Summation of peaks with isobaric MRM transitions in LC-ms / ms

Abstract

Systems and methods of quantifying a target analyte including two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The method includes obtaining a set of output data from the LC-MS / MS for a sample including the target analyte, integrating each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks, and summing the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating. The method further includes generating a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte and determining a concentration of the target analyte in the sample using the calibration curve.

Description

SUMMATION OF PEAKS WITH ISOBARIC MRM TRANSITIONS IN LC-MS / MSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is being filed as a PCT International Patent Application that claims priority to and the benefit of U.S. Provisional Application No. 63 / 626,726, filed on January 30, 2024, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) plays a major role in the analysis of complex biological samples. This versatile method integrates the separation capabilities of liquid chromatography with the sensitivity and specificity of tandem mass spectrometry, allowing researchers to explore the intricate molecular landscape of biological samples with precision.

[0003] In LC-MS / MS, the process begins with the separation of complex mixtures of biomolecules using liquid chromatography, where the sample is passed through a stationary phase to separate its components based on their chemical properties. The eluted compounds are then introduced into the mass spectrometer, where they undergo ionization, fragmentation, and subsequent detection. Tandem mass spectrometry involves multiple stages of mass analysis, typically employing two mass analyzers in tandem, providing enhanced selectivity and sensitivity.

[0004] The application of LC-MS / MS to the analysis of complex biological samples is particularly key in the fields of proteomics, metabolomics, and pharmacokinetics. Proteomic studies involve the identification and quantification of proteins within a given sample, enabling insights into cellular functions and disease mechanisms. Metabolomics focuses on the comprehensive analysis of small molecules, shedding light on metabolic pathways and biomarker discovery. LC-MS / MS is also heavily employed in pharmacokinetic studies, facilitating the measurement of drug concentrations in biological fluids to understand absorption, distribution, metabolism, and excretion. The ability of LC-MS / MS to handle complex mixtures, achieve high sensitivity, and provide accurate structural information makes it a central tool in modem analytical chemistry.SUMMARY

[0005] Examples presented herein relate to a method of quantifying a target analyte comprising two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The method includes obtaining a set of output data from the LC-MS / MS for a sample including the target analyte, integrating each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks, and summing the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating. The method further includes generating a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte and determining a concentration of the target analyte in the sample using the calibration curve.

[0006] In other aspects presented herein, the method further includes receiving, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte. In further aspects presented herein, the selection of the two or more chromatographic peaks associated with the target analyte comprises retention times.In still other aspects presented herein, generating the calibration curve for the target analyte as the regression curve comprises using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard. In further aspects presented herein, the area associated with the internal standard is determined by analyzing one or more solutions of the internal standard, integrating two or more chromatographic peaks associated with the internal standard, summing the two or more chromatographic peaks associated with the internal standard, and storing the sum of the two or more chromatographic peaks associated with the internal standard as the area associated with the internal standard.

[0007] In further aspects presented herein, the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte from the internal standard have a second ratio between a height of each of the two or more chromatographic peaks. In yet further aspects presented herein, the first ratio is different from the second ratio. In other further aspects presented herein, the sample is a first sample and two or more chromatographic peaksassociated with the target analyte from a second sample have a third ratio and the first ratio is different from the third ratio.

[0008] In yet other aspects presented herein, at least one intervening peak lies between the two or more chromatographic peaks. In other aspects presented herein, the two or more chromatographic peaks are associated with a multiple reaction monitoring (MRM) transition. In still other aspects presented herein, each of the two or more chromatographic peaks is associated with an isomer of the target analyte. In yet still other aspects presented herein, the regression curve is a linear regression curve.

[0009] Other examples presented herein relate to a system for quantifying a target analyte comprising two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS). The system includes a least one processor and a memory in communication with the processor and including instructions. The instructions, when executed by the processor, cause the processor to obtain a set of output data from the LC-MS / MS for a sample including the target analyte, integrate each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks, sum the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating, generate a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte, and determine a concentration of the target analyte in the sample using the calibration curve.

