Time-of-Flight Mass Spectrometer System

Abstract

To provide a method of calibrating a Time of Flight (TOF) mass analyser.SOLUTION: The present method includes performing a plurality of calibration analyses of calibrant ions using a TOF mass analyser. Each calibration analysis includes measuring the flight times of the calibrant ions using the TOF mass analyser that has an associated instrument parameter having an effect on the flight times of the calibrant ions. Each calibration analysis also includes determining a reference calibration curve for the TOF mass analyser associated with the instrument parameter for the respective calibration analyses based on the known mass to charge ratios of the calibrant ions and the respective flight times. A value of the instrument parameter is different for each analysis. The present method includes determining a calibration curve for use in a TOF mass analysis, the calibration curve being determined based on the plurality of reference calibration curves and the instrument parameter.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This disclosure relates to Time of Flight (TOF) mass spectrometry and TOF mass analyzers. [Background technology]

[0002] Typically, a TOF mass analyzer determines the mass-to-charge ratio of an ion by measuring the time of flight of the ion between two points. To improve the accuracy of the mass-to-charge ratio determination, a TOF mass analyzer may be calibrated by measuring the time of flight of ions of known mass-to-charge ratio. Such ions may generally be referred to as calibrant ions.

[0003] One method for calibrating a TOF mass analyzer involves measuring the time of flight of calibrant ions (e.g., from a calibration mixture) and fitting the measured times to a polynomial. Typically, a second order polynomial function of the form: m / z=At 2 +Bt+C may be used.

[0004] While such an approach can achieve an acceptable level of accuracy, there are various processes occurring within a TOF mass analyzer that can cause the flight times of ions to deviate from the calibration curve. Some deviations are m / z dependent and / or highly non-linear, which can be particularly difficult to capture using a calibration curve.

[0005] One known approach is to use higher order polynomials to fit the calibration curve, however such an approach has little to do with the physics underlying time-of-flight mass analyzers.

[0006] GB-A-2426121 discloses a method of interpolating between measured calibration points which requires that calibration points in a wide m / z range are available.

[0007] Calibration functions with parameters informed by simulation or measurement are also known, e.g., U.S. Pat. No. 6,437,325 (B1), and Christian, NP, Arnold, RJ, & Reilly, JP (2000), "Improved calibration of time-of-flight mass spectra by simplex optimization of electrostatic ion calculations." Such methods do not account for the complex m / z-dependent behavior that arises from electronics or variations between different models.

[0008] Against this background, the present disclosure seeks to provide an improved, or at least a commercially relevant, alternative method of calibrating time-of-flight mass analyzers. Summary of the Invention

[0009] According to a first aspect, a method of calibrating a time-of-flight (TOF) mass analyzer is provided. The method includes performing a plurality of calibration analyses of calibrant ions using the TOF mass analyzer. Each calibration analysis includes measuring a time-of-flight of the calibrant ions using the TOF mass analyzer, the TOF mass analyzer having associated instrumental parameters that affect the time-of-flight of the calibrant ions. Each calibration analysis also includes determining a reference calibration curve for the TOF mass analyzer based on known mass-to-charge ratios of the calibrant ions and their respective times-of-flight, the reference calibration curve being associated with an instrumental parameter of the TOF mass analyzer for the respective calibration analysis. For each of the plurality of calibration analyses, a value of the instrumental parameter of the TOF mass analyzer is different. The method also includes determining a calibration curve for use in the TOF mass analysis performed by the TOF mass analyzer, where the calibration curve is determined based on a plurality of reference calibration curves and instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

[0010] By determining multiple reference calibration curves for different values ​​of the instrument parameter, the relationship between the instrument parameter and the measured time of flight can be explained. In particular, multiple reference calibration curves can more accurately explain any non-linear behavior of the TOF mass analyzer that is difficult to explain using a single calibration measurement.

[0011] By determining a calibration curve for a TOF mass analyzer based on a plurality of reference calibration curves and instrument parameters used in subsequent analysis, the method of the first aspect may provide calibration for the TOF mass analyzer with improved accuracy.

[0012] In some embodiments, the calibrant ions are initially stored in an ion preparation device, preferably an ion trap, connected to a TOF mass analyzer, and measuring the time of flight of the calibrant ions includes injecting the calibrant ions from the ion trap into the TOF mass analyzer. In some embodiments, the calibrant ions are initially stored in the ion trap by applying an RF trapping voltage. In some embodiments, the instrument parameter associated with each reference calibration curve may be the amplitude or frequency of the RF trapping voltage. Thus, in some embodiments, the amplitude and / or frequency of the RF trapping voltage may affect the measured time of flight of the ions. Thus, by calibrating the TOF mass analyzer to account for the RF trapping voltage settings used with each TOF mass analyzer, the accuracy of the analysis may be improved.

[0013] In some embodiments, the time-of-flight shift model may be determined based on the measured time-of-flight of each of the multiple calibration analyses, the known m / z of the calibrant ion for each of the multiple analyses, and the amplitude of the RF trapping voltage for each of the multiple calibration analyses. Thus, the amplitude of the RF trapping voltage may cause a shift in the observed time-of-flight for the calibrant ion of known m / z. That is, the observed time-of-flight may be shifted to (i.e., different from) the expected time-of-flight of the calibrant ion based on its known m / z. This shift in time-of-flight may be characterized by a model (e.g., a polynomial) that is fitted to the observed time-of-flight shift from the calibration analyses. In some embodiments, each reference calibration curve may be determined based on the known mass-to-charge ratio of the calibrant ion, the time-of-flight shift model, and the respective time-of-flight. Thus, the time-of-flight shift model may be utilized to improve the accuracy of the reference calibration curves determined under different RF voltage amplitudes.

[0014] In some embodiments, the time of flight used to determine each reference calibration curve is based on a time of flight shift model and a measured time of flight. Thus, in some embodiments, if the instrument parameters differ from the amplitude of the RF trapping voltage, the effect of the amplitude of the RF trapping voltage can be accounted for by correcting the time of flight used to determine each reference calibration curve.

[0015] In some embodiments, the multiple calibration analyses include a first calibration analysis in which the calibrant ions are first calibrant ions having a first set of mass to charge ratios, and a second calibration analysis in which the calibrant ions are second calibrant ions having a second set of mass to charge ratios different from the first set of mass to charge ratios of the first calibrator ions. Thus, each of the first and second calibration analyses may cover a different mass to charge range of interest, and each of the first and second calibration analyses is performed with different instrument parameters.

[0016] In some embodiments, the plurality of calibration analyses includes a first calibration analysis in which a first reference calibration curve is determined, the first reference calibration curve being defined between a first mass-to-charge value and a second mass-to-charge value. The plurality of calibration analyses may also include a second calibration analysis in which a second reference calibration curve is determined, the second reference calibration curve being defined between a third mass-to-charge value and a fourth mass-to-charge value, the fourth mass-to-charge value being outside the range defined by the first and second mass-to-charge values. Thus, in some embodiments, the mass-to-charge range defined by the third and fourth mass-to-charge values ​​may partially overlap with the mass-to-charge range defined by the first and second mass-to-charge values. In some embodiments, the mass-to-charge range defined by the third and fourth mass-to-charge values ​​may not overlap with the mass-to-charge range defined by the first and second mass-to-charge values. It will be appreciated that when multiple calibration analyses are provided, the mass-to-charge range of one or more of the calibration analyses may overlap with one or more of the other calibration analyses. Each of the calibration analyses may have different instrument parameter settings. Thus, the determined multiple reference calibration curves may provide information regarding the range of different instrument parameter settings and the nonlinearities associated with the instrument parameters for different mass-to-charge ranges.

