Automatic online extraction-mass spectrometry detection system and method based on integrated micro-fluidic chip

Abstract

The invention discloses an automatic online extraction-mass spectrometric detection system and method based on an integrated micro-fluidic chip, and relates to the field of micro-fluidic processing and mass spectrometric analysis, and an automatic integrated micro-fluidic chip unit is bonded by multiple layers of PDMS and is packaged by glass. The on-line solid-phase extraction desalting unit is used for carrying out automatic on-line extraction desalting operation, so that mass spectrometric detection on target components in different solutions is realized. The mass spectrum on-line detection unit is connected with the mass spectrum spray needle through a capillary tube to realize mass spectrum on-line real-time detection. According to the invention, multi-step automatic operation of extraction is realized only through the chip, and the sample consumption is lower. The mass spectrometric detection sample has high-throughput, high-selectivity and high-sensitivity analysis, and online quantitative analysis of a biological sample can be realized. Compared with a traditional detection method, the method has the advantages of automatic sample extraction and desalination treatment, high efficiency, rapidness, reduction of manual operation errors, high flux and shorter analysis time, and meets the requirement of immediate high-flux analysis of temporary biological samples.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic processing and mass spectrometry analysis, and more specifically to an automated online extraction-mass spectrometry detection system and method based on an integrated microfluidic chip. Background Art

[0002] At present, microfluidics is a system science and technology that focuses on manipulating trace amounts of fluids, and its core implementation platform is the microfluidic chip. The main feature of a microfluidic chip is that the structure used to contain the fluid reaches the nanoscale in at least one dimension. With the widespread application of semiconductor micromachining technology in the manufacture of microfluidic chips, and the development of new processes such as multilayer construction of elastic materials, a variety of functional elements and structures can be highly integrated into tiny chips. This chip with multiple functions, complex structure, and precise and controllable operation is called an "integrated microfluidic chip."

[0003] Integrated microfluidic chips, especially those with high density, large scale, high throughput and multifunctional characteristics, have shown important value in the fields of chemistry and biology. Compared with traditional macroscopic experimental devices, microfluidic technology not only significantly reduces sample consumption, but also increases the surface area of ​​the fluid, thereby improving reaction efficiency and reducing environmental pollution caused by waste generation. At the same time, its operational parallelism advantage can achieve high throughput and automated control of experiments. In addition, through the precise control of microstructures such as microvalves and micropumps, microfluidic chips can flexibly improve the temporal and spatial resolution of life science research, showing irreplaceable technical advantages.

[0004] Mass spectrometry detection technology occupies an important position in the detection of proteins, signaling molecules, drugs and metabolites in cell-related analysis due to its wide detection range, rich molecular information, ability to process complex samples and provide molecular structure information. With the rapid development of biomedical technology, the demand for new drug development continues to grow, and drug toxicity testing and metabolic analysis have become an indispensable part of the development process. Compared with traditional animal models and two-dimensional cell culture, in vitro models are more suitable for large-scale screening of compounds due to their advantages such as low sample consumption, short experimental cycle, accurate dose response and high throughput. In the mass spectrometry detection process, the traditional pretreatment process requires the removal of salt from the sample solution to be tested, usually using solid phase microextraction technology. However, traditional solid phase extraction microfluidic chips rely on manual labor and cannot be fully automated. They consume a lot of samples, the steps are cumbersome and the injection relies on syringe pumps, which are large in size and inefficient.

[0005] Therefore, how to realize automated online solid phase extraction desalination, real-time online mass spectrometry detection, and meet the detection of target substances such as drugs, protein factors, nucleic acids, etc. in different biological sample solutions is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] In view of this, the present invention provides an automated online extraction-mass spectrometry detection system and method based on an integrated microfluidic chip, which realizes automated online solid phase extraction desalination and real-time online mass spectrometry detection, meets the detection of target substances such as drugs, protein factors, nucleic acids, etc. in different biological sample solutions, and provides convenience for clinical and drug research and development.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] An automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip, comprising: an automated integrated microfluidic chip unit, an online solid phase extraction and desalination unit, an automated control unit, a pressure unit, and an online mass spectrometry detection unit;

[0009] The automated integrated microfluidic chip unit has multiple layers of PDMS, each layer having different functions, specifically including a fluid layer, a control layer, and a glass packaging layer; the fluid layer is provided with a solid phase extraction column interface;

[0010] The online solid phase extraction desalination unit is provided by inserting a tip having a C 18 The gun tip of the solid phase extraction column, the other end of the gun tip is connected to the automation control unit through a rubber tube, and the control layer of the automated integrated microfluidic chip unit is connected to the automation control unit;

[0011] The automation control unit is connected to the pressure unit;

[0012] The mass spectrometry online detection unit is connected to the automated integrated microfluidic chip unit.

