Compound identification by mass spectrometry

Abstract

A mass spectrum of the compound of interest is received over time. From the mass spectrum, a primary XIC of a primary product ion of the compound is calculated, as well as a secondary XIC of a secondary product ion. From the intensities of one or more points of the primary peak of the primary XIC corresponding to the one or more times, a primary value is calculated, and from the intensities of one or more points of the secondary peak of the secondary XIC corresponding to the one or more times, a secondary value is calculated. Alternatively, the primary value is calculated as the area of a time window within the primary peak, and the secondary value is calculated as the area of the time window within the secondary peak. From the primary value and the secondary value, a ratio is calculated.

Description

[0001] Related US applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 107911, filed October 30, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The teachings of this article involve operating separation devices and mass spectrometers to identify one or more compounds of interest. Background Technology

[0004] primary and secondary peak ratio

[0005] Liquid chromatography (LC) is a well-known technique for separating and analyzing compounds from mixtures of samples. LC is typically coupled with mass spectrometry (LC-MS) or with tandem mass spectrometry (LC-MS / MS). As described below, a series of mass spectra generated from LC-MS or LC-MS / MS over time form a chromatogram. For example, the chromatogram generated for a compound of interest is called an extracted ion chromatogram (XIC).

[0006] A chromatogram and XIC consist of one or more chromatographic peaks. These peaks are intensity versus retention time, where retention time refers to the time since separation from LC. LC-MS or LC-MS / MS peaks are used to identify or quantify compounds in a sample mixture. Compounds are quantified or measured by calculating peak areas. This calculation of peak areas is often called peak integration.

[0007] When performing LC-MS or LC-MS / MS quantization, in many applications, ion ratios are calculated and reported to confirm that the correct LC peak is integrated and that it is relatively undisturbed. The ion ratio is the ratio of the integrated LC peak of the main or quantitative peak of the same compound to the integrated LC peak of the secondary or qualitative peak. For example, multiple reaction monitoring (MRM) scans, predetermined MRM scans, or triggered MRM scans are used to acquire XICs of the main ion and one or more secondary ions. These MRM methods are described below.

[0008] If the ion ratio of a particular sample matches the expected value, this confirms that the reported result is likely accurate and corresponds to the correct compound. Note that the expected value is typically determined experimentally based on reliable standards. Ion ratio calculations are very common and effective. They are also essential for many workflows, including clinical and environmental screening applications. Exemplary clinical applications include, but are not limited to, determining the concentration of a particular vitamin in a blood sample. Exemplary environmental screening applications include, but are not limited to, screening samples from a batch of vegetables for a large number of possible pesticides.

[0009] Unfortunately, conventional methods for calculating ion ratios can pose at least two challenges or problems for LC-MS or LC-MS / MS. First, calculating ion ratios can double or triple the number of chromatographic peaks requiring manual inspection. For example, manual inspection doubles when analyzing only one secondary ion, and triples when analyzing two. These manual inspections, in turn, lead to a corresponding increase in the number of peaks with integration problems requiring manual correction. While features such as "exception checks" and improvements to core peak-finding algorithms can alleviate this burden, the inspection and correction process remains labor-intensive. In other words, calculating ion ratios significantly increases the amount of time required for manual analysis and correction of LC-MS or LC-MS / MS data.

[0010] Second, conventional methods limit the number of compounds of interest that can be monitored simultaneously because mass spectrometers continuously monitor both the main ion and (one or more) secondary ions for each compound of interest. Newer methods for performing LC-MS or LC-MS / MS analyses (e.g., using triggered MRM scans) increase the number of compounds that can be monitored in screening experiments by continuously monitoring only the main ion of each compound of interest and then switching to secondary ion acquisition when the main ion exceeds a specified threshold. However, in this case, if the trigger threshold is set too high, it may be impossible to obtain the entire chromatographic peak of the secondary ion. Therefore, the peak integral of the secondary ion may be very inaccurate, which also reduces the accuracy of ion ratios, potentially leading to incorrect ion ratio reports. Therefore, very low trigger thresholds are often set to ensure the capture of the complete secondary ion peak. Unfortunately, low trigger thresholds result in a greater number of false alarms where the measured intensity of the main ion is above the threshold but not because the main ion was actually detected. This slows down the instrument's duty cycle and defeats the purpose of monitoring only the main ion.

[0011] Figure 2 An exemplary XIC graph 200 of the main ion and secondary ion illustrates how triggering the MRM of the secondary ion by the MRM of the main ion can result in an incomplete chromatographic peak for the secondary ion, upon which various embodiments can be implemented. The XIC 210 of the main ion includes a main peak 211. When the intensity 212 of the main ion is detected to be higher than a threshold intensity 213, the MRM of the secondary ion is triggered, resulting in the XIC 220 of the secondary ion. The XIC 220 of the secondary ion includes a secondary peak 221.

[0012] Compared to the main peak 211, the secondary peak 221 does not have a similar peak shape. More specifically, because the MRM of the secondary ion begins after the leading edge of the main peak 211, the points from the leading edge of the secondary peak 221 are missing. Because of the missing points from the leading edge of the secondary peak 221, the integral of the secondary peak 221 will be inaccurate, and any ratio calculated from the integral of the secondary peak 221 will also be inaccurate.

[0013] Therefore, additional systems and methods are needed to calculate ion ratios to reduce the amount of time required for manual analysis and correction of LC-MS or LC-MS / MS data, and to be used in conjunction with new methods for performing LC-MS or LC-MS / MS, such as triggered MRM.

