A computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking

Abstract

A computer-implemented method for calibrating a customer mass spectrometry instrument (118) for a quantifier-to-verifier ratio check is proposed, the method comprising the following steps: a) at least one manufacturer site standardization step, wherein a set of subject samples and a set of calibrator samples are measured in multiple replicates on multiple mass spectrometric instruments (114), each measurement comprising multiple reaction monitoring with quantifier and identifier transitions for an analyte and an internal standard, and wherein at least three adjustment factors are determined from the measurements of the set of subject samples and the set of calibrator samples, a first adjustment factor α being dependent on the difference between the analyte and the internal standard, a second adjustment factor β being dependent on the difference between the subject sample and the calibrator sample for the analyte quantifier-identifier ratio, and a third adjustment factor γ being dependent on the difference between the subject sample and the calibrator sample for the internal standard quantifier-identifier ratio; b) at least one transfer step, in which the adjustment factor is electronically transferred to a client mass spectrometry instrument (118); c) at least one customer site calibration step, the customer site calibration including at least one calibration measurement, wherein a set of calibrator samples is measured on the customer mass spectrometric instrument (118) from which quantifier-to-identifier ratios are determined, and target values ​​for the quantifier-to-identifier ratios of the analyte and the internal standard are set by applying an adjustment factor to the determined quantifier-to-identifier ratios; Includes.

Description

Technical Field

[0001] The present invention relates to a computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking, a computer-implemented method for quantifier-verifier ratio checking, a computer program, and a mass spectrometry system. This method can be used for in vitro diagnostic assays.

Background Art

[0002] The quantifier-verifier peak area ratio, called the QQ ratio, is known to be an important quality control measure for checking peak identity and interference in all measured patient samples. The use of the QQ ratio is an established approach for liquid chromatography-mass spectrometry (LC-MS) assays and is considered by several guidelines, for example, the Clinical&Laboratory Standards Institute (CLSI) C62-A, guidelines from the 「Society of Toxicological and Forensic Chemistry」 (GTFCh) 「Guideline fwor kuality control in forensic-toxicological analyses」. The target value of the QQ ratio for checking peak identity and interference can be set during any of assay development, validation, or assay calibration. The setting of the target value is performed on a specific instrument. Each acceptance criterion can either be defined during assay development or simply set according to the recommendations of the guidelines.

[0003] For example, the use of the QQ ratio as a quality measure for checking peak identity and interference is described in US Patent Application Publication No. 2017 / 0108478, International Publication No. 2018 / 207228 Pamphlet, International Publication No. 2018 / 136825 Pamphlet, and US Patent Application Publication No. 20120318970.

[0004] This ratio is specific to the analyte, but robustness deficiencies can be observed, especially when compared to the peak area ratio between the analyte and the internal standard. Furthermore, distinct differences can exist between different instruments and temporal drift and shift. In the case of laboratory equipment, the target value of this ratio is usually defined after development or during the validation or verification of a specific instrument. To overcome the lack of robustness, a wide tolerance range can be applied, or the target needs to be adjusted for each batch calibration. In fully automated in vitro diagnostics performed on multiple instruments, this approach may not be feasible and appropriate.

[0005] An overview of the normal adjustment and calibration routines for mass spectrometers is given by Fabio Garofolo: "LC-MS Instrument Calibration: Chan / Analytical Validation" In: "Analytical Method Validation and Instrument Performance Verification", John Wiley & Sons, Inc., Hoboken, NJ, USA, ISBN: 978-0-471-25953-4, pages 197 - 220, DOI: 10.1002 / 0471463728.ch13. An overview of the qualification and verification of equipment is given by Ludwig Huber et al: "Equipment Qualification and Computer System Validation: Chan / Analytical Validation" In: "Analytical Method Validation and Instrument Performance Verification", January 15, 2004, John Wiley & Sons, Inc., Hoboken, NJ, USA, ISBN: 978-0-471-25953-4, pages 255 - 276, DOI: 10.1002 / 0471463728.ch17. Problems to be solved

[0006] Accordingly, it is an object of the present invention to provide a method and an apparatus for quantifier-verifier ratio checking that avoid the above-mentioned drawbacks of known methods and apparatuses. In particular, the method and apparatus shall enable a reliable and fully automated analysis of samples using a mass spectrometer. SUMMARY OF THE INVENTION

[0007] This problem is addressed by a computer-implemented method, a computer program, and a mass spectrometry system having the features of the independent claims. Advantageous embodiments, which can be implemented alone or in any combination, are described in the dependent claims as well as throughout the specification.

[0008] When used hereinafter, the terms "having", "comprising", or "including" or any grammatical variations thereof are used in a non-exclusive manner. Thus, these terms may refer to both situations where no further features exist in the entity described in this context in addition to the features introduced by these terms, and situations where one or more additional features exist. As an example, the expressions "A has B", "A comprises B", and "A includes B" all refer to both the situation where no other elements exist in A besides B (i.e., the situation where A consists of B alone and exclusively), and the situation where one or more additional elements such as element C, elements C and D, or even further elements exist in entity A besides B.

[0009] Furthermore, it should be noted that terms such as "at least one", "one or more", indicating that a feature or element can be present once or multiple times, are usually only used once when introducing each respective feature or element. In the following, 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 can be present once or more than once.

[0010] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "in particular", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with any feature without limiting the possibility of alternatives. Thus, the features introduced by these terms are any features and are not intended to limit the claims in any way. The present invention may be practiced, as will be recognized 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 any features without any limitation regarding alternative embodiments of the present invention, without any limitation regarding the scope of the present invention, and without any limitation regarding the possibility of combining features introduced in such a way with any other optional or non-optional features of the present invention.

[0011] In a first aspect of the present invention, a computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking is disclosed.

[0012] As used herein, the term "computer-implemented method" 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 customized meaning. Specifically, but not limited to, this term can refer to a method that includes at least one computer and / or at least one computer network. The computer and / or computer network can comprise at least one processor configured to execute at least one of the method steps of the method according to the present invention. Preferably, some of the method steps may be executed by the computer and / or computer network. The method may be executed partially or fully automatically, specifically without interaction with a user. As used herein, the term "automatically" 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 customized meaning. Specifically, this term can refer to, among other things, a process that is fully executed by at least one computer and / or computer network and / or machine without the need for manual operations and / or interaction with a user, but is not limited thereto.

[0013] The terms "calibration" and "calibrate" are broad terms and should be given their ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, but not limited to, these terms can refer to the operation or process of determining the relationship between the measured value supplied by a device and the measured value of a calibration standard, specifically the calibration function. In particular, calibration may be the relationship between a measured value and a target value. Calibration can be the relationship between the target value of the quantifier-validator ratio from the measured value determined using a customer mass analyzer and the target value of the quantifier-validator ratio of the calibration standard, particularly determined at the manufacturer site.

[0014] As used herein, the term "mass spectrometry (MS) instrument" 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 customized meaning. Specifically, but not limited to, this term can refer to a mass spectrometry instrument configured to detect at least one analyte based on the mass-to-charge ratio. The mass spectrometry instrument can be, or can include, at least one quadrupole mass spectrometer. The MS instrument can be a tandem mass spectrometry (MS / MS) instrument or a triple quadrupole MS / MS. Specifically, the mass spectrometry instrument can be configured for multiple reaction monitoring (MRM).

[0015] The mass spectrometer may specifically be, or may include, a liquid chromatography mass spectrometer. The term "liquid chromatography mass spectrometer" as used herein 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 customized meaning. Specifically, without limitation, this term can refer to a combination of liquid chromatography and mass spectrometry. 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 can include a liquid chromatography (LC) device and a mass spectrometry (MS) device, and the LC device and the MS are coupled via at least one interface. The interface connecting the liquid chromatography device and the MS can include at least one ionization source configured to generate molecular ions and transfer the molecular ions to the gas phase. The term "liquid chromatography (LC) device" as used herein 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 customized meaning. Specifically, without limitation, this term can refer to an analysis module configured to separate one or more analytes of interest in a sample from other components of the sample for detection by a mass spectrometer. The LC device may be based on any separation principle considered appropriate by those skilled in the art. In an embodiment, the LC device may be reverse phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or chiral chromatography. In a further embodiment, the LC device is reverse phase chromatography. The LC device can include 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 can have a stationary phase, and for the separation and / or elution and / or transfer of the analyte of interest, the mobile phase is pumped through the stationary phase.

[0016] As used herein, the term "multiple reaction monitoring", also referred to as multiple transition monitoring, 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 customized meaning. This term can specifically, but not limited to, 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 "monitored" is a broad term and should be given its ordinary customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to the determination and / or detection of multiple product ions.