[0017] In some embodiments, the calibration curve is determined based on a reference calibration curve among a plurality of reference calibration curves that has an associated instrument parameter that is closest to the instrument parameter of the TOF mass analyzer used in the TOF mass analysis. Thus, in some embodiments, the instrument parameter used in the TOF mass analysis may not correspond to the instrument parameter for which the reference calibration curve exists. In such a case, the TOF mass analyzer may determine the calibration curve based on a reference calibration curve that has an associated instrument parameter that is closest to the instrument parameter of interest. Thus, the calibration of the TOF mass analyzer may take into account the effect of the instrument parameter on the measured flight time, thereby improving the accuracy of the calibration of the TOF mass analyzer.

[0018] In some embodiments, the calibration curve is determined by interpolating between two of the reference calibration curves having associated instrument parameters that bound the instrument parameters used in the TOF mass spectrometry. By using interpolation to determine the calibration curve, the method can take into account multiple reference calibration curves, thereby improving the accuracy of the calibration of the TOF mass analyzer.

[0019] It will be appreciated that the method of the first aspect relates to determining a plurality of reference calibration curves and determining a calibration curve for a given instrument parameter setting. Such a method may be performed at the set-up of a TOF mass analyzer such that the determined calibration curves are available for use by a user of the TOF mass analyzer. Thus, the method of the first aspect relates to the calibration of a TOF mass analyzer, including performing a plurality of calibration analyses using calibrant ions. Once a reference calibration curve has been determined using calibrant ions, a calibration curve may be determined for ions of a sample to be analyzed.

[0020] Thus, according to a second aspect of the present disclosure, there is provided a method of time-of-flight mass spectrometry for a mass to charge ratio range of interest using a TOF mass analyzer. The method of the second aspect comprises: providing a plurality of ions having a mass to charge ratio within a mass to charge ratio range of interest; measuring the time of flight of ions using a TOF mass analyzer, the TOF mass analyzer having associated instrument parameters that are used to measure the time of flight; Obtaining a calibration curve for a TOF mass analyzer, the calibration curve comprising: a plurality of reference calibration curves, each reference calibration curve being associated with a different value of an instrumental parameter of the TOF mass analyzer; and based on instrumental parameters of a TOF mass analyzer used to measure the time of flight of the ions. applying the calibration curve to the measured flight times of the ions to determine the mass to charge ratios for the ions.

[0021] Thus, the method of the second aspect relates to performing a method of TOF mass spectrometry utilizing the reference calibration curve determined according to the first aspect. It will be understood that it is not necessary to obtain the reference calibration curve every time TOF mass spectrometry is performed. However, in some embodiments, the applied calibration curve may be determined from the reference calibration curve every time TOF mass spectrometry is performed based on the instrument parameters.

[0022] In some embodiments, the ions are first stored in an ion preparation device, preferably an ion trap, connected to a TOF mass analyzer, and measuring the time of flight of the ions comprises injecting the ions from the ion trap into the TOF mass analyzer. In some embodiments, the ions are first stored in the ion trap by applying an RF trapping voltage, and the instrument parameter associated with each reference calibration curve is the amplitude or frequency of the RF trapping voltage. Thus, in some embodiments, the amplitude and / or frequency of the RF trapping voltage may affect the measured time of flight of the ions. Thus, by calibrating the TOF mass analyzer to account for the RF trapping voltage setting used with each TOF mass analysis, the accuracy of the analysis may be improved.

[0023] In some embodiments, the mass-to-charge range of interest and associated instrument parameters are specified by a user. Thus, in some embodiments, the instrument parameters may be selected based on the mass-to-charge range of interest specified by the user. For example, for injection of ions into a TOF mass analyzer, one or more instrument parameters associated with the injection of ions may be specified based on the mass-to-charge ratio of the ions to be analyzed, for example, to improve the efficiency of injection of ions into the TOF mass analyzer. However, such instrument parameters may also affect the calibration of the TOF mass analyzer. The method according to the second aspect provides a method for determining a calibration curve that describes the instrument parameters selected to improve the accuracy of the analysis.

[0024] In some embodiments, the mass-to-charge range of interest includes a first subrange having an associated first instrument parameter and a second subrange having an associated second instrument parameter, the first and second instrument parameters being different. In some embodiments, the method of TOF mass spectrometry includes measuring the time of flight of ions having a mass-to-charge ratio in the first subrange using the first instrument parameter and measuring the time of flight of ions having a mass-to-charge ratio in the second subrange using the second instrument parameter. In some embodiments, a first calibration curve is determined for the first subrange and a second calibration curve is determined for the second subrange. Thus, the method of mass spectrometry may vary the instrument parameters over the mass-to-charge range of interest, and the calibration curve used is also updated according to the variation of the instrument parameters.

[0025] According to a third aspect of the present disclosure, a time-of-flight (TOF) mass analyzer is provided. The TOF mass analysis system includes a time-of-flight (TOF) mass analyzer and a controller. The TOF mass analysis system has instrument parameters controlled by the controller, the instrument parameters affecting the time-of-flight of the calibrant ions. The controller is configured to cause the TOF mass analyzer to perform a plurality of calibration analyses of the calibrant ions. Each calibration analysis includes measuring the time-of-flight of the calibrant ions using the TOF mass analyzer, and determining a reference calibration curve for the TOF mass analyzer based on known mass-to-charge ratios of the calibrant ions and their respective times-of-flight, the reference calibration curve being associated with an instrument parameter of the TOF mass analyzer for the respective calibration analysis. For each of the plurality of calibration analyses, a value of the instrument parameter of the TOF mass analyzer is different. The controller is also configured to determine a calibration curve for use in the TOF mass analysis performed by the TOF mass analyzer, where the calibration curve is determined based on the plurality of reference calibration curves and instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

[0026] Thus, there may be provided a TOF mass spectrometry system, such as a TOF mass analyser, configured to carry out the method of the first aspect. It will be appreciated that the TOF mass spectrometry system of the third aspect may be configured to carry out any of the optional features discussed above in relation to the first aspect (and also the second aspect).

[0027] In some embodiments, the TOF mass analysis system may include an ion preparation device, preferably an ion trap, configured to store calibrant ions and inject the calibrant ions into the TOF mass analyzer. For each calibration analysis, the controller may be configured to cause the ion preparation device to inject the calibrant ions from the ion trap into the TOF mass analyzer. In some embodiments, the ion trap is configured to apply an RF trapping voltage to store the calibrant ions in the ion trap. In some embodiments, the instrument parameter associated with each reference calibration curve is the amplitude or frequency of the RF trapping voltage. Thus, in some embodiments, the amplitude and / or frequency of the RF trapping voltage may affect the measured flight time of the ions. Thus, by calibrating the TOF mass analyzer to account for the RF trapping voltage settings used with each TOF mass analysis, the accuracy of the analysis may be improved.

