solution is 40 pg / mL, the norepinephrine concentration is 200 pg / mL, the dopamine concentration is 40 pg / mL, the homovanillyl acid concentration is 4000 pg / mL, the vanillyl mandelic acid concentration is 4000 pg / mL, and the creatinine concentration is 40000 pg / mL;

[0018] The adrenaline concentration in the calibrator C3 working solution is 100 pg / mL, the norepinephrine concentration is 500 pg / mL, the dopamine concentration is 100 pg / mL, the homovanillyl acid concentration is 10000 pg / mL, the vanillyl mandelic acid concentration is 10000 pg / mL, and the creatinine concentration is 100000 pg / mL;

[0019] The adrenaline concentration in the calibrator C4 working solution is 400 pg / mL, the norepinephrine concentration is 2000 pg / mL, the dopamine concentration is 400 pg / mL, the homovanillyl acid concentration is 40000 pg / mL, the vanillyl mandelic acid concentration is 40000 pg / mL, and the creatinine concentration is 400000 pg / mL;

[0020] The adrenaline concentration in the calibrator C5 working solution is 1000 pg / mL, the norepinephrine concentration is 5000 pg / mL, the dopamine concentration is 1000 pg / mL, the homovanillyl acid concentration is 100000 pg / mL, the vanillyl mandelic acid concentration is 100000 pg / mL, and the creatinine concentration is 1000000 pg / mL;

[0021] The adrenaline concentration in the calibrator C6 working solution is 2000 pg / mL, the norepinephrine concentration is 10000 pg / mL, the dopamine concentration is 2000 pg / mL, the homovanillyl acid concentration is 200000 pg / mL, the vanillyl mandelic acid concentration is 200000 pg / mL, and the creatinine concentration is 2000000 pg / mL.

[0022] (2) series of quality control products:

[0023] The series of quality control products are series of quality control working solutions with three different concentration levels, which are quality control working solutions with three different concentration levels diluted by a first solvent from a matrix working solution with a known concentration;

[0024] The three different concentration levels of the quality control working solution are:

[0025] The concentration of epinephrine in the quality control LQC working solution is 80 pg / mL, the concentration of norepinephrine is 400 pg / mL, the concentration of dopamine is 80 pg / mL, the concentration of homovanillyl acid is 8000 pg / mL, the concentration of vanillyl mandelic acid is 8000 pg / mL, and the concentration of creatinine is 80000 pg / mL;

[0026] The concentration of epinephrine in the quality control MQC working solution is 500 pg / mL, the concentration of norepinephrine is 2500 pg / mL, the concentration of dopamine is 500 pg / mL, the concentration of homovanillyl acid is 50000 pg / mL, the concentration of vanillyl mandelic acid is 50000 pg / mL, and the concentration of creatinine is 500000 pg / mL;

[0027] The concentration of epinephrine in the quality control HQC working solution is 1500 pg / mL, the concentration of norepinephrine is 7500 pg / mL, the concentration of dopamine is 1500 pg / mL, the concentration of homovanillyl acid is 150000 pg / mL, the concentration of vanillyl mandelic acid is 150000 pg / mL, and the concentration of creatinine is 1500000 pg / mL;

[0028] (3) Internal standard working solution: The concentration of epinephrine-d6 and dopamine-d4 in the internal standard working solution is 2 ng / mL, the concentration of norepinephrine-d6 is 10 ng / mL, the concentration of vanillyl mandelic acid-d3 and homovanillyl acid-d5 is 200 ng / mL, and the concentration of creatinine-d3 is 2000 ng / mL;

[0029] (4) Precipitant: The precipitant is acetonitrile;

[0030] (5) Sample conditioning solution: The sample conditioning solution is composed of a sample conditioning agent and water; the sample conditioning agent is any one or a combination of several of triethylamine, sodium carbonate and sodium bicarbonate; the concentration of the sample conditioning agent in the sample conditioning solution is 0.02 g / mL to 0.10 g / mL;

[0031] (6) Derivatization reagent: The derivatization reagent is benzoyl chloride;

[0032] (7) Mobile phase A: aqueous solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate;

[0033] (8) Mobile phase B: methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate, or acetonitrile solution containing 0.1 vt% formic acid.

[0034] In some embodiments, the known concentration of the matrix working solution is prepared as follows:

[0035] Adrenaline is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL; noradrenaline is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL; dopamine is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL; vanillyl mandelic acid is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL; homovanillic acid is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL; creatinine is weighed and mixed with methanol to prepare a standard stock solution with a concentration of 100 μg / mL;

[0036] The above standard stock solutions are mixed, and a first solvent is used to prepare a matrix working solution; the concentration of adrenaline in the matrix working solution is 2 ng / mL, the concentration of noradrenaline is 10 ng / mL, the concentration of dopamine is 2 ng / mL, the concentration of vanillyl mandelic acid is 200 ng / mL, the concentration of homovanillic acid is 200 ng / mL, and the concentration of creatinine is 2000 ng / mL.

[0037] In some embodiments, the first solvent is 0.001 g / mL-0.040 g / mL aqueous bovine serum albumin solution, i.e., 0.1% g / mL-4% g / mL aqueous bovine serum albumin solution.

[0038] In some embodiments, the first solvent is 0.010 g / mL aqueous bovine serum albumin solution, i.e., 1% g / mL aqueous bovine serum albumin solution.

[0039] In some embodiments, the internal standard working solution is prepared as follows:

[0040] The epinephrine-d6 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL; the norepinephrine-d6 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL; the dopamine-d4 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL; the vanillylmandelic acid-d3 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL; the homovanillic acid-d5 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL; and the creatinine-d3 is weighed and mixed with methanol to prepare a standard internal standard stock solution with a concentration of 100 μg / mL;

[0041] The above standard internal standard stock solutions are mixed, and then mixed with methanol to prepare an internal standard working solution; the concentration of epinephrine-d6 in the internal standard working solution is 2 ng / mL, the concentration of norepinephrine-d6 is 10 ng / mL, the concentration of dopamine-d4 is 2 ng / mL, the concentration of vanillylmandelic acid-d3 is 200 ng / mL, the concentration of homovanillic acid-d5 is 200 ng / mL, and the concentration of creatinine-d3 is 2000 ng / mL.

