Method and apparatus for multi-transition monitoring

Abstract

A method for multiple transition monitoring of at least one analyte in a sample using a quadrupole mass analyzer (110) is disclosed. The method comprises the following steps: a) at least one voltage application step (112), wherein in the voltage application step a direct current (DC) voltage and a radio frequency (AC) voltage are applied between two pairs of electrodes (114) of at least one mass filter (116) of the quadrupole mass analyzer (110), wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC , wherein a supplementary AC voltage is superimposed on the AC voltage and the DC voltage, wherein the amplitude AV DC of the supplementary AC voltage is V DC,最大 , wherein V DC,最大 is the maximum voltage output of the DC voltage and b is the bit size of at least one electronic board (118) of the mass filter (116) of the quadrupole mass analyzer (110); b) at least one measurement step (122), wherein at least one transition of the analyte is determined with at least one detector (120) of the quadrupole mass analyzer (110).

Description

TECHNICAL FIELD

[0001] The present invention relates to a method and an apparatus for multiple transition monitoring using mass spectrometry, in particular liquid chromatography and mass spectrometry. BACKGROUND

[0002] Quadrupole mass analyzers are known for multiple transition monitoring (MRM) of at least one analyte in a sample. As described for example in “Massenspektrometrie” Jürgen H. Gross, Springer Spektrum, DOI 10.1007 / 978-3-8274-2981-0 pages 162 to 168, typically four cylindrical electrode rods are used as mass filter which extend parallel along a z-axis and are arranged in a square manner in an xy-plane. Each pair of opposite rods is held at the same potential which consists of an alternating current (AC) voltage and a direct current (DC) voltage. An attractive force acts on the ions which enter the quadrupole rod from one of the rods having a charge opposite to the charge of the ion in z-direction. The sign of the charge of the rods changes periodically. Only ions in a certain mass-to-charge ratio m / z range have the possibility to have a stable trajectory while all other ions have an unstable trajectory. The trajectory of the ions can be described by Mathieu differential equations. Ions having a stable trajectory are fed to a detector and measured by the detector. The detector determines a so-called mass spectrum which is a two-dimensional representation of signal intensity versus m / z, wherein the signal intensity corresponds to the abundance of the respective ion.

[0003] US 9,099,286 B2 describes a mass spectrometer and a method for measuring information related to a sample using mass spectrometry.

[0004] EP 938743 B1 describes a method of operating a mass spectrometer having a first rod set having: at least two pairs of poles; and an exit end, the method comprising: directing ions into or forming ions in the first rod set; transmitting ions from the exit end of the first rod set as transmitted ions; applying an RF voltage to the first rod set; aligning some of the transmitted ions with one of the pairs of poles, the number of transmitted ions aligned with the one pair of poles being greater than the number of transmitted ions not so aligned; and ejecting ions aligned with the one pair of poles from the exit end with greater kinetic energy than ions not so aligned.

[0005] WO 2012 / 120300 A1 describes a mass spectrometry method comprising: during an experimental run or acquisition, automatically correcting the mass or mass-to-charge ratio resolution of a quadrupole mass filter or mass analyzer one or more times based on a measurement, determination or estimation of the mass or mass-to-charge ratio resolution of one or more reference ions observed in the mass spectrum, or based on mass spectral data acquired during the same experimental run or acquisition or during a previous experimental run or acquisition.

[0006] JP 03404849 B2 describes generating a target ion to be determined by an ion source and introducing it into a first stage quadrupole. A bias voltage V1 is applied to the ion to select a target parent ion having a mass Mp. The selected parent ion is collided with a collision gas by applying a bias voltage V2 to a second stage quadrupole in a collision chamber to decompose the parent ion into a daughter ion having a mass Md1, Md2, and bring the daughter ion to a third stage quadrupole. At this time, only the daughter ion having the mass Md1 can pass by applying a bias voltage V3, and based on a ratio of the mass Mp of the prescribed parent ion and the mass Md1 of the prescribed daughter ion, the bias voltage applied to the ion lens is changed, and the target daughter ion converges and is transmitted to the ion detector as much as possible.

[0007] Ernst P. Sheretov et al. in “Modulation parametric resonances and their influence on stability diagram structure”, International Journal of Mass Spectrometry 184 (1999) 207-216 describe a theory of parametric resonances in a hyperbolic mass spectrometer, which occur if the parameters of the rf signal are periodically modulated.

[0008] US 5,227,629 A describes a small perturbation AC voltage applied to the electrodes of a quadrupole mass spectrometer in addition to the normal DC voltage and AC voltage. The perturbation AC voltage generates unstable bands in the triangular stable region and cuts off the edges of the peak profiles of each mass, which improves the resolution of the masses in the mass spectrum and increases the reliability of the measurement results.

[0009] US 2012 / 305762 describes a method in which predetermined ions are separated and left in an ion trap when performing mass spectrometry using the ion trap. In order to have high ion separation accuracy and shorten the time required for ion separation, a first time in which ions having a mass smaller than the ions to be left are separated is set to be shorter than a second time in which ions having a mass greater than the ions to be left are separated.

[0010] The power supply of the mass filter is usually performed using at least one digital-to-analog converter. The available adjustment of the digital-to-analog converter can limit the step resolution of the resulting mass spectrometer peak of the mass spectrum. The step refers to the width of the m / z range during ion detection. In principle, this step can be further optimized, however, this is very expensive and reaches technical limits. Furthermore, the resolution of the step can be important when comparing the analyte and the internal standard peak. The signal related to the analyte and the signal related to the internal standard can be located on different steps. This can lead to a huge variation of the area ratio. This variation can easily occur over time when the mass axis is shifted, for example, due to a change of environmental conditions such as temperature.

[0011] Problem to be solved

[0012] It is therefore an object of the present application to provide a method and a device for multi-transition monitoring which avoids the above-mentioned drawbacks of the known methods and devices. In particular, the method and the device shall improve the signal stability and the reliability of the area ratio of the multi-transition monitoring. SUMMARY

[0013] This problem is solved by a method and a device for multi-transition monitoring having the features of the independent claims. Preferred embodiments of the present application are disclosed in the dependent claims, which can be realized in the individual way or in any arbitrary combination.

[0014] As used hereinafter, the terms "have", "comprise" or "include" or any arbitrary grammatical variations thereof will be used in a non-exclusive way to mean that the entity referred to thereby can include, but is not limited to, one or more of the features mentioned in connection therewith, optionally together with further features. As used hereinafter, the term "exemplary" will mean "serving as an example, instance, or illustration". Any representation of the disclosure with the term "exemplary" does not preferentially imply that the described feature is preferred over other possible features.

[0015] Further, as used herein, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or like terms are used to describe optionally featured embodiments and not to limit alternatives. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. As will be apparent to a person skilled in the art, the application can be performed by using alternative features. Similarly, features introduced by "in an embodiment of the application" or like expressions are intended to be optional features and do not limit alternative embodiments of the application in any way, nor the scope of the application, and also not the possibility to combine features introduced in such way with other optional or non-optional features of the application.

[0016] In a first aspect of the application, a method for multiple transition monitoring using a quadrupole mass analyzer is disclosed.

[0017] As used herein, the term "multiple transition monitoring", also denoted as multiple reaction monitoring (MRM), 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 can refer, without limitation, to a method used in mass spectrometry, in particular in tandem mass spectrometry, wherein multiple product ions from one or more precursor ions are monitored. As used herein, the term "monitoring" 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 can refer, without limitation, to the determination and / or detection of multiple product ions.

