Gain calibration procedure

Abstract

A method for gain calibration for an ion detector operating at a detector voltage, the method comprising the following steps: Generation of single ions; Determining a parameter of a first relationship between a detector output of the ion detector and a number of ions for a first detector voltage by detecting individual ions at the ion detector; Detecting an ion peak at the ion detector using the first detector voltage to determine a number of ions in the ion peak based on the parameter of the first relationship; adjusting the detector voltage to reduce a ratio between the detector output and a noise level and thereby obtain a second detector voltage at which the detector output for the ion peak remains above the noise level; Determining a parameter of a second relationship between the detector output and the number of ions for the second detector voltage based on the determined number of ions in the ion peak.

Description

Field of the invention

[0001] The present disclosure relates to a method for gain calibration, particularly for use in a mass spectrometer. Background of the invention

[0002] Mass spectrometry is an analytical technique that can be used to measure the mass-to-charge (m / z) ratio of ions. A mass spectrometer typically consists of three main parts: an ion source for generating ions, a mass analyzer for separating ions by m / z, and an ion detector for detecting the separated m / z ions.

[0003] Ion detectors measure the induced charge or current generated when an ion passes by or impacts an ion detector's surface. Since individual particles generally produce effects too small to be directly detected at nonrelativistic energies, ion detectors generally amplify the signal generated by a passing / indicating ion so that it can be measured. Typically, this is done by converting an incoming ion into secondary electrons.

[0004] The gain of the ion detector (the number of electrons generated per incident ion) is typically controlled by adjusting the voltage across an electron multiplier stage as well as the energy of the incident ions. It is generally preferable for a detector to operate with sufficient gain to reliably detect single ions, thus maximizing sensitivity. However, excessive gain reduces the detector's dynamic range (the ratio between the largest and smallest values ​​that can be detected), which is a key factor in performance. Excessive gain also accelerates aging, which affects the detector's lifetime. In addition, aging and short-term effects, such as water desorption, lead to a perceptible change in gain, even over the course of a day.

[0005] In addition to the need for reliable ion detection, the current output from a detector allows the measurement of the number of ions in the signal, thus providing quantitative information about an analyte. Mass spectrometers incorporating ion traps also typically require the measurement of ion current to regulate the number of ions trapped in the trap during analysis to prevent harmful space charge effects, in a process known as "automatic gain control." Therefore, regular gain calibration is important to accurately determine the number of ions in a signal.

[0006] Conventional mass spectrometers generally include routines for measuring and calibrating detector gain that must be performed regularly. These routines are typically too slow to monitor and / or recalibrate detector gain during or between experimental runs and are instead run as part of daily and / or weekly calibration routines.

[0007] Time-of-flight (TOF) mass analyzers operating at repetition rates of 10–30,000 Hz typically use micro-channel plate (MCP)-based detectors, for example, as described in US 2004 / 0206911 A1, due to their uniform detection surface and sub-ns timing, which is best for high resolution. A standard detector incorporating a pair of Chevron MCPs 101 for enhanced gain is described with reference to Fig. 1, as briefly explained below.

[0008] A microchannel plate 101 is made of a high-resistivity material and includes an array of channels extending between a first surface and a second surface of the microchannel plate 101. The channels are parallel to each other and are typically at a small angle to a normal of the MCP input surface (e.g., about 8°). Fig. In the pair of chevron MCPs 101 shown in Figure 1, the channels of the two plates 101 are angled relative to each other to form a chevron (V-like) shape.

[0009] Ion packets 102 impact the MCP 101. A particle 102 entering one of the channels is guaranteed to hit the channel wall due to the angled channel and emit secondary electrons 103. The secondary electrons 103 are accelerated within the resistive channel of the MCP 101 and emit further secondary electrons 103 when they hit the channel walls, amplifying the original signal in cascades of electron-to-electron conversion. Electrons exiting the first plate 101 initiate another cascade in the second plate 101.

[0010] The gap between the MCPs 101 in Fig. 1 allows charge spreading across multiple channels, increasing gain. Alternatively, the MCPs could have no gap between them to preserve spatial resolution.

[0011] The Fig. The Chevron Dual MCP 100 shown in Figure 1 is characterized by a gain of 1E+6 to detect single ions and a lifetime of less than one coulomb (1C), measured at the output of the second MCP 101. The Chevron Dual MCP 100 can alternatively have an operating lifetime of 3C or 10C. This may be gain-dependent, and the lifetime may increase inversely proportional to the gain.

[0012] Chevron MCPs tend to suffer from limited dynamic range, although fast detector schemes capable of linear detection from single ions up to more than 1000 ions per pulse are also described, for example, dynode chains with magnetic electron focusing as described in US 6,982,428 B2 and MCPs / dynodes connected to scintillator / photomultiplier combinations to add additional amplification stages in US 7,180,060 B2.

[0013] A standard procedure for detector calibration (e.g. for the Fig. 1) is to measure the response of individual ions at different detector gain settings. Measuring the intensity or range of individual ion pulses provides the most direct measurement, while measuring the rate of individual ion pulses provides a measurement of the ion current, and a plateau of a count rate versus detector voltage plot represents an optimal voltage setting for efficient single-ion detection. An example of this plot is shown in Fig. 2, based on a similar drawing in Prohaska et al., Sector Field Mass Spectrometry for Elemental and Isotopic Analysis, Royal Society of Chemistry, 2015.

[0014] As in Fig. As can be seen in Figure 2, increasing the detector voltage initially leads to a steep increase in the count rate. However, there is a plateau region in the curve where increasing the detector voltage has only a minor effect on the count rate. For efficient single-ion detection, it is advantageous to work in this plateau region. As shown in Fig. As shown in Figure 2, the count rate begins to rise steeply again when the detector voltage is further increased beyond the plateau region.

[0015] The problem arises that a reliable measurement of the single-ion region at the minimum gain level required to detect such ions is difficult, as they are located around the noise level. Instead, such values ​​can be derived from a calibration of the trend of individual ion regions measured at a higher gain range. US 9,564,301 B2 shows another example of a single-ion measurement in time-of-flight instruments, where detection optimization is performed by comparing single-ion and noise peaks.

[0016] The main difficulty with single-ion measurements is avoiding the simultaneous detection of multiple ions. The instrument can be detuned to attenuate the ion beam and reduce the likelihood of multiple ion impacts, but this results in many blank acquisitions. For instruments with kHz-level acquisition rates, such as orthogonal time-of-flight analyzers, this is not a major problem, but for instruments incorporating ion traps and operating at lower acquisition rates, i.e., significantly below 1 kHz, the calibration process becomes longer or unreliable.

[0017] Ion trap instruments can instead incorporate ion population measurements based on the statistical variation of peak intensities of multiple ion populations over many acquisitions, as described in US Pat. No. 7,109,474 B2. This method eliminates the need for single-ion measurements and achieves highly consistent results. However, it is relatively slow and suffers from large systematic errors caused by additional sources of noise in the peak intensity, such as the number of secondary electrons / ions produced by an incident analyte ion.

[0018] US 2021 / 0013019 A1 describes applying a voltage to an electrode such that ions with the same m / z, simultaneously ejected from an ejector, are scattered in a temporal direction, resulting in multiple low peaks corresponding to individual ions being observed in a profile spectrum. The peak height of each peak in the profile spectrum is determined, and the median of these values ​​is determined. If the mean peak height is not within a predetermined reference range, the detector voltage is increased by a predetermined amount. The process is repeated until the determined median of the peak heights is within the reference range, and then the detector voltage at that point is selected as the optimal voltage.

