Magnetic bead extraction unit and application thereof in catecholamine pre-packaging extraction

Abstract

The invention provides a magnetic bead extraction unit, a magnetic bead extraction system and application of the magnetic bead extraction unit in catecholamine pre-packaging extraction, and designs the magnetic bead extraction unit which is pre-filled with a magnetic bead treatment reagent of a single sample and can be used for completing magnetic bead extraction before mass spectrometry of the single sample; according to the number of samples needing to be detected, a corresponding number of magnetic bead extraction units are selected and placed on the test tube rack, so that a magnetic bead extraction instrument can be matched to complete magnetic bead extraction of the required number of samples; a pre-packaging kit for extracting catecholamine in a sample is designed, and a magnetic bead activating agent, a washing solution and an eluent which are not easy to volatilize and higher in stability are screened. The magnetic bead extraction unit is simple in structure and convenient to use, the problem of waste of magnetic bead treatment reagents is avoided, the magnetic bead treatment process is more flexible and efficient, the pre-subpackaged reagents are stable in performance, not prone to volatilization and long in quality guarantee period, and the magnetic bead extraction unit is suitable for large-scale application and popularization.

Description

[0001] This application claims priority to a prior Chinese application, application number 2024105588691, filed on May 8, 2025, all of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, and more specifically, relates to a magnetic bead extraction unit and its application in the pre-dispensing extraction of catecholamines. Background Technology

[0003] Magnetic bead extraction is a method for separating and enriching target molecules from samples, widely used in bioscience, medical research, and industrial production. It utilizes the specific binding of magnetic particles to target molecules, achieving separation and concentration with the aid of a magnetic field.

[0004] The principle of magnetic bead extraction is mainly based on the specific binding between immunomagnetic beads and target molecules. The surface of the magnetic beads is modified with antibodies or other chemically modified groups, which can specifically bind to target molecules (such as proteins and nucleic acids). Under the influence of a magnetic field, the magnetic beads bound to the target molecules aggregate together. After elution, the target molecules are separated from the magnetic beads. Magnetic rods are then used to adsorb and remove the magnetic beads, thus achieving the separation and concentration of the target molecules. The remaining sample after magnetic bead extraction (containing the target molecules) is intended to be directly used for detection by liquid chromatography-tandem mass spectrometry.

[0005] Existing magnetic bead extraction methods typically employ a 96-well plate (16 or 96 test units) placed in a magnetic bead extraction instrument to complete the relevant operations. The 96-well plate is pre-loaded with magnetic bead activator, magnetic bead eluent, and magnetic bead elution buffer. In a 16-test 96-well plate, each reagent needs to be pre-loaded and sealed in a row. In other words, a 96-well plate pre-loads all the reagents needed for magnetic bead extraction of 16 samples. Therefore, each magnetic bead extraction operation requires exactly 16 samples or an integer multiple thereof. If there are 15 samples, the magnetic bead processing reagents (including magnetic bead solution, magnetic bead activator, magnetic bead eluting agent, magnetic bead eluent, etc.) for one sample will be wasted because once the plate is placed in the magnetic bead extractor, the corresponding magnetic rod will perform all magnetic bead extraction operations regardless of whether a sample is in the well. If there are 17 samples, two 96-well plates are needed: the first plate processes 16 samples, and the second plate processes 1 sample, resulting in a waste of magnetic bead processing reagents for 15 samples in the second plate. Therefore, reagent handling often leads to significant waste of magnetic bead processing reagents, increasing the cost of testing reagents and making the calculation of testing fees difficult.

[0006] Therefore, there is an urgent need to find a way to flexibly complete the magnetic bead extraction operation for any number of samples without wasting magnetic bead processing reagents, and at the same time, require fewer operations. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a magnetic bead extraction unit, a system, and its application in the pre-dispensing extraction of catecholamines. A magnetic bead extraction unit is designed, pre-filled with magnetic bead processing reagents for a single sample, which can be used to extract magnetic beads from a single sample before mass spectrometry analysis. Depending on the number of samples to be analyzed, the appropriate number of magnetic bead extraction units can be selected and placed on a test tube rack to match the magnetic bead extractor, completing the extraction of the required number of samples. A pre-dispensing kit for extracting catecholamines from samples is also designed, screening out activators, buffers, eluents, and elution buffers that are less volatile and more stable. The magnetic bead extraction unit has a simple structure and is easy to use, avoiding the waste of magnetic bead processing reagents and making the magnetic bead processing process more flexible and efficient. Furthermore, the pre-dispensed reagents are stable, less volatile, and have a long shelf life, making them suitable for large-scale application.

[0008] On one hand, the present invention provides a magnetic bead extraction unit, which includes an integral structure composed of a component for accommodating magnetic beads, a component for accommodating samples, and a component for processing samples with magnetic beads, and can be used independently to complete the magnetic bead extraction of a single sample.

[0009] Existing magnetic bead extraction instruments typically require the use of specific plate models for magnetic bead extraction. For example, an instrument designed for 96-well plates can only use 96-well plates for extraction. Each 96-well plate is pre-loaded with magnetic bead extraction reagents for 16 samples. Therefore, each extraction operation requires exactly 16 samples. Processing fewer or more than 16 samples leads to waste of reagents, increased testing costs, and varying average testing costs per sample across batches due to different sample quantities, making calculation difficult and complicating both magnetic bead processing and testing.

[0010] The magnetic bead extraction unit provided by this invention can be used to complete the magnetic bead extraction of a single sample. Therefore, the number of magnetic bead extraction units can be selected according to the number of samples. For example, if 12 samples need to be tested, 12 magnetic bead extraction units can be selected and placed into the magnetic bead extractor for testing. This is similar to "movable type printing", making the magnetic bead extraction process more flexible and controllable, allowing for flexible selection and processing, and completely solving the problem of waste of magnetic bead processing reagents in the prior art.

[0011] The term "independent use" as used in this invention means that the magnetic bead extraction unit can be used alone or in flexible combination with one or more other magnetic bead extraction units. It is understood that the magnetic bead extraction unit is primarily used in conjunction with a magnetic bead extractor to automatically complete the magnetic bead extraction process; however, the extraction can also be performed manually.

[0012] Furthermore, the components for processing samples with magnetic beads include components for containing magnetic bead activator, components for containing washing liquid, and components for containing elution liquid.

