Method for multi-reaction monitoring using mass spectrometry device

Abstract

A method for multi-reaction monitoring using a mass spectrometry device (106) is presented. The method comprises the steps of: i) (128) measuring a multi-reaction monitoring transition of quantitative and qualitative factors of both an internal standard and an analyte using staggered multi-reaction monitoring comprising at least three sets of multi-reaction monitoring channels by using the mass spectrometry device (106), wherein one of the sets of multi-reaction monitoring channels is measured at respective theoretical m / z values for the quantitative and qualitative factors of both the internal standard and the analyte, and the other two sets of multi-reaction monitoring channels are measured at respective m / z values offset to higher and lower values by a predefined level; ii) (130) comparing, for at least two groups, at least two of the quantitative factor / qualitative factor ratios of the multi-reaction monitoring transitions of the internal standard substance with a reference value from a database (126) by using at least one processing device (120), where the comparing comprises determining a deviation between the quantitative factor / qualitative factor ratios and the reference value; iii) (132) determining a measurement from a measured multi-reaction monitoring transition of the analyte and the internal standard by using the processing device (120) if the deviation for at least one of the quantitative / qualitative factor ratios is within at least one predefined tolerance range, in other cases, the measured multi-reaction monitoring transition is rejected (136).

Description

Technical Field

[0001] The present invention discloses a method and a device for performing multiple reaction monitoring using mass spectrometry, in particular liquid chromatography and mass spectrometry. Background Art

[0002] Mass spectrometry (MS) systems are widely used in the analysis of biological samples due to their high resolution and ability to analyze relatively small sample volumes relative to some other analytical methods. As part of the analytical workflow, mass spectrometry systems can be connected to liquid chromatography (LC) separation systems. Complex samples such as body fluids can be injected into the LC separation system and separated into sequentially eluted components, which are then analyzed on the MS system. The combination of LC separation and MS-based selective analysis enables quantitative analysis of different samples.

[0003] Mass axis instability, i.e., drift in mass accuracy and resolution, can be caused by changes in one or more of temperature and humidity, MS contamination, and drift of the mass axis over long periods of time between mass axis calibrations. This can hamper LC-MS methods by reducing sensitivity due to lower ion transmission and detection of target analytes, and reducing selectivity due to relatively higher ion transmission and detection of sample matrix components with similar physicochemical properties to the analytes (i.e., similar LC retention times and multiple reaction monitoring (MRM) transitions). A common strategy to maintain the sensitivity of LC-MS methods to mass axis instability is to reduce MS resolution. However, this strategy can reduce method selectivity by compromising the ability of the method to distinguish signals from sample matrix components from analyte signals.

[0004] WO 2021 / 140178 A1 describes a system for analyzing a biological sample, the system comprising a separation unit configured to separate a component from the biological sample; an ionization unit configured to generate a plurality of ions from the component; an adjustable mass selective filter element; a detector configured to detect ions passing through the mass selective filter element; and a controller connected to the mass selective filter element and to the detector. The controller is configured so that during operation of the system, the controller adjusts the mass selective filter element and activates the detector to measure at least three different ion signals corresponding to the plurality of ions, and determines the mass axis offset of the system based on the at least three different ion signals.

[0005] WO 2021 / 239692 A1 describes a computer-implemented method for calibrating a customer mass spectrometer for a quantifier-qualifier ratio calibration. The method comprises the following steps: a) at least one manufacturer field standardization, wherein a set of samples from a subject and a set of calibrator samples are measured on a plurality of mass spectrometer instruments in multiple replicates, wherein each measurement comprises multiple reaction monitoring of quantifier and qualifier transitions for an analyte and an internal standard, wherein at least three adjustment factors are determined from the measurement results of the set of samples from the subject and the set of calibrator samples, wherein a first adjustment factor depends on the difference between the analyte and the internal standard, wherein a second adjustment factor depends on the difference between the subject's sample and the calibrator sample for the quantifier-qualifier ratio for the analyte, and wherein a third adjustment factor depends on the difference between the subject's sample and the calibrator sample for the quantifier-qualifier ratio for the internal standard; b) at least one transfer step, wherein the adjustment factors are electronically transferred to a customer mass spectrometer instrument; c) at least one customer field calibration, wherein the customer field calibration comprises at least one calibration measurement, wherein a set of calibrator samples are measured on a customer mass spectrometer instrument and quantifier-qualifier ratios are determined therefrom, wherein target values ​​of the quantifier-qualifier ratios for the analyte and for the internal standard are set by applying the adjustment factors to the determined quantifier-qualifier ratios.

[0006] Issues to be resolved

[0007] Therefore, the object of the present invention is to provide a method and a device for multi-transition monitoring which avoids the above-mentioned disadvantages of the known methods and devices. In particular, the method and the device will improve the following: Maintaining the sensitivity and selectivity of the LC-MS method to mass axis instability. Summary of the invention

[0008] This problem is solved by a method and a device for multi-transition monitoring having the features of the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims, which can be realized individually or in any arbitrary combination.

[0009] As used below, the terms "having", "comprising" or "including" or any grammatical variations thereof are used in a non-exclusive manner. Thus, these terms may refer to the absence of other features in the entity described in this context in addition to the features introduced by these terms, or the presence of one or more other features. As an example, the expressions "A has B", "A includes B" and "A contains B" may refer to the absence of other elements in A in addition to B (i.e., the presence of A solely and exclusively consisting of B); or to the presence of one or more other elements in entity A in addition to B (such as element C, element C and element D, or even other elements).

[0010] Further, as used hereinafter, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features without limiting the possibilities of alternatives. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As the skilled person will recognize, the present invention may be carried out using alternative features. Similarly, features introduced by "in one embodiment of the present invention" or similar expressions are intended to be optional features without any limitation on alternative embodiments of the present invention, without any limitation on the scope of the present invention, and without any limitation on the possibility of combining features introduced in this manner with other optional or non-optional features of the present invention.

[0011] In a first aspect of the present invention, a method for performing multi-transition monitoring using a mass spectrometry apparatus is disclosed.

[0012] The method comprises the following steps, which may be performed in a given order as an example. However, it should be noted that different orders are possible. Further, one or more method steps may be performed once or repeatedly. Further, two or more method steps may be performed simultaneously or in a timely overlapping manner. The method may include other method steps not listed.

[0013] The method comprises the following steps:

[0014] i) measuring multiple reaction monitoring transitions of quantitative factors and qualitative factors of both the internal standard and the analyte using staggered multiple reaction monitoring by using a mass spectrometry device, wherein the staggered multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, wherein one of the multiple reaction monitoring channel groups is measured at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups are measured at corresponding m / z values ​​shifted to higher and lower values ​​by predefined levels;

[0015] ii) comparing at least two quantifier / qualifier ratios of multiple reaction monitoring transitions of the internal standard with reference values ​​from a database by using at least one processing device, wherein the comparison includes determining a deviation between the quantifier / qualifier ratios and the reference values;

[0016] iii) determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device in case the deviation for at least one of the quantifier / qualifier ratios is within at least one predefined tolerance range and rejecting the measured multiple reaction monitoring transitions in other cases.

[0017] As described above, the method can use staggered multiple reaction monitoring. This can allow the sensitivity and selectivity of the LC-MS method to be maintained to the mass axis instability.

[0018] The method may be computer-implemented. As used herein, the term "computer-implemented method" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a method involving at least one computer and / or at least one computer network. The computer and / or computer network may include at least one processor configured to perform at least one of the method steps of the method according to the present invention. Preferably, each method step is performed by a computer and / or a computer network. The method may be performed completely automatically (particularly, without user interaction). As used herein, the terms "automatically" and "automatically" are broad terms and should be given their common and customary meanings to a person of ordinary skill in the art, and should not be limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a process performed entirely with the aid of at least one computer and / or at least one computer network and / or at least one machine, in particular, without the need for manual operation and / or interaction with a user.

