A sample pretreatment method and kit for extracting 8 catecholamines and their metabolites from urine

Abstract

The present invention provides a sample pretreatment method and kit for extracting 8 catecholamines and their metabolites from urine. The sample pretreatment method adopts a magnetic bead extraction method in combination with a fully automatic extractor for automatic extraction. The magnetic bead working solution in the magnetic bead extraction method is a suspension of magnetic beads, water and isopropanol, and the magnetic beads are a mixture of carboxyl magnetic beads and amino magnetic beads. The sample pretreatment method comprises: S100, the fully automatic extractor sequentially performs sample loading, rinsing and elution steps to obtain a solution to be tested; S200, the solution to be tested is used for LC-MS / MS detection. The technical problem solved by the present invention is that in the prior art, when the magnetic bead extraction method uses a single magnetic bead extraction, the detection of 8 catecholamines and their metabolites requires two sample pretreatments and two tests to complete, resulting in low throughput, poor recovery rate, and cumbersome operation, making it difficult to meet the needs of clinical high throughput.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sample pretreatment method and a kit for extracting eight catecholamines and their metabolites from urine. Background Art

[0002] Detection of catecholamines (dopamine, epinephrine, norepinephrine) and their metabolites (metanephrine, normetanephrine, 3-methoxytyrosine, vanillylmandelic acid, and homovanillic acid) is crucial for the early screening and clinical diagnosis of diseases such as pheochromocytoma, paraganglioma, and neuroblastoma. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is crucial for the detection of catecholamines and their metabolites in urine, as their concentrations are low and they present numerous interferences. Therefore, effective sample preparation procedures, such as derivatization, protein precipitation, liquid-liquid extraction, solid-phase extraction, or magnetic bead extraction, are essential for LC-MS / MS analysis. In the existing technology, the sample pretreatment methods have the following defects: the derivatization method has the disadvantages of strict reaction conditions, long time consumption, and uneven derivatization biochemistry; the protein precipitation method has low sensitivity and serious matrix interference; the liquid-liquid extraction method has complicated steps, including organic reagent extraction, centrifugation, nitrogen blowing, and re-dissolution. Organic reagents are not friendly to humans and the environment, and the recovery rate of catecholamines and their metabolites is low; the solid-phase extraction method has complicated steps, requiring activation, equilibration, loading, washing, elution and other steps, which is time-consuming, about 30-60 minutes, and manual operation is prone to introduce errors and poor repeatability. Among them, it is greatly affected by the filler batch, flow rate control, and operator experience, and relies on equipment such as positive pressure or negative pressure equipment, centrifuges, vortex mixers, nitrogen blowing instruments, nitrogen bottles or nitrogen generators.

[0003] Compared with the above-mentioned sample pretreatment methods, the magnetic bead extraction method has the advantages of high degree of automation, high purity of the extracted target, simple steps, strong sample adaptability, greater environmental protection and safety. It has become a more popular sample pretreatment method in recent years.

[0004] The traditional magnetic bead extraction method uses a single magnetic bead extraction method. Among them, carboxyl magnetic beads can only adsorb neutral alkaline metabolites (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine), and cannot effectively capture strongly acidic metabolites (such as vanillylmandelic acid and homovanillic acid). The detection of 8 catecholamines and their metabolites requires two sample pretreatments and two tests to complete. It has low throughput, poor recovery rate, and cumbersome operation, which makes it difficult to meet the clinical high-throughput needs. Summary of the Invention

[0005] The technical problem solved by the present invention is that in the existing technology, when the magnetic bead extraction method uses a single magnetic bead extraction, the detection of 8 catecholamines and their metabolites requires two sample pretreatments and two tests to complete, with low throughput, poor recovery rate, and cumbersome operation, which is difficult to meet the clinical high-throughput requirements.

[0006] To solve the above problems, the present invention provides a sample pretreatment method for extracting eight catecholamines and their metabolites from urine. The sample pretreatment method adopts a magnetic bead extraction method in conjunction with a fully automatic extractor for automatic extraction. The magnetic bead working solution in the magnetic bead extraction method is a suspension of magnetic beads, water and isopropanol, and the magnetic beads are a mixture of carboxyl magnetic beads and amino magnetic beads. The sample pretreatment method comprises: S100, the fully automatic extractor sequentially performs sample loading, rinsing and elution steps to obtain a test solution; S200, using the test solution for LC-MS / MS detection.

