Methods for evaluating the presence of interferants in a target-substance in mass spectrometry

Abstract

The present invention relates to novel systems and methods estimating contamination of a target substance using Mass Spectrometry.

Description

[0001] International Patent Application Klinikum der Universitaet Muenchen SWK Ref. KUM18409PCT

[0002] Methods for evaluating the presence of interferants in a target-substance in mass spectrometry

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the right of priority of European patent application EP24214725 filed with the European Patent Office on 22 November 2024, the entire content of which is incorporated herein for all purposes.

[0005] De-tuning ratio analysis

[0006] The present invention relates to the use of methods to evaluate the presence of interferants in a sample comprising a target-substance in mass spectrometry, especially in liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0007] Background

[0008] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is a highly specific technology suitable for quantitative analyses of small or large molecules in complex biological samples. In LC-MS / MS, MS-chromatograms based on specific mass transitions of a target-substance are produced. In principle, the hybrid technology (chromatography for material separation plus mass spectrometry (MS) for analyte Detection) is very specific. However, in the quantification as well as in the qualitative analysis of a target-substance, interference with signals caused by further (unknown) substances (interferants) eluting at the same time may lead to falsely MS-analysis results. Therefore, the detection of the presence of potential interferants / contaminations potentially causing a change in a peak signal (e.g., intensity of one or more MS-peak(s) of a target-substance in a mass spectrometric spectrum (MS-spectrum) or peak area in an MS-chromatogram) is important (see e.g. Clin Toxicol (Phila). 2006; 44(2): 147-53 in: Allen KR. Interference by venlafaxine ingestion in the detection of tramadol by liquid chromatography linked to tandem mass spectrometry for the screening of illicit drugs in human urine; and Sauvage FL, et al. Pitfalls and prevention strategies for liquid chromatography-tandem mass spectrometry in the selected reaction-monitoring mode for drug analysis. Clin Chem. 2008 Sep;54{9}: 1519-27).

[0009] WO 2014 / 096915 describes systems and methods are provided for compound identification using multiple spectra that are a function of a variable instrument parameter that affects the intensity of fragment ions, i.e., the document describes a method in which fragments of selected ions are measured by changing the collision energy to identify a compound, but the document does neither disclose nor teach a method to evaluate the presence or absence of one or more interferants affecting quantification of a targetsubstance.

[0010] The state of the art procedure for checking peak purity of a liquid volume of a targetsubstance eluting in a peak (e.g., in liquid chromatography) has so far been checked using the “ion ratio”, which, however, can only be used if at least two mass transitions with similar ion yields can be found for the respective target-substance. With this method simultaneous chromatograms (based on two (or more) different mass transitions of each substance) are recorded. For liquid chromatography coupled tandem mass spectrometry (LC-MS / MS) spectra, the fragmentation pattern depends on the collision energy (CE) used and can vary somewhat between instruments.

[0011] Accordingly, there is a need for an additional system that goes beyond the state of the art method for checking peak purity in MS-analytic, such as in LC-MS / MS-based (bio)analytics.

[0012] This problem is addressed by the methods according to the invention in which a new assessment variable is assigned to a chromatographic peak in MS-analysis, such as LC-MS / MS analysis, the de-tuning ratio. Surprisingly, it was found the presence of interferants can be determined using a de-tuning ratio even if the target-substance does not exhibit two suitable mass transitions with similar ion yield. This is important because the ion ratio method used in the art to check peak purity can only be applied to targetsubstances with at least two similar mass transitions which does not apply to all substances. The method described herein for the first time (de-tuning ratio) can be combined with the already used ion ratio method and thus increases the safety of the analysis in terms of detection of possible interferences caused by unknown (co-eluting) substances (interferants), but it is also more sensitive than the ion ratio method and it can be used for substances having only one distinct mass transition in their MS- spectrum. Moreover, it is not necessary to know the structure or concentration of an interferant to evaluate its disturbing presence in a sample. It is also possible to de-tune more than one setting parameter in an analysis in the sense of a multi-dimensional detuning.

[0013] Figures

[0014] The skilled person will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0015] Figure 1 shows a cortisone breakdown curve (A) (in form of ion yield (given in peak area) as a function of the collision energy (CE)) with a peak maximum occurring at the CE between 35 and 40 V for the cortisone quantifier transition (361 .1 — >163.1 ). The collision energy breakdown-curve (B) of the prednisolone quantifier transition (361.1 — >147.1 ) exhibits a high degree of similarity to that of cortisone. However, prednisolone and cortisone breakdown-curves with the same mass transition (361 .1 — >163.1 ) differ (Figure 1A, 1 C). A comparison of Figures 1A and 1 C reveals a notable decline in the chromatographic peak area of cortisone at a CE = 20 V, showing a reduction of approximately 85% in comparison to the optimum at CE = 35 V. Conversely, the chromatographic peak area of prednisolone demonstrates an increase at the same mass transition at a CE = 20 V in contrast to CE = 35 V. Therefore, a parameter setting with a CE value of 20 V was defined as a de-tuned parameter setting while a parameter setting with a CE value of 35 V was defined as a tuned parameter setting.

[0016] Figure 2 shows the peak area (A1 ) based on the use of a tuned ion yield parameter set (see upper MS-chromatogram) and the peak area (A2) based on the use of a de-tuned ion yield parameter set (see lower MS-chromatogram). To evaluate the de-tuning ratio, the peak area A1 is divided by the peak area A2 resulting in the de-tuning ratio of a liquid volume which elutes from an LC-column at a retention time z and is detected via MS / MS with two specific parameter setting which differentiate by the value of (CE (tuning parameter).

[0017] Figure 3A shows a CID spectrum of cortisone with the product ion peak (1 ) at a m / z value of 163.1 which represents the most intense fragment ion, and which is used for the quantification of cortisone; and with the precursor ion peak of cortisone (2); and in Figure 3B a CID spectrum of prednisolone with a peak (3) at the m / z ratio at which also the most intense fragment ion peak of cortisone (see Figure 3A) is found. A sample with a cortisone content of 29.57 ng / ml at a spiked prednisolone level of 250 ng / ml (see Sample 5 in Figure 4) therefore showed an increased cortisone result, which is around 20.93% higher than in a sample which is not spiked with prednisolone. This means that in ignorance of the presence of prednisolone a falsely high cortisone result would have been reported.

[0018] Figure 4 discloses results for samples having the same amount of cortisone (29.57 ng / mL), but which are spiked with six different concentrations of prednisolone (0 ng / mL (not spiked, pure target-substance), 5 ng / mL, 20 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL, respectively) (x-axis). Each sample was measured 20 times with the respective parameter setting. The left y-axis shows the ion-ratio / detuning ratio; while the right y- axis shows the measured cortisone concentration. The graph with triangles shows the ion-ratio; the graph with squares shows the detuning ratio, respectively, based on the comparison of the results of the measurements at a CE of 20 V (using the de-tuned parameter setting) compared to the results of the measurements at a CE of 35 V (using the tuned parameter setting). The graph with circles shows the measured cortisone amounts at a CE of 35 V. The de-tuning ratio was determined using the MS-spectra based on the parameter setting with CE=35 V (tuned) and with CE=20 V (de-tuned), respectively.

[0019] Figure 5: Total Ion Current (TIC) MS-chromatograms of (A) cortisone + prednisolone; (B) Cortisone; and (C) prednisolone. X-axis shows time in min, y-axis shows the intensity of the signals.

[0020] Figure 6 shows a block diagram illustrating a computer system 100 which includes a bus 102 or other communication mechanism for communicating information; a processor 104 coupled with bus 102 for processing information; a memory 106 coupled to bus 102 for storing instructions to be executed by processor 104; a read only memory (ROM) 108 or other static storage device coupled to bus 102 for storing static information and instructions for processor 104; a storage device 110 coupled to bus 102 for storing information and instructions; a display 112; an input device 114; optionally another user input device 116 (e.g. a cursor control). Figure 7 shows a schematic diagram showing a system 200 for compound identification using multiple spectra that are a function of a variable instrument parameter that affects the ion yield in accordance with the embodiments of the present invention. System 200 comprises MS-device 210, processor 220, and database 230.

[0021] Figure 8 shows a O-desmethylvenlafaxine breakdown curve (9A) (in form of ion yield (given in peak area) as a function of the collision energy (CE)) with a peak maximum occurring at the CE value 40 V for the O-desmethylvenlafaxine quantifier transition (264.1 ^57.8). O-desmethylvenlafaxine and tramadol HCI (see 9B) breakdown curves (for these graphs, CXP was kept at 1V) with a similar mass transition (264.2^58.2 for tramadol HCI) differ.

[0022] Figure 9 discloses results for samples having each the same amount of O- desmethylvenlafaxine (208.7 ng / mL) with six different concentrations of cis-tramadol HCI (0 ng / mL (not spiked, pure target-substance), 5 ng / mL, 20 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL, respectively) (x-axis). Each sample was measured 20 times with the respective parameter setting. The left y-axis shows the ion-ratio / detuning ratio; while the right y-axis shows the measured O-desmethylvenlafaxine concentration. The graph with triangles shows the ion-ratio and the graph with squares shows the detuning ratio, respectively, based on the comparison of the results of the measurements at a CE of 10 V (detuned) compared to the results of the measurements at a CE of 50 V (tuned). The graph with circles shows the measured O-desmethylvenlafaxine amounts at a CE of 40 V. The de-tuning ratio was determined using the MS-spectra based on the parameter setting with CE=40 V (tuned) and with CE=10 V (de-tuned), respectively.

[0023] Figure 10(a) shows a CID spectrum of O-desmethylvenlafaxine with the product ion peak (1 ) at m / z 57.8 which represents the most intense fragment ion of O- desmethylvenlafaxine, and which interferes with the most intense fragment ion of tramadol HCI at m / z 58.1 (see peak (2) in Figure 10(b); indeed, peak (2) demonstrates the only major mass transition of tramadol HCI). Peak (1 ) is usually used for the quantification of O-desmethylvenlafaxine. The tuned parameter setting for the spectrum 10(a) was performed with a CE of 40 V. The tuned parameter setting for the spectrum 10(b) was performed with a CE of 50 V. The settings were chosen (see also the breakdown curves in Figure 8) to demonstrate which fragment patterns appear in the MS-spectra of the two components. Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings.

[0024] Definitions

[0025] Notably, all embodiments, irrespective if the embodiments are named embodiments, preferred embodiments, more preferred embodiments, most preferred embodiments or otherwise named embodiments of a method in accordance with the invention, a system in accordance with the invention, or computer implemented system in accordance with the invention, respectively, can be combined with each other unless such a combination would contravene a law of nature.

[0026] The skilled person is aware that aspects of the present invention and their various embodiments, irrespective if identified “only” as embodiments or “preferred”, “more preferred”, “even more preferred”, or “most preferred” embodiments, can be combined with each other (e.g. a preferred embodiment or part thereof can be combined with a more preferred embodiment or part thereof), unless such a combination would violate a law of nature.

[0027] The term “a” as used herein is the indefinite article and has the meaning of “one or more than one”. The meaning of “only one” is explicitly stated in form of “one” in this application.

[0028] “Collision cell exit potential” (CXP) as used herein refers to the potential applied at the exit of the collision chamber (Q2) of an MS / MS device to ensure successful ion acceleration out of Q2 and into the second quadrupole (Q3).

[0029] “Collission energy” (CE) as used herein refers to the kinetic energy transferred to ions during a collision process. In an MS / MS device, CE refers to the kinetic energy transferred to ions during a collision process within Q2 of a mass spectrometer.

[0030] “Cone Voltage” (CV) (also known as Declustering Potential) as used herein (e.g. in electrospray ionization (ESI)) refers to the potential to remove solvent molecules attached to an ion in an MS-device without fragmenting the ion. "De-tuning ratio” as used herein refers to the quotient of a tuned signal strength divided by a de-tuned signal strength (see, e.g., Figure 2).

[0031] The term “disturbs quantification” of a target-substance as used herein refers to the presence of one or more interferants which distort the measurement result of the quantification of target-substance / a target substance comprised in a sample. Based on the methods of the present invention, such interferant(s) can be detected even if its amount / concentration and / or structure is not known. The identification of the presence of at least on interferant which disturbs quantification is based on the observation whether a value of a sample comprising a target-substance differs by more than 10% from the value of the de-tuning ratio of the pure target-substance.

[0032] The term “evaluating the presence of at least one interferant” OR “evaluating the presence of one or more interferant(s) in an amount in a sample comprising a targetsubstance which amount disturbs quantification of said target-substance” which is indicated by a difference of the value of de-tuning ratio (2) (ratio of a signal strength of a chosen major peak using a tuned parameter setting vs using a de-tuned parameter setting in accordance with the present invention of a sample comprising the targetsubstance) and de-tuning ratio (1 ) (ratio of a signal strength of a chosen major peak using an tuned parameter setting vs using a de-tuned parameter setting in accordance with the present invention of a sample comprising the pure target substance (the standard) of more than 10% based on the value of de-tuning ratio (1 ).

[0033] An “interferant” as used herein refers to one or more substances which produce in an MS-spectrum a mass peak at a m / z value which peak interferes with a major peak of a target-substance in an MS-spectrum or MS-. This is especially important, if such one (or more) interferant also elutes at the same time in an MS-chromatogram as the targetsubstance and interferes with the MS-detection of the target-substance.

[0034] “Ion yield” as used herein refers to the ionization efficiency, defined as the fraction of sputtered atoms / molecules that become ionized. Ion yields vary over many orders of magnitude for the various elements / molecules. The ion yield for a method in accordance with the present invention can be determined by looking at the signal strength of a mass peak of interest (e.g. a major peak of an MS-spectrum or as defined herein). The MS- spectrum can be used as such or is part of multiple MS-spectra which form the basis for an MS-chromatogram). The signal strength depends on the choice of the parameter setting used to prepare the various MS-spectra and / or MS-chromatograms based on said MS-spectra.

[0035] The “main peak” of an MS-spectrum or MS-chromatogram, respectively, refers to the peak of said MS-spectrum or MS-chromatogram, respectively, with the highest signal strength in a given parameter setting.