[0019] As used herein, the term "multiple reaction monitoring" (MRM), also denoted as multiple transition monitoring, is a broad term and will be given its ordinary and customary meaning to one of ordinary skill in the art, and is not limited to a special or customary meaning. The term specifically may refer to, but is not limited to, a method used in mass spectrometry, particularly in tandem mass spectrometry, in which multiple product ions from one or more precursor ions are monitored. As used herein, the term "monitoring" is a broad term and will be given its ordinary and customary meaning to one of ordinary skill in the art, and is not limited to a special or customary meaning. The term specifically may refer to, but is not limited to, the determination and / or detection of multiple product ions.

[0020] As used herein, the term "mass spectrometer" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, an analytical technique for determining the mass-to-charge ratio of ions. Mass spectrometry may be performed using at least one mass spectrometer. As used herein, the term "mass spectrometer," also referred to as "mass analyzer," is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, an analyzer configured to detect at least one analyte based on a mass-to-charge ratio.

[0021] The mass spectrometer can be or can include at least one quadrupole analyzer. As used herein, the term "quadrupole mass analyzer" is a broad term and is given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term can specifically refer to, but is not limited to, a mass analyzer including at least one quadrupole as a mass filter. A quadrupole mass analyzer may include a plurality of quadrupoles. For example, a quadrupole mass analyzer may be a triple quadrupole mass spectrometer. For example, a mass spectrometer may include: an ionization source, a skimmer, and three quadrupole stages Q1, Q2, and Q3, and a detector. Each of the quadrupole stages Q1, Q2, and Q3 includes a quadrupole.

[0022] As used herein, the term "mass filter" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a device configured to select ions injected into the mass filter according to the mass-to-charge ratio m / z of the ions. The mass filter may include two pairs of electrodes. The electrodes may be rod-shaped, particularly cylindrical. Ideally, the electrodes may be hyperbolic. The electrodes may be designed to be identical. The electrodes may be arranged to extend in parallel along a common axis, such as the z-axis. The quadrupole mass analyzer may include at least one power supply circuit, which is configured to apply at least one direct current (DC) voltage and at least one alternating current (AC) voltage between the two pairs of electrodes of the mass filter. The power supply circuit may be configured to keep each relative electrode pair at the same potential. The power supply circuit system may be configured to periodically change the charge polarity of the electrode pair so that only ions within a certain mass-to-charge ratio m / z may have a stable trajectory. The trajectory of the ions in the mass filter may be described by the Mathieu differential equation. In order to measure ions with different m / z values, the DC voltage and the AC voltage may be varied in time so that ions with different m / z values ​​may be transmitted to the detector of the mass spectrometer.

[0023] The mass spectrometer may further include at least one ionization source. As used herein, the term "ionization source", also referred to as "ion source" or "ionizer", is a broad term and will be given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically may refer to, but is not limited to, a device configured to generate molecular ions, for example, from a gas, liquid or solid sample. The ionization source may be or may include at least one source selected from the group consisting of the following: at least one gas phase ionization source, such as at least one electron ionization (EI) source or at least one chemical ionization (CI) source; at least one desorption ionization source, such as at least one plasma desorption (PD) source, at least one fast atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LD) source, and at least one matrix-assisted laser desorption ionization (MALDI) source; at least one spray ionization source, such as at least one thermal spray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI) and at least one atmospheric pressure ionization (API) source.

[0024] The mass spectrometer may include at least one detector. As used herein, the term "detector" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a device configured to detect input ions. The detector may be configured to detect charged particles. The detector may be or may include at least one electron multiplier. The mass spectrometer, in particular the detector and / or at least one processing unit of the mass spectrometer, may be configured to determine at least one mass spectrogram of the detected ions. As used herein, the term "mass spectrogram" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a two-dimensional representation of signal intensity compared to mass-to-charge ratio m / z, wherein the signal intensity corresponds to the abundance of the corresponding ions. The mass spectrogram may be a pixelated image. In order to determine the resulting intensity of the mass spectrogram pixel, the signal detected by the detector within a certain m / z range may be integrated. The analyte in the sample may be identified by the processing unit. In particular, the processing unit may be configured to associate a known mass with an identified mass or by a characteristic fragmentation pattern.

[0025] The mass spectrometer may be or may include a liquid chromatography mass spectrometer. The mass spectrometer may be connected to and / or may include at least one liquid chromatograph, also represented as a liquid chromatography (LC) device. The liquid chromatograph may be used as sample preparation for the mass spectrometer. Other embodiments of sample preparation are also feasible, such as at least one gas chromatograph. As used herein, the term "liquid chromatography mass spectrometer" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a combination of liquid chromatography and mass spectrometry. The mass spectrometer may include at least one liquid chromatograph. The liquid chromatography mass spectrometer may be or may include at least one high performance liquid chromatography (HPLC) device or at least one microfluidic liquid chromatography (μLC) device. The liquid chromatography mass spectrometer may include a liquid chromatography (LC) device and a mass spectrometer (MS) device, in the present case a mass filter, wherein the LC device and the mass filter are coupled via at least one interface. The interface coupling the LC device and the MS device may include an ionization source configured to generate molecular ions and transfer the molecular ions to the gas phase. The interface may further include at least one ion migration module disposed between the ionization source and the mass filter. For example, the ion migration module may be a high-field asymmetric waveform ion mobility spectrometry (FAIMS) module.

[0026] As used herein, the term "liquid chromatography (LC) device" is a broad term and is given its ordinary and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, an analytical module that is configured to separate one or more target analytes of a sample from other components of the sample for use in detecting one or more analytes using a mass spectrometer. The LC device may include at least one LC column. For example, the LC device may be a single-column LC device or a multi-column LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped to separate and / or elute and / or transmit the target analyte. The liquid chromatography-mass spectrometer device may further include a sample preparation station for automated pretreatment and preparation of samples, each sample including at least one target analyte.

[0027] The mass spectrometer can be configured to perform an end-to-end workflow (also expressed as a sample measurement workflow), in which a sample is injected into the inlet of a liquid chromatography column, the sample is separated into components on the column, and each component is eluted from the column. The eluted components are directed to a mass spectrometer, where they are ionized and analyzed. The mass spectrometer measures the ion fragmentation pattern associated with each component. Each ion fragmentation pattern consists of one or more peaks corresponding to ion fragments with a specific m / z ratio. The peak pattern of a specific analyte (e.g., the m / z ratio and intensity of the peak) effectively serves as the "fingerprint" of the analyte. Due to the complexity of the fragmentation pattern, a variety of components can be identified and quantified based on these measurements. Typically, identification is performed by comparing the measured ion fragmentation pattern with reference information (e.g., a previously measured or simulated ion fragmentation pattern of a known component). The identification of a specific component can also be performed based on the time interval between the initial introduction of the sample (e.g., injection into the inlet of the LC-MS system) and the elution of the component from the LC column, or the time interval between the initial introduction of the sample and the measurement of the component ion fragmentation pattern in the mass spectrometer. Certain components can migrate through an LC column at specific rates, and the elapsed time interval can be used as an indicator of the identity of the component. As with the ion fragmentation pattern, the elapsed time interval can be compared to reference information (e.g., previously measured migration and / or measurement times of known components) to determine the identity of the component.

[0028] The mass spectrometer can be operated in random access mode. As used herein, the term "random access mode" is a broad term and will be given its common and customary meaning for those of ordinary skill in the art, and is not limited to a special or customized meaning. The term specifically may refer to, but is not limited to, a mode of operation in which a sample is randomly and continuously loaded. The random access mode may include loading a sample fully automatically. The random access mode may further include a thorough and / or fully automatic sample measurement workflow on the mass spectrometer. This may allow a significant increase in throughput.

