Highly sensitive and selective mass spectrometric method and mass spectrometer system for detecting an analyte in a biological sample

Abstract

Mass spectrometric method (100) for detecting an analyte in a biological sample solution comprising the steps: providing (101) a constant stream of ionized sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard including electrospray ionization (ESI), and / or nano-electrospray ionization (nano-ESI); performing (102a) a spatial accumulation of the analyte and the analyte-specific internal standard and performing (102b) at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing (103) at least partially a second separation of the analyte and the analyte-specific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting (104) the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; providing (105) a stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on a detection mass spectrometer; and recording (106) with the detection mass spectrometer at least two pre-summed mass-resolved spectra and summing (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

Description

[0001] Highly sensitive and selective mass spectrometric method and mass spectrometer system for detecting an analyte in a biological sample

[0002] Field of the Invention

[0003] The present invention relates to a mass spectrometric (MS) method and a mass spectrometer system for detecting an analyte in a biological sample as well as a computer-program product for carrying out the method in the field of In Vitro Diagnostics (IVD). MS already provides a high degree of selectivity and, in addition, the invention can provide an improved degree in sensitivity. Further, the invention can provide a very robust method and system and a higher degree in sustainability, as the amount of solvents may be reduced compared to prior art MS methods and systems.

[0004] Background of the Invention

[0005] MS is widely used for the qualitative and quantitative analysis of chemical substances ranging from small molecules to macromolecules.

[0006] Often, MS is combined with chromatographic techniques, particularly gas and liquid chromatography (GC and LC) such as e.g. HPLC. In that case, the analyzed molecule (analyte) of interest is separated chromatographically and is individually subjected to MS analysis (Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 p. 181- 190). For example, Meier F, et al. “Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer”. Mol Cell Proteomics. 2018 Dec; 17(12):2534-2545. doi: 10.1074 / mcp.TIRl 18.000900. Epub 2018 Nov 1. PMID: 30385480; PMCID: PMC6283298 (denoted herein “Meier F, et al.”) describes the use of a timsTOF instrument (Trapped Ion Mobility Spectrometry combined with time of flight spectrometry) in combination with liquid chromatography to introduce online serial fragmentation (PASEF). Therefore, molecules in the sample are separated using liquid chromatography prior to ionization and introduction into the mass spectrometer system. The timsTOF instrument corresponds therein to a quadrupole time-of-flight (QTOF) mass spectrometer equipped with a second generation dual TIMS analyzer in a first vacuum stage.

[0007] In general, separation via GC and HPLC prior to MS, as described in Meier F, et al. offers many advantages in terms of sample preparation and technical options that can be used in the separation (e.g. different columns and solvents). Furthermore, there is the advantage of a separation in front of, i.e. upstream of the mass spectrometer such that the analysis may be pre-enriched and may be generally eluted as a "peak" from the stationary phase and is therefore ionized and detected in an enriched manner. The prior enrichment is an advantage but it also has a disadvantage regarding the very limited time span for the measurement of the "enriched analyte as a peak". Further, this combination has inherent problems of analyte - surface contact and the need for stationary phases and liquid solvents. Furthermore, the mechanical parts underlie an inherent stress by being moved and this may result in a deterioration of the equipment. Therefore, the systems require intensive maintenance due to the mechanical parts, which are used. Stationary phases, which are typically used, for example in GC and / or HPLC need to be exchanged on a regular basis due to deterioration and / or chemical blockage. Therefore, chromatography is cumbersome to use in clinical diagnostics, which seeks to achieve a high throughput in sample analysis and is run by few technicians, who might not be trained in the complicated maintenance of chromatographic systems. Waiting for service technicians to fix problems and / or take care of the maintenance of the systems may result in long downtimes of the MS and in such a waiting time, no sample analysis may be performed.

[0008] In general, MS is a versatile tool over all disciplines in science. For environmental analyses, it can determine traces of pollutants, in mineralogy, it can assess chemical compositions and in chemistry, it can reflect pathways of reactions by measuring certain compounds qualitatively as well as quantitatively. Moreover, MS provides a high degree of selectivity with a sufficient analyte-dependent sensitivity, allowing even for the analysis of complex biological, for example (e.g. serum, plasma, urine), environmental and / or clinical samples. However, for several analytes, especially if analyzed from complex biological matrices such as a serum, the sensitivity turns out to be challenging. To provide a broad applicability in diagnostics, i.e. the possibility to analyze a wide variety of biomolecules at a high selectivity, the sensitivity requirements must be met. Marginal improvements in sensitivity have proven to be only achieved through complex technical measures. In order to make clinically relevant analytes measurable, e.g. for neurodegenerative diseases, an approach is required that allows increased sensitivity ranges.

[0009] In clinical diagnostics, MS has proven to be a versatile tool with the potential to meet different aspects in terms of sample types (e.g. liquid (e.g. serum / blood), solid (e.g. cross sections of tissue) or gaseous (e.g. diagnostic breath analytic)) as well as analytical tasks e.g. qualitative or quantitative.

[0010] MS which is used in clinical diagnostics / analysis may further fulfill the needs for dealing with low mass analytes e.g. alkali metals (Na, K, etc.) or small acids / bases e.g. valproic acid as well as medium size analytes (e.g. testosterone) and large molecular mass analytes e.g. biopolymers like proteins. All of the analytes need to be addressed quantitatively as well as qualitatively, specifically at a large “dynamic range”.

[0011] Additionally, there is growing interest for the implementation of MS in the clinical diagnostics, specifically in large diagnostic laboratories that provide analytic service to numerous customers, subjects and / or patients. Further, the number of published methods related to MS especially for the detection of small molecules in therapeutic drug monitoring or drug of abuse testing is increasing.

[0012] Some ready to use kits for pre-validated clinical MS applications are becoming commercially available. The use of MS, even in connection with such kits, may however not be regulatory approved for clinical diagnostics. This is mostly because of lack of standardized procedures, except for very few analytes, and because of the still large number of user-dependent factors, e.g. due to a number of manual steps that are still conducted and the diversity of hardware components that may be used and combined, and that play a role in delivering reliable and reproducible results of clinical relevance. In particular, sample preparation is typically a manual and tedious procedure. Protein precipitation with subsequent centrifugation is the most popular method to remove unwanted and potentially disturbing sample matrix. The use of kits may in part facilitate sample preparation that can at least partially be automated. Kits are however available only for a limited number of analytes of interest and the entire process from sample preparation to separation and detection remains complex, requiring the attendance of highly trained laboratory personnel to run highly sophisticated complex and / or high-maintenance instruments.

[0013] In addition, typically, a batch approach is followed, where a batch of samples prepared in advance under the same preparation conditions undergo consecutive separation runs under the same separation conditions. This approach however does not enable high throughput and is not flexible, e.g. does not allow re-scheduling (changing a pre-defined processing sequence) in view for example of incoming emergency samples that have higher priority and hence have to be processed first.

[0014] Summary of the Invention

[0015] It is therefore desirable to provide an MS method and / or MS system and / or an analyzer based on MS each being adapted to the specific requirements in diagnostic analysis, specifically in diagnostic analysis of large laboratories with high throughput. Moreover, it is desirable to increase the sensitivity of MS such that it can be broadly used in a versatile manner in diagnostic analysis. It is further desirable to achieve, maintain and / or increase the degree in selectivity of MS. In general, it is desirable to provide an MS clinical diagnostic method and / or MS system, which allows for a comfortable, low maintenance, reliable, sensitive, selective and / or fast detection of a wide variety of analytes from complex biological matrices. Moreover, it is desirable to provide to health care workers, medical lab technicians and other users with little or no experience in MS and / or other techniques (such as chromatography) a user-friendly method based on MS which is simple, safe and / or convenient to use, without the need of many manual steps and requiring low maintenance. It is also desirable to provide a very robust MS method and / or MS system. Moreover, it is desirable to increase the degree in sustainability, specifically by reducing the amount of solvents used to perform MS.

[0016] At least one of these objects is overcome by the subject matter of the independent claims. Specific embodiments with optional features are subjected to the dependent claims.

[0017] An MS method and an MS system rendering the use of MS more sensitive and therefore suitable for clinical diagnostics are described herein. In particular, high-throughput, e.g. up to 100 samples / hour or more with random access for analytes and sample concentrations / matrix components sample preparation can be achieved at a high sensitivity and a high selectivity. Moreover, the process can be fully automated increasing the walk-away time and decreasing the level of skills required by an operator.

[0018] The invention according to aspects and according to embodiments are described as follows in more detail.

[0019] According to a first aspect, a mass spectrometric method for detecting an analyte in a biological sample solution comprises the steps: providing a constant stream of ionized sample molecules comprising the analyte, i.e. ionized analyte molecules, from the biological sample solution and an analyte-specific internal standard, i.e. ionized analytespecific internal standard molecules, wherein the providing of the constant stream of ionized sample molecules and ionized analyte-specific internal standard molecules includes atmospheric pressure ionization, e.g. electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI); performing a spatial accumulation of the analyte and the analyte-specific internal standard and performing at least partially a first separation of the analyte (i.e. ionized analyte molecules) and the analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing at least partially a second separation of the analyte (i.e. ionized analyte molecules) and the analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from remained other components (i.e. other ionized molecules) of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting the ionized analyte (also denoted “ionized analyte precursor molecules” or just “precursor molecules”) into ionized analyte fragments (also denoted “ionized analyte fragment molecules”, “ionized analyte daughter molecules / ions” or just “daughter molecules / ions”) and the analyte-specific internal standard into ionized analytespecific internal standard fragments (also denoted “ionized internal standard fragment molecules”) by collision of the analyte with gas molecules, i.e. by collision with particles of a gas phase (i.e. gas being introduced into a chamber of a fragmenter module); providing a stream (which may be a pulsed or a (quasi)constant stream for a certain time at least) comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on a detection mass spectrometer; and recording with the detection mass spectrometer at least two firstly accumulated mass-resolved spectra, also denoted as “digitally pre-summed mass-resolved spectra” and accumulating the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum, also denoted as “digitally summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum”. The method according to the first aspect is based on MS and is adapted to at least one specific requirement in diagnostics, specifically in diagnostic analysis of large laboratories with high throughput. The method according to the first aspect is specifically suited to increase and / or improve the sensitivity of MS such that it may be broadly used in a versatile manner in diagnostics to better determine the level of the analyte of interest. The method according to the first aspect may either achieve, maintain and / or increase a high degree in selectivity, specifically when being compared to existing MS. The method according to the first aspect may allow for a comfortable, low maintenance, reliable, sensitive, selective and / or fast detection of a wide variety of analytes from complex biological matrices. The method according to the first aspect provides a more user-friendly way of performing MS in diagnostics, being accessible to health care workers and / or other users with little to no experience and / or knowledge in / of MS and / or other techniques (such as chromatography). The MS method may be very robust and allows increasing the degree in sustainability, specifically by reducing the amount of solvents used to perform MS. For example, the amount of Acetonitrile required to perform MS may be reduced, specifically when using Nano-ESI.

[0020] Alternatively or in addition a mass spectrometric method for detecting an analyte of interest in a biological sample solution may comprise the steps: providing to an ionizer a constant stream of sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard by means of a direct inlet approach; at least partially ionizing with the ionizer the sample molecules from the constant stream that comprise the analyte and the analyte-specific internal standard and thereby providing to a first selectivity enhancer module a constant stream of ionized sample molecules comprising the (ionized) analyte and the (ionized) analyte-specific internal standard (i.e. ionized analyte and ionized snalyte-specific internal standard), the ionizer including a plasma-based ambient ionization technique, such as Direct analysis in real time (DART) and / or atmospheric pressure ionization, such as electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI); performing with the first selectivity enhancer module a spatial accumulation of the ionized sample molecules (comprising the ionized analyte and the ionized analyte-specific internal standard) and performing at least partially a first separation of the (ionized) analyte (i.e. ionized analyte molecules) and the (ionized) analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing, specifically with a second selectivity enhancer module, at least partially a second separation of the (ionized) analyte (i.e. ionized analyte molecules) and the (ionized) analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from remained other components (i.e. other ionized molecules) of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting, specifically with a fragmenter module, the ionized analyte (also denoted “ionized analyte precursor molecules” or just “precursor molecules”) into ionized analyte fragments (also denoted “ionized analyte fragment molecules”, “ionized analyte daughter molecules / ions” or just “daughter molecules / ions”) and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments (also denoted “ionized internal standard fragment molecules”) by collision of the (ionized) analyte with gas molecules, i.e. by collision with particles of a gas phase (i.e. gas being introduced into a chamber of a fragmenter module); providing a stream (which may be a pulsed or a (quasi)constant stream for a certain time at least) comprising the ionized analyte fragments and the ionized analytespecific internal standard fragments on / to a detection mass spectrometer; and recording with the detection mass spectrometer at least two pre-summed (i.e. firstly accumulated) mass-resolved spectra and summing (i.e. secondly accumulating) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum, wherein the final mass-resolved spectrum may serve for detecting / determining whether or not the analyte of interest is present in a biological sample solution and specifically for qualitatively and / or quantitatively estimating its (absolute or relative) amount if present.

[0021] This method is based on MS and is adapted to at least one specific requirement in diagnostics, specifically in diagnostic analysis of large laboratories with high throughput. This method is specifically suited to increase and / or improve the sensitivity of MS such that it may be broadly used in a versatile manner in diagnostics to better determine the level of the analyte of interest. This method may either achieve, maintain and / or increase a high degree in selectivity, specifically when being compared to existing MS. This method may allow for a comfortable, low maintenance, reliable, sensitive, selective and / or fast detection of a wide variety of analytes from complex biological matrices. This method provides a more user-friendly way of performing MS in diagnostics, being accessible to operators, health care workers and / or other users with little to no experience and / or knowledge in / of MS and / or other techniques (such as chromatography). This MS method may be very robust and may allow increasing the degree in sustainability, specifically by reducing the amount of solvents used to perform MS. For example, the amount of Acetonitrile required to perform MS may be reduced, specifically when using Nano-ESI and / or avoiding the use of a chromatographic technique.

[0022] Both methods, specifically the method according to the first aspect may be free of any type of chromatography and may to a high / to an improved extent exploit the potential of ion separation provided by the MS separation steps performed after the ionization.

[0023] Importantly, the disadvantages regarding the very limited time span for the measurement of the "enriched analyte as a peak" may be avoided in the embodiment that uses a direct inlet approach for providing to an ionizer a constant stream of sample molecules to be then selected by mass spectrometry without chromatography. In other words, the temporal limitations dictated by the chromatography may be overcome by using a direct inlet approach instead of a chromatography. The required separation of ions may substantially be performed by the mass spectrometry steps of the methods in combination with ion mobility. If a systematic and / or automated and / or synchronized pre-processing of the sample is desired or needed, this may be performed by methods other than chromatography, which do not contradict the direct inlet approach, such as for example magnetobead separation and / or magnetobeads-capturing. As no chromatography is required, the method according to the first aspect reduces the degree of maintenance and therefore increases the user-friendliness. It also allows reducing the amount of solvents, which would be typically required to perform chromatography such as for example HPLC. The problems arising for the analyte - surface contact and the need for stationary phases and liquid solvents may be avoided. Furthermore, inherent stress of mechanical parts resulting in a deterioration of the equipment may be avoided. Therefore, the methods may be simple and easy to apply in clinical diagnostics, while achieving a high throughput in sample analysis and being run by only few technicians, who are not required to be trained in the complicated maintenance of chromatographic systems. Waiting time for service technicians to fix problems and / or take care of the maintenance of the systems and downtimes may be tremendously reduced.

[0024] The methods may allow providing to a first selectivity enhancer module a constant / quasi-constant stream of ionized sample molecules, which are then spatially accumulated by the first selectivity enhancer module, e.g. collected in a chamber before being further selected and provided to a detector. Specifically in the case of the direct inlet approach and the constant / quasi-constant stream of ionized sample molecules provided in the system, a high number of data sets for generating multiple native and / or pre-summed mass-resolved spectra may be detected and recorded. Specifically, the multiple steps, i.e. providing a constant stream of ionized sample molecules by using ESI and / or nano-ESI, performing a first and a second separation as well as a spatial accumulation and recording multiple presummed mass-resolved spectra, interact together in such a way that a synergistic effect is given that multiple (presummed = already accumulated) mass-resolved spectra may be summed (i.e. added up and / or digitally accumulated), such that an excellent Signal-to-noise ratio (SNR) can be achieved.

[0025] The stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments onto the detection mass spectrometer may correspond to a stream only consisting of the ionized analyte fragments and / or the ionized analyte-specific internal standard fragments but substantially not comprising any fragments of other molecules. In other words, the stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments may be highly purified due to multiple steps of separation from fragments of other molecules and therefore a high selectivity may be achieved.

[0026] The stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments onto the detection mass spectrometer may be a pulsed stream, a (quasi-) constant stream for a certain time / period and / or a repeated stream. Herein, a pulsed stream may be considered a stream of ions provided on the detection mass spectrometer that takes less than 5 s of providing ions thereon before a break occurs which may, without limitation, also take 5s. The break may alternatively be longer or shorter but preferably in the short second range, e.g. between about 0,1s and 15s. A quasi-constant stream may be considered a stream of ions provided on the detection mass spectrometer that takes longer than 5s and less than 15s of providing ions thereon before a break occurs which may also take about 5s to 15s. The break may alternatively be longer or shorter but preferably in the second range, e.g. between about 0.5s and 30s. Herein, a constant stream may be considered a stream of ions provided on the detection mass spectrometer that takes longer than 15s and more specifically longer than about 30s of providing ions thereon. Typically, there is no break and only one “shot” of a constant stream of ions is provided on the detection mass spectrometer for one measurement.

[0027] The number of pre-summed mass-resolved spectra used to generate and / or obtain the final mass-resolved spectrum is substantially only limited by the amount of sample solution provided per time, specifically in the case of the direct inlet approach for providing to the ionizer the constant stream of sample molecules. Therefore, it is possible to sum / add up the multiple pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum at an increased sensitivity, which may result in revealing sufficiently clear peaks for molecules, which would otherwise be hardly detectable. Contrary, using typical MS technologies of the known art, usually only one pre-summed mass-resolved spectrum can be generated. In other words, according to typical conventional techniques, only one “shot” (or in some cases only a very limited number of shots) per time event can be recorded due to the choice of method steps. Specifically the chromatography combined with MS techniques limits the detection to a single “shot” or in some cases only a very limited number of shots of sample material that can be detected.

[0028] In general, any kind of spectrum generated from multiple summed spectra provides an improved Signal-to-noise ratio (SNR) compared to a single spectrum alone. The SNR is proportional to the root of n, i.e. / n. where n is the number of accumulated spectra. Therefore, the present invention allows detecting very low concentrations of analytes, i.e. determine a low level of the analyte of interest, such as approximately lOOpg / ml while obtaining sufficiently strong signals for identification of such analytes. This is important for a broad applicability in medical diagnostics, where often, small concentrations of analytes need to be detected. At the same time, other analytes and / or the same analytes in other samples may be present at a high concentration. Therefore, the “dynamic range” for detecting a broad range of different concentrations and / or different analytes is large and makes the method very versatile. Providing to an ionizer a constant stream of sample molecules and / or providing the constant stream of ionized sample molecules and the analyte-specific internal standard may refer to providing a flow of molecules which are present in the biological sample, mixed with the analyte-specific internal standard solution, specifically at a substantially constant density for a certain time span. Providing to the first selectivity enhancer module the constant stream of ionized sample molecules may comprise providing the ionized analyte molecules and the ionized analyte-specific internal standard molecules. The ionized analyte molecules and / or the ionized analyte-specific internal standard molecules may at least partially be permanently ionized and / or ionized by a chemical reaction and / or may already be naturally present as ions. In other words, at least some of the molecules (analyte molecules and / or analyte-specific internal standard molecules) may already be ionized before the ESI and / or nano-ESI. At latest the ESI and / or nano- ESI ionizes the molecules.

[0029] Besides the ionized analyte molecules and the ionized analyte-specific internal standard molecules, the biological sample solution may comprise many other molecules before the separation steps which may start with the first selectivity enhancer module / first selectivity enhancement, for example other biological molecules, solvent and / or matrix molecules from which the ionized analyte molecules should be separated before the measurement and / or recording of the spectrum by means of separation (also denoted “selectivity enhancement”). A relatively impure sample may in some cases be provided to the ionizer. The full potential of the provided selectivity enhancement steps may be exploited for purification and to provide (ionized) fragments of the analyte and the anaylte-specific standard at a high purity to the detection mass spectrometer.

[0030] The direct inlet (DI) approach used in some cases allows the biological sample to be introduced directly into the ionizer, i.e. the ion source (here atmospheric pressure ionization, e.g. electrospray ionization (ESI) and / or nanoelectrospray ionization (nano-ESI)) of the mass spectrometer without the need for prior separation by any sort of chromatography. This means that all molecules (analyte molecules, analyte-specific internal standard molecules, solvent, impurities etc.) provided / fed into the ionizer are ionized at the same time together. If no purification was performed previously all sample molecules are subjected to the ionization which is not the case for the chromatographic approach as described in the prior art where only purified samples are subjected to ionization. The DI approach is hence based on a chromatography-free method. This method may specifically be useful for compounds that may decompose or react during chromatographic separation. If desired, a pre-processing of the biological sample by means of magnetobeads-capturing and / or other immunoassay methods may be provided before the direct inlet. The DI approach provides a high degree of simplicity and may bypass complex sample preparation or separation techniques such as chromatography. The technique further allows for a very rapid analysis as the sample is inserted directly to the ionizer / ion source. Often, minimal preparation of the sample is required, which reduces analysis time. Moreover, direct introduction can enhance the detection sensitivity for certain compounds because there are fewer stages where sample loss can occur. In the present case, via direct inlet, a constant / quasi-constant stream of (already ionized at least partially or not ionized) sample molecules is provided to the ionizer. Therefore, in case of a preprocessing, the sample molecules before being provided to the ionizer, regardless of whether being already ionized or not, may correspond to and / or comprise pre-processed sample molecules from a pre-processed sample solution. The providing of the constant stream of sample molecules comprising the analyte from the biological sample solution and the analyte-specific internal standard by means of the direct inlet approach to an ionizer and / or the providing of the constant stream of ionized sample molecules and the analyte-specific internal standard may comprise - besides the addition of the analyte-specific internal standard - at least one step of pre-processing of the sample molecules prior to ionization and / or after ionization, specifically the ionization using ESI and / or nano-ESI. In line with the above statement, the at least one step of pre-processing the sample molecules may comprise an additional step of ionizing the sample molecules in addition to the ESI and / or the nano-ESI. As indicated before, the biological sample solution may already comprise at least partially ionized sample molecules such as ionized analyte molecules and / or other ionized molecules.

