Method and device for monitoring and controlling the performance of an ion source

Abstract

The invention relates to a method and a device for monitoring and controlling the performance of an ion source, said method comprising: (a) using a laser to ionise a control sample, the composition of which is substantially known and which is sampled before, in parallel with, or after an analytical sample; (b) generating items of control sample spectral data from the ionised control sample in an ion analyser connected to the ion source; (c) repeating steps (a) and (b) over a plurality of control samples in order to collect a plurality of items of control sample spectral data and analyse them such that items of spectral data of individual control samples have a low weight and a performance trend appears in the analysis; (d) if the performance trend enters a range outside a predefined performance interval, adjusting an operating parameter of the laser of the ion source in order to adjust the laser to conform to the interval; and (e) repeating steps (a) to (d) in order to continuously monitor and control the ion source.

Description

Method and device for performance monitoring and control of an ion source Field of the invention

[0001] The invention relates to the performance monitoring and control of an ion source operating with laser-assisted ionization, in particular with MALDI ionization. More specifically, the invention relates to an ion spectrometry system comprising an ion analyzer, an ion source connected to the ion analyzer, and a processor unit communicating with the ion analyzer and the ion source. Background of the invention

[0002] The prior art is explained below with reference to a specific aspect. However, this should not be understood as a limitation. Useful developments and modifications of what is known from the prior art may also be applicable beyond the comparatively narrow scope of this introduction and will be readily apparent to experienced practitioners in this field after reading the disclosure of the invention following this introduction.

[0003] In MALDI time-of-flight mass analysis (MALDI, matrix-assisted laser desorption and ionization), there are several operating parameters that are important for a usable measurement. These operating parameters include, among others, the spectral baseline, various electrical voltages at accelerating electrodes along the flight path, the detector voltage, and the laser energy density of the ion source, to name just a few. Typically, these parameters are checked and adjusted for optimal results either in the mass spectrometer manufacturer's test facility or in the user's laboratory by qualified service personnel from the manufacturer or a contracted partner. However, these measures are isolated events, so the mass spectrometer must operate unmonitored for an extended period after setup or readjustment.

[0004] However, it is known and based on experience that some parameters change over the operating and service life of the mass spectrometer. This can also be referred to as aging or wear. Of the aforementioned parameters, the detector voltage is particularly directly affected, and the laser energy density is indirectly affected.

[0005] Detector aging is particularly evident in the decrease in the gain factor of, for example, a secondary electron multiplier with progressive operation. This decrease is typically counteracted by increasing the detector voltage, i.e., the voltage gradient over which the secondary electrons are accelerated. In practice, this need for adjustment of the detector voltage builds up over a relatively long period of time and can be determined largely automatically, e.g., during calibration phases in which single-ion signals are observed, as described in patent publication DE 10 2008 010 118 A1 (corresponding to GB 2 457 559 A and US 2009 / 0206247 A1).

[0006] In the case of laser energy density, it can happen that the ion source, and in particular its surfaces, e.g., electrodes or confining housing parts, become contaminated with progressive operation, thus altering the conditions under which the electric fields in the ion source are generated. This then manifests itself in a reduced ion yield. Such a performance drop can be counteracted, for example, by semi- or fully automated cleaning, which seeks to eliminate or at least mitigate the disturbance through physical and chemical action. An example of such a cleaning process is presented in patent publication DE 10 2008 008 634 A1 (corresponding to GB 2 457 362 A and US 2009 / 0200457 A1).Another possibility is to increase the laser energy density or fluence, since the ion yield scales with a multiple of the energy density or fluence, as explained in the review article by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425). Depending on the analyzer's use—think of nearly uninterrupted measurement phases daily from morning to night—the need for remedial action due to contamination can build up quite rapidly compared to detector aging.

[0007] Once a mass spectrometer is installed in a user's laboratory, intensive operation, for example, during high-throughput, repeated identification measurements of disease-causing microorganisms in clinical settings, can lead to such degradations in performance that high-frequency maintenance, e.g., weekly or even more frequent, becomes necessary; a significant burden for service personnel and, at the same time, a cost driver for users.

[0008] In the following, some prior art documents that may be relevant to the disclosure are briefly reviewed, without claiming to be exhaustive:

[0009] The technical teaching disclosed in patent publication DE 10 2010 019 857 A1 (corresponding to GB 2 483 322 A and US 2011 / 0272573 A1) is based on recording several series of mass spectra of a mixture of analyte substances with a gradual increase in the energy density in the laser foci, but overcoming the possible resulting problem of signal saturation at the detector by special measures.

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

[0011] The patent publication WO 2017 / 160857 Al relates to systems and methods for real-time monitoring of laboratory analytical instruments for the purpose of quality control.

[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 rotating a polarization beam splitter. The laser light is then directed onto a sample with the adjusted energy.

[0013] Patent publication EP 3 651 184 A1 relates to a mass spectrometer and methods and programs for adjusting the laser light intensity used in the mass spectrometer for MALDI ionization.

[0014] In view of the foregoing explanations, there is a need to provide methods and devices with which the performance monitoring and control of an ion source can be ensured and improved over a longer period of time. Further problems to be solved by the invention will become readily apparent to those skilled in the art upon reading the following disclosure. Summary of the invention

[0015] In a first aspect, the present disclosure relates to a method for monitoring and controlling 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, in parallel 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) over a plurality of control samples in order to collect a plurality of control sample spectral data and to evaluate them in such a way that spectral data from individual control samples have little weight and a performance trend appears in the evaluation, (d) adjusting an operating parameter of the laser of the ion source if the performance trend enters a range outside a predefined performance interval in order to regulate the laser in accordance with the interval, and (e) repeating steps (a) to (d) for the purpose of continuously monitoring and controlling the ion source.

