CN111398450A - Kit for detecting 8 catecholamines and metabolites thereof in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Kit for detecting 8 catecholamines and metabolites thereof in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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Abstract
The invention relates to a kit for detecting 8 catecholamine and metabolites thereof in urine by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which comprises an eluent, a calibrator solution, a mixed internal standard solution, an extraction liquid and a quality control product, wherein the 8 catecholamine and the metabolites thereof are respectively as follows: adrenaline, noradrenaline, dopamine, metaadrenaline, normetaadrenaline, 3-methoxytyramine, homovanillic acid and vanillylmandelic acid. The kit is used for detecting 8 catecholamine and metabolites thereof in urine, the pretreatment process is simple, solid-phase extraction or derivatization reaction is not needed, the cost is low, the sensitivity is high, the specificity is strong, the pretreatment is completed within 10min, the separation and detection of the 8 catecholamine and the metabolites thereof are completed within 4.5min, the matrix effect and the precision basically meet the requirements, and the kit can be used for the clinical quantitative analysis of the 8 catecholamine and the metabolites thereof.
Description
Technical Field
The invention belongs to the technical field of urine detection, and particularly relates to a kit for detecting 8 catecholamine and metabolites thereof in urine by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.
Background
Epinephrine (E), Norepinephrine (NE), and Dopamine (DA) are enzymes that convert tyrosine, and the 3 catecholamines are important neurotransmitters of the central nervous system and play important roles in self-regulation of vascular tone, intestinal and bronchial smooth muscle tone, heart rate and contractility, and glucose metabolism. 3-Methoxytyramine (3-Methoxytyramine, 3MT), Metanephrine (MN) and noradrenaline (NMN) are metabolites of dopamine, epinephrine and norepinephrine, respectively. These metabolites are further metabolized to vanillylmandelic acid (VMA) and Homovanillic acid (HVA).
Pheochromocytoma is a rare, but potentially fatal, adrenal medullary pheochromocytoma that can cause hypertension, palpitations, severe headaches, and sweating ("seizures"). Pheochromocytoma patients may also be asymptomatic, manifested by persistent hypertension or by occasional findings of adrenal tumors. Pheochromocytomas and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastoma) secrete catecholamines (epinephrine, norepinephrine, and dopamine). Pheochromocytoma cells also have the ability to convert methylated catecholamines into metaepinephrine that is secreted into the urinary circulation. Elevated levels of 1 or several catecholamines can also be observed in patients with neuroblastoma and related tumors (ganglionic neuroblastoma and ganglionic neuroma), and occasionally in other neuroectodermal tumors. In patients with high suspicion of pheochromocytoma, the screening is best performed by measuring plasma catecholamines. The 24-hour urinary catecholamine can be used as the first detection of low suspicious cases and can also be used as a verification research of patients with the increase of less than 2 times of the plasma catecholamine. This is highly desirable because the incidence of pheochromocytoma is low in the population (less than 1:100,000 per year), and testing 24h urine catecholamines can avoid subjecting large numbers of patients to unnecessary, expensive, and sometimes dangerous imaging procedures.
At present, the detection types of the urinary catecholamine and the metabolites thereof are basically 3-5, and solid-phase extraction is used for pretreatment, so that the pretreatment is complex and the cost is high. The increase or decrease of the catecholamine and the metabolites thereof in the human body has great relevance to diseases such as pheochromocytoma, sympathoblastoma, idiopathic hypertension, neuroblastoma and the like, and has important reference value for the diagnosis of the diseases, so that the development of the kit capable of detecting 8 catecholamine and the metabolites thereof in urine together has economic and application values.
Disclosure of Invention
The invention aims to provide a kit for detecting 8 catecholamine and metabolites thereof in urine by using an ultra-high performance liquid chromatography tandem mass spectrometry technology on the basis of the prior art.
The invention also aims to provide the application of the kit in detecting 8 catecholamine and metabolites thereof in urine by using an ultra performance liquid chromatography tandem mass spectrometry technology.
The technical scheme of the invention is as follows:
a kit for detecting 8 catecholamine and metabolites thereof in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology,
the 8 catecholamines and metabolites thereof are respectively: epinephrine (E), Norepinephrine (NE), Dopamine (DA), Metaepinephrine (MN), noradrenaline (NMN), 3-methoxytyramine (3-MT), homovanillic acid (HVA) and vanillylic acid (VMA).
The internal standard substances corresponding to the 8 catecholamines and the metabolites thereof are respectively as follows: adrenaline-d 3(E-d3), noradrenaline-d 6(NE-d6), dopamine-d 4(DA-d4), noradrenaline-d 3(MN-d3), noradrenaline-d 3(NMN-d3), 3-methoxytyramine-d 4(3-MT-d4), homovanillic acid-d 3(HVA-d3) and vanillylmandelic acid-d 3(VMA-d 3).
The kit comprises the following reagents:
(1) eluent:
eluent A: 0.05 to 0.1 percent of formic acid aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
a mixed standard stock solution comprising 100 μ g/m L epinephrine, 400 μ g/m L norepinephrine, 2mg/m L dopamine, 200 μ g/m L norepinephrine, 200 μ g/m L norepinephrine, 400 μ g/m L3-methoxytyramine, 5mg/m L homovanillic acid, and 5mg/m L vanillylmandelic acid was formulated with a blank urine matrix solution into calibrator solutions at seven different concentration points, the seven concentration points of the calibrator solution being:
seven concentrations of epinephrine are 0.5ng/m L, 1.0ng/m L, 2.0ng/m L, 5.0ng/m L, 10.0ng/m L, 25.0ng/m L and 50.0ng/m L in sequence;
the concentration of the metaadrenaline and the normetaadrenaline is the same, and the seven concentrations are 1.0ng/m L, 2.0ng/m L, 4.0ng/m L, 10.0ng/m L, 20.0ng/m L, 50.0ng/m L and 100ng/m L in sequence;
the concentrations of the norepinephrine and the 3-methoxytyramine are the same, and seven concentrations are sequentially 2.0ng/m L, 4.0ng/m L, 8.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L and 200.0ng/m L;
seven concentrations of dopamine are sequentially 10.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L, 200.0ng/m L, 500.0ng/m L and 1000.0ng/m L;
the concentration of homovanillic acid and vanillylmandelic acid is the same, seven concentrations are 100.0ng/m L, 200.0ng/m L, 400.0ng/m L, 1000.0ng/m L, 2000.0ng/m L, 5000.0ng/m L and 10000.0ng/m L in sequence;
(3) mixing internal standard liquid:
respectively preparing adrenaline-d 3 with the concentration of 2 mu g/m L, noradrenaline-d 6 with the concentration of 8 mu g/m L, dopamine-d 4 with the concentration of 40 mu g/m L, adrenaline-d 3 with the concentration of 4 mu g/m L, noradrenaline-d 3 with the concentration of 4 mu g/m L, 3-methoxytyramine-d 4 with the concentration of 8 mu g/m L, homovanillic acid-d 3 with the concentration of 800 mu g/m L and vanillylmandelic acid-d 3 with the concentration of 200 mu g/m L by using 30-70% methanol aqueous solution, and then adding 20 mu L of the internal standard solution into 0.24m L diluent to obtain mixed internal standard solution;
(4) extracting liquid:
aqueous formic acid or acetic acid;
(5) quality control product:
the blank urine matrix solution containing catecholamine and metabolites thereof is divided into low and high concentration QC (L) and QC (H), wherein QC (L) is used for diluting the mixed standard stock solution to 1000 times by using the blank urine matrix solution, and QC (H) is used for diluting the mixed standard stock solution to 50 times by using the blank urine matrix solution.
