Increased MRM capacity

Abstract

In one aspect, a method for performing a plurality of MRM measurements is disclosed, which includes ionizing a sample to generate sample ions, selecting at least one precursor ion from among said sample ions. For the selected at least one precursor ion, performing at least two consecutive MRM measurements including a first and a subsequent, second consecutive MRM measurements associated with a plurality of fragment ions generated via dissociation of said at least one precursor ion, where one of the first and the second MRM measurement includes an ion emptying period and the other MRM measurement lacks an ion emptying period.

Description

INCREASED MRM CAPACITYRELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No.63 / 746,337 filed on January 17, 2025, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to mass spectrometry and in particular to methods and systems for performing fast MRM (multi reaction monitoring).BACKGROUND

[0003] Mass spectrometry (MS) is an analytical technique for determining the structure of chemical substances with both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the composition of atomic elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a particular chemical compound in a mixed sample. Mass spectrometers detect chemical entities as ions such that a conversion of the analytes to charged ions must occur.

[0004] Tandem mass spectrometry or MS / MS involves ionization of one or more compounds of interest from a sample, selection of one or more precursor ions of the one or more compounds, fragmentation of the one or more precursor ions into product ions, and mass analysis of the product ions.

[0005] Tandem mass spectrometry can provide both qualitative and quantitative information. The product ion spectrum can be used to identify a molecule of interest. The intensity of one or more product ions can be used to quantitate the amount of the compound present in a sample.

[0006] A large number of different types of experimental methods or workflows can be performed using a tandem mass spectrometer. These workflows can include, but are not limited to, targeted acquisition, information dependent acquisition (IDA) or data dependent acquisition (DDA), and data independent acquisition (DIA).

[0007] In a targeted acquisition method known as Multiple Reaction Monitoring (MRM), a target ion is selected and the selected target ion is subjected to dissociation, e.g., in a collision14925-1617-8568, v. 1cell, to generate a plurality of product ions and the product ions are detected and analyzed.Conventionally, to prevent cross-talk between such analysis of different compounds or different fragments of the same compound, various devices (elements) along the ion path, are emptied of ions between each MRM measurement period. Such emptying of the devices (elements) along the ion path can reduce the speed at which analysis of various compounds in a sample can be performed. Such a reduction in measurement speed can be particularly problematic when a large number of compounds need to be analyzed within a sample.SUMMARY

[0008] In one aspect, a method for performing a plurality of MRM measurements is disclosed, which includes ionizing a sample to generate sample ions and selecting at least one precursor ion from the sample ions. The method further includes performing at least two consecutive MRM measurements associated with a plurality of fragment ions generated via dissociation of said at least one precursor ion, where one of the consecutive MRM measurements includes an ion emptying period and the other of the consecutive MRM measurements lacks an ion emptying period.

[0009] In various embodiments, the selection of the target precursor ion from the sample ions can be achieved by passing the sample ions through a mass filter that is configured to allow selective passage of the fragment ion of interest while preventing other fragment ion(s) from passing through the filter. By way of example, and without limitation, such a mass filter can include a plurality of rods arranged as a quadrupole rod set where application of RF and DC voltages to the rods can allow selective passage of a target fragment ion through the mass filter.

[0010] In various embodiments, the fragment ions associated with the selected precursor ion can be generated using a variety of different ion fragmentation devices. By way of example, and without limitation, in some embodiments, the ion fragmentation device can be a collision cell in which the precursor ion undergoes collisions with a background gas (such as nitrogen) that lead to the collisional dissociation of the precursor ion into a plurality of fragment ions. In other embodiments, the fragmentation of the precursor ion can be achieved via electron activated dissociation (EAD), all by way of example.24925-1617-8568, v. 1

[0011] In various embodiments, the first MRM measurement can be an initial MRM measurement having a temporal duration that includes an ion emptying period, a first pause period and a first dwell period for detection of a first fragment ion of the plurality of the fragment ions and a consecutive second one of the MRM measurements that lacks an ion emptying step. More specifically, the consecutive second MRM measurement period can include a second pause period and a second dwell period. During each of the first and the second pause periods, one or more operating parameters of one or more devices of a mass spectrometer utilized to perform the MRM measurements can be adjusted to allow selection of the first and second fragment ions, respectively. By way of example, the RF voltages applied to the rods of a mass filter employed for the selection of the second fragment ion can be adjusted to select the first fragment ion during the first MRM measurement and to select the second fragment ion during the second MRM measurement.

[0012] During the first and the second dwell periods, ion detection data corresponding to the first and second fragment ions, respectively, can be collected.

[0013] In various embodiments, one or more operating parameters of a first mass filter, a dissociation device positioned downstream of the first mass filter and a second mass filter positioned downstream of the dissociation device are adjusted to cause removal of one or more fragment ions associated with a previous precursor ion (and any remaining unfragmented precursor ions) from the first and the second mass filters and the dissociation device during an ion emptying period of an initial MRM measurement. Subsequently, during a pause period, one or more operating parameters of the first mass filter and a second mass filter positioned downstream of the first mass filter are adjusted to select, respectively, a target precursor ion and a fragment ion (herein referred to as first fragment ion) generated via dissociation of that target precursor ion. This is followed by detecting the selected fragment ion and generating ion detection data corresponding thereto during a dwell period. A subsequent, consecutive MRM measurement corresponding to the other fragment ion is initiated via a pause period without using a previous ion emptying period, where during the pause period one or more operating parameters of the second mass filter are adjusted to configure the second mass filter to allow selective passage of another fragment ion (herein referred to as the second fragment ion) associated with that precursor ion for detection.34925-1617-8568, v. 1

