Solid phase microextraction-electrospray ionization integrated transfer pipette and use method thereof

Abstract

The invention discloses a transfer pipette for integrated operation of solid-phase microextraction and electrospray ionization and a use method of the transfer pipette. The transfer pipette is mainly made of a polymer material, a conductive metal clamping sleeve is embedded in the outer surface of the front end of the transfer pipette, and a triangular porous matrix, an artificially synthesized bead, an adsorbent and a circular porous matrix are sequentially arranged at the inner front end of the transfer pipette. Based on the design of the integrated pipette, a complex sample to be detected can be directly injected into the pipette, and by utilizing the interaction between the sample and the adsorbent, after a spray solvent is added, elution of a sample matrix is effectively inhibited, and meanwhile, rapid desorption of a target compound is promoted. And then, voltage is applied to the conductive metal clamping sleeve at the front end of the pipette, so that the tip of the triangular porous substrate generates electrospray, and in-situ ionization is realized. The method is combined with a mass spectrum technology, and can be used for efficient pretreatment and in-situ, rapid and high-sensitivity mass spectrum analysis of a target compound in a complex sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sample pretreatment and mass spectrometry ionization source technology, in particular to a solid phase microextraction-electrospray ionization integrated pipette and a use method thereof. BACKGROUND

[0002] Solid phase microextraction is a sample preparation technology proposed by Arthur and Pawliszyn of the University of Waterloo in Canada in 1990. The principle is to use adsorbents loaded on the surface or inside of fibers, stirring rods, membranes, microtubes, etc. to interact with samples containing target analytes. When the adsorption equilibrium is reached, the target analytes can be quantitatively extracted or enriched on the surface of the adsorbent.

[0003] Due to the characteristics of sample collection, extraction, concentration, and injection in one, and the need for only a small amount of organic solvent, the preparation process of complex samples is greatly simplified, providing an innovative solution for green, efficient, and low-cost analysis of target compounds, and attracting widespread attention.

[0004] At the same time, solid phase microextraction also shows strong compatibility and can be used in conjunction with gas / liquid chromatography, capillary electrophoresis, mass spectrometry, etc., significantly improving the analysis sensitivity and selectivity of target compounds in complex samples.

[0005] Mass spectrometry technology has been widely used in the detection of target compounds in biological, environmental, and food complex samples due to its high sensitivity and specificity.

[0006] When chromatography technology is combined with mass spectrometry detection, not only can complex samples be efficiently separated, but also the detection sensitivity of mass spectrometry is improved, which is the gold method for analyzing complex samples so far. However, before analyzing the sample in this way, tedious sample pretreatment is required, followed by chromatographic separation and mass spectrometry detection, which cannot achieve high-throughput analysis of samples. Ambient ionization mass spectrometry technology significantly improves the detection efficiency of target compounds in complex samples due to its characteristics of no sample pretreatment, simple operation, and fast analysis speed.

[0007] However, during the ionization process of the target compounds in complex samples using this technology, interfering compounds in the sample matrix will also be ionized, which severely inhibits the mass spectrometry analysis sensitivity of the target compounds.

[0008] In order to overcome the above shortcomings, solid phase microextraction (such as probe type, in-tube enrichment type, blade type, mesh type, surface coating type, filter membrane / filter paper separation type, etc.) is coupled with ambient ionization ionization source, which effectively reduces the interference of the sample matrix on the target compounds.

[0009] Nevertheless, the current method of coupling solid-phase microextraction (SPE) with atmospheric pressure ionization sources basically involves first enriching the target compound in the sample with SPE, then coupling it with an atmospheric pressure ionization source, and finally performing mass spectrometry analysis.

[0010] While this operating mode can significantly improve the sensitivity of mass spectrometry analysis of target compounds in complex samples, it is cumbersome to operate and is not suitable for high-throughput mass spectrometry analysis of trace samples. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a pipette for solid phase microextraction-electrospray ionization integration and its usage method, which aims to integrate solid phase microextraction and atmospheric pressure ionization technology into a low-cost pipette to achieve in-situ coupling of the two.

