Method for quality check of at least one LC-MS measurement

Abstract

A method is proposed for quality checking of at least one liquid chromatography-mass spectrometry (LC-MS) measurement on a sample comprising a substance under test of interest and at least one internal standard in a specified amount. The method comprises the following steps, namely: a) information regarding the analyte signal of the LC-MS measurement TIFF2025524604000079.tif627 and information regarding the internal standard signal TIFF2025524604000080.tif627 are determined (step 120); b) by using at least one processing device (114), at least one monitoring parameter is determined (step 122) that includes at least a minimum of the internal standard signal relative to the analyte signal of the sample, by using the information regarding the analyte signal and the information regarding the internal standard signal; and c) by using the processing device (114), the information regarding the internal standard signal is compared with the monitoring parameter (step 124), wherein the LC-MS measurement is flagged as passing the quality check if the information regarding the internal standard signal is greater than or equal to the monitoring parameter by using the processing device (114), and is flagged as not passing if otherwise.

Description

Technical Field

[0001] The present disclosure relates to a method and a system for quality checking of at least one liquid chromatography-mass spectrometry measurement.

Background Art

[0002] For quality assurance of liquid chromatography-mass spectrometry (LC-MS) assays, usually separate samples are measured with a target compound of known concentration. Usually, quality control (QC) samples are tested at specific frequencies in the analysis run and the calculated results are checked against acceptance ranges. Further, a prescribed amount of internal standard (IStd) is usually added to each sample to enable monitoring of chromatographic parameters such as absolute peak area, retention time, and peak width. Monitoring of the signal of the IStd in the LC-MS assay is a common way to ensure sufficient performance and quality of the measurement and thus the results obtained for the samples. This concept is part of several international guidelines for mass spectrometry assays, such as the guidelines by CLSI (Clinical and Laboratory Standards Institute), EMA (European Medicines Agency), or GTFCh (German society for toxicological and forensic chemistry). Such techniques are described, for example, in U.S. Patent No. 9,606,088, U.S. Patent No. 9,465,911, U.S. Patent No. 7,451,052, U.S. Patent Application Publication No. 2011 / 0101215, and German Patent Application Publication No. 10 2020 200 915.

[0003] Each of the IStd parameters must meet the tolerance requirements for the maximum deviation or a specific cut-off value. The variation of the IStd should ideally cover the variation of the entire assay. Therefore, the IStd can be used, on the one hand, for the normalization of the analyte signal for the purpose of enhancing the accuracy of the results. However, on the other hand, due to these variations, there is a possibility that the false sample flagging rate will increase when the parameters are outside the allowable range, or due to the variations, a wide allowable range may be required for each of the IStd parameters. A high sample flagging rate increases the workload because each sample needs to be reexamined, for example, by an expert. A wide allowable range not only reduces the flagging rate but also the ability to monitor the IStd, and is accompanied by the risk of overlooking defective measurement values. Therefore, alternative or supplementary techniques are desired to fill the above-mentioned gap and avoid frequent manual reexamination of chromatograms by experts for verification.

[0004] German Patent Application Publication No. 10 2020 200915 describes a chromatograph device including a control unit and a storage unit. The storage unit stores diagnostic criteria based on the waveforms W0 to W2 or intensities A0 to A2 of the standard chromatogram CR of the standard sample to be measured. When the measured chromatogram CR of the standard sample whose quantity or ratio is known in advance is measured, the control unit notifies management information regarding the chromatograph device based on the diagnostic criteria regarding the waveforms W0a, W0c to W2c, or intensities A0b to A2b of the measured chromatogram.

Summary of the Invention

[0005] Therefore, an object of the present invention is to provide a method and a system for quality checking of at least one liquid chromatography-mass spectrometry measurement, which avoid the above-mentioned drawbacks of the known methods and devices. In particular, the method and the system must reduce the need for manual reexamination without reducing the ability to monitor parameters for quality assurance purposes.

[0006] This problem is addressed by a method and a system for quality checking of at least one liquid chromatography - mass spectrometry measurement, by a method and a system having the features of the independent claims. Advantageous embodiments, which may be implemented alone or in any combination, are set out in the dependent claims as well as throughout the specification.

[0007] When used hereinafter, the terms "having", "comprising", or "including", or any grammatical variant thereof, are used in a non - exclusive manner. Thus, these terms can refer to both situations where there are no additional features in the entity being described in this context, in addition to the features introduced by these terms, and situations where there are one or more additional features. By way of example, the expressions "A has B", "A comprises B", and "A includes B" can refer to both situations where there are no elements other than B in A (i.e., the situation where A is exclusively composed of only B), and situations where there are one or more additional elements in entity A, such as element C, elements C and D, or yet further elements.

[0008] Furthermore, it should be noted that the term "at least one" or "one or more" or similar expressions indicating that a feature or element may be present one or more times is typically used only once when introducing each respective feature or element. Hereinafter, in most cases, when referring to each respective feature or element, the expressions "at least one" or "one or more" are not repeated, despite the fact that each respective feature or element may be present one or more times.

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

[0010] In a first aspect of the present invention, a method for quality checking of at least one liquid chromatography-mass spectrometry (LC-MS) measurement on a sample comprising a target analyte and a specified amount of at least one internal standard is disclosed.

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

[0012] The method includes the following steps, namely, a) information regarding the analyte signal of the LC-MS measurement

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[0013] The method may be computer-implemented. As used herein, the term "computer-implemented method" is a broad term and should be given its general and ordinary 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 method involving at least one computer and / or at least one computer network. 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 executed completely automatically, specifically without interaction with a user. As used herein, the terms "automatically" and "automation" are broad terms and should be given their general and ordinary 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 process that is fully implemented by at least one computer and / or computer network and / or machine, especially without manual acts and / or interaction with a user.

[0014] LC-MS measurements can be performed by using at least one mass spectrometer configured to perform at least one LC-MS measurement on a sample containing the target analyte and a specified amount of internal standard. As used herein, the term "mass spectrometry" is a broad term and should be given its general and ordinary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term can refer to an analytical technique for determining the mass-to-charge ratio of ions. As used herein, the term "mass spectrometer", also referred to as a "mass analyzer", is a broad term and should be given its general and ordinary meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without limitation, this term can refer to an analyzer configured to detect at least one analyte based on the mass-to-charge ratio.

