A purification separation column for clinical pre-treatment, a pre-treatment device, a pre-treatment method and application

Abstract

The application provides a purification separation column for clinical detection pretreatment, a pretreatment device, a pretreatment method and application, the purification separation column comprises a column pipe with at least one open end, a filter assembly is arranged in the column pipe, a to-be-filtered liquid enters from the outside of the column pipe through the opening, and enters the inner cavity of the column pipe after passing through the filter assembly; the filter assembly comprises a first sieve plate, a water-blocking layer, a filler layer and a second sieve plate arranged in sequence from bottom to top. The purification separation column provided by the application is mainly used for pretreatment of a to-be-tested sample for clinical detection, conventional solid-phase extraction needs to go through multiple steps such as activation, adsorption, washing and elution, and the solution reagents involved are also different, in order to simplify the pretreatment operation steps, the purification separation column provided by the application realizes one-step pretreatment of the to-be-tested sample, greatly reduces the operation process of the pretreatment, and does not need careful operation in the process, so that the operation threshold is reduced.

Description

[0001] The present application claims priority to the Chinese patent application with the application date of May 11, 2022, the application number of 202210513149.4, and the invention name of "A purification separation column for pre-treatment of clinical detection, pre-treatment device, pre-treatment method and application", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of clinical detection, and relates to a purification separation column for pre-treatment of clinical detection, a pre-treatment device, a pre-treatment method and application. BACKGROUND

[0003] Solid phase extraction (SPE) technology is a sample pretreatment method based on chromatographic theory, which uses selective adsorption and selective elution to enrich, separate and purify the sample. Solid phase extraction is to use a solid adsorbent to adsorb the target substance in the liquid sample, so that the target substance is separated from the matrix and interfering compounds in the sample, and then eluted or heated to desorb the adsorbent, so as to separate and enrich the target substance.

[0004] Compared with traditional liquid-liquid extraction, solid phase extraction does not require a large number of mutually insoluble solvents, and does not produce emulsification during processing. Because of the use of high-selectivity adsorbent (stationary phase), solid phase extraction can significantly reduce the amount of solvent used. The pretreatment process of solid phase extraction is relatively simple, has high selectivity, high recovery rate and is easy to automate, and is widely used in water quality detection, pharmaceuticals, environmental analysis, food analysis and tobacco analysis. It is a very common pretreatment method for organic compound testing (including clinical applications), including vitamins D, aldosterone, steroid hormones, bile acids, etc.

[0005] Solid phase extraction operation can be divided into four steps of column pretreatment, sample loading, column washing and analyte elution, as shown in the following figure. Whether each step is designed reasonably will affect the recovery rate of the experiment, resulting in premature penetration of solutes, incomplete washing of interferents, loss of target substances, incomplete elution, etc., and it is also difficult to achieve analyte recovery at this time.

[0006] CN212974266U is a kind of solid phase extraction rapid separation device and its solid phase extraction tube, including pressurizing structure, solid phase extraction tube and filter head, the pressurizing structure includes foot-pedal type air cylinder and buffer air bag, the air outlet of the foot-pedal type air cylinder is connected with the connecting head through pipeline, the buffer air bag is arranged on the pipeline, and the filter head is inserted into the liquid outlet pipe opening of the solid phase extraction tube body. When the adsorbent completely adsorbs the sample liquid in the solid phase extraction tube body, the foot-pedal type air cylinder is used to continuously pressurize the solid phase extraction tube body to press out the treated sample liquid in the solid phase extraction tube body, which flows out of the solid phase extraction tube body through the filter head.

[0007] CN212039361U A centrifugal and pneumatic dual-purpose solid phase extraction and separation device, comprising two components of upper and lower parts, the upper part being a solid phase extraction and separation component, and the lower part being a collection component. The solid phase extraction and separation component comprises a column body, a sealing cover, a sieve plate and a filler, the upper end of the column body is connected to the sealing cover, and the lower part of the column body can be connected to the lower component through a sleeve screwing mode; the sealing cover is provided with an air inlet hole, and an external pressurizing device can be connected to the air inlet hole to increase the air pressure in the column; the sieve plate is located in the middle part of the column body, and two or more sieve plates are fixed at intervals. The collection component is a collection tube, the upper end of which is connected to the sealing cover; the collection tube is provided with an air hole in the middle upper part to ensure that the inside of the tube is in communication with the outside.

[0008] CN202161857U discloses a solid phase extraction pretreatment filter, which is applied to the pretreatment process of a sample for testing. The filter is a funnel-shaped device made of glass, the upper part of which has a trapezoidal structure, and the lower part has a cylindrical structure. The outer diameter of the cylindrical part is consistent with the inner diameter of the solid phase extraction column. In use, the filter is inserted into a filter paper, and then the cylindrical part is sleeved on the upper part of the solid phase extraction column.

[0009] In solid phase extraction, several steps are usually required to purify the pretreatment liquid to obtain the sample, including sample preliminary treatment, filler activation, sample adsorption, one to three times of washing and elution, etc. Because the pores of the solid phase extraction column filler are small, the purity of the sample has special requirements, and there cannot be suspended matter or solid particles, otherwise the column will be blocked and subsequent operations will be difficult to perform. Because several different solutions are involved in the activation, adsorption, washing and elution steps, during the pretreatment operation, a positive pressure or negative pressure device is required to roughly dry the solution in the solid phase extraction column before the next solution is added. However, the drying cannot be excessive, and the wetness of the filler must be maintained to ensure its separation performance. Therefore, it is difficult to maintain the drying degree of the surface of the filler, the channel path formed by the residual liquid and the resistance of the sample liquid during the treatment process, thereby affecting the batch consistency and inter-batch consistency of the sample. SUMMARY

[0010] In view of the deficiencies of the prior art, the purpose of the present application is to provide a purification and separation column for clinical detection pretreatment, a pretreatment device, a pretreatment method and an application, which are mainly used for pretreating the sample to be tested in clinical detection. When conventional clinical detection is pretreated by solid phase extraction, multiple operations such as activation, adsorption, washing and elution are required, and the solution reagents involved are also different. In order to simplify the pretreatment operation steps, the present application provides a purification and separation column to realize one-step pretreatment of the sample to be tested, greatly reducing the operation process of the pretreatment, and without the need for careful operation in the process, thereby reducing the operation threshold.

