Method and apparatus for automatic peak integration

Abstract

The present invention proposes a computer-implemented method for automatic peak integration of at least one chromatogram of at least one sample. The sample contains at least one analyte and at least one chemically related substance. The method comprises the following steps: a) retrieving at least one chromatogram of the chemically related substance and at least one chromatogram of the analyte; (114, 116) b) evaluating the chromatogram of the chemically related substance, wherein the evaluation comprises b1) determining at least one initial value (117) of the analyte retention time by determining the retention time (118) of the chemically related substance and adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor (120), b2) determining the retention time of the chemically related substance by determining the retention time of the chemically related substance; c) evaluating the chromatogram of the analyte, wherein the evaluation comprises c1) at least one position determination step (124), wherein the analyte retention time is determined by taking into account the initial value of the retention time of the chemically related substance; c2) at least one peak integration step (126), wherein at least one fitting analysis is applied to the chromatogram of the analyte by taking into account the initial value of the analyte peak shape parameter and the analyte retention time, thereby determining the analyte peak area and the analyte peak shape.

Description

Technical Field

[0001] The present invention relates to a computer-implemented method, a computer-implementable processing line and a mass spectrometry system for automatic peak integration of at least one chromatogram of at least one sample. Background Art

[0002] Peak finding and evaluation in liquid chromatography-mass spectrometry (LC-MS) or mass spectrometry (MS) usually requires user interaction or correction by an expert user, such as for selecting or assigning the correct peak. For several years, there has been a need for automation of peak finding and evaluation, for example to reduce user interaction and thereby enhance the reliability of the measurements.

[0003] Peaks in LC-MS or mass spectrometry-based chromatography experience time shifts and / or peak shape changes. Such shifts and changes may be caused by matrix effects and system performance losses (e.g., degradation of LC columns). Automated methods for peak picking use fixed retention time targets and fixed retention time windows to specify target peaks that should be identified as signals. Such strategies are implemented in, for example, Multi Quant Sciex software, see https: / / sciex.com / Documents / Downloads / Literature / verify-update-retention-times.pdf. Due to retention time shifts, such methods may lead to errors, forcing users to manually assign the correct peaks.

[0004] Similar problems may occur when fitting analytical peak shape models to calculate peak areas. Known methods (e.g., Ascent software from Indigo bioautomation, see https: / / s3-us-west-2.amazonaws.com / ascent-data / manual / AscentManual.pdf) perform independent shape fitting for analyte and internal standard signals. This can often lead to erroneous results for analyte shape fitting in the presence of low analyte concentrations, complex background signals, high noise levels, or interfering peaks. This drawback makes full automation of peak integration impossible and makes it necessary for expert users to perform corrections and manually adjust the results when performing measurements with complex chromatographic features.

[0005] EP 3 271 715 A1 screening technology, it is used to detect the analyte in biological specimen qualitatively and quantitatively using threshold analyte calibration.Specifically, in EP 3 271 715 A1, it is described to consider that the retention time of internal standard is offset for peak picking.In addition, Waters MassLynx 4.0 manual, http: / / www.ecs.umass.edu / eve / facilities / equipment / Quattromicro / MassLynx%20Version%204.0%20User's%20Guide.pdf suggests to consider other characteristics of internal standard peak, such as peak width and peak asymmetry.However, only considering the offset of internal standard retention time suggested in EP 3271 715 A1 or even the peak width and asymmetry suggested in Waters MassLynx 4.0 manual may not be enough to realize reliable peak discovery and evaluation to allow automation without any user interaction or inspection.

[0006] Issues to be resolved

[0007] It is therefore an object of the present invention to provide a method and apparatus for automatic peak integration which avoids the above-mentioned disadvantages of known methods and apparatuses. In particular, the method and apparatus should allow reliable and fully automatic peak integration, in particular without user interaction. Summary of the invention

[0008] The problem is solved by a computer implemented method for automatic peak integration of at least one chromatogram of at least one sample, a computer implementable processing line and a mass spectrometry system having the features of the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims, which can be implemented individually or in any arbitrary combination.

[0009] As used hereinafter, the terms "having", "including" or "comprising" or any of their arbitrary grammatical variations are used in a non-exclusive manner. Thus, these terms may refer both to situations in which, apart from the features introduced by these terms, no other features are present in the entity described in this context, and to situations in which one or more additional features are present. As an example, the expressions "A has B", "A includes B" and "A contains B" may refer both to situations in which, apart from B, no other elements are present in A (i.e., A consists solely and exclusively of B), and to situations in which, apart from B, one or more other elements (such as element C, element C and element D, or even other elements) are present in entity A.

[0010] Furthermore, as used below, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features without limiting the alternative possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As the skilled person will recognize, the present invention can be carried out by using alternative features. Similarly, the features introduced by "in one embodiment of the present invention" or similar expressions are intended to be optional features without any limitation on alternative embodiments of the present invention, without any limitation on the scope of the present invention, and without any limitation on the possibility of combining the features introduced in this manner with other optional or non-optional features of the present invention.

[0011] In a first aspect of the present invention, a computer-implemented method for automatic peak integration of at least one chromatogram of at least one sample is disclosed.

[0012] As used herein, the term "computer-implemented method" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a method involving at least one computer and / or at least one computer network. The computer and / or computer network may include at least one processor configured to perform at least one of the method steps of the method according to the present invention. Preferably, each method step is performed by a computer and / or a computer network. The method may be performed completely automatically (particularly, without user interaction). As used herein, the term "automatically" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a process performed entirely with the aid of at least one computer and / or at least one computer network and / or at least one machine, in particular, without the need for manual operation and / or interaction with a user.

[0013] As used herein, the term "chromatogram" is a broad term and is given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, the visual results or achievements of the separation process of separating sample components. A chromatogram may refer to an intensity distribution over time generated during at least one chromatographic run. A chromatogram may be or may include a graphical representation of the retention time of sample components on the x-axis and the intensity on the y-axis. A chromatogram may be determined by using at least one mass spectrometry device (e.g., at least one liquid chromatography mass spectrometry device). As used herein, the term "liquid chromatography mass spectrometry device" is a broad term and is given its common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a combination of liquid chromatography and mass spectrometry. A liquid chromatography mass spectrometry device may be or may include at least one high performance liquid chromatography (HPLC) device or at least one microfluidic liquid chromatography (μLC) device. A liquid chromatography mass spectrometry device may include a liquid chromatography (LC) device and a mass spectrometry (MS) device, wherein the LC device and the MS are coupled via at least one interface. As used herein, the term "liquid chromatography (LC) device" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, an analytical module configured to separate one or more target analytes of a sample from other components of the sample for use in detecting the one or more analytes using a mass spectrometer. The LC device may include at least one LC column. For example, the LC device may be a single-column LC device or a multi-column LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped to separate and / or elute and / or transfer the target analyte. As used herein, the term "mass spectrometer" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a mass analyzer configured to detect at least one analyte based on a mass-to-charge ratio. The mass spectrometer may be or may include at least one quadrupole mass spectrometer. The interface coupling the LC device and the MS may include at least one ionization source configured for generating molecular ions and for transferring the molecular ions into a gas phase.

[0014] As used herein, the term "peak" is a broad term and is given its common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, at least one local maximum of a chromatogram. The term "signal peak" may be used to represent an identified peak of a target analyte. As used herein, the term "peak integral" is a broad term and is given its common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, at least one mathematical operation and / or mathematical algorithm for determining the peak area surrounded by the peak of a chromatogram. Specifically, the integration of a peak may include identification and / or measurement of the curve features of a chromatogram. Peak integration may include one or more of the following: peak detection, peak discovery, peak identification, peak fitting, peak evaluation, determination of peak start and / or peak end, determination of background, determination of baseline. Peak integration may allow determination of one or more of the following: peak area, retention time, peak height, and peak width. As used herein, the term "automatic peak integration" is a broad term and is given its ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or customized meaning. The term may specifically refer to, but is not limited to, peak integration that is non-manual but performed by at least one computer and / or at least one computer network and / or at least one machine. In particular, automatic peak integration may be performed without manual operation or interaction with a user. In particular, peak identification and / or peak detection and / or determination of peak area may be performed non-manually and without manual operation or interaction with a user.

[0015] As used herein, the term "sample" is a broad term and is given a common and customary meaning for those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, any sample, such as a biological sample, also referred to as a test sample. For example, a test sample may be selected from a group consisting of the following items: physiological fluids, including blood, serum, plasma, saliva, lens fluid, cerebrospinal fluid, sweat, urine, emulsion, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, etc. The sample may be used directly when obtained from a corresponding source, or may be pre-treated and / or sample preparation workflow. The sample comprises at least one analyte and at least one chemically related substance. For example, in general, a target analyte may be vitamin D, a drug of abuse, a therapeutic drug, a hormone, and a metabolite. As used herein, the term "chemically related substance" is a broad term and is given a common and customary meaning for those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a substance having a mass-to-charge ratio similar to that of a target analyte. For example, the chemically related substance can be at least one internal standard and / or at least one substance used as a quantitative factor. Even for low intensity peaks, high noise levels or frequent interferences, the method according to the present invention also allows reliable peak integration. Specifically, the method proposes the use of a chromatogram for evaluating a target analyte input value or a starting value, which is determined from other chromatograms of chemically related substances of the same measurement, for which sufficiently high intensity peaks, low noise levels and low frequency interferences can be guaranteed. For example, the method can be used to enhance the integration of an analyte chromatogram based on the result obtained from an internal standard chromatogram. For example, the method can be used to enhance the integration of a qualitative factor chromatogram based on the result obtained from a quantitative factor chromatogram. As used herein, the term "internal standard" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically refers to, but is not limited to, an analyte present in a sample at a known concentration. An internal standard sample can be a sample comprising at least one internal standard substance with a known (e.g., predetermined) concentration. An internal standard can be almost the same as an analyte. The internal standard substance may be similar to the target analyte (e.g., a deuterated molecule) or an isotope-labeled form of the target analyte. For more details about samples, reference is made to, for example, EP 3 425 369 A1, the entire disclosure of which is included herein by reference. Other target analytes are also possible.

