A detection method and kit for catecholamines and catecholamine metabolites

Abstract

The present invention discloses a detection method and kit for catecholamines and catecholamine metabolites, including: using creatinine Cre correction, using dansyl chloride derivatization method to convert 8 catecholamines and catecholamine metabolites in urine samples into stable derivatives, using high performance liquid chromatography for separation, and using triple quadrupole tandem mass spectrometer to simultaneously determine the concentrations of creatinine and 8 catecholamines and catecholamine metabolites in urine and process data. The present invention selects random urine samples as detection objects, solves the drawbacks of poor compliance of clinical outpatients and difficulty in collecting samples, does not increase creatinine detection experiments, and 8 catecholamines and catecholamine metabolites react with dansyl chloride to generate stable derivatives, and then using triple quadrupole tandem mass spectrometer to simultaneously determine the concentrations of creatinine and 8 catecholamines and catecholamine metabolites in urine. The detection method of the present invention is simple, fast, accurate and efficient, and has high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and relates to the detection of catecholamines and catecholamine metabolites, and in particular to a method and a kit for detecting catecholamines and catecholamine metabolites in a random urine sample. Background Art

[0002] Hypertension can cause serious events such as stroke, myocardial infarction, heart failure, and death, becoming a major disease threatening human health; secondary hypertension accounts for about 10% of total hypertension; in the diagnosis and evaluation of hypertension, it is necessary to determine the cause of hypertension, distinguish between primary and secondary hypertension, make a differential diagnosis of the cause of hypertension in a timely manner, and actively treat the cause; and newly diagnosed hypertensive patients should undergo common secondary hypertension screening.

[0003] Pheochromocytoma and paraganglioma (PPGL) are one of the main causes of secondary hypertension. Pheochromocytoma accounts for about 1% of the total hypertensive population, at least 2.7 million people, that is, 1.7 people out of every 1,000 are pheochromocytoma patients. On the other hand, there are 3 million new cases of hypertension each year, of which about 30,000 are pheochromocytoma. Pheochromocytoma can cause high blood pressure in patients, resulting in serious complications such as heart, brain, and kidney. Clinical manifestations may include persistent (50%), paroxysmal hypertension (30%), and normal blood pressure (10%). It is often accompanied by the triad of headache, palpitations, and hyperhidrosis, as well as abnormal sugar and lipid metabolism. Patients may suffer from severe heart, brain, and kidney damage due to long-term hypertension or crises due to sudden severe hypertension (multiple organ system dysfunctions such as heart, brain, and kidney, such as myocardial infarction, arrhythmia, cardiogenic shock; pulmonary edema, acute respiratory distress syndrome (ARDS); cerebrovascular accident; renal failure, etc.; severe cases may lead to shock and ultimately death from respiratory and circulatory failure), which is life-threatening. However, if it can be diagnosed and treated in a timely and early manner, it is a curable secondary hypertension. About 90% of pheochromocytomas can be cured through surgery. Therefore, its early diagnosis is very important; however, the autopsy results of Mayo Clinic, a large third-party diagnostic institution in the United States, show that the misdiagnosis rate of early pheochromocytoma is as high as 75%, and about 25% of pheochromocytomas are incidental findings in imaging. Therefore, the screening of pheochromocytoma must use highly sensitive and specific indicators and advanced detection methods.

[0004] The biggest feature of pheochromocytoma is the secretion of large amounts of catecholamines and their metabolites. According to the 8th edition of "Internal Medicine" published by Higher Education Press, the 2018 edition of "Guidelines for the Prevention and Treatment of Hypertension in China", the 2014 edition of "Clinical Practice Guidelines for the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma", the 2020 edition of "Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma" and important journals (such as Lancet, N Engl J Med, JAMA): Monitoring of blood and urine catecholamines and their metabolite concentrations is the preferred biochemical indicator for screening and diagnosis of pheochromocytoma.

