Carryover monitoring

Abstract

The present invention relates to a method for determining analyte carryover from a previous sample to a target sample on a liquid chromatography mass spectrometer (LC-MS) device, the method comprising: (a) determining at least one chromatogram of the target sample on the LC-MS device; (b) determining a background height of the chromatogram; and (c) determining analyte carryover from the previous sample to the target sample based on the background height. The present invention also relates to methods, systems, and computer program products related to the aforementioned methods.

Description

[Technical field]

[0001] The present invention relates to a method for determining analyte carryover from a previous sample to a sample of interest in a liquid chromatography mass spectrometry (LC-MS) device, comprising: (a) determining at least one chromatogram of the sample of interest in the LC-MS device, (b) determining a background height of the chromatogram, and (c) determining the analyte carryover from the previous sample to the sample of interest based on the background height. The present invention also relates to methods, systems, and computer program products related to the aforementioned methods. [Background technology]

[0002] Carryover of analytes from sample to sample is one of the main limitations associated with liquid chromatography, but primary sample handling and sample preparation also contribute. State-of-the-art techniques for assessing the extent or risk of carryover are described by regulatory guidelines such as CLSI C62-A (Lynch (2020), Clinical Chemistry 62(1):24-299). These guidelines suggest evaluating carryover during method development as well as validation and verification stages by using blank samples after high concentration samples. The resulting peak areas of these blank samples are used to calculate potential carryover. Zeng et al. (2006), Rapid Comm Mass Spectrom 20:635 teach estimating the effect of a preceding sample on the peak area of ​​an analyte in a subsequent sample based on the concentration of the analyte in the preceding sample in an HPLC-MS system.

[0003] The assessment of carryover during method development gives a general overview about the risk of carryover and its extent. However, this approach is not suitable to provide information about the actual sample measurement. In batch mode analysis, a reanalysis of the sample following a high concentration sample is usually triggered, which is not feasible in the case of random access where the analytes of the previous sample are different from those of the actual sample. Issues to be resolved

[0004] Thus, there remains a need in the art for means and methods to aid in the detection and possible correction of carryover in LC-MS measurements. Summary of the Invention

[0005] This problem is addressed by a method, a system, a computer program product, a computer or computer network, a computer loadable data structure, a computer program and a storage medium having the features of the independent claims. Advantageous embodiments, which can be realised alone or in any combination, are set out in the dependent claims.

[0006] Accordingly, the present invention provides a method for determining analyte carryover from a previous sample to a sample of interest in a liquid chromatography mass spectrometry (LC-MS) instrument, comprising: (a) providing at least one chromatogram of the sample of interest on the LC-MS instrument; (b) determining the background height of the chromatogram; (c) determining analyte carryover from the previous sample to the sample of interest based on a background height; The present invention relates to a method comprising the steps of:

[0007] The terms used herein are broad terms and should be given their ordinary and customary meanings as assigned to them by those skilled in the art. Thus, the terms used herein are not limited to special or customized meanings unless otherwise specified. When used below, the terms "have", "comprise" or "include" or any grammatical variants thereof are used in a non-exclusive manner. Thus, these terms may refer to both the situation where no further features are present in the entity described in this context in addition to the features introduced by these terms, and the situation where one or more additional features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to the situation where, apart from B, no other elements are present in A (i.e., A is solely and exclusively composed of B), and the situation where, apart from B, one or more further elements are present in entity A, such as element C, elements C and D, and even further elements. Also, as will be appreciated by those skilled in the art, the terms "comprising a" and "comprising an" preferably refer to "comprising one or more," i.e., equivalent to "comprising at least one." Similarly, the term "determining an X" refers to determining one X, as well as determining two or more of X, such as two, three, or four X. Also, the term "plurality" refers to a number, in one embodiment at least two, in a further embodiment at least three, in a further embodiment at least four, and in a further embodiment at least five of the indicated items.

[0008] Furthermore, when used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," "more particularly," "specifically," "more specifically," or similar terms are used in conjunction with any feature without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to constrain the scope of the claims in any manner. The invention may be implemented by using alternative features, as recognized by those skilled in the art. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features without limitations on further embodiments of the invention, without limitations on the scope of the invention, and without limitations on the possibility of combining the feature introduced in such a manner with other optional or non-optional features of the invention.

[0009] As used herein, the term "about" refers to the indicated value with a generally accepted technical precision in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5% of the indicated value. Furthermore, the term "essentially" indicates that there is no deviation affecting the indicated result or use, i.e., potential deviations do not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "essentially consisting of" means including the specified components, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "essentially consisting of" encompasses any known acceptable additives, excipients, diluents, carriers, and the like. Preferably, a composition consisting essentially of a set of components contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of an unspecified component. As referred to herein, measured and calculated parameters are described on an exemplary basis. As will be appreciated by those skilled in the art, the parameters can be modified by standard mathematical operations, in particular multiplication, division, addition, subtraction, reciprocal formation, scaling, and other operations known to those skilled in the art. In one embodiment, the criteria are adjusted accordingly, in particular by applying the same mathematical operations. The measured and calculated parameters may also be used to calculate a score, which may be calculated based on one or more parameter values, which may be optionally weighted, and / or by further mathematical operations, in particular as described above, such as scaling.

[0010] The method for determining carryover is an in vitro method. Moreover, the method may include multiple steps in addition to those explicitly mentioned above. For example, further steps may relate to, for example, providing a sample for step a), or further calculations for step b) and / or c). Moreover, one or more of said steps may be performed by an automated device. In one embodiment, in particular steps b) and c) are performed by a processor, in particular a computer, which may be configured as an evaluation device, as specified elsewhere herein.

[0011] The term "providing" as used herein relates to making available the indicated information or object. Thus, when providing a chromatogram, the chromatogram can be provided as data, for example as a graphical representation, as a list of measurements, as value pairs, such as elution time / quantifier value pairs and / or elution time / qualifier value pairs, or as a mathematical model. The chromatogram can be provided via any medium deemed appropriate by the skilled artisan, in particular via an operable connection between the MS device and the evaluation device, via a data connection such as a data network, or via a data storage medium. In one embodiment, providing is determining as specified herein below.

[0012] The term "determining" is understood by those skilled in the art to refer to establishing, concluding, or confirming facts and / or data. Thus, in one embodiment, the determination relates to a qualitative determination, in a further embodiment to a semi-quantitative determination, and in a further embodiment to a quantitative determination. Thus, "determining carryover" includes establishing the fact that carryover has occurred (qualitative determination), establishing whether the degree of carryover is within acceptable limits (semi-quantitative determination), and establishing the amount of carryover (quantitative determination). In one embodiment, the determination relates to a quantitative determination. Also, "determining a chromatogram" relates to measuring a chromatogram, specifically recording and optionally storing in a suitable storage device data points representing the signal of the LC-MS device recorded over the course of an LC-MS run. As will be appreciated by those skilled in the art, when determining a chromatogram, the determination in one embodiment is quantitative. Similarly, in one embodiment, "determining background height" is quantitatively confirming the value of the background height, as specified elsewhere herein. However, the term can also relate to a semi-quantitative determination of the background height, for example by determining whether the background height exceeds a predefined exclusion criterion.