[0010] In other aspects presented herein, the instructions further cause the processor to receive, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte. In further aspects presented herein, the selection of the two or more chromatographic peaks associated with the target analyte comprises retention times.

[0011] In yet other aspects presented herein, generating the calibration curve for the target analyte as the regression curve comprises using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard. In further aspects presented herein, the area associated with the internal standard is determined by analyzing one or more solutions of the internal standard, integrating two or more chromatographic peaks associated with the internal standard, summing the two or more chromatographic peaks associated with the internal standard, and storing the sum of the two or more chromatographic peaksassociated with the internal standard as the area associated with the internal standard. In further aspects presented herein, the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte from the internal standard have a second ratio between a height of each of the two or more chromatographic peaks.

[0012] In still other aspects presented herein, at least one intervening peak lies between the two or more chromatographic peaks. In yet other aspects presented herein, each of the two or more chromatographic peaks is associated with an isomer of the target analyte.

[0013] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings is as follows:

[0015] FIG. 1 is a schematic depiction of an example a liquid chromatographytandem mass spectrometry (LC-MS / MS) system in which embodiments of the present disclosure are implemented.

[0016] FIG. 2 is E / Z isomers of derivatized testosterone appearing as two chromatographic peaks in the specific MRM transition channel of the product.

[0017] FIG. 3 shows the ratio between the two chromatographic peaks of derivatized testosterone of FIG. 2.

[0018] FIG. 4 is a graph showing Vitamin D isomeric metabolites, with each species appearing as two chromatographic peaks.

[0019] FIG. 5 is an example of the formation of cis / trans isomers of sirolimus and everolimus.

[0020] FIG. 6 is an example workflow demonstrating underlying principles of the present disclosure.

[0021] FIG. 7 is a flowchart of an example method of quantifying a target analyte comprising two or more chromatographic peaks in an output from a LC-MS / MS system.

[0022] FIG. 8 illustrates an example block diagram of a virtual or physical computing system.DETAILED DESCRIPTION

[0023] Liquid chromatography tandem mass spectrometry (LC-MS / MS) quantitation provides high specificity and accuracy of analytical measurements in complex biological samples and, as a result, is becoming a primary method of analysis in the clinical lab. In typical LC-MS / MS quantitation each chromatographic peak represents a compound that is quantified based on its multiple reaction monitoring transition (MRM transition), which as discussed herein refers to the transition from the parent compound to its product ions. However, in highly complex biological samples there are often additional compounds which share the same MRM transition. For example, with metabolites and small molecules, products derived from isobaric compounds often share the same MRM transition.

[0024] In order to rule out their interference in the accurate quantification of the analyte of interest, they must be chromatographically separated, thus the chromatographic profile of a specific MRM transition channel potentially possesses more than one peak. In many cases it is crucial to sum the areas of more than one chromatographic peak, for both of the analyte and its internal standard (e.g., to sum the area of more than one peak of the analyte and to sum the area of more than one peak for the internal standard), to obtain a reliable and accurate quantitation.

[0025] Disclosed herein are improved methods and systems for summing two or more peak areas for analytes. In some embodiment, the corresponding internal standards are also summed, resulting in more accurate quantification for those analytes in patient or surrogate (e.g., calibrators, quality control (QC), blank, spiked samples). Further disclosed herein are novel software features, algorithms, and methods to enable the peak area summation of all chosen chromatographic peaks which possess isobaric MRM transitions. The calculated areas are used, as-is or as ratios of an analytes over an internal standard, for example, to generate calibration curves, assess QC values, and calculate the concentration values of analytes in samples. In embodiments, results are reported (e.g., ng / dL concentrations) based on a peak area ratio of summed analyte peak area and summed internal standard peak area.