In a preferred embodiment, the eluent a is 0.05% to 0.5% aqueous formic acid, preferably 0.1% aqueous formic acid.
The mixed standard stock solution is prepared by preparing 100 mu g/m L, 400 mu g/m L, 2mg/m L, 200 mu g/m L, 200 mu g/m L, 400 mu g/m L, 5mg/m L and 5mg/m L from 30-70% methanol aqueous solution, adding 10 mu L each of the prepared adrenaline solution, noradrenaline solution, dopamine solution, noradrenaline solution and 3-methoxytyramine solution, adding 40 mu g/m L each of homovanillic acid solution and vanillic mandelic acid solution to 0.86m L diluent to obtain the mixed standard stock solution, wherein the concentration of adrenaline (E) is 1 mu g/m L, the concentration of vanillic acid (NM) is equal to 100 mu g/m L, the concentration of vanillic acid (NM) is equal to 200 mu g/m L, and the concentration of vanillic acid (NM) is equal to 8520 mu g/m 634.
In preparing the mixed standard stock solution and the mixed internal standard solution, the diluent used is an aqueous formic acid solution or an aqueous acetic acid solution, such as an aqueous formic acid solution, preferably an aqueous 0.1% to 5% formic acid solution, and more preferably an aqueous 2% formic acid solution.
When preparing the mixed standard substance stock solution and the mixed internal standard solution, the adopted 30-70% methanol aqueous solution contains formic acid, such as 1-5% formic acid-30-70% methanol aqueous solution, preferably 1-5% formic acid-45-55% methanol aqueous solution; more preferably 2% formic acid-50% aqueous methanol.
In one embodiment, the extract solution is a formic acid aqueous solution, preferably a 5% to 20% formic acid aqueous solution, and more preferably a 10% formic acid aqueous solution.
In one embodiment, the blank urine matrix is aqueous formic acid or acetic acid, preferably aqueous formic acid, more preferably 0.1-5% aqueous formic acid, and even more preferably 2% aqueous formic acid.
The concentration of the aqueous formic acid solution or the aqueous acetic acid solution or the aqueous methanol solution referred to in the present invention generally means a volume concentration.
The urine referred to in the present invention is human or animal urine.
In a preferred scheme, the kit for detecting 8 catecholamine and metabolites thereof in urine by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:
(1) eluent:
mobile phase A: 0.05 to 0.5 percent of formic acid aqueous solution; mobile phase B: acetonitrile;
(2) calibration solution:
preparing standard mother liquor with the concentrations of 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 5mg/m L and 5mg/m L by using 1-5% formic acid-30-70% methanol aqueous solution;
then, 1-5% formic acid-30-70% methanol water solution is used for respectively diluting the concentrations of adrenaline, noradrenaline and 3-methoxytyramine to intermediate solutions with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, respectively taking 10 mu L of the intermediate solution and 2mg/m L of dopamine, respectively taking 40 mu L of 5mg/m L of homovanillic acid and 5mg/m L of vanillylmandelic acid, respectively adding the homovanillic acid and the vanillylmandelic acid into 0.86m L aqueous solution containing 0.1-5% formic acid, and obtaining mixed standard product stock solution;
preparing the mixed standard substance stock solution into calibration substance solutions with seven different concentration points by using a blank urine matrix solution;
(3) mixing internal standard liquid:
noradrenaline-d 6 and noradrenaline-d 3 are purchased in the form of solution, the concentration of each is 100 mu g/m L, isotope internal standard noradrenaline-d 3(E-d3), isotope internal standard dopamine-d 4(DA-d4), isotope internal standard noradrenaline-d 3(NMN-d3), isotope internal standard 3-methoxytyramine-d 4(3-MT-d4), isotope internal standard homovanillic acid-d 3(HVA-d3) and isotope internal standard vanillylmandelic acid-d 3(VMA-d3) are dissolved in methanol water solution to prepare the solution, and the specific preparation process is as follows:
the noradrenaline-d and noradrenaline-d are purchased in the form of solution, the concentration of which is 100 mu g/m, the adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid are prepared into isotope internal standard solutions of adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid, the concentration of which is 2mg/m, 1mg/m 0, 1mg/m 1, 3-methoxytyramine-d, 10mg/m 4, respectively, by 1-5% formic acid-30-70% methanol aqueous solution, the noradrenaline-d, adrenaline-d, dopamine-d, adrenaline-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic acid-d, the concentration of which is 1-5-m 1-70% formic acid-2, the concentration of the noradrenaline-d, the concentration of the homovanillic acid-d, the concentration of which is 6 mu g/m, the concentration of the noradrenaline-d, the concentration of the homovanillic mandelic acid-d, the isotopic solution is 1-d, the isotopic solution of which is 1-8.8 mu m, the isotopic solution of the isotopic solution is 2 g/m;
(4) extracting liquid:
5 to 20 percent of formic acid aqueous solution;
(5) quality control product:
preparing the mixed standard substance stock solution into QC (L) and QC (H) with two different concentrations by using 0.1-5% formic acid aqueous solution;
QC (L) contains 1ng/m L adrenaline, 4ng/m L noradrenaline, 20ng/m L dopamine, 2ng/m L noradrenaline, 2ng/m L noradrenaline, 4ng/m L3-methoxytyramine, 200ng/m L homovanillic acid and 200ng/m L vanillylmandelic acid;
QC (H) comprises 20ng/m L adrenaline, 80ng/m L noradrenaline, 400ng/m L dopamine, 40ng/m L noradrenaline, 40ng/m L noradrenaline, 80ng/m L3-methoxytyramine, 4000ng/m L homovanillic acid and 4000ng/m L vanillylmandelic acid.
In a more preferred scheme, the kit for detecting 8 catecholamine and metabolites thereof in urine by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:
(1) eluent:
eluent A: 0.1 aqueous formic acid; eluent B: acetonitrile;
(2) calibration solution:
respectively weighing 3.425mg of adrenaline (E), 3.951mg of Noradrenaline (NE), 3.882mg of Dopamine (DA), 3.768Mg of Noradrenaline (MN), 3.503mg of noradrenaline (NMN), 13.176mg of 3-methoxytyramine (3-MT), 5.212mg of homovanillic acid (HVA) and 4.657mg of vanillylmandelic acid (VMA), respectively adding 2% formic acid-50% methanol aqueous solution of 1.678m L, 1.592m L, 1.536m L, 1.558m L1, 1.402m L, 5.275m L, 1.021m L4 and 0.912m L to prepare noradrenaline with the concentration of 2mg/m L, noradrenaline of 2mg/m L, noradrenaline of 2mg/m L, norepinephrine of 2mg/m L, norepinephrine/m 2, norepinephrine of 2mg/m 6356 mg/m and vanillylic acid of 8653 mg/m 865 to prepare standard vanillylic acid solution of vanillylic acid solution of vanillyl 8653 mg/m 8653;
then 2% formic acid-50% methanol water solution is used for respectively diluting the concentrations of epinephrine, norepinephrine and 3-methoxytyramine to intermediate solution with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, 10 mu L of each intermediate solution and 2mg/m L of dopamine are respectively taken, 5mg/m L of homovanillic acid and 40 mu L of 5mg/m L of vanillylmandelic acid are respectively taken to be added into 0.86m L aqueous solution containing 2% formic acid, and mixed standard substance stock solution is obtained;
the mixed standard stock solution is prepared into calibrator solutions of seven different concentration points by using 2% formic acid aqueous solution, wherein the concentrations of various compounds in the calibrator solutions of the different concentration points are E (0.5ng/m, 1.0ng/m, 2.0ng/m 0, 5.0ng/m 1, 10.0ng/m 2, 25.0ng/m 3 and 50.0ng/m 4), NE and 3-MT (2.0ng/m 5, 4.0ng/m 6, 8.0ng/m 7, 20.0ng/m 8, 40.0ng/m 9, 100.0ng/m and 200ng/m 0), MN and NMN (1.0ng/m 1, 2.0ng/m 2, 4.0ng/m 3, 10.0ng/m 4, 20.0ng/m 5, 50.0ng/m 6 and 100ng/m 7), DA (8.0 ng/m 0, 5000.0ng/m 2, 4.0ng/m 3, 10.0ng/m 4, 20.0ng/m 5, 50.0ng/m 6 and 100ng/m 0, 200.0ng/m 0, 200ng/m and 500 ng/m A (1, 0ng/m 0, 200 g/m) and 500 ng/m).