[0042] In some embodiments, the sample adjusting agent is sodium bicarbonate; and the concentration of the sample adjusting agent in the sample adjusting solution is 0.02 g / mL.

[0043] In some embodiments, the mobile phase B is a methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate.

[0044] The kit described above is also within the protection scope of the present application in the application of preparing a diagnostic reagent for simultaneously detecting catecholamine compounds, metabolites thereof and creatinine in human urine or plasma.

[0045] The kit described above is also within the protection scope of the present application in the application of simultaneously detecting catecholamine compounds, metabolites thereof and creatinine in human urine or plasma by using high performance liquid chromatography tandem mass spectrometry.

[0046] In some embodiments, the catecholamine compounds are epinephrine, norepinephrine and dopamine; and the catecholamine compound metabolites are vanillylmandelic acid and homovanillic acid.

[0047] The steps of detecting catecholamine compounds, metabolites thereof and creatinine in human urine or plasma include:

[0048] ①The internal standard working solution is added to the series of calibration samples, the series of quality control samples, the plasma sample and the urine sample respectively, and mixed well;

[0049] ② Add the precipitant to each sample obtained in ①, mix well, and centrifuge;

[0050] ③ Take the supernatant from ② after centrifugation and place it on a nitrogen blower to dry it with nitrogen;

[0051] ④ Add the sample conditioning solution to each sample after nitrogen blowing and drying in step ③, and mix well;

[0052] ⑤ Add the derivatization reagent to each of the samples obtained in ④ to carry out the derivatization reaction;

[0053] ⑥ After the derivatization reaction is completed, the derivatization reaction solution is taken for LC-MS / MS detection and analysis.

[0054] The human urine needs to be diluted 100 times with deionized water beforehand.

[0055] In some embodiments, the steps for detecting catecholamines and their metabolites and creatinine in human urine or plasma include:

[0056] ① Add 20 μL of the internal standard working solution to each of the following calibrators (50 μL~200 μL), the quality control products (50 μL~200 μL), the plasma sample (50 μL~200 μL), and the urine sample (50 μL~200 μL), and mix well.

[0057] ② Add 200 μL of the precipitant to each sample obtained in ①, mix well, and centrifuge at 10 °C;

[0058] ③ Take 200 μL of the supernatant from step ② after centrifugation and place it on a nitrogen blower to dry it with nitrogen;

[0059] ④ Add 50 μL of the sample conditioning solution to each sample after nitrogen blowing and drying in step ③, and mix well;

[0060] ⑤ Add 20µ of the derivatization reagent to each of the samples obtained in ④ to carry out the derivatization reaction;

[0061] ⑥ After the derivatization reaction is completed, the derivatization reaction solution is taken for LC-MS / MS detection and analysis.

[0062] Furthermore, in some embodiments, the steps for detecting catecholamine compounds and their metabolites, and creatinine in human urine or plasma include:

[0063] ① Add 20 μL of the internal standard working solution to 100 μL of the series of calibrators, 100 μL of the series of quality control products, 100 μL of plasma sample and 100 μL of urine sample respectively, and mix well;

[0064] ② Add 200 μL of the precipitant to each sample obtained in ①, mix well, and centrifuge at 10°C;

[0065] ③ Take 200 μL of the supernatant from step ② after centrifugation and place it on a nitrogen blower to dry it with nitrogen;

[0066] ④ Add 50 μL of the sample conditioning solution to each sample after nitrogen blowing and drying in step ③, and mix well;

[0067] ⑤ Add 20µ of the derivatization reagent to each of the samples obtained in ④ to carry out the derivatization reaction;

[0068] ⑥ After the derivatization reaction is completed, the derivatization reaction solution is taken for LC-MS / MS detection and analysis.

[0069] In some embodiments, the chromatographic conditions for LC-MS / MS detection are as follows:

[0070] High-performance liquid chromatography (HPLC) conditions: The chromatographic column was a Pfim C18 column (2.1 × 50 mm, 2.6 μm), or a Waters HSS T3 column (2.1 × 100 mm, 1.8 μm), or a Pfim PPF column (2.1 × 100 mm, 2.6 μm); mobile phase A was an aqueous solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate; mobile phase B was a methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate, or an acetonitrile solution containing 0.1 vt% formic acid; the acquisition time was 6 min; gradient elution was used, and the specific elution conditions are shown in Table 1 below.

[0071]

[0072] Mass spectrometry conditions: Ion source type: electrospray ion source, positive ion scanning mode, MRM mode scanning; ion source spray voltage: 2000V-5500V; temperature: 500-600℃; curtain gas (CUR): 30-40 psi; collision gas: 8 psi; nebulizing gas (Gas1): 55 psi; auxiliary heating gas (Gas2): 40 psi.

[0073] Furthermore, in some embodiments, the chromatographic conditions for LC-MS / MS detection are as follows:

[0074] High performance liquid chromatography (HPLC) conditions: The chromatographic column was a Pfim C18 column, 2.1 × 50 mm, 2.6 μm; mobile phase A was an aqueous solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate; mobile phase B was a methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate; the acquisition time was 6 min; gradient elution was used, and the specific elution conditions were as shown in Table 1.

[0075] Mass spectrometry conditions: Ion source type: electrospray ion source, positive ion scanning mode, MRM mode scanning; ion source spray voltage: 2000V-5500V; temperature: 500-600℃; curtain gas (CUR): 30-40 psi; collision gas: 8 psi; nebulizing gas (Gas1): 55 psi; auxiliary heating gas (Gas2): 40 psi.

[0076] In some embodiments, the derivatization reaction is carried out at a temperature of 40°C to 60°C and for a reaction time of 10 min to 30 min.

[0077] In some embodiments, the derivatization reaction is carried out at a temperature of 40°C for a time of 15 min.

[0078] Beneficial effects:

[0079] (1) This invention breaks through the bottleneck of synchronous detection: for the first time, it realizes the synchronous detection of catecholamines (prototype + metabolites) and creatinine in plasma and random urine, without the need for multiple operations, and obtains all the core indicators required for clinical diagnosis in one go, which greatly improves the detection efficiency.

