Patent Application
20240201149
This application claims priority from and the benefit of United Kingdom patent application No. 2105778.1 filed on 23 Apr. 2021. The entire contents of this application are incorporated herein by reference.
The present invention relates generally to mass spectrometers and in particular to mass spectrometers that are controlled in a way that may cause a variation in the sensitivity with which the spectrometer is able to detect ions.
Mass and/or ion mobility spectrometers include various electrodes that are used to manipulate ions, such as ion optics. Voltages are applied to these electrodes so as to maintain them at the electrical potentials required for their use. In general, positive ions are repelled from electrodes that are maintained at a positive electric potential, whereas these ions are attracted to electrodes maintained at a negative potential. Similarly, negative ions are generally repelled from electrodes that are maintained at a negative potential, whereas they are attracted to electrodes maintained at a positive potential.
Since electrodes are electrically conductive and connected to a voltage supply, any ions hitting the electrodes will be neutralised and the charge of the ions returned to the voltage supply. The potential on the electrode will be held at the potential of the voltage supply, no matter what ion current impinges upon it.
However, over time, at least some of the electrodes may become contaminated by the samples being analysed by the spectrometer. For example, the analyte, the matrix in which the analyte is provided, or any other additives in the sample may build up on various electrodes of the spectrometer. If this contamination is not electrically conductive then it may form an electrically insulating layer on parts of the electrodes. Any ions hitting these insulating surfaces will not then be electrically discharged and hence a voltage due to the charge of the ions will build up on the insulating layer. Ions passing close to the insulating surface will then be affected by the voltage of the ions on the insulating surface, hence affecting their passage through the mass spectrometer. This may affect the efficiency with which ions are guided to the analyser of the mass spectrometer, and thus may affect the sensitivity of the spectrometer.
Ions on the insulating layer may eventually migrate off the insulating layer so as to directly impact the electrode and thus be discharged. Therefore, the voltage due to ions located on the insulating layer may vary based on the ion current impacting the insulating layer, the capacitance formed by the insulating layer and the rate at which ions leave the insulating layer.
Certain events can cause relatively large ion currents to be directed towards or onto the electrodes. For example, if the electrodes in an ion guiding region are maintained at voltages so as to guide positive ions therethrough, but then the voltages are changed for guiding negative ions, any positive ions remaining in the ion guiding region may hit the electrodes. If one or more electrode of the ion guide is contaminated with an electrically insulating layer then this sudden increase in ion current impinging on the insulating layer may lead to a temporary change in instrument sensitivity (i.e. the efficiency with which ions are transmitted through the spectrometer). The change in sensitivity may then gradually recover to, or towards, its previous value as ions migrate off the insulating layer.
The above described contamination can cause problems with analysing analytes. For example, the above-described changes in sensitivity of the spectrometer can cause problems with quantitation analysis of analytes. For example, conventional approaches to quantitation of an analyte involve the sequential analysis of the analyte and an internal standard, but the above described time-varying change in sensitivity of the spectrometer may affect the analysis of the analyte and internal standard differently. This results in a measurement bias, which may not be well mitigated for, e.g. by use of a calibration curve. Where quality control analysis is being performed, this problem may result in erroneous quality control failures.
The present invention provides a method of mass analysing a single analytical sample comprising: i) transmitting different species of ions through a mass spectrometer; ii) sequentially mass analysing, or otherwise detecting, said different species of ions in a particular sequential order; and then iii) repeating steps i) and ii), wherein the sequential order in which said different species of ions are mass analysed, or otherwise detected, is different when step ii) is repeated.
The present invention recognises that the sensitivity with which the spectrometer is able to detect ions may temporarily vary for a period of time whilst analysing the analytical sample. This variation in sensitivity may be caused by varying the operation of the spectrometer, e.g. by switching an operational parameter thereof. For example, changing the amplitude or polarity of the voltage applied to one or more electrodes may cause this variation in sensitivity. Alternatively, or additionally, a variation in sensitivity of the spectrometer may be caused by a change in another variable, such as being caused by the components of the sample being analysed varying with time. For example, the sample may be separated by chromatography and ionised prior to analysis, and a relatively highly concentrated component may elute from the separation device and be ionised so as to produce a relatively intense ion signal. This intense ion signal may cause the sensitivity of the spectrometer to vary with time, e.g. by the intense ions causing surface charging of electrodes within the spectrometer or by another effect that changes sensitivity.