[0007] Compared with the existing technology, the technical effects achieved by adopting this technical solution are: carboxyl magnetic beads specifically adsorb cationic substances (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine), and amino magnetic beads specifically adsorb anionic / hydrophobic metabolites (such as vanillylmandelic acid and homovanillic acid); by mixing carboxyl magnetic beads and amino magnetic beads, the recovery rate of vanillylmandelic acid and homovanillic acid is increased from 25%-40% of the traditional carboxyl magnetic bead extraction method to 90%-105%, and the extracted catecholamines and their metabolites are increased from 6 to 8. The sample pretreatment method of the present invention adopts a magnetic bead extraction method in conjunction with a fully automatic extractor. A single sample pretreatment can effectively extract eight catecholamines and their metabolites with large polarity differences. The extracted test solution is subjected to an LC-MS / MC detection method, thereby achieving the effect of simultaneously extracting and detecting eight catecholamines and their metabolites in a single sample pretreatment, achieving high throughput, high recovery rate, simple operation, and streamlined equipment to meet the needs of clinical high throughput.

[0008] In one example of the present invention, S100 includes: S110, loading the sample, mixing the sample to be tested, the internal standard working solution, the sample diluent and the magnetic bead working solution, and separating the magnetic beads and the solution; the mass volume content of the carboxyl magnetic beads in the magnetic bead working solution is 0.05 g / mL~0.2 g / mL, and the mass volume content of the amino magnetic beads in the magnetic bead working solution is 0.05 g / mL~0.2 g / mL.

[0009] Compared with existing technologies, this solution achieves the following technical benefits: By optimizing the ratio of carboxyl and amine magnetic beads, the adsorption capacity is balanced, ensuring efficient capture of all eight target compounds (catecholamines and their metabolites). This avoids competitive adsorption of non-target substances (such as lipids and proteins), reduces matrix interference, and prevents magnetic bead saturation and bead carryover, thereby improving recovery and sensitivity. The optimized method increases the recovery of vanillylmandelic acid and homovanillic acid from 25%-40% with conventional methods to 90%-105%, while the recovery of the remaining metabolites remains stable at 95%-110%.

[0010] In one embodiment of the present invention, the volume ratio of water to isopropanol in the magnetic bead working solution is 1:1.

[0011] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: by setting the volume ratio of water and isopropanol to 1:1, the magnetic beads can be evenly suspended in the solution and remain without sedimentation for a short time (30 minutes), making it convenient to accurately transfer the magnetic bead working solution.

[0012] In one example of the present invention, S100 includes: S120, eluting, adding an eluent to the loaded magnetic beads and mixing, separating to obtain the washed magnetic beads; the eluent is a mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, ethanol and water, the concentration of disodium hydrogen phosphate is 50 mmol / L~80 mmol / L, the concentration of sodium dihydrogen phosphate is 10 mmol / L~30 mmol / L, the concentration of sodium chloride is 0.05 mol / L~0.5 mol / L, and the ratio of ethanol to water is 1:1; and / or the pH of the eluent is 6.5~7.0.

[0013] Compared with the existing technology, the technical effects achieved by adopting this technical solution are: removing hydrophobic impurities such as lipids and some proteins through ethanol; preventing the target from being competitively eluted due to excessive salt concentration through sodium chloride; maintaining the charge state of carboxyl magnetic beads (weakly acidic) and amine magnetic beads (strongly alkaline) through disodium hydrogen phosphate and sodium dihydrogen phosphate, preventing the target from desorbing while maintaining ion exchange.

[0014] In one example of the present invention, S100 includes: S130, elution, adding eluent to the washed magnetic beads for step-by-step elution; the eluent includes eluent 1 and eluent 2; S130 includes: S131, adding eluent 1 to the washed magnetic beads and mixing, separating to obtain solution 1 to be tested and magnetic beads; S132, adding eluent 2 to the separated magnetic beads and mixing, separating to obtain solution 2 to be tested.

[0015] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: eluent 1 is used to elute the carboxyl magnetic bead adsorbed substance, and eluent 2 is used to elute the amino magnetic bead adsorbed substance. Through step-by-step elution, the carboxyl magnetic bead adsorbed substance is eluted first, and then the amino magnetic bead adsorbed substance is eluted, so as to avoid the mutual interference of the elution conditions of the two magnetic beads, ensure that each type of target is efficiently released at the optimal pH, and the overall recovery rate is increased to 95%-110%; among them, catecholamines (such as dopamine, epinephrine, and norepinephrine) are more stable under acidic conditions, and preferential elution can reduce oxidation losses. Acidic metabolites such as vanillylmandelic acid and homovanillic acid have high solubility in alkaline environments, and elution in the later stage can ensure their full release.

[0016] In one embodiment of the present invention, eluent 1 is an aqueous solution of formic acid, ascorbic acid, sodium metabisulfite and oxalic acid, and / or eluent 1 includes formic acid with a volume fraction of 1% to 2.5%, sodium metabisulfite with a mass concentration of 0.01% to 0.05%, and oxalic acid with a mass concentration of 0.05% to 0.5%.

[0017] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: the carboxyl magnetic bead adsorbent is eluted under acidic conditions (pH <3), and the ion exchange effect between the negative charge (-COOH) of the carboxyl magnetic beads and the cationic target (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyrosine) can be destroyed by adjusting the pH of the eluent.