[0005] With the continuous advancement of technology, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is known as the gold standard for small molecule quantitative analysis due to its high sensitivity, strong specificity, relatively low interference, and simultaneous analysis of multiple indicators. The consensus of relevant experts and clinical practice guidelines at home and abroad recommend the use of LC-MS / MS to determine catecholamines to reduce experimental costs and increase detection throughput. Therefore, comprehensive detection of catecholamines and metabolites by LC-MS / MS can significantly improve the sensitivity and specificity of pheochromocytoma diagnosis, and efficiently screen for cardiovascular and cerebrovascular diseases such as secondary hypertension caused by it.

[0006] Pheochromocytoma metastasizes slowly in the human body, and the amount of catecholamines released into the blood is low, but catecholamines are eventually excreted in the form of urine through metabolites. The catecholamines and catecholamine metabolites in the patient's urine will increase significantly, which is very helpful for the accurate diagnosis of pheochromocytoma. Due to differences in the patient's drinking water and renal function metabolism, the concentration of catecholamines and catecholamine metabolites in the patient's urine will be affected, so the patient's 24-hour urine is generally used as a test sample in clinical practice.

[0007] Clinically, the 24-hour urine sample retention period is relatively long, and substances such as catecholamines and catecholamine metabolites are extremely easy to oxidize and have poor stability. At the same time, sampling is not convenient, and patient compliance is poor. It is not suitable for outpatient examinations, especially for out-of-town patients in outpatient examinations. Therefore, blood sampling is mainly used in clinical practice to detect catecholamines and catecholamine metabolites in the blood. Random urine sampling is convenient and can improve the clinical sample retention process. Urine samples can be taken at any time, and there is no strict requirement for timing, which shortens the detection cycle and diagnosis time, while reducing medical expenses (such as hospital bed occupancy). Creatinine in urine is often used to correct the concentration of illegal substances in urine excretion. Correcting the concentration of catecholamines and catecholamine metabolites in urine with the content of creatinine in urine can reduce the impact of factors such as renal function and drinking water on the results. The creatinine content in urine is relatively high, measured in mg per mL of urine, while the catecholamine content in urine is at the ng level; creatinine has high polarity and is not retained on ordinary chromatographic columns; in addition, measuring the concentrations of creatinine and catecholamines separately will increase the detection time and cost.

[0008] The Chinese patent application publication number is CN109959738A, and the invention name is "Liquid-mass analysis method for detecting the content of catecholamines in 24-hour urine". It is aimed at the determination of three catecholamines, adrenaline, norepinephrine and dopamine, in urine samples. The patented method uses solid phase extraction technology in the sample pretreatment process, which increases the cost and time of the method, and the detection indicators do not involve catecholamine metabolites and creatinine.

[0009] Chinese patent application publication number CN113917007A, invention name "Kit and method for detecting catecholamines and their intermediate metabolites in human urine" is used to detect 6 catecholamines and their intermediate metabolites in 24H urine. The patented method uses acetaldehyde solution derivatization technology to detect 6 catecholamines and their intermediate metabolites in 24H urine. The patented technology is simple and quick in sample pretreatment, and the catecholamines are more stable after derivatization. However, the patented technology does not include the determination of the final metabolites of catecholamines, vanillylmandelic acid and homovanillic acid, and the trial sample type is a 24H urine sample.

[0010] Chinese patent application publication number CN10746262647A pheochromocytoma diagnostic kit and its use method, the patent uses solid phase extraction method to determine three catecholamine metabolites and creatinine in random urine, after the solid phase extraction column is activated and balanced, the sample solution to be tested is loaded onto the solid phase extraction column, the solid phase extraction column selectively adsorbs the target component, removes the interference by elution, and then elutes the target from the solid phase extraction column with an elution solvent to achieve the purpose of separation and purification, the eluate is vacuum concentrated and spin-dried and redissolved, and then measured by liquid chromatography. Its disadvantages are that the operation steps are cumbersome and time-consuming, including activation, loading, elution, elution, spin-drying and other steps, and no internal standard substance is used for quantification, which has a greater impact on the detection results of catecholamine substances with poor stability, and the detection flux and qualitative ability of liquid chromatography are poorer than those of triple quadrupole mass spectrometry.