(3) Mixing internal standard liquid:
respectively weighing 2.5mg of epinephrine-d, 1mg of dopamine-d, 1mg of noradrenaline-d, mg of 3-methoxytyramine-d, 1mg of homovanillic acid-d and 10mg of vanillylmandelic acid-d, respectively adding 1.225m, 0.795m 0, 0.811m 1, 1.089m 2, 0.999m 3 and 0.994m 4 of 2% formic acid-50% methanol aqueous solution for complete dissolution to prepare 2mg/m 5 of epinephrine-d, 1mg/m 6 of dopamine-d, 1mg/m 7 of noradrenaline-d, 5mg/m 8 of 3-methoxytyramine-d, 1mg/m 9 of homovanillic acid-d, 10mg/m of vanillylmandelic acid-d of internal standard solution, respectively adding 2% of noradrenaline-50% of noradrenaline-d, 0.999/m 8 of 3-methoxytyramine-d, 1mg/m 9 of homovanillylic acid-d, 10mg/m of isotope of vanillylic acid-d into 2mg/m 5 of noradrenaline-d, 0.8 g/m 2% noradrenaline-d, 0.8 g/m 2 g/m of methanol aqueous solution for dilution to obtain 2 g/m of internal standard solution, 2 g/m solution for dilution to obtain 2 g/m solution, 2 g/m of noradrenaline-4 g of noradrenaline-8 g/m solution, 2 g of noradrenaline-8 aqueous solution, 2 g/m solution, and 8 g/m solution for dilution to obtain 2 g of internal standard solution, 2 g of noradrenaline-;
(4) extracting liquid:
10% aqueous formic acid;
(5) quality control product:
and taking the mixed standard stock solution 1 mu L and 20 mu L, and respectively diluting the mixed standard stock solution to 1000 mu L by using a 2% formic acid aqueous solution to obtain QC (L) and QC (H).
Wherein, the corresponding concentration of the quality control products in QC (L) and QC (H) is shown in Table 1;
TABLE 1 corresponding concentration of quality control (unit ng/m L)
The application of the kit in detecting 8 catecholamine and metabolites thereof in urine by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting 8 catecholamine and metabolites thereof in urine by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
detecting the 8 catecholamines and metabolites thereof in the pretreated urine by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating a substance to be detected from a urine matrix by utilizing the ultra-high performance liquid chromatography, establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of the 8 catecholamines and the metabolites thereof, wherein the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.05 to 1 percent of formic acid aqueous solution; mobile phase B: acetonitrile;
the type of the chromatographic column is Agilent Pursuit PFP 2.0 × 150mm,3.0 μm;
adopting a mobile phase A and a mobile phase B as a mixed mobile phase to carry out gradient elution, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 99:1 to 60:40 at a constant speed within 0-1.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 60:40 to 2:98 at a constant speed within 1.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 2:98 within 2.0-2.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 99:1 at a constant speed within 2.5-4.5 minutes.
(2) Mass spectrum conditions:
in an electrospray ionization detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, the spraying voltage is 0.5kV (ESI +) and 2.5kV (ESI-), the desolvation temperature is 150 ℃, the atomization gas temperature is 600 ℃, the atomization gas flow rate is 1000L/h, the cone hole gas flow rate is 150L/h, and all targets and isotope internal standards are simultaneously monitored.
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. In a preferable embodiment, the mobile phase a is 0.05% to 0.5% formic acid aqueous solution, and in the case of not affecting the effect of the present invention, the mobile phase a is preferably 0.1% formic acid aqueous solution.
The invention adopts an ultra-high performance liquid chromatography tandem mass spectrometry technology to detect the 8 catecholamine and metabolites thereof in the pretreated urine, wherein the chromatographic column is a pentafluorobenzene-based column, the selection of the chromatographic column is very important in chromatography, and the requirements on the chromatographic column are high column efficiency, good selectivity, high analysis speed and the like.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The method adopts epinephrine-d 3, norepinephrine-d 6, dopamine-d 4, norepinephrine-d 3, norepinephrine-d 3, 3-methoxytyramine-d 4, homovanillic acid-d 3 and vanillylmandelic acid-d 3 as internal standards, the deuterated internal standards and the substances to be detected have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy of the 8 catecholamines and metabolites thereof in the urine are better.
In a preferable scheme, the flow rate is 0.2-1.0 m L/min, and is preferably 0.4m L/min.
Further, the column temperature is 30-50 ℃, and preferably 40 ℃.
Furthermore, the injection volume is 0.5-2 μ L, preferably 1 μ L.
In a preferred embodiment, when detecting the 8 catecholamines and their metabolites in the pretreated urine by using the ultra-high performance liquid chromatography tandem mass spectrometry technology, the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.1% aqueous formic acid; mobile phase B: acetonitrile;
the type of the chromatographic column is Agilent Pursuit PFP 2.0 × 150mm,3.0 μm;
the gradient elution mode is adopted, and is shown in the table 2;
the flow rate is 0.4m L/min, the column temperature is 40 ℃, and the sample injection volume is 1 mu L;
TABLE 2 mobile phase gradient elution parameters
(2) Mass spectrum conditions:
in an electrospray ionization detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, the spraying voltage is 0.5kV (ESI +) and 2.5kV (ESI-), the desolvation temperature is 150 ℃, the atomization gas temperature is 600 ℃, the atomization gas flow rate is 1000L/h, the taper hole gas flow rate is 150L/h, all targets and isotope internal standards are monitored simultaneously, and the parameters of the cluster-removing voltage, the collision voltage and the like of all the targets are shown in a table 3.
TABLE 3 detection of Mass Spectrometry parameters for catecholamines and their metabolites
The pretreated urine mentioned in the present invention is prepared as follows: and (3) putting the filtered urine into a centrifugal tube, adding a mixed internal standard working solution into the centrifugal tube, and taking supernatant after vortex.
In a preferred embodiment, the pre-treated urine mentioned in the present invention is prepared by taking 20 μ L filtered urine into a 1.5m L centrifuge tube, adding 780 μ L mixed internal standard working solution, and taking 70 μ L supernatant after vortexing.
In a more preferred embodiment, the pre-treated urine of the present invention is prepared by drawing 100 μ L urine through a 0.22 μm nylon filter using a syringe, transferring 20 μ L of the filtrate into a 1.5m L EP tube labeled with a label, adding 780 μ L of the mixed internal standard working fluid, vortexing and mixing for 5 seconds, and finally collecting 70 μ L of the supernatant to be injected.
