CN116908325A - Sample pretreatment liquid, method and kit capable of simultaneously detecting 12 catecholamines, metabolites and creatinine in human urine - Google Patents
Sample pretreatment liquid, method and kit capable of simultaneously detecting 12 catecholamines, metabolites and creatinine in human urine Download PDFInfo
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- CN116908325A CN116908325A CN202310841317.7A CN202310841317A CN116908325A CN 116908325 A CN116908325 A CN 116908325A CN 202310841317 A CN202310841317 A CN 202310841317A CN 116908325 A CN116908325 A CN 116908325A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a sample pretreatment liquid, a method and a kit capable of simultaneously detecting 12 catecholamines, metabolites thereof and creatinine in human urine. The pretreatment liquid consists of diluent I, diluent II, diluent III, diluent IV, eluent I, eluent II, eluent III and eluent IV; the diluent I is 5-50% methanol containing 0.1-5% formic acid; the diluent II is 0.5-5% acetic acid solution; the diluent III is 1X phosphate buffer solution; the diluent IV is 10-100 mM of hydroxyethyl piperazine acetic sulfuric acid solution; the eluent I is methanol; the eluent II is 5-50 mM ammonium acetate solution; the eluent III is 5-50% methanol; the leaching solution IV is 5-50% methanol containing 0.1-5% formic acid. According to the invention, through optimizing and simplifying the sample pretreatment liquid and the condition parameters of the LC-MS/MS method, the method for simultaneously extracting and detecting catecholamine and metabolites thereof and creatinine in urine by using an SPE-LC-MS/MS method is realized, the time can be saved, the cost can be reduced, and the detection homogeneity can be ensured.
Description
Technical Field
The invention belongs to the technical field of preparation of kits, and particularly relates to a sample pretreatment liquid capable of simultaneously detecting 12 catecholamines, metabolites thereof and creatinine in human urine, a pretreatment method and a kit based on the sample pretreatment liquid.
Background
Catecholamines (CA) include Norepinephrine (NE), epinephrine (E), and Dopamine (DA), and CA in the circulatory system is mainly derived from the adrenal medulla and sympathetic nervous system, and has extremely strong physiological activity as an important neurotransmitter or hormone, and participates in the regulation and metabolism of various organ systems such as the cardiovascular system, nervous system, endocrine system, kidney, smooth muscle tissue, and the like of the body. DL-3, 4-dihydroxyphenyl glycol (3, 4-Dihydroxyphenyl glycol, DHPG), 4-hydroxy-3-methoxyphenethylene glycol (4-hydroxy-3-methoxyphenylethylene glycol, MHPG), 3,4-dihydroxyphenylacetic acid (3, 4-dihydroxyphenylacetic acid, DOPAC), cystein DOPA (5-S-cysteinyl DOPA, cys-DOPA), methoxynorepinephrine (NMN), methoxyepinephrine (MN), and 3-methoxytyramine (3-methoxytyramine, 3-MT) are intermediate metabolites of catecholamine, vanillyl mandelic acid (vanillylmandelic acid, VMA), and homovanillic acid (homovanillic acid, HVA) are final end metabolites, and are all finally excreted from urine.
CA and its metabolite levels increase or decrease in association with a number of pathophysiological states, and can be used as important biomarkers for screening, diagnosis, treatment and prognosis of diseases such as tumors (e.g., pheochromocytoma, paraganglioma, neuroblastoma, metastatic melanoma, etc.), metabolic diseases (e.g., dopamine beta hydroxylase deficiency, aromatic amino acid decarboxylase deficiency, mentha's disease, etc.), and autonomic nervous system diseases (e.g., diabetic peripheral neuropathy, familial amyloidogenic nerve damage, orthostatic hypotension, etc.).
More classical is the use for diagnosis of neuroendocrine tumors such as pheochromocytoma, paraganglioma and neuroblastoma. Pheochromocytoma (PCC) originates from the adrenal medulla, and Paraganglioma (PGL) originates from the sympathetic nerve chain outside the adrenal gland, both of which (collectively PPGL) are capable of synthesizing, secreting and releasing a large amount of CA, causing a series of clinical syndromes such as elevated blood pressure and metabolic changes in patients, and can cause serious complications such as heart, brain, kidney, and even death in patients. The non-metastatic PPGL can be cured by surgical excision for early diagnosis, and the metastatic PPGL can be used for early diagnosis and timely surgery for prolonging the life of a patient, so that the early diagnosis and early treatment of the PPGL have great significance for disease prognosis. CA and its metabolite are the main basis for qualitative diagnosis of PPGL, and relevant guidelines at home and abroad recommend detection of blood or urine NE, E, DA, NMN, MN, 3-MT, HVA and VMA concentrations. Neuroblastoma (NB) originates from the adrenal medulla or paraspinal sympathetic nervous system, is the most common extracranial solid tumor for infants and young children, accounts for 8% -10% of malignant tumors in children, has high heterogeneity in clinical manifestation and prognosis, and has high synchronous and obvious increase in urine/serum CA and metabolite levels in infants, and guidelines at home and abroad recommend detection of urine VMA and HVA to assist diagnosis and to evaluate response to treatment.
The determination of CA and its metabolites in blood or urine is also of diagnostic interest for other diseases. Such as a significant elevation in blood or urine Cys-DOPA from patients with metastatic melanoma; the diseases such as dopamine beta hydroxylase deficiency, aromatic amino acid decarboxylase deficiency, mentha disease and the like can all occur with different degrees of blood or urine elevation of DA and DOPAC, and reduction of NE and DHPG; some of the more common autonomic nervous system diseases such as diabetic peripheral neuropathy, familial amyloidogenic nerve damage, orthostatic hypotension, etc. can occur with decreased blood or urine NE and E, increased DHPG; in addition, MHPG is a major metabolite of NE in the hub and has diagnostic significance for some mental diseases.
The detection of CA and its metabolite concentration in blood or urine is characterized. The body position and stress state of a patient have great influence on the CA and metabolite levels in blood when blood is taken, the blood is taken after the posture is fixed for a certain time (sitting position or lying position for at least half an hour), the blood is taken as invasive operation, the operation is inconvenient in clinical practice, meanwhile, the CA and metabolite concentration levels in the blood are extremely low (pg/ml level), the sensitivity requirement on a detection instrument is extremely high, the sample pretreatment is more complicated and time-consuming, the detection cost is high, and the popularization and the clinical application are difficult. CA and its metabolite concentration levels in urine are relatively high (ng/ml and μg/ml levels) and constant, urine is noninvasively available. The random urine is convenient and quick to collect, patient compliance is good, but is easily influenced by concentration degree of urine, and because the generation and the excretion of Creatinine (Cr) of a human body are relatively stable, concentration degree of urine by kidneys can be accurately reflected, and clinical detection of the random urine can be realized by detecting concentration of Creatinine in the random urine to correct concentration of CA metabolite. However, for patients with reduced creatinine production (e.g., those with prolonged bed rest, wasting) or limited excretion (e.g., those with renal insufficiency), it is necessary to combine the 24-hour urine output for quantification,
The existing methods for detecting urinary CA and its metabolites have the following drawbacks:
(1) The detection method has low sensitivity, specificity and convenience. For example, patent applications CN104062388B, CN107462647B, CN114994211a and CN115015426a, high Performance Liquid Chromatography (HPLC), high performance liquid chromatography-electrochemical method (HPLC-ECD) and the like are adopted to detect urine CA and its metabolites, the sensitivity of the HPLC method is limited, and the detection is easily affected by a plurality of interferents in the body, resulting in reduced accuracy; the HPLC-ECD method has the advantages of complicated sample pretreatment, time consumption, long detection time and easy interference of a plurality of substances.