[0011] Therefore, there is an urgent clinical need for a detection method that can simultaneously and rapidly measure creatinine and eight catecholamines and catecholamine metabolites in random urine. Summary of the invention

[0012] In order to solve the above problems, the present invention provides a detection method that can simultaneously and rapidly determine creatinine and 8 catecholamines and catecholamine metabolites in random urine, using dansyl chloride derivatization technology to convert 8 catecholamines and catecholamine metabolites in urine samples into relatively stable derivatives, using high performance liquid chromatography to separate creatinine and 8 catecholamines and catecholamine metabolites, and then using a triple quadrupole mass spectrometer to detect. A series of experiments have proven the reliability and stability of the method.

[0013] The present invention also provides a kit capable of simultaneously and rapidly determining creatinine and 8 catecholamines and catecholamine metabolites in random urine.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] The present invention first provides a method for detecting catecholamines and catecholamine metabolites, which comprises: using creatinine Cre correction, using dansyl chloride derivatization method to convert 8 catecholamines and catecholamine metabolites in a urine sample into stable derivatives, using high performance liquid chromatography to separate creatinine and the 8 catecholamines and catecholamine metabolites, and then using a triple quadrupole tandem mass spectrometer to simultaneously determine the concentrations of creatinine and the 8 catecholamines and catecholamine metabolites in urine and process data; wherein the 8 catecholamines and catecholamine metabolites are: vanillylmandelic acid VMA, homovanillic acid HVA, normetanephrine NMN, normetanephrine MN, 3-methoxytyramine 3-MT, norepinephrine NE, normetanephrine E and dopamine DA.

[0016] As a preferred embodiment of the present invention, the specific steps of the detection method are:

[0017] 1) Preparation of calibration curve: preparing six concentrations of calibrators 1-6, each concentration of which contains the eight catecholamines and catecholamine metabolites and creatinine;

[0018] 2) Preparation of a mixed internal standard working solution: prepare a mixed stock solution of methanol solutions of the eight catecholamines and catecholamine metabolites and a creatinine stock solution, and dilute them with a release agent A to a mixed internal standard working solution; wherein the concentrations of the eight catecholamines and catecholamine metabolites in the mixed internal standard working solution are: 8 μg / mL VMA-d3, 8 μg / mL HVA-d3, 100 ng / mL NMN-d3, 100 ng / mL MN-d3, 500 ng / mL 3-MT-d4, 100 ng / mL NE-d3, 100 ng / mL E-d6 and 500 ng / mL DA-13C6, and the concentration of inositol in the mixed internal standard solution is 50 μg / mL Cre-d3;

[0019] 3) Preparation of quality control products: Add 1 mL of concentrated hydrochloric acid to 99 mL of the tested artificial urine matrix, mix well and use it as the quality control matrix. Add catecholamines and catecholamine metabolite mixed standards to prepare low-concentration and high-concentration quality control products;

[0020] 4) Sample pretreatment: midstream urine sample collection; midstream urine sample, standard samples 1-6 in step 1), mixed internal standard working solution in step 2), low-concentration and high-concentration quality control products in step 3) are placed in a centrifuge tube, vortexed and mixed, centrifuged, and the supernatant is collected; release agent B and derivatization solution are added to the supernatant, vortexed and mixed, shaken at a constant temperature, release agent C is added, vortexed, centrifuged at high speed, and the supernatant is collected;

[0021] 5) The supernatant of step 4) is detected by phase chromatography triple quadrupole tandem mass spectrometry;

[0022] 6) Data processing: The instrument's built-in software MassLynx collects and correctly integrates the chromatograms of the analyte and its internal standard. The isotope internal standard method is used for quantification, with 1 / x as the weight. The ratio of the peak area of ​​each compound to its isotope internal standard is used as the ordinate, and the concentration of each compound is used as the abscissa to draw a calibration working curve. The calibration curve is used to calibrate the samples and quality control products to obtain the concentration of each compound.

[0023] As a preferred embodiment of the present invention, in step 2), the releasing agent A is 0.1% formic acid and 50-100% acetonitrile aqueous solution.

[0024] As a preferred embodiment of the present invention, in step 3), the release agent B is 0.05-0.2% M sodium hydroxide solution, the release agent C is 1-10% formic acid aqueous solution; and the derivatization liquid is 1-10 mg / mL dansyl chloride solution.