[0014] In various embodiments, any of the pause period and the dwell period of two consecutive MRM measurements corresponding to different fragment ions of the same precursor ion can have different temporal durations. By way of example, and without limitation, the pause period associated with an initial MRM measurement can be longer than the pause period associated with a subsequent, consecutive MRM measurement because during the initial MRM measurement the operating parameters of more of the mass spectrometer’s devices need to be set. For example, in various embodiments, during the pause period associated with the initial MRM measurement, the operating parameters of both a first mass filter utilized to select the precursor ion of interest as well as those of a second, downstream mass filter utilized to select the fragment ion of interest need to be adjusted. In contrast, during the subsequent, consecutive MRM measurement the operating parameter(s) of the first mass filter can be unchanged (as the second MRM measurement corresponds to the same precursor ion as the first MRM measurement) and hence only the operating parameter(s) of the second mass filter need to be adjusted to select the second fragment ion. It is noted that in some cases, the operating parameters of a collision cell can be adjusted when transitioning from performing MRM measurements on one precursor ion to a different precursor ion.

[0015] Further, as noted above, in some embodiments, the dwell periods associated with two consecutive MRM measurements associated with the same precursor ion can be different. By way of example, the difference between the dwell periods of the two consecutive MRM measurements can be selected based on at least one of a difference in m / z ratios, a difference in expected signal intensities, a difference in expected signal-to-noise ratios, and a desired limit-of-detection associated with the first and the second fragment ions.

[0016] In various embodiments, a shorter pause period may be used in connection with a longer dwell period to ensure that the total temporal duration of the consecutive MRM measurements remains substantially constant.

[0017] In various embodiments, the MRM measurements are ordered such that the m / z ratio of the fragment ions associated with the MRM measurements increase as the MRM measurements are performed. For example, for two consecutive MRM measurements associated with two fragment ions generated via dissociation of the same precursor ion, the m / z ratio of the fragment ion associated with the temporally first MRM measurement can be less than the44925-1617-8568, v. 1respective m / z ratio of the fragment ion associated with the temporally subsequent MRM measurement.

[0018] In various embodiments, three or more consecutive MRM measurements corresponding to different fragment ions of the same precursor ion can be performed. In some such embodiments, the MRM measurements are ordered so as to minimize a difference between m / z ratios of the fragment ions corresponding to neighboring MRM measurements.

[0019] In various embodiments, the ion detection signal acquired during an MRM measurement corresponding to one of the fragment ions can be used to qualify (i.e., identify) the precursor ion and the ion detection signal corresponding to another one of the fragment ions can be used to quantify the precursor ion. In some such embodiments, the dwell period or the pause period associated with the two MRM measurements can be different, e.g., to obtain a higher intensity signal for quantification or to obtain a higher intensity signal for identification on a weaker qualifier MRM. By way of example, the intensity of qualifier ion signals can be typically less than the respective intensity of quantifier ion signals. As such, in some cases, a user may configure the measurement protocol so as to have a longer dwell period for acquisition of qualifier ion signals for a more robust identification, or alternatively, configure the measurement protocol so as to have a longer dwell period for acquisition of quantifier ion signals for a more accurate quantification.

[0020] As noted above, in various embodiments, an axial electric field can be established within the dissociation device, e.g., a collision cell, to axially accelerate the fragment ions generated within the dissociation device, thereby expediting their passage through the device. In some such embodiments, the axial electric field can be different between at least two consecutive MRM measurements corresponding to different fragment ions of the same precursor ion.

[0021] By way of example, the axial acceleration electric field for one of at least two consecutive MRM measurements that is associated with a fragment ion having a larger m / z ratio can be greater than a respective axial acceleration electric field for the other MRM measurement.

[0022] In a related aspect, a method for performing MRM mass spectrometry is disclosed, which includes ionizing a sample to generate sample ions, configuring a first mass filter to select said at least one precursor ion from the sample ions, using an ion dissociation device to cause54925-1617-8568, v. 1dissociation of said at least one precursor ion to generate a plurality of fragment ions, and configuring a second mass filter to select a first one of said plurality of the fragment ions for detection. Subsequently, the second mass filter can be adjusted to select a second one of the plurality of the fragment ions for detection without prior adjustment of one or more operating parameters of any of the ion dissociation device and the second mass filter for causing removal of the first one of said fragment ions from any of said ion dissociation device and said second mass filter.

[0023] By way of example, in various embodiments, the ion dissociation device can be a collision cell.

[0024] In a related aspect, a method for performing a plurality of MRM measurements is disclosed, which includes ionizing a sample to generate sample ions, and selecting at least one precursor ion from the sample ions. For the selected at least one precursor ion, at least two consecutive MRM ion detection signals are acquired with each corresponding to one of the fragment ions, wherein a transition between acquisition of the two ion detection signals is performed without employing an ion emptying period.

[0025] In various embodiments of the above method, the step of selecting the at least one precursor ion includes using a mass filter configured to allow selective passage of the at least one precursor ion therethrough.