[0018] As used herein, the term “mass analyzer”, also denoted as “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 can refer, without limitation, to an analyzer configured for detecting at least one analyte based on mass-to-charge ratio. As used herein, the term “quadrupole mass analyzer” 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 can refer, without limitation, to a mass analyzer comprising at least one quadrupole as mass filter. A quadrupole mass analyzer can comprise a plurality of quadrupoles. For example, a quadrupole mass analyzer can be a triple quadrupole mass analyzer. As used herein, the term “mass filter” 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 can refer, without limitation, to a device configured for selecting ions injected into the mass filter according to their mass-to-charge ratio m / z. A mass filter comprises two pairs of electrodes. The electrodes can be rod-shaped, in particular cylindrical. In an ideal case, the electrodes can be hyperbolic. The electrodes can be designed to be identical. The electrodes can be arranged to extend in parallel along a common axis, e.g. the z-axis. A quadrupole mass analyzer comprises at least one power supply circuit configured for applying at least one direct current (DC) voltage and at least one alternating current (AC) voltage between the two pairs of electrodes of the mass filter. The power supply circuit can be configured for keeping each pair of opposing electrodes at the same potential. The power supply circuit can be configured for periodically changing the charge sign of the electrode pairs such that only ions within a certain mass-to-charge ratio m / z range can have stable trajectories. The trajectories of ions within the mass filter can be described by Mathieu differential equations. In order to measure ions having different m / z values, the DC voltage and the AC voltage can be changed in time, in particular proportionally such that ions having different m / z values can be transmitted to a detector.

[0019] The quadrupole rod mass analyzer can further comprise at least one ionization source. As used herein, the term "ionization source", also denoted as "ion source", 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 can refer, without limitation, to a device configured for generating ions, e.g. from neutral gas molecules. The ionization source can be or can comprise at least one source selected from the group consisting of at least one gas phase ionization source, such as at least one electron impact (El) source or at least one chemical ionization (CI) source; at least one desorption ionization source, such as at least one plasma desorption (PDMS) source, at least one fast atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source and at least one matrix assisted laser desorption (MALDI) source; at least one spray ionization source, such as at least one thermal spray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI) and at least one atmospheric pressure ionization (API) source.

[0020] The quadrupole rod mass analyzer comprises at least one detector. As used herein, the term "detector" 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 can refer, without limitation, to a device configured for detecting incoming ions. The detector can be configured for detecting charged particles. The detector can be or can comprise at least one electron multiplier. The detector of the quadrupole rod mass analyzer and / or the at least one evaluation device can be configured to determine at least one mass spectrum of the detected ions. As used herein, the term "mass filter" 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 can refer, without limitation, to a two-dimensional representation of signal intensity versus mass-to-charge ratio m / z, wherein the signal intensity corresponds to the abundance of the respective ions. The mass spectrum can be a pixelated image. For determining the resulting intensity of a mass spectrum pixel, the signal detected with the detector within a certain m / z range can be integrated. The analyte in the sample can be identified by the at least one evaluation device. Specifically, the evaluation device can be configured for correlating known masses with the identified masses or by characteristic fragmentation patterns.

[0021] The quadrupole mass analyzer can be or can include a liquid chromatography mass spectrometry device. The quadrupole mass analyzer can be connected to and / or can include at least one liquid chromatograph. The liquid chromatograph can be used as a sample preparation for the quadrupole mass analyzer. Other embodiments of sample preparation are possible, such as at least one gas chromatograph. As used herein, the term “liquid chromatography mass spectrometry device” 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 specifically can refer, without limitation, to a combination of liquid chromatography and mass spectrometry. The quadrupole mass analyzer can include at least one liquid chromatograph. The liquid chromatography mass spectrometry device can be or can include at least one high performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (µLC) device. The liquid chromatography mass spectrometry device can include a liquid chromatography (LC) device and a mass spectrometry (MS) device, in the present case a mass filter, wherein the LC device and the mass filter are coupled via at least one interface. The interface coupling the LC device and the MS device can include an ionization source configured for generating molecular ions and transferring the molecular ions into a gas phase. The interface can further include at least one ion mobility module arranged between the ionization source and the mass filter. For example, the ion mobility module can be a high-field asymmetric waveform ion mobility spectrometry (FAIMS) module.

[0022] As used herein, the term “liquid chromatography (LC) device” 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 specifically can refer, without limitation, to an analytical module configured to separate one or more target analytes of a sample from other components of the sample for detection of the one or more analytes using a mass spectrometry device. The LC device can include at least one LC column. For example, the LC device can be a single column LC device or a multi-column LC device having multiple LC columns. The LC column can have a stationary phase through which a mobile phase is pumped in order to separate and / or elute and / or transfer the target analytes. The liquid chromatography mass spectrometry device can further include a sample preparation station for automated pre-treatment and preparation of samples, each sample including at least one target analyte.

[0023] The term "sample" 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 limited to a special or customized meaning. The term specifically can refer, without limitation, to any test sample, such as a biological sample and / or an internal standard sample. The sample can comprise one or more target analytes. For example, the test sample can be selected from the group consisting of physiological fluids including blood, serum, plasma, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, and the like. The sample can be used directly as obtained from the respective source or can be subjected to a pre-treatment and / or sample preparation workflow. The sample can be pre-treated by adding an internal standard and / or by dilution with another solution and / or by mixing with reagents or the like. For example, generally, the target analytes can be vitamin D, drugs of abuse, therapeutic drugs, hormones, and metabolites. The internal standard sample can be a sample comprising at least one internal standard substance having a known concentration. For further details on 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.

[0024] The method comprises the following steps, which can be carried out in the given order as an example. However, it should be noted that different orders are also possible. Furthermore, one or more method steps can also be carried out once or repeatedly. Furthermore, two or more method steps can be carried out simultaneously or in a timely coinciding manner. The method can comprise further method steps which are not listed.

[0025] The method comprises the following steps:

[0026] a) at least one voltage application step, wherein in the voltage application step a direct current (DC) voltage and a radio frequency (AC) voltage are applied between two pairs of electrodes of at least one mass filter of the quadrupole mass analyzer, wherein the AC voltage has an amplitude V AC and the DC voltage has an applicable voltage V DC wherein a supplementary AC voltage is superimposed on the AC voltage and the DC voltage, wherein the amplitude AV DC of the supplementary AC voltage is wherein V DC,最大 is the maximum voltage output of the DC voltage and b is the bit size of at least one electronic board of the mass filter of the quadrupole mass analyzer;

[0027] b) at least one measurement step, wherein at least one transition of the analyte is determined with at least one detector of the quadrupole mass analyzer.

[0028] As used herein, the term "DC voltage" 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 can refer, without limitation, to a component of the potential applied to the pair of electrodes which is essentially time-independent over the measurement time at a certain m / z value. As used herein, the term "essentially time-independent" refers to a voltage which is completely time-independent over the measurement time at a certain m / z value, wherein deviations of < 1 %, preferably < 0.5 % are possible. For example, the DC voltage can have deviations of 0.1 % to 0.2 % from the time-independent development. As used herein, the term "AC voltage" 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 can refer, without limitation, to a component of the potential applied to the pair of electrodes which periodically changes direction. The AC voltage has an amplitude V AC and a DC voltage has an applicable voltage V DC which are applied to the electrodes of the quadrupole. The amplitude of the AC voltage

[0029]

[0030] wherein V (AC,最大) is the maximum amplitude of the AC voltage applied by the AC voltage generator to the electrodes of the mass filter, c AC is a constant, and m / z is the mass-to-charge ratio. The AC signal can be a radio frequency signal having a frequency in the range of 3 kHz to 300 GHz. The applicable voltage may be described as

[0031]

[0032] wherein V (DC,最大) is the maximum amplitude of the DC voltage, c DC is a constant, and m / z is the mass-to-charge ratio. The term "applicable voltage " can refer to a voltage which can be supplied and / or provided to the electrodes of the mass filter. With regard to further embodiments of the AC voltage and the DC voltage applied to the electrodes, reference is made to US 5,227,629, the content of which is included herein by reference.