[0019] US 10,593,525 B2 describes a method for calibrating a mass spectrum of a time-of-flight mass spectrometer (TOF MS) to account for temperature changes. Ions are introduced into a Fourier transform mass spectrometer (FTMS), and their mass-to-charge ratios are determined. Ions, including calibration ions, are also introduced into a TOF MS, and the m / z ratios of at least the calibration ions are also determined. Specific peaks representing calibration ions are selected and aligned between the TOF-MS and FTMS spectra. The relative position of the matching peaks in each spectrum is then used to determine a temperature correction factor for the TOF-MS data based on the relative independence of the FTMS spectrum with respect to temperature.

[0020] US 2018 / 0286647 A1 relates to a method for operating a mass spectrometer and a mass spectrometer that use a variable or ion-specific detector gain or a variable emission current during mass calibration, resolution tuning, or tuning of ion optics.

[0021] US 2009 / 0108191 A1 concerns the calibration of ion detectors in mass spectrometers, in particular the challenges in measuring and analyzing the pulse height distribution (PHD) of electron multipliers.

[0022] US 2006 / 0080045 A1 relates to a method for dynamically adjusting the control voltage and thus the gain of an ion detector, such as an electron multiplier, in order to optimize its performance, in particular the dynamic range.

[0023] Detector gain fluctuations and instabilities in the conventional methods described above pose challenges for efficient single-ion detection, dynamic range, and quantification of the number of ions detected in a time-of-flight analyzer. The conventional approach also lacks an accurate method for measuring and calibrating the detector that is fast enough to be performed in practice within or between experimental runs, even for relatively slow analyzers containing ion traps.

[0024] A gain calibration method that overcomes these problems is desirable. Brief description of the invention

[0025] According to the present disclosure, a method of gain calibration for an ion detector operating at a detector voltage is provided.

[0026] The procedure includes the following steps: Generation of single ions; Determining a parameter of a first relationship between a detector output of an ion detector and a number of ions for a first detector voltage by detecting individual ions at the ion detector; Detecting an ion peak at the ion detector using the first detector voltage to determine a number of ions in the ion peak based on the parameter of the first relationship; adjusting the detector voltage to reduce a ratio between the detector output and a noise level and thereby obtain a second detector voltage at which the detector output for the ion peak remains above the noise level; Determining a parameter of a second relationship between the detector output and the number of ions for the second detector voltage based on the determined number of ions in the ion peak.

[0027] This method allows for more accurate and rapid determination of the optimal detector voltage. The optimal detector voltage provides sufficient gain to reliably detect single ions, thus maximizing sensitivity. Excessive gain reduces the dynamic range of the detector and accelerates aging, so accurately determining the optimal detector output voltage can improve the detector's lifetime and dynamic range. Advantageously, in some cases, the optimal detector voltage can be determined without the need to create a calibration curve of single ion responses.

[0028] The method may further comprise monitoring the gain calibration of the ion detector. This may involve detecting a second ion peak at the ion detector using a third detector voltage. The third detector voltage may be the same as the first detector voltage used during the initial gain calibration. Alternatively / additionally, the third detector voltage may be a high gain voltage. Monitoring the gain calibration may further comprise steps of determining the number of ions in the second ion peak and determining a second parameter of the second relationship between the detector output of the ion detector and the number of ions for the third detector voltage based on the determined number of ions in the peak. The number of ions may be a single ion or multiple ions.Monitoring may further include comparing the second parameter of the second relationship with an expected value based on the second relationship and determining, based on the comparison, whether a drift in the gain has occurred. The expected value of the second parameter may be determined based on a calculated calibration curve or may be the determined first parameter of the second relationship.

[0029] Therefore, gain calibration can be quickly monitored during or between experimental runs rather than being operated as part of daily or weekly calibration routines.

[0030] Based on the determination that a gain drift has occurred, an adjustment or correction parameter can be calculated to adjust the detector voltage to a level at which the relationship between the detector output and the number of ions corresponds to the expected value of the second parameter of the second relationship, based on the second relationship. The detector voltage can then be adjusted based on the correction parameter / factor.

[0031] This monitoring can be performed without the need to create a calibration curve, as the trends of previously acquired relationships can be assumed to be true, providing rapid gain correction. Ion detector gain drift can thus be quickly corrected between or during experiments following gain monitoring.

[0032] The ion detector can be operated with the second detector voltage. The second voltage advantageously provides sufficient gain to reliably detect single ions and thus maximize sensitivity, while limiting excessive gain, which accelerates aging and reduces the detector's dynamic range.

[0033] The ion detector can be part of a time-of-flight mass spectrometer. In this case, the single ions can be obtained by setting the TOF-MS to operate in a temporally defocused mode, in which ions with the same mass-to-charge ratio arrive at the ion detector at different times.

[0034] Advantageously, this method of generating single ions means that many single-ion pulses can be recorded in a single acquisition, making single-ion calibration accessible even for relatively slow analyzers, such as multi-reflection time-of-flight (MR-TOF) analyzers. Statistical methods based on pulse height variation can be used with such analyzers, but they are slow and can contain significant systematic errors. Defocusing ions to generate single ions also eliminates the need for careful attenuation of an ion beam to remove multiple ion peaks while minimizing empty spectra, which would be required via beam attenuation techniques.

[0035] Detecting the second ion peak may involve setting the TOF-MS to operate in the temporally defocused mode, such that the second ion peak corresponds to a single ion. The single ion response used to determine the parameter of the first relationship can then be directly compared to the single ion response of the second peak, allowing monitoring of drift enhancement to occur more quickly.

[0036] The detector voltage can be adjusted stepwise (i.e., adjusted in small increments). A calibration curve can then be easily calculated based on the detector output generated by detecting ions at the ion detector for each detector voltage step.

[0037] The first relationship and the second relationship can be the same relationship, but the parameters of the first relationship and the parameters of the second relationship are different. This allows a direct comparison between the parameters, enabling faster calibration / monitoring of the gain.

[0038] The ion detector may comprise a first ion detector device and a second ion detector device. The second ion detector device may be part of a time-of-flight (TOF) mass spectrometer (MS) or an ion trap. The steps of determining a parameter of a first relationship and detecting an ion peak at the ion detector may be performed with respect to the first ion detector device. The steps of adjusting the detector voltage and determining a parameter of a second relationship may be performed with reference to the second ion detector device. Thus, the first ion detector device may be used to calibrate the gain of the second ion detector device. This may provide a straightforward gain calibration that is fast and relatively stable. The gain calibration may also advantageously be performed using other cross-calibration methods, e.g.Mass cross calibration between the first and second ion detector devices.

[0039] The first ion detector device can be part of a Fourier transform mass spectrometer. The FTMS can be an orbital trap or Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The output of an FTMS (the position and shape of peaks in a mass spectrum it generates) is relatively stable over time (e.g., with respect to short- and long-term temperature changes) and therefore offers a convenient and stable way to provide gain cross-calibration or gain calibration monitoring.

[0040] Because the behavior of the FTMS is sufficiently similar over time and across other instruments of the same type, the calibration of a particular instrument (e.g., a first orbital trap mass spectrometer) can be applied to another instrument of the same type (e.g., a second orbital trap mass spectrometer). This means that the number of ions producing a particular output (e.g., signal-to-noise ratio) becomes a known property for a class of instruments, rather than something that must be calibrated for each instrument.