[0011] To achieve this purpose, the present application adopts the following technical solutions:

[0012] In a first aspect, the present application provides a purification separation column for pre-treatment of clinical detection, which comprises a column tube with at least one open end, wherein a filter assembly is arranged in the column tube, and a to-be-filtered liquid enters the column tube from outside of the column tube through the open end and then passes through the filter assembly.

[0013] The filter assembly comprises, from bottom to top, a first sieve plate, a water-blocking layer, a filler layer and a second sieve plate.

[0014] The purification separation column provided by the present application is mainly used for pre-treatment of a to-be-tested sample for clinical detection. When a conventional clinical detection is pre-treated by solid-phase extraction, a plurality of operations such as activation, adsorption, washing and elution are required, and different reagents are involved. In order to simplify the pre-treatment operation steps, the present application provides a purification separation column to realize one-step pre-treatment of the to-be-tested sample, greatly simplifying the operation process of pre-treatment, reducing the amount of reagents, improving the sample processing efficiency, and reducing the operation threshold. During the process, careful operation is not required. When used, the to-be-tested sample passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the main tube of the purification separation column. In the process of passing through the filter assembly, the impurities in the to-be-tested sample are preliminarily separated by the first sieve plate. The high-molecular compounds in the water-blocking layer allow the organic phase to pass through smoothly, and swell rapidly when encountering the aqueous phase, thereby separating the organic phase and the aqueous phase while blocking water. The aqueous phase and protein precipitate in the to-be-filtered liquid are blocked below the water-blocking layer, and then the organic phase continues to pass through the filler layer. The filler layer is filled with adsorbent material with specific adsorption function, which selectively adsorbs specific substances in the organic phase to purify the extract. The second sieve plate can filter some layered impurities and particulate impurities leaked from the previous layers to prevent the chromatographic column from being blocked.

[0015] As a preferred technical solution of the present application, the filter assembly is located at the bottom of the inner cavity of the column tube, and the to-be-filtered liquid passes through the filter assembly from the bottom of the column tube and then enters the inner cavity of the column tube.

[0016] Preferably, the filler layer is filled with purification material, and the purification material comprises specific adsorbent material for adsorbing polypeptides and phospholipid compounds.

[0017] Preferably, the purification material comprises any one or a combination of at least two of silica gel, octadecyl-bonded silica gel and its derivatives, octyl-bonded silica gel and its derivatives, diatomite, activated carbon, N-propyl ethylenediamine, quartz sand, molecular sieve, hydrophilic-lipophilic polymeric filler, strong cation exchange resin, weak cation exchange resin, strong anion exchange resin or weak anion exchange resin.

[0018] It should be noted that, considering that the blood sample mainly contains protein, polypeptide, fat (mainly phospholipid compounds), inorganic salt and other objects to be separated and detected, so as to avoid the large matrix effect, in the process of extraction by using acetonitrile, the protein in the sample is first precipitated, and the inorganic salt in the sample remains in the water phase due to the strong water solubility. Therefore, the purification filler used in the present application preferentially selects specific adsorption materials capable of adsorbing polypeptides and phospholipid compounds to further remove impurities from the sample.

[0019] Preferably, the particle size of the purification material is 5-2000 μm, for example, it can be 5 μm, 50 μm, 100 μm, 300 μm, 500 μm, 700 μm, 900 μm, 1000 μm, 1200 μm, 1400 μm, 1600 μm, 1800 μm or 2000 μm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0020] Preferably, the filling mass of the purification material is 10-100 mg, for example, it can be 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg, but not limited to the listed values, and other values not listed in the range are also applicable.

[0021] As a preferred technical solution of the present application, the pore size of the first sieve plate is the same as or different from that of the second sieve plate.

[0022] Preferably, the pore size of the first sieve plate is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0023] Preferably, the pore size of the second sieve plate is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0024] Preferably, the water-blocking layer is a water-blocking film, and the pore size of the water-blocking film is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0025] It should be noted that the water-blocking layer defined in the present application can adopt a sieve plate structure with a certain pore size, and the inner surface of the hole is coated with a polymer compound. The organic phase can pass through the water-blocking layer, and the polymer compound swells after being exposed to water, thereby achieving the effect of hindering the passage of the aqueous phase, and ultimately realizing the separation of the aqueous phase and the organic phase.

[0026] As a preferred technical solution of the present application, a filter layer is further arranged between the first sieve plate and the water-blocking layer.

[0027] In the pretreatment process of the sample to be tested using the purification separation column provided by the present application, an extractant such as acetonitrile needs to be added. For blood samples, which mainly contain proteins and polypeptides, the proteins in the blood sample will form a precipitate after the addition of the extractant. By arranging a filter layer between the first sieve plate and the water-blocking layer, the protein precipitate can be filtered and retained to prevent the water-blocking layer at the rear end from being blocked.

[0028] Preferably, the filter layer comprises at least one filter membrane.

[0029] Preferably, the filter layer comprises at least two filter membranes, and the pore sizes of the filter membranes are the same or different.

[0030] Preferably, the filter layer comprises at least two filter membranes with different pore sizes, and the pore sizes of the filter membranes decrease from bottom to top.

[0031] Preferably, the filter layer comprises a first filter membrane, a second filter membrane, and a third filter membrane stacked in order from bottom to top, and the pore size of the first filter membrane is greater than or equal to the pore size of the second filter membrane, which is greater than or equal to the pore size of the third filter membrane.

[0032] Preferably, the pore size of the first filter membrane is 0.5-2.5 μm, for example, it can be 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.2 μm, 1.5 μm, 2.0 μm, or 2.5 μm, but it is not limited to the listed values. Other values not listed in this range are also applicable.

[0033] Preferably, the pore size of the second filter membrane is 0.2-0.8 μm, for example, it can be 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, or 0.8 μm, but it is not limited to the listed values. Other values not listed in this range are also applicable.