In one embodiment, the mixed internal standard solution is prepared as follows:
the method comprises the steps of purchasing noradrenaline-d and noradrenaline-d in the form of solutions with the concentrations of 100 mu g/m, preparing adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid into isotope internal standard solutions of adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid with the concentrations of 2mg/m, 1mg/m 0, 1mg/m 1, 3-methoxytyramine-d, 10mg/m 4, respectively, by using methanol aqueous solutions with the concentrations of 1-5% formic acid-30-70%, adrenaline-d, dopamine-d, homovanillic acid-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid, diluting the isotope internal standard solutions to obtain an internal standard solution, adding the internal standard solution to the concentration of the mixture to the mixture, and diluting the mixture to obtain a mixed solution, wherein the mixture comprises 2mg/m, 1-m 0.0-m 4 g, 8-m and 8.8-m.
And taking 200 mu L of the mixed internal standard solution, adding 1m L of extract liquor, and diluting with 78.8m L of ultrapure water to obtain the mixed internal standard working solution.
In a preferred embodiment, the mixed internal standard working solution provided by the invention is prepared according to the following method:
norepinephrine-d 6 and norepinephrine-d 3 were purchased as solutions at 100 μ g/m L;
respectively weighing 2.5mg of adrenaline-d 3, 1mg of dopamine-d 4, 1mg of noradrenaline-d 3, 6.841mg of 3-methoxytyramine-d 4, 1mg of homovanillic acid-d 3 and 10mg of vanillylmandelic acid-d 3, respectively adding 2% formic acid-50% methanol aqueous solution of 1.225m L, 0.795m L, 0.811m L0, 1.089m L1, 0.999m L and 0.994m L to completely dissolve the noradrenaline-d 3, respectively preparing adrenaline-d L, 1mg/m L dopamine-d L, 1mg/m L of norvanillylic acid-d L, 5mg/m L of 3-methoxytyramine-d L, 1mg/m L of norvanillylic acid-d L, 10mg/m L of norvanillylic acid-d L, and 10mg of vanillylic acid-d 8672, respectively diluting the noradrenaline-d L, the noradrenaline-72, the noradrenaline-d L, the norvanillylic acid-72, the norvanillylic acid aqueous solution with the concentration of 2/m L, the concentration of 2% noradrenaline-72, the concentration of 2% L, the concentration of 2m L, the concentration of 2 μm L, the concentration of 2 μm L, the concentration of.
Taking 200 mu L of the mixed internal standard solution, adding 1m L of extract (10% formic acid aqueous solution) and then diluting with 78.8m L of ultrapure water to obtain the mixed internal standard working solution.
In one embodiment, the standard solution is prepared as follows:
preparing standard mother liquor with the concentrations of 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 5mg/m L and 5mg/m L by using 1-5% formic acid-30-70% methanol aqueous solution;
then, 1-5% formic acid-30-70% methanol water solution is used for respectively diluting the concentrations of adrenaline, noradrenaline and 3-methoxytyramine to intermediate solutions with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, 10 mu L of each intermediate solution and 2mg/m L of dopamine are respectively used, 40 mu L of homovanillic acid with the concentration of 5mg/m L and 40 mu L of each vanillylmandelic acid with the concentration of 5mg/m L are respectively used for adding the intermediate solutions into 0.86m L aqueous solution containing 0.1-5% formic acid, and thus obtaining the mixed standard stock solution.
Preparing the mixed standard substance stock solution into calibration substance solutions with seven different concentration points by using a blank urine matrix solution;
adding 10 mu L mixed standard stock solution into 190 mu L blank urine matrix solution to serve as a first high-value concentration point, diluting the first high-value concentration point with 4 times of volume of blank urine matrix solution to obtain a third high-value concentration point, diluting the second high-value concentration point with 4 times of volume of blank urine matrix solution to obtain a fourth high-value concentration point, diluting the third high-value concentration point with 4 times of volume of blank urine matrix solution to obtain a fifth high-value concentration point, diluting the fourth high-value concentration point with 4 times of volume of blank urine matrix solution to obtain a sixth high-value concentration point, and diluting the fifth high-value concentration point with 3 times of volume of blank urine matrix solution to obtain a seventh high-value concentration point.
The concentrations of each compound in the calibrator solutions at the different concentration points were E (0.5ng/m, 1.0ng/m, 2.0ng/m 0, 5.0ng/m 1, 10.0ng/m 2, 25.0ng/m 3 and 50.0ng/m 4), NE and 3-MT (2.0ng/m 5, 4.0ng/m 6, 8.0ng/m 7, 20.0ng/m 8, 40.0ng/m 9, 100.0ng/m and 200ng/m 0), MN and NMN (1.0ng/m 1, 2.0ng/m 2, 4.0ng/m 3, 10.0ng/m 4, 20.0ng/m 5, 50.0ng/m 6 and 100ng/m 7), DA (10.0ng/m 8, 20.0ng/m 9, 40.0ng/m, 100.0ng/m 0ng/m 5, 50.0ng/m 6 and 100ng/m 7), VMA (10.0ng/m 8, 20.0ng/m 9, 40.0ng/m, 100.0ng/m, 5000 ng/m, 0ng/m 0, 200.0ng/m, 1000.0ng/m and 1000.0ng/m A).
In a preferred embodiment, the standard solution is prepared as follows:
respectively weighing 3.425mg of adrenaline (E), 3.951mg of Noradrenaline (NE), 3.882mg of Dopamine (DA), 3.768Mg of Noradrenaline (MN), 3.503mg of noradrenaline (NMN), 13.176mg of 3-methoxytyramine (3-MT), 5.212mg of homovanillic acid (HVA) and 4.657mg of vanillylmandelic acid (VMA), respectively adding 2% formic acid-50% methanol aqueous solution of 1.678m L, 1.592m L, 1.536m L, 1.558m L1, 1.402m L, 5.275m L, 1.021m L4 and 0.912m L to prepare noradrenaline with the concentration of 2mg/m L, noradrenaline of 2mg/m L, noradrenaline of 2mg/m L, norepinephrine of 2mg/m L, norepinephrine/m 2, norepinephrine of 2mg/m 6356 mg/m and vanillylic acid of 8653 mg/m 865 to prepare standard vanillylic acid solution of vanillylic acid solution of vanillyl 8653 mg/m 8653;
and then 2% formic acid-50% methanol water solution is used for respectively diluting the concentrations of epinephrine, norepinephrine and 3-methoxytyramine to intermediate solution with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, 10 mu L of homovanillic acid with the concentration of 5mg/m L and 40 mu L of vanillic mandelic acid with the concentration of 5mg/m L are respectively taken from the intermediate solution and 2mg/m L of dopamine, and the intermediate solution and the 2% formic acid-50% methanol water solution are respectively added into 0.86m L aqueous solution containing 2% formic acid to obtain mixed standard substance stock solution.
Preparing the mixed standard substance stock solution into seven calibrator solutions with different concentration points by using a 2% formic acid aqueous solution;
the calibrator solutions with different concentration points comprise:
seven concentrations of epinephrine are 0.5ng/m L, 1.0ng/m L, 2.0ng/m L, 5.0ng/m L, 10.0ng/m L, 25.0ng/m L and 50.0ng/m L in sequence;
the concentration of the metaadrenaline and the normetaadrenaline is the same, and the seven concentrations are 1.0ng/m L, 2.0ng/m L, 4.0ng/m L, 10.0ng/m L, 20.0ng/m L, 50.0ng/m L and 100ng/m L in sequence;
the concentrations of norepinephrine and 3-methoxytyramine are the same, and seven concentrations are sequentially 2.0ng/m L, 4.0ng/m L, 8.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0 and ng/m L200.0.0 ng/m L;
seven concentrations of dopamine are sequentially 10.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L, 200.0ng/m L, 500.0ng/m L and 1000.0ng/m L;
the concentration of homovanillic acid and vanillylmandelic acid is the same, seven concentrations are 100.0ng/m L, 200.0ng/m L, 400.0ng/m L, 1000.0ng/m L, 2000.0ng/m L, 5000.0ng/m L and 10000.0ng/m L in sequence.