(2) Basically, 24 hours urine needs to be collected for detection, which is long in time consumption and easy to make mistakes. Because the concentration degree difference of random urine of a human body is large, the concentration difference of substances in the urine is also large, and the urine volume of 24 hours is always collected for comprehensively quantifying the substances secreted in the urine, the current detection of catecholamine and metabolites thereof is basically carried out by collecting the urine of 24 hours, but the time for collecting the urine of 24 hours is long and is easy to make mistakes, and the operation of an outpatient and a special patient (such as an infant patient and a long-term bedridden patient) is inconvenient, so that the accuracy and the rapidness of auxiliary diagnosis of tumors are affected.
(3) The pretreatment steps of the urine sample are tedious, time-consuming and low in efficiency. For example, in patent applications CN113917007A, CN114441664A, CN109709255A and CN109142594a, the respective detection methods need to perform steps of acidification high-temperature hydrolysis, derivatization reaction, re-dissolution after drying solid phase extraction eluent with nitrogen, etc. when urine is pretreated, which is long in time consumption and not easy to control, increases reagent cost and time cost, and also affects clinical use of the solid phase extraction eluent.
In view of the above, it is very necessary to develop a simple and economical pretreatment solution and pretreatment method for samples, which can detect catecholamine and its metabolites and creatinine in 24 hours urine and random urine of human body simultaneously, and has practical clinical application value.
Disclosure of Invention
The first object of the present invention is to provide a sample pretreatment liquid capable of simultaneously detecting 12 catecholamines, their metabolites and creatinine in human urine, the second object of the present invention is to provide a sample pretreatment method capable of simultaneously detecting 12 catecholamines, their metabolites and creatinine in human urine, and the third object of the present invention is to provide a kit based on a pretreatment liquid capable of simultaneously detecting 12 catecholamines, their metabolites and creatinine in human urine.
The first object of the invention is realized by a sample pretreatment liquid for detecting 12 catecholamines and metabolites thereof and creatinine in human urine, wherein the 12 catecholamines and metabolites thereof are norepinephrine, epinephrine, dopamine, 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid, 5-S-cysteine dopa, methoxynorepinephrine, methoxyepinephrine, 3-methoxytyramine, vanilla mandelic acid and homovanillic acid, the sample is urine, a calibrator and a quality control substance, and the sample pretreatment liquid consists of a diluent and a leaching solution;
the diluent consists of diluent I, diluent II, diluent III and diluent IV; the diluent I is 5-50% methanol containing 0.1-5% formic acid; the diluent II is 0.5-5% acetic acid solution; the diluent III is 1X phosphate buffer solution; the diluent IV is 10-100 mM of hydroxyethyl piperazine acetic sulfuric acid solution;
the leacheate consists of leacheate I, leacheate II, leacheate III and leacheate IV; the leaching solution I is methanol; the leaching solution II is 5-50 mM ammonium acetate solution; the leaching solution III is 5-50% of methanol; the leaching solution IV is 5-50% methanol containing 0.1-5% formic acid.
The second object of the invention is achieved by a sample pretreatment method for detecting 12 catecholamines, metabolites thereof and creatinine in human urine by using the pretreatment liquid, which is characterized by comprising a pretreatment step I and a pretreatment step II;
the pretreatment step I is to directly mix a sample, a diluent and an internal standard solution, and then to centrifuge at a high speed to obtain a supernatant to obtain a treated sample liquid I, wherein the centrifugation speed is 10000-14000 rpm, and the centrifugation time is 5-10 minutes;
the pretreatment step II is to mix a sample, a diluent and an internal standard solution and then carry out solid-phase extraction, and the obtained extraction liquid is a treated sample injection liquid II;
finally, carrying out LC-MS/MS detection on the treated sample liquid I and sample liquid II;
in the first pretreatment step, the preparation method of the series of calibrator/quality control product systems comprises the following steps: respectively taking 20 mu L of calibrator/quality control material, 200 mu L of diluent II and 20 mu L of internal standard solution, and uniformly mixing; the system preparation method of the urine sample comprises the following steps: respectively taking 20 mu L of urine sample, 200 mu L of diluent II and 20 mu L of internal standard solution, and uniformly mixing;
in the second pretreatment step, the preparation method of the series of calibration materials/quality control materials system comprises the following steps: mixing 30 μl of calibrator/quality control, 270 μl of diluent III, 20 μl of internal standard, and 450 μl of diluent IV; the system preparation method of the urine sample comprises the following steps: mu.L of diluent I, 270. Mu.L of urine sample, 20. Mu.L of internal standard and 450. Mu.L of diluent IV were mixed separately.
The third object of the present invention is achieved by a kit for detecting 12 catecholamines and their metabolites and creatinine in human urine, comprising the sample pretreatment liquid.
The sample pretreatment adopts a solid-phase extraction method, selects a proper solid-phase extraction material according to physicochemical properties (such as polarity, molecular weight, pKa value and the like) of target analytes, and adjusts extraction reagents and extraction conditions so as to achieve the purposes of concentrating and purifying biological samples. The filler of the 96-well solid-phase extraction plate is a weak cation exchange material, and is mainly reserved by charge action and/or nonpolar acting force, the pKa of CA and its metabolite is about 10, while the pKa of Cr is 4.8, when the pH of the solution is near neutral, the former is mainly positively charged and is mainly reserved in the filler small column by charge adsorption, and the latter is mainly negatively charged and cannot be reserved by charge action, so the solution is mainly reserved by nonpolar acting force.