[0029] As used herein, the term "sample" is a broad term and is given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, any test sample, such as a biological sample. A mass spectrometer may be configured to measure a variety of biological samples. Examples of such samples include, but are not limited to, physiological fluids, including blood, serum, plasma, urine, sweat, saliva, lens fluid, cerebrospinal fluid, milk, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, lymph, interstitial fluid, cerebrospinal fluid, tissue, cells, etc. A sample may contain one or more target analytes, also referred to as target analytes. Samples may be used directly when obtained from a corresponding source, or may be pretreated and / or sample preparation workflows. For example, the sample may be pretreated by adding an internal standard and / or by diluting with another solution and / or by mixing with a reagent, etc. For example, in general, a target analyte may be vitamin D, a drug of abuse, a therapeutic drug, a hormone, and a metabolite. For respective further details on samples, reference is made, for example, to EP 3 425 369 A1, the entire disclosure of which is included herein by reference. Other target analytes are also possible.

[0030] As used herein, the term "internal standard" (ISTD) is a broad term and will be given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically may refer to, but is not limited to, a known amount of a substance. When experiencing the workflow of a mass spectrometer, an internal standard may exhibit properties similar to the target analyte. The workflow may include any pretreatment, enrichment, and actual detection steps, as described above. For example, an internal standard may be an isotope-labeled variant of the target analyte (comprising markers such as 2H, 13C, or 15N).

[0031] As used herein, the term "quantitative factor", also expressed as quantitative ion, is a broad term and will be given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a transition for quantifying an analyte. The quantitative factor may be the ion with the greatest abundance. As used herein, the term "qualitative factor", also expressed as qualifier ion, is a broad term and will be given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, another transition to confirm measurement. As used herein, the term "quantitative factor / qualitative factor ratio" is a broad term and will be given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a signal intensity ratio or peak area ratio of a quantitative factor and a qualitative factor.

[0032] As used herein, the term "staggered multiple reaction monitoring" (S-MRM) is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically may refer to, but is not limited to, multiple reaction monitoring using a set of staggered m / z values ​​(e.g., theoretical m / z values ​​and m / z values ​​that shift to higher values ​​and lower predefined levels). The predefined level can be specified as half of the maximum expected drift of the mass axis about the relevant m / z range. Additional measurements at other levels are also possible.

[0033] Staggered multiple reaction monitoring includes at least three multiple reaction monitoring channel groups.As used herein, term " multiple reaction monitoring group " is a broad term and will be given its common and customary meaning for those of ordinary skill in the art, and is not limited to special or self-defined meaning.This term specifically can refer to but is not limited to one or more in the MRM transition measured at Q1 and Q3 using the same m / z value.One in the multiple reaction monitoring channel group is measured with the corresponding theoretical m / z value of the quantitative factor and the qualitative factor of both internal standard and analyte, and other two multiple reaction monitoring channel groups are measured with the corresponding m / z value of the predefined level to higher value and lower value offset.For example, S-MRM can use the MS resolution optimized for all MRM transitions of target analyte at Q1 and Q3.The first MRM transition set can be measured, which includes the quantitative factor and the qualitative factor MRM transition of both analyte and internal standard at its corresponding target m / z value. In addition, a second set of MRM transitions shifted to higher m / z values ​​at Q1 and Q3 can be measured, and a third set of MRM transitions shifted to lower m / z values ​​at Q1 and Q3 can be measured. Thus, a total of six MRM transitions of the target analyte and a total of six MRM transitions of the internal standard can be measured. For example, the method can include the following measurements:

[0034]

[0035]

[0036] Step ii) comprises comparing at least two quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values ​​from a database by using at least one processing device.

[0037] As used herein, the term "processing device" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, any logic circuit system configured to perform the basic operations of a computer or system; and / or, in general, a device configured to perform calculations or logical operations. The processing device may be configured to process basic instructions that drive a computer or system. As an example, the processing device may include at least one arithmetic logic unit (ALU), at least one floating point unit (FPU) (such as a math coprocessor or a numerical coprocessor), a plurality of registers (specifically registers configured to supply operands to the ALU and store operation results), and memory (such as L1 and L2 cache memories). The processing device may be a multi-core processor. The processing device may be or may include a central processing unit (CPU). Additionally or alternatively, the processing device may be or may include a microprocessor, so that, in particular, the elements of the processor may be contained in a single integrated circuit (IC) chip. Additionally or alternatively, the processing device may be or may include one or more application specific integrated circuits (ASICs) and / or one or more field programmable gate arrays (FPGAs) and / or one or more tensor processing units (TPUs) and / or one or more chips, such as dedicated machine learning optimization chips, etc. The processing device may be configured (such as by software programming) to perform one or more evaluation operations. The processing device may be configured to perform specified method steps. Therefore, as an example, the processing device may include software code stored thereon, which includes multiple computer instructions. The processing device may provide one or more hardware elements for performing one or more of the indicated operations, and / or may provide one or more processors with software running thereon for performing one or more of the method steps.

[0038] As used herein, the term "database" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, an organized collection of data that is typically stored and accessed electronically from a computer or computer system. A database may include a data storage device, or may consist of a data storage device. A database may include at least one database management system, which includes software running on a computer or computer system that allows interaction with one or more of a user, an application, or the database itself, such as to capture and analyze the data contained in the database. A database management system may further encompass facilities for managing a database. Thus, a database containing data may consist of a database system that, in addition to the data, also includes one or more associated applications.

[0039] The database may be part of the processing device or may be external to the processing device. The processing device may include at least one communication interface. The communication interface may be configured to perform at least one of the following: transmitting data from the processing device or transmitting data to the processing device or transmitting data within the processing device. As used herein, the term "communication interface" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, an item or element that forms a boundary that is configured to transmit information. In particular, the communication interface may be configured to transmit information from a computing device (e.g., a computer), such as sending or outputting information to, for example, another device. Additionally or alternatively, the communication interface may be configured to transmit information to a computing device (e.g., on a computer), such as, in order to receive information. The communication interface may specifically provide a means for transmitting or exchanging information. In particular, the communication interface may provide a data transmission connection, such as Bluetooth, NFC, inductive coupling, etc. As an example, the communication interface may be or may include at least one port, including one or more of a network or Internet port, a USB port, and a disk drive. The communication interface may include at least one Web interface.

[0040] The processing device and / or the database may be at least partially cloud-based. As used herein, the term "cloud-based" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art, and should not be limited to a special or custom meaning. The term may specifically refer to, but is not limited to, outsourcing a processing device or part of a processing device to an at least partially interconnected external device, specifically a computer or computer network with greater computing power and / or data storage capacity. The external device may be arbitrarily spatially distributed. The external device may change over time, specifically on demand. The external devices may be interconnected using the Internet. The external devices may each include at least one communication interface.

[0041] As used herein, the term "reference value" is a broad term and will be given its common and customary meaning for those of ordinary skill in the art, and should not be limited to a special or custom meaning. The term specifically may refer to, but is not limited to, a predefined and / or pre-measured quantitative factor / qualitative factor ratio of an internal standard at a target m / z. The database may include at least one information in the group consisting of: analyte ID, sample matrix ID, instrument ID, method or assay ID, average quantitative factor / qualitative factor ratio of an internal standard. From one or more of these information, the processing device may determine the reference value.

[0042] Step ii) may comprise comparing all quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard measured in step i) with reference values.

