Multiple Reaction Monitoring Method Using a Mass Spectrometer

Abstract

A method for multiple reaction monitoring using a mass spectrometer (106) is proposed. The method comprises i) measuring (128), using a mass spectrometer (106), multiple reaction monitoring transitions of both a quantifier and a qualifier of both an internal standard and an analyte using alternating multiple reaction monitoring, the alternating multiple reaction monitoring comprising at least three multiple reaction monitoring channel groups, one of the multiple reaction monitoring channel groups being measured at the respective theoretical m / z values of both the quantifier and the qualifier of both the internal standard and the analyte, and the two other multiple reaction monitoring channel groups being measured at respective m / z values shifted by a predetermined level higher and lower; ii) comparing (130), for at least two of the groups, 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) using at least one processing device (120), the comparing including determining a deviation between the quantifier / qualifier ratio and the reference value; iii) determining (132), if at least one deviation of the quantifier / qualifier ratio is within at least one predetermined tolerance range, using a processing device (120), a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard, and, if not, rejecting (136) the measured multiple reaction monitoring transitions; and including.

Description

Technical Field

[0001] This disclosure is for methods and apparatus for mass spectrometry techniques, specifically multiple reaction monitoring using liquid chromatography and mass spectrometry.

Background Art

[0002] Mass spectrometry (MS) systems are widely used for the analysis of biological samples due to their high resolution and the ability to analyze relatively small sample volumes compared to certain other analytical methods. As part of an analytical workflow, an MS system can be coupled to a liquid chromatography (LC) separation system. A complex sample, such as a body fluid, is injected into the LC separation system, separated into continuously eluting components, and then analyzed in the MS system. The combination of LC separation and selective MS-based analysis enables the quantitative analysis of a wide variety of different samples.

[0003] Instability of the mass axis, i.e., drift in mass accuracy and resolution, can be caused by temperature and humidity over longer times between mass axis calibrations, MS contamination, and one or more changes among these that result in drift of the mass axis. This can impede the LC-MS method by causing a decrease in sensitivity due to lower ion transmission and detection of the target analyte, and a decrease in selectivity due to relatively higher ion transmission and detection of sample matrix components with physicochemical properties similar to the analyte, i.e., similar LC retention times and multiple reaction monitoring (MRM) transitions. A common strategy for maintaining the sensitivity of the LC-MS method against mass axis instability is to reduce the MS resolution. However, this strategy can reduce the selectivity of the method by impairing its ability to distinguish signals from sample matrix components from analyte signals.

[0004] International Publication No. 2021 / 140178 describes a system for analyzing a biological sample, which includes a separation unit configured to separate components from a biological sample, an ionization unit configured to generate a plurality of ions from the components, an adjustable mass selectivity filtering element, a detector configured to detect ions passing through the mass selectivity filtering element, and a controller connected to the mass selectivity filtering element and the detector. The controller is configured to adjust the mass selection filtering element during operation of the system, activate the detector to measure at least three different ion signals corresponding to the plurality of ions, and determine a mass axis shift of the system based on the at least three different ion signals.

[0005] International Publication No. 2021 / 239692 describes a computer-implemented method for calibrating a customer's mass spectrometry instrument for a quantifier-confirmor ratio check. The method includes the following steps: a) at least one manufacturer site standardization step, in which a set of target samples and a set of calibrator samples are measured in multiple replicates on multiple mass spectrometers, each measurement including multiple reaction monitoring using transitions of the quantifier and confirmor for the analyte and internal standard, at least three adjustment coefficients are determined from the measurements of the set of target samples and the set of calibrator samples, the first adjustment coefficient depending on the difference between the analyte and the internal standard, the second adjustment coefficient depending on the difference between the target sample and the calibrator sample of the quantifier-confirmor ratio of the analyte, and the third adjustment coefficient depending on the difference between the target sample and the calibrator sample of the quantifier-confirmor ratio of the internal standard; b) at least one transfer step in which the adjustment coefficients are electronically transferred to the customer's mass spectrometry instrument; c) at least one customer site calibration step, in which the calibration at the customer site includes at least one calibration measurement, a set of calibrator samples are measured on the customer mass spectrometry instrument, from which the quantifier-confirmor ratio is determined, and the target values of the quantifier-confirmor ratios of the analyte and the internal standard are set by applying the adjustment coefficients to the determined quantifier-confirmor ratio. SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

[0006] Accordingly, an object of the present invention is to provide a method and an apparatus for multiple transition monitoring that avoid the above-mentioned drawbacks of known methods and apparatuses. In particular, the method and apparatus are intended to improve the maintenance of the sensitivity and selectivity of the LC-MS method against mass axis instability.

Means for Solving the Problems

[0007] This problem is solved by a method and an apparatus for multiple transition monitoring having the features of the independent claims. Preferred embodiments of the present invention, which may be implemented alone or in any combination, are described in the dependent claims.

[0008] When used hereinafter, the terms "having", "comprising", or "including", or any grammatical variants thereof, are used in a non-exclusive manner. Thus, these terms may refer to both situations where there are no further features in the entity being described in this context in addition to the features introduced by these terms, and situations where one or more additional features are present. By way of example, the expressions "A has B", "A comprises B", and "A includes B" refer to situations where there are no other elements in A besides B (i.e., situations where A is exclusively composed of B only), and situations where one or more additional elements such as element C, elements C and D, or still further elements are present in entity A.

[0009] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "specifically", "more specifically", "in particular", "even more particularly" or similar terms are used with any feature without limiting the possibility of alternatives. Accordingly, the features introduced by these terms are any features and are not intended to limit the technical scope of the claims in any way. The present invention may be implemented by using alternative features, as would be understood by a person skilled in the art. Similarly, features introduced by "in an embodiment of the present invention" or similar expressions are any features without any limitation regarding alternative embodiments of the present invention, without any limitation regarding the technical scope of the present invention, and without any limitation regarding the possibility of combining such introduced features with any other or non-optional features of the present invention.

[0010] In a first aspect of the present invention, a method for multiple transition monitoring using a mass spectrometer is disclosed.

[0011] The method may include the following steps, which may be performed in a given order by way of example. However, it should be noted that different orders are possible. Furthermore, one or more of the method steps may be performed once or repeatedly. Furthermore, two or more method steps may be performed simultaneously or overlapping in time. The method may include additional method steps not recited.

[0012] The method includes the following steps, namely i) Measuring, by using a mass spectrometer, multiple reaction monitoring transitions of both a quantifier and a qualifier for both an internal standard and an analyte using alternating multiple reaction monitoring, wherein the alternating multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, and wherein one of the multiple reaction monitoring channel groups is measured at the respective theoretical m / z values of both the quantifier and the qualifier for both the internal standard and the analyte, and the two other multiple reaction monitoring channel groups are measured at respective m / z values shifted by a predetermined level higher and lower values; ii) Comparing, by using at least one processing device, at least two quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values from a database, the comparing including determining a deviation between the quantifier / qualifier ratio and the reference value; iii) When at least one deviation of the quantifier / qualifier ratio is within at least one predetermined tolerance range, determining, by using the processing device, a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard, and otherwise rejecting the measured multiple reaction monitoring transitions. Including.

[0013] As described above, the method may use alternating multiple reaction monitoring. This may make it possible to maintain the sensitivity and selectivity of the LC-MS method against mass axis instability.

