Sample processing method for liquid chromatography and mass spectrometry

Abstract

A method of treating a sample for liquid chromatography and mass spectrometry and use of surface-hydrophilic magnetic beads for treating a sample for liquid chromatography and mass spectrometry includes contacting the sample with surface-hydrophilic magnetic beads and a precipitating agent to form precipitate-magnetic bead aggregates, and applying a magnetic field to remove the precipitate-magnetic bead aggregates. The sample treatment method can be used in sample preparation or pretreatment of mass spectrometry or liquid chromatography, especially liquid chromatography tandem mass spectrometry.

Description

Sample Preparation Methods for Liquid Chromatography and Mass Spectrometry Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a method for processing samples for liquid chromatography and mass spectrometry analysis. Background Art

[0002] Biological samples generally refer to animal (including human) body fluids (such as whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, lymph, and other secretions of organisms), muscle, hair, and some tissues and organs (such as liver, lungs, kidneys, brain, stomach, thymus, and pancreas). Biological samples, especially clinical samples, have complex compositions, especially protein components, which can easily interfere with subsequent analysis and testing. Therefore, samples need to be pre-treated to remove protein impurities before analysis and testing.

[0003] Sample pretreatment is an important part of bioanalysis. For liquid chromatography and mass spectrometry, especially liquid chromatography tandem mass spectrometry (LC-MS / MS) methods used for clinical testing, it is very necessary to pretreat the biological samples before sample analysis and detection. The main purpose of sample pretreatment is to separate one or more target analytes from other components in the sample matrix. Sample pretreatment is a very important part of LC-MS / MS method development and optimization. In many cases, sample pretreatment is the bottleneck restricting high-throughput detection and the main source of analytical errors. The failure of LC-MS / MS methods is often due to improper sample pretreatment, which leads to the suppression of the target analyte signal or interference by coexisting substances in the biological matrix. In particular, if the impurities in the sample are not properly handled, they may contaminate the analysis pipeline. For example, insoluble substances such as impurity components (such as proteins) enter the liquid chromatography-mass spectrometry system, which can easily cause blockage of the pipeline or chromatographic column, affecting the analysis.

[0004] The sample pretreatment methods used in the prior art include centrifugation, static standing, protein precipitation (PPT), liquid-liquid extraction (LLE), solid phase extraction (SPE), etc. However, the pretreatment methods in the prior art have the disadvantages of being cumbersome, time-consuming, and unable to be automated.

[0005] For example, currently used centrifugation or static separation methods can remove protein impurities, but they are cumbersome and difficult to automate. Moreover, static separation is time-consuming.

[0006] CN113376270B discloses a pretreatment method for protein precipitation in serum samples using high-performance liquid chromatography-tandem mass spectrometry, including a negative pressure treatment step. This method is simple to operate, but because protein precipitation essentially dilutes the sample, the concentration of the analyte in the sample is relatively low, requiring high sensitivity from the instrument. Furthermore, the method requires negative pressure operation, which hinders full automation of the process.

[0007] CN110779780A discloses a pretreatment method for serum sample detection using high-performance liquid chromatography-tandem mass spectrometry. The method uses ethanol for protein precipitation, n-hexane for liquid-liquid extraction, nitrogen purging to dry the extract, and sample reconstitution before sample analysis. This method involves extraction and nitrogen purging, requiring extensive manual labor and hindering automated processing. Furthermore, the process is cumbersome and requires high labor requirements for the experimenter.

[0008] CN111198238A discloses a method for extracting and detecting serum samples, employing protein precipitation and solid-phase extraction. However, in the experiment, the solid-phase extraction filler swells upon contact with organic reagents, affecting the flow rate of the solution through the filler, resulting in poor uniformity between wells. Furthermore, the nitrogen drying process is not conducive to automated processing.

[0009] Therefore, existing pretreatment methods for removing protein from samples suffer from significant shortcomings, such as excessive manual work and time-consuming processes. This makes it difficult to achieve automated and rapid sample processing, thus failing to meet the demands of testing. Testing practices, especially clinical testing, urgently require automated methods for removing protein impurities from samples.

[0010] Summary of the Invention

[0011] Through extensive experimental research, the inventors unexpectedly discovered that by using hydrophilic magnetic beads (especially silica gel magnetic beads) and a precipitant to contact the sample, impurities in samples for liquid chromatography and mass spectrometry analysis can be effectively removed, which is conducive to the automation of the operation.

[0012] Based on this discovery, in a first aspect of the present invention, a method for processing samples for liquid chromatography and mass spectrometry analysis is provided, 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.

