A magnetic molecular imprinted polymer for pheochromocytoma diagnosis and its detection method

Abstract

The present invention belongs to the field of bioanalysis detection technology, and specifically relates to a magnetic molecular imprinted polymer for diagnosing pheochromocytoma and a detection method thereof. The method of the present invention comprises (1) preparing a magnetic molecular imprinted polymer for urine sample pretreatment; (2) using the magnetic molecular imprinted polymer to pretreat a random urine sample to be tested; (3) injecting the pretreated supernatant into a high performance liquid chromatograph-fluorescence detector / diode array detector to detect 6 catecholamines and their metabolites and creatinine; (4) performing quantitative analysis using an internal standard curve method to calculate the ratio of catecholamine metabolites to creatinine.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis and detection, and particularly relates to a magnetic molecular imprinting polymer for diagnosing pheochromocytoma and a detection method thereof. Background Art

[0002] Pheochromocytomas originate from the adrenal medulla; paragangliomas originate from outside the adrenal glands and are located in the abdomen (most common), chest, pelvis, and neck. According to the guidelines of the Endocrine Society, pheochromocytomas and paragangliomas (PPGLs) are usually classified as PPGLs. At present, the biomarker detection for the diagnosis of PPGLs mainly detects the levels of catecholamines and their metabolites. Normetanephrine (NMN) and metanephrine (MN) in plasma and 24-hour urine are preferred. It is recommended to detect dopamine (DA), epinephrine (Epinephrine), norepinephrine (NE) or 3-methoxytyramine (3-MT) in blood or urine samples. The recommended detection method usually uses solid phase extraction (SPE)-high performance liquid chromatography (HPLC)-electrochemical or mass spectrometry detection.

[0003] However, catecholamines have a short half-life in the blood and are rapidly metabolized. At the same time, plasma collection requires patients to lie still for 30 minutes, which is not conducive to outpatient clinics, especially for children whose compliance is poor. In addition, 24-hour urine collection is also extremely inconvenient and patient compliance is poor. In addition, the SPE operation steps are cumbersome and the detection process usually takes about 3-4 hours. Based on the existing technology, existing literature has proposed that random urine collection is simple and non-invasive, and patient compliance is good, but random urine is easily affected by the degree of urine concentration and affects the accuracy of the test results.

[0004] Mass spectrometry has the characteristics of high sensitivity, specificity and accuracy, but the equipment is expensive and has high maintenance costs. Compared with mass spectrometers, electrochemical detectors are cheaper and simpler to operate. Some studies have also recommended liquid chromatography electrochemical detection of catecholamine metabolites (CAs). However, electrochemical detection requires the addition of ion pair reagents to the mobile phase to enhance the retention of the analyte on the chromatographic column, but the added ion pair reagents will be deposited on the chromatographic column and affect the service life. Therefore, it is necessary to further improve or innovate the detection method. At present, there are no reports on the use of high performance liquid chromatography-fluorescence method / diode array detector method to detect biochemical indicators including epinephrine (E), norepinephrine (NE) and dopamine (DA).

[0005] Magnetic molecularly imprinted polymer (MMIP) is a combination of molecularly imprinted polymers (MIPs) and magnetic nanoparticles (MNPs). It not only has specific selectivity for template molecules, but also has excellent magnetic properties. MMIP can specifically identify and adsorb target molecules, and can be quickly separated under the action of a magnetic field. In addition, due to its small particle size and large specific surface area, MMIP is currently used as an ideal adsorbent for dispersed solid phase extraction method (DSPE) for pretreatment of biological samples.

[0006] The patent with publication number CN111825814A (20201027) and invention name "A magnetic molecular imprinting polymer of catecholamine substances and its preparation method and application" discloses a magnetic molecular imprinting polymer synthesized by thermal free radical polymerization reaction with Fe3O4 as a carrier and a dual template molecule. The magnetic molecular imprinting polymer can adsorb three markers in urine, thereby realizing the diagnosis of pheochromocytoma. However, the patented technology uses dual template synthesis. Since the dual template synthesis method often needs to be carried out under anaerobic conditions, the operation convenience is not high. In addition, the dual templates used in the patented technology are DA and MN, and the price of MN is significantly higher than that of DA, so the overall preparation cost of the patented method is relatively high.