[0036] The term “major peak” of a target substance in an MS-spectrum or MS-chromatogram, respectively, as used herein, refers to any peak with

[0037] - either the highest signal strength (the MS-peak based on a target-substance which has the highest intensity in an MS-spectrum or with the largest area in an MS-chromatogram (the main peak); or

[0038] - any of the peaks based on a target-substance with any one of the nine highest signal strengths of an MS-spectrum or MS-chromatogram, respectively, after the signal strength of the main peak, with the proviso that the signal strength of such a peak is at least 20% of the signal strength of the main peak of the spectrum or chromatogram, respectively.

[0039] For clarity’s sake, if an MS-spectrum or MS-chromatogram comprises more than ten peaks (including the main peak) but only four of them show a signal strength (e.g. an intensity or peak area) of 20% or more compared to the main peak in said MS-spectrum or MS-chromatogram, respectively, such a spectrum or chromatogram, respectively, would only comprise five major peaks. Thus, a person skilled in the art can easily identify any major peak of an MS-spectrum or chromatogram, respectively. The skilled person can usually even calculate due to which modification of the target-substance the existence of said peak is based on (e.g., [M]+(cation with the same mass as the molecular mass, in the following also referred to as M+), conjugates such as [M+Na]+, [M+K]+, fragments such as [M-OH]+(= M-17), [M-H2O]+(M-18), [M-CO]+(= M-28), [M- C2H4]+(M-28), [M-COH]+(M-29), etc.), M+1 (e.g., due to the presence of an isotope of a single atom being one atom mass higher than the other isotope), or M+2 peaks (e.g., due to chlorine or bromine presence). If a skilled person chooses one or more major peaks for a method according to the invention, the skilled person will use these major peaks for all measurements required for carrying out a method according to the invention.

[0040] The terms “major peak of interest” or “main peak of interest” as used herein refer to those peaks in an MS-spectrum (or MS-chromatogram which is based on MS-spectra) which are used by a skilled person to receive qualitative and / or quantitative information about a target substance using a method with an MS-device.

[0041] The term “mass spectrometry” and its abbreviation “MS” refer to the analytical technique to measure the mass-to-charge (m / z) value of ion. The results are presented as mass spectrum, a plot of intensity as a function of mass-to-charge value. In a typical MS procedure, a sample, which may be solid, liquid, or gaseous, is ionized, for example by bombarding it with a beam of electrons. This may cause some of the sample's molecules to break up into positively charged fragments or simply become positively charged without fragmenting. These ions (fragments) are then separated according to their mass- to-charge ratio, for example by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The skilled person is well aware how atoms or molecules in a sample can be identified by correlating known masses (e.g. an entire molecule) to the identified masses or through a characteristic fragmentation pattern. MS also allows a quantification of a target-molecule in a sample.

[0042] An MS-device can be used as a “stand alone” analytical technique of a sample or can be coupled with, e.g. a prior chromatographic step performed by a chromatographic (C) device (coupled C-MS-device), non-limiting examples are a liquid chromatography (LC) device such as high pressure liquid chromatography (HPLC); a gas chromatography (GC) device, a capillary electrophoresis device, or an ion mobility spectrometry device (IMS).

[0043] In case an MS-device is a coupled C-MS-device, a mass spectrometric chromatogram can be used for the presentation of MS data, where the x- axis represents retention time (the retention time of a substance in a chromatogram is the time at which the peak caused by the substance has its maximum height (i.e. , f’(x) = 0, f”(x) < 0) and and the y-axis represents signal intensity or relative signal intensity. The source data contains mass information, i.e. the results are based on MS-spectra; however, it is not graphically represented in a mass chromatogram in favor of visualizing signal intensity versus time. There are many different types of metrics that this intensity may represent, depending on what information is extracted from each mass spectrum. In an extracted-ion chromatogram (EIC or XIC or RIC, e.g. derived from LC-MS analysis), one or more m / z values representing one or more analytes of interest are recovered ('extracted') from the entire data set for a chromatographic run. The total intensity or base peak intensity within a mass tolerance window around a particular analyte's mass-to-charge ratio is plotted at every point in the analysis. The size of the mass tolerance window typically depends on the mass accuracy and mass resolution of the instrument collecting the data. An extracted-ion chromatogram is generated by separating the ions of interest from a data file containing the full mass spectrum over time after the fact; this is different from selected-ion monitoring chromatograms (SIM), in which data is collected only for specific m / z values. SIM is similar to an EIC / XIC, with the exception that the mass spectrometer is operated in SIM mode, such that only preselected m / z values are detected in the analysis. SIM experiments can be performed using mass spectrometry (MS) or tandem mass spectrometry (MS / MS) instruments. This differs from the extracted-ion chromatogram in that only data for the ion(s) of interest are collected in a SIM experiment while for extracted-ion chromatograms, data for an entire mass range are collected during the run and then examined after the completion of the run. Both techniques are suitable for carrying out a method in accordance with the present invention.

[0044] The term “MS-spectrum” as used herein refers to an MS-spectrum measured over the entire bandwidth of an MS-device used in a method in accordance with the present invention or any fraction of said bandwidth as long as this fraction encompasses the m / z value at which a major peak in accordance with the present invention is identified.

[0045] “Parameter setting” as used herein refers to the sum of all parameters which can be altered at an MS-device to receive an MS-spectrum or MS-chromatogram, respectively. The skilled person is aware in which range usually parameter values are required to receive meaningful MS-spectra or MS-chromatograms. In methods according to the invention, all parameters except one or more tuning parameters influencing ion yield are kept constant for the required measurements. The term a “peak interfering with a major peak” (of interest of a target-substance), when talking about signals (in the following, signals in an MS-spectrum are also referred to as peaks) in an MS-spectrum, refers to a peak which has a m / z ratio which is identical to the m / z ratio of a major peak of interest of a target-substance or which m / z ratio has a value which is ±1 unit of the value of the m / z ratio of a major peak of interest of a targetsubstance. When talking about peaks in an MS-chromatogram, the term refers to a peak which peak area has an overlap with the peak area of a major peak (of interest of a target-substance) of at least 1 % of the peak area of said major peak; and said MS- chromatogram peak is based on MS-spectra which comprise a peak which has a m / z ratio which is identical to the m / z ratio of a major peak of interest of a target-substance or which m / z ratio has a value which is ±1 unit of the value of the m / z ratio of a major or main peak of interest of a target-substance.

[0046] A “sample” in accordance with the present invention refers to any volume and formulation consisting of or comprising a target-substance either as a pure targetsubstance or as a target-substance to be analyzed which is comprised in a sample. Notably, a sample comprising a pure target-substance refers to a sample which may comprise the pure target-substance and further known ingredients (such as solvents) which do not exhibit MS-spectra with mass peaks having the same or similar m / z value as a major mass peak in an MS-spectrum of a target-substance. In case of doubt, this can be easily checked by a skilled person by, e.g., measuring MS-spectra of a solvent or a further additive in a sample comprising a pure target-substance.

[0047] A “sample comprising a target-substance” as used herein refers to a sample, wherein the absence or presence of an interferant has to be determined and / or wherein the amount of said target-substance in the sample is not known and has to be evaluated using an MS-device (or even a coupled C-MS-device) while clarifying whether an interferant is present in an amount in a sample comprising a target-substance which amount disturbs quantification of said target-substance.

[0048] The term “signal strength” as used herein refers to the value of a signal (e.g., peak intensity or peak area, respectively, in an MS-spectrum or MS-chromatogram, respectively), which corresponds to the ion yield of said signal. The signal strength of tuned and de-tuned peaks of MS-spectra or MS-chromatograms, respectively, are used to calculate a de-tuning ratio. For example, in case of an MS-spectrum, the signal strength is the intensity of a peak with a specific m / z ratio. In case of an MS- chromatogram, the signal strength is the area of a peak measured at a specific retention time.

[0049] “Simultaneously" as used herein refers to MS-measurements which are performed with the same sample, wherein the time difference between the measurement of a data point with one parameter setting and the measurement of a data point with a different parameter setting is 100 ns or less.

[0050] “Target-substance” as used herein refers to a substance in a sample for which the absence or presence of an interferant should be examined. The target-substance can be quantified in said sample. Usually, such a target-substance is known to a skilled person and also MS-spectra and MS-chromatograms, respectively, with the major peaks and / or fragmentation pattern, and / or retention time from a chromatographic device of said substance are known from the literature or other sources or can be prepared by the skilled person using equipment and methods known in the art. A “pure target-substance” (or standard) as used herein refers to a target-substance, which has a chemical purity of at least 95% (w / w) - and does not comprise or only comprise interferant(s) as impurity in a known amount which is not more than 5% (w / w) based on the total weight of the pure target-substance. In case a pure target-substance is used for preparing a sample of said pure target-substance (e.g. by diluting a specific amount of the pure targetsubstance with a solvent for MS-analysis (preferably LC-MS / MS analysis)), the skilled person is aware to choose a suitable solvent (or any other substance in such a sample) which does not produce a peak in an MS-spectrum with a m / z ratio which would interfere with a major peak of interest of a target-substance).

[0051] Target-substances with only “one major mass transition” as used herein refers to substances such as but not limited to tramadol which exhibit only one MS peak in an MS-spectrum or MS-chromatogram, respectively. “Only one MS peak in an MS- spectrum or MS-chromatogram, respectively,” as used herein refers to an MS spectrum or MS-chromatogram, respectively, wherein more than 98% of the sum of all signal strengths, e.g., the peak intensity or peak area in such an MS-spectrum or MS- chromatogram of such a substance, respectively, is concentrated in the main peak (see, e.g., Figure 10b). “Tuning-parameter” are device parameters which influence the ion yield (reflected in, e.g., the peak height in an MS-spectrum or the peak area in an MS-chromatogram) of a sample. Tuning parameters are, e.g. collision energy (CE) and Collision Cell Exit Potential (CXP).

[0052] “Tuned parameter setting” (also referred to herein as “optimized” or “reference” parameter setting) and “de-tuned parameter setting” as used herein are related to each other. The two settings differ from each other in the value(s) of the one or more defined tuning parameters influencing ion yield. Likewise, spectra, chromatograms or peaks of the latter two, which are measured using a tuned or de-tuned, respectively, parameter setting are characterized as tuned or de-tuned, respectively, spectra, chromatograms or peaks of the latter two.

[0053] Depending on the target-substance and the used analytical devices the skilled person defines a major peak of interest in accordance with the teaching of the present invention.

[0054] When looking at the signal strength of said major peak of interest as the function of the sum of all parameters of a device including the one or more tuning parameter(s) and wherein only the value(s) of the one or more tuning parameter(s) are varied, two of these parameter settings are chosen and identified as tuned and de-tuned parameter settings, when the quotient of the higher value of the two signal strengths of said major peak of interest divided by the lower value of the two signal strengths (de-tuning ratio) results in a value of said de-tuning ratio of 4 or more. The parameter setting resulting in the higher signal strength is defined as tuned parameter setting, the parameter setting resulting in the lower signal strength is defined as de-tuned parameter setting.

[0055] Thus, in case an MS-spectrum is used in a method according to the invention, the signal strength (intensity / height) of a major peak of interest with a specific m / z (mass divided by charge) value in an MS-spectrum at a given tuned parameter setting, wherein said intensity (height) is at least a factor four higher than the intensity (height) of said major peak at the same specific m / z value in an MS-spectrum at a given de-tuned parameter setting; in case an MS-chromatogram is used (e.g., a chromatographic coupled MS- device (e.g. LC-MS / MS)), the tuned ion yield is given by the peak area of the peak at a specific retention time in an MS-chromatogram which is based on a multitude of MS- spectra encompassing the major peak of interest at a given tuned parameter setting; likewise, the de-tuned ion yield is given by the peak area of said peak using a de-tuned parameter setting. The respective parameter settings are defined, when said peak area of the said major MS-chromatogram peak using a tuned- parameter setting is at least a factor four higher than the intensity of said major peak at the same retention time in an MS-chromatogram at a given de-tuned parameter setting. The skilled person is well- aware how to determine the area of a peak of a chromatogram.

[0056] The advantage of the methods according to the invention is the skilled person does not have to know the amount / concentration / number or chemical structure of one (or more) interferants, nor does the skilled person has to identify the signals (peaks) caused by the presence of an interferant. The skilled person only has to compare the value of the de-tuning ratio of a sample comprising a pure target substance with the value of the detuning ratio of a sample comprising said target-substance.

[0057] Summary

[0058] Aspect 1 refers to a method (method 1 ) for evaluating the presence of at least one interferant in a sample comprising a target-substance, preferably evaluating the presence of at least one interferant in said sample in an amount that disturbs quantification of said target-substance using multiple MS-spectra or MS-chromatograms of at least one ion of a pure target-substance, respectively, that are a function of at least one tuning parameter influencing ion yield, comprising: a) receive, from an MS-device at least one MS-spectrum or part thereof of a sample comprising the pure target substance; or from a coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof of a sample comprising the pure target substance using a first parameter setting comprising at least one tuning parameter influencing ion yield; b) identifying at least one major peak of interest of an ion of a pure targetsubstance having a specific m / z ratio in said at least one MS-spectrum or parts thereof comprising said major peak of interest or MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said major peak; c) determine the signal strength of said at least one major peak of interest which is based on the first parameter setting; d) receive, from said MS-device at least one MS-spectrum or part thereof comprising the at least one major peak of interest of said pure targetsubstance; or from said coupled chromatographic-MS-device at least one MS- chromatogram which is based on multiple MS-spectra or parts thereof comprising the at least one major peak of interest of said pure target substance using a second parameter setting wherein the values of all parameters are the same as in the first parameter setting except for the value of the at least one tuning parameter influencing ion yield; e) determine the signal strength of said at least one major peak of interest which is based on the second parameter setting; f) form the quotient of the higher value of the two signal strengths divided by the lower value of the two signal strength (de-tuning ratio): if the value of said de-tuning ratio is 4 or more, then the parameter setting resulting in the higher value of the signal strength of said at least one major peak of interest is defined as tuned parameter setting and the parameter setting resulting in the lower value of the signal strength of said at least one major peak of interest is defined as de-tuned parameter setting: if the value of said quotient is below 4, step a) and / or step d) are repeated with different values of the at least one tuning parameter influencing ion yield until the quotient is 4 or more; g) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the de-tuned parameter setting; h) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the tuned parameter setting; i) form the quotient of the signal strength of said at least one major peak of interest based on the tuned parameter setting divided by the signal strength of said at least one major peak of interest based on the de-tuned parameter setting; j) compare the values the de-tuning ratio of the sample comprising the pure target substance with the value of the de-tuning ratio of the sample comprising the target-substance:

[0059] (i) if the value of the de-tuning ratio of a sample comprising the target-substance differs at most 10% from the value of the de-tuning ratio of a sample comprising the pure targetsubstance, the sample is classified as fulfilling the quality measurements for quantification of the target-substance in the sample;

[0060] (ii) if the value of the de-tuning ratio of a sample comprising the target-substance is higher than 10% of the value of the de-tuning ratio of a sample comprising the pure target-substance, the sample is classified as contaminated.