[0031] The providing of the constant stream of ionized sample molecules may comprise a pre-processing step that corresponds to or comprises a step of collecting the analyte of interest from a sample which may correspond to or comprise a native sample, a pre-processed sample, a component of a sample, a whole blood sample, a serum and / or another component of a body fluid by at least one of the following techniques: magnetobeads-capturing, immunoassay methods, and / or adding the analyte-specific internal standard. Specifically the technique of magnetobeads-capturing is useful to provide a pre-processed sample which may have the analyte of interest (if it is present in the sample) already at a high purity compared with other purification techniques.

[0032] The providing of the constant stream of sample molecules to the ionizer and / or the providing of the constant stream of ionized sample molecules and the analyte-specific internal standard comprises, prior to the ESI and / or nano-ESI, may comprise providing of the biological sample solution as a pre-processed sample solution including separating a serum from a whole blood sample and specifically collecting the analyte from the serum by at least one of the following: magnetobeads-capturing, immunoassay methods, and / or adding the analyte-specific internal standard

[0033] The magnetobeads-capturing approach may provide an additional improvement in purification before the molecules are provided to the ionizer, which is specifically helpful to achieve high purifications without the need for chromatographic techniques. Pre-processing the biological sample may specifically be advantageous for providing a constant stream of fairly purified ions using nano-ESI before spatial accumulation thereof. Further, the yield of analyte from a sample is very high such that the method according to this embodiment is specifically sensitive. Specifically, the providing of the constant stream of ionized sample molecules may comprise, prior to atmospheric pressure ionization (e.g. ESI, nano-ESI, APCI, SESI etc.), specifically prior to the ESI and / or nano-ESI, providing of the sample solution as a pre-processed sample solution including separating a serum from a whole blood sample, collecting the analyte molecule from the serum by at least one of the following: magnetobeads-capturing, immunoassay methods, chromatography and / or and / or adding the analyte-specific internal standard to the analyte. Purification by magnetobeads-capturing may in some cases have the advantages by its own of providing: a high specificity and yield of a target molecule, i.e. the analyte; a simple technique to be used for purification; a fast purification as purification with magnetobeads is generally faster than many traditional methods, such as column chromatography or ultracentrifiigation; easy scalability and flexibility; compatibility with various formats (comprising tubes, microplates, or automated systems, facilitating high-throughput processing and integration with robotic platforms for workflow automation); a gentle purification technique for sensitive and / or instable biomolecules / samples; high versatility in use for different types of samples (including including nucleic acid isolation, protein purification, cell separation, and immunoprecipitation). Overall, the use of magnetobeads purification may in some cases simplify the workflow, increase efficiency, and provide highly reliable and reproducible results.

[0034] The use of nano-ESI may in some cases have the advantage by its own of providing a powerful technique that enhances the capabilities of mass spectrometry, especially when dealing with small sample amounts and complex mixtures. Specifically, the advantages of nano-ESI may in some cases comprise: Increased Sensitivity: NanoESI operates at very low flow rates (typically in the nanoliter per minute range), which results in higher ionization efficiency and increased sensitivity. This is particularly beneficial for detecting low- abundance analytes.

[0035] Reduced Sample Consumption: Due to the low flow rates, nanoESI requires significantly less sample volume, which is advantageous when working with limited or precious samples.

[0036] Enhanced Desolvation: The small droplet size produced by nanoESI promotes more effective desolvation, leading to improved ion generation and more stable electrospray.

[0037] Low Flow Rates: Lower flow rates reduce the need for extensive flow splitting and simplify the system setup. This also minimizes solvent waste.

[0038] Improved Signal-to-Noise Ratio: The combination of increased ionization efficiency and lower background noise often results in a better signal-to-noise ratio, enhancing the quality of the mass spectrometric data.

[0039] Compatibility with Microfluidic Devices: NanoESI is well-suited for integration with microfluidic systems, allowing for high-throughput and automated analyses.

[0040] Reduced Ion Suppression Effects: NanoESI's high efficiency and low flow rates can mitigate ion suppression effects, which are common in complex matrices, thereby improving the reliability of quantitative analyses.

[0041] Enhanced Resolution and Peak Shape: The finer control of the spray and droplets can lead to better chromatographic resolution and sharper peak shapes in mass spectra.

[0042] Direct analysis in real time (DART) refers to an additional or alternative ionization technique used in some cases and is a plasma-based ambient ionization technique that allows a rapid automated analysis, that effectively can ionize a broad range of compounds. DART corresponds to an ambient ionization technique used in mass spectrometry, notable for its ability to analyze samples directly in their native state with minimal or no sample preparation. DART may use a heated gas (typically helium or nitrogen) that is ionized in a high-voltage electric field. The ionized gas may interact with the sample placed near the DART source. This may cause desorption and ionization of molecules from a sample’s surface into the gas phase. The ionized sample may then be introduced into the mass spectrometer for mass-to-charge ratio (m / z) analysis. DART may in some specific cases allow analyzing samples in open air, making the process faster and more straightforward.

[0043] The providing of the constant stream of ionized sample molecules and the ionized analyte-specific internal standard may comprise ionizing at least one portion of the pre-processed sample molecules by ESI and / or nano-ESI and may in some cases comprise ionizing at least one portion of the pre-processed sample molecules prior to the ESI and / or nano-ESI. Therefore, the pre-processed sample solution may already comprise ionized molecules (e.g. permanent positive or permanent negative charged molecules), specifically ionized sample molecules prior to the ESI and / or nano-ESI.

[0044] The pre-processed sample may in some cases be subjected to the atmospheric pressure ionization (e.g. ESI, nano-ESI, APCI, SESI etc.) and / or the plasma-based ambient ionization technique (e.g. DART) by the direct inlet (DI) approach. Providing a step of sample pre-processing may in some cases of sample purification be considered providing an additional step of selectivity enhancement, such that a pre-selected mix of molecules may be subjected to the selectivity enhancement steps using mass spectroscopy. In general, the method according to the first aspect or at an embodiment thereof may allow for surrendering a preprocessing of the sample by chromatography, however, if required for a specific reason, it may still be applied and combined with the method according to the first aspect - specifically for in a timely-uncoupled relation, i.e. performing the chromatography early before the measurement as a complete sample. Specifically, in a clinical approach, the step of at least partially ionizing with the ionizer the sample molecules that comprise the analyte and the analyte-specific internal standard may correspond to completely or only partially ionizing the constant stream of sample molecules comprising the analyte from the biological sample solution and the analyte-specific internal standard. Regardless of whether the sample molecules are completely or only partially ionized, only ionized sample molecules may be subjected to the spatial accumulation, the first and the second separation and the fragmentation, as the ionization and charge carried by the molecules is a prerequisite for performing such steps. Non-ionized molecules are therefore lost. The chromatography-free approach may have the advantage of providing a more reproducible method as compared to a method including chromatography and therefore, the samples may be treated equally over time.

[0045] In general, ionization may be considered the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons to form ions. This process can occur through various means, including: Chemical reactions, where elements react to form compounds, and transfer of electrons leads to the formation of ions; radiation, where high-energy radiation, such as X-rays or gamma rays, ejects electrons from atoms or molecules; electric discharge involving plasmas, where electric fields can strip electrons from atoms; electrospray ionization (ESI).

[0046] For ESI, a liquid sample containing the analyte (and analyte-specific standard) may in some cases be introduced through a fine capillary. A high voltage may be applied to the liquid, which may cause it to form a fine mist of charged droplets (Electrospray Formation). The solvent in the droplets evaporates, assisted by a flow of heated gas, leading to the production of smaller droplets and ultimately individual ionized analyte molecules (Desolvation). The generated ions may then be transferred into the mass spectrometer for detection and analysis. ESI may particularly be useful because it allows for the ionization of large biomolecules (like proteins and peptides) without fragmenting them before injection, making it a powerful tool in proteomics and other areas of biochemical research. Nano-ESI may allow injecting minor amounts of the sample solution to generate a constant stream of ionized molecules over a long time span (longer than with other techniques and / or ESI) at which multiple pre-summed spectra can be summed to generate a high quality final mass-resolved spectrum.

[0047] The providing of the constant stream of ionized sample molecules and ionized analyte-specific internal standard for the spatial accumulation may be performed for, during or in a certain time span, which may be longer than about Is, specifically longer than about 5s, more specifically longer than about 15s and even more specifically longer than about 30s. In other words, the constant stream is characterized in that it is provided for a minimum time span, which may exceed about Is, specifically about 5s, more specifically about 15s, and even more specifically about 30s and potentially even minutes, such as about 2 minutes. The providing of the constant stream of ionized sample molecules and the ionized analyte-specific internal standard may be stopped when the measurement is stopped for example in a case when the SNR has reached a sufficient quality and / or (threshold) value.

[0048] The constant or in some cases pulsed stream of ionized sample molecules and / or the constant or pulsed stream of ionized analyte fragments may be generated for a time span of up to about 30 minutes or even longer, specifically at a unprocessed or a pre-processed sample solution volume of approximately InL / min to lOOOnL / min, specifically about 200nL / min to 500nL / min, more specifically about 250nL / min to 350nL / min, such as 300nL / min. Specifically the step of spatially accumulating the ionized molecules may be configured such that a stream, specifically a pulsed stream of ionized sample molecules and / or ionized fragment molecules (for analyte and standard) may be generated (specifically behind the first selectivity enhancer module and after the spatial accumulation) for a time span of up to about 30 minutes (or even longer) for obtaining a single final mass-resolved spectrum. The method allows using minor analyte solution volumes, analyte concentrations and / or sample solution volumes while still achieving excellent SNR results, specifically in the case when using nano-ESI. In some cases, only the sample solution volume may limits the time span for detection.

[0049] When a predetermined degree of spatial accumulation is achieved, a chamber and / or volume in which the spatial accumulation is performed may be “opened” and / or otherwise triggered to provide a stream of ions to the space where the first separation is performed. The opening and / or triggering may be performed, without limitations, at a predetermined frequency by repeatedly opening and closing and / or triggering the providing for the first separation after a certain first trigger time (which corresponds to a first trigger frequency Fl). For example, after every about 40ms to 300ms of spatial accumulation (corresponding to the first trigger time), a gate between the space for spatial accumulation (e.g. a spatial accumulation tunnel without limitation) may be opened to the space of first separation (e.g. a TIMS tunnel without limitation). This may allow performing the first separation of the (ionized) analyte and the (ionized) analyte-specific internal standard in a pulsed manner over first separation time spans tl, which correspond to the above-described first trigger time.

[0050] The performing of the first separation and spatial accumulation of the (ionized) analyte molecules and (ionized) analyte-specific internal standard molecules from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules may result in and / or allow providing a stream (pulsed and / or (quasi-)constant) of at least partially separated ionized analyte molecules (separated from at least some other molecules, which differ from the ionized analyte molecules and ionized analyte-specific internal standard molecules). The at least partially, by first separation, separated ionized analyte molecules and ionized analyte-specific internal standard molecules may be considered as being at least partially purified and as being spatially bunched together (specifically in at least two dimensions). Moreover, the first separation may be considered a “first selectivity enhancement” (step), in which the (ionized) analyte molecules and (ionized) analyte-specific internal standard molecules are selected and / or spatially extracted from the entity of sample molecules. The step of performing the at least partial first separation and spatial accumulation of the (ionized) analyte molecules and (ionized) analyte-specific internal standard molecules from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules may refer to performing at least partially a first separation of the (ionized) analyte molecules and (ionized) analyte-specific internal standard molecules from other components of the ionized sample molecules and performing a spatial accumulation of the (ionized) analyte molecules, such that at least some other molecules, which differ in size and / or charge from the (ionized) analyte molecules, are not spatially mixed with the (ionized) analyte molecules and are removed and / or separated therefrom. The first separation may be performed in an effective separation length of about 3 cm to 8m, specifically about 4cm to 3m. For example, a TIMS tunnel of a TIMS cartridge as described further below, may have an effective separation length of about 5cm. A drift tube may have an effective separation length of about 1,5m.

[0051] The space where the first separation is performed may be opened at a second trigger frequency F2, i.e. repeatedly after a second trigger time t2 that may preferably be shorter than the first trigger time tl. This second trigger time t2 may be provided for the performing of the second separation of the analyte and the analyte-specific internal standard being therefore performed in a pulsed manner over second separation time spans t2 (second trigger times). The performing of the second separation of the (ionized) analyte molecules and / or (ionized) analyte-specific internal standard molecules from the remained other components of the ionized sample molecules (remained after the first separation) based on the mass to charge ratio of the ionized molecules may also result in providing a stream (specifically pulsed and / or (quasi-)constant) of at least partially separated ionized analyte molecules (separated from at least some other molecules, which are not ionized analyte molecules and / or ionized analyte-specific internal standard molecules). The at least partially, by second separation, separated ionized analyte molecules may be considered as at least partially purified (having a higher degree of purity than before the second separation, specifically than after the first separation if the second separation following i.e. being downstream of the first separation). Moreover, the second separation may be considered a second selectivity enhancement in which the ionized analyte molecules and ionized analyte-specific internal standard molecules are selected from (remaining other portions of) the entity of ionized sample molecules.

[0052] Selectivity enhancement (the first and / or the second separation, i.e. the first and / or the second selectivity enhancement) may refer to an effective and / or efficient separation of the (ionized) analyte and (ionized) analyte-specific internal standard molecules from other components in the sample. In other words, the selectivity enhancement allows for efficiently selecting and / or sorting out the analyte molecules and analyte-specific internal standard molecules from other and / or remaining molecules of the sample such that a signal may be detected that originates solely from the analyte molecules and analyte-specific internal standard molecules.

[0053] The internal standard, which is specific to the analyte, is added to the sample allowing the test result and / or detection result to become a quantitative measure. Such internal standard molecules resemble the analyte molecules in such a way that the selectivity enhancement does not select the analyte molecules from the internal standard molecules. In other words, the steps of first and second separation do not separate the analyte molecules from the internal standard molecules, which are specific to the analyte of interest. If however internal standard molecules specific to a different analyte (which is currently not of interest) are added, they will be sorted out, as they are not of interest for the current measurement of the analyte of current interest. In this way, ideally, spectra are recorded, which only contain signals generated by and / or detected for the analyte molecules and the internal standard molecules. In reality, this may not always be completely achieved and a test result may still contain minor signals from other molecules than the analyte of interest and the corresponding analyte-specific internal standard may be realistic and / or tolerated.

[0054] The performing of the at least partial second separation may refer to performing at least partially a second separation of the (ionized) analyte molecules and (ionized) analyte-specific internal standard molecules from other components of the ionized sample molecules such that at least some other molecules which differ in size and / or charge from the analyte molecules and analyte-specific internal standard molecules are not spatially mixed with the analyte molecules and removed therefrom.

[0055] The performing of at the least partial first separation and spatial accumulation of the (ionized) analyte and the (ionized) analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules may be considered a step of performing the at least partial first separation from other components of the sample based on the size and / or shape of the ionized molecules and spatial accumulation of the (ionized) analyte and (ionized) analyte-specific internal standard. A first spatial separation from other components of the sample is taking place, based on the size and / or shape of the ionized molecules. It is to be noted that the “first separation” may be preceded by other separations and / or separation techniques for example used in the pre-processing of the sample and therefore in some embodiments the “first separation” may technically not correspond to the very first separation of the (ionized) analyte and / or the (ionized) analyte-specific internal standard from other components - the expression of the present “first separation” shall however indicate that it corresponds to a separation technique that differs from the “second separation” technique. “First and second separation” therefore do not necessarily indicate an order of the types of separation. Further, the spatial accumulation may be performed before the first separation (as for example performed in the below described accumulation tunnel of a TIMS cartridge which is positioned in that embodiment upstream of a TIMS tunnel). Alternatively, the first separation may be performed before / in front of the spatial accumulation. Alternatively or in addition, the first separation may be (also) followed by a second spatial accumulation. Alternatively or in addition, the spatial accumulation may be followed by a further first separation. The same statements apply accordingly to the mass spectrometer system described herein. Specifically, the first separation may be performed at first and the second separation may be performed secondly, i.e. after the first separation. Alternatively or in addition, the second separation may be performed first and the first separation may be performed secondly, i.e. after the first separation. Specifically, the second separation may be performed more than once, for example twice. In that case, the second separation may be performed at least once after the first separation and the second separation may either be repeated after the second separation or the second separation may be performed before the first separation. The first separation and / or spatial accumulation of the (ionized) analyte and / or (ionized) analytespecific internal standard from other components of the ionized sample molecules may also be repeated several times. In a specific embodiment, the first separation is performed once and the second separation is performed once right after the first separation and / or the spatial accumulation. Therefore, the terms “first separation” and “second separation” may only refer to the two different techniques of separation without limiting the temporal order of the separation.

[0056] The space, where the second separation is performed may be opened at a third trigger frequency F3, i.e. repeatedly after a third trigger time t3 that may preferably be shorter than the second trigger time t2. This third trigger time t3 may be provided for the performing of the fragmenting of the (ionized) analyte and the (ionized) analyte-specific internal standard being therefore performed in a pulsed manner over fragmenting time spans t3 (third trigger times). The fragmenting of the ionized analyte molecules into ionized analyte fragment molecules and the ionized analytespecific internal standard into ionized analyte-specific internal standard fragment molecules by collision with particles of the gas phase may result in providing a stream (pulsed or quasi-constant) of ionized fragment molecules, i.e. fragments of the ionized analyte molecules and ionized analyte-specific internal standard molecules. This may then result in the providing of the stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on the detection mass spectrometer. The providing of the stream comprising the ionized analyte fragments and ionized analyte-specific internal standard fragment molecules on the detection mass spectrometer may refer to repeatedly and / or quasi-constantly providing ionized analyte fragments on the detector field of the detection mass spectrometer by leading the stream onto the detector for a certain time span.

[0057] The recording with the detection mass spectrometer of the at least two pre-summed mass-resolved spectra may be performed over seconds (at least Is) up to several minutes (e.g. up to 30 minutes or even more) or hours in a pulsed manner over short recording time spans t4 (fourth trigger times), specifically being shorter than the fragmenting time spans t3, corresponding to a recording frequency F4, which may be considered a fourth trigger frequency F4.

[0058] This may be performed by using a time of flight (TOF) mass spectrometry, in which the ions are pushed into the field free region and the arrival time at the detector field is indicative to the mass to charge ratio and therefore helps identifying the molecule or fraction of a molecule. The recording with the detection mass spectrometer of at least two pre-summed mass-resolved spectra may comprise a step of detecting and / or acquiring and / or collecting of signals and / or mass-resolved spectral data with detection elements of the detection mass spectrometer combined with one or more steps of signal and / or data processing and / or pre-processing and / or generation of mass-resolved spectra and / or accumulation of the generated mass-resolved spectra to generate a plurality of pre-summed mass-resolved spectra, namely the at least two pre-summed mass-resolved spectra.

[0059] The said signal and / or data (pre-)processing, the generation of the mass-resolved spectra from the recorded / acquired signals or signal data and / or the first accumulation to generate each of the at least two pre-summed mass-resolved spectra may be performed by at least one data (pre-)processor (denoted “first data processor” further below), controller and / or machine that is associated with and / or belongs to and / or is integrated in the detection mass spectrometer. In other words, the data pre-processing and / or first data processing of the collected data may be performed by a data processor belonging to the detection mass spectrometer.

[0060] The step of detecting and / or acquiring and / or collecting of signals and / or mass-resolved spectral data from detection elements of the detection mass spectrometer may be obtained for / from the stream of the ionized analyte fragment molecules and ionized analyte-specific standard molecules.

[0061] The step of accumulating the at least two pre-summed mass-resolved spectra to obtain a single final mass-resolved spectrum or several final mass-resolved spectra may be performed in a separated step after the step of generating the at least two pre-summed mass-resolved spectra.

[0062] The step of detecting and / or acquiring and / or collecting of signals and / or mass-resolved spectral data and / or the step of accumulating the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be constantly and / or repeatedly performed, without limitation, between approximately 5 s and 30 minutes or even longer such as Ih, specifically between approximately 15s and 10 minutes and more specifically between approximately 30s and 2 minutes. Accumulating the at least two pre-summed mass-resolved spectra may refer to accumulating a number of between approximately 5 and 500 pre-summed mass-resolved spectra, without limitation.

[0063] The overall time span for the step of summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may resemble or correspond to the time span for the providing (to the ionizer) of the constant stream of analyte from the biological sample solution and analyte-specific internal standard.

[0064] In one specific embodiment of the method, synergistic effect(s) are achieved by providing a constant stream of ionized sample molecules comprising the analyte from a sample solution (and analyte-specific standard) and by the subsequent separations by means of specific selectivity enhancement principles: the first separation step may specifically be performed by means of ion mobility separation (IMS) and / or MS, and the second separation step may specifically be performed by quadrupole MS, e.g. single, dual or triple quadrupole MS. The combination of these separation steps enables not only a selective but also a sensitive analysis. Selectivity may be achieved by means of a full scanning IMS and optional MSMS fragmentation (fragmentation step), whereas sensitivity can be enhanced by increasing the measurement time in contrast to a single event measurement (single shot) which is mostly provided in conventional MS techniques.

[0065] In a conventional measurement with pre-enrichment techniques using HPLC and / or GC, the measurement time of the analyte of interest depends on / is limited by the width of the analytically pre-selected peak. In contrast, a constant analyte supply as provided by the present invention, the measurement time of the analyte of interest only depends on the sample volume and / or the period in which the constant stream of analyte is provided. Therefore, the measurement allows a measurement signal that is far less limited (almost unlimited) in terms of detection time. The concept of summing (pre-summed) spectra as described herein is similar to the Electro-chemiluminescence (ECL) technology, fluorescence detection, etc., in which the measurement signal is also generated by the summing of light from the sample. Latest developments in signal detectors for e.g. MS systems provide a signal even for one event hitting the detector but the dark noise (background) remains an issue. The summing of the pre-summed signal during constant ongoing measurement, as made possible by an embodiment of the present invention, will vanish the dark noise by statistics while summing the (pre-summed) analyte signal as "real event" vs. "random dark noise".

[0066] Furthermore, it is a surprising technical effect of the invention that the summing of signals still works with spectra that have already been pre-summed, as provided by a time-of-flight (TOF) detector or ion trap systems. When using a TOF detector, the device already provides a pre-processing internally, but this entity is not per se visible to the user. Modern TOF instruments may work at a spectral rate of approx. 5-20 kHz, which the user can accumulate / sum to a maximum of approx. 50 Hz spectra rate (compare Bruker "tims-TOF") as an output.

