Data acquisition method for improving screening sensitivity of mass spectrometer

Abstract

The data acquisition method for improving the screening sensitivity of the mass spectrometer comprises the steps that a database meeting the standard is established in advance, the database comprises multiple sets of configuration parameters, and each set of configuration parameters comprises the precursor ion mass-to-charge ratio and the product ion mass-to-charge ratio of one or more target compounds; and acquiring precursor ion spectrograms and product ion spectrograms of all target compounds in the database by adopting an MPRM mode. The MPRM mode is composed of a plurality of PRM stages which are continuously executed and is time extension of the PRM stages, each PRM stage is responsible for collecting secondary mass spectrograms of a group of target compounds, and operating parameters of the PRM are provided by a database. According to the method, the low-concentration additive can be efficiently screened in the complex matrix, and meanwhile, extra operation time is not increased.

Description

Technical Field

[0001] The invention belongs to the technical field of mass spectrometer analysis and relates to a data acquisition method for improving the screening sensitivity of a mass spectrometer. Background Art

[0002] Mass spectrometer is an important tool for analyzing chemical substances. It can achieve qualitative and quantitative analysis of sample components by measuring the mass-to-charge ratio (m / z) of ions. Mass spectrometry analysis usually includes sample ionization, ion separation by a mass analyzer, and signal recording by a detector to generate a mass spectrum. Due to its high sensitivity and high throughput, mass spectrometry technology has been widely used in life sciences, drug development, and environmental analysis.

[0003] Data-Dependent Acquisition (DDA) is a commonly used screening mode for mass spectrometers. Its working principle is based on the ion signal intensity detected in real time in the full scan (MS1) spectrum, and the ions with the strongest signal are preferentially selected for fragmentation (MS2) analysis. The DDA mode has strong specificity and can generate high-quality fragmentation spectra, making it an important means of compound screening. However, the DDA mode is overly dependent on ion signal intensity. In complex samples, low-abundance ions may be ignored due to ion competition effects, resulting in insufficient screening sensitivity and omission of potential information.

[0004] In contrast, Data-Independent Acquisition (DIA) avoids selection bias and improves component coverage by synchronously fragmenting all ions within a predetermined mass-to-charge ratio window. However, the fragmentation spectra generated by the DIA mode are often more complex and difficult to interpret, which is not conducive to rapid screening. Therefore, the DDA mode is still the first choice in many screening tasks, but how to further improve its sensitivity remains an important challenge. Summary of the invention

[0005] To solve the above technical problems, the present invention aims to provide a data acquisition method for improving the screening sensitivity of a mass spectrometer, which can efficiently screen low-concentration additives in complex matrices without increasing additional operating time.

[0006] The present invention provides a data acquisition method for improving the screening sensitivity of a mass spectrometer, comprising:

[0007] A database meeting the standards is established in advance, wherein the database includes multiple groups of configuration parameters, and each group of configuration parameters includes the mass-to-charge ratio of precursor ions and the mass-to-charge ratio of product ions of one or more target compounds;

[0008] The MPRM mode was used to acquire the precursor ion spectra and product ion spectra of all target compounds in the database.

[0009] Furthermore, the MPRM mode consists of a plurality of continuously executed PRM stages, which is a temporal extension of the PRM stage. Each PRM stage is responsible for collecting a set of secondary mass spectra of target compounds, and the operating parameters of PRM are provided by a database.

[0010] Furthermore, two front automatic gain control stages AGC were introduced before each PRM stage to dynamically adjust the injection time, and both AGC stages adopted the selected ion monitoring mode SIM of the mass spectrometer.

[0011] Furthermore, the two automatic gain control stages AGC are respectively the AGC-1 stage and the AGC-2 stage;

[0012] In the AGC-1 stage, according to the configuration parameter grouping in the database, the mass spectrometer performs SIM at a fixed injection time to obtain the precursor ion spectrum of the group of target compounds in the AGC-1 stage, and calculates the injection time of the AGC-2 stage according to the number of target precursor ions obtained in the AGC-1 stage;

[0013] In the AGC-2 stage, according to the configuration parameter grouping in the database, the mass spectrometer performs SIM with the calculated injection time to obtain the AGC-2 stage precursor ion spectrum of the group of target compounds, and compares the mean of the total number of target precursor ions obtained in the AGC-2 stage with the preset threshold. If the mean of the total number of target precursor ions is lower than the preset threshold, the corresponding PRM stage is automatically skipped and it is determined that the group of target compounds does not exist; otherwise, the PRM stage is executed to obtain the product ion spectrum.