[0014] Mass Spectrometry Background

[0015] As described above, mass spectrometers are typically coupled with separation devices, such as chromatographic devices, to identify and characterize compounds of interest from a sample or to analyze multiple samples. In such a coupled system, the eluting or injected solvent is ionized, and a series of mass spectra are obtained from the eluting solvent at specified time intervals, known as retention times. These retention times range, for example, from 1 second to 100 minutes or more. This series of mass spectra forms a chromatogram, or extracted ion chromatogram (XIC).

[0016] For example, peaks detected in XIC are used to identify or characterize known peptides or compounds in a sample. More specifically, the retention time and / or area of ​​a peak are used to identify or characterize (quantify) known peptides or compounds in a sample. In cases where multiple samples are provided over time by a sample introduction device, the peak retention time is used to align the peak with the correct sample.

[0017] In traditional separation-coupled mass spectrometry (SCMS) systems, fragments or product ions of known compounds are selected for analysis. Tandem mass spectrometry (MS / MS) scans are then performed at each separation interval for a mass range including the product ions. The intensities of the product ions detected in each MS / MS scan are collected over time and analyzed as a spectral aggregate or, for example, by XIC.

[0018] Tandem mass spectrometry, or MS / MS, is a well-known technique for analyzing compounds. Tandem mass spectrometry involves the ionization of one or more compounds from a sample, the selection of one or more precursor ions of one or more compounds, the fragmentation of one or more precursor ions into fragments or product ions, and the mass analysis of the product ions.

[0019] Tandem mass spectrometry can provide both qualitative and quantitative information. Product ion spectra can be used to identify molecules of interest. The intensities of one or more product ions can be used to quantify the amount of compounds present in a sample.

[0020] A wide variety of experimental methods or workflows can be performed using tandem mass spectrometers. These workflows fall into three main categories: targeted acquisition, information-dependent acquisition (IDA) or data-dependent acquisition (DDA), and data-independent acquisition (DIA).

[0021] In targeted acquisition methods, one or more transitions from precursor ions to product ions are predefined for the compound of interest. When the sample is introduced into a tandem mass spectrometer, these one or more transitions are interrogated or monitored during each of multiple time periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ion for each transition and performs targeted mass analysis only on the product ion of that transition. As a result, an intensity (product ion intensity) is generated for each transition. Targeted acquisition methods include, but are not limited to, multiple reaction monitoring (MRM) and selected reaction monitoring (SRM).

[0022] In targeted acquisition methods, the list of transformations is typically queried during each cycle. To reduce the number of transformations queried at any given time, some targeted acquisition methods have been modified to include a retention time or retention time range for each transformation. A specific transformation will only be queried at that retention time or within that retention time range. One type of targeted acquisition method that allows specifying retention times for transformations is called predefined MRM.

[0023] In a pre-defined MRM, a retention time or retention time range is also assigned to each MRM transition to be analyzed during the experiment. Then, during the experiment, MRM transitions are added to or removed from the list of transitions to be performed during each cycle of the tandem mass spectrometer based on their retention time or retention time range. In this way, the number of transitions performed within any single cycle is reduced.

[0024] Unfortunately, in some cases, the compound of interest may not be isolated from the sample at the retention time specified in the predetermined MRM experiment. For example, the predetermined MRM experiment may be performed by different laboratories or under different experimental conditions. Furthermore, predetermined MRM depends on the accuracy and absolute value of the retention time used for each transition.

[0025] Therefore, a method for triggering MRM transitions that is not based on retention time was developed. In this method, one MRM transition triggers one or more other MRM transitions to be analyzed. This MRM method is called triggered MRM.

[0026] In the IDA method, users can specify standards for performing non-targeted mass analysis of product ions while introducing the sample into the tandem mass spectrometer. For example, in the IDA method, a precursor ion or mass spectrometry (MS) survey scan is performed to generate a precursor ion peak list. Users can select standards to filter the peak list to obtain a subset of precursor ions on the peak list. MS / MS is then performed on each precursor ion in the precursor ion subset. Product ion spectra are generated for each precursor ion. MS / MS is repeated on the precursor ions of the precursor ion subset as the sample is introduced into the tandem mass spectrometer.

[0027] However, in proteomics and many other sample types, the complexity and dynamic range of compounds are extremely high. This poses a challenge to traditional targeting and IDA methods, requiring very high-speed MS / MS acquisition to deeply interrogate samples in order to both identify and quantify a wide range of analytes.

[0028] Therefore, a third major class of tandem mass spectrometry methods, the DIA method, was developed. These DIA methods have been used to improve the reproducibility and comprehensiveness of data collected from complex samples. The DIA method can also be called a non-specific fragmentation method. In traditional DIA methods, the role of the tandem mass spectrometer does not change between MS / MS scans based on data acquired in previous precursor ion or product ion scans. Instead, a precursor ion mass range is selected. A precursor ion mass selection window is then stepped across this precursor ion mass range. All precursor ions within this precursor ion mass selection window are fragmented, and all product ions of all precursor ions within this precursor ion mass selection window are mass-analyzed.

[0029] The precursor ion mass selection window used to scan the mass range can be very narrow, making it unlikely that multiple precursors will be present within the window. For example, this type of DIA method is called MS / MS. ALL In MS / MS ALL In this method, a precursor ion mass selection window of approximately 1 amu is scanned or stepped across the entire mass range. Product ion spectra are generated for each 1 amu precursor mass window. The time required to analyze or scan the entire mass range once is called a scan cycle. However, scanning a narrow precursor ion mass selection window across a wide precursor ion mass range during each cycle is impractical for some instruments and experiments.

