Method and apparatus for performance monitoring and performance adjustment of ion sources

Abstract

The present invention relates to a method and apparatus for performance monitoring and conditioning of an ion source, comprising: (a) laser-assisted ionization of a control sample, the composition of which is substantially known and which is sampled before, simultaneously with or after an analytical sample, (b) generating control sample spectral data from the ionized control sample in an ion analyzer connected to an ion source, (c) repeating steps (a) and (b) over a plurality of control samples in order to collect and evaluate a plurality of control sample spectral data such that the spectral data for a single control sample has a small weight and shows a performance trend in the evaluation, (d) if the performance trend enters a range outside the predetermined performance interval, adjusting operating parameters of a laser of the ion source in order to adjust the laser to meet interval requirements, and (e) repeating steps (a) to (d) in order to continuously monitor and adjust the ion source.

Description

Technical Field

[0001] The present invention relates to performance monitoring and performance adjustment of ion sources operating with laser assisted ionization, in particular with MALDI ionization. More specifically, the present invention relates to an ion spectrometry system comprising an ion analyzer, an ion source connected to the ion analyzer, and a processing unit in communication with the ion analyzer and the ion source. Background Art

[0002] The prior art is explained below for specific aspects. However, this should not be construed as limiting. Useful improvements and variations known from the prior art can also be applied outside the relatively narrow scope of this introduction, and will be apparent to those skilled in the art after reading the disclosure after this introduction of the present invention.

[0003] In MALDI time-of-flight mass analysis (MALDI, matrix-assisted laser desorption and ionisation matrix-assisted laser desorption and ionisation), there are some important operating parameters for available measurements. These operating parameters include, but are not limited to, spectral baseline, various voltages at the accelerating electrode of the flight path, detector voltage and the laser energy density of the ion source, only a few examples are cited here. Usually, these parameters are either detected in the test area of ​​the mass spectrometer manufacturer or in the user's laboratory by the qualified service personnel of the manufacturer or contractor and set for the best possible result. However, these measures are isolated events, so that the mass spectrometer must be operated without monitoring in a long period of time after successfully establishing or resetting.

[0004] However, it is known and consistent with experience that some parameters change with the operation and use time of the mass spectrometer. It can also be said to be aging or wear. Among the above parameters, in particular, it is directly related to the detector voltage and indirectly to the laser energy density.

[0005] Detector aging is particularly manifested in that the amplification factor, for example the amplification factor of the secondary electron multiplier, decreases with continued operation. This decrease is usually dealt with by increasing the detector voltage, i.e. the voltage gradient, by which the secondary electrons are accelerated. This adjustment requirement of the detector voltage is established over a considerable period of time in practice and can be determined to a large extent automatically, for example in the calibration phase of observing single ion signals described in patent publication DE102008010118A1 (corresponding to GB2457559A and US2009 / 0206247A1).

[0006] What may happen in the case of laser energy density is that the ion source, and in particular the surface thereof, for example, the surface of the electrode or the housing part limited therein, is contaminated during continuous operation, and the conditions for generating an electric field in the ion source are thereby changed. This then manifests as a reduced ion output. This efficiency drop can be dealt with, for example, by semi-automatic or fully automatic cleaning, which is intended to eliminate or at least reduce the interference caused by physicochemical effects. An example of such cleaning is proposed in patent disclosure DE102008008634A1 (corresponding to GB2457362A and US2009 / 0200457A1). Another possibility is to increase the laser energy density or laser flux, because the ion output is scaled with multiple powers of energy density or flux, as described in the overview article of Klaus Dreisewerd (Chem.Rev.2003,103,395-425). Depending on the use of the analyzer, considering the uninterrupted measurement phase from morning to night almost every day, the maintenance requirements caused by contamination may accumulate faster than detector aging.

[0007] Once a mass spectrometer is first installed in a user's laboratory, intensive operation (e.g., repeated identification and measurement of clinical pathogenic microorganisms at high throughput) may cause such performance degradation to occur so rapidly that high-frequency maintenance, such as weekly or even more frequent, is required; this is a huge expense in terms of service personnel and at the same time a source of cost for the user.

[0008] In the following, some prior art documents that may be relevant to the present disclosure will be briefly introduced without claiming completeness:

[0009] The technical teaching disclosed in patent publication DE 10 2010 019 857A1 (corresponding to GB2483322A and US2011 / 0272573A1) is based on recording multiple series of mass spectra of a mixture of analyte substances by gradually increasing the energy density in the laser focus, but special measures are taken to overcome the problem of signal saturation on the detector that may result from this.

[0010] Patent publication WO 2014 / 140625 A1 discloses a method for ion imaging, the method comprising testing a first sample portion by automatically changing one or more laser parameters, and manually or automatically determining one or more optimal or preferred laser parameters from the first sample portion. Then, a second sample portion is analyzed using the one or more optimal or preferred parameters.

[0011] Patent publication WO 2017 / 160857 A1 relates to a system and method for real-time monitoring of laboratory analytical devices for quality control purposes.

[0012] Patent publication EP 3 806 135 A1 describes a MALDI ion source in which laser light from a laser light source is reflected by a mirror and the energy of the laser light is adjusted by a rotating polarization beam splitter. The laser light with the adjusted energy is then directed to the sample.

[0013] Patent publication EP 3 651 184 A1 relates to a mass spectrometer and a method and a procedure for adjusting the intensity of laser light for MALDI ionization in a mass spectrometer. Summary of the invention

[0014] Based on the above description, it is necessary to provide a method and apparatus for ensuring and improving the performance monitoring and adjustment of an ion source over a long period of time. Other objects to be achieved by the present invention will be easily recognized by those skilled in the art after reading the following disclosure.