[0012] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a pipette for solid-phase microextraction-electrospray ionization integration, comprising a polymer tube and a conductive metal sleeve nested at the front end of the polymer tube;

[0013] The conductive metal sleeve is further fitted with a triangular porous matrix at its front end. Artificial beads are provided inside the triangular porous matrix near its front end. The inner surface of the triangular porous matrix is ​​also loaded with an adsorbent.

[0014] The adsorbent also has a circular porous matrix located inside the polymer tube at its rear end.

[0015] Preferably, the polymer tube is a tapered sample tube open at both ends:

[0016] The outer diameter of the front end is 0.8mm to 2.0mm, and the inner diameter is 0.5mm to 1.5mm;

[0017] The outer diameter of the rear end is 7mm to 10mm, and the inner diameter is 5mm to 8mm;

[0018] The length is 40mm to 50mm.

[0019] Preferably, the triangular porous matrix is ​​an isosceles triangle with a apex angle of 30° to 35° and a length of 6 mm to 15 mm, and the material is selected from any one of paper, porous polymer or fiber.

[0020] Preferably, the diameter of the synthetic beads is 0.5mm to 1.2mm, and the material is glass, metal or polymer.

[0021] Preferably, the adsorbent is a porous inorganic material, an organic material, or a composite material thereof, loaded in the form of a solution with a concentration of 0.15 g / mL to 0.45 g / mL, and the loading volume is 10 μL to 35 μL.

[0022] Preferably, the circular porous matrix is ​​in the shape of a disc with a diameter of 0.8 mm to 1.4 mm, and the material is the same as that of the triangular porous matrix.

[0023] Preferably, the conductive metal sleeve has a conical structure that matches the shape of the front end of the polymer tube, and its material is any one of stainless steel, aluminum or copper.

[0024] The conductive metal ferrule has an outer diameter of 0.7mm to 2.2mm and an inner diameter of 0.5mm to 2.0mm at the front end.

[0025] The outer diameter of the rear end is 5.4mm to 8.4mm, and the inner diameter is 5.2mm to 8.2mm;

[0026] The length ranges from 0.4mm to 2.0mm.

[0027] Another aspect of the present invention discloses a preparation method comprising the following steps:

[0028] S1: Cut the tip of a commercial pipette into a tapered polymer tube with openings at both ends;

[0029] S2: Insert the conductive metal ferrule into the outer surface of the front end of the polymer tube;

[0030] S3: Insert the triangular porous matrix from the rear end of the polymer tube, so that its tip extends 1mm to 5mm beyond the front end of the polymer tube;

[0031] S4: Place the synthetic beads into the front end of the polymer tube, so that they are located inside the triangular porous matrix and the front end is flush with the end face of the polymer tube.

[0032] S5: Add the adsorbent solution from the rear end of the polymer tube to the inner surface of the triangular porous matrix, and let it dry to form an adsorbent layer;

[0033] S6: Insert the circular porous matrix from the rear end of the polymer tube, so that it is perpendicular to the rear side of the adsorbent.

[0034] Another aspect of the present invention discloses a method of use, comprising the following steps:

[0035] S1: Add 1 μL to 5 μL of the sample solution to be tested to the rear end of the polymer tube and dry it in the air for more than 2 hours;

[0036] S2: Immerse the front end of the polymer tube in the spray solvent and draw in 15μL to 30μL of spray solvent through self-absorption.

[0037] S3: Apply a DC voltage of 3000V to 5000V to the conductive metal sleeve and perform electrospray ionization for 10s to 420s;

[0038] S4: The generated electrospray stream is directly introduced into the mass spectrometer for detection.