[0026] In a related aspect, a mass spectrometer system is disclosed, which includes an ion source for receiving a sample and generating sample ions. The mass spectrometer can further include a first mass filter operating under control of a controller that can be configured to select at least one precursor ion from the sample ions. An ion dissociation device, such as a collision cell, positioned downstream of the first mass filter and operating under control of the controller can receive the selected precursor ion and cause its fragmentation into a plurality of fragment ions. The mass spectrometer can further include a second mass filter positioned downstream of the collision cell for receiving the fragment ions and selecting a target fragment ion for detection. A mass analyzer, such as a time-of-flight mass analyzer, can be positioned downstream of the second mass filter for detecting the selected fragment ion.64925-1617-8568, v. 1

[0027] The controller can be programmed to configure at least the first and the second mass filter to select a precursor ion of interest and to perform a plurality of consecutive MRM measurements corresponding to that precursor ion according to various embodiments of the present teachings. For example, for analysis of each precursor ion, the controller can be programmed so as to adjust one or more operating parameters of the first and second mass filters and the collision cell to cause removal of fragment ions associated with a previously -analyzed precursor ion (and also any remaining ones of the previously-analyzed precursor ion) during an initial MRM measurement period to prepare the mass spectrometer for performing MRM measurements on the precursor ion of interest. The controller can also be programmed to configure the first and second mass filters and the collision cell, during a pause period, to select the precursor ion of interest and the fragment ion to be analyzed. By way of example, the controller can communicate with one or more voltage sources, such as an RF and / or a DC voltage source, to cause those voltage sources to apply the appropriate voltages to the mass filters and the collision cell (typically to a set of rods of these components). Further, the controller can provide a trigger signal to a mass analyzer to begin acquisition of ion detection data during a dwell period having a predefined temporal duration, which can also be set by the controller. The controller can further be programmed to initiate a subsequent MRM measurement, e.g., after completion of the initial MRM measurement, by causing, during a subsequent pause period, the adjustment of the operating parameters of the mass filter used to select the fragment ion of interest, to allow passage of that fragment ion of the precursor ion for analysis. The controller can then initiate, during a dwell period, the acquisition of ion detection data by the mass analyzer.

[0028] Further understanding of various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 schematically depicts a conventional MRM workflow,74925-1617-8568, v. 1

[0030] FIG.2 schematically depicts a modified version of the workflow shown in FIG. 1A in which ion emptying periods between configuring and acquiring data for different fragment ions associated with the same precursor ion are eliminated,

[0031] FIG.3 is a flow chart depicting various steps in an embodiment of a method for performing a plurality of MRM measurements according to an embodiment of the present teachings,

[0032] FIG.4A schematically depicts a mass spectrometer according to an embodiment of the present teachings for performing MRM measurements,

[0033] FIG.4B schematically depicts a collision cell employed in the mass spectrometer of FIG. 4A as well as an ion lens positioned in proximity of the inlet of the collision cell,

[0034] FIG. 5A shows an MRM transition of Methamidophos obtained using a conventional MRM workflow,

[0035] FIG. 5B shows the same MRM transition as that in FIG 5A, but obtained using an MRM workflow according to an embodiment of the present teachings,

[0036] FIG.6A shows an MRM transition of Methabenzthiazuron obtained using a conventional MRM workflow,

[0037] FIG.6B shows the same MRM transition as that depicted in FIG. 6A, but obtained using an MRM workflow according to an embodiment of the present teachings,

[0038] FIG. 7A shows MRM transitions of a plurality of pesticides, where the data was obtained using a conventional MRM workflow,

[0039] FIG. 7B shows the same data as that presented in FIG. 7A, but obtained using an MRM workflow according to an embodiment of the present teachings,

[0040] FIG. 8 depicts variation of ion detection signal intensity as a function of time for several MRM transitions, illustrating that the elimination of an ion emptying period allows fast switching between acquisition of ion detection data corresponding to two different fragment ions of the same precursor ion,84925-1617-8568, v. 1

[0041] FIG.9 depicts variation of ion detection signal intensity as a function of time for several MRM transitions, illustrating that in various embodiments the application of an axial acceleration field to the collision cell of mass spectrometer can enhance data acquisition speed,

[0042] FIGS. 10A and 10B show ion detection signal intensity as a function of time for an MRM transition acquired at different collision energies employed for causing fragmentation of a precursor ion in a collision cell, and

[0043] FIG. 11 depicts an example of an implementation of a controller utilized in various embodiments of the present teachings.DETAILED DESCRIPTION

[0044] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0045] As used herein, the terms "about" and "substantially equal" refer to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the terms "about" and "substantially" as used herein mean 10% greater or less than the value or range of values stated or the complete condition or state. For instance, a concentration value of about 30% or substantially equal to 30% can mean a concentration between 27% and 33%. The terms also94925-1617-8568, v. 1refer to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art.

[0046] As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0047] The terms “ion emptying period” and “emptying period” are used herein interchangeably to refer to a temporal period in which, via active adjustment of one or more parameters of one or more devices of a mass spectrometer (such as mass filters), ions contained in those devices are removed to prepare the mass spectrometer for collecting MRM ion detection data corresponding to a precursor ion.

[0048] In scheduled MRM methods, a common problem is that the retention time of a target compound eluting from an LC (liquid chromatography) column may shift outside the scheduled MRM data acquisition window for that compound. One solution is to increase the size of the data acquisition windows. However, increasing the size of the data acquisition windows would require increasing the rate at which MRM measurements are acquired in order to analyze the same number of compounds within the same time period. Acquiring more MRM measurements per second using conventional techniques may, however, lead to poor ion counting statistics. Thus, there is a need for methods and systems that allow performing MRM measurements at a fast rate while ensuring that signal-to-noise ratio of the collected data is not compromised.

[0049] As noted above, in conventional approaches, to prevent cross-talk between consecutive MRM measurement periods associated with different compounds and / or different fragment ions associated with a single compound, various devices (elements) along the ion path are emptied from ions prior to initiation of data acquisition period associated with each MRM measurement. FIG. 1A schematically depicts an example of such a conventional MRM workflow in which four MRM measurement periods, namely, MRM Al, MRM A2, MRM Bl and MRM B2, are depicted corresponding to two different fragment ions Al and A2 generated via fragmentation of a precursor ion A and two different fragment ions Bl and B2 generated via fragmentation of a precursor ion B.