[0080] (2) The present invention improves the reliability of detection: the optimal derivatization reagent can enhance the mass spectrometry response of the target product, reduce the detection limit of low concentration catecholamines in plasma, and has fewer side reactions and less interference, thus improving the accuracy of detection.

[0081] (3) Simplify clinical procedures: Support random urine sample testing, eliminating the need to collect 24-hour urine samples; single-dose derivation + simultaneous testing replaces multiple rounds of operation, shortens the testing cycle, improves patient cooperation, and reduces operational errors.

[0082] (4) The present invention takes adrenaline, noradrenaline, dopamine, homovanillic acid, vanillylmandelic acid and creatinine standards as research objects, and adopts derivatization method to systematically optimize their LC-MS / MS (liquid chromatography-tandem mass spectrometry) detection conditions (including chromatographic separation parameters, mass spectrometry ion source parameters, collision energy, etc.) to ensure that the derivatized products can be effectively separated and stably detected.

[0083] (5) This invention comprehensively compares the application effects of derivatization reagents from two dimensions: detection sensitivity (with the mass spectrometry response intensity of the derivatized product as the core indicator) and operational convenience (covering reagent solubility, complexity of derivatization reaction steps, controllability of reaction conditions, etc.) to evaluate the effect of derivatization and non-derivatization on improving the sensitivity of 6 compounds.

[0084] (6) Further optimize the derivatization reaction conditions for the derivatization reagent, including key parameters such as reagent dosage, reaction temperature, reaction time, and pH value, in order to maximize the efficiency and specificity of the derivatization reaction, reduce side reaction interference, and ensure the stability and consistency of the derivatized products.

[0085] (7) Based on the optimized derivation method and LC-MS / MS detection conditions, the present invention conducts systematic methodological validation on plasma samples and urine samples respectively. The validation content includes linear range, detection limit, quantitation limit, precision (including intra-day precision and inter-day precision), accuracy, matrix effect and stability, etc., to comprehensively evaluate the applicability and reliability of the detection method in different biological samples. Attached Figure Description

[0086] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0087] Figure 1 This is a schematic diagram illustrating the principle of using benzoyl chloride to derivatize adrenaline, noradrenaline, dopamine, vanillylmandelic acid, homovanillic acid, and creatinine.

[0088] Figure 2 Extracted ion chromatograms of six compounds in calibrator C1 sample (limit of quantitation sample) processed by non-derivative methods.

[0089] Figure 3 Extracted ion chromatograms of six compounds in calibrator C1 sample (limit of quantitation sample) processed by derivatization method.

[0090] Figure 4 Extraction ion chromatograms of six compounds from human plasma samples treated with derivatization methods.

[0091] Figure 5 Extraction ion chromatograms of six compounds in human urine samples treated with derivatization methods. Detailed Implementation

[0092] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0093] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0094] Example 1:

[0095] 1. Preparation of reagents

[0096] 1.1 The preparation of stock solutions of catecholamine compounds, catecholamine metabolites and creatinine standards, as well as stock solutions of internal standards, are as follows.

[0097] (1) Preparation of standard stock solutions: Weigh 1 mg each of adrenaline (E), norepinephrine (NE), dopamine (DA), vanillylmandelic acid (VMA), homovanillic acid (HVA) and creatinine (Cr) standards, place them in 15 mL centrifuge tubes, add 10 mL of methanol solution to each, and prepare single standard stock solutions with a concentration of 100 μg / mL.

[0098] (2) Preparation of standard internal standard stock solutions: Weigh 1 mg of each of the following standards: adrenaline-d6 (E-d6), norepinephrine-d6 (NE-d6), dopamine-d4 (DA-d4), vanillylmandelic acid-d3 (VMA-d3), homovanillic acid-d5 (HVA-d5) and creatinine-d3 (Cr-d3), place them in 15 mL centrifuge tubes, add 10 mL of methanol solution to each tube, and prepare a single standard internal standard stock solution with a concentration of 100 μg / mL.

[0099] 1.2 Preparation of mixed working solution: Transfer 10 μL of each of the following standard stock solutions: adrenaline stock solution, norepinephrine stock solution, dopamine stock solution, vanillylmandelic acid stock solution, homovanillic acid stock solution, and creatinine stock solution, and mix them in a 15 mL centrifuge tube. Dilute with methanol to prepare the mixed working solution. The concentrations of adrenaline, norepinephrine, dopamine, vanillylmandelic acid, homovanillic acid, and creatinine in the mixed working solution are all 100 ng / mL.

[0100] 1.3 Preparation of mixed internal standard working solution: Transfer 10 μL of each of the following internal standard stock solutions: adrenaline-d6, norepinephrine-d6, dopamine-d4, vanillylmandelic acid-d3, homovanillic acid-d5, and creatinine-d3. Mix them in a 15 mL centrifuge tube and dilute with methanol to prepare the mixed internal standard working solution. The concentrations of adrenaline-d6, norepinephrine-d6, dopamine-d4, vanillylmandelic acid-d3, homovanillic acid-d5, and creatinine-d3 in the mixed internal standard working solution are all 100 ng / mL.

[0101] 1.4 Derivatization reagent: Take 1.0 mL of benzoyl chloride and place it in a 1.5 mL centrifuge tube for later use.

[0102] 1.5 Preparation of sample conditioning solution: Weigh 2 g of sodium bicarbonate into a reagent bottle, add 100 mL of water to dissolve, and prepare a 2% (w / v) sodium bicarbonate aqueous solution for later use. Where w / v is g / mL.

[0103] 2. Optimization of LC-MS / MS conditions for derivatives

[0104] The principle of using benzoyl chloride to derivatize adrenaline, noradrenaline, dopamine, vanillylmandelic acid, homovanillic acid, and creatinine is as follows: Figure 1 As shown.