[0007] The document "Preparation of a Magnetic Molecular Imprinting Polymer and Its Application in the Diagnosis of Pheochromocytoma" (Chongqing Medical University, 20220501) discloses a magnetic molecular imprinting polymer synthesized using DA as a single template and a preparation method. The magnetic molecular imprinting polymer prepared in this document can only detect NMN and MN in random urine from patients, which cannot meet the growing clinical needs.

[0008] In summary, it is necessary to propose new methods or strategies to supplement the shortcomings of existing technologies. Summary of the invention

[0009] In view of this, the purpose of the present invention is to provide a new magnetic molecular imprinted polymer for the diagnosis of pheochromocytoma and a method for detecting random urine samples using the new magnetic molecular imprinted polymer in combination with liquid chromatography, so as to make up for or partially make up for the deficiencies in the prior art. The specific technical scheme is as follows.

[0010] A method for detecting pheochromocytoma biochemical markers and creatinine in a random urine sample, wherein the pheochromocytoma biochemical markers include catecholamine metabolites, and the catecholamine metabolites include norepinephrine, epinephrine, methylnorepinephrine, dopamine, methylepinephrine and / or 3-methoxytyramine, and the detection steps are as follows:

[0011] S01: preparing a magnetic molecular imprinted polymer for urine sample pretreatment, wherein the preparation method of the magnetic molecular imprinted polymer comprises modifying and preparing large-sized Fe3O4 magnetic nanoparticles using dopamine as a single template under aerobic conditions, wherein the particle size of the large-sized Fe3O4 magnetic nanoparticles ranges from 100 to 200 nm;

[0012] S02: uniformly mixing the magnetic molecular imprinting polymer with the random urine sample to be tested and an internal standard solution, wherein the internal standard solution is 3,4-dihydroxybenzylamine hydrobromide, further adding a phosphate solution to obtain a mixed magnetic suspension, pre-treating the random urine sample to be tested in the mixed magnetic suspension, and then obtaining a supernatant by magnetic separation;

[0013] S03: The supernatant in S02 was detected by high performance liquid chromatography-fluorescence method / diode array detector method, and methanol was used as mobile phase A and 70mmol / L sodium dihydrogen phosphate solution was used as mobile phase B for gradient elution; the gradient elution conditions were:

[0014] 0.0min: A%-B%: 3.0%-97.0%;

[0015] 8.0min: A%-B%: 10.0%-90.0%;

[0016] 10.5min: A%-B%: 10.0%-90.0%;

[0017] 10.6min: A%-B%: 3.0%-97.0%;

[0018] 12.0min: A%-B%: 3.0%-97.0%.

[0019] Furthermore, the pretreatment operation in S02 includes allowing the mixed magnetic suspension to stand at room temperature for 4-8 minutes, then performing magnetic separation, discarding the supernatant, and obtaining a first pretreatment product.

[0020] Furthermore, the pretreatment operation in S02 includes adding 2% acetic acid solution to the first pretreatment product, placing it in a constant temperature water bath at 30-35°C for 8.0-12 minutes for elution, and then performing magnetic separation to obtain a supernatant for detection.

[0021] Furthermore, the pH value of the phosphate solution added to the S02 is 9.0.

[0022] Furthermore, 0.04 mg of the magnetic molecular imprinting polymer is added to every 1 μL of the random urine sample to be tested and the internal standard solution in S02; the volume ratio of the random urine sample to be tested and the internal standard solution is 9:1.

[0023] Furthermore, in S03, the high performance liquid chromatograph is equipped with a fluorescence detector and a diode array detector, the chromatographic column used is a Shim-Pack VP-ODS chromatographic column, the injection volume is 15-20 μL, and the mobile phase flow rate is 0.4-0.8 mL / min.

[0024] Preferably, the injection volume is 20 μL, and the mobile phase flow rate is 0.8 mL / min.

[0025] Furthermore, the fluorescence detector (used to detect norepinephrine, epinephrine, methylnorepinephrine, dopamine, methylepinephrine or 3-methoxytyramine and internal standard) has an excitation wavelength of 278 nm and an emission wavelength of 320 nm; the diode array detector (used to detect creatinine) has a detection wavelength of 235 nm.

[0026] Furthermore, the internal standard curve method is used for quantification to calculate the ratio of the catecholamine metabolite to the creatinine.

