Sample pretreatment method, sample detection method and kit

Abstract

A sample pretreatment method, a sample detection method and a kit. The sample pretreatment method comprises: preparing a releasing agent working solution, the releasing agent working solution comprising an activated magnetic bead suspension and a precipitant; and placing a sample to be treated and the releasing agent working solution in a fully automated sample preparation instrument for sample pretreatment. Compared with the traditional liquid-liquid extraction method which requires prolonged standing and bulky instruments such as centrifuges for protein precipitation, the present method using magnetic beads for protein adsorption can separate proteins simply by applying a magnetic field; moreover, such steps facilitate self-assembly and workflow configuration in automated devices, and, after assembly, the automated workflow enables simplified preparation of metabolomics samples, reducing human intervention, shortening the total time for sample preparation, and effectively increasing sample preparation throughput.

Description

Sample pretreatment methods, sample detection methods and reagent kits

[0001] Priority information:

[0002] This disclosure claims priority and benefits to patent application (2025102023468) filed with the China National Intellectual Property Administration on February 24, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biotechnology, specifically, it relates to sample pretreatment methods, sample detection methods, and reagent kits. Background Technology

[0004] Metabolomics, due to its ability to directly reflect the real-time state of biological systems, its close correlation with genotype and phenotype, its sensitive response to external changes, and its strong clinical applicability, is considered one of the omics research methods closest to phenotype. With the continuous development of metabolomics and the increasing number of large clinical cohort samples, the demand for developing highly accurate and fully automated stable metabolomics sample preparation methods is constantly growing. Based on research objectives, metabolomics is divided into targeted metabolomics and non-targeted metabolomics. Targeted metabolomics involves the extraction and accurate qualitative and quantitative analysis of a specific set of known metabolites from a sample; while non-targeted metabolomics broadly explores all metabolites in a sample, using relative quantification to understand changes in the levels of known or unknown metabolites between different samples.

[0005] Body fluids are widely used biological samples in metabolomics research due to their ease of collection. Currently, liquid-liquid extraction is a common method for pretreatment of body fluid samples in metabolomics, which involves using organic reagents to precipitate large protein molecules in the body fluid, followed by high-speed centrifugation to separate the supernatant and protein precipitate. However, liquid-liquid extraction typically requires a certain amount of high-speed centrifugation and repeated transfer of the supernatant, and suffers from drawbacks such as difficulty in automation, high degree of manual intervention, time consumption, easy formation of emulsions, and low sample preparation throughput, which seriously hinder the development of large-scale metabolomics research. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems in related technologies. To this end, this application proposes an automated non-targeted metabolomics sample preprocessing method that reduces manual intervention, shortens the total sample preparation time, minimizes emulsion formation, and effectively improves sample preparation throughput.

[0007] Specifically, this application provides the following technical solution:

[0008] In a first aspect, this application proposes a sample pretreatment method. According to an embodiment of this application, the method includes: preparing a release agent working solution, the release agent working solution comprising: an activated magnetic bead suspension and a precipitant; and placing the sample to be treated and the release agent working solution in a fully automated sample preparation instrument for sample pretreatment.

[0009] The aforementioned method uses magnetic beads with specific chemical modifications on their surfaces to adsorb impurities such as proteins precipitated by a precipitant in the sample. A magnetic field is applied to control the aggregation of the adsorbed magnetic beads, thereby separating the supernatant containing the target molecules from the proteins and other impurities. Compared to traditional liquid-liquid extraction methods that require long periods of settling and the use of large instruments like centrifuges to settle proteins, the magnetic bead-based protein adsorption method only requires the application of a magnetic field to separate proteins. This step is easily integrated into automated equipment, allowing for streamlined process setup. Once assembled, the automated process simplifies the preparation of metabolomics samples, reduces manual intervention, shortens the total sample preparation time, and effectively increases sample preparation throughput.

[0010] In some examples of this application, the volume ratio of the activated magnetic bead suspension to the precipitant in the release agent working solution is selected as 1:(8-9). In some preferred examples of this application, the volume ratio of the activated magnetic bead suspension to the precipitant is 1:8.75. The release agent working solution prepared based on the aforementioned volume ratio can effectively remove impurities in the sample and improve the recovery rate of the target molecule.

[0011] In some examples of this application, the volume ratio of the sample to be treated to the working solution of the release agent is selected from 10:(35-40), and optionally 10:35, 10:36, 10:37, 10:38, 10:39, or 10:40. In some preferred examples of this application, the volume ratio of the sample to be treated to the working solution of the release agent is selected from 10:39. Based on the aforementioned ratio of sample to working solution, impurities in the sample can be effectively removed, and the dilution of target molecules in the sample can be avoided, thus preventing the impact on the subsequent detection sensitivity of target molecules.

