Method for detecting catecholamine and metabolites thereof in urine

Abstract

The invention provides a method for detecting catecholamine and metabolites thereof in urine, and belongs to the technical field of substance detection. The method for detecting catecholamine and metabolites thereof in urine comprises the following steps: providing pretreated to-be-detected urine; loading a sample of urine to be detected into the activated and balanced solid-phase extraction column, carrying out pressurized extraction, washing with a methanol aqueous solution and water, and eluting with a methanol solution of formic acid in sequence to obtain an eluent; directly detecting the eluent by using pulse direct-current spray tandem mass spectrometry to obtain mass spectrum signals of the six catecholamines and metabolites thereof in the urine to be detected; and substituting the mass spectrum signals into a standard curve of the catecholamine and the metabolites thereof to obtain the concentrations of the six catecholamines and the metabolites thereof. The detection method has the advantages of high detection speed, high sensitivity, good stability and the like.

Description

Technical Field

[0001] The present invention belongs to the field of substance detection, and particularly relates to a method for detecting catecholamines and their metabolites in urine. Background Art

[0002] Six catecholamines and their metabolites, including dopamine (DA), epinephrine (E), norepinephrine (NE) and their O-methylated metabolites (normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT)), are important clinical diagnostic markers for pheochromocytoma and paraganglioma (PPGL). There are various analytical methods for measuring catecholamines and metanephrines, and the most common ones include immunoassay, fluorescence detection, electrochemical detection and mass spectrometry. However, most of these methods have certain limitations, such as insufficient sensitivity, susceptibility to interference, cumbersome operation, long analysis time and the need for a large sample volume. Summary of the Invention

[0003] In view of this, the present invention provides a method for detecting catecholamines and their metabolites in urine, in order to at least partially solve the above technical problems. For this, the technical solutions provided by the present invention are as follows.

[0004] The present invention provides a method for detecting catecholamines and their metabolites in urine, comprising: providing pretreated urine to be tested; loading the urine to be tested into an activated and balanced solid-phase extraction column, and after pressurized extraction, washing successively with methanol aqueous solution and water, and eluting with a methanol solution of formic acid to obtain an eluate; directly detecting the eluate by pulsed direct current electrospray tandem mass spectrometry to obtain mass spectrometry signals of six catecholamines and their metabolites in the urine to be tested; substituting the mass spectrometry signals into the standard curves of catecholamines and their metabolites to obtain the concentrations of six catecholamines and their metabolites.

[0005] Based on the above technical solutions, the method for detecting catecholamines and their metabolites in urine provided by the present invention has at least one of the following beneficial effects.

[0006] (1) The present invention combines solid-phase extraction (SPE) pretreatment and pulsed direct current electrospray tandem mass spectrometry (pulsed-dc-ESI-MS / MS) direct detection technology. In addition to having the advantages of both technologies, it also has the advantages of simple operation, fast analysis speed and high analysis sensitivity.

[0007] (2) Before detecting by using the pulsed-dc-ESI-MS / MS technology, the present invention improves the solid-phase extraction pretreatment process of the urine to be detected, so that it can simultaneously enrich six kinds of catecholamines and their metabolites. Combining with pulsed-dc-ESI-MS / MS can realize the rapid and synchronous detection of the mass spectrometry signals of six kinds of catecholamines and their metabolites in the eluate. Bringing the obtained mass spectrometry signals into the established standard curve of catecholamines and their metabolites can correspondingly obtain the concentrations of six kinds of catecholamines and their metabolites.

[0008] (3) The present invention combines solid-phase extraction and the direct detection technology of pulsed-dc-ESI-MS / MS, which can make the detection limits of methoxyepinephrine (MN) and normetanephrine (NMN) in urine significantly lower than the upper limit of the normal reference value; at the same time, the detection limits of DA, NE and E can also effectively distinguish healthy individuals from patients, meeting the requirements of practical applications. Description of the Drawings

[0009] Figure 1 It is a schematic flow chart for detecting catecholamines and their metabolites in urine in the embodiment of the present invention;

[0010] Figure 2 It is a schematic flow chart for determining catecholamines and their metabolites in the urine to be detected by combining solid-phase extraction and pulsed direct current electrospray tandem mass spectrometry in the embodiment of the present invention;

[0011] Figure 3 It is a mass spectrometry diagram of six kinds of catecholamines and their metabolites in artificial urine in Embodiment 1 of the present invention;

[0012] Figure 4A It is a microscopic diagram of pulsed-dc-ESI-MS / MS detecting real urine without solid-phase extraction pretreatment in Scheme 1) of Embodiment 2 of the present invention;

[0013] Figure 4B It is a microscopic diagram of pulsed-dc-ESI-MS / MS detecting the eluate after solid-phase extraction pretreatment in Scheme 2) of Embodiment 2 of the invention;

[0014] Figure 5 It is a tandem mass spectrometry signal intensity diagram of the solution obtained in each step during the solid-phase extraction process with NMN as the representative in Embodiment 3 of the present invention;

[0015] Figure 6 It is a recovery rate diagram of the solid-phase extraction of six kinds of catecholamines and their metabolites in Embodiment 3 of the present invention;

[0016] Figure 7AIt is the tandem mass spectrometry signal intensity diagram of ammonia-ammonium chloride buffer solution at different pH values during the solid-phase extraction process represented by NMN in Example 4 of the present invention;

[0017] Figure 7B It is the tandem mass spectrometry signal intensity diagram of the urine sample to be tested after solid-phase extraction with different pH pre-treatments represented by NMN in Example 4 of the present invention;

[0018] Figure 7C It is the tandem mass spectrometry signal intensity diagram of different formic acid contents in the elution solution during the solid-phase extraction process represented by NMN in Example 4 of the present invention;

[0019] Figure 8A It is the mass spectrometry signal standard curve diagram of NMN in six catecholamines and their metabolites spiked in artificial urine in Example 5 of the present invention;

[0020] Figure 8B It is the mass spectrometry signal standard curve diagram of MN in six catecholamines and their metabolites spiked in artificial urine in Example 5 of the present invention.

