Gain calibration method

Abstract

Gain calibration is performed for an ion detector operating at a detector voltage. A single ion is generated. Parameters of a first relationship between a detector output of the ion detector and a number of ions for a first detector voltage are determined by detecting the single ion at the ion detector. 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 parameters of the first relationship. The detector voltage is adjusted 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. Parameters of a second relationship between the detector output and the number of ions for the second detector voltage are determined based on the determined number of ions in the ion peak.

Description

Technical Field

[0001] This disclosure relates to a method for gain calibration, specifically for use in a mass spectrometer. Background Technology

[0002] Mass spectrometry is an analytical technique 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 based on their m / z, and an ion detector for detecting the separated m / z ions.

[0003] Ion detectors measure the charge or current induced when ions pass through or collide with the detector's surface. Since the effect produced by a single particle is generally too small to be directly detected at non-relativistic energies, ion detectors typically amplify the signal generated by passing / indicating ions, making the signal measurable. This is usually accomplished by converting the incident ion into a secondary electron.

[0004] The gain of an ion detector (the number of electrons generated per incident ion) is typically controlled by setting the voltage of the electron multiplication phase and the energy of the incident ion. It is generally preferred that the detector operate with sufficient gain to reliably detect single ions and thus maximize sensitivity. However, excessive gain reduces the detector's dynamic range (the ratio between the maximum and minimum detectable values), a critical factor in performance. Excessive gain also accelerates aging, which shortens the detector's lifespan. Furthermore, aging and short-term effects (e.g., water desorption) can cause significant changes in gain, even within a day.

[0005] In addition to requiring reliable ion detection, the detector's current output allows for the measurement of the number of ions in the signal, thus providing quantitative information about the analyte. Mass spectrometers incorporating ion traps typically also require the measurement of ion currents to regulate the number of ions stored within the trap during analysis, preventing harmful space charge effects in a process known as "automatic gain control." Therefore, routine gain calibration is crucial for accurately determining the number of ions in the signal.

[0006] Conventional mass spectrometers typically do not have routines for measuring and calibrating detector gain, which must be run periodically. These routines are often too slow to monitor and / or recalibrate detector gain during or between experimental runs, and are instead operated as part of daily and / or weekly calibration routines.

[0007] Time-of-flight (TOF) quality analyzers operating at repetition rates from 10 Hz to 30,000 Hz typically use, for example, microchannel plate (MCP)-based detectors as described in US2004 / 0206911, due to their detection surface and sub-nanosecond time response, which are optimal for high resolution. A standard detector, a pair of herringbone MCP101 detectors for enhanced gain, is shown in Figure 1 and will be briefly described below.

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

[0009] Ion packets 102 collide with MCP 101. Due to the angled channels, particles 102 entering one of the channels are ensured to impact the channel wall and emit secondary electrons 103. These secondary electrons 103 are accelerated within the resistive channels of MCP 101 and emit more secondary electrons 103 upon impact with the channel wall, thus amplifying the original signal in an electron-to-electron conversion cascade. Electrons leaving the first plate 101 begin another cascade in the second plate 101.

[0010] The gaps between the MCPs 101 in Figure 1 allow charge to diffuse across multiple channels, which increases gain. Alternatively, there can be no gaps between the MCPs to maintain spatial resolution.

[0011] The herringbone dual MCP 100 shown in Figure 1 is characterized by a single-ion gain of 1E+6 detected at the output of the second MCP 101 and a lifetime of less than one coulomb (1C). The herringbone dual MCP 100 can alternatively have an operating lifetime of 3C or 10C. This may depend on the gain, and the lifetime may increase inversely with the gain.

[0012] Chevron MCPs are often limited by their dynamic range, although fast detector schemes capable of linear detection from single ions to more than 1,000 ions per pulse have also been described, such as the multiplier electrode chain with magnetoelectronic focusing described in US 6,982,428 and the MCP / multiplier electrode combination combining scintillator / photomultiplier tubes to add an additional gain stage in US 7,180,060.

[0013] The standard method for detector calibration (e.g., the MCP detector shown in Figure 1) is to measure the response of a single ion at different detector gain settings. Measurements of the single-ion pulse intensity or area provide the most direct measurement, while measurements of the single-ion pulse rate provide a measurement of the ion current, and the plateau period of the count rate versus detector voltage curve indicates the optimal voltage setting for efficient single-ion detection. An example of this curve is shown in Figure 2, based on a similar plot from Prohaska et al.'s "Sector Field Mass Spectrometry for Elemental and Isotopic Analysis" published in the Royal Society of Chemistry in 2015.

[0014] As shown in Figure 2, increasing the detector voltage initially leads to a sharp 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. Operating within this plateau region is advantageous for efficient single-ion detection. As shown in Figure 2, as the detector voltage is further increased beyond the plateau region, the count rate begins to increase sharply again.

[0015] One problem arises that the single-ion area is difficult to measure reliably at the minimum gain level required for detecting such ions because it is near the noise level. Alternatively, such values ​​can be inferred from calibration of the trend of single-ion areas measured over a higher gain range. US 9,564,301 shows another example of single-ion measurement in a time-of-flight instrument where detection optimization is performed by comparing single-ion and noise peaks.

[0016] The main challenge in single-ion measurements lies in avoiding the simultaneous detection of multiple ions. Instruments may be detuned to attenuate the ion beam and reduce the likelihood of multiple ion bombardments, but this comes with the problem of generating many empty acquisitions. This is not a major issue for instruments with kHz-level acquisition rates, such as orthogonal time-of-flight analyzers (for example), but for instruments with ion traps operating at lower acquisition rates (i.e., significantly less than 1 kHz), the calibration process becomes lengthy or unreliable.

[0017] As described in US 7,109,474, ion trap instruments can alternatively incorporate measurements of ion clusters based on the statistical variation of peak intensities across multiple acquisitions. This method eliminates the need for single-ion measurements and produces very consistent results. However, it is relatively slow and has significant systematic errors due to other noise sources in the peak intensities, such as the number of secondary electrons / ions generated by the incident analyte ions.

[0018] US2021 / 0013019 describes applying a voltage to an electrode such that ions with the same m / z, simultaneously ejected from an ejector, are dispersed in the temporal direction, resulting in the observation of multiple low peaks corresponding to each ion in a distribution spectrum. The peak height of each peak in the distribution spectrum is determined, and the median of these values ​​is determined. If the median peak height is not within a predetermined reference range, the detector voltage is increased by a predetermined amount. This process is repeated until the determined median peak height is within the reference range, and then the detector voltage at that point is selected as the optimal voltage.

[0019] US10,593,525 describes a method for calibrating a time-of-flight mass spectrometer (TOF MS) to account for temperature variations. Ions are introduced into a Fourier transform mass spectrometer (FTMS), and their mass-to-charge ratios are determined. Additionally, ions containing calibration ions are introduced into the TOF MS, and at least the m / z ratio of the calibration ions is determined. Specific peaks representing the calibration ions are selected and matched between the TOF MS and FTMS spectra. Then, based on the relative independence of the FTMS spectra with respect to temperature, the relative positions of the matching peaks in each spectrum are used to determine a temperature correction factor for the TOF MS data.

[0020] The detector gain variations and instabilities in the aforementioned conventional methods present challenges in quantifying efficient single-ion detection, dynamic range, and the number of ions detected within a time-of-flight analyzer. Conventional methods also lack accurate methods for measuring and calibrating detectors quickly enough to be performed during or between experimental runs, even for relatively slow analyzers with ion traps.