[0013] As used herein, the term "sample", also referred to as "test sample", relates to any type of composition of matter. Thus, the term can refer to any sample, such as, but not limited to, a biological sample. In one embodiment, the sample is a liquid sample, and in a further embodiment, an aqueous sample. In one embodiment, the test sample is selected from the group consisting of physiological fluids, including blood, serum, plasma, saliva, ocular lens fluid, tears, cerebrospinal fluid, sweat, urine, milk, peritoneal fluid, mucus, synovial fluid, peritoneal fluid, and amniotic fluid, lavage fluids, tissues, cells, and the like. However, the sample may also be a natural or industrial liquid, in particular surface or ground water, sewage, industrial wastewater, process fluids, soil leachates, and the like. In one embodiment, the sample contains or is suspected to contain at least one compound of interest, i.e., a chemical to be determined, referred to as an "analyte". The sample may contain one or more further chemical compounds, which are not to be determined, commonly referred to as a "matrix". The sample may be used directly as obtained from the respective 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, contact with binding and / or detection reagents, and / or any other method deemed appropriate by the skilled artisan. 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. The sample may be added with an internal standard. 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 normal operating conditions of the mass spectrometer. The concentration of the internal standard is predetermined and may be significantly higher than the concentration of the analyte.

[0014] As mentioned above, the term "analyte" as used herein relates to any compound or group of compounds to be determined in a sample. In one embodiment, the analyte is a macromolecule, i.e. a compound with a molecular mass of more than 1000u (i.e. more than 1kDa). In a further embodiment, the analyte is a biological macromolecule, in particular a polypeptide, a polynucleotide, a polysaccharide, or a fragment of any of the above. In one embodiment, the analyte is a small molecule compound, i.e. a compound with a molecular mass of up to 1000u (1kDa). In a further embodiment, the analyte is a chemical compound that is metabolized by the body of a subject, in particular a human subject, or a compound that is administered to a subject to induce a change in the subject's metabolism. Thus, in one embodiment, the analyte is a drug of abuse or a metabolite thereof, such as amphetamine, cocaine, methadone, ethyl glucuronide, ethyl sulfate, an opiate, particularly buprenorphine, 6-monoacatylmorphine, codeine, dihydrocodeine, morphine, morphine-3-glucuronide, and / or tramadol, and / or an opioid, particularly acetylfentanyl, carfentanyl, fentanyl, hydrocodone, norfentanyl, oxycodone, and / or oxymorphone.

[0015] In one embodiment, the analyte is a therapeutic agent, such as valproic acid, clonazepam, methotrexate, voriconazole, mycophenolic acid (total), mycophenolic acid-glucuronide, acetaminophen, salicylic acid, theophylline, digoxin, immunosuppressants, particularly cyclosporine, everolimus, sirolimus, and / or tacrolimus, analgesics, particularly meperidine, normeperidine, tramadol, and / or O-desmethyl-tramadol. , antibiotics, in particular gentamicin, tobramycin, amikacin, vancomycin, piperacillin (tazobactam), meropenem, and / or linezolid, antiepileptic drugs, in particular phenytoin, valporic acid, free phenytoin, free valproic acid, levetiracetam, carbamazepine, carbamazepine-10,11-epoxide, phenobarbital, primidone, gabapentin, zonisamide, lamotrigine, and / or topiramate. In one embodiment, the analyte is a hormone, in particular cortisol, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, aldosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), dihydrotestosterone, and / or cortisone. In one embodiment, the sample is a serum or plasma sample and the analytes are cortisol, DHEA-S, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, aldosterone, DHEA, dihydrotestosterone, and / or cortisone. In one embodiment, the sample is a saliva sample and the analytes are cortisol, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, androstenedione, and / or cortisone. In one embodiment, the sample is a urine sample and the analytes are cortisol, aldosterone, and / or cortisone.In one embodiment, the analyte is a vitamin, in one embodiment vitamin D, in particular ergocalciferol (vitamin D2) and / or cholecalciferol (vitamin D3) or derivatives thereof, such as 25-hydroxy-vitamin-D2, 25-hydroxy-vitamin-D3, 24,25-dihydroxy-vitamin-D2, 24,25-dihydroxy-vitamin-D3, 1,25-dihydroxy-vitamin-D2 and / or 1,25-dihydroxy-vitamin-D3. In a further embodiment, the analyte is a metabolite of the subject.

[0016] The term "carry-over" relates in principle to any transfer of at least one component from one sample to a second sample, in one embodiment, the transfer of a component from one sample to a second sample. In the context of this specification, said transfer is the transfer of a sample component from a sample processed at a first time in an analytical device, in particular an LC-MS device as specified elsewhere in this specification, to a second sample processed at a second, later time in said analytical device, in the embodiment processed at said analytical device immediately after the first sample. As will be understood, said second sample is the sample for which the carry-over is determined. At least said second sample is the sample for which the analyte is determined. Consistent with the above, said first sample is also called the "previous sample" and said second sample is also called the "sample of interest". As will be understood by the skilled person, in an LC-MS measurement, carry-over can occur before the LC step, which essentially results in an undesired increase in analyte peaks caused by analytes from the previous sample (peak carry-over). However, carryover can also occur after the LC step, causing an increase in the background in the chromatogram (background carryover). Surprisingly, as found in the studies underlying the present invention, the two types of carryover are correlated, in particular directly proportional, so that the measurement of the background carryover can be used to determine and, if necessary, correct the peak carryover.

[0017] The term "liquid chromatography mass spectrometry device", abbreviated as "LC-MS device", is understood by those skilled in the art. In one embodiment, the term relates to a device configured to perform a combination of liquid chromatography (LC) and mass spectrometry (MS). Thus, the device, in one embodiment, comprises at least one LC unit and at least one MS unit, the LC unit and the MS unit being coupled via at least one interface. As used herein, the term "liquid chromatography (LC) unit" relates to an analytical module, in one embodiment, configured to separate one or more analytes of interest of a sample from other components of the sample via liquid chromatography, in one embodiment, for detecting one or more analytes using a mass spectrometry device. The LC may be based on any separation principle considered appropriate by the skilled artisan. In one embodiment, the LC is reversed-phase chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, or chiral chromatography. In a further embodiment, the LC is reversed-phase chromatography. The LC device may comprise at least one LC column. For example, the LC device may be a single-column LC device or a multi-column LC device having multiple LC columns. The LC column may have a stationary phase through which a mobile phase is pumped to separate and / or elute and / or transport the analytes of interest. The LC unit may be or comprise at least one high performance liquid chromatography (HPLC) unit and / or at least one micro liquid chromatography (μLC) device. As used herein, the term "mass spectrometry unit" refers, in one embodiment, to a mass spectrometry device configured to detect at least one analyte based on the mass-to-charge ratio of the analyte or a fragment thereof. The mass spectrometry unit may be or comprise at least one quadrupole mass spectrometry device.The interface coupling the LC unit and the MS unit may comprise at least one ionization source configured to generate molecular ions and transfer the molecular ions to a gas phase. In one embodiment, the MS unit is a tandem mass spectrometry (MS / MS) unit, in a further embodiment a triple quadrupole MS / MS, in a further embodiment a multiple reaction monitoring (MRM) mode.