[0061] Aspect 2 refers to a method (method 2) for quantifying the amount of a target-substance in a sample using multiple MS-chromatograms, respectively, that are a function of at least one tuning parameter influencing ion yield of a tuning parameter setting, comprising: Steps a) to j) according to claim 1 , wherein steps a), b), d), g), and h) are carried out using MS-chromatograms; and further comprising step k) calculate the amount of the target substance in the sample by comparing.

[0062] A preferred embodiment of method 2 refers to a method 2, wherein step k) comprises providing the linear area of a calibration curve using different amounts of pure targetsubstance in samples for preparing tuned MS-chromatograms, based on the tuned parameter setting in accordance with step f).

[0063] A preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, using a processor for at least one step. Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein the at least one tuning parameter is one tuning parameter.

[0064] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein a tuning parameter influencing ion yield is selected from the group consisting of CE and CXP.

[0065] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein a major peak in step b) is the peak with either the highest signal strength (main peak) in an MS-spectrum or MS-chromatogram, respectively, or any of the nine peaks with the highest signal strengths after the signal strength of the main peak in an MS-spectrum or MS-chromatogram, respectively, with the proviso that the signal strength of such a peak is at least 20% of the signal strength of the main peak of said spectrum or chromatogram, respectively.

[0066] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein the at least one major peak is one major peak.

[0067] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein the factor in step f) is 4.5 or more.

[0068] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein the MS-device is a time of flight (TOF) mass spectrometer, a triple quadrupole (tandem or (MS / MS)) mass spectrometer, a magnetic sector mass spectrometer, an ion mass trap mass spectrometer, an orbitrap mass spectrometer, a MS 3 mass spectrometer, a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometer, a combined TOF and quadrupole mass spectrometer (Q-TOF), an inductively coupled plasma mass spectrometer, and a direct analysis in real time mass spectrometer; more preferably MS / MS, MS 3; even more preferably MS / MS and the signal strength is the peak intensity in an MS-spectrum.

[0069] Another preferred embodiment of method 1 , 2 and their embodiments disclosed herein, refers to any of these methods, wherein the MS-device is a coupled chromatographic- MS-device, preferably a coupled liquid chromatographic (LC)-MS-device or a gas chromatographic (GC)-MS-device, an LC-MS / MS-device, an LC-MS3-device, a GC-MS- device, a GC-MS / MS-device, or a GC-MS3-device and the signal strength is the peak area in an MS-spectrogram.

[0070] Aspect 3 refers to a system for evaluating the presence of an interferant of a targetsubstance comprising: an MS-device or coupled C-MS-device that is configured to analyze a sample comprising a target-substance by measuring, preferably simultaneously, signal strengths of peaks in an MS-spectra or MS- chromatograms, respectively, each MS-spectrum or MS-chromatogram based on a specific parameter settings at the MS-device, to produce at least one tuned and at least one de-tuned MS-spectrum or MS- chromatogram, respectively, each based on the signal strength of a selected major peak of said MS-spectrum or MS-chromatogram, respectively, which are a function of at least one tuning parameter influencing ion yield; a database comprising data of at least one pure target-substance, such data include a de-tuning ratio (de-tuning ratio 1 ) of at least one tuned and at least one de-tuned MS-spectrum or MS-chromatogram, respectively, based on a major peak of interest and each based on a defined tuned and de-tuned, respectively, parameter setting; and a processor in communication with the MS-device or coupled C-MS-device, respectively and the database that is configured to: receive the at least one tuned and at least one de-tuned MS-spectrum or MS- chromatogram, respectively, for the at least one ion of a target-substance of a sample comprising said target-substance, from the MS-device, wherein the tuned and de-tuned parameter settings are the same as for evaluating detuning ratio 1 ; calculate the de-tuning ratio (de-tuning ratio 2) between the signal strength of said major peak of interest of said tuned spectrum and the signal strength of said major peak of interest of said de-tuned spectrum; compare de-tuning ratio (1 ) with said de-tuning ratio (2); if the value of the de-tuning ratio 2 of a sample comprising said targetsubstance differs at most 10% from the value of the de-tuning ratio 1 , the sample is classified as interferant-free and optionally the processor is configured to quantify the target-substance if said sample is classified as interferant-free, if the value of the de-tuning ratio 2 differs by more than 10% from the value of the de-tuning ratio 1 , classify and mark the sample is contaminated, optionally using the linear area of a calibration curve, if the difference between the value of ratio 2 and ratio 1 is 10% or less based on the value of ratio 1 .

[0071] A preferred embodiment of Aspect 3 and its embodiments refers to the system, wherein the mass spectrometer is configured to analyze the sample using tandem mass spectrometry (MS / MS or MS2), or mass spectrometry / mass spectrometry / mass spectrometry (MS3).

[0072] Another preferred embodiment of Aspect 3 and its embodiments refers to the system, wherein the target-substance is characterized in having only one intensive mass transition peak in an MS-spectrum.

[0073] A further Aspect 4 refers to a computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method according to any one of claims 1 to 11 , wherein the method comprises providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise a measurement module and an analysis module.

[0074] Detailed description of the invention

[0075] In MS spectroscopy, e.g., in the atmospheric pressure ionization (API) of mass spectrometric analysis systems, the ion yield typically depends on several device setting parameters (tuning parameters). These tuning parameters are usually optimized to achieve a maximum ion yield for a quantitative analysis of a defined target substance. With (chromatography coupled) MS-devices, distinct MS-spectra (MS-chromatograms) for a substance can be produced, preferably simultaneously, recorded using different tuning parameters. It was surprisingly found; the use of de-tuned parameter settings allows to provide qualitative information regarding purity and / or amount of a target substance in a sample. For example, the impact of a tuning parameter on the ion yield can be represented graphically in the form of breakdown curves (see, e.g., Figures 1 and 9). The ratio of the ion yield (e.g., detected as peak area in MS-chromatograms or peak intensity in MS- spectra) that occurs at fixed, different parameter settings for, preferably simultaneously, recorded MS-spectra or MS-chromatograms, respectively, can be observed (see, e.g., Figure 2). The de-tuning ratio for a specific chromatographic-mass spectrometric parameter setting is device dependent, but is surprisingly reproducible and typical on an individual device.

[0076] Thus, the invention refers in one aspect to the use of a de-tuning ratio (1 ) of a standard of a target-substance (pure target-substance) and a de-tuning ratio (2) of a sample comprising said target-substance, e.g. to quantify a target-substance in a sample via a coupled C-MS-device, while clarifying the quality of the measured quantity. Moreover, the invention also refers to the use of a de-tuning ratio (1 ) of a standard of a targetsubstance (pure target-substance) and a de-tuning ratio (2) of a sample comprising said target-substance to evaluate the presence of one or more interferant(s) of a targetsubstance in a sample via an MS-device or a coupled C-MS-device. More specifically, the invention also refers to the use of a de-tuning ratio (1 ) of a standard of a targetsubstance (pure target-substance) and a de-tuning ratio (2) of a sample comprising said target-substance to evaluate the presence of one (or more) interferant(s) in a sample, wherein the (combined) amount of interferant(s) in a sample comprising a targetsubstance which amount disturbs quantification of said target-substance. Notably, it is not required to identify the one or more interferants or quantify the one or more interferants. The methods and uses in accordance with the present invention enables the skilled person to make a statement in regard of the qualitative and, e.g., in case of coupled C-MS-devices, quantitative quality of a sample.

[0077] The invention refers in a further aspect to a method (method 1 )

[0078] A method for evaluating the presence of at least one interferant in a sample comprising a target-substance, preferably evaluating the presence of at least one interferant in said sample in an amount that disturbs quantification of said target-substance, using multiple MS-spectra or MS-chromatograms of at least one ion of a pure target-substance, respectively, that are a function of at least one tuning parameter influencing ion yield, comprising: a) receive, from an MS-device at least one MS-spectrum or part thereof of a sample comprising the pure target substance; or from a coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof of a sample comprising the pure target substance using a first parameter setting comprising at least one tuning parameter influencing ion yield; b) identifying at least one major peak of interest of an ion of a pure targetsubstance having a specific m / z ratio in said at least one MS-spectrum or parts thereof comprising said major peak of interest or MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said major peak; c) determine the signal strength of said at least one major peak of interest which is based on the first parameter setting; d) receive, from said MS-device at least one MS-spectrum or part thereof comprising the at least one major peak of interest of said pure targetsubstance; or from said coupled chromatographic-MS-device at least one MS- chromatogram which is based on multiple MS-spectra or parts thereof comprising the at least one major peak of interest of said pure target substance using a second parameter setting wherein the values of all parameters are the same as in the first parameter setting except for the value of the at least one tuning parameter influencing ion yield; e) determine the signal strength of said at least one major peak of interest which is based on the second parameter setting; f) form the quotient of the higher value of the two signal strengths divided by the lower value of the two signal strength (de-tuning ratio): if the value of said quotient is 4 or more, then the parameter setting resulting in the higher value of the signal strength of said at least one major peak of interest is defined as tuned parameter setting and the parameter setting resulting in the lower value of the signal strength of said at least one major peak of interest is defined as de-tuned parameter setting: if the value of said quotient is below 4, step a) and / or step d) are repeated with different values of the at least one tuning parameter influencing ion yield until the quotient is 4 or more; g) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the de-tuned parameter setting; h) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the tuned parameter setting; i) form the quotient of the signal strength of said at least one major peak of interest based on the tuned parameter setting divided by the signal strength of said at least one major peak of interest based on the de-tuned parameter setting; j) compare the values the de-tuning ratio of the sample comprising the pure target substance with the value of the de-tuning ratio of the sample comprising the target-substance:

[0079] (i) if the value of the de-tuning ratio of a sample comprising the targetsubstance differs at most 10% from the value of the de-tuning ratio of a sample comprising the pure target-substance, the sample is classified as fulfilling the quality measurements for quantification of the target-substance in the sample;

[0080] (ii) if the value of the de-tuning ratio of a sample comprising the targetsubstance differs more than 10% from the value of the de-tuning ratio of a sample comprising the pure target-substance, the sample is classified as contaminated.

[0081] For clarification’s sake, the information of step c) is extracted from an MS-spectrum or MS-chromatogram received in step a); while the information of step e) is extracted from an MS-spectrum or MS-chromatogram received in step d). Notably, the order of execution of steps a) (followed by step c)), step b) and step d) (followed by step e)) is arbitrary. The skilled person is well aware how to determine a major peak of interest for a (pure) target substance. The information which peak of an MS-spectrum or MS- chromatogram is suitable for carrying out this invention can either be received from an MS-spectrum or MS-chromatogram received in step a) or d), respectively, or can be received from literature or other information sources referring to the target-substance. For clarification’s sake, the value of the de-tuning ratio of a sample comprising the pure target-substance is used as basis to evaluate whether the value of the de-tuning ratio of a sample comprising the target-substance differs at most 10% or more than 10% from said value of the de-tuning ratio of a sample comprising the pure target-substance.

[0082] A further aspect refers to a method (method 2) for quantifying the amount of a targetsubstance in a sample using multiple MS-chromatograms, respectively, that are a function of at least one tuning parameter influencing ion yield of a tuning parameter setting, comprising: Steps a) to j) according to method 1 , wherein steps a), b), d), g), and h) are carried out using MS-chromatograms; and further comprising step k): calculate the amount of the target substance in the sample by comparing.

[0083] One preferred embodiment refers to method 2, wherein step k) comprises providing the linear area of a calibration curve using different amounts of pure target-substance in samples for preparing tuned MS-chromatograms, based on the tuned parameter setting in accordance with step f).

[0084] In one preferred embodiment, at least one information (.e.g., parameter setting(s), an MS-spectrum, an MS-chromatogram or a de-tuning ratio) from steps a) to i) is saved in a database (e.g. 230 in Figure 7).

[0085] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiment, using a processor for at least one step.

[0086] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiment, wherein the at least one tuning parameter is one tuning parameter. One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiment, wherein a tuning parameter influencing ion yield is selected from the group consisting of CE, CXP.

[0087] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiment, wherein a major peak in step b) is a peak with either the highest signal strength (main peak) or any of the nine highest signal strengths after the signal strength of the main peak in an MS-spectrum or MS-chromatogram, respectively, with the proviso that the signal strength of such a peak is at least 20% of the signal strength of the main peak of said spectrum or chromatogram, respectively.

[0088] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiments, wherein the at least one major peak is one major peak.

[0089] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiments, wherein the factor in step b) is 4.5 or more.

[0090] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiments, wherein the MS-device is a time of flight (TOF) mass spectrometer, a triple quadrupole (tandem or (MS / MS)) mass spectrometer, a magnetic sector mass spectrometer, an ion mass trap mass spectrometer, an orbitrap mass spectrometer, a MS 3 mass spectrometer, a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometer, a combined TOF and quadrupole mass spectrometer (Q-TOF), an inductively coupled plasma mass spectrometer, and a direct analysis in real time mass spectrometer; more preferably MS / MS, MS 3; even more preferably MS / MS and the signal strength is the peak intensity in an MS-spectrum.

[0091] One preferred embodiment refers to method 1 or method 2 and any of their preferred embodiments, wherein the MS-device is a coupled chromatographic-MS-device, preferably a coupled liquid chromatographic (LC)-MS-device or a gas chromatographic (GC)-MS-device, an LC-MS / MS-device, an LC-MS3-device, a GC-MS-device, a GC- MS / MS-device, or a GC-MS3-device and the signal strength is the peak area in an MS- spectrogram.