[0012] In some examples of this application, the activated magnetic bead suspension is prepared by the following steps: mixing magnetic beads with an activation reagent at a predetermined mass-volume ratio to obtain the activated magnetic bead suspension. This step activates the magnetic beads to more effectively adsorb and separate impurities in the sample, reducing the interference of impurities on metabolite detection and improving the efficiency and purity of target molecule extraction.

[0013] It is understood that the magnetic beads in this application may be selected from commercially available ones (such as those from Ingenic) or customized based on the type of impurities adsorbed.

[0014] In some examples of this application, the activating agent is selected from methanol, preferably pure methanol (such as analytical grade methanol). Choosing pure methanol as the activating agent for magnetic beads can enhance the surface activity of the magnetic beads, improve dispersibility, and remove surface impurities.

[0015] In some examples of this application, the predetermined mass-to-volume ratio is selected from (5-15):1, and optionally is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1. In some preferred examples of this application, the predetermined mass-to-volume ratio is 10:1.

[0016] In some examples of this application, the precipitant is selected from a mixture of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is selected from 1:(3-5), optionally 1:3, 1:4, or 1:5. In some preferred examples of this application, the volume ratio of methanol to acetonitrile is 1:4. The precipitant in this application is water-free to avoid emulsion formation and to prevent interference with the suspension of magnetic beads. Furthermore, by optimizing the volume ratio of methanol to acetonitrile, its ability to adsorb proteins is ensured, and impurities are avoided from interfering with the sensitivity of subsequent target molecule detection.

[0017] In some examples of this application, the release agent working solution further includes an isotope internal standard solution. Adding an isotope internal standard solution can effectively improve the accuracy of target molecule quantification, correct for losses and biases in sample processing, reduce errors caused by changes in experimental conditions, and enhance the stability and repeatability of the data.

[0018] In some examples of this application, the isotope internal standard solution is prepared by the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13 C3-Progesterone is mixed at a predetermined concentration ratio to obtain the isotope internal standard solution. By adding the isotope internal standard, deviations in sample processing and detection can be corrected, thereby improving the accuracy and stability of target molecule quantification.

[0019] In some examples of this application, the predetermined concentration ratio of each isotope internal standard is selected from (1-20):(1-3):1:1, and optionally 1:2:1:1, 2:2:1:1, 3:2:1:1, 4:2:1:1, 5:2:1:1, 6:2:1:1, 7:2:1:1, 8:2:1:1, 9:2:1:1, 10:2:1:1, 11:2:1:1, 12:2:1:1, 13:2:1:1, 14:2:1:1, 15:2:1:1, 16:2:1:1, 17:2:1:1, 18:2:1:1, 19:2:1:1, and 20:2:1:1. Based on the aforementioned predetermined concentration ratio of isotope internal standards, a wide range of target molecule concentrations can be covered, avoiding interference from the sample matrix on the detection of target molecules. In some preferred embodiments of this application, the predetermined concentration ratio of the isotopic internal standards is 20:2:1:1. Based on cost considerations, this concentration ratio achieves the same effect but at a lower cost.

[0020] In some examples of this application, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is selected from 10:(80-90):1, and optionally 10:80:1, 10:81:1, 10:82:1, 10:83:1, 10:84:1, 10:85:1, 10:86:1, 10:87:1, 10:88:1, 10:89:1, or 10:90:1. In some preferred examples of this application, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is 10:87.5:1. The release agent working solution prepared based on the aforementioned volume ratio can effectively remove impurities in the sample and improve the recovery rate of the target molecule.

[0021] In some examples of this application, the step of placing the sample to be treated and the releasing agent working solution in a fully automated sample preparation instrument for sample pretreatment includes: 1) setting a first predetermined parameter to mix the sample to be treated and the releasing agent working solution separately; 2) setting a second predetermined parameter to pretreatment the mixed sample to be treated and the releasing agent working solution to obtain a pretreated sample. The first predetermined parameter mixes the sample to be treated and the releasing agent working solution to ensure uniform distribution; the second predetermined parameter ensures thorough mixing of the sample to be treated and the releasing agent working solution, improving precipitation and adsorption efficiency. The instrument's predetermined parameter settings reduce inconsistencies caused by human operation, improving experimental repeatability and stability. Furthermore, the sample pretreatment method based on automated instruments allows for simultaneous sample pretreatment in multiple channels, effectively improving sample pretreatment efficiency and throughput. It also reduces human intervention and improves the stability of the sample preparation process.

[0022] In some examples of this application, the aforementioned fully automated sample preparation instrument may be model MSP-400 (manufacturer: Wuhan BGI Genomics Co., Ltd.).