[0013] The surface-hydrophilic magnetic beads described in the present invention are magnetic particles having a magnetic core and a coating material coated on the surface of the magnetic core, and the surface coating material is hydrophilic. The surface coating material can be silica gel or modified silica gel (for example, amino-modified silica gel, carboxyl-modified silica gel, or silica gel (i.e., unmodified bare silica gel)). In this article, the term "silica gel magnetic beads" refers to magnetic beads comprising a silica gel layer wrapped around the outside of the magnetic core. In this article, "surface-hydrophilic magnetic beads" are, for example, surface-hydrophilic silica-based magnetic beads. Silica gel magnetic beads may include magnetic beads whose silica surface is further functionally modified, for example, they may be amino-modified silica gel magnetic beads, carboxyl-modified silica gel magnetic beads, hydroxyl-modified silica gel magnetic beads, etc.; magnetic beads that have not been functionally modified as described above are the "unmodified bare silica gel magnetic beads" described herein.

[0014] Preferably, the magnetic beads have an appropriate particle size, for example, they have 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.

[0015] In the present invention, the "sample" means the product form from which the component to be detected is derived. In this article, the sample may refer to a biological sample, such as a clinical sample, which is preferably selected from whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid and lymph. The sample may comprise a biological material selected from whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid and lymph. In this article, the sample also includes the above-mentioned biological material after treatment, such as treated whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid and lymph.

[0016] In the present invention, the magnetic core of the magnetic beads comprises magnetic particles, which can be composed of any magnetic substance, preferably selected from iron, cobalt, and nickel oxides, more preferably ferrosoferric oxide. As known in the art, the magnetic core can be prepared by any suitable method.

[0017] In the present invention, the contacting of the hydrophilic surface magnetic beads and the precipitant with the sample may include the following methods:

[0018] (1) contacting the sample with a precipitant to form a precipitate, and then contacting the precipitate with magnetic beads having a hydrophilic surface to form a precipitate-magnetic bead aggregate; or

[0019] (2) Magnetic beads with hydrophilic surfaces and a precipitant are brought into contact with a sample to form a precipitate-magnetic bead aggregate.

[0020] Preferably, the step of bringing the hydrophilic magnetic beads and the precipitant into contact with the sample is achieved by bringing the hydrophilic magnetic beads and the precipitant into contact with the sample together.

[0021] In this article, "making the hydrophilic magnetic beads and the precipitant contact the sample together" means exposing the hydrophilic magnetic beads and the precipitant to the sample together, wherein there is no obvious difference in the order of exposure time points of the magnetic beads and the sample, and the precipitant and the sample; in a more specific embodiment, the magnetic beads, the precipitant and the sample are contacted basically synchronously, but it is not required to be in contact at the same time in a strict sense.

[0022] Herein, "magnetic beads having a hydrophilic surface" are preferably silica-based magnetic beads having a hydrophilic surface.

[0023] Herein, the step of contacting the sample with the hydrophilic magnetic beads and the precipitant further comprises the steps of mixing, shaking, oscillating, vortexing and / or stirring the mixture of the sample, the magnetic beads and the precipitant.

[0024] Without being limited by theory, in the present invention, when the precipitant is brought into contact with the sample, impurities (e.g., proteins) in the sample form a precipitate; when magnetic beads are present in the system, the precipitate can simultaneously or sequentially combine with the hydrophilic magnetic beads, encapsulating the magnetic beads therein to form a precipitate-magnetic bead aggregate.

[0025] 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 that can precipitate proteins in a sample, including substances that dehydrate and precipitate proteins, such as organic solvents such as acetonitrile, methanol, ethanol, etc., preferably acetonitrile; optionally, the protein precipitant may also include, for example, an organic solvent that is immiscible with water (such as alkanes / cycloalkanes, ethyl acetate). Those skilled in the art may also use other known suitable precipitants. Without being limited by theory, in the present invention, the purpose of adding a precipitant is to prevent insoluble substances from entering the system during LC / MS analysis of the sample, thereby causing blockage of the pipeline and chromatographic column, affecting the analysis.

[0026] The inventors have further discovered that the formed precipitate (precipitate-magnetic bead aggregate) can be removed by applying a magnetic field. For example, applying a magnetic field allows for rapid and efficient transfer of samples, particularly liquids (including solutions or suspensions), while also preventing problems such as residual magnetic material from clogging chromatographic columns during transfer. This discovery makes it possible to automate the removal of precipitated impurities under an applied magnetic field.

