Method and apparatus for monitoring and controlling ion source performance

Abstract

A method and apparatus for monitoring and controlling the performance of an ion source is provided. [Solution] The method and apparatus include the steps of: (a) performing laser-assisted ionization of a control sample having a substantially known composition and 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 the ion source; (c) collecting control sample spectral data for multiple control samples and repeating steps (a) and (b) for multiple control samples to evaluate the spectral data for multiple control samples so that the weight of the spectral data for each individual control sample is small and performance trends emerge in the evaluation; (d) adjusting the operating parameters of the ion source laser if the performance trend falls outside a predefined performance interval to adjust the laser to fit within the interval; and (e) repeating steps (a) to (d) for continuous monitoring and control of the ion source.

Description

[Technical Field]

[0001] The present invention relates to monitoring and controlling the performance of an ion source operating in laser-assisted ionization, particularly MALDI ionization. The present invention particularly relates to an ion spectroscopy system having an ion analyzer, an ion source connected to the ion analyzer, and a processor unit in communication with the ion analyzer and the ion source. [Background technology]

[0002] The prior art will now be described with reference to certain specific embodiments, which should not be understood as limiting. Useful advances and variations from what is known in the prior art are applicable beyond the relatively narrow scope of this introduction and will be readily apparent to those skilled in the art upon reading the disclosure of the invention which follows this introduction.

[0003] In MALDI time-of-flight mass spectrometry (MALDI, matrix-assisted laser desorption / ionization), several operating parameters are critical for useful measurements. These include the spectral baseline, the various voltages between the accelerating electrodes in the flight path, the detector voltage, and the laser energy density of the ion source, to name a few. These parameters are typically tested and set for best results by the manufacturer's or contractor's qualified service personnel in the mass spectrometer manufacturer's testing facility or the user's laboratory. However, because these measurements are one-off, the mass spectrometer must function unsupervised for extended periods after setup or reconditioning.

[0004] However, it is known and consistent with experience that some parameters change with the operation and use of the mass spectrometer, also known as aging or wear. Among these parameters, the detector voltage is directly affected and the laser energy density is indirectly affected.

[0005] Detector aging is manifested, for example, by a decrease in the amplification factor of the secondary electron multiplier as operation progresses. This decrease is usually compensated for by an increase in the detector voltage, i.e., an increase in the potential gradient along which the secondary electrons are accelerated. In practice, the need for such detector voltage adjustment accumulates over a fairly long period of time and can be determined in a largely automated manner during a calibration phase in which single ion signals are observed, as described, for example, in patent document 1 (corresponding to GB 2 457 559 A and US 2009 / 0206247 A1).

[0006] In the case of laser energy densities, the ion source, especially its internal surfaces, such as the surfaces of electrodes or boundary housing components, can become contaminated as operation progresses, resulting in changes in the conditions under which the electric field is generated within the ion source. This can manifest itself as a decrease in ion yield. This performance degradation can be counteracted, for example, by semi-automated or fully automated cleaning to eliminate or at least reduce perturbations due to physicochemical interactions. An example of such a cleaning procedure is shown in Patent Document 2 (corresponding to GB 2 457 362 A and US 2009 / 0200457 A1). Another option is to increase the laser energy density or laser fluence, since ion yield is proportional to the power of the energy density or fluence, as explained in the review by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425). Depending on the analyzer's usage, given the virtually uninterrupted measurement phase from early to late each day, the need for repair due to contamination can increase very rapidly compared to detector aging.

[0007] As soon as a mass spectrometer is first installed in a user's laboratory, it can be subject to intensive operation (e.g., high-throughput repeated identification of pathogenic microorganisms in clinical settings), and this performance degradation can occur very quickly, requiring frequent maintenance work, for example, weekly or even more frequently, which is both a significant burden for service personnel and a cost driver for users.

[0008] Although not exhaustive, the following provides a brief description of some prior art documents that may be relevant to the present disclosure.

[0009] The technical teaching disclosed in patent application 3 (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 analytes with stepwise increasing energy density at the laser focus, while at the same time dealing with the problem of possible detector signal saturation by special measures.

[0010] Patent Document 4 discloses a method of 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] US Patent No. 5,929,633 relates to a system and method for real-time monitoring of laboratory analyzers for quality control purposes.

[0012] Patent Document 6 describes a MALDI ion source in which laser light from a laser source is reflected by a mirror and the energy of the laser light is adjusted by rotating a polarizing beam splitter, and the energy-adjusted laser light is then irradiated onto a sample.

[0013] US Patent No. 5,949,999 relates to a mass spectrometer and a method and program for adjusting the intensity of a laser light used in a mass spectrometer for MALDI ionization.

[0014] In view of the above, there is a need to provide a method and apparatus that can ensure and improve the monitoring and control of ion source performance over time. Further objectives achieved by the present invention will become readily apparent to those skilled in the art upon reading the following disclosure. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] German Patent Application Publication No. 10 2008 010 118 A1 [Patent Document 2] German Patent Application Publication No. 10 2008 008 634 A1 [Patent Document 3] German Patent Application Publication No. 10 2010 019 857 A1 [Patent Document 4] International Patent Application Publication No. 2014 / 140625 A1 [Patent Document 5] International Patent Application Publication No. 2017 / 160857 A1 [Patent Document 6] European Patent Application Publication No. 3 806 135 A1 [Patent Document 7] European Patent Application Publication No. 3 651 184 A1 Summary of the Invention

[0016] In a first aspect, the present disclosure (sometimes referred to herein as the "disclosure") relates to a method for monitoring and controlling the performance of an ion source, the method comprising: (a) laser-assisted ionization of a control sample having a substantially known composition, 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 the ion source; (c) collecting control sample spectral data for multiple control samples and repeating steps (a) and (b) for multiple control samples to evaluate the spectral data for each individual control sample so that the spectral data has minor weight and performance trends are evident in the evaluation; (d) adjusting operating parameters of a laser of the ion source if the performance trend falls outside a predefined performance interval to adjust the laser to fit within the interval; and (e) repeating steps (a) through (d) for continuous monitoring and control of the ion source.