[0014] The method may be computer-implemented. As used herein, the term "computer implemented method" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to a method in which at least one computer and / or at least one computer network is involved. The computer and / or computer network may comprise at least one processor configured to execute at least one of the method steps of the method according to the invention. Preferably, each of the method steps is executed by a computer and / or a computer network. The method may be performed completely automatically, specifically without interaction with a user. The terms "automatically" and "automated" as used herein are broad terms and should be given their ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to a process that is carried out completely by at least one computer and / or computer network and / or machine, particularly without manual operations and / or interaction with a user.

[0015] As used herein, the term "multiple reaction monitoring" (MRM), also referred to as multiplexed transition monitoring, is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. This term specifically, but not exclusively, can refer to a method used in mass spectrometry, specifically 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 should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. This term specifically, but not exclusively, can refer to the determination and / or detection of multiple product ions.

[0016] As used herein, the term "mass spectrometry" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. This term specifically, but not exclusively, can refer to an analytical technique for determining the mass-to-charge ratio of ions. Mass spectrometry may be performed using at least one mass spectrometry device. As used herein, the term "mass spectrometry device", also referred to as a "mass analyzer", is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. This term specifically, but not exclusively, can refer to an analyzer configured to detect at least one analyte based on the mass-charge ratio.

[0017] The mass spectrometer can be or include at least one quadrupole analyzer. As used herein, the term "quadrupole mass analyzer" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not limited to, this term may refer to a mass analyzer having at least one quadrupole as a mass filter. The quadrupole mass analyzer may include a plurality of quadrupoles. For example, the quadrupole mass analyzer may be a triple quadrupole mass analyzer. For example, the mass spectrometer may include an ionization source, a skimmer, three quadrupole stages Q1, Q2, and Q3, and a detector. Each of the quadrupole stages Q1, Q2, and Q3 includes a quadrupole.

[0018] As used herein, the term "mass filter" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not limited to, this term may refer to an apparatus configured to select ions injected into the mass filter according to the mass-to-charge ratio m / z. The mass filter may comprise two pairs of electrodes. The electrodes may be rod-shaped, particularly cylindrical. Ideally, the electrodes may be hyperbolic. The electrodes may be designed identically. The electrodes may be arranged to extend parallel to each other along a common axis, for example, the z-axis. A quadrupole mass analyzer may comprise at least one power supply circuit 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 hold each pair of opposing electrodes at the same potential. The power supply circuit may be configured to periodically change the polarity of the voltage of the electrode pairs such that stable orbits are possible only for ions within a certain mass-charge ratio m / z. The orbits of the ions in the mass filter may be described by Mathieu's differential equation. The DC and AC voltages may be varied over time such that ions having different m / z values can be transmitted to the detector of the mass spectrometer for measuring ions having different m / z values.

[0019] The mass spectrometer may further include at least one ionization source. As used herein, the term "ionization source", also sometimes referred to as "ion source" or "ionizer", is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or particular meaning. This term may refer, without limitation, for example, to an apparatus configured to generate molecular ions from a gaseous, liquid, or solid sample. The ionization source may be at least one source selected from the group consisting of 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 thermospray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI) source, and at least one atmospheric pressure ionization (API) source, or it may include or be provided with such.

[0020] The mass spectrometer may comprise at least one detector. As used herein, the term "detector" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to a device configured to detect incoming ions. The detector may be configured to detect charged particles. The detector may be or may comprise at least one electron multiplier. The mass spectrometer, particularly the detector of the mass spectrometer and / or at least one processing unit, may be configured to determine at least one mass spectrum of the detected ions. As used herein, the term "mass spectrum" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may also refer to a two-dimensional representation of signal intensity versus mass-to-charge ratio (m / z), where the signal intensity corresponds to the abundance of each ion. The mass spectrum may be a pixelated image. To determine the obtained intensity of the pixels of the mass spectrum, the signal detected by the detector within a specific m / z range may be integrated. The analyte in the sample may be identified by the processing unit. Specifically, the processing unit may be configured to correlate a known mass to an identified mass or via a characteristic fragmentation pattern.

[0021] The mass spectrometer may be or may include a liquid chromatography mass spectrometer. The mass spectrometer may be connected to at least one liquid chromatograph, also referred to as a liquid chromatography (LC) device, and / or may include at least one liquid chromatograph. The liquid chromatograph may be used for sample preparation for the mass spectrometer. Other embodiments of sample preparation may be possible, such as at least one gas chromatograph. As used herein, the term "liquid chromatography mass spectrometry device" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, but not limited to, this term may refer to a combination of liquid chromatography and mass spectrometry. The mass spectrometer may include at least one liquid chromatograph. The liquid chromatography mass spectrometry device may be or may include at least one high performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (μLC) device. The liquid chromatography mass spectrometry device may include a liquid chromatography (LC) device and, in this case, a mass spectrometry (MS) device that is a mass filter, and the LC device and the mass filter are connected via at least one interface. The interface connecting 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 mobility module disposed between the ionization source and the mass filter. For example, the ion mobility module may be a high-field asymmetric waveform ion mobility spectrometry (FAIMS) module.

[0022] As used herein, the term "liquid chromatography (LC) device" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to an analytical module configured to separate one or more analytes of interest in a sample from other components of the sample for detection of the one or more analytes using a mass spectrometer. The LC device may comprise at least one LC column. For example, the LC device may be a single-column type LC device or a multi-column type LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped for separation and / or elution and / or migration of the analyte of interest. The liquid chromatography mass spectrometer may further comprise a sample preparation station for automatic pretreatment and preparation of a sample each comprising at least one analyte of interest.

[0023] The mass spectrometer may be configured to perform an end-to-end workflow (also referred to 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 the individual components are 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 having a specific m / z ratio. The pattern of peaks for a particular analyte (e.g., the m / z ratio and intensity of the peaks) effectively functions as the “fingerprint” of the analyte. Due to the complex nature of the fragmentation pattern, a wide variety of components can be identified and quantified based on such measurements. Typically, identification is performed by comparing the measured ion fragmentation pattern to reference information (e.g., ion fragmentation patterns previously measured or simulated for known components). Identification of a particular component can be based on the time interval from the initial introduction of the sample (e.g., injection into the inlet of an LC-MS system) to the elution of the component from the LC column, or the time interval from the initial introduction of the sample to the measurement of the fragmentation pattern of the component ions in the mass spectrometer. A particular component may move through the LC column at a specific rate, and the elapsed time interval can be used as an indicator of the identity of the component. Similar to the ion fragmentation pattern, the elapsed time interval can be compared to reference information (e.g., previously measured travel and / or measurement times for known components) to determine the identity of the component.

[0024] The mass spectrometer may be operated in a random access mode. As used herein, the term "random access mode" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. This term may refer, without limitation, to an operating mode in which samples are loaded randomly and continuously. The random access mode may include fully automatically loading the samples. The random access mode may further include fully and / or completely automatically executing a sample measurement workflow on the mass spectrometer. This may potentially significantly improve throughput.

[0025] As used herein, the term "sample" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, this term may refer, without limitation, to any test sample such as a biological sample. The mass spectrometer may be configured to measure a wide variety of biological samples. Examples of such samples include, but are not limited to, physiological fluids such as blood, serum, plasma, urine, sweat, saliva, aqueous humor, cerebrospinal fluid, milk, ascitic fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, lymph fluid, interstitial fluid, cerebrospinal fluid, tissue, cells, etc. The sample may comprise one or more analytes of interest, also referred to as target analytes. The sample may be used directly as obtained from each source, or may be subjected to a pretreatment and / or sample preparation workflow. For example, the sample may be pretreated by addition of an internal standard and / or dilution with another solution and / or mixing with a reagent. For example, the analytes of interest may generally be vitamins D, drugs of abuse, therapeutic agents, hormones, and metabolites. For further details regarding the sample, reference may be made, for example, to European Patent Application Publication No. 3425369A1, the entire disclosure of which is incorporated herein by reference. Other analytes of interest are possible.

