Liquid phase method for simplifying clinical mass spectrum sample pretreatment

Abstract

The invention discloses a liquid phase method for simplifying clinical mass spectrum sample pretreatment, and relates to the technical field of sample pretreatment. The liquid phase method for simplifying the pretreatment of the clinical mass spectrum sample is based on the following system which comprises two liquid phase pumps, a first six-way valve, a sample injector module, an online solvent mixer and a chromatographic column. Based on the system and switching the interface connection mode of the first six-way valve, two sample injection modes can be realized: mobile phases output by the first liquid phase pump and the second liquid phase pump are mixed by the on-line solvent mixer and then enter the sample injector module to transfer a sample to a chromatographic column, and the mode is suitable for sample injection of a weak solvent dissolved sample; the second mobile phase and the second mobile phase transfer a sample to an online solvent mixer through a sample injector module to be mixed with the first mobile phase, then the sample enters a chromatographic column, and the mode is suitable for sample injection of a strong solvent dissolved sample. According to the invention, two sample introduction modes can be automatically switched according to requirements, and the method has great application and popularization values in clinical tandem mass spectrum quantitative detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sample pretreatment, in particular to a liquid phase method for simplifying clinical mass spectrometry sample pretreatment. BACKGROUND

[0002] Clinical tandem mass spectrometry detection has become one of the important means for clinical laboratories to achieve accurate quantitative detection, but the complex and tedious clinical mass spectrometry sample pretreatment process seriously limits the popularization and application expansion of clinical tandem mass spectrometry technology.

[0003] Blood drug concentration monitoring is one of the applications of clinical tandem mass spectrometry. At present, the mainstream sample pretreatment method adopted is protein solvent precipitation method, and the basic processing steps thereof include precipitating proteins in blood plasma with a cooled organic phase (such as methanol), centrifuging to obtain supernatant, removing the organic phase by nitrogen blowing, re-dissolving in water phase, and on-machine detection, wherein the nitrogen blowing step is a key rate-limiting step. At present, automation is mainly used to reduce the labor input in sample pretreatment, but these steps cannot be omitted. In addition, there are many studies in the field of liquid chromatography aiming to realize direct strong solvent on-machine detection, such as trap-and-elute, on-column dilution, and in-column dilution, but these methods all require in-depth understanding of chromatographic retention behavior and hardware modification, which are difficult to apply to actual clinical tests. SUMMARY

[0004] The main purpose of the present application is to provide a liquid phase method for simplifying clinical mass spectrometry sample pretreatment, aiming to solve the problems of complicated process and time-consuming in the application of tandem mass spectrometry to clinical blood drug concentration monitoring.

[0005] To achieve the above-mentioned purpose, the present application provides a liquid phase method for simplifying clinical mass spectrometry sample pretreatment, which is used for blood drug concentration monitoring, and the liquid phase method for simplifying clinical mass spectrometry sample pretreatment is based on the following liquid chromatography injection system, which comprises: a liquid delivery system comprising a first liquid phase pump and a second liquid phase pump, wherein the first liquid phase pump is used for pumping a first mobile phase, and the second liquid phase pump is used for pumping a second mobile phase; a sample injection system comprising a first six-way valve and a sample injector module, wherein six interfaces of the first six-way valve are ①, ②, ③, ④, ⑤ and ⑥ in sequence, and the interface communication mode of the first six-way valve comprises a first communication mode or a second communication mode; an online solvent mixer; and a separation and detection system comprising a chromatographic column; The first liquid phase pump is connected to the inlet end of the online solvent mixer, the second liquid phase pump is connected to the ⑥ interface of the first six-way valve, the ⑤ interface of the first six-way valve is connected to the inlet end of the sample injector module, the outlet end of the sample injector module is connected to the ② interface of the first six-way valve, the ① interface of the first six-way valve is connected to the inlet end of the online solvent mixer, the outlet end of the online solvent mixer is connected to the ④ interface of the first six-way valve, and the ③ interface of the first six-way valve is connected to the chromatographic column; Based on the above liquid chromatography sample injection system, the simplified liquid phase method for clinical mass spectrometry sample pretreatment comprises the following steps: S10, starting the first liquid phase pump and the second liquid phase pump; injecting the sample to be tested through the sample injector module; S20, the first liquid phase pump pumps the first mobile phase to the online solvent mixer by pumping, and the second liquid phase pump pumps the second mobile phase to the ⑥ interface of the first six-way valve by pumping.

[0006] In an embodiment, the second mobile phase has a stronger elution ability for the separation target than the first mobile phase.

[0007] In an embodiment, the first mobile phase is selected from a solvent that is a weak elution solvent based on the chromatographic retention behavior of the chromatographic column, and the second mobile phase is selected from a solvent that is a strong elution solvent based on the chromatographic retention behavior of the chromatographic column.