Seven different calibrator solutions 20. mu. L samples were taken from each concentration point and dispensed into different centrifuge tubes, 780. mu. L mixed internal standard working solution was added to the tubes, and after vortexing for a few seconds, 70. mu. L supernatant was removed and transferred to chromatography vials for loading for L C-MS/MS analysis.
In one scheme, the quality control product is prepared according to the following method:
the blank urine matrix solution containing catecholamine and metabolites thereof is divided into low and high concentration QC (L) and QC (H), wherein QC (L) is used for diluting the mixed standard stock solution to 1000 times by using the blank urine matrix solution, and QC (H) is used for diluting the mixed standard stock solution to 50 times by using the blank urine matrix solution.
In a preferred embodiment, the quality control product is prepared according to the following method:
and taking the mixed standard stock solution 1 mu L and 20 mu L, and respectively diluting the mixed standard stock solution to 1000 mu L by using a 2% formic acid aqueous solution to obtain QC (L) and QC (H).
QC (L) contains 1ng/m L adrenaline, 4ng/m L noradrenaline, 20ng/m L dopamine, 2ng/m L noradrenaline, 2ng/m L noradrenaline, 4ng/m L3-methoxytyramine, 200ng/m L homovanillic acid and 200ng/m L vanillylmandelic acid;
QC (H) comprises 20ng/m L adrenaline, 80ng/m L noradrenaline, 400ng/m L dopamine, 40ng/m L noradrenaline, 40ng/m L noradrenaline, 80ng/m L3-methoxytyramine, 4000ng/m L homovanillic acid and 4000ng/m L vanillylmandelic acid.
Quality control solution QC (L) and QC (H) 20 mu L are respectively taken in a centrifuge tube of 1.5m L, 780 mu L mixed internal standard working solution is added into the centrifuge tube, 70 mu L supernatant is taken after vortex for several seconds and transferred into a chromatographic bottle for sampling for L C-MS/MS analysis.
By adopting the technical scheme of the invention, the advantages are as follows:
the kit is used for detecting 8 catecholamine and metabolites thereof in urine, the pretreatment process is simple, solid-phase extraction and derivatization reaction are not needed, the cost is low, the sensitivity is high, the specificity is strong, the pretreatment is completed within 10min, the separation and detection of the 8 catecholamine and the metabolites thereof are completed within 4.5min, the matrix effect and the precision basically meet the requirements, the kit can be used for clinically quantitatively analyzing the 8 catecholamine and the metabolites thereof, and a reliable detection method is provided for clinically evaluating or diagnosing the risks of diseases such as pheochromocytoma, sympathoblastoma, neuroblastoma and the like.
Drawings
FIG. 1 is an extracted ion flow chromatogram of 8 catecholamines and their metabolites and an internal standard;
figure 2 is an extracted ion flow chromatogram of 8 catecholamines and their metabolites and internal standards in urine.
Detailed Description
The kit of the present invention is further illustrated by the following examples in conjunction with the drawings, but these examples do not limit the present invention in any way.
Example 1:
first, experimental material and instrument
1. Material
Methodology samples from the study were obtained from urine samples collected from the clinic in 2019, month 5 of the heart disease hospital, wuhan asia.
(1) The instrument comprises a Xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation), an UP L C I-Class ultra-high performance liquid chromatography system (matched with an autosampler, Waters Corporation), a SCI L OGEX D2012 high-speed table centrifuge (USA), an ultrapure water instrument (E L GA L abWater, UK), a multi-tube Vortex mixer (Vortex generator 2, USA), an adjustable pipettor (Eppendorf 0.5-10 mu L, 10-100 mu L, 100-1000 mu L), a glass instrument, a measuring cylinder and the like.
(2) Reagent supplies MS grade acetonitrile (Fisher, USA), MS grade formic acid (Merck, USA), chromatographic column Agilent Pursuit PFP (2.0 × 150mm,3.0 μm) (Agilent Technology), 0.22 μm nylon filter (Bonajier), 1m L syringe (gold tower, China).
(3) And (3) standard substance: adrenaline, noradrenaline, dopamine, metaadrenaline, noradrenaline, homovanillic acid and vanillylmandelic acid and corresponding internal standards thereof are purchased from TRC company, 3-methoxytyramine is purchased from Mecline company, and the purity is more than or equal to 98 percent.
(4) The quality control product is blank urine matrix solution containing catecholamine and metabolite molecules thereof, and the low concentration and the high concentration are QC (L) and QC (H) respectively, which are shown in Table 1.
The upper and lower peripheries of the kit are coated with a film, the kit is shockproof and heat-insulated, 10 × 1m L ampoules are placed on the left, which are respectively a calibrator solution, a quality control product and a mixed internal standard solution, the eluates (eluent A and eluent B) are placed on the upper right, and the extract liquid of 2m L is placed on the lower right.
Second, liquid condition
(1) Chromatographic conditions were 0.1% aqueous formic acid as mobile phase A, 3.0 μm acetonitrile as mobile phase B, Agilent Pursuit PFP (2.0 × 150mm,3.0 μm) as column model, and the gradient elution was carried out as detailed in Table 2, at a flow rate of 0.4m L/min, a column temperature of 40 ℃ and a sample injection volume of 1 μ L.
(2) The mass spectrum conditions are that a mass spectrum scanning mode of multi-reaction monitoring is adopted in an electrospray ionization detection mode, the spraying voltage is 0.5kV (ESI +) and 2.5kV (ESI-), the solvent removing temperature is 150 ℃, the atomizing temperature is 600 ℃, the atomizing airflow speed is 1000L/h, the taper hole airflow speed is 150L/h, and all targets and isotope internal standards are monitored at the same time, and the details are shown in Table 3.
Third, the experimental process
(1) Preparing a standard substance:
weighing epinephrine 3.425mg, noradrenaline 3.951mg, dopamine 3.882mg, noradrenaline 3.768mg, noradrenaline 3.503mg, 3-methoxytyramine 13.176mg, homovanillic acid 5.212mg and vanillylmandelic acid 4.657mg respectively, adding 2% formic acid-50% methanol aqueous solution of 1.678m L, 1.592m L, 1.536m L0, 1.558m L1, 1.402m L2, 5.275m L3, 1.021m L4 and 0.912m L5 respectively to prepare standard vanillylic acid mother liquor of vanillylic acid with the concentration of 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 3-methoxylamine, 5mg/m L and 5mg/m of vanillylic acid L mg;
and then 2% formic acid-50% methanol water solution is used for respectively diluting the concentrations of epinephrine, norepinephrine and 3-methoxytyramine to intermediate solution with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, 10 mu L of homovanillic acid with the concentration of 5mg/m L and 40 mu L of vanillic mandelic acid with the concentration of 5mg/m L are respectively taken from the intermediate solution and 2mg/m L of dopamine, and the intermediate solution and the 2% formic acid-50% methanol water solution are respectively added into 0.86m L aqueous solution containing 2% formic acid to obtain mixed standard substance stock solution.
Wherein the concentration of adrenaline (E) is 1 mu g/m L, the concentrations of adrenaline (MN) and noradrenaline (NMN) are both 2 mu g/m L, the concentrations of Noradrenaline (NE) and 3-methoxytyramine (3-MT) are both 4 mu g/m L, the concentration of Dopamine (DA) is 20 mu g/m L, and the concentrations of homovanillic acid (HVA) and vanillylmandelic acid (VMA) are both 200 mu g/m L.