[0018] In one embodiment of the present invention, the second eluent is a sodium hydroxide aqueous solution, and / or the second eluent includes sodium hydroxide with a mass concentration of 0.1% to 0.35%.

[0019] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: the amine-based magnetic bead adsorbent is eluted under alkaline conditions (pH>9), and the positive charge (-N + Ion exchange interaction between (CH3)3) and anionic metabolites (such as vanillylmandelic acid and homovanillic acid).

[0020] In one embodiment of the present invention, S130 includes: S133, combining the first test solution and the second test solution and mixing them evenly to obtain the test solution; the ratio of combining the first test solution and the second test solution is (1-3):1.

[0021] Compared with existing technologies, this technical solution achieves the following technical benefits: direct injection of the test solution avoids the loss of easily oxidized metabolites (dopamine, epinephrine, and norepinephrine), and sample pretreatment time is shortened to 10 minutes. Traditional elution methods require further nitrogen purging and reconstitution of the eluate, taking a total of 30 minutes. This improves sample pretreatment efficiency by 200% compared to traditional methods for treating target substances. By adjusting the combined ratio of test solution one and test solution two to maintain an acidic environment, catecholamines (such as dopamine, epinephrine, and norepinephrine) are more stable at low pH, inhibiting the oxidation of catechol hydroxyl groups and preventing target substance loss. Acidic metabolites (such as vanillylmandelic acid and homovanillic acid) exist in molecular form at low pH, resulting in higher sensitivity. Furthermore, the low pH environment is compatible with LC-MS / MS mobile phases (such as 0.05% acetic acid in water), reducing the risk of column damage.

[0022] In one embodiment of the present invention, the sample diluent is an aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, the concentration of disodium hydrogen phosphate is 50 mmol / L to 80 mmol / L, the concentration of sodium dihydrogen phosphate is 10 mmol / L to 30 mmol / L, and / or the pH of the sample diluent is 6.5 to 7.0.

[0023] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: by adjusting the pH of the sample diluent to 6.5~7.0, the target object is ionized, and the zwitterionic properties of the target object are utilized to be adsorbed by two magnetic beads at the same time.

[0024] On the other hand, the present invention further provides a kit, which is used in the sample pretreatment method as described in any of the above examples.

[0025] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: applying the kit to the sample pretreatment method as in any of the above examples can achieve the technical effect corresponding to any of the above examples, which will not be repeated here.

[0026] After adopting the technical solution of the present invention, the following technical effects can be achieved:

[0027] (1) By mixing carboxyl magnetic beads and amino magnetic beads, the recovery rate of vanillylmandelic acid and homovanillic acid was increased from 25%-40% in the traditional single carboxyl magnetic bead method to 90%-105%;

[0028] (2) The number of catecholamines and their metabolites extracted increased from 6 to 8, and all 8 target compounds with large polarity differences were effectively extracted;

[0029] (3) After the test solution 1 and the test solution 2 are mixed, the sample is directly injected, which is simple to operate and omits the steps of nitrogen blowing, concentration and re-dissolution, thus avoiding the loss of easily oxidized metabolites (dopamine, epinephrine and norepinephrine);

[0030] (4) Sample pretreatment time is shortened to 10 minutes. After elution by the traditional method, the eluent needs to be further nitrogen-purged and redissolved, which takes a total of 30 minutes. Compared with the traditional method for processing target substances, the sample pretreatment time is increased by 200%;

[0031] (5) Specificity and sensitivity can be improved by optimizing the ratio of carboxyl magnetic beads to amine magnetic beads and step-by-step elution;

[0032] (6) The magnetic bead extraction method is combined with a fully automatic extraction instrument, which is simple to operate, reduces manual operation errors, improves the consistency and comparability of experimental results, and shortens the sample pretreatment process with high throughput. The entire pretreatment process does not require other auxiliary equipment, the equipment is streamlined, and laboratory costs and laboratory floor space are saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings to be used in describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive efforts.

[0034] Figure 1 Chromatograms of norepinephrine and norepinephrine-d6 provided in embodiments of the present invention;

[0035] Figure 2 A standard curve of norepinephrine provided in an embodiment of the present invention;

[0036] Figure 3 Chromatograms of epinephrine and epinephrine-d6 provided in embodiments of the present invention;

[0037] Figure 4 A standard curve for epinephrine provided in an embodiment of the present invention;

[0038] Figure 5 Chromatograms of normetanephrine and normetanephrine-d3 provided in embodiments of the present invention;

[0039] Figure 6 A standard curve of normetanephrine provided in an embodiment of the present invention;

[0040] Figure 7 Chromatograms of dopamine and dopamine-d4 provided in embodiments of the present invention;

[0041] Figure 8 A standard curve for dopamine provided in an embodiment of the present invention;

[0042] Figure 9 Chromatograms of metanephrine and metanephrine-d3 provided in embodiments of the present invention;

[0043] Figure 10 A standard curve for metanephrine provided in an embodiment of the present invention;