[0015] In a first aspect, the present disclosure relates to a method for performance monitoring and performance adjustment of an ion source, comprising: (a) laser-assisted ionization of a control sample, the composition of which is substantially known and which is sampled before, simultaneously with, or after an analytical sample, (b) generating control sample spectral data from the ionized control sample in an ion analyzer connected to the ion source, (c) repeating steps (a) and (b) on a plurality of control samples so as to collect a plurality of control sample spectral data and evaluate them so that the spectral data of a single control sample has a small weight and a performance trend emerges in the evaluation, (d) if the performance trend enters a range outside a predetermined performance interval, adjusting operating parameters of a laser of the ion source so as to adjust the laser to meet the interval requirements, and (e) repeating steps (a) to (d) so as to continuously monitor and adjust the ion source.

[0016] In different embodiments, in step (a), the control sample can be sampled in the same measurement process as the analytical sample, and in step (c), steps (a) and (b) can be repeated on multiple measurement processes and control samples. According to the meaning of the present disclosure, a measurement process can be understood as particularly relating to the operating mode of an ion spectrometry system. For example, a measurement process can include all measurements recorded from a (control) sample on a sample carrier, as long as the sample carrier is spatially located in the ion source. An example is the introduction of a sample carrier into a low-pressure region of an ion source, provided that the ion source operates at low pressure, such as in vacuum-MALDI. If the sample carrier is removed from the ion source, for example from the low-pressure region, and subsequently another sample carrier with a new (control) sample is introduced, all measurements of the new (control) sample on the subsequent sample carrier can be regarded as independent measurement processes.

[0017] In addition to the preparation of analytical samples, multiple control sample preparations can also be arranged on the sample carrier for laser assisted ionization. If both control samples and analytical samples are present on the sample carrier, for example 2-10 technical control sample copies, in particular 4-8 technical control sample copies, all control sample spectral data prepared by these preparations can be used to determine performance trends and / or their temporal variation trends. In order to generate control sample spectral data, a control sample preparation on the sample carrier can be sampled once or multiple times, in principle only limited by the coverage (Ergiebigkeit) of the control sample. Ultimately, the control sample spectral data can include scattered and independent spectra. Repeated sampling of the same control sample preparation improves the basis of the control sample spectral data for subsequent statistical evaluation, and reduces the influence of interferences that occur, especially very briefly, in the measurement process, which may cause abnormal value measurements.

[0018] Sampling the control sample before, in parallel with or after the analytical sample can in particular mean that the control sample and the analytical sample are studied in the same measurement process. The sampling of the control sample can in particular be carried out directly before or directly after the analytical sample. The measurement mode or measurement settings of the ion spectrometry system can be consistent or different for the sampling of analytical samples and control samples. As mentioned above, in the case where the measurement process includes recording the spectral data of all (control) samples on the sample carrier, this can include that the sample carrier has a plurality of marked sample positions, for example on the known AnchorChip or MBT Biotarget 96 carrier of Bruker or on the sample point area on a steel plate, and some of these sample positions are prepared with a predetermined number of control samples on the one hand, while some or all of the remaining sample positions are occupied by analytical samples. The control samples and the analytical samples can then be sampled in a manner separate from each other in the block (e.g., first all control samples and then all analytical samples or vice versa), or the control samples and the analytical samples can also be sampled alternately (e.g., control samples => analytical samples => control samples => analytical samples and so on). In an ion source with multiple laser beams, if the connected ion analyzer is configured and arranged to handle a multiplexed ion flow, or if there is more than one ion analyzer, the analytical samples and the control samples can also be measured in parallel by the same sample carrier if necessary.

[0019] In different embodiments, the ion analyzer can be constructed as a mass analyzer, which in particular classifies ionized control samples according to the time-of-flight (TOF) dispersion principle. The mass analyzer separates charged molecules or molecular ions according to the mass-to-charge ratio (usually expressed as m / z) of the charged molecules or molecular ions. In addition to the time-of-flight analyzer mentioned in the introduction (for which not only a linear structure but also a reflector structure and / or a structure with axial or orthogonal acceleration of ions into the flight path can be set), in principle, other types of mass dispersion separators can also be used, such as quadrupole mass filters (single quads), triple quadrupole analyzers ("Tripel-Quads"), ion cyclotron resonance cells (ion cyclotron resonance, ICR), such as Kingdon analyzer (Thermo Fisher Scientific) etc.

[0020] In different embodiments, ion analyzer can also be a mobility analyzer or a combined mobility mass analyzer. Mobility analyzer separates charged molecules or molecular ions according to the collision cross-section charge ratio (sometimes referred to as Ω / z or σ / z) of charged molecules or molecular ions. Its basis is the interaction of ion species with the electric field coupled to the charge of ions, and also the effect of the buffer gas acting on the average cross-sectional area of ​​ions. It is especially known that there is a drift tube mobility separator with an electrostatic field gradient, which pushes ions through substantially stationary gas, wherein the drift velocity of ion species is produced by the forward driving force of the electric field and the deceleration force colliding with gas particles. It is also common to have a storage ion mobility separator (trapped ion mobility separators, TIMS) with a continuous laminar airflow that pushes ions forward, which reacts to the electric field gradient that changes step by step, and the electric field gradient has a braking force that changes accordingly. It can also be mentioned that traveling wave (travelling wave) mobility separator. Of course, analysers of the type described above may be coupled to allow the separation of ion species multi-dimensionally, ie according to more than one physicochemical property, such as m / z, Ω / z or σ / z.

[0021] In various embodiments, the control sample may include a preparation of a microorganism, such as a prokaryotic organism, in particular a preparation of a bacterial species. Microbial cells with precisely known taxonomic affiliation and defined soluble molecular content, such as ribosomal proteins and peptides, are commercially available. Such an embodiment may be expected to provide well-predicted and robust analyzer and detector responses, such as mass signal curves or abundance, in the case of standardized preparations. An example is the Bruker company's MALDI Bacterial Test Standard for MALDI Axial Linear Time-of-Flight Mass Spectrometry Systems; this standard includes a typical Escherichia coli DH5α-peptide and protein distribution as well as additional proteins and is also suitable for large-volume calibration.