[0016] In various 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 over a plurality of measurement processes and control samples. A measurement process within the meaning of the present disclosure can be understood in particular with regard to the mode of operation of the ion spectrometry system. For example, a measurement process can comprise all measurements taken from (control) samples on a sample carrier, as long as it is spatially located in the ion source. An example would be the introduction of the sample carrier into a negative pressure region of the ion source, provided that it operates in negative pressure, such as in vacuum MALDI. If the sample carrier is removed from the ion source, e.g.the negative pressure area, and then another sample carrier with new (control) samples is reinserted, all measurements from the new (control) samples on this next sample carrier can be regarded as a separate measurement process.

[0017] In addition to analytical sample preparations, several control sample preparations can also be arranged on a sample carrier for laser-assisted ionization. If there are both control samples and analytical samples on the sample carrier, e.g., 2-10 technical control sample replicates, in particular 4-8 technical control sample replicates, all control sample spectral data from these preparations can be used to determine the performance trend and / or its temporal course. A control sample preparation on the sample carrier can be sampled once or several times to generate control sample spectral data, limited in principle only by the yield of the control sample. As a result, the control sample spectral data can include isolated and separate spectra. Repeated sampling of the same control sample preparation increases the basis of the control sample spectral data for a subsequent statistical evaluation and reduces the influence of particularly short-term disturbances in the measurement process, which can cause outlier measurements.

[0018] Sampling the control sample before, in parallel with, or after an analytical sample can, in particular, mean that the control sample and the analytical sample are analyzed in the same measurement process. Sampling the control sample can, in particular, take place immediately before or immediately after an analytical sample. The measurement modes or measurement settings of the ion spectrometry system can be the same or different for sampling analytical samples and control samples. If a measurement process includes recording spectral data of all (control) samples on a sample carrier, as previously explained, this can mean that the sample carrier has a plurality of designated sample locations, e.g.an array of sample points such as on the well-known Anchor Chip or MBT Biotarget 96 carriers from Bruker or also on a steel plate, and some of these sample locations are prepared with a predetermined number of control samples, whereas some or all of the remaining sample locations are occupied with analytical samples. Control samples and analytical samples can then be sampled separately in blocks (e.g., first all control samples, then all analytical samples, or vice versa) or alternately (e.g., control sample => analytical sample => control sample => analytical sample, etc.). In ion sources with multiple laser beams, analytical samples and control samples can also be measured in parallel from the same sample carrier if the connected ion analyzer is designed and equipped to process multiplexed ion currents or if more than one ion analyzer is present.

[0019] In various embodiments, the ion analyzer can be designed as a mass analyzer that sorts the ionized control sample, in particular according to the principle of time-of-flight (TOF) dispersion. A mass analyzer separates charged molecules or molecular ions according to their mass-to-charge ratio, usually referred to as m / z. In addition to the time-of-flight analyzers already mentioned in the introduction, for which both linear and reflector setups and / or those with axial or orthogonal acceleration of ions into the flight path can be provided, other types of mass-dispersing separators can also be used, e.g., quadrupole mass filters (single quads), triple quadrupole analyzers (triple quads), ion cyclotron resonance (ICR) cells, Kingdon-type analyzers such as the Orbitrap® (Thermo Fisher Scientific), and others.

[0020] In various embodiments, the ion analyzer can also be a mobility analyzer or combined mobility-mass analyzer. A mobility analyzer separates charged molecules or molecular ions according to their collision cross-section-to-charge ratio, sometimes referred to as Q / z or o / z. This is based on the interaction of the ion species with an electric field that couples to the charge of the ions, with the simultaneous exposure of a buffer gas that acts on the average cross-sectional area of ​​the ion. Drift tube mobility separators with a static electric field gradient are particularly known. These drive ions through an essentially static gas, with the drift velocity of an ion species resulting from the propulsive force of the electric field and the decelerating force of the collisions with the gas particles.Also common are trapped ion mobility separators (TIMS), which use a steady laminar gas flow that propels the ions, counteracted by a gradually changing electric field gradient with a correspondingly variable stopping force. Traveling wave mobility separators are also worth mentioning. It is understood that analyzers of the aforementioned types can be coupled to separate ion species multidimensionally, i.e., according to more than one physicochemical property such as m / z, δ / z, or δ / z.

[0021] In various embodiments, the control sample can comprise a preparation of a microorganism, e.g., a prokaryotic organism, in particular a preparation of a bacterial species. Microorganism cells with a precisely known taxon and a defined content of soluble molecules, e.g., ribosomal proteins and peptides, can be purchased commercially. With such a configuration, a standardized preparation can be expected to yield a well-predictable and consistent analyzer and detector response, e.g., with respect to mass signal profile or abundance. One example is the Bacterial Test Standard for the MALDI Biotyper® from Bruker, a MALDI axial-linear time-of-flight mass spectrometry system; the standard comprises a typical Escherichia coli DH5 alpha peptide and protein profile, as well as additional proteins, suitable, among other things, for mass calibration.

[0022] Preferably, the plurality of control samples are congruent or identical; in particular, control samples that are congruent or identical are used across the plurality of measurement processes. In the case of a microorganism preparation as the control sample, the measurements can be recorded over many preparations and over a long period of time from biological and / or technical replicates of the microorganism.