[0039] The advantages of this invention compared with the prior art are: this invention integrates solid phase microextraction and electrospray ionization functions into a low-cost polymer pipette, realizing in-situ pretreatment and direct ionization of complex samples;

[0040] This integrated design eliminates the need for sample transfer, avoiding the loss of target analytes; it utilizes adsorbents to selectively retain matrix interferences, significantly improving the sensitivity of mass spectrometry detection.

[0041] A conductive metal sleeve is embedded at the tip of the pipette, which induces the internal spray solvent to be stably electro-sprayed at the tip of the triangular porous matrix by an external voltage, avoiding direct contact between the electrode and the sample and reducing the risk of contamination.

[0042] Meanwhile, the triangular porous matrix, synthetic beads and circular porous matrix in the structure work together to prevent adsorbent detachment and ensure rapid solvent penetration and efficient desorption of target substances.

[0043] The overall preparation is simple and inexpensive, making it suitable for high-throughput, rapid mass spectrometry analysis of trace samples. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the integrated pipette for solid-phase microextraction-electrospray ionization according to the present invention.

[0045] Figure 2 This is a schematic diagram of the fabrication process of the integrated solid-phase microextraction-electrospray ionization pipette of the present invention.

[0046] Figure 3 The mass spectrometry ion chromatograms (metal beads, ZnO adsorbent) obtained by performing five parallel experiments on a serum sample (containing amitriptyline, clozapine, amisulpride, quetiapine, risperidone and aripiprazole) with a concentration of 1 μg·mL⁻¹ using the integrated solid-phase microextraction-electrospray ionization pipette of the present invention are shown.

[0047] Figure 4 The mass spectrometry ion chromatograms (glass beads, ZnO adsorbent) obtained by performing five parallel experiments on a serum sample (containing amitriptyline, clozapine, amisulpride, quetiapine, risperidone and aripiprazole) with a concentration of 1 μg·mL⁻¹ using the integrated solid-phase microextraction-electrospray ionization pipette of the present invention are shown.

[0048] Figure 5The mass spectrometry ion chromatograms (glass beads, ZrO2 adsorbent) obtained by performing five parallel experiments on a serum sample (containing amitriptyline, clozapine, amisulpride, quetiapine, risperidone and aripiprazole) with a concentration of 1 μg·mL⁻¹ using the integrated solid-phase microextraction-electrospray ionization pipette of the present invention are shown.

[0049] Figure 6 The mass spectrometry ion chromatograms (glass beads, SiO2 adsorbent) obtained by performing five parallel experiments on a serum sample (containing amitriptyline, clozapine, amisulpride, quetiapine, risperidone and aripiprazole) with a concentration of 1 μg·mL⁻¹ using the integrated solid-phase microextraction-electrospray ionization pipette of the present invention are shown.

[0050] Illustration:

[0051] 1. Polymer pipette, 2. Conductive metal ferrule, 3. Triangular porous matrix, 4. Synthetic beads, 5. Adsorbent, 6. Circular porous matrix. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to the accompanying drawings.

[0053] The following specific examples will provide further explanation.

[0054] A pipette for solid-phase microextraction-electrospray ionization integration, such as Figure 1 As shown, it includes:

[0055] The polymer pipette 1 has a conductive metal sleeve 2 embedded on its outer front surface for applying the electrospray voltage. Inside the front of the polymer pipette 1, there are triangular porous matrix 3, synthetic beads 4, adsorbent 5, and circular porous matrix 6. The tip of the triangular porous matrix 3 generates the electrospray flow, while the middle and rear ends of the triangular porous matrix 3 are used to load the adsorbent 5. The synthetic beads 4 are loaded inside the front of the triangular porous matrix 3 within the polymer pipette 1 to prevent the adsorbent 5 from falling off the front end of the polymer pipette and to ensure that the sprayed solvent can penetrate from the porous channels of the triangular porous matrix 3 into the middle and rear ends of the triangular porous matrix 3 during self-absorption. The adsorbent 5 is loaded above the synthetic beads 4 and below the circular porous matrix 6 to remove matrix interference in the sample matrix and improve the desorption efficiency of the target analyte. The circular porous matrix 6 is located above the adsorbent 5 and the triangular porous matrix 3 to prevent the adsorbent from falling off the rear end of the polymer pipette 1 during transport or vibration of the solid-phase microextraction-electrospray ionization integrated pipette.