[0026] The systems and method of the present disclosure provide a number of benefits over conventional means of LC-MS / MS quantitation. For example, concentration results will be readily generated with greater accuracy. Greater accuracy in concentration results carries forward into more reliable applications, such as a more reliable clinical diagnosis. Automated peak summation software enables accurate data processing of a high number of samples which may be derivatized, in embodiments, with particular reagents for signal enhancement. In addition, laboratories that develop their own tests could benefit for any summation of compounds that possess isobaric MRM transitions. Existing quantitation accuracy problems are solved for the isomers that separate in chromatography or derivatized compounds that form different derivatives which separate chromatographically, that currently cannot be easily resolved.

[0027] FIG. 1 is a schematic depiction of an example LC-MS / MS system 100 in which embodiments of the present disclosure are implemented. The components of such systems are generally known in the art, but are described here to provide further context to the disclosure.

[0028] The LC-MS / MS system 100 includes one or more solvent reservoirs 102 that contain solvents) to be used as a moving fluid stream (the so-called “mobile phase”). A variety of solvents that may be utilized and are well-known in the art. The solvent(s) are drawn from the one or more reservoirs 102 by a solvent delivery system in the form of a high-pressure liquid chromatography pump 104. Samples are then delivered to the mobile phase at an autosampler 106, which injects the samples into the solvent. Discrete samples are then carried by the mobile phase to the head of a chromatographic column 108. The mobile phase and injected samples enter the column 108 and pass through a particle bed therein. The particle bed separates the sample into individual analyte bands based on the affinity of compounds for a stationary phase. In embodiments, an integrated detector (not shown) monitors the eluent from the column, generating signals indicative of the presence of separated compounds.

[0029] The separated mixture passes to a detector 110, which may be a mass analysis device such as a mass spectrometry (MS) detector, such as a tandem mass spectrometer (MS / MS). Example system 100 includes an ion source 120, a first mass filter 122, a fragmentation device 124, and a second mass separator or a mass analyzer 126.

[0030] One or more compounds of interest from a sample are introduced to ion source 120 over time. Sample introduction may be performed using techniques thatinclude, but are not limited to, direct injection, liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility.

[0031] Mass filter 122 and fragmentation device 124 are shown as different stages of a quadrupole and mass analyzer 126 is shown as a time-of-flight (TOF) device. Those of ordinary skill in the art will appreciate that either of mass filter 122 and mass analyzer 126 may include other types of mass separator and analysis devices including, but not limited to, ion traps, orbitraps, ion mobility devices, time-of-flight (TOF) devices, or Fourier transform ion cyclotron resonance (FT-ICR) devices.

[0032] Ion source device 120 transforms a sample or compounds of interest from a sample into an ion beam. Ion source device 120 can perform ionization techniques that include, but are not limited to, matrix assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI).

[0033] Mass filter 122 receives the ion beam. In embodiments, mass filter 122 is configured by a user for a particular precursor ion transmission window based on the experimental goals for the sample being run. In many tandem mass spectrometry (MS / MS) experiments, the precursor ions are first selected based on their m / z (mass-to- charge ratio) in order to isolate a specific ion of interest for further analysis or fragmentation. The precursor ion selection process employs a mass filter or a specific set of voltages that allow only ions within a certain m / z range (the precursor ion transmission window) to pass through to the next stage.

[0034] Fragmentation device 124 of tandem mass spectrometer 110 fragments or transmits the precursor ions transmitted by mass filter 122. Fragmentation device 124 fragments the precursor ions when a collision energy high enough to fragment ions is used. Fragmentation device 124 transmits the precursor ions when a collision energy low enough not to fragment ions is used. As a result, the resulting product ions can include precursor ions.

[0035] Mass analyzer 126 of tandem mass spectrometer 110 detects intensities or counts for each of the one or more resulting product ions for each overlapping window of the series that form mass spectrum data for each overlapping window of the series.

[0036] A controller in the form of a computer 112 may be used to process, analyze, display, etc., the results received from the detector 110, as well as control the various other components within the LC-MS / MS system 100. Thus, sample compounds that include multiple analytes that otherwise could not be distinguished from one another bystandard MS systems may have those analytes first separated in an LC column, prior to introduction, analysis, and identification in the MS system.