[0041] Detecting the second ion peak at the ion detector may be performed relative to the first ion detector device, and the step of determining a parameter of a third relationship may be performed relative to the second ion detector device. Thus, the more stable first ion detector device may be used to monitor the gain calibration of the second ion detector device over time.

[0042] The parameter or factor of the first relationship can be a signal-to-noise (S / N) ratio. The S / N ratio of an instrument (e.g., an FTMS) can be proportional to the number of ions in a detected ion peak and can thus be used to determine how many ions are in a subsequently detected ion peak.

[0043] An FTMS can be used to detect multiply charged single ions at the first ion detector device and calculate the S / N ratio of the multiply charged single ions. The S / N ratio of a singly charged single ion can then be determined based on the calculated S / N ratio of the multiply charged single ions. The S / N ratio of the singly charged single ion can then be used as the proportionality constant between the number of ions in a detected ion peak and the S / N ratio of the detected ion peak.

[0044] A correction factor can be applied to the specific number of ions in the ion peak to account for the transmission of ions to the second ion detector device. The correction factor can be between 30% and 50%. Applying the correction factor increases the accuracy of the gain calibration during cross-calibration between two ion detector devices.

[0045] Single ions can be generated by beam attenuation, fragmentation, ion scattering with background gas, or using an electrospray ionization (ESI) source. Thus, gain calibration can be performed on a wide variety of instruments.

[0046] The methods described above may be implemented as a computer program comprising instructions for operating a computer or computer system. The computer program may be stored on a computer-readable medium.

[0047] The computer system may include a processor, such as a central processing unit (CPU). The processor may execute logic in the form of a software program. The computer system may include memory that includes a volatile and a non-volatile storage medium. A computer-readable medium may be included to store the logic or program instructions. The various parts of the system may be connected using a network (e.g., wireless networks and wired networks). The computer system may include one or more interfaces. The computer system may include a suitable operating system, such as UNIX (including Linux), Windows (RTM).

[0048] The above methods may be implemented in a system comprising a mass spectrometer assembly and a controller configured to operate the mass spectrometer assembly.

[0049] It should be understood that each feature described above may be used with any particular aspect or embodiment of the invention. Furthermore, the combination of any specific device, structure, or method features is also contemplated, even if such combination is not expressly disclosed. Brief description of the drawings

[0050] The present invention may be put into practice in many ways and embodiments will now be described by way of example only with reference to the accompanying drawings in which: Fig. Figure 1 shows a detector incorporating a pair of chevron MCPs; Fig. Figure 2 illustrates a plot of count rate versus detector voltage for detector calibration over a single ion counting plateau; Fig. 3A shows a flowchart method for calibrating ion gain; Fig. 3B shows a flowchart method for monitoring ion gain calibration; Fig. Figure 4 illustrates a multiple reflection time of flight that can be used with the methods described herein to calibrate ion gain; Fig. Figure 5 shows an example of single ion scattering obtained by temporally defocusing a TOF detector as described herein; Fig. Figure 6A shows a flowchart describing a detector gain calibration method based on direct single ion measurement; Fig. 6B illustrates a flowchart describing a fast gain correction method suitable for interleaving in trial runs; Fig. 7 illustrates a flowchart showing an indirect detector gain calibration method combining a temporally defocused single ion method and a focused multiple ion method; Fig. 8 shows calibration diagrams generated by repeated experiments with indirect single-ion measurement and direct single-ion measurement as well as hand measurements; Fig. Figure 9 shows a comparison of a statistical method for determining a single ion range and the indirect single ion measurement described herein; and Fig. 10 illustrates an exemplary hybrid orbital trap mass spectrometer and TOF instrument that may be used with the methods described herein;

[0051] Please note that the figures are presented schematically for simplicity and are not necessarily drawn to scale. Identical features are designated by identical reference numerals. Detailed description of preferred embodiments

[0052] Fig. Figure 3A shows a flowchart of a gain calibration method for an ion detector. At step 301, single ions are generated to measure a single ion response. The single ions can be generated by breaking down a concentrated ion packet that would arrive at the detector at a specific time into single ion arrivals at many different times.

[0053] The step 301 of generating individual ions can be achieved by various methods. For example, a temporal defocusing method can be used, which is described herein with reference to Fig. 4. Alternatively, fragmentation can be used to convert a mass of ions into multiple (or many) fragments. This scatters ions across many different m / z values ​​with a much wider range of possible flight times, greatly increasing the probability that these ions arrive as single ions. Any fragmentation method could be used to achieve this, such as collision-induced dissociation (CID), surface-induced dissociation (SID), electron-transfer dissociation (ETD), photodissociation, in-source fragmentation, and so on.

[0054] Another method for generating single ions in step 301 that can be used is scattering ions with background gas. In this method, ions impact the gas molecules and exit with an energy and time difference compared to other ions of the same mass. The time and energy difference is likely large enough that the system cannot refocus the ions, and the scattered ions arrive at very different times, allowing single ions to be detected.

[0055] Single ions could also be generated by beam attenuation. Alternatively, single ions can be generated using an electrospray ionization (ESI) source. In general, the ion current stability for commercial electrosprays is approximately 4% on a shot-to-shot basis for a calibration mixture (i.e., the ion current does not vary greatly between scans). Consistency between the ion current measurement (ion number) and the presence of a similar current in immediately subsequent measurements is an important prerequisite for critical processes such as ion population control (e.g., automatic gain control (AGC)), which is widely used to optimize the number of ions delivered in orbital trap mass spectrometers and other ion trap instruments.Therefore, this tolerance is more than sufficient for accurate calibration using the methods disclosed herein.

[0056] The single ions are detected at an ion detector operating at a first detector voltage. In step 302, a parameter of a first relationship between an output of the ion detector (e.g., a current or a voltage) and a number of ions for the first detector voltage is determined by detecting the single ions at the ion detector. For example, an ion detector at a first detector voltage (e.g., 1800 V) might produce an average 5 mV peak for a single ion. The parameter could therefore be a factor of 5 mV per ion. That is, the detector output can be considered a factor between the number of ions and the detector output. Alternatively, the parameter could be an S / N response of a single charge, since the S / N ratio can be proportional to the number of ions in an ion peak.To use the S / N response as a parameter, the detector's S / N ratio should be relatively stable over time. For example, the S / N ratio of a single charge might be 4.0 ± 0.4, so the parameter is a factor of 4.0 per ion.

[0057] The method may continue with step 303, in which an ion peak is detected at the ion detector using the first detector voltage to determine a number of ions in the ion peak based on the parameter of the first relationship. The ion peak may be a multiple ion peak (i.e., the number of ions in the ion peak determined in step 303 should be greater than one). Based on the factor of 5 mV per ion stated above with respect to step 302, a detected ion peak with an intensity of 200 mV may correspond to a determination that the ion peak contains 40 ions. In the case where the S / N response factor of 4.0 per ion is used, an intensity peak of 200 mV may correspond to a determination that the ion peak contains 50 ions.

[0058] At step 304, the detector voltage is adjusted (increased or decreased) to reduce a ratio between the detector output and a noise level, thereby obtaining a second detector voltage at which the detector output for the ion peak remains above the noise level. A signal at least 10 times the noise level is sufficient, and preferably the signal is 30 or 100 times the noise level. If the signal is much larger, a single ion pulse may be large enough to saturate the detector. The detector can be adjusted stepwise, and steps 302-304 can be repeated until the ratio between the detector output and the noise level is sufficiently reduced and / or the ion peak remains above the noise level.The single ion response and voltage data acquired during these steps can be used to plot a curve of the relationship between the detector output of the ion detector and the number of ions for a first detector voltage (e.g., as shown in ). Fig. 2 or Fig. 8). This can be called a calibration curve.