[0008] In an embodiment, the first six-way valve is a two-position six-way switching valve.

[0009] In an embodiment, the interface connection mode of the first six-way valve adopts a first connection mode: The second mobile phase flows from the ⑥ interface of the first six-way valve to the ① interface of the first six-way valve, and then flows from the ① interface of the first six-way valve to the inlet end of the online solvent mixer; The first mobile phase and the second mobile phase are mixed in the online solvent mixer to obtain a mixed mobile phase, the mixed mobile phase flows out of the outlet end of the online solvent mixer and flows to the ④ interface of the first six-way valve, and then flows from the ④ interface of the first six-way valve to the ⑤ interface of the first six-way valve, and then flows from the ⑤ interface of the first six-way valve to the inlet end of the sample injector module, forming a mixture containing the first mobile phase, the second mobile phase and the sample to be tested; The mixture containing the first mobile phase, the second mobile phase and the sample to be tested flows out of the outlet end of the sample injector module and flows to the ② interface of the first six-way valve, and then flows from the ② interface of the first six-way valve to the ③ interface of the first six-way valve, and then flows from the ③ interface of the first six-way valve to the chromatographic column.

[0010] In an embodiment, the interface connection mode of the first six-way valve adopts a second connection mode: The second mobile phase flows from port 6 of the first six-way valve to port 5 of the first six-way valve, and then from port 5 of the first six-way valve to the inlet of the injector module. The second mobile phase dissolves the sample to be tested and forms a mixture of the second mobile phase and the sample to be tested. Then it flows out from the outlet end of the injector module and flows to the interface ② of the first six-way valve. Then it flows from the interface ② of the first six-way valve to the interface ① of the first six-way valve, and then from the interface ① of the first six-way valve to the inlet end of the online solvent mixer. The first mobile phase and the mixture of the second mobile phase and the sample to be tested are mixed in an online solvent mixer to form a mixture containing the first mobile phase, the second mobile phase and the sample to be tested. The mixture containing the first mobile phase, the second mobile phase and the sample to be tested flows out from the outlet of the online solvent mixer and flows to the port ④ of the first six-way valve, then flows from the port ④ of the first six-way valve to the port ③ of the first six-way valve, and then flows from the port ③ of the first six-way valve to the chromatographic column.

[0011] In one embodiment, the injector module includes an injector, which includes either a quantitative loop injector or a flow-through needle (FTN) injector.

[0012] In one embodiment, the sample to be tested is a plasma sample or a serum sample.

[0013] In one embodiment, the injector module further includes a second six-way valve, the six ports of which are a, b, c, d, e and f respectively.

[0014] In one embodiment, the second six-way valve is a two-position six-way switching valve.

[0015] The present invention provides a simplified clinical mass spectrometry sample pretreatment method based on a liquid chromatography injection system. The liquid chromatography injection system includes an infusion system, an injection system, an online solvent mixer, and a separation and detection system. The infusion system includes a first liquid pump and a second liquid pump. The injection system includes a first six-way valve and an injector module. The six ports of the first six-way valve are ①, ②, ③, ④, ⑤, and ⑥, and the port connection method of the first six-way valve includes a first connection method or a second connection method. The separation and detection system includes a chromatographic column. Based on the above system, and by switching the interface connection method of the first six-way valve, two injection modes can be achieved: First, the mobile phase output from the two liquid phase pumps is mixed via an online solvent mixer, and then enters the injector module to transfer the sample to the chromatographic column. This mode is suitable for injecting samples dissolved in weak solvents. Second, the second mobile phase transfers the sample via the injector module to an online solvent mixer, then mixes with the first mobile phase, and subsequently enters the chromatographic column. This mode is suitable for injecting samples dissolved in strong solvents, allowing the analyte to be dissolved in a strong solvent and directly injected without significant peak breakthrough. Using the liquid chromatography method provided by this invention to process the sample facilitates switching between the two injection modes. Furthermore, when using the second injection mode, the step of drying with strong solvent nitrogen in conventional pretreatment methods can be eliminated, while also exhibiting good sensitivity. This method has significant application and promotion value in clinical tandem mass spectrometry quantitative detection. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 A schematic diagram of the connection of the liquid chromatography injection system for the simplified clinical mass spectrometry sample pretreatment method provided by the present invention when the first connection mode is adopted; Figure 2 A schematic diagram of the connection of the liquid chromatography injection system for the simplified clinical mass spectrometry sample pretreatment method provided by the present invention when the second connection mode is adopted; Figure 3 A schematic diagram of the connection of the liquid chromatography injection system in the first embodiment of the simplified clinical mass spectrometry sample pretreatment liquid chromatography method provided by the present invention when the liquid chromatography injection system adopts the first connection mode; Figure 4 A connection diagram of the liquid chromatography injection system in the second embodiment of the simplified clinical mass spectrometry sample pretreatment liquid chromatography method provided by the present invention when the liquid chromatography injection system adopts the second connection mode; Figure 5 A schematic diagram of the connection of the liquid chromatography injection system in the third embodiment of the liquid chromatography method for simplified clinical mass spectrometry sample pretreatment provided by the present invention; Figure 6 The image shows the results of detecting antibacterial drugs using the simplified clinical mass spectrometry sample pretreatment liquid chromatography method provided by this invention.