The invention optimizes and simplifies the extraction reagent and extraction condition of sample pretreatment, so that the solid phase extraction can directly sample and analyze in five steps, and the invention relates to the following principle: (1) the activation aims at infiltrating the solid phase, so that the solid phase is activated in a liquid phase environment and is prepared for the subsequent steps; (2) balancing, namely enabling the filler to be in a proper state capable of adsorbing target analytes, and using a buffer solution (pH 6-7) with a certain concentration to reach an equilibrium state; (3) in order to ensure that most of target analytes are reserved in the solid-phase filler to achieve a certain recovery rate, the pH value of a sample loading system is maintained to be about 7, and the pH value of the sample loading system is regulated by using a hydroxyethyl piperazine ethyl sulfate acid solution with a certain concentration because the pH value of urine of a clinical patient has a large change range; (4) the leaching is used for washing away impurities or interferents adsorbed in the filler as much as possible under the condition that the retention of target analytes is not influenced, and during the development process, we find that Cr retained in the filler is easy to be washed away by an organic reagent (a conventional leaching step) and the nonpolar acting force is presumed to be easy to be damaged by the organic reagent, so that the leaching after the sample application cannot be conventionally washed by using a pure organic reagent, but a large proportion of water phase is used instead, and most of water-soluble impurities in urine can be washed away and the retention of Cr is not influenced; (5) the eluting solution is required to not only elute and retain the target analyte of [1] on the filler, but also meet the detection requirement of LC-MS/MS. The eluent needs to be added with a certain amount of acid to reduce the pH value and contain a certain amount of organic reagent to destroy the binding force of the target analyte and the filler so as to elute the target analyte, but the ratio of the pH value to the organic reagent needs to be controlled within a certain range, otherwise, the LC-MS/MS detection is influenced. Furthermore, the amount of eluent should not be too great, otherwise the analyte is excessively diluted and the concentration is reduced, and a small portion of the clinical sample itself having a lower concentration may be below the minimum quantification limit. The eluent developed by the invention has specific formic acid addition amount, methanol proportion and volume, and the concentration of the object to be detected in the eluent can reach the lowest quantitative limit by only eluting once.
The invention optimizes the liquid phase condition and mass spectrum parameter of LC-MS/MS. In the aspect of mass spectrometry conditions, because CA and metabolites thereof form sulfate binding products in urine, the binding rate IS 80% -90%, the free type accounts for 10% -20% (the free type concentration IS detected by the method), the concentration IS relatively low, and mass spectrometry parameters are optimized to maximize the signal response of each analyte, so that parameters such as spray voltage (IS), ion source Temperature (TEM), gas curtain gas (CUR) and collision gas (CAD) are optimized and adjusted to maximize the signal response of the analytes. NE, E, DA, DHPG, MHPG, DOPAC, cys-DOPA, NMN, MN, 3-MT and Cr respond well in positive ion mode, VMA and HVA respond well in negative ion mode, so that detection is performed in two modes separately, and time is saved if mass spectrum capable of switching positive and negative ions in the same method is used.
In terms of liquid phase conditions, the choice of analytical column is critical. The detected CA and the metabolite thereof are aromatic compounds, creatinine is a heterocyclic compound, the molecular weight is small, the water solubility is high, the polarity is strong, the pentafluorophenyl reversed phase chromatographic column takes ultrapure silica gel as a matrix surface to bond with pentafluorophenyl ethyl as a filler, the group can generate dipole induction effect, and has stronger separation capability on easily polarized substances such as aromatic rings, heterocyclic compounds and the like, so that the detected substances can achieve better retention and separation effects, and meanwhile, the service life of the analytical column can be prolonged by being provided with a protective column. In LC-MS/MS methods, the mobile phase is the basis for ensuring that each target analyte can be effectively separated and detected, and generally includes mobile phase a (aqueous phase) and mobile phase B (organic phase), and in most cases, a certain amount of additives are added, and an appropriate flow rate and column temperature are selected in cooperation with an analytical column, and an optimal ratio gradient is explored to achieve the objective of optimizing the chromatographic behavior of the analyte (including appropriate retention time, symmetrical and smooth peak shape, narrower peak width, etc.). In the method, a certain amount of formic acid is added to adjust the pH value of the mobile phase, so that the protonizing capacity of 13 objects to be detected can be effectively improved, the response is enhanced, the peak shape is improved, a large proportion of water phase (phase A) is used as the initial proportion of the mobile phase, the proportion of the organic phase is increased in a gradient manner, the timely elution and separation of the analytes are facilitated, and the final gradient elution scheme is shown in a table 3. In addition, the method uses an internal standard method for quantification, and an ideal internal standard substance has basically consistent physicochemical properties, chromatographic behavior and response characteristics with the analyzed compound, can offset systematic errors and matrix effects, achieves more accurate quantitative analysis, and is an ideal internal standard substance. The method of the invention uses the isotope internal standard corresponding to each analyte, has the same retention time and matrix effect as the analyte, and enhances the detection accuracy and anti-interference capability.
The invention optimizes the liquid phase condition and mass spectrum parameters, further shortens the detection time, improves the sensitivity and the specificity, and realizes the simultaneous detection of 12 CA and metabolites thereof and Cr.
The beneficial effects of the invention are as follows:
1. the pretreatment liquid for the urine sample, developed by the invention, overcomes the systematic defect in the prior art, effectively separates and enriches the object to be detected in the urine sample, further simplifies the solid-phase extraction step of the urine sample, is easy to elute, and can directly carry out on-machine sample injection analysis without the time-consuming and complicated steps of nitrogen blowing, re-dissolving and the like. The method has the advantages of improving the detection efficiency, reducing the cost, improving the detection sensitivity, improving the clinical disease diagnosis efficiency by popularization and application, and reducing the burden and inconvenience of patients.
2. The sample pretreatment method based on the treatment fluid can simultaneously extract and separate catecholamine and metabolites thereof and creatinine in urine, further realizes the simultaneous detection of catecholamine and metabolites thereof and creatinine by one LC-MS/MS method through the optimization of liquid phase conditions and mass spectrum parameters, does not need to independently treat and independently use other methods or equipment to extract and detect creatinine, further shortens the detection time by more than 50%, improves the efficiency, reduces the cost and ensures the detection homogeneity.
3. The kit based on the sample pretreatment liquid can be used for simultaneously detecting 12 catecholamine substances (namely NE, E, DA, DHPG, MHPG, DOPAC, cys-DOPA, NMN, MN and 3-MT, VMA, HVA) in human urine samples, expands the disease range of auxiliary diagnosis, and can assist diagnosis including but not limited to pheochromocytoma, paraganglioma, neuroblastoma, metastatic melanoma, some metabolic diseases and autonomic nervous system diseases.
4. The urine detection kit disclosed by the invention has high detection sensitivity, and the random urine sample detection result can meet the clinical diagnosis requirement, so that the defect that the detection method in the prior art needs to collect a 24-hour (accumulated) urine sample for detection can be used for diagnosis is overcome. In clinical practice, the inventor finds that 24-hour urine for examination is easy to have the conditions of unrecorded 24-hour urine volume, error taking and the like, and the 24-hour urine taking is long in time and easy to pollute, which can lead to poor patient detection compliance. In addition, it is difficult for some special patients such as infants and long-term bedridden patients to leave 24-hour urine. In contrast, the detection technology of the invention only needs to collect random urine samples, is convenient and quick, has better patient compliance, and has evidence at present that the diagnosis efficacy of 24-hour urine and random urine for pheochromocytoma and paraganglioma is equivalent, so that the detection of the random urine is clinically required, and the kit and the detection method can solve the clinical problem.