[0059] Because the detector voltage has been adjusted, the signal intensity of the ion peak detected by the ion detector for the same number of ions has also changed. This means that the determined parameter of the first relationship does not necessarily have to be an accurate representation of the relationship between the detector output and the number of ions for the second detector voltage. However, the determined number of ions in the ion peak is known from step 303. Therefore, in step 305, a parameter of a second relationship between the detector output and the number of ions for the second detector voltage is determined based on the determined number of ions in the ion peak (step 303).

[0060] For example, an ion peak with an intensity of 300 mV (if the ion detector voltage was increased in step 304), corresponding to 40 ions (as determined in steps 302 and 303), could result in a factor of 7.5 mV per ion for the particular parameter of the second relationship. If the ion detector voltage were decreased in step 304 instead, the factor would decrease.

[0061] It should be noted that it is not necessary for the ion peak measured at the second detector voltage to contain exactly the same number of ions as the ion peak measured at the first detector voltage. As long as the number of ions is approximately the same, accurate calibration can be achieved using the methods disclosed herein. For example, even a 10% difference in the number of ions in detected ion peaks is more than sufficient to achieve accurate calibration.

[0062] It is also possible to apply a correction factor to account for ion losses in the ion detector device and / or fluctuations in the number of ions in a detected ion peak.

[0063] Another option is to average the number of ions over several scans, which can be useful when an ion source is particularly noisy. Significant source drift is unlikely to occur over the course of these scans, and if it does occur, it can be corrected. Correction may involve, for example, varying the order of the detector voltage scan steps or performing a single-ion versus multiple-ion comparison multiple times (e.g., as described with reference to Fig. 7) and calculating a mean.

[0064] In any case, a multi-ion peak may comprise many hundreds of ions, so ion statistics do not play a significant role. For averaged measurements of multiple scans, especially with hundreds or thousands of ions, the statistical variation in the number of ions between two measurements or the signal generated by each ion becomes even less significant. Thus, using the methods disclosed herein, sufficiently accurate calibration is possible even if the number of ions in the detected ion peaks is not exactly the same.

[0065] The parameters of the first and second relationships do not have to represent the same relationship. Preferably, when using the S / N factor as the parameter of the first relationship, the second parameter represents a different relationship. For example, using the values ​​obtained using the S / N factor of 4.0, a 300 mV intensity peak corresponding to 50 ions can correlate with the parameter of the second relationship being a factor of 6 mV per ion.

[0066] The gain calibration of the ion detector can also be monitored over time by Fig. The method shown in Figure 3B is used.

[0067] At step 311, a second ion peak may be detected at the ion detector using a third detector voltage. This voltage may be the same as the second detector voltage or may be a different voltage (e.g., a high gain voltage). The second ion peak may correspond to a single ion or multiple ions. In the case of single ions, single ions may be generated using any of the methods discussed above, but most preferably using the temporal defocusing method described with reference to Fig. 4 is described.

[0068] Step 312 involves determining the number of ions in the second peak. For a multi-ion peak, this can be determined based on the parameter of the first relationship, which can be an S / N ratio for a single charge. S / N ratios can be proportional to the number of ions detected in an ion peak and can be relatively stable over time, such that they do not need to be calculated more than once.

[0069] At step 313, a second parameter of the second relationship between the detector output of the ion detector and the number of ions may be determined based on the determined number of ions in the second ion peak. The second parameter may therefore be a factor between the number of ions and the detector output.

[0070] In step 314, the second parameter of the second relationship is compared to an expected value based on the second relationship. This may be a direct comparison between the first parameter of the second relationship and the second parameter of the second relationship. For example, the first parameter determined in step 205 as 7.5 mV per ion may be considered the expected value and compared to the second parameter determined in step 313 (e.g., 6 mV per ion). Alternatively, the expected value of the second relationship may be determined based on a calibration curve, which may be obtained, for example, during steps 302-304. In step 315, it may be determined based on the comparison whether a drift in the gain has occurred.For example, if a value of the first parameter of the second relationship differs from a value of the second parameter of the second relationship, it can be determined that a gain drift has occurred. Alternatively, if the expected value of the second relationship from the calibration curve differs from the second parameter of the second relationship, a gain drift may have occurred. There may be a tolerance level for this determination, below which it is not determined that a gain drift has occurred. This can prevent excessive gain calibration correction.

[0071] After determining that a gain drift has occurred, an adjustment parameter can be calculated to adjust the detector voltage to correct the gain drift. The adjustment parameter can be calculated to adjust the detector voltage to a level where the relationship between the detector output and the number of ions is determined by the first parameter of the second relationship. The adjustment parameter can only be calculated based on the calibration curve.

[0072] The calibration curve describes a change in the signal waveform for a specific voltage change. The assumption is that the shape of the curve does not change when the detector gain decreases, although the proportions may change. This means that if the single-ion region is re-measured and the result shows that the detector gain has decreased, the calibration curve can be used to determine a proportional voltage (or alternatively, an absolute voltage change) that will return the gain to the expected level. For example, if the gain has decreased by 50%, the expected gain can be achieved by determining a proportional voltage that would result in a 100% increase on the calibration curve. The detector voltage can then be adjusted to a new voltage based on this adjustment parameter.

[0073] As briefly discussed above, single ions are generated in step 301, which can be achieved by various methods. One such method involves temporal defocusing of a TOF analyzer.

[0074] TOF analyzers typically use an ion mirror to focus the arrival time of ions with the same m / z to the detector as narrowly as possible. This allows resolutions of 10–90 K to be achieved, depending on the flight path length and focus quality.

[0075] Multi-reflection time-of-flight (MR-TOF) instruments contain opposing ion mirrors between which a packet of analyte ions can undergo multiple reflections, thus traveling a greatly extended flight path within a small analyzer volume. An example of an MR-TOF analyzer is shown in Fig. 4. Ions are accumulated using a pulsed extraction ion trap 401, which serves as the ion source, and ejected into the analyzer via a pair of deflectors 405. The deflectors 405 enable an optimal injection angle and alignment of the ion packet's focal plane with the analyzer 400. The ions are reflected back and forth between two elongated ion mirrors 403 and drift along the direction of mirror extension. The two ion mirrors 403 are tilted relative to each other, establishing an increasing average potential in the drift dimension, which slows the ion drift velocity and causes the ions to be reflected back into the drift dimension and focused onto a detector 404.Time-of-flight errors caused by tilting the mirrors are corrected by “strip electrodes” 406 that run the length of the drift space and allow for a tight temporal focus.

[0076] In such an analyzer (which may, for example, have a trajectory of 20 m), a small change in the power supplies between the ion mirrors 403, e.g., <5%, can be sufficient to completely temporally defocus even relatively strong ion packets at the detector into single ions. For example, ions with an m / z of 500, which could normally be focused onto a <4 ns peak, might instead be scattered over about a microsecond. That is, ions with the same m / z reach the detector at different times instead of converging in time. This allows the signal to be decomposed into about 1000 individual peaks, each about 1 ns wide. A signal from 100 ions is split into 100 single ion peaks, with a small probability that some of these peaks will overlap. Therefore, recording single ion signals is two orders of magnitude faster than attenuating a focused peak onto a single ion.This is within the control of the automatic ion trap gain control and eliminates the need for beam attenuation by detuning the ion optics. The average time of flight of the defocused single ions of approximately 700 µs remains similar to that of 500 m / z ions in normal operation, and the level spread of a few µs allows for a rough retention of the m / z assignment, which can be improved by appropriate calibration. Ideally, the deflector and / or strip electrode voltages would also be adjusted to maintain optimal transmission of ions to the detector, although this is not necessary.

