Patent Description:
Multiple transition monitoring (MRM) during one liquid chromatography run reduces dwell times for each transition with increasing number of observer MRM. Known methods such as scheduled MRMs use only the times frames in a liquid chromatography run that is relevant to a certain transition. For example, analyte X with MRM Y is resulting in peak Z at time x and, thus, only a certain time of relevance such as x-<NUM> to x+ <NUM> may be recorded. Usually a certain time frame is defined with respect to a certain time before and after the peak maximum of the peak, usually referred to as the retention time.

<CIT> describes a chromatograph mass spectrometer including: an MSn-<NUM> analysis setter for setting an analysis execution period for performing an MSn-<NUM> analysis, an execution time for the analysis and a loop time; an analysis period divider for dividing the analysis period into segments according to a change in number or analysis condition of MSn-<NUM> analyses to be performed within the same time window; an MSn analysis setter for performing MSn-<NUM> analysis to obtain mass spectrum data and for scheduling MSn analysis, an ion corresponding to a peak satisfying a set condition being designated as a precursor ion; an MSn analysis execution time allotter for allotting, in each segment, a time period for execution of the MSn analysis, the time period being calculated by subtracting an event execution time from the loop time; and an analysis executer for repeatedly performing MSn-<NUM> analysis and MSn analysis in each segment.

<CIT> describes an analysis schedule which is pre-created such that streams of a plurality of liquid chromatograms can operate in parallel and a mass spectrometer can collect data at the timing of each component elution. A control unit controls so as to: divide the time required to analyze each sample in a plurality of liquid chromatogram systems into pre-collection time, time during collection, and post-collection time; search and allocate time positions in which the time during collection in the liquid chromatogram units do not overlap; determine start times for the plurality of liquid chromatogram units to thereby create an analysis schedule; and thereafter perform analysis. The control unit further stores parameter sets for varying component elution times, adjusts analysis parameters so as to make data collection timings suitable for creating an analysis schedule, and changes the component elution times.

<CIT> describes prior to multiple reaction monitoring measurement condition optimization, an analysis operator which prepares, for each precursor ion of an objective compound, two lists on a product-ion selection condition setting screen, i.e. a list which shows ions to be preferentially selected as product ions for which the optimization needs to be performed and a list which shows ions to be excluded from the optimization. When a measurement is performed, a product-ion scan measurement for the precursor ion of the objective compound is performed and a spectrum is obtained. Among the ions extracted from this spectrum, any ion registered in the excludable-ion list is excluded, while any ion registered in the preferred-ion list is preferentially selected as a product ion. For each combination of the m/z values of the precursor ion and the product ions thus determined, optimum conditions of the MRM measurement are searched for.

<CIT> describes mass spectrometry systems and methods which utilize a dynamic a data acquisition/instrument control methodology. These systems and methods employ artificial intelligence algorithms to greatly increase quantitative and/or identification accuracy during data acquisition. In an embodiment, the algorithms can adapt the instrument methods and systems during data acquisition to direct data acquisition resources to increase quantitative or identification accuracy of target analytes, such as proteins, peptides, and peptide fragments.

<CIT> describes systems and methods for identifying actual XIC peaks of compounds of interest from samples. In one system, an actual XIC peak is identified using standard samples. The ratio of the quantity of the compound of interest in any two different samples is known, so this ratio is compared to the intensities of the XIC peak calculated in the two samples to identify an actual XIC peak. In another system, an actual XIC peak is identified using information about other compounds of interest in a plurality of samples. It is known that the XIC peaks of compounds of interest in the same samples have a similar distribution of retention times across those samples, so the distributions of retention times of XIC peaks are compared to identify actual XIC peaks.

<CIT> describes creation of a mass calibration table. After a specimen is injected into a specimen vaporization chamber of GC, before the lapse of the time for completely eluting a specimen solvent from a column, mass analysis is not performed and data are not collected, either. Thus, mass calibration is prevented from being performed by erroneously utilizing an analysis result of the solvent. Thereafter, collection of data is started by scan measurement and when a signal strength of TIC becomes equal to or higher than a threshold value, it is recognized that the elution of components for mass calibration is started. Then, the scan measurement is executed while changing a scan speed, a mass spectrum of the different scan speed with respect to the component for mass calibration is acquired and based on the mass spectrums, a mass calibration table is created.

<NPL> describes selected reaction monitoring (SRM) for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC-MS experiment. Lange et al. explain the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions.