5. The kit based on the sample pretreatment liquid has higher sensitivity, and when 5 indexes (NMN, MN, 3-MT, VMA and HVA) in random urine are used for jointly diagnosing the cytoma and the paraganglioma, the detection sensitivity and the specificity reach 100%, the AUC value reaches 1, and the detection limit can meet the detection of almost all clinical urine samples, so that the kit can be widely applied to various level type liquid chromatograph tandem mass spectrometers; all detection indexes are corrected by using corresponding isotope internal standards, so that the specificity is high, and the detection anti-interference capability is high.
In conclusion, the sample pretreatment liquid kit capable of simultaneously detecting 12 catecholamines, metabolites thereof and creatinine in urine can meet the clinical detection of random urine and 24-hour urine, can rapidly, simply and noninvasively assist in diagnosing diseases such as pheochromocytoma, paraganglioma or neuroblastoma, and the like, enables patients to be effectively and correctly diagnosed in time, is beneficial to improving the diagnosis and treatment rate of the diseases, saves medical cost, and has important clinical significance and economic value.
Drawings
FIG. 1 is a LC-MS/MS plot (blank matrix) of VMA and HVA and corresponding internal standards in example 2;
FIG. 2 is a LC-MS/MS diagram (urine sample (analyte)) of VMA and HVA and corresponding internal standard in example 2;
FIG. 3 is a LC-MS/MS diagram of VMA and HVA and corresponding internal standard (urine sample (test object internal standard)) in example 2;
FIG. 4 is a LC-MS/MS diagram (blank matrix) of Cr, NMN, MN, 3-MT, cr, etc. and corresponding internal standards in example 2;
FIG. 5 is a LC-MS/MS diagram (urine sample (analyte)) of Cr, NMN, MN, 3-MT, cr, etc. and corresponding internal standard in example 2;
FIG. 6 is a LC-MS/MS diagram of Cr, NMN, MN, 3-MT, cr, etc. and corresponding internal standard (urine sample (internal standard of test object)) in example 2;
fig. 7 is a ROC graph of random urine (fig. 7A) versus 24 hours urine (fig. 7B).
Detailed Description
The invention is further illustrated, but is not limited in any way, by the following examples, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The invention relates to a sample pretreatment liquid capable of simultaneously detecting 12 catecholamines and metabolites thereof and creatinine in human urine, wherein the 12 catecholamines and the metabolites thereof are norepinephrine, epinephrine, dopamine, 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxyl phenethyl glycol, 3, 4-dihydroxyphenylacetic acid, 5-S-cysteine dopa, methoxyl norepinephrine, methoxyl epinephrine, 3-methoxyl tyramine, vanillyl mandelic acid and homovanillic acid, the sample is urine, a calibrator and a quality control product, and the sample pretreatment liquid consists of a diluent and a leaching solution;
The diluent consists of diluent I, diluent II, diluent III and diluent IV; the diluent I is 5-50% methanol containing 0.1-5% formic acid; the diluent II is 0.5-5% acetic acid solution; the diluent III is 1X phosphate buffer solution; the diluent IV is 10-100 mM of hydroxyethyl piperazine acetic sulfuric acid solution;
the leacheate consists of leacheate I, leacheate II, leacheate III and leacheate IV; the leaching solution I is methanol; the leaching solution II is 5-50 mM ammonium acetate solution; the leaching solution III is 5-50% of methanol; the leaching solution IV is 5-50% methanol containing 0.1-5% formic acid.
The calibrator consists of norepinephrine, epinephrine, dopamine, 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid, 5-S-cysteine dopa, methoxynorepinephrine, methoxyepinephrine, 3-methoxytyramine, vanillin, homovanillic acid and creatinine;
the concentration range of norepinephrine, epinephrine and dopamine is 12.5-800 ng/ml, the concentration range of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxyl phenethyl glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 0.3-20 mug/ml, the concentration range of vanillyl mandelic acid and homovanillic acid is 0.6-40 mug/ml, the concentration range of methoxynorepinephrine, methoxyepinephrine and 3-methoxyl tyramine is 6.3-400 ng/ml, and the concentration range of creatinine is 63-4000 mug/ml.
The quality control product consists of a low quality control product and a high quality control product;
wherein, the concentration of norepinephrine, epinephrine and dopamine in the low quality control product is 60 ng/ml, the concentration of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxyl phenethyl glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 2 mug/ml, the concentration of vanillyl mandelic acid and homovanillic acid is 3 mug/ml, the concentration of methoxyl norepinephrine, methoxyl epinephrine and 3-methoxyl tyramine is 30 ng/ml, and the concentration of creatinine is 300 mug/ml;
the concentration of norepinephrine, epinephrine and dopamine in the high quality control is 600 ng/ml, the concentration of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 18 mug/ml, the concentration of vanillyl mandelic acid and homovanillic acid is 30 mug/ml, the concentration of methoxynorepinephrine, methoxyepinephrine and 3-methoxytyramine is 300 ng/ml, and the concentration of creatinine is 3000 mug/ml.
The invention also provides a sample pretreatment method for detecting 12 catecholamines, metabolites thereof and creatinine in human urine based on the sample pretreatment liquid, which comprises a pretreatment step I and a pretreatment step II;
The pretreatment step I is to directly mix a sample, a diluent and an internal standard solution, and then to centrifuge at a high speed to obtain a supernatant to obtain a treated sample liquid I, wherein the centrifugation speed is 10000-14000 rpm, and the centrifugation time is 5-10 minutes;
the pretreatment step II is to mix a sample, a diluent and an internal standard solution and then carry out solid-phase extraction, and the obtained extraction liquid is a treated sample injection liquid II;
finally, carrying out LC-MS/MS detection on the treated sample liquid I and sample liquid II;
in the first pretreatment step, the preparation method of the series of calibration materials/quality control materials system comprises the following steps: respectively taking 20 mu L of calibrator/quality control material, 200 mu L of diluent II and 20 mu L of internal standard solution, and uniformly mixing; the system preparation method of the urine sample comprises the following steps: mu.L of urine sample, 200 mu.L of diluent II and 20 mu.L of internal standard solution are respectively taken and uniformly mixed.
In the second pretreatment step, the preparation method of the series of calibration materials/quality control materials system comprises the following steps: mixing 30 μl of calibrator/quality control, 270 μl of diluent III, 20 μl of internal standard, and 450 μl of diluent IV; the system preparation method of the urine sample comprises the following steps: mu.L of diluent I, 270. Mu.L of urine sample, 20. Mu.L of internal standard and 450. Mu.L of diluent IV were mixed separately.