[0037] In operation, samples undergo careful preparation, including extraction and purification steps, to ensure precision and reproducibility. Subsequently, the LC system separates compounds as they traverse the chromatographic column. The ionization source in the MS / MS system then converts these separated compounds into ions. The mass analyzer facilitates precise mass measurements, enhancing specificity. The tandem mass spectrometry process, in embodiments, involves subjecting selected ions to collision-induced dissociation (CID) in the collision cell, resulting in the generation of fragments that offer valuable structural insights into the compounds. The detector records the abundances of these fragmented ions. Data analysis software processes this information, enabling the identification and quantification of compounds present in the sample.

[0038] There is presently no known efficient way to quantify the targeted analytes if the isomers exist together in the matrices and only overall concentration is known. Users must go through extensive manual calculation or queries to combine the results before quantitation.

[0039] For example, the geometrical (E / Z) isomers of derivatized testosterone are considered. FIG. 2 is E / Z isomers of derivatized testosterone appearing as two chromatographic peaks in the specific MRM transition channel of the product. Upon derivatization of testosterone, the derivatized product elutes as a double peak due to the formation of positional isomers which are baseline resolved under the given LC chromatographic conditions.

[0040] FIG. 3 shows the ratio between the two chromatographic peaks of derivatized testosterone of FIG. 2. FIG. 3 demonstrates that the two (or more) peaks of an analyte, seen in graph 302, are not always similar to that of the internal standard, seen in graph 304. In this example, in graph 302 of the analyte, first peak 322 has an area of 6.834e4 and second peak 324 is 4.153e4. In contrast, in graph 304 of the internal standard (IS) first peak 342 has an area of 7.844e4 and second peak 344 has an area of 3.865e4. While some variation is measured values is expected, the difference in ratio between the two peaks is particularly significant. In the example chromatogram of FIG. 3 the peak area ratio of the analyte to internal standard varies according to how calculations are performed. For example, the ratio is 1.87 when both peaks are integrated, 1.65 when only the first peak is integrated, and 2.02 when the second peak is integrated. Since the ratiobetween the two peaks is not constant and moreover, sometimes this ratio does not follow a consistent pattern which can be set by the internal standard, as is the case in the example of FIG. 3, for an accurate determination of analyte concentration it is critical to sum both peaks together.

[0041] In another example, quantification of vitamin D metabolite species are considered. The following isomers of vitamin D3 will resolve chromatographically with an MRM transition of 401383 but appear as a separate peaks:- 25(OH)D3, 3-epi-25(OH)D3- 24,25(OH)2D, 1 ,25(OH)2D3 3-epi 24,25(OH)2D 3-epi-24,25(OH)D3- 4S,25(OH)2D 24R,25(OH)2D 23R,25(OH)2D.Therefore, depending on the research purpose, quantification of certain species could require summation of isomeric peaks. For some of those vitamin D metabolites species the endogenous concentrations are too low and derivatization is required.

[0042] FIG. 4 is a graph showing Vitamin D isomeric metabolites, with each species appearing as two chromatographic peaks. In FIG. 4, the several species of vitamin D metabolites noted above are derivatized and show two geometrical peaks for each isomeric species. Peak area summation of each geometrical isomeric pair is necessary for accurate quantification.

[0043] In yet another example, isomers of immunosuppressant drugs are considered.FIG. 5 is an example of the formation of cis / trans isomers of sirolimus and everolimus. When the LC gradient elution of immunosuppressant drugs is shallow and extended to longer time, the cis and trans isomers of each drug are separated. The most accurate quantification can be achieved by integration of both. Systems and method are disclosed herein to automatically sum the peak areas of such desired isomers.

[0044] FIG. 6 is an example workflow 600 demonstrating underlying principles of the present disclosure. In the example of workflow 600 samples with known testosterone concentrations are analyzed using the peak summation method as disclosed herein, and discussed in greater detail below, and the calculated concentrations are compared to those calculated via manual integration of both peaks. In this example, an acquisition method uses the same MRM transition for each of the analyte and the IS.