[0027] Accordingly, in the method of the present invention, it includes applying a magnetic field to remove the precipitate-magnetic bead aggregates. More specifically, the application of a magnetic field to remove the precipitate-magnetic bead aggregates can be achieved by placing a magnet on the outside of the wall of a container containing a sample so that the magnetic beads bound to the precipitate are attracted to the inside of the container wall when the magnetic field is applied (for example, at this time, the sample and the magnetic beads bound to the precipitate can be separated by absorbing the sample solution or suspension, etc.); or it can be achieved by inserting a magnet into a container containing a sample to adsorb the magnetic beads bound to the precipitate and removing the magnet. In some embodiments, the application of a magnetic field to remove the precipitate-magnetic bead aggregates 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.

[0028] The magnet can be a permanent magnet or an electromagnet, preferably an electromagnet, which can conveniently control the presence and absence of a magnetic field by turning the power on and off. In the case of an electromagnet, the power 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.

[0029] During specific operation, the magnet can be placed or inserted into a solution or suspension containing a sample.

[0030] In one embodiment, the application of a magnetic field to remove the precipitate-magnetic bead aggregates is achieved by placing a magnet outside the wall of the container containing the sample so that the magnetic beads bound to the precipitate (i.e., the precipitate-magnetic bead aggregates) are attracted to the inner wall of the container when the magnetic field is applied. In a more specific embodiment, the magnet can be present in two positions: a side suction position and a bottom suction position. For the magnet located at the side suction position, the magnet is located outside the side wall of the container containing the solution or suspension of the sample so that when the magnetic field is applied (e.g., by turning on the power of the electromagnet or moving the permanent magnet), the magnetic beads bound to the precipitate are attracted to the inner wall of the container; for the magnet located at the bottom suction position, the magnet is located below the bottom wall of the container containing the solution or suspension of the sample so that when the magnetic field is applied (e.g., by turning on the power of the electromagnet or moving the permanent magnet), the magnetic beads bound to the precipitate are attracted to the bottom wall of the container. In some embodiments, there may be only magnets at the side suction position; in other embodiments, there may be only magnets at the bottom suction position; in other embodiments, there may be magnets at both the side suction position and the bottom suction position.

[0031] By switching the position and / or on / off state of the magnet, in conjunction with the operation of transferring liquid (such as solution), the automated operation of transferring liquid (such as solution) between different containers can be effectively achieved.

[0032] Furthermore, the present invention utilizes a "side suction" magnet to better achieve sufficient liquid transfer by placing a magnet outside the side wall of a container containing a sample solution or suspension so that when a magnetic field is applied, the precipitated magnetic beads are attracted to the inner side wall of the container to remove precipitated impurities (e.g., proteins) under an external magnetic field. Compared to a "bottom suction" method in which the magnet is located below the bottom wall of the container, the side suction magnet can more effectively avoid liquid loss caused by the bottom suction magnetic field (which sometimes results in insufficient bottom liquid absorption) and magnetic material residue and / or chromatographic column clogging caused by the magnetic material being absorbed. Furthermore, the transfer operation is facilitated, making it easier to automate.

[0033] Based on the above findings, in the method described in the present invention, the application of a magnetic field to remove the precipitate-magnetic bead aggregates is achieved by placing a magnet on the outside of the wall of a container containing a solution or suspension of the sample (preferably outside the side wall), so that when the magnetic field is applied, the magnetic beads bound to the precipitate are attracted to the inside of the container wall (preferably inside the side wall), or by inserting a magnet (such as a magnetic rod) into a solution or suspension containing the sample to adsorb the magnetic beads bound to the precipitate and removing the magnet.

[0034] In the method of the present invention, the container can be a multi-well plate or a separate tube, and the multi-well plate is, 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 can have a U-shaped or V-shaped bottom shape.

[0035] In some embodiments of the present invention, the method of transferring (moving and / or separating) a sample is achieved by aspirating a liquid (or fluid) in one container and releasing the liquid (or fluid) into another container.

[0036] In the method of the present invention, samples can be transferred between different containers by moving a pipette needle up and down within the container. Preferably, the pipette needle base is provided with an elastic element, allowing the pipette needle to elastically retract when it moves downward to contact the bottom of the container. Preferably, the pipette needle base is provided with an adjustment structure, allowing the stopping position of the pipette needle tip to be adjusted to be as close to the bottom of the container as possible without damaging the pipette needle and / or the container.