[0016] The sample can be pre-treated by adding a chemically related substance (e.g., an internal standard) and / or by diluting with another solution and / or by mixing with a reagent, etc. The method may include at least one sample preparation step. In the sample preparation step, the at least one chemically related substance (e.g., an internal standard) may be added to the sample of the analyte. The sample containing the analyte may be doped with a chemically related substance. For example, a chemically related substance may be added to the sample at a predefined concentration. The chemically related substance may be selected so that it can be easily identified under normal operating conditions of a mass spectrometer. The concentration of the chemically related substance (e.g., an internal standard) may be predetermined and significantly higher than the concentration of the analyte.

[0017] The method comprises the following steps, which can be performed in a given order as an example. However, it should be noted that different orders are also possible. In addition, one or more method steps can also be performed once or repeatedly. In addition, two or more method steps can be performed simultaneously or in a timely overlapping manner. The method may include other method steps not listed.

[0018] The method comprises the following steps:

[0019] a) searching at least one chromatogram of a chemically related substance and at least one chromatogram of an analyte;

[0020] b) evaluating the chromatogram of chemically related substances, wherein the evaluation comprises

[0021] b1) determining at least one initial value for the retention time of the analyte by determining the retention time of the chemically related substance and adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor,

[0022] b2) determining at least one initial value of a peak shape parameter of the analyte by determining a peak shape parameter of at least one peak of a chromatogram of a chemically related substance;

[0023] c) evaluating the chromatogram of the analyte, wherein the evaluating comprises

[0024] c1) at least one position determination step, wherein the analyte retention time is determined by taking into account initial values ​​of the retention times of chemically related substances;

[0025] c2) at least one peak integration step, wherein at least one fitting analysis is applied to the chromatogram of the analyte by taking into account initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape.

[0026] Method steps a) to c) can be performed fully automatically. For example, the method steps can be performed by at least one computer-implementable processing line which will be outlined in detail below.

[0027] Additional embodiments of the method include determining the error interval of the peak retention time and the peak shape parameters. In detail, the additional embodiment includes the following steps:

[0028] a) As described above

[0029] b) As described above, wherein the determination of the initial value of the analyte retention time comprises the determination of an error interval thereof.

[0030] c) as described above. In addition, the position determination step c1) comprises the additional requirement that the analyte position may deviate from the initial value by less than the analyte retention time error interval determined in step b1).

[0031] In addition, the peak integration step includes the analyte peak shape parameters that can deviate from the initial values ​​by less than step

[0032] Additional requirements for the error intervals of the peak shape parameters determined in b2).

[0033] As used herein, the term "retrieve at least one chromatogram" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, receiving, downloading, accessing, determining, detecting, and recording one or more of the at least one chromatogram. For example, the chromatogram may be retrieved by downloading and / or accessing the chromatogram from at least one database (such as a database of a detector or a database of a cloud). For example, the chromatogram may be retrieved by performing at least one chromatographic run. The chromatogram of a chemically related substance may be a chromatogram comprising at least one signal corresponding to a chemically related substance (e.g., an internal standard). The chromatogram of an analyte may be a chromatogram comprising at least one signal corresponding to a target analyte.

[0034] The method includes evaluating a chromatogram of chemically related substances. The method may include at least one preprocessing step. The preprocessing may include one or more of the following: selecting at least one target region in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, a Savitzky-Golay smoothing, and a Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0035] This assessment includes determining at least one initial value of the analyte retention time by determining the retention time of chemically related substances. For each sample, the peak retention time of the chemically related substances and / or additional peak shape parameters, such as peak width and / or peak asymmetry, can be determined. Due to the measurement principle of mass spectrometry, the retention time of the analyte can be the same as its corresponding chemically related substance, or can be calculated according to the chemically related substance, such as by adding a constant difference and / or multiplying by a constant factor. Therefore, even if the retention time is shifting, the analyte peak can also be correctly identified using the chemically related substance retention time and (if applicable) some constant shifts and / or factors.

[0036] Chemically related substances added to the sample at predefined concentrations can be readily identified under normal operating conditions of the mass spectrometer. The method may include performing at least one error check in which the chemically related substance signal is tested. The error check can be used to ensure that the correct peak is interpreted as a chemically related substance signal.

[0037] As used herein, the term "initial value of analyte retention time" is a broad term and is given the ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or customized meaning. The term may specifically refer to, but is not limited to, a starting value and / or input value for determining the analyte retention time from a chromatogram of the analyte.

[0038] In an additional embodiment of the method, the initial value of the analyte retention time may also include a corresponding error interval. The term "error interval" is a broad term and is given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or customary meaning. The term specifically may refer to, but is not limited to, a confidence interval for the analyte retention time determination result.

[0039] In order to determine the initial value of the analyte retention time, the retention time of chemical related substances can be determined first. This can include determining at least one local intensity maximum in a predefined retention time interval. The predefined retention time interval for determining the retention time of chemical related substances can depend on one or more of the following items: the expected retention time of chemical related substances, the chromatographic peak width of chemical related substances, the chemical similarity level of chemical related substances and target analyte (such as whether deuterated, C13 labeled or other chemical related substances are used) and the chromatographic noise level. The retention time of the determined local intensity maximum can be determined from the chromatogram and can be used as the initial value of the analyte retention time after adding a constant offset and / or multiplying by a constant factor. The determination of the local intensity maximum can be performed by considering the predefined interval of the quantitative factor and the qualitative factor ratio at the maximum position. Specifically, the local maximum value corresponding to the peak related to the target analyte is identified due to the quantitative factor and the qualitative factor ratio having a characteristic value close to the target analyte. As used herein, the term "quantitative factor and qualitative factor ratio" is a broad term and is given to ordinary and customary meanings for those of ordinary skill in the art, and is not limited to special or customized meanings. The term specifically refers to, but is not limited to, the ratio of the signal of the ion used for quantitative and the signal of the ion used for identity confirmation. Additionally or alternatively, the determination of the retention time of chemically related substances may include determining a pattern of multiple local maxima within a predefined retention time interval, wherein the pattern has a predefined distance between the maxima. Such peak patterns may be features of certain target analytes measured with a given LC-MS measurement scheme, and therefore may be used to distinguish peaks associated with the target analyte from peaks associated with other substances. The determination of the pattern of multiple local maxima may be performed considering the predefined interval of the quantitative factor and qualitative factor ratio at the maximum position.

[0040] The evaluation includes adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor. As used herein, the term "constant offset" is a broad term and is given a common and customary meaning to one of ordinary skill in the art and is not limited to a special or customary meaning. The term specifically may refer to, but is not limited to, an additional retention time added to the determined retention time of the chemically related substance. For example, the retention time of the chemically related substance may be t1, where the initial value of the analyte retention time t ini Can be obtained by ini =t1+t off To determine, where t off is a constant offset. In one embodiment of the method, the initial value of the analyte retention time t ini Can be obtained by ini =t1+t off ±ε to determine, where toff is a constant offset, and ε represents the error interval of the analyte retention time.

[0041] The constant offset can be predetermined or predefined by comparing the determined retention time of a chemically related substance with at least one predicted or expected retention time of the chemically related substance. The predicted or expected retention time of a chemically related substance can be a known retention time of the chemically related substance (e.g., a predefined or predetermined retention time under certain conditions, particularly temperature conditions). For example, the comparison can include subtracting the determined retention time of the chemically related substance from the predicted or expected retention time. The deviation of the predicted or expected retention time from the determined retention time of the chemically related substance can be used as a constant offset. As used herein, the term "constant factor" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customized meaning. The term specifically may refer to, but is not limited to, an additional retention time, which is applied to the determined retention time of the chemically related substance by multiplication. For example, the retention time of a chemically related substance can be t1, where the initial value t of the analyte retention time is t2. ini Can be obtained by ini =t1*t fac To determine, where t fac is a constant factor. In one embodiment of the method, the initial value of the analyte retention time t ini Can be obtained by ini =(t1*t fac )±ε, where t fac is a constant factor, and ε represents the error interval of the analyte retention time.

[0042] Can be determined by comparing the determined retention time of chemical related material with at least one prediction or expected retention time of chemical related material to predetermine or predefine constant factor.The prediction or expected retention time of chemical related material can be the known retention time of chemical related material (for example, under certain conditions, particularly the predefined or predetermined retention time under temperature conditions).For example, this comparison can include generating at least one ratio of the determined retention time of chemical related material and the predicted or expected retention time.The ratio of the predicted or expected retention time and the determined retention time of chemical related material can be used as constant factor.For example, due to external factors (such as the limited accuracy of the gradient setting in the instrument temperature change or chromatographic method) and the small retention time offset that occurs, using constant offset may obtain satisfactory results.For the effect (such as LC column aging) that may cause larger retention time offset, compared with the application constant offset, using constant factor may provide more superior performance.As outlined above, determining the initial value of analyte retention time can include determining at least one local intensity maximum in the predefined retention time interval. The initial value of the analyte retention time may be determined by adjusting the predefined retention time interval according to the determined local maximum corrected by a predetermined or predefined constant offset and / or a predetermined or predefined constant factor.