[0021] A gain calibration method is needed to overcome these problems. Summary of the Invention

[0022] According to this disclosure, a method for gain calibration of an ion detector operating at a detector voltage is provided.

[0023] The method includes the following steps:

[0024] Generate single ions;

[0025] A parameter used to determine the first relationship between the detector output of the ion detector and the number of ions at the first detector voltage by detecting a single ion at the ion detector;

[0026] The ion peak is detected at the ion detector using the first detector voltage, and the number of ions in the ion peak is determined based on the parameters of the first relationship.

[0027] The detector voltage is adjusted to reduce the ratio between the detector output and the noise level, thereby obtaining a second detector voltage at which the detector output of the ion peak remains above the noise level.

[0028] Based on the determined number of ions in the ion peak, a parameter is used to determine the second relationship between the detector output and the number of ions at the second detector voltage.

[0029] This method allows for accurate and faster determination of the optimal detector voltage. The optimal detector voltage provides sufficient gain for reliable single-ion detection, thus maximizing sensitivity. Excessive gain reduces the detector's dynamic range and accelerates aging; therefore, accurately determining the optimal output detector voltage improves detector lifetime and dynamic range. Advantageously, in some cases, the optimal detector voltage can be determined without plotting calibration curves of the single-ion response.

[0030] The method may further include monitoring the gain calibration of the ion detector. This may include 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 initial gain calibration. Alternatively / additionally, the third detector voltage may be a high-gain voltage. Monitoring the gain calibration may further include the steps of: determining the number of ions in the second ion peak and, based on the determined number of ions in the peak, determining a second parameter of a second relationship between the detector output of the ion detector and the number of ions in the third detector voltage. 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 gain drift has occurred. The expected value of the second parameter may be determined based on a calculated calibration curve, or may be the first parameter determined by the second relationship.

[0031] Therefore, gain calibration can be monitored rapidly during or between experimental rounds, rather than being performed as part of a daily or weekly calibration routine.

[0032] Based on the determination that gain drift has occurred, adjustment or correction parameters can be calculated based on a second relationship to adjust the detector voltage to a level where the relationship between the detector output and the number of ions corresponds to the expected value of the second parameter of the second relationship. The detector voltage can then be set based on the correction parameters / factors.

[0033] This monitoring can be performed without plotting calibration curves because the trend of the previously obtained relationship can be assumed to be true, thus providing rapid gain correction. Gain drift in the ion detector can therefore be rapidly corrected between or during experiments following gain monitoring.

[0034] The ion detector can be operated at a second detector voltage. The second voltage advantageously provides sufficient gain to reliably detect single ions and thus maximize sensitivity, while limiting excessive gain that accelerates aging and reduces the detector's dynamic range.

[0035] The ion detector can form part of a time-of-flight mass spectrometer. In this case, single ions can be obtained by setting the TOF MS to operate in time-defocus mode, where ions with the same mass-to-charge ratio arrive at the ion detector at different times.

[0036] Advantageously, this method of generating single ions means that many single-ion pulses can be recorded in a single acquisition, enabling single-ion calibration even with relatively slow analyzers, such as multiple reflection time-of-flight (MR-TOF) analyzers. Statistical methods based on pulse height variations can be used with such analyzers, but they are slow and can contain significant systematic errors. Defocusing the ions to generate single ions also avoids the need for careful attenuation of the ion beam to remove multiple ion peaks, instead minimizing the empty spectrum required by beam attenuation methods.

[0037] Detecting the second ion peak may involve setting the TOF MS to operate in time-defocused mode, such that the second ion peak corresponds to a single ion. The single-ion response of the parameters used to determine the first relationship can then be directly compared with the single-ion response of the second peak, allowing for faster monitoring of drift gain.

[0038] The detector voltage can be adjusted incrementally (i.e., in smaller steps). Therefore, a calibration curve can be easily calculated based on the detector output generated by detecting ions at the ion detector for each detector voltage step.

[0039] The first and second relations can be the same, where the parameters of the first relation differ from those of the second relation. This allows for direct comparison between parameters, enabling faster gain calibration / monitoring.

[0040] The ion detector may include a first ion detector device and a second ion detector device. The second ion detector device may form part of a time-of-flight mass spectrometer (TOF) or an ion trap. Steps of determining parameters of a first relationship and detecting ion peaks at the ion detector can be performed relative to the first ion detector device. Steps of adjusting the detector voltage and determining parameters of a second relationship can be performed relative to the second ion detector device. Therefore, the first ion detector device can be used to calibrate the gain of the second ion detector device. This provides rapid and relatively stable direct gain calibration. Gain calibration can also be advantageously performed using other cross-calibration methods, such as mass cross-calibration between the first and second ion detector devices.

[0041] The first ion detector device can form 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 the FTMS (the position and shape of the peaks in the resulting mass spectrum) is relatively stable over time (e.g., relative to short-term and long-term temperature variations), and therefore provides a convenient and stable way to provide gain cross-calibration or monitor gain calibration.

[0042] Furthermore, since the behavior of FTMS is sufficiently similar over time to that of other instruments of the same type, the calibration of one particular instrument (e.g., a first orbital capture mass spectrometer) can be applied to another instrument of the same type (e.g., a second orbital capture mass spectrometer). In other words, the number of ions that produce a particular output (e.g., signal-to-noise ratio) becomes a known property of a class of instruments, rather than something that must be calibrated for each instrument individually.

[0043] The steps of detecting a second ion peak at the ion detector can be performed relative to the first ion detector device, and the steps of determining parameters of a third relationship can be performed relative to the second ion detector device. Therefore, a more stable first ion detector device can be used to monitor the gain calibration of the second ion detector device over time.

[0044] The parameter or factor for the first relationship can be the signal-to-noise ratio (S / N). The S / N ratio of an instrument (e.g., FTMS) can be proportional to the number of ions in the detected ion peak, and thus can be used to determine how many ions are in subsequent detected ion peaks.

[0045] FTMS can be used to detect multi-charged single ions at a first ion detector and calculate their S / N ratios. The S / N ratio of a single-charged single ion can then be determined based on the calculated S / N ratio of the multi-charged single ions. The S / N ratio of the single-charged single ion can then be used as a proportionality constant between the number of ions in the detected ion peak and the S / N ratio of the detected ion peak.

[0046] A correction factor can be applied to determine the number of ions in the ion peak to facilitate ion transport to a second ion detector. The correction factor can be between 30% and 50%. Applying a correction factor improves the accuracy of gain calibration when performing cross-calibration between two ion detectors.

[0047] Single ions can be generated through beam attenuation, fragmentation, ion scattering with background gas, or by using an electrospray ionization (ESI) source. Therefore, gain calibration can be performed on a variety of instruments.

[0048] The above method can be implemented as a computer program, which includes instructions for operating a computer or computer system. The computer program can be stored on a computer-readable medium.

[0049] A 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, which contains volatile and non-volatile storage media. It may include computer-readable media to store logic or program instructions. Different parts of the system may be connected using a network (e.g., wireless and wired networks). The computer system may include one or more interfaces. For example, the computer system may contain a suitable operating system, such as UNIX (including Linux) or Windows (RTM).

[0050] The above method can be implemented in a system that includes a mass spectrometer apparatus and a controller configured to operate the mass spectrometer arrangement.