[0044] Figure 11 Chromatograms of vanillylmandelic acid and vanillylmandelic acid-d3 provided in the embodiments of the present invention;

[0045] Figure 12 A standard curve for vanillylmandelic acid provided in an embodiment of the present invention;

[0046] Figure 13 The chromatogram of 3-methoxytyramine and 3-methoxytyramine-d3 provided by the embodiment of the present invention;

[0047] Figure 14 The standard curve of 3-methoxytyramine provided by the embodiment of the present invention;

[0048] Figure 15 Chromatograms of homovanillic acid and homovanillic acid-d5 provided in the embodiments of the present invention;

[0049] Figure 16 This is the standard curve of homovanillic acid provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0050] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0051] To solve the above problems, the present invention provides a sample pretreatment method for extracting eight catecholamines and their metabolites from urine. The sample pretreatment method adopts a magnetic bead extraction method in conjunction with a fully automatic extractor for automatic extraction. The magnetic bead working solution in the magnetic bead extraction method is a suspension of magnetic beads, water and isopropanol, and the magnetic beads are a mixture of carboxyl magnetic beads and amino magnetic beads. The sample pretreatment method comprises: S100, the fully automatic extractor sequentially performs sample loading, rinsing and elution steps to obtain a test solution; S200, using the test solution for LC-MS / MS detection.

[0052] Furthermore, the magnetic bead extraction method uses a mixture of carboxyl magnetic beads and amine magnetic beads. The surface of the carboxyl magnetic beads contains carboxyl groups (-COOH), which are negatively charged under acidic or neutral conditions and adsorb positively charged compounds through electrostatic interaction. The surface of the amine magnetic beads contains quaternary ammonium groups (-N +(CH3)3), which is positively charged under neutral or alkaline conditions and adsorbs negatively charged compounds; in some embodiments of the present invention, carboxyl magnetic beads specifically adsorb cationic substances (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine); amine magnetic beads specifically adsorb anionic / hydrophobic metabolites (such as vanillylmandelic acid and homovanillic acid); by mixing carboxyl magnetic beads and amino magnetic beads, the recovery rate of vanillylmandelic acid and homovanillic acid is increased from 25%-40% in the traditional carboxyl magnetic bead extraction method to 90%-105%, and the extracted catecholamines and their metabolites are increased from 6 to 8. By using the magnetic bead extraction method in conjunction with a fully automatic extractor, eight catecholamines and their metabolites can be extracted simultaneously in one sample pretreatment. The extracted test solution is then subjected to the LC-MS / MC detection method, achieving the effect of simultaneously extracting and detecting eight catecholamines and their metabolites in one sample pretreatment, achieving high throughput, high recovery rate, simple operation, and streamlined equipment to meet the needs of clinical high throughput.

[0053] In a specific embodiment of the present invention, S100 includes: S110, loading the sample, mixing the sample to be tested, the internal standard working solution, the sample diluent and the magnetic bead working solution, and separating the magnetic beads and the solution; the mass volume content of the carboxyl magnetic beads in the magnetic bead working solution is 0.05g / mL~0.2g / mL, and the mass volume content of the amino magnetic beads in the magnetic bead working solution is 0.05g / mL~0.2g / mL.

[0054] Furthermore, in some embodiments, the mass volume content of carboxyl magnetic beads in the magnetic bead working solution is 0.1 g / mL, and the mass volume content of amine magnetic beads is 0.125 g / mL. By optimizing the ratio of carboxyl magnetic beads to amine magnetic beads, the adsorption capacity is balanced, ensuring efficient capture of all eight targets. This avoids competitive adsorption of non-target substances (such as lipids and proteins), reduces matrix interference, and prevents magnetic bead saturation and bead residue, thereby improving recovery and sensitivity. Specifically, the targets are catecholamines and their metabolites. After optimization, the recovery of vanillylmandelic acid and homovanillic acid increased from 25%-40% in the traditional method to 90%-105%, while the recovery of the remaining metabolites remained stable at 95%-110%.

[0055] In a specific embodiment of the present invention, the volume ratio of water to isopropanol in the magnetic bead working solution is 1:1.

[0056] Furthermore: the volume ratio of water and isopropanol in the magnetic bead working solution is 1:1; by setting the volume ratio of water and isopropanol to 1:1, the magnetic beads can be evenly suspended in the solution and remain without sedimentation for a short period of time (30 minutes), making it convenient to accurately pipette the magnetic bead working solution.

[0057] In a specific embodiment of the present invention, S100 includes: S120, eluting, adding an eluent to the loaded magnetic beads and mixing, separating to obtain the washed magnetic beads; the eluent is a mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, ethanol and water, the concentration of disodium hydrogen phosphate is 50 mmol / L~80 mmol / L, the concentration of sodium dihydrogen phosphate is 10 mmol / L~30 mmol / L, the concentration of sodium chloride is 0.05 mol / L~0.5 mol / L, and the ratio of ethanol to water is 1:1; and / or the pH of the eluent is 6.5~7.0.