[0043] The evaluation of the chromatogram of chemically related substances includes: by determining the peak shape parameters of at least one peak of the chromatogram of chemically related substances, at least one initial value of the analyte peak shape parameters is determined. Similar to the peak retention time, the measurement principle of the mass spectrometer may cause the analyte peak shape to be similar to the chemically related substance peak shape, due to the similarity of the two compounds. As outlined above, the chemically related substance signal is well controlled because the compound is added to the sample at a predetermined and relatively high concentration, thereby ensuring reliable peak shape fitting results. The result of the chemically related substance shape can be used as an input for subsequent shape determination of the target analyte, and due to the known similarity of the chemically related substances and the analyte peak shape, a tight constraint can be imposed on the deviation from the input. As used herein, the term "initial value of the analyte peak shape parameter" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically refers to, but is not limited to, the starting value and / or input value for determining the analyte peak shape from the chromatogram of the analyte.

[0044] In additional embodiments of the method, the initial value of the initial value of the analyte peak shape parameter may also include a corresponding error interval. The term "error interval" is a broad term and is given its ordinary and customary meaning to one of ordinary skill in the art and is not limited to a special or customary meaning. The term specifically may refer to, but is not limited to, a confidence interval for determining the initial value of the analyte peak shape parameter.

[0045] Determining at least one initial value of the analyte peak shape parameter may include fitting at least one peak of a chemically related substance chromatogram. The fitting may include using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc. For example, at least one exponentially corrected Gaussian function may be used to fit a chromatogram of a chemically related substance. For example, the fitting may include using at least one fitting function, such as "Mathematical functions for the representation of chromatographic peaks", Valerio B. DiMarco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1–30. Using one or more of these curve fitting techniques, a peak is fitted for a chemically related substance chromatogram. According to the curve fitting technique, peak parameters of a chemically related substance peak may be determined. Peak parameters may include one or more of peak width, peak asymmetry, peak position, baseline, etc. Peak parameters determined from chromatograms of chemically related substances can be used to estimate peak parameters of analyte peaks by extending the peak parameters of the analyte peaks to an interval having a so-called tolerance interval.

[0046] The method may include determining at least one tolerance interval for the corresponding peak parameter. The tolerance interval p ± ε may be given by the corresponding determined initial value p and the error interval ε of the analyte peak shape parameter. The error interval may depend on the resolution of the detector, the fitting error, and the level of chemical similarity between the chemically related substance and the target analyte, such as whether deuterated, C13 labeling or other chemically related substances are used. The tolerance intervals for different peak shape parameters may be different.

[0047] In one embodiment of the method, the error interval ε of the analyte peak shape parameters may be set to the error interval of the peak shape parameters of the chemically related substances.

[0048] The method includes evaluating a chromatogram of an analyte. The evaluation of the chromatogram of the analyte may include at least one preprocessing step. The preprocessing may include one or more of the following: selecting at least one target region in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, a Savitzky-Golay smoothing, and a Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, a discrete or continuous wavelet basis background determination, and determining a moving average minimum. The preprocessing may be the same or different from the preprocessing applied to the chromatogram of the chemically related substance.

[0049] The evaluation includes at least one position determination step, wherein the analyte retention time is determined by considering the initial value of the retention time of the chemically related substances. The analyte retention time can be determined by using at least one curve fitting technique. The initial value of the analyte retention time can be used as the starting value of the curve fitting. Given the initial value of the analyte retention time determined from the chromatogram of the chemically related substances, the analyte retention time can be determined, for example, by using at least one curve fitting technique. The fitting may include using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc. For example, the fitting may include using at least one fitting function, such as "Mathematical functions for the representation of chromatographic peaks", Valerio B. DiMarco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30. The analyte retention time can be determined using a narrow tolerance interval near the initial value of the retention time. For example, the tolerance interval t ± ε t The initial value t and the error interval ±ε are determined by the corresponding retention time of the analyte peak. t To give.

[0050] In one embodiment of the method, the error interval ε of the analyte retention time t Can be set as an error interval for the retention time of chemically related substances. The error interval may depend on the resolution of the detector, the chromatographic peak widths of the target analyte and the chemically related substances, the level of chemical similarity between the chemically related substances and the target analyte (e.g. whether deuterated, C13 labeled or other chemically related substances are used), and the chromatographic noise level. The width of the allowed interval t±ε t Typically this may be smaller than the chromatographic peak width of the target analyte.

[0051] The evaluation further includes at least one peak integration step, wherein at least one fitting analysis is applied to the chromatogram of the analyte by considering the initial values ​​of the analyte peak shape parameters and the analyte retention time, thereby determining the analyte peak area and the analyte peak shape. Using the determined analyte retention time, and the initial values ​​of the analyte peak shape parameters, a curve can be fitted to the chromatogram of the analyte to determine the analyte peak area and peak shape. In the peak integration step, at least one exponentially corrected Gaussian function can be used to fit the analyte chromatogram. For example, the fitting can include using at least one fitting function, such as "Mathematical functions for the representation of chromatographic peaks", Valerio B. Di Marco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30. The analyte peak area and the analyte peak shape can be determined using the tolerance interval p ± ε near the initial values ​​of the corresponding analyte peak shape parameters, as described above.

[0052] Using retention time and peak shape parameters of chemically related substances for evaluating analyte chromatograms may lead to more accurate analyte peak area determinations, especially in cases where analyte concentrations are low, backgrounds are complex, noise levels are high, and interfering peaks are present. Thus, the method according to the present invention enables reliable automation of peak integration without the need for expert user corrections and manual adjustments.

[0053] In addition to using internal standard chromatograms, other fragment parameters (such as quantitation factors of the analyte and / or internal standard) can also be used for quantitation and / or error checking.

[0054] On the other hand, a computer program is disclosed, which includes computer executable instructions for executing a method according to any one of the embodiments described herein (specifically method steps a) to c)) when the program is executed on a computer or a computer network (specifically on a processor).

[0055] Therefore, in general, a computer program is disclosed and proposed herein, which includes computer executable instructions for executing the method according to the present invention in one or more embodiments attached herein when the program is executed on a computer or a computer network. Specifically, the computer program can be stored on a computer readable data carrier. Therefore, specifically, one, more than one or even all method steps as indicated above can be performed by using a computer or a computer network, preferably by using a computer program. Specifically, the computer can be fully or partially integrated into a mass spectrometer, and the computer program can be specifically embodied as software. However, alternatively, at least a portion of the computer can also be located outside the mass spectrometer.

[0056] This document further discloses and proposes a computer program product with program code means, so that when the program is executed on a computer or a computer network, the method according to the present invention, such as one or more method steps mentioned above, is performed in one or more embodiments attached hereto. Specifically, the program code means can be stored on a computer readable data carrier.

[0057] The present invention further discloses and proposes a data carrier having a data structure stored thereon, which, after being loaded into a computer or a computer network, such as into a working memory or a main memory of the computer or the computer network, can execute a method according to one or more embodiments disclosed herein, specifically one or more method steps mentioned above.

[0058] This article further discloses and proposes a computer program product with a program code tool stored on a machine-readable carrier, so that when the program is executed on a computer or a computer network, the method according to one or more embodiments disclosed herein, specifically one or more method steps mentioned above, is executed. As used herein, a computer program product refers to a program as a tradable product. The product can generally exist in any format (such as in paper format) or on a computer-readable data carrier. Specifically, the computer program product can be distributed on a data network.

[0059] Finally, this document discloses and proposes a modulated data signal, which contains instructions that can be read by a computer system or a computer network, and is used to execute the method according to one or more embodiments disclosed in this document, specifically one or more method steps mentioned above.

[0060] Specifically, this article further discloses:

[0061] - a computer or a computer network comprising at least one processor, wherein the processor is adapted to execute the method according to one of the embodiments described in this specification,

[0062] - a computer loadable data structure, which is suitable for executing a method according to one of the embodiments described in this specification when the data structure is executed on a computer,

[0063] - a computer program, wherein the computer program is adapted to perform a method according to one of the embodiments described in this description when the program is executed on a computer,

[0064] - a computer program comprising program means for carrying out the method according to one of the embodiments described in the present description when the computer program is executed on a computer or on a computer network,

[0065] a computer program comprising program means according to the aforementioned embodiments, wherein these program means are stored on a computer-readable storage medium,

[0066] a storage medium, on which a data structure is stored and on which the data structure is suitable, after being loaded into a main memory and / or a working memory of a computer or a computer network, for executing a method according to one of the embodiments described in the present description, and

[0067] - A computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium for executing the method according to one of the embodiments described in this description when the program code means are executed on a computer or a computer network.

[0068] In another aspect of the present invention, a computer-implemented processing line for automatic peak integration of at least one chromatogram of at least one sample is disclosed. The sample comprises at least one analyte and at least one chemically related substance. The processing line comprises

[0069] - at least one data collector configured to retrieve at least one chromatogram of chemically related substances and at least one chromatogram of the analyte;

[0070] - at least one evaluation device configured to evaluate the chromatogram of the chemically related substances and the chromatogram of the analyte,

[0071] wherein the evaluation device comprises at least one retention time processor configured to determine at least one initial value for the retention time of the analyte by determining the retention time of chemically related substances,

[0072] wherein the evaluation device comprises at least one adder configured to add the retention time of the chemically related substances to a predetermined or predefined constant offset and / or at least one multiplier configured to multiply the retention time of the chemically related substances by a predetermined or predefined constant factor,

[0073] wherein the evaluation device comprises at least one position determination device configured to determine the analyte retention time by taking into account initial values ​​of retention times of chemically related substances,

[0074] wherein the evaluation device further comprises at least one peak processor configured to determine at least one initial value of the analyte peak shape parameter by determining a peak shape parameter of at least one peak of the chromatogram of the chemically related substances,

[0075] The evaluation device comprises at least one fitting device configured to apply at least one fitting analysis to the chromatogram of the analyte by considering initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape.

