Miniature prefilter for high performance liquid chromatographic column, preparation method and application thereof

Abstract

The invention discloses a miniature pre-filter for a high performance liquid chromatographic column, a preparation method and application thereof, the miniature pre-filter comprises a cylindrical hollow column shell and a three-stage filter assembly mounted in the column shell. The miniature prefilter provided by the invention adopts a three-stage filtering design, so that the occurrence of aggregative blockage is reduced. The miniature prefilter is applied to high performance liquid chromatography detection, the column efficiency and the separating capacity of a chromatographic column are protected to the maximum extent, and the service life of the chromatographic column is remarkably prolonged. All filtering materials are hydrophilic deep filtering materials, the performance of the filtering materials is further verified, and the pore diameter and material selection of the filtering materials are optimized. The miniature prefilter provided by the invention meets the application requirements of clinical blood sample treatment, and the use cost of high-value consumables of high performance liquid chromatography is remarkably reduced.

Description

Technical Field

[0001] The invention relates to a micro prefilter for a chromatographic analysis column, in particular to a micro prefilter for a high performance liquid chromatography column, and belongs to the field of high performance liquid chromatography analysis. Background Art

[0002] High performance liquid chromatography (HPLC) is an analytical method developed in the late 1960s. It is widely used in medical testing and can be used to detect common biomarkers, such as glucose content, glycosylated hemoglobin, uric acid content and serum lipid content, etc. These items have important clinical application value in disease diagnosis, formulation of treatment plans, and disease prognosis estimation.

[0003] The principle of HPLC is to use the differences in adsorption and elution behavior of different substances on the chromatographic column for separation and detection. The performance of the chromatographic column is closely related to the performance of the detection system. For HPLC, solid particulate matter contained in the sample and mobile phase will clog the chromatographic column sieve plate, causing the column pressure to increase and the column efficiency to decrease. In order to protect the chromatographic column and extend its service life, the HPLC detection system uses a pre-column filter, but it is expensive and increases the detection cost.

[0004] It is found in clinical practice that the existing fully automatic glycosylated hemoglobin analyzer using a pre-column filter (i.e., the pre-column filter of the HPLC detection system) will increase or decrease the glycosylated hemoglobin test results of the first 1-2 samples when the instrument stops testing for a period of time and retests the sample (this situation is found in clinical testing and is considered to be related to the filter membrane and the filter membrane filtration stability), resulting in inaccurate test results and affecting the result judgment. Therefore, it is necessary to develop a new micro pre-filter to reduce measurement deviation and extend the life of the chromatographic column.

[0005] The existing HPLC prefilters that use surface filter membranes and single filter structures are prone to clogging and are used less frequently. When particles of different sizes gather on the surface filter membrane at the same time, the plug effect is more likely to cause clogging of the micro prefilter. Using a depth filter membrane and filtering step by step according to the size of the particles can extend the number of times the micro prefilter can be used, maintain the filtering effect, and protect the chromatographic column to the maximum extent. Therefore, the step-by-step filtration design is suitable for the research and development of new micro prefilters. Summary of the invention

[0006] The purpose of the present invention is to provide a micro prefilter for a high performance liquid chromatography column, a preparation method and an application thereof. The micro prefilter of the present invention adopts a three-stage filtration design, which significantly reduces the clogging effect, effectively protects the separation ability and column efficiency of the HPLC chromatographic column, and prolongs the service life of the chromatographic column.

[0007] To achieve the purpose of the present invention, on the one hand, the present invention provides a micro pre-filter for a high performance liquid chromatography column, comprising: a cylindrical, hollow column shell, and a three-stage filtration component installed inside the column shell, that is, a three-stage filtration design is adopted, wherein the first stage filtration adopts a large-pore filter paper, the second stage filtration adopts a large-pore filter membrane, and the third stage filtration adopts a small-pore filter membrane.

[0008] In particular, the filter material used in the three-stage filtration design is a hydrophilic depth filter material, including a hydrophilic filter paper and a hydrophilic filter membrane, and the filter material has a low protein adsorption characteristic.

[0009] In particular, the filter assembly includes a first, second and third filter layers arranged in sequence from the liquid inlet end to the liquid outlet end of the micro pre-filter; the first filter layer is made of two layers of filter paper stacked together; the second and third filter layers are respectively made of one layer of filter membrane; wherein the pore size of the filter membrane of the second filter layer is larger than the pore size of the filter membrane of the third filter layer.

[0010] In particular, it also includes the establishment of a performance evaluation procedure for micro pre-filters, by evaluating the impact of micro pre-filters with different filter materials and pore sizes on detection performance (precision and carryover contamination) and chromatographic columns (retention time, chromatographic column efficiency and number of column uses), in order to optimize the filter membrane material and pore size of the micro pre-filter.

[0011] In the present invention, the first filter layer is one or more of glass fiber filter paper, composite cellulose filter paper and polymer filter paper, the filter paper used has a pore size of 0.22-2.70 μm and a thickness of 0.30-0.40 mm, and different micro pre-filters are prepared by fixing the filter materials of the second and third filter layers and changing the filter materials of the first filter layer for HPLC glycated hemoglobin (HbA 1c ) detection, based on the evaluation of detection precision and carryover contamination, it is further preferred that the glass fiber filter paper has a pore size of 2.70 μm.