[0056] This invention is based on a solid-phase microextraction-electrospray ionization integrated pipette technology. The main body of the pipette is a polymer pipette 1, with a conductive metal sleeve 2 embedded in its outer front surface. The inner front end is sequentially filled with a triangular porous matrix 3, synthetic beads 4, adsorbent 5, and a circular porous matrix 6. For electrospray ionization analysis of complex samples, 1 μL–5 μL of sample solution is first loaded onto the surface of the circular porous matrix 6 from the rear end of the polymer pipette 1. Utilizing the capillary action of the porous structure, the sample solution penetrates and interacts with the adsorbent 5. After standing for 2 hours at room temperature and pressure, the front end of the polymer pipette 1 is immersed in the spray solvent for 1–10 seconds. At this time, the spray solvent penetrates back through the triangular porous matrix 3 into the upper region of the adsorbent 5, extracting and desorbing the target compounds adsorbed on its surface. During this process, an electrospray voltage of 3000–5000 V is applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette 1, resulting in a stable electrospray formation at the front end of the triangular porous matrix 3. Subsequently, mass spectrometry was used to detect the test compounds released in the spray stream.

[0057] Using the above-mentioned solid-phase microextraction-electrospray ionization integrated pipette and its usage method, such as Figure 2 As shown, it includes the following steps:

[0058] S1. A polymer pipette 1 is prepared by cutting the tip of a commercial pipette according to the above specifications.

[0059] S2. Insert a conductive metal sleeve 2 that matches the front diameter of the polymer pipette 1 into the front end of the pipette 1 to ensure that the conductive metal sleeve and the pipette have a very good fit.

[0060] S3. Insert a porous matrix 3 with a triangular tip into the rear end of the polymer pipette 1, ensuring that the front end of the triangular porous matrix 1 extends 1 mm to 5 mm beyond the front end of the polymer pipette 1.

[0061] S4. Load the synthetic beads 4 of a specific size onto the front end of the pipette 1 containing the triangular porous matrix 2, ensuring that the front end of the synthetic beads 4 is flush with the front end of the polymer pipette 1.

[0062] S5. A certain volume of adsorbent 5 solution of a specific concentration is loaded from the rear end of the polymer pipette 1 onto the inner surface of the triangular porous matrix 2 inside the polymer pipette 1, and then placed in the air to air dry naturally.

[0063] S6. Load a circular porous substrate 6 from the rear end of the polymer pipette 1 to the rear end of the triangular porous substrate 1, ensuring that the direction of the circular paper 6 is perpendicular to the direction of the pipette.

[0064] Furthermore, 1 μL to 5 μL of complex sample solution is loaded into the solid-phase microextraction-electrospray ionization integrated pipette prepared above, and dried in air for more than 2 hours. Then, the pipette is placed into the spray solvent for 1 to 10 seconds through the front end of the polymer pipette 1, and 15 μL to 30 μL of spray solvent is loaded by self-absorption. Finally, an electrospray voltage of 3000V to 4500V is applied for electrospray ionization, and the spraying time is 10 to 420 seconds. The electrospray stream generated by the spray can be directly collected and detected by mass spectrometer.

[0065] This invention not only considers the characteristics of adsorbent 5 in solid-phase microextraction of complex analytes, which can effectively remove interference from complex matrices and improve the direct mass spectrometry analysis sensitivity of target analytes in complex analytes to the 10 pg / mL level; but also uses a cut polymer pipette 1 as a carrier for the conductive metal sleeve 2, triangular porous matrix 3, synthetic beads 4, adsorbent 5, circular porous matrix 6, and spray solvent, which can effectively suppress the volatilization of the spray solvent and significantly delay the electrospraying time, improving it by more than 10 times compared to traditional paper spraying; more importantly, this method uses a self-absorption method to load the spray solvent into the polymer pipette 1, eliminating the need for any device to load the spray solvent, significantly reducing analysis costs; moreover, this technology applies the spray voltage to the polymer pipette 1 through induction, eliminating the need for contact between the analyte and the electrode, reducing sample contamination and dissociation.