[0037] The present invention also relates to a sample detection method, comprising the step of pre-treating the sample using the above-mentioned method. On this basis, the present invention relates to a sample detection method, comprising the step of analyzing the sample after the above-mentioned pre-treatment using 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 the sample analysis method of the present invention, in one embodiment, the sample is first mixed with a solution or suspension of an internal standard, and then the steps of pre-treatment and analysis are performed.

[0038] The present invention also provides a kit for sample pre-detection treatment, which includes magnetic beads with hydrophilic surface (such as silica magnetic beads, especially bare silica magnetic beads without surface modification) and optional reference substances. Specifically, the kit is used for mass spectrometry or liquid chromatography.

[0039] The kit of the present invention can be used to implement the sample pretreatment method or sample detection method of the present invention.

[0040] In another aspect of the present invention, there is provided the use of magnetic beads with hydrophilic surfaces (such as silica magnetic beads, in particular bare silica magnetic beads without surface modification) for removing impurities (including proteins) from a sample.

[0041] By adopting the method of the present invention, the centrifugation and ultrasonic operations commonly used in the prior art can be avoided, and nitrogen blowing treatment is not required. Therefore, the automation of the operation can be greatly improved, manpower can be saved, operation time can be reduced, and processing efficiency can be significantly improved.

[0042] Terminology

[0043] In this document, the terms "magnetic beads", "magnetic particles", "magnetic particles" and "magnetic particles" are used interchangeably to refer to particles with magnetic properties.

[0044] As used herein, "surface-hydrophilic magnetic beads" refer to magnetic particles comprising a magnetic core and a hydrophilic coating material coated on the surface of the magnetic core. The surface coating material can be silica gel or modified silica gel, such as amino-modified silica gel or carboxyl-modified silica gel. Particularly preferred surface-hydrophilic magnetic beads herein are silica gel magnetic beads, particularly unmodified bare silica gel magnetic beads.

[0045] In this article, “making the surface hydrophilic magnetic beads and the precipitant contact the sample” means exposing the sample to the magnetic beads and the precipitant, so that an interaction occurs. The contact may have a sequence, for example, first making the sample contact the precipitant, and then making the precipitate contact the magnetic beads, or there may be no sequence, meaning that they can be contacted together. The inventors unexpectedly discovered that when the surface hydrophilic magnetic beads and the precipitant are contacted with the sample together to form a precipitate-magnetic bead aggregate, the treatment effect is no worse than that of the step-by-step contact (first making the sample contact the precipitant, and then making the precipitate contact the magnetic beads). This simultaneous contact method is more conducive to the automation of the operation and saves processing time.

[0046] In this article, particle size refers to the average particle size of a particle group. Particle size can be measured by sieving, microscopy, sedimentation, electrical resistance, etc.

[0047] In this article, "liquid chromatography" refers to chromatography using a liquid as the mobile phase, although the stationary phase can take a variety of forms. "Mass spectrometry" refers to the use of electric and magnetic fields to separate moving ions (charged atoms, molecules, or molecular fragments, including molecular ions, isotopic ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, and ions generated by ion-molecule interactions) according to their mass-to-charge ratio for subsequent detection. Liquid chromatography or mass spectrometry in this article also includes combinations or coupled methods, such as liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0048] In this article, the terms "silica-based magnetic beads", "silica-based magnetic beads" or "silica magnetic beads" have the same meaning and can be used interchangeably.

[0049] In this document, the term "selected from" means that it can be one of the options listed thereafter, or a combination of one or more of the options.

[0050] As used herein, "sample" refers to the object being tested, and may be a biological sample, such as a clinical sample, including whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph, etc., or a biological material selected from whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph, or any combination thereof. The sample may be in liquid or fluid form, or even in solid form. Solid samples may optionally be processed, such as by homogenization, before testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG1 is a schematic diagram of a magnet of the present invention located at a side suction position.

[0052] FIG2 is a schematic diagram of the magnet of the present invention being located at the bottom suction position.

[0053] In Figure 1, magnet 4 is located outside the side wall of container 1 (plate well), and sample solution or suspension 2 is contained in container 1. Under the action of the external magnetic field of magnet 4 at the side suction position, magnetic beads 3 are aggregated and fixed to the side wall of container 1, thereby not affecting the transfer operation of solution or suspension 2, and ensuring that the liquid in container 1 is fully aspirated.

[0054] In Figure 2, the magnet 4 is located below the bottom wall of the container 1 (plate well), and there is a sample solution or suspension 2 in the container 1. 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.

[0055] FIG3 is a photograph showing the impurity removal effect of different types of magnetic beads, wherein the numbers marked on the containers represent the types of magnetic beads.