[0027] Furthermore, the preparation method of the magnetic molecular imprinting polymer in S01 also includes reacting large-sized Fe3O4 magnetic nanoparticles with isobutylene glycol trimethoxysilane to obtain large-sized magnetic nanoparticles with a coating structure, and 10-15 mg of the Fe3O4 magnetic nanoparticles are added to every 1 mL of isobutylene glycol trimethoxysilane; or, the preparation method also includes using dopamine as a single template and methacrylic acid as a functional monomer to react in ultrapure water and acetonitrile, and the molar ratio of dopamine to methacrylic acid is 1:5 to obtain a prepolymer reaction solution.

[0028] Furthermore, the large-sized magnetic nanoparticles with a coating structure react with the prepolymer reaction solution at a ratio of 2:5 to 4:5; then a mixed solution of ethylene glycol dimethacrylate and azobisisobutyronitrile is added to react, and finally the magnetic molecular imprinted polymer is prepared, and the magnetic molecular imprinted polymer is Fe3O4@MPS@MIP.

[0029] Furthermore, the method is not for diagnostic purposes. It is understandable that the detection method provided by the present invention is more for scientific research purposes rather than directly for the diagnosis of diseases.

[0030] Beneficial technical effects

[0031] (1) The present invention provides a method that uses high performance liquid chromatography-fluorescence method / diode array detector method to simultaneously meet the detection requirements of linearity, precision, and recovery rate of 6 catecholamine substances and their methods. Compared with other detection methods, the detection cost is greatly reduced, the operation is convenient and fast, and the sensitivity, specificity and accuracy of the detection method can be guaranteed at the same time, which is of great significance for the detection of catecholamines and their metabolites. Creatinine (Cr) is also added to the detection method provided by the present invention. Through creatinine correction, the influence of random urine volume is eliminated, and the accuracy of the method and the credibility of the detection results are improved.

[0032] (2) The detection method provided by the present invention adopts a newly synthesized large-scale magnetic molecular imprinting polymer, which can simultaneously extract and separate 6 catecholamine substances (NE, E, NMN, DA, MN, 3-MT) in human urine, and is easy to precipitate and more convenient to collect. Therefore, it not only overcomes the inconvenience of the existing detection method that requires the collection of plasma or 24-hour urine samples for detection, but is also more suitable for the detection of large quantities of samples. The large-scale magnetic molecular imprinting polymer provided by the present invention is a single template synthesis, and adopts the means of pre-polymerization with functional monomers. The synthesis process is carried out under aerobic conditions, so the synthesis conditions are convenient and the cost is lower.

[0033] (3) The high performance liquid chromatography-fluorescence method / diode array detector method (HPLC-FLD / DAD) used in the method of the present invention combines the high sensitivity of fluorescence detection with the qualitative ability of diode array detection. Compared with mass spectrometry and liquid chromatography electrochemical detection, it is simple to operate and convenient for clinical promotion. This method is used to jointly detect four indicators (NE, E, NMN, MN) in random urine. The sensitivity of the entire detection method is 90%, the specificity is 98%, and the AUC value is 97%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0035] Figure 1 This is an infrared spectrum of a magnetic molecularly imprinted polymer (Fe3O4@MPS@MIP) prepared in one of the embodiments of the present invention;

[0036] Figure 2 This is a scanning electron micrograph of a magnetic molecularly imprinted polymer (Fe3O4@MPS@MIP) prepared in one of the embodiments of the present invention (scale bar 5 μm);

[0037] Figure 3 A transmission electron microscopy image of Fe3O4 used in one of the embodiments of the present invention (scale bar 50 nm);

[0038] Figure 4 This is a transmission electron microscopy image of a magnetic molecular imprinted polymer (Fe3O4@MPS@MIP) prepared in one of the embodiments of the present invention (scale bar 50 nm);

[0039] Figure 5 Effect of Fe3O4@MPS@MIP with different particle sizes prepared in one of the embodiments of the present invention on the adsorption of MNs in random urine ( n=3, A is urine sample from healthy people, B is urine sample from PPGLs patients);

[0040] Figure 6 HPLC chromatograms of Examples 3.1 and 3.2 of the present invention;

[0041] Figure 7The chromatograms are optimized for screening isocratic and gradient elution conditions of CAs at different buffer concentrations in one embodiment of the present invention;

[0042] Figure 8 This is a chromatogram for optimizing the isocratic elution and gradient elution conditions of Cr at different buffer concentrations according to another embodiment of the present invention;

[0043] Fig. 9 Chromatograms of random urine extracted from a healthy person (a) and a pheochromocytoma patient (b) for Fe3O4@MPS@MIP (A is catecholamine metabolites detected by fluorescence detector, B is creatinine detected by diode array detector);

[0044] Fig.10 The ROC curve of random urine CAs for detecting PPGLs in one embodiment of the present invention (A is c CAs / c Cr Concentration ratio, B is A CAs / A Cr peak area ratio). DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0047] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0048] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0049] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0050] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0051] Example 1

[0052] This embodiment provides a specific example of a method for preparing a magnetic molecular imprinted polymer. It should be understood that the method shown in this embodiment is only an example and not a limitation.