[0058] Furthermore: In some embodiments, the concentration of disodium hydrogen phosphate is 80 mmol / L, the concentration of sodium dihydrogen phosphate is 30 mmol / L, the concentration of sodium chloride is 0.15 mol / L, and the ethanol is 50% ethanol. Hydrophobic impurities such as lipids and some proteins are removed by 50% ethanol; 0.15 mol / L sodium chloride is used to prevent the target from being competitively eluted due to excessive salt concentration; disodium hydrogen phosphate and sodium dihydrogen phosphate are used to maintain the charge state of carboxyl magnetic beads (weakly acidic) and amine magnetic beads (strongly alkaline) to prevent desorption of the target while maintaining ion exchange.

[0059] In a specific embodiment of the present invention, S100 includes: S130, elution, adding eluent to the washed magnetic beads for step-by-step elution; the eluent includes eluent 1 and eluent 2; S130 includes: S131, adding eluent 1 to the washed magnetic beads and mixing, separating to obtain solution 1 to be tested and magnetic beads; S132, adding eluent 2 to the separated magnetic beads and mixing, separating to obtain solution 2 to be tested.

[0060] Furthermore: In the existing technology, the use of carboxyl magnetic beads alone can only adsorb neutral and alkaline metabolites (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine), and cannot effectively capture strongly acidic metabolites (such as vanillylmandelic acid and homovanillic acid). Single acid elution causes some target substances (such as vanillylmandelic acid and homovanillic acid) to remain on the magnetic beads, and the recovery rate is less than 40%. The present invention uses mixed magnetic beads of carboxyl magnetic beads and amine magnetic beads. Through step-by-step elution, the carboxyl magnetic bead-adsorbed substances are eluted first, and then the amino magnetic bead-adsorbed substances are eluted, avoiding mutual interference between the elution conditions of the two magnetic beads, ensuring that each type of target is efficiently released at the optimal pH, and the overall recovery rate is improved to 95%-110%; in addition, catecholamines (such as dopamine, epinephrine, and norepinephrine) are more stable under acidic conditions, and preferential elution can reduce oxidation losses. Acidic metabolites such as vanillylmandelic acid and homovanillic acid have high solubility in alkaline environments, and elution in the later stage can ensure their full release.

[0061] In a specific embodiment of the present invention, eluent 1 is an aqueous solution of formic acid, ascorbic acid, sodium metabisulfite and oxalic acid, and / or eluent 1 includes formic acid with a volume fraction of 1% to 2.5%, sodium metabisulfite with a mass concentration of 0.01% to 0.05%, and oxalic acid with a mass concentration of 0.05% to 0.5%.

[0062] Further: In some embodiments, the pH of eluent 1 is less than 3. Specifically, eluent 1 is an aqueous solution composed of 2% formic acid by volume, 0.02% sodium metabisulfite by mass, and 0.15% oxalic acid by mass; the carboxyl magnetic bead adsorbent can be eluted under acidic conditions (pH <3), and by adjusting the pH of eluent 1, the ion exchange effect between the negative charge (-COOH) of the carboxyl magnetic beads and the cationic target (such as dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyrosine) can be destroyed.

[0063] In a specific embodiment of the present invention, the second eluent is a sodium hydroxide aqueous solution, and / or the second eluent comprises sodium hydroxide with a mass concentration of 0.1% to 0.35%.

[0064] Further: In some embodiments, the pH of the second eluent is greater than 9. Specifically, the second eluent is an aqueous solution of sodium hydroxide with a mass concentration of 0.25%. The amine-based magnetic bead adsorbent can be eluted under alkaline conditions (pH>9). By adjusting the pH of the second eluent, the positive charge (-N + Ion exchange interaction between (CH3)3) and anionic metabolites (such as vanillylmandelic acid and homovanillic acid).

[0065] In a specific embodiment of the present invention, S130 includes: S133, combining the test solution 1 and the test solution 2 and mixing them to obtain the test solution; the ratio of combining the test solution 1 and the test solution 2 is (1-3):1.

[0066] Furthermore, in the prior art, after the elution step, nitrogen purging, concentration, and redissolution are required. This is a cumbersome process that can easily lead to target loss. It also requires separate treatment for acidic and alkaline targets, resulting in a long detection cycle (>0.5 hours). The present invention directly injects the sample by mixing the eluted test solution 1 and test solution 2, eliminating the nitrogen purging, concentration, and redissolution steps. This avoids the loss of easily oxidized metabolites (dopamine / epinephrine and norepinephrine). The sample pretreatment time is shortened to 10 minutes. After elution using the traditional method, the eluate requires further nitrogen purging and redissolution, taking a total of 30 minutes. Compared to the traditional method for treating target substances, the sample pretreatment time is 200% more efficient. By adjusting the ratio of the combined test solution 1 and test solution 2, the mixed solution maintains an acidic environment. In some embodiments, the combined ratio of the test solution 1 and test solution 2 is 2:1, resulting in a pH of <3 for the test solution. Among them, catecholamines (such as dopamine, epinephrine, and norepinephrine) are more stable in a low pH environment, which can inhibit the oxidation reaction of catechol hydroxyl groups and avoid the loss of target substances; acidic metabolites (such as vanillylmandelic acid and homovanillic acid) exist in molecular form in a low pH environment and have higher sensitivity; and the low pH environment is compatible with LC-MS / MS mobile phases (such as 0.05% acetic acid water), reducing the risk of chromatographic column damage.