It is known to analyse different species of ions in sequence and then repeat this sequence of analysis. However, if said different species of ions are sequentially transmitted through the spectrometer in the same order each time, during each period in which the sensitivity of the spectrometer varies, then any given one of these different species of ions will experience substantially the same level of the time-varying sensitivity every time it is analysed. This is problematic, for example, for the comparison of the ion signals for the different species of ions. Embodiments of the present invention enable the sensitivity of the spectrometer to vary on multiple different occasions, but in contrast to known techniques, the order in which the different species of ions are sequentially transmitted is varied so as to be different for said multiple different occasions. This ensures that each of the different species of ions is subjected to multiple different levels of the time-varying sensitivity, thereby mitigating the above problem.
The sensitivity with which the mass spectrometer is able to detect ions may vary for a period of time, and step ii) and iii) may be performed during said period of time.
The method may comprise varying the operation of the mass spectrometer in a manner that causes the sensitivity with which the spectrometer is able to detect ions to vary for said period of time.
Said step of varying the operation of a mass spectrometer may correspond to varying the operational parameter of the mass spectrometer so as to control the mass spectrometer to operate differently. The step of varying the operation of the mass spectrometer may comprise switching the operational parameter between different, discrete values, thus causing the sensitivity with which the spectrometer is able to detect ions to vary for said period of time (after each switch).
Accordingly, said step of varying the operation of the mass spectrometer may comprise switching the voltage applied to at least one electrode of the mass spectrometer that controls the transmission of ions therethrough to a different voltage; and step i) may comprise subsequently transmitting said different species of ions through the spectrometer and passed said at least one of said electrodes.
Said step of varying the operation of the mass spectrometer may comprise switching the polarity of the voltage to a different polarity.
For example, the at least one electrode may be maintained at a first polarity (e.g. positive) in order to transmit ions of the opposite polarity (e.g. negative) through the mass spectrometer, and the step of varying the operation of the mass spectrometer may comprise switching the polarity of that at least one electrode to a second, opposite polarity (e.g. negative) in order to transmit ions of the opposite polarity (e.g. positive) through the mass spectrometer. Steps i) and ii) may then be performed so as to analyse the different species of ions. The method may then comprise switching the at least one electrode back to the first polarity, e.g. in order to analyse different species of ions. The method may then be repeated by switching back to the at least one electrode back to the second polarity etc.
The step of varying the operation of the mass spectrometer may comprise alternating the mass spectrometer between a first mode of operation in which it generates and transmits positive ions and a second mode of operation in which it generates and transmits negative ions, and/or vice versa.
Each time step ii) is performed, it may comprise mass filtering said different species of ions using a mass filter such that only a single species of ion is transmitted to an ion detector at any one time, and the mass filter may be controlled so as to change the species of ion that is transmitted to the detector at different times, thereby defining said sequential order in which the different species of ions are mass analysed.
A first time that step ii) is performed, the mass filter may transmit only a first of said different species of ions to the detector at a first time, and subsequently may transmit only a second of the different species of ions to the detector at a second, subsequent time. A subsequent time that step ii) is performed, the mass filter may transmit only the second of said different species of ions to the detector at one time, and may subsequently transmit only the first of the different species of ions to the detector at a later time.
It will be appreciated that the invention is not limited to transmitting only two species of ions at two respective times, each time step ii) is performed, but that further species of ions might also be transmitted at further respective times. For example, each time step ii) is performed, the mass filter may also transmit only a third species of ion at a third time and optionally may also transmit a fourth (or further) species of ion at a fourth (or further) time.
