A liquid chromatography-mass spectrometer with an electrospray ion source device

Abstract

The invention provides a liquid chromatography-mass spectrometer containing an electrospray ion source device, wherein the electrospray ion source device comprises a disk atomizer, a ring electrode group and a repeller electrode, wherein the disk atomizer can generate a plurality of Taylor cone structures by controlling the voltage intensity, thereby greatly improving the atomization charging effect; at the same time, it can be compatible with a larger flow rate, and can improve the atomization charging effect of the compound under a high flow rate when the voltage is increased; an annular electrode is applied under the spray needle to converge and focus the charged particles diffused by the spray; finally, an electrode is arranged on the opposite side of the ion inlet to push the charged compound from the ion inlet into the vacuum chamber, thereby increasing the number of ions that can be analyzed and significantly improving the mass spectrometry sensitivity, and also providing technical support and new ideas for improving the sensitivity of the mass spectrometer, and having good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of liquid chromatography-mass spectrometry, and in particular relates to a liquid chromatography-mass spectrometry instrument containing an electrospray ion source device. Background Art

[0002] At present, most of the electrospray ion sources of liquid chromatography-tandem mass spectrometry adopt a single nozzle design. The mobile phase carries the target compound into the ion source through the liquid phase pipeline. The tip of the ion source probe is conical, and the tip of the spray needle extends from the middle of the probe. It carries high voltage. The droplets flowing out of the spray needle are dispersed into conical mist droplets under the action of the electric field, which is called the Taylor cone effect. The solvent of the droplets gradually evaporates, and its surface tension is lower than the gradually increasing charge repulsion inside. Coulomb explosion occurs, and charged particles are generated. They collide with the target compound to make the target compound charged. The target compound is pushed by the potential difference inside the mass spectrometer and enters the vacuum area where the mass analyzer is located. Neutral particles and some impurities that cannot be ionized will enter the exhaust pipe and be discharged along the spray direction.

[0003] The solvent atomization effect of a single nozzle is related to the liquid flow rate in the needle. A flow rate of nanoliters can often achieve a better atomization effect. As the flow rate increases, the spray effect will weaken, and the droplets produced will be larger. During the movement, the solvent cannot evaporate in time and fails to cause a Coulomb explosion, but directly enters the exhaust pipe. On the other hand, the current design of the ion inlet is not suitable for multi-nozzle probes. The excessive spray range of the multi-nozzles makes it impossible for some charged particles to be effectively attracted by the electric field into the ion source.

[0004] The Chinese invention patent with the authorization announcement number CN 110681505 B discloses an electrospray device, which relates to the field of electrospray technology and can generate multiple electrospray sources without increasing the number of electrospray nozzles, thereby increasing the output of electrospray. However, in the application of liquid-mass spectrometer, the excessive spray range of multiple electrospray sources makes it impossible for some charged particles to be effectively attracted by the electric field into the ion source, thereby reducing the number of ions that can be analyzed.

[0005] The Chinese invention patent with the authorization announcement number CN 114475015 B discloses a focusing electric field structure electrostatic jet direct writing system and direct writing method, which relates to the field of electrostatic jet technology. A focusing electrode structure is added between the nozzle and the jet substrate, wherein the control electrode group includes a plurality of mutually parallel and coaxial annular electrodes, and the potential connected thereto gradually decreases in the direction away from the nozzle to form a pull-down electric field that gradually focuses toward the central axis, thereby controlling the direction of the liquid jet to be only along the central axis, and any deflection will be constrained by the focusing electric field and suppressed, thereby strictly controlling the landing point of the electrostatic jet or droplet. However, in the application of liquid-mass spectrometer, this single structure limits the direction of the liquid flow, making it impossible for the droplet to deflect and only fall vertically at the landing point, so that the liquid flow cannot effectively enter the ion inlet, significantly reducing the target compounds entering the vacuum chamber.

[0006] Therefore, there is an urgent need to find a liquid chromatography-mass spectrometer containing an electrospray ion source device, which can significantly improve the atomization effect while being compatible with a larger flow rate, while also improving the efficiency of charged particles entering the vacuum chamber and increasing the number of ions that can be analyzed. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a liquid chromatography-mass spectrometer containing an electrospray ion source device, wherein the liquid chromatography-mass spectrometer adopts an electrospray ion source device, wherein the electrospray ion source device includes a disk atomization device, a ring electrode group and a repelling electrode. Among them, the disk atomization device can generate multiple Taylor cone structures by controlling the voltage intensity, greatly improving the atomization charging effect; at the same time, it can be compatible with a larger flow rate, and can improve the atomization charging effect of the compound at a high flow rate when the voltage is increased; an annular electrode is applied under the spray needle to converge and focus the charged particles diffused by the spray; finally, an electrode is set on the opposite side of the ion inlet to push the charged compound from the ion inlet into the vacuum chamber, thereby increasing the number of ions that can be analyzed.

[0008] In order to achieve the above object, the present invention adopts the following scheme:

[0009] On the one hand, the present invention provides a liquid chromatography-mass spectrometer containing an electrospray ion source device, wherein the liquid chromatography-mass spectrometer adopts an electrospray ion source device, and the electrospray ion source device includes a disk atomization device, a ring electrode group and a repeller electrode; wherein the ring electrode group is arranged below the disk atomization device, and the repeller electrode is arranged at the lower left of the ring electrode group.

[0010] Furthermore, the disc atomization device includes an atomization electrode, a spray disc and an injection tube; the spray disc has a middle opening connected to the injection tube, and liquid can be connected to the injection tube from the outside, flow out from the middle opening, and cover the entire surface of the spray disc.

[0011] Furthermore, an atomizing electrode is arranged outside the spray disc, the distance between the edge of the spray disc and the atomizing electrode is 10 mm, and the inner diameter of the atomizing electrode is 6-10 mm; a voltage is applied between the atomizing electrode and the injection tube, and the voltage is 4-11 kV.

[0012] In some embodiments, the distance between the edge of the disk and the electrode in the disk atomization device is screened, and the results show that the distance between the edge of the disk and the electrode ranges from 6 to 14 mm. Within this range, the optimal distance between the edge of the disk and the electrode is finally determined to be 10 mm based on experimental data. At this time, the peak area of ​​the compound reaches the highest correspondingly.

[0013] In some embodiments, the voltage between the electrode and the metal tube in the disc atomization device is screened, and the results show that for the mobile phase used in the present invention, the voltage between the electrode and the injection tube in the disc atomization device ranges from 4 to 11 kV, and the number of Taylor cones is positively correlated with the square of the voltage. As the voltage increases, the ion atomization efficiency continues to increase; at the same time, the preferred voltage is 8 kV. When the voltage is 8 kV, the response of the compound in the instrument reaches the highest, which significantly improves the sensitivity and accuracy of the detection.

[0014] Furthermore, the spray disc is made of stainless steel, with a diameter of 5 mm and a thickness of about 50 μm; the inner diameter of the injection tube is 0.1-2 mm.

[0015] In some embodiments, the disc spray device is compatible with a larger flow rate. Even at a larger flow rate, within the voltage range of 4 to 11 kV, more Taylor cones are generated as the voltage increases, thereby significantly improving the atomization charging effect of the compound and improving the ion atomization efficiency. At the same time, it is compatible with a wider range of flow rates, which facilitates the establishment of analytical methods and the selection of chromatographic columns.

[0016] Furthermore, the annular electrode group includes 3 to 5 annular electrodes, the vertical distance between the first annular electrode below the spray disk and the disk is 10 mm, and the diameter of the annular electrode gradually decreases from the position close to the disk atomization device to a diameter of 10 to 50 mm.