[0067] In a specific embodiment of the present invention, the sample diluent is an aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, the concentration of disodium hydrogen phosphate is 50 mmol / L~80 mmol / L, the concentration of sodium dihydrogen phosphate is 10 mmol / L~30 mmol / L, and / or the pH of the sample diluent is 6.5~7.0.

[0068] Furthermore: In some embodiments, the concentration of disodium hydrogen phosphate is 80 mmol / L, and the concentration of sodium dihydrogen phosphate is 30 mmol / L; by adjusting the pH of the sample diluent to 6.5-7.0, the target is ionized and the zwitterionic properties of the target are utilized to simultaneously adsorb the target by the two magnetic beads.

[0069] On the other hand, the present invention further provides a kit, which is used in the sample pretreatment method as described in any of the above examples.

[0070] Furthermore: In some embodiments, the kit includes: Box A: calibrator, quality control product, internal standard working solution, magnetic bead working solution; Box B: sample diluent, eluent, eluent 1, eluent 2, mobile phase aqueous phase, mobile phase organic phase; Box A is stored at 2°C-8°C, and Box B is stored at room temperature.

[0071] Specifically, the composition of the kit is shown in Table 1:

[0072] Table 1 Kit composition

[0073]

[0074] The preparation of the components of the kit includes:

[0075] (1) Preparation of calibrators: Prepare mixed solutions of dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyramine, vanillylmandelic acid, and homovanillic acid standards at seven different concentration levels using artificial urine containing 0.5% ascorbic acid. The concentrations of the calibrators are shown in Table 2:

[0076] Table 2 Calibrator concentrations

[0077]

[0078] (2) Preparation of quality control products: Collect human urine and add 0.5% ascorbic acid after mixing. Then add standard solutions of dopamine, epinephrine, norepinephrine, metanephrine, norepinephrine, 3-methoxytyramine, vanillylmandelic acid and homovanillic acid to prepare two urine solutions with different concentration levels. After packaging, freeze-dry and assign the freeze-dried powder with the value. The concentration of quality control products is shown in Table 3:

[0079] Table 3 Concentration of quality control products

[0080]

[0081] (3) Preparation of internal standard working solution: Prepare a mixed solution of dopamine-IS, epinephrine-IS, norepinephrine-IS, metanephrine-IS, normetanephrine-IS, 3-methoxytyramine-IS, vanillylmandelic acid-IS, and homovanillic acid-IS standards using a 0.5% ascorbic acid aqueous solution. The concentrations of the internal standard working solution are shown in Table 4:

[0082] Table 4 Concentration of internal standard working solution

[0083]

[0084] (4) Preparation of other reagents

[0085] Preparation of magnetic bead working solution: Take 1 mL of water, 1 mL of isopropanol, 200 mg of WCX magnetic beads, and 250 mg of MAX magnetic beads to prepare magnetic bead working solution.

[0086] Preparation of mobile phase (aqueous phase): Take 250 μL of acetic acid and 500 mL of ultrapure water to prepare 0.05% acetic acid water.

[0087] Preparation of mobile phase (organic phase): Take 250 μL of acetic acid and 500 mL of methanol respectively to prepare 0.05% acetic acid in methanol.

[0088] Sample diluent: Take 1.135 g of disodium hydrogen phosphate, 0.36 g of sodium dihydrogen phosphate, and 100 mL of ultrapure water to prepare sample diluent.

[0089] Eluent: Prepare the eluent by taking 1.135 g of disodium hydrogen phosphate, 0.36 g of sodium dihydrogen phosphate, 17.5 mg of sodium chloride, 50 mL of ultrapure water, and 50 mL of ethanol.

[0090] Eluent 1: Prepare eluent 1 by taking 2.5 mL of formic acid, 20 mg of sodium metabisulfite, 150 mg of oxalic acid, and 97.5 mL of ultrapure water.

[0091] Eluent 2: Take 250 mg of sodium hydroxide and 100 mL of ultrapure water to prepare eluent 2.