[0023] If the control sample contains a microorganism, it is preferably sterilized. Sterilization can involve exposing the microorganism to a metabolism-inhibiting liquid, e.g. an alcohol such as ethanol or isopropanol or an acid such as formic acid, and / or energy exposure, e.g. using heat or high-energy radiation (particularly ultraviolet light). Sterilization is understood in particular to mean that the microorganism loses the ability to reproduce even under favorable conditions. In this way, the biological hazards associated with unintentional / uncontrolled spread in an analytical laboratory can be avoided. Under certain conditions, it may not be necessary to sterilize the microorganism in a control sample, for example when working in an analytical laboratory with biological safety level 2 or higher.It can be assumed that adequately trained specialist personnel will be employed there.

[0024] In various embodiments, the ion source can operate according to the MALDI principle. The principle of matrix-assisted laser desorption and ionization (MALDI) has been described in detail elsewhere; see, for example, the article by Klaus Dreisewerd mentioned in the introduction. In a common variant of MALDI sample preparation, soluble molecules, e.g., ribosomal proteins of a microorganism or bacterium, are inserted into a structure of matrix crystals that exhibit a high absorption capacity for laser light. Small organic molecules that strongly absorb energy at the laser wavelength used, e.g., solid-state Nd:YAG at a frequency-tripled wavelength of 355 nanometers, are selected as matrix substances. Examples include sinapic acid, 2,5-dihydroxybenzoic acid, α-cyanohydroxycinnamic acid, or 2,4,6-trihydroxyacetophenone.When the matrix crystal structure is exposed to pulses of laser radiation, it evaporates explosively, releasing the embedded molecules. During this high-energy ablation process, molecules are also ionized. These are then accessible for subsequent ion spectrometric analysis. For this analysis, the ions are typically accelerated in electric fields to different velocities depending on their mass. After traversing a long, largely field-free flight path, they impinge on a detector with a secondary electron multiplier in a time-resolved manner. The time spans from the laser desorption of the sample material or from an acceleration pulse into the flight path of the mass analyzer to the reception of the various ion current signals at the detector are converted to the charge-related masses m / z of the ions.

[0025] Example of setting up a MALDI measurement. When creating a new MALDI measurement method, some operating parameters are ideally defined using a standard that has been produced under controlled conditions and contains the most defined number and concentration of target molecules in the desired mass range (control sample). This standard, e.g., Bacterial Test Standard from Bruker, is freshly prepared and measured in the instrument. Before measuring this standard, other operating parameters, e.g., a leveling voltage for setting a largely field-free space in the desorption region of a MALDI sample carrier (called back bias), the detector voltage, or baseline, can also be defined separately. An important parameter in a MALDI measurement is the laser energy density. If the laser energy density is too low, hardly any or no ions are generated.If the laser energy density is too high, the generated ion cloud will be populated with too many charge carriers, and the mass resolution in the connected analyzer will reach its limits. Therefore, it is preferable to find and define a so-called "desorption threshold." This laser energy density threshold defines the point at which, after gradually increasing the laser energy density from a low initial value, the first ion signals can be detected at the detector. Starting from this desorption threshold, the laser energy density is then preferably further increased for the initial instrument setup to achieve a compromise between the number and intensity of signals and the deterioration in mass resolution. Typically, such a preferred laser energy density lies 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 and high analyte concentrations can be measured. This means that the laser energy density is varied by a few percentage points during the measurement of analytical samples. For example, after the first measurement attempt, the energy density can be increased or decreased in order to obtain signals that can be optimally analyzed. 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. One consequence of this procedure in practice, however, can be that a suboptimally adjusted laser or a contaminated ion source remains undetected for a long time because the laser energy density is allowed to be constantly increased during the measurement. This represents a measurement disadvantage, for example, for samples with a low analyte content.Therefore, according to current practice, a MALDI measurement of analytical samples is only partially capable of monitoring and adjusting the initially optimally set laser energy density, as the MALDI measurement itself can be highly variable and dependent on many parameters. A declining laser power or a dirty laser can be a potential problem. Ion sources are factors that can cause an increase in laser energy density. The invention presented here is essentially based on the frequent measurement of a defined standard or control sample, whose composition is substantially known, with a laser energy density set for performance monitoring measurements, with the goal of finding, in particular, an expected signal intensity. Since a single MALDI measurement can nevertheless be highly variable, these measurements are averaged over a longer period of time to suppress the influence of short-term or "day-to-day"-dependent variances. If the expected signal intensity is no longer found, a laser energy density baseline value for the measurements of analytical samples can be readjusted and serves as a baseline upon which the previously explained single-measurement-based variation for analytical samples is based.

[0027] In various embodiments, the performance trend can be derived from an abundance or intensity of ions detected in the analyzer; in particular, an average abundance or intensity can be used. For example, the total amount of detected ion currents in the spectral data can be used as an intensity measure or abundance measure; this is sometimes called total ion count (TIC). The TIC can be recorded over the entire spectral range or over a limited subrange, for example, a range from the spectral data of 30,000 or more atomic mass units (amu), 20,000 or more amu, 10,000 or more amu, 5,000 or more amu, or 1,000 amu or more. It is also possible to calculate an average intensity over the entire spectral range or a section of it, for example a range from the spectral data of 30,000 or more atomic mass units (amu), 20,000 or more amu, 10.000 or more amu, 5,000 or more amu, or 1,000 amu or more should be used as a basis. A broad database ensures statistical stability, i.e., low weighting of outliers when considering multiple control samples, control sample sampling, or measurement processes.