[0017] In some embodiments, the number of ring electrodes in the ring electrode group is screened, and the results show that when the number of ring electrodes is preferably 3 to 5, within this range, as the number increases, the response of the compound in the instrument continues to increase, further improving the sensitivity of detection, and when the number of ring electrodes is 5, the compound response reaches the highest.

[0018] Furthermore, direct current with the same direction is applied to each annular electrode, and different voltages are applied between different annular electrodes to generate a potential difference, so that the charged particles gather and accelerate toward the cone plate, which is arranged at the lower right of the annular electrode group.

[0019] Furthermore, the repeller electrode is arranged at the lower left side of the annular electrode group, and the distance between the two is: 10 mm from the bottom coil of the annular electrode in the vertical direction, and 30 mm from the center of the annular electrode in the horizontal direction; an ion inlet is arranged on the opposite side of the repeller electrode, and charged particles enter the vacuum chamber by the combined action of the electromagnetic field of the repeller electrode and the potential difference in the ion inlet, and neutral particles and other excess gases enter the exhaust pipe along the direction of movement; the exhaust pipe is arranged at the lower left side of the ion inlet and directly below the annular electrode group.

[0020] On the other hand, the present invention provides a use of an electrospray ion source device for improving compound detection limit and analysis efficiency in liquid chromatography-mass spectrometry detection.

[0021] In some embodiments, the use of the electrospray ion source provided by the present invention improves the response of the compound in the instrument, significantly increases the number of compounds that can be analyzed in a single injection, and does not require the establishment of different detection methods due to different compound responses, nor does it require the use of a complex pretreatment process. The compound can achieve the response value after being processed by a single method, which significantly saves the consumables cost and experimental time of the pretreatment process and improves detection efficiency.

[0022] In another aspect, the present invention provides an electrospray ion source device for improving the detection sensitivity of a liquid chromatography-mass spectrometer and increasing the types of detected compounds.

[0023] In some embodiments, the present invention provides an electrospray ion source, comprising a disk atomizer, a ring electrode group and a repeller electrode, wherein the disk atomizer generates more Taylor cones within a certain voltage range to improve ionization efficiency. The ring electrode group focuses the charged particles dispersed by the spray, and works together with the repeller electrode to push more ions into the vacuum chamber, increase the number of ions that can be analyzed, and improve the response of the compound in the instrument, thereby improving the sensitivity and accuracy of the detection.

[0024] In another aspect, the present invention provides a use of an electrospray ion source device in liquid chromatography-mass spectrometry detection to reduce the pre-treatment process and reduce the sample volume required for analysis.

[0025] On the other hand, the present invention provides an electrospray ion source device, and the electrospray ion source device is as described in any one of the above technical solutions.

[0026] The beneficial effects of the present invention are:

[0027] 1. The present invention provides a liquid chromatography-mass spectrometer containing an electrospray ion source device, wherein the electrospray ion source device includes a disk atomization device, a ring electrode group and a repeller electrode; wherein the disk atomization device increases the number of Taylor cones generated by several times or even dozens of times compared with the traditional electrospray ion source, and the increase in Taylor cones greatly increases the number of atomized droplets, and the number of compounds ionized with the assistance of desolvation gases on both sides will also be effectively increased, which also brings better ionization effect to high-flow liquids and significantly improves ionization efficiency; at the same time, the ring electrode group focuses the charged particles dispersed by the spray, and works together with the repeller electrode to push more ions into the vacuum chamber, increasing the number of ions that can be analyzed. These ions are screened by the mass analyzer and enter the detector to be converted into electrical signals, which are finally reflected in a significant improvement in the sensitivity of the mass spectrometer. This will bring the following advantages:

[0028] 1) Improvement of compound detection limits: compounds that could not be accurately quantified due to low concentrations can now be accurately quantified;

[0029] 2) The number of detectable compounds has increased, and compounds that could not be determined by this method due to low response can be detected;

[0030] 3) Improved analysis efficiency. The number of compounds that can be analyzed in a single injection is increased. There is no need to establish different detection methods due to different compound responses, or even to go through different pre-treatment processes to complete sample preparation;

[0031] 4) Simplification of pretreatment methods. No complicated pretreatment process is required to concentrate and enrich the compounds. The samples can be injected after extraction or even simple dilution.

[0032] 5) Reduce the sample volume required for analysis. Some samples are expensive to prepare or difficult to obtain. Increased sensitivity reduces the amount of sample injection required to complete the analysis, which can reduce the cost of obtaining samples;

[0033] 6) The compatibility of the analytical method with high flow rates is improved. With the increase of flow rate, the atomization efficiency of the traditional ion source will gradually decrease. In this scheme, the number of Taylor cones generated can increase with the increase of voltage, and the atomization efficiency can be maintained at a high level. It is compatible with a wider range of flow rates, which facilitates the establishment of analytical methods and the selection of chromatographic columns.

[0034] 2. The present invention provides an electrospray ion source device, including a disk atomization device, a ring electrode group and a repeller electrode, which provides technical support and new ideas for improving the sensitivity of a mass spectrometer and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1It is a schematic diagram of the structure of a liquid chromatography-mass spectrometer containing the electrospray ion source device of the present invention.

[0036] Figure 2 It is a front view of the ionization component structure of the present invention.

[0037] Figure 3 It is a side view of the ionization component structure of the present invention.

[0038] Figure 4 This is an exploded view of the electrospray ion source assembly of the present invention.

[0039] Figure 5 Exploded views of the electrospray ion source assembly of the present invention at different angles (the atomization assembly hanging ring is hidden).

[0040] Figure 6 This is an exploded view of the ion source atomization and focusing assembly of the present invention.

[0041] Figure 7 It is an overall exploded view of the ion source assembly of the present invention.

[0042] Figure 8 Schematic diagram of the disc atomization device structure.

[0043] Fig. 9 Different numbers of Taylor cones are generated for different voltages (V=7.6 kV).

[0044] Fig.10 Different numbers of Taylor cones were generated for different voltages (V=8.0 kV).

[0045] Fig.11 This is a diagram of a common ion source structure.

[0046] Fig.12 This is a front view of a common ion source.

[0047] Fig.13 This is a side view of a common ion source.

[0048] Fig.14 This is the principle of electrospray ionization (taking positive ion mode as an example).

[0049] Fig.15 are the peak areas of each compound detected by the traditional electrospray ion source and the present invention, respectively.

[0050] Fig.16 This is the injection result of a sample with a concentration of about 3.5 ng / mL (near the detection limit) after being treated with protein precipitation method.

[0051] Fig.17 These are the chromatograms of four samples after 1000 injections of the same sample.

[0052] Fig.18 The chromatograms of tylosin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the electrospray ion source combined method of the present invention respectively.

[0053] Fig.19 The chromatograms are of 20 μg / L roxithromycin after using the traditional ion source single method, the traditional ion source combined method and the electrospray ion source combined method of the present invention respectively.

[0054] Fig. 20 The chromatograms of kitasamycin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the electrospray ion source combined method of the present invention respectively.

[0055] Fig.21 The chromatograms of Tilmicosin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the electrospray ion source combined method of the present invention respectively.