[0015] The mass spectrometer may be at least one quadrupole analyzer or may comprise at least one quadrupole analyzer. As used herein, the term "quadrupole mass analyzer" is a broad term and should be given its general and ordinary 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 mass analyzer comprising at least one quadrupole as a mass filter. The quadrupole mass analyzer may comprise a plurality of quadrupoles. For example, the quadrupole mass analyzer may be a triple quadrupole mass spectrometer. For example, the mass spectrometer may also comprise an ionization source, a skimmer, three quadrupole stages Q1, Q2, and Q3, and a detector. Each of the quadrupole stages Q1, Q2, and Q3 comprises a quadrupole.

[0016] As used herein, the term "mass filter" is a broad term and should be given its ordinary and common 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 an apparatus configured to select ions injected into the mass filter according to their mass-to-charge ratio m / z. The mass filter may comprise two pairs of electrodes. The electrodes may be rod-shaped and may in particular be 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, such as 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 sign of the charge of the electrode pairs such that stable orbits are possible only for ions within a specific range of mass-to-charge ratios m / z. The orbits of the ions in the mass filter can be described by the Mathieu differential equation. In order to measure ions of different m / z values, the DC and AC voltages may be varied over time so that ions having different m / z values can be conveyed to the detector of the mass spectrometer.

[0017] The mass spectrometer may further include at least one ionization source. As used herein, the term "ionization source", which is also referred to as "ion source" or "ionizer", is a broad term and should be given its ordinary and common meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, but not limited thereto, this term may refer to, for example, an apparatus configured to generate ions from neutral gas molecules. The ionization source may be at least one ionization source selected from the group consisting of at least one gas-phase ionization source such as at least one electron impact (EI) source or at least one chemical ionization (CI) source, at least one plasma desorption (PDMS) source, at least one fast atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source, and at least one matrix-assisted laser desorption (MALDI) source, or may include such an ionization source.

[0018] 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 and general 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 at least one electron multiplier tube or may comprise at least one electron multiplier tube. The mass spectrometer, particularly the detector of the mass spectrometer and / or at least one processing device, 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 and general meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, without being limited thereto, this term may refer to a two-dimensional representation of the signal intensity versus the charge-to-mass ratio m / z, where the signal intensity corresponds to the abundance of each ion. The mass spectrum may be an image composed of pixels. To determine the intensity obtained for 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 can be identified by the processing device. Specifically, the processing device may be configured to correlate known masses to the identified masses or may be configured by characteristic fragmentation patterns.

[0019] LC-MS measurement may be at least one measurement by using a mass spectrometer, or may include at least one measurement by using a mass spectrometer. The mass spectrometer may comprise at least one liquid chromatograph, also called a liquid chromatography (LC) device. The mass spectrometer may be a liquid chromatography mass spectrometer. The liquid chromatograph may be used for sample preparation for the mass spectrometer. As used herein, the term "liquid chromatography (LC) device" is a broad term and should be given its general and ordinary 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 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 by 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, and the mobile phase is sent through the stationary phase for separation and / or elution and / or transfer 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 containing at least one analyte of interest. As used herein, the term "liquid chromatography mass spectrometer" is a broad term and should be given its general and ordinary 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 combination of liquid chromatography and mass spectrometry. The liquid chromatography mass spectrometer may be at least one high performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (μLC) device, or may comprise these.A liquid chromatography mass spectrometer may comprise a liquid chromatography (LC) device and, in this case, a mass spectrometry (MS) device which 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 comprise an ionization source configured to generate molecular ions and transfer the molecular ions into the gas phase. The interface may further comprise 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.

[0020] A 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 in 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 may measure the ion fragmentation pattern for each component. Each ion fragmentation pattern consists of one or more peaks corresponding to ion fragments having a particular m / z ratio. The pattern of peaks (e.g., the m / z ratio and intensity of the peaks) for a particular analyte effectively functions as a “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 also be performed based on the time period between the initial introduction of the sample (e.g., injection into the inlet of an LC-MS system) and the elution of the component from the LC column, or the time period between the initial introduction of the sample and the measurement of the component ion fragmentation pattern in the mass spectrometer. Since a particular component may move through the LC column at a particular rate, the elapsed time period can be used as an indicator of the identity of the component. Similar to the ion fragmentation pattern, the elapsed time period can be compared to reference information (e.g., previously measured migration and / or measurement times for known components) to determine the identity of the component.

[0021] As used herein, the term "sample" 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 any test sample, such as a biological sample. A 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, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, lymphatic fluid, interstitial fluid, cerebrospinal fluid, tissue, cells, and the like.

[0022] A sample may contain one or more analytes of interest, also referred to as target analytes. A sample may be used directly as obtained from its respective source or may be subjected to a pretreatment and / or sample preparation workflow. For example, a sample may be pretreated by addition of an internal standard and / or dilution in another solution and / or mixing with a reagent. For example, analytes of interest may generally be vitamins D, drugs of abuse, therapeutic agents, hormones, and metabolites. For further details regarding samples, see, for example, European Patent Application Publication No. 3 425 369, the entire disclosure of which is incorporated herein by reference. Other analytes of interest are possible.

[0023] 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 may specifically, but not by way of limitation, refer to a known amount of a substance. An internal standard may exhibit characteristics similar to the analyte of interest when subjected to a workflow using a mass spectrometer. The workflow may include any pre-treatment, concentration, and actual detection steps. For example, the internal standard may be an isotope-labeled variant of the analyte of interest (e.g., containing labels such as 2H, 13C, or 15N). The method may include adding at least one internal standard substance to the sample.

[0024] The term "quantifier" as used herein, also referred to as "quantitative ion" when used herein, 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 by way of limitation, refer to a transition necessary for quantification of the analyte. The quantifier may be the most abundant ion. The term "qualifier" as used herein, also referred to as "confirmation ion" when used herein, 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 by way of limitation, refer to additional transitions for confirming 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 may specifically, but not by way of limitation, refer to the signal intensity ratio or peak area ratio of the quantifier and qualifier.