[0013] Optionally, the fluid layer has multiple sample inlets and multiple sample outlets and a solid phase extraction column interface, and the control layer is provided with multiple valves at different positions, and the valves are connected to an automated control unit.

[0014] Optionally, the automated integrated microfluidic chip unit has a three-layer structure; the upper layer is the fluid layer, the middle layer is the control layer, and the lower layer is the glass packaging layer. The materials of the upper and middle layers are PDMS, and the material of the lower layer is glass; the upper and middle layers are bonded by PDMS in different proportions, the PDMS ratio of the upper layer is 5:1, and the PDMS ratio of the middle layer is 20:1, and the PDMS curing method is heating at 80°C for 20 minutes; the middle layer and the lower layer are bonded by plasma, using a plasma cleaning machine, vacuuming for 150S, air intake for 50S, surface hydrophilic treatment for 90S, and brushing the glass clean before bonding.

[0015] Optionally, the automation control unit includes a computer, a controller, and a solenoid valve, which are connected in sequence through a circuit; one end of the solenoid valve is connected to the controller, and the other end is connected to the automation integrated microfluidic chip unit through a plastic hose.

[0016] Optionally, the pressure unit is one of a steel cylinder and an air pump; it provides pressure for the automated integrated microfluidic chip unit, with the control layer pressure being 20 psi and the fluid layer pressure being 5 psi.

[0017] Optionally, the mass spectrometry online detection unit comprises a capillary with an outer diameter of 669 μm, a 360 μm spray needle, a liquid chromatography two-way PEEK reducer of 1 / 16' to 1 / 32' and a mass spectrometer; the automated integrated microfluidic chip unit is connected to the capillary, and the capillary is connected to the mass spectrometry spray needle via a two-way reducer.

[0018] Optionally, the sample collection process is to use gas pressure to elute the sample from the solid phase extraction column at the end of the gun tip into the capillary, the sample passes through the capillary and forms a spray after the mass spectrometer needle is energized, and finally completes the mass spectrometer online detection.

[0019] An automated online extraction-mass spectrometry detection method based on an integrated microfluidic chip comprises the following steps:

[0020] Sample preparation: First, prepare a 1 M stock solution of capecitabine, the solvent is DMSO, 500 μM capecitabine is diluted from the stock solution using complete medium, and 0.1% formic acid is added to make its pH <4;

[0021] Activate the solid phase extraction column: 100% acetonitrile reservoir is pressurized to enter through injection port 1, valves ①②⑦ are opened, and other valves are closed to fill the channel with acetonitrile, then ①⑦③ are opened, and other valves are closed, and acetonitrile enters the gun tip loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10μL, stop injecting; valves ③⑦ are opened, and other valves are closed. The pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid. Repeat the above operation three times;

[0022] Equilibrate the solid phase extraction column: apply pressure to the 0.1% formic acid reservoir to allow it to enter through injection port 2, open valves ⑤②⑦, and close other valves to fill the channel with formic acid, then open valves ⑤②③, and close other valves, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, stop injecting; open valves ③⑦, and close other valves, the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and repeat the above operation three times;

[0023] Loading: Apply pressure to the sample reservoir to allow the sample to enter through the injection port 4, open valve ④, close other valves, and allow the sample to enter the gun head loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10 μL, stop injecting the sample; open valves ③⑦, close other valves, and allow the pressure to push the solution in the gun head into the fluid channel and through the waste liquid collection port to recover the waste liquid. Repeat the above operation 7-10 times;

[0024] Cleaning the solid phase extraction column: 0.1% formic acid reservoir is applied with pressure to allow it to enter through injection port 2, valves ⑤②⑦ are opened, and other valves are closed, so that the channel is filled with formic acid, then valves ⑤②③ are opened, and other valves are closed, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, the injection is stopped; valves ③⑦ are opened, and other valves are closed, and the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and the above operation is repeated three times;

[0025] Sample elution: 1% formic acid / 50% methanol reservoir is given pressure to allow it to enter through injection port 3, valves ⑥②⑦ are opened, and other valves are closed to fill the channel with eluent, then valves ⑥②③ are opened, and other valves are closed, and the eluent enters the gun tip loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10μL, the injection is stopped; valves ③⑦⑧ are opened, and other valves are closed. The pressure pushes the solution in the gun tip into the fluid channel and through the mass spectrometer injection port, and enters the mass spectrometer needle through the two-way valve. After the mass spectrometer needle is energized, an electrospray is formed, thereby directly performing mass spectrometer online detection.