[0022] Preferably, the multiple control samples are congruent or identical; in particular, congruent or identical control samples are used in multiple measurement processes. In the case of microbial preparations as control samples, measurements can be recorded from biological and / or technical copies of the microorganisms on multiple preparations and over a long period of time.

[0023] If the control sample contains microorganisms, it is preferably sterilized. Sterilization can include exposing the microorganism to a metabolically inhibited liquid, such as an alcohol (such as ethanol or isopropanol), or an acid (such as formic acid), and / or energy effects, such as using heat or high-energy radiation (particularly ultraviolet light). Sterilization refers in particular to making the microorganism lose its ability to proliferate under conditions favorable to it. In this way, the biological risks associated with unintentional / uncontrolled transmission in the analytical laboratory can be avoided. Under certain conditions, such as when working in an analytical laboratory at a biosafety level 2 or higher, it is not necessary to sterilize the microorganisms of the control sample. It can be considered that fully trained professionals are provided there.

[0024] In different embodiments, the ion source can work according to the MALDI principle. The principle of matrix-assisted laser desorption and ionization (MALDI) has been described in detail elsewhere, for example, see the article of Klaus Dreisewerd mentioned in the introduction. In the common variant of MALDI sample preparation, soluble molecules, such as ribosomal proteins of microorganisms or bacteria, are inserted into the structure of a matrix crystal with high absorption of laser light. Small organic molecules that strongly absorb energy at the wavelength of the laser used, such as solid Nd:YAG at a wavelength of triple frequency of 355 nanometers, are selected as matrix substances. Examples thereof are sinapinic acid, 2,5-dihydroxybenzoic acid, α-cyanohydroxybenzoic acid or 2,4,6-trihydroxyacetone. When the matrix crystal structure is exposed to laser radiation, they evaporate explosively and release the embedded molecules. In addition, the molecules are ionized during this energy-enriched removal process. Therefore, these molecules are accessible for subsequent ion spectroscopic analysis studies. For this study, the ions are usually accelerated to different velocities in an electric field depending on their mass and, after a long, largely field-free flight distance, impact in a time-resolved manner on a detector with a secondary electron multiplier. The time interval from laser desorption of the sample material or from the acceleration pulse in the flight path of the mass analyzer to the receipt of the different ion current signals at the detector gives the mass per unit charge m / z of the ion by conversion.

[0025] Example of establishing a MALDI measurement. When a new MALDI measurement method is created, some operating parameters are ideally defined with the aid of a standard substance, which is manufactured in a controlled manner and contains the target molecule (control sample) in the mass range that is sought in a quantity and concentration that is as determined as possible. Such a standard substance, such as the Bacterial Test Standard of the company Bruker, is freshly prepared and measured in the device. Before the measurement of this standard substance, other operating parameters can also be defined separately, such as the leveling voltage (called Back-Bias in English) for creating a basically field-free space in the desorption area of ​​the MALDI sample carrier, the detector voltage or the baseline. An important parameter for the MALDI measurement is the laser energy density. If the laser energy density is too low, no or almost no ions are generated. If the laser energy density is too high, the generated ion cloud is occupied by too many charge carriers, and the mass resolution in the connected analyzer reaches its limit. Therefore, it is preferred to find and define the so-called "desorption threshold". This threshold value of the laser energy density defines the point at which the first ion signal can be detected at the detector after gradually increasing the laser energy density from a low initial value. Starting from this desorption threshold, the laser energy density is then preferably further increased for initial instrument adjustment in order to achieve a compromise between the number of signals and the degradation of signal intensity and mass resolution. Typically, this preferred laser energy density is a few percentage points above the desorption threshold.

[0026] The laser energy density for subsequent measurements of analytical samples is set so that both samples with low analyte concentrations and samples with high analyte concentrations can be measured. That is, during the measurement of analytical samples, the laser energy density changes by a few percentage points, for example, the energy density can be increased or decreased after the first measurement test, so as to obtain a signal that can be best evaluated. If the analyte concentration in the sample is too high, the initial laser power is reduced. If the analyte content of the sample is too low, the laser power is increased. However, in practice, the result of these methods may be that a suboptimal laser or a contaminated ion source remains undetected for a long time, because during the measurement, the laser energy density is allowed to increase continuously, which is a measurement disadvantage for samples with low analyte content, for example. Therefore, according to current practice, the MALDI measurement of analytical samples can only conditionally monitor and track the laser energy density of the initial optimal setting, because the MALDI measurement itself may be very variable and depends on many parameters. The declining laser power or the contaminated ion source are factors that contribute to the increase in laser energy density. The core of the invention presented here is essentially that defined standard substances or reference samples, whose composition is essentially known, are frequently measured with a laser energy density that is fixedly set for performance monitoring measurements in order to find, in particular, the expected signal intensity. Since there can still be large variations in the individual MALDI measurements, these measurements are averaged over a longer period of time in order to suppress the influence of short-term variations or variations that are influenced by "diurnal patterns". If the expected signal intensity can no longer be found, the initial value of the laser energy density used to measure the analytical sample can be recalibrated and used as a basis, to which the previously described variations based on individual measurements for the analytical sample are added.