[0105] The procedure for derivatizing catecholamines, their metabolites, and creatinine using benzoyl chloride is as follows: Take 50 μL of the prepared mixed working solution and place it in a 1.5 mL centrifuge tube. Add 20 μL of the mixed internal standard working solution, 50 μL of benzoyl chloride, and 20 μL of sample conditioning solution (2% (w / v) sodium bicarbonate aqueous solution, where w / v is g / mL). Incubate at 60 °C for 30 min to carry out the derivatization reaction. After the reaction, add 2 μL of formic acid to quench the reaction. Analyze the derivatized reaction solution using a Jasper HPLC / SCIEX 4500 liquid chromatography-tandem triple quadrupole mass spectrometer. This experiment only optimized the liquid chromatography and mass spectrometry conditions for six compounds and their derivatized products; the derivatization conditions only needed to produce the derivatized products. The specific chromatographic conditions are as follows.

[0106] High performance liquid chromatography (HPLC) conditions: The chromatographic column was a Fenomex C18 column (2.1×50mm, 2.6μm); mobile phase A was an aqueous solution containing 0.1 vt% formic acid and 2 mM ammonium acetate; mobile phase B was a methanol solution containing 0.1 vt% formic acid and 2 mM ammonium acetate; the injection volume was 10 μL; the acquisition time was 6 min; gradient elution was used, and the specific elution parameters are shown in Table 2.

[0107]

[0108] Mass spectrometry conditions: The ion source parameters of the SCIEX 4500 mass spectrometer are as follows: ion source type is electrospray ionization (ESI), positive ion scanning mode, MRM mode scanning; ion source spray voltage is 2000V-5500V; temperature is 500-600℃; curtain gas (CUR) is 30-40 psi; collision gas is 8 psi; nebulizing gas (Gas1) is 55 psi; auxiliary heating gas (Gas2) is 40 psi.

[0109] The MRM ion pair parameters of the above-mentioned catecholamine compounds, catecholamine metabolites, and creatinine and benzoyl chloride derivatization products are shown in Table 3. The experimental results show that catecholamine compounds, catecholamine metabolites, and creatinine and benzoyl chloride derivatization products can be detected simultaneously on a conventional C18 reactor in MRM positive ion mode.

[0110]

[0111] 3. LC-MS / MS parameters of non-derivative products

[0112] A non-derivative method was used to detect adrenaline, noradrenaline, dopamine, vanillylmandelic acid, homovanillic acid, and creatinine. The specific experimental procedures are as follows.

[0113] Take 50 μL of the prepared mixed working solution and place it in a 1.5 mL centrifuge tube. Add 20 μL of the mixed internal standard working solution and 70 μL of deionized water, mix well, and then analyze in a Jasper HPLC / SCIEX 4500 liquid chromatography-tandem triple quadrupole mass spectrometer. The specific chromatographic conditions are as follows.

[0114] High performance liquid chromatography (HPLC) conditions: The chromatographic column was a PPF column (2.1×100mm, 2.6μm); mobile phase A was an aqueous solution containing 0.1 vt% formic acid; mobile phase B was a methanol solution containing 0.1 vt% formic acid; the injection volume was 20 μL; the acquisition time was 4 min; gradient elution was used, and the specific elution parameters are shown in Table 4.

[0115]

[0116] Mass spectrometry conditions: Since vanillylmandelic acid (VMA) and homovanillic acid (HVA) are acidic compounds, and the other compounds are basic compounds, the SCIEX 4500 mass spectrometer requires positive and negative ion switching scanning when simultaneously detecting 6 compounds. The ion source type is electrospray ionization (ESI), and the scanning mode is MRM. The ion source spray voltage is 1250V-4500V; the temperature is 600℃; the curtain gas (CUR) is 30 psi; the collision gas is 8 psi; the nebulizing gas (Gas1) is 55 psi; and the auxiliary heating gas (Gas2) is 55 psi.

[0117] The MRM ion pair parameters of the above-mentioned catecholamines, catecholamine metabolites, and creatinine are shown in Table 5. The experimental results show that: when detecting catecholamines and metabolites using the non-derivative method, switching between positive and negative ions is required; when using reagents, since the sample processing methods for VMA and HVA are incompatible with other catecholamines, two separate methods are required for detection.

[0118]

[0119] 4. Optimization of sample processing conditions

[0120] (1) The processing of samples by the derivation method is as follows.

[0121] ① Accurately pipette 100 μL of plasma or urine sample (urine sample pretreatment: dilute with deionized water 100 times and take 100 μL) into different 1.5 mL centrifuge tubes, add 20 μL of internal standard solution (i.e. the mixed internal standard working solution prepared above), and vortex mix for 30 seconds.

[0122] ② Add 200 μL of precipitant acetonitrile to the above plasma sample system or urine sample system respectively, vortex at 2500 rpm for 5 minutes, and then centrifuge at 13000 rpm for 5 minutes at 10℃;

[0123] ③ Take 200 μL of the supernatant after centrifugation into a 96-well plate, and place the 96-well plate on a nitrogen blower to blow nitrogen for 10 minutes;

[0124] ④ Take 50 μL of pH adjuster - 2% (w / v) sodium bicarbonate aqueous solution (where w / v is g / mL) and add it to the 96-well plate after nitrogen blowing, and shake at 1000 rpm for 2 minutes.

[0125] ⑤ Transfer 20µL of the derivatization reagent—benzoyl chloride, and add it to the above 96-well plate. Seal the plate and place it in a 96-well plate constant temperature shaker. Incubate at 40℃ and 1000rpm for 15 minutes to carry out the derivatization reaction.

[0126] ⑥ After the derivatization reaction is complete, remove the centrifuge tube, transfer the sample to a 96 plate, and perform LC-MS / MS detection and analysis. The chromatographic conditions are the same as those in "2. Optimization of LC-MS / MS conditions for derivatized products".

[0127] (2) The processing of samples from non-derivative methods is as follows.

[0128] Since vanillylmandelic acid (VMA) and homovanillic acid (HVA) are acidic compounds, while the other four compounds (adrenaline, noradrenaline, dopamine, and creatinine) are basic compounds, the six compounds cannot be extracted and purified in a single sample processing.

[0129] (2-1) When detecting adrenaline (E), norepinephrine (NE), dopamine (DA), and creatinine (Cr), the sample processing method is as follows.