The internal standard solution is prepared from norepinephrine-D6, epinephrine-D3, dopamine-D4, 3, 4-dihydroxyphenyl glycol 13 C. 4-hydroxy-3-methoxyphenylethylene glycol-D3, 4-dihydroxyphenylacetic acid-D5, 5-S-cysteine dopa-D3, methoxynorepinephrine-D3, methoxyepinephrine-D3, 3-methoxytyramine- 13 C. Vanilla mandelic acid-D3, homovanillic acid-D3 and creatinine-D3; the concentration of norepinephrine-D6, epinephrine-D3 and dopamine-D4 is 160 ng/ml, and the concentration of 3, 4-dihydroxyphenyl glycol is 160- 13 C. 4-hydroxy-3-methoxyphenylethylene glycol-D3, 4-dihydroxyphenylacetic acid-D5,5-S-cysteine dopa-D3 at a concentration of 5 μg/ml, vanilloid-D3, homovanilloid-D3 at a concentration of 5 μg/ml, methoxynorepinephrine-D3, methoxyepinephrine-D3, 3-methoxytyramine- 13 The concentration of C was 80 ng/ml and the concentration of creatinine-D3 was 1000. Mu.g/ml.
In the pretreatment step II, the solid phase extraction is carried out by adopting a 96-hole solid phase extraction column through a positive pressure device, the filler is a weak cation exchange material, and the specific solid phase extraction method is realized according to the following steps:
1) Activating: adding 300 mu L of eluent I into each hole, and passing through a column;
2) Balance: adding 300 mu L of eluent II into each hole, and passing through a column;
3) Loading: loading all the prepared system into a column;
4) Eluting impurities: adding 500 mu L of eluent III for leaching;
5) Eluting: and adding 100 mu L of eluent IV for pressurized elution to a 96-well collection plate, and directly injecting and analyzing the obtained eluent.
In LC-MS/MS detection, the liquid chromatographic analysis column is a pentafluorophenyl column and a corresponding protection column, the mobile phase A is 0.1-1% formic acid aqueous solution, the mobile phase B is 0.1-1% formic acid acetonitrile or methanol solution, the column temperature is 40 ℃, and the flow rate is 0.45 ml/min; mass spectrometer using ESI mode, MRM negative ion mode and positive ion mode detection;
the LC-MS/MS detection result calculation method comprises the following steps: the concentration of the analyte is on the abscissa and the ratio of the peak area of the analyte to the internal standard is on the ordinate, weighted (1/x 2 ) And (3) performing linear regression analysis, and establishing a calibration curve to obtain a linear fitting equation. Taking the ratio of the peak area of each analyte in the urine sample to be detected to the peak area of the internal standard substance into a corresponding linear fitting equation, and calculating to obtain the concentrations of catecholamine metabolites and creatinine in the urine sample to be detected;
if the urine is random urine, the detection result is obtained by dividing the measured concentration of each analyte by the creatinine concentration of the same urine;
if the urine is 24 hours urine, the measured concentration of each analyte is multiplied by the 24 hour urine volume.
The invention further provides a kit comprising the sample pretreatment liquid, which can detect 12 catecholamines, metabolites thereof and creatinine in human urine at the same time.
The application of the kit is that the kit is applied to auxiliary diagnosis of pheochromocytoma, paraganglioma, neuroblastoma, metastatic melanoma, metabolic diseases or autonomic nervous system diseases through human urine detection results.
Example 1
1. Urine sample collection
Collecting 1-3 mL of random urine, or collecting 24 hours of urine, uniformly mixing, taking 1-3 mL, placing into a urine cup or a plastic tube, and preserving at-20 ℃ to be tested.
2. Sample pretreatment
Detecting the day, taking out the urine sample, the calibrator, the quality control product and the internal standard liquid from the refrigerator and putting the urine sample, the calibrator, the quality control product and the internal standard liquid to room temperature; taking a 1 mL urine sample, and centrifuging at a high speed (12000 rpm,5 minutes) for later use; a standard curve series working fluid was prepared and the calibration was diluted to a series of concentration points using diluent I (i.e. calibration 7 to calibration 1, see table 1).
Pretreatment step one: the system was prepared in the amounts shown in Table 2, and after mixing, the mixture was centrifuged at high speed (12000 rpm,5 minutes) and the supernatant was sampled in 100. Mu.L to 96-well plates for analysis.
Pretreatment step two: the system was prepared in the amount shown in table 2, and was subjected to solid phase extraction by a positive pressure apparatus using 96 Kong Guxiang extraction column, according to the following steps: (1) activating, wherein 300 mu L of eluent I is added to each hole and then the mixture is passed through a column; (2) balancing, adding 300 mu L of eluent II into each hole, and passing through a column; (3) loading the sample, namely loading all the prepared system into a column; (4) eluting impurities, adding 500 mu L of eluent III for eluting, (5) adding 100 mu L of eluent IV for pressurized eluting to a 96-well collection plate, and finally carrying out sample injection analysis. The liquid is led to pass through the column by using a positive pressure device in the steps, and the dropping speed is controlled to be 2-3 seconds per drop.
Table 1 calibrator, quality control and internal standard concentrations
Table 2 pretreatment system preparation method
3. Liquid chromatography tandem mass spectrometry
The mass spectrometer uses ESI mode, MRM negative ion mode and positive ion mode detection, the sample liquid obtained in the first pretreatment step adopts negative ion mode detection, and the sample liquid obtained in the second pretreatment step adopts positive ion mode detection. The quantitative ion pairs for each analyte, the corresponding isotopic internal standard ion pairs, and the associated mass spectral parameters are shown in table 3. The ion source parameters and liquid phase conditions are shown in table 4, using analytical column (Kinetex ® 2.6 Mu m F5A) is provided with guard posts (security guard) TM ULTRA cards) was analyzed for a total of 10 minutes per sample.
Table 3 mass spectrometry parameter settings
Note that: DP cone voltage, CE collision energy.
TABLE 4 ion Source parameters and liquid phase conditions
Note that: curtain Gas (CUR), collision Gas (CAD), ion spray Voltage (IS), temperature (TEM), ion Source Gas1 (GS 1), ion Source Gas1 (GS 2).
4. Calculation result
Using an internal calibration method, taking the concentration of the analyte as an abscissa [ ]X) The peak area ratio of the analyte to the internal standard is taken as the ordinateY) With weighting (1-x 2 ) And (3) performing linear regression analysis, and establishing a calibration curve to obtain a linear fitting equation. And (3) taking the ratio of the peak area of each analyte to the peak area of the internal standard substance in the urine sample into a corresponding linear fitting equation, and calculating to obtain the concentration of catecholamine metabolite and creatinine in the urine sample to be detected. If the urine is random urine, the detection result Dividing the measured concentration of each analyte by the concentration of creatinine in the same urine; in the case of 24-hour urine, the measured concentration of each analyte is multiplied by the 24-hour urine volume.