Scheduled MRM in particular works well for very defined measurements and/or analyte mixtures with high concentrations. Despite the advantages of scheduled MRM, however, there is growing interest for maximizing column lifetimes, which may go hand in hand with significant peak shifts such that scheduled MRM with a fixed time frame for peak recording may lead to non reliable and incorrect results.

It is therefore an objective of the present invention to provide a method and a device for multiple transition monitoring, which avoid the above-described disadvantages of known methods and devices. In particular, the method and the device shall allow reliable and correct multiple transition monitoring even in case of retention time shifts due to column aging, capillary exchanges, solvent composition inaccuracies, and other factors. Moreover, the method and the device shall allow lowering limit of quantification, specifically for critical analytes.

This problem is solved by a method and a device for multiple transition monitoring, having the features of the independent claims. Preferred embodiments of the invention, which may be realized in an isolated way or in any arbitrary combination, are disclosed in the dependent claims.

In a first aspect of the present invention, a method for multiple transition monitoring using a liquid chromatography mass spectrometry device is disclosed.

The term "multiple transition monitoring", also denoted multiple reaction monitoring (MRM), as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a method used in mass spectrometry, specifically in tandem mass spectrometry, in which multiple product ions from one or more precursor ions are monitored. As used herein, the term "monitored" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to determining and/or detecting of multiple product ions.

As used herein, the term "liquid chromatography mass spectrometry device" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a combination of liquid chromatography with mass spectrometry. The liquid chromatography mass spectrometry device may be or may comprise at least one high-performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (µLC) device. The liquid chromatography mass spectrometry device may comprise 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 to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an analytical module configured to separate one or more analytes of interest of a sample from other components of the sample for detection of the one or more analytes with the mass spectrometry device. The LC device may comprise at least one LC column. For example, the LC device may be a single-column LC device or a multi-column LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped in order to separate and/or elute and/or transfer the analytes of interest. The LC column may be exchangeable, for example after a predefined or pre-determined time and/or number of runs, and/or other suitable counters. For example, the LC column may be exchanged if one or more thresholds of one or more of a volume of solvent, a number of switching event of valves, a number of runs, a number of injections, a number of samples, a number of samples of a certain type, an LC pressure/curves are reached. As used herein, the term "mass spectrometry device" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a mass analyzer configured for detecting at least one analyte based on mass to charge ratio. The mass spectrometry device may be or may comprise at least one quadrupole mass spectrometry device. The interface coupling the LC device and the MS may comprise at least one ionization source configured for generating of molecular ions and for transferring of the molecular ions into the gas phase.

As used herein, the term "sample" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary sample such as a biological sample, also called test sample, a quality control sample, an internal standard sample. The sample may comprise one or more analytes of interest. For example, the test sample may be selected from the group consisting of: a physiological fluid, including blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells or the like. The sample may be used directly as obtained from the respective source or may be subject of a pretreatment and/or sample preparation workflow. For example, the sample may be pretreated by adding an internal standard and/or by being diluted with another solution and/or by having being mixed with reagents or the like. For example, analytes of interest may be vitamin D, drugs of abuse, therapeutic drugs, hormones, and metabolites in general. The quality control sample may be a sample that mimics the test sample, and that contains known values of one or more quality control substances. The quality control substance may be identical to the analyte of interest or may be an analyte which generates by reaction or derivatization an analyte identical to the analyte of interest and/or may be an analyte of which the concentration is known and/or may be a substance which mimics the analyte of interest or that can be otherwise correlated to a certain analyte of interest. The internal standard sample may be a sample comprising at least one internal standard substance with a known concentration. For further details with respect to the sample, reference is made e.g. to <CIT>.

Other analytes of interest are possible.

The method comprises the following steps which, as an example, may be performed in the given order. It shall be noted, however, that a different order is also possible. Further, it is also possible to perform one or more of the method steps once or repeatedly. Further, it is possible to perform two or more of the method steps simultaneously or in a timely overlapping fashion. The method may comprise further method steps which are not listed.

As used herein, the term "data set" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to stored and/or deposited information about at least one previous MRM measurement such as from previous runs. The information about the previous MRM measurement may comprise at least one chromatogram and/or at least one information evaluated from the chromatogram such as peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor, and/or any type of peak fitting and filtering. The tailing factor T may be determined by T= W<NUM>/(2d), wherein W<NUM> is the peak width at <NUM> of the peak height and d is a distance between a perpendicular line through the peak maximum and a leading edge of the peak at <NUM> of the peak height. As used herein, the term "data base" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a collection of data comprising the at least one data set. The data base may comprise at least one table and/or at least one look-up table in which the at least one data set is stored. The data base may comprise at least one storage unit configured to store the data set. As used herein, the term "determining at least one data set from at least one data base" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to access the data base and to retrieve the data set from the data base.