[0050] More specifically, FIG. 1A shows that an initial MRM measurement period corresponding to the fragment ion Al of the precursor ion A includes an initial ion emptying104925-1617-8568, v. 1period, followed by a pause period in which the system is configured for the selection of the precursor ion A and the detection of the fragment ion Al. This is followed by a dwell period during which ion detection data corresponding to fragment ion Al is acquired. Subsequent to the acquisition of ion detection data corresponding to the fragment ion Al, another MRM measurement period is started in which during an ion emptying period, the fragment ion Al is emptied from the system, e.g., from a mass filter that was configured to detect Al, followed by a pause period in which the system is configured for the detection of fragment ion A2. After completion of acquisition of ion data corresponding to fragment ion A2, during an ion emptying period of a subsequent MRM measurement period, the system is emptied of fragment ion A2 (and remaining unfragmented precursor ion A) to prepare the system for performing MRM measurement of the fragment ion Bl of the other precursor ion B. By way of example, during this pause period, one or more operating parameters of a mass filter used for precursor ion selection are adjusted to switch the selection of the precursor ion from A to B. Further, one or more operating parameters of another mass filter used for fragment ion selection are adjusted to detect fragment ion Bl. Subsequently, the acquisition of the ion detection data corresponding to fragment ion Bl is performed during the dwell period. Similarly, the following MRM measurement period corresponding to the fragment ion B2 includes an ion emptying period, a pause period and a dwell period.

[0051] In contrast, in various embodiments, this disclosure provides MRM workflows that eliminate an ion emptying period when switching between MRM measurement periods of fragment ions corresponding to the same precursor ion.

[0052] By way of illustration, FIG. IB shows a modification of the workflow described above in connection with FIG. 1A based on various embodiments of the present teachings.Specifically, the workflow depicted in FIG. 1A lacks an ion emptying period during MRM measurements periods corresponding to measuring the fragment ions A2 and B2. In other words, the workflow shown in FIG. 1 A has been modified to eliminate the ion emptying periods between successive MRM measurements of different fragment ions generated via dissociation of the same precursor ion. As discussed in more detail below, in various embodiments, such elimination of the ion emptying periods between consecutive MRM measurements periods114925-1617-8568, v. 1corresponding to different fragment ions of the same precursor ion can significantly shorten the time required for MRM analysis of a plurality of compounds within a sample.

[0053] FIG. 3 is a flow chart depicting various steps of a method according to an embodiment for performing a plurality of MRM measurements, which includes ionizing a sample, thereby generating sample ions corresponding to one or more target analytes. The ionization of the target analyte(s) can be achieved using a variety of ion sources known in the art. Some examples of suitable ion sources can include, without limitation, an electrospray ionization device, a nebulizer assisted electrospray device, a chemical ionization device, a nebulizer assisted atomization device, a chemical ionization device, an atmospheric pressure chemical ionization (APCI) device, a heated nebulizer device, a thermal desorption ion source, a matrix-assisted laser desorption / ionization (MALDI) ion source, a photoionization device, a laser ionization device, a thermospray ionization device, an inductively coupled plasma (ICP) ion source, a sonic spray ionization device, a glow discharge ion source, and an electron impact ion source, among others.

[0054] In various embodiments, a sample can be introduced into a liquid chromatography (LC) column and the eluate exiting the LC column can be introduced into the ion source to ionize one or more target analytes in the eluate.

[0055] At least one precursor ion is selected from the sample ions and at least two consecutive MRM measurements are performed on the selected precursor ion, where each of the MRM measurements is associated with one of at least two fragment ions generated via dissociation of the at least one precursor ion. By way of example, and without limitation, in various embodiments, the precursor ion can be introduced into a collision cell to undergo fragmentation via collisions with gas molecules within the collision cell. The collisional fragmentation of the precursor ion leads to the generation of at least two fragment ions, each of which can be detected and analyzed during one of the MRM measurement periods. As discussed in more detail below, in various embodiments, a DC voltage applied between an ion lens and the collision cell’s electrodes can generate an acceleration electric field that causes axial acceleration of the precursor ion as it enters the collision cell to impart a desired collisional energy to the precursor ion. By way of example, and without limitation, in various embodiments, the collision cell can include a set of quadrupole rods to which RF voltages can be applied for providing124925-1617-8568, v. 1radial confinement of ions within the collision cell and a lens electrode can be positioned in proximity of an entrance of the collision cell such that the application of a DC voltage between the lens electrode and the quadrupole rods can generate a voltage for accelerating the precursor ion, thereby imparting a desired kinetic energy to the ions. As discussed in more detail below, the collision energy can be adjusted to optimize the signal intensity associated with the fragment ions generated via collisional dissociation of the precursor ion.

[0056] Moreover, in various embodiments, in addition to the quadrupole rods, the collision cell can further include a plurality of LINAC electrodes that are interposed between the quadrupole rods, where the application of DC voltage(s) to the LINAC electrodes can generate an axial electric field within at least a portion of the collision cell for accelerating the fragment ions generated within the cell and hence increasing the speed by which the MRM measurements are performed, i.e., enhancing the rate of performing the MRM measurements.

[0057] Of particular note, and with continued reference to the flow chart of FIG. 3, in such a method for performing a plurality of MRM measurements, one of the MRM measurement periods lacks an ion emptying period. More specifically, unlike conventional methods of performing MRM measurements in which each MRM measurement period includes an ion emptying period in which ions are removed, a pause period in which one or more operating parameters of one or more devices are set followed by a dwell period in which ion detection data is collected, in various embodiments, at least one MRM measurement period associated with the plurality of MRM measurements of fragment ions generated via dissociation of the same precursor ion includes only a pause period and a dwell period without an ion emptying period that would precede the pause period.