[0012] The hydrophilic depth filter membrane used in the third filter layer is one or more of a polyethersulfone filter membrane (PES filter membrane), a mixed cellulose membrane (MCE filter membrane), a hydrophilic polytetrafluoroethylene membrane (PTFE filter membrane) or a perchlorethylene filter membrane (PVC filter membrane), a nitrocellulose filter membrane (NC filter membrane), and a polyvinylidene fluoride filter membrane (PVDF filter membrane). The pore size of the filter membrane is 0.22 or 0.45 or 0.80 μm, and the thickness is 0.30-0.40 mm. According to the HbA 1c The evaluation of detection precision and carryover contamination, as well as the impact on chromatographic column retention time, chromatographic column efficiency and the number of times the chromatographic column is used, further preferably, the MCE filter membrane, hydrophilic PTFE filter membrane and PES filter membrane have a pore size of 0.22 μm.

[0013] In another aspect, the present invention provides a method for preparing a micro prefilter for a high performance liquid chromatography column, comprising:

[0014] 1) Cut the filter material into discs with a diameter that matches the inner diameter of the column shell;

[0015] 2) placing a first filter layer, a second filter layer, a third filter layer, and a metal mesh in order from the liquid inlet end to the liquid outlet end inside the column shell;

[0016] 3) The column shell is buckled and sealed to obtain the micro pre-filter.

[0017] The micro prefilter for high performance liquid chromatography column of the present invention adopts a three-stage filtration design, and its main design is that the first filter layer (primary filtration) adopts large-pore filter paper, the second filter layer (secondary filtration) adopts a large-pore filter membrane, and the third filter layer (final filtration) adopts a small-pore filter membrane. The filter materials used in the three-stage filtration design are all hydrophilic depth filter materials, and the filter materials have low protein adsorption characteristics. The preparation of the micro prefilter adopts 2 layers of filter paper in the first filter layer, 1 layer of large-pore filter membrane in the second filter layer, and 1 layer of small-pore filter membrane in the third filter layer. The primary filtration removes cells, cell membranes, cell fragments or particulate matter from the mobile phase, the secondary filtration is used to remove polymer particles such as fat, phospholipids, lipids, etc. generated during hemolysis, and the final filtration is used to filter small particles of surface-activated polymers, small-molecule serum proteins, fats and membrane phospholipids from the mobile phase, so as to protect the separation ability and column efficiency of the chromatographic column to the maximum extent and extend the service life of the chromatographic column.

[0018] The micro prefilter for a high performance liquid chromatography column of the present invention is used in high performance liquid chromatography analysis and detection; the prefilter is placed at the front end of the chromatographic column to protect the column efficiency and separation ability of the chromatographic column; and can also be used in the field of high performance liquid chromatography tandem mass spectrometry analysis.

[0019] Another aspect of the present invention provides a micro prefilter for a high performance liquid chromatography column, comprising: a cylindrical column shell with a hollow interior, a filter assembly for graded filtration fixedly installed inside the column shell, and a metal mesh supporting the filter assembly.

[0020] Wherein, the filter assembly includes the first, second and third filter layers, and the first, second and third filter layers are superimposed on the metal mesh, and the third filter layer, the second filter layer and the first filter layer are arranged on the metal mesh in sequence.

[0021] In particular, the filter material of the filter assembly is a hydrophilic depth filter material, including a hydrophilic filter paper and / or a filter membrane.

[0022] In particular, the first filter layer is made of two layers of filter paper stacked together; the second and third filter layers are made of one layer of filter membrane respectively; wherein the pore size of the filter membrane of the second filter layer is larger than the pore size of the filter membrane of the third filter layer.

[0023] In particular, the filter paper is glass fiber filter paper, composite fiber filter paper or polymer filter paper, preferably glass fiber filter paper; the second filter layer membrane is a polyethersulfone filter membrane (PES filter membrane), a mixed cellulose membrane (MCE filter membrane), a hydrophilic polytetrafluoroethylene membrane (PTFE filter membrane) or a perchlorethylene filter membrane (PVC filter membrane), a nitrocellulose filter membrane (NC filter membrane), a polyvinylidene fluoride filter membrane (PVDF filter membrane), preferably a polyethersulfone filter membrane (PES filter membrane); the third filter layer membrane is a polyethersulfone filter membrane (PES filter membrane), a mixed cellulose membrane (MCE filter membrane), a hydrophilic polytetrafluoroethylene membrane (PTFE filter membrane) or a perchlorethylene filter membrane (PVC filter membrane), a nitrocellulose filter membrane (NC filter membrane), a polyvinylidene fluoride filter membrane (PVDF filter membrane), preferably a polyethersulfone filter membrane (PES filter membrane), a mixed cellulose membrane (MCE filter membrane) or a hydrophilic polytetrafluoroethylene membrane (PTFE filter membrane).