[0028] In various embodiments, the performance interval may include deviations of up to a predefined percentage, in particular ± 30 percent or less, ± 25 percent or less, ± 20 percent or less, ± 15 percent or less, or ± 10 percent or less, from a performance benchmark. If an intensity measure is used to evaluate the performance, it is possible - enabled by the substantial knowledge of the molecular content of the control sample - to determine, for example, an intensity benchmark that is set in an interval whose boundary to higher intensities is determined by the This results in a detector saturation avoidance, and its limit at lower intensities is derived from experience as to how high an ion current signal must be to allow detectable and reliable spectral data evaluation. The interval boundaries can be arranged equidistantly or non-equidistantly from the performance benchmark. In one embodiment, an upper interval boundary can be closer to the performance benchmark than a lower interval boundary; in another embodiment, a lower interval boundary can be closer to the performance benchmark than an upper interval boundary.

[0029] In alternative embodiments, the performance trend can also be derived from the quality of the characterization of the molecular content of the substantially known control sample. For example, if a microorganism is used as a control sample, the highest similarity measure (also called log(score)) of the relevant reference spectral data from an ion spectrometry system designed to identify the taxon, such as the Bruker MALDI Biotyper®, can serve as a performance benchmark. Since the taxon of the microorganism in the control sample is substantially known, the so-called log(score) of the MALDI Biotyper® should be significantly above 2.00. If the trend leads to the lower interval limit of 2.00 being undercut over a longer observation period, and thus independent of outliers, it may be necessary to adjust an operating parameter of the laser to counteract this performance drop.

[0030] In various embodiments, the evaluation in step (c) can subject the plurality of control sample spectral data to averaging, in particular by calculating the mean or median. The averaging can be based, in particular, on the arithmetic mean. By recording control sample spectral data across a plurality of control samples, control sample samplings, or measurement processes, a moving average can be calculated, i.e., a time-varying subset of a set of measurements can be used for averaging, e.g., by considering control sample spectral data from a shifting time window, possibly across a plurality of measurement processes.

[0031] In various embodiments, the averaging may be based on a predetermined number of (i) control samples, e.g. 50-250 control samples, (ii) measurement processes, e.g. 10-30 measurement processes, (iii) laser activations, e.g. over 10 5 -10 7Laser shots for single spectral data acquisition, or (iv) a number of control samples or measurements from a predetermined period, e.g., over a period of 7-21 days, 1-4 weeks, or 1-2 months. It is also possible to apply the averaging to a predetermined number of to apply individual control sample samplings, for example, 400-2000 individual control sample samplings. All embodiments of the present disclosure have in common that the need to adjust the laser operating parameter is not dependent on a single control sample, and certainly not on a single control sample sampling, but is based on a plurality of control samples and a plurality of control sample samplings.

[0032] In various embodiments, the laser energy density, laser fluence, laser power, and / or laser intensity can be changed to adjust the operating parameter during laser-assisted ionization. In particular, the laser energy density, laser fluence, laser power, and / or laser intensity can be increased by a preset percentage if a performance trend deviation is detected. An increase can be in the range of substantial single-digit percentage values, e.g., 1-10 percent, in particular approximately 1 percent. In MALDI ionization, the ion yield scales approximately with the sixth to eighth power of the laser fluence, so that an increase of 1 percent results in an increase in detectable ions of approximately 6-8 percent. Such a measure can be sufficient to counteract a negative performance trend and adjust the laser to the interval. The adjustment of the laser's operating parameter can alternatively be a predefined increment, e.g.,a certain percentage; however, it can also be calculated as a function of the trend and / or the absolute deviation from the interval boundary, e.g. with a fixed increment (possibly a percentage) as a basis, which is corrected by a trend-dependent term in order to avoid over-modulation during the adjustment and to make the adjustment routine less dependent on outliers.

[0033] In various embodiments, a notification and / or flag can be generated if the number of adjustments to the operating parameter reaches or exceeds a predetermined value, e.g., 2-10 adjustments, in particular 3-5 adjustments. The notification can be in the form of an entry in the log file of the ion spectrometry system used. Messages for immediate user notification are also possible, e.g., a pop-up window with a text or image message in the graphical user interface on the computer of the ion spectrometry system, or an automatically generated email or SMS with content that summarizes the result of the spectral data analysis. Such a text / image message, email, or SMS can be sent directly to a recipient address of the ion spectrometry system manufacturer or a service partner to automatically issue a maintenance order. In particular, marking can include an entry in a comment field of the metadata for the spectral data acquired under the modified laser conditions. This allows the user to track the automated laser adjustment at a later time, e.g., when evaluating the spectral data from the analytical samples that were acquired alongside the spectral data from the control samples.

[0034] In various embodiments, the composition of the analytical sample may be substantially unknown. The control sample and the analytical sample differ primarily in that the control sample, its molecular content, and its behavior during laser-assisted ionization are very well known and characterized, which is why it is used as a controlled variable. In contrast, information about the analytical sample must first be determined from the corresponding spectral data, possibly through sophisticated post-processing. In the case of MALD-L ionization, only the matrix substance is well known during the preparation of the analytical sample; however, the molecular content and ionization behavior of the analyte molecules are not verified and cannot be determined.The analytical sample can, for example, be a preparation believed to contain a microorganism and / or extracted molecules thereof, with the aim of determining the taxon of the microorganism down to the species and / or resistance to an antimicrobial substance. The analytical sample can also be a preparation containing cells, e.g., eukaryotic cells, whose behavior and / or response to exposure to a specific stimulus is to be investigated, e.g., exposure to a toxicologically and / or pharmacologically active substance.