[0017] As used herein, the term "customer" is a broad term and should be given its ordinary customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to an owner or operator of a mass spectrometry instrument obtained from one or more of a seller, vendor, or supplier. As used herein, the term "customer mass spectrometry instrument" is a broad term and should be given its ordinary customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to a customer's mass spectrometry instrument.

[0018] As used herein, the term "quantifier", also referred to as 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 customized meaning. This term can specifically, but not exclusively, refer to an ion that characterizes the compound of interest. Generally, the most abundant and / or most reliably detected transition or fragment is used to quantify the compound. Specifically, the quantifier ion can have a peak of maximum signal intensity on the mass spectrum of the compound. As used herein, the term "confirmator", also shown as confirmator 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 customized meaning. This term can specifically, but not exclusively, refer to another ion that characterizes the compound of interest and has a different mass-to-charge ratio compared to the quantifier ion. The confirmator can be used to confirm the identity of the compound. Generally, a second transition or fragment is used as the confirmator. As used herein, the term "quantifier-confirmator ratio", also referred to as quantifier-confirmator peak area 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 customized meaning. This term can specifically, but not exclusively, refer to the ratio of the signal intensity of the peak of the confirmator ion to the signal intensity of the peak of the quantifier ion and / or the ratio of the peak area of the peak of the quantifier ion to the peak area of the peak of the confirmator ion.

[0019] As used herein, the term "quantifier - confirmatory ratio check" 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 customized meaning. This term can refer, without limitation, to quality control measures for checking the identity and interference of peaks within a sample. The use of the quantifier - confirmatory ratio is an established approach for liquid chromatography - mass spectrometry assays and is considered by several guidelines, such as the Clinical&Laboratory Standards Institute (CLSI) C62 - A, and guidelines from the "Society of Toxicological and Forensic Chemistry" (GTFCh), "Guideline for quality control in forensic - toxicological analyses". In the case of a quantifier - confirmatory ratio check, the determined quantifier - confirmatory ratio is compared to a target value for the quantifier - confirmatory ratio and is verified considering at least one acceptance criterion.

[0020] The term "quality control" as used herein is known to one of ordinary skill in the art. In embodiments, quality control is a process that ensures that processes performed and / or goods produced by an entity meet predetermined quality standards. In further embodiments, quality control in sample measurement, particularly in the measurement of medical samples such as patient samples, e.g., in clinical diagnostics and / or clinical chemistry, includes ensuring that the analytical results obtained by a particular measurement method correspond to the results obtained by a gold standard method and thus, in embodiments, correspond to results that would theoretically be obtained within a predetermined range.

[0021] 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 also possible. Furthermore, it is also possible to perform one or more method steps once or repeatedly. Furthermore, it is possible to perform two or more method steps simultaneously or in a timely and overlapping manner. The method may include additional method steps not listed herein. As used herein, the term "step" 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 customized meaning. Specifically, but not limited to, this term can refer to a work step, a process step, or a stage of an operation or procedure.

[0022] The method includes the following steps, namely, a) At least one manufacturer site standardization step, wherein a set of subject samples and a set of calibrator samples are measured in multiple replicates on multiple mass spectrometers, each measurement including multiple reaction monitors using quantifier and confirmatory transitions for the analyte and internal standard, at least three adjustment factors are determined from the measurements of the set of subject samples and the set of calibrator samples, the first adjustment factor α depends on the difference between the analyte and the internal standard, the second adjustment factor β depends on the difference between the subject sample and the calibrator sample for the analyte quantifier-confirmatory ratio, and the third adjustment factor γ depends on the difference between the subject sample and the calibrator sample for the internal standard quantifier-confirmatory ratio; at least one manufacturer site standardization step; b) At least one transfer step, wherein the adjustment factor is electronically transferred to the customer mass spectrometer; at least one transfer step; c) At least one customer site calibration step, wherein the customer site calibration includes at least one calibration measurement, a set of calibrator samples is measured on the customer mass spectrometer, and the quantifier-confirmatory ratio is determined therefrom, and by applying the adjustment factor to the determined quantifier-confirmatory ratio, the target value of the quantifier-confirmatory ratio of the analyte and the internal standard is set; at least one customer site calibration step; and includes.

[0023] To perform a quality check based on the quantifier - confirmator ratio, at least one target value against which the measured quantifier - confirmator ratio is compared is used. However, the target value can vary or change for each mass spectrometry instrument. Thus, it can be advantageous to use for the quality check target value of a particular customer's mass spectrometry instrument. Even a change over time is possible. Thus, it can be advantageous to use for the quality check target value that can be repeatedly adjusted or adapted in - time at the customer site. Further, for calibration, usually a small number of calibration samples are measured and the composition of these calibration samples may be different compared to the sample of the subject. Thus, the quality check based on the quantifier - confirmator ratio is strongly affected by measurement inaccuracies and biased by the matrix difference between the calibrator and the sample of the subject. To overcome these problems, the present invention proposes a data transfer method. At the manufacturer's site, the set of samples of the subject and the calibrator samples can be measured in multiple replicates on multiple instruments during step a). Three adjustment factors are determined in step a) and can be electronically transferred to the customer mass spectrometry instrument in step b). At the customer site, in step c), calibration measurements can be performed on the calibrator samples and initial target values of the quantifier - confirmator ratios of the analyte and the internal standard can be determined. The adjustment factors are applied to the initial target values to calculate the adjusted target values of the analyte and the internal standard quantifier - confirmator ratio. The adjusted target values are instrument - specific, can track the sample of the subject, and have better accuracy for the multiple data points used. Due to the assay - specific calibration frequency, temporal drifts and shifts can be periodically corrected. For subsequent sample analysis, at least one acceptance criterion is used to verify the measured quantifier - confirmator ratio. The acceptance criterion or acceptance criteria can be determined during assay development. The at least one acceptance criterion may not be instrument - specific and may change over time. The at least one acceptance criterion may be electronically transferred to the customer instrument by an application parameter file.

[0024] As used herein, the term "manufacturer" 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 customized meaning. Specifically, but not limited to, this term can refer to at least one manufacturer of a mass spectrometry instrument. The term "manufacturer" can further refer to a single manufacturer that manufactures all parts of a mass spectrometry instrument and / or multiple manufacturers such as suppliers of specific components of a mass spectrometry instrument. The manufacturer can be the final manufacturer that provides the final product for use by the customer. As used herein, the term "manufacturer site" 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 customized meaning. Specifically, but not limited to, this term can refer to all processes performed by the manufacturer before providing the mass spectrometry instrument to the customer. All reagents, columns, calibrators, system reagents, disposables can be manufactured by or for the manufacturer. Conversely, at the customer's site, the customer can place the subject's sample and control sample on the instrument as non-manufacturer components.

[0025] As used herein, the term "standardization" 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 customized meaning. Specifically, but not limited to, this term can refer to a process of determining the inaccuracy of a measured quantifier-verifier ratio and estimating bias due to matrix differences between a calibrator and a subject's sample, and providing a correction therefor. Standardization can include determining a chromatogram for each measurement of a sample. The term "chromatogram" is known to those of ordinary skill in the art. In embodiments, this term relates to a correlation plot of a quantitative measure of a signal obtained from a sample and determined by an MS instrument and the progress of chromatographic separation, and in one embodiment a correlation plot over time, for example with respect to retention time and / or elution volume. In embodiments, the quantitative measure of the signal correlates with the concentration of at least a portion of the sample components, particularly the analyte. Thus, the quantitative measure of the signal can in particular be the signal intensity. The chromatogram can be an MS chromatogram, and in further embodiments an MS / MS chromatogram. As will be understood by those of ordinary skill in the art, the foregoing representation need not necessarily be, but can be, a graphical representation. However, the representation can be provided, for example, as a list of value pairs, such as elution time / quantifier value pairs and / or elution time / qualifier value pairs, or as a mathematical model. The quantitative measure of the signal can include analyte signal intensity and / or internal standard signal intensity. The quantitative measure of the signal can include an analyte quantifier, an internal standard quantifier, an analyte qualifier, and / or an internal standard qualifier. Thus, in embodiments, particularly when the MS is tandem MS, determining at least one chromatogram includes, as described above, measuring at least one of an analyte quantifier, an internal standard quantifier, an analyte qualifier, and / or an internal standard qualifier over time and / or over elution time. As will be understood by those of ordinary skill in the art, the elution time can be replaced by, in particular, the elution volume or the retention time, or any other measure of the progress of the LC considered appropriate by those of ordinary skill in the art.