[0018] Explanation of icon numbers: 1. Infusion system; 11. First liquid phase pump; 12. Second liquid phase pump; 2. Injection system; 21. First six-way valve; 22. Injector module; 221. Second six-way valve; 3. Online solvent mixer; 4. Separation and detection system; 41. Chromatographic column; 5. Detection system; 51. Detector.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0022] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0023] Clinical tandem mass spectrometry has become one of the important means for clinical laboratories to achieve accurate quantitative detection. However, the complex and cumbersome sample pretreatment process for clinical mass spectrometry has severely limited the promotion and application of clinical tandem mass spectrometry technology.

[0024] Blood drug concentration monitoring is one of the applications of tandem mass spectrometry in clinical practice. Currently, the mainstream sample pretreatment method is protein solvent precipitation. Its basic steps include precipitating proteins from plasma with a cooled organic phase (such as methanol), centrifuging to obtain the supernatant, nitrogen drying to remove the organic phase, reconstitution in aqueous phase, and detection. The nitrogen drying step is the critical rate-limiting step. Currently, automation is mainly used to reduce manual labor in sample pretreatment, but these steps cannot be omitted. Furthermore, conventional liquid chromatography-tandem mass spectrometry (LC-MS) uses a high-flow-rate, low-proportion mobile phase to directly carry samples dissolved in a strong solvent. This injection method is prone to the precipitation of retained analytes (those with low water solubility). In addition, many studies in the field of liquid chromatography aim to achieve direct detection of strong solvents on the instrument, such as trap-and-elute, on-column dilution, and in-column dilution. However, these methods all require a deep understanding of chromatographic retention behavior and hardware modifications. In clinical practice, clinical laboratories often lack the ability to actively modify the liquid chromatography flow path connections. Therefore, existing technologies are still difficult to apply to actual clinical trials.

[0025] Based on the above background, please refer to Figure 1 This invention proposes a simplified liquid chromatography method for clinical mass spectrometry sample pretreatment for blood drug concentration monitoring. This simplified liquid chromatography method is based on a liquid chromatography injection system comprising: The infusion system 1 includes a first liquid phase pump 11 and a second liquid phase pump 12, wherein the first liquid phase pump 11 is used to pump in a first mobile phase and the second liquid phase pump 12 is used to pump in a second mobile phase. The injection system 2 includes a first six-way valve 21 and an injector module 22. The six ports of the first six-way valve 21 are ①, ②, ③, ④, ⑤ and ⑥ respectively. The port connection mode of the first six-way valve 21 includes a first connection mode or a second connection mode. Online solvent mixer 3; and, Separation and detection system 4, including chromatographic column 41; The first liquid phase pump 11 is connected to the inlet end of the online solvent mixer 3, the second liquid phase pump 12 is connected to the ⑥ port of the first six-way valve 21, the ⑤ port of the first six-way valve 21 is connected to the inlet end of the injector module 22, the outlet end of the injector module 22 is connected to the ② port of the first six-way valve 21, the ① port of the first six-way valve 21 is connected to the inlet end of the online solvent mixer 3, the outlet end of the online solvent mixer 3 is connected to the ④ port of the first six-way valve 21, and the ③ port of the first six-way valve 21 is connected to the chromatographic column 41. Based on the above liquid chromatography injection system, the simplified liquid chromatography method for clinical mass spectrometry sample pretreatment includes the following steps: S10. Turn on the first liquid phase pump 11 and the second liquid phase pump 12; inject the sample to be tested through the injector module 22. S20, the first liquid phase pump 11 pumps the first mobile phase into the online solvent mixer 3 by pumping, and the second liquid phase pump 12 pumps the second mobile phase into the port ⑥ of the first six-way valve 21 by pumping.

