Method for processing samples for liquid chromatography and mass spectrometry

Abstract

The present invention relates to a method for preparing a sample for liquid chromatography and mass spectrometry, and the use of hydrophilic surface magnetic beads for preparing a sample for liquid chromatography and mass spectrometry. The method includes contacting hydrophilic surface magnetic beads and a precipitant with a sample to form precipitate-magnetic bead aggregates, and applying a magnetic field to remove the precipitate-magnetic bead aggregates. This sample processing method can be used for preparing or pretreating samples for mass spectrometry or liquid chromatography, particularly liquid chromatography-tandem mass spectrometry.

Description

[Technical Field]

[0001] The present invention belongs to the field of biology, and specifically relates to a method for processing samples for liquid chromatography and mass spectrometry. [Background technology]

[0002] Biological samples generally refer to animal (including human) body fluids (e.g., whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, lymph, and other secretions of the body), muscles, hair, and some tissues and organs (e.g., liver, lung, kidney, brain, stomach, thymus, pancreas, etc.). Biological samples, especially clinical samples, have complex compositions, and the protein components therein are particularly likely to interfere with subsequent analytical detection. Therefore, the samples need to be pretreated to remove protein impurities before analytical detection.

[0003] Sample pretreatment is a critical step in bioanalysis. For clinical laboratory liquid chromatography and mass spectrometry, especially liquid chromatography-tandem mass spectrometry (LC-MS / MS), biological sample pretreatment is crucial before analytical detection. The primary purpose of sample pretreatment is to separate one or more target analytes from other components in the sample matrix. Sample pretreatment is a crucial step in the development and optimization of LC-MS / MS methods. In many cases, sample pretreatment is a bottleneck in high-throughput detection and a major source of analytical error. In LC-MS / MS methods, improper sample pretreatment often leads to failure due to suppression of target analyte signals or interference from coexisting substances in the biological matrix. In particular, improper sample treatment can contaminate the analytical pipeline. For example, insoluble substances such as impurities (e.g., proteins) can easily clog the pipeline and column if they enter the LC-MS / MS system, potentially affecting the analysis.

[0004] Sample pretreatment methods used in conventional techniques include centrifugation, settling, protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE), etc. However, the pretreatment methods in conventional techniques have drawbacks such as complicated and time-consuming operations and the inability to automate them.

[0005] For example, protein impurities can be removed by centrifugal separation or static separation, which are currently commonly used, but these procedures are cumbersome and difficult to automate, and static separation takes a long time.

[0006] CN113376270B discloses a pretreatment method for detecting protein precipitates in serum samples using high-performance liquid chromatography-tandem mass spectrometry, which includes a step of treating the sample using a negative pressure device. While this method is simple to operate, the protein precipitate essentially dilutes the sample, resulting in a low concentration of the analyte in the sample on the instrument, which requires high sensitivity from the measuring instrument. Furthermore, the method requires negative pressure operation, which is disadvantageous for achieving full automation of the operation flow.

[0007] CN11079780A discloses a pretreatment method for detecting serum samples using high-performance liquid chromatography-tandem mass spectrometry, which involves protein precipitation using ethanol, liquid-liquid extraction using n-hexane, drying of the extract using nitrogen spray, and reconstitution of the sample before analysis. The experimental process of this method involves steps such as extraction and nitrogen spraying, which require a large amount of manual labor, making it unsuitable for automation. Furthermore, the complicated operation process places increased demands on the experimental staff.

[0008] CN1111198238A discloses a method for extracting and detecting samples in serum. This method utilizes protein precipitation and solid-phase extraction processing. However, when the solid-phase extraction packing comes into contact with organic reagents during the experiment, swelling occurs, affecting the flow rate of the solution passing through the packing and resulting in poor pore size uniformity. Furthermore, the drying process using nitrogen spraying is also disadvantageous for automation.

[0009] Therefore, the pretreatment methods for removing proteins from samples in the prior art have obvious drawbacks, such as requiring too much manual work and taking too long a time, making it difficult to realize automated sample processing and unable to meet the needs of testing.In detection practice, especially in clinical detection, there is an objective need to provide an automated processing method that can remove protein impurities from samples. Summary of the Invention

[0010] As a result of extensive experimental research, the inventors have surprisingly discovered that impurities in samples for liquid chromatography and mass spectrometry can be effectively removed by contacting the sample with hydrophilic surface magnetic beads (particularly silica gel magnetic beads) and a precipitant, and that this is advantageous for automating the operation.

[0011] Based on this finding, in a first aspect of the present invention, there is provided a method for processing a sample for liquid chromatography and / or mass spectrometry, comprising the steps of contacting hydrophilic surface magnetic beads and a precipitant with the sample to form precipitate-magnetic bead aggregates, and applying a magnetic field to remove the precipitate-magnetic bead aggregates.