[0026] 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 customized meaning. This term can specifically, but not limited to, refer to any sample, such as a biological sample. In an embodiment, the sample is a liquid sample, and in a further embodiment, it is an aqueous sample. In an embodiment, the sample is selected from the group consisting of physiological fluids, cleaning fluids, tissues, cells, etc. including blood, serum, plasma, saliva, aqueous humor, tears, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, and amniotic fluid. However, the sample may be a natural or industrial liquid, particularly surface water or groundwater, sewage, industrial wastewater, treatment fluid, soil eluate, etc. In an embodiment, the sample contains or is suspected of containing at least one target compound, i.e., a determined chemical substance called an "analyte". The sample may contain one or more additional chemical compounds, which are not determined and are generally called "matrix". The sample may be used directly as obtained from each source or may be subjected to one or more pretreatment and / or sample preparation steps. Thus, the sample can be pretreated by physical and / or chemical methods, in one embodiment, centrifugation, filtration, mixing, homogenization, chromatography, precipitation, dilution, concentration, binding and / or contact with detection reagents, and / or any other method considered appropriate by a deceased person. In the sample preparation step, i.e., before, during, and / or after the sample preparation step, one or more internal standards can be added to the sample. An internal standard may be added to the sample. For example, the internal standard may be added to the sample at a predetermined concentration. The internal standard can be selected so that it can be easily identified under the normal operating conditions of the mass spectrometry instrument. The concentration of the internal standard is predetermined and may be significantly higher than the concentration of the analyte.

[0027] As used herein, the term "internal standard" relates, in one embodiment, to an analyte present at a defined concentration in a sample. Thus, in an embodiment, the concentration of the internal standard is known. However, while the concentration of the standard is unknown, it is also contemplated that it will be the same for at least the subject's sample and at least one calibration sample. In such cases, in an embodiment, the concentration of the internal standard is the same for all samples being analyzed. The internal standard is, in an embodiment, structurally similar to the analyte and, in a further embodiment, is structurally identical to the analyte. In particular in the latter case, in an embodiment, the internal standard is an isotopically labeled molecule, in particular an isotopically labeled version of the analyte, such as 2 H (deuterated), 15 N and / or 13 C labeled derivatives. The internal standard sample can be a sample containing at least one internal standard substance having a known, for example, predetermined concentration. For further details regarding standard samples, see, for example, EP 3 425 369.

[0028] As used herein, the term "subject" 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 customized meaning. Specifically, but not limited thereto, this term can refer to a mammal. In an embodiment of the present invention, the subject is a human. Specifically, the subject can be a patient. A patient according to the present invention is typically a person who may be suffering from a disease or is suspected of suffering from a disease, that is, a person who may already exhibit some or all of the negative symptoms associated with the disease. As used herein, the term "sample of a subject" 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 customized meaning. Specifically, but not limited thereto, this term can refer to a biological sample of a subject to be tested. A set of patient samples can include a plurality of different samples of at least one subject. A set of patient samples can be a set of representative samples. Typically, a set of patient samples can include 5 to 30 samples. However, a set of patient samples can include more than 30 samples.

[0029] The term "calibrator 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 customized meaning. Specifically, but not limited thereto, this term can refer to any sample having a known concentration of the substance of the calibrator sample. For example, the concentration value of the calibrator sample can be determined by a reference laboratory. For example, the calibrator sample can be at least one commercially available calibrator. A set of calibrator samples can include a plurality of different calibrator samples. The calibrator sample can be a sample having an assigned target value or can include it. For example, a set of calibrator samples can include 2 to 3 calibrator samples. A set of calibrator samples can include at least one calibrator sample. A set of calibrator samples can include 4 to 10 calibrator samples.

[0030] The term "adjustment factor" 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 customized meaning. Specifically, but not limited to, this term can refer to a factor for correcting the initial target value of the quantifier - qualifier ratio of an analyte and / or internal standard determined on a customer mass spectrometry instrument due to measurement inaccuracies and biases resulting from matrix differences between the calibrator and the subject's sample.

[0031] This method includes determining at least three adjustment factors from the measured values of a set of the subject's samples and a set of calibrator samples. This method can include evaluating the determined chromatogram and determining the quantifier - qualifier ratio therefrom. Evaluating can include determining the peak area of the quantifier peak of the chromatogram and determining the peak area of the qualifier peak of the chromatogram. Evaluating can include determining the ratio of the peak area of the quantifier peak to the peak area of the qualifier peak. Step a) can include, for each of a plurality of mass spectrometry instruments, determining the median of the quantifier - qualifier ratio of the analyte and the internal standard of the calibrator sample and the subject's sample. The adjustment factor can be determined by using the between - instrument means. The first adjustment factor α depends on the difference between the analyte and the internal standard, particularly the relationship. In step a), the first adjustment factor α is

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[0032] The term "transfer" 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 customized meaning. Specifically, but not limited to, this term can mean one-way or two-way information exchange, especially data exchange. Transfer can include, for example, transferring information from a computing device, such as a computer, in order to transmit or output the information to another device. The transfer may be performed via at least one communication interface. The term "communication interface" as used herein 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 customized meaning. Specifically, but not limited to, this term can refer to an item or element that forms a boundary configured to transfer information. In particular, a communication interface can be configured to transfer information from a computing device, such as a computer, for example, to transmit or output the information to another device. Additionally or alternatively, a communication interface may be configured to transfer information to a computing device, such as a computer, for example, to receive the information. Specifically, a communication interface can provide means for transferring or exchanging information. In particular, a communication interface can provide a data transfer connection, such as Bluetooth (R), NFC, inductive coupling, etc. By way of example, a communication interface can be or include at least one port including one or more of a network or Internet port, a USB port, and a disk drive. A communication interface can be at least one web interface. The term "transfer electronically" 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 customized meaning. Specifically, but not limited to, this term can refer to a transfer that uses at least one electronic data transfer technology, especially a transfer that uses at least one transmission protocol.Specifically, the electronic transfer can include at least one download of at least one parameter file from a dedicated database. The transfer may include the customer mass spectrometry obtaining information from the manufacturer. As used herein, the term "obtaining" is a broad term and should be given its ordinary customary meaning to those of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to receiving data and / or downloading data from a data server or the like.

[0033] The term "customer site" calibration is a broad term and should be given its ordinary customary meaning to those of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to calibration performed by the customer. Thus, the calibration may be performed without the manufacturer. However, the manufacturer may provide support to the customer as needed.

[0034] The present invention proposes to split the calibration into two parts. In the first part, standardization may be performed at the manufacturer's site, and in the second part, calibration is performed on the customer's mass spectrometry instrument. The standardization may be performed before the calibration at the customer site. The customer site calibration includes at least one calibration measurement, and a set of calibrator samples is measured on the customer's mass spectrometry instrument. The set of calibrator samples used for calibration at the customer site may be the same as the set of calibrator samples used for standardization at the manufacturer's site. The set of calibrator samples may be provided by the manufacturer. The calibration measurement can include multiple measurements on multiple calibrator samples and multiple replicates of the set of calibrator samples. The calibration measurement can include multiple reaction monitoring using transitions of quantifiers and confirmers for analytes and / or internal standards. The calibration measurement can include determining at least one chromatogram for each measurement of the sample. The calibration measurement can include determining an initial target value for the quantifier-confirming ratio of the analyte and the internal standard. The initial target value can be determined by evaluating the chromatogram and determining the quantifier-confirming ratio therefrom.

[0035] The target value of the quantifier-confirming ratio of the analyte and the internal standard, also called the initial target value, is set by applying an adjustment factor to the determined quantifier-confirming ratio. In step c), all the quantifier-confirming ratios determined during the calibration measurement can be used to set the target value. In particular, all the calibrator levels and all the calibrator replicates of the analyte and the internal standard can be used to set the target value. The target value of the quantifier-confirming ratio of the analyte quantifier AQN and the analyte confirmator AQL

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[0036] Here, R is the quantifier - confirmator peak area ratio of a single measurement value, IQN is the internal standard quantifier, IQL is the internal standard confirmator, and N is the total number of quantifier - confirmator ratios used in the calculation. The target values of the quantifier - confirmator ratios of the internal standard quantifier IQN and the internal standard confirmator IQL

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[0037] Here, R is the quantifier - confirmator peak area ratio of a single measurement value, AQN is the analyte quantifier, AQL is the analyte confirmator, and N is the total number of quantifier - confirmator ratios used in the calculation. These target values can be made instrument - specific based on native patient samples and can have better accuracy due to the multiple data points used. Due to the assay, the temporal changes in specific calibration frequencies can be corrected periodically.