[0025] As a preferred embodiment of the present invention, in step 5), the chromatographic parameters of the liquid chromatography are: chromatographic column: watersBEH C18 chromatographic column + online filter; mobile phase: mobile phase A, mobile phase B; liquid chromatography elution gradient: 0-1.4min2%B, 1.4-2.0min 2-40%B, 2.0-3.0min 40-55%B, 3.0-4.0min 55-65%B, 4.0-5.0min 65-80%B, 5.0-8.0min 80-95%B, 9.0-9.1min 95-2%B, 2.5-2.6min 80%B, 2.6-3min 80%B; flow rate: 0.4mL / min; column temperature: 40°C; injection: 10μL.

[0026] As a preferred embodiment of the present invention, the mobile phase A is an aqueous solution of 10% formic acid and 1M ammonium acetate; the mobile phase B is an acetonitrile solution of 10% formic acid.

[0027] As a preferred embodiment of the present invention, in step 5), the mass spectrometry parameters are: mass spectrometry model: H-class Waters TQD; ionization mode: positive ion mode; electrospray capillary voltage: 3.2 kV; ion source temperature: 150°C; cone voltage: 40 V; cone gas flow rate: 150 L / Hr; desolvation temperature: 550°C; desolvation flow rate: 950 L / Hr.

[0028] As a preferred embodiment of the present invention, in step 3), the low quality control values ​​of 8 catecholamines and catecholamine metabolites are: VMA is 1.5 μg / mL, HVA is 1.5 μg / mL, NMN is 15 ng / mL, MN is 15 ng / mL, 3-MT is 37.5 ng / mL, NE is 15 ng / mL, E is 1.5 ng / mL and DA is 75 ng / mL, and the low quality control value of creatinine is 0.375 mg / mL;

[0029] The high quality control values ​​of 8 catecholamines and catecholamine metabolites are: VMA is 12μg / mL, HVA is 12μg / mL, NMN is 120ng / mL, MN is 120ng / mL, 3-MT is 300ng / mL, NE is 120ng / mL, E is 36ng / mL and DA is 600ng / mL. The high quality control value of creatinine is 3.0mg / mL.

[0030] As a preferred embodiment of the present invention, in step 4), the conditions for the constant temperature oscillation reaction are: temperature 60° C., rotation speed 1000 rpm, and reaction time 10 min.

[0031] The second aspect of the present invention provides a kit for catecholamines and catecholamine metabolites, comprising a calibrator solution, a quality control product, a mixed internal standard working solution, a releaser A, a releaser B, a releaser C and a derivative solution, wherein the preparation methods of the calibrator solution, the quality control product and the mixed internal standard working solution are as described above.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention selects random urine samples as detection objects, solves the problems of poor compliance of clinical outpatients and difficulty in collecting samples, adopts creatinine correction to solve the influence of factors such as patients' renal function metabolism and drinking water differences on the detection results, does not increase the creatinine detection experiment, and 8 catecholamines and catecholamine metabolites react with dansyl chloride to generate stable derivatives, and then a triple quadrupole tandem mass spectrometer is used to simultaneously determine the concentrations of creatinine and 8 catecholamines and catecholamine metabolites in urine. The detection method of the present invention is simple, rapid, accurate and efficient, and has high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is the mass spectrum of Cre and its isotope internal standard.

[0035] Figure 2 It is the mass spectrum of VMA and its isotope internal standard.

[0036] Figure 3 It is the mass spectrum of HVA and its isotope internal standard.

[0037] Figure 4 This is the mass spectrum of NMN and its isotope internal standard.

[0038] Figure 5 This is the mass spectrum of MN and its isotope internal standard.

[0039] Figure 6 This is the mass spectrum of 3-MT and its isotope internal standard.

[0040] Figure 7 This is the mass spectrum of NE and its isotope internal standard.

[0041] Figure 8 This is the mass spectrum of E and its isotope internal standard.

[0042] Fig. 9 It is the mass spectrum of DA and its isotope internal standard.

[0043] Fig.10 is the creatinine (Cre) calibration curve.