[0130] ① Activation: Take out the WCX type SPE plate, add 0.2 mL of methanol to each target well, place it on a 96-well plate (this 96-well plate is used for waste liquid collection), and place it in a positive pressure solid phase extraction instrument. Adjust the pressure so that the methanol flows out at a rate of 2~3 seconds / drop until the liquid has completely flowed out.

[0131] ② Equilibration: Add 0.4 mL of 1×PBS buffer (pH=7.4) or water to each target well, adjust the pressure to allow the buffer to flow out at a rate of 2~3 seconds / drop until the liquid has completely flowed out.

[0132] ③ Sample loading: Mix 300 μL of plasma or 300 μL of urine sample (urine sample is diluted 100 times with deionized water beforehand) with 20 μL of internal standard solution (i.e., the mixed internal standard working solution prepared above) by vortexing for 30 seconds; transfer to a well-equilibrated solid-phase extraction plate, adjust the pressure, add eluent so that the eluent flows out at a rate of 2~3 seconds / drop until the liquid flows out completely. The specific rinsing operation is as follows.

[0133] ④ Wash 1: Add 0.4 mL of 1×PBS buffer (pH=7.4) to the target well, adjust the pressure, and let the buffer flow out at a rate of 2~3 seconds / drop until the liquid has completely flowed out.

[0134] ⑤ Rinsing 2: Add 0.4 mL of acetonitrile to the target well again, adjust the pressure so that the acetonitrile flows out at a rate of 2-3 seconds / drop until the liquid flows out completely (the pressure can be increased to make the rinsing solution flow out completely).

[0135] ⑥ Elution: Place the SPE plate on an unused 96-well plate and add 0.1 mL of acetonitrile solution containing 1.0 vt% formic acid to each target well. Adjust the pressure to allow the eluent to flow out at a rate of about 2-3 drops per second until the liquid has completely flowed out (the pressure can be increased to allow the eluent to completely drain).

[0136] ⑦ Nitrogen blowing and sample reconstitution: Place the eluent in the 96-well plate in a nitrogen blower and blow it dry at 20~30℃. Then add 100μL of 10vt% acetonitrile aqueous solution to reconstitute the eluent, shake and mix for 10min, centrifuge, and then use it for LC-MS / MS detection and analysis. The chromatographic conditions are the same as those in "3. LC-MS / MS parameters of non-derivative products".

[0137] (2-2) When detecting vanillylmandelic acid (VMA) and homovanillic acid (HVA), the sample processing method is as follows.

[0138] ① Take 20 μL of plasma or 20 μL of diluted urine sample (the urine sample was diluted 100 times with deionized water beforehand), add 20 μL of internal standard solution (i.e. the mixed internal standard working solution prepared above), and mix well.

[0139] ② Add 100 μL of acetonitrile to each target well, vortex centrifuge for 10 min, take out 20 μL of the test solution, add 180 μL of deionized water, mix well and use for LC-MS / MS detection and analysis. The chromatographic conditions are the same as those in "3. LC-MS / MS parameters for non-derivative products".

[0140] (3) Comparative analysis of the effects of derivatization methods and non-derivatization methods

[0141] (3-1) Prepare calibrator C1, i.e., the limit of quantitation sample, according to the methods and procedures in "5.1, Preparation of Calibrators and Quality Controls". Process the limit of quantitation sample (i.e., calibrator C1), fresh human plasma sample, and fresh human urine sample according to the methods in "4, Optimization of Sample Processing Conditions" for "Processing of Samples Using Derivatization Methods" and "Processing of Samples Using Non-Derivatization Methods" (VMA and HVA need to be detected separately in the non-derivatization method sample processing method). Analyze the processed samples using a SCIEX 4500 liquid chromatography-tandem mass spectrometer, and perform the chromatographic analysis under the conditions specified in the respective sample processing methods. Results are as follows: Figures 2-5 As shown.

[0142] Figure 2 Extracted ion chromatograms of six compounds in calibrator C1 sample (limit of quantitation sample) processed by non-derivative methods. Figure 3 Extracted ion chromatograms of six compounds in calibrator C1 sample (limit of quantitation sample) treated with derivatization methods. From Figure 2 and Figure 3 It can be seen that in the derivatization method, the signal-to-noise ratio (S / N) of NE, E, and DA is significantly higher than that of the corresponding substances in the non-derivatization method, and the retention time is also longer. In addition, VMA and HVA have good responses in both derivatization and non-derivatization methods, but the derivatization method can achieve simultaneous detection with other compounds, while the non-derivatization method requires separate detection of VMA and HVA.

[0143] Figure 4 Extraction ion chromatograms of six compounds from human plasma samples treated with derivatization methods. Figure 5 Extraction ion chromatograms of six compounds from a human urine sample treated with derivatization methods. Figure 4 and Figure 5 It can be seen that non-derivative methods can achieve good results for both plasma and urine samples.

[0144] 5. Preparation of the reagent kit

[0145] 5.1 Preparation of calibrators and quality control samples

[0146] ① Preparation of 1% (w / v) BSA (w / v, g / mL): Weigh 10 g of bovine serum albumin (BSA), place it in a reagent bottle, add 1000 mL of water to make up to volume, dissolve, and prepare 1% (w / v) BSA for later use.

[0147] ② Preparation of matrix working solution: Take appropriate volumes of the epinephrine standard stock solution, norepinephrine standard stock solution, dopamine standard stock solution, vanillylmandelic acid standard stock solution, homovanillic acid standard stock solution, and creatinine standard stock solution (all with a concentration of 100 μg / mL) prepared in "1. Reagent Preparation" above, mix them, and dilute to 250 mL with 1% (w / v) BSA. Mix well to prepare the matrix working solution. The concentrations of the epinephrine and dopamine standards in the matrix working solution are 2 ng / mL each, the norepinephrine standard is 10 ng / mL, the vanillylmandelic acid and homovanillic acid standards are 200 ng / mL each, and the creatinine standard is 2000 ng / mL. See Table 6 for detailed preparation procedures.

[0148] The abbreviations in the table are: adrenaline (E), noradrenaline (NE), dopamine (DA), vanillylmandelic acid (VMA), homovanillic acid (HVA), and creatinine (Cr).