[0066] The following specific examples will provide further explanation.

[0067] Example 1

[0068] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0069] First, a polymer pipette 1 is prepared, with an inner diameter of 0.5 mm and an outer diameter of 0.8 mm at the front end, and an inner diameter of 5 mm and an outer diameter of 7 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the outer surface of the polymer pipette, with an inner diameter of 0.5 mm and an outer diameter of 0.7 mm at the front end, an inner diameter of 5 mm and an outer diameter of 5.2 mm at the rear end, and a length of 0.5 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 6 mm and a tip angle of 30°, and 2 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a metal bead 4 with a diameter of 0.5 mm is loaded into the front end of the polymer pipette 1. Then, 10 μL of a 0.15 g / mL ZnO aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0070] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn in by self-absorption for 2 seconds. Next, a 3000V electrospray voltage was applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. The results are as follows. Figure 3 As shown in the figure, parallel experiments were conducted on five groups of serum test samples at a concentration of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0071] Example 2

[0072] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0073] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.3 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 6 mm and an outer diameter of 6.2 mm at the rear end, and a length of 0.7 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 7 mm and a tip angle of 30°, and 2 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a glass bead 4 with a diameter of 0.5 mm is loaded into the front end of the polymer pipette 1. Then, 10 μL of a 0.15 g / mL ZnO aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0074] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then aspirated via self-absorption for 2 seconds. A 3000V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. Parallel experiments were performed on five groups of serum samples containing 1 μg / mL of amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole. The results are as follows. Figure 4 As shown, very stable experimental results can be obtained.

[0075] Example 3

[0076] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0077] First, a polymer pipette 1 is prepared, with an inner diameter of 1.5 mm and an outer diameter of 1.8 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the front outer surface of the polymer pipette, with an inner diameter of 1.5 mm and an outer diameter of 1.7 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.0 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 7 mm and a tip angle of 30°, and 2 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, glass beads 4 with a diameter of 1.5 mm are loaded into the front end of the polymer pipette 1. Then, 10 μL of a 0.15 g / mL ZrO2 aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0078] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then aspirated via self-absorption for 2 seconds. A 3000V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. Parallel experiments were performed on five groups of serum samples containing 1 μg / mL of amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole. The results are as follows. Figure 5 As shown, very stable experimental results can be obtained.

[0079] Example 4

[0080] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0081] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.0 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 9 mm and a tip angle of 35°, and 2 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a glass bead 4 with a diameter of 1.0 mm is loaded into the front end of the polymer pipette 1. Then, 10 μL of a 0.15 g / mL SiO2 aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0082] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then aspirated via self-absorption for 2 seconds. A 3000V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. Parallel experiments were performed on five groups of serum samples containing 1 μg / mL of amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole. The results are as follows. Figure 6 As shown, very stable experimental results can be obtained.

[0083] Example 5

[0084] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0085] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the front outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.0 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 9 mm and a tip angle of 35°, and 3 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a metal bead 4 with a diameter of 1.0 mm is loaded into the front end of the polymer pipette 1. Then, 15 μL of a 0.15 g / mL ZrO2 aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0086] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn up by self-absorption for 2 seconds. A 3000V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. Parallel experiments were conducted using this method on five groups of serum samples at concentrations of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0087] Example 6