[0024] In particular, the pore size of the filter paper of the first filter layer is 0.22 to 2.70 μm, preferably 2.70 μm; the thickness is 0.30 to 0.40 mm; the pore size of the filter membrane of the second filter layer is 0.22 to 0.80 μm, preferably 0.80 μm; the thickness is 0.10 to 0.20 mm; the pore size of the filter membrane of the third filter layer is 0.22 to 0.80 μm, preferably 0.22 or 0.45 or 0.80 μm, further preferably 0.22 μm; the thickness is 0.1 to 0.2 mm.

[0025] In particular, the material and pore size of the first filter layer are screened and obtained by the following method: the micro pre-filter of the first filter layer with different materials / pore sizes is combined with a high performance liquid chromatography column to measure HbA in whole blood samples. 1c Concentration, evaluate the effect of different first filter materials on HbA 1c For the precision and carryover of concentration detection, select filter materials with a precision of less than 1% and a carryover rate of less than 0.5%.

[0026] In particular, the second filter layer of the micro pre-filter is a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is a MCE filter membrane with a pore size of 0.45 μm.

[0027] In particular, the material and pore size of the third filter layer membrane are screened and obtained by the following method: the micro pre-filter of the third filter layer with different materials / pore sizes is combined with a high performance liquid chromatography column to measure HbA in whole blood samples. 1c Concentration, evaluate the effect of different third filter materials on HbA 1c For the precision and carryover of concentration detection, select filter materials with a precision of less than 1% and a carryover rate of less than 0.5%.

[0028] That is, filter materials with a coefficient of variation less than 1% are screened.

[0029] In particular, the first filter layer of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm.

[0030] In particular, the first filter layer filters and removes cells, cell membranes, cell fragments or particulate matter in the mobile phase; the second filter layer filters and removes polymer particles such as fats, phospholipids, lipids, etc. in the mobile phase; and the third filter layer filters and removes small particles of surface-activated polymers, small molecule serum proteins, fats and membrane phospholipids in the mobile phase.

[0031] Particularly, the metal mesh is a nickel mesh or a stainless steel mesh, which plays the role of supporting and distributing the liquid flow.

[0032] Particularly, the metal mesh has an aperture of 100.0 to 400.0 meshes, preferably 200 meshes, and a thickness of 0.1 to 0.5 mm, preferably 0.3 mm.

[0033] In particular, the column shell is made of one or more of high pressure resistant and corrosion resistant polycarbonate (PC) materials, polyethylene (PE) materials, and Teflon materials.

[0034] In particular, the column housing comprises a column body and an upper cover and a lower cover for sealing the upper and lower bottom surfaces of the column body, wherein the upper cover and the lower cover are circular, and their diameters match the diameter of the column body.

[0035] In particular, the column body is a hollow cylinder; the upper cover is integrally connected to the column body.

[0036] In particular, the central position (center position) of the upper cover is set at the liquid inlet, which is sealed with the liquid inlet pipe; the central position (center position) of the lower cover is set at the liquid outlet, which is sealed with the liquid outlet pipe.

[0037] In particular, the filter assembly is circular, having the same shape as the upper and lower bottom surfaces of the column body, that is, the first, second and third filter layers and the metal mesh are circular structures, and the diameter matches the inner diameter of the column body.

[0038] In another aspect, the present invention provides a method for preparing the micro prefilter for a high performance liquid chromatography column as described above, comprising the following steps:

[0039] 1) Cut the filter paper of the first filter layer and the filter membranes of the second and third filter layers into discs with diameters matching the inner diameter of the column shell;

[0040] 2) Cut the metal mesh into discs with a diameter matching the inner diameter of the column shell;

[0041] 3) placing the first filter layer, the second filter layer, the third filter layer, and the metal mesh in the column housing in the order from the upper cover to the lower cover;

[0042] 4) The lower cover is fastened on one end of the column shell close to the metal mesh, and the upper cover is fastened on the other end to obtain the micro pre-filter.

[0043] Another aspect of the present invention provides a use of the micro prefilter for a high performance liquid chromatography column as described above in high performance liquid chromatography analysis and detection.

[0044] In another aspect, the present invention provides a use of the micro prefilter for a high performance liquid chromatography column as described above in high performance liquid chromatography analysis and detection of glycated hemoglobin and hemoglobin variants.

[0045] The invented micro pre-filter is a micro pre-filter used in the chromatographic column in the field of high performance liquid chromatography detection to protect the column efficiency and separation ability of the chromatographic column, and is mainly used in the detection of glycated hemoglobin by high performance liquid chromatography.

[0046] The micro pre-filter of the present invention is placed at the front end of a high performance liquid chromatography column to protect the chromatography column. The micro pre-filter of the present invention can also be used in the field of high performance liquid chromatography tandem mass spectrometry analysis.