[0034] Preferably, the third filter membrane has a pore size of 0.1-0.5 μm, for example, 0.1 μm, 0.15 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm or 0.5 μm, but not limited to the listed values, and other values not listed in the range are also applicable.

[0035] In the present application, the pore sizes of the first filter membrane, the second filter membrane and the third filter membrane are different, and decrease from bottom to top, so that the purification separation column can adapt to the filtration of different to-be-filtered liquids, thereby expanding the application range of the purification separation column; at the same time, the pore sizes of the filter membranes are different, so that the separation process of the to-be-filtered liquid is progressive, and the separation effect of the to-be-filtered liquid is enhanced.

[0036] In a second aspect, the present application provides a pre-treatment device for clinical detection, which comprises a liquid storage tube and the purification separation column for pre-treatment of clinical detection.

[0037] The liquid storage tube and the purification separation column are used in cooperation, the extraction reagent is pre-packaged in the liquid storage tube, the to-be-tested sample is added into the liquid storage tube, the to-be-tested sample is mixed with the extraction reagent and then is left to stratify, the purification separation column is pressed into the liquid storage tube, and the upper organic phase passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube of the purification separation column.

[0038] As a preferred technical solution of the present application, the inner cavity of the column tube of the purification separation column is divided into a first liquid storage cavity and a second liquid storage cavity from bottom to top.

[0039] Preferably, the outer wall diameter of the column tube corresponding to the first liquid storage cavity is smaller than the outer wall diameter of the column tube corresponding to the second liquid storage cavity, so that an annular step surface is formed at the outer wall of the column tube between the first liquid storage cavity and the second liquid storage cavity; after the purification separation column is pressed into the liquid storage tube, the opening end surface of the liquid storage tube is in contact with the annular step surface.

[0040] As a preferred technical solution of the present application, the outer wall of the column tube at the position corresponding to the filter assembly is provided with a sealing ring in the circumferential direction.

[0041] In the present application, the sealing ring is sleeved on the outer wall of the column tube in the circumferential direction, after the purification separation column is pushed into the liquid storage tube, the sealing ring can seal the gap between the purification separation column and the liquid storage tube, prevent the to-be-tested sample from flowing out of the gap between the purification separation column and the liquid storage tube, and ensure that the to-be-tested sample stored in the liquid storage tube can only enter from the bottom opening of the purification separation column.

[0042] Preferably, a cutout is arranged at the opening of the top end of the column tube of the purification separation column, and during the process of pressing the purification separation column into the liquid storage tube, the air in the liquid storage tube is discharged through the cutout.

[0043] In a third aspect, the present application provides a pretreatment method for clinical detection, which uses the pretreatment device for clinical detection in the second aspect to pretreat a sample to be detected, and the pretreatment method comprises the following steps:

[0044] The sample to be detected is added into the liquid storage tube, and the sample to be detected is mixed with the extraction reagent previously added and left to stand, and after the water phase and the organic phase are preliminarily separated, the purification separation column is pressed into the liquid storage tube, the organic phase passes through the filter assembly from the bottom of the purification separation column into the inner cavity of the column tube of the purification separation column, and the water phase is isolated by the filter assembly, and the organic phase entering the inner cavity is the pretreated sample to be detected, which is detected on a machine or is redissolved on a machine after nitrogen blowing.

[0045] In addition to the redesign of the structure of the purification separation column, the operation process of the pretreatment device is also improved, and compared with the traditional dispersive solid-phase extraction and solid-phase extraction, the pretreatment method provided by the present application is simple in operation and good in purification effect, and one-step purification treatment is realized. Specifically,

[0046] (1) Compared with the dispersive solid-phase extraction method, the conventional operation process of the dispersive solid-phase extraction method comprises: adding an extraction reagent into a sample to be detected to form a mixed solution, adding a separation material package into the mixed solution, then vortex mixing, then performing first centrifugation, transferring the upper organic phase to another container after the water phase and the organic phase are separated, then adding a purification agent for second vortex mixing, transferring the obtained organic phase to another container after standing or centrifugation and using a filter membrane, and finally taking the organic phase to a machine for detection or nitrogen blowing and redissolving for machine detection. As can be seen, the dispersive solid-phase extraction method needs to transfer the organic phase multiple times, and in addition, it involves complex operations such as two times of centrifugation and two times of vortex mixing. In addition, the purification agent used in the dispersive solid-phase extraction method only relies on its adsorption for purification, while the present application designs the purification agent filler into a bed structure and fills it into the column tube, so that the extraction liquid is purified by the adsorption of the purification agent while part of the impurities is intercepted when passing through the filler layer, and the supernatant after purification is discharged.

[0047] (2) Compared with the solid phase extraction method, the solid phase extraction method is complex in process, and most samples need to be pretreated before the solid phase extraction method is used, such as adding a regulator, a diluent or a precipitant to the sample to be tested to pre-precipitate proteins to ensure that the column is not blocked. In addition, the solid phase extraction column needs to be completed under the action of a positive pressure or negative pressure device to complete the two-step activation, multiple washing and elution processes, and the degree of suction needs to be controlled to ensure that the packing is not affected by the column efficiency, and a large amount of waste liquid is generated. When eluted, an additional collection plate needs to be replaced to collect the eluent, and the operator is required to be higher. The pretreatment device provided by the application only needs to press the purification separation column into the liquid storage pipe, which saves the complex operations such as activation, washing, elution and replacement of the collection plate required by the traditional solid phase extraction method, greatly reduces the operation difficulty and saves the operation time. At the same time, the method of the application basically does not generate waste liquid, and is more environmentally friendly than the solid phase extraction method.

[0048] As a preferred technical solution of the application, the extraction reagent comprises a salt and an extractant.

[0049] Preferably, the salt comprises any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride or potassium chloride.

[0050] Preferably, the extractant comprises acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile or a mixture of acidified acetonitrile and methanol.

[0051] Preferably, the extraction reagent further comprises a purification agent.