[0033] As used herein, the term "supplemental AC voltage" 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 can refer, without limitation, to an additional AC voltage superimposed on both the DC voltage and the AC voltage. Typically, it is known that a further AC voltage in addition to the AC component and the DC component has certain influences on the measurements with a quadrupole mass spectrometer or a quadrupole mass filter. For example, US 5,227,629 A describes the use of a small AC voltage in addition to the AC component and the DC component of a quadrupole, in particular to avoid or compensate manufacturing tolerances. Further, it is described therein that this additional small AC voltage can cause ions to have unstable trajectories. However, the present invention proposes to use a supplemental AC voltage in order to enhance the robustness against mass axis drift and / or shift, in particular for multi- transition monitoring.

[0034] With regard to embodiments of the supplemental AC voltage, reference is made to US 5,227,629 A, the content of which is included herein by reference.

[0035] The supplemental AC voltage can be a triangular signal or a sinusoidal signal. As used herein, the term "triangular signal" refers to a perfect triangular signal, wherein it is possible that the triangular signal peaks are rounded or curved due to non-ideal electronics. In case a triangular supplemental AC voltage is applied, all resulting data points of the mass spectrum can have the same weight. However, applying a sinusoidal signal can introduce a weighting of the data points. In order to compensate this influence, the method can comprise applying a predetermined and / or predefined weight to the supplemental AC voltage and / or the weighted measurement data.

[0036] The supplemental AC voltage can have a frequency different from the frequency of the AC voltage. The supplemental AC voltage can have a frequency v of wherein n is the number of repetitions and t d is the dwell time. As used herein, the term "dwell time" 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 custom- ary meaning. The term specifically can refer, without limitation, to the time duration in which each m / z ion signal is detected. For example, the dwell time can refer to the time range in which the mass filter is kept in a certain configuration and / or setting. For example, for a typical dwell time of 2 ms, at least 30 repetitions can be used, resulting in a frequency v of 15 kHz.

[0037] The supplemental AC voltage can be small, i.e. have a small amplitude compared to the amplitude of the AC voltage. It was surprisingly found that superimposing a small wave on top of the AC voltage and the DC voltage, the measured data becomes smooth, in particular averaged, during the measurement and thus less dependent on the step size. For example, a well-controlled small wave is imposed by the supplemental AC voltage. The electronics board of the filter can be configured for providing the AC voltage and the DC voltage to the filter, such as to the electrodes of the filter. The term "electronics board of the filter" 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 limited to a special or customized meaning. The term specifically can refer, without limitation, to a device comprising and / or mechanically supporting at least one electronic component. The electronics board can comprise, for example, at least one printed circuit board on which the at least one electronic component is arranged. The electronics board can comprise at least one digital-to-analog converter. The digital-to-analog converter can be configured for converting a continuously applied voltage signal, such as from a power supply, e.g. from an AC voltage generator and a DC voltage generator, into at least one discrete voltage signal. For measuring ions having different m / z values, the DC voltage value and the AC voltage value can be adjusted over time such that ions having different m / z values can be transmitted to the detector. For operating the filter, the AC voltage and the DC voltage can be adjusted such that the filter is passable or open for a desired mass. The selectable mass cannot be selected continuously but only in discrete steps, because the digital-to-analog converter can divide the voltage range in b discrete steps, wherein b is also denoted as bit size. In particular, the bit size can refer to the total number of bits. This also has the effect that the observable mass range is divided in b steps, wherein the larger the number of steps of the digital-to-analog converter, the finer the m / z steps in the mass range. For a given voltage, a certain mass can be filtered, in particular selected, by the filter and transmitted to the detector. The detector can determine the intensity, in particular the frequency, of the impinging ions. The detected intensity can depend on the dwell time and the difference between the actual mass of the ions and the filtered mass. In particular due to variations in temperature, the filtered mass can shift. Due to the discretization of the filter, the shift can not be visible as a continuous change in intensity but as a jump in intensity. The supplemental AC voltage can have a maximum amplitude in the range of the width of a statistical bin of the mass spectrum. The amplitude of the supplemental AC voltage ΔV DC For wherein V DC,最大is the maximum voltage output for the DC voltage, and b is the bit size of the electronics board of the mass filter of the quadrupole mass analyzer. As used herein, the term "bit size" 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 can refer, without limitation, to the number of bits provided by a digital-to-analog converter. As outlined above, due to the discretization of the mass filter, the displacement of the filtered mass can be observed as a jump in intensity. It was surprisingly found that a smoothing effect can be achieved in case the amplitude of the supplementary AC voltage is lower than the statistical pile size of the mass filter. As used herein, the term "statistical pile width" Δ(m / z), also denoted as "statistical pile size", 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 can refer, without limitation, to the resolution limit at which m / z values can be adjusted. For determining the resulting intensity of a mass spectrum pixel, the detected signal over a certain m / z range can be integrated. If the intensity values of the signal belong to the same statistical pile, the intensity values of the signal can be accumulated for the mass spectrum pixel. The statistical pile width can depend on or can be chosen in accordance with the number of bits available for discretizing the total mass range. The statistical pile width Δ(m / z) can be defined as

[0038]

[0039] where the mass range is the mass range possible for the applied DC voltage, in particular the total mass range, and b is the bit size of the electronics board of the mass filter of the quadrupole mass analyzer. The amplitude of the supplementary AC voltage can be

[0040]

[0041] The AC voltage superimposed with the supplementary AC voltage can be

[0042]

[0043] where V AC,最大 is the maximum amplitude of the AC voltage, c AC is a constant, and m / z is the mass-to-charge ratio. The DC voltage superimposed with the supplementary AC voltage can be

[0044]

[0045] where a is a constant, where is the Matthieu equation, V DC,最大 is the maximum amplitude of the AC voltage, c AC is a constant, and m / z is the mass-to-charge ratio.

[0046] Superimposing a supplementary AC voltage on top of the DC voltage can result in a quadrupole mass analyzer being less prone to small fluctuations, such as caused by electronics or slight changes of the mass axis. Measuring MRM with the method according to the present application can reduce errors that are produced with more stable and / or more reliable areas and / or area ratios. The proposed solution can be implemented quite cost-effectively and quite simply compared to the implementation of better resolving electronics. As will be outlined in more detail below, the method can be implemented using additional components, which can be switched off, if desired, for example for full scan mode. The proposed method can be used during measurement and is thus time neutral and universally applicable to all MRM.

[0047] The method comprises the at least one measuring step, wherein at least one transition of an analyte is determined with a detector of the quadrupole mass analyzer. The measuring step can be triggered by a user, for example by inputting at least one input into at least one human-machine interface of the quadrupole mass analyzer. The method can comprise detecting ions that have passed the mass filter with the detector. The method can comprise evaluating data recorded with the detector. The evaluation can comprise determining a mass spectrum. The evaluation can comprise identifying an analyte, such as by correlating known masses with identified masses or by characteristic fragmentation patterns. The evaluation can be performed using the at least one evaluation device. The evaluation can 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 evaluation can comprise one or more of the following: pre-processing, smoothing, background reduction or removal, peak detection, peak integration.

[0048] The method steps a) and b) can be performed by using at least one computer. In particular, the control of the voltage application in step a) can be performed completely automatically. Further, the data acquisition and evaluation in step b) can be performed completely automatically. In particular, the method can be completely or partially computer-implemented, in particular on a computer, such as a processor, of the quadrupole mass analyzer.

[0049] In a further aspect, a computer program is disclosed, the computer program comprising computer executable instructions for performing the method according to any one of the embodiments described herein, in particular method steps a) to b), when the program is executed on a computer or computer network, in particular on a processor of a quadrupole mass analyzer for multiple transition monitoring.