[0047] Compared with the prior art, the present invention has the advantages of:

[0048] 1. The micro pre-filter of the present invention adopts a multi-stage filtration design, which avoids the disadvantage that the micro pre-filter with a single filtration structure is prone to clogging (because when particles of different sizes are simultaneously gathered on the same filter membrane surface, the micro pre-filter is more likely to be clogged due to the clogging effect during filtration), so as to reduce the occurrence of aggregation clogging. The three-stage filtration structure design of the present invention can increase the number of times the micro pre-filter can be used;

[0049] 2. The micro pre-filter of the present invention adopts hydrophilic depth filter material, which can achieve the filtration of high-concentration particulate matter compared with surface filter material;

[0050] 3. The micro pre-filter of the present invention protects the separation ability and column efficiency of the chromatographic column to the maximum extent and prolongs the service life of the chromatographic column;

[0051] 4. Compared with large pore depth filter materials, small pore filter materials show better performance.

[0052] 5. The micro prefilter of the present invention has a simple preparation process, low production cost, good chromatographic column protection efficiency, meets the application requirements of clinical blood sample processing, and significantly reduces the use cost of high-value consumables for high-performance liquid chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the structure of the micro pre-filter of the present invention;

[0054] Figure 2 The effect of using micro pre-filters of different materials and pore sizes as the third filter layer on the theoretical pedal number of the chromatographic column.

[0055] Description of Reference Numerals

[0056] 1. Column body; 11. Upper cover; 12. Lower cover; 13. Liquid inlet pipe; 14. Liquid outlet pipe; 15. Liquid inlet; 16. Liquid outlet; 2. First filter layer; 3. Second filter layer; 4. Third filter layer; 5. Metal mesh. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0058] Example 1 Micro prefilter

[0059] The micro pre-filter for a high performance liquid chromatography column of the present invention adopts a three-stage filtration design, comprising:

[0060] A cylindrical hollow shell, a filter assembly for three-stage filtration fixedly installed inside the cylindrical shell, and a metal mesh supporting the filter assembly, wherein:

[0061] The column housing includes a cylindrical column body 1, an upper cover 11 and a lower cover 12 for sealing the upper and lower bottom surfaces of the column body; the upper cover and the lower cover are circular and have the same shape as the upper and lower bottom surfaces of the column body; the size of the upper cover and the lower cover matches the diameter of the column body. The upper cover is integrally connected to the column body, and the upper cover and the lower cover seal the two ends of the column housing to form a sample filtering space.

[0062] The center of the upper cover (i.e., the center of the circle of the upper cover) is located at the liquid inlet 15, which is sealed and connected to the liquid inlet pipe 13, and the solution to be tested is introduced into the micro pre-filter through the liquid inlet pipe;

[0063] The center of the lower cover (i.e., the center of the circle of the lower cover) is provided with a liquid outlet 16 which is sealed and connected to the liquid outlet pipe 14, and the solution to be tested after being filtered by the micro pre-filter is led out and then enters the high performance liquid chromatography column for detection;

[0064] The column shell is made of polycarbonate (PC), polyethylene (PE) and Teflon materials. The column shell has high pressure resistance and corrosion resistance.

[0065] The metal mesh is arranged at the lower part of the filter assembly to support the filter assembly. The metal mesh is a nickel mesh or a stainless steel mesh, which plays the role of supporting and distributing the liquid flow.

[0066] The metal mesh is circular in shape, which is the same as the shape of the bottom surface of the column body; the diameter of the metal mesh layer matches the inner diameter of the column body (the shape is the same as the cross-section of the cavity inside the column body, and the size of the cross-section matches that).

[0067] The metal mesh has an aperture of 100.0 to 400.0 meshes, preferably 200 meshes; and a thickness of 0.1 to 0.5 mm, preferably 0.3 mm.

[0068] The filter assembly comprises a first, a second and a third filter layer, and the first, the second and the third filter layer are superimposed on the metal mesh, and the third filter layer, the second filter layer and the first filter layer are arranged on the metal mesh in sequence.

[0069] The first, second and third filter layers are circular in shape, the same as the bottom surface of the column body; the diameters of the first, second and third filter layers match the inner diameter of the column body (they have the same shape as the cross section of the cavity inside the column body and the size of the cross section matches that of the column body).

[0070] The filter assembly is made of hydrophilic depth filter material (hydrophilic filter paper, filter membrane).

[0071] The first filter layer is made of two layers of hydrophilic filter paper stacked together; the filter paper is made of glass fiber filter paper, composite fiber filter paper or polymer filter paper, the filter paper pore size of the first filter layer is 0.22-2.70 μm, preferably 2.70 μm; the thickness of the first filter layer is 0.60-0.80 mm.

[0072] In the specific implementation manner of the present invention, the first filter layer uses glass fiber filter paper with a pore size of 2.70 μm and a thickness of 0.35 mm as an example for description.

[0073] The second and third filter layers are respectively made of a layer of hydrophilic filter membrane; the pore size of the filter membrane of the second filter layer is larger than the pore size of the filter membrane of the third filter layer.

[0074] The second filter layer is made of a polyethersulfone filter membrane (PES filter membrane), the pore size of the filter membrane of the second filter layer is 0.22-0.80 μm, preferably 0.80 μm, and the thickness is 0.10-0.20 mm.

[0075] The second filter layer is preferably a polyethersulfone filter membrane (PES filter membrane) with a pore size of 0.8 μm and a thickness of 0.2 mm.