As used herein, the term "reference measurement" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to at least one previous MRM measurement such as a MRM measurement of a previous run and/or at least one prediction about a MRM measurement. The reference measurement may comprise or may be at least one known MRM transition. The reference measurement may be at least one measurement of at least one quality control sample acquired during a previous quality control run and/or at least one measurement of at least one internal standard sample acquired during a previous internal standard sample run and/or at least one measurement of the test sample acquired during a previous run. The reference measurement may be at least one measurement acquired and/or determined and/or measured in the same way and under the same or at least similar and/or comparable conditions as the measurement of the actual test sample. The reference measurements and the measuring of the transition of the analyte may be performed under substantially the same conditions. For example, the reference measurement and the measuring of the transition of the analyte may be performed under constant chromatographic conditions, specifically with the same LC column and eluents. The method may apply to known compounds and well-known conditions. The method may however also comprise predicting at least one reference measurement, specifically in case of changes of gradients. The predicting may comprise considering aging of LC column, capillary exchanges, solvent composition inaccuracies, and other factors.

As used herein, the term "reference peak information" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to at least one information of a peak, i.e. a local maximum, of the chromatogram corresponding and/or relating to the analyte of interest of the reference measurement which is suitable to limit the relevant time frame for measurement of the analyte of interest. The reference peak information may comprise one or more of: peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor and/or any type of peak fitting and filtering. The determining of the reference peak information may comprise evaluating the reference measurement. The evaluating may comprise performing at least one data analysis comprising performing at least one peak finding algorithm and/or performing at least one peak fitting algorithm. The evaluating may comprise one or more of checking of raw data, preprocessing, smoothening, background reduction or removal, peak detection, peak integration.

As used herein, the term "measurement window" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a time frame in which the measurement of the analyte of interest is performed. The measurement window is defined by a time frame of retention times. Limiting the measurement of the analyte of interest to a certain pre-defined time frame is generally known. As used herein, the term "setting" of the measurement window is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to values for one or both limits of the measurement window. Specifically, the setting of the measurement window may comprise one or both of a value for a lower limit of the measurement window, i.e. a retention time at which the measurement starts, and a value for an upper limit of the measurement window, i.e. a retention time at which the measurement stops.

The term "initial setting" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a setting of the measurement window which is used for determining the reference peak information. The initial setting may be pre-determined and/or pre-defined. For example, in case of a first measurement after change of a column of the LC device, the initial setting may be a default setting which may be deposited in the data base. The method may comprise at least one initial calibration step, wherein in the initial calibration step the reference measurement and/or the initial setting may be determined. The initial calibration step may be performed during and/or subsequent to at least one quality control run of the liquid chromatography mass spectrometry device and/or during and/or subsequent to at least one internal standard samples run. A start of the initial calibration step may be triggered by changing of a column of the liquid chromatography mass spectrometry device and/or after a predefined or pre-determined time and/or after a predefined or predetermined number of runs, or other suitable counters. The term "trigger" as used herein, may refer to either an automatic procedure that is initiated and executed automatically or a warning generated for and prompting a user to manually set the setting to the initial setting. In case of performing the method repeatedly, the initial setting may be a setting of the measurement window determined from the measurement of at least one prior run or a plurality of prior runs, such as a mean value for the limits of the time frame.

The term "actual setting" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a setting of the measurement window determined by considering the reference peak information determined in step b). In addition, to considering the reference peak information of only the preceding measurement, the actual setting may be determined considering the preceding measurement or a plurality of preceding measurements. The determining of the actual setting may comprise evaluating the reference peak information and thereby determining a lower and/or an upper limit of the measurement window. Specifically, at least one automated analysis of retention times may be performed. Moreover, the measurement window may be automatically reassigned. The actual setting may be determined and/or calculated based on expected retention time and tailing factor. The actual setting may be determined by comparing peak, in particular signal intensity, and background. For example, signal intensity and background may be compared by defining and/or using at least one threshold value at which the peak starts and/or at least one threshold value at which the peak ends. The actual setting may be determined by making a prediction based on a plurality of datasets.

