High-stability liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage

Abstract

The invention discloses a high-stability liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage, which comprises the following steps of: acquiring electrospray voltage required by a liquid chromatograph operating in a stable conical injection mode under different organic phase and water phase proportions at an interval of 5 percent, and then calculating the electrospray voltage according to the liquid chromatography gradient of the liquid chromatograph set by an experiment to obtain the high-stability liquid chromatography-mass spectrometry data of the high-stability liquid chromatography-mass spectrometry data of the high-stability liquid chromatography-mass spectrometry data of the high-stability liquid chromatography-mass spectrometry data of the high-stability liquid chromatography-mass spectrometry. Gradually recording the ratio of the organic phase to the water phase, the time corresponding to the ratio of the organic phase to the water phase and the optimal electrospray voltage in the set liquid chromatogram gradient at an interval of 5% to obtain variable electrospray voltage setting data of the mass spectrometer, and starting to collect data of the liquid chromatogram-mass spectrometer after the variable electrospray voltage is set; the device has the advantages that the liquid chromatograph-mass spectrometer can always run in a stable conical injection mode in the whole liquid chromatography elution process, so that a mass spectrum signal with high strength, high stability and good multi-needle experiment result repeatability can be obtained.

Description

Technical Field

[0001] The invention relates to a highly stable liquid chromatography-mass spectrometry data acquisition method, in particular to a highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage. Background Art

[0002] Liquid chromatography-mass spectrometry has become an important research equipment in the field of life sciences with its high resolution, high sensitivity, and high throughput, and has played an irreplaceable role in research in disciplines such as proteomics, metabolomics, clinical medicine, and biomarker exploration. The electrospray ion source is currently the standard ion source technology for liquid chromatography-mass spectrometry. The electrospray ion source will not destroy the chemical bonds of the sample to be tested during the ionization process, so the ions generated by the electrospray ion source can maintain their original molecular structure without generating fragment ions, thereby better analyzing the mass of the intact molecule. In addition, the use of an electrospray ion source can make the sample to be tested carry multiple charges, thereby effectively reducing the mass-to-charge ratio of the analyte ion, which enables mass spectrometers with a limited mass-to-charge ratio detection range to be applied to the detection of important biological macromolecules such as nucleic acids and proteins.

[0003] Although the liquid chromatography-mass spectrometer, which consists of a liquid chromatograph and a mass spectrometer with an electrospray ion source, provides great advantages for the analysis of complex biological samples, it also introduces new problems. Under different voltage conditions, electrospray will present different spray modes, including sputtering mode, spindle spray mode, intermittent cone spray mode, stable cone spray mode, twisted cone spray mode and bifurcation mode. Among all the spray modes, when the electrospray is operated in the stable cone spray mode, the mass spectrometry signal intensity is the strongest and the repeatability and stability are the best. In other spray modes, the mass spectrometry signal intensity will be significantly reduced and serious random fluctuations will occur, resulting in reduced repeatability and stability of the mass spectrometry signal. Although the electrospray ion source can be operated in the cone spray mode within a certain voltage range, this voltage range will change significantly with the change of the ratio of organic phase to aqueous phase in the liquid chromatograph mobile phase. During the liquid phase gradient elution process, the mobile phase composition will continue to change over time. However, existing electrospray ion sources all operate at a fixed electrospray voltage, which means that the electrospray ion source can only operate in a stable conical spray mode at a specific ratio of organic phase to aqueous phase, but cannot operate in a stable conical spray mode during the entire liquid phase gradient elution process of the liquid chromatograph. Therefore, the liquid chromatography-mass spectrometer consisting of a liquid chromatograph and a mass spectrometer with an electrospray ion source is prone to problems such as low mass spectrometry signal intensity, poor stability, and poor repeatability of multi-needle experimental results. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage, which can enable a liquid chromatography-mass spectrometer to always operate in a stable conical injection mode during the entire liquid chromatography elution process, thereby obtaining a mass spectrometry signal with high intensity, high stability and good repeatability of multi-needle experimental results.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage, characterized in that the electrospray voltage required for the liquid chromatograph to operate in a stable conical injection mode at different organic phase and aqueous phase ratios is first collected at intervals of 5%, and then, according to the liquid chromatography gradient of the liquid chromatograph set experimentally, the organic phase and aqueous phase ratio, the time corresponding to the organic phase and aqueous phase ratio and the optimal electrospray voltage in the set liquid chromatography gradient are recorded successively at intervals of 5%, to obtain the variable electrospray voltage setting data of the mass spectrometer, and after setting the variable electrospray voltage, the liquid chromatography-mass spectrometry data is started to be collected.