[0053] S01: Preparation of large-sized magnetic nanoparticles with a coating structure: Fe3O4 magnetic nanoparticles and methacryloyloxypropyltrimethoxysilane are placed in ultrapure water and ethanol for reaction modification to obtain large-sized magnetic nanoparticles with a coating structure; the particle size range of the Fe3O4 magnetic nanoparticles is 100-200nm; 10-15mg of the Fe3O4 magnetic nanoparticles are added to every 1mL of methacryloyloxypropyltrimethoxysilane.

[0054] S02: preparing a prepolymer of a single template and a functional monomer: using dopamine as a single template and methacrylic acid as a functional monomer to react in ultrapure water and acetonitrile, wherein the molar ratio of the dopamine to the methacrylic acid is 1:5, to obtain a prepolymer reaction solution.

[0055] S03: reacting the large-sized magnetic nanoparticles with a coating structure obtained in S01 with the prepolymer reaction solution obtained in S02 under aerobic conditions to obtain an intermediate reaction solution, wherein the ratio of the large-sized magnetic nanoparticles with a coating structure to the prepolymer reaction solution is 2:5 to 4:5. Subsequently, a mixed solution of ethylene glycol dimethacrylate and azobisisobutyronitrile is further added to the intermediate reaction solution, wherein the ratio of the ethylene glycol dimethacrylate to the azobisisobutyronitrile is 50:3 to 50:9, to obtain a crude magnetic molecular imprinted polymer.

[0056] S04: placing the crude magnetic molecular imprinted polymer obtained in S03 in a water bath at 80-90°C and eluting with 10.0% acetic acid for at least 4 days. After elution, collecting and drying to obtain the magnetic molecular imprinted polymer, the magnetic molecular imprinted polymer is Fe3O4@MPS@MIP.

[0057] In the above preparation method, the Fe3O4 magnetic nanoparticles are commercially available magnetic beads, the coating material is methacryloyloxypropyltrimethoxysilane, the template molecule is dopamine, the functional monomer is methacrylic acid, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

[0058] As a specific optional operation method, the following steps are included:

[0059] S01: 100.0 mg Fe3O4 magnetic beads (particle size 100 nm) were added to 4.0 mL ultrapure water, 4.0 mL ethanol and 8.0 mL methacryloyloxypropyltrimethoxysilane (MPS) in sequence, vortexed for 2.0 min, and then placed on a shaker at 40 ° C and 250 rpm for 12 h to prepare large-sized magnetic nanoparticles with a coating structure. The surface of the magnetic nanoparticles has carbon-carbon double bonds, see Figure 1 , providing sites for subsequent reactions.

[0060] S02: Using dopamine as a single template, 0.2 mmol dopamine was added to 2.0 mL ultrapure water and vortexed, followed by the addition of 18.0 mL acetonitrile and 1.0 mmol methacrylic acid (MAA). After ultrasonication for 15 min, the mixture was placed at room temperature for 12 h to obtain a prepolymer reaction solution.

[0061] S03: After S01 reacted for 12 hours, the supernatant was discarded, leaving the large-sized magnetic nanoparticles with a coating structure. After rinsing the reaction container with 10.0 mL acetonitrile twice, the prepolymer reaction solution prepared by S02 was added to prepare an intermediate reaction solution, in which the ratio of large-sized magnetic nanoparticles with a coating structure to the prepolymer reaction solution was 4:5. Subsequently, 2.0 mmol of ethylene glycol dimethacrylate (EGDMA) and 20.0 mg of azobisisobutyronitrile (AIBN) were further added to the intermediate reaction solution in a ratio of 50:3. After vortexing for 2.0 minutes, it was placed on a 60°C shaker at 250 rpm for 24 hours to obtain the crude Fe3O4@MPS@MIP.