[0028] In some embodiments, the controller is configured to cause the TOF mass analyzer to perform a plurality of calibration analyses, including causing the TOF mass analyzer to perform a first calibration analysis in which the calibrant ions are first calibrant ions having a first set of mass to charge ratios, and causing the TOF mass analyzer to perform a second calibration analysis in which the calibrant ions are second calibrant ions having a second set of mass to charge ratios that are different from the first set of mass to charge ratios of the first calibrator ions. Thus, each of the first and second calibration analyses may cover a different mass to charge range of interest, and each of the first and second calibration analyses is performed with different instrument parameters.

[0029] In some embodiments, the controller is configured to cause the TOF mass analyzer to perform a plurality of calibration analyses, the plurality of calibration analyses being configured to cause the TOF mass analyzer to perform a first calibration analysis, where the controller determines from the first calibration analysis a first reference calibration curve, the first reference calibration curve being defined between a first mass-to-charge value and a second mass-to-charge value. The controller may also be configured to cause the TOF mass analyzer to perform a second calibration analysis, where the controller determines from the second calibration analysis a second reference calibration curve, the second reference calibration curve being defined between a third mass-to-charge value and a fourth mass-to-charge value, the fourth mass-to-charge value being outside the range defined by the first and second mass-to-charge values. Thus, in some embodiments, the mass-to-charge range defined by the third and fourth mass-to-charge values ​​may overlap with the mass-to-charge range defined by the first and second mass-to-charge values. In some embodiments, the mass-to-charge range defined by the third and fourth mass-to-charge values ​​may not overlap with the mass-to-charge range defined by the first and second mass-to-charge values. It will be appreciated that when multiple calibration analyses are provided, the mass-to-charge range of one or more of the calibration analyses may overlap with one or more of the other calibration analyses. Each of the calibration analyses may have different instrument parameter settings. Thus, the determined multiple reference calibration curves may provide information regarding the range of different instrument parameter settings and the nonlinearity associated with the instrument parameters for different mass-to-charge ranges.

[0030] In some embodiments, the controller is configured to determine the calibration curve based on a reference calibration curve from a plurality of reference calibration curves having associated instrument parameters that are closest to instrument parameters of a TOF mass analyzer used in the TOF mass analysis.

[0031] In some embodiments, the controller is configured to determine the calibration curve by interpolating between two of the reference calibration curves having associated instrument parameters that bound the instrument parameters used in the TOF mass spectrometry. By using interpolation to determine the calibration curve, the method can take into account multiple reference calibration curves, thereby improving the accuracy of the calibration.

[0032] According to a fourth aspect, there is provided a TOF mass spectrometry system configured to mass analyze a plurality of ions having a mass to charge ratio range of interest. The TOF mass spectrometry system comprises a TOF mass analyzer and a controller. The controller is configured to cause the TOF mass analyzer to measure the time of flight of a plurality of ions having a mass to charge ratio within the mass to charge range of interest, the TOF mass analyzer performing the time of flight measurements using associated instrumental parameters of the TOF mass spectrometry system. The controller is also configured to determine a calibration curve for the TOF mass analyzer. The controller determines the calibration curve based on a plurality of reference calibration curves, each reference calibration curve being associated with a different value of an instrumental parameter of the TOF mass analyzer, the instrumental parameters of the TOF mass spectrometry system being used to measure the time of flight of the ions. The controller is also configured to apply the calibration curve to the measured time of flight of the ions to determine the mass to charge ratio of the ions.

[0033] It will therefore be appreciated that the TOF mass spectrometry system of the fourth aspect may be configured to carry out the method of the second aspect of the present disclosure. In some embodiments, the TOF mass spectrometry system of the fourth aspect may also be capable of carrying out the method of the first aspect.

[0034] In some embodiments, the TOF mass spectrometry system of the fourth aspect may include an ion preparation device, preferably an ion trap. In some embodiments, the ions to be measured are first stored in the ion preparation device connected to the TOF mass analyzer, and the controller is configured to cause the ion preparation device to inject the ions from the ion trap into the TOF mass analyzer for measuring the time of flight of the ions. In some embodiments, the ion preparation device is configured to apply an RF trapping voltage to store the ions in the ion trap, and the instrument parameter associated with the measurement of the ions is the amplitude or frequency of the RF trapping voltage.

[0035] In some embodiments, the mass-to-charge range of interest and instrument parameters are specified by the user.

[0036] In some embodiments, the mass-to-charge range of interest includes a first subrange having an associated first instrument parameter and a second subrange having an associated second instrument parameter, the first and second instrument parameters being different. In some embodiments, the controller is configured to cause the TOF mass analyzer to measure a time-of-flight of an ion for each of the first and second subranges using the respective first and second instrument parameters. In some embodiments, the controller is configured to determine a first calibration curve for the first subrange and a second calibration curve for the second subrange, the controller being configured to apply the first calibration curve to the measured time-of-flight of the ion over the first subrange and the second calibration curve to the measured time-of-flight of the ion over the second subrange to determine the mass-to-charge ratio for the ion.

[0037] According to a fifth aspect of the present disclosure there is provided a computer program comprising instructions for causing a TOF mass spectrometry system of the third or fourth aspect to carry out steps of the method according to the first and / or second aspects of the present disclosure.

[0038] According to a sixth aspect of the present disclosure, there is provided a computer readable medium storing the computer program of the fifth aspect. The present disclosure will now be described with reference to the following figures: [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 is a schematic diagram of a TOF mass spectrometry system according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram of a TOF mass analyzer according to the present disclosure. [Diagram 3] 1 is a graph of a standard calibration curve. [Figure 4] 1 is a graph of the mass error obtained from subsequent measurements of the same calibrant ions using a TOF mass analyzer. [Diagram 5] 1 is a graph showing the variation of m / z shift depending on the RF trapping voltage. [Figure 6] 13 shows oscilloscope traces of signals applied to electrodes of an ion preparation device during a pulse extraction process. [Figure 7] 1 is a block diagram of a method for calibrating a TOF mass analyzer according to the present disclosure. [Figure 8] FIG. 1 is a block diagram of a method for TOF mass spectrometry. [Figure 9a] 1 shows a graph of measured m / z shifts of sample ions of known m / z for TOF mass spectrometry under different instrument parameter settings. [Figure 9b] 1 shows a graph of measured m / z shifts of sample ions of known m / z for TOF mass spectrometry under different instrument parameter settings. [Figure 9c] 1 shows a graph of measured m / z shifts of sample ions of known m / z for TOF mass spectrometry under different instrument parameter settings. [Figure 10] 1 is a graph of the time-of-flight shift in RF trapping amplitude for calibrant ions of different m / z. [Figure 11]1 is a graph showing the variation of residual mass error for different RF trapping voltages over a range of m / z values. [Figure 12] FIG. 12a shows a graph of the time-of-flight shift in RF trap amplitude for calibrant ions of different m / z, and FIG. 12b shows a graph of the residual mass error (in ppm) for the same calibrant ions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] According to one embodiment of the present disclosure, there is provided a TOF mass spectrometry system 1. A schematic diagram of the TOF mass spectrometry system is shown in Figure 1. The TOF mass spectrometry system 1 includes an ion source 2, an ion preparation device 4, a controller 6, and a TOF mass analyzer 10.