For example, the first ions may be ions of (or derived from) an analyte of interest. The second ions may be ions of (or derived from) an internal standard for the analyte of interest. The third ions may be confirmatory ions for confirming the analyte of interest is present. The fourth ions may be secondary confirmatory ions for confirming the analyte of interest is present.
Step iii) may comprise performing an analytical sequence that consists of repeating steps i) and ii) a plurality of times, wherein the sequential order in which said different species of ions are mass analysed, or otherwise detected, during these plurality of times is different each and every time step ii) is performed within the analytical sequence.
The method may comprise performing said analytical sequence multiple times, such as an integer number of times.
The method may comprise comparing the ion signal detected for one of said different species of ions with the ion signal detected for another of said different species of ions.
For example, said different species of ions may comprise ions of, or derived from, an analyte of interest and also ions of, or derived from, an internal standard. The ion signal for the ions of, or derived from, the analyte of interest may be compared to the ion signal for ions of, or derived from, the internal standard, e.g. in order to quantify the ions of, or derived from, the analyte of interest.
The user may input into the mass spectrometer the species of ions that are said different species of ions, so that the user selects the species of ions that are analysed in different sequential orders in step iii) (of claim 1). The species of ions may be input into the spectrometer by inputting their mass to charge ratios.
Accordingly, the spectrometer may comprise a user interface and, prior to step ii), the method may comprise selecting the species of ions that are to be said different species of ions and inputting these selected species of ions into the user interface so that the mass spectrometer performs steps ii) and iii) on these ions.
Optionally, only the species of ions that are input into the user interface are mass analysed or otherwise detected during steps ii) and iii).
The species of ions that are input into the spectrometer as said different species of ions are preferably species of ions whose intensities are directly related to each other (at a constant spectrometer sensitivity).
All of the steps described above, or elsewhere herein, may be performed within a single experimental run. In other words, the method described herein may be performed whilst analysing a single analytical sample (rather than analysing different analytical samples in different experiments, or analysing different replicates of an analytical sample in different experiments). For example, the method described herein may be performed during the continuous introduction of an analytical sample into the mass spectrometer (e.g. during the continuous elution of a sample from a liquid chromatography device).
The method may comprise fragmenting or reacting ions of an analyte of interest, and optionally ions of a corresponding internal standard, so as to form fragment or product ions; wherein said different species of ions comprise multiple different ones of the fragment or product ions.
For the avoidance of doubt, the step of reacting ions comprises reacting the ions with another species of ion or molecule so as to form product ions that are different from the ions being reacted.
Said different species of ions that are sequentially analysed may comprise a first fragment or product ion of the analyte of interest, a second fragment or product ion of the analyte of interest, and a fragment or product ion of the internal standard.
It may be desirable to analyse multiple different analytes of interest in the sample.
The method may comprise separating a sample that comprises a plurality of different analytes of interest using a chromatography device such that said different analytes of interest elute from the chromatography device over different respective time periods, and ionising the sample eluting from the chromatography device so as to provide ions of said analytes of interest. Optionally, the sample also comprises an internal standard corresponding to one or more of said different analytes of interest, wherein each internal standard elutes from the chromatography device over substantially the same time period as its respective analyte of interest and is then ionised.
For example, the chromatography device may be a liquid chromatography device or a gas chromatography device.
Steps i) to iii) may be performed during each time period that each analyte of interest is expected to elute from the chromatograph device.
Where multiple different analytes of interest elute from the chromatography device over time periods that partially overlap with each other, performing steps i) to iii) may comprise: a) performing the method of analysis so as to trigger a period of time in which the sensitivity with which the mass spectrometer is able to detect ions varies; and then b) fragmenting or reacting ions of a first analyte of interest, and optionally ions of a corresponding internal standard, so as to form fragment or product ions, and sequentially mass analysing or otherwise detecting these fragment or product ions; and then c) performing the method of analysis so as to trigger a period of time in which the sensitivity with which the mass spectrometer is able to detect ions varies; and then d) fragmenting or reacting ions of a second analyte of interest that elutes from the chromatography device over a time period that partially overlaps with that of the first analyte of interest, and optionally also fragmenting or reacting ions of a corresponding internal standard, so as to form different fragment or product ions, and sequentially mass analysing or otherwise detecting these fragment or product ions; and e) repeating steps a) to d) during the period that the first and second analytes of interest co-elute from the chromatography device.