[0088] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0089] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the front outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.0 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 9 mm and a tip angle of 35°, and 3 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a glass bead 4 with a diameter of 1.0 mm is loaded into the front end of the polymer pipette 1. Then, 35 μL of a 0.125 g / mL SiO2 aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 1.0 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0090] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn up by self-absorption for 2 seconds. A 3000V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 10 seconds. Parallel experiments were conducted using this method on five groups of serum samples at concentrations of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0091] Example 7

[0092] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0093] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the front outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.0 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 9 mm and a tip angle of 35°, and 3 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a polymer bead 4 with a diameter of 1.0 mm is loaded into the front end of the polymer pipette 1. Then, 15 μL of an Al₂O₃ aqueous solution with a concentration of 0.15 g / mL is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 0.8 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 2 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0094] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn up by self-absorption for 5 seconds. A 3500V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 20 seconds. Parallel experiments were conducted using this method on five groups of serum samples at concentrations of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0095] Example 8

[0096] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0097] First, a polymer pipette 1 is prepared, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 10 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the outer surface of the polymer pipette, with an inner diameter of 1.0 mm and an outer diameter of 1.2 mm at the front end, an inner diameter of 7 mm and an outer diameter of 7.2 mm at the rear end, and a length of 1.2 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 9 mm and a tip angle of 35°, and 3 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, a polymer bead 4 with a diameter of 1.0 mm is loaded into the front end of the polymer pipette 1. Then, 10 μL of a 0.45 g / mL ZnO aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 1.2 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 5 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0098] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn up by self-absorption for 2 seconds. A 3500V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 60 seconds. Parallel experiments were conducted using this method on five groups of serum samples at concentrations of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0099] Example 9

[0100] The solid-phase microextraction-electrospray ionization integrated method for mass spectrometry analysis of serum test samples using the above-mentioned integrated solid-phase microextraction-electrospray ionization pipette includes the following steps:

[0101] First, a polymer pipette 1 is prepared, with an inner diameter of 1.5 mm and an outer diameter of 1.8 mm at the front end, and an inner diameter of 6 mm and an outer diameter of 8 mm at the rear end. Next, a conductive metal sleeve 2 is embedded in the front outer surface of the polymer pipette, with an inner diameter of 1.8 mm and an outer diameter of 2.0 mm at the front end, and an inner diameter of 8 mm and an outer diameter of 8.2 mm at the rear end, and a length of 1.5 mm. Then, a triangular porous matrix 3 is loaded into the polymer pipette 1, with a length of 8 mm and a tip angle of 30°, and 2 mm of the triangular porous matrix 3 protruding from the front end of the polymer pipette 1. Next, polymer beads 4 with a diameter of 1.2 mm are loaded into the front end of the polymer pipette 1. Then, 35 μL of a 0.15 g / mL ZnO aqueous solution is loaded into the middle and rear ends of the triangular porous matrix 3 in the polymer pipette 1, and allowed to air dry at room temperature. Finally, a circular porous matrix with a diameter of 1.2 mm is vertically loaded into the rear end of the triangular porous matrix 3 in the polymer pipette 1. To test the performance of the solid-phase microextraction-electrospray ionization integrated pipette prepared in this invention, 5 μL of a diagnostic drug compound with a concentration of 1 μg / mL was loaded into the polymer pipette prepared above. After air drying at room temperature for 2 hours, the pipette was tested using an automated electrospray ionization platform-mass spectrometry technique.

[0102] The polymer pipette 1 prepared above was then loaded onto an automated electrospray ionization source platform. The spray solvent was then drawn up by self-absorption for 2 seconds. A 3500V electrospray voltage was then applied to the conductive metal sleeve 2 on the outer surface of the polymer pipette for 60 seconds. Parallel experiments were conducted using this method on five groups of serum samples at concentrations of 1 μg / mL for amitriptyline, clozapine, amisulpride, quetiapine, risperidone, and aripiprazole, yielding very stable experimental results.

[0103] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0104] The advantage of this invention is that the technology can complete the electrospray ionization of the analyte compound while pretreating complex samples (especially micro-volume samples), thereby significantly simplifying the analysis process.