[0056] Fig. 22 The chromatograms are of lincomycin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the electrospray ion source combined method of the present invention respectively. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with the examples. It should be pointed out that the examples described below and the materials used therein are intended to facilitate the understanding of the present invention and do not have any limiting effect thereon.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0060] Example 1 A liquid chromatography-mass spectrometer containing an electrospray ion source device provided by the present invention

[0061] This embodiment provides a liquid chromatography-mass spectrometer containing an electrospray ion source device, specifically as follows Figure 1 As shown, Figure 1 Schematic diagram of the structure of a liquid chromatography-mass spectrometer containing an electrospray ion source device of the present invention. Figure 1It can be seen that in a liquid chromatography-mass spectrometer provided by the present invention, a solvent tray 28, a binary high-pressure pump 29, an automatic sampler 30, a column oven 31, and a triple quadrupole mass spectrometer 32 are included. In the triple quadrupole mass spectrometer 32, an electrospray ion source device 33 provided by the present invention is installed, and the specific structure is as described in Example 2. The advantages of installing an electrospray ion source in the liquid chromatography-mass spectrometer are: the disc atomization device in the electrospray ion source can produce multiple Taylor cone structures by controlling the voltage intensity, greatly improving the atomization charging effect and ionization efficiency; at the same time, it can be compatible with a larger flow rate, and when the voltage is increased, the atomization charging effect of the compound at a high flow rate can be improved; a group of annular electrodes are applied under the spray needle to converge and focus the charged particles diffused by the spray, so that more target compounds can enter the mass analyzer in the vacuum chamber; finally, a repeller electrode is set on the opposite side of the ion inlet to push more ions into the vacuum chamber and increase the number of ions that can be analyzed.

[0062] Example 2 An electrospray ion source device provided by the present invention

[0063] This embodiment provides an electrospray ion source device, specifically as follows Figures 2 to 22 As shown, Figure 2 It is a front view of the ionization component structure of the present invention; Figure 3 A side view of the ionization component structure of the present invention; Figure 4 An exploded view of an electrospray ion source assembly of the present invention; Figure 5 Exploded views of the electrospray ion source assembly of the present invention at different angles (with the atomization assembly hanging ring hidden); Figure 6 An exploded view of the ion source atomization and focusing assembly of the present invention; Figure 7 It is an overall exploded view of the ion source assembly of the present invention; Figure 8 It is a schematic diagram of the structure of the disc atomization device; Fig. 9 Different numbers of Taylor cones are generated for different voltages (V = 7.6 kV); Fig.10 Different numbers of Taylor cones were generated for different voltages (V = 8.0 kV); Fig.11 This is a diagram of a common ion source structure; Fig.12 This is a front view of a common ion source; Fig.13 This is a side view of a common ion source; Fig.14 It is the principle of electrospray ionization (taking positive ion mode as an example); Fig.15 are the peak areas of each compound detected by the conventional electrospray ion source and the present invention, respectively; Fig.16 This is the injection result of a sample with a concentration of about 3.5 ng / mL (near the detection limit) after being treated with protein precipitation method; Fig.17 These are the results of four chromatograms after 1000 consecutive injections of the same sample; Fig.18The chromatograms of tylosin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the ion source combined method of the present invention respectively; Fig.19 The chromatograms of roxithromycin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the ion source combined method of the present invention respectively; Fig. 20 The chromatograms of kitasamycin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the ion source combined method of the present invention respectively; Fig.21 The chromatograms of Tilmicosin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the ion source combined method of the present invention respectively; Fig. 22 The chromatograms are of lincomycin 20 μg / L after using the traditional ion source single method, the traditional ion source combined method and the ion source combined method of the present invention respectively.

[0064] from Figures 1 to 10 It can be seen that an electrospray ion source device of the present invention includes a disc atomization device 1, a ring electrode group 2 and a repelling electrode 3, and the atomization focusing assembly 25 includes a disc atomization device 1 and a ring electrode group 2, two heating rods 10 are arranged on both sides, and the sampling tube 6 is a metal tube, the upper end of which is a liquid inlet, and one side is provided with a vertical adjustment micrometer 11 (unit mm) and a horizontal adjustment micrometer 12 (unit mm). In the disc atomization device 1, the spray disc 5 is cut from stainless steel, with a diameter of 5mm and a thickness of about 50μm, a hole in the middle, and a sampling tube 6 with an inner diameter of about 0.1 to 2mm is connected, and the sampling tube 6 is perpendicular to the spray disc 5. The liquid can be connected to the sampling tube 6 from the outside, and the pump outside the ion source provides power to the liquid, and at a controllable pump flow rate (0.1 to 1.0mL / min), the liquid flows out from the middle opening and covers the entire surface of the spray disc 5. The outside of the spray disk 5 is the atomizing electrode 4, and the inner diameter of the atomizing electrode 4 is about 6 to 10 mm. The edge of the spray disk 5 and the atomizing electrode 4 maintain a fixed distance of 10 mm. This distance affects the voltage value required to generate a Taylor cone. The closer the distance, the smaller the voltage required. However, if the distance is too close, the droplets generated by the spray will directly touch the atomizing electrode, causing losses. Therefore, at this distance, the best atomization effect is achieved. A voltage of 4 to 11 kV is applied between the atomizing electrode 4 and the sample injection tube 6. Under the action of the electric field, the liquid is radially sprayed at the edge of the spray disk 5 to form a Taylor cone structure. The number of Taylor cones increases with the increase of voltage. According to relevant experimental data, the number of Taylor cones generated is positively correlated with the square of the voltage. Figures 8 to 10 In, V d represents the voltage applied between the atomizing electrode 4 and the sample injection tube 6, and N represents the number of Taylor cones. When the square of the voltage increases, the number of Taylor cones increases obviously.

[0065] 3 to 5 ring-shaped electrode groups 2 with gradually decreasing diameters are arranged below the disk atomization device 1. When the number of ring-shaped electrodes is 5, the effect is best. The diameter of the first ring-shaped electrode below the spray disk 5 is about 50 mm, and the vertical distance between the electrode and the spray disk 5 is about 10 mm; the diameter of the second layer of ring-shaped electrodes is about 40 mm, and the vertical distance between the second layer of electrodes and the first layer of electrodes is about 2.5 mm. The diameter of each electrode decreases by 10 mm downwards, and the vertical distance between each layer of electrodes is 2.5 mm. The diameter of the bottom layer of electrodes is 10 mm, and the radius of the bottom opening is 2.5 mm. A conductor is arranged on one side of each ring-shaped electrode for supplying power to the ring-shaped electrodes, and direct current with the same direction is applied thereto. Different voltages are applied between different ring-shaped electrodes to generate a potential difference. The electromagnetic field formed by the annular electrode group 2 causes the charged particles generated by the spray of the spray disk 5 to gradually converge and move toward the annular center, and a potential difference is formed between different annular electrodes, so that the charged particles can be accelerated to move toward the cone plate 13 (the cone hole of the cone plate 13 is the ion inlet 7), so as to avoid staying in the ion source for a long time and colliding with other molecules and being broken down. A repelling electrode 3 is set on the opposite side of the cone plate 13. The horizontal distance between the repelling electrode and the sampling cone hole is about 50mm. The repelling electrode body is a metal body of about 20×20mm, which is set at the lower left of the annular electrode group 2. The distance between the two is 10mm from the bottom layer of the annular electrode coil in the vertical direction and 30mm from the center of the annular electrode in the horizontal direction. After the charged particles are gathered by the above structure, they enter the vacuum chamber 9 (the vacuum chamber 9 is on the right side of the ion inlet 7, Figure 1 The neutral particles and other excess gases follow the direction of movement and enter the exhaust pipe 8, which is arranged at the lower left of the cone plate 13 and directly below the annular electrode group 2, and the vertical distance from the lowest coil of the annular electrode 2 is 45 mm.

[0066] Electrospray ion source is the most common interface for liquid chromatography-mass spectrometry. Figures 11 to 13 As shown in the figure, its working principle is to dissolve the sample to be tested in a solvent, and under the action of a high-voltage electric field, the solution is formed into extremely fine droplets. When these droplets pass through the ion source, they will undergo a process of drying and charge transfer, and finally form charged gaseous ions, which enter the mass spectrometry analysis. This process includes three stages: the formation of charged droplets, desolvation, and the generation of gas-phase ions. Fig.14 shown.