[0030] Therefore, a large precursor ion mass selection window is used, stepping across the entire precursor mass range, or a selection window with a large width. For example, this type of DIA method is called SWATH acquisition. In SWATH acquisition, the precursor ion mass selection window, stepping across the precursor mass range in each cycle, can have a width of 5-25 amu or even greater. (Compared to MS / MS) ALL The method is similar; all precursor ions in each precursor ion mass selection window are fragmented, and all product ions of all precursor ions in each mass selection window are mass analyzed. Summary of the Invention

[0031] This application relates to systems and methods for identifying compounds of interest by mass spectrometry. More specifically, in some embodiments, systems and methods are provided to calculate the ratio of the sum or average intensity of one or more points of a main chromatographic peak to the sum or average intensity of one or more corresponding points of a secondary chromatographic peak. Additionally, systems and methods are provided to calculate the ratio of the area of ​​a time window within the main peak to the area of ​​the same time window within a secondary peak.

[0032] The systems and methods disclosed herein can be combined with processors, controllers, microcontrollers, or computer systems (such as...) Figure 1 (The computer system) is used to execute.

[0033] A system, method, and computer program product are disclosed for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest. The system may include a separation device, an ion source device, a mass spectrometer, and a processor.

[0034] The separation device separates and fragments or isolates only the compound of interest from the sample at multiple different times. An ion source device ionizes the separated fragments of the compound or only the compound, thereby generating an ion beam. A mass spectrometer analyzes the product ions of the compound from the ion beam at these multiple different times, or selects and fragments the compound and analyzes the product ions by mass. Multiple mass spectra are generated for the compound.

[0035] The processor can use the multiple mass spectrometers to calculate the primary XIC of the main product ion of the compound and the secondary XIC of the secondary product ion of the compound. In a first method, the processor can calculate the primary value based on a combination of the intensities of the primary peak corresponding to one or more times but fewer than all points in the primary XIC, and the secondary value based on a combination of the intensities of the secondary peak corresponding to the same one or more times in the secondary XIC. Alternatively, in a second method, the processor can calculate the primary value as the area of ​​a time window within the primary peak that is smaller than the peak width of the primary peak, and the secondary value as the area of ​​that time window within the secondary peak. The processor can then calculate the ratio between the primary and secondary values.

[0036] These and other characteristics of the applicant's teachings are described in this article. Attached Figure Description

[0037] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0038] Figure 1 This is a block diagram illustrating a computer system on which embodiments of the present teachings can be implemented.

[0039] Figure 2This is an exemplary XIC graph of the primary ion and the secondary ion, which illustrates how the triggering of the primary ion's MRM to the secondary ion's MRM results in an incomplete chromatographic peak for the secondary ion, upon which various embodiments can be implemented.

[0040] Figure 3 yes Figure 2 An exemplary graph of XIC illustrates how, according to various embodiments, ion ratios can be calculated using only a few points from the main ion peak and the secondary ion peak.

[0041] Figure 4 This is an exemplary table showing the average ion ratio and standard deviation of the ion ratio calculated for the primary and secondary ions of vitamin D for 140 samples, using the peak area and the sum of the intensities from points 1, 3, 5, and 7 on each peak, according to various embodiments.

[0042] Figure 5 This illustrates how, according to various embodiments, the area of ​​a narrow time window between the main ion peak and the secondary ion peak is used to calculate the ion ratio. Figure 2 An example graph of XIC.

[0043] Figure 6 This is a schematic diagram of a system for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest, according to various embodiments.

[0044] Figure 7 This is a flowchart illustrating a method, according to various embodiments, for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest.

[0045] Figure 8 This is a schematic diagram of a system comprising one or more different software modules according to various embodiments, the one or more different software modules performing a method for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest.

[0046] Before describing one or more embodiments of this teaching in detail, those skilled in the art will understand that the application of this teaching is not limited to the details of the construction, the arrangement of components, and the arrangement of steps set forth in the following detailed description or illustrated in the accompanying drawings. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be construed as limiting. Detailed Implementation

[0047] Computer-implemented systems

[0048] Figure 1This is a block diagram illustrating a computer system 100 on which embodiments of the present teachings may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for transmitting information, and a processor 104 coupled to the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be random access memory (RAM) or other dynamic storage device, coupled to the bus 102 for storing instructions to be executed by the processor 104. The memory 106 may also be used to store temporary variables or other intermediate information during the execution of instructions to be executed by the processor 104. The computer system 100 also includes a read-only memory (ROM) 108 or other static storage device coupled to the bus 102 for storing static information and instructions for the processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to the bus 102 for storing information and instructions.

[0049] Computer system 100 can be coupled to display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), via bus 102 for displaying information to the computer user. Input device 114, including alphanumeric keys and other keys, is coupled to bus 102 for transmitting information and command selections to processor 104. Another type of user input device is cursor control 116, such as a mouse, trackball, or cursor arrow keys for transmitting directional information and command selections to processor 104 and for controlling cursor movement on display 112. This input device typically has two degrees of freedom on two axes, a first axis (x) and a second axis (y), to allow the device to specify a position in a plane.

[0050] Computer system 100 can execute this teaching. Consistent with certain implementations of this teaching, the result is provided by computer system 100 in response to processor 104 executing one or more sequences of one or more instructions contained in memory 106. These instructions may be read into memory 106 from another computer-readable medium, such as storage device 110. Execution of the sequence of instructions contained in memory 106 causes processor 104 to perform the procedures described herein. Alternatively, this teaching may be implemented using hardwired circuitry instead of or in combination with software instructions. Therefore, implementations of this teaching are not limited to any particular combination of hardware circuitry and software.