[0044] Fig.11 This is the calibration curve of vanillylmandelic acid (VMA).

[0045] Fig.12 This is the calibration curve for homovanillic acid (HVA).

[0046] Fig.13 is the calibration curve of normetanephrine (NMN).

[0047] Fig.14 is the metanephrine (MN) calibration curve.

[0048] Fig.15 This is the calibration curve of 3-methoxytyramine (3-MT).

[0049] Fig.16 is the norepinephrine (NE) calibration curve.

[0050] Fig.17 is the adrenaline (E) calibration curve.

[0051] Fig.18 is the dopamine (DA) calibration curve. DETAILED DESCRIPTION

[0052] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0053] Example

[0054] 1. Preparation of calibration curves for creatinine, catecholamines and their metabolites

[0055] Table 1. Composition of random urine catecholamine and catecholamine metabolite standards

[0056] raw material Preparation volume / mL Solvents Calculated weight / mg Creatinine (Cre) 10 water 1000 Vanillylmandelic acid (VMA) 10 Methanol 10 Homovanillic acid (HVA) 10 Methanol 10 Normetanephrine (NMN) 10 Methanol 0.1M HCl 10 Normetanephrine (MN) 10 Methanol 0.1M HCl 10 3-Methoxytyramine (3-MT) 10 Methanol 0.1M HCl 10 Norepinephrine (NE) 10 Methanol 0.1M HCl 10 Normetanephrine (E) 10 Methanol 0.1M HCl 10 Dopamine (DA) 10 Methanol 0.1M HCl 10

[0057] Take the raw materials in Table 1, weigh the corresponding weights and put them into clean beakers, dissolve them with the corresponding solvents, transfer them to a 10mL volumetric flask and dilute to 10mL, mix well. Prepare the mother solutions of each standard. Use 0.1M hydrochloric acid water to prepare the mixed standard curve in Table 2.

[0058] Table 2. Calibration curve concentrations of catecholamines and catecholamine metabolites

[0059]

[0060]

[0061] Table 3. Preparation method of internal standard stock solution of catecholamines and catecholamine metabolites

[0062] Abbreviation Raw material usage / mg Solvents Preparation volume / mL Mother solution concentration mg / mL Cre-d3 10 water 2 5.0 VMA-d3 1 Methanol 1 1.0 HVA-d3 1 Methanol 1 1.0 NMN-d3 1 Methanol 0.1M HCl 1 1.0 MN-d3 1 Methanol 0.1M HCl 1 1.0 3-MT-d4 1 Methanol 0.1M HCl 1 1.0 NE-d6 0.1 Methanol 0.1M HCl 1 0.1 E-d6 0.1 Methanol 0.1M HCl 1 0.1 DA-13C6 1 Methanol 0.1M HCl 1 1.0

[0063] Table 4. Concentration of mixed internal standards of catecholamines and catecholamine metabolites

[0064]

[0065]

[0066] Prepare the internal standard stock solutions of catecholamines and catecholamine metabolites according to Table 3. When used, dilute the internal standard stock solutions of catecholamines and catecholamine metabolites 20 times with releaser A according to the concentrations in Table 4 to prepare the catecholamine and catecholamine mixed internal standard working solutions.

[0067] II. Configuration of Catecholamine and Catecholamine Metabolite Substance Control Products

[0068] The tested artificial urine matrix was added with 1 mL of concentrated hydrochloric acid according to 99 mL of artificial urine, and the mixture was used as the quality control matrix. The mixed standards of catecholamines and catecholamine metabolites were added to prepare low- and high-concentration quality control products, as shown in Table 5. All quality control products were packaged in 1.5 mL centrifuge tubes, 80 μL per tube, and placed in 100-well cryoboxes and stored at -80°C.

[0069] Table 5. Table of target values ​​for catecholamines and catecholamine metabolites

[0070]

[0071] 3. Mobile Phase Preparation

[0072] Mobile phase A: 10% formic acid 1M ammonium acetate aqueous solution: Take 0.77g ammonium acetate and add 9mL pure water and 1mL formic acid. Dilute 100 times with pure water before use.