[0021]

Description of the reference numerals

[0022] 1 - Sample collection bottle, 2 - Solid-phase extraction column, 3 - Collection tube, 4 - Capillary spray needle, 5 - Conductive metal wire, 6 - Mass spectrometry port. Detailed implementation manners

[0023] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in detail with reference to specific embodiments.

[0024] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, obviously, one or more embodiments can also be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0025] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the gold standard for catecholamines and metanephrines in urine due to its high sensitivity and the accurate identification ability of the mass spectrometry detector. However, before using liquid chromatography for detection, the samples need to be pretreated (such as extraction, derivatization, etc.). In the actual application process, the chromatographic separation process of each sample is relatively cumbersome and time-consuming. In addition, the concentrations of catecholamines and metanephrines in biological fluids are low and the urine matrix is complex, so it is very difficult to directly analyze catecholamines and metanephrines in urine. Therefore, developing an efficient extraction strategy to separate catecholamines and metanephrines from complex urine samples is crucial for their detection. In this regard, the present invention proposes to combine solid-phase extraction technology with direct detection by pulsed-dc-ESI-MS / MS, which can quickly and sensitively detect the concentrations of six catecholamines and their metabolites in urine.

[0026] Figure 1 It is a schematic flow chart for detecting catecholamines and their metabolites in urine in the embodiments of the present invention.

[0027] As Figure 1 shown, the present invention provides a method for detecting catecholamines and their metabolites in urine, including: step S1-step S4.

[0028] Step S1: Provide the pretreated urine to be tested.

[0029] Step S2: Load the urine to be tested into the activated and balanced solid-phase extraction column. After pressurized extraction, wash it successively with methanol aqueous solution and water, and elute it with methanol solution of formic acid to obtain the eluate.

[0030] Step S3: Directly detect the eluate by pulsed direct current electrospray tandem mass spectrometry to obtain the mass spectrometry signals of six catecholamines and their metabolites in the urine to be tested.

[0031] Step S4: Substitute the mass spectrometry signals into the standard curves of catecholamines and their metabolites to obtain the concentrations of six catecholamines and their metabolites.

[0032] In an embodiment of the present invention, pretreating the urine to be tested can remove impurities and interfering factors in the urine, such as protein precipitation, cell debris, bacteria, etc., to ensure the accuracy of the test results. Subsequently, the pretreated urine to be tested is loaded into a solid-phase extraction column. While further removing interfering impurities in the urine through the solid-phase extraction column, catecholamines and their metabolites in the urine to be tested are extracted and enriched to obtain an eluate. The eluate can be directly detected by pulsed direct current electrospray tandem mass spectrometry to obtain the mass spectrometry signals of six catecholamines and their metabolites in the urine to be tested. Finally, according to the corresponding relationship between the concentration and the mass spectrometry signal in the standard curve of catecholamines and their metabolites, the concentrations of six catecholamines and their metabolites in the urine to be tested can be determined. Compared with the traditional liquid chromatography-tandem mass spectrometry detection method, it greatly simplifies the processing steps of the urine to be tested, shortens the detection time, and has the advantages of high sensitivity and stability.

[0033] According to an embodiment of the present invention, in step S1, the pretreatment includes: performing acid hydrolysis on the urine to be tested; and adjusting the pH of the urine to be tested after acid hydrolysis to be alkaline. Specifically, the pretreatment includes: adding an acidic reagent to the urine to be tested and heating to obtain the urine to be tested after acid hydrolysis; after cooling, adding an alkaline reagent to adjust the pH of the urine to be tested after acid hydrolysis to be alkaline and removing the precipitate, that is, adjusting the pH of the urine to be tested to 7-9.5. Further, during the acid hydrolysis process, the acidic reagent used can be hydrochloric acid with a concentration of 0.3M / L, the volume ratio of hydrochloric acid to the urine to be tested is 2:3, the heating temperature is 100°C, and the heating time is 20 min. During the process of adjusting the pH of the urine to be tested after acid hydrolysis, the alkaline reagent used can be 28% ammonia water, and preferably the pH of the urine to be tested after acid hydrolysis is adjusted to 8-9.5. By performing acid hydrolysis on the urine to be tested, impurities and interfering factors in the urine can be effectively removed, ensuring the purity of the urine to be tested and making the test results more reliable. During the adjustment of the pH of the urine to be tested, since the pKa of catecholamines and their metabolites is in the range of 9-10, controlling the pH of the urine to be tested about 2 pH units above or below the pKa of catecholamines and their metabolites can significantly contribute to retaining the compounds.

[0034] According to an embodiment of the present invention, internal standards corresponding to six catecholamines and their metabolites are added to the urine to be tested. Among them, the six catecholamines and their metabolites include: dopamine, epinephrine, norepinephrine, 3-methoxytyramine, normetanephrine, and metanephrine. Further, the internal standards corresponding to dopamine, epinephrine, and norepinephrine are dopamine-d4; the internal standards corresponding to 3-methoxytyramine, normetanephrine, and metanephrine are metanephrine-d3.