[0052] Preferably, the purification agent comprises any one or a combination of at least two of N-propyl ethylenediamine, activated carbon or C18.

[0053] In a fourth aspect, the application provides an application of the pretreatment device of the second aspect, which is used for pretreating a sample to be tested before clinical detection.

[0054] The numerical range of the application includes not only the above-mentioned point values, but also any point values between the above-mentioned numerical ranges which are not mentioned, and for the sake of brevity and simplicity, the application does not enumerate the specific point values included in the range.

[0055] Compared with the prior art, the application has the following beneficial effects:

[0056] The purification separation column provided by this invention is mainly used for pretreatment of samples for clinical testing. Conventional clinical testing using solid-phase extraction requires multiple steps such as activation, adsorption, washing, and elution, involving various solutions and reagents. To simplify the pretreatment process, this invention provides a purification separation column that enables one-step pretreatment of samples, greatly simplifying the pretreatment process, reducing reagent usage, and improving sample processing efficiency. The process requires no careful handling, lowering the operational threshold; and it generates virtually no waste liquid, making it environmentally friendly. In use, the sample to be tested passes through the filter assembly from the bottom of the purification separation column and enters the main chamber of the purification separation column. During the process of passing through the filter assembly, the first sieve plate performs preliminary separation of impurities in the sample. The high molecular weight compounds in the water-blocking layer allow the organic phase to pass smoothly. When it encounters the aqueous phase, it expands rapidly, blocking water and separating the organic and aqueous phases at the same time. The aqueous phase and protein precipitate in the filtrate are blocked below the water-blocking layer. Subsequently, the organic phase continues to pass through the packing layer, which is filled with adsorbent material with specific adsorption function. The selective adsorption of specific substances in the organic phase by the adsorbent material purifies the extract. The second sieve plate can filter out some layered impurities and particulate impurities missed by the previous layers to prevent clogging of the chromatographic column. Attached Figure Description

[0057] Figure 1 A schematic diagram of the structure of a purification and separation column provided in a specific embodiment of the present invention;

[0058] Figure 2 A schematic diagram of the pretreatment device provided in a specific embodiment of the present invention;

[0059] Wherein, 1-column tube; 2-second sieve plate; 3-packing layer; 4-water barrier layer; 5-filter layer; 51-first filter membrane; 52-second filter membrane; 53-third filter membrane; 6-first sieve plate; 7-sealing ring; 8-cut; 9-liquid storage tube; 10-organic phase; 11-aqueous phase. Detailed Implementation

[0060] It should be understood that in the description of this invention, the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0061] It should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "set," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0062] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0063] In one specific embodiment, the present invention provides a purification and separation column for pretreatment in clinical testing, such as... Figure 1 As shown, the purification and separation column includes a column tube 1 with at least one open end. A filter assembly is provided inside the column tube 1. The liquid to be filtered enters from the outside of the column tube 1 through the opening, passes through the filter assembly, and enters the inner cavity of the column tube 1.

[0064] The filter assembly includes a first screen plate 6, a water-blocking layer 4, a filler layer 3, and a second screen plate 2 arranged sequentially from bottom to top.

[0065] The purification and separation column provided by this invention is mainly used for pretreatment of samples to be tested in clinical tests. Conventional clinical tests using solid-phase extraction for pretreatment require multiple steps such as activation, adsorption, washing, and elution, and the solutions and reagents involved vary. In order to simplify the pretreatment operation steps, this invention provides a purification and separation column that realizes one-step pretreatment of samples to be tested, which greatly simplifies the pretreatment operation process, reduces reagent consumption, and improves sample processing efficiency; moreover, no careful handling is required during the process, lowering the operation threshold. In use, the sample to be tested passes through the filter assembly from the bottom of the purification separation column and enters the main chamber of the purification separation column. During the process of passing through the filter assembly, the impurities in the sample to be tested are initially separated by the first sieve plate 6. The high molecular weight compounds in the water-blocking layer 4 allow the organic phase 10 to pass through smoothly. When it encounters the aqueous phase 11, it expands rapidly, blocking water and separating the organic phase 10 and the aqueous phase 11 at the same time. The aqueous phase 11 and protein precipitate in the filtrate are blocked below the water-blocking layer 4. Subsequently, the organic phase 10 continues to pass through the packing layer 3, which is filled with adsorbent material with specific adsorption function. The specific substances in the organic phase 10 are adsorbed by the selective adsorption of the adsorbent material, so that the extract is purified. The second sieve plate 2 can filter some layered impurities and particulate impurities missed by the previous layers to prevent clogging of the chromatographic column.

[0066] Furthermore, the filter assembly is located at the bottom of the inner cavity of the column tube 1, and the filtrate enters the inner cavity of the column tube 1 after passing through the filter assembly from the bottom of the column tube 1.

[0067] Furthermore, the filler layer 3 is filled with a purification material, which includes a specific adsorption material for adsorbing polypeptides and phospholipids.

[0068] Furthermore, the purification material includes any one or a combination of at least two of the following: silica gel, octadecyl bonded silica gel and its derivatives, octadecyl bonded silica gel and its derivatives, diatomaceous earth, activated carbon, N-propylethylenediamine, quartz sand, molecular sieve, hydrophilic-lipophilic polymeric filler, strong cation exchange resin, weak cation exchange resin, strong anion exchange resin, or weak anion exchange resin.

[0069] It should be noted that, considering that blood samples mainly contain proteins, peptides, fats (mainly phospholipids), and inorganic salts, which need to be separated from the target analytes to avoid affecting subsequent detection and to minimize matrix effects, proteins in the sample precipitate first during the extraction process using acetonitrile (or a mixture of acetonitrile and methanol). Inorganic salts in the sample remain in the aqueous phase 11 due to their strong water solubility. Therefore, the purification packing material used in this invention preferentially selects specific adsorption materials that can adsorb peptides and phospholipids to further remove impurities from the sample.

[0070] Furthermore, the particle size of the purification material is 5–2000 μm.

[0071] Furthermore, the filling mass of the purification material is 10-100 mg.