[0027] In different embodiments, performance trends can be inferred from the abundance or intensity of ions detected in the analyzer; In particular, average abundance or intensity can be considered. As an intensity metric or abundance metric, for example, the total amount of ion currents detected in spectral data can be used; This is sometimes referred to as total ion count (TIC). TIC can be obtained over the entire spectral range or over a defined sub-range, for example, over the following range of spectral data: 30000 or more atomic mass units (amu), 20000 or more amu, 10000 or more amu, 5000 or more amu or 1000amu or more. It can also be based on the average intensity of the entire spectral range or its local, for example, based on the average intensity of the range of 30000 or more atomic mass units (amu), 20000 or more amu, 10000 or more amu, 5000 or more amu or 1000amu or more spectral data. Here, a wide range of data bases ensures statistical stability, i.e., the low weight of outliers in the observation across multiple control samples, control sample sampling or measurement processes.

[0028] In various embodiments, the performance interval may include deviations from the performance standard value up to a predefined percentage, in particular ±30% or less, ±25% or less, ±20% or less, ±15% or less, or ±10% or less. For example, when using an intensity metric to assess performance, the determination of the intensity standard value may be achieved by a basic understanding of the molecular content of the control sample, the intensity standard value being set within an interval whose higher intensity limit is obtained by the requirement to avoid detector saturation and whose lower intensity limit is empirically derived, i.e., how high the ion current signal must be in order to achieve a demonstrable and reliable spectral data analysis. The interval limits may be arranged equidistantly or unequally relative to the performance standard value. In one embodiment, the upper interval limit may be closer to the performance standard value than the lower interval limit; in another embodiment, the lower interval limit may be closer to the performance standard value than the upper interval limit.

[0029] In alternative embodiments, performance trends can also be derived from characterizations of the molecular content of essentially known control samples. For example, if microorganisms are used as control samples, an ion spectrometry system suitable for identifying taxonomic affiliation (e.g., MALDI from Bruker) can be used. ) is used as the performance criterion value. Since the taxonomic affiliation of the microorganisms in the control sample is basically known, MALDI The so-called log(score) should be significantly above 2.00. If the trend leads to a drop below the lower interval limit of 2.00 over a longer observation period and thus independently of outliers, the operating parameters of the laser may need to be adjusted to counteract this performance degradation.

[0030] In various embodiments, the evaluation in step (c) may average a plurality of control sample spectral data, in particular forming an average value or a median value. Forming the average value may in particular be based on an arithmetic mean. By recording the control sample spectral data over a plurality of control samples, control sample samplings or measurement processes, a sliding average may be formed, i.e., a temporally varying subset is introduced over a series of measurements, e.g. by taking into account the control sample spectral data obtained by a moving time window, if necessary across a plurality of measurement processes, for the formation of the average value.

[0031] In various embodiments, the average can be applied over a predetermined number of (i) control samples, e.g., 50-250 control samples, (ii) measurement runs, e.g., 10-30 measurement runs, and (iii) laser activity, e.g., for a single spectral data record of more than 10 5 -10 7 The invention relates to a method for averaging a laser operating parameter, wherein the average is applied to a predetermined number of individual test sample samplings, such as 400-2000 individual test sample samplings, or (iv) the number of control samples or measurement processes within a predetermined time period, such as a time period of 7-21 days, 1-4 weeks or 1-2 months. It is also possible to apply the average to a predetermined number of individual test sample samplings, such as 400-2000 individual test sample samplings. All embodiments of the present disclosure have in common that the need for adjustment of laser operating parameters is not based on a single control sample and not even on a single control sample sampling, but relies on multiple control samples and multiple control sample samplings.

[0032] In various embodiments, in order to adjust the operating parameters, the laser energy density, laser flux, laser power and / or laser intensity may be varied during laser-assisted ionization. In particular, if a performance trend deviation is identified, the laser energy density, laser flux, laser power and / or laser intensity may be increased by a preset percentage. The increase may vary within a range of substantially single-digit percentage values, such as 1-10%, in particular about 1%. In MALDI ionization, the ion yield is roughly proportional to the sixth to eighth power of the laser flux, so that each 1% increase thereof results in an increase of about 6-8% in detectable ions. Such measures may already be sufficient to counteract negative performance trends and adjust the laser to meet interval requirements. Alternatively, the adjustment of the operating parameters of the laser may include a predefined increment, such as a specific percentage; however, it may also be calculated based on trend changes and / or absolute deviations from interval limits, such as based on a fixed increment (possibly a percentage), which is corrected by a term related to the trend to avoid overcorrection in the adjustment and to make the adjustment process less dependent on outliers.

[0033] In different embodiments, a notification and / or flag may be generated once the number of adjustments of an operating parameter reaches or exceeds a predetermined value, such as 2-10 adjustments, in particular 3-5 adjustments. The notification may be in the form of an entry recorded in a log file of the ion spectroscopy system used. It may also be a message for directly informing the user, such as a pop-up window with text or image information in a graphical user interface on the ion spectroscopy system computer, or an automatically generated email or text message with the content of the comprehensive spectral data evaluation results. Such text / image message, email or text message may be sent directly to a receiving address of the ion spectroscopy system manufacturer or service partner to automatically issue a maintenance order. In particular, the flag may include an entry in a note field of metadata of spectral data recorded under varying laser conditions. In this way, the user is enabled to track the automatic laser readjustment process at a later point in time (for example, when evaluating spectral data of analytical samples, which are obtained in addition to spectral data of control samples).

[0034] In different embodiments, the composition of the analytical sample can be virtually unknown. The difference between the control sample and the analytical sample is especially that the control sample, its molecular content and its behavior during laser-assisted ionization are very well known and characterized, so they are used as adjustment variables. In contrast, the information about the analytical sample must be determined by the corresponding spectral data through a post-processing that is fine if necessary. In the case of MALDI ionization, when preparing the analytical sample, only the matrix material is well known, while the molecular content and ionization behavior of the analytical molecules are not verified and are to be determined. The analytical sample can be, for example, a preparation, which is assumed to contain microorganisms and / or molecules extracted therefrom, in order to determine the classification of microorganisms to their types / species and / or resistance behavior to antimicrobial substances. The analytical sample can also be a preparation comprising cells, such as eukaryotic cells, whose behavior and / or the reaction to being exposed to a specific stimulus should be studied, such as being exposed to toxicological and / or pharmacologically active substances.