[0025] As used herein, the term "quality" is a broad term and should be given its ordinary and common meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. Specifically, but not by way of limitation, this term may refer to a measure of the reliability of measurement results obtained by using a mass spectrometer. The measurement results may be quantitative and / or qualitative measurements, such as the presence and / or concentration of an analyte in a sample. Quality may depend on several factors such as measurement conditions, background, interference, instrument performance, retention time shift, peak width, peak height, and peak area.

[0026] As used herein, the term "quality check" is a broad term and should be given its ordinary and common 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 at least one process for testing whether at least one predetermined quality requirement is met after data acquisition. Quality checks can be used to distinguish reliable measurement results from unreliable measurement results. As used herein, the term "reliable" is a broad term and should be given its ordinary and common 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 measurement results that are considered to be one or more of reliable, acceptable, or "good quality". A reliable measurement result may be a measurement result in which the corresponding internal standard signal meets at least one predetermined requirement. A reliable measurement result may be a measurement result having an accuracy and / or precision within a predetermined tolerance range. As used herein, the term "unreliable" is a broad term and should be given its ordinary and common 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 measurement results that are considered to be one or more of unreliable, unacceptable, or "not good quality". An unreliable measurement result may be a measurement result in which the corresponding internal standard signal does not meet at least one predetermined requirement. An unreliable measurement result may be a measurement result having an accuracy and / or precision outside the tolerance range.

[0027] For example, quality checks may be performed for each measurement. For example, quality checks may be performed at a predetermined frequency, such as after a predetermined period of time, after a new calibration, after a column replacement, or after one or more of the maintenance activities of the instrument.

[0028] As used herein, the term "analyte signal" is a broad term and should be given its ordinary and common meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term may refer, without limitation, to signals from LC-MS measurements related to the analyte, such as peaks in the chromatogram and / or mass spectrum of the analyte. The term "internal standard signal" is a broad term as used herein and should be given its ordinary and common meaning to one of ordinary skill in the art and should not be limited to a special or particular meaning. This term may refer, without limitation, to signals from LC-MS measurements related to the internal standard, such as peaks in the chromatogram and / or mass spectrum of the internal standard. Information regarding the analyte signal may be at least one of peak area, peak height, peak width, or retention time. Information regarding the internal standard signal may be at least one of peak area, peak height, peak width, or retention time.

[0029] The determination of information regarding the analyte signal and information regarding the internal standard signal may include one or more of measurement, acquisition, or reading of measurement data.

[0030] For example, information regarding the analyte signal and information regarding the internal standard signal may be determined by performing at least one LC-MS measurement using a mass spectrometer. For example, step a) may include performing at least one LC-MS measurement on a sample. In step a), at least one liquid chromatography mass spectrometer may be used.

[0031] For example, information regarding the analyte signal and information regarding the internal standard signal may be determined by, for example, reading measurement data via at least one communication interface of a processing device. The measurement data read by the communication interface may be acquired by using a mass spectrometer and transmitted to the processing device. For example, the measurement data may be read by the communication interface from at least one database. For example, the determination may include the processing device reading at least one chromatogram and / or mass spectrum via, for example, its communication interface. As used herein, the term "communication interface" is a broad term and should be given its general and ordinary 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 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 example, for the purpose of transmitting or outputting information to another device. In addition to or instead of this, the communication interface may be configured to transfer information to a computing device such as a computer, for example, for receiving information. 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 (registered trademark), NFC, inductive coupling, etc. 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 be at least one web interface.

[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, without limitation, this term may 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 may be constituted by a data storage device. A database may further comprise at least one database management system that comprises 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 containing data may be constituted by a database system that also includes one or more related applications in addition to the data. A database may be part of a processing device or may be external to the processing device. 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, without limitation, this term may refer to outsourcing a processing device or a part of a processing device to at least partially interconnected external devices, specifically, a computer or computer network having greater computing power and / or data storage capacity. The external devices may optionally be spatially distributed. The external devices may be, in particular, on-demand and vary over time. The external devices may be interconnected by use of the Internet. The external devices may each comprise at least one communication interface.

[0033] The determination may further include processing the measured or read measurement data using a processing device to determine information regarding the analyte signal and information regarding the internal standard signal. For example, the determination may include applying at least one peak fitting modeling to a chromatogram and / or a mass spectrum using a processing device. As used herein, the term "processing device" is a broad term and should be given its ordinary and general meaning to those skilled in the art and should not be limited to a special or particular meaning. Specifically, but not limited thereto, 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 a numeric processor, a plurality of registers, specifically registers configured to supply operands to the ALU and store the results of operations, and memories 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 a CPU. In addition to or instead of this, the processing device may be a microprocessor or may include a microprocessor, and thus, specifically, the elements of the processor may be included in a single integrated circuit (IC) chip. In addition to or instead of this, the processing device may be 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 graphics processing units (GPUs) and / or one or more chips such as a dedicated machine learning optimization chip, or may include these. The processing device may be configured to perform one or more evaluation operations, such as by software programming.The processing device may be configured to execute the specified method steps. Thus, by way of example, the processing device may store software code including several computer instructions. The processing device may provide one or more hardware elements for executing one or more of the shown operations, and / or may provide one or more processors for operating software for executing one or more of the method steps.

[0034] As used herein, the term "monitoring parameter" is a broad term and should be given its ordinary and general 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, for example, readout parameters, measured quantities, or measurable parameters suitable as a basis for quality checks for distinguishing reliable measurement results from unreliable measurement results. As outlined above, the concept of quality checks is known from several international guidelines regarding mass spectrometry assays, such as the guidelines by CLSI (Clinical and Laboratory Standards Institute), EMA (European Medicines Agency), or GTFCh (German Society of Toxicological and Forensic Chemistry). The monitoring parameters according to the present invention may be advanced monitoring parameters. The monitoring parameters according to the present invention may make it possible to supplement known common parameters, for example when they lack reliability due to their high variability. By using this monitoring parameter, it may be possible to reduce the need for manual re-examination of peaks by experts due to false sample flagging without degrading the ability to monitor parameters for quality assurance purposes.

[0035] Determining the monitoring parameter includes using, by a processing device, information regarding the analyte signal and information regarding the internal standard signal. The monitoring parameter includes a minimum of the internal standard signal relative to the analyte signal of the sample. Determining the monitoring parameter may include a processing device that calculates the monitoring parameter using information regarding the analyte signal and information regarding the internal standard signal. The monitoring parameter may be determined by combining the value of the minimum IStd signal limit with the dependence of the analyte signal measured for each sample. The determination of the monitoring parameter may be performed by executing a software algorithm.