[0041] The ion source 2 is configured to provide a source of ions for analysis by the TOF mass analyzer 10. The ion source 2 may be configured to ionize sample molecules to produce sample ions that are analyzed by the TOF mass analyzer 10. In some embodiments, the ion source 2 may be an electrospray ionization source. In some embodiments, the ion source 2 may be configured to ionize a stream of sample molecules provided from a chromatographic device (not shown in FIG. 1 ).

[0042] The ion preparation device 4 is configured to receive ions from the ion source 2. The ions may be accumulated (stored) in the ion preparation device 4. The ion preparation device 4 may comprise a plurality of electrodes arranged to define an ion storage volume in which the ions are stored. The ion preparation device 4 may be configured to apply one or more voltages to the electrodes to receive, trap and release ions from the ion storage volume. The ion preparation device 4 is configured to release the stored ions from the ion preparation device (directly) to the TOF mass analyzer 10. In some embodiments, the ion preparation device 4 may be an ion trap. For example, the ion trap may be a linear ion trap or a curved ion trap (C-trap).

[0043] The ion preparation device 4 may be configured to store ions received from the ion source 2 until a desired number of ions are accumulated in the ion preparation device 4 or until a predetermined period of time has elapsed, after which the ion preparation device 4 may release the stored ions into the TOF mass analyzer 10. The ion preparation device 4 may be configured to store ions by applying an RF trapping voltage to electrodes of the ion preparation device 4. The applied RF trapping voltage has an RF voltage amplitude and an RF voltage frequency. The trapped ions may be released from the ion preparation device 4 by applying an release voltage to electrodes of the ion preparation device 4.

[0044] In some embodiments, other ion transport devices (not shown in FIG. 1 ) may be provided as part of the TOF mass spectrometry system 1. For example, the TOF mass spectrometry system may also include a fragmentation chamber configured to fragment (precursor) ions to generate product ions. The TOF mass spectrometry system may also include a mass selection device (e.g., a quadrupole mass selector) configured to select the mass-to-charge ratio of the ions provided to the ion preparation device 4. Fragmentation chambers and mass selection devices are well known to those skilled in the art and will not be described further herein.

[0045] The TOF mass analyzer 10 is configured to receive the calibrant ions from the ion preparation device 4. In some embodiments, the TOF mass analyzer 10 may be a multi-reflecting TOF mass analyzer (MR-TOF). A schematic diagram of a MR-TOF mass analyzer 10a is shown in FIG.

[0046] The MR-ToF 200 comprises a first focusing ion mirror 11 and a second focusing ion mirror 12. The first and second focusing ion mirrors 11, 12 are arranged opposite each other to define an ion trajectory with multiple reflections between the first and second focusing ion mirrors 11, 12. As further shown in FIG. 2, ions are input to the MR-ToF 10a from an ion preparation device 4. The ions travel from the ion preparation device 4 through a first out-of-plane lens 13, a first deflector 14, a second out-of-plane lens 15 and a second deflector 16 before traveling between the focusing ion mirrors 11, 12. Ions leaving the MR-ToF 10a are captured by an ion detector 17. Further details of the MR-TOF 10a can be found at least in US Pat. No. B-9136101.

[0047] In the following description of the calibration of a TOF mass analyzer 10, the calibration of the MR-TOF 10a of Figure 2 is considered, however, it will be understood that the method may be applied to other TOF mass analyzers 10. Thus, the method of calibrating a TOF mass analyzer 10 according to the present disclosure may be applied to any TOF mass analyzer 10.

[0048] The TOF mass analyzer 10 is typically capable of determining the m / z ratio of ions within an accuracy of about 5 ppm (parts per million), more preferably up to about 1 ppm.

[0049] In essence, the ion flight time t can be expressed as a function of the effective path length L, the energy induced per charge by the accelerating voltage U in electron volts, and the mass / charge ratio m / z of the ion.

[0050]

number

[0051] Therefore, m / z~t 2As such, one method for calibrating a TOF mass analyzer involves measuring the time of flight of multiple calibrant ions of known m / z. The calibrant ions may be provided as part of a calibration mixture (e.g., Pierce™ Flexmix™) that is ionized by ion source 2. The measured times of flight may then be used to fit parameters from a second order polynomial calibration function. m / z=At 2 +Bt+C

[0052] The polynomial-based calibration method has been found to be suitable for TOF mass analyzers, including MR-TOFs such as the MR-TOF of FIG. 2. In these instruments, the significance of measurement effects due to ion source variations (e.g., voltage variations during ion injection) is reduced due to a larger percentage of the total flight time spent within the main analyzer volume. FIG. 3 shows a calibration curve fitted to the measured flight times of calibrant ions obtained from the MR-ToF analyzer 10a of known m / z. FIG. 4 shows the m / z errors obtained from subsequent measurements of the same calibrant ions using the MR-TOF mass analyzer 10a and the determined calibration curve. The solid lines show the average m / z error for multiple measurements of each calibrant ion of known m / z. As shown in FIG. 4, the m / z accuracy for most measurements is less than 1 ppm, but measurements are made where shot-to-shot jitter or ion statistical uncertainty add some excess error.

[0053] The calibration curve obtained in FIG. 3 can calibrate the TOF mass analyzer 10 to have a mass accuracy of about 1 ppm, but the calibration is most accurate when the TOF mass analyzer 10 is operated under the same conditions as the calibration measurement. Thus, variations in one or more instrument parameters of the TOF mass analysis system 1 can cause deviations from a simple calibration curve. This is because the calibration curve is a de facto approximation of the often very complex ion flight path, incorporating regions of acceleration / deceleration, focusing, and considerable deviations in ion energy and trajectory. Some of these deviations are quite m / z dependent. For example, the rise time of the pulsed extraction voltage induces an m / z dependence of ion energy as lighter ions move further across the acceleration field during their rise. Ripples during pulse extraction also cause similar problems. Another example is that RF trapping and space charge cause high m / z ions to be more widely distributed in the ion source, resulting in a larger energy spread after pulse extraction. Thus, although the following discussion of calibration of TOF mass spectrometry system 1 focuses on the effects of RF trapping voltage, it will be understood that the calibration techniques discussed herein may be applied to account for any nonlinear effects associated with any instrumental parameter of TOF mass spectrometry system 1.

[0054] The inventors have realised that for a TOF mass spectrometry system comprising an ion preparation device 4 for RF trapping and pulse-extracting ions into a TOF mass analyser 10, the variations in the instrument parameters associated with the ion preparation device can be highly m / z dependent. Thus, the inventors have realised that variations in the trapping RF amplitude of the ion preparation device 4 produce very substantial shifts in the measured m / z. Furthermore, the variations in the measured m / z with the trapping RF amplitude are highly non-linear and m / z dependent, making it difficult to capture the variations with a single calibration curve.