[0012] The hydrophilic surface magnetic beads according to the present invention are magnetic particles having a magnetic core and a coating material coated on the surface of the magnetic core, the coating material being hydrophilic. The coating material can be silica gel or modified silica gel (e.g., amino-modified silica gel, carboxyl-modified silica gel, or unmodified bare silica gel). The term "silica gel magnetic beads" used herein refers to magnetic beads having a silica gel layer coated on the outer surface of the magnetic core. The term "hydrophilic surface magnetic beads" used herein refers to, for example, silica gel-based magnetic beads having hydrophilic surfaces. The silica gel magnetic beads include magnetic beads whose silica surface has been further functionalized, such as amino-modified silica gel magnetic beads, carboxyl-modified silica gel magnetic beads, and hydroxyl-modified silica gel magnetic beads. Magnetic beads without the functionalization are referred to herein as "unmodified bare silica gel magnetic beads."

[0013] Preferably, the magnetic beads have an appropriate particle size, for example, an average particle size of 0.1 to 300 μm, 0.2 to 250 μm, 0.3 to 200 μm, 0.4 to 150 μm, 0.5 to 100 μm, 0.6 to 80 μm, 1 to 200 μm, 10 to 100 μm, or 30 to 50 μm.

[0014] In the present invention, the term "sample" refers to a product form derived from a component to be detected. In the present specification, the sample is a biological sample, which may be, for example, a clinical sample, but is preferably selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph. The sample may contain a biological material selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph. In the present specification, the sample further includes the above-mentioned processed biological material, for example, processed whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph.

[0015] In the present invention, the magnetic core of the magnetic beads comprises magnetic particles. The magnetic particles are made of any magnetic material, preferably selected from oxides of iron, cobalt, and nickel, and more preferably triiron tetroxide. It is known in the art that the magnetic core can be produced by any suitable method.

[0016] In the present invention, contacting the hydrophilic magnetic beads and the precipitant with the sample includes: (1) contacting the sample with a precipitant to form a precipitate, and then contacting the precipitate with hydrophilic magnetic beads to form a precipitate-magnetic bead aggregate; or The method may include a method (2) in which hydrophilic surface magnetic beads and a precipitant are brought into contact with the sample together to form a precipitate-magnetic bead aggregate.

[0017] Preferably, contacting the hydrophilic surface magnetic beads and the precipitant with the sample is achieved by contacting the hydrophilic surface magnetic beads and the precipitant with the sample together.

[0018] As used herein, "contacting the hydrophilic surface magnetic beads and the precipitant with the sample together" means exposing the hydrophilic surface magnetic beads and the precipitant to the sample together, with no clear difference in priority between the time points of exposure of the magnetic beads and the sample, and the time points of exposure of the precipitant and the sample. In a more specific embodiment, the magnetic beads and the precipitant are contacted with the sample essentially at the same time, but do not necessarily have to be contacted at the same time in the strict sense.

[0019] In this specification, "magnetic beads with hydrophilic surfaces" are preferably silica gel-based magnetic beads with hydrophilic surfaces.

[0020] As used herein, contacting the hydrophilic surface magnetic beads and the precipitant with the sample further includes mixing, shaking, vibrating, vortexing and / or stirring the mixture of the sample, magnetic beads and the precipitant.

[0021] Without being limited by theory, in the present invention, when a precipitant is brought into contact with a sample, a precipitate is formed from impurities (e.g., proteins) in the sample. If magnetic beads are present in the system, the precipitate can bind to the magnetic beads, which have hydrophilic surfaces, so as to envelop the magnetic beads, simultaneously with or after their formation, forming a precipitate-magnetic bead aggregate.

[0022] In some embodiments, the precipitant includes a protein precipitant. As is well known to those skilled in the art, a protein precipitant is a substance capable of precipitating proteins in a sample, including substances that dehydrate and precipitate proteins, such as organic solvents such as acetonitrile, methanol, and ethanol, preferably acetonitrile. The protein precipitant may optionally include, for example, a water-immiscible organic solvent (e.g., an alkane / cycloalkane, ethyl acetate). Other suitable precipitants known to those skilled in the art may also be used. Without being limited by theory, in the present invention, the precipitant is added to prevent insoluble substances from entering the system during LC / MS analysis, clogging the pipelines and columns and adversely affecting the analysis.

[0023] As a result of further investigation, the present inventors have found that the application of a magnetic field makes it possible to remove the formed precipitate (precipitate-magnetic bead aggregates). For example, by applying a magnetic field, it is possible to rapidly and sufficiently move a sample, particularly a liquid (including a solution or suspension), while also avoiding problems such as clogging of a column due to residual magnetic material from the transfer. Based on this finding, it is possible to automatically remove impurities that have precipitated under the application of a magnetic field.