[0035] In a second aspect, the present disclosure relates to an ion spectrometry system comprising an ion analyzer, an ion source connected to the ion analyzer, and a processor unit communicating with the ion analyzer and the ion source and configured and programmed to coordinate and execute a method as previously explained and described. Short description of the figures

[0036] For a better understanding of the invention, reference is made to the following figures. The elements in the figures are not necessarily drawn to scale, but are primarily intended to illustrate the principles of the invention (mostly schematically). In the figures, corresponding elements are identified by the same reference numerals in the different views.

[0037] Figure 1 schematically illustrates the principle of an axial-linear time-of-flight mass spectrometry system with laser-assisted ionization; for example, the micro-flex® LT / SH from Bruker.

[0038] Figure 2 shows a schematic of a typical MALDI spot sample preparation process.

[0039] Figure 3 schematically illustrates the principle of performance monitoring and control in the sense of the present disclosure.

[0040] Figure 4 shows schematically and by way of example an ion spectrometry system with which the methods described in the present disclosure can be implemented. Detailed description

[0041] While the invention has been shown and explained in terms of a number of embodiments, it will be appreciated by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the technical teachings defined in the appended claims.

[0042] Using individual control sample measurements to monitor and control the performance of an ion source can lead to confusing results, as a single control sample itself may be flawed, e.g., due to oversights during preparation. For MALDI preparation, a poorly crystallized matrix substance is conceivable, e.g., if the ratio of matrix substance to molecule to be embedded (normally -10,000-5,000:1) does not meet the specifications. Particularly in clinical applications such as the Bruker MALDI Biotyper®, it may happen that the analysis system is operated by a person with only the most basic training in its operation. For this reason, this operator may not be able to detect any incorrect preparation of individual control samples.As already mentioned in the introduction, regular inspections by trained specialists from the manufacturers or their service partners can provide a remedy, but only at high cost and with enormous personnel expenditure. Control sample preparations that do not meet the requirements can also be caused by the age of the control sample substrate itself or by other factors. Inferiority of the reagents used for control sample preparation, e.g. if the user overlooks or ignores expiration dates.

[0043] The goal is to have the ion spectrometry system optimally adjusted once by specialist personnel after delivery to the user, and to reduce the number of maintenance procedures required by specialist personnel or users during ongoing operation, or at least keep them as low as possible. This reduces labor and costs. This goal can be achieved, in addition to other measures such as the ion source cleaning or detector voltage control mentioned above, which are not the subject of this disclosure but can only serve as a supplement, with a (quasi-)automated readjustment of an operating parameter of the laser for laser-assisted ionization.

[0044] A method for monitoring and controlling the performance of an ion source comprises the following steps: (a) laser-assisted ionization of a control sample whose composition is substantially known and which is sampled, preferably in the same measurement process, before, parallel to 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) over a plurality of control samples and, if necessary,Measurement processes to collect a large number of control sample spectral data and evaluate them in such a way that spectral data of individual control samples have little weight and a performance trend appears in the evaluation, (d) adjusting an operating parameter of the laser of the ion source if the performance trend enters a range outside a predefined performance interval in order to regulate the laser in accordance with the interval, and (e) repeating steps (a) to (d) for the purpose of continuous monitoring and control of the ion source.

[0045] 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 over a plurality of measurement processes and control samples. A measurement process within the meaning of the present disclosure can be understood in particular with respect to the mode of operation of the ion spectrometry system. For example, a measurement process can include all measurements taken from (control) samples on a sample carrier, as long as it is spatially located within the ion source. An example would be the introduction of the sample carrier into a negative pressure region of the ion source, provided that the ion source operates in negative pressure. especially in vacuum MALDI. If the sample carrier is removed from the ion source, e.g., the negative pressure area, and then another sample carrier with new (control) samples is reinserted, all measurements of the new (control) samples on this next sample carrier can be considered a separate measurement process.

[0046] In addition to analytical sample preparations, several control sample preparations can also be arranged on a sample carrier for laser-assisted ionization. If both control samples and analytical samples are present on the sample carrier, e.g., 2-10 technical control sample replicates, in particular 4-8 technical control sample replicates, all control sample spectral data from these preparations can be used to determine the performance trend and / or its temporal course. A control sample preparation on the sample carrier can be sampled once or multiple times to generate control sample spectral data, limited only by the yield of the control sample. As a result, the control sample spectral data can include isolated and separate spectra.Repeated sampling of the same control sample preparation increases the basis of the control sample spectral data for subsequent statistical evaluation and reduces the influence of particularly short-term disturbances in the measurement process that can cause outlier measurements.

[0047] Sampling the control sample before, in parallel with, or after an analytical sample can, in particular, mean that the control sample and the analytical sample are analyzed in the same measurement process. Sampling the control sample can, in particular, take place immediately before or immediately after an analytical sample. The measurement modes or measurement settings of the ion spectrometry system can be the same or different for sampling analytical samples and control samples. If a measurement process includes recording spectral data of all (control) samples on a sample carrier, as previously explained, this can mean that the sample carrier has a plurality of designated sample locations, e.g.an array of sample points, such as those on Bruker's well-known Anchor Chip or MBT Biotarget 96 carriers, or even on a steel plate, and some of these sample locations are prepared with a predetermined number of control samples, while some or all of the remaining sample locations are occupied by analytical samples. Control samples and analytical samples can then be sampled separately in blocks (e.g., first all control samples, then all analytical samples) or alternately (e.g., control sample => analytical sample => control sample => analytical sample, etc.). In ion sources with multiple laser beams, it is possible, if necessary, to sample several samples at a time. Analytical samples and control samples can also be measured in parallel from the same sample carrier if the connected ion analyzer is designed and set up to process multiplexed ion streams or if more than one ion analyzer is present.