Example 2 test kit for 12 catecholamines and their metabolites and creatinine in urine of the present invention Performance verification
The kit comprises the diluents, leaches, eluents, calibrators, quality controls and internal standard solutions mentioned in the examples. The detection method of the kit is as in example 1. The kit and the detection method thereof are verified by methodology according to the verification guidelines of the biological sample quantitative analysis method of four parts of Chinese pharmacopoeia 2020 edition. The specific verification items are as follows:
(1) Selectivity and residue detection
PBS (1X) was used as a blank urine matrix. Taking PBS, PBS containing analyte and corresponding internal standard, analyte and corresponding internal standard reference solution, treating urine of six different patients according to a given method, and performing sample detection analysis. The VMA and HVA results are shown in FIGS. 2-3, with VMA and its isotope internal standard, HVA and its isotope internal standard retention times of 1.3min, 2.3 min, respectively; the rest of the analyte results are shown in figures 5-6, the retention time of Cr, NMN, MN, 3-MT and the respective isotope internal standard is respectively 1.2min, 1.7min, 1.8min, 2.2min, NE, E, DA, DHPG, MHPG, DOPAC, cys-DOPA and the respective isotope internal standard is respectively 3.6min, 3.9min, 4.0min, 4.8min, 5.0 min, 4.7 min and 5.2 min, the retention time is relatively stable, the peak shapes of the analyte and the internal standard are good, the endogenous components of the matrix have no obvious interference on the determination of the target analyte and the internal standard, and the selectivity meets the requirements.
After 10 consecutive injections of the high concentration sample (calibration 7), the blank sample was analyzed to evaluate the residue, and the results are shown in fig. 1 and 4, indicating that there was almost no residue in the blank sample after the high concentration sample, so that the accuracy and precision of the subsequent sample were not affected.
(2) Linear and quantitative lower limit detection
Serial calibrators were prepared at the concentrations shown in table 1, treated according to the established method, and subjected to sample detection analysis. Concentration of analyte is taken as abscissaX) The peak area ratio of the analyte to the internal standard is taken as the ordinateY) With weighting (1-x 2 ) Linear regression analysis was performed and the results are shown in Table 5, indicating that each analyte was within the respective standard curverAll are larger than 0.99, and the linearity meets the detection requirement.
5 parts of the lowest concentration point are prepared in parallel for calibration 1 (namely lower limit of quantification, LOQ), and sample injection detection is carried out after the same treatment. The results are shown in Table 5, the calculated concentrations of the analytes LOQ are within +/-20% of the marked values, the precision is within +/-20%, and the detection requirements are met.
TABLE 5 Linear and quantitative lower limit
(3) Accuracy and precision detection
The low and high 2 concentration quality control samples (prepared using separately prepared stock solutions) were prepared at the concentrations shown in table 1, treated according to the predetermined method, and subjected to sample detection analysis. Intra-batch accuracy and precision were examined by one analytical batch, and inter-batch accuracy and precision were examined by 3 analytical batches (performed in 3 days). Calculating the measured concentration of the analyte by using the same batch of accompanying standard curve, and comparing the measured concentration with the marked concentration to calculate the accuracy, wherein the accuracy is expressed as: (measured value/labeled value) ×100% and the results are shown in table 6, which indicate that the average value of accuracy of each analyte is within ±15% of the labeled value of the quality control sample, and meets the detection requirements. Precision is expressed as Coefficient of Variation (CV) of each analyte measurement, and the results are shown in Table 6, which indicate that each analyte has a coefficient of variation within the batch of not more than 15% and a coefficient of variation between batches of not more than 15% and meets the detection requirements.
TABLE 6 accuracy and precision
(4) Extraction recovery rate and matrix effect detection
Taking 6 different urine, processing according to a set method, and performing sample detection analysis to obtain peak area A1 and measured concentration C1; taking 540 mu L of each six parts of urine of 6 different individuals, adding 60 mu L of low-concentration standard working solution into three parts of the six parts, uniformly mixing, adding 60 mu L of high-concentration standard working solution into the other 3 parts of the six parts, uniformly mixing, assuming that the theoretical concentration is C0, carrying out detection analysis after the treatment by the same method, and obtaining the measured concentration C2; taking six urine of 6 different individuals to be treated by the same method to obtain supernatant (for measuring VMA and HVA) and eluent (for measuring other analytes), wherein three parts of the supernatant are added with low-concentration standard working solution, and the other three parts of the supernatant are added with high-concentration standard working solution, and sample injection detection and analysis are carried out to obtain peak area A3; and similarly, six parts of PBS are treated by the same method to obtain supernatant and eluent, three parts of the supernatant and the eluent are added with low-concentration standard working solution, the other 3 parts of the supernatant and the eluent are added with high-concentration standard working solution, and the peak area A4 is obtained through sample injection detection and analysis. The analyte extraction recovery was (C2-C1)/C0×100%, and the absolute matrix effect of the analyte was (A3-A1)/A4×100%, and the results are shown in Table 7. The extraction recovery rate and the variation coefficient of matrix effect are not more than 15 percent.
TABLE 7 extraction recovery and matrix Effect
(5) Stability detection
1. Stability detection of analyte working fluid
And (3) newly-prepared low-concentration and high-concentration standard working solutions (n=3), examining the stability of the working solutions after being placed at room temperature for 8 hours and stored for 7 months at-20 ℃ and repeatedly frozen and thawed for three times, analyzing each batch of working solutions and the newly-prepared working solutions in the same batch, and calculating the ratio of the area (A1) of the analyte and the internal standard peak in each batch of working solutions to the area (A2) of the analyte and the internal standard peak in the newly-prepared working solutions, namely A1/A2×100%.
Results: as shown in Table 8, the difference between the results of each analyte before and after the placement is within 20%, which indicates that each analyte working solution has good stability after being placed at room temperature for 8 hours and stored at-20 ℃ for 7 months and repeatedly frozen and thawed for three times, and daily detection is not affected.
TABLE 8 stability of analyte working fluid
2. Stability detection of urine samples
The stability of each analyte in the urine matrix was examined by storage at room temperature (25-28 ℃) for 8 hours and at 2-8℃for 7 days, respectively. 3 fresh urine samples (n=3) from different individuals were taken separately, each sample was analyzed along with a standard curve prepared by the current, and the ratio of the analyte concentration (C1) in each sample to the analyte concentration (C2) in the fresh urine sample, i.e., C1/C2×100%, was calculated.
Results: as shown in Table 9, the difference between the results of the analytes before and after the storage is within 15%, which indicates that the urine has good stability after being stored at room temperature for 8 hours and at 2-8 ℃ for 7 days. Therefore, in daily detection, clinical samples are sent to be detected within 8 hours after being collected, and the samples are stored at 2-8 ℃ in time after being collected in a laboratory for detection.