[0051] In various embodiments, computer system 100 can be connected across a network to one or more other computer systems (such as computer system 100) to form a networked system. The network can include a private network or a public network (such as the Internet). In a networked system, one or more computer systems can store data and provide data to other computer systems. In a cloud computing scenario, one or more computer systems that store and provide data can be referred to as servers or the cloud. For example, one or more computer systems can include one or more web servers. For example, other computer systems that send data to and receive data from servers or the cloud can be referred to as clients or cloud devices.

[0052] As used herein, the term "computer-readable medium" refers to any medium that participates in providing instructions to processor 104 for execution. Such media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical discs or magnetic disks, such as storage device 110. Volatile media include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wires, and optical fibers, including the lines constituting bus 102.

[0053] Common forms of computer-readable media or computer program products include, for example, floppy disks, flexible disks, hard disks, magnetic tapes or any other magnetic media, CD-ROMs, digital video discs (DVDs), Blu-ray discs, any other optical media, thumb drives, memory cards, RAM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, or any other tangible media from which a computer can read.

[0054] Various forms of computer-readable media may involve carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on a disk of a remote computer. The remote computer may load the instructions into its dynamic memory and transmit the instructions over a telephone line using a modem. A modem local to computer system 100 may receive data over the telephone line and convert the data into an infrared signal using an infrared transmitter. An infrared detector coupled to bus 102 may receive the data carried in the infrared signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 before or after execution by processor 104.

[0055] According to various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium may be a means of storing digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) known in the art for storing software. The computer-readable medium is accessed by a processor adapted to execute the instructions configured to be executed.

[0056] For illustrative and descriptive purposes, various implementations of this teaching have been presented below. This is not exhaustive and does not limit this teaching to the exact form disclosed. Modifications and variations are possible based on the above teaching, or can be derived from the practice of this teaching. Furthermore, the described implementations include software, but this teaching can be implemented as a combination of hardware and software or as hardware alone. This teaching can be implemented using both object-oriented and non-object-oriented programming systems.

[0057] Ion ratios from several points or narrow time windows

[0058] As described above, in many applications, when performing LC-MS or LC-MS / MS quantification, ion ratios are calculated and reported to confirm that the correct LC peak is integrated and that it is relatively undisturbed. An ion ratio is the ratio of the integrated LC peak of the main or quantitative peak of the same compound to the integrated LC peak of the secondary or qualitative peak. In this application, the term "main ion" refers to the representative or target ion of interest. In some embodiments, the main ion may be the highest-intensity fragment or product ion produced after fragmentation. In other cases, "main ion" may include fragments or fragment ions of interest with intensities lower than the highest-intensity product ion. The term "secondary ion" refers to one or more confirmatory ions that provide additional confirmation or assurance that the expected precursor ion has produced a fragment or daughter ion comprising the main ion and (one or more) secondary ions.

[0059] The purpose of assessing the ion ratio is to confirm the expected relative presence of the primary and secondary ions, which provides further assurance regarding the expected precursor ion's role in generating the primary and secondary ions. Therefore, in conjunction with other relevant information (such as retention time, one or more mass filters, etc.), the ion ratio confirms the detection of the expected fragment.

[0060] Examples of systems and methods for calculating ion ratios are presented, which reduce the amount of time required for manual analysis and correction of LC-MS or LC-MS / MS data, and can be used in conjunction with new methods for performing LC-MS or LC-MS / MS, such as triggered MRM.

[0061] In some embodiments, a system and method for compound identification can be provided, wherein a mass spectrometer is configured to monitor whether the intensities of a plurality of main product ions are higher than a predetermined intensity threshold, and if the intensity of one of the plurality of main product ions is detected to be higher than the threshold, the mass spectrometer is operable to activate monitoring of one or more different secondary ions. If the mass spectrometer detects the one or more different secondary ions, the mass spectrometer is operable to calculate a plurality of ion ratios based at least on the plurality of different secondary ions. In some cases, the mass spectrometer may further calculate at least one of the plurality of ion ratios based on the main ion.

[0062] In various embodiments, the ion ratio is calculated using only a few points from the main and secondary ion peaks, or using a narrow time window within the main and secondary ion peaks. Neither of these methods significantly increases the amount of time required for manual analysis and correction of LC-MS or LC-MS / MS data, and can be used in conjunction with newer methods for performing LC-MS or LC-MS / MS, such as triggered MRM.

[0063] In some embodiments, the boundary of the time window coincides with the sampling point of at least one product ion among the product ions. In some embodiments, the width of the time window is an integer multiple of the sampling interval of at least one product ion among the product ions captured within the window. In some embodiments, the width of the time window may include the same number of sampling points for each product ion among the product ions captured within the window.

[0064] Both methods can optionally continue to integrate the entire peak area of ​​the main ion peak to construct a calibration curve, thereby quantifying the compound of interest. However, not using the entire peak area of ​​the secondary ion peak significantly reduces the amount of time required for manual analysis and correction of LC-MS or LC-MS / MS data, and enables these methods to be used with newer methods for performing LC-MS or LC-MS / MS, such as triggered MRM.

[0065] More specifically, in one embodiment, the intensities measured at one or more points (i.e., sampling points) corresponding to one or more retention times on the chromatographic peak of the main ion are summed or averaged to produce the main peak. Similarly, the intensities measured at one or more points corresponding to the same one or more retention times on the chromatographic peak of the secondary ion are summed or averaged to produce the secondary peak. The ratio of the main ion to the secondary ion is then calculated by dividing the main peak by the secondary peak.