[0076] The processing line may be configured to perform the method according to any of the aforementioned embodiments.For embodiments, terms used herein and possible definitions, reference may be made to the description of the above method.

[0077] As used herein, the term "computer-implemented processing line" is a broad term and is given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or customized meaning. The term may specifically refer to, but is not limited to, a set of sequential operations and / or devices (such as computing devices, processors, etc.) to perform automatic peak integration. The processing line may be configured to perform the method according to the present invention. The processing line may be configured to fully automatically perform method steps a) to c).

[0078] As used herein, the term "data collector" is a broad term and is given a common and customary meaning to one of ordinary skill in the art and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, at least one database configured to receive and / or store at least one chromatogram of a chemically related substance and at least one chromatogram of an analyte. The receiving and / or storing of the chromatograms of the chemically related substance and the analyte may be performed simultaneously or sequentially.

[0079] As further used herein, the term "evaluation device" generally refers to any device suitable for performing the method steps as described above, preferably by using at least one data processing device, and more preferably by using at least one processor and / or at least one application specific integrated circuit. Thus, as an example, the at least one evaluation device may include at least one data processing device having software code stored thereon, the software code comprising a plurality of computer commands. The evaluation device may provide one or more hardware elements for performing one or more specified operations, and / or may provide one or more processors with software for running thereon for performing one or more method steps.

[0080] As outlined above, the evaluation device includes a retention time processor. As used herein, the term "retention time processor" is a broad term and is given to ordinary and customary meanings for those of ordinary skill in the art, and is not limited to special or custom meanings. The term may specifically refer to, but is not limited to, a processor that is configured to perform the determination of the retention time of a chemically related substance, particularly the determination of the local maximum value in the chromatogram of a chemically related substance. The retention time processor may be configured to determine at least one local intensity maximum value in a predefined retention time interval for determining the retention time of a chemically related substance. For example, the retention time processor may be configured to determine the local intensity maximum value by considering the predefined interval of the quantitative factor at the maximum position and the qualitative factor ratio. Additionally or alternatively, the retention time processor may be configured to determine the pattern of multiple local maximum values ​​in the predefined retention time interval for determining the retention time of a chemically related substance, wherein the pattern has a predefined distance between the maximum values. The retention time processor may be configured to determine the pattern of multiple local maximum values ​​by considering the predefined interval of the quantitative factor at the maximum position and the qualitative factor ratio, as described in detail above about the method.

[0081] As further outlined above, the evaluation device includes at least one adder and / or at least one multiplier, wherein the at least one adder is configured to add the retention time of the chemically related substances to a predetermined or predefined constant offset, and the at least one multiplier is configured to multiply the retention time of the chemically related substances by a predetermined or predefined constant factor. The adder and / or multiplier may be configured as a digital circuit or may be implemented as software.

[0082] The evaluation device includes a position determination device, which is configured to determine the analyte retention time by considering the initial value of the retention time of the chemically related substances. As used herein, the term "position determination device" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or customized meaning. The term may specifically refer to, but is not limited to, a device such as a processor, which is configured to determine the analyte retention time from the chromatogram of the analyte. The position determination device may be arranged after the retention time processor and the adder and / or multiplier. Additionally or alternatively, the position determination device may be integrated with the retention time processor and the design and / or evaluation device may include at least one processor, which is configured to perform two operations: determining the initial value of the analyte retention time and the analyte retention time. The position determination device may be configured to determine the analyte retention time by using at least one curve fitting technique, wherein the initial value of the analyte retention time is used as the starting value of the curve fitting. The curve fitting technique may include using at least one fitting selected from the group consisting of the following items: polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc.

[0083] As used herein, the term "peak processor" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, a processor configured to determine peak shape parameters of at least one peak. The peak processor may be configured to perform at least one fitting program. The peak processor may be configured to fit at least one peak of a chromatogram of a chemically related substance. The fitting may include using at least one curve fitting technique, such as one or more of a polynomial interpolation method, a Gauss-Newton algorithm, a Fourier transform, etc. The peak processor may be configured to determine at least one tolerance interval. The tolerance interval p ± ε may be given by the corresponding determined initial value p and the error interval ± ε of the analyte peak shape parameter.

[0084] The peak processor may be arranged after the retention time processor, the adder and / or the multiplier and the position determination device. Additionally or alternatively, the peak processor may be designed integrally with the retention time processor and / or the position determination device, and / or the evaluation device may include at least one processor configured to perform the following actions: determining an initial value of the analyte retention time and the analyte retention time, determining peak shape parameters.

[0085] As used herein, the term "fitting device" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, at least one processor configured to apply at least one fitting analysis to a chromatogram of an analyte by considering the initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape. For example, the fitting device may be configured to fit an analyte chromatogram using at least one exponentially corrected Gaussian function. For example, the fitting may include using at least one fitting function, as described in "Mathematical functions for the presentation of chromatographic peaks", Valerio B. Di Marco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30.

[0086] The evaluation device may also include at least one preprocessor. The preprocessor may be configured to preprocess the chromatogram of the chemically related substances and / or the chromatogram of the analyte by one or more of the following items: selecting at least one target area in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0087] In another aspect of the present invention, a mass spectrometry system is disclosed. As used herein, the term "system" is a broad term and is given a common and customary meaning to a person of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically may refer to, but is not limited to, a device comprising at least two elements. The elements may interact functionally, for example, for performing a method according to the present invention.

[0088] Mass spectrometry systems include

[0089] - at least one mass spectrometer, said at least one mass spectrometer comprising at least one mass filter and at least one detector;

[0090] - at least one computer implementable processing line according to the invention.

[0091] For embodiments, terms used herein and possible definitions, reference is made to the description of the above-mentioned method and computer-implementable processing line.

[0092] The mass spectrometer may be or may include at least one liquid chromatography mass spectrometer. The mass spectrometer may include at least one chromatograph. As used herein, the term "mass filter" is a broad term and is given a common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, at least one device configured to separate the components of a sample according to the mass of the components of the sample. As used herein, the term "detector" is a broad term and is given a common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term may specifically refer to, but is not limited to, at least one device configured to detect incoming particles and to determine the at least one chromatogram.

[0093] The mass spectrometry system may also include at least one sample preparation device. As used herein, the term "sample preparation device" is a broad term and is given a common and customary meaning to one of ordinary skill in the art, and is not limited to a special or customary meaning. The term may specifically refer to, but is not limited to, a device configured to prepare a sample for subsequent analysis. The sample preparation device may be configured to add at least one chemically related substance to a sample of an analyte.

[0094] The method and apparatus according to the invention may provide a number of advantages over known methods and apparatus for peak integration. In particular, the method and apparatus allow reliable automatic peak integration without the need for expert user correction or manual adjustments.

[0095] To summarize and without excluding further possible embodiments, the following embodiments may be envisaged:

[0096] Embodiment 1: A computer-implemented method for automatic peak integration of at least one chromatogram of at least one sample, wherein the sample comprises at least one analyte and at least one chemically related substance, wherein the method comprises the following steps:

[0097] a) searching at least one chromatogram of a chemically related substance and at least one chromatogram of an analyte;

[0098] b) evaluating the chromatogram of chemically related substances, wherein the evaluation comprises

[0099] b1) determining at least one initial value for the retention time of the analyte by determining the retention time of the chemically related substance and adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor,

[0100] b2) determining at least one initial value of a peak shape parameter of the analyte by determining a peak shape parameter of at least one peak of a chromatogram of a chemically related substance;

[0101] c) evaluating the chromatogram of the analyte, wherein the evaluating comprises

[0102] c1) at least one position determination step, wherein the analyte retention time is determined by taking into account initial values ​​of the retention times of chemically related substances;

[0103] c2) at least one peak integration step, wherein at least one fitting analysis is applied to the chromatogram of the analyte by taking into account initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape.

[0104] Embodiment 2: The method according to any one of the preceding embodiments, wherein method steps a) to c) are performed fully automatically.

[0105] Embodiment 3: The method according to any of the preceding embodiments, wherein the determination of the retention times of chemically related substances comprises determining at least one local intensity maximum within a predefined retention time interval.

[0106] Embodiment 4: The method according to the preceding embodiment, wherein the determination of the local intensity maximum is performed taking into account a predefined interval of the ratio of the quantitative factor to the qualitative factor at the maximum position.

[0107] Embodiment 5: A method according to any one of the preceding embodiments, wherein determining the retention time of chemically related substances comprises determining a pattern of multiple local maxima within a predefined retention time interval, wherein the pattern has a predefined distance between the maxima.

[0108] Embodiment 6: The method according to the preceding embodiment, wherein the determination of the pattern of the plurality of local maxima is performed taking into account a predefined interval of the ratio of the quantitative factor to the qualitative factor at the maximum position.

[0109] Embodiment 7: A method according to any one of the four preceding embodiments, wherein the initial value of the analyte retention time is determined by adjusting a predefined retention time interval according to a determined local maximum corrected by a predetermined or predefined constant offset and / or a predetermined or predefined constant factor.

[0110] Embodiment 8: A method according to any one of the preceding embodiments, wherein the analyte retention time t is determined using a tolerance interval determined from a chromatogram of chemically related substances as a domain, wherein the tolerance interval t±ε of the analyte peak retention time t includes an error interval ε of the analyte peak retention time, which is set to the corresponding error interval of the peak retention time of the chemically related substance.

[0111] Embodiment 9: A method according to any one of the preceding embodiments, wherein determining the at least one initial value of the analyte peak shape parameter comprises fitting at least one peak of a chromatogram of a chemically related substance, wherein the fitting comprises using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc.

[0112] Embodiment 10: The method according to any one of the preceding embodiments, wherein the analyte retention time is determined by using at least one curve fitting technique, wherein the initial value of the analyte retention time is used as a starting value for the curve fitting.