[0048] The ion analyzer can be designed as a mass analyzer that sorts the ionized control sample, particularly according to the principle of time-of-flight (TOF) dispersion. A mass analyzer separates charged molecules or molecular ions according to their mass-to-charge ratio, commonly referred to as m / z.

[0049] Figure 1 schematically shows the principle of an axial-linear time-of-flight mass spectrometry system. Upper diagram: A laser bombards a (control) sample on the sample carrier 2, which has been prepared, for example, with a MALDI matrix substance. An assembly of electrodes 4 accelerates the ions created by the laser bombardment, possibly after a delay time, into a flight tube 6 of the time-of-flight analyzer along a straight trajectory 8. Since the different molecular species created by the laser bombardment have different masses and carry largely uniform charges, so that they are imparted with approximately the same kinetic energy during acceleration, different velocities result. Light molecules, shown as small black circles, thus travel faster than heavy molecules, shown as circles with a larger circumference, and arrive at the detector 10 sooner, which, for example,It can be configured as a dynode array or as a multichannel plate (MCP). This temporal dependence of arrival at the detector allows the assignment of charge-related masses (m / z) to the detection events; lower diagram.

[0050] In addition to time-of-flight analyzers, which can be designed with axial-linear setups as indicated in Figure 1, as well as reflector setups and / or those with orthogonal acceleration of ions into the path of flight (OTOF), other types of mass-dispersing separators can also be used, e.g., quadrupole mass filters (single quads), triple quadrupole analyzers (triple quads), ion cyclotron resonance (ICR) cells, Kingdon-type analyzers such as the Orbitrap® (Thermo Fisher Scientific), and others. It is also conceivable to design the ion analyzer as a mobility analyzer or a combined mobility mass analyzer, as previously discussed.

[0051] The control sample may comprise a preparation of a microorganism, e.g. a prokaryotic organism, in particular a preparation of a bacterial species. Microorganism cells with a precisely known taxon and a defined content of soluble molecules, such as ribosomal proteins and peptides, can be purchased commercially. With standardized preparation, such a design can be expected to yield a predictable and consistent analyzer and detector response, for example, regarding mass signal profile or abundance. One example is the Bacterial Test Standard for the MALDI Biotyper® from Bruker, a MALDI axial-linear time-of-flight mass spectrometry system; the standard includes a typical Escherichia coli DH5 alpha peptide and protein profile, as well as additional proteins suitable for mass calibration, among other things.

[0052] Preferably, the plurality of control samples are congruent or identical; in particular, control samples that are congruent or identical are used across the plurality of measurement processes. In the case of microorganism preparation as a control sample, the measurements can be recorded over a long period of time using biological and / or technical replicates of the microorganism.

[0053] If the control sample contains a microorganism, it is preferably sterilized. Sterilization can involve exposing the microorganism to a metabolism-inhibiting liquid, e.g. an alcohol such as ethanol or isopropanol or an acid such as formic acid, and / or the application of energy, e.g. heat or high-energy radiation (possibly ultraviolet light). Sterilization is understood in particular to mean that the microorganism loses the ability to reproduce even under favorable conditions. In this way, the biological hazards associated with unintentional / uncontrolled spread in an analytical laboratory can be avoided. Under certain conditions, it may not be necessary to sterilize the microorganism in a control sample, for example when working in an analytical laboratory with biological safety level 2 or higher.It can be assumed that adequately trained specialist personnel will be employed there.

[0054] As previously explained, the ion source can operate according to the MALDI principle. Figures 2A-C schematically show a MALDI process from the preparation of the (control) samples to the acquisition of spectral data. In Figure 2A, (control) samples such as a microorganism suspension 12 or a suspension containing soluble molecules extracted from a microorganism are applied to the MALDI sample carrier 2* using a pipette 14 or another suitable dispensing device. For example, the AnchorChip or MBT Biotarget 96 carriers from Bruker or steel plates can be used as MALDI sample carriers. After excess fluid has been removed from the suspension has been removed, a matrix substance solution can be applied to the fluid-depleted cell layer and / or layer of soluble molecules, e.g., using a suitable tool 16 such as another pipette, illustrated here with tile hatching. After embedding, drying, and crystallization of the matrix, the individual (control) samples 18 are ready for sampling with a laser beam 20. The resulting ions 22 can then, for example, be accelerated into the flight path of a time-of-flight analyzer, as schematically indicated in Figure 1.

[0055] The performance trend can be derived from the abundance or intensity of ions detected in the analyzer; in particular, an average abundance or intensity can be used. For example, the total amount of detected ion currents in the spectral data can be used as an intensity measure or abundance measure; this is sometimes called the total ion count (TIC). The TIC can be recorded over the entire spectral range or over a limited subrange, for example, a range from the spectral data of 30,000 or more atomic mass units (amu), 20,000 or more amu, 10,000 or more amu, 5,000 or more amu, or 1,000 amu or more. It is also possible to calculate an average intensity over the entire spectral range or a portion of it, for example a range from the spectral data of 30,000 or more atomic mass units (amu), 20,000 or more amu, 10,000 or more amu, 5,000 or more amu, or 1.000 amu or more as a basis. A broad database ensures statistical stability, i.e., low weighting of outliers across multiple control samples, control sample sampling, or measurement processes.