[0018] The term "chromatogram" is known to the skilled person. In one embodiment, the term relates to a correlation plot between a quantitative measure of a signal obtained from a sample and determined by an MS detector and the progress of a chromatographic separation, in one embodiment over time, for example retention time and / or elution volume. In one embodiment, said quantitative measure of a signal correlates with the concentration of at least some of the sample components, in particular the analytes. The quantitative measure of a signal can therefore in particular be a signal intensity. Thus, in one embodiment, the chromatogram is an MS chromatogram, and in a further embodiment an MS / MS chromatogram. As will be appreciated by the skilled person, said representation may be a graphical representation, although this does not necessarily have to be the case. However, the representation may for example be provided 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. In one embodiment, said quantitative measure of a signal comprises an analyte signal intensity and / or an internal standard signal intensity. In one embodiment, said quantitative measures of the signal include an analyte quantifier, an internal standard quantifier, an analyte qualifier and / or an internal standard qualifier. Thus, in one embodiment, particularly when the MS is a tandem MS, determining at least one chromatogram includes 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 elution time, as described above. As the skilled person will appreciate, elution time can be replaced by any other measure of the progress of the LC considered appropriate by the skilled person, particularly by elution volume or retention time.

[0019] The terms "quantifier" and "qualifier" are understood by the skilled artisan in the context of this specification. In one embodiment, the terms relate to the signals of fragments of an analyte generated in tandem MS, generally the most abundant and / or most reliably detected fragment is used to quantify the analyte (analyte quantifier), and the second transition (fragment) is used to confirm the identity of the analyte (analyte qualifier). The corresponding terms are often abbreviated as AQN (analyte quantifier) ​​and AQL (analyte qualifier). The values ​​of the two parameters are usually determined as the signal intensity of the ions with the corresponding m / z value, in particular as the peak area after peak integration, for example. These values ​​can be correlated with the progress of the LC to result in the chromatograms of this specification. As will be understood by the skilled artisan, the terms "qualifier" and "quantifier" and their values ​​are used in the art to refer not only to the raw values ​​measured as signal intensity, but also to the values ​​derived therefrom, in particular by peak integration. Thus, unless otherwise indicated, the terms quantifier and qualifier are used for measured signal intensities, including values ​​derived by standard mathematical operations, in particular multiplication, ratio calculation, curve smoothing, or average calculation, but not including their derived integral values, referred to as "integrated qualifier" and "integrated quantifier", respectively. Thus, in one embodiment, the values, in particular when used to calculate the quantifier / qualifier ratio, are single cycle values, i.e., intensities determined in one scan cycle, selected ion monitoring (SIM) cycle, or multiple reaction monitoring (MRM) cycle. The expression "determining the qualifier to qualifier ratio" relates to calculating the ratio by dividing the qualifier value by the quantifier value. Similarly, the expression "determining the integrated qualifier to integrated qualifier ratio" relates to calculating the ratio by dividing the integrated qualifier value by the integrated quantifier value. In one embodiment, the integrated AQN / AQL ratio is an analyte compound-specific and measurement method-specific parameter. As will be appreciated, the above applies mutatis mutandis to internal standard quantitative and qualitative measurements.

[0020] In one embodiment, step b) comprises the steps of: b1) identifying a peak in the chromatogram corresponding to the analyte; and b2) determining an upper and lower peak boundary for said peak. In a further embodiment, step b) comprises the steps of: b3) determining at least one minimum signal intensity value within the upper and lower peak boundaries.

[0021] The term "peak" as used herein refers to at least one local maximum of a chromatogram. Correspondingly, peak integration refers to at least one mathematical operation and / or mathematical algorithm for determining a peak area enclosed by a peak of a chromatogram. In particular, peak integration may include identification and / or measurement of curve characteristics of a chromatogram. Peak integration may include one or more of peak detection, peak finding, peak identification, peak fitting, peak evaluation, determination of peak start and / or peak end, background determination, and baseline determination. Peak integration may allow for the determination of one or more of peak area, retention time, peak height, and peak width. In one embodiment, peak integration is automatic peak integration, i.e., peak integration performed by at least one computer and / or computer network and / or machine. In particular, automatic peak integration may be performed without manual action or user interaction.

[0022] The term "background" is known to those skilled in the art. In one embodiment, the term relates to a signal measured by a detection device, e.g. an MS device, that is not caused by the peaks mentioned herein above. The term "background height" therefore relates to a quantitative measure of the background in a chromatogram. The term background height can therefore also relate to the value of the background height. Those skilled in the art can in principle determine the background height, e.g. by determining the offset of the chromatogram graph from the zero line, by determining the area under the baseline, etc. In one embodiment, the background height is determined as at least one minimum signal intensity value in the vicinity of the analyte peak, in particular within the upper and lower peak boundaries of the analyte peak. In a further embodiment, the background height is determined immediately upstream of the upper peak boundary and / or immediately downstream of the lower peak boundary, in which case the term "immediately" refers to up to 1 peak width upstream or downstream, in one embodiment up to 1 / 2 peak width, and in a further embodiment up to 1 / 4 peak width, respectively. In one embodiment, the background height is determined as part of a peak integration process, which is preferably performed by an automated device. In one embodiment, background height is determined as the average of values, for example, as the median, mean, or another parameter that indicates the average as deemed appropriate by the skilled artisan.In one embodiment, at least 2, in a further embodiment at least 5, and in a further embodiment at least 10 single cycle values ​​are used in said average calculation.In one embodiment, from 2 to 1000, in a further embodiment from 5 to 250, and in a further embodiment from 10 to 100, single cycle background values ​​are used in said average calculation, and the term "single cycle" refers to a single cycle of signal intensity measurement by the MS unit that generates the recorded signal.

[0023] According to step c) of the method, determining the carryover of the analyte from at least one previous sample to the sample of interest is based on the background height, i.e. the value of the background height determined in step b). Thus, in one embodiment, the background height determined as described herein above is correlated with the carryover. In one embodiment, the correlation is a quantitative relationship, in one embodiment a direct proportionality, and in a further embodiment, comprises at least one linear function, at least one quadratic function, at least one rational function, and / or at least one sigmoid function. Thus, the correlation can be represented by any mathematical, graphical, or other representation that reflects the above correlation. In one embodiment, the relationship is linear above a lower threshold of the background height. Methods for determining the aforementioned correlation and establishing a suitable representation thereof are described herein below, in particular in the examples. In one embodiment, the peak area of ​​the analyte peak is determined after step a), i.e. after establishing the specified chromatogram. In such a case, a correlation between the peak area and the background height and the corrected peak area or a value derived therefrom can be established, and the corrected analyte value can be directly established from such a three-parameter correlation.