[0026] The present invention provides a simplified clinical mass spectrometry sample pretreatment method based on a liquid chromatography injection system. The liquid chromatography injection system includes an infusion system 1, an injection system 2, an online solvent mixer 3, and a separation and detection system 4. The infusion system 1 includes a first liquid pump 11 and a second liquid pump 12. The injection system 2 includes a first six-way valve 21 and an injector module 22. The six ports of the first six-way valve 21 are ①, ②, ③, ④, ⑤, and ⑥, and the port connection mode of the first six-way valve 21 includes a first connection mode or a second connection mode. The separation and detection system 4 includes a chromatographic column 41. Based on the above system, and by switching the interface connection method of the first six-way valve, two injection modes can be achieved: First, the mobile phase output from the two liquid phase pumps is mixed via the online solvent mixer 3, and then enters the injector module 22 to transfer the injected sample to the chromatographic column 41. This mode is suitable for the injection of samples dissolved in weak solvents. Second, the second mobile phase is transferred via the injector module 22 to the online solvent mixer 3 and mixed with the first mobile phase, and then enters the chromatographic column 41. This mode is suitable for the injection of samples dissolved in strong solvents, allowing the analyte to be dissolved in a strong solvent and directly injected without significant chromatographic peak breakthrough. Using the liquid chromatography method provided by this invention to process the sample requires only a software click (or setting an automatic program) to switch the injection mode, facilitating the switching between the two injection modes. It also has good sensitivity, and when using the second injection mode, the step of drying with strong solvent nitrogen in conventional pretreatment methods can be eliminated. This method has significant application and promotion value in clinical tandem mass spectrometry quantitative detection.

[0027] The simplified liquid chromatography method for clinical mass spectrometry sample pretreatment provided by this invention allows for direct injection of samples dissolved in strong solvents. In scenarios where samples are directly injected, a low-proportion (flow rate) second mobile phase carries the sample to an online solvent mixer, where it mixes with a high-proportion (flow rate) first mobile phase. This significantly reduces the solvent strength entering the column, allowing analytes with high retention coefficients to accumulate at the column head, forming narrow sample bands and resulting in sharp peaks (improved sensitivity and resolution). Compared to traditional injection methods that use a high-flow-rate, low-proportion second mobile phase to directly carry samples dissolved in strong solvents, the injection method provided by this invention is more effective in avoiding the precipitation of highly retained analytes (with low water solubility).

[0028] In an embodiment of the present invention, the second flow has a stronger elution capacity relative to the target analyte than the first flow phase.

[0029] Furthermore, in embodiments of the present invention, the first mobile phase is selected from solvents that are weak eluting solvents in terms of chromatographic retention behavior based on the chromatographic column, and the second mobile phase is selected from solvents that are strong eluting solvents in terms of chromatographic retention behavior based on the chromatographic column. Selecting two mobile phases with different eluting capabilities allows for adaptation to samples of different types and complexities; moreover, utilizing the different solubilities and interaction forces of different chemical substances in solvent mixtures of varying proportions is beneficial for improving the separation effect between components in complex samples.

[0030] It should be noted that in this article, "weak eluting solvent" means that on a specific chromatographic column, it has a low elution ability for the target compound, and the sample component is difficult to elute from the chromatographic column, i.e., it has strong retention; "strong eluting solvent" means that on a specific chromatographic column, it has a strong elution ability for the target compound, and the sample component is easily eluted from the chromatographic column, i.e., it has weak retention.

[0031] In an embodiment of the present invention, the first six-way valve 21 is a two-position six-way switching valve. The two-position first six-way valve can switch between two states, and in each state, the connection method between the six ports is different. The technical solution of the present invention uses a two-position six-way switching valve commonly used in the field of liquid chromatography, which allows for flexible control of the fluid direction and path.

[0032] Please see Figure 1 In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts a first connection method: The second mobile phase flows from port 6 of the first six-way valve 21 to port 1 of the first six-way valve 21, and then flows through port 1 of the first six-way valve 21 to the inlet end of the online solvent mixer 3. The first mobile phase and the second mobile phase are mixed in the online solvent mixer 3 to obtain a mixed mobile phase. The mixed mobile phase flows out from the outlet end of the online solvent mixer 3 and flows to the port ④ of the first six-way valve 21. Then it flows from the port ④ of the first six-way valve 21 to the port ⑤ of the first six-way valve 21, and then from the port ⑤ of the first six-way valve 21 to the inlet end of the injector module 22, forming a mixture containing the first mobile phase, the second mobile phase and the sample to be tested in the injector module 22. The mixture containing the first mobile phase, the second mobile phase, and the sample to be tested flows out from the outlet of the injector module 22 and flows to the interface ② of the first six-way valve 21, then flows from the interface ② of the first six-way valve 21 to the interface ③ of the first six-way valve 21, and then flows from the interface ③ of the first six-way valve 21 to the chromatographic column 41.