[0149]

[0150] ③ Preparation of calibrators and quality control samples: The matrix working solutions prepared above were mixed with 1% (w / v) BSA (w / v, g / mL) at a certain volume ratio to prepare calibrators C1, C2, C3, C4, C5, C6, and quality control samples LQC, MQC, and HQC, respectively. Detailed preparation procedures are shown in Table 7. After preparation, 200 μL of each calibrator and quality control sample was aliquoted into 1.5 mL centrifuge tubes and stored at -20℃.

[0151]

[0152] 5.2 Preparation of Internal Standard Working Solution

[0153] Take appropriate volumes of the following internal standard stock solutions (each with a concentration of 100 μg / mL): epinephrine-d6, norepinephrine-d6, dopamine-d4, vanillylmandelic acid-d3, homovanillic acid-d5, and creatinine-d3 (all with a concentration of 100 μg / mL), mix them, and dilute with methanol to prepare the internal standard working solution. The concentrations of epinephrine-d6 and dopamine-d4 in the internal standard working solution are 2 ng / mL each, norepinephrine-d6 is 10 ng / mL, vanillylmandelic acid-d3 and homovanillic acid-d5 are 200 ng / mL each, and creatinine-d3 is 2000 ng / mL. After preparation, aliquot 2 mL of the internal standard working solution into brown glass bottles and store at -20°C. The detailed preparation process is shown in Table 8.

[0154] The specific abbreviations in the table are: adrenaline-d6 (E-d6), norepinephrine-d6 (NE-d6), dopamine-d4 (DA-d4), vanillylmandelic acid-d3 (VMA-d3), homovanillic acid-d5 (HVA-d5), and creatinine-d3 (Cr-d3).

[0155]

[0156] 5.3 Preparation of precipitant: Acetonitrile is preferred as the precipitant; dispense 20 mL of precipitant into brown reagent bottles.

[0157] 5.4 Preparation of sample conditioning solution: In a preferred embodiment, the sample conditioning agent is any one or a combination of several of triethylamine, sodium carbonate and sodium bicarbonate, more preferably sodium bicarbonate.

[0158] Preparation of sample conditioning solution: Weigh 2 g of sodium bicarbonate into a reagent bottle, add 100 mL of water to dissolve, and prepare a 2% (w / v) sodium bicarbonate aqueous solution. This is the sample conditioning solution, which is then ready for use. (w / v refers to g / mL.) Dispense 5 mL portions of the sample conditioning solution into brown reagent bottles.

[0159] 5.5 Preparation of derivatization reagent: Benzoyl chloride is preferred as the derivatization reagent; dispense 2 mL of the derivatization reagent into brown reagent bottles.

[0160] 5.6 Preparation of Mobile Phase A and Mobile Phase B: In a preferred embodiment, mobile phase A is an aqueous solution containing 0.1 vt% formic acid and 2 mM ammonium acetate, and mobile phase B is a methanol solution containing 0.1 vt% formic acid and 2 mM ammonium acetate, or an acetonitrile solution containing 0.1 vt% formic acid. In a more preferred embodiment, mobile phase A is an aqueous solution containing 0.1 vt% formic acid and 2 mM ammonium acetate, and mobile phase B is a methanol solution containing 0.1 vt% formic acid and 2 mM ammonium acetate. Mobile phase A and mobile phase B are dispensed into 250 mL aliquots into brown reagent bottles.

[0161] 6. Sample processing procedure

[0162] ① Accurately pipette 100 μL of each calibrator, 100 μL of plasma sample to be tested, or 100 μL of urine sample to be tested (the urine sample to be tested is diluted 100 times with deionized water beforehand) into different 1.5 mL centrifuge tubes. Add 20 μL of internal standard working solution to each sample and vortex mix for 30 seconds.

[0163] ② Add 200 μL of precipitant acetonitrile to each of the above samples, vortex at 2500 rpm for 5 minutes, and then centrifuge at 13000 rpm for 5 minutes at 10℃;

[0164] ③ Take 200 μL of the supernatant after centrifugation into a 96-well plate, and place the 96-well plate on a nitrogen blower to blow nitrogen for 10 minutes;

[0165] ④ Add 50 μL of 2% (w / v) sodium bicarbonate aqueous solution (w / v is g / mL) to each of the 96-well plates after nitrogen blowing, and shake at 1000 rpm for 2 minutes.

[0166] ⑤ Transfer 20µL of benzoyl chloride to each of the above 96-well plates, seal the plates, place them in a 96-well plate constant temperature shaker, and incubate at 40℃ and 1000 rpm for 15 min to carry out the derivatization reaction.

[0167] ⑥ After the derivatization reaction is complete, remove the centrifuge tube, transfer the sample to a 96 plate, and perform LC-MS / MS analysis; the chromatographic conditions for LC-MS / MS detection are as follows:

[0168] High performance liquid chromatography (HPLC) conditions: The chromatographic column was a Phenomena C18 column (2.1×50mm, 2.6μm); mobile phase A was an aqueous solution containing 0.1vt% formic acid and 2mM ammonium acetate; mobile phase B was a methanol solution containing 0.1vt% formic acid and 2mM ammonium acetate; the injection volume was 10 μL; the acquisition time was 6 min; gradient elution was used, and the specific elution parameters are shown in Table 2 under “2. Optimization of LC-MS / MS conditions for derivatized products”.

[0169] Mass spectrometry conditions: The ion source parameters of the SCIEX 4500 mass spectrometer are as follows: ion source type is electrospray ionization (ESI), positive ion scanning mode, MRM mode scanning; ion source spray voltage is 2000V-5500V; temperature is 500-600℃; curtain gas (CUR) is 30-40 psi; collision gas is 8 psi; nebulizing gas (Gas1) is 55 psi; auxiliary heating gas (Gas2) is 40 psi.

[0170] 7. Reagent kit performance validation

[0171] The method performance of benzoyl chloride derivatization for the simultaneous determination of six compounds was evaluated through experimental verification of the method parameters, including limit of quantitation, linearity, accuracy (recovery), and precision. All verification results were satisfactory. The specific experimental results are as follows: The six compounds and their abbreviations are: adrenaline (E), noradrenaline (NE), dopamine (DA), vanillylmandelic acid (VMA), homovanillic acid (HVA), and creatinine (Cr).