[0050] Accordingly, in general terms, a computer program is disclosed and proposed herein, which comprises computer-executable instructions for performing the method according to the present application in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. In particular, the computer program can be stored on a computer-readable data carrier. Thus, in particular, one, more than one or even all of the method steps as indicated above can be performed by using a computer or computer network, preferably by using the computer program. In particular, the computer can be fully or partially integrated into the quadrupole mass analyzer and the computer program can in particular be embodied as software. However, alternatively, at least a part of the computer can also be located outside the quadrupole mass analyzer.

[0051] Further disclosed and proposed herein is a computer program product having program code means, in order to perform the method according to the present application, for example one or more of the method steps mentioned above, in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. In particular, the program code means can be stored on a computer-readable data carrier.

[0052] 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, can perform the method according to one or more of the embodiments disclosed herein, in particular one or more of the method steps mentioned above.

[0053] Further disclosed and proposed herein is a computer program product having 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, in particular one or more of the method steps mentioned above, when the program is executed on a computer or computer network. As used herein, a computer program product refers to a program as a tradable product. The product can generally exist in any format, such as in paper format, or on a computer-readable data carrier. In particular, the computer program product can be distributed on a data network.

[0054] Finally, a modulated data signal is disclosed and proposed herein, 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, in particular one or more of the method steps mentioned above.

[0055] In particular, it is further disclosed herein:

[0056] - a computer or computer network comprising at least one processor, wherein the processor is adapted to perform the method according to one of the embodiments described in this specification,

[0057] - a computer loadable data structure adapted to perform a method according to one of the embodiments described in this description when the data structure is executed on a computer,

[0058] - 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,

[0059] - a computer program comprising program means for performing a method according to one of the embodiments described in this description when the computer program is executed on a computer or on a computer network,

[0060] - a computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a computer readable storage medium,

[0061] - a storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform a method according to one of the embodiments described in this description after being loaded into the main memory and / or the working memory of a computer or of a computer network, and

[0062] - a computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium, for performing a method according to one of the embodiments described in this description, in case the program code means are executed on a computer or on a computer network.

[0063] In a further aspect of the application, a quadrupole mass analyzer for multiple transition monitoring of at least one analyte in a sample is disclosed.

[0064] The quadrupole mass analyzer comprises:

[0065] at least one mass filter comprising two pairs of electrodes and at least one detector configured for determining at least one transition of the analyte, wherein the mass filter further comprises at least one electronic board;

[0066] at least one direct current (DC) voltage generator configured for generating a DC voltage and at least one alternating current (AC) voltage generator configured for generating an AC voltage, wherein the AC voltage has an amplitude V AC and the DC voltage has an applicable voltage V DC ;

[0067] - at least one power supply circuit configured for applying the DC voltage and the AC voltage between the two pairs of electrodes of the filter;

[0068] - at least one supplemental AC voltage generator configured for generating a supplemental AC voltage having an amplitude AV DC with V DC,最大 being a maximum voltage output of the DC voltage and b being a bit size of the electron board of the filter of the quadrupole mass analyzer;

[0069] - at least one supplemental power supply circuit configured for superimposing the supplemental AC voltage on top of the AC voltage and the DC voltage.

[0070] The quadrupole mass analyzer can be configured to perform the method according to any of the preceding embodiments. For most of the terms used herein, possible definitions and embodiments, reference can be made to the description of the method above.

[0071] As used herein, the term “DC voltage generator” 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 can refer, without limitation, to an electronic device configured to generate at least one DC voltage signal. The DC voltage generator can be configured to adjust the DC voltage signal according to the m / z range to be measured. As used herein, the term “AC voltage generator” 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 can refer, without limitation, to an electronic device configured to generate at least one AC voltage signal. The AC voltage generator can comprise at least one frequency generator.

[0072] As used herein, the term “power supply circuit” 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 can refer, without limitation, to at least one electronic circuit connecting the DC voltage generator and the AC voltage generator to the pairs of electrodes of the filter. The power supply circuit can comprise a plurality of power supply lines and / or further electrical devices and components.

[0073] The filter comprises the at least one electronic board. The electronic board can be configured for providing the AC voltage and the DC voltage to the filter, such as to the electrodes of the filter. The electronic board can comprise at least one digital-to-analog converter. The digital-to-analog converter can be configured for converting the continuously applied voltage from the AC voltage generator and / or the DC voltage generator and / or the supplemental AC voltage generator into at least one discrete voltage signal.

[0074] As used herein, the term "supplemental AC voltage generator" 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 can refer, without limitation, to at least one electronic device configured to generate a supplemental AC voltage signal. The superimposition of the AC voltage and the DC voltage with the supplemental AC voltage is performed by using the at least one supplemental power supply circuit. As used herein, the term "supplemental power supply circuit" 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 can refer, without limitation, to at least one electronic circuit configured for superimposing the AC voltage and the DC voltage with the supplemental AC voltage. The supplemental power supply circuit can be electrically connected with the power supply circuit.

[0075] The supplemental AC voltage can be superimposed on the AC voltage and the DC voltage by feeding the supplemental AC voltage into the power supply circuit before applying the DC voltage and the AC voltage to the electrodes of the filter. The supplemental AC voltage can be superimposed on the AC voltage and the DC voltage by feeding the AC voltage and the DC voltage together with the supplemental AC voltage into the power supply circuit. For example, the supplemental AC voltage generator can be embodied as integrated with one or both of the AC voltage generator and the DC voltage generator. For example, the quadrupole mass analyzer can comprise two supplemental AC voltage generators. For example, one supplemental AC voltage generator can be embodied as integrated with the AC voltage generator, and the other supplemental AC voltage generator can be embodied as integrated with the DC voltage generator. In case the supplemental AC voltage generator is embodied as integrated with one or both of the AC voltage generator and the DC voltage generator, the AC voltage signal and / or the DC voltage signal can be directly superimposed with the supplemental AC voltage during or after generation, and can be fed together within the power supply circuit.

[0076] The superimposition can be embodied capacitively resulting in a sinusoidal signal, or by using at least one operational amplifier resulting in a triangular wave. The supplemental power supply circuit can comprise one or more of at least one capacitor, at least one operational amplifier. The supplemental power supply circuit can be implemented using electronic components in addition to existing and known power supply circuits for quadrupole mass analyzers. If desired, these additional components can be switched off, for example for full scan mode.

[0077] The quadrupole mass analyzer can comprise at least one control unit configured for controlling one or more of the DC voltage, the AC voltage and the supplemental AC voltage. As used herein, the term "control unit" 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 can refer, without limitation, to an electronic and / or logic unit configured to control the operation of one or more of the DC voltage, the AC voltage and the supplemental AC voltage, in particular the output signal of one or more of the AC voltage generator, the DC voltage generator and the supplemental AC voltage generator and / or the power supply circuit and / or the supplemental power supply circuit.

[0078] The quadrupole mass analyzer can comprise the at least one evaluation device configured for evaluating the at least one detector signal of the detector for determining the transition of the analyte. As used further 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 can comprise at least one data processing device having software code stored thereon, the software code comprising a plurality of computer commands. The evaluation device can provide one or more hardware elements for performing one or more specified operations, and / or can provide one or more processors with software running thereon for performing one or more method steps.

[0079] Compared to known methods and devices for multiple transition monitoring, the method and device according to the present application can provide a number of advantages. Thus, in particular, superimposing a supplemental AC voltage on top of the AC voltage and the DC voltage can result in a quadrupole mass analyzer being less prone to small fluctuations, such as caused by slight changes of electronics or mass axis. Measuring MRM with the method according to the present application can reduce errors with more stable and / or more reliable areas and / or area ratios. The proposed solution can be implemented rather cost-efficiently and rather simply compared to implementations with better resolving electronics. The method can be implemented using additional components, which can be switched off, if desired, for example for full scan mode. The proposed method can be used during measurement and thus is time neutral and universally applicable for all MRM.