[0017] In various embodiments, in step (a), the control sample may be sampled in the same measurement run as the analytical sample, and in step (c), steps (a) and (b) may be repeated for multiple measurement runs and control samples. A measurement run in the context of the present disclosure may be considered to be an operation particularly related to the operating mode of the ion spectroscopy system. For example, a measurement run may include all measurements recorded from a (control) sample on a sample carrier while the sample is present in the space within the ion source. One example, for example, in the case of vacuum MALDI, is introducing a sample carrier into a reduced pressure region of an ion source while the ion source is operating under reduced pressure. If a sample carrier is removed from the ion source, e.g., from the reduced pressure region, and then a further sample carrier containing a new (control) sample is reintroduced, all measurements of the new (control) sample on this next sample carrier may be considered a separate measurement run.

[0018] In addition to analytical sample preparations, it is also possible to place multiple control sample preparations on the sample carrier for laser-assisted ionization. When both control and analytical samples are present on the sample carrier, e.g., 2-10 technical control sample replicates, especially 4-8 technical control sample replicates, the control sample spectral data for all of these preparations can be used to confirm performance trends and / or their progression over time. Control sample preparations on the sample carrier can be sampled once or multiple times to generate control sample spectral data; in principle, this is limited only by the amount of control sample. As a result, the control sample spectral data can contain individualized, separate spectra. Repeated sampling of the same control sample preparation increases the base of control sample spectral data for subsequent statistical evaluation and, in particular, can mitigate the effects of very brief perturbations in the measurement sequence, which may result in outlier measurements.

[0019] In particular, sampling a control sample before, simultaneously with, or after an analytical sample may mean that the control sample and analytical sample are examined in the same measurement operation. In particular, the control sample may be sampled immediately before or immediately after the analytical sample. The measurement mode or measurement settings of the ion spectroscopy system may correspond to or be different from the sampling of analytical and control samples. As mentioned above, when the measurement operation includes recording spectral data of all (control) samples on a sample carrier, this may mean that the sample carrier has a number of designated sample sites (e.g., sample spot sites on a steel plate such as the well-known AnchorChip from Bruker or MBT Biotarget 96 carrier), and some of these sample sites are initially prepared with a predetermined number of control samples, while some or all of the remaining sample sites are occupied by analytical samples. The control samples and analytical samples can then be sampled separately from each other (e.g., first all control samples, then all analytical samples, or vice versa) or alternately in blocks (e.g., control samples → analytical samples → control samples → analytical samples, etc.). In an ion source with multiple laser beams, it is also optionally possible to measure analytical and control samples in parallel from the same sample carrier if the connected ion analyzers are designed and configured to handle multiplexed ion currents, or if there are multiple ion analyzers.

[0020] In various embodiments, the ion analyzer may be specifically designed as a mass analyzer for selecting the ionized control sample by the principle of time-of-flight (TOF) dispersion. Mass analyzers separate charged molecules or molecular ions according to their mass-to-charge ratio, usually referred to as m / z. As well as the time-of-flight analyzers already mentioned in the introduction, which may provide both linear and reflector structures and / or structures with axial or orthogonal acceleration of ions into the flight path, it is also possible in principle to use other types of mass-dispersive separators, such as quadrupole mass filters (single quad), triple quadrupole analyzers (triple quad), ion cyclotron resonance (ICR) cells, Kingdon analyzers such as Orbitrap® (Thermo Fisher Scientific), etc.

[0021] In various embodiments, the ion analyzer may also be a mobility analyzer or a combined mobility / mass analyzer. Mobility analyzers separate charged molecules or molecular ions according to their collision cross-section-to-charge ratio, sometimes referred to as Ω / z or σ / z. Their basis is the interaction of ion species with an electric field that couples the ion charge under the simultaneous action of a buffer gas acting on the ion's average cross-section. In particular, drift tube mobility analyzers with a static electric field gradient are known, which drive ions through an essentially stationary gas, obtaining the drift velocity of the ion species through the driving force of the electric field and the deceleration force of collisions with gas particles. Similarly, trapped ion mobility separators (TIMS) are known, which have a constant laminar gas flow that drives ions forward, offset by a stepped electric field gradient with a correspondingly varying deceleration force. One may also refer to traveling wave mobility separators. It will be apparent that the aforementioned types of analyzers may be combined to enable the separation of ion species in multiple dimensions, i.e., according to multiple physicochemical properties such as m / z, Ω / z, or σ / z.

[0022] In various embodiments, the control sample may include a preparation of a microorganism, e.g., a prokaryotic organism, particularly a bacterial species. Microbial cells of precisely known taxonomic groups and defined content of soluble molecules, e.g., liposomal proteins and peptides, are commercially available. This configuration, in the case of a standardized preparation, is expected to result in readily predictable and consistent analyzer and detector responses, e.g., in terms of mass signal profile or abundance. One example is the Bacterial Test Standard for the Bruker MALDI Biotyper®, a MALDI axial linear time-of-flight mass spectrometry system. This standard sample consists of a typical Escherichia coli DH5α peptide and protein profile and additional proteins appropriate for the application, including mass calibration.