[0036] Such monitoring parameters may make it possible to minimize false result flagging (false positives) and overlooking of bad measurements (false negatives). The monitoring parameter may be superior compared to a simple absolute minimum IStd signal limit. The rationale may be based on the required accuracy for the assay and the fact that the accuracy depends on the signal. Generally, a decrease in the signal usually also leads to a decrease in accuracy. The analyte signal may be normalized by the IStd signal. Thus, the final measurement result may depend on the signals of both the analyte and the IStd. After determining the parameters required in one system, the minimum of the IStd signal required to achieve the required accuracy for the assay in a particular sample may be calculated individually for each analyte signal of all other systems.

[0037] The monitoring parameter may be determined by the following formula.

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[0038] The derivation of the monitoring parameter will be described below.

[0039] When the signal ratio of the analyte signal and the IStd signal, which is represented by the area ratio of the analyte quantifier (aqn) and the IStd quantifier (iqn) below, is measured, the accuracy represented by CV below may be estimated by Equation 1.

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[0040] The correlation between the area values of aqn and iqn may not be known in advance. However, for example, since a certain degree of similarity is generally required in the physicochemical behavior of the analyte and the IStd, a negative correlation is not assumed (Assumption 1). Therefore, only a positive correlation can be assumed, while no correlation represents the worst-case scenario (Assumption 2). The positive correlation between the area values of aqn and iqn can result in a decrease, i.e., an improvement, in the resulting CV(aqniqn) when the area ratio aqn / iqn is calculated. Assumptions 1 and 2 are explained below. If a correlation exists, it may be necessary to implement the respective coefficients appropriately in the equations. The dependence of the signals on the areas of aqn and iqn and their respective CVs can follow one of two function types. It can follow Equation 2 for aqn and Equation 3 for iqn, or Equation 4 for aqn and Equation 5 for iqn.

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[0041] Below, Formula 2 and Formula 3 are shown. Furthermore, the behaviors of the test substance and the internal standard should be extremely similar with respect to MS measurement. In these cases, CV(aqn) ≒ CV(iqn) can also be assumed, and thus the parameters c = a and d = b (Assumption 3). Below, Assumption 3 is shown. The combination of Formula 1 with Formula 2 and Formula 3 yields Formula 6 given below.

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[0042] The accuracy acceptance criteria for the assay are given by CV(assay). The actual accuracy of the result CV(result) needs to meet this criterion according to Formula 7.

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[0043] The main part of the poor accuracy is based on the signal itself, i.e., CV(aqniqn), and not on the calibration that transmits the signal to the concentration and thus to the result. Therefore, below, CV(result) ≒ CV(aqniqn) is assumed (Assumption 4). From this, Formula 8 is derived from Formula 7. Combining Formula 6 with Formula 8 gives Formula 9.

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[0044] Solving Formula 9 for peakArea(iqn) gives the following Formula 10 and the monitoring parameters.

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[0045] Equation 10 may represent the minimum basis of the IStd signal required for the measured analyte signal to achieve the required accuracy for the measurement result. This calculation can be based on the assumption that the accuracy of the measurement result depends mainly on the measurement signal, regardless of its root cause. This means that it does not matter whether the signal loss is due to, for example, low sample concentration or low MS performance (assumption 5). Further factors of MS performance loss, such as an increase in background or an increase in noise, can also have an impact on the measurement result.

[0046] This method may include considering the discontinuity of the monitoring parameters using at least one check rule. Assume that a > 0, b > 0, peakArea(aqn) > 0, and CV(assay) > 0. The check rule may use multiple criteria, and the criteria are as follows:

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[0047] Equation i may enable checking the denominator of Equation 10 that results in a negative peakArea(iqn) value. Equations ii and iii may enable checking peakArea(iqn) values below the discontinuity in peakArea(aqn). Equation iv may enable checking for negative roots in the denominator.

[0048] Parameters a and b can be determined by performing a plurality of LC-MS measurements on a sample. For each measurement, information regarding the analyte signal can be determined. The method may include, by using a processing device, determining the median or average value of the information regarding the analyte signal and its CV(aqn), and calculating parameters a and b by using at least one regression fit according to the following.

Number

Number

[0049] The method includes comparing information regarding the internal standard signal with a monitoring parameter by using a processing device. The comparison may include at least one mathematical operation. The comparison may include determining the deviation between the information regarding the internal standard signal and the monitoring parameter. The LC-MS measurement is flagged as meeting the quality check if the information regarding the internal standard signal is greater than or equal to the monitoring parameter by using a processing device, and otherwise, i.e., if the information regarding the internal standard signal is less than the monitoring parameter, it is flagged as not meeting. The comparison may include considering a predetermined tolerance range. The predetermined tolerance range may be a deviation of ±20%, preferably ±15%, more preferably ±10% from the monitoring parameter. The predetermined tolerance range may be fixed or may be adjustable, for example, by a user. The comparison may be performed by executing a software algorithm.

[0050] The method may further include providing a quality check result, such as whether the measurement result is flagged or not. Providing may include displaying the result to the user. The display may be performed by using at least one user interface. As used herein, the term "user interface" is a broad term and should be given its ordinary and general meaning to those skilled in the art and should not be limited to a special or particular meaning. This term may refer to an element or device configured to interact with the surrounding environment for the purpose of exchanging information, such as in one or two directions, for example, to exchange one or more data or commands. For example, the user interface may be configured to share information with the user and receive information from the user. The user interface may be a function that visually interacts with the user, such as a display, or a function that aurally interacts with the user. As an example, the user interface may include one or more of a graphical user interface, a data interface such as a wireless and / or wired data interface. The method may include, for example, requiring manual review of the measurement result, repetition of the measurement, or performance of equipment maintenance when the measurement result is flagged as not meeting the quality check. For example, the method may include the following operations, for example, in a given order, when the measurement result is flagged as not meeting the quality check. 1. Re-measurement of the sample 2. Investigation of substances (such as the expiration date of the lot) 3. Calibration of the analyzer 4. Monitoring of the analyzer (maintenance, failure, etc.) 5. Monitoring of the environment (humidity, temperature, etc.) 6. Additional investigation

[0051] Method steps a) to c) can be executed using at least one computer. Specifically, for example, the determination of information regarding the analyte signal and the internal standard signal by performing measurements can be executed completely automatically. Further, the determination and comparison of the monitoring parameters in steps b) and c) can be executed completely automatically. This method can be implemented completely or partially on a computer, specifically, on a computer such as a processor, in particular.