[0024] Accordingly, the method of the present invention includes applying a magnetic field to remove the precipitate-magnetic bead aggregate. More specifically, applying a magnetic field to remove the precipitate-magnetic bead aggregate may be achieved by placing a magnet on the outer wall of a container containing the sample and attracting the magnetic beads bound to the precipitate to the inner wall of the container when the magnetic field is applied (e.g., the sample can be separated from the magnetic beads bound to the precipitate by sucking in the sample solution or suspension), or by inserting a magnet into the container containing the sample to attract the magnetic beads bound to the precipitate, and then removing the magnet. In some embodiments, applying a magnetic field to remove the precipitate-magnetic bead aggregate is achieved by removing the sample solution from the container containing the sample and the magnetic beads, or by removing the magnetic beads bound to the precipitate from the container containing the sample and the magnetic beads.

[0025] The magnet may be a permanent magnet or an electromagnet, and is preferably an electromagnet because it is easy to control the presence or absence of a magnetic field by turning it on and off. In the case of an electromagnet, the on and off of the electromagnet can also be controlled by a programmable logic controller (PLC) to meet the needs of fully automated sample processing.

[0026] In a specific operation, a magnet can be placed or inserted into a solution or suspension containing a sample.

[0027] In one embodiment, applying a magnetic field to remove the precipitate-magnetic bead aggregates is achieved by placing a magnet on the outer wall of a container containing a sample, and attracting the magnetic beads bound to the precipitate (i.e., the precipitate-magnetic bead aggregates) to the inner wall of the container when a magnetic field is applied. In a more specific embodiment, the magnet can be located at two positions: a side suction position and a bottom suction position. The magnet located at the side suction position is located on the outer surface of the side wall of a container containing a sample solution or suspension, so that the magnetic beads bound to the precipitate are attracted to the inner wall of the container when a magnetic field is applied (e.g., by turning on an electromagnet or moving a permanent magnet). On the other hand, the magnet located at the bottom suction position is located below the bottom wall of a container containing a sample solution or suspension, so that the magnetic beads bound to the precipitate are attracted to the bottom wall of the container when a magnetic field is applied (e.g., by turning on an electromagnet or moving a permanent magnet). In some embodiments, there may only be magnets in the side attraction position, in other embodiments there may only be magnets in the bottom attraction position, and in other embodiments there may be magnets in both the side attraction position and the bottom attraction position simultaneously.

[0028] By combining switching of the magnet position and / or the electrical current / current state with the liquid (e.g., solution) transfer operation, automatic transfer of liquid (e.g., solution) between different containers can be effectively realized.

[0029] Furthermore, in the case of removing precipitated impurities (e.g., proteins) under the application of a magnetic field by placing a magnet on the outer wall surface of a container containing a sample solution or suspension so that magnetic beads bound to the precipitate are attracted to the inner wall surface of the container when a magnetic field is applied, the present invention can more effectively achieve sufficient liquid movement by using a magnet in the "side suction position." Compared to the "bottom suction position" in which the magnet is located on the bottom wall of the container, a magnet in the side suction position can more effectively avoid disadvantages such as liquid loss due to the magnetic field in the bottom suction position (liquid may not be sufficiently suctioned from the bottom), magnetic material remaining due to suction, and / or clogging of the chromatography column, and also makes the transfer operation more convenient and easier to automate.

[0030] Based on the above findings, in the method of the present invention, the removal of the precipitate-magnetic bead aggregates by applying a magnetic field can be achieved by placing a magnet on the outer wall surface (preferably the outer surface of the side wall) of a container containing a sample solution or suspension so that the magnetic beads bound to the precipitate are attracted to the inner wall surface (preferably the inner surface of the side wall) of the container when a magnetic field is applied, or by inserting a magnet (e.g., a magnetic rod) into a container containing a sample solution or suspension, adsorbing the magnetic beads bound to the precipitate, and then removing the magnet.

[0031] In the method of the present invention, the container may be a multiwell plate or a separation tube. The multiwell plate may be, for example, a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. The container may have a U-shaped or V-shaped bottom.

[0032] In some embodiments of the present invention, the transfer (movement and / or separation) of the sample in the method is achieved by drawing a liquid (or fluid) from one container and expelling the liquid (or fluid) into another container.

[0033] In the method of the present invention, sample transfer between different containers can be achieved by moving the pipette up and down within the container. Preferably, the base of the pipette is provided with an elastic element that allows the pipette to resiliently retract when it moves downward and contacts the bottom of the container. Preferably, the base of the pipette is provided with an adjustment structure that allows the stop position of the pipette tip to be adjusted to maximally approach the bottom of the container without damaging the pipette and / or the container.