[0092] A further aspect refers to a system for evaluating the presence of an interferant of a target-substance comprising: an MS-device or coupled C-MS-device that is configured to analyze a sample comprising a target-substance by measuring, preferably simultaneously, signal strengths of peaks in an MS-spectra or MS- chromatograms, respectively, each MS-spectrum or MS-chromatogram based on a specific parameter settings at the MS-device, to produce at least one tuned and at least one de-tuned MS-spectrum or MS- chromatogram, respectively, each based on the signal strength of a selected major peak of said MS-spectrum or MS-chromatogram, respectively, which are a function of at least one tuning parameter influencing ion yield; a database comprising data of at least one pure target-substance, such data include a de-tuning ratio (de-tuning ratio 1 ) of at least one tuned and at least one de-tuned MS- spectrum or MS-chromatogram, respectively, based on a major peak of interest and each based on a defined tuned and de-tuned, respectively, parameter setting; and a processor in communication with the MS-device or coupled C-MS-device, respectively and the database that is configured to: receive the at least one tuned and at least one de-tuned MS-spectrum or MS- chromatogram, respectively, for the at least one ion of a target-substance of a sample comprising a target-substance, from the MS-device, wherein the tuned and de-tuned parameter settings are the same as for evaluating ratio 1 ; calculate the de-tuning ratio (de-tuning ratio 2) between the signal strength of said major peak of interest of a tuned spectrum and the signal strength of said major peak of a de-tuned spectrum; compare ratio (1 ) with a ratio (2); if the value of the de-tuning ratio 2 of a sample comprising the targetsubstance differs at most 10% from the value of the de-tuning ratio 1 of a sample comprising the pure target-substance, the sample is classified as interferant-free of and then optionally quantifying the target-substance, if the value of the de-tuning ratio 2 differs by more than 10% from the value of the de-tuning ratio 1 , classify and mark the sample is contaminated. optionally using the linear area of a calibration curve, if the difference between the value of ratio 2 and ratio 1 is 10% or less based on the value of ratio 1 .

[0093] One preferred embodiment refers to the system, wherein the mass spectrometer is configured to analyze the sample using tandem mass spectrometry (MS / MS or MS2), or mass spectrometry / mass spectrometry / mass spectrometry (MS3).

[0094] One preferred embodiment refers to the system, or any of its other preferred embodiments, wherein the target-substance is characterized in having only one intensive mass transition peak in an MS-spectrum.

[0095] A further aspect refers to a computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method according to the invention, wherein the method comprises providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise a measurement module and an analysis module.

[0096] Detailed description of methods according to the invention

[0097] As described above, trying to evaluate the purity / evaluate the presence of contaminations (one or more interferants) of a target-substance, preferably also targetsubstances or a target substance with only one intensive mass transition (e.g. Tramadol), often produce ambiguous results. This can be due to the difficulty in scoring comparisons of the acquired spectra to a library or database of stored spectra for known compounds. In addition, some tandem mass spectrometry methods, such as liquid chromatography coupled tandem mass spectrometry (LC-MS / MS), produce spectra that depend heavily on a variable instrument parameter. As a result, it is difficult to compare the results from one instrument across different instruments or laboratories.

[0098] The use of tuned vs de-tuned ratios improves the consistency of results across different instruments or laboratories. The method according to the invention using de-tuned ratios can be carried out in various ways.

[0099] In a preferred embodiment, all MS-spectra or all MS-chromatograms, respectively, in a method according to the invention are measured on the same MS-device or coupled C- MS-device, respectively. Optionally, MS-spectra or MS-chromatograms, respectively, are measured using this device and are stored to be compared with MS-spectra or MS- chromatograms, respectively, of future measurements on the same device.

[0100] In one preferred embodiment, the method for evaluating the presence of at least one interferant in a sample comprising a target-substance further comprises the quantification of a target-substance in said sample if the difference between the value of a de-tuning ratio of a sample comprising the pure target-substance and the value of a de-tuning ratio of a sample comprising a target substance is at most 10%.

[0101] In another preferred embodiment, a sample comprising the target substance is classified as fulfilling the quality measurements for quantification of the target-substance in the sample if the difference between the value of a de-tuning ratio of a sample comprising the pure target-substance and the value of a de-tuning ratio of a sample comprising a target substance is at most 9%, more preferably at most 8%, even more preferably at most 7%. Likewise, a sample comprising the target substance is classified as contaminated if the value of the de-tuning ratio of a sample comprising the targetsubstance is higher than 11 % of the value of the de-tuning ratio of a sample comprising the pure target-substance (if the threshold for a sample fulfilling the quality measurements is at most 9%); is classified as contaminated if the value of the de-tuning ratio of a sample comprising the target-substance is higher than 12% of the value of the de-tuning ratio of a sample comprising the pure target-substance (if the threshold for a sample fulfilling the quality measurements is at most 8%); is classified as contaminated if the value of the de-tuning ratio of a sample comprising the target-substance is higher than 13% of the value of the de-tuning ratio of a sample comprising the pure targetsubstance (if the threshold for a sample fulfilling the quality measurements is at most 7%),

[0102] Thus, also all embodiments and combinations of the various embodiments described herein also refer to a method for quantifying the amount of a target-substance in a sample, which methods comprises the steps for evaluating whether or whether not at least one interferant is present in a sample, which influences the signal strength of at least one major peak (i.e. the at least one major peak, which was chosen according to the methods in accordance with this invention for quantifying the amount of a targetsubstance in a sample) and quantify a target-substance based on its MS-spectra or MS- chromatograms, respectively, if no presence of an interferant is evaluated.

[0103] In another preferred embodiment, the method according to the invention is carried out on a MS-device and the evaluation is carried out using MS-spectra.

[0104] In another preferred embodiment, the method according to the invention is carried out on a coupled C-MS-device, preferably an LC-MS / MS-device and the evaluation is carried out using MS-chromatograms.

[0105] In a further preferred embodiment, the method for evaluating the presence of at least one interferant in a sample comprising a target-substance further comprises the quantification of a target-substance in said sample, further comprising the steps of providing the linear area of a calibration curve using different amounts of pure targetsubstance in samples for preparing further tuned MS-spectra or MS-chromatograms comprising the at least one major peak of interest based on the tuned parameter setting in accordance with the present invention. Optionally these information are stored in a database.

[0106] Such a method can therefore also comprise the step of calculating the amount of the target substance in the sample by comparing the signal strength of the said major peak using the tuned parameter setting with the saved information of the data referring to the amounts of the pure target-substance in a sample.

[0107] The skilled person is well aware how to prepare a calibration curve based on measurements of samples with different and known amounts of a target-substance. Preferably, the skilled person identifies the area of a calibration curve, which is essentially linear. The skilled person is ware of the fact that the amounts of a targetsubstance in which a calibration curve is linear is dependent on the target-substance, also the slope of the linear area of a calibration curve is dependent on the targetsubstance. The preparation of calibration curves and subsequent comparison of MS- data of a sample comprising said target-substance with the data used for the preparation of a calibration curve is a well-established and well-known method to quantify a targetsubstance. Moreover, a skilled person is also aware how to dilute a sample comprising a target-substance to receive signal strength results which are in the same range as the signal strength of the known amounts of a target-substance in the samples which were used to prepare the calibration curve.

[0108] MS-device-system

[0109] One preferred embodiment refers to a method, an MS-device-system and / or computer implemented system comprising an MS-device according to the invention, wherein the MS-device is a time of flight (TOF) mass spectrometer, a magnetic sector mass spectrometer, an ion mass trap mass spectrometer, an orbitrap mass spectrometer, a tandem mass spectrometer (also named MS2 or MS / MS), a triple quadrupole mass spectrometer (TQMS - a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass-resolving) radio frequency (RF)-only quadrupole between them for collision-induced dissociation), a MS3 mass spectrometer, a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometer, a combined TOF and quadrupole mass spectrometer (Q-TOF), an inductively coupled plasma mass spectrometer, and a direct analysis in real time mass spectrometer; more preferably MS / MS-, MS3- and TQMS-; even more preferably MS / MS-mass spectrometer.

[0110] In another preferred embodiment, an MS-device is coupled with one or more, preferably one, chromatographic (C) device which is / are located prior to the mass spectrometer (coupled C-MS-device), preferably, the MS-device in a coupled C-MS-device is selected from the group consisting of a TOF-, a magnetic sector-, an ion mass trap-, an orbitrap mass-, an MS2-, a TQMS-, an MS3-, an MALDI-TOF-, a Q-TOF, an inductively coupled plasma-, and a direct analysis in real time-mass spectrometer; more preferably an MS2- , an MS3- and a TQMS-; even more preferably an MS2-mass spectrometer.

[0111] In another preferred embodiment, the chromatographic device of a coupled C-MS- device is selected from the group consisting of a liquid chromatography device (LC) such as high pressure liquid chromatography (HPLC); a gas chromatography device (GC), a capillary electrophoresis device, or an ion mobility spectrometry device (IMS), more preferably LC or GC. In one preferred embodiment, a coupled MS device of a method, an MS-device-system and / or computer implemented system comprising an MS-device according to the invention, is an LC-MS / MS-device.

[0112] In one preferred embodiment, a coupled MS-device suitable for carrying out a method according to the invention, an MS-device-system and / or computer implemented system as described herein can simultaneously measure two or more, such as five, five or more, ten, ten or more up to 20, up to 50 or up to 100 (or even more) MS-spectra or MS- chromatograms.

[0113] In one preferred embodiment, an MS-chromatogram is generated by separating the data of ions of interest (i.e. being responsible for one or more major peaks in an MS-spectrum) from a data file containing the full MS-spectrum.

[0114] In another preferred embodiment, an MS-chromatogram is generated by collecting data only for specific m / z values.

[0115] In yet another preferred embodiment, an MS-chromatogram is generated by an MS- device in SIM mode in that only preselected m / z values are detected in the analysis.

[0116] In yet another preferred embodiment, SIM is performed using a mass spectrometry device (MS).

[0117] In yet another preferred embodiment, SIM is performed using a tandem mass spectrometry device (MS / MS).

[0118] Notably, all ratios which are calculated in a method according to the present invention are based on measurements from the same MS-device.

[0119] Simultaneously

[0120] In one preferred embodiment, a coupled MS device of a method, an MS-device-system and / or computer implemented system can simultaneously measure two or more MS- spectra or MS-chromatograms, Wherein the time period between two measurements is 100 ns or less , more preferably 20 ns or less, such as 10 ns or less or 8 ns or less, or even more preferably 5 ns or less, such as 2 ns or less or even 1 ns or less. Pure target-substance

[0121] A “pure target-substance” (a standard) as used herein refers to a substance, which has a chemical purity of at least 95% (w / w) when directly measured with an MS-device or used for the preparation of a sample comprising said pure target-substance. In the latter case, the skilled person is aware such a sample will contain only further substances (such as solvents) which MS-peaks do not interfere with major MS-peaks of a target substance in an MS-spectrum or an MS-chromatogram, respectively (i.e. such solvents are no interferants). Notably, a suitable solvent does not produce a peak in an MS- spectrum with the same m / z ratio or a m / z ratio with a value ±1 unit of the value of the m / z ratio as a major peak of interest of a target-substance.

[0122] Most preferably, a pure target-substance does not comprise interferants which would produce one or more MS-peaks which have, e.g., the same m / z ratio or a m / z ratio with a value ±1 unit of the value of the m / z ratio of a major peak of interest of a targetsubstance as a major peak of the pure target-substance and would, thus, interfere with the said major peak of the target-substance).

[0123] A further preferred embodiment refers to a pure target-substance having as chemical purity of at least 98% (w / w), more preferably 99% (w / w).

[0124] Thus, the term “pure” target-substance refers to a substance which does not or comprise only minor amounts of other substances which could interfere with major MS-peaks of the target substance by producing MS-peaks with the same m / z value as MS-peaks of a target-substance, but a sample comprising a pure target substance may comprise further compounds (such as solvents) which do not exhibit MS-spectra with MS-peaks haven the same or similar (±1 unit) m / z value as major peaks of a target-substance or which do not exhibit the same or similar elution times from a chromatographic devise so the elution peaks could interfere with the elution peak of a target substance and whereof the resulting MS-spectra comprise MS-peaks haven the same or similar (±1 unit) m / z value as major peaks of a target-substance.

[0125] Target-substance

[0126] A target-substance is any substance if interest which can be subject of an MS-analysis (i.e., providing cationic ions in an MS-device). One preferred embodiment refers to a method according to the invention, wherein the target-substance is a compound which structure comprises at least one covalent carbon hydrogen bond.

[0127] In one preferred embodiment, a target-compound is a pharmaceutical drug, a (poly)carbohydrate, a steroid, a peptide, a protein, a metal protein, a metal, inorganic metal compounds.

[0128] Preferred target-compounds are identical with the preferred pure target-compounds listed herein.

[0129] In one preferred embodiment, a method in accordance with the present invention is carried out with a target substance having only one distinct mass transition (i.e. only one major peak as defined herein).

[0130] In one preferred embodiment, a method in accordance with the present invention is carried out with an interferant having only one distinct mass transition (i.e. only one major peak as defined herein).

[0131] • In one preferred embodiment, a target substance is selected from the group consisting of tramadol (2-[(dimethylamino)methyl]-1 -(3- methoxyphenyl)cyclohexan-1 -ol e.g. in form of tramadol HCI), Cortisone (17- hydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-1 ,2, 6, 7, 8, 9, 12,14,15,16- decahydrocyclopenta[a]phenanthrene-3, 11 -dione), prednisolone (11 ,17- dihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-7,8,9, 11 , 12, 14, 15, 16-octahydro- 6 / - / -cyclopenta[a]phenanthren-3-one), O-desmethylvenlafaxine (4-[(1 S)-2- (dimethylamino)-l -(1 -hydroxycyclohexyl)ethyl]phenol), morphine (3-methyl- 2,4,4a,7,7a,13-hexahydro-1 / - / -4,12-methanobenzofuro[3,2-e]isoquinoline-7,9- diol), hydromorphone ((4R,4aR,7aR,12bS)-9-hydroxy-3-methyl- 1 ,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinolin-7-one), nicotine (1 -methylpyrrolidin-2-yl]pyridine), anabasine (3-[-piperidin-2-yl]pyridine), norcotinine (5-pyridin-3-ylpyrrolidin-2-one), cortisol (11 (3, 17a, 21 - Trihydroxypregn-4-ene-3, 20-dione), and fenofibrate (propan-2-yl 2-[4-(4- chlorobenzoyl)phenoxy]-2-methylpropanoate). In another preferred embodiment, the target-substance is cortisone and at least one interferant is prednisolone.