[0026] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses an automated online extraction-mass spectrometry detection system and method based on an integrated microfluidic chip, which has the following beneficial effects:

[0027] 1. The multi-layer structure of the integrated microfluidic chip of the present invention is combined with electromagnetic valve control, and the multi-step automated operation of extraction can be realized only through the chip, and the sample consumption is lower.

[0028] 2. The present invention adopts integrated microfluidic chip for online extraction-mass spectrometry. After the chip is automatically processed, it directly enters the tip of the mass spectrometer through instructions. The tip is powered on, and the sample forms an electrospray. The mass spectrometry detection sample has high throughput, high selectivity and high sensitivity analysis, which can realize online quantitative analysis of biological samples.

[0029] 3. Compared with traditional detection methods, the present invention automates sample extraction and desalination, is efficient and rapid, eliminates manual labor, reduces manual operation errors, has high throughput, and shortens analysis time, thus meeting the needs of real-time high-throughput analysis of clinical biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 A schematic diagram of the structure of the detection system provided by the present invention;

[0032] Figure 2 A schematic diagram of the structure of the automated integrated microfluidic chip unit provided by the present invention;

[0033] Figure 3 This is an online mass spectrometry detection diagram of the drug capecitabine provided by the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The embodiment of the present invention discloses an automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip, such as Figure 1 As shown, it includes: an automated integrated microfluidic chip unit, an online solid phase extraction desalination unit, an automated control unit, a pressure unit, and a mass spectrometry online detection unit;

[0036] The automated integrated microfluidic chip unit has multiple layers of PDMS, each layer has different functions, specifically including a fluid layer, a control layer, and a glass packaging layer; the fluid layer is provided with a solid phase extraction column interface;

[0037] The online solid phase extraction desalting unit is connected by inserting the tip of the solid phase extraction column into the interface. 18 The gun tip of the solid phase extraction column, the other end of the gun tip is connected to the automation control unit through a rubber tube, and the control layer of the automated integrated microfluidic chip unit is connected to the automation control unit;

[0038] The automation control unit is connected to the pressure unit;

[0039] The mass spectrometry online detection unit is connected to the automated integrated microfluidic chip unit.

[0040] In a specific embodiment, Figure 2As shown, the automated integrated microfluidic chip unit includes three layers, namely, the fluid layer, the control layer, and the glass packaging layer. The fluid layer has 4 injection ports, 2 outlets and a solid phase extraction column interface. Injection ports 1 to 3 are 100% acetonitrile, 0.1% formic acid, and 1% formic acid / 50% methanol, respectively. Injection port 4 is the biological sample (capecitabine) injection port. The outlets are the waste liquid collection port and the mass spectrometry injection port. The solid phase extraction column interface is selected to contain C 18 The gun tip of the solid phase extraction column. The control layer contains 8 valves, that is, 8 injection ports. The control valves ①②③④⑤⑥⑦⑧ are all set in the fluid channel part to block the flow of the fluid.

[0041] In a specific embodiment, the automated integrated microfluidic chip unit has a three-layer structure; the upper layer is a fluid layer, the middle layer is a control layer, and the lower layer is a glass packaging layer. The materials of the upper and middle layers are PDMS, and the material of the lower layer is glass; the upper and middle layers are bonded by PDMS in different proportions, the PDMS ratio of the upper layer is 5:1, and the PDMS ratio of the middle layer is 20:1. The PDMS curing method is heating at 80°C for 20 minutes; the middle and lower layers are plasma bonded, using a plasma cleaning machine, vacuuming for 150S, air intake for 50S, surface hydrophilic treatment for 90S, and brushing clean before glass bonding.

[0042] In a specific embodiment, the automation control unit includes a computer, a controller, and a solenoid valve, which are connected in sequence through a circuit; one end of the solenoid valve is connected to the controller, and the other end is connected to the automation integrated microfluidic chip unit through a plastic hose. The solenoid valve is connected to the computer through a multi-channel controller, and the computer issues instructions to the multi-channel controller through a program, and finally the solenoid valve implements the opening and closing of the gas path valve.

[0043] In a specific embodiment, the pressure unit is a steel cylinder or an air pump; it provides pressure for the automated integrated microfluidic chip unit, with a pressure of 20 psi for the control layer and 5 psi for the fluid layer. Two flow meters are required to control the pressure of the control layer and the fluid layer. Importantly, before the experiment, the eight control valves of the control layer need to be filled with solution. When the pressure is provided to the control valve, the solution in the control valve chamber impacts the control layer film and arches it upward, thereby compressing the fluid layer channel, thereby achieving the left and right of the barrier fluid.