Preparing the mixed standard substance stock solution into seven calibrator solutions with different concentration points by using a 2% formic acid aqueous solution;
the method comprises the steps of adding 10 mu L mixed standard substance stock solution into 190 mu L2% formic acid aqueous solution to serve as a first high-value concentration point, diluting the first high-value concentration point with 4 times of volume of 2% formic acid aqueous solution to obtain a third high-value concentration point, diluting the second high-value concentration point with 4 times of volume of 2% formic acid aqueous solution to obtain a fourth high-value concentration point, diluting the third high-value concentration point with 4 times of volume of 2% formic acid aqueous solution to obtain a fifth high-value concentration point, diluting the fourth high-value concentration point with 4 times of volume of 2% formic acid aqueous solution to obtain a sixth high-value concentration point, and diluting the fifth high-value concentration point with 3 times of volume of 2% formic acid aqueous solution to obtain a seventh high-value concentration point.
The calibrator solutions with different concentration points comprise:
seven concentrations of epinephrine are 0.5ng/m L, 1.0ng/m L, 2.0ng/m L, 5.0ng/m L, 10.0ng/m L, 25.0ng/m L and 50.0ng/m L in sequence;
the concentration of the metaadrenaline and the normetaadrenaline is the same, and the seven concentrations are 1.0ng/m L, 2.0ng/m L, 4.0ng/m L, 10.0ng/m L, 20.0ng/m L, 50.0ng/m L and 100ng/m L in sequence;
the concentrations of the norepinephrine and the 3-methoxytyramine are the same, and seven concentrations are sequentially 2.0ng/m L, 4.0ng/m L, 8.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L and 200.0ng/m L;
seven concentrations of dopamine are sequentially 10.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L, 200.0ng/m L, 500.0ng/m L and 1000.0ng/m L;
the concentration of homovanillic acid and vanillylmandelic acid is the same, seven concentrations are 100.0ng/m L, 200.0ng/m L, 400.0ng/m L, 1000.0ng/m L, 2000.0ng/m L, 5000.0ng/m L and 10000.0ng/m L in sequence.
(2) Preparation of mixed internal standard liquid
Noradrenaline-d 6 and noradrenaline-d 3 are purchased in the form of solution, the concentration of each is 100 mu g/m L, isotope internal standard noradrenaline-d 3(E-d3), isotope internal standard dopamine-d 4(DA-d4), isotope internal standard noradrenaline-d 3(NMN-d3), isotope internal standard 3-methoxytyramine-d 4(3-MT-d4), isotope internal standard homovanillic acid-d 3(HVA-d3) and isotope internal standard vanillylmandelic acid-d 3(VMA-d3) are dissolved in methanol water solution to prepare the solution, and the specific preparation process is as follows:
respectively weighing 2.5mg of adrenaline-d 3, 1mg of dopamine-d 4, 1mg of noradrenaline-d 3, 6.841mg of 3-methoxytyramine-d 4, 1mg of homovanillic acid-d 3 and 10mg of vanillylmandelic acid-d 3, respectively adding 2% formic acid-50% methanol aqueous solution of 1.225m L, 0.795m L, 0.811m L0, 1.089m L1, 0.999m L and 0.994m L to completely dissolve the noradrenaline-d 3, respectively preparing adrenaline-d L, 1mg/m L dopamine-d L, 1mg/m L of norvanillylic acid-d L, 5mg/m L of 3-methoxytyramine-d L, 1mg/m L of norvanillylic acid-d L, 10mg/m L of norvanillylic acid-d L, and 10mg of vanillylic acid-d 8672, respectively diluting the noradrenaline-d L, the noradrenaline-72, the noradrenaline-d L, the norvanillylic acid-72, the norvanillylic acid aqueous solution with the concentration of 2/m L, the concentration of 2% noradrenaline-72, the concentration of 2% L, the concentration of 2m L, the concentration of 2 μm L, the concentration of 2 μm L, the concentration of.
(3) Preparation of quality control product
And taking the mixed standard stock solution 1 mu L and 20 mu L, and respectively diluting the mixed standard stock solution to 1000 mu L by using a 2% formic acid aqueous solution to obtain QC (L) and QC (H).
QC (L) contains 1ng/m L adrenaline, 4ng/m L noradrenaline, 20ng/m L dopamine, 2ng/m L noradrenaline, 2ng/m L noradrenaline, 4ng/m L3-methoxytyramine, 200ng/m L homovanillic acid and 200ng/m L vanillylmandelic acid;
QC (H) comprises 20ng/m L adrenaline, 80ng/m L noradrenaline, 400ng/m L dopamine, 40ng/m L noradrenaline, 40ng/m L noradrenaline, 80ng/m L3-methoxytyramine, 4000ng/m L homovanillic acid and 4000ng/m L vanillylmandelic acid.
(4) Sample processing
1) And (3) standard substance treatment, namely taking 200 mu L of mixed internal standard solution, adding 1m L of extract (10% formic acid aqueous solution) firstly, then diluting with 78.8m L of ultrapure water to obtain mixed internal standard working solution, taking 20 mu L of samples of each concentration point of seven different calibration substance solutions, subpackaging in different centrifuge tubes, adding 780 mu L of mixed internal standard working solution, then carrying out vortex for several seconds, taking 70 mu L supernatant, transferring the supernatant into a chromatographic bottle, and carrying out L C-MS/MS analysis.
2) Pretreatment of a urine sample, namely, 100 mu L urine is filtered through a 0.22 mu m nylon filter membrane, 20 mu L urine is accurately transferred into a 1.5m L centrifuge tube, 780 mu L mixed internal standard working solution is added into the centrifuge tube, 70 mu L supernatant is transferred into a chromatographic bottle after being vortexed for a plurality of seconds, and L C-MS/MS analysis is carried out on the supernatant.
3) And (3) pretreatment of the quality control product, namely, respectively taking 20 mu L of quality control product solution QC (L) and QC (H) into a centrifuge tube of 1.5m L, and then, conforming to the pretreatment method of the urine sample, and the method is not repeated herein.
The components of the assay kit are shown in Table 4.
TABLE 4 preparation of catecholamines and their metabolite analysis kit Components (100 persons)
Fourth, method verification
1. Extracting an ion current chromatogram: the 8 catecholamine and metabolite and isotope internal standard extraction ion current chromatograms are shown in fig. 1 and fig. 2, and the 8 catecholamine and metabolite and isotope internal standard extraction ion current chromatograms are shown in the urine.
2. And (3) adopting an isotope internal standard quantitative method, establishing a calibration curve by using Target L ynx software and taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of the substance to be detected in the urine, wherein the linear fitting equations of 8 catecholamines and metabolites thereof in respective concentration ranges have good linearity, the correlation coefficient is more than 0.998, and the quantitative requirements are met, and the table 5 shows.
TABLE 58 linear regression equations and linear correlation coefficients for catecholamines and their metabolites
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. One sample of human plasma was randomly selected, 1 of which was not added with the standard, and the other 3 were added with the low, medium and high 3 concentrations of the standard, respectively, and the same procedure was repeated and measured 5 times, and the results were calculated as shown in table 6. The results show that the standard recovery rate of 8 catecholamines and metabolites thereof is between 92.7% and 106.9%, and the RSD of 5 times of repeated experiments is in the range of 0.47% to 9.02%.
TABLE 68 catecholamines and their metabolites normalized recovery results (unit ng/m L)
4. And (3) precision test: taking normal human urine samples, repeatedly processing 6 batches within one day, quantitatively measuring the concentrations of 8 catecholamine and metabolites thereof by using an isotope internal standard method, wherein the internal batch precision is 0.67-11.18%, processing 3 batches within three days, and calculating the batch precision to be 2.40-9.44%, and the results are shown in Table 7.