[0024] Depending on the result of the determination of step c), as shown below, the method for determining carryover may further comprise the step of marking the result of step a) as unreliable and optionally initiating a rerun of said sample of interest if the result of step c) indicates a carryover of the analyte from said at least one previous sample above a predefined threshold. Thus, the LC-MS device may not provide a determination of the analyte, may indicate that the result of the determination is unreliable and / or may trigger a repetition of the chromatogram determination, in one embodiment, all of steps a) to c) of the method for the affected sample. In one embodiment, said repetition is performed immediately after a control sample lacking the analyte or immediately after a sample with a known low concentration of the analyte. In a further embodiment, the obtained amount or concentration determined from the chromatogram established in step a) can be corrected for the carryover of the analyte from said at least one previous sample. As mentioned above, step b) of the method provides a background height that correlates with carryover, allowing the skilled person to correct the amount or concentration of the analyte determined from the chromatogram established in step a) for the carryover that has occurred. Thus, a corrected amount or concentration of the analyte can be calculated. Exemplary methods for quantifying such correlations are described elsewhere herein.

[0025] In one embodiment, the method further comprises at least one confirmation step, which comprises determining the reliability of the background height. That is, in one embodiment, the method further comprises a step of providing an assessment of whether the increased background value is caused by carryover. As will be appreciated, the confirmation step is optional, since for quality control purposes it may be sufficient to establish carryover that likely confounds the measured value for the analyte. In one embodiment, determining the reliability of the background height comprises analyzing the quantifier signal and / or qualifier signal of the background data point in one embodiment of the background data point used to determine the background height of the chromatogram. In a further embodiment, determining the reliability of the background height comprises analyzing the quantifier signal and qualifier signal, in particular the ratio of the quantifier signal to the qualifier signal, i.e. the quantifier / qualifier ratio of the background data point in one embodiment of the background data point used to determine the background height of the chromatogram. In one embodiment, determining the confidence of the background height comprises determining a quantifier / qualifier ratio immediately upstream of the upper peak boundary and / or immediately downstream of the lower peak boundary, in an embodiment immediately upstream of the upper peak boundary, and in a further embodiment immediately downstream of the lower peak boundary, the term "immediately" having the meaning specified herein above. In one embodiment, the values, particularly when used in calculating the quantifier / qualifier ratio, are single cycle values, as described herein above.

[0026] In one embodiment, determining the reliability of the background height comprises determining a reliability parameter from the aforementioned signals and / or parameters. As a result of the increase in carryover, the determined values ​​for the analyte quantifier (AQN) and analyte qualifier (AQL) are increased in the background measurement, and it has been found that the value of the quantifier / qualifier ratio approaches the reference quantifier / qualifier ratio, said reference quantifier / qualifier ratio being analyte-specific and system-specific, i.e. assay-specific. In one embodiment, the reference quantifier / qualifier ratio is a quantifier / qualifier ratio determined in an essentially pure analyte and / or carryover-free analytical run, and in a further embodiment, the reference quantifier / qualifier ratio is an integrated quantifier / qualifier ratio determined in an essentially pure analyte and / or carryover-free analytical run. Thus, in one embodiment, the reliability of the background height determination is confirmed when the value of the quantifier / qualifier ratio approaches the reference quantifier / qualifier ratio.

[0027] It has also been found that as a result of the increased carryover, the variability of the values ​​determined for the analyte quantifier (AQN) and analyte qualifier (AQL) in the background measurements decreases, so that in an embodiment, in an embodiment specified elsewhere in this specification, the reliability of the background height determination is confirmed if the variability of the quantifier / qualifier ratio values ​​is below a criterion. Thus, in an embodiment, determining the reliability of the background height comprises determining the average of the background data points, in an embodiment the average of the quantifier / qualifier ratios, in particular the median, arithmetic mean or geometric mean, in an embodiment of the background data points used to determine the background height of the chromatogram, which can be a reliability parameter. In an embodiment, said average value is compared to a predefined criterion. In a further embodiment, a measure of the variability of the background data points is provided as a reliability parameter, in particular of the quantifier signal, the qualifier signal and / or of the quantifier / qualifier ratio. Such a measure of the variability of the background data points can be any variability parameter considered appropriate by the skilled person, in particular the coefficient of variation. However, it is also envisaged that, for example, the standard deviation or an equivalent parameter is determined.

[0028] The term "reference" as used herein refers to a discriminator that allows to evaluate the reliability of the carryover determination. Such a discriminator may be a value of a reliability parameter, e.g., the mean or variability parameter specified elsewhere herein, that indicates carryover. In one embodiment, the reference is a quantitative indicator, and in a further embodiment, a value, e.g., a threshold value, a range, e.g., a range of values ​​that indicates a reliable carryover determination, a score, or any other value or range deemed appropriate by the skilled artisan. In one embodiment, depending on the type of reference selected, the reference is an analyte-specific, device-specific, and / or method-specific parameter, and in a further embodiment, an analyte-specific parameter. Methods for establishing suitable references are exemplarily provided elsewhere herein. In view of the above, in one embodiment, the reference is determined using the same type of LC-MS device as used in the method specified herein. In one embodiment, if the quantifier signal and / or qualifier signal of background data points are analyzed, the reference is a value of a quantitative indicator of the variability of the quantifier signal and / or qualifier signal and / or the deviation of the quantifier signal and / or qualifier signal from the expected value. Thus, when the quantifier / qualifier ratio is determined, the reference can be the quantifier / qualifier ratio or the integrated quantifier / qualifier ratio determined in a pre-purified or essentially pure analyte and / or a carry-over-free analysis. A person skilled in the art can determine an appropriate reference value, for example, during assay development. Illustratively, in one embodiment, the reference is, for example, a threshold or reference range when the quantifier / qualifier ratio is determined. Thus, when the average, particularly the median, is determined, the reference can be ± up to 10 times, in a further embodiment ± up to 2 times, in a further embodiment ± up to 1.5 times, in a further embodiment ± up to 1 times, in a further embodiment ± up to 0.5 times, in a further embodiment ± up to 0.1 times the (internal) quantifier / qualifier ratio of the value determined in an essentially pure analyte and / or a carry-over-free analysis run.Thus, the reference range can be, for example, a (internal) quantifier / qualifier ratio determined for an essentially pure analyte and / or carryover-free analytical run of ±1000%, in one embodiment ±100%, in a further embodiment ±50%, in a further embodiment ±20%, in a further embodiment ±10%. In such a case, in one embodiment, a value below or corresponding to a threshold value or within the reference range indicates a reliable carryover determination. In a further exemplary embodiment, if the coefficient of variation is determined as the variation parameter, the criterion is a threshold value of up to 0.5, in one embodiment up to 0.4, in a further embodiment up to 0.3, or a mathematically corresponding value. In such a case, a value below the threshold value indicates, in one embodiment, a reliable carryover determination.

[0029] The term "quality control" as used herein is known to those skilled in the art. In one embodiment, quality control is a process of ensuring that processes performed and / or goods produced by an entity comply with predefined quality standards. In a further embodiment, quality control in sample measurements, in particular in measurements of medical samples such as patient samples, for example in clinical diagnostics and / or clinical chemistry, involves ensuring that analytical results obtained with a particular measurement method correspond to results obtained with a gold standard method, and thus, in one embodiment, correspond to results theoretically obtained within predefined ranges. Thus, in one embodiment, the method for determining carryover comprises one or more of the following further steps depending on the determination result of step c): i) if the result of step b) indicates a background height above a predefined threshold, marking the result of step a) as unreliable and, optionally, initiating a re-run of the sample of interest; ii) if the result of step c) indicates a carryover of analyte from said at least one previous sample above a predefined threshold, marking the result of step a) as unreliable and, optionally, initiating a re-run of said sample of interest; and iii) in embodiments specified elsewhere in this specification, correcting the result of step a) for carryover of analyte from said at least one previous sample.