[0113] Embodiment 11: The method according to the preceding embodiment, wherein the curve fitting technique comprises using at least one fitting selected from the group consisting of: polynomial interpolation, Gauss-Newton algorithm, Fourier transform, and the like.

[0114] Embodiment 12: The method according to any of the preceding embodiments, wherein the method comprises determining at least one tolerance interval, wherein the tolerance interval p±ε is given by the corresponding determined initial value p of the analyte peak shape parameter and the error interval ±ε.

[0115] Embodiment 13: The method according to the above embodiment, wherein the error interval ±ε of the peak shape parameters of the analyte is set to the error interval of the peak shape parameters of the chemically related substances.

[0116] Embodiment 14: The method according to any one of the preceding embodiments, wherein in the peak integration step, at least one exponentially modified Gaussian function is used to fit the analyte chromatogram.

[0117] Embodiment 15: A method according to any one of the preceding embodiments, wherein the evaluation of the chromatograms of chemically related substances and / or the chromatograms of analytes includes at least one preprocessing step, wherein the preprocessing includes one or more of the following items: selecting at least one target area in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0118] Embodiment 16: The method according to any one of the preceding embodiments, wherein the method comprises at least one sample preparation step, wherein the at least one chemically related substance is added to a sample of the analyte.

[0119] Example 17: A computer-implemented processing line for automated peak integration of at least one chromatogram of at least one sample, wherein the sample comprises at least one analyte and at least one chemically related substance, wherein the processing line comprises

[0120] - at least one data collector configured to retrieve at least one chromatogram of chemically related substances and at least one chromatogram of the analyte;

[0121] - at least one evaluation device configured to evaluate the chromatogram of the chemically related substances and the chromatogram of the analyte,

[0122] wherein the evaluation device comprises at least one retention time processor configured to determine at least one initial value for the retention time of the analyte by determining the retention time of chemically related substances,

[0123] wherein the evaluation device comprises at least one adder configured to add the retention time of the chemically related substances to a predetermined or predefined constant offset and / or at least one multiplier configured to multiply the retention time of the chemically related substances by a predetermined or predefined constant factor,

[0124] wherein the evaluation device comprises at least one position determination device configured to determine the analyte retention time by taking into account initial values ​​of retention times of chemically related substances,

[0125] wherein the evaluation device further comprises at least one peak processor configured to determine at least one initial value of the analyte peak shape parameter by determining a peak shape parameter of at least one peak of the chromatogram of the chemically related substances,

[0126] The evaluation device comprises at least one fitting device configured to apply at least one fitting analysis to the chromatogram of the analyte by considering initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape.

[0127] Embodiment 18: The processing line according to the preceding embodiment, wherein the processing line is configured to perform a method according to any of the preceding embodiments relating to methods.

[0128] Embodiment 19: A processing line according to the preceding embodiment, wherein the processing line is configured to fully automatically perform method steps a) to c).

[0129] Embodiment 20: The processing line according to any of the preceding embodiments relating to processing lines, wherein the retention time processor is configured for determining at least one local intensity maximum within a predefined retention time interval for determining retention times of chemically related substances.

[0130] Embodiment 21: The processing line according to the preceding embodiment, wherein the retention time processor is configured to determine the local intensity maximum by considering a predefined interval of the ratio of the quantifier to the qualifier at the position of the maximum.

[0131] Embodiment 22: A processing line according to any one of the preceding embodiments involving processing lines, wherein the retention time processor is configured to determine a pattern of multiple local maxima within a predefined retention time interval for determining the retention time of chemically related substances, wherein the pattern has a predefined distance between the maxima.

[0132] Embodiment 23: The processing line according to the preceding embodiment, wherein the retention time processor is configured to determine the pattern of the plurality of local maxima by considering a predefined interval of the ratio of the quantifier to the qualifier at the maximum position.

[0133] Embodiment 24: A processing line according to the preceding embodiments, wherein the evaluation device is configured to determine an initial value of the analyte retention time by adjusting a predefined retention time interval according to a determined local maximum corrected by a predetermined or predefined constant offset and / or a predetermined or predefined constant factor.

[0134] Embodiment 25: A processing line according to any one of the preceding embodiments involving processing lines, wherein the peak processor is configured to fit at least one peak of a chromatogram of a chemically related substance, wherein the fitting comprises using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc.

[0135] Embodiment 26: The processing line according to any of the preceding embodiments relating to processing lines, wherein the position determination device is configured for determining the analyte retention time by using at least one curve fitting technique, wherein the initial value of the analyte retention time is used as a starting value for the curve fitting.

[0136] Embodiment 27: The processing line according to the preceding embodiment, wherein the curve fitting technique comprises using at least one fitting selected from the group consisting of: polynomial interpolation, Gauss-Newton algorithm, Fourier transform, and the like.

[0137] Embodiment 28: A processing line according to any of the preceding embodiments relating to processing lines, wherein the peak processor is configured to determine at least one tolerance interval, wherein the tolerance interval p±ε is given by the corresponding determined initial value p of the analyte peak shape parameter and the error interval ±ε.

[0138] Embodiment 29: The processing line according to any of the preceding embodiments relating to processing lines, wherein the fitting device is configured for fitting the analyte chromatogram using at least one exponentially modified Gaussian function.

[0139] Embodiment 30: A processing line according to any one of the foregoing embodiments involving processing lines, wherein the evaluation device includes at least one preprocessor, wherein the preprocessor is configured to evaluate the chromatograms of preprocessed chemical-related substances and / or the chromatograms of analytes by one or more of the following items: selecting at least one target area in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0140] Embodiment 31: A mass spectrometry system comprising

[0141] - at least one mass spectrometer, said at least one mass spectrometer comprising at least one mass filter and at least one detector;

[0142] - At least one computer implementable processing line according to any of the preceding embodiments relating to a processing line.

[0143] Embodiment 32: The mass spectrometry system according to the preceding embodiment, wherein the mass spectrometry system further comprises at least one sample preparation device, wherein the sample preparation device is configured to add at least one chemically related substance to the sample of the analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Preferably in conjunction with the dependent claims, further optional features and embodiments will be disclosed in more detail in the subsequent description of embodiments. Wherein, as will be appreciated by those skilled in the art, each optional feature can be implemented in a separate manner and in any arbitrary feasible combination. The scope of the present invention is not limited by the preferred embodiments. Embodiments are schematically depicted in the accompanying drawings. Wherein, the same reference numerals in these drawings refer to the same or functionally equivalent elements.

[0145] In the attached picture:

[0146] Figure 1 A comparison of methods known in the art for automatic peak integration and integration using manual inspection is shown;

[0147] FIG. 2A to FIG. 2C Chromatograms of the analytes without and with retention time adjustment are shown ( Figure 2A and 2B ) and chromatograms of chemically related substances ( Figure 2C ) peak integration;

[0148] Figure 3 A flowchart of a computer-implemented method according to the present invention is shown;

[0149] FIG. 4A to FIG. 4C The use of chemically related substance peak features for analyte quantification is shown;

[0150] Figure 5A and Figure 5B The chromatograms of chemically related substances with fitted peak shapes are shown ( Figure 5A ) and the resulting peak fits of the analyte chromatograms ( Figure 5B );as well as

[0151] Figure 6 An embodiment of a mass spectrometry system according to the present invention is shown. DETAILED DESCRIPTION

[0152] Figure 1 A comparison of the method for automatic peak integration known in the art using a fixed retention time window (box) and true peak integration using manual inspection is shown. For lower analyte concentrations, the automated method using a fixed retention time window can lead to incorrect quantification. The erroneous results are caused by the integration of incorrect peaks due to sample-specific retention time shifts. In addition, Figure 1 A comparison of the method for automatic peak integration according to the invention and the true peak integration using manual inspection (circles) is shown. The method according to the invention is used to ensure that the correct signal is picked up and the area ratio is quantified correctly.

[0153] Figures 2A to 2C An example of the effect of adjusting the retention time (RT) of an internal standard (ISTD) based on its RT is shown. Figure 2A and 2B 1 shows the peak integration of an exemplary analyte chromatogram, with retention time RT plotted on the x-axis and intensity I plotted on the y-axis. Line 112 is at Figure 2A and 2B The pre-processed chromatogram of the analyte is shown in Figure 2CThe pre-processed chromatogram of the ISTD is shown in FIG. Line 110 represents the integrated area. The vertical dashed line 113 represents the correct position of the analyte peak. Although the integration without retention time adjustment cannot capture the correct peak, as shown in FIG. Figure 2A , but using the internal standard retention time as input for adjusting the analyte retention time results in correct integration of the analyte peak, see Figure 2B .

[0154] Figure 3 A flow chart of an exemplary embodiment of a computer-implemented method for automatic peak integration of at least one chromatogram of at least one sample according to the present invention is shown. The method can be performed completely automatically (specifically, without user interaction). The method includes the following steps, which can be performed in a given order as an example. However, it should be noted that different orders are also possible. In addition, one or more method steps can also be performed once or repeatedly. In addition, two or more method steps can be performed simultaneously or in a timely overlapping manner. The method may include other method steps that are not listed.