[0066] Figure 3 This illustrates how, according to various embodiments, ion ratios can be calculated using only a few points from the main ion peak and secondary ion peak. Figure 2 An example graph of XIC 300. Figure 3This includes the XIC 210 of the main ion and the XIC 220 of the secondary ion. Similarly, the XIC 210 of the main ion includes the main peak 211, and the XIC 220 of the secondary ion includes the secondary peak 221. The ratio of the main ion to the secondary ion is not calculated based on the area of ​​the main peak 211 and the secondary peak 221, but rather using the intensity at multiple points on each peak.

[0067] In a preferred embodiment, the highest intensity point of the main peak 211 is selected. For example, points 311, 312, and 313 of the main peak 211 are selected. The intensities measured at points 311, 312, and 313 on the main peak 211 corresponding to retention times 301, 302, and 303 are summed or averaged to produce the main peak value. Similarly, the intensities measured at points 321, 322, and 323 on the secondary peak 221 corresponding to the same retention times 301, 302, and 303 are summed or averaged to produce the secondary peak value. The ratio of the main ion to the secondary ion is then calculated by dividing the main peak value by the secondary peak value.

[0068] In various embodiments, a timing window is used to select points 311, 312, and 313. For example, the timing window used is centered at time 302 of vertex 312 and has a width extending from time 301 to time 303. Points 311, 312, and 313 include vertex 312. In various embodiments, the size of the timing window or summation window is user-specified. In various alternative embodiments, the size of the timing window or summation window is automatically determined based on the width in points. Note that in this case, for example, each point corresponds to an MRM transition scan or a TOF full scan. TOF data does not have the MRM triggering problem but still benefits from minimizing user inspection.

[0069] exist Figure 3 In this model, neither XIC 210 nor XIC 220 includes a baseline offset. In other words, these XICs are not shifted along the y-axis to achieve a baseline intensity offset. However, in some cases, these XICs may include a baseline offset. Therefore, in various embodiments, the baseline offset can be determined based on the minimum intensity of each XIC. The baseline offset of the primary XIC 210 is then subtracted from the primary peak value, and the baseline offset of the secondary XIC 220 is then subtracted from the secondary peak value; these values ​​are then used to calculate the ratio.

[0070] like Figure 3 As shown, it is not necessary to run the peak-finding algorithm on the secondary XIC 220. Nor is manual checking, correction, or integration of the secondary peak 221 required. Furthermore, Figure 3 As shown, in the case of MRM triggered by MRM, only a few points near the vertex or even along the trailing edge of the main peak 211 are selected, which means that the missing leading edge points of the secondary peak 221 are not used.

[0071] To confirm that this method uses only a few points from the main and secondary ion peaks in the calculation of ion ratios, it was compared with a conventional method that uses peak areas. A large number of clinical samples were obtained and analyzed. For each sample, the ratio was calculated using peak area, peak height of each peak, and the sum of one or more intensities from one or more points on each peak.

[0072] Figure 4 This is an exemplary table 400 showing the average ion ratio and standard deviation of the ion ratio calculated for the primary and secondary ions of vitamin D for 140 samples, using peak area and the sum of intensities from points 1, 3, 5, and 7 on each peak, according to various embodiments. Please note that in editing... Figure 5 The data did not use the integral, which is problematic for the peak.

[0073] Figure 4 The results show that the standard deviations of all methods except those using the intensity at a single point are better than those of the conventional method that uses the peak area to calculate the ratio. Figure 4 It was also shown that increasing the number of points used in the method of summing the intensities from points at each peak improved the standard deviation. However, when the number of points increased to more than three, the standard deviation did not improve significantly. In other words, Figure 4 The results show that using the sum of intensities from only three points provides an average ion ratio comparable to conventional methods using peak areas, and even offers an improvement over this conventional method in terms of standard deviation.

[0074] In the second embodiment, the area of ​​a narrow time window within the chromatographic peak of the main ion is calculated to generate the main peak value. Similarly, the area of ​​the same narrow time window within the chromatographic peak of the secondary ion is calculated to generate the secondary peak value. The ratio of the main ion to the secondary ion is then calculated by dividing the main peak value by the secondary peak value.

[0075] Figure 5 This illustrates how, according to various embodiments, the area of ​​a narrow time window between the main ion peak and the secondary ion peak is used to calculate the ion ratio. Figure 2 Example graph 500 of XIC. Figure 5 This includes the XIC 210 peak of the main ion and the XIC 220 peak of the secondary ion. Similarly, the XIC 210 peak of the main ion includes the main peak 211, and the XIC 220 peak of the secondary ion includes the secondary peak 221. The ratio of the main ion to the secondary ion is not calculated based on the area of ​​the main peak 211 and the secondary peak 221, but rather using the area of ​​a narrower time window for each peak.

[0076] In a preferred embodiment, the time window is centered on the retention time 502 of the apex of the primary peak 211. The width of the time window is smaller than the width of the primary peak 211 and extends, for example, from time 501 to time 503. The area 510 of the narrow time window of the primary peak 211 between time 501 and time 503 is calculated to generate the primary peak value. Similarly, the area 520 of the same narrow time window of the secondary peak 221 between time 501 and time 503 is calculated to generate the secondary peak value. The ratio of primary ions to secondary ions is then calculated by dividing the primary peak value by the secondary peak value.