[0033] The above injection method is suitable for injecting samples that are easily soluble in the first mobile phase. By setting the interface connection mode of the first six-way valve 21 to the first connection mode, the mobile phases output from the two liquid phase pumps are first mixed via the online solvent mixer 3, and then enter the injector module 22 to transfer the injected sample to the chromatographic column for separation and analysis. Adjusting the mixing ratio of the first and second mobile phases helps improve the separation efficiency and analytical accuracy of the sample.

[0034] In an embodiment of the present invention, when the interface connection mode of the first six-way valve 21 adopts the first connection mode, in the step of the first liquid phase pump 11 pumping the first mobile phase into one inlet end of the online solvent mixer 3 by pumping, the pumping speed of the first mobile phase is 0.05~0.8mL / min.

[0035] In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts a first connection method, and in the step of the second liquid phase pump 12 pumping the second mobile phase into the interface ⑥ of the first six-way valve 21 by pumping, the pumping speed of the second mobile phase is 0.1~0.8mL / min.

[0036] In an embodiment of the present invention, the interface connection mode of the first six-way valve 21 adopts the first connection mode, and the injection volume is 1~10µL.

[0037] When the interface connection mode of the first six-way valve 21 adopts the first connection mode, the flow rate, injection volume, and mixing time are set according to the particle size, column size, and methodology of the chromatographic column. The technical solution of this invention does not impose specific limitations. In one embodiment of the invention, ultrapure water containing 0.1% formic acid is used as the first mobile phase, and methanol containing 0.1% formic acid is used as the second mobile phase. The volume ratio of the first mobile phase to the second mobile phase is 10% / 90%, the flow rate of the first mobile phase is 0.05 mL / min, the flow rate of the second mobile phase is 0.45 mL / min, and the injection volume is 5 µL.

[0038] Please see Figure 2 In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts a second connection method: The second mobile phase flows from port 6 of the first six-way valve 21 to port 5 of the first six-way valve 21, and then from port 5 of the first six-way valve 21 to the inlet end of the injector module 22. The second mobile phase dissolves the sample to be tested and forms a mixture of the second mobile phase and the sample to be tested. Then it flows out from the outlet end of the injector module 22 and flows to the interface ② of the first six-way valve 21. Then it flows from the interface ② of the first six-way valve 21 to the interface ① of the first six-way valve 21, and then flows through the interface ① of the first six-way valve 21 to the inlet end of the online solvent mixer 3. The mixture of the first mobile phase, the second mobile phase, and the sample to be tested is mixed in the online solvent mixer 3 to form a mixture containing the first mobile phase, the second mobile phase, and the sample to be tested. The mixture containing the first mobile phase, the second mobile phase, and the sample to be tested flows out from the outlet end of the online solvent mixer 3 and flows to the port ④ of the first six-way valve 21. Then it flows from the port ④ of the first six-way valve 21 to the port ③ of the first six-way valve 21, and then from the port ③ of the first six-way valve 21 to the chromatographic column 41.

[0039] The above-described injection method is suitable for processing samples that are readily soluble in the second mobile phase. By setting the interface connection of the first six-way valve 21 to the second connection mode, the sample to be tested is first dissolved separately using the second mobile phase and transferred to the online solvent mixer 3. Then, it is thoroughly mixed with the first mobile phase in the online solvent mixer 3 to ensure sufficient solvent dilution before entering the chromatographic column for enrichment at the column head. This injection mode ensures sufficient dissolution of the sample in the strong solvent, avoids breakthrough phenomena on the chromatographic column due to excessively high sample molecular concentrations, and simplifies the time-consuming and labor-intensive nitrogen drying and sample reconstitution steps in sample pretreatment, thereby improving sample pretreatment efficiency and ensuring the accuracy of the analytical results.

[0040] In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts the second connection method, and in the step of the first liquid phase pump 11 pumping the first mobile phase into one inlet end of the online solvent mixer 3 by pumping, the pumping speed of the first mobile phase is 0.05~0.8mL / min.

[0041] In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts the second connection method, and in the step of the second liquid phase pump 12 pumping the second mobile phase into the interface ⑥ of the first six-way valve 21 by pumping, the pumping rate of the second mobile phase is 0.01~0.2mL / min.

[0042] In an embodiment of the present invention, the interface connection method of the first six-way valve 21 adopts the second connection method, and the injection volume is 1~10µL.

[0043] When the interface connection mode of the first six-way valve 21 adopts the second connection mode, the flow rate, injection volume, and mixing time are set according to the particle size, column size, and methodology of the chromatographic column. The technical solution of this invention does not impose specific limitations. In one embodiment of the invention, ultrapure water containing 0.1% formic acid is used as the first mobile phase, and methanol containing 0.1% formic acid is used as the second mobile phase. The volume ratio of the first mobile phase to the second mobile phase is 10% / 90%, the flow rate of the first mobile phase is 0.05 mL / min, the flow rate of the second mobile phase is 0.45 mL / min, and the injection volume is 5 µL.