[0055] FIG. 5 is a graph of the measured m / z variation for three different calibrant ions with RF trapping voltage amplitudes varying from 400V to 1800V. Three calibrant ions with known m / z of 195, 524, and 1422, respectively, were mass analyzed using TOF mass spectrometry system 1, using different RF trapping voltage amplitudes. The measured m / z of each calibrant ion was compared to the known m / z to determine the mass shift (in ppm) for different RF trapping voltages. As shown in FIG. 5, the variation in instrument parameters causes a mass shift of about 10 ppm over the range of RF trapping voltages analyzed, which is about an order of magnitude higher than the desired mass accuracy of 1 ppm. Furthermore, it will be appreciated that the nature of the mass shift is highly m / z dependent (e.g., near an RF trapping voltage of about 1000V) and highly nonlinear. Instrument parameter dependent mass shifts of up to about 60 ppm have been observed in other TOF mass spectrometry systems. It will be appreciated that the multi-dimensional non-linear relationships between these parameters mean that it is difficult to calibrate a TOF mass analyzer to account for these parameters using a single calibration measurement.

[0056] In the case of the RF trapping voltage, the cause of the mass shift is believed to be due to interference from residual RF interfering with the pulse extraction process. Figure 6 shows an oscilloscope trace of the signal applied to the electrodes of the ion preparation device 4 during the pulse extraction process, which operates in a manner described in detail by Hock in US Patent No. B-9548195. The RF is first quenched near the zero crossing point (optionally at different points for different phases), there is an optional short delay, and then a pulse extraction voltage of 900V is applied to the electrodes.

[0057] As shown in FIG. 6, the applied opposing RF trapping voltages are applied to the electrodes of the ion preparation device 4 during the ion storage period. During the ion release process, the ions stored in the ion preparation device 4 are injected into the MR-TOF mass analyzer 10a. It is believed that the overshoot and associated ringing introduce mass errors that are m / z dependent. In particular, ringing on the extraction waveform, as well as relatively small shifts in the rise time and start point of the extraction waveform, are observed to vary with the amplitude of the RF trapping voltage. Notably, these variations are observed despite quenching of the RF trapping voltage. Thus, even with advanced RF power supply design techniques to reduce RF overshoot and ringing (such as quenching), it is extremely difficult to completely eliminate the relationship between the RF trapping voltage and the extraction voltage. Since different RF trapping amplitudes are used by the ion preparation device to trap ions of different m / z ranges, it is not feasible to simply use a single value for the RF trapping voltage amplitude. For example, in the TOF mass analysis system 1, the RF trapping amplitude is usually adjusted from scan to scan according to the m / z ranges of interest for different analytes. Therefore, to improve the m / z accuracy of a TOF mass spectrometry system 1, a method of calibrating the TOF mass spectrometry system 1 that takes into account the instrument parameters used in a particular TOF mass spectrometry scan is desired.

[0058] According to an embodiment of the present disclosure, there is provided a method 100 for calibrating the TOF mass analyzer 10 of Figure 1. Figure 7 provides a block diagram of the method 100 according to an embodiment of the present disclosure.

[0059] According to the method 100, multiple calibration analyses of calibrant ions are performed using the TOF mass analyzer 10. For each calibration analysis, the TOF mass analyzer 10 is used to measure the time of flight of the calibrant ions, the TOF mass analyzer having associated instrument parameters that affect the time of flight of the calibrant ions.

[0060] For example, in method 100, the instrument parameter is the RF trapping voltage amplitude of the ion preparation device 4. Thus, in steps 101, 102, 103 and 104 of method 100, multiple calibration analyses are performed using different RF trapping voltage amplitudes.

[0061] Thus, in step 101, an RF trapping voltage amplitude (instrument parameter) is selected from the list of RF trapping voltages to be measured. In general, the calibration should be performed over the range of instrument parameter values ​​for which the TOF mass spectrometry system 1 will be operated. For example, for TOF mass analyzer 10a, the calibration may be performed for RF trapping voltage amplitudes from about 400V to about 1800V, for example in intervals of about 50V. Thus, at least 10, preferably at least 20, calibration analyses may be performed for the instrument parameter to be calibrated.

[0062] In step 102 , the calibrant ions are analyzed using the TOF mass analyzer 10 and selected instrument parameters are applied to the TOF mass spectrometry system 1 .

[0063] In step 103, a reference calibration curve is determined for the TOF mass analyzer 10 based on the known mass-to-charge ratios of the calibrant ions and their respective times of flight. In some embodiments, the reference calibration curve determined may be a polynomial calibration curve. For example, the reference calibration curve may be: m / z=At 2 +Bt+C The compound may be in the form:

[0064] Thus, in some embodiments, the reference calibration curve may be a polynomial of at least order 2, 3, or 5. Determining the reference calibration curve may include fitting parameters A, B, and C such that measured m / z ratios of the calibrator ions fit known m / z ratios of the calibrator ions.

[0065] In step 104 of the method, the TOF mass spectrometry system 1 may evaluate whether reference calibration curves have been obtained for all instrument parameter settings of interest. Thus, steps 101, 102, and 103 may be repeated with different instrument parameter settings to obtain multiple reference calibration curves as desired.

[0066] In some embodiments, each calibration analysis may be performed using the same calibrant ions over the same m / z range.

[0067] In some embodiments, it may be desirable to calibrate the TOF mass spectrometry system 1 over a relatively wide m / z range so that different calibrant ions and / or different m / z ranges can be used for the calibration analysis.

[0068] For example, in some embodiments, one or more first calibration analyses may be performed in which the calibrant ions are first calibrant ions having a first set of mass to charge ratios. In some embodiments, one or more second calibration analyses may also be performed in which the calibrant ions are second calibrant ions having a second set of mass to charge ratios that are different from the first set of mass to charge ratios of the first calibrator ions. Thus, multiple calibration analyses may use different calibrant ions (or different subsets of calibrator ions) to build a library of reference calibration curves over a relatively wide m / z range with improved accuracy.

[0069] In some embodiments, the first calibration analysis may be performed over a first m / z range, and the second calibration analysis may be performed over a second m / z range. Thus, the first calibration analysis may be used to determine a respective first reference calibration curve defined between a first mass-to-charge value and a second mass-to-charge value. One or more second calibration analyses may be used to determine a respective second reference calibration curve defined between a third mass-to-charge value and a fourth mass-to-charge value, the fourth mass-to-charge value being outside the range defined by the first and second mass-to-charge values. Thus, the first and second reference calibration curves may cover different m / z ranges. In some embodiments, the first and second reference calibration curves may at least partially overlap in m / z. Thus, multiple calibration analyses may cover different m / z ranges to build a library of reference calibration curves over a relatively wide m / z range with improved accuracy.

[0070] In step 105, the reference calibration curve may be stored for later use by the TOF mass spectrometry system 1. In some embodiments, as part of step 105, the TOF mass spectrometry system 1 may determine a calibration curve for use in the TOF mass analysis performed by the TOF mass analyzer 10. The calibration curve may be determined based on a plurality of reference calibration curves and instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

[0071] For example, in some embodiments, the calibration curve is determined based on a reference calibration curve among a plurality of reference calibration curves having associated instrument parameters that are closest to the instrument parameters of the TOF mass analyzer used in the TOF mass spectrometry. Thus, the reference calibration curve obtained under the instrument parameter settings that are most similar to the instrument parameter settings used in the TOF mass spectrometry can be selected.