[0067] The present invention therefore allows enhancing detection limits as well as SNR ratios. Further, broader full scan mass-to-charge segments provide the possibility to record several transitions of the precursor ion simultaneously. These signals from daughter ions or parent ion as pseudo MS / MS, i.e. ionized analyte fragment molecules, can be summed up together in order to even further increase the sensitivity.

[0068] The methods specifically the method according to the first aspect may not require / may be free of gas chromatography (GC) and / or liquid chromatography (LC). The methods may in some cases not requires chromatography due to the direct inlet and may therefore be completely free thereof. The methods may specifically be free of an HPLC preparation and / or separation step and / or the methods specifically the method according to the first aspect may be free of a column preparation and / or separation step and / or the method may be free of a chromatographic preparation and / or separation step. This is described in more detail, further below.

[0069] The avoidance of chromatography separation techniques upstream of the mass spectrometer system, such as GC and / or LC, specifically HPLC, offers an advantage in terms of the speed, at which samples can be fed into the MS system, since there is no need to establish an equilibrium between the analyte and the separation unit.

[0070] The methods of this disclosure are not per se incompatible with chromatography, if, for example, in advance, high amounts / volumes of a pre-processed sample is collected and then provided as a pre-processed sample. In other words, if the chromatography method is performed beforehand to collect a pre-selected / pre-purified sample but not being timely coupled to the following steps in that the following method steps depend on the width of the chromatographic analyte peak, a sample subjected beforehand to chromatography may be provided. In that case, the time constants valid for the selection steps of the system are not dependent from the chromatography and / or the chromatography is not a part of the system, specifically operating in an automated manner. In that case, the term “direct inlet” may still be considered chromatography-free as it does not include the chromatography in the system itself - only the sample may have been subjected to chromatography before being provided to the system. In that case, the chromatography is not considered systematic and / or automated and / or synchronized in or with the system and / or method described herein.

[0071] However, this may in some cases contradict the fast and efficient performance, the user-friendliness and eco- friendliness of the method described herein. Therefore, the clinical approach preferably avoids chromatography. In general, the invention is suited to make clinically relevant analytes measurable, e.g. for neurodegenerative diseases, using a different approach than provided in the prior art and allows for increased sensitivity ranges beyond the preenrichment of samples, their purification and separation.

[0072] The step of providing of the constant stream of ionized sample molecules comprising the analyte from the biological sample solution may in some cases be performed constantly over a providing time span that may range between approximately Is and 5 hours or even more, specifically between approximately 3s and 120 minutes, more specifically between 15s and 30 minutes and more specifically between 2 minutes and 20 minutes and the analyte-specific internal standard. In some cases, the methods may be considered a targeted diagnostic approach, which may be reflected by the detection of known analytes and / or the use of an analyte-specific internal standard and / or a (fine) adjustment of parameters to the features of the known analyte. In a non-targeted approach, the analytes may be unknown and the parameters may be therefore be scanned instead of being adjusted.

[0073] The providing, to the ionizer, of the constant stream of sample molecules comprising the analyte from the biological sample solution and the analyte-specific internal standard by means of the direct inlet approach may be free of a generation and / or a providing of a pulsed stream of ionized sample molecules. The providing, to the first selectivity enhancer module, of the constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard may be free of a generation and / or a providing of a pulsed stream of ionized sample molecules

[0074] The MS method, specifically the step of providing the constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard may be free of a generation and / or a providing of a pulsed stream of ionized sample molecules.

[0075] The “constant feeding” of the ionizer and / or the first selectivity enhancer module may allow to constantly generate and provide a pulsed stream of ionized fragments to the detection MS and to constantly detect signals that can be presummed and finally summed. Specifically, up to the step of spatially accumulating the ionized molecules, there is no pulsed stream of sample molecules (ionized or not ionized) generated and / or provided, even if the spatial accumulation is preceded by a first and / or a second separation and / or some other separation technique. Specifically, no pulsed stream of ionized sample molecules (sample molecules and / or analyte-specific internal standard molecules) may be used in the mass spectrometric method upstream of the spatial accumulation, specifically in the step of providing the constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard before the spatial accumulation and the first separation. If however a pulsed stream of ionized sample molecules should be used (specifically in the said step of providing the molecules), each pulse may not be shorter than about 5s, specifically not shorter than about 15s. The mass spectrometric method may therefore specifically be free of MALDI-TOF MS, as MALDI-TOF MS uses or provides a pulsed stream of ionized molecules, specifically in the upstream direction and / or upstream portions of an MS system.

[0076] Providing a constant non-pulsed stream of ionized sample molecules (specifically prior to spatial accumulation) allows to constantly spatially accumulate the ions and hence constantly record and digitally sum up mass-resolved spectra to obtain a final mass-resolved spectrum such that a good SNR can be achieved in a short time.

[0077] Each accumulation, i.e. pre-summing of the at least two firstly accumulated, i.e. pre-summed mass-resolved spectra may be performed by a first processor. Specifically, the step of recording of the pre-summed mass-resolved spectra may comprise a pre-summing of native mass-resolved spectra to obtain the pre-summed mass-resolved spectra, which is performed by a first data processor. In other words, the pre-summing, that may be a first data processing and / or a data pre-processing of the spectra or spectral data to obtain the pre-summed mass-resolved spectra may be performed by a first data processor, specifically a data (pre-)processor that is associated with the detection mass spectrometer (that may be bought together with the detection mass spectrometer).

[0078] The step of recording each of the at least two pre-summed mass-resolved spectra may comprise: detecting a plurality of mass-resolved spectral data sets; and generating from the plurality of mass-resolved spectral data sets a plurality of native mass-resolved spectra. In other words, to obtain one single pre-summed mass-resolved spectrum, mass-resolved spectral data sets corresponding to a plurality of n native mass-resolved spectra may be recorded and from these mass- resolved spectral data sets the n native mass-resolved spectra may be generated and / or a single pre-summed mass- resolved spectrum may be generated.

[0079] The pre-summing of each of the native mass-resolved spectra to obtain each of the at least two pre-summed mass- resolved spectra may be performed by means of a first data processor, specifically comprising an internal data preprocessor and / or spectra pre-processor connected and / or belonging to the detection mass spectrometer.

[0080] The step of summing of the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed by a second data processor different from the first data processor. Specifically, the first data processor may correspond to a data processor, specifically a data pre-processor comprised by the detection mass spectrometer and the second data processor may correspond to an external data processor. Therefore, the method may be realized by using a standard commercial detection mass spectrometer, such as a TOF MS spectrometer as a ready- to-use entity combined with an external data processor and / or controller. The methods, specifically the method according to the first aspect or any of the described embodiments may be controlled, for example, at least partially by this external data processor and / or controller. This makes it easy to realize the invention without developing a complete new instrument. The system that is controlled when using the methods, specifically the method according to the first aspect may therefore be based on several (at least two) commercially available units that are combined with each other.

[0081] The step of summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed after the generation of all / each pre-summed mass-resolved spectra or “on the fly”, i.e. whenever a pre-summed mass-resolved spectrum is generated, it is accumulated with the previously generated pre-summed mass- resolved spectrum. In other words, the step of summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed online (i.e. “on the fly”). In other words, the step of summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed subsequently to the pre-summing but at least partially while data for generating another pre-summed mass-resolved spectrum are detected and / or recorded and / or the measurement is ongoing. In that way the final mass-resolved spectrum may be continuously built up / summed up. The time span for detection of spectral data and the time span for summing the pre-summed spectra therefore may overlap. Again, in other words, the summing of the pre-summed mass-resolved spectra may be performed (already) during the ongoing measurement to consecutively build up / generate the final mass-resolved spectrum. This may allow a fast process to obtain a result, i.e. a mass-resolved spectrum quickly and it may specifically be useful to quickly detect the moment when a coefficient of variance and / or an S / N ratio of the summed mass-resolved spectrum (while summing is in progress) reaches a pre-defined threshold value that indicates a satisfying and / or sufficiently precise result for stopping the measurement. Such a threshold value may for example correspond to about 20% + / - 5%. Altematively, the step of summing the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed subsequently and after all pre-summed mass-resolved spectra are recorded and / or generated.

[0082] At least one of the at least two pre-summed mass-resolved spectra may be recorded by the first data processor / data pre-processor at a frequency between about 20kHz (corresponding to t4 of 25 ps, i.e. one cycle every 50 microseconds) and about 100Hz (corresponding to t4 of 0,005s, i.e. one cycle every 0,01s), specifically between about 5kHz (corresponding to t4 of 0,1ms, i.e. one cycle every 0,2ms) and about 100Hz. In other words, the at least two presummed mass-resolved spectra may each be and / or correspond to pre-summed spectra recorded at a frequency, without limitation, between approximately 20kHz and 100Hz. Each of the at least two pre-summed mass-resolved spectra may be considered a pre-processed spectrum.

[0083] The pre-processed spectra, specifically pre-summed mass-resolved spectra may be generated and / or provided at a rate of approximately 0,1Hz to 20Hz, without limitation. Typical digitizers used in MS systems can have an internal recording frequency of approximately 20kHz, without limitation. Therefore, the pre-processed spectra are typically output at a rate between approximately 0, 1Hz and 20Hz, without limitation.

[0084] The step of recording the at least two pre-summed mass-resolved spectra may be repeatedly performed over a total time span (including the entire / integrated time for all cycles that are required to record all pre-summed mass-resolved spectra) of approximately 5s to Ih, more specifically during a time span of approximately 10s to 20 minutes, more specifically during a time span of approximately 15s to 10 minutes and more specifically between approximately 30s to 2 minutes. Alternatively or in addition, the at least two pre-summed mass-resolved spectra may correspond to and / or comprise 5 to 500 pre-summed mass-resolved spectra. One pre-summed mass-resolved spectrum may correspond to about 2 to 2000 accumulations of native mass-resolved spectra. The scan time may correspond to about 50-100ms and the recording rate may correspond to 20kHz.

[0085] In some embodiments / cases, the data pre-processing may comprise other steps than pre-summing: Data preprocessing may involve a series of steps to prepare raw spectral data for analysis. This may be useful to improve the accuracy and reliability of the results. Common pre-processing steps may in some cases comprise: Removing background noise or baseline drift to ensure that the true signal is accurately represented (Baseline Correction); Using algorithms like Savitzky-Golay filter to reduce noise while preserving important spectral features (Smoothing); Adjusting the spectra to a common scale to facilitate comparison which can involve techniques like area normalization or max-min normalization (Normalization); Reducing random noise in the spectral data using methods like wavelet transform (De-noising); Adjusting the spectral data based on calibration standards to correct for instrument variability and improve quantification accuracy (Calibration); Applying mathematical transformations (e.g., Fourier transform, principal component analysis) to emphasize certain features or reduce dimensionality (Spectral Transformation); Choosing specific spectral regions that are most relevant for the analysis (Wavelength Selection).

[0086] The step of summing the at least two pre-summed mass-resolved spectra to obtain the final mass-resolved spectrum may be completed, stopped and / or considered sufficiently sensitive when a coefficient of variance and / or an SNR reaches a pre-defined threshold value, specifically the threshold value may correspond to about 20% specifically to about 20%+ / - 5%. A user may customize and / or pre-define a quality measure that defines / determines the moment, at which the generation of the final mass-resolved spectrum may be completed and / or sufficiently precise, ft may be that the user considers a certain quality of the final mass-resolved spectrum sufficient to decide that the measurement can be completed. The pre-defined quality measure may also correspond to an acceptable trade-off between quality and a time span required to achieve this quality.

[0087] The performing of the first separation may comprise ion mobility spectrometry (or ion mobility separation).

[0088] Ion mobility spectrometry (IMS), as used herein in one or more embodiments may, without limitation, comprise at least one of the following: Drift tube ion mobility spectrometry (DTIMS), Traveling wave ion mobility spectrometry (TWIMS), Trapped Ion Mobility Spectrometry (TIMS), atmospheric pressure IMS techniques, such as field asymmetric waveform ion mobility spectrometry (FAIMS), differential mobility spectrometry (DMS) and differential ion mobility spectrometry (DIMS), Differential mobility analyzers (DMA). "(High-)field asymmetric-waveform ionmobility spectrometry (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. Experiments have shown feasibility for at least some of the above listed ion mobility spectrometry methods, for example for TIMS combined with TOF MS, i.e. timsTOF MS (or “tims- TOF”). ft is believed that aspects and embodiments of the present disclosure are not combinable and / or compatible with the so-called “structures for loss lesion manipulation” (SLIM), specifically due to the requirements of the present invention with regards to the frequencies and / or trigger times at which the modules are operated, the separation length of the ions and / or the spatial accumulation performance. Based on printed circuit-board (PCB) technology with printed electrodes thereon, SLIM uses arrays of mirror-image electrodes patterned on closely spaced surfaces of the PCBs. Applying electric potentials to the electrodes on the surfaces creates electric fields defining a path in the space between the surfaces where ions can be moved along. SLIM is a specific IMS / TWIMS technique using moving electric fields (so-called traveling waves) to rapidly transport ions in a lossless fashion so that they can be separated. The ions do not touch the surfaces, so theoretically none is lost. Further, SLIM guides ions onto curved trajectories to realize long separation lengths for the ions in a compact module. As it does not appear feasible to combine the present invention with SLIM, the present disclosure neither discloses such a combination as an embodiment nor does it indicate such a combination as being realizable. Therefore, all aspects and embodiments described herein, specifically the ion mobility spectrometry of the present invention and entire disclosure is free of the “structures for loss lesion manipulation” (SLIM) technology, which means that the ion mobility spectrometry described herein is neither based on SLIM, nor does it contain SLIM, nor is it connected or connectable to SLIM. In other words, the separation of ions performed by ion mobility spectrometry appears to be realizable for any technology known in the art but SLIM, which is explicitly excluded from the scope of the present invention and disclosure. Therefore, the separation of ions performed by ion mobility spectrometry as described herein is free of a separation technology, which applies electric fields to electrodes printed on conventional parallel printed circuit board surfaces (specifically stacks of such PCBs with printed electrodes) creating ion conduits that can separate and move ions on curved trajectories without losses according to the SLIM technology.

[0089] The performing of the second separation may comprise quadrupole mass spectrometry (QMS). The performing of the second separation of the ionized analyte molecules and ionized analyte-specific standard from remained other components of the ionized sample molecules and / or the fragmenting of the ionized analyte molecules into ionized analyte fragment molecules may therefore be based on and / or performed by quadrupole mass spectrometry. The first separation and / or the second separation may be understood in the sense of a spatial selection such that a high spatial concentration of (ionized) analyte and / or analyte-specific standard molecules with a high purity can be achieved.

[0090] The mass spectrometer system and / or the detection mass spectrometer being used for performing the methods described herein, specifically the method according to the first aspect or an embodiment thereof may comprise and / or may be based on at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry, sector field MS, specifically wherein the detection mass spectrometer is combined with an ionization unit comprising at least one of the following: NanoSpray (ESI), APCI and in specific cases also SESI and / or PTR ionization. The detection mass spectrometer may be free of El, Laser Desorption to avoid a vacuum and / or the requirement for a pulsed technique, respectively it is however not required to exclude these techniques from all possible embodiments. At least, a vacuum and / or a pulsed technique upstream of the spatial accumulation is not required to realize the invention.

[0091] In one embodiment, spectra may be recorded by means of a TOF-spectrometer combined with Nano-ESI, for example a TIMS TOF spectrometer.

[0092] The step of performing the second separation may be repeated at least one time. Specifically, the step of performing at least partially the second separation of the (ionized) analyte molecules and (ionized) analyte-specific standard molecules from remained other components of the ionized sample molecules may be repeated at least one time. Specifically, in some cases, the second separation or the same type of the second separation - if performed multiple times - may either be performed by guiding the stream through the same second selectivity enhancer module or by providing one or more additional second selectivity enhancer modules. Repeating the second separation of the (ionized) analyte molecules from remained other components of the ionized sample molecules may enhance the selectivity and add a further selectivity enhancement.

[0093] The spatial accumulation of the (ionized) analyte and (ionized) analyte-specific standard may be performed in a section of the first selectivity enhancer module for performing the spatial accumulation of the (ionized) analyte and the (ionized) analyte-specific internal standard that has a volume of approximately 1cm3to 70cm3, specifically of approximately 5cm3to 50cm3and more specifically of approximately 10cm3to 45cm3. For example, the spatial accumulation of the (ionized) analyte may be performed in a volume of approximately 12cm3. This volume may correspond to a physical volume that is defined by the physical size of the section of the first selectivity enhancer module for performing the spatial accumulation of the (ionized) analyte and the (ionized) analyte-specific internal standard. The entire volume of the first selectivity enhancer module may have approximately 1.5 - 5 times, specifically twice the size of the section of the first selectivity enhancer module for performing the spatial accumulation. In this embodiment, the spatial accumulation and the first separation are specifically performed in one shared housing. Alternatively, the spatial accumulation and the first separation may be performed in at least two different and / or separated housings, which are connected with each other, for example via a tube. The two or more housings may still form the first selectivity enhancer module but comprise two or more housings.

[0094] In general, the spatial accumulation refers to a step of bunching together charged particles, i.e. charged molecules / ions in a confined space and / or volume, i.e. a space that is confined for example by electric fields, which are applied to trap the charged particles in one dimension (in a defined plane), in two dimensions (on a defined path and / or line) or in three dimensions (in a defined volume), for example in an accumulation tunnel and / or an accumulation chamber. The spatial accumulation allows the invention to constantly provide (fragmented) ions to the detection mass spectrometer for a longer time span and to allow summing of the pre-summed spectra. It is an advantage to provide a space for trapping the ions, which is not too small due to the repulsive coulomb forces of the charge carried by the ions and / or minimizing the probability of gas phase reactions of the respective ions. As an example, without limiting the scope of the invention, the spatial accumulation may correspond to an accumulation of about up to 107ions or even more.

[0095] The spatial accumulation may be performed in one specific embodiment by means of Trapped Ion Mobility Spectrometry (TIMS), specifically Trapped Ion Mobility Spectrometry combined with time of flight spectrometry (timsTOF). Therefore, a TIMS cartridge may comprise a section with an accumulation tunnel for the step of spatial accumulation and which comprises the volume that may correspond to the trap volume and / or the confined volume being confined for the ions by applying fields. The TIMS cartridge may further comprise a section with a TIMS tunnel for performing the actual ion mobility spectrometry, specifically the first separation. The accumulation tunnel may be positioned directly in front of the TIMS tunnel, i.e. upstream of the TIMS tunnel being in direct contact therewith. Further, the TIMS cartridge may comprise a first funnel in front of the accumulation tunnel, i.e. upstream of the accumulation tunnel and in direct contact therewith. Further, the TIMS cartridge may comprise a second funnel behind the TIMS tunnel, i.e. downstream of the TIMS tunnel and in direct contact therewith.

[0096] Alternatively or in addition, the spatial accumulation may be performed by means of an ion trap and / or Orbi Trap in all known variations.

[0097] The timsTOF platform may be considered in embodiments as an advanced mass spectrometry system that combines ion mobility spectrometry (IMS) with time-of-flight (TOF) mass analysis. The timsTOF platform may bring several key advantages, especially for a (targeted) diagnostic approach (but also for other applications in the field of proteomics and metabolomics research):

[0098] Enhanced Sensitivity and Resolution: improved sensitivity due to its efficient ion trapping and focusing capabilities. This may result in better detection limits for low-abundance analytes. The integration of ion mobility with TOF may allow for high-resolution mass analysis, offering greater accuracy in mass determination and improved separation of ions.

[0099] High Speed and Throughput: performing rapid measurements, enabling high-throughput analyses. This is particularly beneficial for large numbers of samples and / or a high number of analytes of interest.

[0100] Overall, timsTOF may offer significant advantages in terms of sensitivity, resolution, throughput, and data quality.

[0101] One specific embodiment of the methods (and / or the respective system) being specifically powerful in achieving high a selectivity and sensitivity, outperforming systems that are based on chromatography, uses the timsTOF setup in combination with a sample pre-processor being based on magnetobeads-capturing, specifically being configured to collect the analyte molecules from the serum by at least one of the following: magnetobeads-capturing. The combination of timsTOF with magnetobeads-capturing, providing to an ionizer, specifically being based on nano-ESI, a constant stream of sample molecules by means of a direct inlet approach and summing of the pre-summed spectra, specifically online summing, allows for a very efficient and fast analysis that is simple and uncomplicated to handle for a user and that requires only very low maintenance. In other words, a very powerful embodiment of the method and the respective system relies on the combination of the following principles: magnetobeads-capturing (sample preprocessing), direct inlet approach (providing a constant stream of analyte and analyte-specific standard to an ionizer), nano-ESI (ionizing the analyte and analyte-specific standard), timsTOF (mass spectrometry for selection and detection) and (online) summing of pre-summed mass-resolved spectra (final data processing). Instead of purifying the analyte by means of chromatography, the potential of the tims cartridge may be exploited by a higher degree and separation may be performed in the tims cartridge. The magnetobeads-capturing approach provides further purification if required or desired. Instead of purifying the analyte by means of chromatography, therefore, the separation of the analyte from other molecules of the sample may be performed by means of the magnetobeads- capturing approach combined with direct inlet into an ionizer and the tims cartridge. This specific combination is particularly strong in outperforming the chromatography approach.

[0102] The providing of the constant stream of the analyte and the analyte-specific internal standard may be performed over a providing time span tOa. The performing of the spatial accumulation of the analyte and the analyte-specific internal standard may be performed over an accumulation time span tOb; The performing of the spatial accumulation of the analyte and the analyte-specific internal standard may be performed constantly; The performing of the first separation of the analyte and the analyte-specific internal standard may be carried out / performed in a pulsed manner over first separation time spans tl; the performing of the second separation of the analyte and the analyte-specific internal standard may be carried out / performed in a pulsed manner over second separation time spans t2; the fragmenting of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analytespecific internal standard fragments may be carried out / performed in a pulsed manner over fragmenting time spans t3; and the recording with the detection mass spectrometer at least two pre-summed mass-resolved spectra may be carried out / performed in a pulsed manner over recording time spans t4. Specifically the following relation between the time spans may apply: tOa or t5> tl > t2 = t3 > t4 or tOa or t5> tl > t2 > t3 > t4. This relation may allow that the loss of ions may be reduced, possibly substantially reduced as the downstream processes become faster. The overall measurement time span t5 may in some cases correspond to the sum of the single recording time spans t4 (in which for example each of the pre-summed spectra and / or the native spectra are recorded and / or generated) for the entire measurement needed to collect the data required for generating the final mass-resolved spectrum. The providing time span tOa and / or the overall measurement time span t5 may range between 15s and 30 minutes, the accumulation time span tOb may be in the ms-time range (and tOa + tOb « tOa tOa » tOb). each of the first separation time spans tl (previously denoted first trigger times) may range between about 40ms and 300ms, each of the second separation time spans t2 (previously denoted second trigger times) may range between about 1ms and 40ms, each of the fragmenting time spans t3 (previously denoted third trigger times) may range between about 1ms and 40ms and / or each of the recording time spans t4 (previously denoted fourth trigger times) may be less than about 1ms, in the ps time range. The providing time span tOa may limit the measurement time span t5. The providing time span tOa may have the same or a similar length as the measurement time span t5 or may at least be roughly in the same time range.