[0080] Summarizing and without excluding further possible embodiments, the following embodiments can be envisaged:

[0081] Embodiment 1 : A method for multiple transition monitoring of at least one analyte in a sample using a quadrupole mass analyzer, the method comprising the steps of:

[0082] a) at least one voltage application step, wherein in the voltage application step a direct current (DC) voltage and a radio frequency (AC) voltage are applied between two pairs of electrodes of at least one mass filter of the quadrupole mass analyzer, wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC , wherein a supplementary AC voltage is superimposed on the AC voltage and the DC voltage, wherein the amplitude AV DC of the supplementary AC voltage is , wherein V DC,最大 is the maximum voltage output of the DC voltage and b is the bit size of at least one electronic board of the mass filter of the quadrupole mass analyzer;

[0083] b) at least one measurement step, wherein at least one transition of the analyte is determined with at least one detector of the quadrupole mass analyzer.

[0084] Embodiment 2: The method according to the preceding embodiment, wherein the supplementary AC voltage is a triangular signal or a sinusoidal signal.

[0085] Embodiment 3: The method according to the preceding embodiment, wherein the supplementary AC voltage is a sinusoidal signal, wherein the method comprises applying a predetermined and / or predefined weight to the supplementary AC voltage and / or a weighting of the measurement data determined in step b).

[0086] Embodiment 4: The method according to any one of the preceding embodiments, wherein the supplementary AC voltage has a frequency of , wherein n is the number of repetitions and t d is the dwell time, wherein the frequency ≤ 15 kHz.

[0087] Embodiment 5: The method according to any one of the preceding embodiments, wherein the AC voltage to which the supplementary AC voltage is superimposed has an amplitude of

[0088]

[0089] wherein V AC,最大 is the maximum amplitude of the AC voltage, c ACwhere a is a constant, and m / z is the mass to charge ratio.

[0090] Embodiment 6: The method according to any of the preceding embodiments, wherein the DC voltage where a is a constant, and m / z is the mass to charge ratio.

[0091]

[0092] where a is a constant, and m / z is the mass to charge ratio. , V AC,最大 where a is a constant, and m / z is the mass to charge ratio. AC where a is a constant, and m / z is the mass to charge ratio.

[0093] Embodiment 7: The method according to any of the preceding embodiments, wherein the method steps a) and b) are performed by using at least one computer.

[0094] Embodiment 8: A quadrupole mass analyzer for multiple transition monitoring of at least one analyte in a sample, comprising:

[0095] at least one mass filter comprising two pairs of electrodes and at least one detector configured for determining at least one transition of the analyte, wherein the mass filter further comprises at least one electronics board;

[0096] at least one direct current (DC) voltage generator configured for generating a DC voltage and at least one alternating current (AC) voltage generator configured for generating an AC voltage, wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC ;

[0097] at least one power supply circuit configured for applying the DC voltage and the AC voltage between the two pairs of electrodes of the mass filter;

[0098] at least one supplementary AC voltage generator configured for generating a supplementary AC voltage having an amplitude AV DC , wherein V DC,最大 is the maximum voltage output of the DC voltage, and b is the bit size of the electronics board of the mass filter of the quadrupole mass analyzer;

[0099] at least one supplementary power supply circuit configured for superimposing the supplementary AC voltage on top of the AC voltage and the DC voltage.

[0100] Embodiment 9: The quadrupole mass analyzer according to any of the preceding embodiments, wherein the quadrupole mass analyzer comprises at least one control unit configured for controlling one or more of the DC voltage, the AC voltage and the supplemental AC voltage.

[0101] Embodiment 10: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the supplemental power supply circuit comprises one or more of at least one capacitor, at least one operational amplifier.

[0102] Embodiment 11: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the supplemental power supply circuit is electrically connected with the power supply circuit.

[0103] Embodiment 12: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the supplemental AC voltage is superimposed on the AC voltage and the DC voltage by feeding the supplemental AC voltage into the power supply circuit prior to applying the DC voltage and the AC voltage to the electrodes of the mass filter.

[0104] Embodiment 13: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the supplemental AC voltage is superimposed on the AC voltage and the DC voltage by feeding the AC voltage and the DC voltage together with the supplemental AC voltage into the power supply circuit.

[0105] Embodiment 14: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the quadrupole mass analyzer comprises at least one evaluation device configured for evaluating at least one detector signal of the detector for determining the transition of the analyte.

[0106] Embodiment 15: The quadrupole mass analyzer according to any of the preceding embodiments relating to a quadrupole mass analyzer, wherein the quadrupole mass analyzer is configured to perform a method according to any of the preceding claims relating to a method. BRIEF DESCRIPTION OF DRAWINGS

[0107] Further optional features and embodiments will be disclosed in more detail in the subsequent embodiment description, preferably in combination with the dependent claims. Therein, individual optional features can be realized in a separate manner as well as in any arbitrary feasible combination, as will be appreciated by the skilled person. The scope of the present application is not limited to the preferred embodiments. Embodiments are schematically depicted in the accompanying drawings. Therein, identical reference signs refer to identical or functionally equivalent elements in these drawings.

[0108] In the drawings:

[0109] Fig. 1 shows a flow chart of a method according to the present application;

[0110] Fig. 2 shows a schematic embodiment of a quadrupole mass analyzer according to the present application;

[0111] Figs. 3A to C show a visualization of MRM measurements with superimposed supplemental AC voltage;

[0112] Fig. 4 shows results of a simulation of signal averaging effects; and

[0113] Fig. 5 shows an embodiment of a power supply circuit according to the present application. DETAILED DESCRIPTION

[0114] Fig. 1 shows a flow chart of a method for multiple transition monitoring of at least one analyte in a sample using a quadrupole mass analyzer 110 according to the present application. The sample can be any test sample, such as a biological sample and / or an internal standard sample. The sample can comprise one or more target analytes. For example, the test sample can be selected from the group consisting of physiological fluids including blood, serum, plasma, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, etc. The sample can be used directly as obtained from the respective source or can be subjected to a pre-treatment and / or a sample preparation workflow. The sample can be pre-treated by adding an internal standard and / or by dilution with another solution and / or by mixing with a reagent or the like. For example, generally, the target analytes can be vitamins D, drugs of abuse, therapeutic drugs, hormones and metabolites. The internal standard sample can be a sample comprising at least one internal standard substance having a known concentration. For further details regarding the sample, reference is made to e.g. EP 3 425 369 A1, the entire disclosure of which is incorporated herein by reference. Other target analytes are possible as well.

[0115] The method comprises the following steps:

[0116] The method comprises the following steps, which can be carried out in the given order as an example. However, it should be noted that different orders are also possible. Furthermore, one or more method steps can also be carried out once or repeatedly. Furthermore, two or more method steps can be carried out simultaneously or in a timely overlapping manner. The method can comprise further method steps which are not listed.

[0117] a) at least one voltage application step 112, wherein in the voltage application step 112 a direct current (DC) voltage and a radio frequency (AC) voltage are applied between two pairs of electrodes 114 of at least one mass filter 116 of the quadrupole mass analyzer 110, wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC , wherein a supplementary AC voltage is superimposed on the AC voltage and the DC voltage, wherein the amplitude AV DC of the supplementary AC voltage is , wherein V DC,最大 is the maximum voltage output of the DC voltage, and b is the bit size of at least one electron plate 118 of the mass filter 116 of the quadrupole mass analyzer 110;

[0118] b) at least one measurement step 122, wherein at least one transition of the analyte is determined with at least one detector 120 of the quadrupole mass analyzer 110.