[0038] In a further aspect, a computer - implemented method for quantifier - confirmator ratio checking in a customer mass spectrometry instrument is disclosed. The method includes performing a calibration of a customer mass spectrometry instrument according to a computer - implemented method for calibrating a customer mass spectrometry instrument for quantifier - confirmator ratio checking according to the present invention. Thus, for the definition and embodiments of the method for quantifier - confirmator ratio checking, reference is made to the definition and embodiments of the method for calibrating a customer mass spectrometry instrument for quantifier - confirmator ratio checking according to the present invention, as described in more detail above or below.

[0039] The method includes at least one sample measurement using a customer mass spectrometry instrument. The method further includes at least one sample analysis step, during which, during the sample analysis of each sample measurement, the quantifier - confirmator ratio of the analyte and / or internal standard is determined and compared to a target value, taking into account at least one acceptance criterion.

[0040] As used herein, the term "sample measurement" 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 customized meaning. Specifically, without limitation, this term can refer to the process of measuring a sample under test using a mass spectrometry instrument. As used herein, the term "sample analysis" 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 customized meaning. Specifically, without limitation, this term can refer to the process of evaluating the signals of a mass spectrometry instrument. Sample analysis can include determining at least one chromatogram. Sample analysis can include evaluating the chromatogram and determining at least one quantifier - confirmatory ratio.

[0041] As used herein, the term "acceptance criterion" is a broad term and should be given its ordinary customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term can refer to any criterion that characterizes a quantifier-verifier ratio as acceptable or rejectable. An acceptance criterion or acceptance criteria can be determined during assay development. At least one acceptance criterion may not be instrument specific and may vary over time. At least one acceptance criterion may be electronically transferred to a customer instrument by an application parameter file. An acceptance criterion can include at least one acceptance limit or acceptance range. An acceptance criterion can be used for peak identity checking. An acceptance criterion can be used to distinguish quantifier ions from interferences. An acceptance criterion can characterize whether the quantifier-verifier ratio measured by a customer mass spectrometry instrument is appropriate. A quantifier-verifier ratio below an acceptance limit or within an acceptance range can be verified. The method can further include flagging each sample measurement value that does not meet the acceptance criterion. A quantifier-verifier ratio above an acceptance limit or outside an acceptance range is flagged and may require further review by an operator or user of the customer mass spectrometry instrument.

[0042] Further disclosed and proposed herein is a computer program product having program code means, wherein when the program code means is executed on a computer or computer network, it calibrates a customer mass spectrometry instrument for a quantifier-verifier ratio check according to the present invention and / or executes a computer-implemented method for a quantifier-verifier ratio check on a customer mass spectrometry instrument according to the present invention, and the program code means can be stored in a storage medium or is stored in a storage medium, which is a computer program product.

[0043] Further disclosed and proposed herein is a computer program product having program code means which, when executed on a computer or computer network, calibrate a customer mass spectrometry instrument for a quantifier - confirmator ratio check according to the present invention and / or execute a computer - implemented method for a quantifier - confirmator ratio check on a customer mass spectrometry instrument according to the present invention, and the program code means can be stored or are stored in a storage medium, which is a computer program product. Specifically, the program code means can be stored in a computer - readable data carrier and / or a computer - readable storage medium.

[0044] As used herein, the terms "computer - readable data carrier" and "computer - readable storage medium" can specifically refer to non - transitory data storage means such as a hardware storage medium in which computer - executable instructions are stored. The computer - readable data carrier or storage medium can specifically be or include a storage medium such as random access memory (RAM) and / or read - only memory (ROM).

[0045] Accordingly, specifically, one, more than one, or all of the method steps a) to c) as described above can be executed using a computer or computer network, preferably using a computer program.

[0046] Further disclosed and proposed herein is a data carrier in which a data structure is stored, which, after being loaded into a computer or computer network, for example, into a working memory or the main memory of a computer or computer network, can execute one or both of the methods according to one or more of the embodiments disclosed herein.

[0047] Further disclosed and proposed herein is a computer program product having program code means stored on a machine-readable carrier for executing one or both of the methods according to one or more of the embodiments disclosed herein when the program is executed on a computer or computer network. 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 on a computer-readable data carrier and / or computer-readable storage medium. Specifically, the computer program product may be distributed over a data network.

[0048] Finally, further disclosed and proposed herein is a modulated data signal containing instructions readable by a computer system or computer network for executing one or both of the methods according to one or more of the embodiments disclosed herein.

[0049] Referring to the computer-implemented aspects of the present invention, one or more, or even all, of the method steps of one or both of the methods according to one or more of the embodiments disclosed herein can be implemented using a computer or computer network. Thus, generally, any method step, including those that generally involve data provision and / or manipulation, can be executed by using a computer or computer network. Generally, these method steps can include any method step, usually excluding method steps that require manual work, such as specific ways of providing samples and / or performing actual measurements.

[0050] In a further aspect of the present invention, a mass spectrometry system for determining the concentration of at least one analyte in a sample is disclosed. The mass spectrometry system is - A manufacturer site calibration system comprising a plurality of mass spectrometry instruments configured to measure a set of subject samples and a set of calibrator samples in a plurality of replicates, each of the mass spectrometry instruments being configured for a plurality of reaction monitoring using quantifier and qualifier transitions for an analyte and an internal standard, the manufacturer site calibration system comprising at least one processing unit configured to determine at least three adjustment factors from measurement values of a set of subject samples and a set of calibrator samples, a first adjustment factor α depending on the difference between the analyte and the internal standard, a second adjustment factor β depending on the difference between the subject samples and the calibrator samples for the analyte quantifier-qualifier ratio, and a third adjustment factor γ depending on the difference between the subject samples and the calibrator samples for the internal standard quantifier-qualifier ratio, the manufacturer site calibration system; - At least one communication interface configured to electronically transfer the adjustment factors from the manufacturer site calibration system to at least one customer mass spectrometry instrument; - At least one customer mass spectrometry instrument, the customer mass spectrometry instrument being configured to perform at least one calibration measurement in which a set of calibration samples is measured on the customer mass spectrometry instrument, the customer mass spectrometry instrument comprising at least one evaluation device configured to determine a quantifier-qualifier ratio from the calibration measurement, the evaluation device being configured to set target values for the quantifier-qualifier ratios of the analyte and the internal standard by applying the adjustment factors to the determined quantifier-qualifier ratio, at least one customer mass spectrometry instrument; Comprising.

[0051] The mass spectrometry instrument can be a liquid chromatography mass spectrometry (LC-MS) device.

[0052] The customer mass spectrometry instrument can be configured to perform at least one sample measurement. The evaluation device can be configured to perform at least one sample analysis, during which the quantifier-qualifier ratios of the analyte and / or the internal standard are determined and compared with the target values taking into account at least one acceptance criterion during the sample analysis of all sample measurements.

[0053] As used herein, the term "processing unit" 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 customized meaning. Specifically, but not limited thereto, in embodiments, by using at least one data processing device, and in further embodiments, by using at least one processor and / or at least one application specific integrated circuit, it can refer to any device adapted to perform the method steps described above. Thus, by way of example, at least one processing unit can comprise at least one data processing unit in which software code including several computer commands is stored. The processing unit can provide one or more hardware elements for performing one or more of the indicated operations, and / or can provide software to be executed by one or more processors for performing one or more of the method steps.

[0054] As used herein, the term "evaluation 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 customized meaning. Specifically, but not limited thereto, in embodiments, by using at least one data processing device, and in further embodiments, by using at least one processor and / or at least one application specific integrated circuit, it can refer to any device adapted to perform the method steps described above. Thus, by way of example, at least one evaluation device can comprise at least one data processing unit in which software code including several computer commands is stored. The evaluation device can provide one or more hardware elements for performing one or more of the indicated operations, and / or can provide software to be executed by one or more processors for performing one or more of the method steps.

[0055] This system can be configured to execute a computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking according to the present invention and / or a computer-implemented method for quantifier-verifier ratio checking for a customer mass spectrometry instrument according to the present invention. Therefore, for the definition and embodiments of the system, reference is made to the embodiments and definitions of the method according to the present invention as described above and in more detail below.

[0056] In summary, the following embodiments can be envisioned without excluding further possible embodiments.