[0014] Furthermore, the ion isolation signals in the AGC-1 stage, the AGC-2 stage and the PRM stage adopt broadband ion excitation signals with real-time generation capability, and the signals are generated in real time by a field programmable gate array, which can shorten the detection time and improve the screening throughput.

[0015] A data acquisition method for improving the screening sensitivity of a mass spectrometer according to the present invention has the following beneficial effects:

[0016] The traditional DDA mode usually selects the ion with the highest signal intensity in the full spectrum for fragmentation analysis, but this method is difficult to guarantee the screening effect when the target concentration is low or the sample background is complex. To solve this problem, the present invention proposes a data acquisition method for improving the screening sensitivity of a mass spectrometer, and designs a targeted data-dependent acquisition mode (targeted Data-Dependent Acquisition, tDDA). When performing target screening, the pre-established target database is automatically read, and the mass spectrometer is controlled to scan the secondary mass spectra of the target one by one according to the database content. The database contains multiple groups of configuration parameters, each group of configuration parameters contains one or more target precursor ions that are isolated and targeted, and their existence is verified through a PRM stage. If the target precursor ion exists, its corresponding secondary spectrum is further scanned, otherwise the relevant steps are skipped to save running time.

[0017] When screening each target compound, the method of the present invention will run two pre-AGC stages (AGC-1 and AGC-2) in advance, and automatically calculate the injection time of the AGC-2 stage based on the intensity of the target ion in the AGC-1 stage to avoid overcorrection that may be caused by a single AGC, thereby improving the accuracy of the screening results.

[0018] In the method of the present invention, isolated signals such as broadband ion excitation signals that have real-time generation capability or very short generation time are used in both the AGC stage and the PRM stage. The rapid generation of isolated signals ensures that the acquisition process is completed quickly, and the running time will not be too long due to the increase in the number of targets in the database. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a flow chart of a data acquisition method for improving the screening sensitivity of a mass spectrometer according to the present invention;

[0020] Figure 2a is the calibration curve of Zolpidem (m / z 308) obtained using an ion trap mass spectrometer in Example 1;

[0021] Figure 2b is a full scan mass spectrum of a pure sample of Zolpidem at the detection limit concentration in Example 1;

[0022] Figure 2c are the precursor ion spectrum and product ion spectrum of the pure Zolpidem sample under the tDDA mode at the detection limit concentration in Example 1;

[0023] Figure 2d is a full scan mass spectrum of a blood sample pretreated by adding a pure sample of Zolpidem (m / z 308) to a blood sample in Example 1;

[0024] Figure 2eThe precursor ion spectrum and product ion spectrum obtained in the tDDA mode when pure Zolpidem sample (m / z 308) was added to the blood sample in Example 1;

[0025] Figure 2f is a full scan mass spectrum of the pretreated blood sample in which the three target substances are added to the blood sample at the same time in Example 1;

[0026] Figure 2g The precursor ion spectrum and product ion spectrum obtained in the tDDA mode when the three target substances in Example 1 are added to the blood sample at the same time;

[0027] Figure 3a is the experimental result of three tDDA mode screenings in Example 2;

[0028] Figure 3b is the full scan mass spectrum of the saliva sample in Example 2;

[0029] Figure 3c This is the spectrum of the AGC-1 stage using the tDDA mode in Example 2;

[0030] Figure 3d This is the spectrum of the AGC-2 stage using the tDDA mode in Example 2;

[0031] Figure 3e This is the spectrum of the PRM stage using the tDDA mode in Example 2. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings.

[0033] like Figure 1 As shown, a data acquisition method for improving the screening sensitivity of a mass spectrometer of the present invention comprises:

[0034] A database that meets the standards is pre-established, and the database contains multiple groups of configuration parameters, each group of configuration parameters containing precursor ion mass-to-charge ratios and product ion mass-to-charge ratios of one or more target compounds.

[0035] The MPRM mode is used to collect the precursor ion spectra and product ion spectra of all target compounds in the database. The MPRM mode consists of multiple PRM stages that are executed continuously, which is an extension of the PRM stage in time, similar to the extension of the SRM to MRM mode on a triple quadrupole. Each PRM stage is responsible for collecting a set of secondary mass spectra of target compounds, and the operating parameters of PRM are provided by the database.

[0036] Through the above-mentioned logical improvements, the tDDA mode designed in the present invention avoids the reliance on full-spectrum scanning in the traditional DDA mode, significantly improves the targeting and efficiency of screening, and at the same time improves the detection sensitivity for low-abundance targets.