[0051] It should be noted that any of the above features may be used in conjunction with any particular aspect or embodiment of the invention. Furthermore, combinations of any particular device, structural, or method features are provided, even if such combinations are not explicitly disclosed. Attached Figure Description

[0052] The invention can be practiced in many ways, and embodiments will now be described by way of example only and with reference to the following drawings, in which:

[0053] Figure 1 shows a detector with a pair of herringbone MCPs;

[0054] Figure 2 shows a graph of the count rate versus detector voltage during detector calibration via the single-ion count plateau.

[0055] Figure 3A A flowchart method for calibrating ion gain is described;

[0056] Figure 3B This document demonstrates a flowchart method for monitoring ion gain calibration.

[0057] Figure 4 This illustrates the multi-reflection time of flight that can be used in conjunction with the methods described herein to calibrate ion gain;

[0058] Figure 5 An example of single-ion diffusion obtained by time-defocusing the TOF detector described herein is presented;

[0059] Figure 6A A flowchart illustrating the detector gain calibration process based on direct single-ion measurement is shown;

[0060] Figure 6B A flowchart describing a fast gain correction method suitable for insertion into experimental runs is shown;

[0061] Figure 7A flowchart is shown illustrating an indirect detector gain calibration method that combines a single-ion method with time-defocused and a multi-ion method with focusing.

[0062] Figure 8 The calibration curves are shown, generated from repeated indirect and direct single-ion measurements, as well as manual measurements.

[0063] Figure 9 This paper presents a comparison between statistical methods for determining single-ion area and the indirect single-ion measurements described herein; and Figure 10 Examples of hybrid orbital capture mass spectrometers and TOF instruments that can be used with the methods described herein are shown;

[0064] It should be noted that the figures are shown for simplicity and are not necessarily drawn to scale. Similar features are provided using the same reference numerals. Detailed Implementation

[0065] Figure 3A A flowchart illustrating a method for gain calibration of an ion detector is provided. At step 301, single ions are generated to measure the single-ion response. Single ions can be generated by breaking down a concentrated ion packet that will arrive at the detector at a specific time into single ions that arrive at many different times.

[0066] Step 301, which generates a single ion, can be achieved through a variety of methods. For example, the method described in this article can be used. Figure 4 The time-defocusing method will be described in more detail. Alternatively, fragmentation can be used to break down a single-mass ion into several (or many) fragments. This diffuses the ion over a wider range of possible time-of-flight times to many different m / z values, which greatly increases the probability that those ions arrive as single ions. This can be achieved using any fragmentation method, such as collision-induced dissociation (CID), surface-induced dissociation (SID), electron transfer dissociation (ETD), photodissociation, intrasource fragmentation, etc.

[0067] Another method for generating single ions that can be used in step 301 is to scatter ions using a background gas. In this method, ions collide with gas molecules and exhibit energy and time deviations compared to other ions of the same mass. The time and energy differences may be large enough that the system cannot refocus the ions, and the arrival times of the scattered ions are significantly different, thus enabling the detection of single ions.

[0068] Furthermore, single ions can be generated through beam attenuation. Alternatively, single ions can be generated using an electrospray ionization (ESI) source. Typically, for calibration mixtures, the ion current stability of commercially available ESI sources is approximately 4% per spray (i.e., the ion current varies little across scans). Consistency between the measured ion current (number of ions) and the presence of a similar current in subsequent measurements is a critical prerequisite for critical processes such as ion swarm control (e.g., automatic gain control, AGC), which are widely used to optimize the number of ions delivered to orbital trap mass spectrometers and other ion trap instruments. Therefore, this tolerance is more than sufficient for accurate calibration using the methods disclosed herein.

[0069] A single ion is detected at an ion detector operating at a first detector voltage. In step 302, a parameter is determined by detecting a single ion at the ion detector to establish a first relationship between the output of the ion detector (e.g., current or voltage) and the number of ions at the first detector voltage. For example, an ion detector at the first detector voltage (e.g., 1800 V) may produce a peak with an average value of 5 mV for a single ion. Therefore, the parameter may be a factor of 5 mV per ion. That is, the detector output may be considered as a factor between the number of ions and the detector output. Alternatively, the parameter may be the S / N response of a single charge, since the S / N ratio may be proportional to the number of ions in the ion peak. To use the S / N response as a parameter, the detector's S / N ratio should be relatively stable over time. The S / N ratio of a single charge may be, for example, 4.0 ± 0.4, such that the parameter is a factor of 4.0 per ion.

[0070] The method can proceed to step 303, where an ion peak is detected at the ion detector using a first detector voltage to determine the number of ions in the ion peak based on parameters of a first relationship. The ion peak can be a multi-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 given above regarding step 302, a detected ion peak with an intensity of 200 mV corresponds to the presence of 40 ions in the determined ion peak. Using an S / N response factor of 4.0 per ion, a 200 mV intensity peak corresponds to the presence of 50 ions in the determined ion peak.

[0071] At step 304, the detector voltage is adjusted (increased or decreased) to reduce the ratio between the detector output and the noise level, thereby obtaining a second detector voltage, whose detector output for the next ion peak remains above the noise level. A signal at least 10 times higher than the noise level is sufficient, and preferably, the signal is 30 or 100 times the noise level. If the signal is much larger, the single-ion pulse may be large enough to saturate the detector. The detector can be gradually adjusted, and steps 302 to 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 obtained in these steps can be used to plot a curve showing the relationship between the detector output of the ion detector and the number of ions at the first detector voltage (e.g., as shown in Figure 2 or...). Figure 8 (As shown). This can be called a calibration curve.

[0072] Because the detector voltage has been adjusted, the signal intensity of the ion peak detected by the ion detector will also change for the same number of ions. That is, the parameters of the determined first relationship may not necessarily be an accurate representation of the relationship between the detector output and the number of ions at the second detector voltage. However, the determined number of ions in the ion peak is known from step 303. Therefore, in step 305, based on the determined number of ions in the ion peak (step 303), the parameters of the second relationship between the detector output and the number of ions at the second detector voltage are determined.

[0073] For example, an ion peak with an intensity of 300 mV corresponding to 40 ions (as determined from steps 302 and 303) (if the ion detector voltage is increased in step 304) can produce a factor of 7.5 mV per ion for the parameters of the determined second relationship. If the ion detector voltage is actually reduced in step 304, the factor will decrease.

[0074] It should be noted that the ion peak measured at the second detector voltage does not need to include the exact 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 method disclosed herein. For example, even a 10% difference in the number of ions in the detected ion peaks is sufficient to achieve accurate calibration.

[0075] In addition, correction factors can be applied to explain ion loss in ion detector devices and / or changes in the number of ions in detected ion peaks.

[0076] Another option is to average the number of ions over several scans, which is applicable when the ion source is particularly noisy. Significant source drift is unlikely to occur during these scans, and if it does, it can be corrected. For example, correction may include changing the sequence of steps in the detector voltage scan, or performing single-ion versus multiple-ion comparisons multiple times (e.g., as referenced). Figure 7 (As discussed) and calculate the average value.

[0077] In any case, multiple ion peaks may include hundreds of ions, making ion statistics insignificant. For average measurements from multiple scans, especially with hundreds or thousands of ions, the statistical change in the number of ions or the signal produced by each ion between two measurements becomes even more negligible. Therefore, sufficiently accurate calibration can be performed using the methods disclosed herein, even when the number of ions in the detected ion peaks is not exactly the same.

[0078] The parameters of the first and second relations do not need to represent the same relationship. Preferably, when the S / N factor is used as the parameter of the first relation, the second parameter represents a different relation. For example, using a value obtained by using a 4.0 S / N factor, a 300 mV intensity peak corresponding to 50 ions can be correlated with the parameter of the second relation, which is a factor of 6 mV per ion.