[0057] Embodiment 1: A computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking, comprising: a) At least one manufacturer site standardization step, wherein a set of subject samples and a set of calibrator samples are measured in multiple replicates on multiple mass spectrometry instruments, each measurement including multiple reaction monitors using quantifier and verifier transitions for the analyte and internal standard, at least three adjustment factors are determined from the measurements of the set of subject samples and the set of calibrator samples, the first adjustment factor α depends on the difference between the analyte and the internal standard, the second adjustment factor β depends on the difference between the subject sample and the calibrator sample for the analyte quantifier-verifier ratio, and the third adjustment factor γ depends on the difference between the subject sample and the calibrator sample for the internal standard quantifier-verifier ratio; at least one manufacturer site standardization step; b) At least one transfer step, wherein the adjustment factor is electronically transferred to the customer mass spectrometry instrument; at least one transfer step; c) At least one customer site calibration step, wherein the customer site calibration includes at least one calibration measurement value, a set of calibrator samples is measured on the customer mass spectrometry instrument, and the quantifier-verifier ratio is determined therefrom, and by applying the adjustment factor to the determined quantifier-verifier ratio, the target values of the quantifier-verifier ratios of the analyte and the internal standard are set; at least one customer site calibration step; A method comprising

[0058] Embodiment 2: The method according to the preceding embodiment, wherein in step c), all quantifier - confirmator ratios determined during the calibration measurement are used to set a target value, and all calibrator levels and all calibrator replicates of the analyte and internal standard are used to set the target value.

[0059] Embodiment 3: The target value of the quantifier - confirmator ratio of the analyte quantifier AQN and the analyte confirmator AQL

Number

Number

[0060] Embodiment 4: The target value of the quantifier - confirmator ratio for the internal standard quantifier IQN and the internal standard confirmator IQL

Number

Number

[0061] Embodiment 5: In step a), the first adjustment coefficient α is

Number

[0062] Embodiment 6: In step a), the second adjustment coefficient β is [Number] determined by, where [Number] is the average of the analyte quantifier - confirmer ratios of the plurality of measurements of the sample of the subject in step a), [Number] is the average of the analyte quantifier - confirmer ratios of the plurality of measurements of the calibrator sample in step a), the method according to any one of the preceding embodiments.

[0063] Embodiment 7: In step a), the second adjustment coefficient γ is [Number] determined by, where [Number] is the average of the internal standard quantifier - confirmer ratios of the plurality of measurements of the sample of the subject in step a), [Number] The method according to any one of the preceding embodiments, which is the average of the quantifier - confirmator ratios of the internal standard for a plurality of measurements of the calibrator sample in step a).

[0064] Embodiment 8: The method according to any one of the preceding embodiments, wherein step a) includes determining, for each of a plurality of mass spectrometers, the median of the quantifier - confirmator ratio of the analyte, and the internal standard of the calibrator sample and the sample of the subject.

[0065] Embodiment 9: The method according to the preceding embodiments, wherein the adjustment factor is determined by using an inter - instrument means.

[0066] Embodiment 10: The method according to any one of the preceding embodiments, wherein the mass spectrometer used in steps a) and c) is a liquid chromatography - mass spectrometry (LC - MS) device.

[0067] Embodiment 11: A computer - implemented method for quantifier - confirmator ratio checking on a customer mass spectrometer, the method including performing the calibration of the customer mass spectrometer according to a computer - implemented method for calibrating the customer mass spectrometer for quantifier - confirmator ratio checking according to any one of the preceding embodiments, the method including performing at least one sample measurement using the customer mass spectrometer, the method further including at least one sample analysis step, during the sample analysis of all sample measurements, the quantifier - confirmator ratio of the analyte and / or the internal standard is determined and compared with a target value considering at least one acceptance criterion.

[0068] Embodiment 12: The method according to the preceding embodiments, further including flagging each of the sample measurement values that do not meet the acceptance criteria.

[0069] Embodiment 13: The method according to the preceding embodiments, further including reviewing the flagged sample measurement values.

[0070] Embodiment 14: A computer program product having program code means which, when executed on a computer or a computer network, refers to a method for calibrating a customer mass spectrometry instrument, a computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking according to any one of the preceding embodiments, and / or a computer-implemented method for quantifier-verifier ratio checking in a customer mass spectrometry instrument according to any one of the preceding embodiments which refers to a method for quantifier-verifier ratio checking, wherein the program code means can be stored or is stored on a storage medium, a computer program product.

[0071] Embodiment 15: A computer program product having program code means which, when executed on a computer or a computer network, refers to a method for calibrating a customer mass spectrometry instrument, a computer-implemented method for calibrating a customer mass spectrometry instrument for quantifier-verifier ratio checking according to any one of the preceding embodiments, and / or a computer-implemented method for quantifier-verifier ratio checking in a customer mass spectrometry instrument according to any one of the preceding embodiments which refers to a method for quantifier-verifier ratio checking, wherein the program code means can be stored or is stored on a storage medium, a computer program product.

[0072] Embodiment 16: A mass spectrometry system for determining the concentration of at least one analyte in a sample, - A manufacturer site calibration system comprising a plurality of mass spectrometers configured to measure a set of subject samples and a set of calibrator samples in a plurality of replicates, each of the mass spectrometers being configured for a plurality of reaction monitoring using quantifier and confirmatory transitions for an analyte and an internal standard, the manufacturer site calibration system comprising at least one processing unit configured to determine at least three adjustment factors from the measured values of the set of subject samples and the set of calibrator samples, a first adjustment factor α depending on the difference between the analyte and the internal standard, a second adjustment factor β depending on the difference between the subject sample and the calibrator sample for the analyte quantifier-confirmatory ratio, and a third adjustment factor γ depending on the difference between the subject sample and the calibrator sample for the internal standard quantifier-confirmatory ratio, the manufacturer site calibration system; - At least one communication interface configured to electronically transfer adjustment factors from the manufacturer site calibration system to at least one customer mass spectrometer; - At least one customer mass spectrometer, the customer mass spectrometer being configured to perform at least one calibration measurement in which a set of calibration samples is measured on the customer mass spectrometer, the customer mass spectrometer comprising at least one evaluation device configured to determine a quantifier-confirmatory ratio from the calibration measurement, the evaluation device being configured to set target values for the quantifier-confirmatory ratios of the analyte and the internal standard by applying adjustment factors to the determined quantifier-confirmatory ratio, at least one customer mass spectrometer; Comprising.

[0073] Embodiment 17: A computer-implemented method for calibrating a customer mass spectrometer for quantifier-confirmatory ratio checking according to any one of the preceding embodiments, wherein the system refers to a method for calibrating the customer mass spectrometer, and / or a computer-implemented method for quantifier-confirmatory ratio checking on a customer mass spectrometer according to any one of the preceding embodiments, wherein the system is configured to execute the method for calibrating the customer mass spectrometer for quantifier-confirmatory ratio checking according to any one of the preceding embodiments, the system according to the preceding embodiments.

[0074] Embodiment 18: The system according to any one of the preceding embodiments referring to a system in which the mass spectrometer is a liquid chromatography mass spectrometer (LC-MS) device.

Brief Description of the Drawings

[0075] Any further optional features and embodiments are preferably disclosed in more detail in the subsequent description of the embodiments, in conjunction with the dependent claims. Here, each optional feature may be implemented in an independent manner and in any practicable combination, as will 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 signs in these figures refer to the same or functionally equivalent elements. The figures are as follows.

[0076]

Figure 1

Figure 2

Figure 3A

Figure 3B

Modes for Carrying Out the Invention

[0077] FIG. 1 shows an embodiment of a mass spectrometry system 110 according to the present invention. The mass spectrometry system 110 includes a manufacturer site calibration system 112 having a plurality of mass spectrometry instruments 114 configured to measure a set of subject samples and a set of calibrator samples in a plurality of replicates. Each of the mass spectrometry instruments 114 is configured for multiple reaction monitoring using quantifier and confirmatory transitions of analytes and internal standards. The mass spectrometry (MS) instrument 114 can be a mass spectrometer configured to detect at least one analyte based on a mass-to-charge ratio. Each of the mass spectrometry instruments 114 may be at least one quadrupole mass spectrometer or may include at least one quadrupole mass spectrometer. The MS instrument 114 can be a tandem mass spectrometry (MS / MS) instrument or a triple quadrupole MS / MS. Specifically, the mass spectrometry instrument 114 can be configured for multiple reaction monitoring (MRM).