[0056] FIG4 is a photograph showing the effect of different sample addition orders on the magnetic bead removal efficiency, wherein the numbers marked on the containers represent the types of magnetic beads.

[0057] FIG5 shows the consistency of the measurement results of vitamin B1 content in 10 serum samples determined by the magnetic bead method of the present invention and the conventional precipitation method in Example 3;

[0058] FIG6 shows the consistency of the measurement results of vitamin B1 content in plasma samples determined by the magnetic bead method of the present invention and the conventional precipitation method in Example 4;

[0059] FIG7 shows the consistency of the measurement results of the vitamin B1 content in the whole blood sample determined by the magnetic bead method of the present invention and the conventional precipitation method in Example 4. DETAILED DESCRIPTION

[0060] In the following examples, unless otherwise specified, the reagents and instruments used are commonly used in the art and can be purchased from chemical or biological product / preparation companies; the methods used in the following examples are conventional methods in the art, and those skilled in the art can undoubtedly know the operating procedures of these experiments and obtain the corresponding results based on the existing technology or the operating manuals provided by the manufacturers.

[0061] Example

[0062] The materials, reagent sources, and instrument models used in the present invention are shown in the table below.

[0063] Table 1. Instrument brands and models

[0064] The following magnetic beads were used in the examples herein:

[0065] Impurity-removing magnetic beads 1 and impurity-removing magnetic beads 2: purchased from Suzhou Ying Ruicheng Biochemical Technology Co., Ltd., both are superparamagnetic materials with a core-shell structure, the core is superparamagnetic ferroferric oxide, and the shell is a silica layer with functional functional groups modified on the surface. The silica layer is coupled with functional functional groups for surface modification, and the functional functional groups include amino and carboxyl groups. Among them, impurity-removing magnetic beads 1 are mixed magnetic beads of amino magnetic beads and carboxyl magnetic beads, in which the ratio of amino and carboxyl groups is 6:4; impurity-removing magnetic beads 2 increase the proportion of primary and secondary amine groups in the mixed magnetic beads compared with impurity-removing magnetic beads 1. The impurity-removing magnetic beads 1 and impurity-removing magnetic beads 2 used in this embodiment were purchased from Suzhou Ying Ruicheng Biochemical Technology Co., Ltd., and the models were MSi050 / FLD01 and MSi050 / FLD02, respectively.

[0066] Silica-based carboxyl magnetic beads: magnetic beads with a superparamagnetic ferroferric oxide core, a silica shell, and carboxyl groups modified on the silica surface; the silica-based carboxyl magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., model MSi050 / WCX; they are hydrophilic polymer magnetic beads, 1.5 μm, with a sandwich structure, a porous polymer microsphere core inside, and a polymer coating of different materials on the outside to meet different application requirements. The magnetic material fills the pores between the two, model / item number: MPHCC-150.

[0067] Silica-based amino magnetic beads: These are magnetic beads with a superparamagnetic ferroferric oxide core, a silica shell, and amino groups modified on the silica surface. The silica-based amino magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., model MSi050 / MAX.

[0068] Agarose-based carboxyl magnetic beads: These are magnetic beads with a superparamagnetic ferroferric oxide core, an agarose shell, and surface modified with carboxyl groups. For their preparation, see CN113181883A. The agarose-based carboxyl magnetic beads used in this example were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., model MAgr100K / WCX.

[0069] Bare silica magnetic beads without surface modification: magnetic beads with a superparamagnetic ferroferric oxide core, a silica shell and no surface modification; the bare silica magnetic beads without surface modification used in the embodiment were purchased from Suzhou Yingruicheng Biochemical Technology Co., Ltd., model EM001.

[0070] Example 1. Effect of different types of magnetic beads on protein removal

[0071] Type of magnetic beads used:

[0072] (1) Remove impurity magnetic beads 1;

[0073] (2) removing impurity magnetic beads 2;

[0074] (3) agarose matrix carboxyl magnetic beads;

[0075] (4) silica-based carboxyl magnetic beads;

[0076] (5) Silica-based amino magnetic beads;

[0077] (6) Bare silica beads without surface modification;

[0078] (7) PS magnetic beads (polystyrene, divinylbenzene, purchased from Suzhou Ajie Boya Bioelectronic Technology Co., Ltd.; neutral styrene / divinylbenzene reversed-phase adsorption magnetic extraction material; model / product number: BNMA12300010-0);

[0079] (8) C18 magnetic beads (silica gel matrix, 20-40 μm, purchased from Suzhou Ajie Boya Bioelectronics Technology Co., Ltd.; model / product number: BNMS9300001-0)

[0080] A. Protein precipitation agent: acetonitrile

[0081] Sample processing:

[0082] Add 200 μL of acetonitrile precipitant to 100 μL of serum sample (three parallels for each sample), vortex mix, then add 50 μL of magnetic bead working solution (containing 1 mg of magnetic beads, 50% methanol solution), vortex mix, apply an external magnetic field to allow the precipitate to adhere to the magnetic beads, aspirate the supernatant solution and add it to the microplate reader, and read the absorbance at a wavelength of 620 nm.