[0023] In some examples of this application, the first predetermined parameter includes at least one of the following: number of pipetting / mixing cycles, pipetting volume, forward and reverse oscillation time, magnetic rod side-mounted magnetic adsorption time, magnetic ring bottom-mounted magnetic adsorption time, and volume of liquid transferred per transfer. The aforementioned first predetermined parameter ensures that the sample to be treated and the releasing agent working solution are evenly distributed, reducing magnetic bead loss and avoiding insufficient subsequent protein adsorption. Furthermore, double magnetic adsorption and automated liquid aspiration ensure sufficient acquisition of the sample containing the target molecules and effectively reduce the difficulty of transferring the supernatant.

[0024] In some examples of this application, the second predetermined parameter includes at least one of: mixing aspiration rate, mixing injection rate, mixing aspiration height, mixing injection height, and mixing immersion depth. The aforementioned second predetermined parameter ensures thorough mixing of the sample to be treated with the releasing agent working solution, enhancing the precipitation effect of the precipitant and the adsorption efficiency of the magnetic beads, thereby improving the thoroughness of impurity removal and the purity of target molecule extraction. Furthermore, it avoids the generation of air bubbles that could affect the accuracy of target molecule quantification. The standardized settings of the aforementioned parameters better adapt to automated operating procedures, reducing human intervention and improving experimental efficiency and throughput.

[0025] In some examples of this application, the samples are selected from metabolomics samples. It is understood that metabolomics can be targeted metabolomics and untargeted metabolomics.

[0026] In some examples of this application, the sample type is a bodily fluid sample, such as whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymphatic fluid. In this document, the aforementioned samples also include the above-mentioned biological materials after processing, such as processed whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymphatic fluid.

[0027] In this application, the container for storing the sample to be processed can be a multi-well plate or a separate tube, including a 96-well plate, a 1.5 mL centrifuge tube or a 2 mL centrifuge tube; the container can have a U-shaped or V-shaped bottom.

[0028] Secondly, this application proposes a sample detection method. According to an embodiment of this application, the method includes: preprocessing the target sample using the method described in the first aspect. The sample detection method based on the aforementioned preprocessing steps not only reduces the interference of impurities on subsequent detection and improves the sensitivity and accuracy of mass spectrometry detection, but also significantly increases the throughput and efficiency of sample processing by adapting to automated workflows. Furthermore, standardized preprocessing steps reduce human error and ensure the stability and repeatability of sample detection, thereby providing reliable technical support for large-scale metabolomics studies, and is particularly suitable for high-throughput detection and analysis of large clinical cohort samples.

[0029] In some examples of this application, the aforementioned method further includes: analyzing the pretreated target sample using the following analytical method, wherein the analytical method is selected from mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, or liquid chromatography-tandem mass spectrometry.

[0030] Thirdly, this application proposes a kit for the pretreatment of untargeted metabolomics samples. According to embodiments of this application, the kit includes a release agent working solution, which comprises an activated magnetic bead suspension and a precipitant. The aforementioned kit can be used for the pretreatment of untargeted metabolomics samples, effectively removing impurities from the samples and improving the recovery rate of target molecules. Its standardized formulation enhances the stability and reproducibility of experiments, while also being compatible with automated operations, increasing sample processing throughput.

[0031] In some examples of this application, the volume ratio of the activated magnetic bead suspension to the precipitant in the release agent working solution is selected from 1:(8-9). In some preferred examples of this application, the volume ratio of the activated magnetic bead suspension to the precipitant is 1:8.75. The release agent working solution prepared based on the aforementioned volume ratio can effectively remove impurities in the sample and improve the recovery rate of the target molecule.

[0032] In some examples of this application, the activated magnetic bead suspension is prepared by the following steps: mixing magnetic beads with an activation reagent at a predetermined mass-volume ratio to obtain the activated magnetic bead suspension. This step activates the magnetic beads to more effectively adsorb and separate impurities in the sample, reducing the interference of impurities on metabolite detection and improving the efficiency and purity of target molecule extraction.

[0033] In some examples of this application, the activating agent is selected from methanol, preferably pure methanol (such as analytical grade methanol). Choosing pure methanol as the activating agent for magnetic beads can enhance the surface activity of the magnetic beads, improve dispersibility, and remove surface impurities.

[0034] In some examples of this application, the predetermined mass-to-volume ratio is selected from (5-15):1, and optionally is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1. In some preferred examples of this application, the predetermined mass-to-volume ratio is 10:1.

[0035] In some examples of this application, the precipitant is selected from a mixture of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is selected from 1:(3-5), optionally 1:3, 1:4, or 1:5. In some preferred examples of this application, the volume ratio of methanol to acetonitrile is 1:4. The precipitant in this application is water-free to avoid emulsion formation and to prevent interference with the suspension of magnetic beads. Furthermore, by optimizing the volume ratio of methanol to acetonitrile, its ability to adsorb proteins is ensured, and impurities are avoided from interfering with the sensitivity of subsequent target molecule detection.