[0072] Furthermore, the aperture of the first sieve plate 6 may be the same as or different from the aperture of the second sieve plate 2.

[0073] Furthermore, the aperture of the first sieve plate 6 is 5 to 50 μm, and the aperture of the second sieve plate 2 is 5 to 50 μm.

[0074] Furthermore, the water-blocking layer 4 is a water-blocking membrane with a pore size of 5–50 μm.

[0075] It should be noted that the water-blocking layer 4 defined in this invention can adopt a sieve plate structure with a certain pore size. The inner surface of the pores is coated with a polymer compound. The organic phase 10 can pass through the water-blocking layer 4, and the polymer compound absorbs water and expands after encountering water, thereby achieving the effect of preventing the aqueous phase 11 from passing through, and finally realizing the separation of the aqueous phase 11 and the organic phase 10.

[0076] Furthermore, a filter layer 5 is also provided between the first sieve plate 6 and the water-blocking layer 4.

[0077] In the pretreatment process of the sample to be tested using the purification separation column provided by the present invention, an extractant, such as acetonitrile, needs to be added. For blood samples, which mainly contain proteins and peptides, the proteins in the blood sample will form precipitates after the extractant is added. By setting a filter layer 5 between the first sieve plate 6 and the water-blocking layer 4, the protein precipitates can be filtered and retained to avoid clogging the water-blocking layer 4 at the back end.

[0078] Furthermore, the filter layer 5 includes at least one filter membrane.

[0079] Furthermore, the filter layer 5 includes at least two filter membranes, the filter membranes having the same or different pore sizes.

[0080] Furthermore, the filter layer 5 includes at least two filter membranes with different pore sizes, the pore sizes of which decrease sequentially from bottom to top.

[0081] Furthermore, the filter layer 5 includes a first filter membrane 51, a second filter membrane 52 and a third filter membrane 53 stacked sequentially from bottom to top, wherein the pore size of the first filter membrane 51 is greater than or equal to the pore size of the second filter membrane 52 and the pore size of the third filter membrane 53.

[0082] Specifically, the pore size of the first filter membrane 51 is 0.5 to 2.5 μm, the pore size of the second filter membrane 52 is 0.2 to 0.8 μm, and the pore size of the third filter membrane 53 is 0.1 to 0.5 μm.

[0083] In this invention, the pore sizes of the first filter membrane 51, the second filter membrane 52, and the third filter membrane 53 are different, decreasing sequentially from bottom to top, so that the purification separation column can adapt to the filtration of different liquids to be filtered, thereby expanding the applicable range of the purification separation column; at the same time, the different pore sizes of each filter membrane make the separation process of the liquid to be filtered progressive, enhancing the separation effect of the liquid to be filtered.

[0084] In another specific embodiment, the present invention provides a pretreatment device for clinical testing, such as... Figure 2 As shown, the pretreatment device includes a liquid storage tube 9 and the purification and separation column described above for pretreatment in clinical testing.

[0085] The storage tube 9 and the purification separation column are used together. The storage tube 9 is pre-filled with extraction reagent. The sample to be tested is added to the storage tube 9. After the sample to be tested and the extraction reagent are mixed, they are allowed to stand and separate into layers. The purification separation column is pressed into the storage tube 9. The upper organic phase 10 passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube 1 of the purification separation column.

[0086] Furthermore, the inner cavity of the column tube 1 of the purification separation column is divided into a first liquid storage chamber and a second liquid storage chamber from bottom to top.

[0087] Furthermore, the outer wall diameter of the column tube 1 corresponding to the first liquid storage chamber is smaller than the outer wall diameter of the column tube 1 corresponding to the second liquid storage chamber, so that an annular step surface is formed on the outer wall of the column tube 1 between the first liquid storage chamber and the second liquid storage chamber; after the purification separation column is pressed into the liquid storage tube 9, the open end face of the liquid storage tube 9 contacts the annular step surface.

[0088] Furthermore, a sealing ring 7 is provided circumferentially on the outer wall of the column tube 1 at the corresponding position of the filter assembly.

[0089] In this invention, a sealing ring 7 is circumferentially fitted on the outer wall of the column tube 1. After the purification separation column is pushed into the liquid storage tube 9, the sealing ring 7 can seal the gap between the purification separation column and the liquid storage tube 9, preventing the test sample from flowing out from the gap between the purification separation column and the liquid storage tube 9, and ensuring that the test sample stored in the liquid storage tube 9 can only enter from the bottom opening of the purification separation column.

[0090] Furthermore, a slit 8 is provided at the top opening of the column tube 1 of the purification separation column. During the process of the purification separation column being pressed down into the liquid storage tube 9, the air in the liquid storage tube 9 is discharged through the slit 8.

[0091] In another specific embodiment, the present invention provides a pretreatment method for clinical testing, which uses the above-mentioned pretreatment device for clinical testing to pretreat the sample to be tested. The pretreatment method includes:

[0092] Add the sample to be tested into the storage tube 9. Mix the sample with the pre-placed extraction reagent and let it stand. After the aqueous phase 11 and organic phase 10 have initially separated into layers, press the purification separation column into the storage tube 9. The organic phase 10 passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube 1 of the purification separation column. The aqueous phase 11 is isolated by the filter assembly. The organic phase 10 that enters the inner cavity is the pretreated sample to be tested. The pretreated sample to be tested is then tested on the instrument or reconstituted after nitrogen blowing and tested on the instrument.