[0034] The present invention also relates to a sample detection method comprising a step of pretreating a sample by the above-mentioned method. Furthermore, the present invention relates to a sample detection method comprising a step of analyzing the pretreated sample by an analytical method. Preferably, the analytical method is selected from mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). In one embodiment, the sample analysis method of the present invention comprises a step of mixing the sample with a solution or suspension of an internal standard substance, followed by pretreatment and analysis.

[0035] The present invention also provides a kit for pre-detection treatment of a sample to be used in mass spectrometry or liquid chromatography, which kit comprises magnetic beads with hydrophilic surfaces (e.g., silica gel magnetic beads, particularly bare silica gel magnetic beads that are not surface-modified) and, optionally, a reference substance.

[0036] The kit of the present invention can be used to carry out the sample pretreatment method or sample detection method described in the present invention.

[0037] According to another aspect of the present invention, there is provided the use of hydrophilic surface magnetic beads (e.g., silica gel magnetic beads, particularly bare silica gel magnetic beads without surface modification) to remove impurities (including proteins) from a sample.

[0038] By using the method of the present invention, it is not necessary to perform centrifugation or ultrasonic operations that are commonly used in conventional techniques, and nitrogen spraying treatment is also not required, so it is possible to achieve a significant improvement in automation of operations, labor savings, reduction in working hours, and a significant improvement in efficiency.

[0039] Terminology In this specification, the terms "magnetic beads," "magnetic granules," "magnetic particles," and "magnetic grains" refer to particles that have magnetic properties and are used interchangeably.

[0040] As used herein, "hydrophilic surface magnetic beads" refers to magnetic particles having a magnetic core and a hydrophilic coating material coated on the surface of the magnetic core. The surface coating material may be silica gel or a modified silica gel, such as silica gel modified with an amino group or silica gel modified with a carboxyl group. As used herein, hydrophilic surface magnetic beads are silica gel magnetic beads, and unmodified bare silica gel magnetic beads are particularly preferred.

[0041] As used herein, "contacting a sample with hydrophilic surface magnetic beads and a precipitant" means exposing the sample to the magnetic beads and the precipitant to cause an interaction between them. The contact may be prioritized, e.g., the sample may be contacted with the precipitant first, and then the precipitate may be contacted with the magnetic beads, or there may be no priority, i.e., the two may be contacted simultaneously. The inventors have surprisingly discovered that the treatment effect of contacting a sample with hydrophilic surface magnetic beads and a precipitant together to form a precipitate-magnetic bead aggregate is not inferior to the effect of contacting the sample in a stepwise manner (first contacting the sample with the precipitant, then contacting the precipitate with the magnetic beads), and that such simultaneous contact is more advantageous for automating operations and reducing processing time.

[0042] In this specification, particle size refers to the average particle size of a group of particles. Methods for measuring particle size include sieving, microscopy, sedimentation, and resistance methods.

[0043] As used herein, "liquid chromatography" refers to chromatography that uses a liquid as the mobile phase but has multiple forms of stationary phase. "Mass spectrometry" refers to a method that uses electric and magnetic fields to separate and detect moving ions (including charged atoms, molecules or molecular fragments, molecular ions, isotope ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, and ions resulting from ion-molecule interactions) based on their mass-to-mass ratio. As used herein, liquid chromatography or mass spectrometry also includes combined or combined methods such as liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0044] As used herein, the terms "silica gel matrix magnetic beads," "silica gel-based magnetic beads," or "silica gel magnetic beads" have the same meaning and are used interchangeably.

[0045] As used herein, the term "selected from" means that it may be any of the options listed before it, or a combination of one or more of the options.

[0046] As used herein, the term "sample" refers to a detection target, and may be a biological sample, such as a clinical sample of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, or lymph, or may be a biological material selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph. The sample may be any combination of the samples. The sample may be in a liquid or fluid form, or in a solid form. A solid sample may be optionally subjected to a treatment such as homogenization before detection. [Brief explanation of the drawings]

[0047] [Figure 1] 1 is a schematic diagram of a magnet placed at a side attraction position in accordance with the present invention. FIG. [Figure 2] FIG. 1 is a schematic diagram of a magnet disposed at a bottom attraction position in the present invention. [Figure 3] This is a photograph showing the impurity removal effect of different types of magnetic beads, where the numbers written on the containers indicate the type of magnetic beads. [Figure 4] Photographs showing the effect of different sample addition orders on the impurity removal effect of magnetic beads, where the numbers written on the containers indicate the type of magnetic beads. [Figure 5] In Example 3, the consistency of the results obtained by measuring the vitamin B1 content in 10 serum samples using the magnetic bead method of the present invention and the conventional precipitation method is shown. [Figure 6] In Example 4, the consistency of the results of measuring the vitamin B1 content in plasma samples using the magnetic bead method of the present invention and the conventional precipitation method is shown. [Figure 7] In Example 4, the consistency of the results of measuring the vitamin B1 content in whole blood samples using the magnetic bead method of the present invention and the conventional precipitation method is shown.