[0045] At operation 602, an area of a first analyte peak is integrated. At operation 604, an area of a second analyte peak is integrated. At operation 606, a sum of the integrated areas for the two analyte peaks is calculated. At operation 608, an area of a first IS peak is integrated. At operation 610, an area of a second IS peak is integrated. Atoperation 612, a sum of the integrated areas for the two IS peaks is calculated. At 614, an area ratio of the two sums, from operations 606 and 612, are used to generate a linear regression curve 616.

[0046] Example results are presented in Table 1 below:Table 1

[0047] While it is possible to sum the peaks by manual integration, e.g., manually forcing the integration to include both adjacent peaks, this is not an effective solution for users as it is labor intensive and unreliable. Moreover, if the related peaks are not adjacent this manual summation is not an option. Further, trials to co-elute the isomeric peaks by speeding up the LC gradient elution may ease integration by conventional methods, but this is not a viable option for precise and accurate results as other interferences could coelute as well. For a reliable clinical assay all components in the chromatogram must be resolved, which can increase the number of peaks associated with a target as isomers elute separately.

[0048] The present disclosure provides systems and methods to improve the quantitation calculation accuracy as compared with using only one isomer’s results for each quantitation. User’s manual calculations are reduced and more automatic, rapid, and accurate quantitation is provided.

[0049] FIG. 7 is a flowchart of an example method 700 of quantifying a target analyte including two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. In embodiments, the LC-MS / MS system is system 100 of FIG. 1. Method 700 may be executed by a controller 112 or may be executed by a separate or integrated processing system independent of controller 112.

[0050] At operation 702, a set of output data from the LC-MS / MS for a sample including the target analyte is obtained. LC-MS / MS output data may encompass various parameters forming a comprehensive set of information for analyzing the composition of a sample. Output may include mass spectra offering insights into the masses and structures of ions present. Chromatograms, such as the Total Ion Chromatogram (TIC) and Selected Ion Chromatogram (SIC), visually represent ion intensities over time, aiding in the identification of compounds. Retention time indicates how long it takes for a compound to traverse the chromatography column, aiding in precise identification. Quantitative data, derived from peak areas or intensities, allows for the quantification of specific compounds. Additional details like molecular formulas, fragmentation patterns, and metadata such as sample information and instrument parameters contribute to a comprehensive understanding of the sample composition.

[0051] At operation 704, each of the two or more chromatographic peaks associated with the target analyte are integrated to yield an area for each of the two or more chromatographic peaks. In embodiments, the two or more chromatographic peaks associated with the target analyte are determined by user selection. For example, the system receives, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte.

[0052] In embodiments, the selection of the two or more chromatographic peaks associated with the target analyte includes retention times. For example, each target chromatographic peak may be defined by a particular retention time. Additional steps of the method may be automated once peak selection is entered by the user or, in embodiments, the system may be configured to determine peaks associated with a target analyte automatically as well.

[0053] In some cases, at least one intervening peak lies between the two or more chromatographic peaks. Whereas conventional methods may prevent integration of multiple peaks in situations where the peaks are not directly adjacent, the present method 700 supports integration of multiple peaks in any arrangement.

[0054] The two or more chromatographic peaks may be associated with a same MRM transition. MRM transitions provide for precise and sensitive quantification of selected substances. A specific precursor ion may be isolated in a first quadrupole based on its mass-to-charge ratio and then undergo controlled fragmentation through CID in a collision cell. This fragmentation generates distinct product ions, which are selectively monitored and quantified. This method provides heightened sensitivity and selectivity asit focuses on monitoring only the transitions relevant to the analytes of interest. MRM is applicable in quantitative analyses, particularly in fields like pharmaceuticals and environmental monitoring, where accurate measurement of specific compounds is critical for reliable results. Researchers can fine-tune MRM transitions to optimize sensitivity and precision based on the unique characteristics of each analyte. In embodiments, each of the two or more chromatographic peaks is associated with an isomer of the target analyte.

[0055] At operation 706, the area, based on the integrating, of each of the two or more chromatographic peaks associated with the target analyte are summed. At operation 708, a calibration curve is generated for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte. In embodiments, the regression curve is a linear regression curve.