[0172] (1) Linearity verification

[0173] Calibrators C1 through C6 were used as linear validation samples. They were processed and tested according to the pretreatment method described in "6. Sample Processing" above. The test results were recorded. Each concentration was measured three times, and the results were recorded. A linear regression equation was performed between the measured values ​​and the theoretical values ​​of the samples. The acceptance criteria were: correlation coefficient (r) ≥ 0.99, deviation of the linear low concentration within ±20%, and deviation of other concentration samples within ±15%. The test results are shown in Tables 9 through 14 below.

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] (2) Validation of the lower limit of quantitation

[0181] Using calibrator C1 as the limit of quantitation verification sample, the test was repeated 10 times. The pretreatment process was performed according to the method outlined in "6. Sample Processing," and the results were recorded. The acceptance criteria were: the mean deviation of the test results from the theoretical value ≤ ±20%, and CV ≤ 15%. The test results are shown in Table 15 below.

[0182]

[0183] (3) Accuracy verification

[0184] Three normal human plasma or urine samples were used as baseline samples. Each baseline sample was aliquoted into 980 μL portions, and 20 μL of LQC (Low Quality Control) sample was added to each. Two concentrations of spiked plasma or urine samples were prepared (plasma samples were used to verify the recovery rates of E, NE, and DA; urine samples were used to verify the recovery rates of VMA, HVA, and creatinine). These samples were processed and analyzed according to the pretreatment method described in "6. Sample Processing Procedure." Each concentration was measured three times. The recovery rate was calculated as follows: Recovery Rate (RE%) = (Spiked Sample Concentration - Baseline Sample Concentration) / Spiked Sample Concentration × 100%. Acceptable criteria were: deviation of the recovered sample value from the theoretical value ≤ ±15%, CV ≤ 15%. Specific results are shown in Tables 16-21.

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] (4) Precision verification

[0192] Two levels of spiked samples were prepared by adding 20 μL of LQC (Low Quality Control) sample or 20 μL of HQC (High Quality Control) sample to either 980 μL of normal human plasma or urine samples. The samples were then processed and analyzed according to the pretreatment method described in "6. Sample Processing Procedure". Each concentration was measured six times. One analytical batch was tested daily for three consecutive analytical batches. The CV (Continuous Value) of the test results was calculated. The acceptable criteria were: intra-day precision CV ≤ 15%, and inter-day precision ≤ 15%. Specific results are shown in Tables 22-27.

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] This invention provides a kit and method for the simultaneous detection of catecholamines and their metabolites, as well as creatinine, in human urine or plasma based on derivatization. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A kit for the simultaneous detection of catecholamines and their metabolites and creatinine in human urine or plasma based on derivatization, characterized in that, The catecholamine compounds are adrenaline, noradrenaline, and dopamine; The metabolites of the catecholamine compounds are vanillylmandelic acid and homovanillic acid; The kit contains the following reagents: (1) Series of calibrators: The series of calibrators consists of a series of calibrator working solutions with six different concentration levels. The series of calibrator working solutions are calibrator working solutions with six different concentration levels prepared by diluting a matrix working solution of known concentration with a first solvent. The six different concentration levels of the calibrator working solution are as follows: The working solution of calibrator C1 contains 20 pg / mL of adrenaline, 100 pg / mL of norepinephrine, 20 pg / mL of dopamine, 2000 pg / mL of homovanillic acid, 2000 pg / mL of vanillylmandelic acid, and 20000 pg / mL of creatinine. The working solution of calibrator C2 contains 40 pg / mL of adrenaline, 200 pg / mL of norepinephrine, 40 pg / mL of dopamine, 4000 pg / mL of homovanillic acid, 4000 pg / mL of vanillylmandelic acid, and 40000 pg / mL of creatinine. The working solution of calibrator C3 contains 100 pg / mL of adrenaline, 500 pg / mL of norepinephrine, 100 pg / mL of dopamine, 10,000 pg / mL of homovanillic acid, 10,000 pg / mL of vanillylmandelic acid, and 100,000 pg / mL of creatinine. The working solution of calibrator C4 contains 400 pg / mL of adrenaline, 2000 pg / mL of norepinephrine, 400 pg / mL of dopamine, 40000 pg / mL of homovanillic acid, 40000 pg / mL of vanillylmandelic acid, and 400000 pg / mL of creatinine. The working solution of calibrator C5 contains 1000 pg / mL of adrenaline, 5000 pg / mL of norepinephrine, 1000 pg / mL of dopamine, 100000 pg / mL of homovanillic acid, 100000 pg / mL of vanillylmandelic acid, and 1000000 pg / mL of creatinine. The working solution of calibrator C6 contains 2000 pg / mL of adrenaline, 10000 pg / mL of norepinephrine, 2000 pg / mL of dopamine, 200000 pg / mL of homovanillic acid, 200000 pg / mL of vanillylmandelic acid, and 2000000 pg / mL of creatinine. (2) Series of quality control products: The series of quality control products are a series of quality control working solutions with three different concentration levels. The series of quality control working solutions are quality control working solutions with three different concentration levels prepared by diluting a matrix working solution of known concentration with a first solvent. The three different concentration levels of the quality control working solution are as follows: The LQC working solution for quality control contains 80 pg / mL of adrenaline, 400 pg / mL of norepinephrine, 80 pg / mL of dopamine, 8000 pg / mL of homovanillic acid, 8000 pg / mL of vanillylmandelic acid, and 80000 pg / mL of creatinine. The quality control MQC working solution contains 500 pg / mL of adrenaline, 2500 pg / mL of norepinephrine, 500 pg / mL of dopamine, 50000 pg / mL of homovanillic acid, 50000 pg / mL of vanillylmandelic acid, and 500000 pg / mL of creatinine. The HQC working solution for quality control contains 1500 pg / mL of adrenaline, 7500 pg / mL of norepinephrine, 1500 pg / mL of dopamine, 150000 pg / mL of homovanillic acid, 150000 pg / mL of vanillylmandelic acid, and 1500000 pg / mL of creatinine. (3) Internal standard working solution: The concentrations of adrenaline-d6 and dopamine-d4 in the internal standard working solution are both 2 ng / mL, the concentration of norepinephrine-d6 is 10 ng / mL, the concentrations of vanillylmandelic acid-d3 and homovanillic acid-d5 are both 200 ng / mL, and the concentration of creatinine-d3 is 2000 ng / mL; (4) Precipitator: The precipitant is acetonitrile; (5) Sample conditioning solution: The sample conditioning solution is composed of a sample conditioning agent and water; the sample conditioning agent is any one or a combination of several of triethylamine, sodium carbonate and sodium bicarbonate; the concentration of the sample conditioning agent in the sample conditioning solution is 0.02 g / mL to 0.10 g / mL; (6) Derivatizing reagent: The derivatizing reagent is benzoyl chloride; (7) Mobile phase A: An aqueous solution containing 0.1 wt% formic acid and 2 mmol / L ammonium acetate; (8) Mobile phase B: a methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate, or an acetonitrile solution containing 0.1 vt% formic acid.