[0077] like Figure 5 As shown, it is not necessary to run the peak-finding algorithm on the secondary XIC 220. Nor is manual checking, correction, or integration of the secondary peak 221 required. Furthermore, Figure 5 As shown, for the case of MRM triggered by MRM, choosing a narrow time window around the vertex or even along the trailing edge means not using the missing leading edge point of secondary peak 221.

[0078] Figure 5 This illustrates a general case where the time window selects regions within the primary peak 211 and the secondary peak 221. In some embodiments, the time window can be located based on at least one sampling point, such that at least one boundary of the time window coincides with a sampling point of the primary peak 211 or the secondary peak 221. In some aspects, a first boundary of the time window may coincide with a sampling point of one of the primary peak 211 and the secondary peak 221, and a second boundary of the time window.

[0079] In some embodiments, the width of the time window may include the same number of sampling points for each of the primary peak 211 and the secondary peak 221. In some embodiments, the width of the time window may include a different number of sampling points for each of the primary peak 211 and the secondary peak 221. Figure 5 In one example, the time window includes a single sample point from each of the primary peak 211 and the secondary peak 221. In other embodiments, the time window may include additional sample points from the primary peak 211 and the secondary peak 221. In some embodiments, the width of the time window may be an integer multiple of the sampling interval between sample points.

[0080] System for calculating the ratio of primary to secondary peaks

[0081] Figure 6 This is a schematic diagram 600 of a system for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of a compound of interest, according to various embodiments. The system includes a separation device 610, an ion source device 620, a mass spectrometer 630, and a processor 640.

[0082] The separation device 610 separates and fragments or separates only the compound of interest 601 from the sample at multiple different times 611. For example, a gas chromatography (GC) device separates and fragments the compound of interest, while an LC device separates only the compound of interest. The separation device 610 is controlled, for example, by a processor 640. The separation device 610 is shown as an LC separation device. In various alternative embodiments, the separation device can perform one of a wide variety of separation techniques, including but not limited to GC, flow injection analysis (FIA), and capillary electrophoresis (CE).

[0083] Ion source device 620 ionizes a separated fragment of compound 601 or only compound 601, thereby generating an ion beam. Ion source device 620 is controlled, for example, by processor 640. Ion source device 620 is shown as a component of mass spectrometer 630. In various alternative embodiments, ion source device 620 is a standalone device. Ion source device 620 may be, but is not limited to, an electrospray ionization (ESI) source or a chemical ionization (CI) source, such as an atmospheric pressure chemical ionization (APCI) source or an atmospheric pressure photoionization (APPI) source.

[0084] Mass spectrometer 630 performs mass analysis of product ions of compound 601 from an ion beam at multiple different times 611, or selects and fragments compound 601 and performs mass analysis of product ions of compound 601. Multiple mass spectra 631 are generated for compound 601. For example, mass spectrometer 630 is controlled by processor 640.

[0085] exist Figure 6 In the system, mass spectrometer 630 is shown as a triple quadrupole device. Those skilled in the art will understand that any component of mass spectrometer 630 may include other types of mass spectrometry devices, including but not limited to ion traps, orbital traps, time-of-flight (TOF) devices, ion mobility devices, or Fourier transform ion cyclotron resonance (FT-ICR) devices.

[0086] Processor 640 uses multiple mass spectrometers 631 to calculate the primary XIC 641 of the main product ion of compound 601 and the secondary XIC 642 of the secondary product ion of compound 601. In method 643, processor 640 calculates a primary value 645 based on a combination of the intensities of the primary peak of primary XIC 641 corresponding to one or more times but fewer than all points, and calculates a secondary value 646 based on a combination of the intensities of the secondary peak of secondary XIC 642 corresponding to one or more times. Alternatively, in method 644, processor 640 calculates the primary value 645 as the area of ​​a time window within the primary peak that is smaller than the peak width of the primary peak, and calculates the secondary value 646 as the area of ​​that time window within the secondary peak. Processor 640 calculates a ratio 647 based on the primary value 645 and the secondary value 646.

[0087] The processor 640 can be as follows Figure 6 The individual device shown may be the processor or controller of the separation device 610 or the mass spectrometer 630. The processor 640 may be, but is not limited to, a controller, a computer, or a microprocessor. Figure 1 A computer system or any device capable of sending and receiving control signals and data and capable of analyzing data.

[0088] In various embodiments, one or more points of the primary peak include a single point, and one or more points of the secondary peak include a single point.

[0089] In various embodiments, one or more points of the primary peak may include five or fewer points, and one or more points of the secondary peak may include five or fewer points.

[0090] In various embodiments, the combination of intensities at one or more points of the primary peak includes a sum, and the combination of intensities at one or more points of the secondary peak includes a sum.

[0091] In various embodiments, the intensity combination of one or more points of the primary peak includes an average value, and the intensity combination of one or more points of the secondary peak includes an average value.

[0092] In various embodiments, one or more points of the main peak include the apex of the main peak.

[0093] In various embodiments, one or more points of the main peak include only points at or after the time of the peak's apex.

[0094] In various embodiments, the center of the time window corresponds to the time of one or more points on the main peak. For example, this point is the vertex of the main peak.

[0095] In various embodiments, before calculating the ratio 647, the processor 640 subtracts the primary baseline intensity or area value from the primary value 645 and subtracts the secondary primary baseline intensity or area value from the secondary value 646. For example, the primary baseline intensity or area value is calculated based on one or more minimum intensity points of the primary peak, and the secondary baseline intensity or area value is calculated based on one or more minimum intensity points of the secondary peak.

[0096] In various embodiments, the processor 640 receives from the user the number of one or more points of the main peak or the width of a time window within the main peak as input.