[0103] In line with the above, the modules further downstream may operate faster than the modules further upstream. The trigger times may therefore be reduced from upstream to downstream modules.

[0104] The providing of the constant stream of the analyte and the analyte-specific internal standard and the performing of the spatial accumulation of the analyte and the analyte-specific internal standard may be performed constantly. Alternatively, only the providing of the constant stream of the analyte and the analyte-specific internal standard may be performed constantly and therefore constantly feeding the spatial accumulation of the analyte and the analytespecific internal standard; the performing of the first separation of the analyte and the analyte-specific internal standard may be carried out / performed at a frequency of about 25Hz to 3Hz; the performing of the second separation of the analyte and the analyte-specific internal standard may be carried out / performed at a frequency of about 1kHz to 25Hz; the fragmenting of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments may be performed at a frequency of about 1kHz to 25Hz; and the recording with the detection mass spectrometer at least two pre-summed mass-resolved spectra may be carried out / performed at a frequency of more than 1kHz.

[0105] The performing of the first separation and / or the second separation of the (ionized) analyte and the (ionized) analytespecific internal standard from other components of the ionized sample molecules may be performed for the (ionized) analyte and the (ionized) analyte-specific internal standard simultaneously.

[0106] The fragmenting of the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase may be performed simultaneously for the ionized analyte and the ionized analyte-specific internal standard. This may be the case in general and specifically in the above case of simultaneous separation(s).

[0107] The providing of the stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on a detection mass spectrometer may be based on a stream that has both, the ionized analyte fragments and the ionized analyte-specific internal standard fragments at the same time. This may be the case in general and specifically in the above case of simultaneous separation(s) and / or simultaneous fragmentation.

[0108] Alternatively to the above described simultaneous separation(s), in all described embodiments, the performing of the first separation and / or the second separation of the (ionized) analyte and the (ionized) analyte-specific internal standard from other components of the ionized sample molecules may be performed consecutively for the (ionized) analyte and the (ionized) analyte-specific internal standard, i.e. in a temporal switched manner (switching back and forth between analyte and analyte-specific internal standard in the ms time range), such that the (ionized) analyte is separated first and / or secondly at a different time than the (ionized) analyte-specific internal standard. The system may therefore switch between the separation of the (ionized) analyte and the (ionized) analyte-specific internal standard. Therefore, the performing of the first separation of the (ionized) analyte and the (ionized) analyte-specific internal standard from other components may be understood as a performing of the first separation of the (ionized) analyte from other components and separate performing of the first separation of the (ionized) analyte-specific internal standard from other components. The spatial accumulation may be performed for the analyte and the analyte-specific internal standard together, i.e. at the same time.

[0109] Alternatively to the above described simultaneous fragmentation, the fragmenting of the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase may be performed consecutively for the (ionized) analyte and the (ionized) analyte-specific internal standard. This may be the case in general and specifically in the above case consecutive separation(s).

[0110] Alternatively to the above described simultaneous providing, the separate providing of the ionized analyte stream may result in a providing of an ionized analyte stream comprising the ionized analyte fragments on the detection mass spectrometer and the separate providing of an ionized analyte-specific standard stream may result in a providing of an ionized analyte-specific internal standard fragments on the detection mass spectrometer, in a case when the ionized analyte fragments and the ionized analyte-specific internal standard fragments are not mixed in the same stream at the same time. This may be the case in general and specifically in the above case of consecutive separation(s) and / or consecutive fragmentation.

[0111] In general, i.e., in all cases regardless of simultaneous or consecutive actions as described above, the recording with the detection mass spectrometer of the at least two pre-summed mass-resolved spectra and the summing of the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may result in a final mass-resolved spectrum that shows in a positive test result of the analyte, at least one analyte-specific signal (peak) and at least one internal standard-specific signal (peak) at the same time, wherein the two peaks are clearly distinguishable. In a negative test result of the analyte, the final mass-resolved spectrum only shows the at least one internal standardspecific signal (peak) but no signal corresponding to the analyte.

[0112] In general, i.e., in all cases regardless of simultaneous or consecutive actions as described above, the signals may be filtered such that - in a positive test result of the analyte - one final mass-resolved spectrum is recorded and / or generated that only shows the at least one signal of the analyte (fragment(s)) and one final mass-resolved spectrum is recorded and / or generated that only shows the at least one signal of the analyte-specific internal standard (fragment(s)). In general, i.e., in all cases regardless of simultaneous or consecutive actions as described above, the step recording and summing the recorded spectra may refer to: recording with the detection mass spectrometer for the analyte (when positively testing for an analyte) at least two pre-summed mass-resolved spectra of the ionized analyte fragments and summing the at least two pre-summed mass-resolved spectra of the ionized analyte fragments to obtain a final mass- resolved spectrum of the ionized analyte fragments, i.e., having at least one signal of the ionized analyte fragments and recording with the detection mass spectrometer for the analyte-specific standard at least two pre-summed mass- resolved spectra of the ionized analyte-specific internal standard fragments and summing the at least two pre-summed mass-resolved spectra of the ionized analyte-specific internal standard fragments to obtain a final mass-resolved spectrum of the ionized analyte-specific internal standard fragments, i.e., having at least one signal of the ionized analyte-specific internal standard fragments.

[0113] The method according to the first aspect may further comprise a step of quantitatively analyzing the final mass- resolved spectrum of the ionized analyte fragments based on a comparison of the at least one signal of the ionized analyte fragments with the at least one signal of the ionized analyte-specific internal standard fragments, wherein both are present in one “shared” / common spectrum or both are present in separate spectra, as outlined above. Based on a comparison of the signals with each other, the presence (quantitative analysis) and the amount and / or concentration (qualitative analysis) of analyte in the biological sample solution may be derived and / or determined. The quantitative and / or qualitative analysis may be performed automatically, i.e. using a machine, a data processor and / or a controller, wherein specifically, without limitations, the quantitative and / or qualitative analysis may be based on using artificial intelligence, neuronal networks, deep learning, and / or the like.

[0114] According to a second aspect, a computer-program product contains machine-readable instructions which when loaded and run on a machine, a computer or a system respectively cause the machine, the computer or the system to perform the previously described method or an embodiment thereof, i.e. the method according to the first aspect.

[0115] According to a third aspect, a mass spectrometer system for detecting an analyte in a biological sample solution comprises: a sample supply module, comprising a sample pre-processor and an ionizer, the sample supply module being configured to provide a constant stream of ionized sample molecules including the analyte from a biological sample solution and an analyte-specific internal standard, wherein the ionizer is based on electrospray ionization (ESI), and / or nano-electrospray ionization (nano-ESI) and may therefore comprise ESI and / or nano-ESI and / or may be connected to ESI and / or nano-ESI; a first selectivity enhancer module configured to perform a spatial accumulation of the analyte and the analyte-specific internal standard and to perform at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules, specifically the analyte and the analyte-specific internal standard; a second selectivity enhancer module configured to perform at least partially a second separation of the analyte and the analytespecific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; a fragmenter module (also denoted “fragmenter”) configured to fragment the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; a detection module comprising a detection mass spectrometer with a first data processor and being configured to record at least two pre-summed mass-resolved spectra from a stream of the ionized analyte fragments and the ionized analyte-specific internal standard fragments; and a controller module comprising a second data processor configured to sum the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

[0116] Alternatively or in addition a mass spectrometer system for detecting an analyte in a biological sample solution may comprise: a sample supply module, comprising a sample pre-processor and an ionizer, wherein the sample preprocessor is configured to provide to the ionizer a constant stream of sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard by means of a direct inlet approach and wherein the ionizer includes a plasma-based ambient ionization technique, such as Direct analysis in real time (DART) and / or atmospheric pressure ionization, such as electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI) and / or the ionizer is based on electrospray ionization (ESI) and / or nano-electrospray ionization (nano- ESI) and configured to at least partially ionize the constant stream of the sample molecules and thereby provide, specifically to the first selectivity enhancer module, a constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard; a first selectivity enhancer module configured to receive the constant stream of ionized sample molecules from the ionizer and perform a spatial accumulation of the (ionized) analyte and the (ionized) analyte-specific internal standard of the ionized sample molecules and to perform at least partially a first separation of the (ionized) analyte and the (ionized) analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules, specifically the analyte and the analyte-specific internal standard; a second selectivity enhancer module configured to perform at least partially a second separation of the (ionized) analyte and the (ionized) analyte-specific internal standard of the ionized sample molecules from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; a fragmenter module (also denoted “fragmenter”) configured to fragment the ionized analyte of the ionized sample molecules into ionized analyte fragments and the ionized analyte-specific internal standard of the ionized sample molecules into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; a detection module comprising a detection mass spectrometer with a first data processor and configured to record at least two pre-summed mass-resolved spectra from a stream of the ionized analyte fragments and the ionized analyte-specific internal standard fragments; and a controller module comprising a second data processor configured to sum the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

[0117] The mass spectrometer systems may be configured to perform the methods, specifically the method according to the first aspect and specifically according to the embodiments described herein. The mass spectrometer systems, specifically the system according to the third aspect is or may be adapted to at least one specific requirement in diagnostics. The mass spectrometer system according to the third aspect is specifically well-suited to increase and / or improve the sensitivity of MS such that it may be broadly used in diagnostics. The mass spectrometer system according to the third aspect may either maintain or further increase the degree in selectivity of existing MS. The mass spectrometer system according to the third aspect allows a comfortable, low maintenance, reliable, sensitive, selective and / or fast detection of a wide variety of analytes from complex biological matrices. In general, all advantages and technical effect listed for the above method may apply accordingly to the mass spectrometer system (which may also be denoted “IVD analyzer” herein). The mass spectrometer system may be considered a clinical diagnostics system or may be an element of a clinical diagnostics system.

[0118] The ionizer may be configured to ionize at least a portion of the pre-processed sample molecules comprising the analyte molecules and the analyte-specific internal standard. The molecules, which are ionized, may be subjected to the following steps but not any of the molecules, which are not ionized. The ionizer may be configured to generate a constant stream of ionized sample molecules before the spatial accumulation. The ionizer may comprise at least one of the following: ESI and / or nano-ESI, APCI, El, Laser Desorption.

[0119] The expression “detection mass spectrometer” is chosen as this MS unit differs from other MS units of the entire MS system in that it serves (in addition to a separation / selection) for the actual detection of the mass spectra while the other mass spectrometer elements / units upstream of the detection mass spectrometer may exclusively be used for selection or selectivity enhancement. Therefore, the last mass spectrometer in the system is denoted “detection mass spectrometer”.

[0120] The mass spectrometer system may specifically be free of a chromatography unit, such as an HPLC unit and / or a column and / or any other chromatograph. Chromatography may be cumbersome for a user at it is often very high- maintenance and requires technical knowledge for operation and / or maintenance. For example, a HPLC unit comprises columns, pipes, connectors, hoses and / or other elements, which operate under very high pressures. If an element is blocked, it needs to be replaced immediately. The proper connection after replacement of elements needs to be assured by a technician as otherwise the high pressures being applied to the elements may damage the entire system or at least elements thereof. Therefore, many hands-on actions may be required when using chromatography, specifically HPLC. Hence, it is difficult to implement chromatography in a fully-automated MS system, that should be simple to use, user-friendly and that should not require extensive maintenance. Therefore, a mass spectrometer system that does not comprise and require chromatography as a technique to pre-process a sample, may be very user- friendly, less intensive in hands-on actions and / or maintenance, and may be partially or even fully-automated. Such a mass spectrometer may further allow a high throughput of tests and a low downtime.

[0121] At least one of the sample supply module (comprising the sample pre-processor and the ionizer), the first selectivity enhancer module, the second selectivity enhancer module, the fragmenter module, the detection module and the controller module may be commercially available and provided as an entity in and / or for the mass spectrometer system, for example comprising a timsTOF instrument. The sample supply module may be configured as one element and / or module, specifically having one housing. Alternatively, the sample supply module may be configured as more than one element and / or module. For example, the sample supply module may be configured as two elements and / or modules having a sample pre-processor module and an ionizer module as separate entities, specifically having multiple housings. Alternatively or in addition, at least two of the above listed elements and / or modules may be provided as a combined single element, module and / or having a shared housing, for example the second selectivity enhancer module may be combined with the fragmenter module to form an entity. A module may be characterized in that it houses all elements in one single housing; and / or all elements of the module are controlled by one single controller of the module; and / or is provided commercially as one single element.

[0122] The fragmenter module may be configured to directly or indirectly provide the stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on the detection mass spectrometer of the detection module.

[0123] The controller module may correspond to a work station, an external data processor, a server and / or a controller on which machine-readable instructions are stored and / or provided by means of a computer-program product, which when loaded and run on a machine, a computer and / or a system, respectively, cause the machine, the computer or the system to perform the method of any one of the preceding claims.

[0124] The mass spectrometer system may comprise more than one of the same module types of the listed modules. For example, the mass spectrometer system may comprise two or more first selectivity enhancer modules, which may be arranged in a row after / behind each other or which may be arranged in front and behind / after the second selectivity enhancer module. This may also apply to other modules.

[0125] The sample pre-processor may be configured to add the analyte-specific internal standard. Alternatively or in addition, the sample pre-processor may be configured to collect the analyte molecules from the serum by at least one of the following: magnetobeads-capturing and / or immunoassay methods. The sample pre-processor may therefore be configured to provide, prior to the ESI and / or nano-ESI, the sample solution with or without the analyte-specific internal standard as a pre-processed sample solution including the said methods and thereby separate the analyte molecules and possibly the analyte-specific internal standard from the matrix. The sample pre-processor may therefore be considered a matrix-analyte separation unit.

[0126] The sample supply module, which is configured to provide the constant stream of ionized sample molecules comprising ionizing at least one portion of the pre-processed sample molecules by the ionizer using ESI and / or nano- ESI, may in some cases comprise ionizing at least one portion of the pre-processed sample molecules prior to the ESI and / or nano-ESI by means of the sample pre-processor. Therefore, the pre-processed sample solution may already comprise ionized molecules, specifically ionized sample molecules prior to the ESI and / or nano-ESI.

[0127] The sample pre-processor may in some cases be configured to separate a serum from a whole blood sample, specifically if a centrifuge is involved or comprised by the sample pre-processor. The sample pre-processor may therefore be configured to provide, prior to the ESI and / or nano-ESI, the sample solution as a pre-processed sample solution including separating a serum from a whole blood sample.

[0128] The first selectivity enhancer module is configured to perform at least partially a spatial accumulation and at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules, wherein the spatial accumulation may be followed by the first separation, i.e. downstream thereof. The spatial accumulation may alternatively be performed upstream of the first separation.

[0129] The first selectivity enhancer module may comprise an ion mobility spectrometer / selector. The first selectivity enhancer module may comprise a TIMS cartridge or may correspond to a TIMS cartridge.

[0130] The second selectivity enhancer module and / or the fragmenter may comprise at least one quadrupole mass spectrometer and / or a gas-filled chamber. Specifically, the second selectivity enhancer module may correspond to or comprise a quadrupole mass spectrometer and the fragmenter may correspond to or comprise a gas-fdled chamber. The combination of such elements is specifically efficient and commercially available as ready-to-use modules. Therefore, it is efficient and convenient to combine an ion mobility spectrometer / selector with a quadrupole mass spectrometer and a gas-filled chamber.

[0131] The detection mass spectrometer may comprise at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry. The detection mass spectrometer may be equipped and / or combined with an ionization unit comprising at least one of the following: NanoSpray, ESI, nano-ESI, APCI, El, Laser Desorption.

[0132] The first selectivity enhancer module may provide an effective length configured for the first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules of about 3cm to 8m, specifically of about 4cm to 3m, more specifically of about 5cm or of 1,5m, specifically wherein the first selectivity enhancer module may comprise a TIMS cartridge with an accumulation tunnel and a TIMS tunnel, wherein the TIMS tunnel may provide the effective length configured for the first separation. The first separation may therefore effectively be performed in a length of about 3 cm to 8m, specifically of about 4cm to 3 m, more specifically of about 5cm or of 1,5m, for example in a tunnel that has this length.

[0133] The first selectivity enhancer module may provide a section for performing the spatial accumulation of the analyte and the analyte-specific internal standard that has a (housing) volume of approximately 1cm3to 70cm3, specifically of approximately 5cm3to 48cm3and more specifically of approximately 10cm3to 45cm3. For example, the spatial accumulation of the analyte may be performed in a volume of approximately 12cm3. The entire volume of the first selectivity enhancer module may larger, for example twice the size of the section for performing the spatial accumulation. In one other embodiment, the first selectivity enhancer module comprises a funnel and an accumulation tunnel and provides a section for performing the spatial accumulation of the analyte and the analyte-specific internal standard, which has dimensions of 5cm x 3cm x 3cm, i.e. 45cm3. This volume may correspond to a physical volume that is defined by the physical size of the section of the first selectivity enhancer module for performing the spatial accumulation of the (ionized) analyte and the (ionized) analyte-specific internal standard. The first selectivity enhancer module may provide the section for performing the spatial accumulation and another section to perform the at least partial first separation of the (ionized) analyte and the (ionized) analyte-specific internal standard from other components of the ionized sample molecules - these two sections may, without limitations, have dimensions of 10cm x 3cm x 3cm, i.e. 90cm3. The section to perform the at least partial first separation may comprise in one specific embodiment a TIMS tunnel.

[0134] Alternatively or in addition, the first selectivity enhancer module may provide a tunnel-like space that has a cross section diameter (in case of a round tunnel) or side length (in case of a square-shaped tunnel) of approximately 0,5cm to 5cm, specifically of approximately 1cm to 4cm configured for efficient and / or sufficient spatial accumulation of the analyte to achieve good results in terms of an embodiment of the invention.

[0135] The described method or any embodiment thereof and / or the mass spectrometer system or any embodiment thereof may perform partially, half- or fully automated to increase efficiency, reduce hands-on / manual operation by a user, increase reliability, reduce errors and / or increase the degree of user-friendliness. Therefore, a user who is not specifically trained in MS may safely and reliably use the mass spectrometer system and apply the method both being described herein. In the following, some general definitions are provided, which apply to the description given herein.

[0136] The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term “including” and “comprising” can be used interchangeable.

[0137] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0138] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4% to 20 %" should be interpreted to include not only the explicitly recited values of 4 % to 20 %, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20 % and sub-ranges such as from 4-10 %, 5-15 %, 10-20%, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0139] The term “about” or “approximately” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

[0140] A “mass spectrometer system”, an “IVD analyzer” and / or a “clinical diagnostics system” is preferably a laboratory- automated apparatus dedicated to the analysis of samples for IVD. The mass spectrometer system may have different configurations according to the need and / or according to the desired laboratory workflow. Additional configurations may be obtained by coupling a plurality of stations and / or apparatuses and / or modules and / or units together.

[0141] A “unit” and / or a “module” is a work cell, typically smaller in size than the entire mass spectrometer system, which has a dedicated function. This function can be analytical but can be also pre-analytical or post analytical or it can be an auxiliary function to any of the pre-analytical function, analytical function or post-analytical function. In particular, a unit and / or module can be configured to cooperate with one or more other modules or units for carrying out dedicated tasks of a sample processing workflow, e.g. by performing one or more pre-analytical and / or analytical and / or post- analytical steps. The terms “unit” and / or “module” and “device” can be used interchangeably. In particular, the mass spectrometer system may comprise one or more analytical apparatuses, designed to execute respective workflows that are optimized for certain types of analysis, e.g. clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Thus, the mass spectrometer system / clinical diagnostic system may comprise one analytical apparatus or a combination of any of such analytical apparatuses with respective workflows. Each of the analytical apparatuses may comprise at least one station and / or at least one unit, preferably a pre-analytical and / or post analytical unit. The mass spectrometer system / clinical diagnostics system may comprise functional units such as liquid handling units for pipetting and / or pumping and / or mixing of samples and / or reagents and / or system fluids, and also functional units for sorting, storing, transporting, identifying, separating, detecting.

[0142] “Detecting an analyte” may refer to the mere qualitative detection of presence of at least one analyte of interest. “Detecting an analyte” may specifically comprise the meaning of quantitative determination of the level of the analyte, as mostly used herein. The term "determining" the level of the analyte of interest, as used herein refers to the quantification or qualification of the analyte of interest, e.g. to determining or measuring the level of the analyte of interest in the pre-treated sample.

[0143] In this context “presence of at least one analyte of interest” encompass the qualification of the analyte of interest by its absolute value and / or its relative signal value to an internal standard and / or reference and / or level of other analyte and / or a limit of analyte concentration which is matched with its concentration.

[0144] In this context "level" or "level value" encompasses the absolute amount, the relative amount or concentration as well as any value or parameter that correlates thereto or can be derived therefrom.

[0145] In the context of the present disclosure, the term “analyte”, “analyte molecule(s)”, or “analyte(s) of interest” are used interchangeably referring the chemical species to be analyzed via mass spectrometry. Chemical species suitable to be analyzed via mass spectrometry, i.e. analytes, can be any kind of molecule present in a living organism, include but are not limited to nucleic acid (e.g. DNA, mRNA, miRNA, rRNA etc.), amino acids, peptides, proteins (e.g. cell surface receptor, cytosolic protein etc.), metabolite or hormones (e.g. testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g. Vitamin D), molecules characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl-residues on genomic DNA) or a substance that has been internalized by the organism (e.g. therapeutic drugs, drugs of abuse, toxins, etc.) or a metabolite of such a substance. Such analytes may serve as biomarkers. In the context of the present invention, the term “biomarker” refers to a substance within a biological system that is used as an indicator of a biological state of said system.

[0146] The term "are connected to each other", as used herein may specifically refer or refers to a physical contact by either a stationary unit e.g. tube connection or a semi physical connection by e.g. a pipetting unit. Furthermore, physical can mean a contact in solid / liquid (e.g. aqueous droplet ejection or piezo driven droplet ejection) or gaseous (e.g. ion optic with an ion mirror, an S-lens or a multipole). Therefore, the term "are connected to each other" may also correspond to a fluidic connection such that a substance specifically a gas, aerosols, liquids, ions, charges and / or other substances with flow behavior may flow between fluidicly connected modules and / or elements.