[0052] In a further aspect, when executed on a computer or computer network, specifically on a processor, a computer program is disclosed that includes computer-executable instructions for executing a method according to any one of the embodiments described herein, specifically method steps a) to c). Specifically, the computer program may be stored on a computer-readable data carrier and / or a computer-readable storage medium.

[0053] 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 random access memory (RAM) and / or read-only memory (ROM), or may comprise such a storage medium.

[0054] Therefore, specifically, one, a plurality, or even all of the method steps i) to iii) shown above can be implemented by using a computer or computer network, preferably by using a computer program.

[0055] In this specification, a computer program product is further disclosed and proposed, and 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.

[0056] A data carrier storing a data structure capable of executing a method according to one or more of the embodiments disclosed herein after being loaded into a computer or a computer network, such as a working memory or a main memory of the computer or the computer network, is further disclosed and proposed herein.

[0057] A non-transitory computer-readable medium including instructions for causing one or more processors to execute a method according to one or more of the embodiments disclosed herein when executed by the one or more processors is further disclosed and proposed herein.

[0058] A computer program product storing program code means on a machine-readable carrier for executing a method according to one or more of the embodiments disclosed herein when the program is executed on a computer or a computer network is further disclosed and proposed herein. As used herein, a computer program product refers to a program as a tradable product. The product can generally exist in any format such as a paper format, or can 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.

[0059] Finally, a modulated data signal containing instructions readable by a computer system or computer network for performing a method according to one or more of the embodiments disclosed herein is disclosed and proposed herein.

[0060] Regarding the computer-implemented aspects of the present invention, one or more or all of the method steps of the method according to one or more of the embodiments disclosed herein may be performed by using a computer or a computer network. Thus, generally, any method step, including those involving the provision and / or manipulation of data, may be performed by using a computer or a computer network. Generally, these method steps may include any method step, typically excluding those method steps that require manual work, such as specific manners of providing samples and / or performing actual measurements.

[0061] Specifically, herein, - A computer or computer network comprising at least one processor, the processor being configured to perform a method according to one of the embodiments described herein, - A computer-loadable data structure configured to perform a method according to one of the embodiments described herein when executed on a computer, - A computer program configured to perform a method according to one of the embodiments described herein when executed on a computer, - A computer program comprising program means for performing 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 a preceding embodiment, the program means being stored on a computer-readable 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 a main memory and / or a 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 is executed on a computer or a computer network is further disclosed.

[0062] In a further aspect of the present invention, a system is disclosed. As used herein, the term "system" is a broad term and should be given its ordinary and general 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 any set of interacting or interdependent components that form a whole. Specifically, the components may interact with each other to perform at least one common function. The system may comprise at least two components, and the at least two components may be handled independently, or may be combined or connectable.

[0063] This system is - at least one mass spectrometer configured to perform at least one liquid chromatography-mass spectrometry (LC-MS) measurement on a sample containing a target analyte and a specified amount of an internal standard, and - information regarding the analyte signal of the LC-MS measurement

Number

Number

[0064] The system may be configured to execute the method according to the present invention. Regarding the definition and embodiments of the system, the definition and embodiments of the above method are referred to.

[0065] The system may further comprise at least one sample preparation unit configured to add at least one internal standard substance to the sample.

[0066] In summary, without excluding the possibility of further embodiments, the following embodiments may be envisioned.

[0067] Embodiment 1. A method for quality checking of at least one liquid chromatography - mass spectrometry (LC - MS) measurement for a sample containing a target analyte and a specified amount of at least one internal standard, comprising a) determining information regarding the analyte signal of the LC - MS measurement

Number

Number

[0068] Embodiment 2. The method according to the preceding embodiment, wherein the information regarding the analyte signal is at least one of peak area, peak height, peak width, or retention time, and the information regarding the internal standard signal is at least one of peak area, peak height, peak width, or retention time.

[0069] Embodiment 3. The monitoring parameter is determined by the following formula:

Number

Number

[0070] Embodiment 4. The method includes considering discontinuities of the monitoring parameter by using at least one check rule, the check rule uses a plurality of criteria, and the criteria are as follows:

Number

Number

Number

Number

Number

[0071] Embodiment 5. Parameters a and b are determined by performing a plurality of LC-MS measurements on a sample. For each measurement, information regarding the analyte signal is determined. The method includes determining the median or average value of the information regarding the analyte signal and its CV(aqn) using a processing device, and calculating parameters a and b by using at least one regression fit according to the following equation, including the method according to any one of the preceding two embodiments.

Number

Number

[0072] Embodiment 6. Step a) includes performing at least one LC-MS measurement on a sample, including the method according to any one of the preceding embodiments.

[0073] Method according to the preceding embodiments, wherein in step a) at least one liquid chromatography mass spectrometer is used.

[0074] Embodiment 8. A method according to any one of the preceding embodiments, wherein the method comprises adding at least one internal standard to the sample.

[0075] Embodiment 9. A method according to any one of the preceding embodiments, wherein the method is computer-implemented.

[0076] Embodiment 10. A computer program comprising instructions which, when the program is executed by a processing device, cause the processing device to execute a method according to any one of the preceding embodiments.

[0077] Embodiment 11. A computer-readable storage medium comprising instructions which, when executed by a processing device, cause the processing device to execute a method according to any one of the preceding embodiments.

[0078] Embodiment 12. A non-transitory computer-readable medium comprising instructions which, when executed by one or more processors, cause the one or more processors to execute a method according to any one of the preceding embodiments.

[0079] Embodiment 13. - At least one mass spectrometer configured to perform at least one liquid chromatography-mass spectrometry (LC-MS) measurement on a sample comprising a test substance of interest and a predetermined amount of an internal standard, - Information regarding the test substance signal of the LC-MS measurement

Number

Number

[0080] Embodiment 14. The system according to the previous embodiments, configured to execute the method according to any one of the embodiments regarding the method.