[0058] By way of example, in some such embodiments, an initial MRM measurement period in a series of MRM measurement periods associated with different fragment ions of a precursor ion, can include an ion emptying period to remove (or at least substantially remove) fragment ions associated with a different, preceding precursor ion (and any remaining unfragmented precursor ions). This is then followed by a pause period during which the system is configured for detection of an initial fragment ion of interest followed by acquisition of the respective ion detection data. For detection and analysis of a subsequent fragment ion, the one or more operating parameters of one or more devices (such as a collision cell and a mass filter employed134925-1617-8568, v. 1for selection of the subsequent fragment ion) are adjusted without first removing the previous fragment ions from the system (e.g., from the mass filter) and the ion detection data is acquired for the subsequent fragment ion.

[0059] In various embodiments, the fragmentation of a precursor ion, e.g., via collisional dissociation, may lead to production of more than two fragment ions. In some such embodiments, the first MRM measurement period can include an ion emptying period and the subsequent MRM measurement periods can lack such an ion emptying period.

[0060] The ordering of the MRM measurements associated with different fragment ions of the same precursor ion can be done based on a variety of criteria. By way of example, in some embodiments, the MRM measurements are ordered so as to minimize the m / z difference between fragment ions associated with neighboring MRM measurements. This can advantageously allow faster reconfiguration of the system parameters as the MRM measurement is transitioned from the detection of one fragment ion to that of a subsequent fragment ion. Other ways of ordering the MRM measurements can also be employed. For example, the MRM measurements can be ordered based on the m / z ratios of the fragment ions, e.g., in a decreasing order of m / z ratios.

[0061] In various embodiments, one MRM measurement may be utilized for quantification of the amount of the precursor ion and another MRM measurement may be utilized for identification of the precursor ion.

[0062] In various embodiments, at least two MRM measurement periods can have different durations for their pause and / or the dwell periods. By way of example, the pause period associated with an initial MRM measurement can be longer than the respective pause period associated with the subsequent MRM measurement(s). For example, in some embodiments, during the pause period associated with the initial MRM measurement, the operating parameter(s) of two mass filters, one of which selects the precursor ion and the other selects a fragment ion of interest, need to be set whereas for the subsequent MRM measurements only the operating parameter(s) of the filter used for the selection of a particular fragment ion need to be set. Accordingly, the pause period for the initial MRM measurement may be selected to be longer than the pause periods for the subsequent MRM measurement(s).144925-1617-8568, v. 1

[0063] Further, in some cases, it may be desirable to provide a longer dwell period during which ion detection data is accumulated for one or more MRM measurements relative to the others. For example, for a fragment ion expected to have a lower ion intensity, a longer dwell time may advantageously allow accumulation of more data, leading to an improved signal-to-noise ratio. By way of example, in some cases, a longer dwell time on a fragment ion associated with a precursor ion may be used to achieve a better limit of quantification for the precursor ion.

[0064] In various embodiments in which the pause period and / or the dwell period vary between two or more of the MRM measurements corresponding to different fragment ions associated with the same precursor ion, the pause and / or the dwell periods can be selected so as to ensure that the different MRM measurements exhibit substantially identical temporal periods. By way of example and as noted above, in some embodiments, the initial MRM measurement may include an ion emptying period, a pause period and a dwell period while the remaining MRM measurements may include only a pause period and a dwell period. In some such embodiments, the temporal durations of all MRM measurements are substantially the same. For example, in some such cases, the MRM measurement(s) lacking an ion emptying step may have a dwell period that is longer than that of the MRM measurements having an ion emptying step while the total duration of each MRM measurement may be nonetheless substantially the same as that of any of the other MRM measurements. An advantage of such an approach is that it can make the integration of peaks easier.

[0065] Without any loss of generality, in the following description it is assumed that an MRM measurement is performed using a mass spectrometer that includes an ion source that can receive a sample, e.g., from an LC column, to ionize one or more compounds of interest within the sample, thereby generating a plurality of precursor ions. A first mass filter (herein also referred to as QI), such as a quadrupole mass filter, can be configured to select one target precursor ion at a time. The selected target precursor ion is transmitted to a collision cell (herein also referred to as Q2) in which the target precursor ion can undergo fragmentation to generate a plurality of fragment ions. A downstream mass filter (herein also referred to as Q3) can then select a fragment ion of interest for detection and analysis.

[0066] In such a mass spectrometer, to perform MRM measurements corresponding to different fragment ions of the same precursor ion, the RF and DC voltages applied to the rods of154925-1617-8568, v. 1QI and Q3 mass filters and Q2 collision cell are adjusted to empty these components of fragment ions corresponding to a previously-analyzed precursor ion (as well as any unfragmented previously-analyzed fragment ions) during an initial MRM measurement period.

[0067] During a subsequent pause period, the RF and DC voltages applied to the rods of QI, Q2 and Q3 can be adjusted to configure QI for selection of the precursor ion of interest, to configure Q2 for causing fragmentation of that precursor ion into a plurality of fragment ions, and to configure Q3 for selection of a first fragment ion. This is followed by acquisition of ion detection signals corresponding to the first fragment ion during a dwell period. To initiate acquisition of ion detection signals corresponding to the second fragment ion, without performing an ion emptying step, the RF and DC voltages applied to Q3 mass filter are adjusted, during a second pause period, to configure Q3 for the detection of a second fragment ion corresponding to the precursor ion. This is then followed by acquisition of ion detection signals corresponding to the second fragment ion.