The method may comprise isolating each analyte of interest (or its respective internal standard) before fragmenting or reacting it. This may be performed by using a further mass filter arranged between the ion source and fragmentation or reaction region so as to transmit only a single analyte of interest (or internal standard) at a time. The method may therefore use a tandem quadrupole (or triple quadrupole) mass spectrometer.
The method may comprise performing steps i) to iii) over the period that an analyte of interest elutes from the chromatography device so as to obtain the intensity of the ion signal detected for one of said different species of ions for each time that the steps are repeated; producing a first set of data that comprises the intensity of the ion signal as a function of detection time; smoothing the first set of data so as to obtain a second set of smoothed data; and determining if maintenance of the spectrometer is required based on a comparison of the first and second sets of data.
Each of the first and second sets of data represent an ion signal peak for said one of the different species of ions. The step of comparing the first and second sets of data may comprise comparing the profiles of the peaks represented by the first and second sets of data. If these differ then it may indicate that the electrodes of the spectrometer may have become contaminated, for example.
The method may comprise controlling a display screen to indicate that maintenance of the spectrometer is required if the first and second sets of data do not match or differ by a predetermined or threshold amount.
Step iii) may comprise repeating step ii) in a manner such that the first species of ion mass analysed, or otherwise detected, any given time that step ii) is performed differs from the last species of ion that was mass analysed, or otherwise detected, the preceding time that step ii) was performed.
This helps to reduce the jitter of the ion signal.
The present invention also provides a mass spectrometer comprising: a mass analyser; and a controller having electronic circuitry configured to control the spectrometer to: i) transmit different species of ions through the mass spectrometer; ii) sequentially mass analyse, or otherwise detect, said different species of ions in a particular sequential order; and then iii) repeat steps i) and ii), wherein the sequential order in which said different species of ions are mass analysed, or otherwise detected, is different when step ii) is repeated.
The spectrometer is configured to perform these steps whilst analysing a single analytical sample, i.e. during a single experimental run.
The spectrometer may be configured to perform any of the methods described herein.
For example, the mass spectrometer may comprise a user interface for inputting the species of ions that are to be said different species of ions into the mass spectrometer, and the mass spectrometer may be configured to perform steps ii) and iii) on these inputted species of ions.
The concept described above of detecting whether or not maintenance is required is new in its own right.
Accordingly, the present invention also provides a method of mass spectrometry comprising: i) operating a mass spectrometer during a period of time in which the sensitivity with which the mass spectrometer is able to detect ions varies; ii) using the mass spectrometer to determine the intensity of the ion signal of an ion of interest within said period of time; iii) repeating steps i) and ii) as the ion of interest, or an analyte from which it is derived, elutes from a separation device; wherein when step ii) is repeated, it is performed at different times after said period of time begins; iv) producing a first set of data that comprises the intensity of the ion signal as a function of detection time; v) smoothing the first set of data so as to obtain a second set of smoothed data; and vi) determining if maintenance of the spectrometer is required based on a comparison of the first and second sets of data.
For example, the method may determine that maintenance is required if the first and second sets of data do not match or differ by a predetermined or threshold amount.
The method may further comprise controlling a display screen to indicate that maintenance of the spectrometer is required, e.g. if the first and second sets of data do not match or differ by a predetermined or threshold amount.