[0132] In yet another preferred embodiment, the target-substance is prednisolone and at least one interferant is cortisone.

[0133] In yet another preferred embodiment, the target-substance is tramadol and at least one interferant is O-Desmethylvenlafaxine.

[0134] In yet another preferred embodiment, the target-substance is O-Desmethylvenlafaxine and at least one interferant is tramadol.

[0135] In yet another preferred embodiment, the target-substance is morphine and at least one interferant is hydromorphone.

[0136] In yet another preferred embodiment, the target-substance is hydromorphone and at least one interferant is morphine.

[0137] In yet another preferred embodiment, the target-substance is nicotine and at least one interferant is selected from the group consisting of anabasine, norcotinine, and a combination of anabasine and norcotinine.

[0138] In yet another preferred embodiment, the target-substance is anabasine and at least one interferant is norcotinine, nicotine, and a combination of anabasine and norcotinine.

[0139] In yet another preferred embodiment, the target-substance is norcotinine and at least one interferant is nicotine, anabasine, and a combination of nicotine and abanasine.

[0140] In another preferred embodiment, the target-substance is cortisol and at least one interferant is fenofibrate.

[0141] In another preferred embodiment, the target-substance is fenofibrate and at least one interferant is cortisol. Tuning parameters

[0142] The skilled person is aware how to optimize parameter settings on an MS-device or coupled C-MS-devise, respectively, to receive MS-spectra or MS-chromatograms with a high ion yield of a (pure) target-compound (i.e. , a reasonable and high signal strength of the peaks of a target substance in its MS-spectra or MS-chromatograms.

[0143] In the course of the de-tuning according to the invention, tuning parameters influencing the ion yield (e.g., CE, CXP, or CV), are adjusted on an MS device, preferably a coupled chromatography (for material separation) and MS (for analyzation), more preferably an LC-MS / MS device, in such a way that they lead to a de-tuned ion yield (see, e.g., breakdown corves in figure 1 ).

[0144] The de-tuning ratio for a specific chromatographic-mass spectrometric setting (recorded for, e.g., a chromatographic peak) is device dependent, but surprisingly reproducible and typical for a target-substance on said device when using the same parameter setting.

[0145] Surprisingly, these different ratios can be used to check whether in a chromatographic peak only the target-substance elutes, or whether an unidentified, unknown substance co-elutes which interferes with the signal recorded to quantify a target-substance in an MS-chromatogram. In this latter case, a deviation of the observed de-tuning ratio (2) from the tuning ratio (1 ), recorded for a pure peak (i.e. a target-substance without an interferant), occurs.

[0146] In accordance with the methods according to the present invention, the skilled person can, after choosing a tuning parameter setting resulting in a reasonable and high ion yield which procedure is well-established practice in the art (resulting in an tuned MS- spectrum or MS-chromatogram, respectively), identify (chose) one or more major peaks of such an MS-spectrum or MS-chromatogram, respectively (i.e. either the main peak (the peak with the highest signal strength in the spectrum or chromatogram) and / or a peak having a signal strength of at least 20% of the signal strength of the main peak) as a reference peak (a major peak of interest in accordance with the present invention), alter one (or more) tuning parameter(s) of said tuned parameter setting, preferably selected from the group consisting of CE, CXP, and CP, more preferably CE and CXP, even more preferably CE, to receive at least one MS-spectrum or MS-chromatogram having a different value of the signal strength of said major peak of interest, wherein the quotient (de-tuning ratio) of the higher value of the signal strength of said major peak(s) of interest (nominated as tuned signal strength) divided by the lower value of the signal strength of said major peak of interest (nominated de-tuned) is 4 or more, more preferably 5 or more (see, e.g., Fig. 4 showing a de-tuning ratio of 6.7). Depending on the (pure) target-substance and the choice of the value of a tuning parameter, such a de-tuning ratio can also range up to 50 or more (see, e.g. Fig. 8 showing a de-tuning ratio of 52),

[0147] In one preferred embodiment of a method according to the invention, the MS-spectra or MS-chromatograms, respectively, are a function of one, two or three tuning parameters influencing ion yield, preferably selected from the group consisting of CE, CXP and CP.

[0148] In a more preferred embodiment of a method according to the invention, the MS-spectra or MS-chromatograms, respectively, are a function of one or two tuning parameters influencing ion yield, preferably selected from the group consisting of CE, CXP and CP, more preferably selected from the group consisting of CE, and CXP.

[0149] In another more preferred embodiment of a method according to the invention, the MS- spectra or MS-chromatograms, respectively, are a function of one tuning parameter influencing ion yield, preferably selected from the group consisting of CE, CXP and CP, more preferably selected from the group consisting of CE and CXP, most preferably the tuning parameter is CE.

[0150] More preferably, all other tuning parameters of an MS-device or coupled C-MS-device of a tuning parameter setting (i.e. the sum of all tuning parameters of a device) are kept constant, except the one, two or three tuning parameters influencing ion yield, more preferably the one or two tuning parameters influencing ion yield, even more preferably the one tuning parameter influencing ion yield.

[0151] In a preferred embodiment of a method according to the invention, the at least one tuning parameter influencing ion yield is selected from the group consisting of CE, CXP and CP, more preferably selected from the group consisting of CE and CXP, most preferably the at least one tuning parameter is CE.

[0152] In a preferred embodiment of a method according to the invention, the MS-spectra or MS-chromatograms, respectively, are a function of one tuning parameter influencing ion yield selected from the group consisting of CE and CXP. In other words, only one tuning parameter at a devise is altered, more preferably the one tuning parameter is selected from the group consisting of CE and CXP, to determine a tuned and de-tuned MS- spectrum as used in a method according to the invention.

[0153] The ratio of the changes of ion yield of a sample comprising a target-substance with (a sample comprising a) pure target-substance as a function of one (or more) tuning parameter(s) can be monitored in a variety of different ways. For example, ion yield (e.g. plotted in form of intensity of an MS-peak in an MS-spectrum or peak area of a peak of an MS-chromatogram) versus a tuning parameter can be plotted for every product ion. The shape of these resulting breakdown curves can be compared or measured against stored data or simultaneously acquired data. In another embodiment, the acquired data can be converted into an image and / or stored.

[0154] Although the examples herein describe a method, an MS-device-system and a computer implemented system comprising an MS-device that identify a compound from tandem mass spectrometer data by using the de-tuning ratios according to the invention, various embodiments are of course not limited to tandem mass spectrometry or CE, CXP and / or CP as tuning parameter.

[0155] In one preferred embodiment, a, preferably the only, tuning parameter is CE and the value of CE to produce tuned and a de-tuned MS-spectrum is in the range from 1 V to 200 V, preferably in the range from 5 V to 180 V.

[0156] In another preferred embodiment, a, preferably the only, tuning parameter is CXP and the value of CXP to produce tuned and a de-tuned MS-spectra is in the range from 0 V to 100 V, preferably in the range from 0 V to 75 V, such as in the range from 0 V to 55 V or 1 V to 55 V.

[0157] In another preferred embodiment, a, preferably the only, tuning parameter is CP and the value of CP to produce tuned and a de-tuned MS-spectra is in the range from 0 V to 200V, such as from 0 V to 150 V. Identifying a major peak

[0158] Major peak(s) and MS-spectra

[0159] Identifying at least one major peak means: after a peak in an MS-spectrum, which is based on a (preferably pure) target-substance or fragment thereof, having a specific m / z value is identified as a major peak in accordance with the methods of the present invention, this peak and the corresponding peak with said m / z value but measured with different parameter settings are then used for preparing an MS-chromatogram (in case of a coupled C-MS-device) and / or for calculating the various tuning and de-tuning ratios in accordance with the methods of the invention.

[0160] Notably, when using an MS-spectrum (or a part thereof) in a method in accordance with the present invention, this MS-spectrum or this part thereof has to comprise the major peak of interest, i.e. encompass the m / z value at which the major peak is detected. For example, the major peak of interest for O-Desmethylvenlafaxine has a m / z value of 57.8. Thus, any MS-spectrum or part thereof used in a method according to the invention encompasses the m / z value of 57.8.

[0161] In one embodiment, an MS-spectrum encompasses m / z values from 0 to 2500, in another embodiment, an MS-spectrum encompasses m / z values from 5 to 2000, in yet another embodiment, an MS-spectrum encompasses m / z values from 10 to 1800, such as from 15 to 1800, 20 to 1500, or 30 to 1500.

[0162] In another preferred embodiment, an MS-spectrum encompasses the m / z value of a major peak of interest and the m / z values between 10 and said m / z value on the one hand; and at least the m / z value of said major peak of interest+5, more preferably at least the m / z value of said major peak of interest +10, even more preferably at least the m / z value of said major peak of interest +20, such as m / z values up to said m / z value of a major peak of interest+30, +50, +100, +200 or the m / z values up to the upper limit of the measuring range of the MS-device, on the other hand. For example, for the major peak of interest for O-Desmethylvenlafaxine having a m / z value of 57.8, an MS-spectrum having the range (or part of an MS-spectrum having the range) from 10 to 67.8 (the m / z value of 57.8+10) is used in a method in accordance with the present invention. The skilled person understands for evaluating a major peak of interest, it may be advantageous to prepare an MS-spectrum over the full bandwidth of the measuring breadth of an MS-device, but such a range can of course also be limited, e.g., if the major peak of interest of a target-substance is already known from the prior art; or after the major peak of interest and its m / z value is identified. In another preferred embodiment, an MS-spectrum used in a method in accordance with the present invention encompasses the m / z value of a major peak of interest and the values ±5 of said m / z value, more preferably the m / z value of a major peak of interest and the values ±10 of said m / z value, such as ±20 of said m / z value, ±50 of said m / z value or ±100 of said m / z value. Notably, the skilled person is aware the lower limit of the m / z values cannot be lower than 0. Moreover, the skilled person also understands any m / z value of such an MS-spectrum cannot be higher than the upper limit of the measuring width of the MS-device used in a method in accordance with the present invention.

[0163] The MS-spectrum of a pure target-substance in accordance with the present invention is usually known to the skilled person, i.e. the skilled person is aware which peaks in an MS-spectrum of a pure target-substance results from, e.g., the M+ ion (typically the peak with the highest intensity out of the peaks in the higher m / z value cluster of a targetsubstance), a conjugate or a fragmentation of the target-substance. The skilled person is aware fragmentation of a target-substance may lead to various fragments (which can again fragment), depending on the kind of bond cleavage pattern (for example but not limited to sigma bond cleavage, radical site-initiated fragmentation, charge site-initiated cleavage and rearrangement reactions). Moreover, also conjugates of ion fragments of a target-substance are well-known in mass spectrometry.

[0164] In one preferred embodiment, an identified major peak is identified from an MS-spectrum of a pure-target substance.

[0165] Notably, all identified major peaks of an MS-spectrum used for a method in accordance with the invention have to be a result of the target-substance and not from any other substance in a sample, e.g., with the pure target-substance, i.e., the identified major peaks result from the M+ ion, a conjugate or fragment of said target-substance. Thus, an MS-spectrum of a target-substance only refers to those peaks which originate from the target substance (preferably the pure-target substance) and not from any other substance (in a sample). Thus, one preferred embodiment refers to a method, wherein, step b) identifying at least one at least one major peak in an MS-spectrum or MS-chromatogram (which is based on MS-spectra or specific parts of MS-spectra), respectively, refers to identifying at least one major peak with a specific m / z value in an MS-spectrum or an MS-spectrum of an MS-chromatogram, respectively, preferably the tuned MS-spectrum, and use said at least one major peak with said specific m / z value and its corresponding peaks in other MS-spectra with this m / z value for all further evaluations, wherein said at least one major peak of the target-substance has either the largest signal strength (e.g., the highest intensity = the main peak) in said MS-spectrum or is a peak of the target-substance having a signal strength in said MS-spectrum of at least 20% of the signal strength of the main peak of the said target-substance (preferably the pure-target substance).

[0166] In one preferred embodiment, a method in accordance with the invention is carried out using the signal strengths of three identified major peaks for the evaluation, more preferably with the signal strengths of two identified major peaks for the evaluation, even more preferably with the signal strength of one identified major peak for the evaluation.

[0167] In one preferred embodiment, an identified major peak is an MS-peak of an MS- spectrum of the target-substance and the signal strength of said peak is the intensity (height of the MS-peak in the MS-spectrum).

[0168] In another preferred embodiment, an identified major peak is the main MS-peak of the target-substance.

[0169] In one more preferred embodiment, a method according to the invention is carried out with one identified major peak and said identified major peak is the main peak of an MS- spectrum of the target-substance (preferably the pure-target substance).

[0170] In yet another preferred embodiment, a method according to the invention is carried out with one identified major peak and said identified major peak is a peak having an intensity of at least 20% of the intensity of the main peak of an MS-spectrum of the target-substance (preferably the pure-target substance).

[0171] In another preferred embodiment, a method in accordance with the invention is carried out using the signal strengths of one identified major peak and the identified major peak is the main MS-peak of the target-substance with the highest intensity of the MS- spectrum of the target-substance (preferably the pure-target substance).

[0172] Thus, in another preferred embodiment, a major peak is the main MS-peak of the targetsubstance with the highest intensity of the MS-spectrum of the target-substance (preferably the pure-target substance) resulting from the M+ ion.

[0173] In another preferred embodiment, an identified major peak is an MS-peak having an intensity of at least 20% of the intensity of the main peak, more preferably at least 50% of the intensity of the main peak, even more preferably at least 70% of the intensity of the main peak, of the MS-spectrum of the target-substance (preferably the pure-target substance).