[0068] By way of example, FIG. 4A schematically depicts a mass spectrometer 400 according to an embodiment that includes an LC column 402 that can receive a sample and can separate a plurality of analytes in the sample based on their elution time from the LC column. The mass spectrometer 400 further includes an ion source 404 that receives an eluate exiting the LC column and ionizes one or more analytes contained in the eluate to generate a plurality of precursor ions. In many implementations, one or more ion guides 406 receive the precursor ions and provide focusing of the ions to generate an ion beam that is received by a mass filter 408 (herein also referred to as the “first mass filter 408”). By way of example, the ion guide(s) can include a plurality of rods (not visible) arranged in a quadrupole configuration to which RF and DC voltages generated by an RF voltage source 410 and a DC voltage source 412 operating under control of a controller 418 can be applied in a manner known in the art to provide radial confinement of the received ions.

[0069] The mass filter 408 provides an ion transmission window that allows transmission of ions having m / z values within an m / z range through the mass filter. By way of example, the mass filter 408 can include a plurality of rods arranged in a quadrupole configuration to which RF voltages as well as a resolving DC voltage can be applied via an RF voltage source 420 and a164925-1617-8568, v. 1DC voltage source 422 operating under control of the controller 418 to generate an ion transmission window to allow selection of a precursor ion of interest.

[0070] In this implementation, the selected precursor ion passing through the mass filter 408 is received by a collision cell 414 that causes fragmentation of the precursor ion to generate a plurality of fragment ions (herein also referred to as product ions). As discussed in more detail below, in this embodiment, the collision cell 414 includes a set of rods (not visible in this figure) that is arranged in a quadrupole configuration as well as a set of LINAC electrodes (not visible in this figure), where the LINAC electrodes are interspersed between the quadrupole rods. An RF voltage source 424 applies RF voltages to the quadrupole rods of the collision cell to cause radial confinement of ions within the collision cell and a DC voltage source 426 applies DC voltage(s) to the LINAC electrodes to generate an axial electric field for accelerating fragment ions generated within the collision cell.

[0071] The fragment ions are received by a second mass filter 416, which is configured to allow passage of one of the fragment ions to a downstream stage of the mass spectrometer for detection and analysis. In this embodiment, similar to the first mass filter 408, the second mass filter 416 includes a plurality of rods (not visible in the figure) to which RF voltages and a DC resolving voltage can be applied via an RF voltage source 428 and a DC voltage source 430 operating under control of the controller 418 to generate a transmission window to allow selective transmission of one of the fragment ions of interest generated via dissociation of the precursor ion to a downstream mass analyzer 432, such as a time-of-flight (ToF) mass analyzer for detection and analysis. In some other embodiments, the mass spectrometer may not include the mass analyzer 432 downstream of the mass filter 416 (which may a quadrupole mass filter), or alternatively, the mass spectrometer may not include the mass filter 416 upstream of the mass analyzer 432.

[0072] With reference to FIG. 4B, in this embodiment, an ion lens 434 is positioned in proximity of an inlet of the collision cell 414 and the collision cell includes, in addition to a quadrupole rod set 436 (only two of the rods are shown in this figure), a set of LINAC electrodes 438 that are interposed between the quadruple rods. The DC voltage source 426 (or a different DC voltage source) can apply a DC voltage to the lens to generate a DC voltage offset between the ion lens and the quadrupole rods of the collision cell to impart a desired kinetic energy to the174925-1617-8568, v. 1precursor ions. It has been found that the kinetic energy of the precursor ions can impact the MRM signal intensity. In various embodiments, the collision energy of the precursor ions can be in a range of 0 to about 180 eV, and more typically in a range about 10 to 180 eV.

[0073] Further, in various embodiments, the application of DC voltages to the LINAC electrodes, e.g., via the DC voltage source 426, can generate an axial electric field within the collision cell that can help accelerate the fragment ions generated within the collision cell. Such acceleration of the fragment ions can expedite their passage through the collision cell and hence enhance the speed at which the MRM measurements can be performed. By way of example, a voltage in a range of 100 V to 500 V can be applied to the LINAC electrodes.

[0074] The mass spectrometer can also include a plurality of RF and / or DC lenses positioned between various components in a manner known in the art for guiding and / or focusing ions, e.g., as they transition from one component to another downstream component.

[0075] The controller 418 can be programmed to perform MRM measurements on one or more precursor ions generated via ionization of one or more target analytes in a sample. For example, for MRM analysis of a precursor ion, the controller 418 can be programmed to cause the RF and the DC voltage sources to apply suitable RF and DC voltages to the first mass filter 408, the collision cell 414 and the second mass filter 416 (and optionally other components such as ion lenses) to cause removal of fragment ions associated with a previously-analyzed precursor ion (and any of the previously-analyzed precursor ions that remained unfragmented) during an ion emptying period of an initial MRM measurement period. The controller can then cause, during a pause period, the RF and the DC voltage sources to apply suitable voltages to the first and the second mass filters as well as the collision cell so as to configure the first and the second mass filters to select, respectively, the target precursor ion and the target fragment ion generated via dissociation of that precursor ion and to configure the collision cell for causing the dissociation of the target precursor ion. By way of example, the controller 418 can send control signals to the RF and DC voltage sources to apply RF voltages and a DC resolving voltage to the quadrupole rods of the first and the second mass filters in a manner known in the art such that the first mass filter provides an ion transmission window that allows selective transmission of the target precursor ion and the second mass filter provides an ion transmission window that allows selective transmission of the target fragment ion.184925-1617-8568, v. 1

[0076] A controller utilized in the practice of the present teachings, such as that discussed above, can be implemented using software, firmware, and hardware in a manner known in the art and as informed by the present teachings. By way of example, FIG. 11 schematically depicts an example of implementation of such a controller 1100 having a digital processing unit 1102 that can communicate via one or more communications buses 1103 with a random-access memory (RAM) module 1104 and a permanent memory 1105. By way of example, various instructions for performing MRM measurements according to various embodiments can be stored in the permanent memory and can be accessed during runtime via the digital processing unit 1102 to perform methods according to various embodiments. By way of example, such instruction can include the amplitudes and / or the phases of RF voltages generated by various RF voltage sources operating under the control of the controller and / or DC voltage generated by DC voltage sources.