The present invention also provides a mass spectrometer comprising: a separation device for separating ions, or analytes; a mass analyser; and a controller having electronic circuitry configured to control the mass spectrometer to: i) operate during a period of time in which the sensitivity with which the mass analyser is able to detect ions varies; ii) determine the intensity of the ion signal of an ion of interest detected by the mass analyser within said period of time; iii) repeat steps i) and ii) as the ion of interest, or an analyte from which it is derived, elutes from the separation device; wherein when step ii) is repeated, it is performed at different times after said period of time begins; iv) produce a first set of data that comprises the intensity of the ion signal as a function of detection time; v) smooth the first set of data so as to obtain a second set of smoothed data; and vi) determine if maintenance of the spectrometer is required based on a comparison of the first and second sets of data.
The spectrometer may have a display screen and electrical circuitry that controls the display screen based on the above comparison in order to display when maintenance of the spectrometer is required.
Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:
Conventional approaches for quantifying an analyte use both the ion signal for the analyte and also the ion signal from an internal standard. Internal standards are required as different instruments may have different sensitivities, or even the same instrument may have different sensitivities at different times, e.g. due to different set-ups or drift. The use of an internal standard enables the response of the instrument to be calibrated for an analyte.
Best practice dictates that for the quantification of any given analyte, a corresponding internal standard is used that has very similar chemical properties to the analyte but a different mass to charge ratio value. For example, the internal standard may be an analogue of the analyte which is the same as the analyte except that some atoms have been replaced by atoms of the same element but a different isotope. For example, hydrogen atoms of the analyte may be replaced by deuterium atoms when forming the internal standard. In this example the analogue would have substantially the same chemical properties as the analyte, but would be N neutrons heavier, where N is the number of hydrogen atoms replaced by deuterium atoms.
In order to assist in the understanding of the present invention, an example of a conventional quantitation analysis will now be described. During sample preparation a fixed amount of one or more internal standards is introduced into the sample for one or more respective analytes of interest. The sample is then injected into a liquid chromatography mass spectrometer (LCMS) system. The liquid chromatography then separates (in time) the analyte(s) of interest from other components in the sample. However, as internal standards typically have substantially the same chemical properties as their corresponding analytes, an internal standard typically co-elutes with its respective analyte. The components eluting from the chromatography device are then ionised. The mass spectrometer filters ions such that only ions of a selected mass to charge ratio are transmitted to the detector at any given time. The detector detects the ion current for the transmitted ions. The spectrometer may be set so as to only transmit ions having a mass to charge ratio corresponding to a first analyte of interest to the detector, such that the ion current of the first analyte is detected. The spectrometer may then be set so as to only transmit ions having a mass to charge ratio corresponding to a first internal standard for the first analyte of interest, such that the ion current of the first internal standard is detected.
The above-described processes of analysing the first analyte of interest and the first internal standard may be repeated within the period of time that the first analyte and first internal standard are expected to elute from the chromatographic device (e.g. at the expected elution time, plus or minus a tolerance time). If more than one analyte of interest is to be quantified, then the above process may be repeated for each analyte of interest that elutes and its corresponding internal standard.
A tandem mass spectrometer may be used to perform the above method.
During the above process, the ion current detected at the detector is determined as a function of time. This data is used to determine the presence of any chromatographic peaks for the ions from the analytes of interest and their internal standards. The area of the chromatographic peak for ions from each analyte of interest is determined and the area of the chromatographic peak for the ions from their corresponding internal standard is determined. The ratio of the analyte peak area to the peak area of its corresponding internal standard is then determined. This quantity is referred to hereon as the Analyte Response. The amount of any given analyte can then be determined by a calculation that includes multiplying the Analyte Response by the amount of its corresponding internal standard that was added during the sample preparation. This ratiometric method is intended to ensure that changes in instrument sensitivity have little effect on the sample quantitation reported by the assay.
As well as using an internal standard, it may also be desired to detect one or more species of confirmatory ion. This is used to confirm that the correct analyte of interest is being analysed. Suitable confirmatory ions may, for example, be one or more species of fragment ion of the analyte of interest.