[0013] Furthermore, the number of components containing the washing liquid is one or more.

[0014] Furthermore, the components containing magnetic beads, magnetic bead activator, sample, washing solution, and elution solution are all reagent tubes.

[0015] Furthermore, each reagent tube is interconnected to form a row of interconnected tubes.

[0016] Furthermore, the magnetic bead extraction unit has a structure of three-tube, four-tube, five-tube, six-tube, seven-tube, or eight-tube.

[0017] The number of reagent tubes included in the magnetic bead extraction unit can be flexibly selected according to the magnetic bead extraction steps.

[0018] In some embodiments, the magnetic bead extraction unit is a six-tube assembly. The first tube contains the magnetic bead solution, the second tube contains the activator, the third tube contains the sample, the fourth tube contains the first eluent, the fifth tube contains the second eluent, and the sixth tube contains the elution solution.

[0019] Furthermore, the component containing the sample contains a diluent and a buffer solution, wherein the sample is for the detection of catecholamines.

[0020] Furthermore, the magnetic bead activator comprises an aqueous ethanol solution, the diluent comprises aqueous phosphoric acid, the buffer comprises ammonium acetate and sodium hydroxide, the washing solution comprises methanol and acetonitrile, and the elution solution comprises a methanol formic acid and ammonium formic acid solution.

[0021] Preferably, the magnetic bead activator is a 10% aqueous ethanol solution, the diluent is 0.2% phosphate water, the buffer solution is 50mM ammonium acetate and 100mM sodium hydroxide, the washing solution includes a first elution solution of 10% methanol and a second elution solution of acetonitrile, and the elution solution includes 1% formic acid methanol and 0.1M ammonium formate solution.

[0022] Existing magnetic bead extraction methods typically use 96-well plates, in which reagents such as magnetic bead activators, buffer solutions, eluents, and elution solutions are added or pre-filled. Because the 96-well plate has a 12×8 arrangement, the wells are closely packed together, and the tube walls are thick. Furthermore, the outer ring of the tubes is wrapped in a plastic shell, with the inner tubes not exposed to air, resulting in a smaller contact area with air, and the outermost tubes only exposed to air on two sides, further preventing the volatilization or degradation of pre-filled reagents. The magnetic bead extraction unit provided by this invention has a six-tube structure with thinner walls, and each well is exposed to the environment without any obstruction. Therefore, the reagents inside the tubes are more prone to volatilization or degradation due to improper storage or changes in environmental conditions. Thus, reagents that can be stored long-term in a 96-well plate and then aliquoted into the magnetic bead extraction unit provided by this invention may not necessarily have the same long-term stability. Therefore, in some embodiments, the present invention has verified through comparative experiments that the magnetic bead activator, buffer solution, rinsing solution and elution solution suitable for long-term storage of 96-well plates do not have long-term stability when dispensed into the magnetic bead extraction unit.

[0023] The magnetic bead solution for catecholamine detection provided by this invention primarily consists of magnetic beads. Magnetic nanoparticles serve as the solid-phase material, and surface-modifying materials on these particles are used to separate, extract, and further enrich the target analytes from the sample. An ethanol activator for catecholamine detection is used to clean and balance the magnetic beads, enabling better adsorption of analytes. The sample diluent primarily consists of phosphoric acid water, used to dilute clinical samples and reduce the influence of the sample matrix on subsequent extraction processes. The buffer solution primarily consists of ammonium acetate and sodium hydroxide, which form a buffer system to regulate the pH of the sample solution to be extracted. The first eluent for catecholamine detection primarily consists of 10% methanol-water, and the second eluent primarily consists of acetonitrile. Using a low concentration of methanol maximizes the removal of water-soluble interfering substances and also acts as a preservative; acetonitrile removes lipid-soluble interfering substances and purifies the substances adsorbed on the surface of the magnetic beads. The eluent for catecholamine detection mainly consists of formic acid and methanol, which have a strong elution ability, effectively desorbing the target compound from the magnetic beads for further detection. The activator, buffer, eluent, and elution solution, dispensed into the magnetic bead extraction unit, maintain their initial detection performance even after long-term storage, demonstrating excellent detection stability.

[0024] In some embodiments, the present invention verified the necessity of using a 10% ethanol aqueous solution as a magnetic bead activator. Results showed that, compared to a 10% ethanol aqueous solution, ammonium acetate activator exhibited bacterial growth after 6 months of storage, affecting detection efficiency. Using 0.1% or 0.2% formic acid solution or 10% or 50% methanol solution as activators resulted in significantly lower catecholamine response values ​​compared to the 10% ethanol aqueous solution, with a significant decrease in detection values ​​after one year of storage. Furthermore, increasing the ethanol-water concentration to 50% also led to a decrease in detection efficiency. Therefore, only using a 10% ethanol aqueous solution as a magnetic bead activator can effectively improve the extraction efficiency of the pre-packaged magnetic bead extraction method.

[0025] In some embodiments, this invention verified the necessity of a combination of ammonium acetate and sodium hydroxide as a sample buffer. Results showed that after one year of storage, the catecholamine response values ​​in samples extracted and detected using buffers containing ammonium acetate and potassium hydroxide, sodium hydroxide, or ammonium bicarbonate and sodium hydroxide were significantly reduced. This is presumably because, after a certain storage period, the ammonium salts or potassium hydroxide in these combinations undergo significant volatilization and deterioration, producing new substances that affect the stability of the buffer and thus fail to provide a suitable pH environment for the sample, leading to reduced extraction efficiency. The combined buffer of ammonium acetate and sodium hydroxide showed the highest catecholamine response values ​​both after one year of storage and during the initial preparation, indicating its optimal effectiveness.