[0105] This integrated design takes into account the advantages of adsorbents in pretreatment of complex samples, and uses micro-spray solvent to rapidly elute target compounds from the adsorbent surface in situ, directly performing electrospray ionization, effectively avoiding dilution and loss of target substances, and improving the sensitivity of mass spectrometry detection.

[0106] In addition, the present invention has a metal sleeve at the front end of the pipette. By directly applying voltage to the sleeve, the sprayed solvent inside the pipette tip is induced to generate an electrospray, which avoids direct contact between the metal electrode and the sample. This not only reduces the risk of sample contamination, but also provides a feasible path for high-throughput analysis of complex samples.

[0107] The establishment of a solid-phase microextraction-electrospray ionization integrated pipette preparation method not only provides an experimental basis for high-throughput analysis of target compounds in complex samples, but also provides an effective strategy for the analysis of target compounds in complex samples such as biological, food, and environmental samples, thereby promoting the establishment and development of high-throughput mass spectrometry detection methods.

[0108] The present invention provides an integrated pipette for solid-phase microextraction-electrospray ionization, using a conical polymer pipette as a carrier for loading triangular porous matrices, synthetic beads, adsorbents, and circular porous matrices. Using a polymer pipette as a carrier is cost-effective and provides good insulation. Its conical structure not only facilitates the self-absorption of the subsequent sprayed solvent, eliminating the need for any flow pump to load the sprayed solvent and significantly reducing costs, but also, during the electrospray process, the conical polymer facilitates electric field focusing, making it easier to generate a stronger electric field at its tip, which helps to produce a stable and continuous electrospray flow at the tip of the triangular porous matrix.

[0109] Synthetic beads are not only inexpensive but also possess perfect symmetry, allowing for closer contact with the surrounding porous matrix and preventing the adsorbent from detaching from the front end of the conical polymer pipette. Thirdly, because the synthetic beads have a single-point contact with the porous matrix, while preventing the adsorbent solid from detaching from the inside of the pipette, it ensures that the sprayed solvent quickly penetrates through the pores of the porous matrix into the rear end of the pipette during the self-absorption process, dissolving and eluting the target compounds in the sample on the adsorbent surface. Fourthly, compared to the polymer sieve plates used in traditional solid-phase microextraction, synthetic beads have excellent rolling properties, making them easier to load onto the inner front end of the porous matrix of the conical polymer pipette. The adsorbent, used to carry the sample, can more strongly adsorb the matrix in complex samples onto its surface, while having a weaker adsorption performance for target compounds. This ensures that when the sprayed solvent comes into contact with the adsorbent, the target compounds can be desorbed from the adsorbent surface, thus enabling rapid analysis of complex samples via electrospray filtration.

[0110] The circular porous matrix is ​​used to prevent the adsorbent from falling off the polymer pipette after loading. At the same time, it has better flexibility and porosity than traditional solid-phase microextraction sieves. It can overcome the clogging of the rear end of the triangular porous matrix in the pipette by the solid-phase microextraction sieve with small pore size, and promote the rapid penetration of the spray solution and sample solution into the adsorbent surface from the front and rear ends of the triangular paper matrix in the pipette, thereby achieving solid-phase microextraction.

[0111] This invention enables the fabrication of an integrated solid-phase microextraction-electrospray ionization pipette, from the cutting of the polymer pipette to the loading of triangular porous matrix, synthetic beads, adsorbent, and circular porous matrix, and the embedding of conductive metal sleeves. This pipette simultaneously performs the functions of sample pretreatment and electrospray ionization, and features readily available raw materials, simple fabrication, and the ability to be mass-produced.

[0112] This pipette not only takes into account the characteristics of adsorbent pretreatment for complex samples, effectively removing interference from the sample matrix and improving the purification efficiency of target compounds, but can also be directly used for electrospray ionization-mass spectrometry analysis, avoiding losses during sample transfer and improving the sensitivity of mass spectrometry analysis.