[0174] Main Peak(s) and MS-chromatograms

[0175] In yet another preferred embodiment, a major peak is an MS-peak with a specific m / z value of multiple MS-spectra of the target-substance which signal strength is used to prepare an MS-chromatogram (preferably resulting from an LC-MS / MS device) of the target-substance. In an MS-chromatogram, the signal strength is the peak area of the eluted volume comprising the detected ion of the target-substance, fragment or conjugate, thereof.

[0176] Notably, a skilled person is aware how to calculate the peak area of a peak of an MS- chromatogram. The area of a peak of an MS-chromatogram is proportional to the ion yield of the MS-signal, which was used to prepare this chromatogram, e.g., a M+ ion MS-signal, a conjugate MS-signal or fragment MS-signal of said target-substance.

[0177] For example, the area of a main peak can be calculated by treating the peak as a triangle. The area of a triangle is calculated by multiplying the height of the peak times its width at half height. Alternatively, peak areas can also be calculated by commercially available software programs.

[0178] Thus, in one embodiment, an MS-chromatogram is based on the MS-signal of a major peak of the underlying MS-spectra, and the major peak is the main peak.

[0179] In one preferred embodiment, a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of three identified major peaks of the underlying MS-spectra for the evaluation, more preferably with the signal strengths of two identified major peaks of the underlying MS-spectra for the evaluation, even more preferably with the signal strengths of one identified major peak of the underlying MS-spectra for the evaluation.

[0180] In another preferred embodiment, a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of an identified major peak being an MS-peak of an MS-spectrum of the target-substance and the signal strength of said peak is the intensity (height of the MS-peak in the MS- spectrum).

[0181] In another preferred embodiment, a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of an identified major peak being the main MS-peak of the target-substance with the highest intensity of the MS-spectrum of the target-substance (preferably the pure-target substance).

[0182] In one more preferred embodiment, a method according to the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of one identified major peak and said identified major peak is the main peak of an MS-spectrum of the target-substance (preferably the pure-target substance).

[0183] In yet another preferred embodiment, a method according to the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of one identified major peak and said identified major peak is a peak having an intensity of at least 20% of the intensity of the main peak of an MS-spectrum of the target-substance (preferably the pure-target substance).

[0184] In another preferred embodiment, a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of one identified major peak and the identified major peak is the main MS-peak of the target-substance with the highest intensity of the MS-spectrum of the target-substance (preferably the pure-target substance).

[0185] Thus, in another preferred embodiment, a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of one identified major peak and the identified major peak is the main MS- peak of the target-substance with the highest intensity of the MS-spectrum of the targetsubstance (preferably the pure-target substance) resulting from the M+ ion.

[0186] In another preferred embodiment a method in accordance with the invention is carried out by comparing MS-chromatograms which are based on the MS-signal strengths of one identified major peak and the identified major peak is an MS-peak having an intensity of at least 20% of the intensity of the main peak, more preferably at least 50% of the intensity of the main peak, even more preferably at least 70% of the intensity of the main peak, of the MS-spectrum of the target-substance (preferably the pure-target substance).

[0187] Identifying of tuned and de-tuned MS-spectrum / MS-chromatogram pairs

[0188] The parameter setting used for the tuned and de-tuned MS-spectra or MS- chromatograms, respectively (which are selected based on the signal strength of identified major peak(s)) as explained above), are called the tuned parameter setting and the de-tuned parameter setting, respectively.

[0189] Thus, one preferred embodiment, refers to a method according to the invention, wherein all parameters are kept constant for all measurements required to carry out the method according to the invention except of one tuning parameter influencing ion yield, which is modified to produce at least one tuned and at least one de-tuned MS-spectrum or MS- chromatogram, respectively.

[0190] Another preferred embodiment refers to a method according to the invention, wherein all parameters are kept constant for all measurements required to carry out the method according to the invention except of two tuning parameters influencing ion yield, which are modified to produce at least one tuned and one de-tuned MS-spectrum or MS- chromatogram, respectively.

[0191] Another preferred embodiment refers to a method according to the invention, wherein a combination of one tuned and one de-tuned parameter setting is chosen from the measurements of a pure target-substance (or sample comprising a pure targetsubstance) which result in a tuned and a de-tuned MS-spectrum or MS-chromatogram, respectively, of a pure target-substance, and further use at least these two settings for the measurements of a sample comprising a target-substance which result in one or more tuned and one or more de-tuned MS- spectra or MS-chromatograms, respectively, of a target-substance.

[0192] The skilled person will understand that for identifying the correct combination of a tuned and a de-tuned parameter setting, a plurality of parameter settings may be used to prepare respective MS-spectra and / or chromatograms to then choose a combination of a tuned and one or more, preferably one, de-tuned parameter setting in accordance with the present invention.

[0193] A further preferred embodiment refers to a method according to the invention, wherein a combination of one tuned parameter setting (resulting in at least one tuned MS- spectrum or MS-chromatogram, respectively, of a pure target-substance) and one detuned parameter setting (resulting in at least one de-tuned MS-spectrum or MS- chromatogram, respectively, of a pure target-substance) is chosen for further use in accordance with a method according to the invention, wherein the selection is based on the signal strength(s) of one or more, preferably one, identified major peak(s) of an MS- spectrum; or the signal strength of a peak of an MS-chromatogram which is based on the MS-signal strengths of one or more, preferably one, identified major peak(s) of a plurality of MS-spectra; using one of the two parameter settings resulting in a higher ion yield (the tuned parameter setting), compared to the signal strength(s) of one or more, preferably one, identified major peak(s) of an MS-spectrum; or the signal strength of a peak of an MS-chromatogram which is based on the MS-signal (MS-peak) of one or more, preferably one, identified major peak(s) of a plurality of MS-spectra using the other one of the two parameter settings resulting in a lower ion yield (the de-tuned parameter setting), wherein the value of the tuned signal strength(s) of a major peak of interest in the MS-spectrum or in an respective MS-chromatogram, respectively, resulting from the use of the one of the two parameter settings resulting in a higher ion yield (the tuned parameter setting) has to be at least a factor 4 higher than the signal strength of said peak of interest in the de-tuned MS-spectrum or de-tuned MS-chromatogram.

[0194] In one preferred embodiment, in case an interferant is known to the skilled person, the value of the at least one tuning parameter (de-tuned tuning parameter) of the parameter setting resulting in the lower ion yield of a major peak of interest of a pure target- substance in a breakdown curve (plotting ion yield (y-axis) vs value of tuning parameter (x-axis)) is chosen

[0195] - if the ion yield of the major peak of interest of said pure target-substance (e.g., cortisone) is at least four times lower than the value of the ion yield of said major peak of interest of said pure target-substance using a parameter setting, wherein a different value of said at least one tuning parameter is used (tuned tuning parameter); and

[0196] - if the ion yield value of the breakdown curve of the interferant at the value of the de-tuned tuning parameter is 5% or more, more preferably 7% or more, even more preferably 8% or more, of the ion yield value of its breakdown curve at the value of the tuning parameter which corresponds to the value of the tuned tuning parameter of the target-substance; and the ion yield value of the breakdown curve of the pure target-substance at the value of the de-tuned tuning parameter compared is 0,1 % or less, more preferably 0,05% or less, even more preferably 0,001 % or less, of the ion yield value of the breakdown curve of the pure target-substance at the value of said tuned tuning parameter of the target-substance.

[0197] In a more preferred embodiment, in case an interferant is known to the skilled person, the value of the at least one tuning parameter (de-tuned tuning parameter) of the parameter setting resulting in the lower ion yield of a major peak of interest of a pure target-substance in a breakdown curve (plotting ion yield (y-axis) vs value of tuning parameter (x-axis)) is chosen

[0198] - if the ion yield of the major peak of interest of said pure target-substance (e.g., cortisone) is at least four times lower than the value of the ion yield of said major peak of interest of said pure target-substance using a parameter setting, wherein a different value of said at least one tuning parameter is used (tuned tuning parameter); and

[0199] - if the breakdown curve of the interferant (e.g., prednisolone) has an ion yield value of the peak interfering with the major peak of interest of the targetsubstance which is higher than the ion yield value of the breakdown curve of the interferant at the value of tuned tuning parameter. Generally, the skilled person can easily identify suitable values of the de-tuned tuning parameter if the interferant is known and if the ion yield value in a breakdown curve of a target-substance is strongly decreasing while the ion yield of the breakdown curve of the interferant is at such tuning parameter values either higher than the ion yield value of the breakdown curve of the interferant at the value of the tuning parameter corresponding to the tuned tuning parameter (preferably the ion yield maximum in a breakdown curve of a pure target-substance or a value close to the ion yield maximum of said breakdown curve), or the decrease of the ion yield value compared to the ion yield value at the value of the tuned tuning parameter is notably lower (preferably at least a factor 100, more preferably a factor 1000) than the decrease in the ion yield value of the breakdown curve of the pure target-substance at these two values of the tuning parameter.

[0200] Computer-implemented system comprising an MS-device

[0201] As outlined above, Figure 6 shows a block diagram illustrating a computer system comprising an MS-device 100 which includes a bus 102 or other communication mechanism for communicating information, a processor 104 coupled with bus 102 for processing information, a memory 106, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus 102 for storing instructions to be executed by processor 104. Memory 106 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 104. Computer system comprising an MS-device 100 further includes a read only memory (ROM) 108 or other static storage device coupled to bus 102 for storing static information and instructions for processor 104. A storage device 110, such as a magnetic disk, optical disk, or a connection to a cloud or other online storage properties, is provided and coupled to bus 102 for storing information and instructions.

[0202] In one preferred embodiment, (1 ) more than one, preferably all, single datapoints leading to an MS-spectrum or MS-chromatogram of one or more sample comprising a pure target-substance used in a method according to the invention are stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive).

[0203] In another preferred embodiment, (2) at least one MS-spectrum or MS-chromatogram, respectively, used in a method according to the invention is stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive).

[0204] In another preferred embodiment, (3) at least one tuned parameter setting and at least one de-tuned parameter setting is stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive).

[0205] In another preferred embodiment, (4) data referring to the m / z value and / or retention time, signal strength and tuning parameter setting leading to these m / z value and / or retention time and signal strength of said identified major peak are stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive).

[0206] In another preferred embodiment, (5) ratio (1 ) is stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive).

[0207] In yet another preferred embodiment, at least information (5) and (4) are stored either in a temporary memory 106 or other dynamic storage device such as cloud storage which is accessible from a computer system comprising an MS-device in accordance with the present invention with an internet connection or a read only memory (ROM) 108 or other static storage device (such as a hard drive). In one embodiment, the computer system comprising an MS-device 100 may be coupled via bus 102 to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to bus 102 for communicating information and command selections to processor 104. Another type of user input device is cursor control 116, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 104 and for controlling cursor movement on display 112. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

[0208] A computer system comprising an MS-device 100 can perform parts or the whole method according to the invention and any of its embodiments. Consistent with certain implementations of the present teachings, results are provided by computer system comprising an MS-device 100 in response to processor 104 executing one or more sequences of one or more instructions contained in memory 106. Such instructions may be read into memory 106 from another computer-readable medium, such as storage device 110. Execution of the sequences of instructions contained in memory 106 causes processor 104 to perform the process described herein. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0209] The term "computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 104 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 110. Volatile media includes dynamic memory, such as memory 106. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102, receiving devices for a network or even a wireless local area network.

[0210] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH- EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0211] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system comprising an MS-device 100 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 102 can receive the data carried in the infra-red signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 either before or after execution by processor 104. In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0212] The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings.

[0213] In one embodiment, the described implementation includes software, but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

[0214] MS-device-System for performing a method according to the invention

[0215] Figure 7 is a schematic diagram showing an MS-device-system 200 for evaluating contamination of a target-compound in an MS sample using multiple spectra that are a function of a variable instrument parameter that affects the intensity of fragment ions. The MS-device-system 200 comprises a MS-device 210, a processor 220, and a database 230.

[0216] In one embodiment, a processor (220 in Figure 7) is selected from the group consisting of a computer, microprocessor, or any device capable of sending and receiving control signals and data to and from MS-device (210 in Figure 7) and processing data. A processor (220 in Figure 7) is in communication with MS-device (210 in Figure7).

[0217] A database (230 in Figure 7) can include magnetic or electronic storage as well as a cloud or other storage space accessible through a (wireless) network. Database (230 in Figure 7) can be part of a memory for processor (220 in Figure 7), or it can be a separate memory. A database (230 in Figure 7) can include software components in addition to hardware components.

[0218] A database (230 in Figure 7) can store inter alia fragment ion spectra including tuned and / or de-tuned fragment ion spectra of one or more pure target-substances that are a function of at least one tuning parameter influencing ion yield and fragment ion spectra including tuned and / or de-tuned fragment ion spectra of samples comprising a targetcompound. If contaminations of a target-substance are known and accessible, a database can also store inter alia fragment ion spectra including tuned and / or de-tuned fragment ion spectra of contaminants.

[0219] In one embodiment, a database (230 in Figure 7) comprises for one or more, preferably one, major peak (resulting from a fragment ion of a pure target-substance) a plurality of fragment ion spectra that are a function of at least one tuning parameter influencing ion yield.

[0220] In one embodiment, a processor (220 in Figure 7) identifies the tuned and de-tuned spectra of a pure target-substance and the tuned and de-tuned spectra of a sample comprising the target-substance in accordance with the methods of the present invention.

[0221] In another embodiment, a processor (220 in Figure 7) calculates the ratio (1 ) between the tuned and de-tuned spectra of a pure target-substance in accordance with the methods of the present invention. In yet another embodiment, a processer (220 in Figure 7) calculates the ratio (2) between the tuned and de-tuned spectra of a sample comprising the target-substance in accordance with the methods of the present invention.

[0222] In yet another embodiment, a processer (220 in Figure 7) compares ratio (1 ) and ratio (2) with each other.

[0223] In yet another embodiment, a processer (220 in Figure 7) compares ratio (1 ) and ratio (2) with each other and quantifies the amount of a target-substance in a sample comprising said target-substance, if the value of ratio (2) differs from the value of ratio

[0224] (1 ) by 10% or less based on the value of ratio (1 ), more preferably 5% or less based on the value of ratio (1 ).