[0026] In some embodiments, this invention also verified the necessity of using 10% methanol-water as the first eluent, acetonitrile as the second eluent, and 1% formic acid-methanol and 0.1M ammonium formate solution as the elution solution. Results showed that when the first eluent was ethanol-water or formic acid-water and the second eluent was acetonitrile, or when the first eluent was formic acid-water and the second eluent was acetic acid-water, the extracted and detected catecholamine response values ​​were significantly lower than those of the methanol and acetonitrile combination. This indicates that these three eluent combinations cannot guarantee the adsorption of catecholamines by the magnetic beads and cannot effectively remove other impurities from the sample. Furthermore, experiments revealed that when the formic acid content in the formic acid-methanol elution solution was less than 1%, the catecholamine response values ​​extracted and detected by the magnetic bead extraction unit were significantly lower than those of the 1% formic acid-methanol and 0.1M ammonium formate solution. The detection effect further decreased after one year of reagent storage, and the extraction effect using a 2% formic acid-methanol and 0.1M ammonium formate solution was also inferior to that of a 1% formic acid-methanol and 0.1M ammonium formate solution. Therefore, only by using 10% methanol-water as the first eluent, acetonitrile as the second eluent, and a solution of 1% formic acid-methanol and 0.1M ammonium formate as the elution solution can the extraction and detection effects of the magnetic bead extraction unit and the pre-packaged kit be effectively improved, and the long-term stability of the reagents be guaranteed.

[0027] On the other hand, the present invention provides a magnetic bead extraction system, the system comprising a magnetic bead extraction unit as described above, and a test tube rack for placing the magnetic bead extraction unit.

[0028] Furthermore, an appropriate number of magnetic bead extraction units can be placed on the test tube rack as needed; after the magnetic bead extraction units are combined with the test tube rack, they can be matched with a magnetic bead extractor to complete the magnetic bead extraction of the required number of samples.

[0029] Furthermore, it may also include a well plate with multiple reagent tubes fixed thereon, and the magnetic bead extractor can be used to extract magnetic beads from the samples in the well plate. The size of the magnetic bead extraction unit combined with the test tube rack matches the size of the well plate.

[0030] Furthermore, the perforated plate includes any one or more of a 32-well plate, a 96-well plate, and a 384-well plate.

[0031] In some embodiments, the well plate is a 96-well plate with 8 rows × 12 columns, each row containing 12 reagent tubes, equivalent to two six-tube strips, which can be used for magnetic bead extraction of two samples. Therefore, the 96-well plate can be used for magnetic bead extraction of 8 rows × 2 = 16 samples.

[0032] In some configurations, the combined size of the magnetic bead extraction unit and the test tube rack matches the size of a 96-well plate, and the test tube rack can hold six tubes of 1 to 16 magnetic bead extraction units.

[0033] In some methods, the magnetic bead extractor can extract magnetic beads from 1 to 2 or 1 to 4 96-well plates at a time. Therefore, after placing the combination of magnetic bead extraction units and test tube racks into the magnetic bead extractor, 1 to 3 96-well plates can be placed simultaneously. This allows the selection of the number of 96-well plates and the number of magnetic bead extraction units on the test tube racks based on the number of samples to be tested. For example, when the number of samples is 10, only 10 magnetic bead extraction units need to be selected and placed on the test tube rack before being placed in the magnetic bead extractor for extraction; when the number of samples is 25, one 96-well plate and a test tube rack containing 9 magnetic bead extraction units need to be selected and placed together in the magnetic bead extractor for extraction; when the number of samples is 38, two 96-well plates and a test tube rack containing 6 magnetic bead extraction units need to be selected and placed together in the magnetic bead extractor for extraction, and so on.

[0034] In another aspect, the present invention provides a pre-packaged kit for catecholamine extraction, comprising the magnetic bead extraction unit as described above.

[0035] In another aspect, the present invention provides a magnetic bead extraction method, wherein the method uses the magnetic bead extraction unit or the magnetic bead extraction system described above to extract target molecules from a sample.

[0036] Furthermore, the combination of the magnetic bead extraction unit and the test tube rack is placed inside the magnetic bead extractor, and the magnetic beads are extracted by the magnetic bead extractor.

[0037] Furthermore, the method includes the following steps:

[0038] (1) Adsorb magnetic beads from the magnetic bead extraction unit into the magnetic bead activator in the magnetic bead activator;

[0039] (2) The activated magnetic beads are added to the component containing the sample, and the magnetic beads bind to the target molecules in the sample;

[0040] (3) Take out the magnetic beads from step (2) and put them into the component containing the washing liquid for washing;

[0041] (4) Take out the washed magnetic beads and add them to the component containing the eluent for elution, so that the target molecules are separated from the magnetic beads;

[0042] (5) Remove the magnetic beads from step (4), and use the remaining solution for detection.

[0043] Furthermore, the magnetic bead extraction unit and the test tube rack can be combined and placed together with the well plate inside the magnetic bead extractor for magnetic bead extraction.

[0044] In some embodiments, the perforated plate is a 96-well plate, which is equivalent to a structure consisting of 16 magnetic bead extraction units fixed together.

[0045] On another aspect, the present invention provides a magnetic bead extraction unit for use in preparing a magnetic bead extraction system that can complete the extraction of any number of samples as needed without wasting reagents. The magnetic bead extraction unit or magnetic bead extraction system needs to be used in conjunction with a magnetic bead extractor to complete the magnetic bead extraction.

[0046] In another aspect, the present invention provides the use of formic acid methanol and formic acid ammonium for preparing the eluent in a pre-filled kit for catecholamine extraction, the pre-filled kit for catecholamine extraction including the magnetic bead extraction unit as described above.

[0047] The present invention has the following beneficial effects:

[0048] (1) A solution of magnetic bead activator in ethanol aqueous solution, diluent in phosphate water, buffer in ammonium acetate and sodium hydroxide, first elution in methanol water and second elution in acetonitrile, and elution in formic acid in methanol and ammonium formate was selected for the detection of catecholamines in samples. The reagents are not volatile, have higher stability and longer shelf life, which ensures the long-term stable detection of catecholamines in samples.

[0049] (2) The number of magnetic bead extraction units can be selected according to the number of samples. The structure is simple and easy to use, completely avoiding the problem of waste of magnetic bead processing reagents.

[0050] (3) It is used with a magnetic bead extractor and has a high level of automation;

[0051] (4) Makes the magnetic bead processing of samples more flexible and efficient, reduces costs, and is suitable for large-scale promotion and application. Attached Figure Description

[0052] Figure 1 This is a structural diagram of the magnetic bead extraction unit provided in Example 1;

[0053] Figure 2 This is a structural diagram of the magnetic bead extraction system provided in Example 1;

[0054] Figure 3 This is a structural diagram of the magnetic bead extraction unit and test tube rack provided in Example 1;

[0055] Figure 4 A schematic diagram of the structure of the 96-well plate provided in Example 1;

[0056] Figure 5 This is a schematic diagram showing the combination of the pre-packaged catecholamine extraction reagent and the 6-connected U-shaped bottom tube base in Example 3;

[0057] Figure 6 This is a schematic diagram of the magnetic bead extraction operation in Example 3;

[0058] Figure 7 This is a schematic diagram of the combination of the receiving plate and the microporous filter plate in Example 3.