[0043] Additionally or alternatively, in step ii), selection can be made. Step ii) can include selecting two quantitative factors / qualitative factors ratios of the multiple reaction monitoring transitions of the internal standard according to the signal intensity of the multiple reaction monitoring transitions of the internal standard quantitative factor. Step ii) can include comparing the selected quantitative factors / qualitative factors ratios of the multiple reaction monitoring transitions of the internal standard with a reference value. Step ii) can include comparing the signal intensity of the multiple reaction monitoring transitions of the comparative internal standard quantitative factor. For example, step ii) can include comparing the signal intensity of three MRM transitions of the comparative internal standard quantitative factor. The comparison of signal intensity can be performed by executing a software algorithm.

[0044] In the case where the mass axis is stable (i.e., minimal or no drift), the MRM channel set will produce the highest signal intensity when measured using the corresponding m / z values ​​for the target analyte and ISTD. In the case where the mass axis drifts significantly to higher or lower m / z values, one of the other MRM channel sets will produce the highest signal intensity.

[0045] Step ii) may comprise rejecting the multiple reaction monitoring transition with the lowest signal intensity. For example, step ii) may comprise selecting two of the three internal standard MRM transitions with the highest signal intensity and rejecting the MRM transition with the lowest signal intensity.

[0046] The comparison of the quantitative factor / qualitative factor ratio of the multiple reaction monitoring transition of the internal standard comprises determining the deviation between the quantitative factor / qualitative factor ratio and the reference value. Step ii) may comprise comparing the quantitative factor / qualitative factor ratio of the remaining multiple reaction monitoring transitions of the internal standard with the reference value. The comparison may comprise at least one mathematical operation.

[0047] According to the result of the comparison in step ii), different actions can be performed in step iii). In the case where the deviation for at least one of the quantitative factor / qualitative factor ratios is within at least one predefined tolerance range, step iii) includes determining the measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device. The predefined tolerance range can be ±15% of the reference value, preferably ±10%, and more preferably ±5%. The determination of the measurement result can be performed by executing a software algorithm. The measurement result can be or include at least one quantitative information, such as a value for an analyte in a sample. The measurement result may be a final patient result. According to the determined deviation, such as a sign, the measurement result may further include quality information about the stability of the mass axis.

[0048] In other cases, i.e., in the case where no deviation of any quantifier / qualifier ratio is within a predefined tolerance range, step iii) comprises rejecting the measured multiple reaction monitoring transition. Step iii) may further comprise marking the data as an outlier. Step iii) may comprise verifying the quantifier / qualifier ratios of the two remaining MRM transitions of the internal standard and rejecting any that deviate from the reference value beyond at least one predefined tolerance range.

[0049] For example, if the deviation from only one of the quantifier / qualifier ratios of the remaining multiple reaction monitoring transitions is within a predefined tolerance range, step iii) comprises determining the measurement result from the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel set corresponding to the quantifier / qualifier ratio. For example, a single MRM transition set of the analyte and the internal standard of the multiple reaction monitoring channel set that satisfies the condition may be used to determine the measurement result by MRM / ISTD MRM Step iii) may include rejecting any MRM channel group whose deviation from the reference value exceeds a predefined tolerance range.

[0050] For example, if the deviations of the two quantitative factor / qualifier ratios for the remaining multiple reaction monitoring transitions are within the predefined tolerance range, step iii) can include determining the measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions. In order to determine the measurement result, the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel group corresponding to the quantitative factor / qualifier ratio that meet the condition can be used. For example, the analyte and internal standard transitions of the monitoring channel group that meet the condition can be recorded as the first analyte MRM , the second analyte MRM , First ISTD MRM and the second ISTD MRM The sum can be determined by

[0051]

[0052] The sum of the two staggered MRM transition sets may result in a higher sensitivity.Step iii) may comprise rejecting any set of MRM transitions that deviate from the reference value beyond a predefined tolerance range.

[0053] For example, in the absence of a selection and if the deviations of each of the quantitative factor / qualifier ratios for the multiple reaction monitoring transitions are within a predefined tolerance range, step iii) may include determining the measurement result from the analyte and the internal standard using the sum of the multiple reaction monitoring transitions. Thus, to determine the measurement result, the multiple reaction monitoring transitions of the analyte and the internal standard of all multiple reaction monitoring channel sets may be used. For example, the analyte and internal standard transitions of the monitoring channel set may be represented as the first analyte MRM , the second analyte MRM , the third analyte MRM , First ISTD MRM and the second ISTD MRM 、Third ISTD MRM The sum can be determined by

[0054]

[0055] The sum of three staggered MRM transition sets can yield higher sensitivity.

[0056] Method steps i) to iii) can be performed by using at least one computer. In particular, the control and performance of the measurements in step i) can be performed fully automatically. In addition, the data acquisition and evaluation in steps ii) and iii) can be performed fully automatically. In particular, the method can be fully or partially computer-implemented, in particular implemented on a computer (such as a processor).

[0057] On the other hand, a computer program is disclosed, comprising computer executable instructions for performing, when the program is executed on a computer or a computer network, in particular a processing device, a method according to any one of the embodiments described herein, in particular method steps i) to iii). In particular, the computer program can be stored on a computer-readable data carrier and / or a computer-readable storage medium.

[0058] As used herein, the terms "computer-readable data carrier" and "computer-readable storage medium" may specifically refer to a non-transitory data storage device, such as a hardware storage medium having computer-executable instructions stored thereon. A computer-readable data carrier or storage medium may specifically be or may include a storage medium such as a random access memory (RAM) and / or a read-only memory (ROM).

[0059] Thus, in particular, one, more than one or even all of the method steps i) to iii) as indicated above may be performed by using a computer or a computer network, preferably by using a computer program.

[0060] This article further discloses and proposes a computer program product with program code means, so that when the program is executed on a computer or computer network, the method according to the present invention is performed in one or more embodiments attached hereto. Specifically, the program code means can be stored on a computer-readable data carrier and / or on a computer-readable storage medium.

[0061] The present invention further discloses and proposes a data carrier having a data structure stored thereon, which, after being loaded into a computer or a computer network, such as after being loaded into a working memory or a main memory of the computer or the computer network, can execute a method according to one or more embodiments disclosed herein.

[0062] Further disclosed and proposed herein is a non-transitory computer-readable medium comprising instructions, which, when executed by one or more processors, cause the one or more processors to perform a method according to one or more of the embodiments disclosed herein.

[0063] This article further discloses and proposes a computer program product having a program code tool stored on a machine-readable carrier so that when the program is executed on a computer or computer network, a method according to one or more embodiments disclosed herein is performed. As used herein, a computer program product refers to a program that is a tradable product. The product can generally exist in any format (such as in paper format) or on a computer-readable data carrier and / or on a computer-readable storage medium. Specifically, the computer program product can be distributed on a data network.

[0064] Further disclosed and proposed herein is a modulated data signal containing instructions readable by a computer system or a computer network for performing a method according to one or more embodiments disclosed herein.

[0065] With reference to the computer-implemented aspects of the present invention, one or more method steps or even all method steps of the method according to one or more embodiments disclosed herein may be performed using a computer or a computer network. Therefore, generally speaking, any method step including providing and / or manipulating data may be performed using a computer or a computer network. Generally speaking, these method steps may include any method step other than method steps that typically require manual operation (such as providing samples and / or performing certain aspects of actual measurement).

[0066] Specifically, this article further discloses the following:

[0067] - a computer or a computer network comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification,

[0068] - a computer-loadable data structure adapted to carry out a method according to one of the embodiments described in this specification when the data structure is executed on a computer,

[0069] - a computer program, wherein the computer program is adapted to carry out a method according to one of the embodiments described in the present description when the program is executed on a computer,

[0070] - a computer program comprising program means for carrying out a method according to one of the embodiments described in the present description when the computer program is executed on a computer or on a computer network,

[0071] - a computer program comprising program means according to the preceding embodiment, wherein the program means is stored on a computer-readable storage medium,

[0072] a storage medium, on which a data structure is stored and on which the data structure is suitable, after being loaded into a main memory and / or a working memory of a computer or a computer network, for carrying out a method according to one of the embodiments described in the present description, and

[0073] A computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium for carrying out the method according to one of the exemplary embodiments described in the present description if the program code means is executed on a computer or a computer network.