Brief Description of the Drawings

[0081] Further optional features and embodiments are disclosed in more detail in the subsequent description of the embodiments, preferably in conjunction with the dependent claims. Among them, each optional feature may be implemented in an independent manner and in any feasible combination, as can be understood by those skilled in the art. The scope of the present invention is not limited by the preferred embodiments. The embodiments are schematically shown in the figures. Here, the same reference numerals in these figures refer to the same or functionally equivalent elements.

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 3C

Best Mode for Carrying Out the Invention

[0082] FIG. 1 shows, in a very schematic way, an embodiment of a system 110 according to the present invention.

[0083] System 110 includes at least one mass spectrometer 112 configured to perform at least one liquid chromatography - mass spectrometry (LC - MS) measurement on a sample containing a target analyte and a specified amount of an internal standard. The mass spectrometer 112 may be at least one quadrupole analyzer or may include at least one quadrupole analyzer. The quadrupole mass analyzer may include a plurality of quadrupoles. For example, the quadrupole mass analyzer may be a triple quadrupole mass spectrometer. 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. The mass spectrometer 112 may further include at least one ionization source. The ionization source may be at least one gas - phase ionization source such as at least one electron impact (EI) source or at least one chemical ionization (CI) source, at least one plasma desorption (PDMS) source, at least one fast atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source, and at least one matrix - assisted laser desorption (MALDI) source, etc., at least one desorption ionization source, 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, etc., at least one spray ionization source, and may be at least one ionization source selected from the group consisting of such ionization sources or may include such an ionization source. The mass spectrometer 112 may include at least one detector. The detector may be configured to detect charged particles. The detector may be at least one electron multiplier or may include at least one electron multiplier. The mass spectrometer 112, particularly the detector of the mass spectrometer and / or at least one processing device, may be configured to determine at least one mass spectrum of the detected ions. The LC - MS measurement may be at least one measurement by using the mass spectrometer 112 or may include at least one measurement by using the mass spectrometer 112.The mass spectrometer 112 may be a liquid chromatography mass spectrometer. The mass spectrometer 112 may include at least one liquid chromatograph, which is also referred to as a liquid chromatography (LC) device. The liquid chromatograph may be used for sample preparation for the mass spectrometer. The LC device may include at least one LC column. For example, the LC device may be a single-column type LC device, or may be a multi-column type LC device having a plurality of LC columns. The LC column may have a stationary phase, and the mobile phase is sent through the stationary phase for separation and / or elution and / or transfer of the target analyte. The liquid chromatography mass spectrometer may further include a sample preparation station for automatic pretreatment and preparation of samples each containing at least one target analyte. The liquid chromatography mass spectrometer 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 spectrometer 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.

[0084] The mass spectrometer 112 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 in 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 112 may measure the ion fragmentation pattern for 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 (e.g., the m / z ratio and intensity of the peaks) for a particular analyte 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 also be performed based on the time period between the initial introduction of the sample (e.g., injection into the inlet of an LC-MS system) and the elution of the component from the LC column, or the time period between the initial introduction of the sample and the measurement of the component ion fragmentation pattern in the mass spectrometer. Since a particular component can move through the LC column at a specific rate, the elapsed time period can be used as an indicator of the identity of the component. Similar to the ion fragmentation pattern, the elapsed time period can be compared to reference information (e.g., previously measured migration and / or measurement times for known components) to determine the identity of the component.

[0085] The mass spectrometer 112 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, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, lymphatic fluid, interstitial fluid, cerebrospinal fluid, tissue, cells, and the like.

[0086] The sample may contain one or more target analytes, also referred to as the analytes of interest. 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, see, for example, European Patent Application Publication No. 3 425 369, the entire disclosure of which is incorporated herein by reference. Other analytes of interest are possible.

[0087] System 110 may be configured to perform at least one quality check. Quality may refer to a measure of the reliability of measurement results obtained by using mass spectrometer 112. The measurement results may be quantitative and / or qualitative measurements, such as the presence and / or concentration of analytes in the sample. Quality may depend on several factors such as measurement conditions, background, interference, instrument performance, retention time shift, peak width, peak height, and peak area. The quality check may be at least one process that tests whether at least one predetermined quality requirement is met after data acquisition, or may include such at least one process. The quality check may be used to distinguish between reliable and unreliable measurement results. Reliable measurement results may be measurement results in which the corresponding internal standard signal meets at least one predetermined requirement. Reliable measurement results may be measurement results having an accuracy and / or precision within a predetermined tolerance range. Unreliable measurement results may be measurement results in which the corresponding internal standard signal does not meet at least one predetermined requirement. Unreliable measurement results may be measurement results having an accuracy and / or precision outside the tolerance range.

[0088] For example, quality checks may be performed for each measurement. For example, quality checks may be performed at a predetermined frequency, such as one or more of after a predetermined period, after new calibration, after column replacement, or after maintenance activities of the instrument.

[0089] System 110 comprises at least one processing device 114 configured to obtain and further / or read information regarding the analyte signal of the LC-MS measurement

Number

Number

[0090] The analyte signal may be a signal of an LC-MS measurement regarding the analyte, such as a peak in the chromatogram and / or mass spectrum of the analyte.

[0091] The internal standard may exhibit characteristics similar to the target analyte when subjected to the workflow using the mass spectrometer 112. The workflow may include any pretreatment, concentration, and actual detection steps. For example, the internal standard may be an isotope-labeled variant of the target analyte (including labels such as 2H, 13C, or 15N). System 110 may be configured to add at least one internal standard substance to the sample. The internal standard signal may be a signal of an LC-MS measurement regarding the internal standard, such as a peak in the chromatogram and / or mass spectrum of the internal standard. The information regarding the analyte signal may be at least one of peak area, peak height, peak width, or retention time. The information regarding the internal standard signal may be at least one of peak area, peak height, peak width, or retention time.

[0092] Determination of information regarding the analyte signal and information regarding the internal standard signal may include one or more of measurement, acquisition, or reading of measurement data.

[0093] For example, information regarding the analyte signal and information regarding the internal standard signal may be determined by performing at least one LC-MS measurement using the mass spectrometer 112. For example, the determination may include performing at least one LC-MS measurement on the sample, for example, by using at least one liquid chromatography mass spectrometer 112.