[0037] Since the intensity of the target precursor ions cannot be determined in advance, using a fixed injection time may result in the target ion intensity after isolation being too high or too low. Too low precursor ion intensity may result in insufficient product ion intensity, resulting in a false negative result; while too high precursor ion intensity may result in changes in peak shape or peak position shift, which is also prone to false negatives.

[0038] To solve the above problems, the tDDA mode designed in the present invention introduces two pre-stage automatic gain control stages AGC before each PRM stage to dynamically adjust the injection time and ensure that the number of target precursor ions is maintained within a suitable range. Both AGC stages use the selected ion monitoring mode SIM of the mass spectrometer. In specific implementation, the two automatic gain control stages AGC are AGC-1 stage and AGC-2 stage respectively.

[0039] In the AGC-1 stage, the mass spectrometer performs SIM at a fixed injection time according to the grouping of configuration parameters in the database to obtain the AGC-1 stage precursor ion spectrum of the group of target compounds. The injection time of the AGC-2 stage is calculated based on the number of target precursor ions obtained in the AGC-1 stage.

[0040] However, since the concentration of the target and its ionization efficiency may vary significantly, the signal intensity of the AGC-1 stage usually has a large uncertainty and is often not optimal. The introduction of the AGC-2 stage is intended to avoid the overcorrection of a single gain control and ensure the optimal number of precursor ions in the ion trap during the PRM stage through the second gain control.

[0041] In the AGC-2 stage, according to the configuration parameter grouping in the database, the mass spectrometer performs SIM at the calculated injection time to obtain the AGC-2 stage precursor ion spectrum of the target compound in the group, and compares the mean of the total number of target precursor ions obtained in the AGC-2 stage with the preset threshold. If the mean of the total number of target precursor ions is lower than the preset threshold, it is determined that the target compound in the group does not exist, and the corresponding PRM stage is automatically skipped to avoid unnecessary waste of running time and improve screening efficiency. Otherwise, the PRM stage is executed to obtain the product ion spectrum.

[0042] Since two SIM scans with AGC are required for each target compound in the database in the tDDA mode, the AGC in the tDDA mode is called targeted AGC, which ensures the quality and consistency of the secondary mass spectra in all PRM stages.

[0043] In specific implementation, the ion isolation signals in the AGC-1 stage, AGC-2 stage and PRM stage adopt broadband ion excitation signals with real-time generation capability. The signals are generated in real time by a field programmable gate array, which can shorten the detection time and improve the screening throughput.

[0044] The specific application embodiments of the present invention are further described below.

[0045] Embodiment 1:

[0046] Figure 2a The calibration curve for Zolpidem (m / z 308) was obtained using an ion trap mass spectrometer, giving a limit of detection (LOD) of 5 ng / ml. Figure 2b This is a full scan mass spectrum of a pure sample of zolpidem at the detection limit concentration. Due to the low concentration of zolpidem, the background noise is more obvious. The 20 peaks with the highest intensity are marked in the full spectrum, including the characteristic peak of the target zolpidem. This shows that in the absence of complex chemical background interference, the traditional DDA mode can still effectively detect the target even at a low concentration. Figure 2c The precursor ion spectrum and product ion spectrum of the pure zolpidem sample under the tDDA mode designed in the present invention at the detection limit concentration are shown.

[0047] In order to verify the ability of the tDDA mode designed in the present invention to screen the target in a complex matrix background, pure zolpidem (m / z 308) was added to the blood sample, and pretreatments such as protein precipitation and solid phase extraction were performed. The concentration of zolpidem was controlled at 5 ng / mL. Figure 2d The full scan mass spectrum of the pretreated blood sample is shown. The 20 peaks with the highest intensity in the full spectrum are highlighted in red in the figure. Due to the complex composition of the blood matrix and significant background noise, the peak-finding algorithm failed to successfully identify the characteristic peaks of zolpidem. This shows that the traditional DDA mode has very limited screening capabilities for low-concentration targets in complex matrices. The tDDA mode designed by the present invention was used to perform targeted screening on the same blood sample. Through the pre-established target database, the tDDA mode performed targeted AGC and PRM scans on the precursor ions of zolpidem, and finally successfully obtained Figure 2e Precursor and product ion spectra are shown.