[0056] In embodiments, generating the calibration curve for the target analyte as the regression curve includes using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard. In embodiments, the area associated with the internal standard is determined by analyzing one or more solutions of the internal standard, and integrating two or more chromatographic peaks associated with the internal standard. The two or more chromatographic peaks, associated with the internal standard, are summed and the sum may be stored as the area associated with the internal standard. The internal standard is a solution of a known concentration and frequently the internal standard is chosen to include a compound related to the analyte, e.g., a chemically modified form of the analyte compound.

[0057] In embodiments, ‘n’ standards, or solutions of the standard, (where n > 1) are used. Then, for each separate solution or standard, the sum of the analyte peak area is determined. Each peak area then defines a point of the one or more points for the calibration curve. Alternatively, if an internal standard is used, then the analyte peak area sum is divided by the internal standard area. The internal standard area can be a sum of two or more internal standard peaks, or may be a single peak.

[0058] In some cases, as discussed in more detail above with reference to FIG. 3, the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte fromthe internal standard have a second ratio between a height of each of the two or more chromatographic peaks.

[0059] The first ratio may be different from the second ratio. In embodiments, the sample is a first sample and two or more chromatographic peaks associated with the target analyte from a second sample have a third ratio and the first ratio is different from the third ratio. In other words, in three analyses of a target analyte including at least one of which is a reference or internal standard of known concentration, a different ratio between isomeric peaks may be returned each time. At operation 710, a concentration of the target analyte in the sample is determined using the calibration curve.

[0060] FIG. 8 illustrates an example block diagram of a virtual or physical computing system 150. One or more aspects of the computing system 150 can be used to implement the systems and methods for constructing data structures for high- dimensionality extraction. In particular, the computing system 150 may be used to implement the controller 112.

[0061] In the embodiment shown, the computing system 150 includes one or more processors 152, a system memory 158, and a system bus 172 that couples the system memory 158 to the one or more processors 152. The system memory 158 includes RAM (Random Access Memory) 160 and ROM (Read-Only Memory) 162. A basic input / output system that contains the basic routines that help to transfer information between elements within the computing system 150, such as during startup, is stored in the ROM 162. The computing system 150 further includes a mass storage device 164. The mass storage device 164 is able to store software instructions and data. The one or more processors 152 can be one or more central processing units or other processors.

[0062] The mass storage device 164 is connected to the one or more processors 152 through a mass storage controller (not shown) connected to the system bus 172. The mass storage device 164 and its associated computer-readable data storage media provide nonvolatile, non-transitory storage for the computing system 150. Although the description of computer-readable data storage media contained herein refers to a mass storage device, such as a hard disk or solid state disk, it should be appreciated by those skilled in the art that computer-readable data storage media can be any available non- transitory, physical device or article of manufacture from which the central display station can read data and / or instructions.

[0063] Computer-readable data storage media include volatile and non-volatile, removable and non-removable media implemented in any method or technology forstorage of information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROMs, DVD (Digital Versatile Discs), other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computing system 150.

[0064] According to various embodiments of the invention, the computing system 150 may operate in a networked environment using logical connections to remote network devices through the network 148. The network 148 is a computer network, such as an enterprise intranet and / or the Internet. The network 148 can include a LAN, a Wide Area Network (WAN), the Internet, wireless transmission mediums, wired transmission mediums, other networks, and combinations thereof. The computing system 150 may connect to the network 148 through a network interface unit 154 connected to the system bus 172. It should be appreciated that the network interface unit 154 may also be utilized to connect to other types of networks and remote computing systems. The computing system 150 also includes an input / output controller 156 for receiving and processing input from a number of other devices, including a touch user interface display screen, or another type of input device. Similarly, the input / output controller 156 may provide output to a touch user interface display screen or other type of output device.