Conventionally, the mass spectrometer is set to automatically analyse an analyte of interest, its internal standard, and its confirmatory ion. This set of analyses may be repeated, but the spectrometer is configured such that each time the set of analyses is repeated the order in which the analyte of interest, its internal standard, and its confirmatory ion are analysed is the same. An example of this will now be described with reference to
More specifically,
The right side of
The mass spectrometer is repeatedly cycled between the three modes of analysing each ion of interest, its confirmatory ion and its internal standard during at least some of the period that the ion of interest is expected to elute from the chromatography device. However, when multiple analytes of interest may elute from the chromatography device with elution times that overlap then it is also necessary to analyse the other analytes of interest in a corresponding manner to that described above in an interleaved manner. An example of this is shown in Tables C and D.
Table C illustrates the analysis of a second analyte of interest that elutes from the chromatograph device over a time period that overlaps with the elution time of the first analyte of interest. As shown in Table C, the mass spectrometer is operated in a positive (electrospray) ion mode so as to generate positive parent ions. The first row in Table C indicates that the first mass filter of the tandem mass spectrometer filters the parent ions so as to transmit only parent ions having a mass to charge ratio (m/z) of 722. The parent ions transmitted by the first mass filter are then fragmented and transmitted to a second mass filter that is set so as to transmit only fragment ions having m/z=334. These fragment ions are detected by a detector and the resulting ion current determined. As shown in the second row of Table C, the mass spectrometer then switches the second mass filter so as to transmit only fragment ions having a m/z=352. This corresponds to the mass to charge ratio of confirmatory ions for the second analyte of interest. As shown in the third row of Table C, the mass spectrometer then switches the first mass filter so as to transmit only parent ions having m/z=756, which corresponds to the mass to charge ratio that ions of the internal standard for the second analyte of interest have. The parent ions transmitted by the first mass filter are then fragmented and transmitted to the second mass filter. The second mass filter is set so as to transmit only fragment ions having m/z=374 (which may be the analogue of the fragment ion having m/z=334). These fragment ions are detected by the detector and the resulting ion current determined.
Similarly, Table D illustrates the analysis of a third analyte of interest that elutes from the chromatograph device over a time period that overlaps with the elution time of the first and second analytes of interest. As shown in Table D, the mass spectrometer is operated in a negative electrospray ion mode (because these molecules are more efficiently ionised in negative mode) so as to generate negative parent ions. The first row in Table D indicates that the first mass filter of the tandem mass spectrometer filters the parent ions so as to transmit only parent ions having a mass to charge ratio (m/z) of 317. The parent ions transmitted by the first mass filter are then fragmented and transmitted to a second mass filter that is set so as to transmit only fragment ions having m/z=131. These fragment ions are detected by a detector and the resulting ion current determined. As shown in the second row of Table D, the mass spectrometer then switches the second mass filter so as to transmit only fragment ions having a m/z=175. This corresponds to the mass to charge ratio of confirmatory ions for the third analyte of interest. As shown in the third row of Table D, the mass spectrometer then switches the first mass filter so as to transmit only parent ions having m/z=335, which corresponds to the mass to charge ratio that ions of the internal standard for the third analyte of interest have. The parent ions transmitted by the first mass filter are then fragmented and transmitted to the second mass filter. The second mass filter is set so as to transmit only fragment ions having m/z=185 (which may be the analogue of the fragment ion having m/z=130). These fragment ions are detected by the detector and the resulting ion current determined.
As described above, if multiple different analytes of interest elute from the chromatography device during overlapping time periods, then the mass spectrometer repeatedly cycles through analysing the different analytes of interest during the period that they co-elute. In the example shown in
If different analyte ions of interest have different polarities, then when the mass spectrometer switches between analysing those ions it will switch the polarity of voltages applied to certain ones of its electrodes. In the example of
As described previously, the ratio of the area of the ion signal derived from the analyte of interest to the area of the ion signal derived from the internal standard is used to quantify the amount of analyte of interest present in the sample. This ratio is known as the Analyte Response and is shown in
In the embodiment of
Analysing ions A, C and I in different orders after different polarity switches (i.e. analysing each type of ion at different times after different polarity switches) results in the sensitivity with which each of the ions is analysed with being varied, such that each type of ion is analysed with relatively high and low sensitivities. In the example shown in
For illustrative purposes it has been assumed that analyte A and its internal standard I co-elute with a perfectly square profile and that the ion currents for the ions A. C and I are all identical. The tables shown in the Figures only show the resultant readings by the spectrometer during the period that analyte A elutes.