[0023] Preferably, multiple control samples are matched or identical, and in particular matched or identical control samples are used over multiple measurement runs. In the case of a microbial preparation as a control sample, measurements can be recorded from biological and / or technical replicates of the microorganism, for multiple preparations, and over a long period of time.

[0024] If the control sample contains microorganisms, the control sample is preferably sterilized. Sterilization may involve exposing the microorganisms to a metabolic inhibitor (e.g., an alcohol such as ethanol or isopropanol, or an acid such as formic acid) and / or to energy (e.g., heat or high-energy radiation, especially ultraviolet light). In particular, sterilization means that the microorganisms lose their ability to replicate even under conditions favorable to the microorganisms. In this way, biological risks associated with unintended / uncontrolled spread in analytical laboratories can be avoided. Under certain conditions, for example, when working in analytical laboratories with biosafety level 2 or higher, it may not be necessary to sterilize the microorganisms in the control sample. This presupposes that well-trained and skilled personnel are employed.

[0025] In various embodiments, the ion source may operate according to the MALDI principle. The principles of matrix-assisted laser desorption / ionization (MALDI) have already been described in detail elsewhere. See, for example, the paper by Klaus Dreisewerd mentioned in the introduction. In a wide variety of MALDI sample preparations, soluble molecules, such as microbial or bacterial liposomal proteins, are intercalated into a matrix crystalline structure with a high absorption capacity for laser light. The matrix material chosen is a small organic molecule that strongly absorbs energy at the laser wavelength used, such as solid Nd:YAG with a tripled wavelength of 355 nanometers. Examples of these are sinapinic acid, 2,5-dihydroxybenzoic acid, α-cyanohydroxycinnamic acid, or 2,4,6-trihydroxyacetophenone. When the matrix crystalline structure is exposed to a pulse of laser radiation, it undergoes explosive evaporation, releasing the embedded molecules. Furthermore, the molecules are ionized during this high-energy ablation process, enabling subsequent ion spectroscopy analysis. In this analysis, ions are accelerated in an electric field at rates generally dependent on their mass, and after traveling a relatively long, nearly field-free flight path, arrive in a time-resolved manner at a detector containing a secondary electron multiplier. The time from laser desorption of the sample material or from the acceleration pulse into the mass analyzer flight path to receiving the different ion current signals at the detector provides, via conversion, the mass (m / z) of the ion based on its charge.

[0026] Example of Establishing a MALDI Measurement. Ideally, when establishing a new MALDI test method, several operating parameters are defined using a standard, which contains a standard substance manufactured in a controlled manner and contains a very clearly defined number and concentration of target molecules in the desired mass range (control sample). This standard, e.g., Bruker's Bacterial Test Standard, is prepared fresh and analyzed in the instrument. Prior to the measurement of this standard, other operating parameters can be separately defined, such as the leveling voltage (called back bias) for establishing a nearly field-free space in the desorption region of the MALDI sample carrier, the detector voltage, or the baseline. A critical parameter for MALDI measurements is the laser energy density. If the laser energy density is too low, very few ions are generated, if at all. If the laser energy density is too high, the ion cloud generated will contain too many charge carriers, exceeding the mass resolution of the associated analyzer. Therefore, it is desirable to find and define the so-called "desorption threshold." This laser energy density threshold defines the point at which the first ion signal can be identified using the detector after gradually increasing the laser energy density from a low initial value. Proceeding from this desorption threshold, the laser energy density is then preferably further increased relative to the initial instrument settings to achieve a compromise between the number and intensity of signals and a decrease in mass resolution, such preferred laser energy densities are typically a few percentage points above the desorption threshold.

[0027] The laser energy density for subsequent measurements of analytical samples is adjusted to allow for the analysis of both low and high analyte concentrations. In other words, the laser energy density is varied by several percentage points during the measurement of an analytical sample. For example, the energy density can be increased or decreased after the first measurement attempt to obtain the optimal usable signal. If the analyte concentration in the sample is too high, the initial laser power is reduced. If the analyte content in the sample is too low, the laser power is increased. However, in practice, this procedure results in the laser energy density being constantly increased during the measurement, meaning that an under-adjusted laser or a contaminated ion source may remain undetected for a long time, which is a measurement disadvantage, for example, for samples with low analyte content. Because MALDI measurements themselves are highly variable and dependent on many parameters, current practice is to monitor and readjust the initially optimized laser energy density only to a limited extent. A decrease in laser power or a contaminated ion source are factors that can cause an increase in laser energy density. The present invention is essentially based on frequently measuring a defined standard or control sample of substantially known composition at a fixed laser energy density for performance monitoring measurements, with the aim of, among other things, finding the expected signal intensity. However, because single MALDI measurements are highly variable, these measurements are averaged over a long period of time to reduce the effects of short-term or intraday variations. If the expected signal intensity is not found, the starting laser energy density value for the measurement of the analytical sample can be readjusted, serving as a basis for building the single-measurement-based variations for the analytical sample discussed above.