3. Stability detection of post-treatment samples
The stability of the treated sample (i.e., sample fluid) was examined after 8 hours of placement in an autosampler (22-25 ℃). Taking fresh urine samples (n=3) of 3 different individuals for treatment, and immediately carrying out sample injection analysis to obtain a peak area A1; after 8 hours of standing in an autosampler, sample again analysis was performed to obtain the peak area A2. The stability (A2/IS)/(A1/IS). Times.100% of each analyte was calculated,
results: as shown in Table 9, the difference in results for each analyte was within.+ -. 10% before and after placement, indicating good stability of the treated sample after 8 hours of placement in the autosampler.
TABLE 9 stability of urine samples and treated samples
Example 3 clinical sample testing
Clinical tests were carried out in the kunming university of medical science, first affiliated hospital ethical committee approved (clinical study trial (2022) renjin No. L257) using the kit for detecting 12 catecholamines and their metabolites in human urine of the present invention. A total of 1127 cases (including 912 cases of 24-hour urine and 215 cases of randomized urine) were examined from 1.6.1.2021 to 1.11.2022, with clinical diagnosis of 20 cases in pheochromocytoma patients and 2 cases in paraganglioma patients.
The diagnostic efficacy of the two samples for the diagnosis of PPGL was compared by analyzing clinical test data of randomized urine and 24 hours urine. The inclusion criteria were: (1) 18 to 65 years old; (2) the sex is unlimited; (3) patients with pheochromocytoma or paraganglioma are clinically diagnosed, and the clinical diagnosis needs to be supported by pathological results; (4) non-PPGL patients. The rejection criteria were: (1) dietary factors, long-term drinking, smoking, eating caffeine-containing foods; (2) disease factors, other diseases that produce CA such as neuroblastoma, congestive heart failure, acute coronary syndrome, acute myocardial infarction, renal failure, some malignancies, etc.; (3) a drug or therapeutic factor, any treatment that may affect CA metabolism or administration of a related drug is performed within 72 hours prior to sampling; (4) for 24 hour urine samples, urine volume 500 mL and below and 24 hour urine leave patients with errors; (5) for random urine samples, chronic kidney disease patients are bedridden for periods 2-5 and long periods.
Random urine samples were included in 218 cases, 101 cases in men, 117 cases in women, and had an average age of 40.3.+ -. 12.7 years. The 24 hour urine samples were included in 760 cases, 438 cases in men, 322 cases in women, and the average age was 43.9.+ -. 11.8 years. The diagnostic value (i.e. area under ROC curve, AUC), sensitivity, specificity and optimal Cut-off value (i.e. Cut-off value) of each index to PPGL are obtained through the analysis of the working characteristic curve (receiver operating characteristic curve, ROC) of the test subject, and the combined diagnostic performance of each index is analyzed. The ROC curve is shown in fig. 7, and the diagnostic performance index is shown in table 10. From the results, except the specificity of NMN/Cr and the sensitivity of MN/Cr, the sensitivity and the specificity of each index of random urine diagnosis PPGL are slightly higher than those of urine in 24 hours; the AUC of each index of the random urine is slightly greater than 24 hours urine; in both randomized and 24 hour urine, the combined indicators had better diagnostic performance than the individual indicators, with higher diagnostic value (AUC > 0.9). The above results demonstrate that the diagnostic properties of randomized urine and 24 hour urine were comparable to PPGL. The Cut-off values corresponding to the respective indices in Table 10 were used as clinical diagnosis thresholds.
TABLE 10 diagnostic properties of various indicators in random urine and 24 hour urine on PPGL
EXAMPLE 4 Chamber interstitial assessment
The kit for detecting 12 catecholamines and metabolites thereof in human urine participates in the room interstitial evaluation of catecholamine and metabolites thereof detection (NCCL-C-38 is coded by planning) initiated by the national health commission clinical test center in 2022 and 5 months, the results of all indexes are shown in table 11, wherein the evaluation results of NMN, MN and 3-MT are all "pass", VMA and HVA are investigation projects, the results are not evaluated, and the actual measurement value in the laboratory is still in the evaluation range.
Table 11 results of chamber interstitial assessment
In conclusion, each verification index of the kit meets the requirements, and the kit also participates in the room interstitial evaluation of catecholamine and the metabolite detection thereof initiated by the clinical inspection center of the national health commission of 2022, and the evaluation indexes pass the evaluation. In addition, the kit increases the quantity (up to 12) of catecholamine substances detected, and expands the disease range of diagnosis or auxiliary diagnosis. The kit is capable of satisfying diagnosis or aiding diagnosis of diseases including but not limited to those in Table 12, as shown in Table 12.
TABLE 12 disease and corresponding index changes
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Claims (10)
1. A sample pretreatment liquid capable of simultaneously detecting 12 catecholamines and metabolites thereof and creatinine in human urine, wherein the 12 catecholamines and metabolites thereof are norepinephrine, epinephrine, dopamine, 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid, 5-S-cysteine dopa, methoxynorepinephrine, methoxyepinephrine, 3-methoxytyramine, vanilla mandelic acid and homovanillic acid, and the sample pretreatment liquid is characterized by comprising urine, a calibrator and a quality control material;
The diluent consists of diluent I, diluent II, diluent III and diluent IV; the diluent I is 5-50% methanol containing 0.1-5% formic acid; the diluent II is 0.5-5% acetic acid solution; the diluent III is 1X phosphate buffer solution; the diluent IV is 10-100 mM of hydroxyethyl piperazine acetic sulfuric acid solution;
the leacheate consists of leacheate I, leacheate II, leacheate III and leacheate IV; the leaching solution I is methanol; the leaching solution II is 5-50 mM ammonium acetate solution; the leaching solution III is 5-50% of methanol; the leaching solution IV is 5-50% methanol containing 0.1-5% formic acid.
2. The sample pretreatment fluid of claim 1, wherein the calibrator consists of norepinephrine, epinephrine, dopamine, 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid, 5-S-cysteine dopa, methoxynorepinephrine, methoxyepinephrine, 3-methoxytyramine, vanillylmandelic acid, homovanillic acid, and creatinine;
the concentration range of norepinephrine, epinephrine and dopamine is 12.5-800 ng/ml, the concentration range of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxyl phenethyl glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 0.3-20 mug/ml, the concentration range of vanillyl mandelic acid and homovanillic acid is 0.6-40 mug/ml, the concentration range of methoxynorepinephrine, methoxyepinephrine and 3-methoxyl tyramine is 6.3-400 ng/ml, and the concentration range of creatinine is 63-4000 mug/ml.