[0030] As used herein, the term "internal standard" in one embodiment relates to an analyte present in a sample at a defined concentration. Thus, in one embodiment, the concentration of the internal standard is known. However, it is also envisaged that the concentration of the standard is unknown but is the same for at least the sample of interest and at least one calibration sample. In such a case, in one embodiment, the concentration of the internal standard is the same for all samples analyzed. The internal standard is in one embodiment structurally similar to the analyte, and in a further embodiment structurally identical to the analyte. In particular in the latter case, in one embodiment, the internal standard is an isotopically labeled molecule, in particular an isotopically labeled version of the analyte, e.g. 2 H (deuterated), 15 N and / or 13 The internal standard sample is a C labeled derivative. The internal standard sample can be a sample that contains at least one internal standard substance having a known, for example, predetermined, concentration. For further details regarding the standard sample, see, for example, EP 3 425 369 A1.

[0031] Advantageously, in the study underlying the present invention, it was found that the degree of carryover between two samples can be determined by measuring the background height as described herein, so that samples affected by an unacceptable degree of carryover can be detected by determining the background height. Furthermore, it was found that there is a proportional relationship between peak carryover and background height, and that quantitative correction of carryover is possible by determining the background height. Furthermore, it was found that background height data, particularly single cycle background data points, can be used to verify the reliability of carryover determination.

[0032] The above definitions apply mutatis mutandis below. The additional definitions and explanations further provided below also apply mutatis mutandis to all embodiments described herein. The present invention further provides a system for determining the concentration of at least one analyte in a sample, comprising: (I) at least one liquid chromatography mass spectrometry (LC-MS) device, the LC-MS device configured to measure analytes in a sample and determine at least one chromatogram; (II) at least one evaluation device configured to determine a background height of the chromatogram and to determine carryover based on the background height; The present invention relates to a system comprising:

[0033] The term "system" as used herein relates to different means operatively linked to each other. Said means may be implemented in a single physical unit or may be physically separated units operatively linked to each other. Suitable components and their properties are described elsewhere herein below and herein above in the context of a method for determining carryover. Thus, the method of the present invention can be implemented by the system specified herein. Thus, in one embodiment, the device is configured to perform a method for determining the carryover of an analyte as specified elsewhere herein, a method for performing at least one measurement as specified elsewhere herein, and / or a method for determining the relationship between the background height of an analyte and the carryover as specified elsewhere herein. The system may comprise further devices or units, in particular a data collector, an output unit, a communication interface, and / or any other device or unit considered appropriate by the skilled person.

[0034] The LC-MS apparatus and means and methods for determining at least one chromatogram are described herein above in the context of methods for determining carryover.

[0035] The term "evaluation device" generally refers to any device adapted to perform the above-mentioned method steps by using in one embodiment at least one data processing device, and in further embodiments at least one processor and / or at least one application specific integrated circuit. Thus, by way of example, at least one evaluation device may comprise at least one data processing unit in which a software code comprising several computer commands is stored. The evaluation device may provide one or more hardware elements for performing one or more of the indicated operations and / or may provide one or more processors with software to be executed for performing one or more of the method steps.

[0036] As used herein, the term "data collector" relates to any storage unit configured to store data, in particular data points determined by the LC-MS device, chromatograms, determined background heights, results of carryover determinations, and / or recommendations provided, and / or decisions taken in proceeding further with respect to the sample. In one embodiment, the data collector comprises at least one database configured to receive and / or store at least one chromatogram. In one embodiment, the data collector comprises at least one database comprising one or more reference values. In one embodiment, the data collector comprises at least one database comprising data on the correlation between background heights and carryover.

[0037] The term "output unit" as used herein relates to any unit configured for the transfer of information from the system to another entity, which may be a further data processing device and / or a user. An output unit may thus comprise a user interface, such as a suitably configured display, or may be a printer. However, an output unit may also be an indicator, e.g. an indicator lamp, for indicating the detection of a carryover.

[0038] The term "communication interface" is understood by those skilled in the art to relate to any interface configured for the exchange of information, in particular data. Such data exchange can be achieved by a permanent or temporary physical connection, such as coaxial, fiber, optical fiber or twisted pair, 10Base-T cable, storage unit connectors such as USB, Firewire, and similar connectors. Alternatively, it may be achieved by a temporary or permanent wireless connection, for example using radio waves, such as Wi-Fi, LTE, LTE Advanced, or Bluetooth.

[0039] The present invention also provides a method for performing at least one measurement of an analyte in a sample using at least one liquid chromatography mass spectrometry device, comprising: (a) performing at least one first measurement on a first sample; (b) performing at least one second measurement on a second sample; (c) determining carryover from the first sample to the second sample by a method for determining carryover according to the method for determining carryover of the present invention; The present invention relates to a method comprising the steps of:

[0040] The method for performing at least one measurement is an in vitro method. Moreover, it may include steps in addition to the specifically mentioned steps. The further steps may relate, for example, to a step of sample pretreatment before step (A), a step of quality control as specified herein above, or a step of correcting the result of the analyte measurement for carryover as specified herein above. Furthermore, one or more steps may be assisted by an automated instrument. However, it is also envisaged that the complete method is carried out in an automated manner, for example in a high-throughput analysis setting.

[0041] The term "measurement of an analyte in a sample" is understood by those skilled in the art. In one embodiment, the term relates to a qualitative, semi-quantitative or quantitative determination of the amount of an analyte in a sample, and in one embodiment, to a quantitative determination of the amount of an analyte in a sample. As used herein, the term "amount" of an analyte relates to any quantitative measure of the analyte and is equivalent to other corresponding measures such as mass fraction and concentration that can be calculated from the amount when the sample mass or sample volume is known. Thus, the measurement result of an analyte in a sample can be expressed in any unit, weight, mass fraction, concentration, or other measurements, or measurements derived therefrom, such as international units according to a given definition, as deemed appropriate by the skilled artisan. Thus, the expressions "performing at least one first measurement" and "performing at least one second measurement" relate to measuring an analyte in a first and second sample, respectively, as described above.

[0042] Methods for determining the amount of an analyte by LC-MS are known in principle to the skilled person. In one embodiment, the method comprises quantitatively determining the amount of an analyte in a sample by determining the peak area of ​​the analyte (analyte peak area). In a further embodiment, the peak area of ​​an internal standard (IS peak area) is further determined, and the ratio of the analyte peak area to the IS peak area is determined, in one embodiment, said ratio is compared to a calibration function, thereby determining a concentration value. Corresponding methods are known to the skilled person. In a further embodiment, said amount of an analyte is corrected by carrying out at least steps b) and c) of a method for determining carryover, in one embodiment by correcting the determined amount for the determined carryover by subtracting the determined carryover from the amount of the analyte determined for the second sample.