[0119] The DC voltage can be a component of the potential applied to this pair of electrodes 114 which is essentially time-independent over the measurement time of a certain m / z value. The AC voltage can be a component of the potential applied to this pair of electrodes 114 which periodically changes direction. The AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC , which are applied to the electrodes 114 of the mass filter 116. The amplitude V of the AC voltage can be described as

[0120]

[0121] wherein V AC,最大 is the maximum amplitude of the AC voltage applied to and / or supplied to and / or provided to the electrodes 114 of the mass filter, c AC is a constant, and m / z is the mass-to-charge ratio. The AC signal can be a radio frequency signal having a frequency in the range of 3 kHz to 300 GHz. The applicable voltage V can be described as

[0122]

[0123] wherein V DC,最大 is the maximum amplitude of the DC voltage, c DC is a constant, and m / z is the mass to charge ratio. For further embodiments of the AC voltage and the DC voltage applied to the electrodes, reference is made to US 5,227,629, the content of which is included herein by reference.

[0124] Generally, it is known that further AC voltages in addition to the AC component and the DC component have certain influences on measurements with a quadrupole mass spectrometer or a quadrupole mass filter. For example, US 5,227,629 A describes the use of a small AC voltage in addition to the AC component and the DC component of a quadrupole, in particular in order to avoid or compensate manufacturing tolerances. Further, it is described therein that this additional small AC voltage can cause ions to have unstable trajectories. However, the present invention proposes the use of a supplementary AC voltage in order to enhance the robustness against mass axis drift and / or shift, in particular for multiple transition monitoring.

[0125] The supplementary AC voltage can be an additional AC voltage superimposed on both the DC voltage and the AC voltage. The supplementary AC voltage can be a triangular signal or a sinusoidal signal. In case a triangular supplementary AC voltage is applied, all resulting data points of the mass spectrum can have the same weight. However, the application of a sinusoidal signal can introduce a weighting of the data points. In order to compensate this influence, the method can comprise applying a predetermined and / or predefined weight to the supplementary AC voltage and / or the weighted measurement data.

[0126] The supplementary AC voltage can have a frequency different from the frequency of the AC voltage. The supplementary AC voltage can have a frequency v of wherein n is the number of repetitions, and t d is the dwell time. For example, for a typical dwell time of 2 ms, at least 30 repetitions can be used, resulting in a frequency v of 15 kHz.

[0127] The supplemental AC voltage can be small, i.e. have a small amplitude compared to the amplitude of the AC voltage. It was surprisingly found that superimposing a small wave on top of the AC voltage and the DC voltage, the measured data becomes smooth, in particular averaged, during the measurement and thus less dependent on the step size. For example, a well-controlled small wave is imposed by the supplemental AC voltage. The electronics board 118 of the filter 116 can be configured for providing the AC voltage and the DC voltage to the filter 116, such as to the electrodes 114 of the filter 116. The electronics board 118 can comprise at least one digital-to-analog converter. The digital-to-analog converter can be configured for converting a continuously applied voltage signal, such as from a power supply, e.g. from an AC voltage generator and a DC voltage generator, into at least one discrete voltage signal. For measuring ions having different m / z values, the DC voltage value and the AC voltage value can be adjusted over time, such that ions having different m / z values can be transmitted to the detector. For operating the filter 116, the AC voltage and the DC voltage can be adjusted, such that the filter is passable or open for a desired mass. The selectable mass cannot be selected continuously but only in discrete steps, because the digital-to-analog converter can divide the voltage range in b discrete steps, wherein b is also denoted as bit size. In particular, the bit size can refer to the total number of bits. This also has the effect that the observable mass range is divided in b steps, wherein the greater the number of steps (bits) of the digital-to-analog converter, the finer the m / z steps in the mass range. For a given voltage, a certain mass can be filtered, in particular selected, by the filter 116 and transmitted to the detector 120. The detector 120 can determine the intensity, in particular the frequency, of the impinging ions. The detected intensity can depend on the dwell time and the difference between the actual mass of the ions and the filtered mass. In particular due to variations in temperature, the filtered mass can shift. Due to the discretization of the filter, the shift can not be visible as a continuous change in intensity but as a jump in intensity. The supplemental AC voltage can have a maximum amplitude in the range of the width of a statistical bin of the mass spectrum. The amplitude ΔV of the supplemental AC voltage DC For wherein V DC,最大Let be the maximum output voltage of the DC voltage, and b be the bit size of the electronic board 118 of the mass filter 116 of the quadrupole mass analyzer 110. The bit size can be the number of bits provided by the digital-to-analog converter. As outlined above, due to the discretization of the mass filter 116, the displacement of the filtered mass can be observed as a jump in intensity. Surprisingly, a smoothing effect can be achieved when the amplitude of the supplementary AC voltage is lower than the statistical stack size of the mass filter 116. The statistical stack width can be the resolution limit where the m / z value can be adjusted. To determine the obtained intensity of the mass spectrometer pixel, the detected signal can be integrated over a certain m / z range. If the intensity values ​​of the signals belong to the same statistical stack, the intensity values ​​of the signals accumulated by the mass spectrometer pixel can be used. The statistical stack width can depend on the number of bits available for discretizing the total mass range or can be selected based on the number of bits available for discretizing the total mass range. The statistical stack width Δ(m / z) can be defined as...

[0128]

[0129] The mass range refers to the possible mass range for the applied DC voltage, specifically the total mass range, and b is the bit size of the electronic board 118 of the mass filter 116 of the quadrupole mass analyzer 110. The amplitude of the AC voltage is also considered. Can be

[0130]

[0131] AC voltage with supplementary AC voltage superimposed Can be

[0132]

[0133] in V AC,最大 c is the maximum amplitude of the AC voltage. AC It is a constant, and m / z is the mass-to-charge ratio. A DC voltage is superimposed with a supplementary AC voltage. Can be

[0134]

[0135] Where a is a constant, (Matthieu equation). V AC,最大 c is the maximum amplitude of the AC voltage. AC It is a constant, and m / z is the mass-to-charge ratio.

[0136] Superimposing the supplementary AC voltage on top of the AC voltage and the DC voltage can result in a quadrupole mass analyzer 110 that is less prone to small fluctuations, such as fluctuations caused by electronics or slight changes of the mass axis. Measuring MRM with the method according to the present application can reduce errors that are caused by more stable and / or more reliable areas and / or area ratios. The proposed solution can be implemented quite cost-effectively and quite simply compared to implementations with better resolving electronics. The method can be implemented using additional components, which can be switched off, if desired, for example for full scan mode. The proposed method can be used during measurement and is thus time-neutral and universally applicable to all MRMs.

[0137] The method comprises the at least one measuring step 122, wherein at least one transition of an analyte is determined with a detector 120 of the quadrupole mass analyzer 110. The measuring step 122 can be triggered by a user, for example by entering at least one input into at least one human-machine interface of the quadrupole mass analyzer 110. The method can comprise detecting ions that have passed the mass filter 116 with the detector 120. The method can comprise evaluating data recorded with the detector. The evaluation can comprise determining a mass spectrum. The evaluation can comprise identifying an analyte, such as by correlating known masses with identified masses or by characteristic fragmentation patterns. The evaluation can be performed using the at least one evaluation device 124. The evaluation can 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 evaluation can comprise one or more of the following: pre-processing, smoothing, background reduction or removal, peak detection, peak integration.

[0138] The method steps a) and b) can be performed by using at least one computer. In particular, the control of the voltage application in step a) can be performed completely automatically. Furthermore, the data acquisition and evaluation in step b) can be performed completely automatically. In particular, the method can be completely or partially computer-implemented, in particular on a computer, such as a processor, of the quadrupole mass analyzer.