[0077] A similar result can be achieved with a single-reflection ToF analyzer with a smaller trajectory (e.g., 2 meters), but the degree of defocus (difference in the power supply of the ion mirrors) would have to be much higher or the number of ions much lower.

[0078] An advantage of defocusing ions in this way is that many single-ion pulses can be recorded in a single acquisition, making single-ion calibration achievable even for relatively slow analyzers, such as MR-TOF analyzers. Statistical methods based on pulse height variation are compatible with such analyzers, but are slow and potentially contain significant systematic errors. Another advantage of defocusing ions to generate single ions is that it eliminates the need for careful beam attenuation to remove multiple ion peaks, while minimizing empty spectra that would be required via beam attenuation methods.

[0079] An example of a propagation of single ions with an m / z of 524 is shown in Fig. 5 as recorded by a mass analyzer used in the manner described with reference to Fig. 4 and in which the ion mirror was set to a defocusing mode. Peaks 520 indicate an ion arriving at the detector. As indicated by peaks 520 in Fig. As shown in Figure 5, single ions with the same m / z arrive at the detector at different recorded times due to the temporal defocusing mode of the analyzer.

[0080] Fig. Figure 6A shows a flowchart procedure for calibrating the detector gain using defocused single ions. This procedure is similar to the more general procedure described in Fig. 3A is similar.

[0081] In step 601, the ion mirror voltages are adjusted to disrupt a focus of the TOF analyzer such that ions with the same m / z do not arrive at the detector simultaneously. In step 602, a detector voltage / gain may be increased from its initial value. Alternatively, the detector voltage / gain may be decreased from its initial value. Single-ion responses (if detectable at this level) may then be measured in step 603 such that a parameter of a first relationship between an ion detector output (e.g., a current or voltage) and a number of ions for the first detector voltage can be determined. The single-ion responses are then compared to a preset upper limit (step 604). The preset upper limit may be a limit above a noise level. For example, the preset upper limit may be 10 times, 30 times, or 100 times the noise level.Alternatively, the preset upper limit can be a signal area / intensity. Providing the preset upper limit can ensure that the ions remain within the detector's dynamic range.

[0082] If the preset upper limit is not reached (step 604 proceeds with No), the detector voltage is increased (stepped up) and measured again until it is reached (steps 602-604). Finally, in step 605, the acquired single ion and voltage data can be used to fit / calculate a curve representing a relationship between the detector output and the number of ions for the first detector voltage. From this curve, an appropriate detector voltage can be extrapolated, and the detector voltage can be adjusted to that value.

[0083] This method requires one or a few acquisitions for each voltage step, so it can be a relatively fast process for an analyzer operating at 100 Hz. In comparison, state-of-the-art statistical methods require hundreds of acquisitions for each step and take several minutes to complete.

[0084] The with reference to Fig. The method described in Figure 6A can vary in many ways. For example, the detector voltage scan range can be set absolutely or relative to the starting value, or voltages can be randomly selected within a preset range to reduce the influence of ion source fluctuations.

[0085] The process of Fig. 6A is faster than conventional methods, but too slow to be used for detector monitoring within a single experiment. A second method suitable for such monitoring is described in Fig. 6B. At step 611, the ion mirror(s) is / are set to the defocused mode, which is described with reference to Fig. 4. Then, in steps 612 and 613, the detector voltage is set to a voltage that produces a high gain (i.e., an ion striking the detector produces a large number of electrons), and a single-ion response is measured. This response is compared with the single-ion response to a previous measurement. The previous measurement can be performed using the method of Fig. 3, Fig. 6A or any other method described herein (e.g., with reference to Fig. 10) can be obtained.

[0086] At step 614, the gain drift and an adjustment parameter for correcting the gain drift may be calculated. The drift may be calculated either by adjusting (increasing / decreasing) the detector voltage based on a previously measured calibration curve (e.g., as described by the method of Fig. 3 or Fig. 6A) or for small gain changes, for example, by adjusting peak intensities, by performing a short scan with small voltage steps until a suitable gain value is reached. Since these measurements are very fast, many can be performed during an experiment, such as a chromatographic separation, and a moving average can be calculated.

[0087] The choice of analyte ions for this step can come from the sample itself, be dynamically selected, or be a background or calibration sample provided by a primary or secondary ion source.

[0088] The calculated optimal detector voltage for the detector can be set in step 615.

[0089] Since an approximate m / z for temporally defocused ions can still be determined using the methods described above, a measurement of multiple single ions with different m / z ratios can be performed simultaneously. This can improve the statistical accuracy of the measurement and allow calibration of the detector response to m / z. Accuracy can be further improved by calculating a specific time-m / z calibration for the temporally defocused ions.

[0090] Likewise, the response of different charge states of analyte ions can be measured and calibrated, provided they are present in the sample or calibration mixture. A regular mass spectral measurement can be performed prior to the defocused single-ion measurement to determine which ions are present in the sample. Quadrupole isolation of desired ions can be performed for measurement.

[0091] In the case of Fig. 6B advantageously eliminates the need to redraw calibration curves due to the assumption that the trends are derived from a previously acquired curve (e.g., using the method of Fig. 6A). Using the method of Fig. 6A, the optimal gain for detecting single ions is lower than would be preferred to properly measure a single ion range, since small single ion peaks are nested in the noise and often represent only a single sample point on a 1-2 gigasamples per second (GS / s) digitizer. Therefore, the selection of the detector voltage is usually based on an extrapolation from one edge of the calibration curve (e.g., as shown in Fig. 8 shown).

[0092] Therefore, as in Fig. As shown in Figure 7, after reaching a suitable detector voltage for a single-ion measurement, an ion packet can be refocused to generate a multi-ion response. This can then be used as a known number of ions for gain calibration at a lower range of detector voltages. This procedure involves a reversal of the Fig. 6A (with multiple ion peaks instead of single ion peaks) until a lower threshold enhancement is determined.

[0093] Steps 701-704 correspond to steps 601-604 of Fig. 6A. When the threshold intensity or plateau of counts per second (cps) is reached at step 704, the ion mirror is placed in a focused mode at step 705. That is, ions with the same m / z ratio arrive at the detector at the same time (i.e., they converge in time). Therefore, ion peaks detected by the detector will comprise multiple ions.

[0094] Steps 701-704 could be replaced by another method described herein (i.e., steps 705-710 could be used in combination with another method described herein). The only requirement is that a parameter of a first relationship is determined prior to step 705. Therefore, steps 705-710 could, for example, be used in combination with a method described with reference to Fig. 10 is described.

[0095] At step 706, the peak intensity of the detected peak is measured, and the number of ions is calculated based on the determined parameter of the first relationship. That is, the previously determined single-ion response can be used to determine the number of ions in the detected peak that includes multiple ions.

[0096] In step 707, the voltage of the detector is adjusted. In the case where this step is preceded by steps 701-704 (which correspond to steps 601-604), the detector voltage is stepped down / decreased. However, in the case where steps 705-710 are used in combination with other methods described herein, the detector voltage could instead be stepped up / increased. For example, if the detector voltage is adjusted in combination with the method described with reference to Fig. 10 is used, it could be increased or decreased in step 707.