[0083] in conclusion

[0084] Magnetic bead types (1)-(2) and (4)-(6) can effectively capture and remove precipitates (mainly including proteins), while magnetic bead (3) (agarose matrix carboxyl magnetic beads) has poor precipitate capture results.

[0085] B. Protein precipitant: Acid precipitant 1 (10% TCA), acid precipitant 2 (10% TCA: pure methanol = 6:4)

[0086] Sample processing:

[0087] To 250 μL of serum sample, add 250 μL of acid precipitant (Acid Precipitant 1 or Acid Precipitant 2), vortex to mix, add 40 μL (4 mg) of magnetic bead working solution, vortex to mix, magnetically aspirate, and collect the supernatant. The magnetic bead types (1), (4), (5), (7), and (8) listed above were used.

[0088] The results show that impurity removal magnetic beads 1, silica-based amino magnetic beads, and silica-based carboxyl magnetic beads exhibit significant protein adsorption. After adding two precipitants, the resulting precipitate-bead aggregates were magnetically attracted to the sidewalls of the beads, resulting in a clear solution. The supernatant was transferred to the chromatography column without clogging, and the column pressure remained normal before and after transfer. Centrifugation of the supernatant (4000g, 10 min) revealed no visible precipitate, and the solution remained clear with no protein formation, confirming the protein adsorption efficacy.

[0089] After adding acid precipitant 1 or acid precipitant 2 to PS magnetic beads, the precipitate-magnetic bead aggregates formed swelled, and some precipitate-magnetic bead aggregates were magnetically attracted to the side wall. The liquid clarity was slightly poor, and the adsorption effect was poor.

[0090] After adding the above-mentioned acid precipitant 1 or acid precipitant 2, C18 magnetic beads did not form precipitate-magnetic bead aggregates, white protein and magnetic beads did not aggregate, and the adsorption effect was poor.

[0091] The above results show that magnetic beads with poor surface hydrophilicity (such as PS magnetic beads and C18 magnetic beads) are not ideal for protein precipitation; while silica-based magnetic beads with hydrophilic surfaces can effectively form precipitate-magnetic bead aggregates, resulting in effective protein precipitation.

[0092] Example 2. Effect of different sample addition orders on protein capture by magnetic beads

[0093] Type of magnetic beads used:

[0094] Same magnetic bead types (1)-(6) as in Example 1.

[0095] Protein precipitation agent: acetonitrile

[0096] Example 2-A (adding protein precipitant first and then adding magnetic beads):

[0097] Add 200 μL of acetonitrile precipitant to 100 μL of serum sample (three parallels for each sample), vortex mix, add 50 μL of magnetic bead working solution (containing 1 mg of magnetic beads, 50% methanol solution), vortex mix, apply an external magnetic field to allow the precipitated protein to adhere to the magnetic beads, aspirate the supernatant solution and add it to the microplate reader, and read the absorbance at a wavelength of 420 nm.

[0098] Example 2-B (adding simultaneously):

[0099] Add 250 μL of acetonitrile solution containing magnetic beads to 100 μL of serum sample (three parallels for each sample), vortex to mix, apply an external magnetic field to allow the precipitated protein to adhere to the magnetic beads, aspirate the supernatant solution and add it to the microplate reader, and read the absorbance at a wavelength of 420 nm.

[0100] Experimental results

[0101] Table 2. Effects of different sample addition orders on the results

[0102] Experimental Conclusion

[0103] 1. Magnetic bead types (1)-(2) and (4)-(6) can effectively remove proteins, but magnetic bead (3) (agarose matrix carboxyl magnetic beads) has a relatively poor protein removal effect.

[0104] 2. The above results demonstrate that adding the precipitant and magnetic beads together does not significantly differ from adding the beads and precipitant separately to the sample. In other words, adding them together achieved the same results as adding them separately, a surprising result. Compared to adding them separately, adding them together is more effective in improving processing efficiency and facilitating automation.