[0092] Example 1

[0093] The kit provided by the present invention is applied to sample pretreatment methods and LC-MS / MS detection methods:

[0094] 1. Sample pre-treatment methods include:

[0095] (1) Sample loading: Take 50 μL of the sample to be tested, add 20 μL of internal standard working solution, 550 μL of sample diluent and 20 μL of magnetic bead working solution in sequence, mix at 1000 rpm for 120 s, and separate the magnetic beads and solution;

[0096] (2) Elution: Add 200 μL of eluent to the magnetic beads separated in (1), mix at 1000 rpm for 30 s, and separate the magnetic beads and the solution;

[0097] (3) Elution: Add 50 μL of eluent 1 to the magnetic beads separated in (2), mix at 1000 rpm for 30 s, and separate the magnetic beads and the solution (test solution 1); add 50 μL of eluent 2 to the magnetic beads, mix at 1000 rpm for 30 s, and separate the magnetic beads and the solution (test solution 2);

[0098] (4) Mixing: Combine test solution 1 and test solution 2 and mix at 1000 rpm for 30 s to obtain the test solution for LC-MS / MS detection.

[0099] The fully automatic extractor automatically performs the extraction process of the test substance, including the steps of sample loading, washing and elution. The extraction procedure of the fully automatic extractor is shown in Table 5:

[0100] Table 5 Extraction procedure of fully automatic extractor

[0101]

[0102] 2. The extracted substance to be tested is detected by LC-MS / MS detection method. The LC-MS / MS detection method includes mass spectrometry conditions and liquid phase conditions:

[0103] (1) Mass spectrometry conditions:

[0104] Mass spectrometry source parameters

[0105] Table 6 Mass spectrometry source parameters

[0106]

[0107] Target compound and internal standard collection

[0108] Table 7 Target compounds and internal standard collection list

[0109]

[0110] (2) Liquid phase conditions:

[0111] Chromatographic conditions

[0112] Table 8 Chromatographic conditions

[0113]

[0114] Liquid phase gradient

[0115] Table 9 Liquid phase gradient

[0116]

[0117] (3) Calibration procedure

[0118] The liquid chromatography-tandem mass spectrometry system analysis software can automatically draw a calibration curve based on the calibration results. Usually, the ratio of the labeled concentration of the calibrator to the concentration of the internal standard is used as the horizontal axis (x), and the ratio of the actual measured peak area of the calibrator to the peak area of each internal standard is used as the vertical axis (y). The least squares method is used for linear regression to automatically draw the standard curve. The regression equation can be obtained: y = ax + b, where y is the vertical axis, x is the horizontal axis, a is the slope, and b is the intercept, and the r (correlation coefficient) value is calculated.

[0119] Through the above sample pretreatment method and LC-MS / MS detection method, 8 catecholamines and their metabolites were extracted and detected at one time. The standard curves of the 8 catecholamines and their metabolites are shown in Figure 2. Figure 2 、 4 , 6, 8, 10, 12, 14, 16; chromatograms of 8 catecholamines and their metabolites, see Figure 1 、 3 , 5, 7, 9, 11, 13, 15.

[0120] Validation results of the standard curves for eight catecholamines and their metabolites: Analysis of the linear relationships of the eight catecholamines and their metabolites showed that dopamine was within the range of (3, 1500) μg / L, epinephrine was within the range of (0.2, 100) μg / L, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine were within the range of (0.5, 250) μg / L, and vanillylmandelic acid and homovanillic acid were within the range of (0.2, 100) mg / L. The linear correlation coefficient (r) should be no less than 0.9900. The analysis results are shown in Table 10:

[0121] Table 10 Catecholamine and its metabolite standard curve verification results

[0122]

[0123] As shown in Table 10, within the linear concentration range, the eight catecholamines and their metabolites have a good linear relationship. The present invention has a wide linear range and a lower limit of quantification, which is higher than the linear range of current technologies and meets clinical diagnostic needs.

[0124] Example 2

[0125] Kit performance verification:

[0126] 1. Accuracy: The accuracy of the test results is expressed as recovery rate (%), which should be within the range of 85.0% to 115.0%.

[0127] Within the linear range of the kit, 25 μL of each of the three standard solutions at low, medium, and high concentrations were added to 475 μL of urine to prepare spiked samples at low, medium, and high concentrations. The theoretical spiked concentrations of each analyte were calculated for each concentration. 25 μL of 0.1 mol / L hydrochloric acid solution was added to 475 μL of urine to serve as the unspiked sample. The test results are shown in Table 11:

[0128] Table 11 Accuracy test results of catecholamines and their metabolites

[0129]

[0130] As shown in Table 11, the spiked recoveries of all analytes were between 85.0% and 115.0%, which met the quality standards of the kit.

[0131] 2. Precision

[0132] Repeatability: The coefficient of variation (CV) should not be greater than 10.0%; inter-batch precision: The coefficient of variation (CV) should not be greater than 10.0%.

[0133] Take the quality control samples at low and high concentration levels, measure ten copies at each concentration level, measure three batches of kits, and calculate the intra-batch, inter-batch and total CV respectively. The analysis results are shown in Table 12:

[0134] Table 12 Precision test results of catecholamines and their metabolites

[0135]

[0136]

[0137]

[0138] As shown in Table 12, the intra-assay and inter-assay CVs of catecholamines and their metabolites were less than 10%, and the precision met the quality standards of the kit.