If desired, the method of
It is good laboratory practice to sample any given chromatographic peak enough times during that peak to allow accurate reconstruction of the peak shape. The examples given in relation to
The spectrometer can analyse the data obtained for an ion peak and usefully infer from this data the level of contamination on the electrodes of the spectrometer. For example, the spectrometer may sample the ion signal a plurality of times over the peak so as to obtain a first set of data and may then smooth that data so as to obtain a second set of (smoothed) data. The spectrometer may compare the two sets of data to determine the level of contamination on the electrodes. This may be done, for example, by the spectrometer taking each point in the peak and summing the squares of the difference between the smoothed and unsmoothed versions so as to provide an indication of the level of instability of the peak. Alternatively, the mean value may be subtracted prior to squaring. For example, the data points, say A to P, in the first set of data may be used to create the second set of data having corresponding smoothed data points A′ to P′. The noise in the first set of data (and hence an inferred level of contamination) may then be obtained by summing the squares of A-A′ to P-P′. These methods can then be used to estimate the effect that contamination is having on the sensitivity of the spectrometer. The spectrometer can then output (e.g. display) advice to the operator with respect to maintenance, e.g. that is it necessary to clean electrodes such as the ion optics and/or run diagnostics etc.
Although
Although the method has been described as analysing only three types of ions for each analyte of interest (i.e. ions A, ions I and ions C), it is also contemplated that one or more further type of ion may be analysed for each analyte of interest. For example, a second confirmatory ion S may be analysed by setting the second mass filter so as to only transmit ions having a mass to charge ratio corresponding to the second confirmatory ion. It is also contemplated that the method could be applied where only two species of ions are analysed, e.g. ions A and one related species of ion, or a pair of different analytes, or one analyte A and one internal standard I, or one analyte A and one if its fragment ion.
As described above, a plurality of MRM transitions are sampled each time an analyte of interest is monitored. The order in which those MRM transitions are sampled is varied so as to be different for different times that the analyte of interest is monitored. It will be appreciated that the order can be varied in a variety of ways.
The order in which the MRM transitions are sampled may be changed every time that the analyte of interest is analysed, for a plurality of consecutive times that the analyte of interest is analysed, so as to form a sequence of analysis. This sequence may then be repeated one or more times for the consecutive times that the analyte of interest is analysed. The sequence may be repeated an integer number of times over the analyte peak, or a non-integer number of times (i.e. the analyte of interest may only be present for a period of time such that it gives a signal over an incomplete number of cycles).
It may be desirable to select the order of the transitions sampled in the sequence so as to provide the most constant sampling rate (lowest maximum jitter) possible, e.g. as will be described in relation to
The upper plot 10 in
The upper plot 10 in
The upper plot 10 in
Alternatively, or additionally, it may be desirable to select the order of the transitions sampled in the sequence so as to reduce the difference between the responses of the different transitions being sampled when a non-integer number of sequences is used in the analysis of an analyte of interest. For example, having a long sequence can increase the impact on response accuracy when a non-integer number of sequences occurs across the peak for the analyte of interest.
In contrast,
Alternatively, or additionally, it may be desirable to select the order of the transitions sampled in the sequence depending on the algorithm used to detect the cleanliness of the ion optics using the method described in relation to
Ideally, for accurate reconstruction of a peak, the sampling times at which any given ion A,C,I,S is sampled should be evenly spaced apart when sampling across its peak, e.g. as shown in the bottom plot 14 in
In order to minimize the effect of the jitter when reconstructing the peak for the ion being analysed, the peak integration software can use the actual sampling times, rather than using the nominal sampling times. Alternatively, or additionally, the amount of distortion can be reduced by increasing the rate at which the transitions are switched between (i.e. monitoring a greater number of transitions per second) as the number of analytes “simultaneously” being monitored decreases.