[0147] The term "are connected to each other", as used herein may specifically refer or refers to an electrical contact and / or electromagnetic contact between stations and / or units.

[0148] The term "directly", as used herein may mean that no other stations are arranged between the two directly connected stations.

[0149] Analytes may be present in a sample of interest, e.g. a biological sample, preferably a clinical biological sample - in that case, the test result should turn out to be positive, i.e. the sample is tested positively for an analyte of interest if it is present in the sample at detectable amounts / concentrations. The analyte of interest may not be present in a sample and in that case, the test result should turn out to be negative. The term "sample", "biological sample solution", "sample of interest" or “biological sample” are used interchangeably herein, and may refer to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual. Upon analysis, a sample provides information about the tissue status or the health or diseased status of an organ or individual. Samples may include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. Further examples of samples are cell cultures or tissue cultures. The term "serum" as used herein is the clear liquid part of the blood hat can be separated from clotted blood. The term "plasma" as used herein is the clear liquid part of blood, which contains the blood cells. Serum differs from plasma, the liquid portion of normal un-clotted blood containing the red and white cells and platelets. It is the clot that makes the difference between serum and plasma. The term "whole blood" as used herein contains all components of blood, for examples white and red blood cells, platelets, and plasma.

[0150] In the context of the present disclosure, the sample may be derived from an “individual” or “subject” or “patient”. Typically, the subject or patient is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the patient sample is from a human.

[0151] The term "small molecule" as used herein is a molecule or compound having a molecular weight or molar mass of less than 2000 Da preferably less than 1000 Da, more preferably 800 Da.

[0152] A “sample pre-processor” or “sample preparation station” is a pre-analytical module coupled to one or more analytical stations, modules or units designed to execute a series of sample (pre-)processing steps aimed at removing or at least reducing interfering matrix components in a sample and / or enriching analytes of interest in a sample. Such processing steps may include any one or more of the following processing operations carried out on a sample or a plurality of samples, sequentially, in parallel or in a staggered manner: pipetting (aspirating and / or dispensing) fluids, pumping fluids, mixing with reagents, incubating at a certain temperature, heating or cooling, centrifuging, separating, fdtering, sieving, drying, washing, re-suspending, aliquoting, transferring, storing...), and / or in some embodiments magnetobead separation.

[0153] A “reagent” is a substance used for treatment of a sample in order e.g. to prepare a sample for analysis, to enable a reaction to occur, or to enable detection of a physical parameter of the sample or analyte contained in the sample. In particular, a reagent can be a substance that is or comprises a reactant, typically a compound or agent capable e.g. of binding to or chemically transforming one or more analytes present in a sample or an unwanted matrix component of the sample. Examples of reactants are enzymes, enzyme substrates, conjugated dyes, protein-binding molecules, ligands, nucleic acid binding molecules, antibodies, chelating agents, promoters, inhibitors, epitopes, antigens, and the like. However, the term reagent is used to include any fluid that can be added to a sample including a dilution liquid, including water or other solvent or a buffer solution, or a substance that is used for disruption of specific or nonspecific binding of an analyte to a protein, binding proteins or surfaces.

[0154] A sample (sample liquid, sample fluid, sample solution may correspond to the same meaning, namely sample in liquid form) may be provided for example in sample containers such as sample tubes, including primary tubes and secondary tubes, or multi-well plates, or any other sample carrying support. Reagents may be arranged for example in the form of containers or cassettes containing individual reagents or group of reagents and placed in appropriate receptacles or positions within a storage compartment or conveyor. Other types of reagents or system fluids may be provided in bulk containers or via a line supply.

[0155] The term “automatically” or “automated” 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 process, which is performed completely by means of at least one computer and / or computer network and / or machine, in particular without manual action and / or interaction with a user. The term “fully automated” may refer to a process, which is performed completely by means of at least one computer and / or computer network and / or machine, without manual action and / or interaction with a user. The term “partially automated” may refer to a process, which is performed by means of at least one computer and / or computer network and / or machine and with the aid of manual action and / or interaction with a user. Preferably, “partially automated” can mean that the manual action and / or interaction with a user is 50% or 40% or 30% or 20% or 10% or 5% at the maximum of the total process, wherein the rest of the process is performed by means of at least one computer and / or computer network and / or machine. “By means of at least one computer and / or computer network and / or machine” can mean that this process is performed without any manual action and / or interaction with a user.

[0156] Typically, an “internal standard“ (ISTD) (denoted “analyte-specific internal standard” herein) is a known amount of a substance which exhibits similar properties as the analyte of interest when subjected to the mass spectrometric detection workflow (i.e. including any pre-treatment, enrichment and actual detection step). Although the ISTD exhibits similar properties as the analyte of interest, it is still clearly distinguishable from the analyte of interest. Exemplified, during ion mobility separation, the ISTD has the same ion size but different m / z ratio than the analyte of interest. Preferably, the internal standard is in its ion size not distinguishable from the analyte but different in the m / z ratio. Thus, both the analyte and the ISTD enter the mass spectrometer at the same time. The ISTD however, exhibits a different molecular mass than the analyte of interest from the sample. This allows a mass spectrometric distinction between ions from the ISTD and ions from the analyte by means of their different mass / charge (m / z) ratios. Both are subject to fragmentation and provide daughter ions. These daughter ions can be distinguished by means of their m / z ratios from each other and from the respective parent ions. Consequently, a separate determination and quantification of the signals from the ISTD and the analyte can be performed. Since the ISTD has been added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of an ISTD allows for a relative comparison of the amount of analyte detected, and enables unambiguous identification and quantification of the analyte(s) of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not necessarily, the ISTD is an isotopically labelled variant (comprising e.g.2H,13C, or15N etc. label) of the analyte of interest.

[0157] In the context of the present invention, the term “derivatization reagent” or “label” or “derivatization agent” are used interchangeably and refer to a chemical substance having a specific chemical structure. Said compound may comprise one or more reactive groups. Each reactive group may fulfil a different functionality, or two or more reactive groups may fulfil the same function. Reactive groups include but are not limited to reactive units, charged units, and neutral loss units. The derivatization reagent can undergo an atmospheric pressure chemical reaction with the analyte of interest.

[0158] The term “hemolysis reagent“ (HR) refers to reagents which lyse cells present in a sample, in the context of this invention hemolysis reagents in particular refer to reagents which lyse the cell present in a blood sample including but not limited to the erythrocytes present in whole blood samples. A well-known hemolysis reagent is water (H2O). Further examples of hemolysis reagents include but are not limited to deionized water, liquids with high osmolarity (e.g. 8M urea), ionic liquids, and different detergents.

[0159] In the context of the present invention, the term “compound” or “derivatization reagent” or “label” or “derivatization agent” are used interchangeably and refer to a chemical substance having a specific chemical structure. Said compound may comprise one or more reactive groups. Each reactive group may fulfil a different functionality, or two or more reactive groups may fulfil the same function. Reactive groups include but are not limited to reactive units, charged units, and neutral loss units. The derivatization reagent can undergo a chemical reaction with the analyte of interest.

[0160] The term "solid-phase extraction (SPE)" refers to the adsorption of analytes from a matrix, e.g. a biological matrix onto a solid sorbent and subsequent elution of the analytes from the sorbent into a solvent, e.g. an organic solvent. SPE is an efficient tool for the sample preparation in chemistry. Conventional SPE materials are silica-based, carbonbased, and clay-based resins. Many adsorbents for SPE applications are commercially available in different formats in the market such as SPE tubes and pipette tip formats such as Oasis-HLB (produced by Waters), Omix (produced by Agilent), and MonoTips (produced by GL Sciences).

[0161] The basic principles of SPE are similar to liquid-liquid extraction. Both methods involve the distribution of dissolved species between two phases. However, SPE involves the dispersion of the analyte between a liquid (sample medium) and a solid (adsorbent) phase instead of the two liquid phases which cannot be not mixed together as in liquid-liquid extraction. This technique allows the enrichment and purification of the analytes on a solid adsorbent through adsorption from the solution. This technique is known for a skilled person and thus not explained in more detail.

[0162] The term "liquid-liquid extraction (LLE)" refers to a separation process consisting of the transfer of a solute from one solvent to another, the two solvents being immiscible or partially miscible with each other. Frequently, one of the solvents can be water or an aqueous mixture and the other can be a nonpolar organic liquid. As in all extraction processes, liquid-liquid extraction comprises a step of mixing (contacting), followed by a step of phase separation. It is important to consider both steps in the selection of solvents and modes of operation. Thus, while vigorous mixing is favorable to the transfer of the analyte of interest from one solvent to the other, it may also impair the ease of phase separation by forming emulsions. This technique is known for a skilled person and thus not explained in detail.

[0163] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0164] The term “liquid chromatography” or "LC" refers to a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and methods in which the stationary phase is less polar than the mobile phase (e.g., water-methanol mixture as the mobile phase and Cis (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC).

[0165] "High performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and Cis (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Micro LC refers to a HPLC method using a column having a narrow inner column diameter, typically below 1 mm, e.g. about 0,5 mm. “Ultra high performance liquid chromatography" or “UHPLC” refers to a HPLC method using a pressure of 120 MPa (17.405 lbf / in2), or about 1200 atmospheres. Rapid LC refers to an LC method using a column having an inner diameter as mentioned above, with a short length <2cm, e.g. 1cm, applying a flow rate as mentioned above and with a pressure as mentioned above (Micro LC, UHPLC). The short Rapid LC protocol includes a trapping / wash / elution step using a single analytical column and realizes LC in a very short time <1 min.

[0166] Further well-known LC modi include “hydrophilic interaction chromatography” (HILIC), size-exclusion LC, ion exchange LC, and affinity LC.

[0167] LC separation may be single-channel LC or multi-channel LC comprising a plurality of LC channels arranged in parallel. In LC analytes may be separated according to their polarity or log P value, size or affinity, as generally known to the skilled person.

[0168] The term “micro particle” may refer to spherical particles or unregularly shaped particles in a range of lOnm-lOmm. The particles can consist of or comprise inorganic and-or organic material e.g. SiCK TiCL, Polystyrene, PMMA and optionally many more materials. The surface of the particles can be modified by a different chemistry from the supporting microparticle core material e.g. Cis chain, CN, Pentaflurophenyl etc.

[0169] The term “bead” does not necessarily refer to a spherical shape but to a particle having an average size in the nanometer or micrometer range and having any possible shape. A bead may be a solid phase. Suitable solid phases include but are not limited to Solid Phase Extraction (SPE) cartridges. Beads may be non-magnetic, magnetic, or paramagnetic. Beads may be coated differently to be specific for the analyte of interest. The coating may differ depending on the use intended, i.e. on the intended capture molecule. It is well-known to the skilled person which coating is suitable for which analyte. The beads may be made of various different materials. The beads may have various sizes and comprise a surface with or without pores.

[0170] Non-magnetic beads may also be used in combination or instead of magnetobeads. In that case, capturing and releasing may be based on filtration. The sample preparation, the sample pre-processing and / or pre-treatment may be performed in or by a sample preparation station and may further comprise one or more pipetting device or fluid transport device for adding / removing fluids, such as samples, reagents, wash fluids, suspension fluids, into / from the reaction container(s).

[0171] The term “inert gas stripping” may refer to the process of bubbling an inert gas e.g. nitrogen or argon through a liquid which needs to be stripped. Volatile compounds are evaporated due to the equilibrium process of the inert gas with the analyte-supported liquid.

[0172] The term “headspace extraction” may refer to the process of having a liquid sample sealed gastight in a vial, which has air or other gases in the headspace over the liquid. The sealing of the vial is at ambient pressure. The sealed vial is getting heated by external heat source and the analyte of interest evaporates in the headspace while increasing the pressure of the vial. The gaseous portion of the vial if further taken of by a syringe or capillary and directly transferred to the analytical unit.

[0173] The term “gas adsorption supported by liquid and / or solid” may refer to a process in which a gas is adsorbed on the active surface of a liquid or a solid. The analyte can undergo during this process endothermal or exothermal processes as well as chemical reactions. The term “unsteady analyte ion supply unit” may refer to a unit which produces ions time dependency e.g. by a laser shot which is pulsed.

[0174] The term “steady analyte ion supply unit” may refer to a unit which produces ions time independent e.g. by a constant low and power supply during nano-ESI.

[0175] The term "Nano Spray" or " APCI," refers to two independent ionization techniques in which a high electrical current is being used for the ionization.

[0176] The term "electrospray ionization" or "ESI," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0177] A specific type of ESI is nano-ESI, specifically static nano-ESI. The term "nano electrospray ionization" or "nano- ESI" refers to methods typically using flow rates below IpL / min either in static or dynamic mode. Therefore, nano- ESI corresponds to ESI, which may operate at flow rates in the range of approximately 200nL / min to lOOOnL / min while using smaller diameter emitters (approximately 10pm to 100pm), producing smaller droplets than conventional ESI and resulting in more efficient ionization and requiring lower sample solution volumes. Preferably, nano-ESI uses a flow rate of approximately 50nL / min to 500nL / min, e.g. 500nL / min. 500nL / min is equal to 0,5pL / min.

[0178] Nano-ESI may in some embodiments refer to static nano-ESI. The term “static nano-ESI” is used in the context of the present disclosure as a non-continuous flow nano-ESI option. The analysis is typically defined by a discrete sample being loaded by single-use pipette tips into an emitter. In contrast, dynamic nano-ESI mass spectrometry is characterized by a mobile phase pumped at low flow rates through a small diameter emitter.

[0179] The term "atmospheric pressure chemical ionization" or "APCI," refers to mass spectrometry methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated nitrogen gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar entity.

[0180] The term “is arranged before” can refer to the arrangement of the two units, which means that the at least one analyte of interest passes at first the first unit (e.g. first ion fragmentation), which is arranged before the second unit (e.g. ion mobility unit), and then passes the second unit.

[0181] The term “is arranged after” can refer to the arrangement of the two units and / or modules, that two units are arranged after one another. This can mean that the at least one analyte of interest passes at first the first unit (e.g. ion mobility unit) and then the second unit (e.g. second ion fragmentation), which is arranged after the first unit.

[0182] The term “Mass Spectrometry” (“Mass Spec” or “MS”) or “mass spectrometric determination“ relates to an analytical technology used to identify compounds by their mass. MS is a methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to- charge ratio or selecting the charged compounds based on the mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" such as the detection mass spectrometer may include or may be connected to an ionizer and / or an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units. The MS method may be performed either in "negative ion mode", wherein negative ions are generated and detected, or in "positive ion mode" wherein positive ions are generated and detected.

[0183] “Tandem mass spectrometry” or “MS / MS” involves multiple steps of mass spectrometry selection, wherein fragmentation of the analyte occurs in between the stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MSI) (which may correspond to the step of the first separation and / or the first selectivity enhancer module). Ions of a particular mass-to-charge ratio (precursor ions or parent ion) are selected (which may correspond to the step of the second separation and / or the second selectivity enhancer module) and fragment ions (or daughter ions) are created by collision-induced dissociation, ion-molecule reaction, or photodissociation (which may correspond to the step of the fragmentation and / or the fragmenter module). The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2) (which may correspond to the step of the recording with the detection mass spectrometer at least two pre-summed mass-resolved spectra and / or the detection module comprising a mass spectrometer with a first data processor). Since a mass spectrometer separates and / or detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. Mass spectrometry is thus, an important method for the accurate mass determination and characterization of analytes, including but not limited to low-molecular weight analytes, peptides, polypeptides or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can typically be performed without prior knowledge of the amino acid sequence.

[0184] "Multiple reaction mode" or "MRM" is a detection mode for an MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0185] The term "in vitro method" is used to indicate that the method is performed outside a living organism and preferably on body fluids, isolated tissues, organs or cells.

[0186] A "kit" is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a medicament for treatment of a disorder, or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention. Typically, a kit may further comprise carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like. In particular, each of the container means comprises one of the separate elements to be used in the method of the first aspect. Kits may further comprise one or more other reagents including but not limited to reaction catalyst. Kits may further comprise one or more other containers comprising further materials including but not limited to buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device. Moreover, the kit may, comprise standard amounts for the analytes of interest for calibration purposes.

[0187] The terms “magnetobeads-capturing”, “magnetobead-capturing” or “magnetobeads-purification” refer to a purification method of an analyte / a molecule of interest, such as a specific biomolecule, protein, nucleic acid, polymer or the like using magnetic beads (also denoted “magnetobeads”). The technique may be considered a magnetic separation technique. The molecule(s) of interest may therefore be desorbed, adsorbed and / or captured by the magnetic beads and separated from samples, specifically from biological samples using a magnetic force which is applied to the sample. The magnetic beads with the molecule(s) of interest are bunched together and the residual portion of the sample may be removed and / or separated from the molecules(s) of interest, i.e. the remaining sample is removed without removing the beads and / or the molecule(s) of interest. Afterwards, the molecule(s) of interest may be released from the magnetic beads, for example by desorption. The magnetobeads-capturing technique may be applied for DNA and mRNA purification, cell isolation, and protein purification.

[0188] It is to be understood that the present invention is not limited to the particular embodiments and examples described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0189] Detailed Description of the Invention

[0190] In the following, some embodiments will be described in detail, wherein the invention should not be understood to be limited to the following specific embodiments being described. The following embodiments and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. Single features being described in a particular embodiment may be arbitrarily combined, given that they are not excluding each other. In addition, different features which are provided together in the example embodiments are not to be considered restrictive to the invention.

[0191] Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements whereas other elements may have been left out or represented in a reduced number in order to enhance clarity and improve understanding of the aspects of the present disclosure.

[0192] The same reference numerals are used among different embodiments and examples for the same or similar elements or elements that have similar or the same effects.

[0193] Description of the Figures

[0194] Fig. la and lb are flow diagrams of mass spectrometric methods for detecting an analyte in a biological sample solution according to embodiments;

[0195] Fig. 2 is a flow diagram of a mass spectrometric method for detecting an analyte in a biological sample solution according to an embodiment;

[0196] Fig. 3 is a schematic drawing of a mass spectrometer system according to an embodiment;

[0197] Fig. 4 is a schematic drawing of a mass spectrometer system according to an embodiment; Fig. 5 is a constructional drawing of at least a portion of a mass spectrometer system comprising a timsTOF MS system according to an embodiment;

[0198] Fig. 6 a, Fig. 6b and Fig. 6c are measurement results using the method and / or the mass spectrometer system according to an embodiment;

[0199] Fig. 7a and Fig. 7b are measurement results using the method and the mass spectrometer system according to an embodiment;

[0200] Fig. 8a is a portion of the timsTOF MS system according Fig. 5, namely a TIMS cartridge and Fig. 8b is a schematic drawing of the TIMS cartridge of Fig. 8a; and

[0201] Fig. 9 is a graph showing the constant nature of the stream of ionized sample molecules and internal standard molecules measured over a period of more than 100s.

[0202] Fig. la shows a mass spectrometric method 100 for detecting an analyte in a biological sample solution according to an embodiment. The mass spectrometric method 100 may be compatible with and / or performed by using the concepts and / or instruments shown in Fig. 2-5. The method 100 comprises the steps: providing 101 a constant stream of ionized sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard including electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI); performing 102a a spatial accumulation of the analyte and the analyte-specific internal standard and performing 102b at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing 103 at least partially a second separation of the analyte and the analyte-specific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting 104 the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; providing 105 a stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on a detection mass spectrometer; and recording 106 with the detection mass spectrometer 8 at least two pre-summed mass-resolved spectra and summing 107 the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

[0203] Fig. lb shows a mass spectrometric method 100’ for detecting an analyte in a biological sample solution according to another embodiment. The method 100’ may correspond to a more specific embodiment of method 100 or may have steps that are compatible with / may be added to method 100 and / or the method 100’ may have alternative steps to steps of method 100. The mass spectrometric method 100’ may be compatible with and / or performed by using the concepts and / or instruments shown in Fig. 2-5. The method 100’ comprises the steps: Providing 101a to an ionizer 3 (see also Fig. 3 and 4) a constant stream of sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard by means of a direct inlet approach;

[0204] At least partially ionizing 101b with the ionizer 3 the sample molecules from the constant stream that comprise the analyte and the analyte-specific internal standard and thereby providing 101 to a first selectivity enhancer module 4 a constant stream of ionized sample molecules comprising the ionized analyte and the ionized analyte-specific internal standard, the ionizer 3 including atmospheric pressure ionization, such as electrospray ionization (ESI) and / or nanoelectrospray ionization (nano-ESI), and / or the ionizer including a plasma-based ambient ionization technique, such as Direct analysis in real time (DART);

[0205] Performing 102a with the first selectivity enhancer module 4 a spatial accumulation of the ionized sample molecules (comprising the ionized analyte and the ionized analyte-specific internal standard) and performing 102b with the first selectivity enhancer module 4 at least partially a first separation of the (ionized) analyte (i.e. ionized analyte molecules) and the (ionized) analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules;

[0206] Performing 103, specifically with a second selectivity enhancer module 5, at least partially a second separation of the (ionized) analyte (i.e. ionized analyte molecules) and the (ionized) analyte-specific internal standard (i.e. ionized analyte-specific internal standard molecules) from remained other components (i.e. other ionized molecules) of the ionized sample molecules based on the mass to charge ratio of the ionized molecules;

[0207] Fragmenting 104, specifically with a fragmenter module 6, the ionized analyte (also denoted “ionized analyte precursor molecules” or just “precursor molecules”) into ionized analyte fragments (also denoted “ionized analyte fragment molecules”, “ionized analyte daughter molecules / ions” or just “daughter molecules / ions”) and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments (also denoted “ionized internal standard fragment molecules”) by collision of the (ionized) analyte with gas molecules, i.e. by collision with particles of a gas phase (i.e. gas being introduced into a chamber of a fragmenter module);

[0208] Providing 105 a stream (which may be a pulsed or a (quasi)constant stream for a certain time at least) comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on / to a detection mass spectrometer 8; and

[0209] Recording with the detection mass spectrometer 8 at least two pre-summed (i.e. firstly digitally accumulated) mass- resolved spectra (for example using a first data processor 9, i.e. an internal data processor and / or pre-processor contained by the detection mass spectrometer 8) and summing 107 (i.e. secondly digitally accumulating), for example with an external data processor 11, the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum. The final mass-resolved spectrum may then serve for detecting whether or not the analyte of interest is present in a biological sample solution and specifically for qualitatively and / or quantitatively estimating its (absolute or relative) amount if present, i.e. a quantitative and / or qualitative analysis 108 of the final mass-resolved spectrum, which may also be performed by the external data processor 11.