[0062] S04: After the reaction is completed, the supernatant is discarded, and the crude Fe3O4@MPS@MIP is placed in a 90°C water bath and eluted with 10.0% acetic acid solution for 4 days, with the solution changed every 12 hours.

[0063] S05: After the elution of Fe3O4@MPS@MIP is completed, the supernatant is discarded and placed in a 37°C incubator to dry for later use. After the elution is completed, a large number of holes with complementary shapes and sizes to DA appear inside the Fe3O4@MPS@MIP, which can specifically enrich substances with similar structures.

[0064] The Fe3O4@MPS@MIP prepared in this example and the magnetic beads used are shown in Figure 2-4 .

[0065] Example 2

[0066] Based on the method for synthesizing Fe3O4@MPS@MIP in Example 1, this example further optimizes and screens the particle size of Fe3O4 magnetic beads.

[0067] Fe3O4@MPS@MIP (MMIP for short) synthesized with Fe3O4 of 20nm, 100nm and 200nm particle sizes were used to adsorb urine samples from healthy people and PPGLs patients. The results are as follows: Figure 5 As shown, the abscissa represents the particle size (it should be noted that the attached Figure 4 The horizontal axis is not marked according to the standard numerical ratio), and the vertical axis represents the peak area of ​​MN and MN. As the particle size of MMIP increases, its specific surface area decreases, and the adsorption effect of MMIP on MN and NMN gradually decreases. Therefore, Fe3O4 with a particle size of 200nm is not applicable. However, MMIP synthesized by 20nm Fe3O4 is easy to demagnetize, and 100nm magnetic beads are easier to settle. Therefore, 100nm Fe3O4 was selected for subsequent experiments after comprehensive consideration.

[0068] Example 3

[0069] This embodiment provides an example of a detection method for a pheochromocytoma biochemical marker in a biological sample and condition optimization screening. The pheochromocytoma biochemical marker is a catecholamine metabolite.

[0070] 3.1 Detection of pheochromocytoma biochemical markers in biological samples by high performance liquid chromatography.

[0071] (1) The Fe3O4@MPS@MIP prepared in Example 1 was used to enrich and separate catecholamine metabolites in urine samples. The specific implementation steps are as follows:

[0072] Weigh 20.0 mg Fe3O4@MPS@MIP in a 10 mL centrifuge tube, add 450 μL of random urine sample, 50.0 μL, 500.0 μg / L internal standard solution to another clean 10 mL centrifuge tube, pre-treat with 2.5 mL 0.01 mol / L phosphate solution (pH = 9.0), add 20.0 mg Fe3O4@MPS@MIP, mix evenly, vortex for 30 seconds, stand at room temperature for 4.0 minutes, perform magnetic separation, and discard the supernatant. Then add 400 μL 2% acetic acid solution and stand in a 30°C constant temperature water bath for 8.0 minutes to elute the magnetic separation to obtain the supernatant.

[0073] (2) Take 20 μL of the supernatant and inject it into a high performance liquid chromatograph for HPLC analysis. The analytical instrument and experimental parameters are as follows:

[0074] (2-1) Instrument model: Thermo Fisher High Performance Liquid Chromatograph Ultimate 3000

[0075] (2-2) Chromatographic conditions: The chromatographic column was a Shim-Pack VP-ODS column, mobile phase A was methanol, mobile phase B was 70 mmol / L sodium dihydrogen phosphate solution, and the gradient elution program was as shown in Table 1.

[0076] Table 1 Gradient elution parameters

[0077]

[0078] (2-3) When gradient elution was performed using a Shim-Pack VP-ODS column, the flow rate was 0.8 mL / min; the column temperature was 30°C; the FLD excitation wavelength was 278 nm, the emission wavelength was 320 nm; the DAD detection wavelength was 235 nm. The injection volume was 20 μL. The detection time was 12 min.

[0079] (2-4) After the supernatant was detected by HPLC, the internal standard curve method and peak area ratio method were used to achieve quantitative and relative quantification of the analyte, respectively.

[0080] 3.2 Provide a screening comparison of gradient elution chromatography conditions.

[0081] The control gradient elution program is shown in Table 2.

[0082] Table 2 Comparative gradient elution parameters

[0083]

[0084] Results: The HPLC chromatograms of 3.1 and 3.2 are as follows: Figure 6As shown in (a) and (b), it can be seen that only NMN, MN and 3-MT can be detected under the chromatographic condition 3.2; while the chromatographic condition 3.1 optimized in this example can simultaneously detect NMN, MN, 3-MT, DA, NE and E, and the separation of NE, E, MN and 3-MT is greater than 1.5.