[0225] In yet another embodiment, a processer (220 in Figure 7) compares ratio (1 ) and ratio

[0226] (2) with each other and , if the value of ratio (2) differs from the value of ratio (1 ) by 10% or less based on the value of ratio (1 ), more preferably 5% or less based on the value of ratio (1 ) quantifies the amount of a target-substance in a sample comprising said targetsubstance by comparing the signal strength of the at least one major peak using the tuned parameter setting of a sample comprising the target-substance, wherein the value of ratio (2) differs from the value of ratio (1 ) by 10% or less based on the value of ratio (1 ), more preferably 5% or less based on the value of ratio (1 ); with at least one, preferably at least two, signal strength value(s) of the at least one corresponding major peak of sample(s) with known amount(s) of the pure targetsubstance based on the tuned parameter setting, wherein one value is lower, and one value is higher than the value of the sample comprising the target-substance, preferably, wherein these at least two signal strength value(s) of the at least one corresponding major peak of sample(s) with known amount(s) of the pure target-substance are located on a linear area of a calibration curve; and then calculating the amount of the target-sample based on the signal strength value(s) of the at least one corresponding major peak of sample(s) with known amount(s) of the pure target-substance; optionally comprising the step of diluting or concentrating a sample comprising a targetsubstance until the signal strength value of the at least one corresponding major peak of the sample comprising the target-compound is between the signal strength value(s) of the at least one corresponding major peak of sample(s) with known amount(s) of the pure target-substance each MS-spectrum based on the tuned parameter setting.

[0227] In a preferred embodiment, processor (220 in Figure 7) identifies the presence of at least one interferant that disturbs quantification of a target-substance in a sample by comparing the value of the de-tuning ratio (1 ) of the pure target-substance with the value of the de-tuning ratio (2) of a sample comprising the target substance, if the value of ratio (2) is 10% % or less based on the value of ratio (1 ), more preferably 5% or less based on the value of ratio (1 ), wherein no interferant is present in an amount which would alter the result of a quantification.

[0228] In various embodiments, comparing the ratios comprises comparing acquired and, optionally, stored breakdown curves based on identical parameter settings.

[0229] A further aspect of the invention refers to an MS-device-system for evaluating the presence of an interferant disturbing the quantification of a target-substance, preferably for evaluating the absence of an interferant and quantifying a target-substance, using multiple spectra that are a function of at least one tuning parameter influencing ion yield, preferably, said spectra or chromatograms are measured simultaneously, comprising: an MS-device or coupled C-MS-device that is configured to analyze a sample comprising a target-substance by measuring, preferably simultaneously, signal strengths of peaks in an MS-spectra or MS-chromatograms, respectively, each based on different tuning parameter settings at the MS-device, to produce at least one tuned and at least one de-tuned fragment ion MS-spectrum or MS-chromatogram, respectively, each based on the signal strength of a selected major peak of interest of a target-substance in said MS-spectrum or MS-chromatogram, respectively which are a function of at least one tuning parameter influencing the ion yield in accordance with the methods according to the invention; and a database for the pure target-substance that includes for said pure target-substance at least one tuned and one de-tuned MS-spectrum or MS-chromatogram, respectively, each comprising said major peak of interest of the (pure) target-substance and each based on the same tuned parameter setting including the at least one tuning parameter as used in the measurements of the target-substance; and a processor in communication with the MS-device or coupled C-MS-device, respectively and the database that is configured to: receive the at least one tuned and at least one de-tuned fragment ion MS-spectrum or MS-chromatogram, respectively, comprising the major peak of interest of said targetsubstance of a sample comprising a target-substance, from the MS-device, calculating the de-tuning ratio based on the signal strength of the major peak of interest of the tuned and the de-tuned MS-spectrum or MS-chromatogram, respectively, of said sample comprising said target-substance; compare the de-tuning ratio of the pure target-substance with the de-tuning ratio of the sample comprising the target substance; a difference between the values of 10% or less more preferably 5% or less (based on the value of the de-tuning ratio of the pure target-substance) based on the value of ratio 1 indicates the absence of an interferant, a difference of more than 10% indicates the presence of an interferant in an amount which can influence the signal strength of a major peak of a target-substance; optionally, quantify a target-substance, optionally using the linear area of a calibration curve, if the difference between the value of the de-tuning ratio of the pure substance and the value of the de-tuning ratio of the sample comprising the target-substance is 10% or less based on the value of the de-tuning ratio of the pure target-substance.

[0230] In a preferred embodiment refers to the system, wherein the mass spectrometer is configured to analyze the sample using tandem mass spectrometry (MS / MS or MS2), or mass spectrometry / mass spectrometry / mass spectrometry (MS3). Computer product

[0231] A further aspect of the invention refers to a computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method according to the invention, wherein the method comprises providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise a measurement module and an analysis module.

[0232] Method processor

[0233] A “processor” as used herein, can be, but is not limited to, a computer, microprocessor, or any device capable of sending and receiving control signals and data to and from an MS-device and processing data. A processor is in communication with the MS-device.

[0234] In a preferred embodiment, a processor is used in at least one step of a method, a system and / or computer implemented system according to the invention and its / their various embodiments. In a further preferred embodiment, a processor is used at least in one of the steps of identifying an optimized fragment ion MS-spectrum and a de-tuned fragment ion MS-spectrum, more preferably in all steps of identifying an optimized fragment ion MS-spectrum and a de-tuned fragment ion MS-spectrum. In a further preferred embodiment, a processor is used in at least in one of the steps of identifying an optimized fragment ion MS-spectrum and a de-tuned fragment ion MS-spectrum, more preferably in all steps of identifying an optimized fragment ion MS-spectrum and a de-tuned fragment ion MS-spectrum, and in at least one, preferably both, step of forming the ratio 1 and ratio 2, respectively. More preferably, a processor is used in all four of these steps. In a furthermore preferred embodiment, a processor is additionally used in the step of comparing the ratios to estimate whether a sample fulfills the quality requirements to allow a quantification of a target-substance based on the carried out measurement. Even more preferably, a processor is used in all steps of a method according to the invention. Example

[0235] Example 1 Cortisone / Prednisolone

[0236] Chemicals and reagents

[0237] Water, methanol, acetonitrile (ULC / MS - CC / SFC grade) were obtained in UHPLC quality from Biosolve (Valkenswaars, the Netherlands). Cortisone, cortisone- 2, 2, 4, 6, 6, 9, 12, 12-d8, prednisolone and ammonium acetate were obtained by Sigma- Aldrich (St. Louis, Missouri, USA). Anonymized serum samples were material from the clinical routine lab.

[0238] A stock solution of 1000 pg / mL was prepared for cortisone in methanol. Another stock solution of prednisolone with a concentration of 1000 pg / mL was also prepared in methanol. For cortisone-d8 internal Standard (IS), a stock solution of 1000 pg / mL in methanol was prepared. Each stock was further diluted with water to obtain required concentrations for analysis. The concentrations of all levels are given in Table 1 .

[0239] Table 1. Concentration of cortisone calibrators, prednisolone spike solutions and IS cortisone d8.

[0240] Analyte Concentration, ng / mL

[0241] Cortisone call 20

[0242] Cortisone cal2 100

[0243] Cortisone cal3 250

[0244] Prednisolone spikel 30

[0245] Prednisolone spike2 120

[0246] Prednisolone spike3 600

[0247] Prednisolone spike4 1500

[0248] Prednisolone spike5 3000

[0249] Cortisone d8 IS 50 Sample preparation

[0250] A 100 pL aliquot was spiked with 20 pL prednisolone, followed by the addition of 50 pL IS. The sample was vortexed and then shaken on a thermomixer for 10 minutes at 10°C and 1400 rpm. Protein precipitation was performed by adding 300 pl acetonitrile as precipitation solution, followed by vortexing and another 5 min shaking (10 min, 10 °C and 1400 rpm). After centrifugation (15 min, 10°C, 14000 g), 50 pl supernatant were diluted 1 :4 with 150 pl methanol. The diluted supernatant was transferred in a glass vial with micro-insert and placed in an autosampler (10°C sample cooling) for analysis.

[0251] LC and MS parameters

[0252] The sample analyses were performed on an Agilent 1290 Infinity II LC system (Santa Clara, California, USA) coupled to an AB Sciex QTrap 6500+ mass spectrometer (Framingham, Massachusetts, USA). Instrument control and data acquisition were performed using Analyst software 1.7.2 (Sciex) and data processing using Analyst software 1.7 (Sciex). Chromatographic separation / coelution was obtained using a Restek Raptor Biphenyl column (50 x 2.1 mm, 2.7 pm) (Bellefonte, Pennsylvania, USA). The column oven was kept at 40 °C and the injection volume was set to 5 pl. The mobile phases included 5 mM ammonium acetate in water (A) and methanol / acetonitrile- mixture (75 / 25, v / v) (B). A custom injection program was driven for autosampler washing with methanol as washing solvent. The total run time was 5 min for chromatographic separation with a flow rate of 0.35 mL / min. For coelution the total run time was 3 min with a flow rate of 0.3 mL / min. The gradient for chromatographic separation was as follows for eluent B: first gradient was driven starting with 35 % at 0.00 min and increasing to 40 % in 1.00 min, second gradient was driven starting with 40 % at 1.00 min and increasing to 63 % in 1 .20 min, third gradient was driven starting with 63 % at 2.20 min and increasing to 75 % in 0.30 min, fourth gradient was driven starting with 75 % at 2.50 min and increasing to 90 % in 1 .00 min, from 3.50 to 3.60 min the column was re-equilibrated to 35 % and conditioned from 3.60 to 5.00 min at 35 %. For the chromatographic coelution the gradient was as follows for eluent B: first gradient was driven starting with 90 % at 0.00 min and increasing to 95 % in 0.6 min, second gradient was driven starting with 95 % at 0.60 min and increasing to 100 % in 0.40 min, 100 % were held for 0.1 min, from 1 .00 to 1 .30 min the column was re-equilibrated to 90 % and conditioned from 1 .30 to 3.00 min at 90 %. A switching valve allowed the elution to enter the mass spectrometer between 1 .5 to 3.5 min for chromatographic separation and 0.2 to 1 .5 min for coelution.

[0253] MS / MS measurement was performed with positive electrospray ionization (ESI+). The optimal ion source parameters were determined by direct infusion of cortisone, prednisolone, cortisone d8: curtain gas 40 psi, collision gas medium, source temperature 400 °C, ion spray voltage 5500 eV, ion source gas 1 50 psi (atomizing gas) and ion source gas 2 60 psi (heating gas).

[0254] Measured precursor and product ions (resulting in a major peak of interest) of cortisone, prednisolone, cortisone d8 with collision energies (CE), collision cell exit potentials (CXP) are given in Table 2.

[0255] Table 2. Exemplary MS-parameters of measurands.

[0256] Measurand Precursor ion Product ion Collision, V CXP, V

[0257] Cortisone 361.201 163.100 15 1

[0258] 361.202 163.100 25 1

[0259] 361.199 163.100 35 1

[0260] 361 .203 (Quantifier) 163.100 35 18

[0261] 361.204 163.100 20 1

[0262] 361.203 163.100 30 1

[0263] 361.205 163.100 30 18

[0264] 361.206 163.100 40 1

[0265] 361.200 (Qualifier) 121.000 35 18

[0266] 361.207 163.100 35 1

[0267] Prednisolone 361 .197 (Quantifier) 147.100 35 18

[0268] 361.195 171.100 35 1

[0269] 361.200 171.100 35 18

[0270] Cortisone d8 369.200 (Quantifier) 169.100 35 20 Fragment ions typical for cortisone (A1 ) also appear in mass spectra of prednisolone (A2) (see also Figure 3A and B).

[0271] A1 A2

[0272] A de-tuning ratio was evaluated for the recorded peak.

[0273] Several aliquots of a serum sample that physiologically comprises cortisone with a known concentration (29,75 ng / mL) were spiked with increasing amounts of prednisolone (5, 20, 100, 250, and 500 ng / mL). Prednisolone is structurally closely related to cortisone (constitutional isomers). MS disintegration spectrum mass transitions (precursor ion to production) of prednisolone were identified, which were also found for cortisone (so-called isobaric transitions) and which elute in this setting at the same time as the major peak of cortisone (see Figure 3A and B (MS-spectra) and Figure 5 (MS-chromatograms).

[0274] For the samples 1 to 6 which were spiked with increasing amounts of prednisolone, the de-tuning ratios of the respective peaks that elute at the expected retention time of cortisone were recorded (see also Figure 4). For Figure 4, the parameter setting with variable CE and constant CXP (1V) were used. Values with variable CE and CXP different from 1V (data not plotted in Figure 4) resulted in even slightly higher signal strength values.

[0275] Based on these data, it was examined at which level of spiking a deviation in the detuning ratio occurred compared to the de-tuning ratio observed for a pure cortisone peak. In addition, cortisone was quantified in this measuring system for all samples including samples which de-tuning ratio classifies them as contaminated.

[0276] Example 2 Tramadol / O-Desmethylvenlafaxine

[0277] Chemicals and reagents

[0278] Water, methanol, acetonitrile (ULC / MS - CC / SFC grade) were obtained in UHPLC quality from Biosolve (Valkenswaars, the Netherlands). O-Desmethylvenlafaxine, O- Desmethylvenlafaxine-d6, cis-Tramadol HCI and ammonium acetate were obtained by Sigma-Aldrich (St. Louis, Missouri, USA). Anonymized serum samples were from the clinical routine lab.

[0279] Sample preparation

[0280] A stock solution of 1000 pg / mL was prepared for O-Desmethylvenlafaxine in methanol. Another stock solution of cis-Tramadol HCI with a concentration of 1000 pg / mL was also prepared in methanol. For O-Desmethylvenlafaxine-d6 internal Standard (IS), a stock solution of 100 pg / mL in methanol was purchased and diluted with water. Cis-Tramadol HCI stock was further diluted with water to obtain required concentrations for analysis. O-Desmethylvenlafaxine was diluted in analyte free human serum. The concentrations of all levels are given in Table 3.

[0281] Table 3. Concentration of cortisone calibrators, prednisolone spike solutions and IS cortisone d8.