[0139] 3. Limit of quantification: The quantification limit of dopamine should be 3.0 μg / L, the quantification limit of epinephrine should be 0.2 μg / L, the quantification limit of norepinephrine, metanephrine, normetanephrine and 3-methoxytyramine should be 0.5 μg / L, the quantification limit of vanillylmandelic acid and homovanillic acid should be 0.2 mg / L. When the concentration of dopamine is within the range of (3.00±0.60) μg / L, the concentration of epinephrine is within the range of (0.20±0.04) μg / L, the concentration of norepinephrine, metanephrine, normetanephrine and 3-methoxytyramine is within the range of (0.50±0.10) μg / L, and the concentration of vanillylmandelic acid and homovanillic acid is within the range of (0.20±0.04) mg / L, the relative deviation between the measured value and the theoretical value should not exceed ±15.0%, and the coefficient of variation (CV) should not be greater than 20.0%.

[0140] Select samples within the quantitative limit concentration range, repeat the measurement ten times, and calculate the relative deviation and coefficient of variation (CV) between the measured value and the theoretical value. The analysis results are shown in Table 13:

[0141] Table 13 Quantification limit detection results of catecholamines and their metabolites

[0142]

[0143] As shown in Table 13, the relative deviation of the quantification limit of catecholamines and their metabolites was less than 15%, the coefficient of variation (CV) was less than 20%, and the quantification limit met the quality standards of the kit.

[0144] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A sample pretreatment method for extracting eight catecholamines and their metabolites from urine, characterized in that: The sample pretreatment method adopts a magnetic bead extraction method in conjunction with a fully automatic extractor for automatic extraction, wherein the magnetic bead working solution in the magnetic bead extraction method is a suspension of magnetic beads, water and isopropanol, and the magnetic beads are a mixture of carboxyl magnetic beads and amine magnetic beads; The sample pre-treatment method comprises: S100, the fully automatic extractor sequentially performs sample loading, rinsing, and elution steps to obtain a test solution; S200, applying the test solution to LC-MS / MS detection; The S100 includes: S110, loading the sample, mixing the sample to be tested, the internal standard working solution, the sample diluent and the magnetic bead working solution, and separating the magnetic beads and the solution; The mass volume content of the carboxyl magnetic beads in the magnetic bead working solution is 0.05g / mL~0.2g / mL, The mass volume content of the amino magnetic beads in the magnetic bead working solution is 0.05 g / mL to 0.2 g / mL; The volume ratio of the water and the isopropanol in the magnetic bead working solution is 1:1; S120, eluting, adding an eluent to the sampled magnetic beads, mixing, and separating to obtain washed magnetic beads; The eluent is a mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, ethanol and water, the concentration of the disodium hydrogen phosphate is 50 mmol / L to 80 mmol / L, the concentration of the sodium dihydrogen phosphate is 10 mmol / L to 30 mmol / L, the concentration of the sodium chloride is 0.05 mol / L to 0.5 mol / L, and the ratio of the ethanol to the water is 1:1; and / or the pH of the eluent is 6.5 to 7.0; S130, eluting, adding eluent to the washed magnetic beads for step-by-step elution, wherein the eluent includes eluent 1 and eluent 2, wherein the eluent 1 is an aqueous solution of formic acid, ascorbic acid, sodium metabisulfite, and oxalic acid, and the eluent 2 is an aqueous solution of sodium hydroxide; The S130 includes: S131, adding eluent 1 to the washed magnetic beads and mixing, and separating to obtain test solution 1 and magnetic beads; S132, adding eluent 2 to the separated magnetic beads and mixing, separating to obtain test solution 2; S133, combining the first test solution and the second test solution to obtain the test solution.

2. The sample pretreatment method according to claim 1, characterized in that: The eluent 1 includes formic acid with a volume fraction of 1% to 2.5%, sodium metabisulfite with a mass concentration of 0.01% to 0.05%, and oxalic acid with a mass concentration of 0.05% to 0.5%.

3. The sample pretreatment method according to claim 1, characterized in that: The second eluent comprises sodium hydroxide with a mass concentration of 0.1% to 0.35%.

4. The sample pretreatment method according to claim 1, characterized in that: The ratio of the test solution 1 and the test solution 2 combined is (1-3):

1.

5. The sample pre-processing method according to claim 1, characterized in that: The sample diluent is an aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, the concentration of the disodium hydrogen phosphate is 50 mmol / L to 80 mmol / L, the concentration of the sodium dihydrogen phosphate is 10 mmol / L to 30 mmol / L, and / or The pH of the sample diluent is 6.5-7.

0.

6. A kit, characterized in that The kit is used for the sample pretreatment method according to any one of claims 1 to 5.

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