[0026] As used herein, the term "internal standard" (ISTD) is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term can, without limitation, specifically refer to a known quantity of a substance. An internal standard can exhibit similar characteristics as the analyte of interest when subjected to the workflow using a mass spectrometer. The workflow can, as described above, include any pre-treatment, concentration, and actual detection steps. For example, the internal standard can be an isotopically labeled variant of the analyte of interest (e.g., with a label such as 2H, 13C, or 15N).

[0027] As used herein, the term "quantifier", also referred to as a quantifier ion, is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term can, without limitation, specifically refer to a transition used for quantification of an analyte. The quantifier can be the most abundant ion. As used herein, the term "qualifier", also shown as a qualifier ion, is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term can, without limitation, specifically refer to additional transitions for confirmation of the measurement. As used herein, the term "quantifier / qualifier ratio" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term can, without limitation, specifically refer to the signal intensity ratio or peak area ratio of the quantifier and qualifier.

[0028] As used herein, the term "staggered-multiple reaction monitoring" (S-MRM) is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term may specifically, but not limited to, refer to multiple reaction monitoring using a set of staggered m / z values, e.g., theoretical m / z values and m / z values shifted by a predetermined level higher and lower than a given level. The predetermined level may be specified as half of the maximum expected drift of the mass axis for the relevant m / z range. Additional measurements at other levels may also be possible.

[0029] Interactive multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups. As used herein, the term "multiple reaction monitoring channel group" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to one or more of the MRM transitions measured at Q1 and Q3 using the same m / z value. One of the multiple reaction monitoring channel groups measures at the respective theoretical m / z values of the quantifier and qualifier for both the internal standard and the analyte, and the two other multiple reaction monitoring channel groups measure at their respective m / z values shifted by a predetermined level higher and lower. For example, S-MRM may use the MS resolution at Q1 and Q3 optimized for the target analyte for all MRM transitions. The first set of MRM transitions may be measured as comprising quantifier and qualifier MRM transitions for both the analyte and the internal standard at their respective target m / z values. Further, a second set of MRM transitions shifted to higher m / z values at Q1 and Q3 may be measured, and a third set of MRM transitions shifted to lower m / z values at Q1 and Q3 may be measured. Thus, a total of six MRM transitions for the target analyte and a total of six MRM transitions for the internal standard may be measured. For example, the method may comprise the following measured values.

Table 1

[0030] Step ii) includes comparing, by using at least one processing device, at least two quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values from a database.

[0031] As used herein, the term "processing device" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to any logic circuit configured to perform the basic operations of a computer or system, and furthermore / or generally, may refer to a device configured to perform computational or logical operations. The processing device may be configured to process the basic instructions that drive a computer or system. By way of example, the processing device may include at least one arithmetic logic unit (ALU), at least one floating point unit (FPU) such as a numeric co-processor or numeric processor, a plurality of registers, specifically registers configured to supply operands to the ALU and store the 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 a central processing unit (CPU) or may include the same. Additionally or alternatively, the processing device may be or include a microprocessor, and thus, specifically, the elements of the processor may be included in one single integrated circuit (IC) chip. Additionally or alternatively, the processing device may be one or more application specific integrated circuits (ASIC) and / or one or more field programmable gate arrays (FPGA) and / or one or more tensor processing units (TPU) and / or one or more dedicated machine learning optimization chips, or may include the same. The processing device may be configured to execute one or more evaluation operations, such as by software programming. The processing device may be configured to execute specified method steps. Thus, by way of example, the processing device may store software code comprising a number of computer instructions.The processing device may provide one or more hardware elements for performing one or more of the illustrated operations and / or provide software executed to perform one or more of the method steps to one or more processors.

[0032] As used herein, the term "database" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not limited to, this term can generally refer to an organized collection of data that is stored and electronically accessible from a computer or computer system. A database may comprise a data storage device or be constituted by a data storage device. A database may further comprise at least one database management system comprising software executed on a computer or computer system, and the software enables interaction with one or more of a user, an application, or the database itself, for example, to capture and analyze data included in the database. The database management system may further include facilities for managing the database. Thus, a database comprising data may be constituted by a database system that also comprises one or more related applications in addition to the data.

[0033] The database may be part of the processing device or external to the processing device. The processing device may comprise at least one communication interface. The communication interface may be configured to transmit data from, to, or within the processing device. As used herein, the term "communication interface" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term may refer to an item or element that forms a boundary configured to transfer information. In particular, the communication interface may be configured to transfer information from a computing device, such as a computer, for purposes such as transmitting or outputting information to another device, for example. In addition to, or alternatively to, this, the communication interface may be configured to transfer information to a computing device, such as a computer, for purposes such as receiving information, for example. The communication interface may specifically provide means for transferring or exchanging information. In particular, the communication interface may provide a data transfer connection such as, for example, Bluetooth, NFC, inductive coupling, and the like. By way of example, the communication interface may be at least one port comprising one or more of a network or Internet port, a USB port, and a disk drive, or may comprise such at least one port. The communication interface may comprise at least one web interface.

[0034] 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 should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not limited thereto, this term may refer to outsourcing at least a part of the processing device or the processing device to externally connected devices, specifically, a computer or a computer network having a greater computing power and / or data storage capacity. The external devices may optionally be spatially distributed. The external devices may change over time, particularly in response to requests. The external devices may be interconnected using the Internet. Each of the external devices may include at least one communication interface.

[0035] As used herein, the term "reference value" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not limited thereto, this term may refer to a predefined and / or pre-measured quantifier / confirmatory ratio of an internal standard at a target m / z. The database may include at least one piece of information from the group consisting of analyte ID, sample matrix ID, instrument ID, method or assay ID, and average quantifier / confirmatory ratio of the internal standard. The processing device may determine a reference value from one or more of this information.

[0036] Step ii) may include comparing all of the quantifier / confirmatory ratios of the multiple reaction monitoring transitions of the internal standard measured in step i) with the reference value.

[0037] Additionally or alternatively, in step ii), the selection may be performed. Step ii) may include selecting two quantifier / confirmator ratios of the multiple reaction monitoring transitions of the internal standard according to the signal intensities of the multiple reaction monitoring transitions of the internal standard quantifier. Step ii) may include comparing the selected quantifier / confirmator ratio of the multiple reaction monitoring transition of the internal standard with a reference value. Step ii) may include comparing the signal intensities of the multiple reaction monitoring transitions of the internal standard quantifier. For example, step ii) may include comparing the signal intensities of three MRM transitions of the internal standard quantifier. The comparison of the signal intensities may be performed by executing a software algorithm.

[0038] When the mass axis is stable (i.e., the drift is minimal or there is no drift), the group of multiple reaction monitoring channels measured using the m / z values of the target analyte and ISTD respectively generates the highest signal intensity. When the mass axis drifts significantly to higher or lower m / z values, one of the other groups of multiple reaction monitoring channels generates the highest signal intensity.