[0036] In some examples of this application, the release agent working solution further includes an isotope internal standard solution. Adding an isotope internal standard solution can effectively improve the accuracy of target molecule quantification, correct for losses and biases in sample processing, reduce errors caused by changes in experimental conditions, and enhance the stability and repeatability of the data.

[0037] In some examples of this application, the isotope internal standard solution is prepared by the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13 C3-Progesterone is mixed at a predetermined concentration ratio to obtain the isotope internal standard solution. By adding the isotope internal standard, deviations in sample processing and detection can be corrected, thereby improving the accuracy and stability of target molecule quantification.

[0038] In some examples of this application, the predetermined concentration ratio of each isotope internal standard is selected from (1-20):(1-3):1:1, and optionally 1:2:1:1, 2:2:1:1, 3:2:1:1, 4:2:1:1, 5:2:1:1, 6:2:1:1, 7:2:1:1, 8:2:1:1, 9:2:1:1, 10:2:1:1, 11:2:1:1, 12:2:1:1, 13:2:1:1, 14:2:1:1, 15:2:1:1, 16:2:1:1, 17:2:1:1, 18:2:1:1, 19:2:1:1, and 20:2:1:1. Based on the aforementioned predetermined concentration ratio of isotope internal standards, a wide range of target molecule concentrations can be covered, avoiding interference from the sample matrix on the detection of target molecules. In some preferred embodiments of this application, the predetermined concentration ratio of the isotopic internal standards is 20:2:1:1. Based on cost considerations, this concentration ratio achieves the same effect but at a lower cost.

[0039] In some examples of this application, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is selected from 10:(80-90):1, and optionally 10:80:1, 10:81:1, 10:82:1, 10:83:1, 10:84:1, 10:85:1, 10:86:1, 10:87:1, 10:88:1, 10:89:1, or 10:90:1. In some preferred examples of this application, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is 10:87.5:1. The release agent working solution prepared based on the aforementioned volume ratio can effectively remove impurities in the sample and improve the recovery rate of the target molecule.

[0040] Fourthly, this application proposes the application of the method described in the first aspect in metabolomics. Based on the method described in the first aspect, it can be used for the preprocessing of non-targeted metabolomics samples, reducing manual intervention, shortening the total sample preparation time, minimizing emulsion formation, and effectively improving sample preparation throughput.

[0041] Fifthly, this application proposes the application of the method described in the first aspect in the preprocessing of metabolomics samples. Preprocessing metabolomics samples based on the method described in the first aspect reduces human intervention, shortens the total sample preparation time, minimizes emulsion formation, and effectively improves sample preparation throughput.

[0042] When preprocessing metabolomics samples using the first approach, a release agent working solution is first prepared. This working solution includes an activated magnetic bead suspension, a precipitant, and an isotope internal standard solution. The activated magnetic bead suspension rapidly binds to proteins in the sample and promotes their precipitation; the precipitant (such as a mixture of methanol and acetonitrile) further synergizes with the magnetic beads to precipitate large protein molecules.

[0043] In the automated pretreatment process, specific parameters such as pipetting and magnetic separation are set to achieve mixing, separation, and supernatant removal, thereby realizing the efficient release and separation of metabolites in the sample and obtaining the pretreated sample required for subsequent mass spectrometry detection. This method has good throughput, stability, and automation potential, and is suitable for large-scale metabolomics sample processing.

[0044] In some examples of this application, the application includes: programming a fully automated sample preparation instrument to perform sample preprocessing according to a predetermined program; wherein programming the fully automated sample preparation instrument to perform sample preprocessing according to a predetermined program includes: 1) mixing the metabolomics sample with the release agent working solution at a predetermined ratio to obtain a mixed sample; 2) removing the liquid from the mixed sample to obtain a preprocessed metabolomics sample. This method's fully automated processing does not rely on manual mixing, centrifugation, transfer, or other steps, significantly improving preprocessing efficiency and achieving the standardized workflow required for high-throughput sample analysis.

[0045] In some examples of this application, step 1) further includes: 1-1) homogenizing the mixed sample; and 1-2) allowing the homogenized mixed sample to stand. Adding both homogenization and standing stages to the mixing step effectively improves the extraction quality of metabolites.

[0046] Understandably, homogenization processes can include a variety of routine laboratory procedures, such as pipetting, shaking, etc.

[0047] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 is a schematic diagram of the experimental process technical route provided in an embodiment of this application;

[0050] Figure 2 is a schematic diagram of the quantitative stability results provided in one embodiment of this application. Detailed Implementation

[0051] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0052] In the process of describing this invention, the terms used herein have been explained and described. These explanations and descriptions are only for the purpose of facilitating the understanding of the solution and should not be regarded as a limitation on the protection of this invention.