[0043] The present invention further provides a method for determining the relationship between background height and carryover of an analyte on a liquid chromatography mass spectrometry instrument, comprising: (i) performing at least one first measurement on a first sample; (ii) performing at least one second measurement on a second sample and determining at least one chromatogram thereof, said second sample not containing said analyte, which in one embodiment is a control sample, and in a further embodiment is a buffer control sample, a blank control sample, or a matrix control sample; (iii) determining the apparent amount of, or a signal corresponding thereto, of the analyte in the second sample; (iv) determining the background height of the chromatogram; (v) repeating steps (i) through (iv) with at least two additional first samples, wherein said first samples contain non-identical concentrations of said analyte; (vi) correlating the apparent amount of analyte determined in step (iii) or a signal corresponding thereto with the background level determined in step (iv); (vii) thereby determining the relationship between background height and analyte carryover; The present invention relates to a method comprising the steps of:

[0044] The method for determining the relationship is an in vitro method. Moreover, the method may include further steps not specifically mentioned. In particular, the method may include carrying out steps i) to iv) with a first sample that does not contain the analyte. Some or all steps of the method may be assisted or carried out by an automated device.

[0045] The term "apparent amount" of an analyte relates to the amount detected by the LC-MS method. Thus, the apparent amount is the amount measured for the analyte in the absence of correction for carryover.

[0046] The term "determining the relationship between background height and carryover" will be understood by those skilled in the art in light of the present disclosure. In one embodiment, the term relates to establishing a graphical and / or mathematical representation of the correlation between background height and carryover, as specified herein above and in the examples. In one embodiment, the relationship is a linear relationship, and in a further embodiment, the relationship is linear above a threshold value.

[0047] The present invention further discloses and proposes a computer program comprising computer executable instructions for carrying out the method according to the present invention in one or more of the embodiments attached hereto when the program is executed on a computer or a computer network.Specifically, the computer program can be stored in a computer readable data carrier.Therefore, specifically, one, more than one, or even all of the method steps described above can be supported or carried out by using a computer or a computer network, preferably by using a computer program.

[0048] The present invention further discloses and proposes a computer program product having program code means for carrying out the method according to the present invention in one or more embodiments contained herein when the program is executed on a computer or a computer network. In particular, the program code means can be stored on a computer readable data carrier.

[0049] Furthermore, the present invention discloses and proposes a data carrier storing a data structure which, after being loaded into a computer or a computer network, such as for example the working or main memory of a computer or a computer network, is capable of carrying out the method according to one or more of the embodiments disclosed in this specification.

[0050] The present invention further proposes and discloses a computer program product having program code means stored on a machine-readable carrier for performing the method 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 tradeable product. The product can generally be present in any format, such as a paper format, or on a computer-readable data carrier. In particular, the computer program product may be distributed over a data network.

[0051] Additionally, the present invention proposes and discloses a modulated data signal containing instructions readable by a computer system or computer network for carrying out a method according to one or more of the embodiments disclosed herein.

[0052] In one embodiment, referring to computer implementations of the present invention, one or more or all of the method steps of the method according to one or more of the embodiments disclosed herein can be performed by using a computer or a computer network. Thus, in general, any of the method steps involving providing and / or manipulating data can be performed by using a computer or a computer network. In general, these method steps can include any method steps, usually excluding method steps that require manual operations, such as providing a sample and / or certain aspects of performing the actual measurement.

[0053] Specifically, the present invention further discloses the following.

[0054] A computer or computer network comprising at least one processor, the processor being adapted to carry out a method according to one of the embodiments described in this description.

[0055] A computer-loadable data structure adapted to carry out a method according to one of the embodiments described herein while the data structure is executed on a computer.

[0056] A computer program adapted to carry out a method according to one of the embodiments described herein while the program is being run on a computer.

[0057] A computer program comprising program means for carrying out a method according to one of the embodiments described herein while said computer program is being run on a computer or on a computer network.

[0058] A computer program comprising program means according to any preceding embodiment, the program means being stored on a computer readable storage medium.

[0059] A storage medium having a data structure stored therein and adapted to perform a method according to one of the embodiments described herein after the data structure has been loaded into a main storage and / or working storage of a computer or computer network.

[0060] A computer program product having program code means, the program code means being capable of being or being stored on a storage medium for performing a method according to one of the embodiments described herein when the program code means is executed on a computer or a computer network.

[0061] In view of the above, the following embodiments are specifically contemplated.

[0062] Embodiment 1: A method for determining analyte carryover from a previous sample to a sample of interest in a liquid chromatography mass spectrometry (LC-MS) instrument, comprising: (a) providing at least one chromatogram of the sample of interest on the LC-MS instrument; (b) determining the background height of the chromatogram; (c) determining analyte carryover from the previous sample to the sample of interest based on a background height; A method comprising:

[0063] Embodiment 2: The method of embodiment 1, wherein the chromatogram is a representation of the intensity of an analyte-specific signal over elution time, and in one embodiment, a representation of the intensity of an analyte quantifier signal over elution time.

[0064] Embodiment 3: The method of embodiment 1 or 2, wherein the measuring step a) comprises determining and optionally storing in a storage medium data points representative of at least one of the quantifier signal and the qualifier signal, in one embodiment over the elution time.

[0065] Embodiment 4: The method of any one of embodiments 1 to 3, wherein step c) comprises using a predetermined relationship between the background height and a parameter that correlates with carryover.

[0066] Embodiment 5: The method of embodiment 5, wherein the relationship comprises at least one linear function, at least one quadratic function, at least one rational function, and / or at least one sigmoid function.

[0067] Embodiment 6: Step b) comprises: b1) identifying a peak in the chromatogram corresponding to an analyte; b2) determining an upper peak boundary and a lower peak boundary of said peak; 5. The method of any one of embodiments 1 to 4, comprising:

[0068] Embodiment 7: The method of embodiment 6, further comprising the step of: b3) determining at least one minimum signal intensity value within an upper peak boundary and a lower peak boundary.

[0069] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the background height is determined immediately upstream of the upper peak boundary and / or immediately downstream of the lower peak boundary.

[0070] Embodiment 9: The method of any one of embodiments 1 to 8, further comprising at least one verification step, the verification step comprising determining the reliability of the background height.

[0071] Embodiment 10: The method of embodiment 9, wherein the reliability of the background height is determined by calculating a reliability parameter and comparing said reliability parameter to a predetermined criterion.

[0072] Embodiment 11: The method of embodiment 9 or 10, wherein the reliability of the background height is determined by analyzing the quantifier and qualifier signals of background data points, preferably background data points used to determine the background height of the chromatogram.

[0073] Embodiment 12: The method of any one of embodiments 9 to 11, wherein in the confirming step, a quantifier / qualifier ratio of background data points is determined.

[0074] Embodiment 13: The method of any one of embodiments 8 to 12, wherein in the confirming step, the median of the quantifier / qualifier ratio of background data points is determined and compared to a predetermined standard.

[0075] Embodiment 14: The method according to embodiment 13, wherein the carryover determined in step c) is classified as reliable if the median quantifier / qualifier ratio differs from the value of the quantifier / qualifier ratio determined in an essentially pure analyte and / or carryover-free analysis by up to ±100%, in one embodiment by up to ±50%, in one embodiment by up to ±20%.

[0076] Embodiment 15: The method of any one of embodiments 9 to 14, wherein the confirming step comprises determining a coefficient of variation of a background data point, preferably a background data point that is used to determine the background height of the chromatogram.