[0028] In various embodiments, performance trends can be derived from the abundance or intensity of ions detected in the analyzer, and in particular, average abundance or intensity can be used. A measure of intensity or abundance can be, for example, the total amount of ion current detected in the spectral data, sometimes referred to as the total ion count (TIC). The TIC can be detected over the entire spectral range or over a limited subrange, such as over 30,000 atomic mass units (amu), 20,000 amu, 10,000 amu, 5,000 amu, or 1,000 amu of spectral data. Also, average intensities over the entire spectral range or a portion thereof, such as over 30,000 atomic mass units (amu), 20,000 amu, 10,000 amu, 5,000 amu, or 1,000 amu of spectral data, can be used as a basis. An extensive database ensures statistical robustness. That is, outliers are given less weight when considered relative to multiple control samples, control sample sampling, or measurement procedures.

[0029] In various embodiments, the performance interval may include a variation from the performance guide value by 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. When intensity measurements are used to assess performance, substantial knowledge of the molecular content of control samples allows for the determination of intensity guide values ​​located in an interval. In this case, the upper limit is for a high intensity resulting from avoiding detector saturation, and the lower limit is for a low intensity derived from experience regarding the height of the ion current signal necessary to enable detectable and reliable evaluation of the spectral data. The interval limits may be equidistant or non-equidistant from the performance guide value. In one embodiment, the upper interval limit may be located closer to the performance guide value than the lower interval limit, and in another embodiment, the lower interval limit may be located closer to the performance guide value than the upper interval limit.

[0030] In another embodiment, the performance trend may be derived from the quality of characterization of the molecular content of a substantially known control sample. For example, if a microorganism is used as the control sample, the highest similarity index (also called log(score)) of the associated spectral reference data of an ion spectroscopy system designed for taxon identification, such as the Bruker MALDI Biotyper®, may serve as the performance guide value. Because 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 effect of the trend causes the log(score) to fall below the lower interval limit of 2.00 over a long observation period (and thus regardless of outliers), it may be necessary to adjust the laser operating parameters to offset this performance degradation.

[0031] In various embodiments, the evaluation in step (c) may involve averaging, particularly mean or median averaging, of multiple control sample spectral data. The averaging may be based, in particular, on an arithmetic mean. A moving average may be formed by recording control sample spectral data for multiple control samples, control sample samplings, or measurement runs; in other words, a time-varying subset may be presented from a set of measurements, for example, by considering control sample spectral data from a moving time window for averaging, optionally over multiple measurement runs.

[0032] In various embodiments, averaging is performed 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., 10-10 for a single spectral data image. 5 ~10 7Averaging may be applied to (i) multiple control samples or measurement runs from (i) more than one laser shot, or (ii) a predetermined period of time, e.g., 7-21 days, 1-4 weeks, or 1-2 months. Similarly, averaging may be applied to the sampling of a predetermined number of individual control samples, e.g., 400-2,000 individual control samples. A common factor in all embodiments of the present disclosure is that the need to adjust laser operating parameters is not dependent on a single control sample, and certainly not on the sampling of an individual control sample, but rather on multiple control samples and the sampling of multiple control samples.

[0033] In various embodiments, the laser energy density, laser fluence, laser power, and / or laser intensity in laser-assisted ionization can be modified to adjust operational parameters. In particular, the laser energy density, laser fluence, laser power, and / or laser intensity can be increased by a preset percentage if a change in performance trend is observed. The increase can be substantially in the single-digit percentage range, e.g., 1 to 10 percent, particularly about 1 percent. In the case of MALDI ionization, ion yield is proportional to the laser fluence to the 6th to 8th power, and a 1 percent increase in the laser fluence is associated with an increase in detectable ions of about 6 to 8 percent. Such measures may already be sufficient to offset negative performance trends and adapt the laser range. Alternatively, the adjustment of the laser operational parameters can involve predefined increments, e.g., specific percentages. It may also be calculated, for example, based on a fixed increment (optionally a percentage), depending on the trend progression and / or absolute variation from the interval limits, which is corrected with a trend-dependent term to avoid oversteer in the adjustment and make the adjustment routine less dependent on outliers.

[0034] In various embodiments, a notification and / or marking may be generated when the number of adjustments of an operating parameter reaches or exceeds a predetermined value, e.g., 2 to 10 adjustments, particularly 3 to 5 adjustments. The notification may take the form of an entry in a log file of the ion spectroscopy system used. Notifications can also be directed to the user's immediate attention, such as a window opening with a text or pictorial message in the graphic user interface of the ion spectroscopy system's computer, or an automatically generated email or SMS summarizing the results of the spectral data evaluation. Such a text / pictorial message, email, or SMS can be sent directly to a recipient address of the manufacturer or service partner of the ion spectroscopy system to automatically issue a maintenance request. The marking may include, in particular, an entry in the comments field of the metadata of the spectral data recorded under the changed laser conditions. In this way, the user can also track the automated laser readjustment at a later point in time, e.g., in the evaluation of the spectral data of the analytical sample detected together with that of a control sample.

[0035] In various embodiments, the composition of the analytical sample may be substantially unknown. A control sample differs from an analytical sample, particularly in that the control sample's molecular content and its behavior during laser-assisted ionization are well known and characterized, which is why the control sample is used as a control variable. In contrast, information about the analytical sample must first be confirmed from the corresponding spectral data, possibly through careful post-processing. In the case of MALDI ionization, only the matrix material is well known in the analytical sample preparation. In contrast, the molecular content and ionization behavior of the analyte molecules are unknown and must be confirmed. The analytical sample may, for example, be a preparation that is expected to contain microorganisms and / or extracted molecules, the purpose of which is to determine the taxonomic group of the microorganisms down to the species level and / or to determine their resistance behavior to antimicrobial substances. The analytical sample may also be a preparation containing cells, e.g., eukaryotic cells, for investigating their behavior and / or response to exposure to a specific stimulus, e.g., a toxicologically and / or pharmacologically active substance.