[0035] In a second aspect, the invention relates to an ion spectroscopy system comprising an ion analyser, an ion source connected to the ion analyser, and a processor unit in communication with the ion analyser and the ion source and configured and programmed to coordinate and execute the method as described and described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Reference is made to the following drawings for a better understanding of the present invention. The elements in the drawings are not necessarily drawn to scale, but are primarily for illustrating the principles of the present invention (mostly schematically). In the drawings, elements corresponding to each other in different viewing angles are marked by the same reference numerals.

[0037] Figure 1 The principle of an axial linear time-of-flight mass spectrometry system with laser-assisted ionization is schematically shown; for example, Bruker's LT / SH.

[0038] Figure 2 A typical workflow for MALDI spot sample preparation is schematically shown.

[0039] Figure 3 The principle of performance monitoring and regulation in the present disclosure is schematically illustrated.

[0040] Figure 4 An ion spectroscopy system for implementing the method described in the present disclosure is shown schematically and exemplarily. DETAILED DESCRIPTION

[0041] While the invention has been shown and described with respect to several embodiments, it will be noted by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the technical principles defined in the appended claims.

[0042] The use of individual control sample measurements for performance monitoring and adjustment of the ion source can lead to misleading results, since the individual control sample itself may be defective, e.g. due to an oversight during preparation. For MALDI preparations, poorly crystallized matrix material can be envisaged, e.g. if the ratio of matrix material to molecule to be embedded (usually ˜10,000-5,000:

[0043] 1) Not in compliance with regulations. Especially in the field of clinical applications, such as Bruker's MALDI It may happen that the analysis system is operated by a trained person who has only the knowledge necessary to run it. For this reason, it is possible that the operator cannot recognize the possible wrong preparation of a single control sample. As already mentioned in the introduction, periodic inspections by trained technicians of the manufacturer or its service partner can provide a remedy, but can only be achieved with high costs and huge labor costs. Unsatisfactory control sample preparation can also be caused by the aging of the control sample substrate itself used or by the poor quality of the reagents used for the control sample preparation, for example if the user ignores or neglects the expiration of the shelf life data.

[0044] The goal is to adjust the ion spectrometry system to the best possible level once by a professional after delivery to the user and to reduce or maintain at least as low as possible the number of maintenance times required by a professional or user during continuous operation. In this way, the work effort and costs can be reduced. This goal can be achieved by (quasi-) automatic readjustment of the operating parameters of the laser used for laser-assisted ionization, in addition to other measures that are not the subject of the present application but are supplementary (for example, ion source cleaning or detector voltage adjustment mentioned at the beginning).

[0045] A method for performance monitoring and adjustment of an ion source comprises the following steps: (a) laser-assisted ionization of a control sample, the material composition of which is known and which is preferably sampled before, simultaneously with or after an analytical sample in the same measurement process, (b) generating control sample spectral data from the ionized control sample in an ion analyzer connected to the ion source, (c) repeating steps (a) and (b) on multiple control samples and, if necessary, multiple measurement processes so as to collect multiple control sample spectral data and evaluate them so that the spectral data of a single control sample has a small weight and performance trends emerge in the evaluation, (d) if the performance trend enters a range outside a predetermined performance interval, adjusting operating parameters of a laser of the ion source so as to adjust the laser to meet the interval requirements, and (e) repeating steps (a) to (d) so as to continuously monitor and adjust the ion source.

[0046] In step (a), the control sample can be sampled in the same measurement process as the analytical sample, and in step (c), steps (a) and (b) can be repeated on multiple measurement processes and control samples. The measurement process in the sense of the present disclosure can be understood in particular with respect to the operating mode of the ion spectrometry system. For example, the measurement process can include all measurements recorded from the (control) sample on the sample carrier, as long as the sample carrier is spatially located in the ion source. An example is to introduce the sample carrier into the low-pressure region of the ion source, as long as it operates under low pressure, especially in vacuum MALDI. If the sample carrier is removed from the ion source, for example, the low-pressure region, and then another sample carrier with a new (control) sample is introduced, all measurements of the new (control) sample on the subsequent sample carrier can be regarded as a separate measurement process.

[0047] In addition to the preparation of analytical samples, multiple control sample preparations can also be arranged on the sample carrier for laser-assisted ionization. If there are both control samples and analytical samples on the sample carrier, for example 2-10 technical control sample copies, in particular 4-8 technical control sample copies, then all control sample spectral data of these preparations can be used to determine performance trends and / or their temporal trends. In order to generate control sample spectral data, a control sample preparation on the sample carrier can be sampled once or multiple times, in principle only limited by the coverage of the control sample. As a result, the control sample spectral data can include separate and separate spectra. Repeated sampling of the same control sample preparation improves the basis of the control sample spectral data for subsequent statistical evaluation and reduces the influence of interferences, especially very short-term interferences, on the measurement process, which may cause abnormal value measurements.

[0048] Sampling the control sample before, in parallel with or after the analytical sample can in particular mean that the control sample and the analytical sample are investigated in the same measurement process. The sampling of the control sample can in particular be carried out directly before or directly after the analytical sample. The measurement mode or the measurement settings of the ion spectrometry system can be consistent or different for the sampling of analytical samples and control samples. As described above, in the case where the measurement process includes recording the spectral data of all (control) samples on the sample carrier, this can mean that the sample carrier has a plurality of marked sample positions, for example as sample spot areas on the known AnchorChip or MBT BioTarget 96 carrier from Bruker or on a steel plate, and some of these sample positions are on the one hand prepared with a predetermined number of control samples, while some or all of the remaining sample positions are occupied by analytical samples. The control samples and the analytical samples can then be sampled in a manner separate from each other in a block (e.g., first all control samples and then all analytical samples), or they can also be sampled alternately (e.g., control samples => analytical samples => control samples => analytical samples and so on). In an ion source with multiple laser beams, if the connected ion analyzer is configured and arranged to handle a multiplexed ion flow, or if there is more than one ion analyzer, the analytical samples and the control samples can also be measured in parallel by the same sample carrier if necessary.