[0097] In step 708, the intensity of the ion peak detected by the detector is measured. As mentioned above, this ion peak includes multiple ions because the ion mirror was set to a focused mode in step 705.

[0098] In step 709, it is determined whether the target gain has been reached and whether the ion peak remains above the noise level. The signal should be strong enough on average that smaller single-ion results are not lost in the noise. A signal at least 10 times the noise level is sufficient, and preferably the signal is 30 or 100 times the noise level. If the signal is much larger, a large single ion pulse can saturate the detector.

[0099] If it is determined at step 709 that the target gain has not been reached / the ion peak is not sufficiently above the noise level, the method returns to step 707. Otherwise, the method continues to step 710, where the optimal detector voltage is calculated and adjusted. An advantage of this method is that the gain can be quickly measured around the optimal detector voltage / target gain level. A calibration curve can be constructed from the focused multi-ion peak during steps 707-709, but without the need to calculate a calibration curve from single-ion responses (e.g., during steps 702-704). This should improve the accuracy and robustness of the gain calibration.

[0100] There is a risk of saturation at the point where single and multiple ion peaks compare. However, saturated peaks are relatively easy to detect, and for an instrument with an ion trap, it is very easy to proportionally control the ion population by changing the accumulation time.

[0101] High dynamic range detectors can handle thousands of ions in a multi-ion peak (especially when broadened under space charge) without saturation, even if the gain is significantly higher than optimal. In any case, detector overpotential is easily detectable and can be displayed. Because the number of ions in a multi-ion peak can be derived from the number of ions in a temporally defocused scatter (since the two should largely match), it is easy to prevent overloading with respect to the number of ions in the temporally defocused scatter. AGC techniques can also optionally be used to adjust the ion population in the multi-ion shots relative to the single-ion scatters without significant loss of accuracy in the ion number determination.

[0102] Calibration curves of the single ion range versus the detector voltage are shown in Fig. 8. These include repeated measurements of the Fig. 7, a single direct measurement of individual ions (e.g. using the method of Fig. 6A) and a direct measurement, which is performed by manually adjusting the detector voltages and analyzing the resulting spectra. Fig. 8 shows that the values ​​are generally reasonably aligned within 10% of the average, except that at low detector voltages the direct measurement breaks down because individual ions fall into the noise band and can no longer be detected.

[0103] A second practical comparison was made between the indirect single ion measurement method (as described with reference to Fig. 7) and a statistical method based on that described in US 7,109,474 B2. This comparison is shown in Fig. 9. At a fixed detector voltage, scans were performed for both methods, and repeated single ion area (SIA) determinations were performed for several batches with an increasing number of scans. The average SIA and standard deviation for both methods are shown in Fig. 9, which shows that the single-ion method becomes reproducible to within 5% within 50 scans per measurement, but the statistical method does not reach this level even with 300 scans. The single-ion method is approximately an order of magnitude faster than the statistical method in achieving similar reproducibility levels. It is also noteworthy that the single-ion method produces a much lower average single-ion area value than the statistical method (1.1 × 10 -11 Vs instead of 1.8 × 10 -11Vs). This is believed to be due to a systematic error caused by uncontrolled noise sources in the statistical procedure.

[0104] Another gain calibration method is possible for hybrid instruments that combine a charge detection analyzer, such as an orbital trap mass spectrometer, with a second analyzer based on an electron multiplier, such as a TOF or ion trap analyzer. The method could also be applied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer and a TOF analyzer, orbital trap mass spectrometer / ion trap, or FT-ICR / ion trap hybrid instrument. The method involves cross-calibration between the two parts of the hybrid instrument. The cross-calibration method can be used to calibrate the gain and / or to facilitate monitoring and adjustment of the gain over time, as described below.

[0105] Orbital trap mass spectrometers detect current induced by charge packets moving between the two sides of the analyzer, producing consistent signal-to-noise (S / N) peaks relative to the number of ions for more than 100 ion packets. Because orbital trap mass spectrometers are not subject to rapid changes in transmission or aging, the peak signal-to-noise can be used as an ion count measurement against which the detector gain is calibrated or detector aging is monitored.

[0106] Orbital trap mass spectrometer preamplifiers are not sensitive enough to detect singly charged single ions by themselves, but can be used to detect multiply charged single ions (for example, as described in Makarov et al., "Dynamics of Ions of Intact Proteins in the Orbitrap Mass Analyzer," Journal of the American Society for Mass Spectrometry, Volume 20, Issue 8, August 2009, pages 1486-1495). In short, the detection of single ions can be demonstrated when the intensity of peaks in ion spectra follows a quantized S / N ratio distribution (i.e., S / N changes in quantum steps such as 1, 2, 3, etc.). From such a measurement, the S / N response of a singly charged single ion can be inferred, and thus the number of ions in a multi-ion peak can be calculated.

[0107] Orbital trap mass spectrometer analyzers are typically connected to curved ion traps (C-traps) designed to prepare ions for injection. These traps can easily become overfilled and suffer from excessive space charge effects without automatic gain control (AGC) techniques to regulate the accumulation time and ion population.

[0108] A standard automatic gain control method for dual orbital trap mass spectrometer / ion trap analyzer hybrid instruments, such as the Orbitrap Fusion (RTM) manufactured by Thermo Fisher Scientific, Inc., is to measure the ion population at the calibrated electron multiplier detector of the ion trap and use this data to regulate the accumulation time at the C-trap. The method proposed here of using the orbital trap mass spectrometer signal intensity or signal-to-noise ratio to regulate the detector voltage is, in a sense, a reversal of this standard method.

[0109] A hybrid orbital trap mass spectrometer / time of flight mass spectrometer suitable for performing methods according to embodiments of the present disclosure is described in Fig. 10. The TOF analyzer 400 in this figure is similar to the one shown in Fig. 4 shown similar.

[0110] Single ions can be generated using an ESI source 1020. It is understood that other methods, as described with reference to Fig. 3A can be used to generate single ions.

[0111] The individual ions then enter the mass spectrometer 1000 and pass through an RF lens 1030. The ions are focused by the lens 1030 into first and second ion guides 1040. Charged ions are guided along the curved path of the second ion guide 1040, while neutral ions are not guided and do not pass through the mass spectrometer 1000.

[0112] An ion gate 1080 controls the transmission of ions from the curved ion guide 1040 into the mass filter 1090. The mass filter 1090 can pass ions of a selected mass number or mass range while excluding ions of other m / z ratios. Alternatively, the mass filter 1090 can be operated in a pure RF mode, where it allows essentially all ions to pass regardless of m / z.

[0113] The ions are then guided through a third ion guide 1100 into a C-trap 1060 for ion injection into a first mass analyzer 1010 (e.g., an FTMS, such as an orbital trap mass spectrometer). The C-trap (first ion trap) 1060 has curved electrodes extending in a longitudinal direction, which are supplied with RF voltages, and end caps to which DC voltages are applied. Accordingly, a potential well is formed extending along the curved longitudinal axis of the C-trap 1060. In a first mode, the DC end cap voltages are adjusted such that the single ions arriving from the ion guide 1100 are trapped in the potential well of the C-trap 1060, where they are cooled.

[0114] The cooled ions are then ejected from the C-trap 1060 into the first mass analyzer 1010 via a Z-lens 1050 into an off-center injection cavity of the first mass analyzer 1010. The ions are then trapped within the orbital trap mass analyzer by a logarithmic electric field such that their orbital motion around the analyzer axis is (approximately) harmonic. Thus, the ions separate according to their m / z ratio.