[0074] In another aspect of the present invention, a system for multi-transition monitoring of at least one analyte in a sample is disclosed.

[0075] The system includes:

[0076] - at least one mass spectrometer device configured to measure MRM transitions of quantitative factors and qualitative factors of both an internal standard and an analyte using staggered MRM, wherein the staggered MRM comprises at least three MRM channel sets, wherein one of the MRM channel sets measures at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two MRM channel sets measure at corresponding m / z values ​​shifted by predefined levels towards higher and lower values;

[0077] - at least one database configured to store at least one reference value;

[0078] - at least one processing device configured to compare at least two quantifier / qualifier ratios of multiple reaction monitoring transitions of an internal standard with reference values ​​from a database, the comparison comprising determining a deviation between the quantifier / qualifier ratios and the reference values, wherein the processing device is configured to determine a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard if the deviation is within at least one predefined tolerance range, and to reject the measured multiple reaction monitoring transitions in other cases.

[0079] The system may be configured for performing the method for performing multiple reaction monitoring according to the invention.Concerning definitions and examples, therefore, reference is made to the description of the method above or as described in more detail below.

[0080] To summarize and without excluding other possible embodiments, the following embodiments may be envisaged:

[0081] Example 1. A method for performing multiple reaction monitoring using a mass spectrometer, the method comprising the following steps:

[0082] i) measuring multiple reaction monitoring transitions of quantitative factors and qualitative factors of both the internal standard and the analyte using staggered multiple reaction monitoring by using a mass spectrometry device, wherein the staggered multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, wherein one of the multiple reaction monitoring channel groups is measured at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups are measured at corresponding m / z values ​​shifted to higher and lower values ​​by predefined levels;

[0083] ii) by using at least one processing device to convert the quantitative factor of the multiple reaction monitoring transition of the internal standard /

[0084] At least two of the qualifier ratios are compared to reference values ​​from a database, wherein

[0085] The comparison involves determining the deviation of the quantitative factor / qualitative factor ratio from the reference value;

[0086] iii) determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device in case the deviation for at least one of the quantifier / qualifier ratios is within at least one predefined tolerance range and rejecting the measured multiple reaction monitoring transitions in other cases.

[0087] Embodiment 2. The method according to the preceding embodiment, wherein the predefined level is specified as half of the maximum expected drift of the mass axis about the relevant m / z range.

[0088] Embodiment 3. The method according to any one of the preceding embodiments, wherein the predefined tolerance range is ±15%, preferably ±10%, and more preferably ±5% of the reference value.

[0089] Embodiment 4. The method according to any one of the preceding embodiments, wherein step ii) comprises selecting two quantifier / qualifier ratios of the MRM transitions of the internal standard according to the signal intensity of the MRM transitions of the internal standard quantifier.

[0090] Embodiment 5. The method according to the preceding embodiment, wherein step ii) comprises comparing the selected quantifier / qualifier ratio of the multiple reaction monitoring transition of the internal standard with a reference value.

[0091] Embodiment 6. A method according to any one of the first two embodiments, wherein step ii) comprises comparing the signal intensities of the multiple reaction monitoring transitions of the internal standard quantitative factor, wherein step ii) comprises rejecting the multiple reaction monitoring transition with the lowest signal intensity, wherein step ii) further comprises comparing the quantitative factor / qualitative factor ratios of the remaining multiple reaction monitoring transitions of the internal standard with a reference value.

[0092] Embodiment 7. A method according to the preceding embodiment, wherein, when the deviation of one of the quantitative factor / qualitative factor ratios from the remaining multiple reaction monitoring transitions is within a predefined tolerance range, step iii) includes determining the measurement result from the multiple reaction monitoring transitions of the analyte and the internal standard in the multiple reaction monitoring channel group corresponding to the quantitative factor / qualitative factor ratio.

[0093] Embodiment 8. A method according to embodiment 6, wherein step iii) comprises determining the measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions when the deviation of the two quantification factor / qualification factor ratios for the remaining multiple reaction monitoring transitions is within a predefined tolerance range.

[0094] Embodiment 9. The method according to any of the first three embodiments, wherein step iii) comprises otherwise rejecting any deviation from the reference value outside a predefined tolerance range.

[0095] Embodiment 10. The method according to any one of the preceding embodiments, wherein the database comprises at least one information selected from the group consisting of: analyte ID, sample matrix ID, instrument ID, method or assay ID, average quantifier / qualifier ratio of internal standard.

[0096] Embodiment 11. The method according to any one of the preceding embodiments, wherein the mass spectrometry device is operated in a random access mode, wherein samples are loaded randomly and continuously.

[0097] Embodiment 12. The method according to any one of the preceding embodiments, wherein the mass spectrometer is a triple quadrupole mass spectrometer.

[0098] Embodiment 13. A method according to any of the preceding embodiments, wherein the method is computer-implemented.

[0099] Embodiment 14. A system for multi-transition monitoring of at least one analyte in a sample,

[0100] The system includes:

[0101] - at least one mass spectrometer device configured to measure MRM transitions of quantitative factors and qualitative factors of both an internal standard and an analyte using staggered MRM, wherein the staggered MRM comprises at least three MRM channel sets, wherein one of the MRM channel sets measures at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two MRM channel sets measure at corresponding m / z values ​​shifted by predefined levels towards higher and lower values;

[0102] - at least one database configured to store at least one reference value;

[0103] - at least one processing device configured to compare at least two quantifier / qualifier ratios of multiple reaction monitoring transitions of an internal standard with reference values ​​from a database, the comparison comprising determining a deviation between the quantifier / qualifier ratios and the reference values, wherein the processing device is configured to determine a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard if the deviation is within at least one predefined tolerance range, and to reject the measured multiple reaction monitoring transitions in other cases.

[0104] Embodiment 15. A system for performing multi-transition monitoring according to the preceding embodiment, wherein the system is configured to perform a method for performing multiple reaction monitoring according to any one of the preceding method embodiments.

[0105] Embodiment 16. A computer program comprising instructions, when the program is executed by a processing device on a system according to any of the preceding embodiments related to a system, the instructions cause the system to perform a method according to any of the preceding embodiments related to a method.

[0106] Embodiment 17. A computer-readable storage medium comprising instructions, when the instructions are executed by a processing device on a system according to any of the preceding embodiments involving systems, the instructions cause the system to perform a method according to any of the preceding embodiments involving methods.

[0107] Embodiment 18. A non-transitory computer readable medium comprising instructions which, when executed by one or more processors, cause the one or more processors to perform a method according to any of the preceding embodiments relating to methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Other optional features and embodiments will be disclosed in more detail in the subsequent description of embodiments, preferably in conjunction with the dependent claims. Wherein, as will be appreciated by those skilled in the art, each optional feature can be implemented in a separate manner and in any arbitrarily feasible combination. The scope of the present invention is not limited by the preferred embodiments. Embodiments are schematically depicted in the accompanying drawings. Wherein, the same reference numerals in these drawings refer to the same or functionally equivalent elements.

[0109] In the attached picture:

[0110] Figure 1 An embodiment of a system according to the present invention is shown;

[0111] Figure 2 a flow chart showing an embodiment of a method according to the present invention; and

[0112] Figure 3A and Figure 3B The experimental results are shown. DETAILED DESCRIPTION

[0113] Figure 1 An embodiment of a system 100 according to the present invention is shown. In this embodiment, the system 100 includes a liquid chromatography mass spectrometry (LC-MS) device. The system 100 may include an inlet 102 connected to a liquid chromatography column 104. The column 104 may be coupled to an MS device 106 via an optional valve 122 connected to an optional waste container 124. The MS device 106 may include an ionizer 108, an interceptor 110, quadrupole stages Q1 112, Q2 114, and Q3 116, and a detector 118. Each of the components may be optionally connected to a processing device 120.