[0035] According to an embodiment of the present invention, in step S2, obtaining an activated and balanced solid-phase extraction column includes: activating the solid-phase extraction column with pure methanol, where the solid-phase extraction column is selected from a hydrophilic-lipophilic balance (HLB) type solid-phase extraction column, and its packing is mainly divinylbenzene / hydrophilic N-vinylpyrrolidone polymer, with an average particle size of 40-60 μm, an average pore size of 80 Å, and a specific surface area of 600 m 2 / g. Activating the HLB type solid-phase extraction column with pure methanol can effectively dissolve the carbon group chains on the packing, enabling it to function and improving the extraction efficiency. Further, the activated solid-phase extraction column is balanced with an ammonia-ammonium chloride buffer solution, where the pH value of the ammonia-ammonium chloride buffer solution is 8-10, to maximize the retention rate of catecholamines and their metabolites on the solid-phase extraction column. Finally, the urine to be tested is loaded into the activated and balanced solid-phase extraction column for pressurized extraction, shortening the extraction time while enriching the six catecholamines and their metabolites in the urine on the HLB type solid-phase extraction column.

[0036] According to an embodiment of the present invention, in step S2, the HLB type solid-phase extraction column enriched with catecholamines and their metabolites is washed successively with an aqueous methanol solution and water to remove some impurities present in the urine to be tested. Further, in the aqueous methanol solution, the volume ratio of methanol to water is 5:95-10:90. In the final elution step, an organic solvent or a non-polar solvent mixture is used to break the hydrophilic interaction between the analyte and the adsorbent functional groups. Considering the compatibility of the eluent with direct detection by pulsed-dc-ESI-MS / MS, a methanol solution of formic acid is selected as the elution solution for elution to obtain an eluate, where in the methanol solution of formic acid, the volume percentage of formic acid is 6.0%-8.0%.

[0037] According to an embodiment of the present invention, in step S3, directly detecting the eluate by pulsed direct current electrospray tandem mass spectrometry includes: automatically aspirating the eluate with the capillary needle of pulsed direct current electrospray tandem mass spectrometry to complete sample loading; applying a direct current voltage to the capillary needle to generate an ionization spray; detecting the ionization spray by pulsed direct current electrospray tandem mass spectrometry to obtain the mass spectrometry signals of the six catecholamines and their metabolites in the urine to be tested. Among them, the inner diameter size of the capillary needle is 5-6 μm, for example, it can be 5.0 μm, 5.1 μm, 5.2 μm, 5.3 μm, 5.4 μm, 5.5 μm, 5.6 μm, 5.7 μm, 5.8 μm, 5.9 μm, 6.0 μm, etc.; the direct current voltage is 2.5-3.0 kV, and the inner diameter size of the capillary needle and the magnitude of the direct current voltage need to be adapted to each other to achieve a better detection effect. In addition, using the detection method provided by the present invention can shorten the detection time and has high sensitivity.

[0038] According to an embodiment of the present invention, a standard curve of catecholamines and their metabolites is obtained through the following steps: an internal standard is added to the original solutions to be measured of catecholamines and their metabolites at different concentrations to obtain standard solutions of catecholamines and their metabolites at different concentrations; the standard solutions at different concentrations are loaded into a solid-phase extraction column for solid-phase extraction to obtain standard eluents at different concentrations; pulsed direct current electrospray tandem mass spectrometry is used to directly detect the standard eluents at different concentrations to obtain the mass spectrometry signals of catecholamines and their metabolites at different concentrations; a standard curve of catecholamines and their metabolites is established based on the mass spectrometry signals of catecholamines and their metabolites and the concentrations of the standard solutions at different concentrations. In the present invention, calculating the signal intensity ratio of the analyte to the internal standard by adding the internal standard can make the obtained standard curve more accurate, thereby making the detection result more accurate. Using the established standard curve of catecholamines and their metabolites and the mass spectrometry signals of catecholamines and their metabolites in the urine to be measured, the concentrations of catecholamines and their metabolites in the urine to be measured can be determined.

[0039] Thus, the present invention improves the solid-phase extraction pretreatment scheme based on a unified solid-phase extraction column and can simultaneously enrich six catecholamines and their metabolites in urine. Combining pulsed-dc-ESI-MS / MS to directly detect the eluate obtained by solid-phase extraction, the signal intensity of catecholamines and their metabolites in the urine to be measured can be obtained, and substituting it into the standard curve, the contents of six catecholamines and their metabolites in the urine to be measured can be obtained. Using the detection method of the present invention to detect catecholamines and their metabolites in urine, compared with the traditional liquid chromatography-tandem mass spectrometry detection method, under optimized conditions, the entire analysis process only takes 8 minutes, and has the advantages of fast detection speed and high stability. In addition, catecholamines and their metabolites are important markers of PPGL, and the sensitivity of this method meets the diagnostic requirements of the "Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma (2020 Edition)", and has good application prospects in the clinical rapid diagnosis of PPGL.

[0040] The following specifically describes the method for detecting catecholamines and their metabolites in urine of the present invention in combination with specific examples and drawings. It should be noted that the examples provided by the present invention are only for illustration and are not limited thereto.

[0041] Unless otherwise specified, the methods in the following examples are all conventional methods, and the reagents and raw materials used can all be obtained from public commercial channels unless otherwise specified.

[0042] Example

[0043] Figure 2 It is a schematic flow chart of determining catecholamines and their metabolites in urine to be measured by solid-phase extraction combined with pulsed direct current electrospray tandem mass spectrometry in an embodiment of the present invention.

[0044] AsFigure 2 As shown in Figure 2 , the system for determining catecholamines and their metabolites in urine by solid-phase extraction combined with pulsed-dc-ESI-MS / MS includes: a sample collection bottle 1 for collecting the urine to be tested, a solid-phase extraction column 2 for extracting and enriching the urine to be tested, a collection tube 3 for collecting the eluate, a capillary spray needle 4 for aspirating the eluate, a conductive metal wire 5 for ionizing the eluate in the capillary spray needle 4, and a mass spectrometry port 6 for collecting the electrospray of the eluate.