The initial setting may comprise a broader time frame of retention times compared to the actual setting and/or the actual setting may be shifted in retention time compared to the initial setting. In the latter case the width of the measurement window may be maintained. Specifically, the initial setting of the measurement window may be selected so broad such that it is ensured that the peak corresponding to the analyte of interest lies within the time frame. Subsequently the initial setting of the measurement window may be optimized in view of measurement results which allows for reducing width of the measurement window and/or for positioning the measurement window, specifically to take into account changes of the LC column such as due to aging or other changes. The reference measurement may comprise or may be a known MRM transition, wherein the method comprises optimizing their measurement. The term "determining the actual setting" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to adapting and/or changing the initial setting of the measurement window depending on at least one subsequent measurement. The timing of the measurement may be fixed. However, due to variation of the peak position as a result of ongoing analysis, specifically in case of repeatedly performing method steps a) to d), the retention time may shift and may be adapted based on prior measurements. The method may comprise adjustment of the MRM scheduled timing as a function of changing parameters over time. Thus, the optimized retention time may be used to optimize scheduled MRM measurements and thus freeing up time for more MRM transitions.

The method comprises in step d) measuring the transition of the analyte within a sample with the liquid chromatography mass spectrometry device. The measurement may be triggered by a user, e.g. by entering at least one input to at least one human-machine-interface of the liquid chromatography mass spectrometry device.

The actual setting of the measurement window is used for determining the measured peak information of the transition of the analyte. As used herein, the term "measured peak information" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to at least one information of a peak of the chromatogram measured in step d) corresponding and/or relating to the analyte of interest using the actual setting of the measurement window. The measured peak information comprises one or more of: peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor. The determining of the measured peak information may comprise evaluating the actual measurement. The evaluating may comprise performing at least one data analysis comprising performing at least one peak finding algorithm and/or performing at least one peak fitting algorithm.

The method further may comprise updating the data set by adding the measurement of the transition of the analyte to the data set. Specifically, in case the method steps a) to d) are performed repeatedly, the data set may be updated after performing step d) such that the subsequent step a) is performed using an updated initial setting. Thus, the updating may be performed permanently.

Method steps a) to d) may be performed repeatedly. In step a) the most recent measurement may be used as reference measurement. In step b) the reference peak information of the transition of the analyte may be determined using the most recently determined actual setting as initial setting. After a plurality of repetitions of methods steps a) to d), in step a) a mean value of a plurality of preceding measurements may be used as reference measurement. The mean value may be determined by using at least five preceding measurements. Additionally or alternatively, in particular in case of larger changes between runs, a moving average of a plurality of preceding measurements may be used as reference measurement. Additionally or alternatively, a maximum amount of preceding measurements for calculating the mean value may be limited. Using more than one measurement may ensure that the reference data is corrected with respect to outliers or sample influence.

A width of the measurement window may narrow with number of repetitions. Thus, the measurement window becomes even better and/or more beneficial for clearing up more MS detection window. The detection window may be a time frame in which the mass spectrometry device has to perform a measurement of a sample. Specifically, the detection window may be the maximum time possible between two consecutive sample inputs in the LC column.

The method may further comprise at least one in-situ adjustment step. The in-situ adjustment step may be performed during step d). In the in-situ adjustment step intensity of the transition of the analyte may be monitored during the measurement and compared to at least one predetermined or predefined threshold level. The liquid chromatography mass spectrometry device may comprise at least one further data base configured for storing at least one definition of threshold levels and/or threshold values. For example, the at least one threshold level and/or the at least one threshold value may be defined by percentual change of signal intensity to background. Additionally or alternatively, at least one absolute value may be used as threshold for determining exceedance or undershoot. The further data base may be configured to receive input information from the data base such as values for the threshold levels. Thus, the threshold levels may be data driven. If the intensity falls below the predefined threshold level acquisition of the transition may be stopped. The pre-determined or predefined threshold level may be defined by a factor X times the signal-to-noise ratio which is also known from the start. The in-situ adjustment step may be implemented as a feedback loop with automated live adjustment of measurement parameter. For example, a measurement, i.e. a specific MRM, may start at a time known on the basis of previous measurements. A run time of the measurement may be determined on the basis of an actual measured intensity of this MRM. In case the intensity falls below the predetermined or predefined threshold level the acquisition of that MRM may be stopped and may allow to free dwell time for other MRMs running at the same time. This approach may be reliable for well articulated peaks. However, problems may arise when signal to noise is low, e.g. a small peak with high variation in individual signal. For these cases it may be advantageous that the predetermined or predefined threshold level may be given by an end of a fit curve such as a Gaussian curve. The fit curve may allow defining the peak region such as start of the peak and end of the peak. In particular, a certain peak height at the end of the peak, denoted as end of the fit curve, may be used as threshold level. In principle, use of a fit curve during measurement is problematic since the full signal is not present at this stage. However, a fit curve such as a Gaussian curve can be used since the fit parameters of the Gaussian or other fit curve may be determined from the one or more preceding measurements. This may allow determining all parameters of the fit curve with only a single iteration. Additionally or alternatively, the fit curve may be determined from a measurement of an internal standard. This may allow accelerating the fitting procedure. Additionally or alternatively, a combination of fit results of different peaks of the same analyte may be used to enhance robustness of the fit result. The fit curve, in particular of a Gaussian, may be independent or less dependent from background and, thus, advantageous even at high noise.