[0094] For example, information regarding the analyte signal and information regarding the internal standard signal may also be determined by reading measurement data, for example, via at least one communication interface 116 of the processing device 114. The measurement data read by the communication interface 114 may be acquired, for example, by using a mass spectrometer such as the mass spectrometer 112 and transmitted to the processing device 114. For example, the measurement data may be read by the communication interface 114 from at least one database. For example, the determination may further include the processing device 114 reading at least one chromatogram and / or mass spectrum, for example, via its communication interface 116. The processing device 114 and / or the database may be at least partially cloud-based.

[0095] The determination may further include processing the measured or read measurement data to determine information regarding the analyte signal and information regarding the internal standard signal using the processing device 114. For example, the determination may include applying at least one peak fitting modeling to the chromatogram and / or mass spectrum using the processing device 114.

[0096] The processing device 114 is further configured to determine at least one monitoring parameter by using information regarding the analyte signal and information regarding the internal standard signal. The monitoring parameter includes a minimum of the internal standard signal with respect to the analyte signal of the sample.

[0097] The monitoring parameter may be, for example, one or more of a readout parameter, a measured quantity, or a measurable parameter suitable as a basis for quality checks for distinguishing reliable measurement results from unreliable measurement results. As outlined above, the concept of quality checks is known from several international guidelines for mass spectrometry assays, such as guidelines by CLSI (Clinical and Laboratory Standards Institute), EMA (European Medicines Agency), or GTFCh (German Society of Toxicological and Forensic Chemistry). The monitoring parameter according to the present invention may be an advanced monitoring parameter. The monitoring parameter according to the present invention may make it possible to supplement known common parameters when they lack reliability due to, for example, their high variability. By using this monitoring parameter, it may be possible to reduce the need for manual re-examination of peaks by experts due to false sample flagging without degrading the ability to monitor parameters for quality assurance purposes.

[0098] Determining the monitoring parameter includes using, by the processing device 114, information regarding the analyte signal and information regarding the internal standard signal. The monitoring parameter includes a minimum of the internal standard signal with respect to the analyte signal of the sample. Determining the monitoring parameter may include the processing device 114 that calculates the monitoring parameter by using information regarding the analyte signal and information regarding the internal standard signal. The monitoring parameter may be determined by combining the value of the minimum IStd signal limit with the dependency of the analyte signal measured for each sample. The determination of the monitoring parameter may be performed by executing a software algorithm.

[0099] Monitoring parameters can enable minimizing false result flagging (false positives) and overlooking bad measurements (false negatives). The monitoring parameters may be superior compared to a simple absolute minimum IStd signal limit. The rationale can be based on the required accuracy for the assay and the fact that the accuracy is signal-dependent. Generally, a decrease in the signal usually leads to a decrease in accuracy. The analyte signal can be normalized by the IStd signal. Thus, the final measurement result can depend on the signals of both the analyte and the IStd. After determining the parameters required in one system, the minimum of the IStd signal necessary to achieve the required accuracy for the assay in a particular sample may be calculated individually for each analyte signal of all other systems.

[0100] The monitoring parameters may be determined by the following formula.

Number

Number

[0101] The derivation of the monitoring parameters is described below.

[0102] When the signal ratio of the analyte signal and the IStd signal, represented by the area ratio of the analyte quantifier (aqn) and the IStd quantifier (iqn), is measured below, the accuracy represented by CV below may be estimated by Equation 1.

Number

[0103] The correlation between the area values of aqn and iqn may not be known in advance. However, since a certain degree of similarity in the physicochemical behavior of the test substance and IStd is generally required, for example, a negative correlation is not assumed (assumption 1). Therefore, only a positive correlation can be assumed, while no correlation represents the worst-case scenario (assumption 2). A positive correlation between the area values of aqn and iqn can result in a decrease, i.e., an improvement, in the resulting CV(aqniqn) when the area ratio aqn / iqn is calculated. Assumptions 1 and 2 are explained below. If a correlation exists, it may be necessary to implement the respective coefficients into the equations accordingly. The dependencies of the signals on the areas of aqn and iqn and their respective CVs can follow one of two function types. Either according to Equation 2 for aqn and Equation 3 for iqn, or according to Equation 4 for aqn and Equation 5 for iqn.

Number

Number

Number

Number

[0104] In Figure 3A, a graph of Equation 2 is shown as an example. In this exemplary graph of Equation 2, for the sake of explanation, peakArea(aqn) on the x-axis and CV(aqn) on the y-axis are shown.

[0105] Shown below are Formula 2 and Formula 3. Furthermore, the behaviors of the test substance and the internal standard should be very similar with respect to MS measurement. In these cases, CV(aqn) ≒ CV(iqn) can also be assumed, and thus the parameters c = a and d = b (Assumption 3). Shown below is Assumption 3. The combination of Formula 1 with Formula 2 and Formula 3 yields Formula 6 given below.

Number

[0106] The accuracy acceptance criteria for the assay are given by CV(assay). The actual accuracy of the result CV(result) needs to meet this criterion according to Formula 7.

Number

[0107] The main part of the poor accuracy is based on the signal itself, i.e., CV(aqniqn), and not on the calibration that conveys the signal to the concentration and thus to the result. Therefore, below, it is assumed that CV(result) ≒ CV(aqniqn) (Assumption 4). From this, Formula 8 is derived from Formula 7. Combining Formula 6 with Formula 8 gives Formula 9.

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Number

[0108] Solving Formula 9 for peakArea(iqn) gives the following Formula 10 and the monitoring parameters.

Number

[0109] Equation 10 may represent the minimum basis of the IStd signal required for the measured analyte signal to achieve the required accuracy for the measurement results. This calculation can be based on the assumption that the accuracy of the measurement results depends mainly on the measurement signal, regardless of its root cause. This means that it does not matter whether the signal loss is due to, for example, low sample concentration or low MS performance (Assumption 5). Further factors of MS performance loss, such as an increase in background or an increase in noise, can also have an impact on the measurement results.