[0093] In addition to redesigning the structure of the purification separation column, this invention also improves the operation process of the pretreatment device. Compared with traditional dispersion solid-phase extraction and solid-phase extraction, the pretreatment method provided by this invention is simple to operate and has a good purification effect, achieving one-step purification treatment. Specifically:

[0094] (1) Compared with the dispersion solid-phase extraction method, the conventional operation process of the dispersion solid-phase extraction method includes: adding extraction reagent to the sample to form a mixture, adding separation material package to the mixture, then vortexing, followed by the first centrifugation, after the aqueous phase 11 and organic phase 10 separate into layers, transferring the upper organic phase 10 to another container, then adding purification agent and vortexing a second time, allowing it to stand or centrifuging, filtering the obtained organic phase 10 using a filter membrane and transferring it to another container, and finally taking the organic phase 10 for instrumental testing or reconstituted with nitrogen and then for instrumental testing. It can be seen that the dispersion solid-phase extraction method requires multiple transfers of the organic phase 10, and also involves two centrifugations and two vortexing operations. In addition, the purification agent used in the dispersion solid-phase extraction method relies solely on its adsorption effect for purification, while the present invention designs the purification agent packing as a bed structure, filling it into the column tube 1. When the extract passes through the packing layer 3, it is purified by the adsorption effect of the purification agent while also retaining some impurities, thus the purified supernatant flows out.

[0095] (2) Compared with solid-phase extraction (SPE), SPE is a complex process. Most samples require pretreatment before SPE, such as adding regulators, diluents, or precipitants to pre-precipitate proteins to prevent column clogging. Furthermore, the SPE column requires two-step activation, multiple rinsing, and elution processes under positive or negative pressure. The degree of drying must be controlled to ensure the packing material remains moist and does not affect column efficiency, resulting in a large amount of waste liquid. Additionally, the eluent needs to be collected by replacing the collection plate during elution, placing high demands on operators. The pretreatment device provided by this invention only requires pressing the purified separation column into the storage tube, eliminating the complex operations of activation, rinsing, elution, and collection plate replacement required by traditional SPE, greatly reducing operational difficulty and saving time. Moreover, the method of this invention generates virtually no waste liquid, making it more environmentally friendly than SPE.

[0096] Furthermore, the extraction reagent includes salts and extractants.

[0097] Furthermore, the salts include any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride, or potassium chloride.

[0098] Preferably, the extractant comprises acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile, or a mixture of acidified acetonitrile and methanol;

[0099] Furthermore, the extraction reagent also includes a purifying agent.

[0100] Furthermore, the purifying agent includes any one or a combination of at least two of N-propylethylenediamine, activated carbon, or C18.

[0101] In another specific embodiment, the present invention provides an application of the above-described pretreatment device, which is used to pretreatment a sample to be tested before clinical testing.

[0102] Application Example 1

[0103] The detection of vitamin D by liquid chromatography includes the following steps:

[0104] 1. Solution preparation:

[0105] (1) Preparation of 25-hydroxyvitamin D3 stock solution: Accurately weigh 25-hydroxyvitamin D3 powder (e.g., 1.0 mg) into a 10 mL volumetric flask, dissolve it completely in methanol and make up to 10 mL to prepare 25-hydroxyvitamin D3 stock solution B1 (100 mg / L);

[0106] (2) Preparation of 25-hydroxyvitamin D2 stock solution: Accurately weigh 25-hydroxyvitamin D2 powder (e.g., 1.0 mg) into a 10 mL volumetric flask, dissolve it completely in methanol and dilute to 10 mL to prepare 25-hydroxyvitamin D2 stock solution C1 (100 mg / L);

[0107] (3) Preparation of 25-hydroxyvitamin D3-d6 stock solution: Accurately weigh 25-hydroxyvitamin D3-d6 powder (e.g., 1.0 mg) into a 10 mL volumetric flask, dissolve it completely in methanol and make up to 10 mL to prepare 25-hydroxyvitamin D3-d6 stock solution E1 (100 mg / L);

[0108] (4) Preparation of 25-hydroxyvitamin D2-d3 stock solution: Accurately weigh 25-hydroxyvitamin D2-d3 powder (e.g., 1.0 mg) into a 10 mL volumetric flask, dissolve it completely in methanol and dilute to 10 mL to prepare 25-hydroxyvitamin D2-d3 stock solution F1 (100 mg / L);

[0109] (5) Preparation of VD intermediate solution: Add 800 μL of methanol to 100 μL of stock solution B1 and 100 μL of stock solution C1, mix well, and prepare VD mixed intermediate solution I (10 mg / L); add 900 μL of methanol to 100 μL of VD mixed intermediate solution I to prepare VD mixed intermediate solution II (1 mg / L).

[0110] (6) Preparation of VD internal standard intermediate solution: Add 100 μL of stock solution E1 and 100 μL of stock solution F1 to 800 μL of methanol, mix well, and prepare VD internal standard mixed intermediate solution I-IS (10 mg / L); add 50 μL of VD internal standard mixed intermediate solution I-IS to 950 μL to prepare VD internal standard working solution VD-IS (500 ng / mL);

[0111] (7) Prepare the VD working curve stock solution, as shown in Table 1:

[0112] Table 1

[0113]

[0114]

[0115] (8) Prepare VD working solutions (standard curve solutions S1-S6 and spiked sample solutions QL, QM and QH), as shown in Table 2:

[0116] Table 2

[0117]

[0118] 2. Sample processing:

[0119] (1) Purification and separation column method: 200 μL of each standard curve solution S1–S5 and quality control samples (solutions QL, QM, and QH) were pipetted into 2 mL centrifuge tubes (containing 40 mg magnesium sulfate and 10 mg sodium chloride). 10 μL of 25-hydroxyvitamin D internal standard solution VD-IS was added, followed by 200 μL of 0.1% formic acid acetonitrile. The mixture was vortexed for 1 min and centrifuged (14800 rpm, 4℃) for 5 min to separate the layers. The purification and separation column (first sieve plate pore size 30 μm, second sieve plate and water-blocking membrane pore size 10 μm, octadecyl bonded silica gel C18 40 mg, strong cation exchange resin MCX 25 mg, particle size 20-50 μm) was slowly (approximately 0.5 mm / s) inserted into the centrifuge tube until it could no longer be pressed. The filtrate from the purification and separation column was transferred to a 96-well plate for analysis. The linear correlation coefficient R between the VD3 and VD2 standard curve equations was calculated. 2 The values ​​were 0.997 and 0.996 respectively. The preprocessing process took approximately 10 minutes.

[0120] The linear correlation coefficient of the standard curve equation obtained by the above preprocessing method shows that the linearity of the method meets the requirements (>0.99).