[0085] 3.3 Provide screening comparison of elution chromatography conditions.

[0086] The Fe3O4@MPS@MIP prepared in Example 1 was used to enrich and separate catecholamine metabolites in urine samples by isocratic elution. Different isocratic elution procedures are shown in the table below.

[0087] Table 3 Isocratic elution parameters 1

[0088]

[0089]

[0090] Table 4 Isocratic elution parameters 2

[0091]

[0092] Table 5 Isocratic elution parameters 3

[0093]

[0094] Table 6 Gradient elution parameters 4

[0095]

[0096] Table 7 Gradient elution parameters 5

[0097]

[0098] Note: 35mmol / L, 50mmol / L, 70mmol / L represent the buffer salt concentration of sodium dihydrogen phosphate solution used.

[0099] The chromatograms of the above five elution conditions are shown in Figure 7 and Figure 8 As can be seen in the figure, the NMN peak failed to achieve baseline separation and the peak shape was poor.

[0100] In summary, based on the screening results, the optimal chromatographic condition is the gradient elution of 70 mmol / L sodium dihydrogen phosphate buffered saline solution, which can significantly improve the separation effect of NMN.

[0101] Example 4

[0102] This example provides examples of optimization screening of other operating parameters or steps in Example 3.1.

[0103] The Fe3O4@MPS@MIP prepared in Example 1 was used to enrich and separate the catecholamine standard solution to evaluate the adsorption and elution efficiency of Fe3O4@MPS@MIP. The specific implementation steps are as follows:

[0104] 40.0 μL of 3.0 mg / L 3-MT, NE, NMN, DA, E and MN mixed standard solution was added to 360.0 μL 0.01 mol / L phosphate buffer saline (PBS, pH = 9.0), mixed well, added with 20.0 mg Fe3O4@MPS@MIP, vortexed for 30 seconds, allowed to stand at room temperature for 4.0 minutes, and then magnetically separated, and the supernatant was discarded. Then 400 μL of 2% acetic acid solution was added and allowed to stand in a 30°C constant temperature water bath for 8.0 minutes for elution. The supernatant was obtained by magnetic separation.

[0105] Table 8 Effect of buffer pH on MMIP adsorption efficiency ( n=3)

[0106]

[0107] In summary, overall, the overall adsorption efficiency is best when the pH value of the phosphate solution is 9.0.

[0108] Table 9 Effect of adsorption time on MMIP adsorption efficiency ( n=3)

[0109]

[0110]

[0111] In summary, the overall adsorption efficiency was the best after standing for 4 minutes to allow MMIP to adsorb the sample.

[0112] Table 10 Effect of elution time on MMIP elution efficiency ( n=3)

[0113]

[0114] In summary, the overall elution efficiency is highest when elution is allowed to stand in a constant temperature water bath for 8.0 minutes.

[0115] Table 11 Effect of elution temperature on MMIP elution efficiency ( n=3)

[0116]

[0117] In summary, elution in a 30°C constant temperature water bath has the highest overall elution efficiency.

[0118] Example 5

[0119] Methodological evaluation, established random urine dispersive solid phase extraction (DSPE)-HPLC-FLD / DAD method for determination of CAs and creatinine (Cr), using Cr to correct urine volume, using internal standard curve method for quantification and peak area ratio for semi-quantification. Specifically including the following contents.

[0120] Standard curve: In order to reduce the matrix effect and the influence of endogenous CAs in urine, Fe3O4@MPS@MIP prepared in Example 1 was used to adsorb CAs in urine as blank urine. 50 μL of a series of standard solutions containing 500 μg / L internal standard solution were added to 450 μL blank urine after adsorption, and the test results were corrected by subtraction method. The peak area ratio of the analyte to the internal standard was deducted from the peak area ratio of the analyte to the internal standard in blank random urine as Y, and the concentration of the added analyte X was used to draw the standard curve. The results are shown in Table 12: the linear ranges of NE, E and 3-MT were 4.88-1250μg / L, the linear ranges of NMN and MN were 9.77-1250μg / L, the linear range of DA was 78.13-1250μg / L, the linear range of Cr was 19.53-1500mg / L, and the correlation coefficient was >0.99. The minimum detection limits of NE, E, NMN, DA, MN, 3-MT and Cr in random urine were 0.39μg / L, 0.87μg / L, 2.61μg / L, 27.53μg / L, 14.11μg / L, 4.70μg / L and 2.8mg / L, respectively.