[0079] Also available Figure 3B The method described herein is used to monitor the gain calibration of the ion detector over time.

[0080] At step 311, the second ion peak can be detected at the ion detector using a third detector voltage. This voltage can be the same as the second detector voltage, or it can be another voltage (e.g., a high-gain voltage). The second ion peak can correspond to a single ion or multiple ions. In the case of a single ion, any of the methods described above can be used to generate the single ion, but the most preferred method will be referred to [reference needed]. Figure 4 Describes a time-based defocusing method.

[0081] Step 312 involves determining the number of ions in the second peak. For multi-ion peaks, the number of ions can be determined based on a parameter of the first relationship, which can be the S / N ratio of a single charge. The S / N ratio can be proportional to the number of ions detected in the ion peak and can be relatively stable over time, so that the calculation does not need to be performed more than once.

[0082] At step 313, a second parameter of the second relationship between the detector output of the ion detector and the number of ions can be determined based on the determined number of ions in the second ion peak. Therefore, the second parameter can be a factor between the number of ions and the detector output.

[0083] In step 314, a second parameter of the second relation is compared to an expected value based on the second relation. This can be a direct comparison between the first parameter and the second parameter of the second relation. For example, the first parameter, determined to be 7.5 mV per ion in step 205, can be considered an expected value and compared to the second parameter (e.g., 6 mV per ion) determined in step 313. Alternatively, the expected value of the second relation can be determined based on a calibration curve, which may be obtained, for example, during steps 302 to 304. At step 315, it can be determined whether gain drift has occurred based on the comparison. For example, if the value of the first parameter of the second relation differs from the value of the second parameter of the second relation, then gain drift can be determined to have occurred. Alternatively, if the expected value of the second relation from the calibration curve differs from the second parameter of the second relation, then gain drift may have occurred. A tolerance level may exist for this determination, below which gain drift will be uncertain. This avoids overcorrection of the gain calibration.

[0084] Once gain drift is identified, adjustment parameters can be calculated to adjust the detector voltage to correct the gain drift. These adjustment parameters 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 a first parameter of a second relationship. The adjustment parameters can be calculated based solely on the calibration curve.

[0085] A calibration curve describes the change in signal response to a specific voltage variation. It is assumed that the shape of the curve will not change as the detector gain decreases, but the scale may change. That is, if the single-ion area is remeasured and the results show that the detector gain has decreased, the calibration curve can be used to determine the proportional voltage (or, alternatively, an absolute voltage shift) that will bring the gain back to the expected level. For example, if the gain has decreased by 50%, the expected gain can be achieved by determining the proportional voltage that will cause the calibration curve to increase by 100%. The detector voltage can then be adjusted based on this to set the new voltage.

[0086] As briefly discussed above, a single ion is generated in step 301, which can be achieved through various methods. One such method involves time-defocusing the TOF analyzer.

[0087] Time-of-flight (TOF) analyzers typically use ion mirrors to focus the arrival times of ions of the same m / z onto the narrowest possible period at the detector. Depending on the flight path length and focusing quality, this can achieve resolutions from 10K to 90K.

[0088] The multiple reflection time-of-flight (MR-TOF) instrument has a relative ion mirror between which the analyte ion packet can undergo multiple reflections and thus travel a greatly extended flight path within a small analyzer volume. Figure 4An example of an MR-TOF analyzer is shown. Ions are accumulated using a pulsed extraction ion trap 401, which acts as an ion source, and ejected into the analyzer via a pair of deflectors 405. The deflectors 405 allow for an optimal ejection angle and allow alignment of the focal plane of the ion packet with the analyzer 400. Ions are reflected back and forth between two elongated ion mirrors 403 and drift downwards along the direction of mirror extension. The two ion mirrors 403 are tilted relative to each other, which generates an increased average potential in the drift dimension, slowing down the ion drift and causing the ions to be reflected back in the drift dimension and focused onto the detector 404. The time-of-flight error caused by the mirror tilt is corrected by a “strip electrode” 406 extending along the length of the drift space, thus allowing narrow-time focusing.

[0089] In such analyzers (e.g., which may have a flight path of 20 m), a small offset in the power supply between the ion mirrors 403, such as <5%, is sufficient to completely time-defocus even a relatively strong ion pack at the detector into a single ion. For example, an ion with an m / z of 500 that would normally be focused to a peak of <4 ns might instead diffuse over approximately 1 microsecond. That is, ions with the same m / z arrive at the detector at different times, rather than temporarily converging. The signal can thus be split into approximately 1000 individual peaks, each with a width of approximately 1 ns. The signal of 100 ions will be split into 100 single-ion peaks, some of which have a low probability of overlap. Therefore, recording a single-ion signal will be two orders of magnitude faster than attenuating the focused peak to a single ion. This is within the control structure of the ion trap's automatic gain control and does not require beam attenuation through detuning of the ion optics. The average flight time of defocused single ions, approximately 700 μs, remains similar to that of 500 m / z ions during normal operation, and the low μs energy level extension allows for a coarse preservation of the m / z distribution, which can be improved with appropriate calibration. Ideally, the deflector and / or bar electrode voltages would also be adjusted to maintain optimal ion transport to the detector, but this is not necessary.

[0090] Similar results can be achieved with a single-reflection ToF analyzer that has a smaller (e.g., 2 meters) flight path, but the level of defocus (the difference between the power supply and the ion mirror) would have to be much larger, or the number of ions much lower.

[0091] One advantage of defocusing ions in this way is that many single-ion pulses can be recorded in a single acquisition, allowing for single-ion calibration even with relatively slow analyzers, such as MR-TOF analyzers. Statistical methods based on pulse height variations are compatible with such analyzers, but are slower and can contain significant systematic errors. Another advantage of defocusing ions to produce single ions is that it avoids the need for careful beam attenuation to remove multiple ion peaks, instead minimizing the empty spectrum required by beam attenuation methods.

[0092] Figure 5 An example of diffusion of a single ion with an m / z of 524, as recorded by a mass analyzer, is shown in the figure, with reference to... Figure 4 The configuration is as described, where the ion mirror is set to defocus mode. Peak 520 indicates that ions have arrived at the detector. (As...) Figure 5 As shown in peak 520, due to the analyzer's time-defocused mode, single ions with the same m / z arrive at the detector at different recording times.

[0093] Figure 6A A flowchart illustrating a method for calibrating detector gain via defocused single-ion calibration is presented. This method is similar to... Figure 3A The more general approach described in [the document].

[0094] In step 601, the ion mirror voltage is set to disrupt the focus of the TOF analyzer, ensuring that ions with the same m / z do not arrive at the detector simultaneously. In step 602, the detector voltage / gain may be increased from its initial value. Alternatively, the detector voltage / gain may be decreased from its initial value. A single-ion response (if detectable at this level) may then be measured in step 603, allowing parameters to be determined regarding a first relationship between the ion detector output (e.g., current or voltage) and the number of ions at the first detector voltage. The single-ion response is then compared to a preset upper limit (step 604). The preset upper limit may be a limit higher than the 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 may be a signal region / intensity. Providing a preset upper limit ensures that ions remain within the detector's dynamic range.

[0095] If the preset upper limit is not reached (step 604 not performed), the detector voltage is increased and measured again until the preset upper limit is reached (steps 602 to 604). Finally, in step 605, with single ion and voltage data acquired, a curve representing the relationship between the detector output and the number of ions at the first detector voltage can be fitted / calculated. A suitable detector voltage can be derived from this curve, and the detector voltage can be set to this value.