[0282] Analyte Concentration, ng / mL

[0283] O-Desmethylvenlafaxine call 50

[0284] O-Desmethylvenlafaxine cal2 100

[0285] O-Desmethylvenlafaxine cal3 200

[0286] O-Desmethylvenlafaxine cal 4 300

[0287] O-Desmethylvenlafaxine cal 5 400 cis-Tramadol HCI spikel 30 cis-Tramadol HCI spike2 120 cis-Tramadol HCI spike3 600 cis-Tramadol HCI spike4 1500 cis-Tramadol HCI spike5 3000

[0288] O-Desmethylvenlafaxine-d6 IS 400 A 100 pL aliquot was spiked with 20 pL c / s-Tramadol HCI (calibrators were spiked with 20pL water), followed by the addition of 50 pL IS. The sample was vortexed and then shaken on a thermomixer for 10 minutes at 10°C and 1400 rpm. Protein precipitation was performed by adding 300 pl acetonitrile as precipitation solution, followed by vortexing and another 5 min shaking (10 min, 10 °C and 1400 rpm). After centrifugation (15 min, 10°C, 14000 g), 100 pl supernatant were diluted 1 :10 with 900 pl water. The diluted supernatant was transferred in a glass vial with micro-insert and placed in an autosampler (10°C sample cooling) for analysis.

[0289] LC and MS parameters

[0290] The sample analyses were performed on an Agilent 1290 Infinity II LC system (Santa Clara, California, USA) coupled to an AB Sciex QTrap 6500+ mass spectrometer (Framingham, Massachusetts, USA). Instrument control and data acquisition were performed using Analyst software 1.7.2 (Sciex) and data processing using Analyst software 1.7 (Sciex). Chromatographic separation / coelution was obtained using a Restek Raptor Biphenyl column (50 x 2.1 mm, 2.7 pm) (Bellefonte, Pennsylvania, USA). The column oven was kept at 30 °C and the injection volume was set to 5 pl. The mobile phases included 10 mM ammonium acetate in water (A) and methanol / acetonitrile- mixture (75 / 25, v / v) (B). A custom injection program was driven for autosampler washing with methanol as washing solvent. The total run time was 6 min for chromatographic separation with a flow rate of 0.4 mL / min. For coelution the total run time was 3.5 min with a flow rate of 0.6 mL / min. The gradient for chromatographic separation was as follows for eluent B: first gradient was driven starting with 10 % at 0.00 min and increasing to 38 % in 2.90 min, second gradient was driven starting with 38 % at 2.90 min and increasing to 80 % in 0.01 min, third gradient was driven starting with 80 % at 2.91 min and increasing to 85 % in 0.59 min, fourth gradient was driven starting with 85 % at 3.50 min and increasing to 100 % in 0.01 min, 100 % were hold for 0.99 min, from 4.50 to 5.00 min the gradient the column was re-equilibrated to 10 % and conditioned from 5.00 to 6.00 min at 10 %. For the chromatographic coelution the gradient was as follows for eluent B: first gradient was driven starting with 85 % at 0.00 min and increasing to 90 % in 1.00 min, second gradient was driven starting with 90 % at 1.00 min and increasing to 10 % in 0.01 min, 10 % were held for 0.99 min, from 2.00 to 2.01 min the column was re-equilibrated to 85 % and conditioned from 2.01 to 3.50 min at 85 %. A switching valve allowed the elution to enter the mass spectrometer between 2.6 to 4.0 min for chromatographic separation and 0.2 to 2.0 min for coelution.

[0291] MS / MS measurement was performed with positive electrospray ionization (ESI+). The optimal ion source parameters were determined by direct infusion of 0- Desmethylvenlafaxine, c / s-Tramadol HCI, O-Desmethylvenlafaxine-d6: curtain gas 40 psi, collision gas medium, source temperature 400 °C, ion spray voltage 5500 eV, ion source gas 1 50 psi (atomizing gas) and ion source gas 2 60 psi (heating gas). The measurements were performed with a CE value of 40 V for the tuned parameter setting and 10 V for the de-tuned parameter setting (see also Figure 9). Measured precursor and product ions of O-Desmethylvenlafaxine, c / s-T ramadol HCI and O-Desmethylvenlafaxine-d6 with collision energies (CE), collision cell exit potentials (CXP) are given in Table 4.

[0292] Table 4. Exemplary MS-parameters of measurands.

[0293] Measurand Precursor ion Product ion Collision, V CXP, V

[0294] O-Desmethylvenlafaxine 264.100 57.800 15 1

[0295] 264.101 57.800 25 1

[0296] 264.102 57.800 35 1

[0297] 264.103 57.800 20 1

[0298] 264.099 57.800 30 1

[0299] 264.093 57.800 40 1

[0300] 264.098 57.800 40 20

[0301] (Quantifier)

[0302] 264.097 107.200 38 19

[0303] (Qualifier)

[0304] 264.104 57.800 10 1

[0305] 264.096 57.800 45 1

[0306] 264.095 57.800 50 1

[0307] 264.105 57.800 5 1 cis-Tramadol HCI 264.195 58.200 50 20

[0308] (Quantifier)

[0309] 264.188 58.200 50 1

[0310] O-Desmethylvenlafaxine- 270.000 64.100 38 18 d6 (Quantifier)

[0311] 270.000 107.000 39 19

[0312] (Qualifier) Breakdown curves for the tuned and the de-tuned parameter setting are plotted in Figure 8.

[0313] Several aliquots of a serum sample that comprises O-Desmethylvenlafaxine with a known concentration (208.7 ng / mL) were spiked with increasing amounts of tramadol (0, 5, 20, 100, 250, and 500 ng / mL). See Figure 9 and Figure 10 (MS-spectra).

[0314] For the samples 1 to 6 which were spiked with increasing amounts of tramadol, the detuning ratios of the respective peaks that elute at the expected retention time of O- Desmethylvenlafaxine were recorded (see Figure 9). For Figure 9, the parameter setting with variable CE and constant CXP (1V) were used. Values with variable CE and CXP different from 1V (data not plotted in Figure 9) resulted in even slightly higher signal strength values.

[0315] Based on these data, it was examined at which level of spiking a deviation in the detuning ratio occurred compared to the de-tuning ratio observed for a pure O- Desmethylvenlafaxine peak. In addition, O-Desmethylvenlafaxine was quantified in this measuring system for all samples including samples which de-tuning ratio classified them as contaminated.

Claims

Claims1. A method for evaluating the presence of at least one interferant in a sample comprising a target-substance using a processor in at least one step, preferably evaluating the presence of at least one interferant in said sample in an amount that disturbs quantification of said target-substance using multiple MS-spectra or MS-chromatograms of at least one ion of a pure target-substance, respectively, that are a function of at least one tuning parameter influencing ion yield, comprising: a) receive, from an MS-device at least one MS-spectrum or part thereof of a sample comprising the pure target substance; or from a coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof of a sample comprising the pure target substance using a first parameter setting comprising at least one tuning parameter influencing ion yield; b) identifying at least one major peak of interest of an ion of a pure targetsubstance having a specific m / z ratio in said at least one MS-spectrum or parts thereof comprising said major peak of interest or MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said major peak; c) determine the signal strength of said at least one major peak of interest which is based on the first parameter setting; d) receive, from said MS-device at least one MS-spectrum or part thereof comprising the at least one major peak of interest of said pure targetsubstance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising the at least one major peak of interest of said pure target substance using a second parameter setting wherein the values of all parameters are the same as in the first parameter setting except for the value of the at least one tuning parameter influencing ion yield; e) determine the signal strength of said at least one major peak of interest which is based on the second parameter setting;f) form the quotient of the higher value of the two signal strengths divided by the lower value of the two signal strength (de-tuning ratio): if the value of said de-tuning ratio is 4 or more, then the parameter setting resulting in the higher value of the signal strength of said at least one major peak of interest is defined as tuned parameter setting and the parameter setting resulting in the lower value of the signal strength of said at least one major peak of interest is defined as de-tuned parameter setting: if the value of said quotient is below 4, step a) and / or step d) are repeated with different values of the at least one tuning parameter influencing ion yield until the quotient is 4 or more; g) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the de-tuned parameter setting; h) receive, from said MS-device at least one MS-spectrum or part thereof comprising said at least one major peak of interest from a sample comprising the target-substance; or from said coupled chromatographic-MS-device at least one MS-chromatogram which is based on multiple MS-spectra or parts thereof comprising said at least one major peak of interest using the tuned parameter setting; i) form the quotient of the signal strength of said at least one major peak of interest based on the tuned parameter setting divided by the signal strength of said at least one major peak of interest based on the de-tuned parameter setting; j) compare the values the de-tuning ratio of the sample comprising the pure target substance with the value of the de-tuning ratio of the sample comprising the target-substance:(i) if the value of the de-tuning ratio of a sample comprising the target-substance differs at most 10% from the value of the detuning ratio of a sample comprising the pure target-substance, the sample is classified as fulfilling the quality measurements for quantification of the target-substance in the sample;(ii) if the value of the de-tuning ratio of a sample comprising the target-substance is higher than 10% of the value of the detuning ratio of a sample comprising the pure target-substance, the sample is classified as contaminated.

2. A method for quantifying the amount of a target-substance in a sample using multiple MS-chromatograms and using a processor in at least one step, respectively, that are a function of at least one tuning parameter influencing ion yield of a tuning parameter setting, comprising: Steps a) to j) according to claim 1 , wherein steps a), b), d), g), and h) are carried out using MS-chromatograms; and further comprising step k) calculate the amount of the target substance in the sample by comparing.

3. The method according to claim 2, wherein step k) comprises providing the linear area of a calibration curve using different amounts of pure target-substance in samples for preparing tuned MS-chromatograms, based on the tuned parameter setting in accordance with step f).

4. The method according to any one of claims 1 to 3, wherein a processor is used in all steps of the method.

5. The method according to anyone of the preceding claims, wherein the at least one tuning parameter is one tuning parameter.

6. The method according to anyone of the preceding claims, wherein a tuning parameter influencing ion yield is selected from the group consisting of collision energy (CE) and collision cell exit potential (CXP).

7. The method according to anyone of the preceding claims, wherein a major peak in step b) is the peak with either the highest signal strength (main peak) in an MS-spectrum or MS-chromatogram, respectively, or any of the nine peaks withthe highest signal strengths after the signal strength of the main peak in an MS- spectrum or MS-chromatogram, respectively, with the proviso that the signal strength of such a peak is at least 20% of the signal strength of the main peak of said spectrum or chromatogram, respectively.

8. The method according to any one of the preceding claims, wherein the at least one major peak is one major peak.

9. The method according to any one of the preceding claims, wherein the factor in step f) is 4.5 or more.

10. The method according to anyone of the preceding claims, wherein the MS- device is a time of flight (TOF) mass spectrometer, a triple quadrupole (tandem or (MS / MS)) mass spectrometer, a magnetic sector mass spectrometer, an ion mass trap mass spectrometer, an orbitrap mass spectrometer, a MS 3 mass spectrometer, a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometer, a combined TOF and quadrupole mass spectrometer (Q-TOF), an inductively coupled plasma mass spectrometer, and a direct analysis in real time mass spectrometer; more preferably MS / MS, MS 3; even more preferably MS / MS and the signal strength is the peak intensity in an MS-spectrum.11 . The method according to anyone of the preceding claims, wherein the MS- device is a coupled chromatographic-MS-device, preferably a coupled liquid chromatographic (LC)-MS-device or a gas chromatographic (GC)-MS-device, an LC-MS / MS-device, an LC-MS3-device, a GC-MS-device, a GC-MS / MS- device, or a GC-MS3-device and the signal strength is the peak area in an MS- spectrogram.

12. A system for evaluating the presence of an interferant of a target-substance comprising: an MS-device or coupled C-MS-device that is configured to analyze a sample comprising a target-substance by measuring, preferably simultaneously, signal strengths of peaks in an MS-spectra orMS-chromatograms, respectively, each MS-spectrum or MS-chromatogram based on a specific parameter settings at the MS-device, to produce at least one tuned and at least one de-tuned MS-spectrum or MS-chromatogram, respectively, each based on the signal strength of a selected major peak of said MS-spectrum or MS-chromatogram, respectively, which are a function of at least one tuning parameter influencing ion yield; a database comprising data of at least one pure target-substance, such data include a de-tuning ratio (de-tuning ratio 1 ) of said target-substance of at least one tuned and at least one de-tuned MS-spectrum or MS-chromatogram, respectively, based on a major peak of interest and each based on a defined tuned and de-tuned, respectively, parameter setting; and a processor in communication with the MS-device or coupled C-MS-device, respectively and the database that is configured to: receive the at least one tuned and at least one de-tuned MS-spectrum or MS-chromatogram, respectively, for the at least one ion of a targetsubstance of a sample comprising said target-substance, from the MS- device, wherein the tuned and de-tuned parameter settings are the same as for evaluating de-tuning ratio 1 ; calculate the de-tuning ratio (de-tuning ratio 2) between the signal strength of said major peak of interest of said tuned spectrum and the signal strength of said major peak of interest of said de-tuned spectrum; compare de-tuning ratio (1 ) with said de-tuning ratio (2); if the value of the de-tuning ratio 2 of a sample comprising said targetsubstance differs at most 10% from the value of the de-tuning ratio 1 , the sample is classified as interferant-free and optionally the processor is configured to quantify the target-substance if said sample is classified as interferant-free, if the value of the de-tuning ratio 2 differs by more than 10% from the value of the de-tuning ratio 1 , classify and mark the sample is contaminated,optionally using the linear area of a calibration curve, if the difference between the value of ratio 2 and ratio 1 is 10% or less based on the value of ratio 1 .

13. The system of claim 12, wherein the mass spectrometer is configured to analyze the sample using tandem mass spectrometry (MS / MS or MS2), or mass spectrometry / mass spectrometry / mass spectrometry (MS3).

14. The system of claim 12 or 13, wherein the target-substance is characterized in having only one intensive mass transition peak in an MS-spectrum.

15. The system of any one of claims 12 to 14, wherein the detuning ratio is calculated as the quotient of the tuned signal strength divided by the de-tuned signal strength.

16. A computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method according to any one of claims 1 to 11 , wherein the method comprises providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise a measurement module and an analysis module.

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