[0006] Compared with the prior art, the advantage of the present invention is that the electrospray voltage required for the liquid chromatograph to operate in the electrospray stable conical spray mode at different organic phase and aqueous phase ratios is collected at intervals of 5%, and then the variable electrospray voltage is set according to the liquid chromatographic gradient of the liquid chromatograph set in the experiment. The specific standard is to record the organic phase and aqueous phase ratio and the time corresponding to the organic phase and aqueous phase ratio in the set liquid chromatographic gradient in succession at intervals of 5%, and use the time corresponding to each recorded organic phase and aqueous phase ratio as the duration of its optimal electrospray voltage, use the time corresponding to all recorded organic phase and aqueous phase ratios and their optimal electrospray voltage as the variable electrospray voltage setting data of the mass spectrometer, and after setting the variable electrospray voltage, the variable electrospray The mass spectrometry signal data generated by the liquid chromatography-mass spectrometry instrument is collected under a voltage. During the operation of the liquid chromatography-mass spectrometry instrument, since the electrospray voltage changes with time, the optimal electrospray voltage is automatically matched at different times. As a result, the electrospray ion source can operate at the optimal electrospray voltage required for the stable conical spray mode during the entire liquid chromatography-mass spectrometry data collection process, that is, it always operates in the stable conical spray mode. Therefore, the present invention can enable the liquid chromatography-mass spectrometry instrument to always operate in the stable conical spray mode during the entire liquid chromatography elution process, thereby obtaining a liquid chromatography-mass spectrometry instrument signal with high intensity, high stability and good repeatability of multi-needle experimental results, thereby significantly improving the accuracy of liquid chromatography-mass spectrometry qualitative and quantitative analysis.

[0007] Furthermore, a highly stable liquid chromatography mass spectrometry data acquisition method based on a variable electrospray voltage comprises the following steps:

[0008] Step 1: Under different ratios of organic phase to aqueous phase, the optimal voltage required for the electrospray ion source of the mass spectrometer to operate in a stable conical spray mode is collected, wherein the sum of the ratios of the organic phase to the aqueous phase is always 100%. The specific collection process is as follows:

[0009] S1.1, setting the proportion of the aqueous phase in the liquid chromatograph of the liquid chromatography-mass spectrometry instrument to 100%, and the proportion of the organic phase to 0%;

[0010] S1.2. If the electrospray ion source of the mass spectrometer is in positive ion mode, the electrospray voltage of the electrospray ion source is increased from 10V every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage. Then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode;

[0011] If the electrospray ion source of the mass spectrometer is in negative ion mode, the electrospray voltage is decreased from -10V, and is decreased every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained, until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage, then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode;

[0012] S1.3, according to the voltage range required for the electrospray ion source to operate in a stable conical spray mode obtained in step S1.2, obtain the middle value of the voltage range, use the middle value as the optimal electrospray voltage under the current ratio of organic phase to aqueous phase, and record the current ratio of organic phase to aqueous phase and the optimal electrospray voltage under the current ratio of organic phase to aqueous phase;

[0013] S1.4, adjusting the proportion of the aqueous phase and the proportion of the organic phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer at intervals of 5% one by one, reducing the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer by 5% and increasing the proportion of the organic phase by 5% each time the adjustment is made, and repeating steps S1.2 to S1.3 until the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer is reduced to 0% and the proportion of the organic phase is increased to 100%;

[0014] S1.5, saving all currently recorded organic phase and aqueous phase ratios and the optimal electrospray voltage under each organic phase and aqueous phase ratio in an optimal electrospray voltage database;

[0015] Step 2: Determine the change electrospray voltage of the mass spectrometer according to the liquid chromatography gradient of the actual liquid chromatography elution process. The specific process is as follows:

[0016] S2.1. Determine whether the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient is a certain organic phase and aqueous phase ratio stored in the optimal electrospray voltage database. If so, record the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and the time corresponding to the initial ratio of the organic phase and the aqueous phase, obtain the optimal electrospray voltage under the initial ratio of the organic phase and the aqueous phase from the optimal electrospray voltage database for recording, and complete the first recording. If not, select an organic phase and aqueous phase ratio that is closest to the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and stored in the optimal electrospray voltage database, record the selected organic phase and aqueous phase ratio and the time corresponding to the organic phase and aqueous phase ratio, obtain the optimal electrospray voltage under the organic phase and aqueous phase ratio from the optimal electrospray voltage database for recording, and complete the first recording;