[0096] This method requires one or more acquisitions for each voltage step, so it can be a relatively fast process for an analyzer operating at 100 Hz. In contrast, existing statistical methods require hundreds of acquisitions per step and take several minutes to complete.

[0097] refer to Figure 6A The described method can vary in several ways. For example, the detector voltage scan range can be fixed as an absolute value or relative to a starting value, or the voltage can be randomly selected within a preset range to reduce the influence of ion source fluctuations.

[0098] Figure 6A While the process is faster than conventional methods, it is still too slow for detector monitoring in experiments. A second method suitable for this type of monitoring is... Figure 6B As shown. In step 611, the ion mirror is set as a reference. Figure 4 The defocusing mode is described. Subsequently, at steps 612 and 613, the detector voltage is set to produce a high-gain voltage (i.e., ions incident on the detector generate a large number of electrons), and the single-ion response is measured. This response is compared to a previously measured single-ion response. [The following text appears to be unrelated and possibly a separate sentence fragment: "can be used..."] Figure 3A , Figure 3B , Figure 6A The method or another method described herein (e.g., refer to...) Figure 10 To obtain the previous measurement values.

[0099] At step 614, gain drift can be calculated and adjustment parameters can be used to correct the gain drift. This can be done using a calibration curve based on previous measurements (e.g., from...). Figure 3A , Figure 3B or Figure 6A The method involves adjusting (increasing / decreasing) the detector voltage to correct drift, or for small gain shifts, adjusting peak intensity, for example, by performing short scans with small voltage steps, until an appropriate gain value is achieved. Because these measurements are very rapid, many measurements can be performed during experiments, such as chromatographic separations and the generation of moving averages.

[0100] The analyte ions used in this step can be selected from the sample itself, dynamically selected, or from background or calibration samples delivered via primary or secondary ion sources.

[0101] The calculated optimal detector voltage can be set for the detector in step 615.

[0102] Since the approximate m / z of time-defocused ions can still be determined using the method described above, measurements of multiple single ions with different m / z ratios can be performed simultaneously. Therefore, the statistical accuracy of the measurement can be improved, and the detector response to m / z can be calibrated. Accuracy can be further improved by calculating a specific time-m / z calibration for the time-defocused ions.

[0103] Similarly, if different charge states of analyte ions exist in the sample or calibration mixture, their response can also be measured and calibrated. Routine mass spectrometry measurements can be performed prior to defocused single-ion measurements to determine the ion type in the sample. Quadrupole isolation of the desired ion for measurement can be performed.

[0104] Because of the assumption that the trend comes from a previously obtained curve (e.g., using...) Figure 6A The method is valid, therefore refer to Figure 6BThe described fast gain adjustment method advantageously avoids the need to redraw the calibration curve. Using... Figure 6A The optimal gain for detecting single ions is lower than the optimal gain for correctly measuring the single ion area because small single-ion peaks are nested in noise and are typically only a single sampling point on a 1 to 2 gigabits per second (GS / s) digitizer. Therefore, the selection of the detector voltage will usually be based on extrapolation from the edge of the calibration curve (e.g., as...). Figure 8 (As shown).

[0105] Therefore, as Figure 7 As shown, after reaching a suitable detector voltage for single-ion measurements, the ion pack can be refocused to produce a multi-ion response. This can then be used as a known number of ions for gain calibration at lower detector voltages. This method involves... Figure 6A The process shown in the figure is reversed (with multiple ion peaks instead of a single ion peak) until the lower limit threshold gain is determined.

[0106] Steps 701 to 704 correspond to Figure 6A Steps 601 to 604. If a threshold intensity or a plateau in counts per second (cps) is reached in step 704, the ion mirror is set to focusing mode in step 705. That is, ions with the same m / z ratio will arrive at the detector simultaneously (i.e., will temporarily converge). Therefore, the ion peak detected by the detector will include multiple ions.

[0107] Steps 701 to 704 can be replaced by another method described herein (i.e., steps 705 to 710 can be used in conjunction with another method described herein). The only requirement is that the parameters of the first relationship are determined before step 705. Thus, for example, steps 705 to 710 can be combined with [the method described herein]. Figure 10 The methods described are used in combination.

[0108] At step 706, the peak intensity of the detected peak is measured and the number of ions is calculated based on the parameters of the determined 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.

[0109] In step 707, the detector voltage is adjusted. If steps 701 to 704 (corresponding to steps 601 to 604) precede this step, the detector voltage will gradually decrease / reduced. However, when steps 705 to 710 are used in conjunction with other methods described herein, the detector voltage can instead gradually increase / reduced. For example, if compared with a reference... Figure 10 If the described methods are used in combination, the detector voltage can be increased or decreased in step 707.

[0110] At step 708, the intensity of the ion peak detected by the detector is measured. As mentioned above, this ion peak will include multiple ions because the ion mirror is set to focusing mode in step 705.

[0111] In step 709, it is determined whether the target gain has been reached and whether the ion peak remains above the noise level. On average, the signal should be strong enough that smaller single-ion results are not lost in the noise. A signal at least 10 times higher than the noise level is sufficient, and preferably, the signal is 30 or 100 times the noise level. If the signal is much greater than this, larger single-ion pulses may saturate the detector.

[0112] If, at step 709, it is determined that the target gain / ion peak has not yet been reached and is not significantly above the noise level, then the method returns to step 707. Otherwise, the method proceeds to step 710, where the optimal detector voltage is calculated and set. The advantage of this method is that gain can be measured rapidly near the optimal detector voltage / target gain level. The calibration curve can be constructed based on the focused multi-ion peaks during steps 707 to 709, but it is not necessary to calculate the calibration curve based on the single-ion response (e.g., during steps 702 to 704). This should improve the accuracy and robustness of gain calibration.

[0113] There is a risk of saturation when comparing single and multiple ion peaks. However, detecting saturated peaks is relatively simple, and for instruments with ion traps, it is very easy to proportionally control the ion cluster by changing the accumulation time.

[0114] Detectors with high dynamic range can handle thousands of ions in multiple ion peaks even under unsaturation conditions (especially with space charge broadening), even when the gain is far above the optimum. In any case, detector overvoltage is readily detectable and can be flagged. Since the number of ions in multiple ion peaks can be inferred from the number of ions in the time-defocused diffusion (because the two should be closely matched), overload can be easily prevented based on the number of ions in the time-defocused diffusion. The AGC method can also optionally be used to tune the ion population in multiple ion emissions relative to single-ion diffusion without significantly reducing the accuracy of ion number determination.

[0115] Figure 8 The calibration curves of the single-ion area versus the detector voltage are shown. These include... Figure 7 The indirect methods shown in the diagram include repeated measurements and single direct measurements of a single ion (e.g., using...). Figure 6A The method involves both manual setting of the detector voltage and direct measurement by analyzing the resulting spectrum. From Figure 8It can be seen that the values ​​are usually reasonably aligned within 10% of the average value, except that at low detector voltages, direct measurement will be interrupted because a single ion falls into the noise band and cannot be detected.

[0116] In indirect single-ion measurement methods (as referenced) Figure 7 A second practical comparison was performed between the methods described above and those based on the statistical methods described in US 7,109,474. The results of this comparison were... Figure 9 The results are shown in the figure. Scans were performed for both methods at a fixed detector voltage, and the single-ion area (SIA) was repeatedly determined for multiple batches with an increasing number of scans. The mean SIA and standard deviation for both methods are shown in the figure. Figure 9 As shown, this indicates that the reproducibility of the single-ion method is within 5% for each measurement of 50 scans, while the statistical method does not reach this level, even at 300 scans. The single-ion method is approximately an order of magnitude faster than the statistical method to achieve a similar level of reproducibility. It is also noteworthy that the average single-ion area value produced by the single-ion method is significantly lower than that of the statistical method (1.1 x 10⁻⁶, respectively). -11 Vs compared to 1.8x10 -11 Vs). This is considered to be a systematic error caused by uncontrolled noise sources in the statistical method.