[0045] Specifically, the determination of catecholamines and their metabolites in the urine to be tested mainly includes the following steps:

[0046] (1) Pretreatment of the urine to be tested

[0047] Collect the urine sample, immediately centrifuge it twice at 4000 rpm for 10 minutes at 4°C, and then store it at -20°C.

[0048] Add 0.3M / L hydrochloric acid and the urine to be tested to the urine to be tested in the sample collection bottle 1 at a volume ratio of 2:3, and then heat it at 100°C for 20 minutes for acid hydrolysis. After cooling, adjust the pH to 8 - 9.5 (such as pH = 9) with 28% ammonia water, and filter off the precipitate with a 0.22μm aqueous needle filter.

[0049] (2) Solid-phase extraction process

[0050] Activate the HLB type solid-phase extraction column (1mL - 30mg) with 1mL of methanol, and then equilibrate it with 1mL of ammonia-ammonium chloride buffer solution with pH = 8 - 10. Load the 2mL of the pretreated urine sample in step (1) onto the solid-phase extraction column 2 (HLB type solid-phase extraction column) in portions, and extract it by pressurizing with a syringe. Wash the solid-phase extraction column with 1mL of methanol aqueous solution (5:95, v / v) and 1mL of water to complete purification. Finally, elute the target analytes (catecholamines and their metabolites) from the solid-phase extraction column with 200μL of a methanol solution of formic acid (where the volume percentage of formic acid includes 6.5% - 8.0%) to obtain the eluate, and collect this eluate in the collection tube 3.

[0051] (3) Pulsed-dc-ESI-MS / MS detection

[0052] The eluent in the collection tube 3 is loaded into the capillary spray needle 4 by self-suction, and ionization is carried out by applying a voltage of 2.5 kV to the conductive metal wire 5 inserted into the capillary spray needle 4 to generate an ionization spray. The ionization spray is detected by pulsed-dc-ESI-MS / MS to obtain the mass spectrometry signals of six catecholamines and their metabolites in the urine to be tested. Among them, the capillary spray needle 4 is drawn from a borosilicate glass capillary by a laser puller, and the inner diameter size of the capillary spray needle 4 is about 5-6 μm, for example, it can be: 5.0 μm, 5.1 μm, 5.2 μm, 5.3 μm, 5.4 μm, 5.5 μm, 5.6 μm, 5.7 μm, 5.8 μm, 5.9 μm, 6.0 μm. The distance between the tip of the capillary spray needle 4 and the mass spectrometry port 6 is not limited. In this invention, 5 mm is taken as an example, and it can also be adaptively changed according to the needs of mass spectrometry analysis.

[0053] Example 1

[0054] Solid-phase extraction combined with pulsed-dc-ESI-MS / MS for rapid and simultaneous analysis of six catecholamines and their metabolites in urine, including the following specific steps:

[0055] (1) Pretreatment of artificial urine

[0056] Preparation and acid hydrolysis of artificial urine: Urea (416.25 mM / L), sodium chloride (78.70 mM / L), magnesium chloride (6.84 mM / L), calcium chloride (5.86 mM / L), potassium chloride (41.46 mM / L), sodium dihydrogen phosphate (20.57 mM / L), ammonium chloride (18.69 mM / L), creatinine (9.72 mM / L) and trisodium citrate (2.20 mM / L) are dissolved in water to obtain artificial urine.

[0057] Standard substances NMN (2 μM / L), MN (2 μM / L), 3-MT (2 μM / L), DA (10 μM / L), E (10 μM / L), NE (10 μM / L), DA-d4 (10 μM / L), and MN-d3 (2 μM / L) are added to the artificial urine. 0.3 M / L hydrochloric acid is added to the spiked artificial urine at a volume ratio of 2:3, and then heated at 100 °C for 20 minutes for acid hydrolysis. After cooling, the pH is adjusted to 9.0 with 28% ammonia water, and the precipitate is filtered off with a 0.22 μm aqueous needle filter to obtain the spiked artificial urine sample.

[0058] (2) Solid-phase extraction process of spiked artificial urine

[0059] Activate the HLB solid-phase extraction column (1 mL - 30 mg) with 1 mL of methanol, and then equilibrate it with 1 mL of ammonia-ammonium chloride buffer solution at pH = 9.5. Load the 2 mL of the spiked artificial urine sample from step (1) onto the solid-phase extraction column in portions, and extract it by applying pressure with a syringe. Wash the solid-phase extraction column with 1 mL of methanol-water solution (5:95, v / v) and 1 mL of water to complete purification. Finally, elute the target analytes from the solid-phase extraction column with 200 μL of a methanol solution of formic acid (where the volume percentage of formic acid is 7.0%) to obtain the eluate.

[0060] (3)pulsed-dc-ESI-MS detection

[0061] Load the eluate into the capillary needle of pulsed-dc-ESI-MS (opening 5.0 μm) by self-suction, and ionize it by applying a voltage of 2.5 kV to the conductive metal wire inserted into the capillary needle to obtain the first-order mass spectrometry signal map of six catecholamines and their metabolites in artificial urine. The specific test results are as Figure 3 shown.

[0062] Figure 3 This is the mass spectrometry map of six catecholamines and their metabolites in the artificial urine of Example 1 of the present invention.

[0063] As Figure 3 shown, six catecholamines and their metabolites can be observed, preliminarily indicating that the combination of this targeted solid-phase extraction protocol and pulsed-dc-ESI-MS can simultaneously detect six catecholamines and their metabolites in artificial urine.

[0064] Example 2

[0065] Compare the phenomena after applying voltage to the conductive metal wire inserted into the capillary needle of the urine sample loaded before and after solid-phase extraction, including the following specific steps:

[0066] (1)Urine sample treatment

[0067] Collect urine samples, immediately centrifuge them twice at 4000 rpm for 10 minutes at 4 °C, and then store them at -20 °C.