[0210] The step of providing 101a to the ionizer 3 of the constant stream of sample molecules, prior to the ESI and / or nano- ESI, may comprise a step of providing a providing lOlaa and / or a step of sample pre-processing lOlaa of the biological sample solution as a pre-processed sample solution including separating a serum from a whole blood sample and specifically collecting the analyte from the serum by at least one of the following: magnetobeads-capturing, immunoassay methods, and / or adding the analyte-specific internal standard.

[0211] The step of recording 106 each of the at least two pre-summed mass-resolved spectra may comprise:

[0212] Detecting 106a a plurality of mass-resolved spectral data sets; and

[0213] Generating 106b from the plurality of mass-resolved spectral data sets a plurality of native mass-resolved spectra; and Pre-summing 106c the native mass-resolved spectra to obtain each of the at least two pre-summed mass-resolved spectra, specifically by means of a first data processor 9.

[0214] The step of summing 107 the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may specifically be performed by a second data processor 11 different from the first data processor 9, specifically wherein the first data processor 9 corresponds to a data processor, specifically a data pre-processor 9, being comprised by and / or connected to the detection mass spectrometer 7 and the second data processor 11 corresponds to an external data processor 11. The step of summing 107 of the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed online. At least one of the at least two pre-summed mass-resolved spectra may be recorded at a frequency between about 20kHz and 100Hz, specifically between about 5kHz and 100Hz. The step of recording 105 the at least two pre-summed mass-resolved spectra is repeatedly performed during a time span of approximately Is to 2h, specifically 5s to Ih, more specifically during a time span of approximately 10s to 20 minutes, specifically 15s to 10 minutes and even more specifically during a time span of approximately 30s to 2 minutes. The at least two pre-summed mass-resolved spectra correspond to about 5 to 500 pre-summed mass-resolved spectra which are summed 107 to obtain one final mass-resolved spectrum.

[0215] The listed method steps may be performed in this order of the list or may be performed in another order.

[0216] Fig. 2 is a flow diagram of a mass spectrometric method 100’ for detecting an analyte in a biological sample solution according to an embodiment, as for example outlined together with Fig. lb. In this embodiment, the providing 101 of the constant stream of ionized sample molecules having the analyte from the biological sample solution includes a sample pre-processing lOlaa, i.e. a sample preparation step lOlaa together with a direct inlet approach for providing 101a the constant stream of sample molecules and an ionization 101b by means of ESI and / or nano-ESI. In this embodiment, the sample preparation step lOlaa comprises a bead-based sample preparation lOlaa (specifically magnetobeads-capturing) and the ionization step 101b comprises a continuous nano-ESI spray ionization to provide 101 the constant stream of ionized sample molecules to the first selectivity enhancer module 4. The sample preparation / sample pre-processing step lOlaa may comprise a preparative extraction of the analyte molecules from other sample molecules, specifically prior to the ionization, by means of magnetobeads-capturing. The sample pre-processing lOlaa may comprise the addition of the analyte-specific internal standard. Specifically, the preparation step lOlaa may comprise the addition of one or more analyte-specific internal standards (for several analytes of interest), which are measured with the detection mass spectrometer to quantify the amount / concentration of one analyte of interest at a time in the sample by comparing the analyte signal with the corresponding analyte-specific internal standard signal.

[0217] The step of performing 102 at least partially a first separation and spatial accumulation of the ionized analyte of interest from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules comprises filtering and / or extracting the analyte molecules and the analyte-specific internal standard by ion mobility spectrometry, i.e. ion mobility collision cross section (CCS) in this embodiment.

[0218] The steps of performing 103 at least partially a second separation and fragmenting 104 of the ionized analyte and the ionized analyte-specific internal standard are resumed in this scheme under the term “mass spectrometry”, i.e. a “predetection mass spectrometry” in this embodiment and being performed by a quadrupole filter and fragmentation based on the mass to charge ratio before the actual mass-spectrometric measurement.

[0219] The providing 105 of the stream of ions on the detection mass spectrometer and the recording 106 with the detection mass spectrometer of the at least two pre-summed mass-resolved spectra as well as the summing 107 of the at least two pre-summed mass-resolved spectra to obtain the final mass-resolved spectrum are resumed under the term “quantitation” in this scheme. The “quantitation” may also comprise the recording of mass-resolved spectra using for example a TOF detection mass spectrometer. Further, the quantitation may comprise an analysis 108 of the final mass- resolved spectrum to determine the presence or absence of the analyte and - in case the analyte is present indeed - to determine the quantity of the analyte of interest. The analysis may be performed at least partially in an automated manner, for example by a data processor 9, 11 using a suitable algorithm. The analysis 108 may be performed by the first data processor 9 and / or by the second data processor 11, specifically an external data processor 11 not being part of the detection mass spectrometer. Preferably, the analysis may be performed by the second data processor 11, which may perform the summation / summing 107 of the pre-summed spectra and / or the qualitative / quantitative analysis 108 of the final spectrum.

[0220] Fig. 3 is a schematic drawing of a mass spectrometer system 10 according to an embodiment and Fig. 4 is a schematic drawing of a mass spectrometer system 10 according to another but similar embodiment. Both embodiments of the mass spectrometer system 10 may be used to perform one or both of the previously described methods 100, 100’ and comprise in these figures a sample supply module 1, which comprises a sample pre-processor 2 and an ionizer 3. The sample pre-processor 2 and the ionizer 3 are provided together in the sample supply module 1, for example in the same housing. However, the sample pre-processor 2 and the ionizer 3 may alternatively be provided separately from each other as independent entities merely being connected with each other physically, fluidically and / or electrically. In that case, the sample supply module 1 functionally comprises the combination of the sample pre-processor 2 with the ionizer 3 in view of the function of providing 101 the constant stream of ionized sample molecules to other components and / or modules upstream such as MS elements like upstream-selectivity enhancers.

[0221] At least one of the sample pre-processor 2 and / or the ionizer 3 may be commercially available as a separate single module and / or instrument.

[0222] The sample pre-processor 2 may in one embodiment comprise a function and / or generate an effect that is suited to at least partially ionize (chemically and / or physically) the molecules comprising the analyte. The partially ionized molecules then enter the ionizer 3 and other molecules which are not yet ionized in the sample pre-processor 2 may then be ionized 101b in the ionizer 3. In another embodiment, the sample pre-processor 2 does not comprise this feature of ionizing the molecules.

[0223] The sample pre-processor 2 and the ionizer 3 are shown to be connected, i.e. physically connected in an order in which the sample pre-processor 2 is positioned in front, i.e. upstream of the ionizer 3. This direct connection allows realizing the direct inlet 101a of the pre-processed molecules into the ionizer 3 using the direct inlet approach, i.e. without a step of performing chromatography. In other words, the sample pre-processor 2 is positioned upstream from the ionizer 3. Alternatively, the sample pre-processor 2 may be positioned downstream from the ionizer 3 , such that the molecules are ionized 101b at first and pre-processed lOlaa afterwards. In this case, unprocessed or otherwise pre-processed molecules may enter 10 la the ionizer 3 using the direct inlet approach. Further, it may be possible to provide a sample pre-processor 2 upstream, i.e. in front of the ionizer 3 and another (with similar, overlapping or different functionality) sample pre-processor 2 downstream, i.e. behind the ionizer 3. The sample supply module 1 comprises elements, which are pre-MS modules, i.e. elements which do not apply mass spectrometry principles but which may be suited, useful and / or required to perform mass spectrometry. Specifically, at least one method step that is suited for ionizing molecules (if not already ionized) is a pre-requisite for performing mass spectrometry. The sample supply module 1, specifically the sample pre-processor 2, may be configured, as a sample pre-processing 10 laa, to perform a separation of molecules before the first and / or second separation. The sample supply module 1, specifically the sample preprocessor 2, may be configured to perform, as a sample pre-processing lOlaa, a separation of the analyte molecules, the ionized analyte molecules, the analyte-specific internal standard molecules and / or the analyte-specific internal standard molecules from other components, such as biological molecules, matrix molecules or the like.

[0224] The sample supply module 1 is configured to provide 101 a constant stream of ionized sample molecules including the analyte from a sample solution, wherein the ionizer 3 is based on electrospray ionization (ESI) and / or nanoelectrospray ionization (nano-ESI).

[0225] The mass spectrometer system 10 comprises in the embodiments of Fig. 3 and Fig. 4, downstream from the sample supply module 1, a first selectivity enhancer module 4 configured to perform a spatial accumulation 102a and at least partially a first separation 102b of the ionized analyte from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules. The first selectivity enhancer module 4 is shown to be physically connected to components of the sample supply module 1, such as the ionizer 3. The first selectivity enhancer module 4 may alternatively or in addition be fluidically and / or electrically connected to components of the sample supply module 1, such as the ionizer 3. The first selectivity enhancer module 4 may be based on ion mobility CCS / spectrometry / separation.

[0226] The mass spectrometer system 10 comprises in the embodiments of Fig. 3 and Fig. 4, downstream from the first selectivity enhancer module 4, a second selectivity enhancer module 5 configured to perform at least partially a second separation 103 of the analyte from remained other components, i.e. molecules and / or atoms and / or substances of the ionized sample molecules based on the specific mass to charge ratio of the ionized analyte molecules, which differs from the mass to charge ratio of the molecules from which the analyte should be selected. The second selectivity enhancer module 5 may be based on quadrupole filtering. The first selectivity enhancer module 4 comprises an (spatial) accumulation section 4b which may correspond to an (spatial) accumulation tunnel and a separation section 4c which may for example correspond to a TIMS tunnel.

[0227] The mass spectrometer system 10 comprises in the embodiment of Fig. 4, downstream from second selectivity enhancer module 5, a further first selectivity enhancer module 4 (being similar or identical to the more upstream- located first selectivity enhancer module 4) to further enhance and / or increase the degree of selectivity. Alternatively, the (pulsed or quasi-constant) stream of ions may be led from the exit of the second selectivity enhancer module 5 to the entrance of the first selectivity enhancer module 4 and after passing the first selectivity enhancer module 4 a second time, the stream of ions may either pass the second selectivity enhancer module 5 a second time or may bypass the second selectivity enhancer module 5. The embodiment of Fig. 4 only differs from the embodiment of Fig. 3 in that two first selectivity enhancer modules 4 are provided in Fig. 4 and not only one as in Fig. 3. Other elements and / or components are identical to the embodiment of Fig. 3.

[0228] The mass spectrometer system 10 comprises in the embodiment of Fig. 3, downstream from second selectivity enhancer module 5 and, in the embodiment of Fig. 4, downstream from the further / second first selectivity enhancer module 4, a fragmenter module 6 configured to fragment 104 the ionized analyte into ionized analyte fragments by collision with particles of a gas phase. The fragmentation 104 is performed by “shooting” and / or accelerating the at least partially selected ionized molecules in a chamber, tube and / or tunnel filled with gas particles, i.e. gas molecules, atoms and / or ions. The collision between the ionized sample molecules and the gas particles leads to a fragmentation 104 of the ionized sample molecules. Specifically, the fragmentation 104 is pre-defined such that, based on the acceleration, the choice of gas particles, the collision energy and / or other pre-definable parameters, the type of fragments which may be produced, if the analyte is present, can be pre-defined.

[0229] The mass spectrometer system 10 comprises in the embodiments of Fig. 3 and Fig. 4, downstream from the fragmenter module 6, a detection module 7 comprising a mass spectrometer 8 with a first data processor 9, i.e. an internal data processor 9. The detection module 7 is configmed to record 106 at least two pre-summed mass-resolved spectra from the constant stream of the ionized analyte fragments. The mass spectrometer 8 detects and / or records 106a signals, for example, using a TOF MS principle, and the detected and / or recorded signals can be translated by the first data processor 9 into a data set and the data set can be translated 106b by the first data processor 9 into pre-summed mass- resolved spectra. Specifically, mass spectrometer 8 and / or the first data processor 9 may be configured for: detecting 106a a plurality of mass-resolved spectral data sets; and generating 106b from the plurality of mass-resolved spectral data sets a plurality of native mass-resolved spectra; and pre-summing 106c the native mass-resolved spectra to obtain each of the at least two pre-summed mass-resolved spectra, specifically by means of a first data processor 9.

[0230] The detection module 7 is connected to a controller module 11. The connection may rely on a physical and / or an electrical connection and / or a data exchange connection in at least one direction (at least from the detection module 7 to the controller module 11). The data exchange connection may rely on a physical wire connection or a -non-physical wireless connection, e.g. via internet, Bluetooth, or the like. The controller module 11 comprises a second data processor configured to sum the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum. The step of summing 107 the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum is therefore performed by the second data processor 11 different from the first data processor 9. The first data processor 9 corresponds to a data pre-processor 9, being comprised by and / or connected to the detection mass spectrometer 7 and the second data processor 11 corresponds to an external data processor 11. The step of summing 107 the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum may be performed online or after all measurements are completed.

[0231] The controller module 11 may - besides controlling the summing of the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum - also control other components and / or functions of the mass spectrometer system 10. For example, the controller module 11 may control the data analysis 108 from the final mass- resolved spectrum and / or the controller module 11 may control functions of the mass spectrometer system 10, for example an automated operation of the mass spectrometer system 10. Alternatively, at least some of the functions and / or components may be controlled by another controller element and / or entity, which is not the controller module 11 shown in these embodiments.

[0232] At least one of the sample supply module 1, sample pre-processor 2, the ionizer 3, the first selectivity enhancer module 4, the second selectivity enhancer module 5, the fragmenter module 6, the detection module 7 and the controller module 11 may be in physical connection / contact, in fluidic connection and / or electric connection / contact with one another or more, specifically all said elements and / or modules are contact and / or connection as specified above. A fluidic connection is provided in that a fluid and / or a material and / or particles, specifically gas particles and / or the stream of ions and / or molecules can flow between two elements, which are in fluidic connection. In the embodiments of Fig. 3 and Fig. 4 the modules are shown to contact each other in a row, however it may be possible that one module is in physical connection / contact, in fluidic connection and / or electric connection / contact with more than one other remaining listed module. A physical connection / contact and / or a fluidic connection may be established by a direct contact between two modules, a channel, a tube, a pipe, a hose, a capillary, an at least partially housed pathway, a housing or the like connecting two or more modules with each other. A physical contact may also be established in an indirect manner, for example by connecting two modules with each other via a tube. An electric connection / contact may be established via a cable or via contactless induction principles.

[0233] At least one of the sample supply module 1, the sample pre-processor 2, the ionizer 3, the first selectivity enhancer module 4, the second selectivity enhancer module 5, the fragmenter module 6, the detection module 7 and the controller module 11 may comprise a data processor that is associated with the function of the respective module and / or element. At least one of the sample supply module 1, the sample pre-processor 2, the ionizer 3, the first selectivity enhancer module 4, the second selectivity enhancer module 5, the fragmenter module 6, the detection module 7 and the controller module 11 may be configured to exchange data with another and / or one or more of the remaining listed modules.

[0234] The mass spectrometer system 10 may not be restricted to the embodiments of Fig. 3 and Fig. 4. The direct / immediate order of the shown elements may for example differ in other embodiments such that other elements and / or modules are positioned between two modules shown in the embodiments of Fig. 3 and Fig. 4. Further, the order of the modules shown in the embodiments of Fig. 3 and Fig. 4 may differ in other embodiments.

[0235] The providing 101 of the constant stream of the analyte and the analyte-specific internal standard is performed constantly over a providing time span tOa and the performing 102a of the spatial accumulation of the analyte and the analyte-specific internal standard is constantly fed and performed for an accumulation time span tOb, tOa being indicated in Fig. 3 as a quasi-constant, i.e. a constant current over a long time span (expressed as current since the ions carry a charge and move through space).

[0236] When a predetermined degree of spatial accumulation 102a is achieved, the spatial accumulation section 4b is “opened” and / or otherwise triggered to provide a stream of ions to the section 4c where the first separation 102b is performed. The opening and / or triggering may be performed, without limitations, at a predetermined frequency by repeatedly opening and closing and / or triggering the providing for the first separation after a certain first trigger time (which corresponds to a first trigger frequency). For example, after every about 40ms to 300ms of spatial accumulation 102a (corresponding to the first trigger time), a gate between the space for spatial accumulation (e.g. an accumulation tunnel without limitation) may be opened to the space of first separation (e.g. a TIMS tunnel without limitation). This may allow performing the first separation 102b of the analyte and the analyte-specific internal standard in a pulsed manner over first separation time spans tl, which correspond to the above-described first trigger time. The performing 102b of the first separation 102b of the analyte and the analyte-specific internal standard is then performed in a pulsed manner over first separation time spans tl as also indicated in Fig. 3 as a pulsed current over time.

[0237] The section / space 4c where the first separation 102b is performed in the first selectivity enhancer module may be opened at a second trigger frequency, i.e. repeatedly after second trigger times t2 that may preferably be shorter than the first trigger time tl. This second trigger time t2 may be provided for the performing of the second separation 103 of the ionized analyte and the ionized analyte-specific internal standard being therefore performed in a pulsed manner over second separation time spans t2 (second trigger times). Therefore, the performing 103 of the second separation of the analyte and the analyte-specific internal standard is performed in a pulsed manner over second separation time spans t2, which is as also indicated in Fig. 3 as a pulsed current over time having shorter pulses than the previous current plots. The space where the second separation 103 is performed, i.e. the second selectivity enhancer module 5, may be opened at a third trigger frequency, i.e. repeatedly after third trigger time t3 that may preferably be shorter than the second trigger time t2. This third trigger time t3 may be provided for the performing 104 of the fragmenting of the ionized analyte and the ionized analyte-specific internal standard being therefore performed in a pulsed manner over fragmenting time spans t3 (third trigger times) in the fragmenter module 6. Therefore, the fragmenting 104 of the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analytespecific internal standard fragments is performed in the fragmenter module 6 in a pulsed manner over fragmenting time spans t3, which is as also indicated in Fig. 3 as a pulsed current over time having even shorter pulses than the previous current plots.

[0238] The fragmenter module 6 is constantly open to allow passage of the ions constantly. The first qudrupole filters and / or guides away the precursor and switches between analyte and analyte-specific internal standard back and forth at a switching frequency. The last quadrupole filter operates accordingly for the according fragments.

[0239] The recording 106 with the detection module 7 (comprising a detection mass spectrometer) of the at least two presummed mass-resolved spectra may be performed over an overall / integrated time span (also denoted measurement time span t5) of seconds up to several minutes (e.g. up to 30 minutes or even more) in a pulsed manner over short recording time spans t4 (fourth trigger times), specifically being shorter than the fragmenting time spans t3 corresponding to a recording frequency which may be considered a fourth trigger frequency. The overall measurement time span t5 is the sum of the single recording time spans t4 (in which for example each of the pre-summed spectra and / or the native spectra are recorded and / or generated) for the entire measurement needed to collect the data required for generating the final mass-resolved spectrum. Therefore, the recording 106 with the detection module 7 (comprising the detection mass spectrometer) of the at least two pre-summed mass-resolved spectra is performed in a pulsed manner over single recording time spans t4 that sum up to the overall measurement time span t5 which is quasi unlimited or at least long, i.e. in the time range of the providing time span tOa.

[0240] The controller module 11 may accumulate one pre-summed mass-resolved spectrum every about 50ms to 300 ms. Specifically, tOa and / or t5 > tl > t2 = t3 > t4 or tOa and / or t5 > tl > t2 > t3 > t4.

[0241] Fig. 5 is a constructional drawing of a mass spectrometer system 10 comprising a timsTOF MS system (Trapped Ion Mobility Spectrometry Time of Flight Mass Spectrometer), according to an embodiment, to generally demonstrate how one possible embodiment of the invention may be realized, without limitation. In general, a timsTOF MS system comprises as core elements a first selectivity enhancer module 4, which is based on trapped ion mobility spectrometry combined with a detection module 7 in combination with and / or comprising a detection mass spectrometer 8 based on time of flight (TOF) spectrometry. The embodiment of the timsTOF MS system shown herein further comprises a second selectivity enhancer module 5 based on quadrupole MS and a fragmenter module 6 also being based on quadrupole MS.

[0242] The constructional drawing is shown as having a cut along a longitudinal axis that reflects the pathway of the analyte molecules through the mass spectrometer system 10 substantially and at least partially along the axis A shown as a dashed line in the drawing. A transfer capillary la guides the constant stream of ionized and / or pre-processed molecules from the sample supply module (not shown in this figure) to the first selectivity enhancer module 4, which corresponds to the trapped ion mobility spectrometry (TIMS) cartridge in this embodiment. The second selectivity enhancer module 5 is physically connected / contacted with / to the first selectivity enhancer module 4 downstream therefrom via a tube and / or a pathway. The second selectivity enhancer module 5 may correspond to and / or comprise a quadrupole filter, specifically two quadrupole filters in a row, in the present embodiment. The fragmenter module 6 may be realized in the form of a collision cell that contains a gas and that is directly physically connected with / contacted to the second selectivity enhancer module 5. The fragmenter module 6 may correspond to and / or comprise a further quadrupole filter, in a row with the quadrupole filters of the second selectivity enhancer module 5, in the present embodiment. The second selectivity enhancer module 5 together with the fragmenter module 6 constitute a triple quad (QQQ) MS (in a QQQ, the third Quadrupole may correspond to the fragmenter module 6 or a portion thereof). The detection module 7, specifically the detection mass spectrometer 8, which may be realized as a TOF spectrometer, is positioned downstream from the fragmenter module 6 and physically contacted therewith.

[0243] The transfer capillary la, the first selectivity enhancer module 4, the second selectivity enhancer module 5, the fragmenter module 6 and the detection mass spectrometer 8 at least partially form a pathway (here a substantially linear pathway) for guiding the constant stream of ions through the modules for processing and / or selection to the detection elements of the detection mass spectrometer 8. The pathway is not necessarily a substantially linear pathway and can have curves and bends. At least the pathway inside a TOF MS spectrometer contains a curve having a radius, which is indicative for a certain mass to charge ratio.

[0244] Fig. 6 a, Fig. 6b and Fig. 6c correspond to measurement results using the method and / or the mass spectrometer system according to an embodiment of the disclosure. In Fig. 6a, a spectrum is shown for the analyte, which is androstenedione at a biologically relevant concentration of about lOOng / ml in the present case as an example. The spectrum is a plot of summed intensity vs. m / z (mass to charge ratio) for a measurement time t5 for summing 107 presummed spectra of about 20,3 seconds. It refers to a summing 107 of a previously filtered measurement signal via IMS and measurement of the selective transitions over the different points in time for the example androstenedione. In Fig. 6b the according spectrum is shown for an a measurement time t5 for summing 107 pre-summed spectra of about 80,3 seconds and in Fig. 6c the according spectrum is shown for a measurement time t5 for summing 107 presummed spectra of about 120 seconds. As can be seen from the three spectra, the signal sums up faster, i.e. to a stronger degree, than the background noise and therefore, the SNR strongly improves in a short time.