[0113] This functionalized pipette not only lays a solid foundation for high-throughput automated mass spectrometry analysis of target compounds in complex samples, but also provides a good foundation for the rapid analysis of biological, food, and environmental samples.

[0114] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0115] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A pipette for solid-phase microextraction-electrospray ionization integration, characterized in that: Includes a polymer tube and a conductive metal sleeve nested at the front end of the polymer tube; The conductive metal sleeve is further fitted with a triangular porous matrix at its front end. Artificial beads are provided inside the triangular porous matrix near its front end. The inner surface of the triangular porous matrix is ​​also loaded with an adsorbent. The adsorbent also has a circular porous matrix located inside the polymer tube at its rear end.

2. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 1, characterized in that: The polymer tube is a tapered sample tube open at both ends: The outer diameter of the front end is 0.8mm to 2.0mm, and the inner diameter is 0.5mm to 1.5mm; The outer diameter of the rear end is 7mm to 10mm, and the inner diameter is 5mm to 8mm; The length is 40mm to 50mm.

3. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 1, characterized in that: The triangular porous matrix is ​​an isosceles triangle with a apex angle of 30° to 35° and a length of 6 mm to 15 mm. The material is selected from any one of paper, porous polymer or fiber.

4. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 1, characterized in that: The synthetic beads have a diameter of 0.5mm to 1.2mm and are made of glass, metal, or polymer.

5. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 1, characterized in that: The adsorbent is a porous inorganic material, organic material, or a composite material thereof, and is loaded in the form of a solution with a concentration of 0.15 g / mL to 0.45 g / mL, with a loading volume of 10 μL to 35 μL.

6. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 3, characterized in that: The circular porous matrix is ​​in the shape of a disc, with a diameter of 0.8 mm to 1.4 mm, and is made of the same material as the triangular porous matrix.

7. The integrated pipette for solid-phase microextraction-electrospray ionization according to claim 1, characterized in that: The conductive metal sleeve has a conical structure that matches the shape of the front end of the polymer tube, and its material is any one of stainless steel, aluminum or copper. The conductive metal ferrule has an outer diameter of 0.7mm to 2.2mm and an inner diameter of 0.5mm to 2.0mm at the front end. The outer diameter of the rear end is 5.4mm to 8.4mm, and the inner diameter is 5.2mm to 8.2mm; The length ranges from 0.4mm to 2.0mm.

8. A method for preparing an integrated pipette for solid-phase microextraction-electrospray ionization as described in any one of claims 1-7, characterized in that: Includes the following steps: S1: Cut the tip of a commercial pipette into a tapered polymer tube with openings at both ends; S2: Insert the conductive metal ferrule into the outer surface of the front end of the polymer tube; S3: Insert the triangular porous matrix from the rear end of the polymer tube, so that its tip extends 1mm to 5mm beyond the front end of the polymer tube; S4: Place the synthetic beads into the front end of the polymer tube, so that they are located inside the triangular porous matrix and the front end is flush with the end face of the polymer tube. S5: Add the adsorbent solution from the rear end of the polymer tube to the inner surface of the triangular porous matrix, and let it dry to form an adsorbent layer; S6: Insert the circular porous matrix from the rear end of the polymer tube, so that it is perpendicular to the rear side of the adsorbent.

9. A method of using a pipette for solid-phase microextraction-electrospray ionization as described in any one of claims 1-7, characterized in that: Includes the following steps: S1: Add 1 μL to 5 μL of the sample solution to be tested to the rear end of the polymer tube and dry it in the air for more than 2 hours; S2: Immerse the front end of the polymer tube in the spray solvent and draw in 15μL to 30μL of spray solvent through self-absorption. S3: Apply a DC voltage of 3000V to 5000V to the conductive metal sleeve and perform electrospray ionization for 10s to 420s; S4: The generated electrospray stream is directly introduced into the mass spectrometer for detection.

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