[0039] Step ii) may include rejecting the multiple reaction monitoring transition with the lowest signal intensity. For example, step ii) may include selecting two of the three internal standard MRM transitions with the highest signal intensities and rejecting the MRM transition with the lowest signal intensity.

[0040] The comparison of the quantifier / confirmator ratios of the multiple reaction monitoring transitions of the internal standard includes determining the deviation between the quantifier / confirmator ratio and the reference value. Step ii) may include comparing the quantifier / confirmator ratios of the remaining multiple reaction monitoring transitions of the internal standard with the reference value. The comparison may include at least one mathematical operation.

[0041] Depending on the result of the comparison in step ii), different operations may be performed in step iii). If at least one deviation of the quantifier / confirmator ratio is within at least one predetermined tolerance range, step iii) includes determining the measurement result from the multiple reaction monitoring transitions of the analyte and internal standard measurements by using a processing device. The predetermined tolerance range may be ±15%, preferably ±10%, more preferably ±5% from the reference value. The determination of the measurement result may be performed by executing a software algorithm. The measurement result may be, or may comprise, at least one quantitative information, e.g., a value, regarding the analyte in the sample. The measurement result may be the final patient result. The measurement result may further comprise quality information regarding the stability of the mass axis depending on the determined deviation, e.g., a flag.

[0042] Otherwise, i.e., if any deviation of the quantifier / confirmator ratio is not within the predetermined tolerance range, step iii) includes rejecting the measured multiple reaction monitoring transition. Step iii) may further include flagging the data as an outlier. Step iii) may include checking the quantifier / confirmator ratio of the remaining two MRM transitions of the internal standard and rejecting those that deviate by more than at least one predetermined tolerance range from the reference value.

[0043] For example, if the deviation from only one of the quantifier / confirmator ratios of the remaining multiple reaction monitoring transitions is within the predetermined tolerance range, step iii) may include determining the measurement result from the multiple reaction monitoring transitions of the analyte and internal standard of the multiple reaction monitoring channel group corresponding to the quantifier / confirmator ratio. For example, the final patient result may be calculated for the analyte by using a single set of MRM transitions of the analyte and the internal standard of the multiple reaction monitoring channel group that meets the conditions. MRM / ISTD MRM Step iii) may include rejecting any multiple reaction monitoring channel group that deviates by more than a predetermined tolerance range from the reference value.

[0044] For example, if the deviations of both the quantifier / confirmatory ratios of the remaining multiple reaction monitoring transitions are within a predetermined tolerance range, step iii) may include determining measurement results from the analyte and internal standard using the sum of the remaining multiple reaction monitoring transitions. To determine the measurement results, the multiple reaction monitoring transitions of the analyte and internal standard in the multiple reaction monitoring channel groups corresponding to the quantifier / confirmatory ratios that meet the conditions may be used. For example, the transitions of the analyte and internal standard in the monitoring channel groups that meet the conditions may be represented as FirstAnalyte MRM , SecondAnalyte MRM , FirstISTD MRM and SecondISTD MRM . The sum may be determined as follows.

Number

[0045] The sum of two alternating MRM transition sets can provide higher sensitivity. Step iii) may include rejecting any multiple reaction monitoring channel group that deviates from a reference value by more than a predetermined tolerance range.

[0046] For example, if no selection is made and the deviation of each of the quantifier / confirmatory ratios of the multiple reaction monitoring transitions is within a predetermined tolerance range, step iii) may include determining measurement results from the analyte and internal standard using the sum of the multiple reaction monitoring transitions. Thus, to determine the measurement results, the multiple reaction monitoring transitions of the analyte and internal standard in all multiple reaction monitoring channel groups may be used. For example, the transitions of the analyte and internal standard in the monitoring channel groups may be FirstAnalyte MRM , SecondAnalyte MRM , ThirdAnalyte MRM , FirstISTD MRM and SecondISTD MRM , ThirdISTDMRM It may be expressed as. The total may be determined as follows.

Number

[0047] The total of the three alternating MRM transition sets can provide higher sensitivity.

[0048] Method steps i) to iii) may be performed using at least one computer. Specifically, the control and execution of the measurement in step i) may be fully automated. Further, the data acquisition and evaluation in steps ii) and iii) may be fully automated. Specifically, this method may be fully or partially computer-implemented on a computer such as a processor.

[0049] In a further aspect, when a program is executed on a computer or computer network, specifically on a processing device, a computer program is disclosed that includes computer-executable instructions for performing the method according to any one of the embodiments described herein, specifically method steps i) to iii). Specifically, the computer program may be stored on a computer-readable data carrier and / or a computer-readable storage medium.

[0050] As used herein, the terms "computer-readable data carrier" and "computer-readable storage medium" may specifically refer to non-transitory data storage means such as a hardware storage medium storing computer-executable instructions. The computer-readable data carrier or storage medium may specifically be a storage medium such as a random access memory (RAM) and / or a read-only memory (ROM), or may comprise such a storage medium.

[0051] Specifically, therefore, one, a plurality, or even all of the method steps i) to iii) described above can be implemented by using a computer or a computer network, preferably by using a computer program.

[0052] In this specification, a computer program product is further disclosed and proposed. The computer program product has program code means for executing the method according to the present invention in one or more of the embodiments included herein when the program is executed on a computer or a computer network. Specifically, the program code means may be stored in a computer-readable data carrier and / or a computer-readable storage medium.

[0053] A data carrier that stores a data structure capable of executing the method according to one or more of the embodiments disclosed herein after being loaded into a computer or a computer network, such as the working memory or main memory of a computer or a computer network, is further disclosed and proposed in this specification.

[0054] A non-transitory computer-readable medium including instructions that, when executed by one or more processors, cause the one or more processors to execute the method according to one or more of the embodiments disclosed herein is further disclosed and proposed in this specification.

[0055] When the program is executed on a computer or a computer network, a computer program product storing program code means on a machine-readable carrier is further disclosed and proposed herein for carrying out a method according to one or more of the embodiments disclosed herein. As used herein, a computer program product refers to a program as a tradable product. The product generally exists in any format such as a paper format, or may exist on a computer-readable data carrier and / or a computer-readable storage medium. Specifically, the computer program product may be distributed on a data network.

[0056] Finally, a modulated data signal containing instructions readable by a computer system or a computer network for carrying out a method according to one or more of the embodiments disclosed herein is disclosed and proposed herein.

[0057] Regarding the computer-implemented aspects of the present invention, one or more or all of the method steps of a method according to one or more of the embodiments disclosed herein may be carried out by using a computer or a computer network. Thus, generally, any method step may be included, typically excluding method steps that require manual work such as specific ways of providing samples and / or performing actual measurements.

[0058] Specifically, herein, - a computer or a computer network comprising at least one processor, the processor being configured to carry out a method according to one of the embodiments described herein - A computer-loadable data structure configured to execute a method according to one of the embodiments described herein when executed on a computer, - A computer program configured to execute a method according to one of the embodiments described herein when executed on a computer, - A computer program comprising program means for executing a method according to one of the embodiments described herein when executed on a computer or a computer network, - A computer program comprising program means according to the preceding embodiments, wherein the program means are stored on a computer-readable storage medium, - A storage medium storing a data structure, the data structure being configured to execute a method according to one of the embodiments described herein after being loaded into the main memory and / or working memory of a computer or a computer network, and - A computer program product having program code means storable or stored on a storage medium, the method according to one of the embodiments described herein being executed when the program code means are executed on a computer or a computer network, are further disclosed.

[0059] In a further aspect of the present invention, a system for multiplex transition monitoring of at least one analyte in a sample is disclosed.