[0036] In a second aspect, the present disclosure relates to an ion spectroscopy system having an ion analyzer, an ion source connected to the ion analyzer, and a processor unit in communication with the ion analyzer and the ion source, and designed and programmed to coordinate and perform the method as elucidated and described above. [Brief explanation of the drawings]

[0037] For a better understanding of the present invention, reference is made to the following drawings, in which elements are not necessarily to scale and which are intended primarily to illustrate (mostly diagrammatically) the principles of the invention, in which elements corresponding to each other in different figures are identified by the same reference numerals. [Figure 1] 1 is a schematic diagram of the principle of an axially linear time-of-flight mass spectrometry system using laser-assisted ionization, such as the Bruker microflex® LT / SH. [Figure 2] FIG. 1 is a schematic diagram of a typical sequence of MALDI spot sample preparation. [Figure 3] FIG. 1 is a schematic diagram of the principles of performance monitoring and control in the context of the present disclosure. [Figure 4] 1 is a schematic diagram of an ion spectroscopy system capable of implementing the methods outlined in this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0038] Although the present invention has been described and elucidated using numerous embodiments, those skilled in the art will recognize that various changes in form and detail can be made without departing from the scope of the technical teachings defined in the appended claims.

[0039] The use of individual control sample measurements for ion source performance monitoring and control can lead to frustrating results, for example, because the individual control samples themselves may be subject to errors as a result of preparation failures. Regarding MALDI preparations, for example, if the matrix material to embedded molecule ratio (typically ~10,000–5,000:1) does not meet specifications, this can lead to matrix materials that are difficult to crystallize. Particularly in clinical applications, such as the Bruker MALDI Biotyper®, the analytical system may be operated by personnel with only the most relevant training. Therefore, this operator may not be able to recognize incorrect preparation of individual control samples. As already mentioned at the beginning, regular verification by trained staff from the manufacturer or its service partners can provide a remedy, but it requires high costs and significant personnel investment. Control sample preparations that do not meet requirements may be due to the age of the control sample substrate itself or the poor quality of the reagents used in the control sample preparation, for example, if the user does not notice or ignore the expiration date.

[0040] The objective is to optimize the ion spectroscopy system once after delivery to the user by trained personnel and to reduce, or at least minimize as much as possible, the number of maintenance operations required by the trained personnel or the user during the course of operation. In this way, it is possible to reduce effort and costs. This objective can be achieved by a (semi-)automatic closed-loop control of the operating parameters of the laser for laser-assisted ionization, apart from other measures such as ion source cleaning or detector voltage adjustment, mentioned at the beginning, which are not the subject of this disclosure but can serve simply as add-ons.

[0041] A method for monitoring and controlling the performance of an ion source includes the following steps: (a) performing 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, preferably in the same measurement run; (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) for multiple control samples and optional measurement runs to collect multiple control sample spectral data and evaluate the spectral data of each individual control sample so that the evaluation is weighted lightly and performance trends emerge; (d) if the performance trend falls outside a predefined performance interval, adjusting operating parameters of a laser in the ion source to adjust the laser to fit within the interval; and (e) repeating steps (a) through (d) for continuous monitoring and control of the ion source.

[0042] In step (a), the control sample may be sampled in the same measurement run as the analytical sample, and in step (c), steps (a) and (b) may be repeated for multiple measurement runs and control samples. A measurement run in the context of the present disclosure may be considered to be an operation particularly related to the operating mode of an ion spectroscopy system. For example, a measurement run may include all measurements recorded from a (control) sample on a sample carrier while the sample is present in the space within the ion source. One example, particularly in the case of MALDI, is introducing a sample carrier into a reduced pressure region of an ion source while the ion source is operating under reduced pressure. If a sample carrier is removed from the ion source, e.g., from the reduced pressure region, and then a further sample carrier containing a new (control) sample is reintroduced, all measurements of the new (control) sample on this next sample carrier may be considered a separate measurement run.

[0043] In addition to analytical sample preparations, it is also possible to place multiple control sample preparations on the sample carrier for laser-assisted ionization. When both control and analytical samples are present on the sample carrier, e.g., 2-10 technical control sample replicates, especially 4-8 technical control sample replicates, the control sample spectral data for all of these preparations can be used to confirm performance trends and / or their progression over time. Control sample preparations on the sample carrier can be sampled once or multiple times to generate control sample spectral data; this is limited, in principle, only by the amount of control sample. As a result, the control sample spectral data can contain individualized, separate spectra. Repeated sampling of the same control sample preparation increases the base of control sample spectral data for subsequent statistical evaluation and, in particular, can mitigate the effects of very brief perturbations in the measurement sequence, which may result in outlier measurements.