[0072] In some embodiments, the calibration curve may be determined by interpolating between two of the reference calibration curves having associated instrument parameters that bound the instrument parameters of the ion trap used in the TOF mass spectrometry. For example, if the reference calibration curve is defined by multiple parameters (e.g., A, B, C, etc.), the calibration curve may be determined by interpolation of each of the parameters between the two sets of parameters (e.g., parameter A is obtained by interpolation between parameters A1 and A2, etc.).

[0073] Although the above describes a method for calibrating a TOF mass analyzer for variations in RF trapping voltage, it will be appreciated that other instrument parameters may be calibrated using similar methodologies, such as RF trapping voltage frequency, ion population per TOF mass analysis, etc., may also be calibrated using similar techniques.

[0074] In some embodiments, a calibration curve may be determined that takes into account the variation of two (or more) instrument parameters. In such cases, the above-described method of determining a calibration curve may take into account a two-dimensional selection of a reference calibration curve. For example, a reference calibration curve that is most similar to the combination of instrument parameters may be used to determine the calibration curve. In other embodiments, a two-dimensional interpolation of parameters A, B, C may be used to determine the calibration curve.

[0075] Thus, a calibration curve may be obtained that more accurately describes the influence of one or more instrument parameters on the calibration of the TOF mass spectrometry system 1.

[0076] In addition to the method 100 for calibrating a TOF mass analyzer, according to a further embodiment of the present disclosure, there is provided a method 200 of TOF mass spectrometry for a mass to charge ratio range of interest. Figure 8 provides a block diagram of method 200. Method 200 will be discussed with reference to the TOF mass spectrometry system 1 discussed above.

[0077] In step 201, a plurality of ions having mass to charge ratios within a mass to charge ratio range of interest is provided. For example, the plurality of ions may be sample ions to be analyzed. The sample ions may be provided by a chromatographic device.

[0078] In step 202, the time of flight of the ions may be measured using a TOF mass analyser 10, which has an associated instrument parameter that is used to measure the time of flight. For example, if the instrument parameter is an RF trapping voltage, the RF trapping voltage may be selected based on the m / z range of the ions to be analysed. It will be appreciated that steps 201 and 202 of providing and mass analysing ions may be carried out in a similar manner to the calibration analysis discussed above, and therefore these steps will not be discussed in further detail.

[0079] In step 203, a calibration curve for the TOF mass analyzer is obtained. The calibration curve is obtained based on a plurality of reference calibration curves, each reference calibration curve being associated with a different value of an instrumental parameter of the TOF mass analyzer, which is used to measure the time of flight of ions. It will be appreciated that the reference calibration curves may be obtained using the method of calibrating a TOF mass analysis system described above.

[0080] In step 204, the calibration curve is applied to the measured flight times of the ions to determine the mass to charge ratios for the ions.

[0081] In some embodiments, the mass-to-charge range of interest includes a first subrange having an associated first instrument parameter and a second subrange having an associated instrument parameter, the first and second instrument parameters being different. Thus, for TOF analyses in which it is desirable to change instrument parameter settings for different m / z ranges, method 200 may update the calibration curve used accordingly.

[0082] Thus, in some embodiments of method 200, step 202 includes measuring the time of flight of ions having mass to charge ratios within a first sub-range using a first instrument parameter, and measuring the time of flight of ions having mass to charge ratios within a second sub-range using a second instrument parameter. In such embodiments, a first calibration curve is determined for the first sub-range and a second calibration curve is determined for the second sub-range in step 203. The associated calibration curve is then applied in step 204 to determine the m / z of the ions.

[0083] Thus, according to method 200, the calibration curve used for TOF mass spectrometry system 1 can be determined on a measurement-by-measurement basis. Thus, when a TOF mass spectrometry workflow is performed that includes multiple TOF measurements with varying instrument parameters, the calibration of the TOF mass analyzer can be adjusted to compensate for any mass shifts associated with the variations in the instrument parameters.

[0084] 9a, 9b, and 9c show graphs of measured m / z shifts of sample ions of known m / z for TOF mass analysis under different instrument parameter settings. The measurements in 9a, 9b, and 9c were obtained using the TOF mass analysis system 1 described above. In the examples of 9a, 9b, and 9c, the instrument parameter that is varied is the RF trapping voltage. For each data point, the time of flight was measured for the sample ion of interest under a particular RF trapping voltage. A calibration curve was then applied and the determined m / z of the sample ion was compared to the known m / z of the sample ion to determine the mass shift (in ppm). In each of the graphs, sample ions of m / z 195.87652, 524.2649645, and 1421.9778621, corresponding to caffeine 915, MRFA524, and Ultramark1422, were analyzed. Sample ions were measured over a range of RF trapping voltages from 400V to 1800V.

[0085] In the example of Figure 9a, the same calibration curve was used for each measurement. The calibration curve used was obtained using an RF trapping voltage of about 800 V. It will be appreciated that using a single calibration curve for all measurements will result in mass shifts in the range of about +9 ppm to -5 ppm (total range of about 14 ppm) as the RF trapping voltage is varied. The average mass shift error was about 5 ppm. It will be appreciated that the variation of m / z shift with m / z is highly non-linear. The variation of m / z shift with RF trapping voltage is also highly non-linear. It will be appreciated that it would therefore be extremely difficult to provide a single calibration curve that accurately compensates for these effects.

[0086] Referring to Figure 9b, a number of reference calibration curves are provided from which a calibration curve was determined for each TOF measurement. The calibration curves were obtained at RF trapping voltages of 600V, 800V, 1200V, and 1600V.

[0087] In Figure 9b, each calibration curve was determined based on a reference calibration curve of a plurality of reference calibration curves having associated instrument parameters that are closest to the instrument parameters of the TOF mass analyzer used in the TOF mass analysis. It will be appreciated that for the method of Figure 9b, the m / z shift was in the range of about +2 ppm to -2 ppm for a substantial majority of the measurements. A larger m / z shift was evident for the m / z 195 ion at an RF trapping voltage of about 1400 V, reflecting that such a large trapping voltage is not particularly suitable for accurate measurement of relatively low m / z ions.

[0088] In Figure 9c, multiple reference calibration curves are provided from which calibration curves were determined for each TOF measurement using interpolation. For these measurements, the average m / z shift was approximately 1 ppm, a further improvement over the method of Figure 9b.

[0089] As discussed above, in some embodiments, the amplitude of the RF trapping voltage may cause, at least in part, a shift in the determined m / z of an ion. This shift may be observed as a shift in the observed flight time for an ion of known m / z. Thus, in some embodiments, multiple calibration analyses may be used to determine the flight time shift for a given instrument parameter (RF trapping voltage). Figure 10 shows a graph of flight time shift (i.e., the difference between the measured flight time and the expected flight time for an ion of known m / z) for different RF trapping voltages. Figure 10 shows the flight time shift due to RF trapping voltage for ions of different known m / z.