The method steps b) to c) and in step d) the determining of the measured peak information of the transition of the analyte may be performed by at least one computer. Specifically, the method steps b) to c) and in step d) the determining of the measured peak information of the transition of the analyte may be performed fully automatically. The method specifically may fully or partially be computer-implemented, specifically on a computer of a device for multiple transition monitoring, such as a processor.

The method may comprise compensating for column aging and/or for further impacts such as capillary exchanges, solvent composition inaccuracies, and the like.

In a further aspect, a computer program including computer-executable instructions for performing the method according to any one of the embodiments as described herein is disclosed, specifically method steps a) to c) and determining of the measured peak information in step d), when the program is executed on a computer or computer network, specifically a processor of the device for multiple transition monitoring.

Thus, generally speaking, disclosed and proposed herein is a computer program including computer-executable instructions for performing the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one or even all of the method steps as indicated above may be performed by using a computer or a computer network, preferably by using a computer program. The computer specifically may be fully or partially integrated into the device for multiple transition monitoring, and the computer programs specifically may be embodied as a software. Alternatively, however, at least part of the computer may also be located outside the device for multiple transition monitoring.

Further disclosed and proposed herein is a computer program product having program code means, in order to perform the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network, e.g. one or more of the method steps mentioned above. Specifically, the program code means may be stored on a computer-readable data carrier.

Further disclosed and proposed herein is a data carrier having a data structure stored thereon, which, after loading into a computer or computer network, such as into a working memory or main memory of the computer or computer network, may execute the method according to one or more of the embodiments disclosed herein, specifically one or more of the method steps mentioned above.

Further disclosed and proposed herein is a computer program product with program code means stored on a machine-readable carrier, in order to perform the method according to one or more of the embodiments disclosed herein, when the program is executed on a computer or computer network, specifically one or more of the method steps mentioned above. As used herein, a computer program product refers to the program as a tradable product. The product may generally exist in an arbitrary format, such as in a paper format, or on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

Finally, disclosed and proposed herein is a modulated data signal which contains instructions readable by a computer system or computer network, for performing the method according to one or more of the embodiments disclosed herein, specifically one or more of the method steps mentioned above.

Specifically, further disclosed herein are:.

In a further aspect of the present invention, a device for multiple transition monitoring of at least one analyte in a sample is disclosed. The device comprises.

The device may be configured to perform the method according to any one of the preceding embodiments. For most of the terms used herein and possible definitions, reference may be made to the description of the methods above.

As further used herein, the term "evaluation device" generally refers to an arbitrary device adapted to perform 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 comprise at least one data processing device having a software code stored thereon comprising a number of computer commands. The evaluation device may provide one or more hardware elements for performing one or more of the named operations and/or may provide one or more processors with software running thereon for performing one or more of the method steps.

The methods and devices according to the present invention may provide a large number of advantages over known methods and devices for multiple transition monitoring. Thus, specifically, dynamic MRMs on the basis of previous runs may free up valuable channel time and consequently increase sampling rate or time for analytes. This is particularly beneficial for analytes that should be measured with high sensitivity. Influences that may be caused by changes of components over their lifetime, e.g. column aging, and/or further impacts such as capillary exchanges, solvent composition inaccuracies, and other factors, and resulting retention time shifts or changes and impact on spray stabilization, can be compensated within the boundary of the available time window. Deviations arising from column batch to batch variations can be compensated, too. The method does not require hardware changes but can be implemented as fast and powerful in situ algorithms.

Summarizing and without excluding further possible embodiments, the following embodiments may be envisaged:.

<FIG> shows a flow chart of a method for multiple transition monitoring using a liquid chromatography mass spectrometry device <NUM>, an embodiment of which is shown in <FIG>, according to the present invention.

The method comprises in step a) determining at least one data set <NUM> from at least one data base <NUM>. The data set <NUM> comprises at least one reference measurement <NUM> of at least one transition of at least one analyte with the liquid chromatography mass spectrometry device <NUM>. The data set <NUM> may be and/or may comprise stored and/or deposited information about at least one previous MRM measurement such as from previous runs. The information about the previous MRM measurement may comprise at least one chromatogram and/or at least one information evaluated from the chromatogram such as peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor, and/or any type of peak fitting and filtering. The data base <NUM> may comprise at least one table and/or at least one look-up table in which the at least one data set <NUM> is stored. The data base may comprise at least one storage unit configured to store the data set.