[0048] In Figure 1, a magnet 4 is positioned on the outer surface of the side wall of a container 1 (well plate) containing a sample solution or suspension 2, and under the action of the external magnetic field of the magnet 4 in the side suction position, magnetic beads 3 are collected and fixed on the side wall of the container 1. In this way, it is ensured that the liquid in the container 1 is sufficiently sucked up without affecting the transfer operation of the solution or suspension 2.

[0049] In Figure 2, a magnet 4 is positioned on the underside of the bottom wall of a container 1 (well plate) containing a sample solution or suspension 2, and when the magnet 4 is in the bottom suction position, the sample solution or suspension 2 in the container 1 can be stirred or shaken. DETAILED DESCRIPTION OF THE INVENTION

[0050] Unless otherwise specified, all reagents and instruments used in the following examples are commonly used in the art and can be purchased from chemical or biological product / preparation companies. In addition, all methods used in the following examples are conventional methods in the art, and those skilled in the art can easily understand the experimental procedures and obtain the results of these examples according to conventional techniques or manuals provided by manufacturers. [Example]

[0051] The materials, reagent manufacturers, and instrument model numbers used in the present invention are shown in the table below.

[0052] [Table 1]

[0053] In this example, the following magnetic beads were used.

[0054] Impurity removal magnetic beads 1 and 2 were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd. and were superparamagnetic materials with a core-shell structure, consisting of a superparamagnetic triiron tetroxide core and a silica layer modified with functional groups on the surface (wherein the silica layer was modified by coupling with functional groups, including amino and carboxyl groups). Impurity removal magnetic beads 1 were a mixture of magnetic beads modified with amino groups and magnetic beads modified with carboxyl groups, with the ratio of amino groups to carboxyl groups being 6:4. Impurity removal magnetic beads 2 had a higher proportion of beads modified with primary and secondary amino groups than impurity removal magnetic beads 1. Impurity removal magnetic beads 1 and 2 used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd. and had model numbers MSi050 / FLD01 and MSi050 / FLD02, respectively.

[0055] The silica gel matrix carboxyl magnetic beads were magnetic beads with a superparamagnetic triiron tetroxide core and a carboxyl-modified silica shell. The silica gel matrix carboxyl magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd. (model number MSi050 / WCX). They were 1.5 μm-sized hydrophilic magnetic polymer beads with a sandwich structure, consisting of a porous polymer microsphere core and a polymer cladding made of different materials to meet various application needs. The pores between the core and the core were filled with a magnetic material. The model number was MPHCC-150.

[0056] The silica gel matrix amino-based magnetic beads were magnetic beads with a superparamagnetic triiron tetroxide core and a shell layer of silica surface-modified with amino groups. The silica gel matrix amino-based magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., and had the model number MSi050 / MAX.

[0057] The agarose matrix carboxyl magnetic beads were magnetic beads with a superparamagnetic iron tetroxide core and a carboxyl-modified agarose shell. The manufacturing method is described in CN11318833A. The agarose matrix carboxyl magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., model number MAgr100K / WCX.

[0058] The unmodified bare silica gel magnetic beads were magnetic beads with a superparamagnetic iron tetroxide core and unmodified silica shell. The unmodified bare silica gel magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd. and had the model number EM001.

[0059] [Example 1] Protein removal effect of different types of magnetic beads The types of magnetic beads used here are: (1) Magnetic beads for impurity removal 1 (2) Magnetic beads for impurity removal 2 (3) Agarose matrix carboxyl group magnetic beads (4) Silica gel matrix carboxyl group magnetic beads (5) Silica gel matrix amino-group magnetic beads (6) Bare silica gel magnetic beads with no surface modification (7) PS magnetic beads (polystyrene and divinylbenzene matrix, manufactured by Suzhou Aijie Boya Bioelectronics Technology Co., Ltd., neutral styrene / divinylbenzene reversed-phase adsorption magnetic extraction material, model number: BNMA12300010-0) (8) C18 magnetic beads (silica gel matrix, 20-40 μm, manufactured by Suzhou Aijie Boya Bioelectronics Technology Co., Ltd., model number: BNMS9300001-0)

[0060] A. Protein Precipitant: Acetonitrile Sample Processing: 200 μL of acetonitrile precipitant was added to 100 μL of serum sample (each sample was tested in three parallel tests) and mixed well by vortexing. 50 μL of magnetic bead working solution (containing 1 mg of magnetic beads in 50% methanol solution) was then added and mixed well by vortexing. The precipitate was attached to the magnetic beads under the action of an applied magnetic field. The supernatant was aspirated and placed in a microplate reader to measure the absorbance at a wavelength of 620 nm.