[0155] The method comprises the following steps:

[0156] a) retrieving at least one chromatogram of chemically related substances (indicated by reference numeral 114) and at least one chromatogram of the analyte (indicated by reference numeral 116);

[0157] b) evaluating the chromatogram of chemically related substances, wherein the evaluation comprises

[0158] b1) by determining the retention time of chemically related substances (indicated by reference numeral 118)

[0159] and adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor (indicated by reference numeral 120) to determine at least one initial value for the retention time of the analyte (indicated by reference numeral 117),

[0160] b2) determining at least one initial value (indicated by reference numeral 122) of a peak shape parameter of the analyte by determining a peak shape parameter (indicated by reference numeral 121) of at least one peak of the chromatogram of the chemically related substance;

[0161] c) evaluating the chromatogram of the analyte, wherein the evaluating comprises

[0162] c1) at least one position determination step (indicated by reference numeral 124), in which the analyte retention time is determined by taking into account initial values ​​of the retention times of chemically related substances;

[0163] c2) at least one peak integration step (indicated by reference numeral 126), wherein at least one fitting analysis is applied to the chromatogram of the analyte by taking into account initial values ​​of analyte peak shape parameters and analyte retention time, thereby determining analyte peak area and analyte peak shape.

[0164] The chromatogram can be determined by using at least one mass spectrometry device 128 (e.g., at least one liquid chromatography mass spectrometry device). The liquid chromatography mass spectrometry device can be or can include at least one high performance liquid chromatography (HPLC) device or at least one microfluidic liquid chromatography (μLC) device. The liquid chromatography mass spectrometry device can include a liquid chromatography (LC) device and a mass spectrometry (MS) device, wherein the LC device and the MS are coupled via at least one interface. The liquid chromatography (LC) device can be configured to separate one or more target analytes of a sample from other components of the sample for use in detecting the one or more analytes using the mass spectrometry device 128. The LC device can include at least one LC column. The mass spectrometry device 128 can be configured to detect at least one analyte based on a mass-to-charge ratio. The mass spectrometry device 128 can be or can include at least one quadrupole mass spectrometry device. The interface coupling the LC device and the MS can include at least one ionization source configured to generate molecular ions and to transfer the molecular ions to the gas phase.

[0165] Integration of peaks may include identification and / or measurement of curve features of a chromatogram. Peak integration may include one or more of the following: peak detection, peak finding, peak identification, peak fitting, peak evaluation, determining peak start and / or peak end, determining background, determining baseline. Peak integration may allow determination of one or more of the following: peak area, retention time, peak height, and peak width. Peak integration may be non-manual but performed by at least one computer and / or at least one computer network and / or at least one machine. In particular, automatic peak integration may be performed without manual operation or interaction with a user. In particular, peak identification and / or peak detection and / or determination of peak area may be performed non-manually and without manual operation or interaction with a user.

[0166] The sample can be selected from the group consisting of the following items: physiological fluids, including blood, serum, plasma, saliva, lens fluid, cerebrospinal fluid, sweat, urine, emulsion, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, etc. The sample can be used directly when obtained from the corresponding source, or can be pre-treated and / or sample preparation workflow. The sample contains at least one analyte and at least one chemically related substance. For example, in general, the target analyte can be vitamin D, drugs of abuse, therapeutic drugs, hormones and metabolites. The chemically related substance can be a substance with a mass-to-charge ratio similar to the target analyte. For example, the chemically related substance can be at least one internal standard and / or at least one substance used as a quantitative factor. Even for low intensity peaks, high noise levels or frequent interferences, the method according to the present invention also allows reliable peak integration. Specifically, the method proposes the use of a chromatogram for evaluating a target analyte input value or a starting value, which is determined from other chromatograms of chemically related substances of the same measurement, and for these other chromatograms, sufficiently high intensity peaks, low noise levels and low frequency interferences can be guaranteed. For example, the method can be used to enhance the integration of the analyte chromatogram based on the result obtained from the internal standard chromatogram. For example, the method can be used to enhance the integration of the qualitative factor chromatogram based on the result obtained from the quantitative factor chromatogram. The internal standard sample can be a sample comprising at least one internal standard substance with a known (e.g., predetermined) concentration. The internal standard can be almost the same as the analyte. The internal standard substance can be similar to the isotope labeled form of the target analyte (e.g., deuterated molecule) or the target analyte. For more details about the sample, reference is made to, for example, EP 3425369A1, the entire disclosure of which is included herein by reference. Other target analytes are also possible.

[0167] The sample can be pre-treated by adding chemical related substances and / or by diluting with another solution and / or by mixing with reagents etc. The method may include at least one sample preparation step. In the sample preparation step, the at least one chemical related substance may be added to the sample of the analyte. The sample comprising the analyte may be doped with chemical related substances. Chemical related substances may be added to the sample at a predefined concentration. Chemical related substances may be selected so that it can be easily identified under normal operating conditions of a mass spectrometer. The concentration of chemical related substances may be predetermined and significantly higher than the concentration of the analyte.

[0168] Retrieving at least one chromatogram 114 and 116 may include receiving, downloading, accessing, determining, detecting, and recording one or more of the at least one chromatogram. For example, the chromatogram may be retrieved by downloading and / or accessing the chromatogram from at least one database (such as a database of the detector or a database of the cloud). For example, the chromatogram may be retrieved by performing at least one chromatographic run. The chromatogram of the chemically related substance may be a chromatogram that includes at least one signal corresponding to the chemically related substance. The chromatogram of the analyte may be a chromatogram that includes at least one signal corresponding to the target analyte.

[0169] The method may include at least one preprocessing step 130, for example, to preprocess one or both of the chromatogram of the chemically related substance or the chromatogram of the analyte. The preprocessing 130 may include one or more of the following: selecting at least one target region in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0170] In step b), the assessment includes determining at least one initial value 117 of the analyte retention time by determining the retention time 118 of a chemically related substance. For each sample, the peak retention time of the chemically related substance and / or additional peak shape parameters, such as peak width and / or peak asymmetry, may be determined. Due to the measurement principle of the mass spectrometer, the retention time of the analyte may be the same as its corresponding chemically related substance, or may be calculated based on the chemically related substance, for example by adding a constant difference and / or multiplying by a constant factor. Therefore, even if the retention time is shifting, the analyte peak may be correctly identified using the chemically related substance retention time and (if applicable) some constant shifts and / or factors.

[0171] Chemically related substances added to the sample at predefined concentrations can be easily identified under normal operating conditions of the mass spectrometry device 128. The method may include performing at least one error check in which the chemically related substance signal is tested. The error check can be used to ensure that the correct peak is interpreted as a chemically related substance signal.

[0172] The initial value of the analyte retention time can be a starting value and / or input value for determining the analyte retention time 124 from the chromatogram of the analyte. In order to determine the initial value 117 of the analyte retention time, the retention time 118 of the chemically related substance can be determined first. This can include determining at least one local intensity maximum in a predefined retention time interval. The predefined retention time interval for determining the retention time 118 of the chemically related substance can depend on one or more of the following items: the expected retention time of the chemically related substance, the resolution of the detector, and the expected retention time offset range due to the aging of the chromatographic column used for measurement and other factors that may affect the robustness of the chromatographic measurement. The retention time of the determined local intensity maximum can be determined from the chromatogram and can be used as the initial value of the analyte retention time after adding a constant offset and / or multiplying by a constant factor 120. The determination of the local intensity maximum can be performed by considering the predefined interval of the quantitative factor and the qualitative factor ratio at the maximum position. Due to the quantitative factor and the qualitative factor ratio with a characteristic value close to the target analyte, the restriction of such predefined intervals can ensure that the selected local maximum corresponds to the peak related to the target analyte. Additionally or alternatively, the determination 118 of the retention time of chemically related substances may include determining a pattern of multiple local maxima within a predefined retention time interval, wherein the pattern has a predefined distance between the maxima. Such peak patterns may be characteristic of certain target analytes measured with a given LC-MS measurement protocol and may therefore be used to distinguish peaks associated with the target analyte from peaks associated with other substances. The determination of the pattern of multiple local maxima may be performed taking into account a predefined interval of the quantitative factor to qualitative factor ratio at the maximum position.

[0173] The evaluation includes adding the retention time of the chemically related substance to a predetermined or predefined constant offset and / or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor 120. For example, the retention time of the chemically related substance may be t1, where the initial value of the analyte retention time t ini Can be obtained by ini =t1+t off To determine, where t off is a constant offset. The constant offset can be predetermined or predefined by comparing the determined retention time of the chemically related substance with at least one predicted or expected retention time of the chemically related substance. The predicted or expected retention time of the chemically related substance can be a known retention time of the chemically related substance (e.g., a predefined or predetermined retention time under certain conditions, particularly temperature conditions). For example, the comparison may include subtracting the determined retention time of the chemically related substance from the predicted or expected retention time. The deviation of the predicted or expected retention time from the determined retention time of the chemically related substance can be used as a constant offset. For example, the retention time of the chemically related substance can be t1, where the initial value t of the analyte retention time is t2.ini Can be obtained by ini =t1*t fac To determine, where t fac For constant factors. Can be determined in advance or predefined constant factors by comparing the determined retention time of chemical related substances with at least one prediction or expected retention time of chemical related substances. The predicted or expected retention time of chemical related substances can be the known retention time of chemical related substances (for example, under certain conditions, particularly the predefined or predetermined retention time under temperature conditions). For example, this comparison can include generating at least one ratio of the determined retention time of chemical related substances and the predicted or expected retention time. The ratio of the predicted or expected retention time and the determined retention time of chemical related substances can be used as a constant factor. For example, due to external factors (such as instrument temperature changes or the accuracy of gradient settings in chromatographic methods is limited) and the small retention time offset that occurs, using constant offset may obtain satisfactory results. For the effect (such as LC column aging) that may cause a larger retention time offset, compared with the application constant offset, using constant factors may provide more superior performance. As outlined above, determining the initial value of the analyte retention time can include determining at least one local intensity maximum in the predefined retention time interval. The initial value of the analyte retention time may be determined by adjusting the predefined retention time interval according to the determined local maximum corrected by a predetermined or predefined constant offset and / or a predetermined or predefined constant factor.