3. The sample pretreatment fluid of claim 1, wherein the quality control comprises a low quality control and a high quality control;
wherein, the concentration of norepinephrine, epinephrine and dopamine in the low quality control product is 60 ng/ml, the concentration of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxyl phenethyl glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 2 mug/ml, the concentration of vanillyl mandelic acid and homovanillic acid is 3 mug/ml, the concentration of methoxyl norepinephrine, methoxyl epinephrine and 3-methoxyl tyramine is 30 ng/ml, and the concentration of creatinine is 300 mug/ml;
the concentration of norepinephrine, epinephrine and dopamine in the high quality control is 600 ng/ml, the concentration of 3, 4-dihydroxyphenyl glycol, 4-hydroxy-3-methoxybenzene glycol, 3, 4-dihydroxyphenylacetic acid and 5-S-cysteine dopa is 18 mug/ml, the concentration of vanillyl mandelic acid and homovanillic acid is 30 mug/ml, the concentration of methoxynorepinephrine, methoxyepinephrine and 3-methoxytyramine is 300 ng/ml, and the concentration of creatinine is 3000 mug/ml.
4. A sample pretreatment method capable of simultaneously detecting 12 catecholamines, metabolites thereof and creatinine in human urine according to claim 1, characterized in that the sample pretreatment method comprises a pretreatment step one and a pretreatment step two;
The pretreatment step I is to directly mix a sample, a diluent and an internal standard solution, and then centrifuge at a high speed to obtain a supernatant to obtain a treated sample liquid I, wherein the centrifugation speed is 10000-14000 rpm, and the centrifugation time is 5-10 minutes;
the pretreatment step II is to mix a sample, a diluent and an internal standard solution and then carry out solid-phase extraction, and the obtained extraction liquid is treated sample injection liquid II;
finally, carrying out LC-MS/MS detection on the treated sample liquid I and sample liquid II;
in the first pretreatment step, the serial calibrator/quality control material system preparation method comprises the following steps: respectively taking 20 mu L of calibrator/quality control material, 200 mu L of diluent II and 20 mu L of internal standard solution, and uniformly mixing; the system preparation method of the urine sample comprises the following steps: respectively taking 20 mu L of urine sample, 200 mu L of diluent II and 20 mu L of internal standard solution, and uniformly mixing;
in the second pretreatment step, the system preparation method of the series of calibration materials/quality control materials comprises the following steps: mixing 30 μl of calibrator/quality control, 270 μl of diluent III, 20 μl of internal standard, and 450 μl of diluent IV; the system preparation method of the urine sample comprises the following steps: mu.L of diluent I, 270. Mu.L of urine sample, 20. Mu.L of internal standard and 450. Mu.L of diluent IV were mixed separately.
5. The method according to claim 4, wherein the internal standard solution is composed of norepinephrine-D6, epinephrine-D3, dopamine-D4, 3, 4-dihydroxyphenyl glycol- 13 C. 4-hydroxy-3-methoxyphenylethylene glycol-D3, 4-dihydroxyphenylacetic acid-D5, 5-S-cysteine dopa-D3, methoxynorepinephrine-D3, methoxyepinephrine-D3, 3-methoxytyramine- 13 C. Vanilla mandelic acid-D3, homovanillic acid-D3 and creatinine-D3; the concentration of norepinephrine-D6, epinephrine-D3 and dopamine-D4 is 160 ng/ml, and the concentration of 3, 4-dihydroxyphenyl glycol is 160- 13 C. 4-hydroxy-3-methoxyphenylethylene glycol-D3, 4-dihydroxyphenylacetic acid-D5, 5-S-cysteinodopa-D3 at a concentration of 5. Mu.g/ml, vanilloid-mandelic acid-D3, homovanillic acid-D3 at a concentration of 5. Mu.g/ml, methoxynorepinephrine-D3, methoxyepinephrine-D3, 3-methoxytyramine- 13 The concentration of C was 80 ng/ml and the concentration of creatinine-D3 was 1000. Mu.g/ml.
6. The sample pretreatment method according to claim 4, wherein in the pretreatment step two, solid phase extraction is performed by using a 96-well solid phase extraction column through a positive pressure device, and the filler is a weak cation exchange material, and the specific solid phase extraction method is realized by the following steps:
1) Activating: adding 300 mu L of eluent I into each hole, and passing through a column;
2) Balance: adding 300 mu L of eluent II into each hole, and passing through a column;
3) Loading: loading all the prepared system into a column;
4) Eluting impurities: adding 500 mu L of eluent III for leaching;
5) Eluting: and adding 100 mu L of eluent IV for pressurized elution to a 96-well collection plate, and directly injecting and analyzing the obtained eluent.
7. The method according to claim 4, wherein in the LC-MS/MS detection, the liquid chromatography column is a pentafluorophenyl column and a corresponding guard column, mobile phase a is 0.1 to 1% formic acid aqueous solution, mobile phase B is 0.1 to 1% formic acid acetonitrile or methanol solution, the column temperature is 40 ℃, and the flow rate is 0.45 ml/min; mass spectrometer using ESI mode, MRM negative ion mode and positive ion mode detection;
the LC-MS/MS detection result calculation method comprises the following steps: the concentration of the analyte is on the abscissa and the ratio of the peak area of the analyte to the internal standard is on the ordinate, weighted (1/x 2 ) Performing linear regression analysis, and establishing a calibration curve to obtain a linear fitting equation;
taking the ratio of the peak area of each analyte in the urine sample to be detected to the peak area of the internal standard substance into a corresponding linear fitting equation, and calculating to obtain the concentrations of catecholamine metabolites and creatinine in the urine sample to be detected;
if the urine is random urine, the detection result is obtained by dividing the measured concentration of each analyte by the creatinine concentration of the same urine;
If the urine is 24 hours urine, the measured concentration of each analyte is multiplied by the 24 hour urine volume.
8. A kit based on the sample pretreatment liquid according to claim 1, which can detect 12 catecholamines and their metabolites and creatinine in human urine at the same time.
9. Use of the kit according to claim 8 for the assisted diagnosis of pheochromocytoma, paraganglioma, neuroblastoma, metastatic melanoma, metabolic disease or autonomic nervous system disease by human urine detection results.
10. Use according to claim 9, characterized in that the joint diagnostic markers in the random urine assisting in the diagnosis of cytoma and paraganglioma are NMN/Cr, MN/Cr, 3-MT/Cr, VMA/Cr and HVA/Cr, wherein the diagnostic threshold for NMN/Cr is 30 μg/g, for MN/Cr is 63 μg/g, for 3-MT/Cr is 45 μg/g, for VMA/Cr is 5 mg/g, for HVA/Cr is 4 mg/g;
the combined diagnostic markers in urine at 24 hours for aiding in diagnosis of cytoma and paraganglioma are NMN, MN, 3-MT, VMA and HVA, wherein the diagnostic threshold for NMN is 64 μg/24h, the diagnostic threshold for MN is 22 μg/24h, the diagnostic threshold for 3-MT is 78 μg/24h, the diagnostic threshold for VMA is 10mg/24h, and the diagnostic threshold for HVA is 5mg/24h.
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