[0059] Figure 8 This is a schematic diagram of the six test tube wells in the 96-well plate selected in Examples 4-7.

[0060] Figure 9 This is a picture of a 96-well plate. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0064] Example 1: Magnetic bead extraction unit and magnetic bead extraction system provided by the present invention.

[0065] The structure of the magnetic bead extraction unit provided in this embodiment is as follows: Figure 1 As shown, the structure of the magnetic bead extraction system is as follows: Figure 2 As shown in the diagram, the structure of the magnetic bead extraction unit combined with the test tube rack is as follows: Figure 3 As shown in the diagram, the structure of the 96-well plate is as follows: Figure 4 As shown.

[0066] like Figure 1 The magnetic bead extraction unit 1 provided in this embodiment includes an integrated structure composed of a component 2 for containing magnetic beads, a component 3 for containing samples, and a component 4 for processing samples with magnetic beads, which can be used to complete the magnetic bead extraction of a single sample. The component 4 for processing samples with magnetic beads includes a component 5 for containing magnetic bead activator, a component 6 for containing washing liquid, and a component 7 for containing eluent. The number of components 6 for containing washing liquid is one or more.

[0067] Preferably, there are two components 6 for containing the washing liquid, namely component 8 for containing the first washing liquid and component 9 for containing the second washing liquid.

[0068] Preferably, the components 2 (containing magnetic beads), 5 (containing magnetic bead activator), 3 (containing sample), 6 (containing washing solution), and 7 (containing eluent) are all reagent tubes 10. Each reagent tube 10 is interconnected to form a row of connected tubes, such as a triplet, quadruplet, quintuplet, hexaplet, heptaplet, or octuplet structure, etc. The number of reagent tubes 10 included in the magnetic bead extraction unit 1 can be flexibly selected according to the magnetic bead extraction steps.

[0069] Preferred, such as Figure 1 The magnetic bead extraction unit 1 is a six-tube unit 21. The first tube 11 is used to contain the magnetic bead solution, the second tube 12 is used to contain the activator, the third tube 13 is used to contain the sample, the fourth tube 14 is used to contain the first eluent, the fifth tube 15 is used to contain the second eluent, and the sixth tube 16 is used to contain the elution solution.

[0070] like Figure 2 The magnetic bead extraction system 17 includes a magnetic bead extraction unit 1 and a test tube rack 18 for placing the magnetic bead extraction unit 1. A suitable number of magnetic bead extraction units 1 can be placed on the test tube rack 18 as needed. The magnetic bead extraction unit 1 and the test tube rack 18 are combined (…). Figure 3 After that, a magnetic bead extractor can be used to extract the required number of samples.

[0071] Preferably, the magnetic bead extraction system 17 may further include a well plate 19 with multiple reagent tubes fixed thereon. The magnetic bead extractor can be used to extract magnetic beads from samples within the well plate. The dimensions of the magnetic bead extraction unit 1 combined with the test tube rack 10 match the dimensions of the well plate 19. The well plate 19 can be any size well plate, such as a 32-well plate, a 96-well plate, or a 384-well plate.

[0072] Preferably, the well plate 19 is a 96-well plate 20, with 8 rows × 12 columns, each row including 12 reagent tubes, equivalent to two six-tube sets 21, which can be used for magnetic bead extraction of two samples. Therefore, the 96-well plate 20 can be used for magnetic bead extraction of 8 rows × 2 = 16 samples. Figure 4 The combined dimensions of the magnetic bead extraction unit 1 and the test tube rack 10 match the dimensions of the 96-well plate 20. The test tube rack 10 can hold 1 to 16 six-unit tubes 21 of the magnetic bead extraction unit 1.

[0073] Example 2: Method for extracting magnetic beads

[0074] Using the magnetic bead extraction unit 1 or magnetic bead extraction system 17 provided in Example 1, the target molecules in the sample are extracted using magnetic beads. This requires placing the combination of the magnetic bead extraction unit 1 and the test tube rack 10 inside the magnetic bead extractor. The magnetic bead extraction is then performed using the magnetic bead extractor. The main process is as follows:

[0075] (1) Adsorb magnetic beads from the magnetic bead-containing component 2 of the magnetic bead extraction unit 1 and add them to the magnetic bead activator component 5 for magnetic bead activation;

[0076] (2) The activated magnetic beads are added to the sample-containing component 3, and the magnetic beads bind to the target molecules in the sample;

[0077] (3) Take out the magnetic beads from step (2) and put them into the component 6 containing the washing liquid for washing;

[0078] (4) Take out the washed magnetic beads and add them to the component 7 containing the eluent for elution, so that the target molecules are separated from the magnetic beads;

[0079] (5) Remove the magnetic beads from step (4), and use the remaining solution for detection.

[0080] When multiple samples need to be tested simultaneously, the combination of magnetic bead extraction unit 1 and test tube rack 10, along with the well plate 19, can be placed together in the magnetic bead extraction instrument for magnetic bead extraction. For example, if 17 samples need to be tested simultaneously, the 96-well plate 20 can be used for 16 samples. One combination of magnetic bead extraction unit 1 and test tube rack 10, along with one 96-well plate 20, can be placed together in the magnetic bead extraction instrument for magnetic bead extraction. Similarly, when multiple samples need to be tested, the required number of magnetic bead extraction units 1, or the required number of magnetic bead extraction units 1 and 96-well plates 20, can be calculated and then the magnetic beads can be extracted using the magnetic bead extraction instrument.

[0081] Example 3: Pre-packaged catecholamine magnetic bead extraction kit

[0082] Magnetic solid-phase extraction utilizes magnetic nanoparticles as adsorbent materials to efficiently separate target compounds from complex samples. The principle involves using an external magnetic field to separate the magnetic particles from the sample, followed by washing and elution steps to obtain the purified target substance.

[0083] I. Composition of the pre-aliported reagent kit

[0084] The composition of the pre-dispensed magnetic bead extraction kit for catecholamines provided by this invention is shown in Table 1, wherein the tube number and... Figure 6 , Figure 7 The hole positions shown are consistent, starting from the first tube and numbered 1, 2, 3, 4, 5, and 6 in sequence.