[0060] This system is, -At least one mass spectrometer device configured to measure multiple reaction monitoring transitions of both internal standards and analytes using alternating multiple reaction monitoring as both a quantifier and a qualifier, wherein the alternating multiple reaction monitoring comprises at least three groups of multiple reaction monitoring channels, and one of the groups of multiple reaction monitoring channels measures at each of the theoretical m / z values of both the internal standard and the analyte as well as the qualifier, and the two other groups of multiple reaction monitoring channels measure at m / z values shifted by a predetermined level higher and lower, respectively. At least one mass spectrometer device; -At least one database configured to store at least one reference value; -At least one processing device configured to compare at least two quantifier / qualifier ratios of multiple reaction monitoring transitions of the internal standard with reference values from the database, the comparison including determining a deviation between the quantifier / qualifier ratio and the reference value, and the processing device being 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 predetermined tolerance range, and to reject the measured multiple reaction monitoring transitions otherwise. At least one processing device; Comprising.

[0061] The system may be configured to perform a method for multiple reaction monitoring according to the present invention. Thus, for definitions and embodiments, reference is made to the description of the method above or the method described in more detail below.

[0062] In summary, without excluding the possibility of further embodiments, the following embodiments may be envisioned.

[0063] Embodiment 1. A method for multiple reaction monitoring using a mass spectrometer, the method comprising the following steps: i) By using a mass spectrometer, measuring multiple reaction monitoring transitions of both the internal standard and the analyte using alternating multiple reaction monitoring, where the alternating multiple reaction monitoring comprises at least three groups of multiple reaction monitoring channels, and one of the groups of multiple reaction monitoring channels is measured at the respective theoretical m / z values of both the quantifier and the qualifier for both the internal standard and the analyte, and the two other groups of multiple reaction monitoring channels are measured at respective m / z values shifted by a predetermined level higher and lower values; the step of measuring; ii) By using at least one processing device, comparing at least two quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values from a database, the comparison including determining a deviation between the quantifier / qualifier ratio and the reference value; the step of comparing; iii) If at least one deviation of the quantifier / qualifier ratio is within at least one predetermined tolerance range, by using a processing device, determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard, otherwise, including rejecting the measured multiple reaction monitoring transitions; the step of determining, including.

[0064] Embodiment 2. The method according to the previous embodiment, wherein the predetermined level is specified as half of the maximum expected drift of the mass axis for the relevant m / z range.

[0065] Embodiment 3. The method according to any one of the previous embodiments, wherein the predetermined tolerance range is ±15%, preferably ±10%, more preferably ±5% from the reference value.

[0066] Embodiment 4. The method according to any one of the previous embodiments, wherein step ii) includes selecting two quantifier / qualifier 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 quantifier.

[0067] Embodiment 5. The method according to the foregoing embodiment, wherein step ii) includes comparing the selected quantifier / confirmator ratio of the multiple reaction monitoring transition of the internal standard with a reference value.

[0068] Embodiment 6. The method according to any one of the foregoing embodiments, wherein step ii) includes comparing the signal intensities of the multiple reaction monitoring transitions of the internal standard quantifier, step ii) includes rejecting the multiple reaction monitoring transition with the lowest signal intensity, and step ii) further includes comparing the quantifier / confirmator ratio of the remaining multiple reaction monitoring transitions of the internal standard with a reference value.

[0069] Embodiment 7. The method according to the foregoing embodiment, wherein when the deviation from one of the quantifier / confirmator ratios of the remaining multiple reaction monitoring transitions is within a predetermined tolerance range, step iii) includes determining a measurement result from the analyte of the multiple reaction monitoring channel group corresponding to the quantifier / confirmator ratio and the multiple reaction monitoring transitions of the internal standard.

[0070] Embodiment 8. The method according to Embodiment 6, wherein when the deviations of the quantifier / confirmator ratios of both of the remaining multiple reaction monitoring transitions are within a predetermined tolerance range, step iii) includes determining a measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions.

[0071] Embodiment 9. The method according to any one of the foregoing three embodiments, wherein step iii) includes rejecting those that deviate beyond a predetermined tolerance range from the reference value.

[0072] Embodiment 10. The method according to any one of the foregoing embodiments, wherein the database comprises at least one piece of information from the group consisting of analyte ID, sample matrix ID, instrument ID, method or assay ID, and average quantifier / confirmator ratio of the internal standard.

[0073] Method according to any one of the preceding embodiments, wherein the mass spectrometer operates in random access mode and samples are loaded randomly and continuously.

[0074] Method according to any one of the preceding embodiments, wherein the mass spectrometer is a triple quadrupole mass spectrometer.

[0075] Method according to any one of the preceding embodiments, wherein the method is computer-implemented.

[0076] A system for multiple transition monitoring of at least one analyte in a sample, comprising: - at least one mass spectrometer device configured to measure multiple reaction monitoring transitions of both internal standards and analytes using alternating multiple reaction monitoring, wherein the alternating multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, and one of the multiple reaction monitoring channel groups measures at each of the respective theoretical m / z values of the quantifier and the qualifier for both the internal standard and the analyte, and the two other multiple reaction monitoring channel groups measure at respective m / z values shifted by a predetermined level higher and lower values, at least one mass spectrometer device; - at least one database configured to store at least one reference value; - 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 the database, the comparison including determining a deviation between the quantifier / qualifier ratio and the reference value, the processing device being configured to determine measurement results from the multiple reaction monitoring transitions measured from the analyte and the internal standard when the deviation is within at least one predetermined tolerance range, and to reject the measured multiple reaction monitoring transitions otherwise, at least one processing device; A system comprising.

[0077] Embodiment 15. A system for multi-transition monitoring according to the foregoing embodiments, wherein the system is configured to execute a method for multi-reaction monitoring according to any one of the foregoing method embodiments.

[0078] Embodiment 16. A computer program comprising instructions that, when executed by a processing device on a system according to any one of the foregoing embodiments referring to the system, cause the system to execute a method according to any one of the foregoing embodiments referring to the method.

[0079] Embodiment 17. A computer-readable storage medium comprising instructions that, when the instructions are executed by a processing device on a system according to any one of the foregoing embodiments referring to the system, cause the system to execute a method according to any one of the foregoing embodiments referring to the method.

[0080] Embodiment 18. A non-transitory computer-readable medium comprising instructions that, when executed by one or more processors, cause the one or more processors to execute a method according to any one of the foregoing embodiments referring to the method.

[0081] Any further optional features and embodiments are preferably disclosed in more detail in the following description of the embodiments, in conjunction with the dependent claims. In that context, each optional feature may, as will be understood by those skilled in the art, be implemented in an independent manner and in any practicable combination. The scope of the present invention is not limited by the preferred embodiments. The embodiments are schematically illustrated in the figures. Here, the same reference numerals in these figures refer to the same or functionally equivalent elements.

Brief Description of the Drawings

[0082]

Figure 1

Figure 2

Figure 3A

Figure 3B

[0083] FIG. 1 shows one embodiment of a system 100 according to the present invention. In this embodiment, the system 100 includes a liquid chromatography mass spectrometry (LC-MS) apparatus. The system 100 may include an inlet 102 connected to a liquid chromatography column 104. The column 104 may be coupled to an MS apparatus 106 via an optional valve 122 connected to an optional waste reservoir 124. The MS apparatus 106 may include an ionization device 108, a skimmer 110, quadrupole stages Q1 112, Q2 114, and Q3 116, and a detector 118. Each component may optionally be connected to a processing device 120.