[0053] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0054] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0055] The current pretreatment of body fluid samples for non-targeted metabolomics has the following drawbacks: Metabolite extraction using organic solvent liquid-liquid extraction requires 2 hours of low-temperature static extraction at -20°C and 30 minutes of high-speed centrifugation to precipitate proteins, making the entire process time-consuming; manual pipetting to aspirate the supernatant without protein precipitate results in high human intervention and poor result stability; manual pipetting throughout the process prevents simultaneous processing of multiple samples, resulting in low throughput; and after organic solvent protein precipitation, transferring the supernatant is difficult if the sample contains a large amount of precipitate, affecting subsequent non-targeted metabolomics analysis.

[0056] The principle of this application is to use magnetic beads with specific chemical modifications on their surfaces to adsorb impurities such as proteins precipitated by a precipitant in the sample to be treated. By applying a magnetic field, the magnetic beads adsorbing impurities are aggregated and controlled, thereby separating the supernatant containing target molecules (such as metabolites) from the protein and other impurities in the sample to be treated. Compared with traditional liquid-liquid extraction methods that require long periods of settling and the use of large instruments such as centrifuges to settle proteins, the method based on magnetic bead adsorption can separate proteins simply by applying a magnetic field. This step is easy to assemble and configure in automated equipment. After assembly, the automated process can simplify the steps of non-targeted metabolomics, reduce human intervention, allow for multi-channel parallel sample processing, and reduce the difficulty of supernatant transfer through two magnetic adsorption processes. The clean supernatant is then used for subsequent analytical methods (such as mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, or liquid chromatography-tandem mass spectrometry) for detection.

[0057] The following example uses the pretreatment of non-target metabolic fluid samples to illustrate the sample pretreatment method of the application in detail. Those skilled in the art will understand that, based on the sample processing method of this application, the replacement of samples and parameters is also within the scope of protection of this application.

[0058] To facilitate understanding of the technical solution of this application, the sample preprocessing method is described in detail below through a specific example:

[0059] Reagent preparation:

[0060] 1) Magnetic bead activation: Magnetic beads used for preparing several non-target metabolic fluid samples were taken and activated to form a magnetic bead suspension of a certain concentration. The magnetic beads were dispersed in 50% isopropanol, and activation was performed using pure methanol. The activated magnetic beads were then suspended in pure acetonitrile. The ratio of magnetic beads to the activation reagent, pure methanol, was 5 mg: 500 μL, and the concentration of the activated magnetic bead suspension was 10 mg / mL.

[0061] 2) Preparation of precipitant and internal standard solution: Prepare precipitant and internal standard solutions in quantities suitable for several non-target metabolic fluid samples. The precipitant is a methanol:acetonitrile mixture of 1:4; the internal standard solution consists of four isotopic internal standards, namely d... 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13 C3-Progesterone was administered at concentrations of 1 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.05 mg / mL.

[0062] 3) Preparation of the release agent working solution: Thoroughly mix the activated magnetic bead suspension from step 1) and the precipitant prepared in step 2) with the internal standard solution to prepare the release agent working solution. The required volume of magnetic bead suspension is 40 μL, the volume of precipitant is 350 μL, and the volume of internal standard solution is 4 μL for each 100 μL sample.

[0063] Instrument operation:

[0064] 4) Powering on the fully automated sample preparation system: Power on the fully automated sample preparation system (Wuhan BGI Genomics Co., Ltd., MSP-400), submit the experimental application. The experimental application must include the specific number of samples (including preparation QC and Blank), select the experimental method (experimental procedure), confirm the starting well position, and select the automatic barcode scanning function.

[0065] 5) Sample and reagent preparation: Place the release agent working solution prepared in step (3) into the working liquid level of the automated equipment, place the sample to be prepared into the sample rack, and place the consumables into the corresponding positions. Manually check to ensure that there are no errors.

[0066] 6) Automated Sample Preparation Process: Click "Start," and the fully automated sample preparation system will perform fully automated sample preparation according to the set experimental procedure. The experimental procedure specifically includes: consumable self-test → reagent dispensing (mix by pipetting before dispensing) → sample dispensing (mix by pipetting after dispensing) → oscillation → plate transfer → side magnetic suction, settling → pipetting → bottom magnetic suction, settling → pipetting. The key parameters of the experimental procedure are as follows:

[0067] Dispense 390 μL of reagent into each well (mix by pipetting 20 times before the first dispensing, and then 6 times for each pipetting volume of 600 μL).

[0068] Dispense 100 μL of sample into each well (mix well by pipetting after dispensing, pipetting 15 times, each time with a volume of 400 μL);

[0069] Oscillate at 1400 rpm for 90 seconds in both directions;

[0070] Magnetic rod side suction for 2 minutes, 300μL per pipette;

[0071] Magnetic ring bottom magnetic suction for 1 minute, then transfer 250μL of liquid twice.

[0072] 7) Sample processing completed: After the experimental procedure is completed, remove the 96-well deep plate containing the metabolite supernatant and freeze-dry it for 6 hours.