[0044] In a specific embodiment, the mass spectrometer online detection unit includes a capillary with an outer diameter of 669 μm, a 360 μm spray needle, a liquid chromatography two-way PEEK reducer of 1 / 16' to 1 / 32', and a mass spectrometer; the automated integrated microfluidic chip unit is connected to the capillary, and the capillary is connected to the mass spectrometer spray needle through a two-way reducer.

[0045] In a specific embodiment, the sample collection process is to use gas pressure to elute the sample from the solid phase extraction column at the end of the gun tip into the capillary. The sample passes through the capillary and forms a spray after the mass spectrometer needle is energized, and finally completes the mass spectrometer online detection.

[0046] An automated online extraction-mass spectrometry detection method based on an integrated microfluidic chip comprises the following steps:

[0047] Sample preparation: First, prepare a 1 M stock solution of capecitabine, the solvent is DMSO, 500 μM capecitabine is diluted from the stock solution using complete culture medium, and 0.1% formic acid is added to make its pH <4;

[0048] Activate the solid phase extraction column: 100% acetonitrile reservoir is pressurized to enter through injection port 1, valves ①②⑦ are opened, and other valves are closed to fill the channel with acetonitrile, then ①⑦③ are opened, and other valves are closed, and acetonitrile enters the gun tip loaded with the solid phase extraction column through the fluid channel, and the injection is stopped when the solution reaches 10μL; valves ③⑦ are opened, and other valves are closed, and the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and the above operation is repeated three times;

[0049] Equilibrate the solid phase extraction column: apply pressure to the 0.1% formic acid reservoir to allow it to enter through injection port 2, open valves ⑤②⑦, and close other valves to fill the channel with formic acid, then open valves ⑤②③, and close other valves, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, stop injecting; open valves ③⑦, and close other valves, the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and repeat the above operation three times;

[0050] Loading: Apply pressure to the sample reservoir to allow the sample to enter through the injection port 4, open valve ④, close other valves, and allow the sample to enter the gun head loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10 μL, stop injecting the sample; open valves ③⑦, close other valves, and allow the pressure to push the solution in the gun head into the fluid channel and through the waste liquid collection port to recover the waste liquid. Repeat the above operation 7-10 times;

[0051] Cleaning the solid phase extraction column: 0.1% formic acid reservoir is applied with pressure to allow it to enter through injection port 2, valves ⑤②⑦ are opened, and other valves are closed, so that the channel is filled with formic acid, then valves ⑤②③ are opened, and other valves are closed, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, the injection is stopped; valves ③⑦ are opened, and other valves are closed, and the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and the above operation is repeated three times;

[0052] Sample elution: 1% formic acid / 50% methanol reservoir is given pressure to allow it to enter through injection port 3, valves ⑥②⑦ are opened, and other valves are closed to fill the channel with eluent, then valves ⑥②③ are opened, and other valves are closed, and the eluent enters the gun tip loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10μL, the injection is stopped; valves ③⑦⑧ are opened, and other valves are closed. The pressure pushes the solution in the gun tip into the fluid channel and through the mass spectrometer injection port, and enters the mass spectrometer needle through the two-way valve. After the mass spectrometer needle is energized, an electrospray is formed, thereby directly performing mass spectrometer online detection.

[0053] This example mainly performs solid phase extraction and desalting on capecitabine diluted with cell culture medium and performs mass spectrometry detection. The detection results are as follows: Figure 3 As shown, after hydrogenation treatment, the molecular ion peak of capecitabine was detected to be 360.1560, and capecitabine was well detected.

[0054] The present invention can realize sample separation, enrichment and detection online, and has broad application prospects in clinical drug detection.

[0055] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip, characterized in that: include: Automated integrated microfluidic chip unit, online solid phase extraction desalination unit, automated control unit, pressure unit, mass spectrometry online detection unit; The automated integrated microfluidic chip unit has multiple layers of PDMS, each layer having different functions, specifically including a fluid layer, a control layer, and a glass packaging layer; the fluid layer is provided with a solid phase extraction column interface; The online solid phase extraction desalination unit is provided by inserting a tip having a C 18 The gun tip of the solid phase extraction column, the other end of the gun tip is connected to the automation control unit through a rubber tube, and the control layer of the automated integrated microfluidic chip unit is connected to the automation control unit; The automation control unit is connected to the pressure unit; The mass spectrometry online detection unit is connected to the automated integrated microfluidic chip unit.

2. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that: The fluid layer has a plurality of sample inlets, a plurality of sample outlets and a solid phase extraction column interface, and the control layer is provided with a plurality of valves at different positions, and the valves are connected to an automatic control unit.

3. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that: The automated integrated microfluidic chip unit has a three-layer structure; the upper layer is a fluid layer, the middle layer is a control layer, and the lower layer is a glass packaging layer. The materials of the upper and middle layers are PDMS, and the material of the lower layer is glass. The upper and middle layers are bonded by PDMS in different proportions, the PDMS ratio of the upper layer is 5:1, and the PDMS ratio of the middle layer is 20:

1. The PDMS curing method is heating at 80°C for 20 minutes. The middle and lower layers are bonded by plasma, using a plasma cleaning machine, vacuuming for 150S, air intake for 50S, surface hydrophilic treatment for 90S, and brushing clean before glass bonding.

4. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that: The automation control unit comprises a computer, a controller and a solenoid valve, which are connected in sequence through a circuit; one end of the solenoid valve is connected to the controller, and the other end is connected to the automation integrated microfluidic chip unit through a plastic hose.

5. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that: The pressure unit is one of a steel cylinder and an air pump; it provides pressure for the automated integrated microfluidic chip unit, with the pressure of the control layer being 20 psi and the pressure of the fluid layer being 5 psi.

6. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that: The mass spectrometer online detection unit comprises a capillary with an outer diameter of 669 μm, a 360 μm spray needle, a liquid chromatography two-way PEEK reducer of 1 / 16' to 1 / 32' and a mass spectrometer; the automated integrated microfluidic chip unit is connected to the capillary, and the capillary is connected to the mass spectrometer spray needle through a two-way reducer.

7. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 6, characterized in that: The sample collection process is to use gas pressure to elute the sample from the solid phase extraction column at the end of the gun tip into the capillary. The sample passes through the capillary and forms a spray after the mass spectrometer needle is energized, and finally completes the mass spectrometer online detection.

8. An automated online extraction-mass spectrometry detection method based on an integrated microfluidic chip, characterized in that: An automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip as described in any one of claims 1 to 7 comprises the following steps: Sample preparation: First, prepare a 1 M stock solution of capecitabine, the solvent is DMSO, 500 μM capecitabine is diluted from the stock solution using complete culture medium, and 0.1% formic acid is added to make its pH <4; Activate the solid phase extraction column: 100% acetonitrile reservoir is pressurized to enter through injection port 1, valves ①②⑦ are opened, and other valves are closed to fill the channel with acetonitrile, then ①⑦③ are opened, and other valves are closed, and acetonitrile enters the gun tip loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10μL, stop injecting; valves ③⑦ are opened, and other valves are closed. The pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid. Repeat the above operation three times; Equilibrate the solid phase extraction column: apply pressure to the 0.1% formic acid reservoir to allow it to enter through injection port 2, open valves ⑤②⑦, and close other valves to fill the channel with formic acid, then open valves ⑤②③, and close other valves, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, stop injecting; open valves ③⑦, and close other valves, the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and repeat the above operation three times; Loading: Apply pressure to the sample reservoir to allow the sample to enter through the injection port 4, open valve ④, close other valves, and allow the sample to enter the gun head loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10 μL, stop injecting the sample; open valves ③⑦, close other valves, and allow the pressure to push the solution in the gun head into the fluid channel and through the waste liquid collection port to recover the waste liquid. Repeat the above operation 7-10 times; Cleaning the solid phase extraction column: 0.1% formic acid reservoir is applied with pressure to allow it to enter through injection port 2, valves ⑤②⑦ are opened, and other valves are closed, so that the channel is filled with formic acid, then valves ⑤②③ are opened, and other valves are closed, formic acid enters the gun tip loaded with the solid phase extraction column through the fluid channel, and when the solution reaches 10μL, the injection is stopped; valves ③⑦ are opened, and other valves are closed, and the pressure pushes the solution in the gun tip into the fluid channel and through the waste liquid collection port to recover the waste liquid, and the above operation is repeated three times; Sample elution: 1% formic acid / 50% methanol reservoir is given pressure to allow it to enter through injection port 3, valves ⑥②⑦ are opened, and other valves are closed to fill the channel with eluent, then valves ⑥②③ are opened, and other valves are closed, and the eluent enters the gun tip loaded with the solid phase extraction column through the fluid channel. When the solution reaches 10μL, the injection is stopped; valves ③⑦⑧ are opened, and other valves are closed. The pressure pushes the solution in the gun tip into the fluid channel and through the mass spectrometer injection port, and enters the mass spectrometer needle through the two-way valve. After the mass spectrometer needle is energized, an electrospray is formed, thereby directly performing mass spectrometer online detection.

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