[0090] As can be seen from Fig. 10, the time-of-flight shift behavior can be characterized by a polynomial function. For example, in the embodiment of Fig. 10, the time-of-flight shift (t s ) can be found by fitting a model of the following form to the measured data: t s (A)=c0+c1A+c2A 2 +c3A 3 where c0, c1, c2, and c3 are parameters that are fitted to the determined time-of-flight shifts from multiple calibration analyses.

[0091] This RF trapping voltage dependent time-of-flight shift can be incorporated into the process of determining multiple reference calibration curves and / or determining a calibration curve for use in a TOF analysis.

[0092] For example, in some embodiments, m / z=At 2 A reference calibration curve of the form +Bt+C can be determined based on the time-of-flight value t that is updated based on the time-of-flight shift. Thus, the RF amplitude-dependent time-of-flight shift t(A) can be subtracted from the arrival time before finding the RF amplitude-dependent model parameters. This offset can be calculated using the following form: t(m,A)=t m(m)+t s (A) can be found by fitting a model of the ion beam to calibration data obtained using different (singly charged) calibration ion species of known mass m and under known RF amplitude A, where t m is the offset parameter for each known calibrant ion mass, and the model t(A) for the RF amplitude dependent time-of-flight shift is mass independent. In FIG. 10, the model t(A) was chosen as a simple polynomial of degree 3.

[0093] For example, in some embodiments, an initial estimate of the model may be based on a linear fit using calibration data. t(m,A)=a0(tt(A)) 2

[0094] In such an embodiment, the parameters a0, c0, c1, c2, c3 may be initially estimated using a linear fit of the calibration data. In some embodiments, the model may be further refined using a curve-fitting algorithm. For example, a Levenberg Marquardt algorithm (damped least squares) may be used to refine the estimated parameters to improve the model behavior.

[0095] In some embodiments, the updated time-of-flight value t(m,A) is then used to calculate the time of flight of the form m / z=At 2 The associated reference calibration curve for +Bt+C can then be determined. Thus, the reference calibration curve for each calibrant ion can be modeled as follows:

[0096]

number

[0097] Figure 11 shows a graph of the residual mass error (in ppm) for ions of known mass to charge ratio, analyzed according to one or more reference calibration curves having RF dependent parameters a(A), b(A) and c(A). It will be seen that the residual error is less than ±2 ppm over the m / z range of 40 to 1900.

[0098] Alternatively, another possible model for each reference calibration curve to obtain m / z from a given arrival time t and RF amplitude A can be of the form:

[0099]

number

[0100] Figure 12a shows a further graph of the variation of the time-of-flight shift with RF trapping voltage amplitude. As can be seen from Figure 12a, the time-of-flight shift depends on the RF trapping voltage, but also has a (less significant) dependence on the m / z (i.e., time-of-flight) of the calibrant ions. Figure 12b shows the residual mass error (in ppm) for each data point in Figure 12a.

[0101] Thus, in some embodiments, the determination of each reference calibration curve may be a multi-step process in which the time-of-flight data is first shifted to account for the effect of RF trapping voltage on flight time, and then a reference calibration curve is determined that accounts for any m / z-dependent behavior.

[0102] Thus, in some embodiments, the multi-step process for determining each reference calibration curve may take into account multiple instrument parameters. For example, in some embodiments, the RF trap voltage behavior may be accounted for in the step of calculating the time-of-flight shift. The reference calibration curve may then be determined based on different instrument parameters (e.g., RF trap voltage frequency, ion population per TOF mass spectrometry, etc.) using measurements of the time-shifted time-of-flight.

[0103] Thus, according to an embodiment of the present disclosure, a method for calibrating a TOF mass analyzer and performing TOF mass analysis is provided.

Claims

1. 1. A method for calibrating a time-of-flight (TOF) mass analyzer, comprising the steps of: performing a plurality of calibration analyses of calibrant ions using the TOF mass analyzer, each calibration analysis comprising: measuring the time of flight of the calibrant ions using the TOF mass analyzer, the TOF mass analyzer having associated instrument parameters that affect the time of flight of the calibrant ions; determining a reference calibration curve for the TOF mass analyzer based on the known mass to charge ratios of the calibrant ions and their respective times of flight, the reference calibration curve being associated with the instrumental parameters of the TOF mass analyzer for each of the calibration analyses, the value of the instrumental parameters of the TOF mass analyzer being different for each of the plurality of calibration analyses; and determining a calibration curve for use in a TOF mass analysis performed by the TOF mass analyzer, the calibration curve being determined based on a plurality of the reference calibration curves and the instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

2. 2. The method of claim 1, wherein the calibrant ions are initially stored in an ion preparation device, preferably an ion trap, connected to the TOF mass analyzer, and measuring the time of flight of the calibrant ions comprises injecting the calibrant ions from the ion trap into the TOF mass analyzer.

3. The method of claim 2 , wherein the calibrant ions are initially stored in the ion trap by applying an RF trapping voltage.

4. The method of claim 3 , wherein the instrument parameter associated with each reference calibration curve is the amplitude or frequency of the RF trapping voltage.

5. a time-of-flight shift model is determined based on the measured time-of-flight of each of the plurality of calibration analyses, a known mass-to-charge ratio of the calibrant ions for each of the plurality of calibration analyses, and an amplitude of the RF trapping voltage for each of the plurality of calibration analyses; 5. The method of claim 3 or 4, wherein each reference calibration curve is determined based on the known mass to charge ratio of the calibrant ions, the time-of-flight shift model, and the respective times-of-flight.

6. 6. The method of claim 5, wherein the time-of-flight used to determine each reference calibration curve is based on the time-of-flight shift model and the measured time-of-flight.

7. said plurality of calibration analyses comprising: a first calibration analysis, wherein the calibrant ions are first calibrant ions having a first set of mass to charge ratios; a second calibration analysis, wherein the calibrant ions are second calibrant ions having a second set of mass to charge ratios different from the first set of mass to charge ratios of the first calibrant ions.

8. said plurality of calibration analyses comprising: a first calibration analysis in which a first reference calibration curve is determined, the first reference calibration curve being defined between a first mass-to-charge value and a second mass-to-charge value; and a second calibration analysis in which a second reference calibration curve is determined, the second reference calibration curve being defined between a third mass-to-charge value and a fourth mass-to-charge value, the fourth mass-to-charge value being outside the range defined by the first and second mass-to-charge values.

9. 4. The method of claim 1, wherein a calibration curve is determined based on a reference calibration curve from the plurality of reference calibration curves that has associated instrument parameters that are closest to the instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

10. 4. The method of claim 1, wherein a calibration curve is determined by interpolating between two of the reference calibration curves having associated instrument parameters that bound the instrument parameters used in the TOF mass spectrometry.

11. 1. A method of time-of-flight mass spectrometry for a mass-to-charge ratio range of interest using a TOF mass analyzer, comprising: providing a plurality of ions having a mass to charge ratio within the mass to charge ratio range of interest; measuring the time of flight of the ions using the TOF mass analyzer, the TOF mass analyzer having associated instrument parameters that are used to measure the time of flight; obtaining a calibration curve for the TOF mass analyzer, the calibration curve comprising: a plurality of reference calibration curves, each reference calibration curve being associated with a different value of the instrument parameter of the TOF mass analyzer; and an instrumental parameter of the TOF mass analyzer used to measure the time of flight of the ions. applying the calibration curve to the measured time-of-flight of the ions to determine a mass-to-charge ratio for the ions.