[0085] Table 1. Components of the Catecholamine Pre-filled Kit

[0086]

[0087]

[0088] II. Reagent Preparation and Sample Testing

[0089] (1) Preparation of standard products and quality control products

[0090] Epinephrine (E), norepinephrine (NE), dopamine (DA), metanephrine (MN), nometanephrine (NMN), and 3-methoxytyramine (3-MT) standards were prepared into a mixed solution. A blank urine sample containing a stabilizer was used as the matrix to prepare the standard curve and quality control samples. Epinephrine, norepinephrine, dopamine, metanephrine, nometanephrine, and 3-methoxytyramine were available in 10 series concentrations (S1–S10) in the urine calibrator, as shown in Table 2.

[0091] Table 2 Concentration of Urine Standards

[0092]

[0093] Epinephrine, noradrenaline, dopamine, epinephrine, nomethylepinephrine, and 3-methoxytyrosine have two series of concentrations in urine quality control samples: low (L) and high (H), as shown in Table 3.

[0094] Table 3 Concentration of Urine Standards

[0095]

[0096] (2) Preparation of internal standard solution

[0097] Internal standard mixed working solutions were prepared with concentrations of 0.5 ng / mL for epinephrine-IS, 2.5 ng / mL for norepinephrine-IS, 2.50 ng / mL for dopamine-IS, 0.8 ng / mL for epinephrine-IS, 5.0 ng / mL for norepinephrine-IS, and 4.0 ng / mL for 3-methoxytyrosine-IS. For internal standard pretreatment, 50 μL of internal standard was added to 500 μL of sample.

[0098] (3) Sample testing

[0099] ① Place the unopened pre-dispensed catecholamine extraction reagent (refer to the test tube number in Table 1) into the 6-tube U-shaped bottom base and centrifuge at 4000 rpm for 0.5 min for subsequent operations. See below. Figure 5 ;

[0100] ②For example Figure 6 As shown, tear open the seal and transfer 100μL of the sample to be tested (urine, standard, or quality control) to columns 3 and 9.

[0101] ③Automatic extraction process of magnetic beads:

[0102] Place the 6-tube base into the fully automatic extractor and run the extraction method. The steps are shown in Table 4.

[0103] Table 4 Operating Procedures of the Fully Automated Magnetic Bead Extractor

[0104] Extraction steps instruction Mixing time / s Magnetization time / s Hole position 1 activation 60 30 Columns 1 and 7 2 balance 60 30 Columns 2 and 8 3 Sample 150 60 Columns 3 and 9 4 Rinse 1 60 30 Columns 4 and 10 5 Rinse 2 60 30 Columns 5 and 11 6 Washout 150 60 Columns 6 and 12 7 Discarded magnetic beads 10 N / A Columns 1 and 7

[0105] After extraction is complete, transfer the reagents from columns 6 and 12 to the microporous filter plate, and place the receiving plate under the microporous filter plate (e.g., Figure 7 The microporous filter plate and the receiving plate were centrifuged together at 4000 rpm for 5 min. The solution in the receiving plate was used for subsequent liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection. The LC-MS / MS detection method is as follows:

[0106] Liquid chromatography-tandem mass spectrometry system: AB SCIEX Triple Quad 4500MD; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol; Injection volume: 30 μL; Flow rate: 0.5 mL / min; Column temperature: 40℃; Gradient elution program is shown in the table below:

[0107] Table 5 Gradient elution program

[0108]

[0109]

[0110] Electrospray ionization (ESI) was used for positive ion MRM scanning analysis. The Q1 / Q3 ion channels are shown in the table below. The curtain gas (CUR) setting was 25, the temperature was 550℃, and the values ​​for GAS1 and GAS2 were 55. Chromatographic and mass spectrometric conditions can be adjusted according to actual needs. The mass spectrometry parameters are shown below:

[0111] Table 6 Mass Spectrometry Parameters

[0112] compound Q1(amu) Q3 (amu) DA 154.1 91 DA-IS 158.1 95 E 184.1 107 E-IS 187.1 107 NE 152.1 107 NE-IS 158.1 111 3-MT 151 119 3-MT-IS 155 123 MN 198 165 MN-IS 201 168 NMN 166 134 NMN-IS 169 137

[0113] III. Results Analysis

[0114] (1) Linearity: The standard curve was established using the internal standard method, and the linearity verification record is shown in Table 7.

[0115] Table 7. Validation results of the standard curve for urinary catecholamines

[0116]

[0117] The results show that the linear relationship of urine sample detection is good within the concentration range.

[0118] (2) Accuracy and precision

[0119] Urine samples were spiked at two concentrations (low and high, 2% by volume) and the actual values ​​after spiking were measured to examine the accuracy and precision of urinary catecholamine detection. The results are shown in Table 8.

[0120] Table 8. Validation results of the accuracy and precision of urinary catecholamines.

[0121]

[0122] According to Table 8, the accuracy bias (Bias) and coefficient of variation (CV) of the detection of catecholamines and their metabolites in urine samples were all less than 10%, and the accuracy and precision met the acceptance criteria.

[0123] Example 4: Screening and optimization of magnetic bead activators

[0124] In the sample extraction process for catecholamine detection, the long-term stability of the reagents in the pre-aliphatic extraction kit after being dispensed into 6-tube strips is an important evaluation indicator. Current magnetic bead extraction methods typically use 96-well plates placed in a magnetic bead extraction instrument. These 96-well plates are pre-filled with magnetic bead activator, magnetic bead eluent, and magnetic bead elution buffer. Because the wells of the 96-well plate are tightly packed and encased in a plastic shell (such as…),… Figure 9As shown), the reagent remains stable after a certain period of storage, exhibiting good detection performance. To verify whether the magnetic bead activator suitable for 96-well plates also exhibits good long-term stability when used in the magnetic bead extraction unit provided by this invention, this embodiment dispenses the magnetic bead equilibration solution ammonium acetate solution (ammonium acetate content of 38.5%) used in the prior art into a 96-well plate and the magnetic bead extraction unit of Embodiment 1 of this invention, respectively. The 96-well plate uses the middle 6 test tube wells (see...). Figure 8 The remaining reagents are the same as those in Example 3, and the magnetic bead extraction method is basically the same as in Example 2. Since the response sensitivity of morphopeptide, noradrenaline, epinephrine, and noradrenaline to the magnetic bead activator is the same, this example only lists the stability data of morphopeptide and noradrenaline (Table 9).