[0017] S2.2. After the first recording is completed, the ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient and the time corresponding to the ratio of the organic phase to the aqueous phase are recorded one by one at intervals of 5%. Each time the recording is made, the organic phase ratio changes by 5% relative to the previous ratio, and the optimal electrospray voltage under the currently recorded organic phase to aqueous phase ratio is selected from the optimal electrospray voltage database until the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is recorded or the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is less than 5% relative to the currently recorded organic phase to aqueous phase ratio;

[0018] S2.3, taking the time corresponding to each recorded ratio of organic phase to aqueous phase as the duration of its optimal electrospray voltage, and using the time corresponding to all recorded ratios of organic phase to aqueous phase and their optimal electrospray voltage as the variable electrospray voltage setting data of the mass spectrometer;

[0019] Step 3, setting the liquid chromatography gradient of the actual liquid chromatography elution process in the liquid chromatograph, and setting the variable electrospray voltage in the mass spectrometer according to the variable electrospray voltage setting data obtained in step S2.3;

[0020] Step 4: Start the liquid chromatography-mass spectrometer to perform liquid chromatography elution, and collect mass spectrum data through the mass spectrometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a variation trend diagram of the total current signal (i.e., the total mass spectrometry current signal) collected by the liquid chromatography-mass spectrometer under different electrospray voltage conditions when the electrospray ion source of the mass spectrometer is in the positive ion mode;

[0022] Figure 2 The graph is a trend chart of the optimal electrospray voltage value under different ratios of organic phase to aqueous phase;

[0023] Figure 3 is a schematic diagram of the liquid chromatography gradient;

[0024] Figure 4 Schematic diagram of electrospray voltage changing with liquid chromatography gradient;

[0025] Figure 5 The effect diagram of three liquid chromatography mass spectrometry signals acquired using a fixed electrospray voltage;

[0026] Figure 6 The image shows the effect of three liquid chromatography mass spectrometry signals acquired using varying electrospray voltages. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings.

[0028] Embodiment 1: A highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage, firstly, at intervals of 5%, the electrospray voltage required for the liquid chromatograph to operate in a stable conical injection mode at different ratios of organic phase to aqueous phase is collected, and then, according to the liquid chromatography gradient of the liquid chromatograph set experimentally, the ratio of organic phase to aqueous phase in the set liquid chromatography gradient, the time corresponding to the ratio of organic phase to aqueous phase and the optimal electrospray voltage are recorded successively at intervals of 5%, to obtain the variable electrospray voltage setting data of the mass spectrometer, and after setting the variable electrospray voltage, the liquid chromatography-mass spectrometry data is collected.

[0029] In this embodiment, during the operation of the liquid chromatography-mass spectrometry instrument, because the electrospray voltage changes with time, the optimal electrospray voltage is automatically matched at different times, thereby enabling the electrospray ion source to operate at the optimal electrospray voltage required for the stable conical spray mode during the entire liquid chromatography-mass spectrometry data acquisition process, that is, always operate in the stable conical spray mode. Therefore, the present invention enables the liquid chromatography-mass spectrometry instrument to always operate in the stable conical spray mode during the entire liquid chromatography elution process, thereby being able to obtain a liquid chromatography-mass spectrometry instrument signal with high intensity, high stability and good repeatability of multi-needle experimental results, thereby significantly improving the accuracy of liquid chromatography-mass spectrometry qualitative and quantitative analysis.

[0030] Embodiment 2: A highly stable liquid chromatography mass spectrometry data acquisition method based on variable electrospray voltage, comprising the following steps:

[0031] Step 1: Under different ratios of organic phase to aqueous phase, the optimal voltage required for the electrospray ion source of the mass spectrometer to operate in a stable conical spray mode is collected, wherein the sum of the ratios of the organic phase to the aqueous phase is always 100%. The specific collection process is as follows:

[0032] S1.1, setting the proportion of the aqueous phase in the liquid chromatograph of the liquid chromatography-mass spectrometry instrument to 100%, and the proportion of the organic phase to 0%;

[0033] S1.2. If the electrospray ion source of the mass spectrometer is in positive ion mode, the electrospray voltage of the electrospray ion source is increased from 10V every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage. Then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode;