[0245] Fig. 7a and Fig. 7b are measurement results for a sample with a pre-set concentration of 80pg / mL of Testosterone using the at least some steps of at least one of the previously described methods 100, 100’ and / or at least some elements of the previously described mass spectrometer system 10 according to an embodiment. The question is addressed of how much accumulation is needed for a stable area ratio. Therefore, a stepwise integration with testosterone at 80pg / mL is performed.

[0246] Fig. 7a describes the relationship between measuring time / accumulation time (x-axis) and the error of the measurement (y-axis) in accordance with the measured internal standard. By enhancing the signal more over the random noise, the signal of the analyte becomes more intense than the background (i.e. the signal grows faster than the background signal) and the overall signal trueness as ration by the signal for the internal standard increases over time. After a certain point in time, the error remains constant. Before this point in time, the error decreases over time. In this specific case, this time, for which the error remains at a constant low level, is about 20s, as an example and without limitations. (The grey line in Fig. 7a corresponds to the ratio of the qualifier and the black line in Fig. 7a corresponds to the ratio of the quantifier; The graph is a plot of percent absolute difference to full time peak area ratio vs. time accumulated in sec. / measurement time t5 in sec.). Fig. 7b, illustrates the raw signals for qualifier / quantifier of the internal standard and the analyte (corresponding to results shown in Fig. 7a), respectively. In this specific case, the signal displayed is summed / digitally accumulated over 35 sec as an example and without limitations. Intensity vs. mass / charge spectra (corresponding to mass spectra): Qualifier for analyte top left; Quantifier for analyte top right; Qualifier for analyte-specific internal standard bottom left; Quantifier for analyte-specific internal standard bottom right.

[0247] Fig. 8a is a portion of the timsTOF MS system according Fig. 5, namely the TIMS cartridge representing the first selectivity enhancer module 4 and Fig. 8b (shown below Fig. 8a) is a schematic drawing of the TIMS cartridge of Fig. 8a.

[0248] The TIMS cartridge 4 is configured for performing 102b at least partially a first separation and 102a spatial accumulation of the ionized analyte and ionized analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules. Therefore, the TIMS cartridge 4 represents an embodiment of the first selectivity enhancer module 4, which is configured to perform a spatial accumulation 102a, mostly inside the accumulation tunnel 4b and at least partially a first separation of the analyte and analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules, inside the TIMS tunnel.

[0249] In general, TIMS corresponds to an IMS technique in which ions are propelled through a TIMS tunnel 4c (as can be seen in Fig. 8a and Fig. 8b) by a gas flow. An applied electrical field pre-determines the movement of each ion defined by the ion’s mobility, where the impact that it experiences from the gas flow matches the force of the electrical field. Ramping down the electrical field allows selectively releasing ions from the TIMS tunnel 4c according to the mobility. By combining TIMS with a quadrupole time-of-flight (QTOF) mass spectrometer (not shown in Fig. 8a and Fig. 8b but in Fig. 5), ions can be spatially accumulated 102a for a specific period before being released for MS separation and / or analysis. For example, peptide ions may be separated using TIMS, eluted (~ 100 ms) and detected in the QTOF, generating a TIMS MS heat map. TIMS may specifically correspond to an IMS separation technique in gas phase, which may resolve sample complexity with an added dimension of separation in addition to mass spectrometry, increasing peak capacity and confidence in compound characterization. TIMS may also allow to spatially accumulate and concentrate ions of a given mass and mobility (as can be seen in Fig. 8a and Fig. 8b), enabling to increase sensitivity and speed along with the additional dimension of separation. TIMS allows for ions to be spatially accumulated in the front section (upstream), specifically in the (spatial) accumulation tunnel 4b, while ions in the rear section (downstream) are sequentially released depending on their ion mobility, specifically downstream of the TIMS tunnel 4c.

[0250] In other words, the spatial accumulation is performed in the embodiment of Fig. 5 and Fig. 8a and Fig. 8b by means of Trapped Ion Mobility Spectrometry (TIMS), wherein TIMS is combined in this embodiment with time of flight spectrometry (timsTOF). Therefore, the TIMS cartridge comprises in this embodiment a section with the (spatial) accumulation tunnel 4b for the step of spatial accumulation. The accumulation tunnel 4b plus a first funnel 4a comprises in this embodiment a box volume VI which may for example correspond to 5x3x3 cm3as an example without limitation. The box / housing volume of the (spatial) accumulation tunnel 4b plus the first funnel 4a plus the TIMS tunnel 4c plus a focus section 4e may be twice the volume VI, namely V2 corresponding to 5x3x3cm3, as an example without limitation. The (spatial) accumulation tunnel 4b has a diameter dl that is suited to efficiently spatially accumulate ions by the applied fields (electric fields) and it may range between approximately 0,5cm and 5cm, specifically between approximately 0,8cm and 4cm. The TIMS cartridge further comprises in this embodiment a section with the TIMS tunnel 4c for performing the actual ion mobility spectrometry, specifically the first separation 102b. The TIMS tunnel 4c provides an effective length L configured for the first separation 102b of the ionized analyte and the ionized analyte-specific internal standard from other components of the ionized sample molecules of about 3cm to 8m, specifically of about 4cm to 3m, more specifically of about 5cm.

[0251] The (spatial) accumulation tunnel 4b is positioned directly in front of the TIMS tunnel 4c, i.e. upstream of the TIMS tunnel 4c being in direct contact therewith. Further, the TIMS cartridge 4 comprises in this embodiment a first funnel 4a in front of the (spatial) accumulation tunnel 4b, i.e. upstream of the (spatial) accumulation tunnel 4b and in direct contact therewith to funnel the ions towards the (spatial) accumulation tunnel 4b. Moreover, the TIMS cartridge 4 comprises in this embodiment a second funnel 4d behind the TIMS tunnel 4c, i.e. downstream of the TIMS tunnel 4c and in direct contact therewith to funnel the ions towards the exit of the TIMS cartridge 4. An intermediate funnel 4e is positioned between the TIMS tunnel 4c and the second funnel 4d.

[0252] The ions travel in the TIMS cartridge 4 along a central length axis A of the TIMS cartridge 4 (shown in Fig. 8a and Fig. 8b) which may be shared or identical with the central length axis A of the entire TOF MS system (shown in Fig. 5). The ions are led from the sample supply module 1 into the TIMS cartridge 4 via a transfer capillary la and a deflection portion lb onto the central length axis A.

[0253] The path of the ions substantially along the central length axis A is pre-determined and / or defined by the electric fields generated by the multiple electrodes E provided along the central length axis A and defining the shape of the deflection portion lb, the first funnel 4a, the accumulation tunnel 4b, the TIMS tunnel 4c the intermediate funnel 4e and the second funnel 4d. In general, TIMS may use PCBs which are combined with each other for example in the shape of a cube (not directly and / or in a strictly stacked above each other), serving as a substrate for the beam electrodes.

[0254] Fig. 9 is a graph illustrating the constant nature of the stream of ionized sample molecules and internal standard molecules measured over a period of more than 100s. Specifically, Fig. 9 describes the measured pre-summed intensities within the mass spectrometer of the analyte (black) signal in contrast to the respective internal standard (grey) against the measurement time span t5. Each data point represents a summation of raw signals (pre-summed signals) processed by the mass spectrometer internal processing unit 9. The summed intensities refers to the respective intensity of an analyte and an analyte-specific internal standard from pre-summed spectra received from the data preprocessor (not received from a final summed spectrum that would be generated by an external data processor). The measurement time span t5 exceeds in this example over 100 minutes and is far beyond the measurement time span that can be reached using prior art techniques that rely on molecule isolation by chromatography techniques as for example described in previously mentioned publication Meier F, et al.

[0255] In this disclosure many embodiments are described to be realized as system and / or method being based on a timsTOF apparatus / module. However, this shall only serve as one possible way of realizing the invention without limitation. Specifically other IMS techniques, such as the ones listed herein, may be used as well.

[0256] In the following, some specific embodiments are described:

[0257] In embodiments of the invention, the sample supply module 1 and / or the sample pre-processor module 2 (which may also be denoted “a sample preparation station”, “a pre-analytical module” and / or “a sample pre-treatment unit”) may comprise or may be connected to an enrichment handling unit. The enrichment can be performed by concentration of the sample through reducing the volume of solvent by e.g. evaporation or by attaching the analyte on a solid supported microparticles. Another enrichment possibility is a precipitation of the analyte in its surrounding liquid by e.g. changing temperature or solvent exchange by following solid-liquid separation. In embodiments of the invention, the sample pre-treatment unit may comprise the derivatization unit which may consist of or comprise a pipetting unit with heating and cooling capabilities. In this unit, a derivatization reagent or derivatization reagents can be used.

[0258] In embodiments of the invention, the derivatization reagents may be selected from the group comprising: dansylchloride, carbamic acid, N-[2-[[[2-(diethylamino)ethyl]amino]carbonyl]-6-quinolinyl]-, 2,5-dioxo-l- pyrrolidinyl ester (RapiFluor-MS), 4-substituted 1, 2, 4-triazoline-3, 5-diones (Cookson-type reagents), 4-Phenyl- 1,2,4- triazolin-3,5-dion-derivative (Amplifex Diene), 1-propanaminium, 3-(aminooxy)-N,N,N-trimethyl-compound comprising an appropriate counter ion, e.g. bromide, chloride, iodine, etc. (Amplifex Keto), acethydrazide trimethylammonium chloride (Girard T), l-(carboxymethyl)pyridinium chloride hydrazide (Girard P) and pyridiyl amine.

[0259] In embodiments of the invention, tandem mass tags may be used. Tandem Mass Tags (TMT) may refer to a type of chemical tag used in mass spectrometry (MS) to enable relative quantification of proteins in different samples. TMTs comprise an amine-reactive group, a spacer, and a mass reporter. The amine-reactive group allows the tag to bind specifically to the N-terminus and lysine residues of peptides. TMTs are isobaric, meaning that different tags have the same mass, so they co-elute during chromatography and appear as a single peak in the mass spectrometer. During MS / MS fragmentation, the tags produce unique reporter ions whose intensity can be measured to quantify the relative abundance of peptides from different samples. TMTs allow multiplexing, where multiple samples (typically up to 6, 10, or 16, depending on the specific TMT set) can be analyzed simultaneously in a single MS run, greatly increasing throughput and efficiency. TMTs are widely used in proteomics for comparative studies, such as understanding disease states, monitoring response to treatment, and profiling protein expression in different conditions.

[0260] In embodiments of the invention, the sample pre-treatment unit may comprise a solvent evaporation unit. The solvent evaporation unit can be part of an enrichment handling unit.

[0261] In embodiments of the present invention, the addition of an hemolysis reagent can be performed in the sample pretreatment unit.

[0262] In embodiments of the present invention, the addition of enzymatic reagents to the sample, e.g. urine sample, can be performed in the sample pre-treatment unit.

[0263] In embodiments of the present invention, the addition of at least one derivatization reagent to the sample, e.g. urine sample, can be performed in the sample pre-treatment unit.

[0264] In embodiments of the present invention, the functional group may be selected from a group comprising carbonyl group, diene group, hydroxyl group, amine group, imine group, ketone group, aldehyde group, thiol group, diol group, phenolic group, expoxid group, disulfide group, nucleobase group, carboxylic acid group, terminal cysteine group, terminal serine group and azide group.

[0265] In embodiments of the present invention, the analyte molecule may comprise a carbonyl group as functional group which is selected from a group comprising a carboxylic acid group, aldehyde group, keto group, a masked aldehyde, masked keto group, ester group, amide group, and anhydride group. Aldoses (aldehyde and keto) exist as acetal and hemiacetals, a sort of masked form of the parent aldehyde / keto.

[0266] In embodiments of the present invention, the carbonyl group may be an amide group, the skilled person is well aware that the amide group as such is a stable group, but that it can be hydrolyzed to convert the amide group into an carboxylic acid group and an amino group. Hydrolysis of the amide group may be achieved via acid / base catalysed reaction or by enzymatic process either of which is well-known to the skilled person. In embodiments of the present invention, wherein the carbonyl group is a masked aldehyde group or a masked keto group, the respective group is either a hemiacetal group or acetal group, in particular a cyclic hemiacetal group or acetal group. In embodiments of the present invention, the acetal group, is converted into an aldehyde or keto group before reaction with the compound. In embodiments of the present invention, the carbonyl group may be a keto group. In embodiments of the present invention, the keto group may be transferred into an intermediate imine group before reacting with the reactive unit of compounds. In embodiments of the present invention, the analyte molecule comprising one or more keto groups may be a ketosteroid. In particular embodiments of the present invention, the ketosteroid may be selected from a group comprising: testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16- ketoestradiol, 16-alpha-hydroxyestrone, 2-hydroxyestrone- 3 -methylether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, A4-androstenedione, 11-deoxycortisol, corticosterone, 21 -deoxycortisol, 11 -deoxycorticosterone, allopregnanolone and aldosterone.

[0267] In embodiments of the present invention, the carbonyl group may be a carboxyl group. In embodiments of the present invention, the carboxyl group may reacts directly with the compound or it is converted into an activated ester group before reaction with the compound. In embodiments of the present invention, the analyte molecule comprising one or more carboxyl groups may be selected from a group comprising: A8-tetrahydrocannabinolic acid, benzoylecgonin, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepac and penicillin. In embodiments of the present invention, the analyte molecule comprising one or more carboxyl groups may be an amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, tryptophan, alanine, isoleucine, leucine, methionine, phenyalanine, valine, proline and glycine.

[0268] In embodiments of present invention, the carbonyl group may be an aldehyde group. In embodiments of the present invention, the aldehyde group may be transferred into an intermediate imine group before reacting with the reactive unit of compounds. In embodiments of the present invention, the analyte molecule comprising one or more aldehyde groups may be selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin andjosamycin.

[0269] In embodiments of the first aspect of the present invention, the carbonyl group may be an carbonyl ester group. In embodiments of the present invention, the analyte molecule comprising one or more ester groups may be selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine artesunate and pethidine.

[0270] In embodiments of the present invention, the carbonyl group may be an anhydride group. In embodiments of the present invention, the analyte molecule comprising one or more anhydride groups may be selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride and maleic anhydride.

[0271] In embodiments of the present invention, the analyte molecule may comprise one or more diene groups, in particular conjugated diene groups, as functional group. In embodiments of the present invention, the analyte molecule comprising one or more diene groups may be a secosteroid. In embodiments, the secosteroid may be selected from the group comprising: cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol and tacalcitol. In particular, the secosteroid is vitamin D, in particular vitamin D2 or D3 or derivates thereof. In particular embodiments, the secosteroid may be selected from the group comprising: vitamin D2, vitamin D3, 25- hydroxyvitamin D2, 25 -hydroxyvitamin D 3 (calcifediol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitaminD3, 1,25 -dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 (calcitriol), 24,25-dihydroxyvitamin D2, 24,25- dihydroxyvitamin D3. In embodiments of the present invention, the analyte molecule comprising one or more diene groups may be selected from the group comprising: vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus and fidaxomicin.

[0272] In embodiments of the present invention, the analyte molecule may comprise one or more hydroxyl group as functional group. In embodiments of the present invention, the analyte molecule may comprise a single hydroxyl group or two hydroxyl groups. In embodiments wherein more than one hydroxyl group is present, the two hydroxyl groups may be positioned adjacent to each other (1,2-diol) or may be separated by 1, 2 or 3 C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In particular embodiments, the analyte molecule may comprise a 1,2-diol group. In embodiments, wherein only one hydroxyl group is present, said analyte is selected from the group comprising: primary alcohol, secondary alcohol and tertiary alcohol. In embodiments of the present invention, wherein the analyte molecule comprises one or more hydroxyl groups, the analyte may be selected from a group comprising: benzyl alcohol, menthol, L-camitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanic acid, Miglitol, zalcitabine, isoprenaline, aciclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, alphahydroxyalprazolam, alpha-Hydroxytriazolam, lorazepam, oxazepam, Temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3 -glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O- desmethyltramadol, Desmetramadol, dihydroquinidine and quinidine. In embodiments of the present invention, wherein the analyte molecule comprises more than one hydroxyl groups, the analyte may be selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacitidine, ribavirin, floxuridine, Gemcitabine, Streptozotocin, adenosine, Vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, Canagliflozin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, Fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0273] In embodiments of the present invention, the analyte molecule may comprise one or more thiol group (including but not limited to alkyl thiol and aryl thiol groups) as functional group. In embodiments of the present invention, the analyte molecule comprising one or more thiol groups may be selected from a group comprising: thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zofenoprilat, tiopronin, dimercaprol, succimer.

[0274] In embodiments of the present invention, the analyte molecule may comprise one or more disulfide group as functional group. In embodiments of the present invention, the analyte molecule comprising one or more disulfide groups may be selected from a group comprising glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L- cy stine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide and peginesatide. Selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2Se. In embodiments of the present invention, the analyte molecule may comprise one or more epoxide group as functional group. In embodiments of the present invention, the analyte molecule comprising one or more epoxide groups may be selected from a group comprising: Carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandomycin.

[0275] In embodiments of the present invention, the analyte molecule may comprise one or more phenol groups as functional group. In particular embodiments of the present invention, analyte molecules comprising one or more phenol groups may comprise steroids or steroid-like compounds. In embodiments of the present invention, the analyte molecule comprising one or more phenol groups may comprise a steroid or a steroid-like compound having an A-ring which is sp2hybridized and an OH group at the 3 -position of the A-ring. In particular embodiments of the present invention, the steroid or steroid-like analyte molecule may be selected from the group consisting of estrogen, estrogen-like compounds, estrone (El), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16, 17-epiestriol and / or metabolites thereof. In embodiments, the metabolites may be selected from a group comprising estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16, 17-epiestriol (16,17-epiE3), 16-ketoestradiol (16- ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2- hydroxyestrone-3 -methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2- hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (El), estrone sulfate (Els), 17a- estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17a-dihydroequilin (EQa), 17b- dihydroequilin (EQb), Equilenin (EN), 17-dihydroequilenin (ENa), 17a-dihydroequilenin, 17p-dihydroequilenin (ENb) , A8,9-dehydroestrone (dEl), A8,9-dehydroestrone sulfate (dEls), A9-tetrahydrocannabinol, mycophenolic acid. P or b can be used interchangeably, a and a can be used interchangeably.

[0276] In embodiments of the present invention, the analyte molecule comprises an amine group as functional group. In embodiments of the present invention, the amine group is an alkyl amine or an aryl amine group. In embodiments of the present invention, the analyte comprising one or more amine groups is selected from a group comprising proteins and peptides. In embodiments of the present invention, the analyte molecule comprising an amine group is selected from a group comprising 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, Amphetamine, Methamphetamine, N-methyl-l,3-benzodioxolylbutanamine, 7 -aminoclonazepam, 7 -aminoflunitrazepam, 3,4-dimethylmethcathinone, 3 -fluoromethcathinone,

[0277] 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, amfepramone, butylone, ethcathinone, elephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dothiepin, doxepin, imipramine, nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N- acetylprocainamide, procainamide, pregabalin, 2-Methylamino-l-(3,4-methylendioxyphenyl)butan, N-methyl-1,3- benzodioxolylbutanamine, 2-Amino- 1 -(3 ,4-methylendioxyphenyl)butan, 1,3 -benzodioxolylbutanamine, normeperidine, O-Destramadol, desmetramadol, tramadol, lamotrigine, Theophylline, amikacin, gentamicin, tobramycin, vancomycin, Methotrexate, Gabapentin sisomicin and 5-methylcytosine.

[0278] In embodiments of the present invention, the analyte molecule is a carbohydrate or substance having a carbohydrate moiety, e.g. a glycoprotein or a nucleoside. In embodiments of the present invention, the analyte molecule is a monosaccharide, in particular selected from a group comprising ribose, desoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneurominic acid, etc. In embodiments, the analyte molecule is an oligosaccharide, in particular selected from a group comprising a disaccharide, trisaccharid, tetrasaccharide, polysaccharide. In embodiments of the present invention, the disaccharide is selected from a group comprising sucrose, maltose and lactose. In embodiments of the present invention, the analyte molecule is a substance comprising above described mono-, di-, tri-, tetra-, oligo- or polysaccharide moiety.

[0279] In embodiments of the present invention, the analyte molecule comprises an azide group as functional group, which is selected from a group comprising alkyl or aryl azide. In embodiments of the present invention, the analyte molecule comprising one or more azide groups is selected from a group comprising zidovudine and azidocillin.

[0280] Such analyte molecules may be present in biological or clinical samples such as body liquids, e.g. blood, serum, plasma, mine, saliva, spinal fluid, etc., tissue or cell extracts, etc. In embodiments of the present invention, the analyte molecule(s) are present in a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, spinal fluid, and a dried blood spot. In some embodiments of the present invention, the analyte molecules may be present in a sample which is a purified or partially purified sample, e.g. a purified or partially purified protein mixture or extract.

[0281] In embodiments of the present invention, the analyte of interest is a steroid.

[0282] In embodiments of the present invention, the steroid is selected from a group comprising Cortisol, DHEA-S, Estradiol, Progesterone, Testosterone, 17-Hydroxyprogesterone, Aldosterone, Androstendione, DHEA, Dihydrotestosterone and Cortisone.

[0283] In embodiments of the present invention, the analyte of interest is a Vitamin D.

[0284] In embodiments of the present invention, the Vitamin D is selected from a group comprising 25(OH)D2, 25(OH)D3, 24,25(OH)2D2, 24,25(OH)2D3, 1,25(OH)2D2 and 1,25(OH)2D3. A skilled person knows the abbreviations mentioned above for Vitamin D.

[0285] In embodiments of the present invention, the analyte of interest is selected from a group comprising Cyclosporine A, Everolimus, Sirolimus, Tacrolimus, Acetaminophen, Salicylate, Theophylline and Digoxin.

[0286] In embodiments of the present invention, the analyte of interest is selected from a group comprising Phenytoin, Valproic acid, Phenytoin, Levetiracetam, Carbamazepine, Carbamazepine- 10,11-epoxide, Phenobarbital, Primidone, Gabapentin, Zonisamid, Lamotrigine and Topiramateand.

[0287] In embodiments of the present invention, the analyte of interest is selected from a group comprising Gentamicin, Tobramycin, Amikacin, Vancomycin, Piperacilline (Tazobactam), Meropenem and Linezolid.

[0288] In embodiments of the present invention, the analyte of interest is selected from a group comprising Methotrexate, Voriconazole, Mycophenolic acid and Mycophenolic acid-glucuronide.

[0289] In embodiments of the present invention, the analyte of interest is selected from a group comprising Buprenorphine, 6-monoacatylmorphine, Codeine, Dihydrocodeine, Morphine, Morphine-3 -glucuronide and Tramadol.