[0078] The mass spectrometer 114 may specifically be, or may include, a liquid chromatography mass spectrometer. 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 be equipped with these. The liquid chromatography mass spectrometer can include a liquid chromatography (LC) device and a mass spectrometry (MS) device, and the LC device and the MS are coupled via at least one interface. The interface connecting the liquid chromatography device and the MS can include at least one ionization source configured to generate molecular ions and move the molecular ions into the gas phase. The liquid chromatography (LC) device may be configured to separate one or more analytes of interest in a sample from other components of the sample in order to detect the one or more analytes using the mass spectrometer 114. The LC device may be based on any separation principle considered appropriate by those skilled in the art. In an embodiment, the LC device may be reverse phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or chiral chromatography. In a further embodiment, the LC device is reverse phase chromatography. The LC device can include 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 can have a stationary phase, and the mobile phase is pumped through the stationary phase for separation and / or elution and / or transfer of the analyte of interest.

[0079] The manufacturer may be at least one manufacturer of the mass spectrometry instrument 114. The manufacturer may be a single manufacturer that manufactures all parts of the mass spectrometry instrument 114, and / or multiple manufacturers such as suppliers of specific components of the mass spectrometry instrument 114. The manufacturer may be the final manufacturer that provides the final product for customer use. As used herein, the term "manufacturer site" 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 customized meaning. Specifically, but not limited to, this term can refer to all processes performed by the manufacturer before providing the mass spectrometry instrument to the customer. All reagents, columns, calibrators, system reagents, disposable items can be manufactured by or for the manufacturer. Conversely, at the customer's site, the customer can place the subject's sample and the control sample on the instrument as non-manufacturer components.

[0080] The manufacturer site calibration system 112 includes at least one processing unit 116 configured to determine at least three adjustment coefficients from the measurements of a set of subject samples and a set of calibrator samples. The processing unit 116 can be adapted to determine the three adjustment coefficients by using at least one data processing device and, in further embodiments, by using at least one processor and / or at least one application-specific integrated circuit. Thus, by way of example, the at least one processing unit 116 can include at least one data processing unit in which software code including several computer commands is stored. The processing unit 116 can provide one or more hardware elements for performing one or more of the illustrated operations and / or can provide software executed to perform the determination of the adjustment coefficients to one or more processors.

[0081] The adjustment factor can be determined during the standardization of the manufacturer site. Standardization can be, or can include, a process that determines an estimate of the bias due to the inaccuracy of the measured quantifier - confirmator ratio and the matrix difference between the calibrator and the sample of the subject and provides a correction therefor. Standardization can include determining a chromatogram for each measurement of the sample. The chromatogram can be a correlation plot of the quantitative measure of the signal determined by the MS instrument 114 as the chromatography separation progresses, obtained from the sample, and in embodiments can be over time, for example, retention time and / or elution volume. In embodiments, the quantitative measure of the signal correlates with the concentration of at least a portion of the sample components, particularly the analyte. Thus, the quantitative measure of the signal can particularly be the signal intensity. The chromatogram can be an MS chromatogram, and in further embodiments an MS / MS chromatogram. As will be understood by those skilled in the art, the foregoing representation may, but need not necessarily, be a graphical representation. However, the representation may be provided, for example, as a list of value pairs, for example, elution time / quantifier value pairs and / or elution time / qualifier value pairs, or as a mathematical model. The quantitative measure of the signal can include the analyte signal intensity and / or the internal standard signal intensity. The quantitative measure of the signal can include the analyte quantifier, the internal standard quantifier, the analyte confirmator and / or the internal standard confirmator. Thus, in embodiments, particularly when the MS is tandem MS, determining at least one chromatogram includes, as described above, measuring at least one of the analyte quantifier, the internal standard quantifier, the analyte qualifier and / or the internal standard qualifier over time and / or over the elution time. As will be understood by those skilled in the art, the elution time can be replaced by the elution volume or the retention time, or by any other measure of the progress of the LC considered appropriate by those skilled in the art.

[0082] The adjustment factor can be, or can include, a factor for correcting an initial target value of the quantifier - confirmator ratio of the analyte and / or internal standard determined in the customer mass spectrometry instrument 118 due to measurement inaccuracies and biases resulting from matrix differences between the calibrator and the subject's sample. The processing unit 116 may be configured to determine at least three adjustment factors from the measured values of the set of the subject's samples and the set of calibrator samples. The processing unit may be configured to evaluate the determined chromatogram and determine the quantifier - confirmator ratio therefrom. The processing unit 116 may be, or may comprise, at least one evaluation device. Evaluating can include determining the peak area of the quantifier peak of the chromatogram and determining the peak area of the confirmator peak of the chromatogram. Evaluating can include determining the ratio of the peak area of the quantifier peak to the peak area of the confirmator peak. The processing unit 116 may be configured to determine the median of the quantifier - confirmator ratio of the subject's analyte and the calibrator sample and the internal standard of the sample for each of a plurality of mass spectrometry instruments. The adjustment factor can be determined by using an inter - instrument means. The first adjustment factor α depends on the difference between the analyte and the internal standard. The first adjustment factor α can be determined by, where, is the mean of the quantifier - confirmator ratios of the analyte of a plurality of measured values, and is the mean of the quantifier - confirmator ratios of the internal standard of a plurality of measured values. The second adjustment factor β depends on the difference between the subject's sample and the calibrator sample for the analyte quantifier - confirmator ratio. The second adjustment factor β is,

Number

Number

Number

Number

[0083] The mass spectrometry system 110 comprises at least one communication interface 120 configured to electronically transfer calibration coefficients from a manufacturer site calibration system 112 to a customer mass spectrometry instrument 118. In FIG. 1, the communication interface 120 is indicated by two dashed lines. The transfer may be a one-way or two-way information exchange, particularly a data exchange. The transfer may include, for example, transferring information from a computing device, such as a computer, for transmitting or outputting information to another device. The communication interface 120 can form a boundary configured to transfer information. In particular, the communication interface 120 can be configured to transfer information from a computing device, such as a computer, for example, for transmitting or outputting information to another device. Additionally or alternatively, the communication interface 120 may be configured to transfer information to a computing device, such as a computer, for receiving information, etc. The communication interface 120 can specifically provide means for transferring or exchanging information. In particular, the communication interface 120 can provide a data transfer connection, such as Bluetooth, NFC, inductive coupling, etc. By way of example, the communication interface 120 can be or include at least one port including one or more of a network or Internet port, a USB port, and a disk drive. The communication interface 120 can be at least one web interface. The electronic transfer can be performed using at least one electronic data transfer technology, particularly using at least one transmission protocol. Specifically, the electronic transfer can include at least one download of at least one parameter file from a dedicated database. The transfer can include the customer mass spectrometry 118 obtaining information from the manufacturer, such as by receiving data and / or downloading data from a data server, etc.

[0084] The mass spectrometry system 110 includes at least one customer mass spectrometry instrument 118. The customer mass spectrometry instrument 118 is disposed at a customer site 122. The customer mass spectrometry instrument 118 is configured to perform at least one calibration measurement in which a set of calibrator samples is measured in the customer mass spectrometry instrument 118. The customer mass spectrometry instrument 118 includes at least one evaluation device 124 configured to determine a quantifier-confirmator ratio from the calibration measurement. The evaluation device 124 is configured to set a target value for the quantifier-confirmator ratio of the analyte and the internal standard by applying an adjustment factor to the determined quantifier-confirmator ratio.

[0085] The target value for the quantifier-confirmator ratio of the analyte and the internal standard, also referred to as the initial target value, is set by applying an adjustment factor to the determined quantifier-confirmator ratio. All quantifier-confirmator ratios determined during the calibration measurement can be used to set the target value. In particular, all calibrator levels and all calibrator replicates of the analyte and the internal standard can be used to set the target value. The target value for the quantifier-confirmator ratio of the analyte quantifier AQN and the analyte confirmator AQL

Number

Number

[0086] where R is the quantifier-confirmator peak area ratio of a single measurement value, IQN is the internal standard quantifier, IQL is the internal standard confirmator, and N is the total number of quantifier-confirmator ratios used in the calculation. The target value for the quantifier-confirmator ratio of the internal standard quantifier IQN and the internal standard confirmator IQL

Number

Number

[0087] Here, R is the quantifier - confirmator peak area ratio of a single measurement value, AQN is the analyte quantifier, AQL is the analyte confirmator, and N is the total number of quantifier - confirmator ratios used in the calculation. These target values can be made instrument - specific based on native patient samples and can have better accuracy due to the multiple data points used. Due to the assay, the temporal variation of a specific calibration frequency can be corrected periodically.

[0088] The customer mass spectrometry instrument 118 can be configured to perform at least one sample measurement. The evaluation device 124 can be configured to perform at least one sample analysis, and during the sample analysis of all sample measurements, the quantifier - confirmator ratio of the analyte and / or internal standard is determined and compared with the target value considering at least one acceptance criterion.