[0034] If the electrospray ion source of the mass spectrometer is in negative ion mode, the electrospray voltage is decreased from -10V, and is decreased every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained, until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage, then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode;

[0035] S1.3, according to the voltage range required for the electrospray ion source to operate in a stable conical spray mode obtained in step S1.2, obtain the middle value of the voltage range, use the middle value as the optimal electrospray voltage under the current ratio of organic phase to aqueous phase, and record the current ratio of organic phase to aqueous phase and the optimal electrospray voltage under the current ratio of organic phase to aqueous phase;

[0036] S1.4, adjusting the proportion of the aqueous phase and the proportion of the organic phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer at intervals of 5% one by one, reducing the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer by 5% and increasing the proportion of the organic phase by 5% each time the adjustment is made, and repeating steps S1.2 to S1.3 until the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer is reduced to 0% and the proportion of the organic phase is increased to 100%;

[0037] S1.5, saving all currently recorded organic phase and aqueous phase ratios and the optimal electrospray voltage under each organic phase and aqueous phase ratio in an optimal electrospray voltage database;

[0038] Step 2: Determine the change electrospray voltage of the mass spectrometer according to the liquid chromatography gradient of the actual liquid chromatography elution process. The specific process is as follows:

[0039] S2.1. Determine whether the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient is a certain organic phase and aqueous phase ratio stored in the optimal electrospray voltage database. If so, record the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and the time corresponding to the initial ratio of the organic phase and the aqueous phase, obtain the optimal electrospray voltage under the initial ratio of the organic phase and the aqueous phase from the optimal electrospray voltage database for recording, and complete the first recording. If not, select an organic phase and aqueous phase ratio that is closest to the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and stored in the optimal electrospray voltage database, record the selected organic phase and aqueous phase ratio and the time corresponding to the organic phase and aqueous phase ratio, obtain the optimal electrospray voltage under the organic phase and aqueous phase ratio from the optimal electrospray voltage database for recording, and complete the first recording;

[0040] S2.2. After the first recording is completed, the ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient and the time corresponding to the ratio of the organic phase to the aqueous phase are recorded one by one at intervals of 5%. Each time the recording is made, the organic phase ratio changes by 5% relative to the previous ratio, and the optimal electrospray voltage under the currently recorded organic phase to aqueous phase ratio is selected from the optimal electrospray voltage database until the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is recorded or the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is less than 5% relative to the currently recorded organic phase to aqueous phase ratio;

[0041] S2.3, taking the time corresponding to each recorded ratio of organic phase to aqueous phase as the duration of its optimal electrospray voltage, and using the time corresponding to all recorded ratios of organic phase to aqueous phase and their optimal electrospray voltage as the variable electrospray voltage setting data of the mass spectrometer;

[0042] Step 3, setting the liquid chromatography gradient of the actual liquid chromatography elution process in the liquid chromatograph, and setting the variable electrospray voltage in the mass spectrometer according to the variable electrospray voltage setting data obtained in step S2.3;

[0043] Step 4: Start the liquid chromatography-mass spectrometer to perform liquid chromatography elution, and collect mass spectrum data through the mass spectrometer.