[0097] In various embodiments, the processor 640 automatically calculates the number of one or more points of the peak or the width of a time window within the peak based on the width of the peak.

[0098] Methods for calculating the ratio of primary to secondary peaks

[0099] Figure 7This is a flowchart 700 illustrating a method for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest, according to various embodiments.

[0100] In step 710 of method 700, a separation device is used to separate and fragment the sample or to separate only the compound of interest at multiple different times.

[0101] In step 720, an ion source device is used to ionize a separated fragment of the compound or only the compound to generate an ion beam.

[0102] In step 730, a mass spectrometer is used to select and analyze the compound from the ion beam at multiple different times, or to select and fragment the compound from the ion beam and analyze the product ions of the compound, thereby generating multiple mass spectra of the compound.

[0103] In step 740, the processor calculates the major XIC of the major product ion of the compound based on the multiple mass spectra, and calculates the minor XIC of the minor product ion of the compound.

[0104] In step 750, the processor calculates a principal value based on the combination of the intensities of the principal peak of the primary XIC corresponding to one or more times but fewer than all points, and calculates a secondary value based on the combination of the intensities of the secondary peak of the secondary XIC corresponding to one or more points at the same time. Alternatively, the processor calculates the principal value as the area of ​​a time window within the principal peak that is smaller than the peak width of the principal peak, and calculates the secondary value as the area of ​​that time window within the secondary peak.

[0105] In step 760, the processor calculates the ratio based on the major and minor values.

[0106] Computer program products for calculating the ratio of primary to secondary peaks.

[0107] In various embodiments, the computer program product includes a tangible computer-readable storage medium containing a program having instructions that execute on a processor to perform a method for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest. This method is executed by a system comprising one or more different software modules.

[0108] Figure 8 This is a schematic diagram of a system 800 according to various embodiments, comprising one or more different software modules that perform methods for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest. System 800 includes a measurement module 810 and an analysis module 820.

[0109] Measurement module 810 instructs the separation device to separate and fragment or separate only the compound of interest from the sample at multiple different times. Measurement module 810 instructs the ion source device to ionize the separated fragments of the compound or only the compound, thereby generating an ion beam. Measurement module 810 instructs the mass spectrometer to mass analyze the product ions of the compound from the ion beam at these multiple different times, or to select and fragment the compound and mass analyze the product ions of the compound, thereby generating multiple mass spectra.

[0110] Analysis module 820 calculates the main XIC of the main product ion of the compound and the secondary XIC of the secondary product ion of the compound based on the multiple mass spectra. Analysis module 820 calculates the principal value based on the combination of intensities of the main peak of the main XIC corresponding to one or more times but fewer than all points, and calculates the secondary value based on the combination of intensities of the secondary peak of the secondary XIC corresponding to one or more times. Alternatively, analysis module 820 calculates the principal value as the area of ​​a time window within the main peak that is smaller than the peak width of the main peak, and calculates the secondary value as the area of ​​that time window within the secondary peak. Analysis module 820 uses the analysis module to calculate the ratio based on the principal and secondary values.

[0111] While this teaching has been described in conjunction with various embodiments, it is not intended to limit this teaching to such embodiments. Rather, as those skilled in the art will understand, this teaching encompasses various alternatives, modifications, and equivalents.

[0112] Furthermore, in describing various embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, the method or process should not be limited to the specific sequence of steps described herein, as it is not dependent on such a specific order. As will be understood by those skilled in the art, other sequences of steps may be possible. Therefore, the specific sequence of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to performing the steps in the written order, and those skilled in the art will readily understand that the order may vary and still remain within the scope of protection.

Claims

1. A system for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest, comprising: A separation device configured to (i) separate and fragment from a sample at multiple different times or (ii) separate only the compound of interest; An ion source device configured to (i) ionize a separated fragment of a compound or (ii) ionize only the compound, thereby generating an ion beam; A mass spectrometer configured to (i) mass analyze the product ions of a compound from the ion beam at the multiple different times, or (ii) select and fragment the compound and the product ions of the compound for mass analysis, thereby generating multiple mass spectra; as well as Processor, the processor being configured to: 1) Calculate the main extracted ion chromatogram (XIC) of the main product ion of the compound using the aforementioned mass spectrometry, and calculate the secondary XIC of the secondary product ions of the compound. 2a) Receive input from the user or automatically calculate the number of one or more points corresponding to one or more times but less than all points of the main peak based on the width of the main peak of the main XIC, calculate the main value based on the combination of the intensities of the one or more points of the main peak, and calculate the secondary value based on the combination of the intensities of the secondary peaks of the secondary XIC corresponding to the one or more times, or 2b) Receive input from the user or automatically calculate the width of time windows within the main peak that are smaller than the main peak's width based on the main peak's width; calculate the main value as the area of ​​the time window within the main peak and the secondary value as the area of ​​the time window within the secondary peak; and 3) Calculate the ratio based on the principal and secondary values.

2. The system of claim 1, wherein the one or more points of the primary peak comprise a point, and the one or more points of the secondary peak comprise a point.

3. The system of claim 1, wherein the one or more points of the primary peak comprise five or fewer points, and the one or more points of the secondary peak comprise five or fewer points.

4. The system of claim 1, wherein the combination of the intensities of the one or more points of the primary peak comprises a sum, and the combination of the intensities of the one or more points of the secondary peak comprises a sum.

5. The system of claim 1, wherein the combination of the intensities of the one or more points of the primary peak comprises an average value, and the combination of the intensities of the one or more points of the secondary peak comprises an average value.