[0139] Figure 2 shows a schematic embodiment of a quadrupole mass analyzer 110. The quadrupole mass analyzer 110 comprises the at least one mass filter 116 comprising two pairs of electrodes 114 and the at least one detector 120 configured for determining at least one transition of an analyte. The quadrupole mass analyzer 110 can be an analyzer configured for detecting at least one analyte based on mass-to-charge ratio. The quadrupole mass analyzer 110 can comprise at least one quadrupole as mass filter 116. The quadrupole mass analyzer 110 can comprise a plurality of quadrupoles. For example, the quadrupole mass analyzer 110 can be a triple quadrupole mass spectrometer. The mass filter 116 can be configured for selecting ions injected into the mass filter 116 according to their mass-to-charge ratio m / z. The mass filter 116 comprises two pairs of electrodes 114. The electrodes 114 can be rod-shaped, in particular cylindrical. The electrodes 114 can be designed to be identical. The electrodes 114 can be arranged to extend in parallel along a common axis, e.g. the z-axis. The quadrupole mass analyzer 110 comprises at least one power supply circuit 126 configured for applying at least one direct current (DC) voltage and at least one alternating current (AC) voltage between the two pairs of electrodes 114 of the mass filter 116. An embodiment of the power supply circuit 126 is shown in Figure 5. The design of the power supply circuit 126 corresponds to the construction of the mass spectrometer of Figure 1 of US 5,227,629 A, however, according to the present application, it can be designed for multiple transition monitoring. The power supply circuit 126 can be configured for keeping each pair of opposing electrodes 114 at the same potential. The power supply circuit 126 can be configured for periodically changing the charge sign of the electrode pairs 114 such that only ions in a certain range of mass-to-charge ratios m / z can have stable trajectories. The trajectories of ions within the mass filter can be described by Mathieu differential equations. In order to measure ions having different m / z values, the DC voltage and the AC voltage can be changed over time, in particular in proportion such that ions having different m / z values can be transmitted to the detector 120.

[0140] The quadrupole mass analyzer 110 can be further connected to and / or can comprise a sample preparation station (not shown here) for automated pre-treatment and preparation of samples, each sample comprising at least one target analyte.

[0141] The quadrupole mass analyzer includes at least one DC voltage generator 128 configured to generate a direct current voltage and at least one AC voltage generator 130 configured to generate a radio frequency AC voltage. The DC voltage generator 128 can be configurable to adjust the DC voltage signal according to the m / z range to be measured. The AC voltage generator 130 can include at least one frequency generator. The power supply circuit 126 can be at least one electronic circuit connecting the DC voltage generator 128 and the AC voltage generator 130 to the electrode pair 114 of the mass filter 116. The power supply circuit 126 can include a plurality of power supply lines and / or additional electrical devices and components.

[0142] The quadrupole mass analyzer includes at least one supplemental AC voltage generator 132 configured to generate a supplemental AC voltage having an amplitude AV DC where V DC,最大The maximum voltage output is for the DC voltage, and b is the bit size of the electronics board 118 of the mass filter 116. The quadrupole mass analyzer 110 includes at least one supplemental power supply circuit 134 configured for superimposing a supplemental AC voltage on top of the AC voltage and the DC voltage. Superimposing the AC voltage and the DC voltage with the supplemental AC voltage is performed by using the at least one supplemental power supply circuit 134. The supplemental power supply circuit 134 can be electrically connected with the power supply circuit 126. The supplemental AC voltage can be superimposed on top of the AC voltage and the DC voltage by feeding the supplemental AC voltage into the power supply circuit 126 before applying the DC voltage and the AC voltage to the electrodes 114 of the mass filter 116. The supplemental AC voltage can be superimposed on top of the AC voltage and the DC voltage by feeding the AC voltage and the DC voltage together with the supplemental AC voltage into the power supply circuit 126. For example, the supplemental AC voltage generator 132 can be embodied as integrated with one or both of the AC voltage generator 130 and the DC voltage generator 128. For example, the quadrupole mass analyzer 110 can include two supplemental AC voltage generators 132. For example, one supplemental AC voltage generator 132 can be embodied as integrated with the AC voltage generator 130, and the other supplemental AC voltage generator can be embodied as integrated with the DC voltage generator 128. In case the supplemental AC voltage generator 132 is embodied as integrated with one or both of the AC voltage generator 130 and the DC voltage generator 128, the AC voltage signal and / or the DC voltage signal can be directly superimposed with the supplemental AC voltage during or after generation, and can be fed together within the power supply circuit 126.

[0143] The superimposition can be embodied as capacitive to result in a sinusoidal signal, or by using at least one operational amplifier to result in a triangular wave. The supplemental power supply circuit 134 can include one or more of at least one capacitor, at least one operational amplifier. The supplemental power supply circuit 134 can be implemented using electronic components in addition to existing and known power supply circuits for quadrupole mass analyzers. If needed, these additional components can be switched off, for example for full scan mode.

[0144] The quadrupole rod mass analyzer 110 comprises at least one detector 120. The detector 120 can be configured for detecting charged particles. The detector 120 can be or can comprise at least one electron multiplier. The detector 120 of the quadrupole rod mass analyzer 110 and / or the at least one evaluation device 124 can be configured to determine at least one mass spectrum of the detected ions. The mass spectrum can be a pixelated image. For determining the resulting intensities of the mass spectrum pixels, the signals detected with the detector over a certain m / z range can be integrated. The analytes in the sample can be identified by the at least one evaluation device 124. In particular, the evaluation device can be configured for correlating known masses with the identified masses or by characteristic fragmentation patterns.

[0145] The quadrupole rod mass analyzer can comprise the at least one evaluation device 124 configured for evaluating the at least one detector signal of the detector 120 for determining a transition of an analyte. The at least one evaluation device 124 can comprise at least one data processing device having software code stored thereon, the software code comprising a plurality of computer commands. The evaluation device 124 can provide one or more hardware elements for performing one or more specified operations, and / or can provide one or more processors with software running thereon for performing one or more method steps.

[0146] The quadrupole rod mass analyzer 110 can comprise at least one control unit 142 configured for controlling one or more of the DC voltage, the AC voltage and the supplemental AC voltage. The control unit can comprise electronics and / or logic units configured to control the one or more of the DC voltage, the AC voltage and the supplemental AC voltage, in particular the output signals of one or more of the AC voltage generator 130, the DC voltage generator 128 and the supplemental AC voltage generator and / or the power supply circuit 126 and / or the supplemental power supply circuit 134.

[0147] The quadrupole mass analyzer 110 can further comprise at least one ionization source 136. The ionization source 136 can be or can comprise at least one source selected from the group consisting of: at least one gas phase ionization source, such as at least one electron impact (El) source or at least one chemical ionization (CI) source; at least one desorption ionization source, such as at least one plasma desorption (PDMS) source, at least one fast atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source, and at least one matrix assisted laser desorption (MALDI) source; at least one spray ionization source, such as at least one thermal spray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI), and at least one atmospheric pressure ionization (API) source.

[0148] The quadrupole mass analyzer 110 can be or can comprise a liquid chromatography mass spectrometry device. The quadrupole mass analyzer 110 can be connected to and / or can comprise at least one liquid chromatograph 138. The liquid chromatograph 138 can serve as a sample preparation for the quadrupole mass analyzer 110. Other embodiments of sample preparation are possible, such as at least one gas chromatograph. The liquid chromatography mass spectrometry device can be or can comprise at least one high performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (pLC) device. The liquid chromatography mass spectrometry device can comprise a liquid chromatography (LC) device 138 and a mass spectrometry (MS) device, in the present case the mass filter 116, wherein the LC device 138 and the mass filter 116 are coupled via at least one interface 140. The interface 140 coupling the LC device 138 and the MS device can comprise an ionization source 136 configured for generating molecular ions and for transferring the molecular ions into the gas phase. The interface 140 can further comprise at least one ion mobility module arranged between the ionization source 136 and the mass filter 116. For example, the ion mobility module can be a high field asymmetric waveform ion mobility spectrometry (FAIMS) module. The liquid chromatography mass spectrometry device can further comprise a sample preparation station for automated pre-treatment and preparation of samples, each sample comprising at least one target analyte.