[0076] In addition to the PES filter membrane, other mixed cellulose membranes (MCE filter membranes), hydrophilic polytetrafluoroethylene membranes (PTFE filter membranes) or perchlorethylene filter membranes (PVC filter membranes) are also suitable for the present invention.

[0077] The filter membrane of the third filter layer is made of polyethersulfone filter membrane (PES filter membrane), mixed cellulose membrane (MCE filter membrane), hydrophilic polytetrafluoroethylene membrane (PTFE filter membrane), perchlorethylene filter membrane (PVC filter membrane), nitrocellulose filter membrane (NC filter membrane), polyvinylidene fluoride filter membrane (PVDF filter membrane), and the pore size of the filter membrane of the third filter layer is 0.22-0.80 μm, preferably 0.22 or 0.45 μm, and further preferably 0.22 μm; the thickness is 0.10-0.20 mm.

[0078] The third filter layer is preferably an MCE filter membrane, a PES filter membrane or a hydrophilic PTFE filter membrane with a pore size of 0.22 μm and a thickness of 0.2 mm.

[0079] Inside the column body (i.e., the sample filtration space), the first filter layer 2, the second filter layer 3, the third filter layer 4, and the metal mesh layer 5 are arranged in sequence from the upper cover to the lower cover (i.e., the liquid flow direction); the first, second, and third filter layers and the metal mesh layer are circular in shape, which is the same as the shape of the bottom surface of the column shell; the diameters of the first, second, and third filter layers and the metal mesh layer match the inner diameter of the column shell (they have the same shape as the cross-section of the cavity inside the column shell and match the size of the cross-section).

[0080] The first, second and third filter layers are stacked on the metal mesh layer, that is, the third filter layer is stacked on the metal mesh layer, the second filter layer is stacked on the third filter membrane layer; the first filter layer is stacked on the second filter layer

[0081] The first filter layer is used to remove cells, cell membranes, cell debris or particulate matter from the sample to be tested to form a first filtrate;

[0082] The second filter layer is used to remove the polymer particles such as fat, phospholipids, lipids, etc. generated during the hemolysis process of the sample to be tested (i.e., to remove the polymer particles such as fat, phospholipids, lipids, etc. from the first filtrate) to obtain a second filtrate;

[0083] The third filter layer is used to filter small particles of surface-activated polymers, small molecule serum proteins, fats and membrane phospholipids from the second filtrate;

[0084] The sample solution to be tested flows into the micro pre-filter from the liquid inlet pipe, is filtered step by step through the first, second and third filter layers in sequence, is evenly distributed through the metal mesh, flows out from the liquid outlet pipe, enters the high performance liquid chromatography column, and is tested.

[0085] The multi-stage (three-stage) filtration design of the present invention protects the target separation ability and column efficiency of the chromatographic column to the maximum extent and prolongs the service life of the chromatographic column; the dead space volume of the pre-filter should be reduced as much as possible to avoid interference between the test results caused by sample residue (the dead space volume is less than twice the volume of the quantitative loop of the corresponding chromatographic column used in conjunction with the micro pre-filter). Through the carryover contamination rate test, it is confirmed whether the filter will cause an increase in the carryover contamination rate. In addition, it is also necessary to consider that the depth filter membrane should have a certain extensible space to prevent it from tearing under high pressure.

[0086] Example 2 Preparation of micro prefilter

[0087] 1. Preparation of plastic housing for micro pre-filter

[0088] like Figure 1 As shown, a column shell made of polyethylene (PE) material is processed and prepared, the column body is cylindrical, hollow inside, and its inner diameter is 7.9mm (usually 7.8-8.0mm); the outer diameter of the upper cover is the same as the outer diameter of the column body, and its inner diameter matches the inner diameter of the column body, and the upper cover and the column body are connected as a whole; the diameter of the liquid inlet is 1.0mm; the diameter of the lower cover matches the inner diameter of the column body, and the diameter of the liquid outlet is 1.0mm.

[0089] 2. Preparation of filter paper and filter membrane discs

[0090] Use a small die punch to cut the glass fiber filter paper of the first filter layer into discs, the diameter of which matches the inner diameter of the column body;

[0091] Use a small die punch to cut the PES filter membrane of the second filter layer into discs, the diameter of which matches the inner diameter of the column body;

[0092] The hydrophilic PTFE filter membrane of the third filter layer is cut into discs using a small die punch, and the diameter of the filter membrane matches the inner diameter of the column body.

[0093] In this embodiment, glass fiber filter paper with a pore size of 2.70 μm and a thickness of 0.35 mm; a PES filter membrane with a pore size of 0.8 μm and a thickness of 0.2 mm; and a hydrophilic PTFE filter membrane with a pore size of 0.45 μm and a thickness of 0.2 mm are used as examples for description.

[0094] 3. Preparation of metal mesh disc

[0095] A small die punch was used to cut the stainless steel (316L) mesh into discs, the diameter of which matched the inner diameter of the column body.

[0096] The stainless steel wire mesh has an aperture of 200 mesh and a thickness of 0.3 mm.

[0097] 4. Assembly

[0098] The upper cover and the column body are connected as one body and are sealed as one body.