The reference measurement may comprise or may be at least one known MRM transition. The reference measurement may be at least one measurement of at least one quality control sample acquired during a previous quality control run and/or at least one measurement of at least one internal standard sample acquired during a previous internal standard sample run and/or at least one measurement of the test sample acquired during a previous run. The reference measurement may be at least one measurement acquired and/or determined and/or measured in the same way and under the same or at least similar and/or comparable conditions as the measurement of the actual test sample. The reference measurements and the measuring of the transition of the analyte may be performed under substantially the same conditions. For example, the reference measurement and the measuring of the transition of the analyte may be performed under constant chromatographic conditions, specifically with the same LC column and eluent. The method may apply to known compounds and well-known conditions. The method may comprise predicting at least one reference measurement, specifically in case of changes of gradients. The predicting may comprise considering aging of LC column, capillary exchanges, solvent composition inaccuracies, and other factors.

The method comprises in step b) (denoted with reference number <NUM>) determining at least one reference peak information of the transition of the analyte using an initial setting of a measurement window <NUM>, wherein the measurement window <NUM> is defined by a time frame of retention times. Examples of measurement windows <NUM> are shown in <FIG>. The reference peak information may be at least one information of a peak, i.e. a local maximum, of the chromatogram corresponding and/or relating to the analyte of interest of the reference measurement which is suitable to limit the relevant time frame for measurement of the analyte of interest. The reference peak information may comprise one or more of: peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor, and/or any type of peak fitting and filtering. The determining of the reference peak information may comprise evaluating <NUM> the reference measurement. The evaluating <NUM> may comprise performing at least one data analysis comprising performing at least one peak finding algorithm and/or performing at least one peak fitting algorithm. The evaluating may comprise one or more of checking of raw data, preprocessing, smoothening, background reduction or removal, peak detection, peak integration.

The measurement window <NUM> is defined by a time frame of retention times. Limiting the measurement of the analyte of interest to a certain pre-defined time frame is generally known. The setting of the measurement window <NUM> may comprise values for one or both limits of the measurement window <NUM>. Specifically, the setting of the measurement window <NUM> may comprise one or both of a value for a lower limit of the measurement window, i.e. a retention time at which the measurement starts, and a value for an upper limit of the measurement window, i.e. a retention time at which the measurement stops.

The initial setting may be a setting of the measurement window <NUM> which is used for determining the reference peak information. The initial setting may be pre-determined and/or pre-defined. For example, in case of a first measurement after change of a column of the LC device, the initial setting may be a default setting which may be deposited in the data base. The method may comprise at least one initial calibration step, not shown here, wherein in the initial calibration step the reference measurement and/or the initial setting may be determined. The initial calibration step may be performed during and/or subsequent to at least one quality control run of the liquid chromatography mass spectrometry device and/or during and/or subsequent to at least one internal standard samples run. A start of the initial calibration step may be triggered by changing of a column of the liquid chromatography mass spectrometry device <NUM> and/or after a predefined or pre-determined time and/or after a predefined or predetermined number of runs, or other suitable counters. In case of performing the method repeatedly, the initial setting may be a setting of the measurement window <NUM> determined from the measurement of at least one prior run or a plurality of prior runs, such as a mean value for the limits of the time frame.

The method furthermore comprises in step c) determining an actual setting of the measurement window <NUM> considering the reference peak information, wherein the determining comprises adjusting the time frame. The actual setting may be a setting of the measurement window <NUM> determined by considering the reference peak information determined in step b). In addition, to considering the reference peak information of only the preceding measurement, the actual setting may be determined considering the preceding measurement or a plurality of preceding measurements. The determining of the actual setting may comprise evaluating the reference peak information and thereby determining a lower and/or an upper limit of the measurement window. Specifically, at least one automated analysis of retention times may be performed, denoted with reference number <NUM>. Moreover, the measurement window may be automatically reassigned, denoted with reference number <NUM>. The actual setting may be determined and/or calculated based on expected retention time and tailing factor. The actual setting may be determined by comparing peak, in particular signal intensity, and background. For example, signal intensity and background may be compared by defining and/or using at least one threshold value at which the peak starts and/or at least one threshold value at which the peak ends. The actual setting may be determined by making a prediction based on a plurality of datasets.