[0068] Protocol 1): Only one urine sample is subjected to acid hydrolysis treatment, that is: add 0.3 M / L hydrochloric acid to the urine sample at a volume ratio of 2:3, and then heat it at 100 °C for 20 minutes for acid hydrolysis. After cooling, adjust the pH to 9.0 with 28% ammonia water, and filter off the precipitate with a 0.22 μm aqueous needle filter to obtain the urine sample to be tested.

[0069] Scheme 2): Another urine sample is subjected to acid hydrolysis - solid phase extraction: 0.3M / L hydrochloric acid is added to the urine sample at a volume ratio of 2:3, and then acid hydrolysis is carried out by heating at 100°C for 20 minutes. After cooling, the pH is adjusted to 9.0 with 28% ammonia water, and the precipitate is filtered off with a 0.22μm aqueous needle filter.

[0070] Subsequently, solid phase extraction is carried out, that is: activate the HLB type solid phase extraction column (1mL - 30mg) with 1mL of methanol, and then balance it with 1mL of ammonia - ammonium chloride buffer solution with pH = 9.5. Load the 2mL pretreated urine sample in Scheme 2) onto the solid phase extraction column in portions and extract it by pressurizing with a syringe. Wash the solid phase extraction column with 1mL of methanol aqueous solution (5:95, v / v) and 1mL of water to complete purification. Finally, elute the target analyte from the solid phase extraction column with 200μL of methanol solution of formic acid (where the volume percentage of formic acid includes 7.0%) to obtain the eluate.

[0071] (2) Characterization

[0072] Inject the urine sample to be tested in Scheme 1) and the eluate in Scheme 2) into two capillary spray needles with the same opening (opening 5.0μm) by self - suction method respectively. One needle injects the real urine sample that has only undergone acid hydrolysis in Scheme 1), and the other needle injects the eluate after the urine in Scheme 2) is treated by solid phase extraction. Insert a conductive metal wire and apply a voltage of 2.5kv for 40s, and observe the phenomena of the two capillary spray needles through a microscope. The specific observation results are as Figures 4A - 4B shown.

[0073] Figure 4A This is the microscope image of the real urine detected by pulsed - dc - ESI - MS / MS without solid phase extraction pretreatment in Example 2 of the present invention; Figure 4B This is the microscope image of the eluate detected by pulsed - dc - ESI - MS / MS after solid phase extraction pretreatment in Example 2 of the invention.

[0074] As Figure 4A shown, the tip of the capillary spray needle is blocked without solid phase extraction pretreatment; while it is normal after solid phase extraction treatment, as Figure 4B shown. This shows that the pretreatment before solid phase extraction is essential in the detection method of the present invention.

[0075] Example 3

[0076] Evaluate the loading, washing and elution processes in the solid phase extraction process, including the following specific steps:

[0077] (1) Pretreatment of artificial urine sample before spiking

[0078] Standard substances NMN (2 μM / L), MN (2 μM / L), 3-MT (2 μM / L), DA (10 μM / L), E (10 μM / L), NE (10 μM / L), DA-d4 (10 μM / L), and MN-d3 (2 μM / L) were added to artificial urine. Hydrochloric acid at 0.3 M / L was added to the spiked artificial urine at a volume ratio of 2:3, and then acid hydrolysis was carried out by heating at 100 °C for 20 minutes. After cooling, the pH was adjusted to 9.0 with 28% ammonia water, and the precipitate was filtered off with a 0.22 μm aqueous needle filter to obtain a spiked artificial urine sample.

[0079] (2) Solid-phase extraction process

[0080] An HLB type solid-phase extraction column (1 mL - 30 mg) was activated with 1 mL of methanol, and then balanced with 1 mL of ammonia-ammonium chloride buffer solution with pH = 9.5. 2 mL of the spiked artificial urine sample was loaded onto the solid-phase extraction column in portions and extracted by pressurization with a syringe. The solid-phase extraction column was washed with 1 mL of methanol-water solution (5:95, v / v) and 1 mL of water to complete purification. Finally, the target analytes were eluted from the solid-phase extraction column with 200 μL of a methanol solution of formic acid (where the volume percentage of formic acid was 7.0%) to obtain an eluate.

[0081] (3) Evaluate the signal intensity of the analyte in the solutions obtained in each step of loading, washing, and eluting in the solid-phase extraction protocol (taking NMN as an example).

[0082] The solutions obtained in each step of the solid-phase extraction were collected, diluted with methanol at a dilution ratio of 1:100, and the diluted solutions were detected by pulsed-dc-ESI-MS / MS. The mass spectrometry conditions were as follows: the capillary temperature was 450 °C, the ion lens voltage was 60%, the ion injection time was set to 100 ms, and the signal acquisition was the average of 2 microscans. The specific test results are as Figure 5 shown.

[0083] Figure 5 This is the tandem mass spectrometry signal intensity diagram of the solutions obtained in each step of the solid-phase extraction process taking NMN as an example in Example 3 of the present invention.

[0084] As Figure 5 shown, the signal intensity of the eluate obtained in the final elution step was significantly higher than that of the loading and two purification washing steps, indicating that most of the metabolites in the urine to be tested were retained on the solid-phase extraction column during the loading process and were completely eluted by the methanol solution of formic acid in the final elution step and remained in the eluate.