[0049] The ion analyzer can be configured as a mass analyzer, which in particular sorts the ionized control sample according to the time-of-flight (TOF) dispersion principle. A mass analyzer separates charged molecules or molecular ions according to their mass-to-charge ratio (usually expressed as m / z).

[0050] Figure 1The principle of an axial linear time-of-flight mass spectrometry system is schematically shown. Upper figure: The laser bombards a (control) sample on a sample carrier 2, which is prepared, for example, with a MALDI matrix material. A set of electrodes 4 accelerates the ions produced by the laser bombardment along a straight trajectory 8 into the flight tube 6 of the time-of-flight analyzer, if necessary after a delay time. Since the different molecular species produced during the laser bombardment have different masses and carry a largely uniform charge, these molecular species are provided with approximately the same kinetic energy during acceleration, so different speeds are obtained. Therefore, light molecules represented as small black circles fly faster than heavy molecules represented as circles with larger circumferences, and hit the detector 10, which is constructed, for example, as a dynode row or a multichannel plate (MCP), earlier. This time correlation of arrival at the detector enables the charge-related mass m / z to correspond to the detection event; the diagram below.

[0051] In addition to the time-of-flight analyzer (for which not only can the Figure 1 In principle, other types of mass dispersion separators can also be used, such as quadrupole mass filters (single quadrupoles), triple quadrupole analyzers ("triple quadrupoles"), ion cyclotron resonance cells (ion cyclotronresonance, ICR), such as Kingdon-type analysers from Thermo Fisher Scientific, etc. It is likewise conceivable to configure the ion analyser as a mobility analyser or a combined mobility-mass analyser, as already mentioned above.

[0052] Control samples can include preparations of microorganisms, such as prokaryotes, especially preparations of bacterial species. Microbial cells with precisely known taxonomic affiliations and defined soluble molecular contents, such as ribosomal proteins and peptides, are commercially available. Such embodiments can be expected to have well-predicted and robust analyzer and detector responses, for example, in terms of mass signal curves or abundance, in the case of standardized preparations. An example is Bruker's MALDI Bacterial Test Standard for MALDI Axial Linear Time-of-Flight Mass Spectrometry Systems; this standard includes a typical Escherichia coli DH5α-peptide and protein distribution as well as additional proteins and is also suitable for large-volume calibration.

[0053] Preferably, the plurality of control samples are congruent or identical; in particular, congruent or identical control samples are used during a plurality of measurements. In the case of microbial preparations as control samples, the measurements can record biological and / or technical copies of the microorganisms over a long period of time.

[0054] If the control sample contains microorganisms, it is preferably sterilized. Sterilization can include exposing the microorganism to a metabolically inhibited liquid, such as an alcohol (such as ethanol or isopropanol), or an acid (such as formic acid), and / or energy effects, such as by heat or high-energy radiation (particularly ultraviolet light). Sterilization refers in particular to making the microorganism itself lose the ability to proliferate under conditions favorable to it. In this way, the biological risks associated with unintentional / uncontrolled propagation in the analytical laboratory can be avoided. Under certain conditions, such as when working in an analytical laboratory at biosafety level 2 or higher, it is not necessary to sterilize the microorganisms of the control sample. It can be considered that fully trained professionals are provided there.

[0055] As mentioned before, the ion source may operate according to the MALDI principle. Figure 2 AC schematically shows the MALDI process from preparation of (control) samples to recording of spectral data. Figure 2 In A, a (control) sample, such as a microbial suspension 12 or a suspension containing soluble molecules extracted from microorganisms, is applied to a MALDI sample carrier 2 using a pipette 14 or other suitable dispensing device. * On. For example, Bruker's AnchorChip or MBT BioTarget 96 carrier or steel plate can be used as MALDI sample carrier. After removing the excess fluid of the suspension, the matrix substance solution can be applied to the cell layer and / or soluble molecule layer from which the fluid has been removed, for example with the aid of a suitable tool 16, such as another pipette, which is illustrated here with checkered hatching. After embedding, drying and crystallizing the matrix, a single (control) sample 18 is prepared for sampling by a laser beam 20. The ions 22 generated here can then be accelerated, for example, into the flight path of a time-of-flight analyzer, as schematically shown in FIG. Figure 1 As shown in .

[0056] Performance trend can be inferred from the abundance or intensity of ions detected in the analyzer; In particular, average abundance or intensity can be considered. As intensity measurement or abundance measurement, for example, the total amount of ion current detected in spectral data can be used; This is sometimes referred to as total ion count (TIC). TIC can be obtained over the entire spectral range or also over a defined sub-range, for example, the range of spectral data is 30000 or more atomic mass units (amu), 20000 or more amu, 10000 or more amu, 5000 or more amu or 1000 or more amu. It can also be based on the average intensity of the entire spectral range or its local, for example, based on the average intensity of the spectral data range of 30000 or more atomic mass units (amu), 20000 or more amu, 10000 or more amu, 5000 or more amu or 1000 or more amu. Here, a wide range of data bases ensures statistical stability, i.e., the influence of outliers across multiple control samples, control sample sampling or measurement processes is small.

