Urine analysis method based on gas phase ion chromatography

Abstract

The invention provides an analysis method for measuring negative ions in urine based on gas phase ion chromatography, and belongs to the technical field of urine analysis. One of the urine analysis methods based on gas phase ion chromatography can comprise the following steps: injecting a chelating ligand into an analyzer through a continuous sample injector electrospray to obtain a background signal; a to-be-detected urine sample is fed one by one through an online six-way valve automatic sampler, a chelating ligand solution and the urine sample can be fully mixed through an online mixer, electrospray ionization is realized, the mixture enters an analyzer, an ion chromatograph records a sample signal, and a background is deducted to obtain a urine analysis result; wherein the ionization technology is an electrospray ion source. According to the method disclosed by the invention, separation of various ions on the gas phase ion chromatography can be realized; the kit can also be used for qualitatively and quantitatively detecting trace inflammatory factors, trace proteins and other proteins in urine, and can be applied to prevention and treatment of kidney stone.

Description

Technical Field

[0001] The present invention relates to the technical field of urine analysis, and in particular to a urine analysis method based on gas phase ion chromatography. Background Art

[0002] Existing quantitative urine testing technologies fall into three categories: 1. Volatile substances, typically using gas chromatography; 2. Ions and salts, typically using ion chromatography; and 3. Proteins, typically using immunochemiluminescence. Existing semi-quantitative urine testing techniques rely on dry chemical methods, using urine test strips for colorimetry. These methods are unable to distinguish between multiple ions or proteins, and therefore cannot be directly applied to the testing for kidney stones, nephritis, or renal damage.

[0003] Ion mobility (gas-phase ion chromatography) is a technique that separates gas-phase ions based on their size, shape, and charge. First, the molecules or ions being analyzed pass through an ion source, forming gas-phase ions. An ion funnel or ion guide creates an ion flow. Controlled by the electric field of a pulse gate, these ions form an ion pulse and enter the separation region for analysis. The ion pulse, consisting of ions of varying sizes and shapes, is accelerated by the electric field and moves forward. During this motion, they collide with the buffer gas molecules in the drift region, creating resistance that slows them down. The kinetic energy lost during collisions is converted into internal energy, raising the ion temperature. Further collisions transfer this increased internal energy to the gas molecules, restoring the system temperature. Therefore, the temperature and velocity of ions do not remain constant during their motion. Electrostatic attraction and Coulomb repulsion may also exist between ions and between ions and the buffer gas, making the motion of ions in the drift region extremely complex and quantifiable only by their average velocity (i.e., ion mobility) or the time td it takes for an ion to pass through the drift region. If the original ion pulse contains ions with different drift velocities, these ions will separate into multiple peaks, a process similar to chromatography. To enable comparison of measurements under different experimental conditions, ion mobility is often converted to reduced ion mobility in practical applications. This refers to ion mobility at 273K and 760 Torr. The size and shape of ions can be measured using the average available cross-section when ions collide with the buffer gas, also known as the collision cross section (CCS).

[0004] As can be seen from the above, low-field ion mobility separation is mainly based on the shape and size of the ions. Under high-frequency and high-field conditions, it is also related to the polarization and polarizability of the ions. This technology can use different detectors. When using a Faraday cup current detector, separation similar to different retention times in chromatography can be obtained. The usual separation time is microseconds (μs) to seconds (s). Depending on the experimental conditions and the gas used, nitrogen is usually used, which can be converted into standard collision cross-section (CCS) data. It is a simple and effective rapid detector. This technology is synchronized with the mass spectrometer in time. When a mass spectrometer is used as a detector, the peaks of the gas phase ion chromatogram can be further separated by mass-to-charge ratio. Under the detector conditions of the mass spectrometer, qualitative and quantitative analysis can be obtained. The mass spectrometer as a detector effectively improves the qualitative and quantitative analysis capabilities of various complex biological samples. At the same time, the complexity and price of the mass spectrometer are also greatly increased.