2. The reagent kit according to claim 1, characterized in that, The known concentration of the matrix working solution is prepared according to the following method: Weigh and mix adrenaline with methanol to prepare a 100 μg / mL standard stock solution; weigh and mix norepinephrine with methanol to prepare a 100 μg / mL standard stock solution; weigh and mix dopamine with methanol to prepare a 100 μg / mL standard stock solution; weigh and mix vanillic mandelic acid with methanol to prepare a 100 μg / mL standard stock solution; weigh and mix homovanillic acid with methanol to prepare a 100 μg / mL standard stock solution; weigh and mix creatinine with methanol to prepare a 100 μg / mL standard stock solution. The above-mentioned standard stock solutions are mixed and then prepared into a matrix working solution using a first solvent; the matrix working solution contains 2 ng / mL of adrenaline, 10 ng / mL of norepinephrine, 2 ng / mL of dopamine, 200 ng / mL of vanillylmandelic acid, 200 ng / mL of homovanillic acid, and 2000 ng / mL of creatinine.

3. The kit according to claim 1 or 2, characterized in that, The first solvent is an aqueous solution of bovine serum albumin at a concentration of 0.001 g / mL to 0.040 g / mL.

4. The reagent kit according to claim 1, characterized in that, The internal standard working solution is prepared according to the following method: Weigh and mix adrenaline-d6 with methanol to prepare a 100 μg / mL internal standard stock solution; weigh and mix norepinephrine-d6 with methanol to prepare a 100 μg / mL internal standard stock solution; weigh and mix dopamine-d4 with methanol to prepare a 100 μg / mL internal standard stock solution; weigh and mix vanillylmandelic acid-d3 with methanol to prepare a 100 μg / mL internal standard stock solution; weigh homovanillic acid-d5 with methanol to prepare a 100 μg / mL internal standard stock solution; weigh and mix creatinine-d3 with methanol to prepare a 100 μg / mL internal standard stock solution. The above-mentioned internal standard stock solutions were mixed and then prepared into an internal standard working solution with methanol. The internal standard working solution contained adrenaline-d6 at a concentration of 2 ng / mL, norepinephrine-d6 at a concentration of 10 ng / mL, dopamine-d4 at a concentration of 2 ng / mL, vanillylmandelic acid-d3 at a concentration of 200 ng / mL, homovanillic acid-d5 at a concentration of 200 ng / mL, and creatinine-d3 at a concentration of 2000 ng / mL.

5. The reagent kit according to claim 1, characterized in that, The sample conditioning agent is sodium bicarbonate; the concentration of the sample conditioning agent in the sample conditioning solution is 0.02 g / mL; the mobile phase B is a methanol solution containing 0.1 wt% formic acid and 2 mmol / L ammonium acetate.

6. The use of the kit according to any one of claims 1 to 5 in the preparation of a diagnostic reagent for the simultaneous detection of catecholamines and their metabolites and creatinine in human urine or plasma.

7. The kit according to any one of claims 1 to 5 is used for the simultaneous detection of catecholamines and their metabolites and creatinine in human urine or plasma using high performance liquid chromatography-tandem mass spectrometry.

8. The application according to claim 7, characterized in that, The catecholamine compounds are adrenaline, noradrenaline, and dopamine; the metabolites of the catecholamine compounds are vanillylmandelic acid and homovanillic acid; The steps for detecting catecholamines and their metabolites, as well as creatinine, in human urine or plasma include: ① Add the internal standard working solution to the series of calibrators, the series of quality control products, the plasma sample, and the urine sample respectively, and mix well; ② Add the precipitant to each sample obtained in ①, mix well, and centrifuge; ③ Take the supernatant from ② after centrifugation and place it on a nitrogen blower to dry it with nitrogen; ④ Add the sample conditioning solution to each sample after nitrogen blowing and drying in step ③, and mix well; ⑤ Add the derivatization reagent to each of the samples obtained in ④ to carry out the derivatization reaction; ⑥ After the derivatization reaction is completed, the derivatization reaction solution is taken for LC-MS / MS detection and analysis.

9. The application according to claim 8, characterized in that, The chromatographic conditions for LC-MS / MS detection are as follows: High-performance liquid chromatography (HPLC) conditions: The chromatographic column was a Pfim C18 column (2.1 × 50 mm, 2.6 μm), or a Waters HSST3 column (2.1 × 100 mm, 1.8 μm), or a Pfim PPF column (2.1 × 100 mm, 2.6 μm); mobile phase A was an aqueous solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate; mobile phase B was a methanol solution containing 0.1 vt% formic acid and 2 mmol / L ammonium acetate, or an acetonitrile solution containing 0.1 vt% formic acid; the acquisition time was 6 min; gradient elution was used, with specific elution conditions as shown below: ； Mass spectrometry conditions: Ion source type: electrospray ion source, positive ion scanning mode, MRM mode scanning; ion source spray voltage: 2000V-5500V; temperature: 500-600℃; curtain gas (CUR): 30-40 psi; collision gas: 8 psi; nebulizing gas (Gas1): 55 psi; auxiliary heating gas (Gas2): 40 psi.

10. The application according to claim 8, characterized in that, The derivatization reaction is carried out at a temperature of 40℃ to 60℃ and a reaction time of 10 min to 30 min.

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