[0110] This method may include considering the discontinuity of the monitoring parameters using at least one check rule. Assume a > 0, b > 0, peakArea(aqn) > 0, and CV(assay) > 0. The check rule may use multiple criteria, and the criteria are as follows:

Number

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Number

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[0111] Equation i may make it possible to check the denominator of Equation 10 that results in a negative peakArea(iqn) value. Equations ii and iii may make it possible to check peakArea(iqn) values that are below the discontinuity in peakArea(aqn). Equation iv may make it possible to check for negative roots in the denominator.

[0112] Figure 3B shows an exemplary graph of Equation 10 combined with the rules described above. Signal values above or to the right of the curve are within the acceptance criteria for the check, and signal values below or to the left of the curve are not within the acceptance criteria and should be flagged.

[0113] Parameters a and b can be determined by performing a plurality of LC-MS measurements on the sample. For each measurement, information regarding the analyte signal can be determined. The method can include determining the median or average value of the information regarding the analyte signal and its CV(aqn) by using a processing device, and calculating parameters a and b by using at least one regression fit according to the following. [Number] or [Number]

[0114] The processing device 114 is further configured to compare information regarding the internal standard signal with the monitoring parameters. The processing device 114 is configured to flag the LC-MS measurement as meeting the quality check if the information regarding the internal standard signal is greater than or equal to the monitoring parameters, and flag it as not meeting the quality check if otherwise. The comparison may include at least one mathematical operation. The comparison may include determining the deviation between the information regarding the internal standard signal and the monitoring parameters. The LC-MS measurement is flagged as meeting the quality check by using the processing device if the information regarding the internal standard signal is greater than or equal to the monitoring parameters, and flagged as not meeting the quality check if otherwise, i.e., if the information regarding the internal standard signal is less than the monitoring parameters. The comparison may include considering a predetermined tolerance range. The predetermined tolerance range may be a deviation of ±20%, preferably ±15%, more preferably ±10% from the monitoring parameters. The predetermined tolerance range may be fixed or adjustable, for example, by the user. The comparison may be performed by executing a software algorithm.

[0115] The system 110 may include at least one user interface 118. The system 110 may be configured to provide the result of the quality check, such as whether the measurement result is flagged or not. Providing may include displaying the result to the user. Displaying may be performed by using the user interface 118. The user interface 118 may include, for example, one or more of a graphical user interface, a data interface such as a wireless and / or wired data interface. The user interface 118 may be configured to request a manual review of the measurement result, a repetition of the measurement, or the execution of equipment maintenance if, for example, the measurement result is flagged as not meeting the quality check. For example, the method may include the following operations, for example, in a given order, if the measurement result is flagged as not meeting the quality check. 1. Re-measurement of the sample 2. Investigation of substances (such as the expiration date of the lot) 3. Calibration of the analyzer 4. Monitoring of the analyzer (maintenance, failure, etc.) 5. Monitoring of the environment (humidity, temperature, etc.) 6. Additional investigation

[0116] Figure 2 shows a flowchart of an embodiment of the method according to the present invention. The method comprises the following steps, namely, a) determining information regarding the analyte signal and the internal standard signal of the LC-MS measurement

Number

Number

[0117] Method steps a) to c) may be performed using at least one computer. Specifically, for example, determination of information regarding the analyte signal and the internal standard signal by performing measurements can be carried out completely automatically. Further, determination and comparison of the monitoring parameters in steps b) and c) can be carried out completely automatically. The method can be implemented wholly or partly on a computer, specifically on a computer such as a processor.

[0118] For an embodiment of the method, refer to the description of system 110 shown in FIG. 1.

[0119] FIG. 3C shows the experimental results. Estradiol and testosterone were added to serum samples up to 10 different concentrations covering the full measurement range of each assay. The compounds, namely estradiol-13C3 and testosterone-13C3, were added in a fixed amount as IStd prior to sample preparation. Each sample was measured 21 times by LC-triple quadrupole (QqQ)-MS in analytical replicates, and the chromatographic data were integrated to obtain the peak areas of the analyte and the IStd as signals. Then, the central analyte area and its CV(aqn) were determined and plotted. An exemplary graph for estradiol is shown in FIG. 3C.

[0120] A regression fit was performed according to Equation 2 as the best fit compared to Equation 3 to calculate the parameters "a" and "b". The parameter "a" was 2.69 for estradiol and 3.84 for testosterone, and the parameter "b" was 5682 for estradiol and 34659 for testosterone. For further calculations, Assumptions 1 to 5 (see above) were considered. Further, the simple minimum IStd signal limit as a well-established general parameter was optimized and determined for estradiol and testosterone (hereinafter referred to as the simple IStd check).

[0121] For verification, ten serum samples with estradiol and testosterone added at different concentrations were measured in three analytical replicates at two different instruments and four different time points. Between each time point, hundreds of serum samples were run using the system to affect the performance of the system. The required assay accuracy was set at 20% CV(assay). The minimum of the IStd signal was determined for each analyte signal and sample according to the rules given above using Equation 10 and criteria i, ii, iii, iv, and v and the predetermined parameters “a” and “b”. Subsequently, the corresponding measured values were checked against (i) the advanced minimum IStd signal limit (advanced IStd check) calculated for each sample, and (ii) the simple IStd signal limit (simple IStd check). As a reference, CV(aqniqn) was calculated for each analyte and sample based on the analytical replicates, checked against the required CV(assay), and flagged accordingly (assay result check). This assay result check revealed that 77% of all measured values were good and 23% were not good. The relative flagging rates of all analytes and samples for the advanced IStd check and the simple IStd check against the assay result check are summarized in the following tables, respectively.

Table 1

[0122] The table shows that the advanced IStd check correctly flagged 93.6% of all samples (76.9% were flagged as good by both, 16.7% were flagged as not good by both), and the simple IStd check correctly flagged 79.6% (70.1% were flagged as good by both, 9.5% were flagged as not good by both).

[0123] In conclusion, the advanced IStd check correctly flagged +14.0% more samples compared to the well-established simple IStd check. This indicates that the advanced monitoring parameters presented herein can minimize false result flagging (false positives) and overlooking of bad measurements (false negatives), and are superior compared to the well-established monitoring parameters.

Explanation of symbols

[0124] 110 System 112 Mass spectrometer 114 Processing device 116 Communication interface 118 User interface 120 Step a) 122 Step b) 124 Step c)

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