[0099] Place the filter paper and depth filter membrane cut into discs in the direction of liquid flow, i.e., from the inlet end (i.e., liquid inlet end) to the outlet end (liquid outlet end) of the plastic column shell. First, place two layers of glass fiber filter paper discs on the surface of the upper cover to form a first filter layer; place one layer of PES filter membrane disc on the surface of the first filter layer to form a second filter layer; place one layer of hydrophilic PTFE filter membrane disc on the top of the second filter layer to form a third filter layer; that is, from the liquid inlet end to the liquid outlet end, place two layers of glass fiber filter paper discs of the first filter layer, the PES filter membrane disc of the second filter layer, and the hydrophilic PTFE filter membrane disc of the third filter layer in the column shell in sequence;

[0100] Then, a 316L stainless steel mesh disc is placed on the surface of the third filter layer;

[0101] Finally, a lower cover made of PE plastic is buckled onto the other end of the column body and sealed to obtain the micro pre-filter of the present invention.

[0102] Example 3 Screening of the first filter layer material

[0103] The micro pre-filter of the present invention is placed at the front end of the HPLC chromatographic column to detect the whole blood sample and determine the HbA in the whole blood sample. 1c Concentration, evaluate the effect of different first filter materials on HbA 1c Precision and carryover of concentration measurements, including:

[0104] The second filter layer of the filter assembly of the micro pre-filter is a PES filter membrane with a pore size of 0.80 μm; the third filter layer is a 0.45 μm MCE filter membrane; the material of the first filter layer is glass fiber or composite cellulose filter paper;

[0105] Precision was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) Guide for Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guidelines-Second Edition (EP5-A2) methods; Carryover was evaluated according to the CLSI Guide for Preliminary Evaluation of Quantitative Measurement Procedures in Clinical Laboratories; Approved Guidelines-Third Edition (EP10-A3).

[0106] The precision evaluation results show that the precision of the glass fiber filter paper micro pre-filter is less than 1.00%, which is significantly lower than that of the composite cellulose filter paper. The carryover contamination rate of the glass fiber filter paper and the composite cellulose filter paper micro pre-filter is 0.00%. Therefore, the use of glass fiber filter paper has little effect on precision and is the best choice as the primary filter material.

[0107] Example 4 Third filter layer material screening

[0108] According to the same evaluation method as in Example 3, the effects of different third filter layer materials on HbA 1c In order to determine the precision of concentration detection and the influence of carryover contamination, a micro pre-filter made of different filter materials is placed in front of an HPLC column for the third filter layer material of the present invention, and a whole blood sample is tested. The HbA content in the same whole blood sample is repeatedly measured for each filter membrane of the third filter layer material. 1c Concentration, where:

[0109] The material of the first filter layer of the filter assembly of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; the third filter layer is a 0.22 μm MCE filter membrane, a PES filter membrane, and a hydrophilic PTFE filter membrane;

[0110] The precision and carryover assessment methods are the same as those in Example 3.

[0111] After filtering with micro prefilters of different filter membranes, the results of HPLC determination are shown in Table 1. The results of precision (coefficient of variation) evaluation are shown in Table 1. The coefficient of variation reflects the precision. The smaller the coefficient of variation, the better the precision.

[0112] Example 4A Third filter layer material screening

[0113] The micro pre-filter of the present invention was placed at the front end of the HPLC column to test the whole blood sample and evaluate the effects of different third filter layer materials on HbA 1c Precision and carryover of concentration measurements, including:

[0114] The first filter layer of the filter assembly of the micro pre-filter is made of glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; the third filter layer is a 0.45 μm MCE filter membrane, a PES filter membrane, and a hydrophilic PTFE filter membrane;

[0115] The precision and carryover assessment methods are the same as those in Example 3.

[0116] The results of precision evaluation are shown in Table 1.

[0117] Table 1 Effect of micro prefilters with different filter materials and pore sizes on HbA 1c Effect of concentration detection precision

[0118]

[0119] The evaluation results in Table 1 show that all micro prefilters are used for HPLC detection of HbA 1cThe precision of concentration is less than 1%. Carryover experiments show that the carryover rate of all micro pre-filters is 0.00%. The above experiments prove that the performance of depth filter membranes with different filter materials and pore sizes meets the requirements of micro pre-filter design for filter materials.

[0120] Example 4B Third Filter Layer Material Screening

[0121] The materials of the first filter layer of the filter assembly of the micro pre-filter are glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is a 0.22 μm PVC filter membrane, NC filter membrane, or PVDF filter membrane. The rest is the same as in Example 4.

[0122] The results of the precision assessment are shown in Table 1A.

[0123] Table 1A Effect of micro prefilters with different filter media and pore sizes on HbA 1c Effect of concentration detection precision

[0124]

[0125] Example 4C Third filter layer material screening

[0126] Except that the material of the first filter layer of the filter assembly of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is made of a 0.45 μm PVC filter membrane, NC filter membrane, or PVDF filter membrane, the rest is the same as in Example 4;

[0127] The results of the precision assessment are shown in Table 1A.