[0114] MS device 106 is configured to use staggered multiple reaction monitoring (S-MRM) to measure the multiple reaction monitoring transition of quantitative factor and qualitative factor of both internal standard and analyte.Staggered multiple reaction monitoring includes at least three multiple reaction monitoring channel groups.One in the multiple reaction monitoring channel group is measured with the corresponding theoretical m / z value of quantitative factor and qualitative factor of both internal standard and analyte, and other two multiple reaction monitoring channel groups are measured with the corresponding m / z value of predefined level to higher value and lower value offset.Staggered multiple reaction monitoring can include the multiple reaction monitoring using a group of staggered m / z values ​​(such as theoretical m / z value and to higher value and lower offset predefined level m / z value).Predefined level can be specified as half of the maximum expected drift of mass axis about relevant m / z range.Additional measurement under other levels is also possible.

[0115] Staggered multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups.Multiple reaction monitoring channel groups can be or more include one or more in the MRM transitions measured at Q1 and Q3 using the same m / z value.One in the multiple reaction monitoring channel group is measured with the corresponding theoretical m / z value of the quantitative factor and the qualitative factor of both internal standard and analyte, and other two multiple reaction monitoring channel groups are measured with the corresponding m / z value of the predefined level to higher value and lower value offset.For example, S-MRM can use the MS resolution optimized for all MRM transitions of target analyte at Q1 and Q3.The first MRM transition set can be measured, which includes the quantitative factor and the qualitative factor MRM transitions of both analyte and internal standard at its corresponding target m / z value.In addition, the second MRM transition set to higher m / z value offset can be measured at Q1 and Q3, and the 3rd MRM transition set to lower m / z value offset can be measured at Q1 and Q3.Therefore, a total of six MRM transitions of target analyte and a total of six MRM transitions of internal standard can be measured. For example, the method may include the following measurements:

[0116]

[0117]

[0118] The processing device 120 may include at least one electronic processor. The processing device 120 is configured to compare at least two quantitative factor / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values ​​from the database 126. The comparison includes determining the deviation between the quantitative factor / qualifier ratio and the reference value. The processing device 120 is configured to determine the measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard when the deviation is within at least one predefined tolerance range, and reject the measured multiple reaction monitoring transitions in other cases.

[0119] The processing device 120 may include at least one database. The processing device 120 may include at least one display device. The processing device 120 may include at least one communication interface for receiving instructions and data from a user of the system 100. For further details of the processing device 120, reference is made to the description of the method according to the present invention, for example, as described with respect to Figure 2 described.

[0120] During the operation of the system 100, the sample is introduced into the inlet 102, for example, by direct injection. After introduction, the sample can enter the column 104 and be deposited on the column material (for example, a resin material). When one or more solvents flow through the column material, the sample may migrate through the column material. When the sample migrates collectively, different components of the sample migrate at different rates, and therefore arrive at the end of the column at different times. The column 104 can be optionally connected to a valve 122 (as described above), which can be optionally connected to a waste container 124. During the operation of the system 100, the valve 122 can be optionally activated by the processing device 120 to guide the eluent from the column 104 to the waste container 124, or to the MS device 106. Selectively guiding only a portion of the eluent to the MS device 106 can ensure that only the target component in the sample is measured. The MS device 106 can include a detector connected to the processing device 120, and when the components of the sample are eluted from the column 104 and arrive at the detector, the detector generates an electrical signal. The processing device 120 can receive the electrical signal and can determine whether to guide the eluent into the waste container 124 or into the MS device 106. The processing device 120 can be configured to determine the direction of guiding the eluent based on the time between the introduction of the sample at the inlet 102 and the detection of the component appearing from the downstream end of the column 104. The elapsed time can be compared with reference information including the elution time of the known sample components to at least preliminarily identify the generated components. Based on the preliminary identification, the processing device 120 can determine whether the component is a target component (and is therefore directed to the MS device 106), or whether the component is a non-target component (and is directed to the waste container 124). When the sample component is not eluted from the column 104 (for example, in the time interval when only the elution solvent flows out of the column 104), the eluent can also be optionally directed to the waste container 124 instead of the MS device 106.

[0121] Various detectors may be positioned between the column 104 and the MS device 106, before the valve 122, between the valve 122 and the MS device 106, or between the valve 122 and the waste container 124 to facilitate detection of sample components as they elute from the column 104. Examples of suitable detectors include, but are not limited to, optical detectors such as photodiodes, photocells, spectral detectors, and CCDs, and electrical detectors such as conductivity sensors and resistivity sensors.

[0122] The sample components entering the MS device 106 can be received in the ionizer 108, where the sample components are ionized to form ion groups. The ionizer 108 can be implemented as any of a variety of different types of ionizers. Examples of suitable ionizers include, but are not limited to, electrospray ionizers, electron ionizers, atmospheric pressure chemical ionizers, thermal spray ionizers, inductively coupled plasma ionizers, glow discharge ionizers, and photoionizers. The ion groups generated in the ionizer 108 can pass through an interceptor 110, which typically includes a hole of reduced size (relative to the exit hole of the ionizer 108), and the interceptor reduces the ion groups directed to the quadrupole stage of the MS device 106. After passing through the interceptor 110, the ions can be separated and detected in the rest of the MS device 106.

[0123] A variety of different mass spectrometer configurations can be used to separate, detect, and analyze ions generated by sample components. MS device 106 is an example of such a configuration. However, it should be understood that the calibration method described herein can be used with many different configurations of MS device 106 and is by no means limited to Figure 1 Configuration shown.

[0124] For example, the MS device 106 can be implemented as a tandem mass spectrometer (e.g., tandem MS / MS) having three quadrupole stages Q1 112, Q2 114, and Q3 116. In the first quadrupole stage 112, ions passing through the interceptor 110 are filtered to select ions falling within a specific m / z value range for further analysis. Ions falling outside the m / z value range are blocked and do not pass through the quadrupole stage 112. On the contrary, ions falling within the desired m / z value range pass through the quadrupole stage 112 and enter the second quadrupole stage 114. The quadrupole stage 112 may include four electrodes arranged around a central symmetry axis. In order to selectively guide only ions with m / z values ​​falling within a desired range to the second quadrupole stage 114, the processing device 120 may be configured to adjust the potential applied to the four electrodes. When a suitable potential is applied, the four quadrupole electrodes generate an oscillating radio frequency (RF) field, which acts to guide ions from one end to the other end along the quadrupole stage 112. For a particular RF field, ions within a particular range of m / z values ​​are directed out of the exit aperture of the quadrupole stage 112, and ions with m / z values ​​falling outside the range are rejected (e.g., blocked) within the quadrupole stage 112. A subset of ions entering the first quadrupole stage 112 passes through the stage 112 and enters the second quadrupole stage 114. The second quadrupole stage 114 is implemented as a collision cell in which ions entering the stage 114 are fragmented to form a distribution of ions of relatively small molecular mass. This distribution of smaller mass ions (derived from larger mass ions that typically enter the stage 114 from the stage 112) passes through the stage 114 into the third quadrupole stage 116. Within the second quadrupole stage 114, the processing device 120 may apply a potential to one or more electrodes to generate one or more electric fields, establishing a field gradient between the entrance aperture and the exit aperture of the stage 114. Ions entering from the stage 112 are typically accelerated by the field gradient. Atoms or molecules of neutral gas are introduced into stage 114 and collide with accelerated ions entering from stage 112, generating (through collision) ion fragments that pass through stage 116. Various gases can be used for the fragmentation process, including but not limited to hydrogen, nitrogen and inert gases (such as argon). After the distribution of ions of smaller mass (referred to herein as "fragment ions") enters the third quadrupole stage 116, the fragment ions are filtered in a manner similar to the filtering that occurs in stage 112. Specifically, stage 116 may include four electrodes arranged around a central symmetry axis, and the controller 120 adjusts one or more potentials applied to the four electrodes to generate an oscillating RF field within stage 116. The generated field guides a subset of ion fragments (each ion fragment having an m / z falling within a specific range) from one end of stage 116 to the other end and to the detector 118. Ion fragments with m / z values ​​beyond this range are rejected (e.g., blocked) within the quadrupole stage 116. A subset of ion fragments is guided out of the third quadrupole stage 116 and measured by the detector 118.Specifically, a measurement signal corresponding to the fragments is generated by the detector 118 and transmitted to the processing device 120, which records the measured intensity. The detector 118 can incorporate a variety of different detection technologies. In some embodiments, the detector 118 corresponds to an electron multiplier, a Faraday cup, or a microchannel plate detector. In some embodiments, the detector 118 is a detector based on an orbital trap. More generally, the detector 118 can implement any one or more known ion detection technologies.