[0089] The mass spectrometry system 110 may be configured to perform a quality check based on a quantifier-verifier ratio. For a quality check based on the quantifier-verifier ratio, at least one target value against which the measured quantifier-verifier ratio is compared is used. However, the target value can vary or change from mass spectrometry instrument to mass spectrometry instrument. Thus, it may be advantageous to use for the quality check target value of a particular customer mass spectrometry instrument. Even a change over time is possible. Thus, it may be advantageous to use for the quality check target value that can be repeatedly adjusted or adapted in a timely manner at the customer site. Further, for calibration, usually a small number of calibration samples are measured and the composition of these calibration samples may be different compared to the sample of the subject. Thus, the quality check based on the quantifier-verifier ratio is strongly influenced by measurement inaccuracies and biased by matrix differences between the calibrator and the sample of the subject. To overcome these problems, the present invention proposes a data transfer method. At the manufacturer site 112, a set of samples of the subject and calibrator samples can be measured in multiple replicates on multiple instruments. Three adjustment factors may be determined and electronically transferred to the customer mass spectrometry instrument 118. At the customer site 122, a calibration measurement can be performed on the calibrator sample and an initial target value of the quantifier-verifier ratio of the analyte and internal standard can be determined. The adjustment factor is applied to the initial target value to calculate the adjusted target value of the analyte and the internal standard quantifier-verifier ratio. The adjusted target value is instrument specific, can track the sample of the subject, and has better accuracy for the multiple data points used. Due to the assay-specific calibration frequency, temporal drifts and shifts can be periodically corrected. For subsequent sample analysis, at least one acceptance criterion is used to verify the measured quantifier-verifier ratio.

[0090] Acceptance criteria can characterize the quantifier - qualifier ratio as acceptable or rejectable. The acceptance criteria or multiple acceptance criteria can be determined during assay development. At least one acceptance criterion may not be instrument - specific and may change over time. At least one acceptance criterion may be electronically transferred to the customer device by an application parameter file. The acceptance criteria can include at least one tolerance limit or tolerance range. The acceptance criteria can be used for peak identity checking. The acceptance criteria can be used to distinguish quantifier ions from interferences. The acceptance criteria can characterize whether the quantifier - qualifier ratio measured by the customer mass spectrometry instrument 118 is appropriate. A quantifier - qualifier ratio below the tolerance limit or within the tolerance range can be verified. The method can further include flagging each of the sample measurement values that do not meet the acceptance criteria. A quantifier - qualifier ratio above the tolerance limit or outside the tolerance range is flagged and may require further review by the operator or user of the customer mass spectrometry instrument.

[0091] The present invention proposes to split the calibration into two parts. In the first part, standardization may be performed at the manufacturer site 112, and in the second part, calibration is performed at the customer mass spectrometry instrument 118. The standardization may be performed before the calibration at the customer site. The customer site calibration includes at least one calibration measurement, and a set of calibrator samples is measured at the customer mass spectrometry instrument 118. The set of calibrator samples used for the calibration at the customer site may be the same as the set of calibrator samples used for the standardization at the manufacturer site. The set of calibrator samples may be provided by the manufacturer. The calibration measurement can include multiple measurements for multiple calibrator samples and multiple replicates of the set of calibrator samples. The calibration measurement can include multiple reaction monitoring using transitions of quantifiers and confirmers for the analyte and / or internal standard. The calibration measurement can include determining at least one chromatogram for each measurement of the sample. The calibration measurement can include determining an initial target value for the quantifier-confirming ratio of the analyte and internal standard. The initial target value can be determined by evaluating the chromatogram and determining the quantifier-confirming ratio therefrom.

[0092] FIG. 2 shows a computer-implemented method for calibrating the customer mass spectrometry instrument 118 for quantifier-confirming ratio check according to the present invention, and a flowchart of a computer-implemented method for quantifier-confirming ratio check on the customer mass spectrometry instrument 118 according to the present invention. The method includes the following steps, namely, a) At least one manufacturer site standardization step (indicated by reference numeral 126), wherein a set of subject samples and a set of calibrator samples are measured in multiple replicates on a plurality of mass spectrometers 114, each measurement including a plurality of reaction monitoring using quantifier and qualifier transitions for the analyte and internal standard, at least three adjustment coefficients being determined from the measurements of the set of subject samples and the set of calibrator samples, a first adjustment coefficient α depending on the difference between the analyte and the internal standard, a second adjustment coefficient β depending on the difference between the subject samples and the calibrator samples for the quantifier - qualifier ratio of the analyte, and a third adjustment coefficient γ depending on the difference between the subject samples and the calibrator samples for the quantifier - qualifier ratio of the internal standard, at least one manufacturer site standardization step; b) At least one transfer step (indicated by reference numeral 128), wherein the adjustment coefficients are electronically transferred to the customer mass spectrometer 118, at least one transfer step; c) At least one customer site calibration step (indicated by reference numeral 130), wherein the customer site calibration includes at least one calibration measurement, a set of calibrator samples being measured on the customer mass spectrometer 118, from which the quantifier - qualifier ratio is determined, and by applying the adjustment coefficients to the determined quantifier - qualifier ratio, target values for the quantifier - qualifier ratios of the analyte and the internal standard are set, at least one customer site calibration step; including.

[0093] A computer - implemented method for quantifier - qualifier ratio checking on the customer mass spectrometer 118 includes steps a) to c). Further, the method for quantifier - qualifier ratio checking includes at least one sample measurement (indicated by reference numeral 132) using the customer mass spectrometer 118. The method further includes at least one sample analysis step (indicated by reference numeral 134), and during the sample analysis of each sample measurement, the quantifier - qualifier ratio of the analyte and / or internal standard is determined and compared with the target value considering at least one acceptance criterion.

[0094] Figures 3A and 3B show the experimental results of a testosterone test case, particularly for the quantification of testosterone in human serum or plasma by an LCMS assay. Automated sample preparation was performed on a robotic workstation, LC separation was performed on a commercially available HPLC system, and mass spectrometric detection was performed by multiple reaction monitoring using the transitions of the quantifier and confirmator of the analyte and internal standard, respectively. For two days of manufacturer site standardization on two mass spectrometers 114, two replicate calibrators and patient samples were measured. The median of the quantifier-confirmator ratio for the calibrators and patient samples on both mass spectrometers 114 was calculated.

Table 1

[0095] By using the between-instrument means, the adjustment coefficients α, β, γ were determined: - α = 1.001 - β = 1.003 - γ = 0.998.

[0096] At the customer site 122, sample measurements were performed under customer conditions. On the customer's mass spectrometer 118, three replicates of two calibrator levels were measured in one day and used for calibration and setting of the target value. Thirty patient samples were measured in multiple replicates over 10 days, and a quantification-confirmator ratio check was applied.

Table 2

[0097] Figures 3A and 3B show the experimental results in the case of a testosterone test. Figure 3A shows the quantifier-confirmator ratio of the analyte QQ ana as a function of the area ratio. The area ratio is the ratio of the peak areas of the analyte quantifier and the internal standard quantifier. Figure 3B shows the internal standard QQ ISTDShows the quantifier - verifier ratio for. The solid line indicates the target values for analyte 1.15 and internal standard 1.14. Additionally, a tolerance range of ±20% is shown. The target values for analyte 1.15 and internal standard 1.14 determined by the adjustment factor from the manufacturer site standardization are shown to be suitable for the entire measurement range of the customer mass spectrometry instrument.