[0073] Mobile phase B: 10% formic acid in acetonitrile: Mix 9 mL of acetonitrile and 1 mL of formic acid. Dilute 100 times with acetonitrile before use.

[0074] 4. Preparation of the test kit

[0075] Use the Baichen Medical product test kit, the main components of the test kit are as follows:

[0076] 1. Release agent A: 0.1% formic acid, 50-100% acetonitrile aqueous solution;

[0077] 2. Release agent B: 0.05-0.2% M sodium hydroxide solution;

[0078] 3. Release agent C: 1-10% formic acid aqueous solution;

[0079] 4. Derivatization solution: 1-10 mg / mL dansyl chloride solution;

[0080] 5. Mix the internal standard working solution;

[0081] 6. Calibrator solution;

[0082] 7. Quality control products

[0083] 5. Sample pretreatment

[0084] 1) Sample collection: Follow the doctor's advice and do not take any food or drink containing catecholamines before sampling. Do not engage in strenuous exercise, maintain your daily lifestyle, and use a urine collection catheter to collect midstream urine.

[0085] 2) Extraction: Use a pipette to draw the collected midstream urine sample, standards 1-6, and 50 μL of low and high quality controls into a 1.5 mL centrifuge tube, add 450 μL of catecholamine and catecholamine metabolite mixed internal standard working solution respectively, vortex mix for 3 minutes, centrifuge at 11000 rpm for 3 minutes, and take 100 μL of supernatant into another clean 1.5 mL centrifuge tube.

[0086] 3) Derivatization: Add 50 μL of release agent B to a centrifuge tube containing 100 μL of the above extract, then add 50 μL of derivatization solution, vortex mix for 10 seconds; shake the reaction at 60°C 1000 rpm on an oscillator for 10 minutes; then add 30 μL of release agent C, vortex for 10 seconds, and centrifuge at 11000 rpm on a high-speed centrifuge for 3 minutes, then transfer 150 μL of the supernatant to a 96-well plate for detection by liquid chromatography triple quadrupole tandem mass spectrometry.

[0087] 6. Sample Testing

[0088] Chromatographic parameters: 1. Chromatographic column: waters BEH C18 chromatographic column + online filter; 2. Mobile phase: mobile phase A, mobile phase B; 3. Liquid chromatography elution gradient: 0-1.4min 2% B, 1.4-2.0min 2-40% B, 2.0-3.0min 40-55% B, 3.0-4.0min 55-65% B, 4.0-5.0min 65-80% B, 5.0-8.0min 80-95% B, 9.0-9.1min 95-2% B, 2.5-2.6min 80% B, 2.6-3min 80% B. Flow rate: 0.4mL / min; Column temperature: 40℃; Injection: 10μL.

[0089] Mass spectrometry parameters: mass spectrometry model is H-class Waters TQD; ionization mode: positive ion mode; electrospray capillary voltage: 3.2 kV; ion source temperature: 150 ° C; cone voltage: 40 V; cone gas flow rate: 150 L / Hr; desolvation temperature: 550 ° C; desolvation flow rate: 950 L / Hr; Table 6 is the multiple reaction monitoring parameters of each compound.

[0090] Table 6. Multiple reaction monitoring (MRM) of catecholamines and catecholamine metabolites

[0091]

[0092]

[0093] VII. Data Processing

[0094] The instrument's built-in software MassLynx collects the chromatograms of the analyte and its internal standard and integrates them correctly. The isotope internal standard method is used for quantification, with 1 / x as the weight. The ratio of the peak area of ​​each compound to its isotope internal standard is used as the ordinate, and the concentration of each compound is used as the abscissa to draw a calibration working curve. The calibration curve is used to calibrate the samples and quality control products to obtain the concentration of each compound.

[0095] 8. Precision

[0096] The sample concentrations at two levels, low and high, were tested respectively, with three repetitions per day for five consecutive days.