[0044] In an embodiment of the present invention, the injector module 22 includes an injector, which includes any one of a quantitative loop injector and a flow-through needle injector (FTN injector module).

[0045] In an embodiment of the present invention, the injector module 22 further includes a second six-way valve 221, the six ports of the second six-way valve 221 being a, b, c, d, e and f respectively.

[0046] In an embodiment of the present invention, when a sample is drawn, the liquid flows from the sample vial through the interface f→e→b→a→injector, and a sample quantitative loop is formed between e and b.

[0047] In embodiments of the present invention, the method further includes a detection system 5, which includes a detector 51 for analyzing samples separated by a chromatographic column. The present invention specifies the type of detector 51, for example, it may be a mass spectrometer detector.

[0048] In embodiments of the present invention, the sample to be tested is a plasma sample or a serum sample. The technical solution of the present invention can be used for the analysis of target analytes in plasma samples or serum samples.

[0049] In an embodiment of the present invention, the second six-way valve 221 is a two-position six-way switching valve.

[0050] The present invention also provides a first embodiment of a liquid chromatography method for simplifying clinical mass spectrometry sample pretreatment.

[0051] For the specific connection method of the liquid chromatography injection system in the first embodiment, please refer to [link / reference needed]. Figure 3 The injector uses a quantitative loop injector, and the interface connection of the first six-way valve 21 adopts the first connection method. The specific operation is as follows: (1) Provide a sample of plasma containing anti-tuberculosis drug concentration. The sample is precipitated by protein solvent precipitation, the supernatant is separated by centrifugation, the supernatant is dried by nitrogen blowing, and the sample is reconstituted with formic acid aqueous solution. The reconstituted solution is used for instrument detection. Provide ultrapure water (containing 0.1% formic acid) as the first mobile phase and methanol (containing 0.1% formic acid) as the second mobile phase. The volume ratio of the first mobile phase and the second mobile phase is 10% / 90%. The total injection volume is 5μL. The flow rate of the first mobile phase is set to 0.05mL / min and the flow rate of the second mobile phase is set to 0.45mL / min. (2) Sample injection in the traditional mode: The sample to be tested is temporarily stored in the quantitative loop. The first liquid phase pump 11 pumps the first mobile phase into the online solvent mixer 3 by pumping, and the second liquid phase pump 12 pumps the second mobile phase into port 6 of the first six-way valve 21 by pumping. Then the second mobile phase flows from port 6 of the first six-way valve 21 to port 1 of the first six-way valve 21, and then flows through port 1 of the first six-way valve 21 to the online solvent mixer 3. The first mobile phase and the second mobile phase are mixed in the online solvent mixer 3. The mixed mobile phase is obtained by combining the two components. The mixed mobile phase then flows out from the outlet of the online solvent mixer 3 and flows to port ④ of the first six-way valve 21. It then flows from port ④ to port ⑤ of the first six-way valve 21, then from port ⑤ to port c of the second six-way valve 221, from port c to port b, carrying the sample in the quantitative loop to port e, then from port e to port d, and finally flows out from port d and carries the sample into port ② of the first six-way valve 21.

[0052] (3) Elution analysis process: The mixture of the mixed mobile phase and the sample to be tested flows out from the port d of the second six-way valve 221 and flows to the port ② of the first six-way valve. Then it flows from the port ② of the first six-way valve to the port ③ of the first six-way valve, and then from the port ③ of the first six-way valve to the chromatographic column 41 for separation. The separated components can then enter the triple quadrupole mass spectrometer for detection.

[0053] The present invention also provides a second embodiment of a liquid chromatography method for simplifying clinical mass spectrometry sample pretreatment.

[0054] In the second embodiment, please refer to the connection method of the liquid chromatography injection system. Figure 4 The injector uses a quantitative loop injector, and the interface connection of the first six-way valve 21 adopts the second connection method. The specific operation is as follows: (1) Provide a sample of plasma containing anti-tuberculosis drug concentration. The sample is precipitated by protein solvent precipitation, and the supernatant is separated by centrifugation. The supernatant is directly used for instrument detection. Provide ultrapure water (containing 0.1% formic acid) as the first mobile phase and methanol (containing 0.1% formic acid) as the second mobile phase. The volume ratio of the first mobile phase to the second mobile phase is 10% / 90%. The total injection volume is 5μL. Set the flow rate of the first mobile phase to 0.05mL / min and the flow rate of the second mobile phase to 0.45mL / min. (2) Sample injection in strong solvent mode: The sample to be tested is temporarily stored in the quantitative loop. The second liquid phase pump 12 pumps the second mobile phase into the port 6 of the first six-way valve 21 by pumping. The second mobile phase flows from port 6 of the first six-way valve 21 to port 5, then from port 5 to port c of the second six-way valve 221, then from port c to port b, carrying the sample in the quantitative loop from port b to port e, then to port e to port d, and finally flows out from port d and carries the sample into port 2 of the first six-way valve 21.