[0125] Table 9. Detection results of ammonium acetate activator in 96-well plates and magnetic bead extraction units.

[0126]

[0127] As shown in Table 9, the initial detection results of the same ammonium acetate magnetic bead activator after being aliquoted into 96-well plates and magnetic bead extraction units were basically consistent. When aliquoted into 96-well plates and stored for one year, the peak areas of norepinephrine and epinephrine in the samples detected upon reuse were basically consistent with the initial detection results. However, when aliquoted into the magnetic bead extraction unit, the peak areas of these two substances were significantly lower than the initial detection results. This indicates that ammonium acetate solution can be stored for a longer period when used in the 96-well plate extraction system. The difference may be due to the porous structure of the 96-well plate, with its tightly packed wells, reducing the possibility of reagent evaporation and making it easier to preserve the reagent. In contrast, the magnetic bead extraction unit consists of six rows of thin-walled tubes, making it more prone to evaporation or degradation during storage. Furthermore, it was found that ammonium acetate solution exhibited bacterial growth after long-term storage in the magnetic bead extraction unit, further affecting the detection stability. Therefore, the magnetic bead activators suitable for existing 96-well plates cannot meet the requirements for long-term preservation in the magnetic bead extraction unit, and further screening of magnetic bead activators that can be preserved in test tubes of the magnetic bead extraction unit for a long time is needed.

[0128] Therefore, this embodiment screened and optimized the magnetic bead activator packaged in the magnetic bead extraction unit, mainly considering the addition of acidic reagents or organic phases for comparative testing. Using the magnetic bead extraction unit 1 or 17 of Example 1, or the magnetic bead extraction system 17, the catecholamines in the samples were detected according to the magnetic bead extraction methods of Examples 2 and 3. The difference was that the magnetic bead activator in the kit used the seven reagents shown in Table 10, and the detection results are shown in the table below.

[0129] Table 10. Effect of magnetic bead activator on the stability of the catecholamine detection method.

[0130]

[0131] As shown in Table 10, the ammonium acetate-based magnetic bead activating reagent showed bacterial growth after 6 months of storage, and the detection results after one year were significantly lower than the initial detection values. Using formic acid, methanol, or ethanol as activators, no bacterial growth was observed after 6 months of storage, but the initial extraction efficiency varied depending on the concentration of formic acid, methanol, or ethanol. The peak areas of epinephrine and noradrenaline extracted and detected using 0.2% or 1% formic acid magnetic bead activators were significantly lower than those using 10% methanol or ethanol, and the peak areas of epinephrine and noradrenaline detected again after one year were significantly reduced, indicating that these two concentrations of formic acid solutions significantly volatilized or degraded after long-term storage. Furthermore, the extraction effect of 10% or 50% methanol or 50% ethanol was also less than that of 10% ethanol, and the detection values ​​after long-term storage were also lower than the initial results. When a 10% ethanol aqueous solution was used as the activator, the peak area response values ​​of epinephrine and noradrenaline were the highest. Even after long-term storage, the response values ​​of epinephrine and noradrenaline remained consistent with the initial values, indicating that the 10% ethanol aqueous solution can ensure sufficient extraction while also preventing bacterial growth during long-term storage of the activator, demonstrating its long-term stability. Therefore, a 10% ethanol aqueous solution is preferred as the magnetic bead activator.

[0132] Example 5: Screening and Optimization of Sample Buffer

[0133] The sample buffer provides a buffer system to regulate the pH of the sample solution to be extracted, promoting thorough extraction and elution by subsequent rinsing and elution buffers, while protecting catecholamines in the sample from degradation. This embodiment further investigates whether a buffer solution suitable for 96-well plates exhibits good long-term stability when aliquoted into the magnetic bead extraction unit provided by this invention. A combination buffer solution of 50mM ammonium acetate and 100mM sodium hydroxide, currently used, was aliquoted into a 96-well plate and the magnetic bead extraction unit of Example 1 of this invention, respectively. The 96-well plate used six middle test tube wells (see...). Figure 8 The remaining reagents are the same as those in Example 3, and the magnetic bead extraction method is basically the same as in Example 2. Since the response sensitivity of epinephrine, noradrenaline, epinephrine, and noradrenaline to the buffer solution is consistent, this example only lists the stability data for epinephrine and noradrenaline. The stability data for epinephrine and noradrenaline are shown in Table 11.

[0134] Table 11 Detection results of ammonium acetate and sodium hydroxide buffer solutions dispensed into 96-well plates and magnetic bead extraction units.

[0135]

[0136] The test results in Table 11 show that the ammonium acetate and sodium hydroxide buffer solutions initially produced consistent extraction effects in both the 96-well plate and the magnetic bead extraction unit of this invention. However, after being aliquoted into the magnetic bead extraction unit and stored for one year, the results were significantly lower than the initial values. Conversely, after being stored in a 96-well plate for one year, the results were consistent with the initial values. This indicates that the ammonium acetate and sodium hydroxide buffer solutions cannot be stored in the magnetic bead extraction unit for extended periods, and their stability decreases after a certain time. Therefore, it is necessary to screen for buffer solutions suitable for long-term storage in test tubes within the magnetic bead extraction unit.

[0137] Therefore, this embodiment further investigated the differences in the effectiveness of sample buffers with different components in extracting catecholamines from samples using the magnetic bead extraction unit, and compared the effects of the buffers after storage for a certain period of time. Using the magnetic bead extraction unit 1 or 17 of Example 1, or the magnetic bead extraction system 17, the catecholamines in the samples were detected according to the magnetic bead extraction methods of Examples 2 and 3. The difference was that the sample buffers in the kits used the four reagents shown in Table 12. The initial detection results of the prepared sample buffers and the results of the re-detection after one year of storage are shown in the table below.