[0085] (4) Evaluate the solid-phase extraction recovery rate

[0086] The pulsed-dc-ESI-MS / MS was used to detect the solid-phase extraction eluate after spiking artificial urine, and the tandem mass spectrometry signal intensities of six catecholamines and their metabolites were obtained. Then, the pulsed-dc-ESI-MS / MS was used to directly detect the blank eluate (methanol solution containing formic acid, where the volume percentage of formic acid was 7.0%) spiked with NMN (2 μM / L), MN (2 μM / L), 3-MT (2 μM / L), DA (10 μM / L), E (10 μM / L), NE (10 μM / L), DA-d4 (10 μM / L), and MN-d3 (2 μM / L) to obtain tandem mass spectrometry signals. The calculation method for the recovery rate was: the ratio of the signal intensities of the six catecholamines and their metabolites eluted by solid-phase extraction in artificial urine to the signal intensities of the standard compounds (i.e., the six catecholamines and their metabolites) directly detected in the blank eluate at the same concentration.

[0087] Specifically, the tandem mass spectrometry parameters of the six catecholamines and their metabolites, as well as the corresponding internal standard substances, are shown in Table 1.

[0088] Table 1

[0089]

[0090] Figure 6 This is the recovery rate graph of the solid-phase extraction of the six catecholamines and their metabolites in Example 3 of the present invention.

[0091] As Figure 6 shown, the recovery rates of the solid-phase extraction of the six catecholamines and their metabolites all exceeded 74%, indicating that the extraction efficiency of the solid-phase extraction was relatively high.

[0092] Example 4 (taking NMN as an example)

[0093] For the optimization during the solid-phase extraction process, it includes the following specific steps:

[0094] (1) Pretreatment of artificial urine before spiking

[0095] Artificial urine was spiked with NMN (1 μM / L), and the subsequent treatment process of the spiked artificial urine referred to the artificial urine pretreatment method in Example 1.

[0096] (2) Influence of the pH value of the ammonia-ammonium chloride buffer solution

[0097] The elution efficiency of catecholamines and their metabolites is related to the pH value of the NH 3 -NH 4 Cl buffer solution. Therefore, in order to maximize the retention rate of the six catecholamines and their metabolites (i.e., the target analytes) on the solid-phase extraction column, it is necessary to introduce a solution simulating the pH value of the urine sample during the equilibration process of the solid-phase extraction column.

[0098] Referring to the solid-phase extraction processes in Example 1 and Example 3, the pH of the ammonia-ammonium chloride solution in the equilibration step during the solid-phase extraction process was adjusted to 8.0, 8.5, 9.0, 9.5, and 10.0 in sequence, and referring to the mass spectrometry detection process in Example 3, the tandem mass spectrometry signal intensity of NMN after solid-phase extraction with ammonia-ammonium chloride buffers at different pH values was obtained. The specific test results are as Figure 7A shown, where Figure 7A the ordinate represents the tandem mass spectrometry signal intensity of NMN after solid-phase extraction with equilibration solutions at different pH values.

[0099] Figure 7A This is the tandem mass spectrometry signal intensity diagram of the ammonia-ammonium chloride buffer of the equilibration solution at different pH values during the solid-phase extraction process represented by NMN in Example 4 of the present invention.

[0100] As Figure 7A shown, the NMN signal intensity is relatively high at pH = 9 - 10. Therefore, in subsequent experiments, an equilibration solution with pH = 9.5 was selected to obtain the best elution ability of the solid-phase extraction column.

[0101] (3) Influence of the pH of the urine sample to be tested

[0102] Controlling the pH value of the urine sample to be tested is very important for the solid-phase extraction process of ionizable compounds because the ionization state of the compound will significantly change its retention and elution characteristics on the adsorbent of the solid-phase extraction column. Adjusting the sample pH to about 2 pH units above or below the pKa of the compound can significantly contribute to retaining the compound, and the pKa of catecholamines and their metabolites is in the range of 9 - 10.

[0103] Specifically, the pH of the pretreated urine sample to be tested was adjusted to 1.5, 3.0, 5.0, 7.0, 8.0, 8.5, 9.0, 9.5, and 10.0 in sequence. Referring to the solid-phase extraction process and mass spectrometry detection process in Example 1 and Example 3, the specific test results are as Figure 7B shown, where Figure 7B the ordinate represents the tandem mass spectrometry signal intensity of NMN after solid-phase extraction with samples at different pH values.

[0104] Figure 7B This is the tandem mass spectrometry signal intensity diagram of the urine sample to be tested pretreated at different pH values represented by NMN after solid-phase extraction in Example 4 of the present invention.

[0105] As Figure 7B shown, the NMN signal intensity is the highest at pH = 8.5. Therefore, in subsequent experiments, the pH value of the urine sample to be tested after acid hydrolysis was adjusted to 8.5.

[0106] (4) Influence of the formic acid content in the eluent

[0107] The final elution step uses an organic solvent or a non-polar solvent mixture to disrupt the hydrophobic interaction between the analyte and the functional groups of the adsorbent. Meanwhile, adjusting the pH value during elution can usually improve the recovery rate of ionizable compounds. Considering the need to take into account the compatibility of the eluent with direct detection by pulsed-dc-ESI-MS / MS, a methanol solution of formic acid was selected as the elution solution.

[0108] Referring to the solid-phase extraction processes in Example 1 and Example 3, only the percentage of formic acid in the elution solution (methanol solution of formic acid) during the solid-phase extraction process was adjusted to 0%, 2%, 5%, 7%, and 10% in sequence, and referring to the mass spectrometry detection process in Example 3, the specific test results are as Figure 7C shown, where Figure 7C the ordinate is the tandem mass spectrometry signal intensity of NMN after solid-phase extraction with an elution solution (methanol solution of formic acid) containing different percentages of formic acid.

[0109] Figure 7C This is the tandem mass spectrometry signal intensity diagram of different formic acid contents in the elution solution during the solid-phase extraction process represented by NMN in Example 4 of the present invention.