[0055] (3) Elution analysis process: The second mobile phase dissolves the sample to be tested and carries the sample out from port d of the second six-way valve 221 and flows to port ② of the first six-way valve 21. Then it flows from port ② of the first six-way valve 21 to port ① of the first six-way valve 21, and then through port ① of the first six-way valve 21 to the online solvent mixer 3. The first liquid phase 11 pumps the first mobile phase into the online solvent mixer 3 by pumping. The first mobile phase, the second mobile phase and the sample to be tested are mixed by the online solvent mixer 3. Then it flows out from the online solvent mixer 3 and flows to port ④ of the first six-way valve 21. Then it flows through port ④ to port ③ of the first six-way valve. From port ③ of the first six-way valve to the chromatographic column 41 for separation. The separated components can then enter the triple quadrupole mass spectrometer for detection.

[0056] The present invention also provides a third embodiment of a liquid chromatography method for simplifying clinical mass spectrometry sample pretreatment.

[0057] For the connection method of the liquid chromatography injection system in the third embodiment, please refer to [link / reference needed]. Figure 5 In this case, a flow-through needle autosampler is used, and the interface connection of the first six-way valve adopts the second connection method. The specific steps are as follows: (1) Provide a sample of plasma containing anti-tuberculosis drug concentration. After the sample is precipitated by protein solvent precipitation and the supernatant is separated by centrifugation, the supernatant is directly used for detection. Provide ultrapure water (containing 0.1% formic acid) as the first mobile phase and methanol (containing 0.1% formic acid) as the second mobile phase. The volume ratio of the first mobile phase to the second mobile phase is 10% / 90%, the total injection volume is 5μL, and the flow rate of the first mobile phase is set to 0.05mL / min and the flow rate of the second mobile phase is set to 0.45mL / min. (2) Sample injection in strong solvent mode: The sample to be tested is temporarily stored in the quantitative loop. The second liquid phase pump 12 pumps the second mobile phase into the port 6 of the first six-way valve 21 by pumping. The second mobile phase flows from the port 6 of the first six-way valve 21 to the port 5, and then from the port 5 to the port d of the second six-way valve 221. Then it flows from the port d to the port c, carrying the sample in the sampling needle and the buffer tube to the port e. It flows out from the port e and carries the sample into the port 2 of the first six-way valve 21.

[0058] (3) Elution analysis process: The second mobile phase dissolves the sample to be tested and carries the sample out from port d of the second six-way valve 221 and flows to port ② of the first six-way valve 21. Then it flows from port ② of the first six-way valve 21 to port ① of the first six-way valve 21, and then through port ① of the first six-way valve 21 to the online solvent mixer 3. The first liquid phase 11 pumps the first mobile phase into the online solvent mixer 3 by pumping. The first mobile phase, the second mobile phase and the sample to be tested are mixed by the online solvent mixer 3. Then it flows out from the online solvent mixer 3 and flows to port ④ of the first six-way valve 21. Then it flows through port ④ to port ③ of the first six-way valve. From port ③ of the first six-way valve to the chromatographic column 41 for separation. The separated components can then enter the triple quadrupole mass spectrometer for detection.

[0059] The chromatographic analysis results of the antibacterial drugs pyrazinamide and clofazimine under two injection modes were tested according to the method described in the above embodiments. The results are shown in Table 1 and... Figure 6 As shown.

[0060] Table 1 shows the quantitative determination results of two antibacterial drugs in actual clinical plasma samples using two different injection methods. Figure 6 The results show the peak shape and peak height of the antibacterial drug pyrazinamide under two injection modes.

[0061] Table 1. Quantitative determination results of two antibacterial drugs in actual clinical plasma samples using two injection modes.

[0062] Note: When calculating CV%, the traditional model is used as the standard method, and the CV value is calculated by standard deviation / mean.

[0063] As shown in Table 1, strong solvent injection yields results nearly identical to those obtained with traditional injection (CV% < 5%), but with a 40-minute reduction in sample pretreatment time. This is primarily due to the elimination of the nitrogen drying step in strong solvent injection. This methodological reduction in sample pretreatment time significantly improves clinical testing efficiency and shortens TAT ​​(test turnaround time).