[0067] However, the current electrospray ion source has the following disadvantages: 1) Most of them adopt a single nozzle design. The solvent atomization effect of a single nozzle is related to the liquid flow rate in the needle. A nanoliter flow rate can often achieve a good atomization effect. As the flow rate increases, the atomization efficiency will gradually decrease, so it is not compatible with high-flow analysis methods; 2) On the other hand, the current design of the ion inlet is not suitable for multi-nozzle probes. The excessive spray range of the multi-nozzle makes it impossible for some charged particles to be effectively attracted by the electric field to enter the ion source; 3) The detection index requires a low detection limit for the compound. If the concentration of the compound is too low, it cannot be accurately quantitatively determined; 4) The response of the mass spectrometer to different compounds varies greatly. Some compounds cannot be detected because the response is too low in a certain analysis method; 5) Because the response of the compound in different analysis methods is different, different detection methods need to be established, and even different pretreatment processes are required to complete the sample preparation. It is impossible to detect all the analytes in a single injection; 6) A complex pretreatment process is required to concentrate and enrich the compound to improve the response of the compound; 7) Some samples are expensive to prepare or difficult to obtain, and a large number of samples cannot be provided, but at the same time, the sensitivity of the instrument is not enough to detect a small number of samples.

[0068] The present embodiment provides an electrospray ion source, including a disk atomization device, a ring electrode group and a repeller electrode. The disk atomization device increases the number of Taylor cones generated by several times or even dozens of times compared with the traditional electrospray ion source. The increase in Taylor cones greatly increases the number of atomized droplets, and the number of ionized compounds assisted by the desolvation gas on both sides will also be effectively increased, significantly improving the ionization efficiency. At the same time, the present invention provides a group of ring electrodes and repeller electrodes. The ring electrodes focus the charged particles dispersed by the spray, and work together with the repeller electrodes to push more ions into the vacuum chamber, increasing the number of ions that can be analyzed. These ions are screened by the mass analyzer and enter the detector to be converted into electrical signals, which significantly improves the sensitivity of the mass spectrometer.

[0069] Moreover, after many tests, it was found that the accuracy of the electrospray ion source provided in this embodiment in the peak area detection of different compounds can reach 99.8%, while the accuracy of the traditional ion source is only 72.6%, which is significantly lower than the accuracy of the electrospray ion source provided by the present invention. In addition, the detection results of the traditional ion source are accidental, and there is a significant difference between the two.

[0070] Example 3 Test of the sensitivity of the electrospray ion source of the present invention

[0071] In order to test the detection sensitivity of the electrospray ion source in Example 2, the same sample was used in this example to perform comparative tests using a conventional electrospray ion source and the device of the present invention. By comparing the peak areas of each compound, the instrument response signal was evaluated, and the sensitivity was further evaluated. Among them, the standard products of 24 compounds were purchased from Dr. Ehrenstorfer GmbH, and the standard products were dissolved in solvents such as methanol and acetonitrile and diluted with 4% BSA (bovine serum albumin) solution to form a mixed solution of appropriate concentration.

[0072] The specific processing methods are:

[0073] 1) Take 450 μL of the above solution, add 100 μL of precipitant (sulfate solution), and vortex for 2 minutes;

[0074] 2) Pipette 450 μL of the mixed sample into a SLE (solid-liquid extraction) plate and let it stand for 15 minutes under gravity;

[0075] 3) Replace the receiving plate, add 1 mL of ethyl acetate: n-hexane (3:2, v / v) to the SLE plate, and after the flow is complete, add 0.8 mL of ethyl acetate: n-hexane (3:2, v / v) for the second elution. Collect the eluate until no liquid flows out under gravity;

[0076] 4) Take the receiving plate containing the eluent and place it in a nitrogen blower to dry; add 100 μL of the reconstitution solution to re-dissolve, mix well and then test on the machine.

[0077] Analysis method-mass spectrometry conditions are:

[0078] Curtain gas: 25psi; collision gas: 6; spray voltage: 8000V (compared with 5500V for conventional ion source); ion source temperature: 500°C; atomizing gas pressure: 55psi; auxiliary heating gas: 50psi.

[0079] Analytical method-liquid phase conditions are:

[0080] Liquid chromatography system high-pressure pump A mobile phase: aqueous solution containing additives; Liquid chromatography system high-pressure pump B mobile phase: methanol containing additives; Flow rate: 0.5 mL / min; Chromatographic column: Chromatographic column 006 (Phenomenex, Kinetex C18, 2.1 mm*100 mm*2.6 μm); Column temperature: 40 °C

[0081] Gradient conditions:

[0082]

[0083]

[0084] Specific results such as Fig.15 As shown, the final statistics are the data in Table 1 (in the comparative test, the detection process, preparation conditions and analysis methods are the same; the remaining device structures, conditions and parameters are the optimal conditions in Example 1):

[0085] Table 1 Comparative test of compound detection sensitivity between conventional electrospray ion source and electrospray ion source of the present invention

[0086] Compound Name Conventional electrospray ionization source Electrospray ion source of the present invention Increase multiple Progesterone (P) 597077 2108477 3.53 17α-Hydroxyprogesterone (17-OH P) 981506 4891491 4.98 Testosterone (T) 3365380 16046015 4.77 Androstenedione (AD) 2467285 13277158 5.38 Dehydroepiandrosterone (DHEA) 900816 3502102 3.89 Dihydrotestosterone (DHT) 639587 2781217 4.35 Dehydroepiandrosterone Sulfate (DHEAS) 103720 1982429 19.11 Dexamethasone (DXSM) 8922791 19138605 2.14 18-hydroxycortisol (18-OHF) 29648 225713 7.61 18-OXOF 6441 76001 11.80 Prednisolone (PNSL) 160783610 312349389 1.94 Methylprednisolone (MPNSL) 183119302 352187661 1.92 Pregnenolone (P5) 206615 666914 3.23 Corticosterone (Cort) 2697095 8127124 3.01 Deoxycorticosterone (DOC) 2778941 15482501 5.57 Cortisone 21210817 97488198 4.60 Cortisol 3465551 7939816 2.29 11-Deoxycortisol (11DOC) 977147 4162448 4.26 21-Deoxycortisol (21-DOC) 589888 3013258 5.11 Aldosterone (ALD) 609544 331375 0.54 Estrone (E1) 34213 152557 4.46 Estradiol (E2) 22717 98539 4.34 Estriol (E3) 19387 88045 4.54 17-Hydroxypregnenolone (17-OHP5) 298753 772220 2.58

[0087] As can be seen from Table 1, after using the electrospray ion source of the present invention, most compounds have a 2-5 times response improvement, and some compounds have an improvement of more than 10 times. Although there is also one compound whose response is lower than before, overall, the response of most compounds is significantly improved, especially for compounds DHEAS-1 and 18-OXOF-1, the response is increased by about 20 times and 12 times respectively. Although the process of atomization and charging of compounds in the ion source is relatively complicated, there are many influencing factors and it is impossible to accurately quantify and measure, the above test results show that this scheme can bring significant improvement in sensitivity compared with the traditional electrospray ion source, especially for compounds DHEAS-1 and 18-OXOF-1 in the sample, the detection sensitivity is significantly improved.

[0088] It can be seen that, on the one hand, the response of most compounds has been greatly improved, and compounds that could not be measured by this method because of their low response can be detected, thereby significantly increasing the types of detectable compounds; on the other hand, some samples have high preparation costs or are difficult to obtain, and the increase in compound response increases sensitivity, thereby reducing the amount of sample injection required to complete the analysis, reducing the sample volume required during the analysis, and thus reducing the cost of sample acquisition; on the other hand, the detection limit of compounds has been improved, and compounds that could not be accurately quantified due to too low concentrations can now be accurately measured.