[0061] conclusion Magnetic beads (1)-(2) and (4)-(6) were able to effectively capture and remove precipitates (mainly containing proteins), but the results of capturing precipitates using magnetic beads (3) (carboxyl agarose matrix beads) were not good.

[0062] B. Protein Precipitants: Acidic Precipitant 1 (10% TCA), Acidic Precipitant 2 (10% TCA: pure methanol = 6:4)

[0063] Sample Processing: To a 250 μL serum sample, 250 μL of acidic precipitant (Acidic Precipitant 1 or Acidic Precipitant 2) was added, mixed thoroughly by vortexing, and then 40 μL (4 mg) of magnetic bead working solution was added. The mixture was mixed thoroughly by vortexing, and the supernatant was collected. The magnetic beads (1), (4), (5), (7), and (8) listed above were used.

[0064] The results showed that magnetic beads 1 for impurity removal, amino-group magnetic beads with a silica gel matrix, and carboxyl-group magnetic beads with a silica gel matrix had a clear adsorption effect on proteins. After adding two types of precipitants separately, magnetic beads 1 for impurity removal, amino-group magnetic beads with a silica gel matrix, and carboxyl-group magnetic beads formed precipitate-magnetic bead aggregates that were adsorbed to the side wall by magnetic force, resulting in a clear liquid. The supernatant was subjected to chromatography and detected, revealing no clogging in the chromatography column and normal column pressure before and after measurement. The supernatant was centrifuged (4000 g, 10 min). No visible precipitate was observed, the liquid was clear, and no protein was produced, demonstrating effective protein adsorption.

[0065] After adding Acid Precipitant 1 or Acid Precipitant 2 separately, the precipitate-magnetic bead aggregates formed by the PS magnetic beads expanded, and some of the precipitate-magnetic bead aggregates were adsorbed to the side walls by magnetic force, resulting in a slight deterioration in the clarity of the liquid, indicating that the adsorption effect was insufficient.

[0066] After adding the above-mentioned acidic precipitant 1 or acidic precipitant 2 separately, the white protein and magnetic beads did not aggregate, and no precipitate-magnetic bead aggregates were formed, indicating that the adsorption effect of C18 magnetic beads was insufficient.

[0067] These results indicate that magnetic beads with poor surface hydrophilicity (e.g., PS magnetic beads and C18 magnetic beads) were not sufficiently effective in protein precipitation, whereas silica gel matrix magnetic beads with hydrophilic surfaces effectively formed precipitate-magnetic bead aggregates and were effective in precipitating proteins.

[0068] [Example 2] Effect of different addition orders on protein capture efficiency by magnetic beads The types of magnetic beads used here are: These are the same as the magnetic beads types (1) to (6) in Example 1. Protein Precipitant: Acetonitrile

[0069] [Example 2-A] (Protein precipitant added first, then magnetic beads added) 200 μL of acetonitrile precipitant was added to 100 μL of serum sample (each sample was tested in three parallel tests) and mixed well by vortexing. Then, 50 μL of magnetic bead working solution (containing 1 mg of magnetic beads in 50% methanol solution) was added and mixed well by vortexing. The precipitated proteins were attached to the magnetic beads under the action of an applied magnetic field. The supernatant was aspirated and placed in a microplate reader to measure the absorbance at a wavelength of 420 nm.

[0070] [Example 2-B] (added simultaneously) 250 μL of acetonitrile solution containing magnetic beads was added to 100 μL of serum sample (each sample was tested in three parallel tests), and after thorough mixing by vortexing, the precipitated proteins were attached to the magnetic beads under the action of an applied magnetic field. The supernatant was aspirated and placed in a microplate reader to measure the absorbance at a wavelength of 420 nm.

[0071] Experimental results [Table 2]

[0072] Experimental Conclusion 1. Magnetic beads (1)-(2) and (4)-(6) were able to effectively remove proteins, but the protein removal effect of beads (3) (agarose matrix carboxyl group magnetic beads) was not as good. 2. The above results show that there was no significant difference in the results when adding the precipitant and magnetic beads simultaneously compared to adding the magnetic beads and precipitant to the sample separately. In other words, the simultaneous addition method and the separate addition method produced the same effect, which was unexpected. Adding them simultaneously is more advantageous than adding them separately in terms of improving processing efficiency and realizing processing automation.

[0073] [Example 3] Consideration of system durability of the method The durability of the column performance using the sample processing method of the present invention, that is, whether the column can be maintained in a normal operating state without clogging, was examined.