[0115] The ions are detected using an image detector (not shown), which creates a time-domain "transient" containing information about the ion species as they pass through the image detector. A Fast Fourier Transform (FFT) is applied to the transient to convert the data into a series of peaks in the frequency domain. A mass spectrum can then be generated, with axes of ion abundance / ion intensity versus m / z (similar to the one shown in Fig. 5, except that the x-axis is Fig. 5 corresponds to the flight time in µs).

[0116] Although Fig. While Figure 10 shows an FTMS 1010 in which ions are trapped axially and radially by an electrostatic field, it is understood that other forms of FTMS, such as an FT-ICR mass analyzer, are contemplated. In an FT-ICR mass analyzer, ions are trapped axially by an electrostatic field, with radial and azimuthal trapping achieved by applying a magnetic field. The main requirement of the FTMS 110 is that its output (i.e., the position and shape of peaks in a mass spectrum it generates) should be relatively stable over time (e.g., with respect to short- and long-term temperature changes).

[0117] The C-trap 1060 is then switched to a second mode to allow the ions from the FTMS 1010 to pass axially toward the fragmentation chamber 1070. The fragmentation chamber 1070 can serve as an ion guide for the ions (i.e., the ions are not exposed to a collision gas / the energy of the collision gas is insufficient to fragment the precursor ions). The fourth ion guide 1100 then directs the ions from the fragmentation chamber 1070 into the extraction trap 401. The extraction trap 401 collects ions ejected from the fragmentation chamber 1070 prior to injection into the TOF mass analyzer 400.

[0118] According to the present disclosure, the mass spectrum generated by the FTMS 1010 can be used to determine a parameter of a first relationship between the detector output and the number of ions for the first detector voltage. For example, the S / N ratio for multiply charged single ions can be determined as described above, and the S / N response for a singly charged single ion can be calculated. This can then be used as a parameter to determine the number of ions in a multi-ion peak, since the S / N ratio should be proportional to the number of ions in the peak. The multi-ion peak is determined as described above with reference to Fig. 10 described using multiple ions instead of generating single ions.

[0119] The gain of a second analyzer 400 (for example, a TOF analyzer or an ion trap) can be adjusted as described with reference to Fig. 3A and Fig. 6A. That is, the detector voltage of the second analyzer 400 can be adjusted to a second voltage at which the detector output for the ion peak remains above a noise level. Then, a parameter of a second relationship between the detector output and the number of ions for the second detector voltage is determined based on the determined number of ions in the multi-ion peak. The second analyzer can then operate at the second detector voltage.

[0120] A correction to the number of ions would need to be made to account for the transmission of ions into and / or loss of ions in the orbital trap mass spectrometer 1010 (which may be approximately 30-50% loss), and an estimate of the transmission to the detector on the second analyzer 400, which may limit the accuracy of such a calibration. That is, the number of ions in the ion peak calculated using the particular parameter does not directly correspond to the number of ions in the second analyzer 400 and therefore needs to be corrected. The transmission of ions into the orbital trap mass spectrometer 1010 may depend on various instrument factors, including, for example, detector voltages and / or ramp times, ion m / z, and the type of orbital trap mass spectrometer 1010.

[0121] Alternatively / additionally, the parameter dedicated to the FTMS 1010 can be used to monitor / correct gain drift over time, similar to the method described with reference to Fig. 3B and Fig. 6B. For example, the S / N ratio known from the FTMS 1010 can be used to calculate a drift in the gain of the less stable second analyzer 400 and an adjustment parameter to correct the drift in the gain. The drift can then be corrected either by adjusting (increasing / decreasing) the detector voltage of the second analyzer 400 or, for small gain changes, by adjusting the peak intensities.

[0122] In particular, the gain calibration of the ion detector can be monitored by detecting a second peak on the FTMS 1010 using a third detector voltage. The third detector voltage can be the same as the first detector voltage of the FTMS 1010 during the initial gain calibration. Since the S / N ratio of the FTMS 1010 is relatively stable over time, the previously determined S / N ratio can be used to determine the number of ions in the second ion peak. As above, a correction to this number would be required to account for the transmission of ions into the detector in the second analyzer 400.

[0123] Then, a second parameter of the second relationship between the detector output of the ion detector and the number of ions for the third detector voltage can be determined based on the determined number of ions in the second ion peak. For example, the S / N ratio of the FTMS 1010 can lead to a determination (based on the transmission rate) that 100 ions are transferred to the second analyzer 400. Thus, a peak of 200 mV intensity can correspond to a second parameter of 2 mV per ion.

[0124] The second parameter can then be compared to an expected value based on the first relationship. This can be a direct comparison between the first parameter of the second relationship and the second parameter of the second relationship. That is, the expected value of the second parameter can be the value of the first parameter. Alternatively, the expected value of the second relationship can be determined based on a calibration curve, which can be obtained, for example, during steps 302-304 or steps 702-704 and / or 707-709.

[0125] Based on the comparison, it can then be determined whether gain drift has occurred. For example, if the first parameter of the second relationship and the second parameter of the second relationship have different values, it can be determined that gain drift has occurred. Alternatively, if the expected value of the second relationship differs from the calibration curve for the second parameter of the second relationship, gain drift may have occurred. There may be a tolerance level for this difference below which it is not determined that gain drift has occurred. This can avoid excessive gain calibration correction.

[0126] For the cross-calibration procedure, a known sample, such as a standard calibration mixture (e.g., Pierce Flexmix, manufactured by Thermo Fisher Scientific, Inc.), is preferably used. When using an unknown analyte, care must be taken to ensure that it is not multiply charged, as multiply highly charged ions with densely packed isotopes create interference patterns within the orbital trap mass spectrometer, reducing the apparent signal-to-noise and compromising the assessment of the number of ions in a peak.

[0127] An advantage of the cross-calibration procedure described here using an FTMS (such as an orbital trap mass spectrometer) is that a single-ion measurement only needs to be performed once on an FTMS. The behavior is sufficiently similar over time and across other instruments that these instruments do not require their own calibrations (or periodic recalibrations) for a reasonable approximation. It is understood that preamplifier characteristics that cause signal differences between instruments, such as component tolerances, also produce a similar impact on the noise, so that the signal-to-noise ratios become relatively consistent.

[0128] The number of ions producing a given signal-to-noise ratio thus becomes a known property for the instrument class, rather than something that needs to be calibrated for each instrument. A more accurate result can be obtained by calibrating a specific instrument, but such accuracy may not be required, and the result is not expected to change significantly over time.

[0129] The cross-calibration procedure is fast and relatively stable. It can also be advantageously performed in conjunction with other cross-calibration methods (e.g., mass cross-calibration).