[0105] Example 3. System durability study of the method

[0106] The column performance durability of the sample processing method of the present invention is examined, that is, whether the chromatographic column can maintain a normal working state and whether the chromatographic column is guaranteed to be free of clogging.

[0107] Experimental steps:

[0108] Samples: Ten real serum samples were used for the following experiments.

[0109] The test method is to treat serum samples by the magnetic bead method of the present invention (here, the silica-based amino magnetic beads described in Example 1 are used) or the conventional protein precipitation method, and then the treated samples are injected into the liquid chromatography tandem mass spectrometry system (model: AB SCIEX Triple Quad TM 4500MD, Aibocaisi Co., Ltd.)) was tested.

[0110] (1) Magnetic bead method of the present invention (using silica-based amino magnetic beads for treatment)

[0111] Take 250 μL of serum sample, add 250 μL of acid precipitant 1 (10% TCA) (deuterated label of vitamin B1 as internal standard), vortex mix, then add 40 μL of magnetic bead working solution, vortex mix, magnetically absorb, take 150 μL of supernatant, and detect on the machine.

[0112] (2) Control - Conventional protein precipitation method

[0113] Take 250 μL of serum sample, add 250 μL of acid precipitant 1 (10% TCA), (deuterated label of vitamin B1 as internal standard), vortex mix, then add 40 μL of purified water, vortex mix, 12000 rpm min -1 Centrifuge for 10 minutes, take 150 μL of the supernatant and detect it on the analyzer.

[0114] Experimental results and conclusions:

[0115] Table 3. Comparison of the detection results of the magnetic bead method of the present invention and the conventional precipitation method

[0116] Figure 5 shows the consistency of the results of the measurement of vitamin B1 content in 10 serum samples using the magnetic bead method of the present invention and the conventional precipitation method. In Figure 5, the horizontal axis of each point represents the concentration value measured by the conventional precipitation method, and the vertical axis represents the concentration value of the corresponding sample measured by the magnetic bead method of the present invention. Linear regression is performed on the 10 points, and the slope and correlation coefficient (R) of the linear equation are obtained. 2 ) can determine the consistency of the results of the two measurement methods.

[0117] in conclusion:

[0118] 1. Compared with the conventional protein precipitation method, the detection results of the magnetic bead method of the present invention are consistent.

[0119] 2. The magnetic bead method and protein precipitation method of the present invention have no adverse effects on the column efficiency of the chromatographic column. The column performance can be kept stable in both the conventional protein precipitation method and the magnetic bead method of the present invention (after multiple injections), the chromatographic column is not blocked, and the column pressure is stable.

[0120] Example 4. Validation of samples from different sources

[0121] To verify the treatment effect on samples from different sources, plasma, serum or whole blood samples were treated using the magnetic bead method of the present invention (here using the silica-based amino / carboxyl magnetic beads described in Example 1) or the conventional protein precipitation method, and then the treated samples were injected into the HPLC-MS / MS system for detection.

[0122] Serum and plasma samples

[0123] (1) Silica-based amino magnetic beads

[0124] Take 250 μL of serum or plasma sample, add 250 μL of acid precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 is used as internal standard), vortex mix for 1 min, then add 40 μL of magnetic bead working solution (silica-based amino magnetic beads), vortex mix, magnetically absorb, take the supernatant solution, and detect on the instrument.

[0125] (2) Conventional protein precipitation method

[0126] Take 250 μL of serum or plasma sample, add 250 μL of acid precipitant 1 (10% TCA), (vitamin B1 deuterium band marker as internal standard), vortex mix for 1 min, then add 40 μL of purified water, vortex mix for 3 min, 12000 rpm min -1 Centrifuge for 10 minutes, take the supernatant and add it to a 96-well V-shaped plate for detection.

[0127] Whole blood sample

[0128] (1) Amino magnetic beads

[0129] Take a 50μμL whole blood sample and add 10μμL of 0.4M ZnSO4 solution and 150μμL of acid precipitant 1 (10% TCA) (vitamin B1 deuterium labeling as an internal standard). Add 20μμL of magnetic bead working solution. Vortex for 5 minutes, magnetically absorb for 1 minute, remove 60μμL of the supernatant, add 60μμL of purified water, mix well, and transfer to the instrument.

[0130] (2) Conventional protein precipitation method

[0131] Take a 50μL whole blood sample and add 10μL of 0.4M ZnSO4 solution and 150μL of acid precipitant 1 (10% TCA) (deuterium-labeled vitamin B1 as an internal standard). Add 20μL of purified water. Vortex for 5 minutes and centrifuge at 12000 rpm / min⁻¹ for 10 minutes. Collect 60μL of the supernatant, add 60μL of purified water, mix well, and transfer to the centrifuge.