[0290] In embodiments of the present invention, the analyte of interest is selected from a group comprising Acetylfentanyl, Carfentanil, Fentanyl, Hydrocodone, Norfentanyl, Oxycodone and Oxymorphone.

[0291] In embodiments of the present invention, the sample preparation station comprises a sample analytical unit. In embodiments of the present invention, the sample analytical unit may comprise at least one unit or more than one units or a combination of the units selected from the group: a solid-liquid support analyte enrichment unit, solid-liquid support matrix depletion unit, gaseous sample enrichment or matrix separation unit, solid sample on solid support unit.

[0292] In embodiments of the present invention, the solid-liquid support analyte enrichment unit is e.g. SPE (solid phase extraction), SLE (Solid liquid extraction), SPME (Solid Phase Micro Extraction). In embodiments, a matrix may be depleted using a matrix depletion unit. In embodiments a matrix depletion unit and an analyte enrichment unit can be combined. This unit brings the sample in contact to a supporting liquid or uses the liquid in which the sample is already in. Furthermore a solid gets into contact with the sample and due to surface adsorption and or absorption a separation of compounds from the sample is achieved. The solid can be removed and the supernatant or the solid particles can be processed further by e.g. washing and / or extraction. The unit is capable to handle this type of extraction in an automated way from sample in, extraction and or washing followed by transferring the processed sample further.

[0293] In embodiments of the present invention, the solid-liquid support analyte enrichment unit is selected form a group comprising solid-phase extraction (SPE), micro particle, liquid-liquid extraction (LLE) and combinations thereof.

[0294] In embodiments of the present invention, a solid-liquid support matrix depletion unit is provided. This unit is capable in a similar manner to the solid-liquid support analyte enrichment unit to remove unwanted matrix components from the sample by e.g. microparticles or a prepacked column. The unwanted matrix is by this unit bound to the particles or gets washed within the supernatant of the particles.

[0295] In embodiments of the first aspect of the present invention, the solid-liquid support matrix depletion unit is selected form a group comprising solid-phase extraction (SPE), micro particle and liquid-liquid extraction (LLE).

[0296] In embodiments of the present invention, the gaseous sample enrichment or matrix separation unit consist of or comprises a SLE unit or a heating / cooling unit in which analytes and / or matrix compounds within a analytical sample gets separated. Further, more filtering devices or units to separate aerosols e.g. water droplets ranging from 10 nm-10 mm get filtered off.

[0297] In embodiments of the present invention, the solid sample on solid support unit may consist of or may comprise a pipetting unit if liquid samples are handled or a robotic arm for solid samples. The solid support can consist of or can comprise a metal plate or a solid strip. The sample can be dried if a liquid was placed and / or mixed on the solid support with other reagents.

[0298] In embodiments of the present invention, the solid sample on solid support unit is imaging of cross sections and mineral analysis.

[0299] In embodiments of the present invention, the gaseous sample enrichment or matrix separation unit may comprise inert gas stripping or headspace extraction or gas adsorption supported by liquid and / or solid e.g. activated charcoal or combinations thereof.

[0300] Some aspects, embodiments and / or examples, which may refer to embodiments of at least one of the methods 100, 100’ (Fig. la, Fig. lb) and / or which may at least be combined with above disclosed features and / or embodiments and which may not be merely understood in an isolated sense, refer to the following:

[0301] A mass spectrometric method (100, 100’) for detecting an analyte in a biological sample solution comprising the steps: providing (101a) to an ionizer (3) a constant stream of sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard by means of a direct inlet approach; at least partially ionizing (101b) with the ionizer (3) the sample molecules that comprise the analyte and the analytespecific internal standard and thereby providing (101) to a first selectivity enhancer module (4) a constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard, the ionizer (3) including atmospheric pressure ionization, such as electrospray ionization (ESI) and / or nano-electrospray ionization (nano- ESI), and / or the ionizer including a plasma-based ambient ionization technique, such as Direct analysis in real time (DART); performing (102a) with the first selectivity enhancer module (4) a spatial accumulation of the ionized sample molecules and performing (102b) at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing (103) at least partially a second separation of the analyte and the analyte-specific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting (104) the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; providing (105) a stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments to a detection mass spectrometer; and recording (106) with the detection mass spectrometer (8) at least two pre-summed mass-resolved spectra and summing (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

[0302] The mass spectrometric method (100, 100’), wherein the providing (101a) to the ionizer (3) of the constant stream of sample molecules comprising the analyte from the biological sample solution and the analyte-specific internal standard by means of the direct inlet approach is free of a generation and / or a providing of a pulsed stream of ionized sample molecules; and / or wherein the providing (101) to the first selectivity enhancer module (4) of the constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard is free of a generation and / or a providing of a pulsed stream of ionized sample molecules.

[0303] The mass spectrometric method (100, 100’), wherein the step of recording (106) each of the at least two pre-summed mass-resolved spectra comprises: detecting (106a) a plurality of mass-resolved spectral data sets; and generating (106b) from the plurality of mass-resolved spectral data sets a plurality of native mass-resolved spectra; and pre-summing (106c) the native mass-resolved spectra to obtain each of the at least two pre-summed mass-resolved spectra, specifically by means of a first data processor (9); and / or wherein the step of summing (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum is performed by a second data processor (11) different from the first data processor (9), specifically wherein the first data processor (9) corresponds to a data processor, specifically a data pre-processor (9), being comprised by and / or connected to the detection mass spectrometer (7) and the second data processor (11) corresponds to an external data processor (11); and / or wherein the step of summing (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum is performed online; and / or wherein at least one of the at least two pre-summed mass-resolved spectra are recorded at a frequency between 20kHz and 100Hz, specifically between 5kHz and 100Hz; and / or wherein the step of recording (105) the at least two pre-summed mass-resolved spectra is repeatedly performed during a time span of Is to 2h, specifically 5s to Ih, more specifically during a time span of 10s to 20 minutes, more specifically during a time span of 15s to 10 minutes and even more specifically during a time span of 30s to 2 minutes; and / or wherein the at least two pre-summed mass-resolved spectra correspond to 5 to 500 pre-summed mass-resolved spectra which are summed (107) to obtain one final mass-resolved spectrum.

[0304] The mass spectrometric method (100, 100’), wherein the providing (101a) of the constant stream of sample molecules to the ionizer (3) , prior to the ESI and / or nano-ESI, comprises providing (lOlaa) of the biological sample solution as a pre-processed sample solution including separating a serum from a whole blood sample and specifically collecting the analyte from the serum by at least one of the following: magnetobeads-capturing, immunoassay methods, and / or adding the analyte-specific internal standard; and / or wherein the constant stream of ionized sample molecules and the analyte-specific internal standard and / or the the constant or pulsed stream of ionized analyte fragments and ionized analyte-specific internal standard fragments is generated for a time span of up to about 15 minutes, specifically at a pre-processed sample solution volume of InL / min to lOOOnL / min, specifically 200nL / min to 500nL / min, more specifically 250nL / min to 350nL / min.

[0305] The mass spectrometric method (100, 100’), wherein the step of summing (107) the at least two pre-summed mass- resolved spectra to obtain the final mass-resolved spectrum is completed, stopped and / or considered sufficiently sensitive when a coefficient of variance and / or an S / N ratio reaches a pre-defined threshold value, specifically the threshold value corresponds to 20% + / - 5%.

[0306] The mass spectrometric method (100, 100’), wherein the performing of the first separation (102b) comprises ion mobility spectrometry; and / or wherein the performing of the second separation (103) comprises quadrupole mass spectrometry.

[0307] The mass spectrometric method (100, 100’), wherein the detection mass spectrometer (8) comprises and / or is based on at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry, sector field MS, specifically wherein the detection mass spectrometer is combined with an ionization unit comprising at least one of the following: Nano Spray, ESI, APCI.

[0308] The mass spectrometric method (100, 100’), wherein the step of performing (103) the second separation is repeated at least one time.

[0309] The mass spectrometric method (100, 100’), wherein the providing (101) of the constant stream of the analyte and the analyte-specific internal standard is performed over a providing time span (tOa) and the performing (102a) of the spatial accumulation of the analyte and the analyte-specific internal standard is performed over an accumulation time span (tOb); the performing (102b) of the first separation of the analyte and the analyte-specific internal standard is performed in a pulsed manner over first separation time spans (tl); the performing (103) of the second separation of the analyte and the analyte-specific internal standard is performed in a pulsed manner over second separation time spans (t2); the fragmenting (104) of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments is performed in a pulsed manner over fragmenting time spans (t3); and the recording (106) with the detection mass spectrometer at least two pre-summed mass-resolved spectra is performed in a pulsed manner over recording time spans (t4), specifically wherein (tOa) > (tl) > (t2) = (t3) > (t4) or (tOa) > (tl) > (t2) > (t3) > (t4), and / or wherein the providing time span (tOa) ranges between 15s and 30 minutes, the accumulation time span (tOb) is in the ms time range, each of the first separation time spans (tl) ranges between 40ms and 300ms, each of the second separation time spans (t2) ranges between 1ms and 40ms, each of the fragmenting time spans (t3) ranges between ims and 40ms and / or each of the recording time spans (t4) is shorter than ims, specifically ranges between 1ms and 50ps. The mass spectrometric method (100, 100’), wherein the providing (101) of the constant stream of the analyte and the analyte-specific internal standard is performed constantly; the performing (102b) of the first separation of the analyte and the analyte-specific internal standard is performed at a frequency (Fl) of 25Hz to 3Hz; the performing (103) of the second separation of the analyte and the analyte-specific internal standard is performed at a frequency (F2) of 1kHz to 25Hz; the fragmenting (104) of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments is performed at a frequency of (F3) 1kHz to 25Hz; and the recording (106) with the detection mass spectrometer of at least two pre-summed mass-resolved spectra is performed at a frequency (F4) exceeding 1kHz.

[0310] A mass spectrometer system (10) for detecting an analyte in a biological sample solution (3) comprising: a sample supply module (1), comprising a sample pre-processor (2) and an ionizer (3), wherein the sample preprocessor (2) is configured to provide to the ionizer (3) a constant stream of sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard by means of a direct inlet approach and wherein the ionizer is based on electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI) and configured to at least partially ionize (101b) the constant stream of the sample molecules and thereby provide (101) a constant stream of ionized sample molecules comprising the analyte and the analyte-specific internal standard; a first selectivity enhancer module (4) configured to receive the constant stream of ionized sample molecules and perform a spatial accumulation (102a) of the analyte and the analyte-specific internal standard of the ionized sample molecules and to perform at least partially a first separation (102b) of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; a second selectivity enhancer module (5) configured to perform at least partially a second separation (103) of the analyte and the analyte-specific internal standard of the ionized sample molecules from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; a fragmenter module (6) configured to fragment (104) the analyte of the ionized sample molecules into ionized analyte fragments and the analyte-specific internal standard of the ionized sample molecules into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; a detection module (7) comprising a detection mass spectrometer (8) with a first data processor (9) and being configured to record (106) at least two pre-summed mass-resolved spectra from a stream of the ionized analyte fragments and the ionized analyte-specific internal standard fragments; and a controller module (11) comprising a second data processor configured to sum (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

[0311] The mass spectrometer system (10), wherein the sample pre-processor (2) is configured to collect the analyte molecules from the serum by at least one of the following: magnetobeads-capturing an / or immunoassay methods.

[0312] The mass spectrometer system (10), wherein the first selectivity enhancer module (4) comprises an ion mobility spectrometer; and / or the second selectivity enhancer module (5) and / or the fragmenter module (6) comprises a quadrupole mass spectrometer and / or a gas-filled chamber.

[0313] The mass spectrometer system (10), wherein the mass spectrometer system (10) and / or the mass spectrometer (8) comprises at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry.

[0314] The mass spectrometer system (10), wherein the first selectivity enhancer module (4) provides an effective length (L) configured for the first separation (102b) of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules of 3cm to 8m, specifically of 4cm to 3m, more specifically of 5cm or of 1,5m, specifically wherein the first selectivity enhancer module (4) comprises and / or corresponds to a TIMS cartridge with an accumulation tunnel (4b) and a TIMS tunnel (4c), wherein the TIMS tunnel (4c) provides the effective length (L) configured for the first separation (102b).

[0315] Reference list

[0316] 1 sample supply module la transfer capillary lb deflection portion

[0317] 2 sample pre-processor module

[0318] 3 ionizer module

[0319] 4 first selectivity enhancer module, for example a TIMS cartridge

[0320] 4a first funnel

[0321] 4b accumulation tunnel 4c TIMS tunnel

[0322] 4d second funnel

[0323] 4d intermediate funnel

[0324] 4e focus section

[0325] 5 second selectivity enhancer module

[0326] 6 fragmenter module

[0327] 7 detection module

[0328] 8 detection mass spectrometer

[0329] 9 first data processor

[0330] 10 mass spectrometer system

[0331] 11 controller module and / or external and / or second data processor

[0332] 100 mass spectrometric method

[0333] 101 providing a constant stream of ionized sample molecules

[0334] 101, 101a providing to the ionizer of the constant stream of sample molecules, in some embodiment using a direct inlet approach lOlaa sample pre-processing

[0335] 101b ionization

[0336] 102a performing a spatial accumulation of the analyte and the internal standard

[0337] 102b performing at least partially a first separation

[0338] 103 performing at least partially a second separation of the analyte

[0339] 104 fragmenting the ionized analyte

[0340] 105 providing a constant stream comprising the ionized analyte fragments on a detection mass spectrometer

[0341] 106 recording with the detection mass spectrometer at least two pre-summed mass-resolved spectra

[0342] 107 accumulating the at least two pre-summed mass-resolved spectra

[0343] 108 Quantitative and / or qualitative analysis of the final mass-resolved spectrum

[0344] A central length axis of the timsTOF MS system dl diameter of the cross section of the accumulation tunnel d2 diameter of the cross section of the TIMS tunnel

[0345] E electrodes

[0346] L effective length for performing the first separation

[0347] VI volume of the accumulation tunnel plus funnel 1

[0348] V2 volume of the accumulation tunnel plus funnel 1 plus TIMS tunnel

Claims

Patent Claims1. Mass spectrometric method (100) for detecting an analyte in a biological sample solution comprising the steps: providing (101) a constant stream of ionized sample molecules comprising the analyte from the biological sample solution and an analyte-specific internal standard including electrospray ionization (ESI) and / or nanoelectrospray ionization (nano-ESI); performing (102a) a spatial accumulation of the analyte and the analyte-specific internal standard and performing (102b) at least partially a first separation of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; performing (103) at least partially a second separation of the analyte and the analyte-specific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; fragmenting (104) the ionized analyte into ionized analyte fragments and the ionized analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; providing (105) a stream comprising the ionized analyte fragments and the ionized analyte-specific internal standard fragments on a detection mass spectrometer; and recording (106) with the detection mass spectrometer (8) at least two pre-summed mass-resolved spectra and summing (107) the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum.

2. Mass spectrometric method (100) of claim 1, wherein the providing (101) of the constant stream of ionized sample molecules is free of a generation and / or a providing of a pulsed stream of ionized sample molecules.

3. Mass spectrometric method (100) of claim 1 or 2, wherein the step of recording (106) of the pre-summed mass-resolved spectra comprises a pre-summing (107) of native mass-resolved spectra to obtain the pre-summed mass-resolved spectra, which is performed by a first data processor (9); and wherein the step of summing (107) of the at least two pre-summed mass-resolved spectra to obtain a final mass-resolved spectrum is performed by a second data processor (11) different from the first data processor (9), specifically wherein the first data processor (9) corresponds to a data processor comprised by the detection mass spectrometer (7) and the second data processor (11) corresponds to an external data processor (11); and / or wherein at least one of the at least two pre-summed mass-resolved spectra are recorded at a frequency between 20kHz and 100Hz, specifically between 5kHz and 100Hz; and / or wherein the step of recording (105) the at least two pre-summed mass-resolved spectra is repeatedly performed during a time span of 5s to Ih, specifically during a time span of 15s to 10 minutes and more specifically during a time span of 30s to 2 minutes; and / or wherein the at least two pre-summed mass-resolved spectra correspond to 5 to 500 pre-summed mass- resolved spectra which are summed (107) to obtain a final mass-resolved spectrum.

4. Mass spectrometric method (100) of any one of the preceding claims, wherein the providing of the constant stream of ionized sample molecules and the analyte-specific internal standard comprises, prior to the ESI and / or nano-ESI, providing (lOlaa) of the sample solution as a pre-processed sample solution including separating a serum from a whole blood sample, collecting the analyte from the serum by at least one of the following: magnetobeads-capturing, immunoassay methods, and / or adding the analyte-specific internal standard; and / or wherein the constant stream of ionized sample molecules and the analyte-specific internal standard and / or the stream of ionized analyte fragments and ionized analyte-specific internal standard fragments is generated for a time span of up to about 15 minutes, specifically at a pre-processed sample solution volume of InL / min to lOOOnL / min, specifically 200nL / min to 500nL / min, more specifically 250nL / min to 350nL / min.

5. Mass spectrometric method (100) of any one of the preceding claims, wherein the step of summing (107) the at least two pre-summed mass-resolved spectra to obtain the final mass-resolved spectrum is completed, stopped and / or considered sufficiently sensitive when a coefficient of variance and / or an S / N ratio reaches a pre-defined threshold value, specifically the threshold value corresponds to 20% .

6. Mass spectrometric method (100) of any one of the preceding claims, wherein the performing of the first separation (102b) comprises ion mobility spectrometry; and / or wherein the performing of the second separation (103) comprises quadrupole mass spectrometry.

7. Mass spectrometric method (100) of any one of the preceding claims, wherein the detection mass spectrometer (8) comprises and / or is based on at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry, sector field MS, specifically wherein the detection mass spectrometer is combined with an ionization unit comprising at least one of the following: NanoSpray, ESI, APCI.

8. Mass spectrometric method (100) of any one of the preceding claims, wherein the step of performing (103) the second separation is repeated at least one time.

9. Mass spectrometric method (100) of any one of the preceding claims, wherein the providing (101) of the constant stream of the analyte and the analyte-specific internal standard is performed over a providing time span (tOa) and the performing (102a) of the spatial accumulation of the analyte and the analyte-specific internal standard is performed over an accumulation time span (tOb); the performing (102b) of the first separation of the analyte and the analyte-specific internal standard is performed in a pulsed manner over first separation time spans (tl); the performing (103) of the second separation of the analyte and the analyte-specific internal standard is performed in a pulsed manner over second separation time spans (t2); the fragmenting (104) of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments is performed in a pulsed manner over fragmenting time spans (t3); and the recording (106) with the detection mass spectrometer at least two pre-summed mass-resolved spectra is performed in a pulsed manner over recording time spans (t4), specifically wherein (tOa) > (tl) > (t2) = (t3) > (t4) or (tOa) > (tl) > (t2) > (t3) > (t4), and / orwherein the providing time span (tOa) ranges between 15s and 30 minutes, each of the first separation time spans (tl) ranges between 40ms and 300ms, each of the second separation time spans (t2) ranges between 1ms and 40ms, each of the fragmenting time spans (t3) ranges between 1ms and 40ms and each of the recording time spans (t4) is shorter than 1ms, specifically ranges between 1ms and 50ps.

10. Mass spectrometric method (100) of any one of the preceding claims, wherein the providing (101) of the constant stream of the analyte and the analyte-specific internal standard is performed constantly; the performing (102b) of the first separation of the analyte and the analyte-specific internal standard is performed at a frequency (Fl) of 25Hz to 3Hz; the performing (103) of the second separation of the analyte and the analyte-specific internal standard is performed at a frequency (F2) of 1kHz to 25Hz; the fragmenting (104) of the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments is performed at a frequency of (F3) 1kHz to 25Hz; and the recording (106) with the detection mass spectrometer of at least two pre-summed mass-resolved spectra is performed at a frequency (F4) exceeding 1kHz.

11. Mass spectrometer system (10) for detecting an analyte in a biological sample solution (3) comprising: a sample supply module (1), comprising a sample pre-processor (2) and an ionizer (3), the sample supply module (1) being configured to provide (101) a constant stream of ionized sample molecules including the analyte from a sample solution and an analyte-specific internal standard, wherein the ionizer is based on electrospray ionization (ESI) and / or nano-electrospray ionization (nano-ESI); a first selectivity enhancer module (4) configured to perform a spatial accumulation (102a) of the analyte and the analyte-specific internal standard and to perform at least partially a first separation (102b) of the analyte and the analyte-specific internal standard from other components of the ionized sample molecules based on the size and / or shape of the ionized molecules; a second selectivity enhancer module (5) configured to perform at least partially a second separation (103) of the analyte and the analyte-specific internal standard from remained other components of the ionized sample molecules based on the mass to charge ratio of the ionized molecules; a fragmenter module (6) configured to fragment (104) the ionized analyte into ionized analyte fragments and the analyte-specific internal standard into ionized analyte-specific internal standard fragments by collision with particles of a gas phase; a detection module (7) comprising a detection mass spectrometer (8) with a first data processor (9) and being configured to record (106) at least two pre-summed mass-resolved spectra from a stream of the ionized analyte fragments and the ionized analyte-specific internal standard fragments; and a controller module (11) comprising a second data processor configured to sum (107) the at least two presummed mass-resolved spectra to obtain a final mass-resolved spectrum.

12. Mass spectrometer system (10) of claim 11, wherein the sample pre-processor (2) is configured to collect the analyte molecules from the serum by at least one of the following: magnetobeads-capturing and / or immunoassay methods.

13. Mass spectrometer system (10) of claim 11 or 12, wherein the first selectivity enhancer module (4) comprises an ion mobility spectrometer; and / or the second selectivity enhancer module (5) and / or the fragmenter module (6) comprises a quadrupole mass spectrometer and / or a gas-filled chamber.

14. Mass spectrometer system (10) of any one of claims 11 to 13, wherein the mass spectrometer system (10) and / or the mass spectrometer (8) comprises at least one of the following: time-of-flight (TOF), Triple Quad (QQQ), Ion Trap, an Orbi-Trap mass spectrometry.

15. Mass spectrometer system (10) of any one of claims 11 to 14, wherein the first selectivity enhancer module (4) provides an effective length (L) configured for the first separation (102b) of the analyte and the analytespecific internal standard from other components of the ionized sample molecules of 3cm to 8m, specifically of 4cm to 3m, more specifically of 5cm or of 1,5m, specifically wherein the first selectivity enhancer module (4) comprises and / or corresponds to a TIMS cartridge with an accumulation tunnel (4b) and a TIMS tunnel (4c), wherein the TIMS tunnel (4c) provides the effective length (L) configured for the first separation (102b).

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