12. 12. The method of claim 11 , wherein the ions are first stored in an ion preparation device, preferably an ion trap, connected to the TOF mass analyzer, and measuring the time of flight of the ions comprises injecting the ions from the ion trap into the TOF mass analyzer.

13. 13. The method of claim 12, wherein the ions are initially stored in the ion trap by applying an RF trapping voltage, and the instrument parameter associated with each reference calibration curve is the amplitude or frequency of the RF trapping voltage.

14. A method according to any one of claims 11 to 13, wherein the mass-to-charge range of interest and the associated instrument parameters are specified by a user.

15. the mass-to-charge range of interest includes a first subrange having an associated first instrument parameter and a second subrange having an associated second instrument parameter, the first and second instrument parameters being different; The method of time-of-flight mass spectrometry comprises: measuring the time of flight of the ions having mass to charge ratios within the first sub-range using the first instrument parameters; and measuring the time of flight of the ions having mass to charge ratios within the second sub-range using the second instrument parameter; a first calibration curve is determined for the first subrange; The method according to any one of claims 11 to 13, wherein a second calibration curve is determined for the second sub-range.

16. 1. A time-of-flight (TOF) mass spectrometry system, comprising: a time-of-flight (TOF) mass analyzer; A controller, the TOF mass spectrometry system having instrument parameters controlled by the controller, the instrument parameters influencing the time of flight of calibrant ions; The controller: causing the TOF mass analyzer to perform a plurality of calibration analyses of calibrant ions, each calibration analysis comprising: measuring the time of flight of the calibrant ions using the TOF mass analyzer; and determining a reference calibration curve for the TOF mass analyzer based on the known mass-to-charge ratios of the calibrant ions and their respective times of flight, the reference calibration curve being related to the instrument parameters of the TOF mass analyzer for each of the calibration analyses; determining, for each of the plurality of calibration analyses, a value of the instrument parameter of the TOF mass analyzer that is different; 1. A time-of-flight (TOF) mass spectrometry system configured to: determine a calibration curve for use in a TOF mass analysis performed by the TOF mass analyzer, the calibration curve being determined based on a plurality of the reference calibration curves and the instrument parameters of the TOF mass analyzer used in the TOF mass analysis.

17. an ion trap configured to store calibrant ions and to inject the calibrant ions into the TOF mass analyzer; 17. The TOF mass spectrometry system of claim 16, wherein for each calibration analysis, the controller is configured to cause the ion trap to inject the calibrant ions from the ion trap into the TOF mass analyzer.

18. the ion trap is configured to apply an RF trapping voltage to store the calibrant ions within the ion trap; 18. The TOF mass spectrometry system of claim 17, wherein the instrument parameter associated with each reference calibration curve is the amplitude or frequency of the RF trapping voltage.

19. The controller is configured to cause the TOF mass analyzer to perform a plurality of calibration analyses, the plurality of calibration analyses comprising: causing the TOF mass analyzer to perform a first calibration analysis, wherein the calibrant ions are first calibrant ions having a first set of mass to charge ratios; and causing the TOF mass analyzer to perform a second calibration analysis, wherein the calibrant ions are second calibrant ions having a second set of mass to charge ratios different from the first set of mass to charge ratios of the first calibrant ions.

20. The controller is configured to cause the TOF mass analyzer to perform the plurality of calibration analyses, the plurality of calibration analyses comprising: causing the TOF mass analyzer to perform a first calibration analysis, wherein the controller determines a first reference calibration curve from the first calibration analysis, the first reference calibration curve being defined between a first mass-to-charge value and a second mass-to-charge value; 19. The TOF mass analyzer system of claim 16, further comprising: causing the TOF mass analyzer to perform a second calibration analysis, wherein the controller determines a second reference calibration curve from the second calibration analysis, the second reference calibration curve being defined between a third mass-to-charge value and a fourth mass-to-charge value, the fourth mass-to-charge value being outside of a range defined by the first and second mass-to-charge values.

21. 19. The TOF mass spectrometry system of claim 16, wherein the controller is configured to determine a calibration curve based on a reference calibration curve from the plurality of reference calibration curves having associated instrument parameters that are closest to the instrument parameters of the TOF mass analyzer used in the TOF mass spectrometry.

22. 19. A TOF mass spectrometry system according to any one of claims 16 to 18, wherein the controller is configured to determine a calibration curve by interpolating between two of the reference calibration curves having associated instrument parameters that bound the instrument parameters used in the TOF mass spectrometry.

23. 1. A time-of-flight mass spectrometry system configured for mass spectrometry of a plurality of ions having a mass-to-charge ratio range of interest, comprising: A TOF mass analyzer; A controller, The controller: causing the TOF mass analyzer to measure the time of flight of a plurality of ions having a mass to charge ratio within the mass to charge range of interest, the TOF mass analyzer performing the measurements of the time of flight using associated instrument parameters of the TOF mass analysis system; determining a calibration curve for the TOF mass analyzer, the calibration curve comprising: a plurality of reference calibration curves, each reference calibration curve being associated with a different value of the instrument parameter of the TOF mass analyzer; and determining based on the instrument parameters of the TOF mass spectrometry system used to measure the time of flight of the ions. applying the calibration curve to the measured time of flight of the ions to determine the mass to charge ratio of the ions.

24. Further comprising an ion trap, 24. The TOF mass spectrometry system of claim 23, wherein the ions to be measured are initially stored in an ion trap connected to the TOF mass analyzer, and the controller is configured to cause the ion trap to inject the ions from the ion trap into the TOF mass analyzer for measurement of the time of flight of the ions.

25. 25. The TOF mass spectrometry system of claim 24, wherein the ion trap is configured to apply an RF trapping voltage to store the ions in the ion trap, and the instrument parameter associated with the measurement of the ions is the amplitude or frequency of the RF trapping voltage.

26. A TOF mass spectrometry system according to any one of claims 23 to 25, wherein the mass-to-charge range of interest and the instrument parameters are specified by a user.

27. the mass-to-charge range of interest includes a first subrange having an associated first instrument parameter and a second subrange having an associated second instrument parameter, the first and second instrument parameters being different; The controller: causing the TOF mass analyzer to measure the time of flight of the ions for each of the first and second subranges using the first and second instrument parameters, respectively; determining a first calibration curve for the first subrange; determining a second calibration curve for the second subrange; 26. A TOF mass spectrometry system according to claim 23, wherein the controller is configured to apply the first calibration curve to the measured times of flight of the ions over the first sub-range and to apply the second calibration curve to the measured times of flight of the ions over the second sub-range to determine a mass to charge ratio for the ions.

28. A computer program comprising instructions for causing a TOF mass spectrometry system according to claim 16 or 23 to carry out the steps of the method according to claim 1 or 11.

29. 30. A computer readable medium having stored thereon the computer program of claim 28.

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