[0065] As mentioned briefly above, the mass storage device 164 and the RAM 160 of the computing system 150 can store software instructions and data. The software instructions include an operating system 168 suitable for controlling the operation of the computing system 150. The mass storage device 164 and / or the RAM 160 also store software instructions, that when executed by the one or more processors 152, cause one or more of the systems, devices, or components described herein to provide functionality described herein. For example, the mass storage device 164 and / or the RAM 160 can store software instructions that, when executed by the one or more processors 152, cause the computing system 150 to receive and execute managing network access control and build system processes.

[0066] The software instructions further include one or more software applications 166. Software applications may include dedicated systems and algorithms for performing specific tasks or actions or providing specific interfaces. One or more of data processingsystem and / or one or more component of data processing system may be encompassed by software applications 166.

[0067] The LC-MS / MS system represents a robust analytical instrument with applications in diverse fields, including pharmaceuticals, environmental monitoring, and clinical diagnostics. The disclosed system and methods herein provide a comprehensive solution for the accurate and sensitive analysis of complex mixtures, making it a valuable asset in numerous scientific and industrial endeavors.

[0068] Illustrative examples of the systems and methods described herein are provided below. An embodiment of the system or method described herein may include any one or more, and any combination of, the clauses described below.

[0069] Clause 1. A method of quantifying a target analyte comprising two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS), including obtaining a set of output data from the LC-MS / MS for a sample including the target analyte; integrating each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks; summing the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating; generating a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte; and determining a concentration of the target analyte in the sample using the calibration curve.

[0070] Clause 2. The method of clause 1, further including receiving, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte.

[0071] Clause 3. The method of clause 1 or 2, wherein the selection of the two or more chromatographic peaks associated with the target analyte includes retention times.

[0072] Clause 4. The method of any of clauses 1-3, wherein generating the calibration curve for the target analyte as the regression curve includes using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard.

[0073] Clause 5. The method of clause 4, wherein the area associated with the internal standard is determined by: analyzing one or more solutions of the internal standard; integrating two or more chromatographic peaks associated with the internal standard; summing the two or more chromatographic peaks associated with the internalstandard; and storing the sum of the two or more chromatographic peaks associated with the internal standard as the area associated with the internal standard.

[0074] Clause 6. The method of clause 5, wherein the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte from the internal standard have a second ratio between a height of each of the two or more chromatographic peaks.

[0075] Clause 7. The method of clause 6, wherein the first ratio is different from the second ratio.

[0076] Clause 8. The method of clause 6, wherein the sample is a first sample and two or more chromatographic peaks associated with the target analyte from a second sample have a third ratio and the first ratio is different from the third ratio.

[0077] Clause 9. The method of any of clauses 1-8, wherein at least one intervening peak lies between the two or more chromatographic peaks.

[0078] Clause 10. The method of any of clauses 1-9, wherein the two or more chromatographic peaks are associated with a multiple reaction monitoring (MRM) transition.

[0079] Clause 11. The method of any of clauses 1-10, wherein each of the two or more chromatographic peaks is associated with an isomer of the target analyte.

[0080] Clause 12. The method of any of clauses 1-11, wherein the regression curve is a linear regression curve.

[0081] Clause 13. A system for quantifying a target analyte including two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS), the system including a least one processor; and a memory in communication with the processor and including instructions which, when executed by the processor, cause the processor to: obtain a set of output data from the LC-MS / MS for a sample including the target analyte; integrate each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks; sum the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating; generate a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte; and determine a concentration of the target analyte in the sample using the calibration curve.

[0082] Clause 14. The system of clause 13, wherein the instructions further cause the processor to receive, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte.

[0083] Clause 15. The system of clause 14, wherein the selection of the two or more chromatographic peaks associated with the target analyte includes retention times.

[0084] Clause 16. The system of any of clauses 13-15, wherein generating the calibration curve for the target analyte as the regression curve includes using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard.

[0085] Clause 17. The system of clause 16, wherein the area associated with the internal standard is determined by: analyzing one or more solutions of the internal standard; integrating two or more chromatographic peaks associated with the internal standard; summing the two or more chromatographic peaks associated with the internal standard; and storing the sum of the two or more chromatographic peaks associated with the internal standard as the area associated with the internal standard.