[0117] For hybrid instruments that combine a charge detection analyzer (e.g., an orbital trap mass spectrometer) with a second analyzer (e.g., a TOF or ion trap analyzer) dependent on an electron multiplier, another gain calibration method is possible. This method can also be applied with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) and TOF analyzers, orbital trap mass spectrometer / ion trap, or FT-ICR / ion trap hybrid instruments. The method involves cross-calibration between the two parts of the hybrid instrument. Cross-calibration methods can be used for gain calibration over time and / or for simpler monitoring and adjustment of the gain, as described below.

[0118] Orbital capture mass spectrometers detect the current caused by charge packets moving across the analyzer and produce a consistent signal-to-noise (S / N) peak proportional to the number of ions for packets exceeding 100. Because orbital capture mass spectrometers do not change rapidly during transport or aging, the peak signal / noise can be used as a measurement of the number of ions to calibrate detector gain or monitor detector aging.

[0119] The preamplifier in an orbital trap mass spectrometer is not sensitive enough to detect single-charged single ions themselves, but it can be used to detect multi-charged single ions (e.g., Makarov et al., "Ion dynamics of intact proteins in an orbital trap mass analyzer," Journal of the American Society for Mass Spectrometry, Vol. 20, No. 8, August 2009, pp. 1486-1495). In short, if the peak intensity in the ion spectrum follows a quantized S / N ratio distribution (i.e., S / N varies with quantum steps to 1, 2, 3, etc.), the detection of individual ions can be demonstrated. The S / N response of a single-charged single ion can be inferred from such measurements, and thus the number of ions in a multi-ion peak can be calculated.

[0120] Orbit capture mass spectrometer analyzers are typically used in conjunction with curved ion traps (C-traps) to prepare ions for implantation. Without automatic gain control (AGC) to regulate accumulation time and ion clusters, these ions can easily overflow and suffer from excessive space charge effects.

[0121] The standard method for automatic gain control in dual-orbit trap mass spectrometer / ion trap analyzer hybrid instruments (e.g., orbital trap combination (RTM) manufactured by Thermo Fisher Scientific) is to measure the number of ions on the ion trap's calibrated electron multiplier detector and use that data to adjust the accumulation time of the C-trap. The method presented in this paper, which uses the orbital trap mass spectrometer signal strength or signal-to-noise ratio to adjust the detector voltage, is somewhat the opposite of this standard approach.

[0122] Figure 10 The figure illustrates a hybrid orbital capture mass spectrometer time-of-flight mass spectrometer suitable for performing methods according to embodiments of the present disclosure. The TOF analyzer 400 in this figure is similar to... Figure 4 The TOF analyzer shown.

[0123] Single ions can be generated using an ESI source of 1020. It should be understood, for example, that references... Figure 3A The other methods described can be modified to generate single ions.

[0124] The single ion then enters the mass spectrometer 1000 and passes through the RF lens 1030. The ion is focused by the lens 1030 into the first and second ion guides 1040. The charged ion is guided along the curved path of the second ion guide 1040, while no neutral ion passes through the mass spectrometer 1000.

[0125] Ion gate 1080 controls the transport of ions from curved ion guide 1040 to mass filter 1090. Mass filter 1090 allows ions of a selected mass number or mass range to pass through while disallowing ions with other m / z ratios. Alternatively, mass filter 1090 can operate in RF-only mode, in which it transports essentially all ions regardless of m / z.

[0126] Ions are then guided by a third ion director 1100 into a C-trap 1060 for ion implantation into a first mass analyzer 1010 (e.g., FTMS, such as an orbital capture mass spectrometer). The C-trap (first ion trap) 1060 has an arc-shaped electrode extending longitudinally, supplied with an RF voltage, and an end cap supplied with a DC voltage. Thus, a potential well is formed extending along the arc-shaped longitudinal axis of the C-trap 1060. In a first mode, the DC end cap voltage is set such that a single ion from the ion director 1100 is trapped in the potential well of the C-trap 1060, where the single ion is cooled.

[0127] The cooled ions are then ejected from the C-well 1060 through the z-lens 1050 into the first mass analyzer 1010, entering the eccentric injection cavity of the first mass analyzer 1010. The ions are then trapped within an orbital capture mass analyzer by a logarithmic electric field, such that their orbital motion around the analyzer axis is (approximately) harmonic. Therefore, the ions are separated according to their m / z ratio.

[0128] Ions are detected using an image detector (not shown). As the ions pass through the image detector, it generates a "transient" in the time domain containing information about the ion species. 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, where the ion abundance / ion intensity axis is relative to m / z (similar to...). Figure 5 As shown, the difference is Figure 5 The x-axis corresponds to the flight time in μs.

[0129] although Figure 10 The FTMS1010 is shown, which traps ions axially and radially using an electrostatic field; however, it should be understood that other forms of FTMS, such as FT-ICR mass analyzers, are covered. In an FT-ICR mass analyzer, ions are axially trapped by an electrostatic field, where radial and azimuth trapping is achieved by applying a magnetic field. A key requirement for the FTMS110 is that its output (i.e., the position and shape of the peaks in the resulting mass spectrum) should be relatively stable over time (e.g., relative to short-term and long-term temperature variations).

[0130] Next, C-trap 1060 is switched to a second mode to allow ions from FTMS 1010 to pass axially toward pyrolysis chamber 1070. Pyrolysis chamber 1070 acts as an ion guide (i.e., ions do not undergo collision with the gas / the energy of the collision gas is insufficient to pyrolyze the precursor ions). Fourth ion guide 1100 then guides the ions from pyrolysis chamber 1070 into extraction trap 401. Extraction trap 401 accumulates the ions ejected from pyrolysis chamber 1070 and then injects them into TOF mass analyzer 400.

[0131] According to this disclosure, the mass spectrometry generated by the FTMS1010 can be used as a parameter to determine a first relationship between the detector output and the number of ions at the first detector voltage. For example, the S / N ratio for multi-charged single ions can be determined as described above, and the S / N response for single-charged single ions can be calculated. The S / N ratio 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. (Refer to above) Figure 10 The method described above generates multi-ion peaks by using multiple ions instead of generating a single ion.

[0132] For reference Figure 3A and Figure 6A As described, the gain of the second analyzer 400 (e.g., a TOF analyzer or an ion trap) can be calibrated. Specifically, the detector voltage of the second analyzer 400 can be adjusted to a second voltage at which the detector output of the ion peak remains above the noise level. Subsequently, based on the determined number of ions in the multi-ion peak, a parameter of a second relationship between the detector output and the number of ions at the second detector voltage is determined. The second analyzer can then operate at the second detector voltage.

[0133] Correction for the ion count must take into account the incoming and / or outgoing ions in the orbital capture mass spectrometer 1010 (with a loss rate of approximately 30% to 50%) and the estimated ion count transferred to the detector in the second analyzer 400, which can limit the accuracy of such calibration. That is, the number of ions in the ion peak calculated using the determined parameters will not directly correspond to the number of ions in the second analyzer 400, thus requiring correction. Ion transfer to the orbital capture mass spectrometer 1010 can depend on various instrumental factors, including one or more of the following: detector voltage, ramp time, ion m / z, and the type of orbital capture mass spectrometer 1010.