[0044] In particular, sampling a control sample before, simultaneously with, or after an analytical sample may mean that the control sample and analytical sample are examined in the same measurement operation. In particular, the control sample may be sampled immediately before or immediately after the analytical sample. The measurement mode or measurement settings of the ion spectroscopy system may correspond to the sampling of the analytical sample and the control sample, or may be different. As mentioned above, when the measurement operation includes recording the spectral data of all (control) samples on the sample carrier, this may mean that the sample carrier has a number of designated sample sites (e.g., sample spot sites on a steel plate such as the well-known AnchorChip from Bruker or the MBT Biotarget 96 carrier), and some of these sample sites are first prepared with a predetermined number of control samples, while some or all of the remaining sample sites are occupied by analytical samples. The control samples and analytical samples can then be sampled separately from each other (e.g., first all control samples, then all analytical samples) or alternately in blocks (e.g., control samples → analytical samples → control samples → analytical samples, etc.). In an ion source with multiple laser beams, it is also optionally possible to measure analytical and control samples in parallel from the same sample carrier if the connected ion analyzers are designed and configured to handle multiplexed ion currents, or if there are multiple ion analyzers.

[0045] The ion analyzer may be specifically designed as a mass analyzer that selects the ionized control sample by the principle of time-of-flight (TOF) dispersion. Mass analyzers separate charged molecules or molecular ions according to their mass-to-charge ratio, usually referred to as m / z.

[0046] Figure 1 shows a schematic diagram of the principle of an axially linear time-of-flight mass spectrometry system. In the top diagram, a laser bombards a (control) sample on a sample carrier 2, prepared, for example, with a MALDI matrix material. An electrode assembly 4 accelerates the ions formed upon laser bombardment, optionally after a delay, along a linear trajectory 8 into the flight tube 6 of a time-of-flight analyzer. Because different molecular species formed upon laser bombardment have different masses and approximately uniform charges, they are accelerated with approximately the same kinetic energy but at different velocities. Lighter molecules, represented by small black circles, therefore travel faster than heavier molecules, represented by larger circles, and strike the detector 10 sooner. The detector 10 may take the form of, for example, a dynode array and a multichannel plate (MCP). This time-dependent arrival at the detector allows for the assignment of charge-based mass (m / z) values ​​to detected events (bottom diagram of Figure 1).

[0047] In principle, it is also possible to use other types of mass-dispersive separators, such as quadrupole mass filters (single quad), triple quadrupole analyzers (triple quad), ion cyclotron resonance (ICR) cells, Kingdon analyzers such as Orbitrap® (Thermo Fisher Scientific), as well as time-of-flight analyzers, which may provide both axially linear structures (as shown in Figure 1) and reflector structures and / or structures with orthogonal acceleration (OTOF) of ions into the flight path. It is also conceivable to design the ion analyzer as a mobility analyzer or a combined mobility / mass analyzer, as already detailed above.

[0048] The control sample may include a preparation of a microorganism, such as a prokaryotic organism, particularly a bacterial species. Microbial cells of precisely known taxonomic groups and defined content of soluble molecules, such as liposomal proteins and peptides, are commercially available. This configuration, in the case of a standardized preparation, is expected to result in easily predictable and consistent analyzer and detector responses, for example, in terms of mass signal profile or abundance. One example is the Bacterial Test Standard for the Bruker MALDI Biotyper®, a MALDI axial linear time-of-flight mass spectrometry system. This standard sample consists of a typical Escherichia coli DH5α peptide and protein profile and additional proteins appropriate for the application, including mass calibration.

[0049] Preferably, multiple control samples are matched or identical, and in particular matched or identical control samples are used across multiple measurement runs. In the case of a microbial preparation as a control sample, measurements can be recorded over time from biological and / or technical replicates of that microorganism.

[0050] If the control sample contains microorganisms, the control sample is preferably sterilized. Sterilization may involve exposing the microorganisms to a metabolic inhibitor (e.g., an alcohol such as ethanol or isopropanol, or an acid such as formic acid) and / or to energy (e.g., heat or high-energy radiation, optionally ultraviolet light). In particular, sterilization means that the microorganisms lose their ability to reproduce even under conditions favorable for the microorganisms. In this way, biological risks associated with unintended / uncontrolled spread in analytical laboratories can be avoided. Under certain conditions, for example, when working in analytical laboratories with biosafety level 2 or higher, it may not be necessary to sterilize the microorganisms in the control sample. This presupposes that well-trained and skilled personnel are employed.

[0051] As mentioned above, the ion source may operate according to the MALDI principle. Figures 2A-2C show a schematic diagram of the MALDI process from preparation of a (control) sample to recording of spectral data. In Figure 2A, a (control) sample, e.g., a microbial suspension 12, or a suspension containing soluble molecules extracted from microorganisms, is introduced into a MALDI sample carrier 2 using a pipette 14 or other suitable dispensing device. * The MALDI sample carrier used may be, for example, a Bruker AnchorChip or MBT Biotarget 96 carrier, or a steel plate. After removing excess fluid from the suspension, a matrix substance solution can be applied to the cell layer and / or layer of soluble molecules from which the fluid has been removed by a suitable tool 16, e.g., a further pipette, shown here with tiled hatching. After the matrix is ​​incorporated, dried, and crystallized, the individual (control) samples 18 are ready for sampling by a laser beam 20. The resulting ions 22 can then be accelerated, for example, onto the flight path of a time-of-flight analyzer, as shown schematically in FIG. 1.

[0052] Performance trends can be derived from the abundance or intensity of ions detected in the analyzer, and in particular, average abundance or intensity can be used. An example of a measure of intensity or abundance is the total amount of ion current detected in the spectral data, sometimes referred to as the total ion count (TIC). The TIC can be detected over the entire spectral range or over a limited subrange, such as over 30,000 atomic mass units (amu), 20,000 amu, 10,000 amu, 5,000 amu, or 1,000 amu. It is also possible to use the average intensity over the entire spectral range or a portion thereof, such as over 30,000 atomic mass units (amu), 20,000 amu, 10,000 amu, 5,000 amu, or 1,000 amu. An extensive database ensures statistical stability. That is, for multiple control samples, control sample sampling or measurement procedures, outliers are given less weight.