[0084] The MS apparatus 106 is configured to measure multiple reaction monitoring transitions of both internal standards and analytes as quantifiers and confirmers using sequential multiple reaction monitoring (S-MRM). Sequential multiple reaction monitoring includes at least three groups of multiple reaction monitoring channels. One of the groups of multiple reaction monitoring channels measures at the respective theoretical m / z values of both the internal standard and the analyte as quantifiers and confirmers, and the two other groups of multiple reaction monitoring channels measure at respective m / z values shifted by a predetermined level higher and lower. Sequential multiple reaction monitoring may include multiple reaction monitoring using a set of sequential m / z values, such as theoretical m / z values, and m / z values shifted by a predetermined level higher and lower. The predetermined level may be specified as half of the maximum expected drift of the mass axis for the relevant m / z range. Additional measurements at other levels may also be possible.

[0085] Interactive multiple reaction monitoring comprises at least three groups of multiple reaction monitoring channels. A group of multiple reaction monitoring channels may comprise one or more of the MRM transitions measured at Q1 and Q3 using the same m / z value. One of the groups of multiple reaction monitoring channels measures at the respective theoretical m / z values of both the internal standard and the quantifier and qualifier of the analyte, and the two other groups of multiple reaction monitoring channels measure at the respective m / z values shifted by a predetermined level higher and lower. For example, S-MRM may use the MS resolution at Q1 and Q3 optimized for the target analyte of all MRM transitions. The first set of MRM transitions may be measured as comprising the MRM transitions of both the analyte and the internal standard at their respective target m / z values for both the quantifier and the qualifier. Further, a second set of MRM transitions shifted to higher m / z values at Q1 and Q3 may be measured, and a third set of MRM transitions shifted to lower m / z values at Q1 and Q3 may be measured. Thus, a total of six MRM transitions for the target analyte and a total of six MRM transitions for the internal standard may be measured. For example, the method may comprise the following measured values.

Table 2

[0086] The processing device 120 may comprise at least one electronic processor. The processing device 120 is configured to compare at least two quantifier / qualifier ratios of the multiple reaction monitoring transitions of the internal standard with reference values from the database 126. The comparison includes determining a deviation between the quantifier / 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 if the deviation is within at least one predetermined tolerance range, and to reject the measured multiple reaction monitoring transitions otherwise.

[0087] 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, refer to the description of the method according to the present invention, as described, for example, with respect to FIG. 2.

[0088] During operation of the system 100, a sample is introduced into the inlet 102, for example, by direct injection. After introduction, the sample may enter the column 104 and be deposited on the column material (e.g., resin material). The sample may move across the column material as one or more solvents flow across the column material. As the sample moves collectively, different components of the sample move at different rates and thus reach the end of the column at different times. The column 104 may optionally be connected to the valve 122 as described above and thus optionally to the waste reservoir 124. During operation of the system 100, the valve 122 may optionally be actuated by the processing device 120 to direct the eluent from the column 104 to either the waste reservoir 124 or the MS device 106. By selectively directing only a portion of the eluent to the MS device 106, it can be ensured that only the components of interest in the sample are measured. The MS device 106 can include a detector connected to the processing device 120 that generates an electrical signal when the components of the sample elute from the column 104 and reach the detector. The processing device 120 may receive the electrical signal and determine whether to direct the eluent to either the waste reservoir 124 or the MS device 106. The processing device 120 may be configured to determine the direction in which to direct the eluent based on the elapsed time between the introduction of the sample at the inlet 102 and the detection of the components emerging from the downstream end of the column 104. The elapsed time may provide at least a preliminary identification of the components by comparison with reference information including the elution times from known sample components. Based on that preliminary identification, the processing device 120 can determine whether the component is of interest (and thus is directed to the MS device 106) or not of interest (and thus is directed to the waste reservoir 124). If the sample components have not eluted from the column 104 (e.g., during a time interval when only the elution solvent emerges from the column 104), the eluent may optionally be directed to the waste reservoir 124 instead of the MS device 106.

[0089] To facilitate component detection when the sample components are eluted from column 104, various detectors can be arranged between column 104 and MS device 106, either before valve 122, between valve 122 and MS device 106, or between valve 122 and waste reservoir 124. Examples of suitable detectors include, but are not limited to, photodiodes, photocells, spectral detectors, and optical detectors such as CCDs, as well as electrical detectors such as conductivity sensors and resistivity sensors.

[0090] The sample components entering MS device 106 may be received by ionization device 108, where they are ionized to form a population of ions. Ionization device 108 can be implemented as any of a wide variety of different types of ionization devices. Examples of suitable ionization devices include, but are not limited to, electrospray ionization devices, electron ionization devices, atmospheric pressure chemical ionization devices, thermospray ionization devices, inductively coupled plasma ionization devices, glow discharge ionization devices, and photoionization devices. The population of ions generated by ionization device 108 may pass through skimmer 110, which typically includes an aperture that is small (relative to the exit aperture of ionization device 108) and reduces the population of ions directed toward the quadrupole stage of MS device 106. After passing through skimmer 110, the ions may be separated and detected in the remainder of MS device 106.

[0091] A wide variety of different mass spectrometer configurations are used to separate, detect, and analyze the ions generated from the 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 in no way limited to the configuration shown in FIG. 1.

[0092] For example, the MS device 106 may 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, the ions passing through the skimmer 110 are filtered to select ions that fall within a specific range of m / z values for further analysis. Ions outside this m / z value range are blocked and do not pass through the quadrupole stage 112. Conversely, ions within the desired range of m / z values pass through the quadrupole stage 112 and enter the second quadrupole stage 114. The quadrupole stage 112 may comprise four electrodes arranged around a central symmetry axis. To selectively direct only ions having m / z values within the desired range to the second quadrupole stage 114, the processing device 120 may be configured to adjust the potentials applied to the four electrodes. When appropriate potentials are applied, the four quadrupole electrodes generate an oscillating radio frequency (RF) field that functions to guide the ions along the quadrupole stage 112 from one end to the other. For a specific RF field, ions within a specific range of m / z values are guided out of the exit aperture of the quadrupole stage 112, and ions with m / z outside the range are rejected (e.g., blocked) within the quadrupole stage 112. A subset of the 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 the ions entering the stage 114 are fragmented to form a distribution of ions of relatively small molecular mass. Typically, this distribution of smaller mass ions derived from larger mass ions entering from stage 112 to stage 114 passes through stage 114 and enters the third quadrupole stage 116. Within the second quadrupole stage 114, the processing device 120 may establish a magnetic field gradient between the inlet aperture and the outlet aperture of the stage 114 and apply potentials to one or more electrodes to generate one or more electric fields. Ions entering from stage 112 are typically accelerated by the magnetic field gradient. Atoms or molecules of a neutral gas are introduced into stage 114 and collide with the accelerated ions entering from stage 112 to generate ion fragments that pass through stage 116 (via the collision).A variety of gases, including but not limited to hydrogen, nitrogen, and noble gases such as argon, can be used in the splitting process. After the distribution of smaller mass ions (referred to herein as "fragment ions") enters the third quadrupole stage 116, the fragment ions are filtered through the filter in a manner similar to the filtering performed at stage 112. Specifically, stage 116 may comprise four electrodes arranged around the 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 having m / z values that each fall within a specific range from one end of stage 116 to the other end and to the detector 118. Ion fragments having m / z values outside this range are rejected (e.g., blocked) within the quadrupole stage 116. The subset of ion fragments is directed from the third quadrupole stage 116 and measured by the detector 118. Specifically, a measurement signal corresponding to the fragment is generated by the detector 118 and transmitted to the processing device 120 that records the intensity of the measurement signal. The detector 118 may incorporate various different detection techniques. In certain embodiments, the detector 118 corresponds to an electron multiplier, a Faraday cup, or a microchannel plate detector. In some embodiments, the detector 118 is an orbitrap type detector. More generally, the detector 118 may implement any one or more known ion detection techniques.