[0110] As Figure 7C shown, the NMN signal intensity is the highest when the formic acid concentration is 7%, so 7% was subsequently selected as the formic acid content in the eluent.

[0111] Example 5

[0112] Six catecholamines and their metabolites were spiked into artificial urine to prepare a standard curve for method validation, including the following specific steps:

[0113] (1) Pretreatment of artificial urine before spiking

[0114] Referring to the treatment method in Example 1, stock solutions to be measured containing different concentrations of six catecholamines and their metabolites were prepared, and MN-d3 at 800 nM / L and DA-d4 at 8000 nM / L were added as internal standards to the stock solution samples to be measured. Specifically, the concentration gradients of NMN were 20, 100, 600, 1500, and 1800 nM / L; the concentration gradients of MN were 20, 250, 1000, 1500, and 1800 nM / L; the concentration gradients of 3-MT were 20, 100, 600, 1200, and 1800 nM / L; the concentration gradients of DA were 200, 1000, 6000, 12000, and 18000 nM / L; the concentration gradients of NE were 200, 1000, 6000, 12000, and 18000 nM / L; the concentration gradients of E were 200, 1000, 6000, 12000, and 18000 nM / L.

[0115] (2) Solid-phase extraction process and pulsed-dc-ESI-MS / MS detection

[0116] Perform solid-phase extraction treatment according to the preferred determination method of Reference Example 4. Then, perform pulsed-dc-ESI-MS / MS detection on the eluate. Under optimized conditions, each experiment at each concentration was repeated at least 3 times. The specific mass spectrometry detection process and mass spectrometry conditions are the same as those in Example 3. Qualitative and quantitative detection of six catecholamines and their metabolites (i.e., analytes to be measured) in the urine to be measured can be achieved based on the obtained mass spectrometry information.

[0117] (3) Establishment of standard curve

[0118] Taking NMN and MN as examples, the average ratio of the signal intensities of the analytes to be measured and the internal standard in urine was obtained through multiple parallel experiments (MN-d3 is the internal standard for NMN, MN, and 3-MT; DA-d4 is the internal standard for DA, E, and NE). The relationship between this ratio and the concentration of the corresponding analyte was plotted to obtain the standard curve, as specifically shown in Figure 8A 、 Figure 8B Shown. The LOD (limit of detection) and LOQ (limit of quantification) were defined as the analyte concentrations corresponding to signal-to-noise ratios (S / N) of 3 and 10, respectively. The results obtained for the standard curves of the six catecholamines and their metabolites are shown in Table 2.

[0119] Table 2

[0120]

[0121] As shown in Table 2, NMN and MN in the present invention both have strong linear correlations, and the range of the correlation coefficient (R²) is from 0.9951 to 0.9990. Among them, the LOD and LOQ of MNs (NMN, MN) and 3-MT are respectively in the ranges of 0.0746 - 1.704 and 0.2488 - 1.299 nM / L; the LOD and LOQ of DA, NE, and E are respectively in the ranges of 36.24 - 136.7 and 120.8 - 455.8 nM / L, all far lower than the upper limit of the relevant reference values of normal people (NMN 3.0 - 3.8 μmol / L; MN 1.2 - 1.9 μmol / L) (Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma (2020 Edition), Chinese Society of Endocrinology, Chinese Journal of Endocrinology and Metabolism, 2020, 36(09): 737 - 750). At the same time, the detection limits of DA, NE, and E can also effectively distinguish the content of catecholamine substances from the standard regulations (studies have shown that the content of catecholamine substances in the urine of normal people ≤ 0.5 μg / L) (Analysis of epinephrine, norepinephrine, and dopamine in urine samples of hospital patients by micellar liquid chromatography. Anal. Bioanal. Chem., 407 (2015), 9009 - 9018).

[0122] (4)Method reproducibility

[0123] The experimental processes of solid-phase extraction and mass spectrometry detection and the internal standard content all refer to Example 5 of the present invention.

[0124] To evaluate the reproducibility of the solid-phase extraction combined with pulsed-dc-ESI-MS / MS method, the reproducibility of the solid-phase extraction combined with pulsed-dc-ESI-MS / MS method was evaluated by continuously extracting NMN, MN, and 3-MT at a spiked level of 800 nM / L and DA, NE, and E at a spiked level of 8000 nM / L in artificial urine 5 times. The specific test results are shown in Table 2 above.

[0125] As shown in Table 2, the relative standard deviations (RSDs) obtained for NMN, MN, 3-MT, DA, NE, and E range from 5.9% to 9.4%, indicating that this method has high precision in the rapid quantification of catecholamines and their metabolites.

[0126] Example 6

[0127] Recover six catecholamines and their metabolites from spiked real urine, including the following specific steps:

[0128] (1) Pretreatment of Real Urine Spiked Samples

[0129] Dilute real urine 10 times with water, and then refer to the urine pretreatment method in Example 1 to prepare undiluted samples to be measured and samples to be measured spiked with six catecholamines and their metabolites at different concentrations, including one sample to be measured spiked with 250 nM / L of NMN, MN, 3-MT and 2500 nM / L of DA, E, NE, and one sample to be measured spiked with 500 nM / L of NMN, MN, 3-MT and 5000 nM / L of DA, E, NE, for a total of three groups of samples to be measured. Add 600 nM / L of MN-d3 and 6000 nM / L of DA-d4 as internal standards to all three samples to be measured.

[0130] (2) Solid Phase Extraction Process and Pulsed-dc-ESI-MS / MS Detection

[0131] Perform solid phase extraction treatment with reference to the preferred determination method in Example 4, and then conduct pulsed-dc-ESI-MS / MS detection on the eluate. Under optimized conditions, each concentration experiment is repeated at least 5 times. The specific mass spectrometry detection process and mass spectrometry conditions are the same as those in Example 3. Qualitative and quantitative detection of the analytes to be measured in the urine samples to be measured can be achieved based on the obtained mass spectrometry information.