[0138] Table 12 Detection results of sample buffer solutions with different formulations

[0139]

[0140]

[0141] As shown in Table 12, there were significant differences in the detection results after using different buffer formulations for magnetic bead extraction and detection. When using a buffer composed of ammonium bicarbonate and sodium hydroxide for magnetic bead extraction, the peak areas of epinephrine and noradrenaline were significantly lower than in other groups, and the response value of catecholamines decreased. This may be because the buffer system formed by these two formulations could not meet the pH requirements of the samples. Furthermore, except for the buffer composed of ammonium acetate and sodium hydroxide, which showed the highest extraction efficiency both initially and after one year of storage, the peak areas of epinephrine and noradrenaline in the other three buffer groups decreased significantly after one year of storage. This may be because when ammonium acetate is combined with potassium hydroxide, sodium hydroxide, or ammonium bicarbonate with sodium hydroxide, the ammonium salts in the buffer may volatilize or decompose after a certain period, producing new substances that disrupt the stability of the original buffer system. Alternatively, the effective concentration of sodium hydroxide or potassium hydroxide may decrease upon contact with air, weakening the alkalinity of the buffer and leading to pH imbalance. Only when ammonium acetate and sodium hydroxide are used in combination can degradation or volatilization be effectively mitigated or inhibited. Therefore, ammonium acetate and sodium hydroxide are preferred as sample buffers in this invention.

[0142] Example 6: Screening and Optimization of Eluent

[0143] In the magnetic bead extraction method, selecting a suitable eluent can effectively remove other interfering substances in the sample, ensuring that the magnetic beads bind only to the target substance, catecholamine, and that the eluted substances are free of impurities, thereby improving the extraction efficiency. Examples 4 and 5 have verified that existing magnetic bead activators are suitable for 96-well plates, but cannot be stored for long periods after being dispensed into the magnetic bead extraction unit. Therefore, this example also verifies whether the eluent suitable for 96-well plates, after being dispensed into the magnetic bead extraction unit provided by this invention, also has good long-term stability. Acetonitrile, the existing eluent, was dispensed into both a 96-well plate and the magnetic bead extraction unit of Example 1 of this invention. The 96-well plate uses the six middle test tube wells (…). Figure 8 The remaining reagents are the same as those in Example 3, and the magnetic bead extraction method is basically the same as in Example 2. Since the response sensitivity of epinephrine, noradrenaline, epinephrine, and noradrenaline to the eluent is consistent, this example only lists the stability data for epinephrine and noradrenaline. The stability data for epinephrine and noradrenaline are shown in Table 13.

[0144] Table 13 Detection results of acetonitrile eluent dispensed into 96-well plates and magnetic bead extraction units.

[0145]

[0146]

[0147] As shown in Table 13, the initial test results of the acetonitrile eluent in both the 96-well plate and the magnetic bead extraction unit of this invention were basically consistent. However, after being aliquoted into the magnetic bead extraction unit and stored for one year, the results of subsequent tests were significantly lower than the initial values. Conversely, after being stored in a 96-well plate for one year, the results were consistent with the initial values. This indicates that the acetonitrile eluent, when stored in the magnetic bead extraction unit for a certain period, will also undergo volatilization or degradation, and its stability will decrease. Therefore, it is necessary to screen for eluents suitable for long-term storage in test tubes within the magnetic bead extraction unit.

[0148] Therefore, this invention further considers using a two-step elution method with two elution solutions to examine the differences in the effectiveness of different elution solutions in extracting catecholamines from samples through the magnetic bead extraction unit. Using the magnetic bead extraction unit 1 or 17 of Example 1, or the magnetic bead extraction system 17, the catecholamines in the samples were detected according to the magnetic bead extraction methods of Examples 2 and 3. The difference lies in that the elution solutions in the kits used the reagents shown in Table 14. The initial detection results of the prepared elution solutions and the results of the re-detection after one year of storage are shown in the following table.

[0149] Table 14 Detection results of rinsing solutions with different formulations

[0150]

[0151] The results in Table 14 show that only by using methanol-water as the first eluent and acetonitrile as the second eluent can the reagent maintain excellent and stable extraction performance both initially after preparation and after one year of storage. Using eluents of ethanol-water, acetonitrile or formic acid-water, acetonitrile or formic acid-water, and acetic acid-water, the peak areas of extracted and detected epinephrine and noradrenaline were significantly lower than those using the methanol-acetonitrile combination. Furthermore, after one year of storage, the peak areas of epinephrine and noradrenaline were significantly reduced upon re-extraction and detection using these three eluent combinations, indicating that using these reagent combinations for elution reduced the catecholamine response. The effectiveness of using ethanol-water as the first eluent may be limited because it fails to effectively remove impurities from the sample (ethanol is more volatile than methanol, and long-term storage may reduce the ethanol content in the eluent). Formic acid-water or acetic acid-water elutes catecholamine analytes bound to the magnetic beads. Acetonitrile, after a certain storage period, undergoes significant volatilization or degradation, further reducing its effectiveness. Therefore, the volume of eluent 2 should be appropriately increased; this protocol uses 400 μL of acetonitrile. Using methanol-water as the first eluent and acetonitrile as the second eluent, both reagents work together to achieve better results. Therefore, methanol-water is preferred as the first eluent, and acetonitrile as the second eluent.

[0152] Example 7: Optimization of the eluent

[0153] Based on the results of Examples 4, 5, and 6, this invention has verified that the stability of existing magnetic bead activators, sample buffers, and eluents decreases after long-term storage in the magnetic bead extraction unit, resulting in deviations between the detection results and actual values. Therefore, this example also examines whether existing eluents for 96-well plates exhibit good long-term stability after being aliquoted into the magnetic bead extraction unit provided by this invention. Existing aqueous solutions containing 0.2% ammonium acetate, 16% methanol, and 0.1% formic acid were aliquoted into a 96-well plate and the magnetic bead extraction unit of Example 1 of this invention, respectively. The 96-well plate used the six middle test tube wells (see...). Figure 8 The remaining reagents are the same as those in Example 3, and the magnetic bead extraction method is basically the same as in Example 2. Since the response sensitivity of morphogenetic epinephrine, noradrenergic epinephrine, epinephrine, and noradrenergic epinephrine to the eluent is consistent, this example only lists the stability data for morphogenetic epinephrine and noradrenergic epinephrine. The stability data for morphogenetic epinephrine and noradrenergic epinephrine are shown in Table 15.

[0154] Table 15 Results of detection of ammonium acetate, methanol, and formic acid eluents dispensed into 96-well plates and magnetic bead extraction units.