[0057] Figure 3 The feedback of monitoring performance trends over a longer period of time, the identification of performance trend deviations and the resulting changes in operating parameters of the laser for laser-assisted ionization are schematically shown. The chart below shows a time series of (average) ion currents at the detector on the vertical axis for multiple measurement processes on the horizontal axis. The measurement process can include an examination of ion spectrometry of all (control) samples placed on the sample carrier, for example as long as the sample carrier is arranged in the low-pressure area of ​​the ion source. In the embodiment shown, there are correspondingly 9 sample carriers loaded with control samples introduced into the analysis. Here, the number of control samples or control sample samplings for each measurement process evaluated for performance trends is schematically and exemplarily given as three, and is represented in the chart by three bars for each measurement process. Of course, the number of control samples or control sample samplings for each measurement process can also be different from it. For example, 2-10 or 3-5 control samples can be sampled in each measurement process or multiples thereof, such as the number of corresponding technical control sample copies on the sample carrier. In the case where the sample carrier has marked spots for sample handling, such as in the AnchorChip or MBT BioTarget 96, the number of control samples is weighed against the number of analytical samples of unknown molecular content that the user wants to characterize by ion spectrometry. In the case where space on the sample carrier is limited, one may instead consider applying and preparing a large number (ergiebig) of individual control samples with increased quality requirements during the preparation process, and then applying multiple sampling for a single measurement process in order to obtain a comprehensive spectral data base.

[0058] The middle horizontal dotted line 24 in the chart below represents the ion current standard value, which can represent the best setting found by detailed testing and calibration. In a typical MALDI time-of-flight mass spectrum, the ion current can correspond to an average intensity of 25000-30000 counts, particularly 27000 counts. The outer dotted line 26 around the center line 24 represents the interval limit around the ion current standard value, within which the performance of the system consisting of the ion source and the analyzer can be considered to be in line with the requirements or satisfactory. The performance interval can include deviations up to a predetermined percentage, particularly ± 30% or less, ± 25% or less, ± 20% or less, ± 15% or less, or ± 10% or less. The limit to higher ion current can be obtained by avoiding the requirement of detector saturation, and the limit to lower ion current can be derived from experience, i.e., how high the ion current signal must reach, so as to achieve provable and reliable spectral data analysis. The interval limits can be arranged equidistantly or unequally relative to the performance standard value. In one embodiment, the upper spacing limit may be closer to the performance standard value than the lower spacing limit; in another embodiment, the lower spacing limit may be closer to the performance standard value than the upper spacing limit.

[0059] Performance trends can also be derived from characterization of the molecular content of essentially known control samples. For example, if microorganisms are used as control samples, an ion spectrometry system suitable for identifying taxonomic affiliation (such as Bruker's MALDI ) is used as the performance criterion value. Since the taxonomic affiliation of the microorganisms in the control sample is basically known, MALDI The log(score) should be significantly above 2.00. If the trend results in reaching and / or falling below the lower interval limit of 2.00 over a longer observation period and thus independently of outliers, the operating parameters of the laser may need to be adjusted to counteract this performance degradation.

[0060] The evaluation in step (c) may be performed by averaging the spectral data of a plurality of control samples, in particular forming an average value or a median value. Forming the average value may in particular be based on an arithmetic mean. Figure 3 In the figure, the ion current values ​​averaged over three control samples or control sample samples in each measurement process are shown by dashed circles 28. By recording the control sample spectral data over a plurality of control samples, control sample samples or measurement processes, a moving average can be formed, i.e. a temporally varying subset is used for the formation of the average value over a series of measurements, e.g. by taking into account the control sample spectral data obtained by a moving time window, if necessary across a plurality of measurement processes.

[0061] The averaging may be applied over a predetermined number of (i) control samples, e.g., 50-250 control samples, (ii) measurement runs, e.g., 10-30 measurement runs, and (iii) laser activations, e.g., for more than 10 individual spectral data records. 5 -10 7 The invention relates to a method for averaging a laser operating parameter, wherein the average is applied to a predetermined number of individual test sample samplings, such as 400-2000 individual test sample samplings, or (iv) the number of control samples or measurement processes within a predetermined time period, such as a time period of 7-21 days, 1-4 weeks or 1-2 months. It is also possible to apply the average to a predetermined number of individual test sample samplings, such as 400-2000 individual test sample samplings. All embodiments of the present disclosure have in common that the need for adjustment of laser operating parameters is not based on a single control sample, and not even based on a single control sample sampling, but relies on multiple control samples and multiple control sample samplings.

[0062] exist Figure 3 , a trend line 30 connecting different ion current average values ​​is shown, which allows an estimation of how the ion current values ​​of the observed control sample may develop in the further course. In the specific example shown, it was determined in the third measurement process that the ion current average value had reached the lower interval limit 26; for the fourth measurement process it was even found that the ion current average value had left the interval region. This finding led, as a countermeasure, to a change in the operating parameters of the laser in order to counteract this performance degradation, see arrow 32.

[0063] To adjust the operating parameters, the laser energy density, laser flux, laser power and / or laser intensity can be varied during laser-assisted ionization, for example according to Figure 3 As can be seen from the illustration of the vertical axis of the upper diagram of . In particular, if a performance trend deviation is identified, the laser energy density, laser flux, laser power and / or laser intensity can be increased by a preset percentage, see increment #1. The increase can vary within a range of substantially single-digit percentage values, such as 1-10%, in particular about 1%. Alternatively, the adjustment of the operating parameter of the laser can include a predefined increment, such as a specific percentage; however, it can also be calculated based on trend changes and / or absolute deviations from interval limits, for example based on fixed increments (possibly percentages), which are corrected by trend-related terms to avoid overcorrection in the adjustment and to make the adjustment process less dependent on outliers.