[0074] Testing Procedure: Samples: The following experiments were carried out on 10 authentic serum samples. Measurement method: Serum samples were treated with either the magnetic bead method of the present invention (here, silica gel matrix amino group beads described in Example 1 were used) or a conventional protein precipitation method, and then the treated samples were analyzed by a liquid chromatography-tandem mass spectrometry system (model number: AB SCIEX Triple Quad TM The samples were subjected to detection using a 4500MD (manufactured by Aibocais Co., Ltd.).

[0075] (1) The magnetic bead method of the present invention (treatment with silica gel matrix amino group beads) A 250 μL serum sample was collected, and 250 μL of acidic precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as the internal standard) was added and mixed well by vortexing. 40 μL of magnetic bead working solution was then added, mixed well by vortexing, and magnetically aspirated. 150 μL of the supernatant was collected and subjected to measurement by the system.

[0076] (2) Control—Conventional protein precipitation method 250 μL of serum sample was collected, and 250 μL of acid precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as an internal standard) was added and mixed well by vortexing. 40 μL of purified water was then added and mixed well by vortexing. The mixture was then stirred at 12,000 rpm min -1 After centrifugation at 40°C for 10 minutes, 150 μL of the supernatant was collected and subjected to measurement in the system.

[0077] Experimental results and conclusions [Table 3]

[0078] Figure 5 shows the consistency of the vitamin B1 content in 10 serum samples measured using the magnetic bead method of the present invention and the conventional precipitation method. In Figure 5, the abscissa of each point represents the concentration value measured using the conventional precipitation method, and the ordinate represents the concentration value of the corresponding sample measured using the magnetic bead method of the present invention. Linear regression analysis was performed on the 10 data points, and the slope of the linear equation and the correlation coefficient (R 2 ) to determine the consistency of the results from the two measurement methods.

[0079] Conclusion: 1. The magnetic bead method of the present invention produced consistent detection results compared with the conventional protein precipitation method. 2. The magnetic bead method and protein precipitation method of the present invention do not have any adverse effects on the column effect, and both the conventional protein precipitation method and the magnetic bead method of the present invention (after multiple injections) have stable column performance, no column clogging, and stable column pressure.

[0080] [Example 4] Verification of samples from different origins To verify the treatment effect of samples of different origins, plasma, serum, and whole blood samples were treated with either the magnetic bead method of the present invention (here, the amino / silica gel matrix carboxyl group magnetic beads described in Example 1 were used) or the conventional protein precipitation method, and then the treated samples were subjected to measurement using an HPLC-MS / MS system.

[0081] Serum and plasma samples (1) Silica gel matrix amino-group magnetic beads 250 μL of serum or plasma sample was collected, and 250 μL of acidic precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as the internal standard) was added and mixed well with a vortex for 1 minute. After that, 40 μL of magnetic bead working solution (silica gel matrix amino-group magnetic beads) was added and mixed well with a vortex. The sample was then magnetically aspirated, and the supernatant was collected and measured in the system.

[0082] (2) Conventional protein precipitation method 250 μL of serum or plasma sample was collected, and 250 μL of acid precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as the internal standard) was added. The mixture was mixed thoroughly by vortexing for 1 minute, and then 40 μL of purified water was added. The mixture was mixed thoroughly by vortexing for 1 minute and then stirred at 12,000 rpm min -1 After centrifugation at 4°C for 10 minutes, the supernatant was placed in a 96-well V-bottom plate and subjected to measurement in the system.

[0083] Whole blood sample (1) Amino-group magnetic beads A 50 μL sample of whole blood was collected, to which 10 μL of 0.4 M ZnSO4 solution and 150 μL of acidic precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as an internal standard) were added, followed by 20 μL of magnetic bead working solution. The mixture was vortexed for 5 minutes and subjected to magnetic aspiration for 1 minute. 60 μL of the supernatant was collected, supplemented with 60 μL of purified water, mixed well, and then loaded into the system.

[0084] (2) Conventional protein precipitation method A 50 μL sample of whole blood was collected, to which 10 μL of 0.4 M ZnSO4 solution and 150 μL of acid precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 was used as an internal standard) were added, followed by 20 μL of purified water. The sample was vortexed for 5 minutes at 12,000 rpm min -1 The mixture was centrifuged at RT for 10 minutes, and 60 μL of the supernatant was collected and supplemented with 60 μL of purified water. After thorough mixing, the mixture was applied to the system.

[0085] Experimental results Serum samples: The results are shown in Table 3 in Example 3. Plasma sample: The results are shown in Table 4. Figure 6 also shows the consistency of the measurement results for vitamin B1 content in serum samples measured using the magnetic bead method of the present invention and the conventional precipitation method in this example. In Figure 6, the abscissa of each point represents the concentration value measured using the conventional precipitation method, and the ordinate represents the concentration value of the corresponding sample measured using the magnetic bead method of the present invention. Linear regression analysis was performed on the 10 data points, and the slope of the measured linear equation and the correlation coefficient (R 2 ) to determine the consistency of the results from the two measurement methods.