[0132] (3) Calculate Relative Recovery Rate

[0133] Substitute the mass spectrometry signal data of the target analytes (six catecholamines and their metabolites) detected into the corresponding standard curve in Example 5 to calculate the concentration of the target substances in the corresponding samples to be measured.

[0134] Evaluate the relative recovery rate by comparing the amount of catecholamines and their metabolites detected in real urine with their corresponding spiked concentrations. Specifically, first calculate the concentration of the target substances (catecholamines and their metabolites) in the unspiked real urine, and then calculate the spiked concentration by subtracting the concentration of the unspiked real urine from the concentration calculated based on the signal intensity after spiking. The ratio of this spiked concentration to the actual spiked concentration is the relative recovery rate. The specific test results are shown in Table 3.

[0135] Table 3

[0136]

[0137] As shown in Table 3, the detected concentrations of six catecholamines and their metabolites found in real urine (diluted with water at a dilution ratio of 1:10) were 66.5 nM / L (MN), 5539.3 nM / L (NMN), 125.9 nM / L (3-MT), 0.625 nM / L (DA), 3.125 nM / L (E), and 2.714 nM / L (NE), and the RSDs were in the range of 4.59 - 7.55%. Given that the samples were collected from the early morning urine of apparently healthy volunteers, the NMN levels obtained by the method of the present invention may show an increase due to unclear factors, and the other five metabolites were consistent with the relevant reference standards. In addition, the relative recoveries were in the range of 95 - 110.78% (RSDs less than 10.1%), demonstrating the low matrix effect and reliable analytical performance of the method.

[0138] In summary, the present invention enriches six catecholamines and their metabolites in urine through a solid-phase extraction column; combines pulsed-dc-ESI-MS / MS to directly detect the eluate obtained by solid-phase extraction, and obtains the contents of six catecholamines and their metabolites in the urine to be tested. The present invention evaluates and improves each step in the detection method, and finally obtains a detection method with fast detection speed and high stability.

[0139] The specific embodiments described above further elaborate on the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only for the specific embodiments of the present invention and is not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting catecholamines and their metabolites in urine, comprising: Provide pre-treated urine for testing; The urine to be tested is loaded into an activated and balanced solid phase extraction column, and after pressure extraction, it is washed with methanol aqueous solution and water in sequence, and eluted with formic acid methanol solution to obtain an eluate; Directly detecting the eluate by pulsed direct current electrospray tandem mass spectrometry to obtain mass spectrometry signals of six catecholamines and their metabolites in the urine to be tested; The mass spectrometry signal is substituted into the standard curve of catecholamines and their metabolites to obtain the concentrations of the six catecholamines and their metabolites.

2. The method according to claim 1, wherein: The pre-processing comprises: Acid hydrolysis of the urine to be tested; and Adjust the pH of the urine to be tested after acid hydrolysis to alkaline; Wherein, internal standards corresponding to the six catecholamines and their metabolites are added to the urine to be tested.

3. The method according to claim 2, wherein: The acid hydrolysis comprises: adding an acidic reagent into the urine to be tested and heating the urine to obtain the urine to be tested after acid hydrolysis; The pH of the urine to be tested after acid hydrolysis is adjusted to 7-9.

5.

4. The method according to claim 1, wherein: Obtaining the activated and balanced solid phase extraction column comprises: Activating the solid phase extraction column using pure methanol; The activated solid phase extraction column is balanced with an ammonia-ammonium chloride buffer solution; Wherein, the pH value of the ammonia-ammonium chloride buffer solution is 8-10, and the solid phase extraction column is a hydrophilic-lipophilic balanced solid phase extraction column.

5. The method according to claim 1, wherein: In the methanol-water solution, the volume ratio of methanol to water is 5:95-10:90; In the methanol solution of formic acid, the volume percentage of formic acid is 6%-8.0%.

6. The method according to claim 1, wherein: Direct detection of the eluent by pulsed direct current electrospray tandem mass spectrometry includes: The eluent is automatically drawn by the capillary needle of the pulsed direct current electrospray tandem mass spectrometry to complete the sample loading; Applying a DC voltage to the capillary spray needle to generate an ionized spray; The ionized spray is detected by the pulsed direct current electrospray tandem mass spectrometry to obtain mass spectrometry signals of the six catecholamines and their metabolites in the urine to be tested.

7. The method according to claim 6, wherein: The inner diameter of the capillary spray needle is 5-6 μm, and the DC voltage is 2.5-3.0 kV.

8. The method according to claim 1 or 2, wherein: The six catecholamines and their metabolites include: dopamine, epinephrine, norepinephrine, 3-methoxytyramine, normetanephrine, and metanephrine.

9. The method according to claim 8, wherein: The internal standard substance corresponding to dopamine, epinephrine and norepinephrine is dopamine-d4; The internal standard substance corresponding to the 3-methoxytyramine, normetanephrine and methylephrine is metanephrine-d3.

10. The method according to claim 1, wherein: The standard curve of catecholamines and their metabolites is obtained by the following steps: Adding internal standards to the original solutions of catecholamines and their metabolites with different concentrations to obtain standard solutions of catecholamines and their metabolites with different concentrations; The standard solutions of different concentrations are loaded into a solid phase extraction column for solid phase extraction to obtain standard eluents of different concentrations; Directly detecting the standard eluents of different concentrations by using the pulsed direct current electrospray tandem mass spectrometry to obtain mass spectrometry signals of the catecholamines and their metabolites at different concentrations; A standard curve of catecholamines and their metabolites is established according to the mass spectrometry signals of the catecholamines and their metabolites at different concentrations and the concentration of the standard solution.

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