[0089] Example 4 Comparative test of the analysis efficiency of the electrospray ion source of the present invention

[0090] In order to test the detection and analysis efficiency of the electrospray ion source in Example 2, LC-MS (liquid spectrometry) was used to detect vitamin D in this example, and the samples were treated by two different methods, respectively. The specific methods are as follows (in the comparative test, the detection process, analysis method or other conditions not mentioned were the same; the sample was a real human serum sample containing vitamin D):

[0091] 1. Conventional sample processing method: Take 100 μL of serum sample, add 10 μL of internal standard, add sodium hydroxide to adjust the pH, pass the sample through the SLE plate (solid-liquid extraction plate), elute with 300 μL of n-hexane when there is no liquid in the SLE plate wells, repeat the previous step four times, collect the eluate, blow dry with nitrogen, add 60 μL of acetonitrile-polyethylene terephthalate glycol mixture, derivatize at 50°C for 30 minutes, re-dissolve with 50 μL of ethanol, vortex mix and then inject.

[0092] 2. Protein precipitation method for measuring vitamin D: Just add an internal standard (the internal standard is the deuterated isotope of 25-hydroxyvitamin D3: 25-hydroxyvitamin D3-d6) and a precipitant (zinc sulfate) to the serum sample, vortex mix and centrifuge, take the supernatant and perform instrumental analysis.

[0093] Detection conditions - mass spectrometry conditions are:

[0094] Curtain gas: 25.0 psi; Collision gas: 8; Spray voltage: 7300 V (compared to 5500 V for conventional ion source); Ion source temperature: 450°C; Nebulizer gas pressure: 50.0 psi; Auxiliary gas pressure: 60.0 psi;

[0095] Detection conditions - liquid phase conditions are:

[0096] Liquid chromatography system high pressure pump A mobile phase: 0.1% formic acid water

[0097] Liquid chromatography system high pressure pump B mobile phase: 0.1% formic acid 80% methanol-20% isopropanol

[0098] Flow rate: 0.6mL / min

[0099] Chromatographic column: Chromatographic column 004 (Phenomenex, Kinetex C18, 2.1mm*50mm*2.6μm)

[0100] Column temperature: 40°C

[0101] Gradient conditions:

[0102]

[0103]

[0104] Of the two sample processing methods, although the protein precipitation method has a simple processing process, it is generally not effective in removing impurities. Matrix effects are easily generated during sample injection, resulting in a decrease in compound response, making it impossible to accurately quantify low-concentration samples. The sample solution carries a large amount of impurities into the instrument, which easily contaminates the instrument, causing the instrument response to decrease and failing to meet detection needs. However, by using the electrospray ion source designed in the present invention, the increase in compound response due to the improved ionization efficiency offsets the decrease in response caused by the above reasons, greatly improving the usability of the protein precipitation method for determining vitamin D pretreatment. The increase in compound response enables the relatively simple pretreatment process to increase the number of samples that can be processed per unit time, thereby improving detection efficiency. The specific results are as follows: Fig.16 , 17 As shown, Fig.16 This is the injection result of a sample with a concentration of about 3.5 ng / mL (near the detection limit) after being treated with protein precipitation. Fig.17 These are the chromatograms of four samples after 1000 injections of the same sample.

[0105] from Fig.16 It can be seen that the average signal-to-noise ratio is above 200:1, which fully meets the requirements of detection limit (3:1) and quantification limit (10:1). Fig.17 It can be seen that when the same sample is injected continuously for 1000 times, the peak area CV value (equivalent to RSD relative standard deviation, representing the reproducibility of instrument data, that is, the stability of performance; the smaller the data, the higher the reproducibility) is 6.13%, indicating that the instrument response has not decreased significantly after multiple injections.

[0106] It can be seen that the use of the electrospray ion source described in Example 1 significantly improves the response of the compound in the instrument and significantly improves the detection and analysis efficiency.

[0107] Example 5 Comparative test of simplified electrospray ion source pretreatment method of the present invention

[0108] Sulfonamides and macrolides are both common antibiotics. When testing the above two types of ingredients in animal food, the Ministry of Agriculture’s Announcement No. 1025-23-2008 “Liquid Chromatography-Tandem Mass Spectrometry for the Detection of Sulfonamide Residues in Animal Food” is generally followed: weigh 5±0.05g of sample into a 50mL centrifuge tube, add 15mL of ethyl acetate, vortex for 2min, centrifuge at 5000rpm for 10min, take the supernatant into a concentration bottle, extract the residue again using the same method, and combine the ethyl acetate layers. Add 5mL of 0.1mol / L hydrochloric acid to the concentration bottle, and rotary evaporate at 45°C until the ethyl acetate layer is completely volatilized. Transfer the remaining hydrochloric acid layer to a 10mL centrifuge tube, wash the concentration bottle with 2mL of 0.1mol / L hydrochloric acid twice, transfer the washing solution to the same centrifuge tube, wash the concentration bottle with 5mL of n-hexane, and transfer the n-hexane to the centrifuge tube containing hydrochloric acid, vortex mix, centrifuge at 3500rpm for 5min, discard the n-hexane layer, repeat the above operation once with 3mL of n-hexane, take the lower clear liquid, add it to the MCX (strong cation exchange) column treated with 3mL of methanol and 3mL of 0.1mol / L hydrochloric acid in advance, and after the reserve solution has completely passed through the column, wash the small column with 2mL of 0.1mol / L hydrochloric acid and 2mL of V(water):V(methanol):V(acetonitrile)=55:25:20 respectively, and wash with 2mL V(water):V(methanol):V(acetonitrile):V(ammonia water)=75:10:10:5 to elute the drug, collect the eluate and blow dry it in nitrogen in a 45℃ water bath, dilute to 1mL with water, and filter it through a 0.2μm organic filter membrane for instrumental analysis; and SN / T 1777.2-2007 "Detection of macrolide antibiotic residues in animal-derived foods Part 2: High performance liquid chromatography tandem mass spectrometry": weigh 5±0.05g of sample into a 50mL centrifuge tube, add 25mL of acetonitrile, homogenize at 12000rpm for 30s, centrifuge at 3000rpm for 3min, filter the upper acetonitrile extract, collect it in a 50mL volumetric flask, add 20mL of acetonitrile to the residue, repeat the above operation, combine the acetonitrile extracts, and dilute to 50mL with acetonitrile.Take 20mL of acetonitrile extract in a 50mL centrifuge tube, add 20mL of acetonitrile saturated n-hexane solution, mix at 2000rpm for 1min, centrifuge at 3000rpm for 3min, discard the n-hexane layer, add 20mL of acetonitrile saturated n-hexane solution, repeat the above operation, pass the lower acetonitrile layer through anhydrous sodium sulfate column, concentrate to dryness under reduced pressure in a water bath at 50℃, dissolve the residue three times with 15mL of phosphate buffer solution, transfer the solution to a C18 solid phase extraction column pretreated with 5mL of methanol and 5mL of water, wash the concentration bottle with 5mL of water and 5mL of 20% methanol solution in sequence, pass the washing liquid through the solid phase extraction column, discard the effluent, drain under negative pressure, elute with 6mL of methanol, control the flow rate not to exceed 3mL / min, collect all the eluent in a 10mL centrifuge tube, dilute to 10mL with water, and provide instrument analysis. If the dilute solution is turbid, it can be purified with 3mL of n-hexane, and pre-treated and analyzed on the machine respectively.