[0073] In the above exemplary sample pretreatment method, the key liquid parameters involved in step 6) are shown in Table 1-2;

[0074] Table 1. Parameters for reagent dispensing and mixing liquids

[0075] Table 2 Sample Distribution Mixture Parameters

[0076] To facilitate understanding of the technical solution of this application, the sample detection method is described in detail below through a specific example:

[0077] Based on the preprocessed sample described above, the following steps are performed:

[0078] 8) Sample reconstitution and detection preparation: Reconstitute the supernatant of the metabolites that was dried in step 7), add 180 μL of 50% methanol solution, shake and centrifuge, separate the samples and mix the QC samples, and prepare for mass spectrometry detection.

[0079] 9) Mass spectrometry analysis: Dispense the sample obtained in step 8) into injection vials and obtain the corresponding chromatographic and mass spectrometric data by mass spectrometry analysis (such as liquid chromatography-mass spectrometry).

[0080] 10) Data analysis: Perform qualitative and / or quantitative analysis (such as Compound Discoverer 3.3) on the mass spectrometry data obtained in step 9) to obtain the non-targeted metabolomics data of the sample.

[0081] The embodiments of this application will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0082] Example 1: Non-targeted metabolomics analysis of 296 human plasma samples

[0083] 1. Batch preparation of reagent kits

[0084] 1) Preparation of magnetic bead suspension

[0085] First, take the magnetic beads (50 mg / mL, 100 mL) stored in 50% isopropanol, mix thoroughly, and aliquot into 50 mL centrifuge tubes. Then, centrifuge at 4000 rpm for 1-2 minutes at 4°C, and fix the magnetic beads with magnetic adsorption, discarding the supernatant. Redissolve the magnetic beads in 500 mL of pure methanol (the ratio of magnetic beads to methanol is 5 mg: 500 μL), and shake repeatedly to ensure full activation. After magnetic adsorption or centrifugation, discard the supernatant, and then redissolve the magnetic beads in 100 mL of pure acetonitrile to obtain an activated magnetic bead suspension with a concentration of 50 mg / mL, which is stored at -20°C for later use.

[0086] 2) Preparation of precipitant

[0087] Methanol and acetonitrile were mixed evenly at a volume ratio of 1:4 to prepare a precipitant, which was then stored at -20℃ for later use.

[0088] 3) Preparation of internal standard solution

[0089] Take four isotopic internal standards: d 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13C3-Progesterone was prepared into stock solutions at a concentration of 1 mg / mL. These stock solutions were then diluted to different concentrations: 1 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.05 mg / mL. Finally, the four isotopic internal standards were mixed in a 1:1:1:1 ratio to obtain a mixed internal standard solution, which was stored at -20°C for later use.

[0090] 4) Reagent kit preparation

[0091] Prepare the magnetic bead suspension, precipitant, and internal standard solution separately according to the required volume for each sample. For a kit specification of 96 samples, the preparation volume for each kit is:

[0092] Magnetic bead suspension: 4.2 mL

[0093] Precipitant: 36.75 mL

[0094] Internal standard solution: 0.42 mL

[0095] After all reagents were prepared, they were aliquoted to the required specifications for 105 samples. The kit consists of the three components described above and should be stored at -20°C for later use.

[0096] 2. Preparation of the releasing agent working solution

[0097] Take out the prepared magnetic bead suspension, precipitant, and internal standard solution. After ensuring the magnetic bead suspension is fully dispersed by shaking, add the internal standard solution to the precipitant and mix thoroughly. Then, transfer the magnetic bead suspension to the mixture of internal standard and precipitant, and vortex to obtain the working solution of the release agent. To ensure the magnetic beads are equilibrated, it is recommended to let the working solution stand at room temperature for 30 minutes.

[0098] Based on the actual number of samples (296 human plasma samples in this example), four batches of release agent working solution need to be prepared, covering 16 sample preparation QCs and 1 reagent Blank, for a total of 313 samples.

[0099] 3. Experiment Application

[0100] Start the fully automated sample preparation system and log in to the user interface. Enter the experiment application interface, fill in the sample quantity, select the experimental procedure, confirm the starting well position, enable the automatic barcode scanning function, and submit the experiment application. Since each batch supports a maximum of 96 samples, if the number exceeds 96, the experiment application must be repeated sequentially according to the batch.

[0101] 4. Preparation of reagents, samples and consumables

[0102] Place the prepared release agent working solution at the working level of the automated equipment, and place the 296 samples to be prepared on the sample rack. Simultaneously, prepare all necessary consumables (such as D1000 conductive pipette tips and 2mL U-shaped bottom orifice plates). After completing these steps, manually verify that all positions are correct.