[0005] Common positive and negative ions in urine (e.g., Na + ,K + ,Ca 2+ ,Mg 2+ , phosphate, oxalate, citrate, etc.) have not been reported or applied in practice. Experiments have found that the retention time of gas ion chromatography is too short, making it impossible to detect or difficult to separate. Furthermore, because the electrospray efficiency of ions in urine changes with increasing salinity, negative ions are difficult to ionize due to severe ion suppression, resulting in a very narrow linear dynamic range.

[0006] Therefore, it is of great practical significance to provide a urine analysis method that can improve ionization efficiency and simultaneously increase the retention time and retention time difference of gas ion chromatography. Summary of the Invention

[0007] The purpose of the present invention is to provide a urine analysis method based on gas phase ion chromatography, aiming to solve the technical problems in the prior art that the retention time of gas phase ion chromatography is too short, and it cannot be detected or is not easy to separate.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a urine analysis method based on gas phase ion chromatography, comprising the following steps:

[0010] The chelating ligand was electrospray injected into the analyzer through a continuous injector to obtain the background signal;

[0011] The urine samples to be tested are injected one by one through an online six-way valve automatic sampler. The chelating ligand solution and urine sample are fully mixed by an online mixer and electrospray ionized into the analyzer. The ion chromatograph will record the sample signal and obtain the urine analysis result after subtracting the background signal.

[0012] Among them, the ionization technology used was electrospray ionization source.

[0013] Furthermore, the chelating ligand structure is shown in Formula 1, Formula 2 and Formula 3.

[0014] Furthermore, the formula 1 and formula 2 are specifically:

[0015]

[0016] Furthermore, the formula 3 is specifically:

[0017]

[0018] Furthermore, the concentration ratio of the chelating ligand to the urine sample to be tested is 1:0.1-100.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] (1) The present invention forms gas-phase positive ions of complex ions by complexing the negative ions in the sample to be tested with a chelating ligand during electrospraying, thereby greatly increasing the collision cross section of the characteristic ions and the retention time of these ions in gas-phase ion chromatography. At the same time, the retention time of different ions can be increased, thereby achieving the separation of multiple ions in gas-phase ion chromatography;

[0021] (2) The present invention utilizes highly efficient electrospray ionization chelate ligands. By measuring the positive ions of the chelate, the ionization efficiency of these negative ions is greatly increased, overcoming the ion signal suppression effect commonly seen in electrospray ionization of such ions.

[0022] (3) The present invention adopts new derivatization and ionization technologies, and uses a gas phase ion mobility spectrometer to ionize non-volatile substances in urine. For example, the electrospray / atmospheric pressure chemical ionization combined ion source increases the detection range of the gas phase ion mobility spectrometer. In particular, charged particles such as biological macromolecules and salts can be analyzed by the gas phase ion mobility spectrometer.

[0023] (4) The urine analysis method of the present invention can detect metabolic molecules, lipid molecules, trace inflammatory factors, cytokines, trace albumin, and other proteins in urine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the gas ion chromatography mass spectrometer used in the present invention;

[0025] Figure 2 This is a schematic diagram of the urine analysis method of the present invention;

[0026] Figure 3This is a schematic diagram of the results obtained in Example 1 of the present invention;

[0027] Figure 4 This is a schematic diagram of the results obtained in Example 2 of the present invention;

[0028] Figure 5 This is a schematic diagram of the results obtained in Example 3 of the present invention;

[0029] Figure 6 This is a schematic diagram of the results obtained in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The present invention provides a urine analysis method based on gas phase ion chromatography, comprising the following steps:

[0031] The chelating ligand was electrospray injected into the analyzer through a continuous injector to obtain the background signal;

[0032] The urine samples to be tested are injected one by one through an online six-way valve automatic sampler. The chelating ligand solution and urine sample are fully mixed by an online mixer and electrospray ionized into the analyzer. The ion chromatograph will record the sample signal and obtain the urine analysis result after subtracting the background.

[0033] Among them, the ionization technology used was electrospray ionization source.