[0086] [Table 4]

[0087] Whole blood sample: The results are shown in Table 5. Figure 7 also shows the consistency of the measurement results for vitamin B1 content in serum samples measured using the magnetic bead method of the present invention and the conventional precipitation method in this example. In Figure 7, the abscissa of each point represents the concentration value measured using the conventional precipitation method, and the ordinate represents the concentration value of the corresponding sample measured using the magnetic bead method of the present invention. Linear regression analysis was performed on the 10 data points, and the slope of the measured linear equation and the correlation coefficient (R 2 ) to determine the consistency of the results from the two measurement methods.

[0088] [Table 5] As can be seen from the above results, the results of detecting various samples using the magnetic bead method of the present invention and the conventional protein precipitation method were consistent. Therefore, the magnetic bead method of the present invention can be effectively used for the pretreatment of various samples such as serum, plasma, and whole blood.

Claims

1. A method for processing a sample for liquid chromatography and / or mass spectrometry, comprising contacting hydrophilic magnetic beads and a precipitant with the sample to form precipitate-magnetic bead aggregates, and applying a magnetic field to remove the precipitate-magnetic bead aggregates.

2. The method according to claim 1, wherein the hydrophilic surface magnetic beads are silica gel magnetic beads, preferably bare silica gel magnetic beads without surface modification.

3. 3. The method of claim 1 or 2, wherein liquid chromatography and / or mass spectrometry is used to analyze one or more analytes in a sample selected from water-soluble analytes, such as anionic analytes (e.g., analytes having a carboxyl group), cationic analytes (e.g., analytes having an amino group), non-ionic analytes, and lipid-soluble analytes.

4. The method according to any one of claims 1 to 3, wherein the sample is or comprises a biological material selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymphatic fluid.

5. The step of contacting the hydrophilic magnetic beads and the precipitant with the sample comprises: (1) contacting the sample with a precipitant to form a precipitate, and then contacting the precipitate with hydrophilic magnetic beads to form precipitate-magnetic bead aggregates; or The method of any one of claims 1 to 4, comprising (2) contacting a sample with hydrophilic surface magnetic beads and a precipitant to form a precipitant-magnetic bead aggregate.

6. The method according to any one of claims 1 to 5, wherein the step of contacting the hydrophilic surface magnetic beads and the precipitating agent with the sample is achieved by contacting the hydrophilic surface magnetic beads and the precipitating agent together with the sample.

7. The method of any one of claims 1 to 6, wherein the precipitating agent comprises a water-miscible organic solvent (e.g., acetonitrile, methanol, ethanol, preferably acetonitrile).

8. applying the magnetic field to remove the precipitate-magnetic bead aggregates comprises: The method according to any one of claims 1 to 7, wherein the method is realized by placing a magnet on the outer surface of the wall of a container containing the sample, and attracting the magnetic beads bound to the precipitate to the inner wall of the container when a magnetic field is applied, or by inserting a magnet into the container containing the sample to adsorb the magnetic beads bound to the precipitate and then removing the magnet.

9. The method according to any one of claims 1 to 8, wherein the application of the magnetic field is generated by a permanent magnet or an electromagnet, for example, by controlling the on / off flow of a current through an electromagnet.

10. applying the magnetic field to remove the precipitate-magnetic bead aggregates comprises: The method according to any one of claims 1 to 9, wherein the method is realized by removing the sample solution from a container containing the sample and magnetic beads, or by removing the magnetic beads bound to the precipitate from a container containing the sample and magnetic beads.

11. The method according to any one of claims 8 to 10, wherein the container is a multi-well plate, such as a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate, or a separation tube.

12. A method for detecting a sample, comprising pretreating the sample by the method according to any one of claims 1 to 11.

13. 13. The method of claim 12, further comprising analyzing the pretreated sample by an analytical method selected from mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

14. 14. The method of claim 12 or 13, comprising mixing the sample with an internal standard solution before pretreatment and analysis.

15. A kit for pre-detection treatment of a sample, specifically for use in mass spectrometry or liquid chromatography, comprising magnetic beads with hydrophilic surfaces (e.g., silica gel magnetic beads, particularly bare silica gel magnetic beads that are not surface-modified) and, optionally, a reference substance.

16. The kit according to claim 15, which is used to carry out the method according to any one of claims 1 to 14.

17. Use of magnetic beads with hydrophilic surfaces (e.g., silica gel magnetic beads, particularly bare silica gel magnetic beads without surface modification) for pretreating samples for mass spectrometry and / or liquid chromatography analysis, particularly for removing protein impurities in samples for mass spectrometry and / or liquid chromatography analysis.

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