[0077] The following examples are provided for further elucidation of various aspects of the present teachings and are not intended to provide necessarily an optical way of practicing the present teachings and / or optimal results that may be obtained.

[0078] Examples

[0079] Example 1

[0080] FIG. 5A depicts ion detection signals corresponding to the 142.0 / 94 (solid plot) and 142.0 / 125.1 (dotted plot) MRM transitions of Methamidophos obtained employing a conventional MRM workflow, such as that depicted in FIG. 1A in which an ion emptying period is employed in each MRM measurement period associated with one of a plurality of different fragment ions generated via dissociation of the same precursor ion. FIG. 5B in turn shows an ion detection signal corresponding to the same MRM transition using an MRM measurement workflow according to an embodiment, which lacked an ion emptying step between acquisition of ion data for the fragment ions from the same precursor ion. The use of such a workflow allowed increasing the dwell period for collecting the ion detection data, and hence led to a significant increase in the MRM signal intensity.

[0081] Example 2

[0082] FIG. 6A depicts ion detection signal corresponding to MRM transitions 222.1 / 165.2 (solid plot) and 222.1 / 150.3 (dotted plot) of Methabenzthiazuron obtained by employing a 194925-1617-8568, v. 1conventional MRM workflow, such as that depicted in FIG. 1A in which an ion emptying period is employed in each MRM measurement period of fragment ions associated with the same precursor ion. FIG. 6B in turn shows an ion detection signal corresponding to the same MRM transition using an MRM measurement workflow according to an embodiment, which lacked an ion emptying step. The use of such a workflow allowed increasing the dwell period for collecting the ion detection data, and hence led to a significant increase in the MRM signal intensity.

[0083] Example 3

[0084] FIG. 7A shows MRM transition signals corresponding to 206 pesticides using a conventional MRM workflow in which each MRM measurement period included an ion emptying period, a pause period of 0.7 msec and a dwell period of 0.5 msec. FIG. 7B shows MRM transitions signals corresponding to the same pesticides using an MRM workflow according to an embodiment in which for each pesticide, the initial MRM measurement period included an ion emptying period, but the following MRM measurement did not. A comparison of the ion detection signals between FIGS. 7A and 7B shows that the MRM transitions obtained using the workflow according to an embodiment of the present teachings exhibit a higher signal intensity relative to the respective ones obtained using the conventional workflow. In fact, the increase in the signal intensity has resulted in certain MRM transitions that could not be discerned in the conventional ion detection data to be observable in the data obtained using an embodiment of the present teachings. Further, the use of the workflow according to the present teachings has allowed increasing the data acquisition rate from 809 MRM / sec to 1001 MRM / sec. Without being limited to any particular theory, it is believed that the signal increase is a result of a longer duration of pause + dwell for the first transition, allowing more time for the signal to return after the emptying period and a lower duration of pause + dwell on the second transition, where a faster measurable signal can be achieved since there is no emptying period and no need to switch the mass filter (QI).

[0085] Example 4

[0086] FIG. 8 shows that not emptying a mass filter (Q3) used to select a fragment ion of interest between acquisition of ion data in two consecutive MRM measurements corresponding to two different fragment ions of the same precursor ion enables very fast switching between data acquisitions of those fragment ions. One way of emptying a collision cell (Q2) and the mass 204925-1617-8568, v. 1filter (Q3) from ions is to set the resolving mass to a very low value, such as 5 Da. This can ensure that all ions are removed from Q2 and Q3. By not emptying Q2 and Q3 when transitioning between different MRM transitions of the same precursor ion, the switching time is closer to the switching between the solid and dashed plots. The plot depicted by dashed lines corresponds to switching between Q3 = 210 Da (previous MRM) to Q3 = 195 Da. The solid plot corresponds to switching between Q3 = 180 Da (previous MRM) to Q3 = 195 Da. The dotted plot corresponds to switching between empty and Q3 = 5Da to Q3 = 195 Da. The plots show the advantage of avoiding an emptying period (dotted plot vs dashed and solid plots). The plots also show that the ordering of MRM transitions such that the changes between the m / z ratios of the respective fragments is small can be useful in reducing the switching time between the MRM transitions.

[0087] Example 5

[0088] As noted above, in various embodiments, an axial acceleration field within a collision cell of a mass spectrometer in which precursor ions are fragmented into fragment ions can be used to accelerate the fragment ions to the subsequent stages of the mass spectrometer, thereby increasing the MRM data acquisition rate.

[0089] By way of illustration, the plots in FIGS. 9 show the time variation of the intensity of ion detection signals generated by an ion detector of a mass spectrometer, similar to the mass spectrometer illustrated in FIG.4, for the detection of a fragment ion having an m / z ratio of 397 Da corresponding to the same MRM transition for three different collision energies of the precursor ion. The data shows that at voltages of 400 V (dotted plot) and 200 V (dashed plot), the rise time of the ion detection signal is much shorter than the respective rise time at a voltage of 50 V.

[0090] The above descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.214925-1617-8568, v. 1

[0091] Depending on certain implementation requirements, embodiments of the present teachings, the controller can be implemented in hardware, firmware and / or in software.

[0092] In some embodiments, the instructions for operating the optical system can be stored using a non-transitory storage medium such as a digital storage medium, for example a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0093] While various embodiments have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; embodiments of the present disclosure are not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing embodiments of the present disclosure, from a study of the drawings, the disclosure, and the appended claims.