[0044] In this embodiment, when the electrospray ion source of the mass spectrometer is in the positive ion mode, during the process of increasing the electrospray voltage of the electrospray ion source, the total current signal collected by the liquid chromatography-mass spectrometer will first continue to increase, but the signal is highly unstable. When the electrospray voltage is increased to a certain voltage value, within the voltage range formed by the voltage value and a previous voltage value, the total current signal collected by the liquid chromatography-mass spectrometer will be highly stable and the signal will be the strongest. When the electrospray voltage exceeds the voltage value, the total current signal collected by the liquid chromatography-mass spectrometer will decrease again and become unstable. Therefore, by increasing the electrospray voltage of the electrospray ion source, a voltage range in which the total current signal collected by the liquid chromatography-mass spectrometer will be highly stable and the signal will be the strongest can be found, and this voltage range can be used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode. Similarly, when the electrospray ion source of the mass spectrometer is in the negative ion mode, during the process of decreasing the electrospray voltage, the total current signal collected by the liquid chromatography-mass spectrometer will be highly stable and the signal will be the strongest. The current signal will first continue to rise but the signal is highly unstable. When the electrospray voltage drops to a certain voltage value, within the voltage range formed by this voltage value and a previous voltage value, the total current signal collected by the liquid chromatography-mass spectrometry will be highly stable and the signal will be the strongest. When the electrospray voltage is lower than this voltage value, the total current signal collected by the liquid chromatography-mass spectrometry will drop again and become unstable. Therefore, by increasing the electrospray voltage of the electrospray ion source, a voltage range in which the total current signal collected by the liquid chromatography-mass spectrometry will be highly stable and the signal will be the strongest can be found, and this voltage range is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode. When the electrospray ion source of the mass spectrometer is in positive ion mode, a relationship curve between the electrospray voltage and the total current signal is drawn according to the set electrospray voltage of the electrospray ion source and the total current signal collected by the liquid chromatography-mass spectrometry at each electrospray voltage, that is, the change trend of the mass spectrometry total current signal under different electrospray voltage conditions, from Figure 1It can be seen that the total current signal collected by the liquid chromatography-mass spectrometer under the electrospray voltage increased for the third time is greatly improved compared with the total current signal collected by the liquid chromatography-mass spectrometer under the electrospray voltage increased for the second time, and the total current signal collected by the liquid chromatography-mass spectrometer under the electrospray voltage increased for the eighth time is greatly reduced compared with the total current signal collected by the liquid chromatography-mass spectrometer under the electrospray voltage increased for the seventh time. Within the voltage range formed by the electrospray voltage increased for the third time and the electrospray voltage increased for the seventh time, the total current signal collected by the liquid chromatography-mass spectrometer will be highly stable and have the strongest signal, so that the voltage range formed by the electrospray voltage increased for the third time and the electrospray voltage increased for the seventh time can be used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode.

[0045] In this embodiment, a trend diagram of the optimal electrospray voltage value under different organic phase and aqueous phase ratios is drawn, such as Figure 2 As shown, from Figure 2 It can be seen that as the proportion of organic phase in the liquid chromatograph increases, the optimal electrospray voltage will show a linear downward trend accordingly.

[0046] In order to verify the effect of the highly stable liquid chromatography mass spectrometry data acquisition method based on variable electrospray voltage of the present invention, the existing method using fixed electrospray voltage is compared with the highly stable liquid chromatography mass spectrometry data acquisition method based on variable electrospray voltage of the present invention. Figure 3 As shown, Figure 3 Schematic diagram of the change of electrospray voltage during the liquid chromatography gradient change is shown in Figure 4 As shown, the three-time liquid chromatography mass spectrometry signal effect diagram obtained by using a fixed electrospray voltage is shown in Figure 5 As shown, the three liquid chromatography mass spectrometry signal effect diagrams obtained by using the variable electrospray voltage of the present invention are as follows Figure 6 Analysis Figure 5 It can be seen that, using a fixed electrospray voltage, the electrospray ion source cannot always operate in the conical spray mode during the entire liquid chromatography elution process. The reproducibility of the liquid chromatography mass spectrometry data obtained by repeated acquisition is extremely poor, and the total current signal of the mass spectrometry is difficult to repeat, which seriously affects the accuracy of the qualitative and quantitative analysis of the liquid chromatography mass spectrometry data. Figure 6 As shown, by utilizing the variable electrospray voltage proposed in the high-stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage of the present invention, the electrospray ion source almost always operates in the conical spray mode during the entire liquid chromatography elution process. Therefore, the liquid chromatography-mass spectrometry data obtained by multiple acquisitions will be highly repeatable, thereby significantly improving the accuracy of liquid chromatography-mass spectrometry qualitative and quantitative analysis.

[0047] In summary, the highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage of the present invention enables the liquid chromatography-mass spectrometer to always operate in a stable conical injection mode during the entire liquid chromatography elution process, thereby obtaining a mass spectrometry signal with high intensity, high stability and good repeatability of multi-needle experimental results.

Claims

1. A highly stable liquid chromatography mass spectrometry data acquisition method based on variable electrospray voltage, characterized in that First, the electrospray voltage required for the liquid chromatograph to operate in a stable conical injection mode at different organic and aqueous phase ratios is collected at intervals of 5%. Then, according to the liquid chromatographic gradient of the liquid chromatograph set in the experiment, the organic and aqueous phase ratios in the set liquid chromatographic gradient, the time corresponding to the organic and aqueous phase ratios and the optimal electrospray voltage are recorded successively at intervals of 5% to obtain the variable electrospray voltage setting data of the mass spectrometer. After setting the variable electrospray voltage, the liquid chromatography-mass spectrometry data is collected.