[0093] FIG. 2 shows a flowchart of one embodiment of the method according to the present invention.

[0094] The method may include, by way of example, the following steps that may be executed in a given order. However, it should be noted that different orders are also possible. Furthermore, it is also possible to execute one or more of the method steps once or repeatedly. Additionally, it is possible to execute two or more method steps simultaneously or overlapping in time. The method may include additional method steps not listed.

[0095] This method comprises the following steps: i) Measuring, by using a mass spectrometer 106, multiple reaction monitoring transitions of quantifiers and confirmers for both an internal standard and an analyte using alternating multiple reaction monitoring (shown by reference numeral 128), wherein the alternating multiple reaction monitoring comprises at least three groups of multiple reaction monitoring channels, one of the groups of multiple reaction monitoring channels being measured at the respective theoretical m / z values of the quantifiers and confirmers for both the internal standard and the analyte, and the two other groups of multiple reaction monitoring channels being measured at respective m / z values shifted by a predetermined level higher and lower (measuring, shown by reference numeral 128); ii) Comparing, by using at least one processing device 120, at least two quantifier / confirmer ratios of the multiple reaction monitoring transitions of the internal standard with reference values from a database 126 (shown by reference numeral 130), the comparing comprising determining a deviation between the quantifier / confirmer ratio and the reference value (comparing, shown by reference numeral 130); iii) If the deviation of at least one of the quantifier / confirmer ratios is within at least one predetermined tolerance range (shown by reference numeral 134), determining measurement results from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using a processing device (shown by reference numeral 132), and if not, rejecting the measured multiple reaction monitoring transitions (step, shown by reference numeral 136). It includes the above steps.

[0096] As outlined above, for example, S-MRM may use MS resolution at Q1 and Q3 optimized for the target analyte for all MRM transitions. The first MRM transition set may be measured as comprising MRM transitions for both the analyte and the internal standard at their respective target m / z values as quantifiers and confirmers. Further, a second MRM transition set shifted to higher m / z values at Q1 and Q3 may be measured, and a third MRM transition set shifted to lower m / z values at Q1 and Q3 may be measured. Thus, a total of six MRM transitions for the target analyte and a total of six MRM transitions for the internal standard may be measured.

[0097] The reference value used in step ii) may be a predefined and / or pre-measured quantifier / confirmers ratio of the internal standard at the target m / z. The database 126 may comprise at least one piece of information from the group consisting of analyte ID, sample matrix ID, instrument ID, method or assay ID, and the average quantifier / confirmers ratio of the internal standard. The processing device 120 may determine the reference value from one or more of this information.

[0098] Step ii) may include comparing 130 all the quantifier / confirmers ratios of the multiple reaction monitoring transitions of the internal standard measured in step i) with the reference value.

[0099] Additionally or alternatively, in step ii), selection 138 may be performed. Step ii) may include selecting 138 two quantifier / confirmatory ratios of the multiple reaction monitoring transitions of the internal standard according to the signal intensities of the multiple reaction monitoring transitions of the internal standard quantifier. For example, step ii) may include comparing the signal intensities of three MRM transitions of the internal standard quantifier. The comparison of the signal intensities may be performed by executing a software algorithm. When the mass axis is stable (i.e., the drift is minimal or there is no drift), the group of multiple reaction monitoring channels measured using the m / z values of the target analyte and the ISTD respectively generates the highest signal intensity. When the mass axis drifts significantly to higher or lower m / z values, one of the other groups of multiple reaction monitoring channels generates the highest signal intensity.

[0100] Step ii) may include rejecting 136 the multiple reaction monitoring transition having the lowest signal intensity. For example, step ii) may include selecting 138 two of the three internal standard MRM transitions having the highest signal intensities and rejecting 136 the MRM transition having the lowest signal intensity. Accordingly, the comparison 130 in step ii) may include comparing 140 the selected quantifier / confirmatory ratios of the remaining multiple reaction monitoring transitions of the internal standard with a reference value.

[0101] Depending on the result of the comparison in step ii), different operations may be performed in step iii). If at least one deviation of the quantifier / confirmator ratio is within at least one predetermined tolerance range, step iii) includes determining the measurement result 132 from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using the processing device 120. The predetermined tolerance range may be ±15%, preferably ±10%, more preferably ±5% from the reference value. Determining the measurement result 132 may be performed by executing a software algorithm. The measurement result may be or may include at least one quantitative information, such as a value, regarding the analyte in the sample. The measurement result may be the final patient result. The measurement result may further include quality information regarding the stability of the mass axis according to the determined deviation, such as a flag.

[0102] Otherwise, that is, if any deviation of the quantifier / confirmator ratio is not within the predetermined tolerance range, step iii) includes rejecting the measured multiple reaction monitoring transition 136. Step iii) may further include flagging the data as an outlier 142. Step iii) may include checking the quantifier / confirmator ratio of the remaining two MRM transitions of the internal standard and rejecting those that deviate beyond at least one predetermined tolerance range from the reference value.

[0103] For example, if the deviation from only one of the quantifier / confirmator ratios of the remaining multiple reaction monitoring transitions is within the predetermined tolerance range, step iii) includes determining the measurement result 144 from the multiple reaction monitoring transitions of the analyte and the internal standard of the multiple reaction monitoring channel group corresponding to the above quantifier / confirmator ratio. For example, the final patient result is obtained by using a single set of MRM transitions of the analyte and the internal standard of the multiple reaction monitoring channel group that meets the conditions, for the analyte MRM / ISTD MRMcan be calculated by. Step iii) may include rejecting any group of multiple reaction monitoring channels that deviate from a reference value by more than a predetermined tolerance range.

[0104] For example, if the deviations of both the quantifier / confirmator ratios of the remaining multiple reaction monitoring transitions are within a predetermined tolerance range, step iii) may include determining the measurement results from the analyte and internal standard using the sum of the remaining multiple reaction monitoring transitions 146. For determining the measurement results 146, the multiple reaction monitoring transitions of the analyte and internal standard of the group of multiple reaction monitoring channels corresponding to the above quantifier / confirmator ratio that meet the conditions may be used. For example, the analyte and internal standard transitions of the monitoring channel group that meet the conditions may be represented as FirstAnalyte MRM , SecondAnalyte MRM , FirstISTD MRM , and SecondISTD MRM and may be determined as follows.

Number

[0105] Figures 3A and 3B show the experimental results.

[0106] The experimental configuration was as follows. Cyclosporin A (Sigma-Aldrich, 30024), Cyclosporin A-(d10) (produced in-house by Roche), 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 TQ6500+).