[0134] Alternatively / additionally, the parameters determined for the FTMS1010 can be used to monitor / correct gain drift over time, similar to the reference. Figure 3B and 6BThe method described. For example, the S / N ratio known from the FTMS1010 can be used to calculate the gain drift of the less stable second analyzer 400 and the adjustment parameters used to correct the gain drift. The drift can then be corrected by adjusting (increasing / decreasing) the detector voltage of the second analyzer 400 or by adjusting the peak intensity for small gain shifts.

[0135] Specifically, the gain calibration of the ion detector can be monitored by detecting the second peak at the FTMS1010 using the third detector voltage. During the initial gain calibration, the third detector voltage can be the same as the first detector voltage of the FTMS1010. Since the S / N ratio of the FTMS1010 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 mentioned above, this number will need to be corrected for ion transport to the detector in the second analyzer 400.

[0136] Subsequently, a second parameter can be determined based on the number of ions identified in the second ion peak to establish a second relationship between the detector output of the ion detector and the number of ions at the third detector voltage. For example, given the S / N ratio of the FTMS1010, it can be determined (based on the transfer rate) that 100 ions will be transferred to the second analyzer 400. Therefore, a peak with an intensity of 200 mV can correspond to a second parameter of 2 mV per ion.

[0137] The second parameter can then be compared to the expected value based on the first relationship. This can be a direct comparison between the first parameter 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 to 304 or steps 702 to 704 and / or 707 to 709.

[0138] Based on this comparison, it can then be determined whether gain drift has occurred. For example, if the first parameter of the second relation is a different value from the second parameter of the second relation, then gain drift can be determined to have occurred. Alternatively, if the expected value of the second relation from the calibration curve differs from the second parameter of the second relation, then gain drift may have occurred. A tolerance level may exist for this difference; below this tolerance level, gain drift will be uncertain. This avoids overcorrection of the gain calibration.

[0139] For cross-calibration methods, it is preferable to use known samples, such as standard calibration mixtures (e.g., Pierce Flexmix manufactured by Thermo Fisher Scientific). If unknown analytes are used, care must be taken to ensure they are not multicharged, as highly multicharged ions and closely packed isotopes can create interfering patterns within the orbital capture mass spectrometer, reducing apparent signal / noise and affecting the assessment of the number of ions in the peaks.

[0140] The advantage of the cross-calibration method using FTMS (e.g., orbital capture mass spectrometer) described in this paper is that only one measurement with a single ion is required on a single FTMS. The behavior over time is so similar to that of other instruments that those instruments do not need to be self-calibrated (or periodically recalibrated) to a reasonable approximation. It should be understood that the properties of the preamplifiers that cause differences in signal between instruments, such as component tolerances, will also have a similar effect on noise, thus resulting in a relatively consistent signal-to-noise ratio.

[0141] The number of ions that produce a specific signal-to-noise ratio is therefore a known property of a class of instruments, rather than something that must be calibrated for every instrument. More accurate results can be obtained by calibrating a specific instrument, but such accuracy may not be necessary, and the results may be assumed not to change significantly over time.

[0142] Cross-calibration methods are fast and relatively stable. They can also be advantageously combined with other cross-calibration methods (e.g., quality cross-calibration).

[0143] The following clauses describe illustrative embodiments only. These illustrative embodiments can be implemented using any of the embodiments described herein. For example, the method of clause A1 can be used to calibrate the gain, and references can be used. Figure 6B The described method performs gain calibration monitoring. Similarly, the parameters of the first relation and the first parameter of the second relation can be obtained using a reference. Figure 6A The described method is used to determine, in order to monitor gain calibration using the methods described in Clause A2 or A3.

[0144] A1. A method for gain calibration of an ion detector that operates at a detector voltage and forms part of a hybrid mass spectrometer, the method comprising the following steps:

[0145] Generate single ions;

[0146] The parameter determining the first relationship between the detector output of the first ion detector device and the number of ions relative to the first detector voltage is obtained by detecting a single ion at the first ion detector device; and

[0147] The ion peak is detected at the first ion detector device using the first detector voltage, and the number of ions in the ion peak is determined based on parameters of the first relationship.

[0148] The detector voltage of the second ion detector device is adjusted to reduce the ratio between the detector output of the second detector and the noise level, and thus a second detector voltage is obtained at which the detector output of the ion peak detected at the second ion detector device remains above the noise level;

[0149] Based on the determined number of ions in the ion peak, a parameter is used to determine the second relationship between the detector output of the second ion detector device and the number of ions in the second detector voltage.

[0150] A2. The method described in Clause A1 further includes:

[0151] Monitor the gain calibration of the ion detector using the following steps:

[0152] The second ion peak is detected at the first ion detector device using the voltage of the third detector.

[0153] Determine the number of ions in the second ion peak;

[0154] Based on the number of ions determined in the second ion peak, a parameter is used to determine the third relationship between the detector output of the first ion detector device and the number of ions at the third detector voltage.

[0155] Compare the second parameter of the second relation with the expected value based on the second relation; and

[0156] Based on the comparison, it is determined whether gain drift has occurred.

[0157] A3. The method described in Clause A2 further includes:

[0158] Based on the confirmed gain drift:

[0159] Adjustment parameters are calculated based on the second relationship 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; and

[0160] The detector voltage is set based on the adjusted parameters.

[0161] A4. The method according to any of the foregoing clauses, wherein the second ion detector device forms part of a time-of-flight mass spectrometer (TOF MS) or an ion trap.

[0162] A5. The method according to any of the foregoing clauses, wherein the first ion detector device forms part of the Fourier transform mass spectrometer (FTMS).

[0163] A6. The method described in accordance with clause A5, wherein the FTMS is an orbital capture mass spectrometer or a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR).

[0164] A7. The method according to any of the foregoing clauses further comprises:

[0165] The second ion detector device is operated at the second detector voltage.

[0166] A8. The method described in Clause A5, wherein the parameter of the first relationship is the signal-to-noise ratio (S / N) of a single ion.

[0167] A9. The method described in Clause A8 further includes:

[0168] Multicharged single ions are detected at the first ion detector device;

[0169] Calculate the S / N ratio of multi-charged single ions;

[0170] The S / N ratio of a single-charged single ion is determined based on the calculated S / N ratio of the multi-charged single ions.

[0171] A10. The method according to any of the foregoing clauses, wherein

[0172] After determining the number of ions in the ion peak, a correction factor is applied to the determined number of ions to allow the ions to be transported to the second ion detector device.

[0173] A11. The method described in clause A10, wherein the correction factor is between 30% and 50%.

[0174] A12. The method according to any of the foregoing clauses further comprises:

[0175] Single ions are generated by beam attenuation, fragmentation, ion scattering with background gas, or by using an electrospray ionization (ESI) source.

[0176] The methods described herein can be implemented using computer system configurations, including handheld devices, microprocessor systems, microprocessor-based or programmable consumer electronics, microcomputers, mainframe computers, etc. Embodiments can also be practiced in distributed computing environments, where tasks are performed by remote processing devices linked via a network.

[0177] Some embodiments may also be implemented as computer-readable code on a non-transitory computer-readable medium. A computer-readable medium is any data storage device that can store data that can subsequently be read by a computer system. Examples of computer-readable media include hard disk drives, network attached storage (NAS), read-only memory, random access memory, CD-ROM, CD-R, CD-RW, magnetic tape, and other optical and non-optical data storage devices. Computer-readable media may also be distributed across a network-coupled computer system, such that the computer-readable code is stored and executed in a distributed manner.