Precision results for the Table 78 catecholamines and their metabolites
Fifth, discuss
The invention establishes a method for simultaneously measuring 8 catecholamine and metabolites thereof in human urine by ID-UP L C-MS/MS, the use of the kit of the invention for detecting 8 catecholamine and metabolites thereof in urine has the advantages of small urine consumption (only 100 mu L), simple pretreatment, no need of solid phase extraction or derivatization reaction, direct dilution and sample injection after urine filtration, no more than 10min of the whole treatment process, and only 4.5min for analyzing 8 substances by one needle, thus being the simplest and most rapid method for currently making the catecholamine and the metabolites thereof.
The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The accuracy of the method is evaluated by a standard addition recovery rate test, and the result shows that the standard addition recovery rate of 8 catecholamine and metabolites thereof is 92.7-106.9%, the RSD of 5 times of repeated tests is 0.47-9.02%, and the accuracy is good.
The reproducibility result of the method shows that the internal precision of 8 catecholamine and metabolites thereof is 0.67-11.18%, the batch precision is 2.40-9.44%, and the reproducibility of the method is good.
In a word, the detection method disclosed by the invention is high in sensitivity, strong in specificity, accurate and simple in pretreatment process, the pretreatment process is completed within 10min, the separation and detection of the compound are completed within 4.5min, the matrix effect, the extraction recovery rate and the precision meet the requirements, and the method can be used for clinically quantitatively analyzing 8 catecholamines and metabolites thereof in urine and provides a reliable detection method for clinically evaluating or diagnosing the risks of diseases such as pheochromocytoma, sympathogenic neuroma and neuroblastoma.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A kit for detecting 8 catecholamine and metabolites thereof in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology,
the 8 catecholamines and metabolites thereof are respectively: epinephrine, norepinephrine, dopamine, metaepinephrine, normetaepinephrine, 3-methoxytyramine, homovanillic acid, and vanillylmandelic acid;
the kit comprises the following reagents:
(1) eluent:
eluent A: 0.05 to 0.1 percent of formic acid aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
a mixed standard stock solution comprising 100 μ g/m L epinephrine, 400 μ g/m L norepinephrine, 2mg/m L dopamine, 200 μ g/m L norepinephrine, 200 μ g/m L norepinephrine, 400 μ g/m L3-methoxytyramine, 5mg/m L homovanillic acid, and 5mg/m L vanillylmandelic acid was formulated with a blank urine matrix solution into calibrator solutions at seven different concentration points, the seven concentration points of the calibrator solution being:
seven concentrations of epinephrine are 0.5ng/m L, 1.0ng/m L, 2.0ng/m L, 5.0ng/m L, 10.0ng/m L, 25.0ng/m L and 50.0ng/m L in sequence;
the concentration of the metaadrenaline and the normetaadrenaline is the same, and the seven concentrations are 1.0ng/m L, 2.0ng/m L, 4.0ng/m L, 10.0ng/m L, 20.0ng/m L, 50.0ng/m L and 100ng/m L in sequence;
the concentrations of the norepinephrine and the 3-methoxytyramine are the same, and seven concentrations are sequentially 2.0ng/m L, 4.0ng/m L, 8.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L and 200.0ng/m L;
seven concentrations of dopamine are sequentially 10.0ng/m L, 20.0ng/m L, 40.0ng/m L, 100.0ng/m L, 200.0ng/m L, 500.0ng/m L and 1000.0ng/m L;
the concentration of homovanillic acid and vanillylmandelic acid is the same, seven concentrations are 100.0ng/m L, 200.0ng/m L, 400.0ng/m L, 1000.0ng/m L, 2000.0ng/m L, 5000.0ng/m L and 10000.0ng/m L in sequence;
(3) mixing internal standard liquid:
respectively preparing adrenaline-d 3 with the concentration of 2 mu g/m L, noradrenaline-d 6 with the concentration of 8 mu g/m L, dopamine-d 4 with the concentration of 40 mu g/m L, adrenaline-d 3 with the concentration of 4 mu g/m L, noradrenaline-d 3 with the concentration of 4 mu g/m L, 3-methoxytyramine-d 4 with the concentration of 8 mu g/m L, homovanillic acid-d 3 with the concentration of 800 mu g/m L and vanillylmandelic acid-d 3 with the concentration of 200 mu g/m L by using 30-70% methanol aqueous solution, and then adding 20 mu L of the internal standard solution into 0.24m L diluent to obtain mixed internal standard solution;
(4) extracting liquid:
aqueous formic acid or acetic acid;
(5) quality control product:
including QC (L) and QC (h):
QC (L) is that the said mixed standard stock solution is diluted to 1000 times with the blank urine matrix solution;
qc (h) diluted 50-fold for the mixed standard stock with blank urine matrix solution.
2. The kit according to claim 1,
the eluent A is 0.05-0.5% formic acid aqueous solution, preferably 0.1% formic acid aqueous solution.
3. The kit according to claim 1,
the extraction liquid is formic acid aqueous solution; preferably 5% to 20% aqueous formic acid, more preferably 10% aqueous formic acid.
4. The kit according to claim 1,
the blank urine matrix is formic acid aqueous solution or acetic acid aqueous solution; preferably an aqueous formic acid solution; more preferably 0.1 to 5 percent aqueous formic acid solution; particularly preferably 2% aqueous formic acid.
5. The kit according to claim 1, 2, 3 or 4,
the mixed standard stock solution is prepared by preparing 100 mu g/m L, 400 mu g/m L, 2mg/m L0, 200 mu g/m L, 200 mu g/m L, 400 mu g/m L, 5mg/m L and 5mg/m L from 30-70% methanol aqueous solution, and adding 10 mu L from the prepared adrenaline solution, noradrenaline solution, adrenaline solution, noradrenaline solution and 3-methoxytyramine solution respectively, and 40 mu L from the homovanillic acid solution and the vanillic mandelic acid solution into 0.86m L diluent to obtain the mixed standard stock solution.
6. The kit according to claim 5,
the diluent is a formic acid aqueous solution or an acetic acid aqueous solution; preferably an aqueous formic acid solution; more preferably 0.1 to 5 percent aqueous formic acid solution; particularly more preferably 2% aqueous formic acid; the methanol aqueous solution contains formic acid, preferably 1-5% formic acid-30-70% methanol aqueous solution, more preferably 2% formic acid-50% methanol aqueous solution.
7. The kit according to claim 1,
the urine is human or animal urine.