[0121] Table 12 Standard curves of CAs and Cr in random urine (n=3)

[0122]

[0123]

[0124] Preparation of standard stock solution: Accurately weigh 10.0 mg NMN, 10.0 mg MN, 10.0 mg 3-MT, 10.0 mg E, 10.0 mg NE and 10 mg DA and dissolve them in 1.00 mL ultrapure water to prepare a 10.0 g / L solution, and store it in a -20°C refrigerator.

[0125] Preparation of internal standard working solution: 5.0 mg of 3,4-dihydroxybenzylamine hydrobromide was dissolved in 1.00 mL of ultrapure water to prepare a 5.0 g / L solution, which was stored in a -20°C refrigerator.

[0126] Preparation of a series of concentration mixed standard solutions: Take 10 μL of each of the 6 catecholamine standard stock solutions, add 940 μL of ultrapure water, prepare a 100 mg / L standard mixed solution, and store in a -20°C refrigerator.

[0127] Precision: Random urine samples mixed from normal subjects, random urine samples mixed from normal subjects and patients, and random urine samples mixed from patients were prepared as low, medium, and high value samples, respectively. The intra-day and inter-day precision of the analytical method were evaluated for 5 consecutive days. As shown in Table 13, the intra-day and inter-day precision were both <10%. This result indicated that the analytical method used had high repeatability and stability.

[0128] Table 13 Precision of random urine CAs detection ( n=5)

[0129]

[0130]

[0131] Recovery rate: Low, medium and high concentrations of CAs and Cr mixed standards were added to blank urine, and the peak area of ​​the analyte was detected after sample pretreatment. Each concentration was measured 3 times. Relative recovery rate = (analyte concentration in urine after adding standard - analyte concentration in blank urine) / concentration of added standard × 100%. The results are shown in Table 14. The average recovery rates of CAs and Cr were between 90% and 118%, indicating that CAs and Cr achieved satisfactory recovery rates in urine specimens.

[0132] Table 14 CAs and Cr recovery ( n=5)

[0133]

[0134]

[0135] Interference test: Ascorbic acid and uric acid are common endogenous interferences in urine. The interference test was conducted according to the evaluation scheme developed by the National Committee for Clinical Laboratory Standards of the United States. The A CAs / A IS and A Cr / A IS (X C ) and A after adding CAs / A IS and A Cr / A IS (X T ), interference value (X T -X C) within 1.96S (95% confidence range) means no significant interference (indicated by N), and if the interference value exceeds 1.96S, there is significant interference (indicated by I). The results are shown in Table 15. Ascorbic acid within 600mg / L and uric acid within 400mg / L have no effect on the test results, indicating that the method established in this experiment has good anti-interference ability at physiological concentrations.

[0136] Table 15 Interfering substances to A CAs / A IS and A Cr / A IS Impact of test results

[0137]

[0138]

[0139] Example 6

[0140] This embodiment provides a clinical application of a method for extracting and detecting catecholamines.

[0141] A total of 215 random urine specimens were collected, including 30 patients with pheochromocytoma, 44 healthy subjects, 35 patients with essential hypertension, and 106 patients with adrenal masses. The established method was used to detect urine specimens from patients with adrenal masses, essential hypertension, healthy subjects, and pheochromocytoma. Fig. 9 The receiver operating characteristic curve (ROC) was used to evaluate the random urine CAs / c Cr and A CAs / A Cr The diagnostic efficacy of the two methods for PPGLs, such as Fig.10 Table 16. Random urine NE / c Cr 、c E / c Cr 、c NMN / c Cr and c MN / c Cr The detection efficiencies for PPGLs were 0.668, 0.747, 0.950 and 0.874 respectively; random urine A NE / A Cr , A E / A Cr , A NMN / A Cr and A MN / A CrThe detection efficiencies for PPGLs were 0.742, 0.755, 0.963, and 0.868, respectively. The results showed that the peak area ratio of CAs to Cr could achieve similar detection efficiency compared with the internal standard method and creatinine correction. The detection efficiency reached 0.970, the sensitivity was 0.900, and the specificity was 0.984 using the combined detection of NE, E, NMN, and MN.

[0142] Table 16 Method sensitivity and specificity (n=215)

[0143]

[0144] The above method uses the internal standard curve method for quantification and uses the ratio of catecholamine metabolites to creatinine to detect pheochromocytoma, providing assistance for clinical medical treatment.