[0109] According to the above two detection standards, the two methods are processed separately, which is cumbersome and time-consuming. If the scheme is changed to a single pretreatment to complete the extraction and purification of the two types of compounds at the same time, that is, the two methods are combined (weigh 2±0.05g of sample in a 50mL centrifuge tube, add 20mL of acetonitrile solution containing 1% formic acid, vortex mix for 5min, ultrasonic extraction for 10min, centrifuge at 7000rpm for 10min, take 5mL of the upper clear liquid to Prime HLB solid phase extraction column, collect the effluent in a 5mL glass graduated tube, blow dry with nitrogen at 50℃, re-dissolve the residue with 1mL of 50% methanol solution, vortex mix, pass through a 0.2μm filter membrane for instrument analysis), the following problems will be faced: (1) In order to take into account the extraction of two types of compounds, the pretreatment method does not adopt the best extraction reagent and purification process, resulting in a low sample recovery rate; (2) In order to take into account the injection analysis of more than 20 compounds of two types, the instrument parameters are not the best conditions for a single compound, resulting in a lower overall response than the single method. However, macrolide compounds have large molecular weights, are not easily ionized, and have low responses. Such characteristics pose challenges to the combined detection of the two methods, and samples with concentrations near the detection limit are often not detected due to low responses.

[0110] Therefore, in this embodiment, the following three methods were used to detect five macrolide compounds (tylosin, roxithromycin, kitasamycin, tilmicosin, and lincomycin):

[0111] 1. Traditional electrospray ion source combined with pre-treatment;

[0112] 2. Single pretreatment results of traditional electrospray ion source;

[0113] 3. Results of electrospray ion source merging treatment in Example 2 of the present invention;

[0114] Specific test results such as Figures 18 to 22 As shown in, Fig.18 This is the chromatogram of tylosin 20 μg / L. Fig.19 This is the chromatogram of roxithromycin 20 μg / L. Fig. 20 This is the chromatogram of kitasamycin 20 μg / L. Fig.21 This is the chromatogram of Tilmicosin 20 μg / L. Fig. 22 The red color in the figure is the result of the electrospray ion source combined method of the present invention, the blue color is the result of the traditional ion source combined method, and the black color is the result of the traditional ion source single pre-treatment (because the red peak overlaps with the blue peak, the highest point of the blue peak is marked in the figure, and different peak intensities are respectively explained).

[0115] As can be seen from the figure, the response of macrolide compounds injected using the combined method after using the electrospray ion source in Example 2 is significantly improved, and the overall response value is close to the single-class determination method, so that the combined method can meet the detection requirements. The combination of the two methods reduces the pretreatment from two times to one time, and no longer requires a complex pretreatment process to concentrate and enrich the compounds. The sample can be injected after extraction or even simple dilution, which significantly saves the consumables cost and experimental time of the pretreatment process.

[0116] It can be seen that by using the electrospray ion source of the present invention, the compound can reach the response value after being treated by a single method without using a complicated pre-treatment process, which significantly saves the consumables cost and experimental time of the pre-treatment process and improves the detection efficiency.

[0117] Example 6 Comparative test of the sensitivity of the electrospray ion source of the present invention to different samples

[0118] In order to further test the detection sensitivity of the electrospray ion source in Example 2 to different samples, urine was selected as a sample in this embodiment to detect 13 kinds of organic acids in urine. By comparing the conventional electrospray ion source and the device of the present invention to determine the increase in the response of the corresponding compounds, it is shown that the electrospray ion source of the present invention can significantly improve the compound response for different sample types, thereby improving the sample detection sensitivity. At the same time, the detection sensitivity of one of the samples is particularly improved, which greatly improves the accuracy of the results. The specific operation process is: add the internal standard (tropic acid) and the extractant (ethyl acetate) to the urine sample in this embodiment, and take the supernatant after extraction for instrumental analysis. Use the conventional electrospray ion source and the electrospray ion source in Example 2 to detect two different samples, respectively. The specific results of the urine sample detection results are shown in Table 2 (the results of the compound peak area in the serum sample are shown in Example 3):

[0119] Table 2 Peak areas of compounds in urine samples

[0120]

[0121]

[0122] As can be seen from Table 2, after using the electrospray ion source of the present invention, the peak areas of 13 organic acids in the urine sample are significantly increased, and the responses are increased by more than 7 times. The responses of all compounds are significantly improved. At the same time, the sensitivity of instrument detection is also significantly improved, especially for compounds benzoic acid and citric acid, the responses are increased by about 22 times and 30 times respectively, and the detection sensitivity is significantly improved;

[0123] Compared with the detection results of serum samples using the electrospray ion source of the present invention in Example 3, the responses of various organic acids in urine samples in this example were greatly improved, proving that the ion source provided by the present invention can improve the sensitivity for different types of samples and compounds.

[0124] It can be seen that the electrospray ion source provided by the present invention can significantly improve the response of compounds in the sample, thereby improving the detection sensitivity; at the same time, the detection sensitivity of compounds in urine samples is particularly significantly improved, especially for compounds benzoic acid and citric acid. When the electrospray ion source of the present invention is used in the detection of urine samples, the sensitivity and accuracy of the detection can be significantly improved.

[0125] Example 7 Screening of the fixed distance between the edge of the disk and the electrode in the electrospray ion source of the present invention

[0126] In the disk atomization device of Example 2, in order to determine the optimal distance between the edge of the disk and the electrode, this distance is screened in the present embodiment, specifically in the following ways (the other conditions are the same, and the solution preparation, detection process and analysis method are the same as those in Example 3):

[0127] 1. The distance between the edge of the disk and the electrode is 6mm;

[0128] 2. The distance between the edge of the disk and the electrode is 8mm;

[0129] 3. The distance between the edge of the disk and the electrode is 10 mm;

[0130] 4. The distance between the edge of the disk and the electrode is 12mm;

[0131] 5. The distance between the edge of the disk and the electrode is 14mm;

[0132] The specific results are shown in Table 3:

[0133] Table 3 Changes in compound response at different distances between the disk edge and the electrode

[0134] Group (different distances) Compound response (progesterone (P) peak area) 6mm 1261087 8mm 1914832 10mm 2108435 12mm 1083771 14mm 255490

[0135] From Table 3, it can be seen that 1) there is an optimal value in the range of 6 to 14 mm between the edge of the disk and the electrode. Too small or too large a distance will affect the spray effect; and under the same analytical conditions, the distance can be optimized according to the change in the peak area of ​​the same compound on the mass spectrum, and the distance corresponding to the maximum value of the compound peak area is the optimal value; when the distance is 10 mm, the compound peak area response reaches the highest; 2) when the distance is reduced (2 mm), the compound peak area also decreases significantly. This may be because when the distance is too short, the droplets are not completely atomized, the ion atomization efficiency is low, and the number of charged particles entering the vacuum chamber is reduced, thereby reducing the number of ions that can be analyzed, and the peak area is significantly reduced; when the distance increases, the intensity of the electric field between the two gradually weakens, the number of Taylor cones generated decreases, the liquid atomization efficiency decreases, and the compound peak area decreases synchronously.

[0136] It can be seen that in the electrospray ion source of the present invention, in the disk atomization device, the distance between the disk edge and the electrode ranges from 6 to 14 mm. Within this range, the optimal distance between the disk edge and the electrode is finally determined to be 10 mm based on experimental data.