[0056] Figure 3 schematically illustrates the feedback from monitoring a performance trend over a longer period of time, detecting a performance trend deviation, and resulting change in an operating parameter of the laser for laser-assisted ionization. The lower diagram shows a time series of an (average) ion current at the detector on the vertical axis over several measurement processes on the horizontal axis. A measurement process can include the ion spectrometric analysis of all (control) samples placed on a sample carrier, e.g., as long as the sample carrier is located in a negative pressure area of ​​the ion source. In the example shown, nine sample carriers with control sample loading were included in the evaluation.The number of control samples or control sample samples evaluated for the performance trend per measurement process is shown schematically and as an example at three and represented by three bars per measurement process in the diagram. It is understood that the number of Control samples or control sample sampling per measurement process can also vary. For example, 2-10 or 3-5 control samples can be sampled per measurement process, or a multiple of these control sample samplings can be carried out, e.g., using a corresponding number of technical control sample replicates on the sample carrier. In the case of a sample carrier with designated points for sample application, such as the Anchor-orChip or MBT Biotarget 96, the number of control samples must be weighed against the number of analytical samples of unknown molecular content that the user wishes to characterize using ion spectrometric measurement.If space on the sample carrier is limited, an alternative approach could be to apply and prepare a single high-yield control sample under increased quality requirements during preparation, which is then subjected to a large number of samples to obtain a comprehensive spectral data basis for a single measurement process.

[0057] The middle horizontal dashed line 24 in the lower diagram indicates a reference ion current value, which may represent an optimal setting found through extensive testing and calibration. In a typical MALDI time-of-flight mass spectrum, this ion current may correspond to an average intensity of 25,000–30,000 counts, specifically 27,000 counts. The outer dashed lines 26 arranged around this middle line 24 represent the interval boundaries around the reference ion current value, within which the performance of the system consisting of ion source and analyzer can be considered to meet requirements or be satisfactory. The performance interval may encompass deviations up to a predefined percentage, specifically ± 30 percent or less, ± 25 percent or less, ± 20 percent or less, ± 15 percent or less, or ± 10 percent or less.The limit to higher ion currents can be determined by avoiding detector saturation, and the limit to lower ion currents can be derived from experience regarding how high an ion current signal must be to allow detectable and reliable spectral data analysis. The interval boundaries can be arranged equidistantly or non-equidistantly from the performance benchmark. In one embodiment, an upper interval boundary can be closer to the performance benchmark than a lower interval boundary; in another embodiment, a lower interval boundary can be closer to the performance benchmark than an upper interval boundary.

[0058] The performance trend can also be derived from the quality of characterization of the molecular content of the substantially known control sample. For example, if a If a microorganism is used as a control sample, the highest similarity measure (the so-called log(score)) of the relevant reference spectral data from an ion spectrometry system designed to identify the taxon, such as the Bruker MALDI Biotyper®, can serve as a performance benchmark. Since the taxon of the microorganism in the control sample is substantially known, the log(score) of the MALDI Biotyper® should be significantly above 2.00. If the trend leads to the lower interval limit of 2.00 being reached and / or undercut over a longer observation period, and thus independent of outliers, it may be necessary to adjust a laser operating parameter to counteract this performance decline.

[0059] The evaluation in step (c) can subject the large number of control sample spectral data to averaging, in particular by calculating the mean or median. The averaging can in particular be based on the arithmetic mean. In Figure 3, the ion current values ​​averaged over three control samples or control sample samplings per measuring process are illustrated by dashed circles 28. By recording control sample spectral data over a large number of control samples, control sample samplings or measuring processes, a moving average can be formed, i.e. a time-varying subset can be used from a set of measurements for the averaging, e.g. by taking into account control sample spectral data from a shifting time window, if necessary across a large number of measuring processes.

[0060] The averaging can be based on a predetermined number of (i) control samples, e.g. 50-250 control samples, (ii) measurement processes, e.g. 10-30 measurement processes, (iii) laser activations, e.g. over 10 5 - 10 7 Laser shots for individual spectral data acquisitions, or (iv) a number of control samples or measurement processes from a predetermined period, e.g., over a period of 7-21 days, 1-4 weeks, or 1-2 months. It is also possible to apply the averaging to a predetermined number of individual control sample samplings, for example, 400-2000 individual control sample samplings. All embodiments of the present disclosure have in common that the need to adjust the laser operating parameter is not dependent on a single control sample, and certainly not on a single control sample sampling, but is based on a plurality of control samples and a plurality of control sample samplings.

[0061] In Figure 3, a trend line 30 is drawn connecting the various ion current mean values, which allows an estimate of how the observed ion current values ​​of the control samples might develop in the further course. In the shown In this specific example, it is determined in the third measurement process that the mean ion current has reached the lower interval limit 26; for the fourth measurement process, it is even found that the mean ion current has left the interval range. This finding leads to a change in an operating parameter of the laser as a countermeasure to counteract this power drop (see arrow 32).

[0062] To adjust the operating parameter, the laser energy density, laser fluence, laser power and / or laser intensity can be changed during laser-assisted ionization, as shown by the labeling of the vertical axis of the upper diagram in Figure 3. In particular, the laser energy density, laser fluence, laser power and / or laser intensity can be increased by a preset percentage if a performance trend deviation is detected, see increment #1. An increase can be in the range of substantial single-digit percentage values, e.g. 1-10 percent, in particular around 1 percent. The adjustment of the laser operating parameter can alternatively comprise a predefined increment, e.g. a certain percentage, but it can also be calculated depending on the trend and / or the absolute deviation from the interval boundary, e.g. with a fixed increment (if necessary.a percentage) as a basis, which is corrected by a trend-dependent term in order to avoid over-modulation during the adjustment and to make an adjustment routine less dependent on outliers.