[0094] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other processing unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0095] Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the present teachings.224925-1617-8568, v. 1

Claims

What is claimed is:

1. A method for performing a plurality of MRM measurements, comprising:ionizing a sample to generate sample ions,selecting at least one precursor ion from said sample ions,performing at least two consecutive MRM measurements associated with a plurality of fragment ions generated via dissociation of said at least one precursor ion, wherein one of the at least two consecutive MRM measurements includes an ion emptying period and the other of the at least two consecutive MRM measurements lacks an ion emptying period.

2. The method of Claim 1, wherein a first one of said at least two consecutive MRM measurements comprises an emptying period, a first pause period and a first dwell period for detection of a first fragment ion of said plurality of the fragment ions and a consecutive second one of said at least two consecutive MRM measurements corresponding to said other of the at least two consecutive MRM measurements lacking an ion emptying period includes a second pause period and a second dwell period for detection of a second fragment ion of said plurality of the fragment ions.

3. The method of Claim 2, further comprising:during said emptying period, adjusting one or more operating parameters of a first mass filter, a dissociation device positioned downstream of the first mass filter and a second mass filter positioned downstream of the dissociation device to cause removal of any of a previous precursor ion and one or more fragment ions thereof from said first, second mass filters and the dissociation device,during said first pause period, configuring the first mass filter to select said at least one precursor ion and configuring a second downstream mass filter to select said first fragment ion for detection during said first dwell period,234925-1617-8568, v. 1during said second pause period, configuring the second mass filter to select said second fragment ion for detection during said second dwell period.

4. The method of Claim 3, wherein at least one of said first and said second dwell periods and said first and said second pause periods are different.

5. The method of Claim 4, further comprising utilizing ion detection data generated during one of said first and said second dwell periods to identify said at least one precursor ion and utilizing ion detection data generated during the other one of said first and said second dwell periods to quantify said at least one precursor ion.

6. The method of Claim 4, further comprising selecting a difference between said dwell periods based on at least one of a difference in m / z ratios, a difference in expected signal intensities, a difference in expected signal-to-noise ratios, and a desired limit-of-detection associated with said first and said second fragment ions.

7. The method of Claim 4, wherein the dwell period for the detection of the first fragment ion is greater than a respective dwell period for the detection of the second fragment ion.

8. The method of Claim 2, wherein an m / z ratio of the first fragment ion is less than an m / z ratio of the second fragment ion.

9. The method of Claim 1, wherein said at least two consecutive MRM measurements comprise three or more consecutive MRM measurements and the three or more consecutive MRM measurements are temporally ordered so as to minimize a difference between m / z ratios of fragment ions corresponding to neighboring MRM measurements.

10. The method of any one of Claims 1 - 9, further comprising utilizing an ion detection signal associated with one of said first fragment ions and said second fragment ions to quantitate the at least one precursor ion.244925-1617-8568, v.

111. The method of any one of Claims 1 - 9, further comprising utilizing an ion detection signal associated with one of said first fragment ions and said second fragment ions to identify said at least one precursor ion.

12. The method of any one of Claims 3-8, wherein said dissociation device comprises a collision cell.

13. The method of Claim 12, further comprising establishing different axial acceleration electric fields within the collision cell for said at least two consecutive MRM measurements.

14. The method of Claim 11, wherein the axial acceleration electric field for one of said at least two consecutive MRM measurements that is associated with a fragment ion having a larger m / z ratio is greater than a respective axial acceleration electric field for another one of said at least two consecutive MRM measurements.

15. A method for performing MRM mass spectrometry, comprising:ionizing a sample to generate at least one precursor ion,configuring a first mass filter to select said at least one precursor ion, using an ion dissociation device to cause dissociation of said at least one precursor ion to generate a plurality of fragment ions,configuring a second mass filter to select a first one of said plurality of the fragment ions for detection,subsequently, adjusting the second mass filter to select a second one of said plurality of the fragment ions for detection without prior adjustment of one or more operating parameters of any of said ion dissociation device and said second mass filter for causing removal of the first one of said fragment ions from any of said ion dissociation device and said second mass filter.

16. The method of Claim 15, wherein said ion dissociation device comprises a collision cell.254925-1617-8568, v.

117. A method for performing a plurality of MRM measurements, comprising: ionizing a sample to generate sample ions,selecting at least one precursor ion from among said sample ions, for said selected at least one precursor ion, acquiring at least two consecutive MRM ion detection signals each corresponding to one of said fragment ions, wherein a transition between acquisition of the two ion detection signals is performed without employing an ion emptying period.

18. The method of Claim 17, wherein the step of selecting the at least one precursor ion comprises using a mass filter to allow selective passage of said at least one precursor ion therethrough.

19. A mass spectrometer system, comprising:an ion source configured to receive a sample and ionize one or more analytes in the sample to generate sample ions,a controller,a first mass filter configured to operate under control of the controller for selecting at least one precursor ion from among said sample ions,an ion dissociation device positioned downstream of the first mass filter configured to receive the at least one precursor ion and cause fragmentation thereof into a plurality of fragment ions,a second mass filter positioned downstream of the ion dissociation device for selecting one of said plurality of fragment ions, anda mass analyzer for receiving the selected fragment ion and generating ion one or more ion detection signals in response to detection thereof,wherein the controller is programmed to send control signals to said first and second mass filters for performing at least two consecutive MRM measurements associated with said precursor ion such that an MRM measurement period corresponding264925-1617-8568, v. 1to one of the first and the second MRM measurements includes an ion emptying period and an MRM measurement period corresponding to the other MRM measurement lacks an ion emptying period.

20. The mass spectrometer of Claim 19, wherein each of the first and second mass filters includes a set of rods arranged in a multipole configuration to which RF voltages and a resolving DC voltage can be applied to provide a transmission window that would allow selective passage of a target precursor or fragment ion.274925-1617-8568, v. 1

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