[0137] Example 8 Screening of the voltage between the electrode and the metal tube in the electrospray ion source of the present invention

[0138] In the disc atomization device of Example 2, the liquid is connected to the metal tube from the outside, flows out from the middle opening to cover the entire disc surface, and under the action of the electric field, the liquid is radially sprayed at the edge of the disc to form a Taylor cone structure. Therefore, in this embodiment, the voltage range applied between the electrode and the metal tube is screened, specifically in the following ways (the other conditions are the same, and the solution preparation, detection process and analysis method are the same as in Example 3):

[0139] 1. The voltage applied between the electrode and the metal tube is 4kV;

[0140] 2. The voltage applied between the electrode and the metal tube is 5kV;

[0141] 3. The voltage applied between the electrode and the metal tube is 7.6 kV;

[0142] 4. The voltage applied between the electrode and the metal tube is 8kV;

[0143] 5. The voltage applied between the electrode and the metal tube is 10kV;

[0144] 6. The voltage applied between the electrode and the metal tube is 11 kV;

[0145] The specific results are shown in Table 4:

[0146] Table 4 Changes in compound response under different voltages

[0147] Group (different voltage) Taylor cone number N Compound response (progesterone (P) peak area) 4kV N＝1 885417 5kV N＝5 1690879 7.6kV N＝10 1839916 8kV N＝26 2147585 10kV N＝100 2043225 11kV N＝384 937423

[0148] From Table 4, we can see that 1) when the voltage applied between the electrode and the metal tube is in the range of 4 to 11 kV, as the voltage increases, the number of Taylor cones increases significantly, and is positively correlated with the square of the voltage, and the ion source atomization efficiency also increases with the increase of voltage; 2) from the change in peak area, we can see that as the atomization efficiency increases, the compound ionization rate also increases synchronously, reaching a peak value at 8 KV, and a deviation occurs after exceeding 8 KV. It is speculated that the main reason for this phenomenon is that when the voltage is small, the number of Taylor cones generated is small, the ion atomization efficiency is low, resulting in a small number of charged particles entering the vacuum chamber, and the peak area is significantly reduced. When the voltage is too high and exceeds the threshold, the current will break through the medium between the electrodes to produce a tip discharge phenomenon, resulting in a large number of ions being neutralized. At this time, the gain brought by the increase in the number of Taylor cones is not as good as the ion loss caused by the tip discharge, and the number of ions entering the mass analyzer in the vacuum chamber is greatly reduced, resulting in a significant reduction in the compound peak area.

[0149] It can be seen that in the electrospray ion source of the present invention, for the mobile phase used in the present invention, in the disk atomization device, the voltage range between the electrode and the metal tube is 5 to 11 kV (this range is preferred because when the voltage is 4 kV, the compound response is lower), the number of Taylor cones is positively correlated with the square of the voltage, and the ion atomization efficiency increases continuously with the increase of the voltage; at the same time, the preferred voltage is 8 kV. When the voltage is 8 kV, the response of the compound in the instrument reaches the highest, which significantly improves the sensitivity and accuracy of the detection.

[0150] At the same time, in the electrospray ion source of the present invention, in the disk atomization device, the voltage range between the electrode and the metal tube is 5 to 11 kV. The user can optimize this voltage value according to the physicochemical properties of the analyzed compound and the composition of the mobile phase to achieve the best compound response value (the voltage value needs to be optimized according to the main component composition and proportion of the mobile phase, and the analysis of different compounds needs to adjust this voltage value to achieve the best effect).

[0151] Example 9 Screening of the number of annular electrode groups in the electrospray ion source of the present invention

[0152] In the annular electrode group of Example 2, in order to determine the number of annular electrodes, the annular electrodes are screened in this example, specifically in the following ways (the other conditions are the same, and the solution preparation, detection process and analysis method are the same as those in Example 3):

[0153] 1. The number of ring electrodes is 2;

[0154] 2. The number of ring electrodes is 3;

[0155] 3. The number of ring electrodes is 4;

[0156] 4. The number of ring electrodes is 5;

[0157] 5. The number of ring electrodes is 6;

[0158] The specific results are shown in Table 5:

[0159] Table 5 Changes in compound response under different numbers of ring electrodes

[0160] Group (different number of ring electrodes) Compound response (progesterone-1 peak area) 2 1804611 3 1957865 4 2083334 5 2126902 6 --

[0161] It can be seen from Table 5 that 1) when the number of ring electrodes is within the range of 3 to 5, as the number of ring electrodes increases, the peak area of ​​the same compound on the mass spectrum also increases continuously, which indicates that as the number of electrodes increases, the response of the compound in the instrument also increases continuously; 2) when the number of ring electrodes is only 2, the peak area of ​​the compound decreases significantly, which may be because when the number is too small, the effect of ion focusing produced by the ring electrodes is weak, and the ions are more dispersed before entering the vacuum chamber, and some ions collide with the conical baffle to generate losses, thereby reducing the number of charged particles entering the vacuum chamber, reducing the number of ions that can be analyzed, and significantly reducing the peak area; and when the number of ring electrodes is 6, it exceeds the installation range of the ring electrode group.

[0162] It can be seen from this that in the electrospray ion source of the present invention, the number of ring electrodes in the ring electrode group is 3 to 5, and the preferred number is 5. Within this range, as the number increases, the response of the compound in the instrument continues to increase, further improving the detection sensitivity, and when the number of ring electrodes is 5, the compound response reaches the highest.

[0163] Example 10 Verification test of compatible flow rate and atomization efficiency of the electrospray ion source of the present invention

[0164] In the current electrospray ion source, the liquid in the spray needle is easily not fully atomized at high flow rates, and the desolvation efficiency is low, which reduces the ionization efficiency; because the high flow rate solution has a large flow rate, the atomizing gas atomizes the large flow of solution into small droplets with a relatively large volume, and the high-temperature desolvation gas is difficult to volatilize the concentrated solution droplets, resulting in low desolvation efficiency. In this embodiment, in order to verify the compatibility of the electrospray ion source of the present invention with flow rate and atomization efficiency, the atomization efficiency at high flow rate is tested, and the specific process is as follows: the electrospray device described in Example 1, the flow rate of the liquid entering the ion source is set to 0.6mL / min, 0.8mL / min, and 1.0mL / min, respectively, and the sample peak area is detected, and the specific results are shown in Table 6 (the other conditions are the same, the solution preparation, detection process and analysis method are the same as in Example 3; the atomization efficiency is reflected in the response of the final compound peak area):

[0165] Table 6 Changes in compound response at different flow rates

[0166]

[0167] As can be seen from Table 6, both the traditional electrospray ion source and the scheme of the present invention follow the law that the target peak response decreases when the flow rate increases. The reason is that the large flow rate increases the liquid entering the ion source, and the atomization effect becomes worse. At the same time, the solvent of a large number of droplets is not fully evaporated, resulting in a reduction in the situation of Coulomb explosion, which ultimately leads to a reduction in the charged ions of the target compound, affecting the instrument response. However, this scheme produces more Taylor cones than the traditional scheme. Compared with the traditional ion source at the same flow rate, there are more target compounds that successfully complete the charge transfer process and become charged ions entering the mass analyzer. At the same time, in the face of the increase in flow rate, the degree of attenuation of the response signal is also smaller than that of the traditional ion source.

[0168] On the other hand, further decreasing the flow rate may result in a higher peak area. However, in practical applications, the present invention also needs to consider the working flow rate range of the chromatographic column. Experiments have shown that the chromatographic column achieves the best separation effect at a flow rate of 0.6 mL / min.

[0169] It can be seen that the electrospray ion source of the present invention is compatible with a relatively large flow rate. Even at a relatively large flow rate, as the voltage increases, the number of Taylor cones increases significantly, and the atomization charging effect of the compound can be significantly improved, and the ion atomization efficiency and the response of the compound in the instrument can be improved. At the same time, at the optimal flow rate of 0.6 mL / min, the best separation effect is also obtained. Therefore, the electrospray ion source in Example 2 is compatible with a relatively wide range of flow rates. At a relatively high flow rate, the atomization effect can still be improved, the response of the compound can be improved, and the separation effect can be taken into account at the same time, which provides convenience for the establishment of the analytical method and the selection of the chromatographic column.