[0064] exist Figure 3After the laser parameter adjustment between the fourth and fifth measurement processes shown in the upper diagram of , the average ion current is adjusted again to the region within the interval without the need for manual intervention. The ion spectrometry system having an ion analyzer and an ion source monitored and adjusted in this way can then continue to operate and continue to provide measurement performance that meets the requirements or is satisfactory. If it is found during other measurement processes that the performance trend approaches the interval limit again, as is exemplarily shown here in the seventh measurement process, and then falls outside the interval region, as is shown in the eighth measurement process, the operating parameters of the laser can be adjusted again, see adjustment #2, similar to what was explained above. Adjustment #2 can be the same as adjustment #1 here, or can also be implemented with other adjustment increments.

[0065] Once the number of adjustments of the operating parameters in time reaches or exceeds a predetermined value, such as 2-10 adjustments, in particular 3-5 adjustments, a notification and / or a mark can be generated. The notification can be in the form of an entry recorded in a log file of the ion spectroscopy system used. It can also be a message for directly informing the user, such as a pop-up window with text or image information in a graphical user interface on a computer of the ion spectroscopy system, or an automatically generated email or SMS with content summarizing the results of the spectral data evaluation. This text / image message, email or SMS can be sent directly to the receiving address of the manufacturer or service partner of the ion spectroscopy system by telecommunication to automatically issue a maintenance order. In particular, the mark can include an entry in the annotation field of the metadata of the spectral data recorded under changed laser conditions, in this way, enabling the user to track the automatic laser readjustment process at a later point in time (for example, when evaluating the spectral data of analytical samples, which are obtained in addition to the spectral data of control samples).

[0066] The composition of the analytical sample can be virtually unknown. The difference between a control sample and an analytical sample is, in particular, that the control sample, its molecular content and its behavior during laser-assisted ionization are very well known and characterized, so that it is used as a regulating parameter, whereas information about the analytical sample must be determined by a fine post-processing, if necessary. In the case of MALDI ionization, when preparing the analytical sample, only the matrix material is well known, while the molecular content and ionization behavior of the analytical molecules need to be determined. The analytical sample can be, for example, a preparation, which is assumed to contain microorganisms and / or molecules extracted therefrom, in order to determine the classification of the microorganisms to their kind / species and / or the resistance behavior to antimicrobial substances. The analytical sample can also be a preparation comprising cells, such as eukaryotic cells, whose behavior and / or the reaction to exposure to a specific stimulus should be studied, such as exposure to toxicological and / or pharmacologically active substances.

[0067] Figure 4 An ion spectroscopy system 40 is schematically shown, which has an ion analyzer 42, an ion source 44 connected to the ion analyzer 42, and a processing unit 46 in communication with the ion analyzer 42 and the ion source 44, and the processing unit is designed and programmed to perform the method as explained and described above, in particular for Figure 3 The path of ions from the ion source 44 to the ion analyzer 42 is indicated by arrow 48. The control and information receiving communication of the processing unit 46 with the ion source 44 and the ion analyzer 42 is indicated by double arrow 50.

[0068] The present invention has been described above with reference to various specific embodiments. However, it should be understood that various aspects or details of the described embodiments may be changed without departing from the scope of the present invention. In addition, the features and measures disclosed in the context of different embodiments may be combined in any manner as long as this is feasible for those skilled in the art. In addition, the foregoing description is provided only to explain the present invention, rather than to limit the scope of protection, which is defined only by the appended claims and their possible equivalents.

Claims

1. A method for monitoring and adjusting the performance of an ion source, comprising: (a) laser-assisted ionization of a control sample, the composition of which is substantially known and which is sampled before, simultaneously with or after the analytical sample, (b) generating control sample spectral data from the ionized control sample in an ion analyzer connected to an ion source, (c) repeating steps (a) and (b) on a plurality of control samples so as to collect a plurality of control sample spectral data and evaluate such that the spectral data of a single control sample has a small weight and a performance trend emerges in the evaluation, (d) if the performance trend enters a range outside of a predetermined performance interval, adjusting operating parameters of a laser of the ion source so as to adjust the laser to meet the interval requirements, and (e) Repeat steps (a) through (d) to continuously monitor and adjust the ion source.

2. The method of claim 1 , wherein in step (a), the control sample is sampled in the same measurement process as the analytical sample, and in step (c), steps (a) and (b) are repeated on multiple measurement processes and control samples.

3. The method according to claim 1 or 2, wherein the ion analyzer is a mass analyzer, which classifies the ionized control sample in particular according to the principle of time-of-flight dispersion.

4. The method according to any one of claims 1 to 3, wherein the control sample comprises a preparation of a microorganism, in particular a preparation of a bacterial species.

5. The method according to any one of claims 1 to 4, wherein the ion source operates according to the MALDI principle.

6. The method according to any one of claims 1 to 5, wherein: The performance trends are inferred from the abundance or intensity of ions detected in the analyser.

7. The method according to any one of claims 1 to 6, wherein: The performance interval includes deviations from the performance standard value up to a predefined percentage.

8. The method according to any one of claims 1 to 7, wherein: The evaluation in step (c) averages the plurality of control sample spectral data, in particular forms an average value or a median value.

9. The method of claim 8, wherein a predetermined number of (i) control samples, (ii) measurement processes, (iii) laser activities, or (iv) the number of control samples or the number of measurement processes from a predetermined time period are averaged.

10. The method according to any one of claims 1 to 9, wherein: In order to adjust the operating parameters, the laser energy density, the laser flux, the laser power and / or the laser intensity are varied during laser-assisted ionization.

11. The method according to any one of claims 1 to 10, wherein: Whenever the number of adjustments to the operating parameter reaches or exceeds a predetermined value, a notification and / or flag is generated.

12. The method according to any one of claims 1 to 11, wherein the composition of the analytical sample is essentially unknown.

13. An ion spectroscopy system comprising an ion analyser, an ion source connected to the ion analyser, and a processing unit in communication with the ion analyser and the ion source, the processing unit being configured and programmed to coordinate and perform the method according to any one of claims 1 to 12.

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