The initial setting may comprise a broader time frame of retention times compared to the actual setting. Specifically, the initial setting of the measurement window <NUM> may be selected so broad such that it is ensured that the peak corresponding to the analyte of interest lies within the time frame. Subsequently the initial setting of the measurement window <NUM> may be optimized in view of measurement results which allows for reducing width of the measurement window and/or for positioning the measurement window <NUM>, specifically to take into account changes of the LC column such as due to aging or other changes. The reference measurement may comprise or may be a known MRM transition, wherein the method comprises optimizing their measurement. The determining of the actual setting may comprise adapting and/or changing the initial setting of the measurement window depending on at least one subsequent measurement. The timing of the measurement may be fixed. However, due to variation of the peak position as a result of ongoing analysis, specifically in case of repeatedly performing method steps a) to d), the retention time may shift and may be adapted based on prior measurements. The method may comprise adjustment of the MRM scheduled timing as a function of changing parameters over time. Thus, the optimized retention time may be used to optimize scheduled MRM measurements and thus freeing up time for more MRM transitions.

The method comprises in step d) measuring, denoted with reference number <NUM>, the transition of the analyte within a sample with the liquid chromatography mass spectrometry device. The measurement may be triggered <NUM> by a user e.g. by entering at least one input to at least one human-machine-interface of the liquid chromatography mass spectrometry device <NUM>.

The method further comprises in step d) determining a measured peak information, denoted with reference number <NUM>, of the transition of the analyte using the actual setting of the measurement window <NUM>. The measured peak information may be at least one information of a peak of the chromatogram measured in step d) corresponding and/or relating to the analyte of interest using the actual setting of the measurement window <NUM>. The measured peak information comprises one or more of: peak maximum, retention time, peak start time, peak end time, peak width, in particular the full width half maximum, peak shape, tailing factor, and/or any type of peak fitting and filtering. The determining of the measured peak information may comprise evaluating the actual measurement. The evaluating may comprise performing at least one data analysis comprising performing at least one peak finding algorithm and/or performing at least one peak fitting algorithm.

The method further may comprise updating <NUM> the data set <NUM> by adding the measurement of the transition of the analyte to the data set <NUM>. Specifically, in case the method steps a) to d) are performed repeatedly, the data set <NUM> may be updated after performing step d) such that the subsequent step a) is performed using an updated initial setting. Thus, the updating <NUM> may be performed permanently.

A width of the measurement window <NUM> may narrow with number of repetitions. Thus, the measurement window <NUM> becomes even better and/or more beneficial for clearing up more MS detection window. The detection window may be a time frame in which the mass spectrometry device has to perform a measurement of a sample.

<FIG> shows a further flowchart of the method according to the present invention, wherein, in addition to the embodiment shown in <FIG>, the method may further comprise at least one in-situ adjustment step <NUM>. The in-situ adjustment step <NUM> may be performed during step d). In the in-situ adjustment step <NUM> intensity of the transition of the analyte may be monitored during the measurement and compared to at least one predetermined or predefined threshold level. The liquid chromatography mass spectrometry device <NUM> may comprise at least one further data base <NUM> configured for storing at least one definition of threshold levels and/or threshold values. For example, the at least one threshold level and/or the at least one threshold value may be defined by percentual change of signal intensity to background. Additionally or alternatively, at least one absolute value may be used as threshold for determining exceedance or undershoot. The further data base <NUM> may be configured to receive (denoted with reference number <NUM>) input information from the data base <NUM> such as values for the threshold levels. Thus, the threshold levels may be data driven. If the intensity falls below the predefined threshold level acquisition of the transition may be stopped. The predetermined or predefined threshold level may be defined by a factor X times the signal-to-noise ratio which is also known from the start. The in-situ adjustment step may be implemented as a feedback loop <NUM> with automated live adjustment of measurement parameter. For example, a measurement, i.e. a specific MRM, may start at a time known on the basis of previous measurements. A run time of the measurement may be determined on the basis of an actual measured intensity of this MRM. In case the intensity falls below the predetermined or predefined threshold level the acquisition of that MRM may be stopped and may allow to free dwell time for other MRMs running at the same time. This approach may be reliable for well articulated peaks. However, problems may arise when signal to noise is low, e.g. a small peak with high variation in individual signal. For these cases it may be advantageous that the predetermined or predefined threshold level may be given by an end of fit curve such as a Gaussian curve. The fit curve may allow defining the peak region such as start of the peak and end of the peak. In particular, a certain peak height at the end of the peak, denoted as end of the fit curve, may be used as threshold level. In principle, use of a fit curve during measurement is problematic since the full signal is not present at this stage. However, a fit curve such as a Gaussian curve can be used since the parameters of the Gaussian or other fit curve may be determined from the one or more preceding measurements. This may allow determining all parameters of the fit curve with only a single iteration. Additionally or alternatively, the fit curve may be determined from a measurement of an internal standard. This may allow accelerating the fitting procedure. Additionally or alternatively, a combination of fit results of different peaks of the same analyte may be used to enhance robustness of the fit result. The fit curve, in particular of a Gaussian, may be independent or less dependent from background and, thus, advantageous even at high noise.