[0125] Figure 2 A flow chart of an embodiment of a method according to the invention is shown.

[0126] The method comprises the following steps, which may be performed in a given order as an example. However, it should be noted that different orders are possible. Further, one or more method steps may be performed once or repeatedly. Further, two or more method steps may be performed simultaneously or in a timely overlapping manner. The method may include other method steps not listed.

[0127] The method comprises the following steps:

[0128] i) (indicated by reference numeral 128) by using the mass spectrometer device 106, using staggered multiple reaction monitoring to measure multiple reaction monitoring transitions of the quantitative factor and the qualitative factor of both the internal standard and the analyte, wherein the staggered multiple reaction monitoring includes at least three multiple reaction monitoring channel groups, wherein one of the multiple reaction monitoring channel groups is measured at corresponding theoretical m / z values ​​of the quantitative factor and the qualitative factor of both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups are measured at corresponding m / z values ​​shifted by predefined levels to higher and lower values;

[0129] ii) (indicated by reference numeral 130) comparing at least two quantifier / qualifier ratios of multiple reaction monitoring transitions of the internal standard with reference values ​​from a database 126 by using at least one processing device 120, wherein the comparison includes determining a deviation between the quantifier / qualifier ratios and the reference values;

[0130] iii) (indicated by reference numeral 132) determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device in the case where the deviation of at least one of the quantitative factor / qualitative factor ratios is within at least one predefined tolerance range (indicated by reference numeral 134), and rejecting the measured multiple reaction monitoring transitions (indicated by reference numeral 136) in other cases.

[0131] As outlined above, S-MRM can use an MS resolution optimized for the target analyte of all MRM transitions at Q1 and Q3. A first MRM transition set can be measured, which includes quantitative and qualitative MRM transitions of both the analyte and the internal standard at their respective target m / z values. In addition, a second MRM transition set shifted to a higher m / z value at Q1 and Q3 can be measured, and a third MRM transition set shifted to a lower m / z value at Q1 and Q3 can be measured. Therefore, a total of six MRM transitions of the target analyte and a total of six MRM transitions of the internal standard can be measured.

[0132] The reference value used in step ii) can be a predefined and / or pre-measured quantitative factor / qualitative factor ratio of the internal standard at the target m / z. The database 126 can include at least one information in the group consisting of: analyte ID, sample matrix ID, instrument ID, method or assay ID, average quantitative factor / qualitative factor ratio of the internal standard. From one or more of these information, the processing device 120 can determine the reference value.

[0133] Step ii) may include comparing 130 all quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard measured in step i) with reference values.

[0134] Additionally or alternatively, in step ii), selection 138 can be performed.Step ii) can include selecting two quantitative factors / qualitative factors ratios of the multiple reaction monitoring transitions of 138 internal standards according to the signal intensity of the multiple reaction monitoring transitions of the internal standard quantitative factor. For example, step ii) can include the signal intensity of three MRM transitions of the comparative internal standard quantitative factor. The comparison of signal intensity can be performed by executing software algorithm. When the mass axis is stable (i.e., minimum or no drift), the multiple reaction monitoring channel group uses the corresponding m / z value for target analyte and ISTD to measure and will produce the highest signal intensity. When the mass axis significantly drifts to a higher or lower m / z value, one of other multiple reaction monitoring channel groups will produce the highest signal intensity.

[0135] Step ii) may include rejecting 136 the multiple reaction monitoring transition with the lowest signal intensity. For example, step ii) may include selecting 138 two of the three internal standard MRM transitions with the highest signal intensity and rejecting 136 the MRM transition with the lowest signal intensity. Thus, the comparison 130 in step ii) may include comparing 140 the selected quantifier / qualifier ratios of the remaining multiple reaction monitoring transitions of the internal standard with the reference value.

[0136] According to the result of the comparison in step ii), different actions can be performed in step iii). In the case where the deviation for at least one of the quantitative factor / qualitative factor ratios is within at least one predefined tolerance range, step iii) includes determining 132 the measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device 120. The predefined tolerance range can be ±15% of the reference value, preferably ±10%, and more preferably ±5%. The determination 132 of the measurement result can be performed by executing a software algorithm. The measurement result can be or include at least one quantitative information, such as a value about the analyte in the sample. The measurement result may be a final patient result. According to the determined deviation, such as a sign, the measurement result can further include quality information about the stability of the mass axis.

[0137] In other cases, i.e., in the case where none of the quantifier / qualifier ratios have deviations within a predefined tolerance range, step iii) includes rejecting 136 the measured multiple reaction monitoring transition. Step iii) may further include flagging 142 the data as an outlier. Step iii) may include verifying the quantifier / qualifier ratios of the two remaining MRM transitions of the internal standard and rejecting any that deviate from the reference value beyond at least one predefined tolerance range.

[0138] For example, if the deviation of only one of the quantifier / qualifier ratios of the remaining multiple reaction monitoring transitions is within a predefined tolerance range, step iii) includes determining 144 the measurement result from the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel set corresponding to the quantifier / qualifier ratio. For example, a single MRM transition set of the analyte and the internal standard of the multiple reaction monitoring channel set that satisfies the condition may be used to obtain the measurement result by the analyte. MRM / ISTD MRM Step iii) may include rejecting any MRM channel group whose deviation from the reference value exceeds a predefined tolerance range.

[0139] For example, if the deviations of the two quantitative factor / qualifier ratios for the remaining multiple reaction monitoring transitions are within the predefined tolerance range, step iii) can include determining 146 the measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions. To determine 146 the measurement result, the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel group corresponding to the quantitative factor / qualifier ratio that meet the condition can be used. For example, the analyte and internal standard transitions of the monitoring channel group that meet the condition can be recorded as the first analyte MRM , the second analyte MRM , First ISTD MRM and the second ISTD MRM The sum can be determined by

[0140]

[0141] Figure 3A and Figure 3B The experimental results are shown.

[0142] The experimental setup is as follows: Cyclosporine A (Sigma-Aldrich, 30024), Cyclosporine A-(d10) (Roche, in-house production), acetonitrile ULC / MS grade (Biosolve, 012041), Milli-Q water (Merck, Advantage A10), ammonium acetate (Sigma-Aldrich, 73594), LC pump (Agilent 1290 Infinity II), external syringe pump (Harvard Apparatus, PHD Ultra series), T-piece (VWR), ESI-MS / MS (e.g. Sciex TQ 6500+).