[0178] Although embodiments according to this disclosure have been described with reference to specific types of apparatus and applications (specifically mass spectrometers) and said embodiments have particular advantages in this case, as discussed herein, the methods according to this disclosure can be applied to other types of apparatus and / or applications. Specific calibration details of the ion detector are not only potentially advantageous (particularly given known calibration constraints and capabilities) but can also be significantly varied to obtain apparatuses with similar or identical operation. Unless otherwise stated, each feature disclosed in this specification can be replaced by alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise stated, each disclosed feature is merely one example of a series of equivalent or similar attribute features.

[0179] All aspects and / or features disclosed in this specification can be combined in any combination, except for at least some mutually exclusive combinations of such features and / or steps. Specifically, preferred features of this disclosure apply to all aspects and embodiments of this disclosure and can be used in any combination. Similarly, features described in non-essential combinations may be used alone (not in combination).

[0180] It should be understood that there are implicit "approximate" terms before the temperature, concentration, time, pressure, flow rate, cross-sectional area, voltage, current, etc., discussed in the teachings of this invention, which may result in slight and non-substantial deviations within the scope of the teachings of this invention.

[0181] As used herein, unless the context otherwise indicates, the singular form of a term contained in the claims shall be construed as including the plural form, and vice versa. For example, unless the context otherwise indicates, singular references contained herein in the claims, such as “a (a / an)” (e.g., an ion pack), mean “one or more” (e.g., one or more ion packs).

[0182] In the description and claims of this disclosure, the words “comprise,” “comprising,” “having,” and “containing,” as well as variations of these words, such as “comprising” and “comprises,” or similar words, mean “including but not limited to,” and are not intended to exclude other components. Furthermore, the use of “or” is inclusive, such that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true.

[0183] The use of any and all instances or exemplary language provided herein (“for instance,” “as,” “for example,” and similar language) is intended only to better illustrate this disclosure and, unless otherwise required, does not indicate any limitation on the scope of this disclosure. No language in this specification should be construed as indicating any unrequired element necessary for the practice of this disclosure.

[0184] The terms "first" and "second" may be reversed without changing the scope of the invention. That is, an element referred to as a "first" element may instead be referred to as a "second" element, and an element referred to as a "second" element may instead be considered a "first" element.

[0185] Unless otherwise stated or required by the context, any steps described in this specification may be performed in any order or simultaneously. Furthermore, the fact that a step is described as being performed after another step does not preclude intermediate steps being performed.

[0186] It should also be understood that, unless otherwise implied or expressly understood or stated, any possible candidates or alternatives listed for any given component or embodiment described herein may generally be used alone or in combination with each other. It should be understood that any list of such candidates or alternatives is merely illustrative and not restrictive, unless otherwise implied or expressly understood or stated.

[0187] In this detailed description of various embodiments, numerous specific details are set forth for illustrative purposes to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these various embodiments can be practiced with or without these specific details. Furthermore, those skilled in the art will readily appreciate that the particular order in which the methods are presented and performed is illustrative, and that the order is contemplated to be changeable while remaining within the scope of the various embodiments disclosed herein.

[0188] All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, monographs, and internet web pages, are expressly and integrally incorporated herein by reference for any purpose. Unless otherwise described, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the various embodiments described herein pertain.

Claims

1. A gain calibration method for an ion detector operating at a detector voltage, the method comprising the following steps: Generate single ions; A parameter for determining a first relationship between the detector output of the ion detector and the number of ions at the first detector voltage by detecting a single ion at the ion detector; Ion peaks are detected at the ion detector using the first detector voltage to determine the number of ions in the ion peaks based on the parameters of the first relationship. The detector voltage is adjusted to reduce the ratio between the detector output and the noise level, thereby obtaining a second detector voltage at which the detector output of the ion peak remains higher than the noise level. A parameter is used to determine a second relationship between the detector output and the number of ions in the second detector voltage, based on the determined number of ions in the ion peak.

2. The method according to claim 1, further comprising: The gain calibration of the ion detector is monitored by the following operation: The second ion peak is detected at the ion detector using the voltage of the third detector. Determine the number of ions in the second ion peak; Based on the determined number of ions in the second ion peak, a second parameter is used to determine the second relationship between the detector output of the ion detector and the number of ions in the third detector voltage; The second parameter of the second relationship is compared with the expected value based on the second relationship; as well as Based on the comparison, it is determined whether gain drift has occurred.

3. The method according to claim 2, further comprising: Based on the confirmed gain drift: Based on the second relationship, an adjustment parameter is 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. as well as The detector voltage is set based on the adjustment parameters.

4. The method according to claim 1 or 2, further comprising: The ion detector is operated at the second detector voltage.

5. The method according to claim 2 or 3, wherein The ion detector forms part of the time-of-flight mass spectrometer.

6. The method of claim 5, further comprising: The single ion is generated by setting the time-of-flight mass spectrometer to operate in a time-defocus mode, in which ions with the same mass-to-charge (m / z) ratio arrive at the ion detector at different times.

7. The method of claim 6, wherein Detecting the second ion peak includes setting the time-of-flight mass spectrometer to operate in the time-defocus mode such that the second ion peak corresponds to a single ion.

8. The method according to any one of claims 1 to 3, further comprising: The detector voltage is gradually adjusted during the step of adjusting the detector voltage; as well as A calibration curve is calculated based on the detector output generated by detecting a single ion at the ion detector for each detector voltage step.

9. The method according to any one of claims 1 to 3, wherein The first relation and the second relation are identical relations; and The parameters of the first relation are different from the parameters of the second relation.

10. The method according to claim 2 or 3, wherein the ion detector comprises: First ion detector device; And a second ion detector device; Furthermore, the steps of determining parameters of a first relationship and detecting an ion peak at the ion detector are performed relative to the first ion detector device, and the steps of adjusting the detector voltage and determining parameters of a second relationship are performed relative to the second ion detector device.

11. The method of claim 10, wherein the second ion detector device forms part of a time-of-flight mass spectrometer (TOF) or an ion trap.

12. The method of claim 10, wherein the first ion detector device forms part of a Fourier transform mass spectrometer (FTMS).

13. The method of claim 12, wherein the FTMS is an orbital trapping or Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer.

14. The method of claim 10, wherein the step of detecting the second ion peak at the ion detector is performed relative to the first ion detector device, and the step of determining parameters of the third relationship is performed relative to the second ion detector device.

15. The method of claim 10, wherein the parameter of the first relationship is the signal-to-noise ratio (S / N).

16. The method of claim 15, further comprising: Multicharged single ions are detected at the first ion detector device; Calculate the S / N ratio of the multicharged single ion; The S / N ratio of a single-charged single ion is determined based on the calculated S / N ratio of the multi-charged single ion.

17. The method of claim 10, wherein A correction factor is applied to the determined number of ions in the ion peak to facilitate ion transport to the second ion detector device.

18. The method of claim 17, wherein the correction factor is between 30% and 50%.

19. The method according to any one of claims 1 to 3, further comprising: Single ions are generated by beam attenuation, fragmentation, ion scattering with background gas, or by using an electrospray ionization (ESI) source.

20. A computer program comprising instructions that, when executed by a computer, cause the computer to perform the method according to any one of claims 1 to 19.

21. A system comprising a mass spectrometer arrangement and a controller, the controller being configured to operate the mass spectrometer arrangement according to any one of claims 1 to 19.

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