[0048] Zolpidem, pethidine hydrochloride and clozapine were added to the blood sample at the same time. After pretreatment, the concentration of the target substances was controlled to 10 ng / mL. Figure 2fAs shown in the figure, the 20 most intense peaks in the full spectrum are highlighted in red, but the added target was not detected. The broadband ion excitation signal waveform used in the tDDA mode has parallel isolation capabilities, allowing the tDDA mode to screen multiple targets simultaneously in one PRM operation. Figure 2g The precursor ion spectra and product ion spectra obtained in parallel screening using tDDA mode are shown, successfully confirming the presence of the three additives.

[0049] Embodiment 2:

[0050] The tDDA mode operation needs to be based on a pre-established database. In this example, a screening database covering multiple categories such as pesticides, veterinary drugs, psychotropic drugs, natural poisons and illegal drugs was established, containing a total of 200 target compounds. Four samples were selected from the database, namely bupropion (m / z 240), papaverine (m / z 340.2), reserpine (m / z609.3) and erythromycin (m / z 716). The above four samples were added to saliva. After pretreatment, the concentration of each additive was controlled at 100ng / mL. Figure 3a The results of three tDDA mode screening experiments are shown, in which blocks filled with different colors represent the probability of the substance appearing, and the darker the color, the higher the positive probability; the green filled blocks represent the actual added target. It can be clearly seen from the figure that the positive probability of the actual added target is significantly higher than that of other entries in the database, and the results of the three parallel screenings are not less than 50%, showing high stability. The tentative positive threshold of this embodiment is 50%, that is, the test result is judged to be positive if it exceeds 50%. In the three tDDA mode screenings, the false negative rate was 0, and the false positive rate was 3 / 196, 2 / 196 and 2 / 196, respectively. False positives occasionally occur in the test without adding samples, but the false positives are not stably reproduced in the three screening results. Rapid re-examination of suspicious targets and raising the positive threshold can further reduce the probability of false positives. Figure 3b The full scan mass spectrum of the saliva sample. The targeted screening data of papaverine were extracted from the results of the first rapid screening in tDDA mode. The spectra of AGC-1, AGC-2 and PRM stages are shown in Figure 2. Figure 3c , 3d and 3e.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the concept of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A data acquisition method for improving the screening sensitivity of a mass spectrometer, characterized in that: include: A database meeting the standards is established in advance, wherein the database includes multiple groups of configuration parameters, and each group of configuration parameters includes the mass-to-charge ratio of precursor ions and the mass-to-charge ratio of product ions of one or more target compounds; The MPRM mode was used to acquire the precursor ion spectra and product ion spectra of all target compounds in the database.

2. The data acquisition method for improving the screening sensitivity of a mass spectrometer according to claim 1, characterized in that: The MPRM mode consists of a plurality of continuously executed PRM stages, which is an extension of the PRM stage in time. Each PRM stage is responsible for collecting a set of secondary mass spectra of target compounds, and the operating parameters of PRM are provided by a database.

3. The data acquisition method for improving the screening sensitivity of a mass spectrometer according to claim 2, characterized in that: Two front automatic gain control stages AGC were introduced before each PRM stage to dynamically adjust the injection time. Both AGC stages used the selected ion monitoring mode SIM of the mass spectrometer.

4. The data acquisition method for improving the screening sensitivity of a mass spectrometer according to claim 3, characterized in that: The two automatic gain control stages AGC are AGC-1 stage and AGC-2 stage; In the AGC-1 stage, according to the configuration parameter grouping in the database, the mass spectrometer performs SIM at a fixed injection time to obtain the precursor ion spectrum of the group of target compounds in the AGC-1 stage, and calculates the injection time of the AGC-2 stage according to the number of target precursor ions obtained in the AGC-1 stage; In the AGC-2 stage, according to the configuration parameter grouping in the database, the mass spectrometer performs SIM with the calculated injection time to obtain the AGC-2 stage precursor ion spectrum of the group of target compounds, and compares the mean of the total number of target precursor ions obtained in the AGC-2 stage with the preset threshold. If the mean of the total number of target precursor ions is lower than the preset threshold, the corresponding PRM stage is automatically skipped and it is determined that the group of target compounds does not exist; otherwise, the PRM stage is executed to obtain the product ion spectrum.

5. The data acquisition method for improving the screening sensitivity of a mass spectrometer according to claim 4, characterized in that: The ion isolation signals in the AGC-1 stage, AGC-2 stage and PRM stage adopt broadband ion excitation signals with real-time generation capability. The signals are generated in real time by a field programmable gate array, which can shorten the detection time and improve the screening throughput.

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