[0145] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A method for detecting pheochromocytoma biochemical markers and creatinine in a random urine sample, characterized in that: The pheochromocytoma biochemical markers include catecholamine metabolites, and the catecholamine metabolites include norepinephrine, epinephrine, methylnorepinephrine, dopamine, methylepinephrine and / or 3-methoxytyramine. The detection steps are as follows: S01: Preparation of magnetic molecular imprinting polymer for urine sample pretreatment, the preparation method of the magnetic molecular imprinting polymer comprising: (1) adding ultrapure water, ethanol and methyl methacrylate to large-sized Fe3O4 magnetic nanoparticles, vortexing and shaking at 40°C to prepare large-sized magnetic nanoparticles with a coating structure, wherein 10-15 mg of the Fe3O4 magnetic nanoparticles are added to each 1 mL of methyl methacrylate, and the particle size of the large-sized Fe3O4 magnetic nanoparticles is 100 nm; (2) using dopamine as a single template and methacrylic acid as a functional monomer to react in ultrapure water and acetonitrile, and then sonicating. The prepolymer reaction liquid is obtained by placing the prepolymer reaction liquid at room temperature, wherein the molar ratio of dopamine to methacrylic acid is 1:5, and the prepolymer reaction liquid is obtained; (3) the large-sized magnetic nanoparticles with a coating structure are reacted with the prepolymer reaction liquid at a ratio of 2:5 to 4:5, and then a mixed solution of ethylene glycol dimethacrylate and azobisisobutyronitrile is added to react, and the mixture is vortexed and placed at 60° C. to obtain a crude magnetic molecular imprinted polymer; (4) the crude magnetic molecular imprinted polymer is placed in a water bath at 80-90° C. and eluted with 10.0% acetic acid for at least 4 days, collected and dried to obtain the magnetic molecular imprinted polymer, and the magnetic molecular imprinted polymer is Fe3O4@MPS@MIP; S02: uniformly mixing the magnetic molecular imprinting polymer with the random urine sample to be tested and an internal standard solution, wherein the internal standard solution is 3,4-dihydroxybenzylamine hydrobromide, further adding a phosphate solution to obtain a mixed magnetic suspension, and pre-treating the random urine sample to be tested in the mixed magnetic suspension; the pre-treatment operation comprises allowing the mixed magnetic suspension to stand at room temperature for 4-8 minutes, then performing magnetic separation, discarding the supernatant, and obtaining a first pre-treatment product; adding 2% acetic acid solution to the first pre-treatment product, allowing it to stand in a constant temperature water bath at 30-35°C for 8.0-12 minutes for elution, and then performing magnetic separation to obtain a supernatant; S03: The supernatant in S02 was detected by high performance liquid chromatography-fluorescence method / diode array detector method. The chromatographic column was a C18 column. The fluorescence detector was used to detect the catecholamine metabolites with an excitation wavelength of 278 nm and an emission wavelength of 320 nm. The diode array detector was used to detect the creatinine with a detection wavelength of 235 nm. Methanol was used as mobile phase A and 70 mmol / L sodium dihydrogen phosphate solution was used as mobile phase B for gradient elution. The gradient elution conditions were: 0.0min: A%-B%: 3.0%-97.0%; 8.0min: A%-B%: 10.0%-90.0%; 10.5min: A%-B%: 10.0%-90.0%; 10.6min: A%-B%: 3.0%-97.0%; 12.0min: A%-B%: 3.0%-97.0%.

2. The method according to claim 1, characterized in that The pH value of the phosphate solution added to the SO2 is 9.

0.

3. The method according to claim 1, characterized in that In the S02, 0.04 mg of the magnetic molecular imprinting polymer is added to every 1 µL of the random urine sample to be tested and the internal standard solution; the volume ratio of the random urine sample to be tested and the internal standard solution is 9:

1.

4. The method according to claim 1, wherein: In the S03, the high performance liquid chromatograph is equipped with a fluorescence detector and a diode array detector, the chromatographic column used is a Shim-Pack VP-ODS chromatographic column, the injection volume is 15-20 μL, and the mobile phase flow rate is 0.4-0.8 mL / min.

5. The method according to claim 1, characterized in that The internal standard curve method was used for quantification, and the ratio of the catecholamine metabolite to the creatinine was calculated.

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