[0034] The urine analysis method of the present invention may further include the following steps: mixing the urine sample to be tested with the chelating ligand to obtain a mixed sample to be tested, and injecting the mixed sample into an analyzer, wherein the ion source of the analyzer is an electrospray ion source;

[0035] The mixing of the urine sample to be tested and the chelating ligand to obtain the mixed sample to be tested is specifically performed by aspirating the above solution into an automatic sampling syringe, the needle of the syringe is connected to the continuous sampler of the tims-ToF, and the sample is injected into the analyzer at a flow rate of 10-1000 microliters per minute;

[0036] The analyzer automatically injects the mixed sample to be tested, and the continuous injector ionizes the mixed sample to be tested into a spray ion source;

[0037] The spray ion source is detected by gas phase ion chromatography to obtain urine analysis results.

[0038] In the present invention, the structures of the chelating ligands are shown in Formula 1, Formula 2 and Formula 3.

[0039] In the present invention, Formula 1 and Formula 2 are specifically:

[0040]

[0041] In the present invention, the formula 3 is specifically:

[0042]

[0043] In the present invention, the concentration ratio of the chelating ligand to the urine sample to be tested is 1:0.1-100.

[0044] The urine analysis method of the present invention can also be: dissolving the chelating ligand in an electrospray solvent to form a homogeneous solution, continuously electrospraying, connecting six copper valves and a mixer in series in the pipeline, and automatically injecting the unknown urine to be tested.

[0045] In the present invention, unless otherwise specified, the raw materials required for preparation are all commercially available products well known to those skilled in the art.

[0046] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] The chelating ligand was electrospray injected into the analyzer through a continuous injector to obtain the background signal;

[0049] The urine samples to be tested are injected one by one through an online six-way valve automatic sampler. The chelating ligand solution and urine sample are fully mixed by an online mixer and electrospray ionized into the analyzer. The ion chromatograph will record the sample signal and obtain the urine analysis result after subtracting the background.

[0050] Among them, the ionization techniques used were electrospray ionization sources;

[0051] Through the above experiment, the complex ions formed by dihydrogen phosphate and hydrogen phosphate ions and the above chelating agent were detected in artificial urine, with a mass-to-charge ratio of m / z 970 (phosphate @1) and a mass-to-charge ratio of m / z 978 (phosphate @2). The results are as follows Figure 3 shown.

[0052] Example 2

[0053] The same steps as those described in Example 1 were performed. The results were as follows: oxalate ions and the complex ions formed by the above-mentioned chelating agent were detected in the artificial urine, with a mass-to-charge ratio m / z of 962 (oxalic acid @ 1) and a mass-to-charge ratio m / z of 970 (oxalic acid @ 2). Figure 4 shown.

[0054] Example 3

[0055] The same steps as those described in Example 1 were performed. The results were as follows: complex ions formed by citrate ions and the above-mentioned chelating agent were detected in artificial urine, with a mass-to-charge ratio m / z of 1065 (citric acid @ 1) and a mass-to-charge ratio m / z of 1072 (citric acid @ 2). Figure 5shown.

[0056] Example 4

[0057] The same steps as those described in Example 1 were performed. The results were as follows: the complex ions formed by dihydrogen phosphate and hydrogen phosphate ions and the above-mentioned three-clawed chelating agent were detected in the artificial urine, with a mass-to-charge ratio m / z of 605 (phosphate @ 3). Figure 6 shown.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A urine analysis method based on gas phase ion chromatography, characterized in that: The following steps are involved: The chelating ligand was injected into the analyzer through a continuous injector electrospray to obtain the background signal; The urine samples to be tested are injected one by one through an online six-way valve automatic sampler. The chelating ligand solution and urine sample are fully mixed by an online mixer and electrospray ionized into the analyzer. The ion chromatograph will record the sample signal and obtain the urine analysis result after subtracting the background signal. Among them, the ionization technology used was electrospray ionization source.

2. The urine analysis method based on gas phase ion chromatography according to claim 1, characterized in that: The structures of the chelating ligands are shown in Formula 1, Formula 2 and Formula 3.

3. The urine analysis method based on gas phase ion chromatography according to claim 2, characterized in that: The formula 1 and formula 2 are specifically:

4. The urine analysis method based on gas phase ion chromatography according to claim 2, characterized in that: The formula 3 is specifically:

5. The urine analysis method based on gas phase ion chromatography according to claim 1, characterized in that: The ratio of the total concentration of the chelating ligand to the total salt concentration of the urine sample to be tested is 1:0.1-100.

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