[0170] Example 11 Increasing the spray range can still increase the number of charged particles entering the ion source

[0171] The design of the ion inlet of the current electrospray ion source is not suitable for the multi-nozzle probe. The excessive spray range of the multi-nozzle causes some charged particles to disperse and cannot be effectively attracted by the electric field to enter the ion source. Therefore, in this embodiment, in order to test the number of ions entering the vacuum chamber after increasing the spray range, the following methods are set (the other conditions are the same, the solution preparation, detection process and analysis method are the same as in Example 3, and the number of ions entering the vacuum chamber is determined according to the peak area of ​​the compound):

[0172] 1. Traditional electrospray ion source, single nozzle probe;

[0173] 2. Traditional electrospray ion source, multi-nozzle probe;

[0174] 3. The voltage of the electrospray ion source in Example 2 is 8.0 kV;

[0175] The specific results are shown in Table 7:

[0176] Table 7 Effect of different spray ranges on the number of ions entering the vacuum chamber

[0177] Group Compound response (progesterone (P) peak area) Conventional electrospray ion source (single-nozzle probe) 571888 Conventional electrospray ion source (multi-nozzle probe n = 24) 1631417 Electrospray ion source (8.0 kV) in Example 1 2205941

[0178] As can be seen from Table 7, 1) for the conventional electrospray ion source, when the ion inlet is designed as a multi-nozzle probe, the target compound response value is significantly improved compared with the single-nozzle design, proving that when multiple Taylor cones are generated in the ion source, the target compound ionization process is promoted; 2) when replaced with the electrospray ion source in Example 1, the conventional ion source with 24 spray needles is compared with the scheme of the present invention that generates 26 Taylor cones. When the number of Taylor cones is close, the compound response improvement is not as good as that of the present scheme. We infer that the possible reason is that the structure of multiple Taylor cones can better realize the spray ionization process, but compared with the scheme of a single nozzle, the charged ions formed by the addition of Taylor cone spray have a wider diffusion range, and some ions are far away from the ion inlet and are not attracted by the electric field to enter the mass analyzer. Therefore, the present scheme sets up multiple annular electrodes under the disk spray structure to focus the generated ions toward the ion inlet, thereby bringing a higher target compound response.

[0179] It can be seen from this that the electrospray ion source provided by the present invention increases the spray range and can still push more ions into the mass analyzer in the vacuum chamber, thereby increasing the number of ions that can be analyzed.

[0180] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A liquid chromatography-mass spectrometer containing an electrospray ion source device, characterized in that: The liquid chromatography-mass spectrometry instrument adopts an electrospray ion source device, which includes a disk atomization device, a ring electrode group and a repeller electrode; wherein the ring electrode group is arranged below the disk atomization device, and the repeller electrode is arranged at the lower left of the ring electrode group; the disk atomization device includes an atomization electrode, a spray disk and an injection tube; the spray disk has a middle opening connected to the injection tube, and liquid can be connected to the injection tube from the outside, flow out from the middle opening, and cover the entire surface of the spray disk; the ring electrode group includes 3 to 5 ring electrodes, the vertical distance between the first ring electrode below the spray disk and the disk is 10 mm, and the diameter of the ring electrode gradually decreases from the position close to the disk atomization device, and the diameter is 10 to 50 mm.

2. The liquid chromatography-mass spectrometry instrument according to claim 1, characterized in that: An atomizing electrode is arranged outside the spray disc, the distance between the edge of the spray disc and the atomizing electrode is 10 mm, and the inner diameter of the atomizing electrode is 6-10 mm; a voltage is applied between the atomizing electrode and the sample injection tube, and the voltage is 4-11 kV.

3. The liquid chromatography-mass spectrometry instrument according to claim 2, characterized in that: The spray disc is made of stainless steel, with a diameter of 5 mm and a thickness of about 50 μm; the inner diameter of the injection tube is 0.1-2 mm.

4. The liquid chromatography-mass spectrometry instrument according to claim 3, characterized in that: Direct current with the same direction is applied to each annular electrode, and different voltages are applied between different annular electrodes to generate a potential difference, so that charged particles gather and accelerate toward the cone plate, which is arranged at the lower right of the annular electrode group.

5. The liquid chromatography-mass spectrometry instrument according to claim 4, characterized in that: The repeller electrode is arranged at the lower left side of the annular electrode group, and the distance between the two is: 10 mm from the bottom coil of the annular electrode in the vertical direction, and 30 mm from the center of the annular electrode in the horizontal direction; an ion inlet is arranged on the opposite side of the repeller electrode, and charged particles enter the vacuum chamber by the combined action of the electromagnetic field of the repeller electrode and the potential difference in the ion inlet, and neutral particles and other excess gases enter the exhaust pipe along the direction of movement; the exhaust pipe is arranged at the lower left side of the ion inlet and directly below the annular electrode group.

6. An electrospray ion source device for use in liquid chromatography-mass spectrometry to improve compound detection limit and analysis efficiency, characterized in that: The electrospray ion source device comprises a disk atomization device, a ring electrode group and a repeller electrode; wherein the ring electrode group is arranged below the disk atomization device, and the repeller electrode is arranged at the lower left of the ring electrode group; the disk atomization device comprises an atomization electrode, a spray disk and an injection tube; the spray disk has a middle opening connected to the injection tube, and liquid can be connected to the injection tube from the outside, flow out from the middle opening, and cover the entire surface of the spray disk; the ring electrode group comprises 3 to 5 ring electrodes, the vertical distance between the first ring electrode below the spray disk and the disk is 10 mm, and the diameter of the ring electrode gradually decreases from the position close to the disk atomization device, and the diameter is 10 to 50 mm.

7. An electrospray ion source device for improving the detection sensitivity of a liquid chromatography-mass spectrometer and increasing the types of detected compounds, characterized in that: The electrospray ion source device comprises a disk atomization device, a ring electrode group and a repeller electrode; wherein the ring electrode group is arranged below the disk atomization device, and the repeller electrode is arranged at the lower left of the ring electrode group; the disk atomization device comprises an atomization electrode, a spray disk and an injection tube; the spray disk has a middle opening connected to the injection tube, and liquid can be connected to the injection tube from the outside, flow out from the middle opening, and cover the entire surface of the spray disk; the ring electrode group comprises 3 to 5 ring electrodes, the vertical distance between the first ring electrode below the spray disk and the disk is 10 mm, and the diameter of the ring electrode gradually decreases from the position close to the disk atomization device, and the diameter is 10 to 50 mm.

8. An electrospray ion source device is used for reducing the pre-treatment process and reducing the sample volume required for analysis in liquid chromatography-mass spectrometry detection, characterized in that: The electrospray ion source device comprises a disk atomization device, a ring electrode group and a repeller electrode; wherein the ring electrode group is arranged below the disk atomization device, and the repeller electrode is arranged at the lower left of the ring electrode group; the disk atomization device comprises an atomization electrode, a spray disk and an injection tube; the spray disk has a middle opening connected to the injection tube, and liquid can be connected to the injection tube from the outside, flow out from the middle opening, and cover the entire surface of the spray disk; the ring electrode group comprises 3 to 5 ring electrodes, the vertical distance between the first ring electrode below the spray disk and the disk is 10 mm, and the diameter of the ring electrode gradually decreases from the position close to the disk atomization device, and the diameter is 10 to 50 mm.

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