[0053] Figure 3 is a schematic diagram illustrating the monitoring of performance trends over time, the recognition of fluctuations in performance trends, and the resulting feedback triggering of changes in the operating parameters of the laser for laser-assisted ionization. The lower diagram shows multiple measurement runs on the horizontal axis and the time series of the (average) ion current at the detector on the vertical axis. A measurement run may include ion spectroscopy analysis of all (control) samples placed on a sample carrier, for example, when the sample carrier is placed in the reduced pressure region of the ion source. In the example shown, nine sample carriers carrying control samples are accordingly included in the evaluation. The number of control samples or control sample samplings per measurement run evaluated for performance trends is shown here as a schematic and exemplary 3, and is represented in the figure by three bars per measurement run. It will be apparent that the number of control samples or control sample samplings per measurement run may be different. For example, 2 to 10 or 3 to 5 control samples can be sampled per measurement run, or multiple control sample samplings can be performed, for example, for a corresponding number of technical control sample replicates on the sample carrier. For example, in the case of sample carriers with designated spots for sample application, such as the AnchorChip or MBT Biotarget 96, the number of control samples must be compared with the number of analytical samples with unknown molecular content that the user wants to characterize by ion spectroscopy. Considering the limited space on the sample carrier, it is instead possible to consider the application and preparation of numerous individual control samples with high quality requirements, which are then subjected to multiple samplings to obtain a comprehensive spectral database for each measurement operation.

[0054] The central dashed horizontal line 24 in the bottom panel of Figure 3 indicates a guide ion current value that may be the 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 to 30,000 counts, particularly 27,000 counts. The outer dashed lines 26, positioned around this central line 24, represent interval limits around the guide ion current value at which the performance of the ion source and analyzer system can be described as meeting or satisfactory. The performance interval may include variations up to a predefined percentage, particularly ±30 percent, ±25 percent, ±20 percent, ±15 percent, or ±10 percent. The boundary for higher ion currents may arise from avoiding detector saturation, while the boundary for lower ion currents may be derived from experience regarding how high the ion current signal must be to enable detectable and reliable spectral data evaluation. The interval limits may be equidistant or non-equidistant from the performance guide value. In one embodiment, the upper interval limit may be located closer to the performance guide value than the lower interval limit, and in another embodiment, the lower interval limit may be located closer to the performance guide value than the upper interval limit.

[0055] Performance trends may also be derived from the quality of characterization of the molecular content of a substantially known control sample. For example, if a microorganism is used as the control sample, the highest similarity index (called the log(score)) of the associated spectral reference data of an ion spectroscopy system designed for taxon identification, such as the Bruker MALDI Biotyper®, may serve as a performance guide value. Because 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 effect causes the log(score) to be at and / or below the lower interval limit of 2.00 over a long observation period (and thus regardless of outliers), adjustments to the laser operating parameters may be necessary to offset this performance degradation.

[0056] In the evaluation in step (c), the spectral data of multiple control samples may be subjected to averaging, in particular mean or median averaging. The formation of the average may in particular be based on an arithmetic mean. Figure 3 shows the ion current value averaged over the control sample or sampling of the control sample for each measurement run by a dotted circle 28. Formation of a moving average is possible by recording the spectral data of multiple control samples, samplings of the control sample, or measurement runs. In other words, a time-varying subset can be derived from a set of measurements, for example, by considering the spectral data of the control sample from a moving time window for forming the average over an optionally large number of measurement runs.

[0057] The averaging may be performed 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., 10 for a single spectral data image. 5 ~10 7Averaging may be applied to (i) multiple control samples or measurement runs from (i) more than one laser shot, or (ii) a predetermined period of time, e.g., 7-21 days, 1-4 weeks, or 1-2 months. Similarly, averaging may be applied to the sampling of a predetermined number of individual control samples, e.g., 400-2,000 individual control samples. A common factor in all embodiments of the present disclosure is that the need to adjust laser operating parameters is not dependent on a single control sample, and certainly not on the sampling of an individual control sample, but rather on multiple control samples and the sampling of multiple control samples.

[0058] 3 includes trend lines 30 connecting the different ion current averages, which allows one to assess how the ion current values ​​observed for the control sample subsequently change. In the illustrated example, the third measurement run confirmed that the ion current average reached the lower interval limit 26, and the fourth measurement run confirmed that the ion current average even left the interval range. As a result of this finding, a countermeasure was taken to change the laser operating parameters (see arrow 32) to offset this power reduction.

[0059] As is evident from the labels on the vertical axis in the top panel of Figure 3, the laser energy density, laser fluence, laser power, and / or laser intensity in laser-assisted ionization can be modified to adjust the operating parameters. 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 fluctuation is observed (see Increment #1). The increase can be substantially in the single-digit percentage range, e.g., 1 to 10 percent, particularly about 1 percent. Alternatively, the adjustment of the laser operating parameters can include predefined increments, e.g., specific percentages. It can also be calculated based on, for example, a fixed increment (optionally a percentage) depending on the progression of the trend and / or absolute fluctuation from the interval limit, which is compensated for with a trend-dependent term to avoid oversteering in the adjustment and make the adjustment routine less dependent on outliers.