[0128] All prefilters of Examples 4B and 4C were used for HPLC detection of HbA 1c The precision of the concentration is greater than 2%, which is significantly higher than the precision of the filter materials used in Examples 4 and 4A, which may be related to the protein retention of PVC filter membranes, NC filter membranes, and PVDF filter membranes.

[0129] It can be seen from this that the performance of PVC filter membranes, NC filter membranes, and PVDF filter membranes with different pore sizes cannot meet the requirements of HPLC micro pre-filter design for filter materials.

[0130] Example 5 Micro prefilter usage times

[0131] The material of the first filter layer of the filter component of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80μm; and the third filter layer is a 0.22μm MCE filter membrane, a PES filter membrane, and a hydrophilic PTFE filter membrane.

[0132] The micro pre-filter of the present invention is placed at the front end of the HPLC column, and the fresh whole blood sample is subjected to continuous HbA 1c The concentration test was performed to obtain the usage times of the micro pre-filters with different filter materials and pore sizes, see Table 2.

[0133] Table 2 Number of times the micro pre-filter was used

[0134]

[0135] *One-way ANOVA, P>0.05.

[0136] Example 5A Micro prefilter usage times

[0137] The first filter layer of the filter assembly of the micro pre-filter is made of glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is a 0.45 μm MCE filter membrane, a PES filter membrane, and a hydrophilic PTFE filter membrane. The rest is the same as in Example 5.

[0138] The number of times the micro pre-filter is used is counted in the same manner as in Example 5.

[0139] The number of times micro pre-filters with different filter materials and pore sizes were used is shown in Table 2. For the same pore size, there was no significant difference in the number of times micro pre-filters with different filter materials were used (P>0.05), but there was a significant difference in the number of times micro pre-filters with the same filter material and different pore sizes were used (P<0.05). This experiment proved that the micro pre-filter with a large-pore third filter layer filter material was used more times, but from the perspective of chromatographic column protection, further research is needed to study the effect of micro pre-filters with different pore sizes of third filter layer membranes on chromatographic columns.

[0140] Example 5B Micro prefilter usage times

[0141] Except that the material of the first filter layer of the filter assembly of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is made of a 0.22 μm PVC filter membrane, NC filter membrane, or PVDF filter membrane, the rest is the same as in Example 5;

[0142] The number of times the micro pre-filter is used is counted in the same manner as in Example 5.

[0143] The number of times the micro prefilter of this embodiment is used is significantly lower than the number of times the micro prefilter of the membrane material in Example 5 is used. The number of times the micro prefilter using 0.22μm membrane material in the third filter layer is used is only 106±15 times (PVC filter membrane), 81±24 times (NC filter membrane) and 94±21 times (PVDF filter membrane).

[0144] Example 5C Micro prefilter usage times

[0145] The first filter layer of the filter assembly of the micro pre-filter is made of glass fiber filter paper; the second filter layer is made of a PES filter membrane with a pore size of 0.80 μm; and the third filter layer is made of a 0.45 μm PVC filter membrane, NC filter membrane, or PVDF filter membrane. The rest is the same as in Example 5.

[0146] The number of times the micro pre-filter is used is counted in the same manner as in Example 5.

[0147] The number of times the micro prefilter of this embodiment is used is significantly lower than the number of times the micro prefilter of the membrane material in Example 5 is used. The number of times the micro prefilter using 0.45μm membrane material in the third filter layer is used is only 152±25 times (PVC filter membrane), 112±26 times (NC filter membrane) and 137±22 times (PVDF filter membrane).

[0148] Example 6 Optimization of filter membrane pore size

[0149] In this embodiment, the pore size of the filter membrane is optimized by the HPLC column efficiency and the number of times the column is used.

[0150] The material of the first filter layer of the filter component of the micro pre-filter is glass fiber filter paper; the second filter layer is a PES filter membrane with a pore size of 0.80μm; the third filter layer is a 0.22μm MCE filter membrane, PES filter membrane, hydrophilic PTFE filter membrane; or a 0.45μm MCE filter membrane, PES filter membrane, hydrophilic PTFE filter membrane.

[0151] 1. Theoretical plates

[0152] The theoretical plate number is an important parameter for the separation efficiency of a chromatographic column. 1c :4.50%-8.50%[26.00-69.00mmol / mol]) HPLC method for HbA 1c The concentration was tested 20 times to evaluate the effect of different filter materials and sizes of micro prefilters on the theoretical plate number. Figure 2 .

[0153] Depend on Figure 2 It can be seen that there is no significant difference in the theoretical plate number of the micro pre-filters with different filter materials and pore sizes in the third filter layer (P>0.05). MCE filter membrane, PES filter membrane and hydrophilic PTFE filter membrane have good performance in protecting the column efficiency.

[0154] 2. Number of times the column is used

[0155] Confirmation of the number of times the chromatographic column has been used, and HbA in fresh whole blood samples has been measured under the protection of micro pre-filters made of different materials and pore sizes. 1c concentration until the following conditions occur (chromatogram is abnormal, measurement value deviation is greater than 10.0%, pressure is greater than 11.0MPa / cm 2 or column efficiency is less than 300), record the maximum detection times of the chromatographic column.