[0174] The evaluation of the chromatogram of chemically related substances includes: determining at least one initial value 122 of the analyte peak shape parameter by determining the peak shape parameter 121 of at least one peak of the chromatogram of chemically related substances. Similar to the peak retention time, the measurement principle of the mass spectrometer 128 may cause the analyte peak shape to be similar to the chemically related substance peak shape, which is due to the similarity of the two compounds. As outlined above, the chemically related substance signal is well controlled because the compound is added to the sample at a predetermined and relatively high concentration, thereby ensuring reliable peak shape fitting results. The result of the chemically related substance shape can be used as an input for subsequent shape determination of the target analyte, and due to the known similarity of the chemically related substance and the analyte peak shape, a tight constraint can be imposed on the deviation from the input. The initial value of the analyte peak shape parameter can be a starting value and / or input value for determining the analyte peak shape from the chromatogram of the analyte. Determining the at least one initial value of the analyte peak shape parameter may include fitting at least one peak of the chemically related substance chromatogram. The fitting may include using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc. For example, at least one exponentially modified Gaussian function may be used to fit the chromatogram of chemically related substances. For example, the fitting may include using at least one fitting function, as described in "Mathematical functions for the representation of chromatographic peaks", Valerio B. Di Marco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30. Using one or more of these curve fitting techniques, the peak is fitted to the chemically related substance chromatogram. According to the curve fitting technique, the peak parameters of the chemically related substance peak can be determined. The peak parameters may include one or more of peak width, peak asymmetry, peak position, baseline, etc. The peak parameters determined from the chromatogram of chemically related substances can be used to estimate the peak parameters of the analyte peak by extending the peak parameters of the analyte peak to an interval with a so-called tolerance interval.

[0175] The method may include determining at least one tolerance interval for the corresponding peak parameter. The tolerance interval p ± ε may be given by the corresponding determined initial value p of the analyte peak shape parameter and the error interval ε. The error interval may depend on the resolution of the detector, the fitting error, and the chemical similarity level of the chemically related substance and the target analyte, such as whether deuterated, C13 labeled or other chemically related substances are used. For example, using deuterated chemically related substances may require setting a wider error interval than using C13 labeled chemically related substances.

[0176] The method includes evaluating a chromatogram of an analyte. The evaluation of the chromatogram of the analyte may include the at least one preprocessing step 130. The preprocessing may include one or more of the following: selecting at least one target region in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, a Savitzky-Golay smoothing, and a Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, a discrete or continuous wavelet basis background determination, and determining a moving average minimum. The preprocessing may be the same or different from the preprocessing applied to the chromatogram of the chemically related substance.

[0177] The evaluation includes a position determination step 124, in which the analyte retention time is determined by considering the initial value of the retention time of the chemically related substances. The analyte retention time can be determined by using at least one curve fitting technique. The initial value of the analyte retention time can be used as the starting value of the curve fitting. Given the initial value of the analyte retention time determined from the chemically related substance chromatogram, the analyte retention time can be determined, for example, by using at least one curve fitting technique. The fitting may include using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc. For example, the fitting may include using at least one fitting function, such as "Mathematical functions for the representation of chromatographic peaks", Valerio B. DiMarco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30. The analyte retention time can be determined using a narrow tolerance interval near the initial value of the retention time. For example, the tolerance interval t ± εt is determined by the corresponding initial value t and the error interval ± ε of the analyte peak retention time. t The error interval may depend on the resolution of the detector, the chromatographic peak widths of the target analyte and the chemically related substances, the chemical similarity level of the chemically related substances and the target analyte (e.g., whether deuterated, C13 labeled or other chemically related substances are used), and the chromatographic noise level. Chromatograms with high noise levels may, for example, require wider error intervals.

[0178] The evaluation further includes a peak integration step 126, in which at least one fitting analysis is applied to the chromatogram of the analyte by considering the initial values ​​of the analyte peak shape parameters and the analyte retention time, thereby determining the analyte peak area and the analyte peak shape. Using the determined analyte retention time, and the initial values ​​of the analyte peak shape parameters, a curve can be fitted to the chromatogram of the analyte to determine the analyte peak area and peak shape. In the peak integration step, at least one exponentially corrected Gaussian function can be used to fit the analyte chromatogram. For example, the fitting can include using at least one fitting function, such as "Mathematical functions for the representation of chromatographic peaks", Valerio B. DiMarco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30. The analyte peak area and the analyte peak shape can be determined using the tolerance interval p ± ε near the initial values ​​of the corresponding analyte peak shape parameters, as described above.

[0179] Using retention time and peak shape parameters of chemically related substances for evaluating analyte chromatograms may lead to more accurate analyte peak area determinations, especially in cases where analyte concentrations are low, backgrounds are complex, noise levels are high, and interfering peaks are present. Thus, the method according to the present invention enables reliable automation of peak integration without the need for expert user corrections and manual adjustments.

[0180] Figures 4A to 4C The use of chemically related substance peak features for analyte quantification is shown. Figure 4A Shown is a chemically related substance chromatogram, in this case an internal standard chromatogram, which was used to determine peak shape and retention time parameters. Figure 4B A method for peak integration using a fixed retention time window is shown and does not take into account the determined peak shapes and retention times of the chromatograms of chemically related substances.The presence of interfering peaks next to the analyte peak (dashed line) can lead to incorrect results for the analyte peak shape 132. Figure 4C It is shown that the analyte peak 132 can be correctly quantified using the chemically related substance shape parameters even in the presence of interfering peaks.

[0181] Figure 5A Another exemplary chemically related substance chromatogram, in this case a chromatogram of an internal standard, with fitting results is shown. In this example, the fitting results may be a peak width of 2950 and a peak asymmetry of 0.1. These peak shape parameters may be used to estimate the peak parameters of the analyte by extending the peak parameters of the analyte to a so-called tolerance interval, such as a peak width of 2950 ± 20% and a peak asymmetry = 0.1 ± 50%. The retention time derived from this internal standard chromatogram may be 4700. The peak shape parameters may be obtained from Figure 5B In the chromatogram shown, the analyte retention time is determined using a narrow tolerance interval around the initial value of the retention time. For example, the tolerance interval t ± εt is determined by the corresponding initial value t and the error interval ± ε of the analyte peak retention time. t For example, in Figure 5A and 5B In the example shown, the tolerance range may be ±0.1%. Figure 5B The resulting peak fit of the analyte chromatogram using the method according to the present invention is further shown. The analyte fit results may be a peak width of 2890, a peak asymmetry of 0.12, and a peak retention time of 47100.

[0182] Figure 6 An embodiment of a mass spectrometry system 134 according to the present invention is shown in a highly schematic manner. The mass spectrometry system 134 includes at least one mass spectrometry device 128, which includes at least one mass filter 136 and at least one detector 138. The mass spectrometry device may be or may include at least one liquid chromatography mass spectrometry device. The mass spectrometry device 128 may include at least one chromatograph. The mass filter 136 may be configured to separate the components of the sample according to the mass of the components of the sample. The detector 138 may be configured to detect incoming particles and to determine the at least one chromatogram. The mass spectrometry system 134 further includes at least one computer-implemented processing line 140 for automatic peak integration of at least one chromatogram of at least one sample according to the present invention.

[0183] Processing line 140 includes

[0184] - at least one data collector 142 configured to retrieve at least one chromatogram of chemically related substances and at least one chromatogram of the analyte;

[0185] at least one evaluation device 144 configured to evaluate the chromatogram of the chemically related substances and the chromatogram of the analyte,

[0186] wherein the evaluation device 144 comprises at least one retention time processor 146 configured to determine at least one initial value for the retention time of the analyte by determining the retention time of chemically related substances,

[0187] wherein the evaluation device 144 comprises at least one adder 148 and / or at least one multiplier 150, wherein the at least one adder is configured to add the retention time of the chemically related substances to a predetermined or predefined constant offset, and the at least one multiplier is configured to multiply the retention time of the chemically related substances by a predetermined or predefined constant factor,

[0188] wherein the evaluation device 144 comprises at least one position determination device 152 configured to determine the analyte retention time by taking into account initial values ​​of the retention times of chemically related substances,

[0189] wherein the evaluation device 144 further comprises at least one peak processor 154, the at least one peak processor being configured to determine at least one initial value of a peak shape parameter of the analyte by determining a peak shape parameter of at least one peak of the chromatogram of the chemically related substance, wherein the evaluation device 144 comprises at least one fitting device 156, the at least one fitting device being configured to apply at least one fitting analysis to the chromatogram of the analyte by taking into account the initial value of the peak shape parameter of the analyte and the retention time of the analyte,

[0190] From this, the analyte peak area and analyte peak shape are determined.

[0191] The data collector 142 may be or may include at least one database configured to receive and / or store at least one chromatogram of chemically related substances and at least one chromatogram of analytes. The receiving and / or storing of the chromatograms of chemically related substances and analytes may be performed simultaneously or sequentially.

[0192] The evaluation device 144 may include at least one data processing device having software code stored thereon, the software code including a plurality of computer commands. The evaluation device 144 may provide one or more hardware elements for performing one or more specified operations, and / or may provide one or more processors with software running thereon for performing one or more method steps.

[0193] Retention time processor 146 can be a processor, which is configured to perform the determination of the retention time of chemical related substances, particularly the determination of the local maximum value in the chromatogram of chemical related substances. Retention time processor 146 can be configured to determine at least one local intensity maximum value in the predefined retention time interval for determining the retention time of chemical related substances. For example, retention time processor 146 can be configured to determine the local intensity maximum value by considering the predefined interval of the quantitative factor and qualitative factor ratio at the maximum position. Additionally or alternatively, retention time processor 146 can be configured to determine the pattern of multiple local maximum values ​​in the predefined retention time interval for determining the retention time of chemical related substances, wherein the pattern has a predefined distance between the maximum values. Retention time processor 146 can be configured to determine the pattern of multiple local maximum values ​​by considering the predefined interval of the quantitative factor and qualitative factor ratio at the maximum position, as described in detail above about the method.