[0155]

[0156] As shown in Table 15, the initial detection results of eluents containing ammonium acetate, methanol, and formic acid, when aliquoted into 96-well plates and magnetic bead extraction units, were basically consistent. When aliquoted into 96-well plates and stored for one year, the peak areas of norepinephrine and eluent in the samples were largely consistent with the initial detection results. However, when aliquoted into the magnetic bead extraction unit, the peak areas of these two substances were significantly lower than the initial detection results. This indicates that eluents containing ammonium acetate, methanol, and formic acid will also volatilize or degrade after being stored in the magnetic bead extraction unit for a certain period, thus reducing their stability. Therefore, the eluents suitable for existing 96-well plates cannot meet the long-term storage requirements of the magnetic bead extraction unit, and further screening of eluents that can be stored long-term in test tubes within the magnetic bead extraction unit is needed.

[0157] The magnetic bead extraction unit 1 or 17 of Example 1, or the magnetic bead extraction system 17, were used to detect catecholamines in the samples according to the magnetic bead extraction methods of Examples 2 and 3. The difference was that the eluent in the kit was the reagent shown in Table 16. The initial test results of the prepared eluent and the results of the retest after one year of storage are shown in Table 16.

[0158] Table 16 Detection results of eluents with different formulations

[0159]

[0160] Based on the above test results, the peak areas of norepinephrine and epinephrine detected after one year of storage in aqueous solutions containing ammonium acetate, methanol, and formic acid, 1% acetic acid in methanol, 1% acetic acid in ethanol, and formic acid in methanol and ammonium formate solutions with formic acid content below 1% were significantly lower than the initial detection values. This indicates that after these reagents were dispensed into 6-tube strips, the thin walls of the tubes made the components prone to volatility. Furthermore, during the testing process, it was found that the original 0.1% and 0.2% formic acid concentrations evaporated more than methanol over long-term storage. Even with the addition of ammonium formate, the acidity in the eluent decreased, thus affecting the elution efficiency of catecholamine compounds from the cation exchange material (WCX magnetic beads). Furthermore, when using 2% formic acid in methanol and 0.1M formate for elution, although the peak areas of epinephrine and noradrenaline did not change significantly at the initial preparation stage and after one year of storage, the elution effect was still inferior to that of the combined solution of 1% formic acid in methanol and 0.1M formate formate. This indicates that selecting a 1% formic acid in methanol and 0.1M formate formate solution as the eluent can ensure the elution effect at the initial preparation stage, reduce the volatility of formic acid, and maintain a stable elution effect even after one year of storage. Therefore, a 1% formic acid in methanol and 0.1M formate formate solution is preferred as the eluent.

[0161] Example 8: Comparison of pre-packaging and non-pre-packaging methods

[0162] To further verify that the pre-packaged catechol reagent kit provided by this invention can achieve the same detection effect as existing non-pre-packaged methods, this embodiment further uses the magnetic bead extraction unit (pre-packaged method) of the pre-packaged catechol reagent kit in Example 1, and applies the same reagent (non-pre-packaged method) to a 96-well plate for detection. The extraction and detection methods are basically the same as in Examples 2 and 3. The detection results of the two methods are shown in Tables 17 and 18.

[0163] Table 17 Comparison of extraction results between pre-packaging and non-pre-packaging methods

[0164]

[0165] Continued from Table 17

[0166]

[0167] Table 18 Comparison of catecholamine detection values ​​between pre-packaging and non-pre-packaging methods

[0168]

[0169] Continued from Table 18

[0170]

[0171] As can be seen from the table results, the levels of dopamine, adrenaline, noradrenaline, and other substances detected by the pre-packaging kit for catecholamine extraction in the samples were not significantly different from those detected by the non-pre-packaging method. The extraction effect was equivalent to that before pre-packaging, indicating that the pre-packaging kit provided by this invention has excellent extraction effect and is simpler and faster to operate.

[0172] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A magnetic bead extraction unit, characterized in that, It includes an integrated structure consisting of a component for containing magnetic beads, a component for containing samples, and a component for processing samples with magnetic beads, for magnetic bead extraction of a single sample.

2. The magnetic bead extraction unit as described in claim 1, characterized in that, The components for processing samples with magnetic beads include a component for containing magnetic bead activator, a component for containing washing liquid, and a component for containing elution liquid.

3. The magnetic bead extraction unit as described in claim 2, characterized in that, The components containing magnetic beads, magnetic bead activator, sample, washing solution, and elution solution are all reagent tubes. Each reagent tube is connected to the others to form a row of six tubes.

4. The magnetic bead extraction unit as described in claim 3, characterized in that, The component containing the sample contains a diluent and a buffer solution, and the sample is for the detection of catecholamines.

5. The magnetic bead extraction unit as described in claim 4, characterized in that, The magnetic bead activator comprises an aqueous ethanol solution, the diluent comprises aqueous phosphoric acid, the buffer comprises ammonium acetate and sodium hydroxide, the washing solution comprises methanol and acetonitrile, and the elution solution comprises a methanol formic acid and an ammonium formic acid solution.

6. A magnetic bead extraction system, characterized in that, It includes a magnetic bead extraction unit as described in any one of claims 1 to 5, and a test tube rack for placing the magnetic bead extraction unit.

7. A pre-packaged kit for extracting catecholamines, characterized in that, It includes the magnetic bead extraction unit as described in any one of claims 1 to 5.

8. A method for extracting magnetic beads, characterized in that, The magnetic bead extraction unit as described in any one of claims 1 to 5, or the magnetic bead extraction system as described in any one of claims 6, is used to extract target molecules from the sample using magnetic beads.

9. The method as described in claim 10, characterized in that, Includes the following steps: (1) Adsorb magnetic beads from the magnetic bead extraction unit into the magnetic bead activator in the magnetic bead activator; (2) The activated magnetic beads are added to the component containing the sample, and the magnetic beads bind to the target molecules in the sample; (3) Take out the magnetic beads from step (2) and put them into the component containing the washing liquid for washing; (4) Take out the washed magnetic beads and add them to the component containing the eluent for elution, so that the target molecules are separated from the magnetic beads; (5) Remove the magnetic beads from step (4), and use the remaining solution for detection.

10. The use of formic acid, methanol, and ammonium formate in the preparation of the eluent in a pre-filled kit for catecholamine extraction, characterized in that, The catecholamine extraction pre-packaging kit includes the magnetic bead extraction unit as described in any one of claims 1 to 5.

Images