[0063] After adjusting the laser parameters between the fourth and fifth measurement cycles, as shown in the upper diagram of Figure 3, the average ion current is adjusted back to within the interval without human intervention. The ion spectrometry system with the ion analyzer and ion source monitored and adjusted in this way can then continue to operate and continue to deliver measurement performance that meets or satisfies requirements. If, during subsequent measurements, it becomes apparent that the performance trend is again approaching an interval boundary, as seen here in the 7th measurement, and then falls outside the interval range, as indicated in the 8th measurement, the laser's operating parameters can be adjusted again, see Adjustment #2, similar to the previously explained procedure. Adjustment #2 can be identical to Adjustment #1 or can be performed with different adjustment increments.

[0064] A notification and / or flag may be generated if the number of adjustments of the operating parameter over time reaches or exceeds a predetermined value, e.g. 2-10 adjustments, in particular 3-5 adjustments. Notification can take the form of an entry in the log file of the ion spectrometry system in use. Messages for immediate user notification are also possible, e.g., a pop-up window with a text or image message in the graphical user interface on the ion spectrometry system's computer, or an automatically generated email or SMS with content summarizing the results of the spectral data analysis. Such a text / image message, email, or SMS can be transmitted directly to a recipient address of the ion spectrometry system manufacturer or a service partner in order to automatically issue a maintenance order. A marking can, in particular, include an entry in a comment field of the metadata for the spectral data recorded under the changed laser conditions, in order to enable the user to carry out the automated laser adjustment at a later time, e.g.,when evaluating the spectral data from the analytical samples collected alongside the spectral data from the control samples.

[0065] The composition of the analytical sample may be substantially unknown. The control sample and the analytical sample differ primarily in that the control sample, its molecular content, and its behavior during laser-assisted ionization are very well known and characterized, which is why it is used as a controlled variable, whereas information about the analytical sample must first be determined through possibly sophisticated post-processing. In the case of MALDI ionization, only the matrix substance is well known during the preparation of the analytical sample; however, the molecular content and ionization behavior of the analyte molecules must be determined.The analytical sample can, for example, be a preparation believed to contain a microorganism and / or extracted molecules thereof, with the aim of determining the taxon of the microorganism down to the species and / or resistance to an antimicrobial substance. The analytical sample can also be a preparation containing cells, e.g., eukaryotic cells, whose behavior and / or response to exposure to a specific stimulus is to be investigated, e.g., exposure to a toxicologically and / or pharmacologically active substance.

[0066] Figure 4 schematically shows an ion spectrometry system 40 with an ion analyzer 42, an ion source 44 connected to the ion analyzer 42, and a processor unit 46 communicating with the ion analyzer 42 and the ion source 44 and designed and programmed to carry out a method as previously explained and described, particularly with reference to Figure 3. The path of the ions from The connection between the ion source 44 and the ion analyzer 42 is symbolized by an arrow 48. The control and information reception communication of the processor unit 46 with the ion source 44 and the ion analyzer 42 is indicated by double-headed arrows 50.

[0067] The 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 modified without departing from the scope of the invention. Furthermore, the features and measures disclosed in connection with different embodiments may be combined in any way, provided this appears practical to a person skilled in the art. Furthermore, the above description serves only to illustrate the invention and not to limit the scope of protection, which is defined exclusively by the appended claims, taking into account any existing equivalents.

Claims

Claims 1. A method for monitoring and controlling 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, in parallel 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) over a plurality of control samples to collect a plurality of control sample spectral data and evaluate them in such a way that spectral data of individual control samples have little weight and a performance trend appears in the evaluation, (d) adjusting an operating parameter of the laser of the ion source if the performance trend enters a range outside a predefined performance interval in order to adjust the laser interval-compliant, and (e) Repeating steps (a) to (d) for continuous monitoring and control of the ion source.

2. The method according to 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 over a plurality of measurements and control samples.

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

4. Method according to 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. Method according to one of claims 1 to 4, wherein the ion source operates according to the MALDI principle.

6. A method according to any one of claims 1 to 5, wherein the performance trend is derived from an abundance or intensity of ions detected in the analyzer. Method according to one of claims 1 to 6, wherein the performance interval comprises deviations up to a predefined percentage from a performance benchmark. Method according to one of claims 1 to 7, wherein the evaluation in step (c) subjects the plurality of control sample spectral data to averaging, in particular mean or median calculation. Method according to claim 8, wherein the averaging is applied to a predetermined number of (i) control samples, (ii) measurement processes, (iii) laser activations, or (iv) a number of control samples or measurement processes from a predetermined period of time. Method according to one of claims 1 to 9, wherein the laser energy density, laser fluence, laser power, and / or laser intensity are changed during laser-assisted ionization to adapt the operating parameter.A method according to any one of claims 1 to 10, wherein a notification and / or flag is generated if a number of adjustments to the operating parameter reaches or exceeds a predetermined value. A method according to any one of claims 1 to 11, wherein a composition of the analytical sample is substantially unknown. An ion spectrometry system comprising an ion analyzer, an ion source connected to the ion analyzer, and a processor unit communicating with the ion analyzer and the ion source, and configured and programmed to coordinate and execute a method according to any one of claims 1 to 12.

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