[0132] Experimental results

[0133] Serum samples: The results are shown in Table 3 in Example 3.

[0134] Plasma samples: The results are shown in Table 4. In addition, FIG6 shows the consistency of the measurement results of the vitamin B1 content in plasma samples determined by the magnetic bead method of the present invention and the conventional precipitation method in this embodiment. In FIG6, the horizontal axis of each point represents the concentration value measured by the conventional precipitation method, and the vertical axis represents the concentration value of the corresponding sample measured by the magnetic bead method of the present invention. Linear regression is performed on the 10 points, and the slope of the measured linear equation and the correlation coefficient (R 2 ) can determine the consistency of the results of the two measurement methods.

[0135] Table 4. Comparison of the test results of the magnetic bead method of the present invention and the conventional precipitation method (plasma samples)

[0136] Whole blood sample: The results are shown in Table 5. In addition, FIG7 shows the consistency of the measurement results of the vitamin B1 content in the whole blood sample using the magnetic bead method of the present invention and the conventional precipitation method in this embodiment. In FIG7, the horizontal axis of each point represents the concentration value measured by the conventional precipitation method, and the vertical axis represents the concentration value of the corresponding sample measured by the magnetic bead method of the present invention. Linear regression is performed on the 10 points, and the slope of the measured linear equation and the correlation coefficient (R 2 ) can determine the consistency of the results of the two measurement methods.

[0137] Table 5. Comparison of the detection results of the magnetic bead method of the present invention and the conventional precipitation method (whole blood sample)

[0138] The above results show that the magnetic bead method of the present invention and the conventional protein precipitation method produce consistent test results for different samples. Therefore, the magnetic bead method of the present invention can be effectively used for the pretreatment of different samples such as serum, plasma, and whole blood.

Claims

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

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

3. A method according to claim 1 or 2, wherein the liquid chromatography and / or mass spectrometry analyzes one or more analytes in the sample, for example, the analytes include types selected from the following: water-soluble analytes and fat-soluble analytes; water-soluble analytes are, for example, anionic analytes (e.g., analytes with carboxyl groups), cationic analytes (e.g., analytes with amino groups), and non-ionic analytes.

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

5. The method according to any one of claims 1 to 4, wherein the step of contacting the sample with the hydrophilic magnetic beads and the precipitant comprises: (1) contacting a sample with a precipitant to form a precipitate, and then contacting the precipitate with magnetic beads having a hydrophilic surface to form a precipitate-magnetic bead aggregate; or (2) The hydrophilic magnetic beads and the precipitant are brought into contact with the sample to form a precipitate-magnetic bead aggregate. 6 . The method according to claim 1 , wherein the step of bringing the hydrophilic magnetic beads and the precipitant into contact with the sample is achieved by bringing the hydrophilic magnetic beads and the precipitant into contact with the sample together.

7. The method according to any one of claims 1 to 6, wherein the precipitant comprises a water-miscible organic solvent (eg acetonitrile, methanol, ethanol, preferably acetonitrile).

8. A method according to any one of claims 1-7, wherein applying a magnetic field to remove the precipitate-magnetic bead aggregates is achieved by placing a magnet on the outside of the wall of a container containing the sample so that the magnetic beads bound to the precipitate are attracted to the inner wall of the container when the magnetic field is applied, or by inserting a magnet into a container containing the sample to adsorb the magnetic beads bound to the precipitate and removing the magnet.

9. The method according to any one of claims 1 to 8, wherein the applied magnetic field is generated by a permanent magnet or an electromagnet, for example, the electromagnet is controlled by turning on and off an electric current.

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

11. The method according to any one of claims 8 to 10, wherein the container is a multi-well plate or a separate tube, the multi-well plate being 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.

12. A sample detection method, comprising pre-treating the sample using the method according to any one of claims 1 to 11.

13. The method according to claim 12, further comprising analyzing the pre-treated sample using the following analytical method, wherein 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).

14. The method according to claim 12 or 13, comprising the steps of first mixing the sample with an internal standard solution and then performing pretreatment and analysis.

15. A kit for sample pre-detection treatment, comprising magnetic beads with hydrophilic surface (such as silica magnetic beads, especially bare silica magnetic beads without surface modification) and optionally reference substances, specifically the kit is used for mass spectrometry or liquid chromatography.

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

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

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