[0121] (2) Solid-phase extraction method:

[0122] Add 200 μL of 0.2M zinc sulfate aqueous solution to 200 μL of each standard curve solution S1–S5 and quality control samples (solutions QL, QM, and QH), followed by 200 μL of methanol. Shake for 1 min, then centrifuge (14800 rpm, 4℃) for 5 min. Transfer the supernatant to an activated HLB solid-phase extraction column (Waters). After sample eluent, wash with 25% acetonitrile solution, then elute with pure acetonitrile before analysis. The pretreatment process takes approximately 1 hour.

[0123] 3. Instrument conditions:

[0124] Chromatographic column: C18 column (Poroshell 120EC-C18, 2.1×50mm, 2.7μm)

[0125] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol

[0126] Flow rate: 0.35 mL / min

[0127] Column temperature: 35℃

[0128] Injection volume: 5 μL

[0129] The liquid chromatography gradient is shown in Table 3:

[0130] Table 3

[0131]

[0132]

[0133] The mass spectrometry acquisition parameters are shown in Table 4:

[0134] Table 4

[0135] Name Parent ion m / z Daughter ion m / z Dwell (ms) Fragmentor CE Polarity VD3-1 401.4 383.3 60 110 2 Positive VD3-2 401.4 365.3 60 110 6 Positive VD3-d6 407.4 389.4 40 110 4 Positive VD2-1 413.3 395.2 60 110 2 Positive VD2-2 413.3 355.2 60 110 4 Positive VD2-d3 416 398.4 40 110 4 Positive

[0136] The ion source parameters are shown in Table 5:

[0137] Table 5

[0138] Dry gas temperature (°C) 270 Dry gas flow rate (L / min) 9.0 Nebulizer pressure (psi) 40 Sheath gas temperature (°C) 375 Sheath gas flow rate (L / min) 11.0 Capillary voltage (V) 5000 Nebulizer voltage (V) 1000

[0139] 4. Calculate the recovery rate (accuracy):

[0140] VD was detected using the purification separation column method, and the accuracy was calculated based on the detection results. The results are shown in Table 6.

[0141] Table 6

[0142]

[0143] As can be seen from the data in Table 6, the recovery rate (accuracy) (85%–115%) of real serum samples with low, medium and high additive concentrations selected by both pretreatment methods meets the requirements.

[0144] This pretreatment method is simple and easy to operate, with short processing time, low solvent consumption, low operator requirements, and low error rate. Its accuracy has been verified by the recovery rate of real samples, and its linearity also meets the requirements (R>0.99). It is an environmentally friendly pretreatment method with high fault tolerance.

[0145] Application Example 2

[0146] The detection of steroid compounds (cortisone, cortisol, corticosterone, androstenedione, deoxycorticosterone, testosterone, 17-hydroxyprogesterone, and progesterone) was performed by liquid chromatography-tandem mass spectrometry. The specific detection process includes the following steps:

[0147] 1. Testing conditions

[0148] The experimental conditions for liquid chromatography detection are shown in Tables 7, 8, and 9:

[0149] Table 7

[0150]

[0151]

[0152] Table 8

[0153]

[0154] Table 9

[0155]

[0156]

[0157]

[0158] 2. Sample pretreatment:

[0159] (1) Purification and separation column method: Pipette 200 μL of each standard curve solution and quality control samples (QL and QM, concentrations shown in Table 10) into a 2 mL centrifuge tube (containing 40 mg magnesium sulfate and 10 mg sodium chloride), add 20 μL of steroid internal standard solution, and then add 200 μL of methanol-acetonitrile mixed solution (volume ratio 1:9). Vortex for 1 min and centrifuge (14800 rpm, 4℃) for 5 min to separate the layers. Slowly (about 0.5 mm / s) insert the purification and separation column into the centrifuge tube until it can no longer be pressed. The purification and separation columns used for cortisone and cortisol contained 10 mg of diatomaceous earth, 20 mg of strong anion exchange resin MAX, and 20 mg of octaalkyl-bonded silica gel C8. The purification and separation columns used for corticosterone, androstenedione, and deoxycorticosterone contained 30 mg of N-ethyldiethylamine, 10 mg of quartz sand, and 20 mg of hydrophilic-lipophilic polymeric packing HLB. The purification and separation columns used for testosterone, 17-hydroxyprogesterone, and progesterone contained 5 mg of silica gel and 30 mg of weak anion exchange resin WAX. 20 μL of the filtrate from each purification and separation column was transferred to a 96-well plate, 100 μL of purified water was added, and the mixture was shaken well before analysis. The linear correlation coefficient R0 for the standard curve equations of cortisone, cortisol, corticosterone, androstenedione, deoxycorticosterone, testosterone, 17-hydroxyprogesterone, and progesterone was calculated. 2 The values ​​were 0.999, 0.999, 0.997, 0.998, 0.999, 0.998, 0.999, and 0.996, respectively. Each pretreatment process took approximately 10 minutes.

[0160] (2) Solid-phase extraction method:

[0161] Add 200 μL of 0.2M zinc sulfate aqueous solution to 200 μL of each standard curve solution and quality control samples (solutions QL and QM), then add 200 μL of methanol. Shake for 5 min, then centrifuge (14800 rpm, 4℃) for 5 min. Transfer the supernatant to an activated HLB solid-phase extraction column (Waters). After sample eluent, wash successively with 10% acetonitrile solution and n-hexane, then elute with 30 μL of a 1:9 methanol-acetonitrile mixture. Finally, mix with 50 μL of water and analyze. The pretreatment process takes approximately 1 hour.