[0077] Embodiment 16: The method of embodiment 15, wherein the carryover determined in step c) is classified as reliable if the coefficient of variation of the background data points is at most a predefined criterion, in one embodiment ≦0.5.

[0078] Embodiment 17: The method according to any one of embodiments 1 to 16, wherein the at least one previous sample is at least one sample measured immediately before the sample of interest on the liquid chromatography mass spectrometer, preferably the at least one previous sample is a sample measured immediately before the sample of interest on the liquid chromatography mass spectrometer.

[0079] Embodiment 18: The method according to any one of embodiments 1 to 17, wherein method steps b) and c) are performed by a computer.

[0080] Embodiment 19: The method according to any one of embodiments 1 to 17, which is or is included in a quality control method.

[0081] Embodiment 20: A system for determining the concentration of at least one analyte in a sample, comprising: (I) at least one liquid chromatography mass spectrometry (LC-MS) device, the LC-MS device configured to measure analytes in a sample and determine at least one chromatogram; (II) at least one evaluation device configured to determine a background height of the chromatogram and to determine carryover based on the background height; A system comprising:

[0082] Embodiment 21: A system according to embodiment 20, configured to carry out a method for determining carryover of an analyte according to any one of embodiments 1 to 19.

[0083] Embodiment 22: A system according to embodiment 20 or 21, wherein the evaluation device is adapted to carry out a method for determining carryover of an analyte according to any one of embodiments 1 to 9.

[0084] Embodiment 23: A method for performing at least one measurement of an analyte in a sample using at least one liquid chromatography-mass spectrometry device, comprising: (a) performing at least one first measurement on a first sample; (b) performing at least one second measurement on a second sample; (c) determining carryover from the first sample to the second sample using a method for determining carryover according to any one of embodiments 1 to 19; A method comprising:

[0085] Embodiment 24: The method of embodiment 23, wherein if a carryover of the analyte from the first sample to the second sample exceeding a predefined threshold is determined, then for the analyte in the second sample (i) no determination is provided, (ii) the result of the determination is indicated as unreliable, and / or (iii) a repeat of the measurement is initiated.

[0086] Embodiment 25: The method of embodiment 23 or 24, comprising quantifying the amount of the analyte in the second sample.

[0087] Embodiment 26: The method of any one of embodiments 23 to 25, wherein a peak area ratio between the analyte and an internal standard quantifier is determined and the amount of said analyte in the sample is determined by a calibration function.

[0088] Embodiment 27: The method according to any one of embodiments 23 to 26, wherein the amount is corrected by performing at least steps b) and c) of embodiment 1 separately and subtracting a carryover determination value determined from the quantification result of the analyte of the second sample.

[0089] Embodiment 28: A method for determining the relationship between background height and analyte carryover in a liquid chromatography mass spectrometer instrument, comprising: (i) performing at least one first measurement on a first sample; (ii) performing at least one second measurement on a second sample and determining at least one chromatogram thereof, said second sample being free of said analyte, in one embodiment being a control sample, and in a further embodiment being a buffer control sample, a blank control sample, or a matrix control sample; (iii) determining the apparent amount of, or a signal corresponding thereto, of the analyte in the second sample; (iv) determining the background height of the chromatogram; (v) repeating steps (i) through (iv) with at least two additional first samples, wherein said first samples contain non-identical concentrations of said analyte; (vi) correlating the apparent amount of analyte determined in step (iii) or a signal corresponding thereto with the background level determined in step (iv); (vii) thereby determining the relationship between background height and analyte carryover; A method comprising:

[0090] Embodiment 29: A database tangibly embedded on a data carrier comprising one or more reference values ​​for the reliability determination according to the method according to any one of embodiments 9 to 19 and / or data on the correlation between background height and carryover in the embodiments determined according to the method according to embodiment 28.

[0091] Embodiment 30: A database as described in embodiment 29 tangibly embedded on a data collector of a system as described in any one of embodiments 20 to 22.

[0092] Embodiment 31: A computer or computer network comprising at least one processor, the processor adapted to perform at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28.

[0093] Embodiment 32: A computer-loadable data structure adapted to perform at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28 while the data structure is executed on a computer.

[0094] Embodiment 33: A computer program adapted to perform at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28 while said program is running on a computer.

[0095] Embodiment 34: A computer program comprising program means for performing at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28 while the computer program is running on a computer or a computer network.

[0096] Embodiment 35: A computer program comprising the program means of embodiment 34, said program means being stored in a computer readable storage medium.

[0097] Embodiment 36: A storage medium adapted to perform at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28 after a data structure is stored in the storage medium and the data structure is loaded into a primary storage and / or working storage of a computer or computer network.

[0098] Embodiment 37: A computer program product having program code means, which may be stored or are stored on a storage medium, and which, when executed on a computer or a computer network, causes at least steps b) and c) of the method according to any one of embodiments 1 to 19 and / or at least step (vi) of the method according to embodiment 28.

[0099] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein. [Brief description of the drawings]

[0100] [Figure 1]Correlation between the Analyte Quantifier (AQN) peak area of ​​the previous sample (x-axis) and the Analyte Quantifier (AQN) peak area of ​​the blank sample (current sample, y-axis). [Diagram 2] Example of AQN mass traces showing the effect of carryover; 1: blank sample; 2: sample with medium amount of analyte; 3: blank sample; 4: sample with high amount of analyte; 5: blank sample; the arrows indicate that sample 3 was measured immediately after sample 3 and sample 5 was measured immediately after sample 4. The bottom row shows the same chromatogram as the top row but at a higher magnification (different scale on the y-axis). [Diagram 3] Correlation of two types of carryover - background height (x-axis) and AQN peak area (y-axis). [Figure 4] Single cycle ratios used as a confirmatory measure of carryover. Top: Exemplary chromatograms of signal intensity over time (AQN (upper line) and AQL (lower line), respectively); Middle: Single values ​​over time and median AQN / AQL ratios of the data points in the top row; Bottom: Coefficient of variation of the AQN / AQL ratios of the data points in the top row. [Diagram 5] Linear calibration function for testosterone (40 pg / mL-200 ng / mL) in the LC-MS method. [Figure 6] FIG. 11 is the correlation between background height and carryover peak area in testosterone measurements in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] The following examples are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the present invention. Example 1: Correlation between carryover and background height

[0102] In the experiment, LC-MS set data was generated for a high concentration sample followed by a blank sample. As shown in Figure 1, a correlation was observed between the analyte peak area of ​​the blank sample and the analyte peak area of ​​the previous sample. The slope of this function is a measure of the degree of carryover. Analyte carryover typically occurs in two forms: peaks from carryover before the LC column and a broad baseline increase (background signal) from carryover after the LC column (Figure 2).