[0060] As shown in the upper diagram of Figure 3, after adjusting the laser parameters between the fourth and fifth measurement runs, the average ion current returned to within the interval range without any human intervention. The ion spectroscopy system, equipped with an ion analyzer and ion source monitored and controlled in this manner, can then be further operated and continue to provide desired or satisfactory measurement performance. If, during further measurement runs, it becomes apparent that the performance trend again approaches the interval limits, as seen here for example in the seventh measurement run, and then falls outside the interval range, as shown in the eighth measurement run, the laser operating parameters can be adjusted again (see Adjustment #2) in a manner similar to that described above. Adjustment #2 may be the same as Adjustment #1 or may be performed in different adjustment increments.

[0061] If the number of adjustments of an operating parameter reaches or exceeds a predetermined value over time, e.g., 2 to 10 adjustments, particularly 3 to 5 adjustments, a notification and / or marking may be generated. The notification may take the form of an entry in the log file of the ion spectroscopy system used. It may also be a notification intended to attract the user's immediate attention, such as a window opening with a text or picture message in the graphic user interface of the ion spectroscopy system's computer, or an automatically generated email or SMS summarizing the results of the spectral data evaluation. Such a text / picture message, email, or SMS may be sent via telecommunication directly to the recipient address of the manufacturer or service partner of the ion spectroscopy system to automatically issue a maintenance request. The marking may, in particular, include an entry in the comments field of the metadata of the spectral data recorded under the changed laser conditions, allowing the user to track the automated laser readjustment at a later time, e.g., in the evaluation of the spectral data of the analytical sample detected together with that of a control sample.

[0062] The composition of the analytical sample may be substantially unknown. A control sample differs from an analytical sample in that, in particular, its molecular content and its behavior during laser-assisted ionization are very well known and characterized in the control sample, which is why the control sample is used as a control variable. In contrast, information about the analytical sample must first be confirmed, possibly by careful post-treatment. In the case of MALDI ionization, only the matrix material is generally known in the analytical sample preparation. In contrast, the molecular content and ionization behavior of the analyte molecules must be confirmed. The analytical sample may, for example, be a preparation that is expected to contain microorganisms and / or extracted molecules, the purpose of which is to determine the taxonomic group of the microorganism down to the species level and / or to determine its resistance behavior to antimicrobial substances. The analytical sample may also be a preparation containing cells, e.g., eukaryotic cells, for investigating their behavior and / or response to exposure to a specific stimulus, e.g., a toxicologically and / or pharmacologically active substance.

[0063] Figure 4 shows a schematic diagram of an ion spectroscopy system 40 having an ion analyzer 42, an ion source 44 connected to the ion analyzer 42, and a processor unit 46 in communication with the ion analyzer 42 and the ion source 44 and designed and programmed to carry out the method described above, particularly with reference to Figure 3. The path of ions from the ion source 44 to the ion analyzer 42 is represented by arrow 48. Control and information receiving communication between the processor unit 46 and the ion source 44 and the ion analyzer 42 is indicated by double arrow 50.

[0064] The present invention has been described with reference to various specific embodiments. However, it will be apparent that various features or details of the described embodiments can be changed without departing from the scope of the present invention. Furthermore, features and means disclosed in connection with different embodiments may be combined as needed, as would be feasible for a person skilled in the art. Furthermore, this specification is intended merely to illustrate the present invention and not to limit the scope of protection, which is defined solely by the appended claims, with respect to existing equivalents.

Claims

1. 1. A method for monitoring and controlling the performance of an ion source, comprising: (a) performing laser-assisted ionization of a control sample of substantially known composition, sampled before, simultaneously with, or after the analytical sample; (b) generating control sample spectral data from the ionized control sample in an ion analyzer coupled to the ion source; (c) collecting spectral data for multiple control samples and repeating steps (a) and (b) for multiple control samples to evaluate the spectral data of each individual control sample in a manner that minimizes the weight of the data and allows for performance trends to be observed; (d) if the performance trend falls within a range outside a predefined performance interval, adjusting operating parameters of the laser of the ion source to adjust the laser to fit within that interval; (e) repeating steps (a) through (d) for continuous monitoring and control of the ion source; A method comprising:

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

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

4. The method of claim 1 , wherein the control sample comprises a treatment of a microorganism, in particular a treatment of a bacterial species.

5. The method of claim 1 , wherein the ion source operates according to MALDI principles.

6. The method of claim 1 , wherein the performance trends are derived from the abundances or intensities of ions detected in the analyzer.

7. The method of claim 1 , wherein the performance interval comprises a variation of up to a predefined percentage from a performance guide value.

8. 2. The method of claim 1, wherein said evaluation in step (c) comprises subjecting said multiple control sample spectral data to averaging, in particular mean or median averaging.

9. 9. The method of claim 8, wherein the averaging is applied to a predetermined number of (i) control samples, (ii) measurement runs, (iii) laser activations, or (iv) multiple control samples or measurement runs from a predetermined time period.

10. The method of claim 1 , wherein the adjustment of the operating parameters involves changing the laser energy density, laser fluence, laser power and / or laser intensity in the laser-assisted ionization.

11. The method of claim 1 , wherein a notification and / or marking is generated if the number of adjustments of the operating parameter reaches or exceeds a predetermined value.

12. The method of claim 1 , wherein the composition of the analytical sample is substantially unknown.

13. 13. An ion spectroscopy system comprising an ion analyzer, an ion source connected to said ion analyzer, and a processor unit designed and programmed to communicate with said ion analyzer and said ion source and to coordinate and carry out the method of any one of claims 1 to 12.

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