[0097] IX. Correctness Verification

[0098] The actual patient urine was selected as the basic sample for the recovery experiment. 50 μL of low, medium and high standard samples were mixed with 450 μL of the basic sample, as shown in Table 7. The requirements are: the low concentration is 3 times the lower limit of the linear range; the medium concentration is the middle concentration of the linear range; the high concentration is 70% of the upper limit of the linear range. The above "basic sample" and "experimental sample" were repeated and measured in parallel 6 times, and the experimental results were recorded and statistically analyzed for the spiked recovery of the three concentration levels of low, medium and high. The recovery rate = (detection value - basic sample value) / theoretical value × 100%, 85% < recovery rate < 115%.

[0099] Table 7. Spike recovery preparation table

[0100]

[0101]

[0102] 10. Lower limit of quantification

[0103] The lower limit of quantification is the lowest value of the target analyte that can be detected by the LC-MS / MS method under the premise of meeting the laboratory's requirements for accuracy and precision. Three samples with concentrations close to the detection limit were selected for the lower limit of quantification. Each concentration sample was divided into 5 parts for processing. Each sample was repeated 3 times, and 3 batches were measured continuously. The total precision (CV) of each concentration sample and the deviation of its concentration measurement mean from the theoretical concentration were evaluated respectively. The measurement mean of the lowest concentration sample with CV≤20% and bias<15% was taken as the lower limit of quantification of the method.

[0104] Experimental Results

[0105] The mass spectra of creatinine, catecholamines, catecholamine metabolites and their internal standards are shown in Figure 2. Figure 1-Figure 9 shown.

[0106] Calibration curve results of catecholamines and catecholamine metabolites

[0107] The retention time and linear range of creatinine and 8 catecholamines and catecholamine metabolites are shown in Table 8. Figure 10-Figure 18 As shown in the figure, the correlation coefficients between creatinine and catechol compounds are all greater than 0.99, indicating that the linear range is acceptable and meets the actual experimental requirements. This experiment set the linear range based on the results of 120 actual clinical urine samples, but did not set a wider linear range.

[0108] Table 8. Retention time and linearity results of each compound

[0109]

[0110]

[0111] The precision of each compound at the low concentration level is shown in Table 9, and the precision at the high concentration level is shown in Table 10. It can be seen from Tables 9-10 that the intra-batch and inter-batch precision of low and high concentrations are both less than 6.25% (the total allowable error TEa of the experimental item = 25%). The results show that the precision of this experimental method meets the clinical requirements for precision.

[0112] Table 9. Results of low-concentration precision verification of catecholamines and catecholamine metabolites

[0113]

[0114] Table 10. Catecholamines and catecholamine metabolites high concentration precision verification results

[0115]

[0116] The results of the spike recovery experiment are shown in Table 11. The judgment criteria for the recovery experiment are: 80% < low concentration average recovery rate < 120, 85% < medium and high concentration average recovery rates < 115% for correctness verification to be passed. It can be seen from Table 11 that this experimental method is accurate and reliable.

[0117] Table 11. Recovery rates of catecholamines and catecholamine metabolites

[0118]

[0119]

[0120] The quantitative limits of each compound tested by Waters TQD are shown in Table 12. The detection results of each compound in 120 clinical samples are far higher than their quantitative limits, indicating that this experimental method meets the needs of clinical sample detection.

[0121] Table 12. Lower limits of quantification of catecholamines and catecholamine metabolites

[0122]