<FIG>show a comparison of a usual approach, shown in <FIG>, and the method according to the present invention, shown in <FIG>, such as described with respect to <FIG>. Specifically, intensity I in % as a function of retention time RT in seconds is depicted. In <FIG>, the upper plot shows the chromatogram for a new LC column and/or initial conditions and the lower plot shows the chromatogram for an aged LC column and/or potential other changes in the experimental performance of the system. For the usual approach shown in <FIG> large measurement windows are used in order to compensate potential changes (denoted with arrow <NUM>) over lifetime of the LC column. For example, the width Δt of the measurement window may be Δt =<NUM> and the position of the measurement window may be from <NUM> to <NUM>. In <FIG> the measurement window <NUM> can be selected narrower compared to <FIG>, e.g. Δt =<NUM>. Moreover, the measurement window <NUM> can be maintained constant or can be even narrowed due to repeating method steps a) to d) and permanent reassignment (denoted with arrow <NUM>) of the measurement window <NUM> based on prior measurements and in-situ adjustment.

<FIG> and <FIG>show a further comparison of measurement windows of a usual approach, shown in <FIG> and of the method according to the present invention, shown in <FIG>. Specifically, intensity I in % as a function of retention time RT in seconds is depicted. Moreover, in <FIG> seven measurement windows and their width are shown each corresponding to a respective peak in the chromatogram. For the usual approach shown in <FIG> large measurement windows are used in order to compensate potential changes over lifetime of the LC column. In contrast, in <FIG> the measurement windows <NUM> are narrowed compared to <FIG>. Using such narrow measurement windows is possible due to permanent reassignment of the measurement window <NUM> based on prior measurements and in-situ adjustment. No safety margins are necessary. The measurements windows show less overlap. Higher sampling rate per peak is possible resulting in more points per peak.

<FIG> shows highly schematically a device <NUM> for multiple transition monitoring according to the present invention. The device <NUM> comprises the at least one liquid chromatography mass spectrometer device <NUM> configured for multiple transition monitoring. The liquid chromatography mass spectrometry device <NUM> may be or may comprise at least one high-performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (µLC) device. The liquid chromatography mass spectrometry device <NUM> may comprise 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 LC device may comprise at least one LC column. For example, the LC device may be a single-column LC device or a multi-column LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped in order to separate and/or elute and/or transfer the analytes of interest. The LC column may be exchangeable, for example after a predefined or pre-determined time and/or number of runs, and/or other suitable counters. The mass spectrometry device may be or may comprise at least one quadrupole mass spectrometry device. The interface coupling the LC device and the MS may comprise at least one ionization source configured for generating of molecular ions and for transferring of the molecular ions into the gas phase.

The device <NUM> further comprises the data base <NUM> configured for storing the data set <NUM> comprising the at least one reference measurement <NUM> of at least one transition of at least one analyte. With respect to description of the data set <NUM> and data base <NUM> reference is made to the description of <FIG> above.

Claim 1:
Method for multiple transition monitoring using a liquid chromatography mass spectrometry device (<NUM>), the method comprises the following steps:
a) determining at least one data set (<NUM>) from at least one data base (<NUM>), the data set (<NUM>) comprising at least one reference measurement (<NUM>) of at least one transition of at least one analyte with the liquid chromatography mass spectrometry device (<NUM>);
b) determining at least one reference peak information of the transition of the analyte using an initial setting of a measurement window (<NUM>), wherein the measurement window (<NUM>) is defined by a time frame of retention times;
c) determining an actual setting of the measurement window (<NUM>) considering the reference peak information, wherein the determining comprises adjusting the time frame, wherein the actual setting is a setting of the measurement window (<NUM>) determined by considering the reference peak information determined in step b);
d) measuring the transition of the analyte with the liquid chromatography mass spectrometry device (<NUM>) and determining a measured peak information of the transition of the analyte using the actual setting of the measurement window (<NUM>),
said method being characterized in that in step a) a mean value and/or a moving average of a plurality of preceding measurements is used as reference measurement (<NUM>).