[0103] 5. Fully automated sample preparation

[0104] Click "Start," and the fully automated sample preparation system will begin preparing samples according to the predetermined procedure. The experimental workflow is shown in Figure 1, and the entire process includes:

[0105] (1) Consumables self-inspection

[0106] (2) Reagent distribution

[0107] Dispense 390 μL of the release agent working solution into each well, mixing it 20 times by pipetting before dispensing, and then mixing it 6 times each time thereafter. The pipetting volume is 600 μL.

[0108] (3) Sample allocation

[0109] Dispense 100 μL of sample into each well and mix by pipetting 15 times, with each pipetting volume being 400 μL.

[0110] (4) Oscillation

[0111] Set the oscillation speed to 1400 rpm, and oscillate for 90 seconds in both forward and reverse directions.

[0112] (5) Moving the plate

[0113] (6) Side magnetic attraction

[0114] Let it sit magnetically for 2 minutes.

[0115] (7) Pipetting

[0116] Use 300 μL for each pipette.

[0117] (8) Bottom magnetic closure

[0118] Let it sit magnetically for 1 minute.

[0119] (9) Secondary pipetting

[0120] Two 250 μL transfers were made from each well.

[0121] Repeat the above steps for each sample batch to complete the preparation of all samples.

[0122] 6. Dry

[0123] The prepared metabolite supernatant (250 μL) was freeze-dried for 6 hours. If LC-MS analysis could not be performed within 24 hours, the sample was stored at -20°C.

[0124] 7. Reconstitute

[0125] The dried metabolites were reconstituted with 50% methanol solution. 180 μL of the reconstituted solution was added to each sample, and the mixture was shaken at 2500 rpm for 10 minutes until the metabolites were completely dissolved.

[0126] 8. Centrifugation

[0127] Place the 96-well plate in a centrifuge, set the speed to 4000 rpm and the centrifugation temperature to 4℃, and centrifuge for 30 minutes to separate the metabolites.

[0128] 9. Separate board and mixed QC

[0129] Take 50 μL of metabolite sample from each well and transfer it to three different sample loading plates for the detection of positive and negative ions, respectively. One plate is used as a backup plate. Simultaneously, take 20 μL of metabolite supernatant from the remaining sample and mix them to form a QC sample.

[0130] 10. LC-MS / MS on-machine testing

[0131] Data acquisition was performed using an ultra-high performance liquid chromatograph (ACQUITY UHPLC I-Class FL / FTN) and a tandem mass spectrometer (Q-Exactive). The main chromatographic and mass spectrometric parameters were set as follows:

[0132] Column: Waters ACQUITY BEH C18 (100x2.1 mm, 1.7 μm)

[0133] Column temperature: 45℃

[0134] Injection volume: 5 μL

[0135] Flow rate: 0.35 mL / min

[0136] Effective gradient:

[0137] 0-1 min: 98% mobile phase A + 2% mobile phase B

[0138] 1-9 min: Mobile phase A decreased from 98% to 2%, while mobile phase B increased from 2% to 98%.

[0139] 9-12 min: 2% mobile phase A + 98% mobile phase B

[0140] 12-12.1 min: Mobile phase A increases from 2% to 98%, mobile phase B decreases from 98% to 2%.

[0141] 12.1-15 min: 98% mobile phase A + 2% mobile phase B

[0142] Mass spectrometry parameters:

[0143] Data Dependency Acquisition (DDA)

[0144] Primary mass spectrometry scanning range: 70-1050 m / z

[0145] Resolution: 70,000

[0146] Maximum ion implantation time: 100ms

[0147] Secondary mass spectrometry fragmentation mode: HCD

[0148] Shattering Energy: NCE 20, 40, 60

[0149] Secondary mass spectrometry resolution: 17,500

[0150] Maximum ion implantation time: 50ms

[0151] Dynamic exclusion time: 3 seconds

[0152] 11. Non-targeted metabolomics data analysis

[0153] 1) Extraction and identification of metabolite ion peaks

[0154] Mass spectrometry data were analyzed using Compound Discoverer 3.3 software, and metabolites were identified using the BMDB, mzCloud, and ChemSpider online databases. A data matrix of metabolite peak areas and identification results was ultimately obtained.

[0155] 2) Data processing and stability assessment

[0156] Further information analysis was performed based on the data matrix to generate metabolite identification and quantitative stability results for the samples. The identification and quantitative results are shown in Table 4 and Figure 2, indicating that the automated preparation method for non-targeted metabolomics body fluid samples using magnetic beads combined with a fully automated sample preparation system yields excellent identification results. Furthermore, the number of metabolites with a CV < 30% in the mixed QC samples accounts for a significant portion of the total number of metabolites. 比 The value is 91%, demonstrating good stability.

[0157] Table 4

[0158] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0159] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0160] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A sample preprocessing method, characterized in that, include: A release agent working solution is prepared, the release agent working solution comprising: an activated magnetic bead suspension and a precipitant; The sample to be processed and the working solution of the release agent are placed in a fully automated sample preparation instrument for sample pretreatment.