[0162] 3. Calculate the recovery rate (accuracy):

[0163] Take real mixed human serum matrix samples, add the following compounds at the concentrations listed in Table 10 to each sample, calculate the concentration of the spiked sample and the background concentration before spiking according to the standard curve, and calculate the recovery rate using the following formula:

[0164]

[0165] Table 10 Spiked Concentrations of Various Sterol Compounds

[0166] QL (pg / mL) QM (pg / mL) Testosterone 320 1600 Progesterone 400 2000 Cortisol 8000 40000 17-Hydroxyprogesterone 400 2000 Androstenedione 200 1000 Deoxycorticosterone 320 1600 Cortisone 4000 20000 Corticosterone 400 2000

[0167] The experimental procedure is as described in section 2:

[0168] The accuracy calculation results are shown in Table 11:

[0169] Table 11 Recovery rates of various sterol compounds

[0170]

[0171] As can be seen from the data in Table 11, the accuracy of both methods for low and medium concentration spiked samples is between 85% and 115%.

[0172] Therefore, when the filtration method of the liquid-liquid extraction separation device provided by the present invention replaces the original centrifugation method for obtaining supernatant, the linearity (R≥0.99) and recovery rate (accuracy) (85%~115%) both meet the requirements, indicating that the rapid purification separation column method provided by the present invention is a simple, efficient and environmentally friendly pretreatment method.

[0173] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A purification and separation column for pretreatment in clinical testing, characterized in that, The purification and separation column includes a column tube with at least one open end. A filter assembly is provided inside the column tube. The liquid to be filtered enters from the bottom of the column tube through the opening, passes through the filter assembly, and enters the inner cavity of the column tube. The filter assembly includes, from bottom to top, a first sieve plate, a filter layer, a water-blocking layer, a filler layer, and a second sieve plate; The water-blocking layer adopts a sieve plate structure with a certain pore size. The inner surface of the pores is coated with a polymer compound. When the polymer compound comes into contact with water, it absorbs water and expands, which hinders the passage of the water phase and realizes the separation of the water phase and the organic phase. The packing layer is filled with a purification material, which includes a specific adsorption material for adsorbing polypeptides and phospholipids; the filling mass of the purification material is 10~100mg. The filter layer includes a first filter membrane, a second filter membrane, and a third filter membrane stacked sequentially from bottom to top, wherein the pore size of the first filter membrane is greater than or equal to the pore size of the second filter membrane and the pore size of the third filter membrane.

2. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The filter assembly is located at the bottom of the inner cavity of the column tube. The filtrate passes through the filter assembly from the bottom of the column tube and then enters the inner cavity of the column tube.

3. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The purification materials include any one or a combination of at least two of the following: silica gel, octadecyl bonded silica gel and its derivatives, octadecyl bonded silica gel and its derivatives, diatomaceous earth, activated carbon, N-propylethylenediamine, quartz sand, molecular sieve, hydrophilic-lipophilic polymeric filler, strong cation exchange resin, weak cation exchange resin, strong anion exchange resin, or weak anion exchange resin.

4. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The particle size of the purification material is 5~2000μm.

5. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The aperture of the first sieve plate may be the same as or different from that of the second sieve plate.

6. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The pore size of the first sieve plate is 5~50μm.

7. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The pore size of the second sieve plate is 5~50μm.

8. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The pore size of the first filter membrane is 0.5~2.5μm.

9. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The pore size of the second filter membrane is 0.2~0.8μm.

10. The purification and separation column for pretreatment in clinical testing according to claim 1, characterized in that, The pore size of the third filter membrane is 0.1~0.5μm.

11. A pretreatment device for clinical testing, characterized in that, The pretreatment device includes a liquid storage tube and a purification and separation column for clinical testing pretreatment as described in any one of claims 1-10; The liquid storage tube and the purification separation column are used together. The liquid storage tube is pre-filled with extraction reagent. The sample to be tested is added to the liquid storage tube. After the sample to be tested and the extraction reagent are mixed, they are allowed to stand and separate into layers. The purification separation column is pressed into the liquid storage tube. The upper organic phase passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube.

12. The pretreatment device for clinical testing according to claim 11, characterized in that, The inner cavity of the purification and separation column is divided into a first liquid storage chamber and a second liquid storage chamber from bottom to top.

13. The pretreatment device for clinical testing according to claim 12, characterized in that, The outer wall diameter of the column tube corresponding to the first liquid storage chamber is smaller than the outer wall diameter of the column tube corresponding to the second liquid storage chamber, so that an annular step surface is formed on the outer wall of the column tube between the first liquid storage chamber and the second liquid storage chamber; after the purification separation column is pressed into the liquid storage tube, the open end face of the liquid storage tube contacts the annular step surface.

14. The pretreatment apparatus for clinical testing according to claim 11, characterized in that, A sealing ring is provided circumferentially on the outer wall of the column tube at the corresponding position of the filter component.

15. The pretreatment apparatus for clinical testing according to claim 11, characterized in that, The purification and separation column has a slit at the top opening of the column tube. As the purification and separation column is pressed down into the liquid storage tube, the air in the liquid storage tube is discharged through the slit.

16. A pretreatment method for clinical testing, characterized in that, The sample to be tested is pretreated using the pretreatment apparatus for clinical testing according to any one of claims 11-15, the pretreatment method comprising: Add the sample to be tested into the storage tube. Mix the sample with the pre-placed extraction reagent and let it stand. After the aqueous phase and organic phase have initially separated into layers, press the purification separation column into the storage tube. The organic phase passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube. The aqueous phase is isolated by the filter assembly. The organic phase that enters the inner cavity is the pretreated sample to be tested. The pretreated sample to be tested is then tested on the instrument or reconstituted after nitrogen blowing and tested on the instrument.

17. The pretreatment method according to claim 16, characterized in that, The extraction reagents include salts and extractants.

18. The pretreatment method according to claim 17, characterized in that, The salts include any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride, or potassium chloride.

19. The pretreatment method according to claim 17, characterized in that, The extractant includes acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile, or a mixture of acidified acetonitrile and methanol.

20. The pretreatment method according to claim 16, characterized in that, The extraction reagent also includes a purifying agent.

21. The pretreatment method according to claim 20, characterized in that, The purifying agent includes any one or a combination of at least two of N-propylethylenediamine, activated carbon, or C18.

22. The application of the pretreatment apparatus according to any one of claims 11-15, characterized in that, The pretreatment device is used to pretreat the sample to be tested before clinical testing.

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