Description of symbols

[0098] 110 Mass spectrometry system 112 Manufacturer site 114 Mass spectrometry instrument 116 Processing unit 118 Mass spectrometry instrument 120 Communication interface 122 Customer site 124 Evaluation device 126 Manufacturer site standardization 128 Transfer step 130 Customer site calibration 132 Sample measurement 134 Sample analysis step

Claims

1. A computer-implemented method for calibrating a customer mass spectrometer (118) for quantitative ion-confirmation ion ratio checking, comprising: a) at least one manufacturer site standardization step, wherein a set of test samples and a set of calibration samples of a subject are measured in a plurality of replicates on a plurality of mass spectrometers (114), each measurement comprising a plurality of reaction monitors involving transitions of quantitative ions and confirmation ions for an analyte and an internal standard, at least three adjustment coefficients being determined from the measurements of the set of test samples and the set of calibration samples of the subject, a first adjustment coefficient α depending on the difference between the analyte and the internal standard, the first adjustment coefficient α being 【Equation 1】 determined by 【Equation 2】 being the mean of the quantitative ion-confirmation ion ratios of the analyte in the plurality of measurements of step a), 【Equation 3】 being the mean of the quantitative ion-confirmation ion ratios of the internal standard in the plurality of measurements of step a), a second adjustment coefficient β depending on the difference between the test sample and the calibration sample of the subject for the quantitative ion-confirmation ion ratio of the analyte, the second adjustment coefficient β being 【Equation 4】 determined by 【Equation 5】 being the mean of the quantitative ion-confirmation ion ratios of the analyte in the plurality of measurements of the test sample of the subject in step a), 【Equation 6】 being the mean of the quantitative ion-confirmation ion ratios of the analyte in the plurality of measurements of the calibration sample in step a), a third adjustment coefficient γ depending on the difference between the test sample and the calibration sample of the subject for the quantitative ion-confirmation ion ratio of the internal standard, the third adjustment coefficient γ being 【Equation 7】 determined by 【Equation 8】 is the average of the quantitative ion - confirmation ion ratios of the internal standard in the plurality of measurements of the sample of the subject in step a), [Equation 9] at least one manufacturer site standardization step, where is the average of the quantitative ion - confirmation ion ratios of the internal standard in the plurality of measurements of the calibration sample in step a), b) at least one transfer step, wherein the at least three adjustment factors are electronically transferred to the customer mass spectrometer (118), c) at least one customer site calibration step, wherein the customer site calibration includes at least one calibration measurement, a set of calibration samples is measured with the customer mass spectrometer (118), from which the quantitative ion - confirmation ion ratio is determined, and by applying the at least three adjustment factors to the determined quantitative ion - confirmation ion ratio, target values of the quantitative ion - confirmation ion ratios of the analyte and the internal standard are set, and the target value of the quantitative ion - confirmation ion ratio of the analyte quantitative ion AQN and the analyte confirmation ion AQL [Equation 10] is [Equation 11] set by, where R is the quantitative ion - confirmation ion peak area ratio of a single measurement, IQN is the internal standard quantitative ion, IQL is the internal standard confirmation ion, N is the total number of quantitative ion - confirmation ion ratios used in the calculation, and the target value of the quantitative ion - confirmation ion ratio for the internal standard quantitative ion IQN and the internal standard confirmation ion IQL [Equation 12] is [Equation 13] set by, where R is the quantitative ion - confirmation ion peak area ratio of a single measurement, AQN is the analyte quantitative ion, AQL is the analyte confirmation ion, and N is the total number of quantitative ion - confirmation ion ratios used in the calculation, at least one customer site calibration step A method comprising **Claim 2** In step c), all quantitative ion - confirmation ion ratios determined during said calibration measurement are used to set said target value, and all calibration levels and all calibration replicates for the analyte and internal standard are used to set said target value. The method according to claim 1. **Claim 3** Step a) includes determining, for each of said plurality of mass spectrometers, the median of the quantitative ion - confirmation ion ratios of the analyte and internal standard for the calibration sample and the subject's sample, and said at least three adjustment factors are determined by using an inter - instrument means. The method according to claim 1 or 2. **Claim 4** A computer - implemented method for quantitative ion - confirmation ion ratio checking on a customer mass spectrometer (118), said method comprising calibrating said customer mass spectrometer (118) according to a computer - implemented method for calibrating a customer mass spectrometer for quantitative ion - confirmation ion ratio checking according to any one of claims 1 to 3, said method including performing at least one sample measurement using said customer mass spectrometer (118), said method further including at least one sample analysis step, during which the quantitative ion - confirmation ion ratio of the analyte and / or internal standard is determined and compared with a target value considering at least one acceptance criterion. **Claim 5** The method according to claim 4, further including flagging each of the sample measurements that do not meet said acceptance criterion. **Claim 6** The method according to claim 5, further including reviewing the flagged sample measurements. **Claim 7** A computer program product having program code means, wherein when the program code means is executed on a computer or a computer network, it refers to a method for calibrating a customer mass spectrometer (118), the computer-implemented method for calibrating a customer mass spectrometer (118) for quantitative ion-confirmation ion ratio checking according to any one of claims 1 to 3, and / or referring to a method for quantitative ion-confirmation ion ratio checking, the computer program product, wherein the program code means can be stored or is stored in a storage medium, for executing the computer-implemented method for quantitative ion-confirmation ion ratio checking on the customer mass spectrometer (118) according to any one of claims 4 to 6.

8. A mass spectrometry system (110) for determining the concentration of at least one analyte in a sample, - A manufacturer site calibration system (112) comprising a plurality of mass spectrometers (114) configured to measure a set of samples of a subject and a set of calibration samples in a plurality of replicates, each of the mass spectrometers (114) being configured for a plurality of reaction monitoring involving transitions of quantitative ions and confirmation ions of an analyte and an internal standard, the manufacturer site calibration system (112) comprising at least one processing unit (116) configured to determine at least three adjustment coefficients from the measurement of the set of samples of the subject and the set of calibration samples, a first adjustment coefficient α depending on the difference between the analyte and the internal standard, the first adjustment coefficient α being 【Equation 14】 determined by, 【Equation 15】 being the average of the quantitative ion-confirmation ion ratios of the analyte in the plurality of measurements of step a), 【Equation 16】 is the average of the quantitative ion - confirmation ion ratios of the internal standard in the plurality of measurements in step a), and the second adjustment coefficient β depends on the difference between the subject's sample and the calibration sample for the quantitative ion - confirmation ion ratio of the analyte, and the second adjustment coefficient β is [Equation 17] determined by [Equation 18] is the average of the quantitative ion - confirmation ion ratios of the analyte in the plurality of measurements of the subject's sample in step a), [Equation 19] is the average of the quantitative ion - confirmation ion ratios of the analyte in the plurality of measurements of the calibration sample in step a), and the third adjustment coefficient γ depends on the difference between the subject's sample and the calibration sample for the quantitative ion - confirmation ion ratio of the internal standard, and the third adjustment coefficient γ is [Equation 20] determined by [Equation 21] is the average of the quantitative ion - confirmation ion ratios of the internal standard in the plurality of measurements of the subject's sample in step a), [Equation 22] is the average of the quantitative ion - confirmation ion ratios of the internal standard in the plurality of measurements of the calibration sample in step a), a manufacturer site calibration system, - at least one communication interface (120) configured to electronically transfer the at least three adjustment coefficients from the manufacturer site calibration system (112) to at least one customer mass spectrometer (118), - At least one customer mass spectrometry instrument (118), wherein the customer mass spectrometry instrument (118) is configured to perform at least one calibration measurement, in which a set of calibration samples is measured by the customer mass spectrometry instrument (118), and the customer mass spectrometry instrument comprises at least one evaluation device (124) configured to determine a quantitative ion - confirmation ion ratio from the calibration measurement, and the evaluation device (124) is configured to set target values of the quantitative ion - confirmation ion ratios of the analyte and the internal standard by applying the at least three adjustment factors to the determined quantitative ion - confirmation ion ratio, the target values of the quantitative ion - confirmation ion ratio for the analyte quantitative ion AQN and the analyte confirmation ion AQL [Equation 23] is [Equation 24] set by where R is the quantitative ion - confirmation ion peak area ratio of a single measurement, IQN is the internal standard quantitative ion, IQL is the internal standard confirmation ion, N is the total number of quantitative ion - confirmation ion ratios used in the calculation, the target values of the quantitative ion - confirmation ion ratio for the internal standard quantitative ion IQN and the internal standard confirmation ion IQL [Equation 25] is [Equation 26] set by where R is the quantitative ion - confirmation ion peak area ratio of a single measurement, AQN is the analyte quantitative ion, AQL is the analyte confirmation ion, N is the total number of quantitative ion - confirmation ion ratios used in the calculation, at least one customer mass spectrometry instrument (118), and A mass spectrometry system comprising.

9. The computer-implemented method for calibrating the customer mass spectrometry instrument (118) according to any one of claims 1 to 3, wherein the mass spectrometry system (110) refers to a method for calibrating the customer mass spectrometry instrument (118), and / or the computer-implemented method for checking the quantitative ion - confirmation ion ratio in the customer mass spectrometry instrument (118) according to any one of claims 4 to 6, wherein the mass spectrometry system (110) refers to a method for checking the quantitative ion - confirmation ion ratio. The mass spectrometry system (110) according to claim 8, which is configured to execute the method.

10. The mass spectrometry system (110) according to claim 8 or 9, wherein the mass spectrometry instrument (114, 118) is a liquid chromatography mass spectrometer (LC - MS) device, referring to the system.

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