2. The method according to claim 1, characterized in that, In the working solution of the release agent, the volume ratio of the activated magnetic bead suspension to the precipitant is selected from 1:(8-9), preferably 1:8.75; Optionally, the volume ratio of the sample to be treated to the working liquid of the release agent is selected from 10:(35-40), preferably 10:

39.

3. The method according to claim 2, characterized in that, The activated magnetic bead suspension was prepared by the following steps: The magnetic beads and the activating reagent are mixed at a predetermined mass-volume ratio to obtain the activated magnetic bead suspension. Optionally, the activating agent is selected from methanol; Optionally, the predetermined mass-to-volume ratio is selected from (5-15):1, preferably 10:

1.

4. The method according to claim 2, characterized in that, The precipitant is selected from a mixture of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is selected from 1:(3-5); preferably 1:

4.

5. The method according to any one of claims 1-4, characterized in that, The working solution of the release agent further includes: an isotope internal standard solution; Optionally, the isotope internal standard solution is prepared by the following steps: Isotope d 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13 C3-Progesterone was mixed at a predetermined concentration ratio to obtain the isotope internal standard solution; Optionally, the predetermined concentration ratio is selected from (1-20):(1-3):1:1, preferably 20:2:1:1; Optionally, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is selected from 10:(80-90):1, preferably 10:87.5:

1.

6. The method according to claim 1, characterized in that, The step of placing the sample to be processed and the working solution of the release agent in a fully automated sample preparation instrument for sample pretreatment includes: 1) Set the first predetermined parameters to mix the sample to be treated and the working solution of the release agent respectively; 2) Set a second predetermined parameter to pretreat the mixed sample and the release agent working solution to obtain a pretreated sample.

7. The method according to claim 6, characterized in that, The first predetermined parameter includes at least one of the following: number of times of mixing by blowing, volume of blowing, time of forward and reverse oscillation, time of magnetic rod side magnetic attraction, time of magnetic ring bottom magnetic attraction, and volume of liquid transferred each time.

8. The method according to claim 6, characterized in that, The second predetermined parameter includes at least one of the following: mixing and aspiration rate, mixing and injection rate, mixing and aspiration height, mixing and injection height, and mixing and immersion depth.

9. The method according to claim 1, characterized in that, The samples were selected from metabolomics samples; Preferably, the sample type is a biological material such as whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, bile, gastric juice, tissue fluid, and lymph.

10. A sample detection method, characterized in that, include: The target sample is preprocessed using the method described in any one of claims 1-9.

11. The method according to claim 10, characterized in that, Further includes: The pretreated target samples were analyzed using the following analytical methods, which were selected from mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, or liquid chromatography-tandem mass spectrometry.

12. A kit for pretreatment of non-targeted metabolomics samples, characterized in that, include: The release agent working solution comprises: an activated magnetic bead suspension and a precipitant.

13. The reagent kit according to claim 12, characterized in that, The volume ratio of the activated magnetic bead suspension to the precipitant is selected from 1:(8-9), preferably 1:8.75; Optionally, the activated magnetic bead suspension is prepared by the following steps: mixing magnetic beads and an activation reagent at a predetermined mass-volume ratio to obtain the activated magnetic bead suspension; Optionally, the activating agent is selected from methanol; Optionally, the predetermined mass-to-volume ratio is selected from (5-15):1, preferably 10:1; Optionally, the precipitant is selected from a mixture of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is selected from 1:(3-5); preferably 1:4; Optionally, the kit may further include: an isotope internal standard solution; Optionally, the isotope internal standard solution is prepared by the following steps: [The steps involve] mixing isotope d... 3 -Leucine 13 C9-Phenylalanine, d 5 -Tryptophan and 13 C3-Progesterone was mixed at a predetermined concentration ratio to obtain the isotope internal standard solution; Optionally, the predetermined concentration ratio is selected from (1-20):(1-3):1:1, preferably 20:2:1:1; Optionally, the volume ratio of the activated magnetic bead suspension, precipitant, and isotope internal standard solution is selected from 10:(80-90):1, preferably 10:87.5:

1.

14. The application of the method according to any one of claims 1-9 in metabolomics.

15. The application of the method according to any one of claims 1-9 in the preprocessing of metabolomics samples.

16. The application according to claim 15, characterized in that, include: Set up the program for the fully automated sample preparation instrument and perform sample preprocessing according to the predetermined program; The process includes programming the fully automated sample preparation instrument and performing sample preprocessing according to a predetermined program, including: 1) Mix the metabolomics samples with the release agent working solution according to a predetermined ratio to obtain a mixed sample; 2) Remove the liquid from the mixed sample to obtain a pretreated metabolomics sample.

17. The application according to claim 16, characterized in that, Step 1) further includes: 1-1) The mixed samples are homogenized; 1-2) The homogenized mixed sample is left to stand.

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