[0156] The replacement frequency of the micro pre-filter of the present invention is 500 times, that is, the micro pre-filter is replaced with a new micro pre-filter after being used 500 times, and when the replacement frequency of the micro pre-filter is to replace the new micro pre-filter after being used 500 times, the evaluation of each micro pre-filter is tested using 5 chromatographic columns, and the number of times the 5 chromatographic columns are used is recorded. The number of times the chromatographic columns are used is shown in Table 3.

[0157] Table 3 The number of times the chromatographic columns were used using micro pre-filters of different materials and pore sizes

[0158]

[0159] *One-way ANOVA, P>0.05.

[0160] From the measurement results in Table 3, we can see that:

[0161] There was no significant difference in the number of times the chromatographic column was used when the third filter layer used micro pre-filters with the same pore size but different materials (P>0.05). There was a significant difference in the number of times the chromatographic column was used when the micro pre-filters with different pore sizes but the same filter materials were used (P<0.05). The third filter layer filter material with small pore size is the best choice for preparing micro pre-filters, and can extend the life of the chromatographic column by 20%-30% compared with the third filter layer filter material with large pore size.

[0162] The above embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A micro prefilter for a high performance liquid chromatography column, Its characteristics are: include: The cylindrical hollow column shell and the three-stage filter assembly installed inside the column shell adopt a three-stage filtration design, wherein the first stage filtration adopts large-pore filter paper, the second stage filtration adopts large-pore filter membrane, and the third stage filtration adopts small-pore filter membrane.

2. The micro prefilter according to claim 1, Its characteristics are: The filter material is a hydrophilic deep filter material, including a hydrophilic filter paper and a filter membrane, and the filter material has the characteristic of low protein adsorption.

3. The micro prefilter according to claim 1 or 2, Its characteristics are: The filter assembly includes a first, a second and a third filter layer which are sequentially arranged from the liquid inlet end to the liquid outlet end of the micro pre-filter; the first filter layer is made of two layers of filter paper stacked together; the second and the third filter layers are respectively made of one layer of filter membrane; wherein the pore size of the filter membrane of the second filter layer is larger than the pore size of the filter membrane of the third filter layer.

4. The micro pre-filter according to claim 3, Its characteristics are: The filter paper is glass fiber filter paper, composite fiber filter paper or polymer filter paper; the second filter layer membrane is polyethersulfone filter membrane, mixed cellulose membrane, hydrophilic polytetrafluoroethylene membrane or perchloroethylene filter membrane, preferably polyethersulfone filter membrane; the third filter layer membrane is polyethersulfone filter membrane, mixed cellulose membrane, hydrophilic polytetrafluoroethylene membrane, perchloroethylene filter membrane, nitrocellulose filter membrane or polyvinylidene fluoride filter membrane, preferably polyethersulfone filter membrane, mixed cellulose membrane or hydrophilic polytetrafluoroethylene membrane.

5. The micro pre-filter according to claim 3, Its characteristics are: The pore size of the filter paper of the first filter layer is 0.22 to 2.70 μm, preferably 2.70 μm; The thickness is 0.30-0.40mm; the pore size of the filter membrane of the second filter layer is 0.22-0.80μm, preferably 0.80μm; the thickness is 0.10-0.20mm; the pore size of the filter membrane of the third filter layer is 0.22-0.80μm, preferably 0.22 or 0.45 or 0.80μm, further preferably 0.22μm; the thickness is 0.1-0.2mm.

6. The micro pre-filter according to claim 2, Its characteristics are: The first filter layer filters and removes cells, cell membranes, cell debris or particulate matter in the mobile phase; the second filter layer filters and removes polymer particles such as fats, phospholipids, lipids, etc. in the mobile phase; the third filter layer filters and removes small particles of surface-activated polymers, small molecule serum proteins, fats and membrane phospholipids in the mobile phase.

7. The micro pre-filter according to claim 1, Its characteristics are: The metal mesh is a nickel mesh or a stainless steel mesh, which plays the role of supporting and distributing the liquid flow.

8. The micro pre-filter according to claim 1, Its characteristics are: It also includes the establishment of a performance evaluation procedure for micro pre-filters, which optimizes the filter membrane material and pore size of the micro pre-filter by evaluating the effects of pre-filters of different materials and pore sizes on the chromatographic column (retention time, chromatographic column efficiency and number of times the chromatographic column is used).

9. A method for preparing a micro prefilter for a high performance liquid chromatography column according to any one of claims 1 to 8, Its characteristics are: The steps include: 1) Cut the filter material into discs with a diameter that matches the inner diameter of the column shell; 2) placing a first filter layer, a second filter layer, a third filter layer, and a metal mesh in the column housing in order from the liquid inlet end to the liquid outlet end; 3) The column shell is buckled and sealed to obtain the micro pre-filter.

10. Use of the micro prefilter for a high performance liquid chromatography column as claimed in any one of claims 1 to 8 in high performance liquid chromatography analysis and detection; the prefilter is placed at the front end of the chromatographic column to protect the chromatographic column. This patent invention can also be used in the field of high performance liquid chromatography tandem mass spectrometry analysis.

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