[0086] Clause 18. The system of clause 17, wherein the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte from the internal standard have a second ratio between a height of each of the two or more chromatographic peaks.

[0087] Clause 19. The system of any of clauses 13-18, wherein at least one intervening peak lies between the two or more chromatographic peaks.

[0088] Clause 20. The system of any of clauses 13-19, wherein each of the two or more chromatographic peaks is associated with an isomer of the target analyte.

[0089] Having described the preferred aspects and implementations of the present disclosure, modifications and equivalents of the disclosed concepts may readily occur to one skilled in the art. However, it is intended that such modifications and equivalents be included within the scope of the claims which are appended hereto.

Claims

What is claimed is:

1. A method of quantifying a target analyte comprising two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS), the method comprising: obtaining a set of output data from the LC-MS / MS for a sample including the target analyte; integrating each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks; summing the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating; generating a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte; and determining a concentration of the target analyte in the sample using the calibration curve.

2. The method of claim 1, further comprising receiving, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte.

3. The method of claim 1 or 2, wherein the selection of the two or more chromatographic peaks associated with the target analyte comprises retention times.

4. The method of any of claims 1-3, wherein generating the calibration curve for the target analyte as the regression curve comprises using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard.

5. The method of claim 4, wherein the area associated with the internal standard is determined by: analyzing one or more solutions of the internal standard; integrating two or more chromatographic peaks associated with the internal standard;summing the two or more chromatographic peaks associated with the internal standard; and storing the sum of the two or more chromatographic peaks associated with the internal standard as the area associated with the internal standard.

6. The method of claim 5, wherein the two or more chromatographic peaks associated with the target analyte from the sample have a first ratio between a height of each of the two or more chromatographic peaks and the two or more chromatographic peaks associated with the target analyte from the internal standard have a second ratio between a height of each of the two or more chromatographic peaks.

7. The method of claim 6, wherein the first ratio is different from the second ratio.

8. The method of claim 6, wherein the sample is a first sample and two or more chromatographic peaks associated with the target analyte from a second sample have a third ratio and the first ratio is different from the third ratio.

9. The method of any of claims 1-8, wherein at least one intervening peak lies between the two or more chromatographic peaks.

10. The method of any of claims 1-9, wherein each of the two or more chromatographic peaks is associated with an isomer of the target analyte.

11. A system for quantifying a target analyte comprising two or more chromatographic peaks in an output from a liquid chromatography-tandem mass spectrometry (LC-MS / MS), the system comprising: a least one processor; and a memory in communication with the processor and including instructions which, when executed by the processor, cause the processor to: obtain a set of output data from the LC-MS / MS for a sample including the target analyte; integrate each of the two or more chromatographic peaks associated with the target analyte to yield an area for each of the two or more chromatographic peaks;sum the area of each of the two or more chromatographic peaks associated with the target analyte based on the integrating; generate a calibration curve for the target analyte as a regression curve using the summed area of the two or more chromatographic peaks associated with the target analyte; and determine a concentration of the target analyte in the sample using the calibration curve.

12. The system of claim 11, wherein the instructions further cause the processor to receive, via a user interface, a selection of the two or more chromatographic peaks associated with the target analyte, wherein the selection of the two or more chromatographic peaks associated with the target analyte comprises retention times.

13. The system of claim 11 or 12, wherein generating the calibration curve for the target analyte as the regression curve comprises using an area ratio of the summed area of the two or more chromatographic peaks associated with the target analyte and an area associated with an internal standard.

14. The system of claim 13, wherein the area associated with the internal standard is determined by: analyzing one or more solutions of the internal standard; integrating two or more chromatographic peaks associated with the internal standard; summing the two or more chromatographic peaks associated with the internal standard; and storing the sum of the two or more chromatographic peaks associated with the internal standard as the area associated with the internal standard.

15. The system of any of claims 11-14, wherein at least one intervening peak lies between the two or more chromatographic peaks.

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