[0103] Both types of carryover can be measured in samples that do not contain the analyte, such as blank samples, double blanks, or solvent samples, but only the background increase can be measured in actual patient samples. Therefore, the result deviation due to carryover cannot be measured directly. Unexpectedly, it was found that both types of carryover show a fairly clear correlation (Figure 3). Thus, the background signal can be used to monitor the carryover in the measured samples. Using this correlation, the calculation of the effect of carryover on the quantitative results of the patient samples can be estimated. Example 2: Confirmation of carryover measurement

[0104] In principle, background signal increase can have several causes. Typical causes are chemical noise (interference), background interference, instrumental noise, and carryover. In particular, instrumental noise is a function of ion source aging and detector aging / voltage, and therefore changes over time. To distinguish effects due to carryover from non-specific sources of background signal increase, the quantifier / quantifier ratios (single cycle ratios) of every single data point can be used to confirm this finding by comparison with a target value. Furthermore, the coefficient of variation (CV) of the single cycle ratios can also be used for confirmation (Figure 4). Example 3: Application in testosterone measurement

[0105] The concentration of testosterone in patient samples can be determined by reading the concentration values ​​of the measured analyte quantifier and internal standard quantifier peak area ratios (AQN / IQN) by the calibration function (Figure 5).

[0106] Three different samples were compared: a very high sample was measured followed by sample 1 with a very low analyte concentration, a high sample was measured followed by sample 2 with a low analyte concentration, and a very high sample was measured followed by sample 3 with a medium concentration. Using such correlations in Figure 6, measurements can be verified for carryover. The results are summarized in Table 1.

[0107] The second column shows the peak area AQN, the peak area IQN, and the AQN / IQN ratio calculated therefrom, as well as the background height determined for each sample. The third column shows the analyte concentration calculated from the AQN / IQN ratio in column 2.

[0108] The fourth column shows the apparent AQN peak area contributed by carryover, determined based on the background height and the correlation in Figure 6, as well as the contribution of carryover to the absolute concentration and the percentage of this contribution to the calculated concentration. In conclusion, in sample 1, 62% of the measured concentration was indeed caused by carryover.

[0109] The fifth column of Table 1 shows the confirmation scale, where the top row of each shows the median single cycle AQN / AQL ratio calculated for the data points just outside the peak boundaries, the given reference range, and the result of comparing each value with the reference, with OK indicating a measured value that is within the reference range, i.e. confirming carryover, and NO indicating a value that is outside the reference range, i.e. not confirming carryover. The bottom row of each shows the coefficient of variation (cv) of the single cycle AQN / AQL ratio calculated for the data points just outside the peak boundaries, the given reference range, and the result of comparing each value with the reference, OK and NO, with the meanings given above.

[0110] As shown in Table 6, carryover interference is observed in samples 1 and 3, although the interference in sample 3 is minor. In contrast, there is no obvious carryover in the measurement of sample 2. Literature: European Patent Application Publication No. 3 425 369 Lynch (2020), Clinical Chemistry 62(1):24-299 [Table 1]

Claims

1. 1. A method for determining analyte carryover from a previous sample to a subject sample in a liquid chromatography mass spectrometry (LC-MS) instrument, comprising: (a) determining at least one chromatogram of the sample of interest with the LC-MS device; (b) determining the background height of the chromatogram; (c) determining carryover of the analyte from the previous sample to the sample of interest based on the background height; A method comprising:

2. The method of claim 1 , wherein the chromatogram is a representation of the intensity of an analyte-specific signal over elution time.

3. The method of claim 2 , wherein the chromatogram is a representation of the intensity of an analyte quantifier signal over elution time.

4. 4. The method according to claim 1, wherein step c) comprises using a predetermined relationship between the background height and a parameter that correlates with the carryover.

5. Step b) is b1) identifying a peak in the chromatogram corresponding to the analyte; b2) determining an upper peak boundary and a lower peak boundary of said peak; The method of any one of claims 1 to 4, comprising:

6. The method of claim 5 , further comprising the step of: b3) determining at least one minimum signal strength value within said upper peak boundary and said lower peak boundary.

7. The method of claim 5 or 6, wherein the background height is determined immediately upstream of the upper peak boundary and / or immediately downstream of the lower peak boundary.

8. The method according to claim 1 , further comprising at least one validation step, said validation step comprising determining the reliability of said background height.

9. The method of claim 8 , wherein the reliability of the background height is determined by calculating a reliability parameter and comparing the reliability parameter to a predefined criterion.

10. The confidence in the background height is (i) by determining the average quantifier / qualifier ratio of the background data points and comparing said average to a predefined average standard; and / or (ii) determining a variability parameter of the background data points and comparing said variability parameter to a predefined variability standard; The method of claim 9, wherein the

11. 11. The method of claim 10, wherein determining an average quantifier / qualifier ratio of background data points comprises determining an average quantifier / qualifier ratio of background data points used to determine the background height of the chromatogram.

12. The method of claim 10 , wherein determining a variation parameter of a background data point comprises determining a variation parameter of a background data point used to determine the background height of the chromatogram.

13. 13. The method according to any one of claims 1 to 12, wherein the method is or is included in a quality control method.

14. 1. A system for determining a concentration of at least one analyte in a sample, comprising: (I) at least one liquid chromatography mass spectrometry (LC-MS) device, the LC-MS device configured to measure the analytes in the sample and determine at least one chromatogram; (II) at least one evaluation device configured to determine a background height of the chromatogram and to determine carryover based on the background height; Equipped with The system is configured to perform a method for determining carryover of an analyte according to any one of claims 1 to 13.

15. 15. The system of claim 14, wherein the evaluation device is adapted to carry out a method for determining carryover of an analyte according to any one of claims 1 to 13.

16. 1. A method for performing at least one measurement of an analyte in a sample using at least one liquid chromatography mass spectrometry device, comprising: (A) performing at least one first measurement on a first sample; (B) performing at least one second measurement on a second sample; and (C) determining carryover from the first sample to the second sample using a method for determining carryover according to any one of claims 1 to 13; A method comprising:

17. 17. The method of claim 16, wherein if a carryover of the analyte from the first sample to the second sample exceeding a predetermined threshold is determined, for the analyte in the second sample, (i) no determination of the amount in the second sample is provided, (ii) the result of the determination is indicated as unreliable, and / or (iii) a repeat of the measurement is initiated.

18. 18. The method of claim 16 or 17, comprising quantitatively determining the amount of the analyte in the second sample.

19. 19. The method of claim 18, wherein the amount is corrected by performing at least steps a) to c) of any one of claims 1 to 13 and subtracting the determined carryover from the quantitative result of the analyte of the second sample.

20. 1. A method for determining the relationship between background height and analyte carryover in a liquid chromatography mass spectrometry instrument, comprising: (i) performing at least one first measurement on a first sample; (ii) performing at least one second measurement on a second sample and determining at least one chromatogram thereof, said second sample being free of said analyte; (iii) determining the apparent amount of, or a signal corresponding thereto, of the analyte in the second sample; (iv) determining the background height of the chromatogram; (v) repeating steps (i) through (iv) for at least two additional first samples, wherein said first samples contain non-identical concentrations of said analyte; (vi) correlating the apparent amount of analyte or a signal corresponding thereto determined in step (iii) with the background level determined in step (iv); (vii) thereby determining the relationship between background height and analyte carryover; A method comprising:

21. A computer program having program code means, which may be or have been stored on a storage medium, causing the execution of at least steps b) and c) of the method according to any one of claims 1 to 13 and / or at least step (vi) of the method according to claim 20, when said program code means is executed on a computer or a computer network.

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