[0194] The adder 148 and / or the multiplier 150 may be configured as a digital circuit or may be implemented as software.

[0195] The position determining device 152 may be configured to determine the analyte retention time from the chromatogram of the analyte. The position determining device 152 may be arranged after the retention time processor 146 and the adder 148 and / or the multiplier 150. Additionally or alternatively, the position determining device 152 may be integrated with the retention time processor 146. The design and / or evaluation device 144 may include at least one processor configured to perform two operations: determining an initial value of the analyte retention time and the analyte retention time. The position determining device 152 may be configured to determine the analyte retention time by using at least one curve fitting technique, wherein the initial value of the analyte retention time is used as a starting value for the curve fitting. The curve fitting technique may include using at least one fitting selected from the group consisting of: polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc.

[0196] The peak processor 154 may be configured to perform at least one fitting procedure. The peak processor 154 may be configured to fit at least one peak of the chemically related substance chromatogram. The fitting may include using at least one curve fitting technique, such as one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform, etc. The peak processor 154 may be configured to determine an allowable interval. The allowable interval p ± ε may be given by the corresponding determined initial value p of the analyte peak shape parameter and the error interval ± ε.

[0197] The peak processor 154 may be arranged after the retention time processor 146, the adder 148 and / or the multiplier 150, and the position determination device 152. Additionally or alternatively, the peak processor 154 may be designed to be integrated with the retention time processor 146 and / or the position determination device 152, and / or the evaluation device 144 may include at least one processor configured to perform the following actions: determine the initial value of the analyte retention time and the analyte retention time, determine the peak shape parameters.

[0198] The fitting device 156 may be configured to fit the analyte chromatogram using at least one exponentially modified Gaussian function. For example, the fitting may include using at least one fitting function as described in "Mathematical functions for the presentation of chromatographic peaks", Valerio B. Di Marco, G. Giorgio Bombi, Journal of Chromatography A, 931 (2001) 1-30.

[0199] The evaluation device 144 may also include at least one preprocessor. The preprocessor may be configured to preprocess the chromatogram of the chemically related substances and / or the chromatogram of the analyte by one or more of the following items: selecting at least one target area in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step, which includes applying one or more of a moving average filter, a Gaussian filter, a discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step, which includes one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

[0200] Reference numerals list

[0201] 110 Curve

[0202] 112 Curve

[0203] 113 lines

[0204] 114 Searching for chromatograms of chemically related substances

[0205] 116 Search Analyte Chromatogram

[0206] 117 Determine initial value of analyte retention time

[0207] 118 Determining the retention times of chemically related substances

[0208] 120 Addition and / or multiplication

[0209] 121 Determining Peak Shape Parameters for Chemically Related Substances

[0210] 122 Determine initial values ​​for analyte peak shape parameters

[0211] 124 Position determination steps

[0212] 126 Peak Integration Steps

[0213] 128 Mass Spectrometry Device

[0214] 130 Preprocessing Steps

[0215] 132 Analyte Peak Shape

[0216] 134 Mass Spectrometry System

[0217] 136 Mass Filter

[0218] 138 Detector

[0219] 140 Computer-Achievable Processing Lines

[0220] 142 Data Collector

[0221] 144 Evaluation Device

[0222] 146 Retention Time Processor

[0223] 148 Adder

[0224] 150 Multiplier

[0225] 152 Position determination device

[0226] 154 Peak Processor

[0227] 156 Fitting device

Claims

1. A computer-implemented method for automatic peak integration of at least one chromatogram of at least one sample, wherein the sample comprises at least one analyte and at least one chemically related substance, wherein the method comprises the following steps: a) retrieving at least one chromatogram (114) of the chemically related substance and at least one chromatogram (116) of the analyte; b) evaluating said chromatogram of said chemically related substances, wherein said evaluating comprises b1) determining at least one initial value (117) for the retention time of the analyte by determining the retention time (118) of the chemically related substance and one or both of: adding the retention time of the chemically related substance to a predetermined or predefined constant offset, or multiplying the retention time of the chemically related substance by a predetermined or predefined constant factor (120), b2) determining at least one initial value (122) of a peak shape parameter of the analyte by determining a peak shape parameter (121) of at least one peak of a chromatogram of a chemically related substance; c) evaluating said chromatogram of said analyte, wherein said evaluating comprises c1) at least one position determination step (124), wherein said analyte retention time is determined by taking into account an initial value of said retention time of said chemically related substance; c2) at least one peak integration step (126) wherein analyte peak area and analyte peak shape are determined by applying at least one fitting analysis to said chromatogram of said analyte by taking into account initial values ​​of analyte peak shape parameters and said analyte retention time.

2. The method according to claim 1, wherein method steps a) to c) are performed fully automatically.

3. The method according to claim 1 or 2, wherein determining the retention time (118) of the chemically related substances comprises determining at least one local intensity maximum within a predefined retention time interval. 4 . The method according to claim 3 , wherein the determination of the local intensity maximum is performed taking into account a predefined interval of the quantitative factor to the qualitative factor ratio at the location of the maximum.

5. The method of claim 3, wherein determining the retention times (118) of the chemically related substances comprises determining a pattern of a plurality of local maxima within the predefined retention time interval, wherein the pattern has a predefined distance between the maxima. 6 . The method according to claim 5 , wherein said determining of said pattern of a plurality of local maxima is performed taking into account a predefined interval of the quantitative factor to qualitative factor ratio at the location of said maximum value.

7. A method according to claim 3, wherein the initial value of the analyte retention time is determined by adjusting the predefined retention time interval according to the determined local maximum value corrected by one or both of the predetermined or predefined constant offset or the predetermined or predefined constant factor.

8. A method according to claim 1 or 2, wherein the analyte retention time t is determined using a tolerance interval determined from the chromatogram of the chemically related substances as a domain, wherein the tolerance interval t±ε of the analyte peak retention time t includes an error interval ε of the analyte peak retention time, and the error interval is set to the corresponding error interval of the peak retention time of the chemically related substances.

9. The method of claim 1 or 2, wherein determining the at least one initial value (122) of the analyte peak shape parameter comprises fitting at least one peak of the chromatogram of the chemically related substances, wherein the fitting comprises using at least one curve fitting technique: one or more of polynomial interpolation, Gauss-Newton algorithm, Fourier transform.

10. The method of claim 1 or 2, wherein the analyte retention time is determined by using at least one curve fitting technique, wherein the initial value of the analyte retention time is used as a starting value for the curve fitting.

11. The method of claim 10, wherein the curve fitting technique comprises using at least one fit selected from the group consisting of: polynomial interpolation, Gauss-Newton algorithm, or Fourier transform.

12. The method according to claim 1 or 2, wherein the method comprises determining at least one tolerance interval, wherein the tolerance interval p±ε is given by the corresponding determined initial value p of the analyte peak shape parameter and the error interval ±ε.

13. The method according to claim 12, wherein the error interval ±ε of the analyte peak shape parameter is set to the error interval of the peak shape parameter of the chemically related substance.

14. The method according to claim 1 or 2, wherein in the peak integration step (126), at least one exponentially modified Gaussian function is used to fit the analyte chromatogram.

15. The method of claim 1 or 2, wherein the evaluation of one or both of the chromatogram of the chemically related substance or the chromatogram of the analyte comprises at least one preprocessing step (130), wherein the preprocessing (130) comprises one or more of the following: selecting at least one target region in the chromatogram; selecting at least one predefined retention time interval; at least one smoothing step comprising applying one or more of a moving average filter, a Gaussian filter, discrete wavelet denoising, Savitzky-Golay smoothing, and Loess smoothing; at least one background subtraction step comprising one or more of asymmetric weighted least squares fitting regularization, applying a morphological top hat filter, discrete or continuous wavelet basis background determination, and determining a moving average minimum.

16. A computer-implemented processing line (140) for automated peak integration of at least one chromatogram of at least one sample, wherein the sample comprises at least one analyte and at least one chemically related substance, wherein the processing line (140) comprises - at least one data collector (142) configured to retrieve at least one chromatogram of the chemically related substances and at least one chromatogram of the analyte; - at least one evaluation device (144) configured to evaluate the chromatogram of the chemically related substances and the chromatogram of the analyte, wherein the evaluation device (144) comprises at least one retention time processor (146) configured to determine at least one initial value for the retention time of the analyte by determining the retention time of the chemically related substance, wherein the evaluation device (144) comprises one or both of at least one adder (148) configured to add the retention time of the chemically related substances to a predetermined or predefined constant offset, or at least one multiplier (150) configured to multiply the retention time of the chemically related substances by a predetermined or predefined constant factor, wherein the evaluation device (144) comprises at least one position determination device (152) configured to determine the analyte retention time by taking into account an initial value of the retention time of the chemically related substance, wherein the evaluation device (144) further comprises at least one peak processor (154) configured to determine at least one initial value of an analyte peak shape parameter by determining a peak shape parameter of at least one peak of the chemically related substance chromatogram, The evaluation device (144) comprises at least one fitting device (156), wherein the at least one fitting device is configured to determine an analyte peak area and an analyte peak shape by applying at least one fitting analysis to the chromatogram of the analyte by taking into account initial values ​​of analyte peak shape parameters and the analyte retention time.

17. The processing line (140) according to claim 16, wherein the processing line (140) is configured for performing a method according to any of the preceding claims 1-15 concerning methods.

18. A mass spectrometry system (134), comprising - at least one mass spectrometer (128), the at least one mass spectrometer comprising at least one mass filter (136) and at least one detector (138); - at least one computer implementable processing line (140) according to claim 16 or 17.

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