[0107] A 90% acetonitrile (v / v) solution containing 100 ng / mL of cyclosporine A (analyte) and 100 ng / mL of isotopically labeled cyclosporine A-(d10) (ISTD) was injected by an external syringe pump (injection pump) at a flow rate of 40 μL / min. In parallel, an LC pump was operated under uniform concentration conditions using 10% of eluent A (15 mM ammonium acetate) and 90% of eluent B (methanol) (v / v). The flow rate of the LC pump was set at 400 μL / min. Both the injection pump and the LC pump were connected via a T-piece, and a total flow rate of 440 μL / min was directed to the ESI-MS / MS.

[0108] Data were acquired in positive polarity and MRM mode. The transition of the target analyte was m / z 1269.875 / 1184.838 amu. The target ISTD transition was m / z 1229.938 / 1194.901 amu. Further, the target m / z - 0.2 amu and the target m / z + 0.2 amu were measured for both the analyte and the ISTD, obtaining a total of six transitions. The measurement time for each transition was 40 ms, and the total number of cycles was 16. Both quadrupoles of the MS / MS instrument were set to a resolution of 0.8 amu. Data analysis was performed based on the signal intensity in cps units.

[0109] At the top of Figure 3A, the average intensity as a function of mass shift is shown for the internal standard quantifier. At the bottom of Figure 3A, the average quantifier / confirmator ratio as a function of mass shift is shown for the internal standard quantifier. The target m / z, the target m / z - 0.2 amu, and the target m / z + 0.2 amu are shown as vertical solid lines. Further, a predetermined tolerance range of ±10% is shown at the bottom of Figure 3A, which is used in step iii).

[0110] In FIG. 3A, several m / z values in the range of -0.5 to +0.5 Da of the theoretical m / z were measured. However, as described above with respect to FIGS. 1 and 2, only three m / z values per analyte and ISTD can be used for the following data analysis. These are shown in FIG. 3B. At the top of FIG. 3B, the average intensity as a function of the mass shift of the internal standard quantifier is shown for the three m / z values used (target m / z, target m / z - 0.2 amu, and target m / z + 0.2 amu). At the bottom of FIG. 3B, the average quantifier / confirmator ratio as a function of the mass shift is shown for the internal standard quantifier. Further, a predetermined tolerance range of ±10% is shown at the bottom of FIG. 3B, which is used in step iii).

[0111] FIG. 3B further shows the selection 138 of two quantifier / confirmator 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 quantifier. Two of the three ISTD MRMs having the highest signal intensity may be selected. (138) (The selected ISTD MRMs are highlighted in circles). The quantifier / confirmator ratios of the two selected ISTD MRMs may be compared with a reference from the database 126, for example 1.578 in this case. If the ratio of the two selected MRMs is within ±10% compared to the reference, the final patient result may be calculated from the analyte and ISTD by using the sum of the two MRMs as described above.

Explanation of symbols

[0112] 100 System 102 Inlet 104 Liquid chromatography column 106 MS device 108 Ionization device 110 Skimmer 112 Q1 114 Q2 116 Q3 118 Detector 120 Processing device 122 Valve 124 Waste reservoir 126 database 128 To measure 130 To compare 132 To determine 134 Is it a deviation within a predetermined tolerance range? 136 To reject 138 Selection 140 To compare 142 To set a flag 144 To determine the measurement result 146 To determine the measurement result

Claims

1. A method for monitoring multiple reactions using a mass spectrometer (106), wherein the method is i) Step (128) of measuring the multiple reaction monitoring transitions of both the quantifier and confirmator of both the internal standard and the analyte using alternating multiple reaction monitoring by using the mass spectrometer (106), wherein the alternating multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, one of the multiple reaction monitoring channel groups measures at the respective theoretical m / z values ​​of the quantifier and confirmator of both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups measure at the respective m / z values ​​shifted to a predetermined level higher and lower, respectively. ii) A comparison step (130) in which, for at least two groups, using at least one processing device (120), at least two of the quantifier / confirmer ratios of the internal standard multiple reaction monitoring transitions are compared to a reference value from a database (126), wherein the comparison includes determining a deviation between the quantifier / confirmer ratio and the reference value. iii) A determining step (132) which includes determining a measurement result from the measured multiple reaction monitoring transitions of the analyte and the internal standard by using the processing apparatus (120) if at least one of the deviations of the quantitative instrument / confirmer ratio is within at least one predetermined tolerance range, and otherwise rejecting the measured multiple reaction monitoring transition (136), Methods that include...

2. The method according to claim 1, wherein the predetermined level is specified as half of the maximum expected drift of the mass axis for the relevant m / z range.

3. The method according to claim 1, wherein the predetermined tolerance range is ±15%, preferably ±10%, and more preferably ±5% from the reference value.

4. The method according to claim 1, wherein step ii) selects two quantifier / confirmer ratios of the multiple reaction monitoring transition of the internal standard in accordance with the signal intensity of the multiple reaction monitoring transition of the internal standard (138).

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

6. The method according to claim 4, further comprising step ii) comparing the signal intensity of the multiple reaction monitoring transitions of the internal standard quantifier, step ii) rejecting the multiple reaction monitoring transition having the lowest signal intensity (136), and step ii) further comprising comparing the quantifier / confirmer ratio of the remaining multiple reaction monitoring transitions of the internal standard with the reference value (130).

7. The method according to claim 6, wherein if the deviation of the remaining multiple reaction monitoring transitions from one of the quantifier / confirmer ratios is within the predetermined tolerance range, step iii) determines the measurement result from the multiple reaction monitoring transitions of the analyte and the internal standard in the group of multiple reaction monitoring channels corresponding to the quantifier / confirmer ratio (144).

8. The method according to claim 6, wherein if the deviations of the quantifier / confirmer ratios of both of the remaining multiple reaction monitoring transitions are within the predetermined tolerance range, step iii) determines the measurement result from the analyte and the internal standard using the sum of the remaining multiple reaction monitoring transitions (146).

9. The method according to claim 6, wherein if the deviation of the quantifier / confirmer ratio of both of the remaining multiple reaction monitoring transitions is not within the predetermined tolerance range, step iii) rejects any deviation from the reference value that exceeds the predetermined tolerance range.

10. The method according to any one of claims 1 to 9, wherein the method is computer-implemented.

11. A system (100) for multiple transition monitoring of at least one analyte in a sample, At least one mass spectrometer (106) configured to measure the multiple reaction monitoring transitions of both the quantifier and confirmator for both the internal standard and the analyte using alternating multiple reaction monitoring, wherein the alternating multiple reaction monitoring comprises at least three multiple reaction monitoring channel groups, one of which measures at the respective theoretical m / z values ​​of the quantifier and confirmator for both the internal standard and the analyte, and the other two multiple reaction monitoring channel groups measure at their respective m / z values ​​shifted by a predetermined level higher and lower, respectively. 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 quantifier / confirmer ratios of the multiple reaction monitoring transitions of the internal standard with a reference value from the database, wherein the comparison includes determining a deviation between the quantifier / confirmer ratio and the reference value, and 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 predetermined tolerance range, and to reject the measured multiple reaction monitoring transitions otherwise. A system (100) comprising the above.

12. A computer program comprising instructions, wherein when the program is executed by a processing unit on the system (100) described in claim 11, the program comprises instructions causing the system (100) to execute the method described in any one of claims 1 to 9.

13. A computer-readable storage medium comprising instructions, wherein when the instructions are executed by a processing device on the system (100) described in claim 11, the instructions cause the system (100) to execute the method described in any one of claims 1 to 9.

14. A non-temporary computer-readable medium comprising instructions, wherein, when executed by one or more processors, the instructions cause the one or more processors to perform the method according to any one of claims 1 to 9.

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