6. The system of claim 1, wherein the one or more points of the main peak include the apex of the main peak.

7. The system of claim 1, wherein the one or more points of the main peak include only points at or after the time of the peak's apex.

8. The system of claim 1, wherein the processor is configured to receive input from a user or automatically calculate the width of a time window within the main peak that is smaller than the width of the main peak based on the width of the main peak, calculate the main value as the area of ​​the time window within the main peak and calculate the secondary value as the area of ​​the time window within the secondary peak, and wherein the center of the time window corresponds to the time of a point among the one or more points of the main peak.

9. The system of claim 1, wherein the boundary of the time window coincides with the point of at least one product ion in the product ions.

10. The system of claim 1, wherein the width of the time window comprises an integer multiple of the sampling interval of the points.

11. The system of claim 1, wherein the width of the time window comprises the same number of points for each product ion in the product ions.

12. The system of claim 8, wherein the point includes the apex of the main peak.

13. The system of claim 1, wherein before calculating the ratio, the processor is configured to subtract the primary baseline intensity or area value from the primary value and subtract the secondary primary baseline intensity or area value from the secondary value.

14. The system of claim 13, wherein the primary baseline intensity or area value is calculated based on one or more minimum intensity points of the primary peak, and the secondary baseline intensity or area value is calculated based on one or more minimum intensity points of the secondary peak.

15. The system according to any one of claims 1 to 14, wherein the main ion comprises the product ion with the highest strength.

16. The system according to any one of claims 1 to 14, wherein the secondary ions comprise a plurality of different secondary ions, and wherein the system is operable to calculate a plurality of ion ratios based at least on the plurality of different secondary ions.

17. A method for calculating the ratio of the chromatographic peak of the main ion to the chromatographic peak of the secondary ion of a compound of interest, the method comprising: 1) Use separation devices to separate and fragment the sample at multiple different times, or to separate only the compound of interest; 2) An ion source device is used to ionize a separated fragment of a compound or to ionize only the compound, thereby generating an ion beam; 3) Using a mass spectrometer to analyze the product ions of the compound from the ion beam at multiple different times, or selecting and fragmenting the compound and the product ions of the compound for mass analysis, thereby generating multiple mass spectra; 4) Using the processor, calculate the main extracted ion chromatogram (XIC) of the main product ion of the compound based on the multiple mass spectrometers, and calculate the secondary XIC of the secondary product ion of the compound; 5a) Using the processor, receive input from the user or automatically calculate the number of one or more points corresponding to one or more times but less than all points of the main peak based on the width of the main peak of the main XIC, calculate the main value based on the combination of the intensities of the one or more points of the main peak, and calculate the secondary value based on the combination of the intensities of the secondary peaks of the secondary XIC corresponding to the one or more times, or 5b) Using the processor, receive input from the user or automatically calculate the width of a time window within the main peak that is smaller than the width of the main peak based on the width of the main peak, calculate the main value as the area of ​​the time window within the main peak, and calculate the secondary value as the area of ​​the time window within the secondary peak; as well as 6) Use the processor to calculate the ratio based on the primary and secondary values.

18. The method of claim 17, wherein the one or more points of the primary peak comprise a point, and the one or more points of the secondary peak comprise a point.

19. The method of claim 17, wherein the one or more points of the primary peak comprise five or fewer points, and the one or more points of the secondary peak comprise five or fewer points.

20. The method of claim 17, wherein the combination of the intensities of the one or more points of the primary peak comprises a sum, and the combination of the intensities of the one or more points of the secondary peak comprises a sum.

21. The method of claim 17, wherein the combination of the intensities of the one or more points of the primary peak comprises an average value, and the combination of the intensities of the one or more points of the secondary peak comprises an average value.

22. The method of claim 17, wherein the one or more points of the main peak include the vertices of the main peak.

23. The method of claim 17, wherein the one or more points of the main peak include only points at or after the time of the peak's apex.

24. The method of claim 17, wherein the processor is configured to receive input from a user or automatically calculate the width of a time window within the main peak that is smaller than the width of the main peak based on the width of the main peak, calculate the main value as the area of ​​the time window within the main peak and calculate the secondary value as the area of ​​the time window within the secondary peak, and wherein the center of the time window corresponds to the time of a point among the one or more points of the main peak.

25. The method of claim 17, wherein the boundary of the time window coincides with the point of at least one product ion in the product ions.

26. The method of claim 17, wherein the width of the time window comprises an integer multiple of the sampling interval of the points.

27. The method of claim 17, wherein the width of the time window comprises the same number of points for each product ion in the product ions.

28. The method of claim 24, wherein the point includes the vertex of the main peak.

29. The method of claim 17, wherein before calculating the ratio, the processor is configured to subtract a primary baseline intensity or area value from the primary value and subtract a secondary primary baseline intensity or area value from the secondary value.

30. The method of claim 17, wherein the primary baseline intensity or area value is calculated based on one or more minimum intensity points of the primary peak, and the secondary baseline intensity or area value is calculated based on one or more minimum intensity points of the secondary peak.

31. The method according to any one of claims 17 to 30, wherein the main ion comprises the product ion with the highest strength.

32. The method according to any one of claims 17 to 30, wherein the secondary ions comprise a plurality of different secondary ions, and wherein the system is operable to calculate a plurality of ion ratios based at least on the plurality of different secondary ions.

33. A computer program product comprising a non-transitory and tangible computer-readable storage medium, the contents of the computer-readable storage medium comprising a program having instructions which, when executed on a processor, cause the processor to perform the method according to any one of claims 17-32.

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