8. The kit according to claim 6,
the kit comprises the following reagents:
(1) eluent:
eluent A: 0.05 to 0.5 percent of formic acid aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
preparing standard mother liquor with the concentrations of 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 5mg/m L and 5mg/m L by using 1-5% formic acid-30-70% methanol aqueous solution;
then, 1-5% formic acid-30-70% methanol water solution is used for respectively diluting the concentrations of adrenaline, noradrenaline and 3-methoxytyramine to intermediate solutions with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, respectively taking 10 mu L of the intermediate solution and 2mg/m L of dopamine, respectively taking 40 mu L of 5mg/m L of homovanillic acid and 5mg/m L of vanillylmandelic acid, respectively adding the homovanillic acid and the vanillylmandelic acid into 0.86m L aqueous solution containing 0.1-5% formic acid, and obtaining mixed standard product stock solution;
preparing the mixed standard substance stock solution into calibration substance solutions with seven different concentration points by using a blank urine matrix solution;
(3) mixing internal standard liquid:
the noradrenaline-d and noradrenaline-d are purchased in the form of solution, the concentration of which is 100 mu g/m, the adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid are prepared into isotope internal standard solutions of adrenaline-d, dopamine-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic mandelic acid, the concentration of which is 2mg/m, 1mg/m 0, 1mg/m 1, 3-methoxytyramine-d, 10mg/m 4, respectively, by 1-5% formic acid-30-70% methanol aqueous solution, the noradrenaline-d, adrenaline-d, dopamine-d, adrenaline-d, noradrenaline-d, 3-methoxytyramine-d, homovanillic acid-d and vanillic acid-d, the concentration of which is 1-5-m 1-70% formic acid-2, the concentration of the noradrenaline-d, the concentration of the homovanillic acid-d, the concentration of which is 6 mu g/m, the concentration of the noradrenaline-d, the concentration of the homovanillic mandelic acid-d, the isotopic solution is 1-d, the isotopic solution of which is 1-8.8 mu m, the isotopic solution of the isotopic solution is 2 g/m;
(4) extracting liquid:
5 to 20 percent of formic acid aqueous solution;
(5) quality control product:
preparing the mixed standard substance stock solution into QC (L) and QC (H) with two different concentrations by using 0.1-5% formic acid aqueous solution;
QC (L) contains 1ng/m L adrenaline, 4ng/m L noradrenaline, 20ng/m L dopamine, 2ng/m L noradrenaline, 2ng/m L noradrenaline, 4ng/m L3-methoxytyramine, 200ng/m L homovanillic acid and 200ng/m L vanillylmandelic acid;
QC (H) comprises 20ng/m L adrenaline, 80ng/m L noradrenaline, 400ng/m L dopamine, 40ng/m L noradrenaline, 40ng/m L noradrenaline, 80ng/m L3-methoxytyramine, 4000ng/m L homovanillic acid and 4000ng/m L vanillylmandelic acid.
9. The kit according to claim 8, wherein the kit comprises the following reagents:
(1) eluent:
eluent A: 0.1 aqueous formic acid; eluent B: acetonitrile;
(2) calibration solution:
weighing epinephrine 3.425mg, noradrenaline 3.951mg, dopamine 3.882mg, noradrenaline 3.768mg, noradrenaline 3.503mg, 3-methoxytyramine 13.176mg, homovanillic acid 5.212mg and vanillylmandelic acid 4.657mg respectively, adding 2% formic acid-50% methanol aqueous solution of 1.678m L, 1.592m L, 1.536m L0, 1.558m L1, 1.402m L2, 5.275m L3, 1.021m L4 and 0.912m L5 respectively to prepare standard vanillylic acid mother liquor of vanillylic acid with the concentration of 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 2mg/m L, 3-methoxylamine, 5mg/m L and 5mg/m of vanillylic acid L mg;
then 2% formic acid-50% methanol water solution is used for respectively diluting the concentrations of epinephrine, norepinephrine and 3-methoxytyramine to intermediate solution with the concentrations of 100 mu g/m L, 400 mu g/m L, 200 mu g/m L0, 200 mu g/m L and 400 mu g/m L, 10 mu L of each intermediate solution and 2mg/m L of dopamine are respectively taken, 5mg/m L of homovanillic acid and 40 mu L of 5mg/m L of vanillylmandelic acid are respectively taken to be added into 0.86m L aqueous solution containing 2% formic acid, and mixed standard substance stock solution is obtained;
preparing the mixed standard substance stock solution into seven calibrator solutions with different concentration points by using a 2% formic acid aqueous solution;
(3) mixing internal standard liquid:
respectively weighing 2.5mg of adrenaline-d 3, 1mg of dopamine-d 4, 1mg of noradrenaline-d 3, 6.841mg of 3-methoxytyramine-d 4, 1mg of homovanillic acid-d 3 and 10mg of vanillylmandelic acid-d 3, respectively adding 2% formic acid-50% methanol aqueous solution of 1.225m L, 0.795m L, 0.811m L0, 1.089m L1, 0.999m L and 0.994m L to completely dissolve the noradrenaline-d 6372, 1mg/m L dopamine-d L, 1mg/m 366, 5mg/m L, 3-methoxytyramine-d L, 1mg/m L, 10mg/m L, respectively diluting the noradrenaline-d L, the norvanillylic acid-d L and the noradrenaline-72 to obtain a noradrenaline-72/m noradrenaline-d L, and noradrenaline-72 aqueous solution, and a norvanillylic acid-72 solution of 2% noradrenaline-72, a concentration of 2 μm L, a noradrenaline-72, a norvanillylic acid-72, a noradrenaline-72, a norvanillylic acid-72 μm L, a norvanillylic acid-72 μm L, a norvanillylic acid aqueous solution of L, a norvanillylic acid solution of 2 μm L, a norvanillyl acid solution of 2;
(4) extracting liquid:
10% aqueous formic acid;
(5) quality control product:
and taking the mixed standard stock solution 1 mu L and 20 mu L, and respectively diluting the mixed standard stock solution to 1000 mu L by using a 2% formic acid aqueous solution to obtain QC (L) and QC (H).
10. Use of the kit of any one of claims 1 to 9 for detecting catecholamine and its metabolites in urine by ultra high performance liquid chromatography tandem mass spectrometry.
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Cited By (6)
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| CN113702539A (en) * | 2021-09-02 | 2021-11-26 | 北京市心肺血管疾病研究所 | Application of catecholamine substances in preparation of kit for predicting risk of cardiovascular events of high blood pressure high-risk patient |
| CN114441665A (en) * | 2020-11-04 | 2022-05-06 | 中国科学院大连化学物理研究所 | Detection method of catecholamine and metabolite thereof |
| CN115327008A (en) * | 2022-09-13 | 2022-11-11 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
| CN116106466A (en) * | 2023-01-09 | 2023-05-12 | 泽达精准(杭州)生物医药有限公司 | Kit for simultaneously determining multiple causative markers of endocrine hypertension |
| CN116148387A (en) * | 2023-02-14 | 2023-05-23 | 杭州佰辰医疗器械有限公司 | Catecholamine and catecholamine metabolite detection method and kit |
| CN117092263A (en) * | 2023-08-15 | 2023-11-21 | 合肥歆智医疗器械有限公司 | Free catecholamine and metabolite detection kit, determination method and application |
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| CN114441665A (en) * | 2020-11-04 | 2022-05-06 | 中国科学院大连化学物理研究所 | Detection method of catecholamine and metabolite thereof |
| CN113702539A (en) * | 2021-09-02 | 2021-11-26 | 北京市心肺血管疾病研究所 | Application of catecholamine substances in preparation of kit for predicting risk of cardiovascular events of high blood pressure high-risk patient |
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| CN115327008A (en) * | 2022-09-13 | 2022-11-11 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
| CN115327008B (en) * | 2022-09-13 | 2023-05-26 | 北京豪思生物科技股份有限公司 | Method for detecting catecholamine and metabolite thereof in blood plasma and urine |
| CN116106466A (en) * | 2023-01-09 | 2023-05-12 | 泽达精准(杭州)生物医药有限公司 | Kit for simultaneously determining multiple causative markers of endocrine hypertension |
| CN116106466B (en) * | 2023-01-09 | 2023-10-20 | 泽达精准(杭州)生物医药有限公司 | Kit for simultaneously determining multiple causative markers of endocrine hypertension |
| CN116148387A (en) * | 2023-02-14 | 2023-05-23 | 杭州佰辰医疗器械有限公司 | Catecholamine and catecholamine metabolite detection method and kit |
| CN117092263A (en) * | 2023-08-15 | 2023-11-21 | 合肥歆智医疗器械有限公司 | Free catecholamine and metabolite detection kit, determination method and application |
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