[0149] Figures 3A to C show visualizations of MRM measurements with superimposed supplemental AC voltage. Figure 3A shows a regular MRM measurement. The statistical pile with the local maximum is indicated with a solid line and an arrow. For this statistical pile, the detector 120 records a single data point for the corresponding DC voltage. Figure 3B shows a MRM measurement according to the present application with a sinusoidal signal as supplemental voltage. Figure 3C shows a MRM measurement according to the present application with a triangular signal as supplemental voltage. In this visualization, the amplitudes are exaggerated. The superimposition of the supplemental voltage can result in multiple data points being recorded for the same statistical pile, so that smoothing or averaging can be performed when generating the statistical pile of the mass spectrum.

[0150] Figure 4 shows results of a simulation of the signal averaging effect. Different strengths of smoothing are applied to the data. Specifically, Figure 4 shows a bar chart for different smoothing widths (i.e. from 0.01 to 0.10), where for each smoothing width the statistical piles are shown from left to right, the first bar indicates the mean of the constant Q1 and Q3, the second bar indicates the mean of the constant Q1 and the varying Q3, the third bar indicates the mean of the varying Q1 and the constant Q3, the fourth bar indicates the mean of the varying Q1 and Q3 in the same direction, and the fifth bar indicates the mean of the varying Q1 and Q3 in different directions. Q1 and Q3 refer to the quadrupole rods of the quadrupole mass analyzer 110. The filtering is only effective when the smoothing width is below the statistical pile size (0.05 u in this example). Furthermore, Figure 4 shows the error region with solid lines. On the left side of the graph, the opening width of the error region indicates the error without smoothing, while on the right side of the graph, the opening width of the error region indicates the error with smoothing. By producing smoothing with more stable and / or more reliable area / area ratios, the error is significantly reduced.

[0151] List of reference signs

[0152] 110 quadrupole mass analyzer

[0153] 112 voltage application step

[0154] 114 electrode

[0155] 116 mass filter

[0156] 118 electron plate

[0157] 120 detector

[0158] 122 measurement step

[0159] 124 evaluation device

[0160] 126 power supply circuit

[0161] 128 DC voltage generator

[0162] 130 AC voltage generator

[0163] 132 supplemental AC voltage generator

[0164] 134 supplemental power supply circuit

[0165] 136 ionization source

[0166] 138 liquid chromatograph device

[0167] 140 interface

[0168] 142 control unit

Claims

1. A method for multiple transition monitoring of at least one analyte in a sample using a quadrupole mass analyzer (110), the method comprising the following steps: a) at least one voltage application step (112), wherein in the voltage application step a direct current (DC) voltage and a radio frequency (AC) voltage are applied between two pairs of electrodes (114) of at least one mass filter (116) of the quadrupole mass analyzer (110), wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC , wherein a supplementary AC voltage is superimposed on the AC voltage and the DC voltage, wherein the amplitude of the supplementary AC voltage is , wherein V DC,最大 is the maximum voltage output of the DC voltage, and b is a bit size of at least one electronic board (118) of the mass filter (116) of the quadrupole mass analyzer (110), wherein the electronic board (118) is configured for providing the AC voltage and the DC voltage to the electrodes (114) of the mass filter (116), wherein the electronic board (118) comprises at least one digital-to-analog converter, and the bit size represents a number of bits provided by the digital-to-analog converter; b) at least one measurement step (122), wherein at least one transition of the analyte is determined with at least one detector (120) of the quadrupole mass analyzer (110).

2. The method according to claim 1, wherein the supplementary AC voltage is a triangular signal or a sinusoidal signal.

3. The method according to claim 2, wherein the supplementary AC voltage is a sinusoidal signal, wherein the method comprises one or both of the following: applying one or both of a predetermined or predefined weight to the supplementary AC voltage, or weighting of the measurement data determined in step b).

4. The method according to claim 1 or 2, wherein the supplementary AC voltage has a frequency v of where n is the number of repetitions, and t d is the dwell time, wherein the frequency v < 15 kHz.

5. The method according to claim 1 or 2, wherein the AC voltage V AC is superimposed with the supplementary AC voltage where V AC,最大 is the maximum amplitude of the AC voltage, c AC is a constant, and m / z is the mass-to-charge ratio.

6. The method according to claim 1 or 2, wherein the DC voltage V DC is superimposed with the supplementary AC voltage where a is a constant, where V AC,最大 is the maximum amplitude of the AC voltage, c AC is a constant, and m / z is the mass-to-charge ratio.

7. The method according to claim 1 or 2, wherein the method steps a) and b) are performed by using at least one computer.

8. A quadrupole mass analyzer (110) for multiple transition monitoring of at least one analyte in a sample, comprising: at least one mass filter (116) comprising two pairs of electrodes (114) and at least one detector (120) configured for determining at least one transition of the analyte, wherein the mass filter (116) further comprises at least one electronics board (118), wherein the electronics board (118) is configured for providing an AC voltage and a DC voltage to the electrodes (114) of the mass filter (116), wherein the electronics board (118) comprises at least one digital-to-analog converter; - at least one DC voltage generator (128) configured for generating a direct current (DC) voltage and at least one AC voltage generator (130) configured for generating a radio frequency (AC) voltage, wherein the AC voltage has an amplitude V AC , and the DC voltage has an applicable voltage V DC ; at least one power supply circuit (126) configured for applying the DC voltage and the AC voltage between the two pairs of electrodes (114) of the mass filter (116); at least one supplemental AC voltage generator (132) configured for generating a supplemental AC voltage having an amplitude where V DC,最大 is a maximum voltage output of the DC voltage and b is a bit size of the electron plate (118) of the mass filter (116) of the quadrupole mass analyzer (110) and the bit size represents a number of bits provided by the digital-to-analog converter; at least one supplementary power supply circuit (134) configured for superimposing the supplementary AC voltage on top of the AC voltage and the DC voltage.

9. The quadrupole mass analyzer (110) according to claim 8, wherein the quadrupole mass analyzer (110) comprises at least one control unit (142) configured for controlling one or more of the DC voltage, the AC voltage and the supplementary AC voltage.

10. The quadrupole mass analyzer (110) according to claim 8 or 9, wherein the supplementary power supply circuit (134) comprises one or more of at least one capacitor, at least one operational amplifier.

11. The quadrupole mass analyzer (110) according to claim 8 or 9, wherein the supplementary power supply circuit (134) is electrically connected with the power supply circuit (126).

12. The quadrupole mass analyzer (110) according to claim 8 or 9, wherein the supplementary AC voltage is superimposed on top of the AC voltage and the DC voltage by feeding the supplementary AC voltage into the power supply circuit (126) before applying the DC voltage and the AC voltage to the electrodes (114) of the mass filter (116).

13. The quadrupole mass analyzer (1 10) according to claim 8 or 9, wherein the supplemental AC voltage is superimposed on the AC voltage and the DC voltage by feeding the AC voltage and the DC voltage together with the supplemental AC voltage into the power supply circuit (126).

14. The quadrupole mass analyzer (1 10) according to claim 8 or 9, wherein the quadrupole mass analyzer (1 10) comprises at least one evaluation device (124) configured for evaluating at least one detector signal of the detector (120) for determining the transition of the analyte.

15. The quadrupole mass analyzer (1 10) according to claim 8 or 9, wherein the quadrupole mass analyzer (1 10) is configured for carrying out the method according to claim 1 or 2.

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