[0130] The following sections describe illustrative embodiments only. These illustrative embodiments may be implemented with any of the embodiments described herein. For example, the gain may be calibrated using the method of Section A1, and the monitoring of the gain calibration may be performed using the method described with reference to Fig. 6B. Similarly, the parameter of the first relationship and the first parameter of the second relationship can be determined using the method described with reference to Fig. 6A to monitor the gain calibration using the method of Section A2 or A3. A1. A method for gain calibration for an ion detector operating at a detector voltage and forming part of a hybrid mass spectrometer, the method comprising the following steps: Generation of single ions; Determining a parameter of a first relationship between a detector output of a first ion detector device and a number of ions for a first detector voltage by detecting individual ions at the first ion detector device; and Detecting an ion peak at the first ion detector device using the first detector voltage to determine a number of ions in the ion peak based on the parameter of the first relationship, Adjusting a detector voltage of a second ion detector device to reduce a ratio between a detector output of the second detector and a noise level, thereby obtaining a second detector voltage at which the detector output for an ion peak detected at the second ion detector device remains above the noise level; Determining a parameter of a second relationship between the detector output of the second ion detector device and the number of ions for the second detector voltage based on the determined number of ions in the ion peak. A2. Procedures according to Section A1, further comprising: Monitor the ion detector gain calibration by: Detecting a second ion peak at the first ion detector device using a third detector voltage; Determining the number of ions in the second ion peak; Determining a parameter of a third relationship between the detector output of the first ion detector device and the number of ions for the third detector voltage based on the determined number of ions in the second ion peak; Comparing the second parameter of the second relationship with an expected value based on the second relationship; and Determine, based on the comparison, whether a drift in the gain has occurred. A3. Procedures according to Section A2, further comprising: based on the determination that a drift in the gain has occurred: Calculating an adjustment parameter to adjust the detector voltage to a level at which the relationship between the detector output and the number of ions corresponds to the expected value of the second parameter of the second relationship based on the second relationship; and Adjust the detector voltage based on the adjustment parameter. A4. The method of any preceding paragraph, wherein the second ion detector device is part of a time-of-flight mass spectrometer (TOF-MS) or an ion trap. A5. The method according to any one of the preceding sections, wherein the first ion detector device is part of a Fourier transform mass spectrometer (FTMS). A6. The method of paragraph A5, wherein the FTMS is an orbital trap mass spectrometer or a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A7. Method according to any one of the preceding sections, further comprising: Operating the second ion detector device at the second detector voltage. A8. The method of paragraph A5, wherein the parameter of the first relationship is a signal-to-noise (S / N) ratio for a single ion. A9. The method of claim A8, further comprising: Detecting multiply charged single ions at the first ion detector device; Calculate the S / N ratio of the multiply charged single ions; Determine the S / N ratio of a singly charged single ion based on the calculated S / N ratio of the multiply charged single ions. A10. Method according to one of the preceding sections, wherein after determining the number of ions in the ion peak, a correction factor is applied to the determined number of ions, which correction factor is corrected to take into account the transmission of ions to the second ion detector device. A11. Procedure according to section A10, with a correction factor between 30% and 50%. A12. Method according to any one of the preceding sections, further comprising: Generate the individual ions by beam attenuation, fragmentation, scattering of ions with background gas or using an electrospray ionization (ESI) source.

[0131] The methods described herein may be implemented with computer system configurations including handheld devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. Embodiments may also be practiced in distributed computing environments where tasks are performed by remote processing devices connected via a network.

[0132] Certain embodiments may also be embodied as computer-readable code on a non-transitory computer-readable medium. The computer-readable medium is any data storage device capable of storing data that can subsequently be read by a computer system. Examples of computer-readable media include hard disks, network-attached storage (NAS), read-only memory, random-access memory, CD-ROMs, CD-Rs, CD-RWs, magnetic tapes, and other optical and non-optical data storage devices. The computer-readable medium may also be distributed across a network-coupled computer system so that the computer-readable code is stored and executed in a decentralized manner.

Claims

[1] A method for gain calibration for an ion detector operating at a detector voltage, the method comprising the following steps: Generation of single ions; Determining a parameter of a first relationship between a detector output of the ion detector and a number of ions for a first detector voltage by detecting individual ions at the ion detector; Detecting an ion peak at the ion detector using the first detector voltage to determine a number of ions in the ion peak based on the parameter of the first relationship; adjusting the detector voltage to reduce a ratio between the detector output and a noise level and thereby obtain a second detector voltage at which the detector output for the ion peak remains above the noise level; Determining a parameter of a second relationship between the detector output and the number of ions for the second detector voltage based on the determined number of ions in the ion peak. [2] The method of claim 1, further comprising: Monitor the ion detector gain calibration over time by: Detecting a second ion peak at the ion detector using a third detector voltage; Determining the number of ions in the second ion peak; Determining a second parameter of the second relationship between the detector output of the ion detector and the number of ions for the third detector voltage based on the determined number of ions in the second ion peak; Comparing the second parameter of the second relationship with an expected value based on the second relationship; and Determine, based on the comparison, whether a drift in the gain has occurred. [3] The method of claim 2, further comprising: based on the determination that a drift in the gain has occurred: Calculating an adjustment parameter to adjust the detector voltage to a level at which the relationship between the detector output and the number of ions corresponds to the expected value of the second parameter of the second relationship based on the second relationship; and Adjust the detector voltage based on the adjustment parameter. [4] The method of claim 1 or claim 2, further comprising: Operating the ion detector at the second detector voltage. [5] A method according to any one of the preceding claims, wherein the ion detector is part of a time-of-flight (TOF) mass spectrometer (MS). [6] The method of claim 5, further comprising: Generate the single ions by setting the TOF-MS to operate in a temporally defocused mode, in which ions with the same mass-to-charge ratio (m / z) arrive at the ion detector at different times. [7] The method of claim 5 or 6 when dependent on claim 2 or 3, wherein detecting the second ion peak comprises setting the TOF-MS to operate in the temporally defocused mode such that the second ion peak corresponds to a single ion. [8] A method according to any one of the preceding claims, further comprising: gradually adjusting the detector voltage during the detector voltage adjusting step; and Calculating a calibration curve based on the detector output generated by detecting single ions at the ion detector for each detector voltage step. [9] A method according to any one of the preceding claims, wherein the first relationship and the second relationship are the same relationship; and the parameter of the first relationship and the parameter of the second relationship are different. [10] A method according to any one of claims 1-3, wherein the ion detector comprises: a first ion detector device; and a second ion detector device; and wherein the steps of determining a parameter of a first relationship and detecting an ion peak at the ion detector are performed with respect to the first ion detector device, and the steps of adjusting the detector voltage and determining a parameter of a second relationship are performed with respect to the second ion detector device. [11] The method of claim 10, wherein the second ion detector device is part of a time-of-flight (TOF) mass spectrometer (MS) or an ion trap. [12] A method according to claim 10 or 11, wherein the first ion detector device is part of a Fourier transform mass spectrometer (FTMS). [13] The method of claim 12, wherein the FTMS is an orbital trap or Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. [14] A method according to claims 10-13 when dependent on claim 2, wherein the step of detecting the second ion peak at the ion detector is performed with respect to the first ion detector device, and the step of determining a parameter of a third relationship is performed with respect to the second ion detector device. [15] A method according to any one of claims 10-14, wherein the parameter of the first relationship is a signal-to-noise (S / N) ratio. [16] The method of claim 15, further comprising: Detecting multiply charged single ions at the first ion detector device; Calculate the S / N ratio of the multiply charged single ions; Determine the S / N ratio of a singly charged single ion based on the calculated S / N ratio of the multiply charged single ions. [17] A method according to any one of claims 10-16, wherein a correction factor is applied to the determined number of ions in the ion peak to account for the transmission of ions to the second ion detector device. [18] A method according to claim 17, wherein the correction factor is between 30% and 50%. [19] A method according to any one of claims 1-5 or 7-18, further comprising: Generate the individual ions by beam attenuation, fragmentation, scattering of ions with background gas or using an electrospray ionization (ESI) source. [20] A system comprising a mass spectrometer arrangement and a controller configured to operate the mass spectrometer arrangement according to any one of the methods of claims 1-19. [21] A computer program product comprising instructions which, when executed by a computer, cause the computer to carry out the method of claims 1-19 with the system of claim 20.

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