[0064] from Figure 6 It can be seen that when using the two injection modes to detect the antibacterial drug pyrazinamide, the second six-way valve interface connection method, namely the strong solvent direct injection mode (on-column dilution mode), has better peak shape and higher mass spectrometry response intensity. This indicates that compared with the traditional injection mode, the strong solvent injection mode can not only shorten the sample pretreatment time but also has the ability to detect analytes at lower concentrations.

[0065] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples, characterized in that, For blood drug concentration monitoring, the simplified clinical mass spectrometry sample pretreatment liquid chromatography method is based on the following liquid chromatography injection system, which includes: The infusion system includes a first liquid phase pump and a second liquid phase pump, wherein the first liquid phase pump is used to pump in a first mobile phase and the second liquid phase pump is used to pump in a second mobile phase; The injection system includes a first six-way valve and an injector module. The six ports of the first six-way valve are ①, ②, ③, ④, ⑤ and ⑥ respectively. The port connection mode of the first six-way valve includes a first connection mode or a second connection mode. Online solvent mixer; and, Separation and detection system, including chromatographic column; The first liquid phase pump is connected to the inlet end of the online solvent mixer, the second liquid phase pump is connected to the ⑥ port of the first six-way valve, the ⑤ port of the first six-way valve is connected to the inlet end of the injector module, the outlet end of the injector module is connected to the ② port of the first six-way valve, the ① port of the first six-way valve is connected to the inlet end of the online solvent mixer, the outlet end of the online solvent mixer is connected to the ④ port of the first six-way valve, and the ③ port of the first six-way valve is connected to the chromatographic column. Based on the above liquid chromatography injection system, the simplified liquid chromatography method for clinical mass spectrometry sample pretreatment includes the following steps: S10. Turn on the first and second liquid phase pumps and inject the sample to be tested through the injector module; S20. The first liquid phase pump pumps the first mobile phase into the online solvent mixer by pumping, and the second liquid phase pump pumps the second mobile phase into the port ⑥ of the first six-way valve by pumping.

2. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The second mobile phase has a stronger elution capacity for the target analyte than the first mobile phase.

3. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 2, characterized in that, The first mobile phase is selected from solvents that are weak eluting solvents in terms of chromatographic retention behavior based on the column, and the second mobile phase is selected from solvents that are strong eluting solvents in terms of chromatographic retention behavior based on the column.

4. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The first six-way valve is a two-position six-way switching valve.

5. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The interface connection method of the first six-way valve adopts the first connection method: The second mobile phase flows from port 6 of the first six-way valve to port 1 of the first six-way valve, and then from port 1 of the first six-way valve to the inlet of the online solvent mixer. The first mobile phase and the second mobile phase are mixed in an online solvent mixer to obtain a mixed mobile phase. The mixed mobile phase flows out from the outlet end of the online solvent mixer and flows to the port ④ of the first six-way valve. Then it flows from the port ④ of the first six-way valve to the port ⑤ of the first six-way valve, and then from the port ⑤ of the first six-way valve to the inlet end of the injector module, forming a mixture containing the first mobile phase, the second mobile phase and the sample to be tested. The mixture containing the first mobile phase, the second mobile phase, and the sample to be tested flows out from the outlet of the injector module and flows to the port ② of the first six-way valve, then flows from the port ② of the first six-way valve to the port ③ of the first six-way valve, and then flows from the port ③ of the first six-way valve to the chromatographic column.

6. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The interface connection method of the first six-way valve adopts the second connection method: The second mobile phase flows from port 6 of the first six-way valve to port 5 of the first six-way valve, and then from port 5 of the first six-way valve to the inlet of the injector module. The second mobile phase dissolves the sample to be tested and forms a mixture of the second mobile phase and the sample to be tested. Then it flows out from the outlet end of the injector module and flows to the interface ② of the first six-way valve. Then it flows from the interface ② of the first six-way valve to the interface ① of the first six-way valve, and then from the interface ① of the first six-way valve to the inlet end of the online solvent mixer. The first mobile phase and the mixture of the second mobile phase and the sample to be tested are mixed in an online solvent mixer to form a mixture containing the first mobile phase, the second mobile phase and the sample to be tested. The mixture containing the first mobile phase, the second mobile phase and the sample to be tested flows out from the outlet of the online solvent mixer and flows to the port ④ of the first six-way valve, then flows from the port ④ of the first six-way valve to the port ③ of the first six-way valve, and then flows from the port ③ of the first six-way valve to the chromatographic column.

7. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The injector module includes an injector, which may be either a quantitative loop injector or a flow needle injector.

8. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 1, characterized in that, The injector module also includes a second six-way valve, the six ports of which are a, b, c, d, e and f respectively.

9. The simplified liquid chromatography method for pretreatment of clinical mass spectrometry samples as described in claim 8, characterized in that, The second six-way valve is a two-position six-way switching valve.

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