2. The highly stable liquid chromatography-mass spectrometry data acquisition method based on variable electrospray voltage according to claim 1, characterized in that The following steps are involved: Step 1: Under different ratios of organic phase to aqueous phase, the optimal voltage required for the electrospray ion source of the mass spectrometer to operate in a stable conical spray mode is collected, wherein the sum of the ratios of the organic phase to the aqueous phase is always 100%. The specific collection process is as follows: S1.1, setting the proportion of the aqueous phase in the liquid chromatograph of the liquid chromatography-mass spectrometry instrument to 100%, and the proportion of the organic phase to 0%; S1.

2. If the electrospray ion source of the mass spectrometer is in positive ion mode, the electrospray voltage of the electrospray ion source is increased from 10V every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage. Then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode; If the electrospray ion source of the mass spectrometer is in negative ion mode, the electrospray voltage is decreased from -10V, and is decreased every two minutes by 10V each time, and the total current signal collected by the liquid chromatography-mass spectrometer at each electrospray voltage is obtained, until a voltage range consisting of two electrospray voltages is obtained, and the total current signal fluctuation at any two electrospray voltages does not exceed 10%, and the total current signal at the closest electrospray voltage after the voltage range fluctuates by more than 10% relative to the total current signal at the previous electrospray voltage, then the voltage range consisting of the two voltages is used as the voltage range required for the electrospray ion source to operate in a stable conical spray mode; S1.3, according to the voltage range required for the electrospray ion source to operate in a stable conical spray mode obtained in step S1.2, obtain the middle value of the voltage range, use the middle value as the optimal electrospray voltage under the current ratio of organic phase to aqueous phase, and record the current ratio of organic phase to aqueous phase and the optimal electrospray voltage under the current ratio of organic phase to aqueous phase; S1.4, adjusting the proportion of the aqueous phase and the proportion of the organic phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer at intervals of 5% one by one, reducing the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer by 5% and increasing the proportion of the organic phase by 5% each time the adjustment is made, and repeating steps S1.2 to S1.3 until the proportion of the aqueous phase set in the liquid chromatograph of the liquid chromatograph-mass spectrometer is reduced to 0% and the proportion of the organic phase is increased to 100%; S1.5, saving all currently recorded organic phase and aqueous phase ratios and the optimal electrospray voltage under each organic phase and aqueous phase ratio in an optimal electrospray voltage database; Step 2: Determine the change electrospray voltage of the mass spectrometer according to the liquid chromatography gradient of the actual liquid chromatography elution process. The specific process is as follows: S2.

1. Determine whether the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient is a certain organic phase and aqueous phase ratio stored in the optimal electrospray voltage database. If so, record the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and the time corresponding to the initial ratio of the organic phase and the aqueous phase, obtain the optimal electrospray voltage under the initial ratio of the organic phase and the aqueous phase from the optimal electrospray voltage database for recording, and complete the first recording. If not, select an organic phase and aqueous phase ratio that is closest to the set initial ratio of the organic phase and the aqueous phase in the liquid chromatography gradient and stored in the optimal electrospray voltage database, record the selected organic phase and aqueous phase ratio and the time corresponding to the organic phase and aqueous phase ratio, obtain the optimal electrospray voltage under the organic phase and aqueous phase ratio from the optimal electrospray voltage database for recording, and complete the first recording; S2.

2. After the first recording is completed, the ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient and the time corresponding to the ratio of the organic phase to the aqueous phase are recorded one by one at intervals of 5%. Each time the recording is made, the organic phase ratio changes by 5% relative to the previous ratio, and the optimal electrospray voltage under the currently recorded organic phase to aqueous phase ratio is selected from the optimal electrospray voltage database until the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is recorded or the final ratio of the organic phase to the aqueous phase in the set liquid chromatography gradient is less than 5% relative to the currently recorded organic phase to aqueous phase ratio; S2.3, taking the time corresponding to each recorded ratio of organic phase to aqueous phase as the duration of its optimal electrospray voltage, and using the time corresponding to all recorded ratios of organic phase to aqueous phase and their optimal electrospray voltage as the variable electrospray voltage setting data of the mass spectrometer; Step 3, setting the liquid chromatography gradient of the actual liquid chromatography elution process in the liquid chromatograph, and setting the variable electrospray voltage in the mass spectrometer according to the variable electrospray voltage setting data obtained in step S2.3; Step 4: Start the liquid chromatography-mass spectrometer to perform liquid chromatography elution, and collect mass spectrum data through the mass spectrometer.

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