[0143] A solution containing 100 ng / mL cyclosporin A (analyte) and 100 ng / mL isotope-labeled cyclosporin A-(d10) (ISTD) in 90% acetonitrile (v / v) was infused at a flow rate of 40 μL / min by an external syringe pump (infusion pump). At the same time, the LC pump was operated under isocratic conditions using 10% eluent A (15 mM ammonium acetate) and 90% eluent B (methanol) (v / v). The flow rate of the LC pump was 400 μL / min. Both the infusion pump and the LC pump were connected via a T-piece, resulting in a total flow rate of 440 μL / min directed to the ESI-MS / MS.

[0144] Data were acquired in positive polarity and MRM mode. The target analyte transition was m / z 1269.875 / 1184.838amu. The target ISTD transition was m / z 1229.938 / 1194.901amu. In addition, target m / z–0.2amu and target m / z+0.2amu were measured for both the analyte and ISTD, resulting in a total of six transitions. The measurement time for each transition was 40ms, and the total number of cycles was 16. Both quadrupoles of the MS / MS instrument were set to 0.8amu resolution. Data analysis was performed based on signal intensity in cps.

[0145] exist Figure 3A In the upper part of , the average intensity of the internal standard quantification factor is shown as a function of the mass shift. Figure 3AIn the lower part of the graph, the average quantitative factor / qualifier ratio of the internal standard quantitative factor as a function of mass shift is shown. The target m / z, target m / z-0.2amu, and target m / z+0.2amu are shown as vertical solid lines. Figure 3A In the lower part of , a predefined tolerance range of ±10% is depicted, which is used in step iii).

[0146] exist Figure 3A In the , several m / z were measured with a theoretical m / z range of -0.5 to +0.5 Da. However, only three m / z per analyte and ISTD can be used for subsequent data analysis, as previously described for Figure 1 and 2 These are described in Figure 3B In Figure 3B In the upper part of FIG, the average intensity of the internal standard quantification factor as a function of mass shift is shown for the three m / z used (target m / z, target m / z-0.2amu and target m / z+0.2amu). Figure 3B In the lower part of the graph, the average quantitative factor / qualifier ratio of the internal standard quantitative factor as a function of mass shift is shown. Figure 3B In the lower part of , a predefined tolerance range of ±10% is depicted, which is used in step iii).

[0147] Figure 3B Further shown is the signal intensity of the multiple reaction monitoring transition of the internal standard quantitative factor to select two quantitative factors / qualitative factors ratios of the multiple reaction monitoring transitions of 138 internal standards. Two of the three ISTD MRMs with the highest signal intensity can be selected 138 (the selected ISTD MRM is highlighted with a circle). The quantitative factors / qualitative factors ratios of the two selected ISTDMRMs can be compared with the reference (for example, 1.578 in this case) from the database 126. In the case where the ratio of the two selected MRMs is within ± 10% compared to the reference, the final patient result can be calculated from the analyte and ISTD by using the sum of the two 2MRMs as described above.

[0148] Reference numerals list

[0149] 100 Systems

[0150] 102 Entrance

[0151] 104 Liquid chromatography column

[0152] 106 MS Device

[0153] 108 Ionizer

[0154] 110 Interceptor

[0155] 112 Q1

[0156] 114 Q2

[0157] 116 Q3

[0158] 118 Detector

[0159] 120 Processing device

[0160] 122 valve

[0161] 124 Waste Container

[0162] 126 Database

[0163] 128 Measurements

[0164] 130 Comparison

[0165] 132 OK

[0166] 134 Deviation within predefined tolerance?

[0167] 136 Rejection

[0168] 138 Selection

[0169] 140 Comparison

[0170] 142 Logo

[0171] 144 Confirm the measurement results

[0172] 146 Confirm the measurement results

Claims

1. A method for performing multiple reaction monitoring using a mass spectrometry device (106), the method comprising the following steps: i) (128) measuring multiple reaction monitoring transitions of quantitative factors and qualitative factors of both the internal standard and the analyte using staggered multiple reaction monitoring by using the mass spectrometry device (106), wherein the staggered multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, wherein one of the multiple reaction monitoring channel groups is measured at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups are measured at corresponding m / z values ​​shifted by predefined levels to higher and lower values; ii) (130) for at least two groups, comparing at least two of the quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values ​​from a database (126) by using at least one processing device (120), wherein the comparison includes determining a deviation between the quantifier / qualifier ratios and the reference values; iii) (132) determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using the processing device (120) in the case where the deviation for at least one of the quantitative factor / qualitative factor ratios is within at least one predefined tolerance range, and rejecting (136) the measured multiple reaction monitoring transitions in other cases.

2. The method according to the preceding claim, wherein the predefined level is specified as half of the maximum expected drift of the mass axis over the relevant m / z range.

3. Method according to any of the preceding claims, wherein the predefined tolerance range is ±15%, preferably ±10%, more preferably ±5% of the reference value.

4. A method according to any of the preceding claims, wherein step ii) comprises selecting (138) two quantification factor / qualification factor ratios of the multiple reaction monitoring transitions of the internal standard based on the signal intensity of the multiple reaction monitoring transitions of the internal standard quantification factor.

5. The method of claim 4, wherein step ii) comprises comparing (140) the selected quantifier / qualifier ratio of the multiple reaction monitoring transition of the internal standard with the reference value.

6. A method according to any one of the two preceding claims, wherein step ii) comprises comparing the signal intensities of the multiple reaction monitoring transitions of the internal standard quantitative factor, wherein step ii) comprises rejecting (136) the multiple reaction monitoring transition having the lowest signal intensity, wherein step ii) further comprises comparing the quantitative factor / qualitative factor ratio of the remaining multiple reaction monitoring transitions of the internal standard with the reference value (130).

7. A method according to the preceding claim, wherein: In case the deviation from one of the quantifier / qualifier ratios of the remaining multiple reaction monitoring transitions is within the predefined tolerance range, step iii) comprises determining (144) the measurement result from the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel group corresponding to the quantifier / qualifier ratio.

8. The method of claim 6, wherein step iii) comprises determining (146) the measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions, when the deviation of the two quantification factor / qualification factor ratios for the remaining multiple reaction monitoring transitions is within the predefined tolerance range.

9. The method according to any one of the three preceding claims, wherein step iii) comprises otherwise rejecting any deviation from the reference value outside the predefined tolerance range.

10. The method according to any one of the preceding claims, wherein the method is computer implemented.

11. A system (100) for multi-transition monitoring of at least one analyte in a sample, the system comprising: - at least one mass spectrometer device (106) configured to measure MRM transitions of quantitative factors and qualitative factors of both an internal standard and an analyte using staggered MRM, wherein the staggered MRM comprises at least three MRM channel sets, wherein one of the MRM channel sets measures at corresponding theoretical m / z values ​​of the quantitative factors and qualitative factors of both the internal standard and the analyte, and the other two MRM channel sets measure at corresponding m / z values ​​shifted by predefined levels towards higher and lower values; - at least one database (126) configured to store at least one reference value; - at least one processing device (120) configured to compare at least two quantitative factor / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values ​​from the database for at least two groups, the comparison comprising determining a deviation between the quantitative factor / qualifier ratio and the reference value, wherein the processing device is configured to determine a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard if the deviation is within at least one predefined tolerance range, and to reject the measured multiple reaction monitoring transitions in other cases.

12. A computer program comprising instructions which, when executed by a processing device on a system (100) according to any of the preceding claims relating to a system, cause the system (100) to perform a method according to any of the preceding claims relating to a method.

13. A computer-readable storage medium comprising instructions which, when executed by a processing device on a system (100) according to any of the preceding claims relating to systems, cause the system (100) to perform a method according to any of the preceding claims relating to methods.

14. A non-transitory computer readable medium comprising instructions which, when executed by one or more processors, cause the one or more processors to perform a method according to any of the preceding claims relating to a method.

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