Embodiments of the invention improve quantitation accuracy even when a calibration curve is used, as the acquisitions that formed the calibration curve may not have the same abundance of ions as the sample acquisitions. This means that the charging/discharging of any contaminated ion optical elements will not be the same for the calibration curve acquisitions and sample acquisitions. Also it may be that the instrument contamination level has changed since the calibration curve data was acquired.
Although embodiments have been described in which the order of the transitions is changed each time the analyte of interest is monitored, it is recognised that this causes jitter and other effects such as increasing the time between the analysis of ion A and ions I, or causing the analysis of both to happen later. Embodiments are therefore contemplated in which the order in which the ions A,C,I,S are monitored may remain constant for a plurality of consecutive times that the analyte of interest is monitored, and that the order may be varied in another part of the acquisition. For example, in embodiments that use a chromatography device to separate the sample, only a particular range of compounds can elute at any one time. The spectrometer may therefore only sample MRM transitions for an analyte of interest over the time at which it is possible for that analyte of interest to elute. This leads to the MRM transitions that the spectrometer monitors for changing over the elution time. In the example shown in
It is also contemplated that whilst only a single analyte of interest elutes, the spectrometer may automatically switch (e.g. by detecting this) so that order of transitions being monitored remains constant, thereby avoiding any jitter related quantitation inaccuracy.
Embodiments are also contemplated in which a single internal standard is used for multiple analytes of interest. The method may monitor the internal standard transition each time that it analyses each of the multiple analytes that share that internal standard. Alternatively, the transitions for said multiple analytes could be grouped, along with the transition for their common internal standard, so as to form a larger sequence that is repeated.
According to embodiments of the present invention, peak detection fidelity may be improved by summing the current from the ions A and the current(s) from one of more of the confirmation ion(s) C and/or S when determining the start and end of the peak during the peak area measurement method.
Although the present invention has been described with reference to various embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.
For example, although the contamination of ion optics such as ion guides and mass filters has been described, it is contemplated that the problem may arise with contamination of other electrodes in the spectrometer, such as in a collision cell for fragmenting or cooling ions.
Although embodiments have been described herein that reduce measurement bias in the event of a polarity switch, the invention is not limited to this and relates to other events that cause a (reproducible) change in the sensitivity able to be detected. Such events may be caused by, for example, circuit response lag times, ion guide charging, mass switching and other events. Alternatively, or additionally, a variation in sensitivity of the spectrometer may be caused by a change in another variable, such as being caused by the components of the sample being analysed varying with time. For example, the sample may be separated by chromatography and ionised prior to analysis, and a relatively highly concentrated component may elute from the separation device and be ionised so as to produce a relatively intense ion signal. This intense ion signal may cause the sensitivity of the spectrometer to vary with time, e.g. by the intense ions causing surface charging of electrodes within the spectrometer or by another effect that changes sensitivity.
Similarly, although embodiments have been described comprising a tandem quadrupole mass spectrometer, the invention is not limited to such instruments but instead applies to all types of instrument that suffer from time varying sensitivities.
According to a less preferred embodiment, rather than alternating the order in which ions A, C and I are analysed so as to occur in different orders after different polarity changes, the different ions may be analysed in the same order after every polarity change, but a greater number of times per second. This will reduce the change in sensitivity between each MRM transition being monitored, but is not preferred for three reasons. Firstly, it is still not balancing low and high sensitivity between, for example, an analyte and its internal standard. Consequently, this method reduces the effect of contamination less effectively than the other methods described herein. Secondly, between each MRM transition there is a short period where no data should be acquired whilst the electronics/optics are given time to settle. Since this method monitors a greater number of transitions per second, a greater total time will be needed for the electronics/optics to settle and so this limits the time spent detecting the ions and so sensitivity will be lost (i.e. the duty cycle is worse). Thirdly, it will be less easy to determine a figure of merit for contamination.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2105778.1 | Apr 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/051029 | 4/22/2022 | WO |