[0123] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting catecholamines and catecholamine metabolites, characterized in that: The specific steps of the detection method are: 1) Preparation of calibration curve: prepare six concentrations of calibrators 1-6, each of which contains 8 catecholamines and catecholamine metabolites and creatinine; the 8 catecholamines and catecholamine metabolites are: vanillylmandelic acid VMA, homovanillic acid HVA, normetanephrine NMN, normetanephrine MN, 3-methoxytyramine 3-MT, norepinephrine NE, nometanephrine E and dopamine DA; 2) Preparation of mixed internal standard working solution: prepare a mixed stock solution of methanol solutions of the eight catecholamines and catecholamine metabolites and a creatinine stock solution, and dilute them with release agent A to a mixed internal standard working solution; wherein the concentrations of the eight catecholamines and catecholamine metabolites in the mixed internal standard working solution are: 8 μg / mL VMA-d3, 8 μg / mL HVA-d3, 100 ng / mL NMN-d3, 100 ng / mL MN-d3, 500 ng / mL 3-MT-d4, 100 ng / mL NE-d3, 100 ng / mL E-d6 and 500 ng / mL DA-13C6, and the concentration of creatinine in the mixed internal standard solution is 50 μg / mL Cre-d3; 3) Preparation of quality control products: Add 1 mL of concentrated hydrochloric acid to 99 mL of the tested artificial urine matrix, mix well and use it as the quality control matrix. Add catecholamines and catecholamine metabolite mixed standards to prepare low-concentration and high-concentration quality control products; 4) Sample pretreatment: midstream urine sample collection; midstream urine sample, standard samples 1-6 in step 1), mixed internal standard working solution in step 2), low-concentration and high-concentration quality control products in step 3) are placed in a centrifuge tube, vortexed and mixed, centrifuged, and the supernatant is collected; release agent B and derivatization solution are added to the supernatant, vortexed and mixed, shaken at a constant temperature, release agent C is added, vortexed, and centrifuged at high speed to collect the supernatant; the release agent B is 0.05-0.2%M sodium hydroxide solution, and the release agent C is 1-10% formic acid aqueous solution; the derivatization solution is 1-10mg / mL dansyl chloride solution; 5) The supernatant of step 4) was subjected to liquid chromatography triple quadrupole tandem mass spectrometry detection, chromatographic column: waters BEH C18 chromatographic column + online filter; mobile phase A was 10% formic acid 1 M ammonium acetate aqueous solution, diluted 100 times with pure water when used; mobile phase B was 10% formic acid acetonitrile solution, diluted 100 times with acetonitrile when used; liquid chromatography elution gradient; 6) Data processing: The instrument's built-in software MassLynx collects the chromatograms of the analyte and its internal standard and integrates them correctly. The isotope internal standard method is used for quantification, with 1 / x as the weight. The ratio of the peak area of ​​each compound to its isotope internal standard is used as the ordinate, and the concentration of each compound is used as the abscissa to draw a calibration working curve. The calibration curve is used to calibrate the samples and quality control products to obtain the concentration of each compound. In step 5), the elution gradient of liquid chromatography is as follows: 0-1.4 min 2%B, 1.4-2.0 min 2-40%B, 2.0-3.0 min 40-55%B, 3.0-4.0 min 55-65%B, 4.0-5.0 min 65-80%B, 5.0-8.0 min 80-95%B, 9.0-9.1 min 95-2%B; flow rate: 0.4 mL / min; column temperature: 40°C; injection: 10 μL; The mass spectrometry parameters are as follows: mass spectrometry model: H-class Waters TQD; ionization mode: positive ion mode; electrospray capillary voltage: 3.2 kV; ion source temperature: 150°C; cone voltage: 40 V; cone gas flow rate: 150 L / Hr; desolvation temperature: 550°C; desolvation flow rate: 950 L / Hr.

2. The method for detecting catecholamines and catecholamine metabolites according to claim 1, characterized in that: In step 2), the releasing agent A is 0.1% formic acid and 50-100% acetonitrile aqueous solution.

3. The method for detecting catecholamines and catecholamine metabolites according to claim 1, characterized in that: Step 3), the low quality control values ​​of 8 catecholamines and catecholamine metabolites are: VMA is 1.5μg / mL, HVA is 1.5μg / mL, NMN is 15ng / mL, MN is 15ng / mL, 3-MT is 37.5ng / mL, NE is 15ng / mL, E is 1.5ng / mL and DA is 75ng / mL, and the low quality control value of creatinine is 0.375mg / mL; The high quality control values ​​of 8 catecholamines and catecholamine metabolites are: VMA is 12μg / mL, HVA is 12μg / mL, NMN is 120ng / mL, MN is 120ng / mL, 3-MT is 300ng / mL, NE is 120ng / mL, E is 36ng / mL and DA is 600ng / mL. The high quality control value of creatinine is 3.0mg / mL.

4. The method for detecting catecholamines and catecholamine metabolites according to claim 1, characterized in that: In step 4), the conditions for the constant temperature oscillation reaction are: temperature 60° C., rotation speed 1000 rpm, and reaction time 10 min.

Images