Improved signal to noise ratio at the lloq through the reduction of chemical noise

Abstract

Various embodiments relate to the reduction of chemical noise at or near the mass of an analyte of interest prior to selection by a mass filter. The use of a narrow bandpass in the ion optics removes chemical noise that can repopulate ions at or near the mass of an analyte of interest by removing other chemical noise ions that can fragment into the same or similar mass as the mass of an analyte of interest through collision induced dissociation.

Description

IMPROVED SIGNAL TO NOISE RATIO AT THE LLOQ THROUGH THE REDUCTION OF CHEMICAL NOISERelated Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 525,269 filed on July 6, 2023 and U.S. Provisional Application No. 63 / 585,763 filed on September 27, 2023, the contents of both of which are incorporated herein by reference in their entireties.Technical Field

[0002] The present disclosure is generally directed to methods and systems for reducing chemical noise in mass spectrometry.Background

[0003] The present disclosure is generally directed to methods and systems for performing mass spectrometry, and in particular, to such methods and systems for reducing, and preferably eliminating, chemical noise in mass spectra.

[0004] Mass spectrometry (MS) is an analytical technique for determining the structure of test 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.

[0005] Multiple reaction monitoring (MRM) is a tandem mass spectrometry technique in which a precursor analyte ion is selected by a mass filter and dissociated, e.g., in a collision cell, to generate product ions, which can be selected by a second mass filter and analyzed. The presence of non-target ions, such as solvent and analyte / solvent clusters, having an m / z ratio that is the same as or close to the m / z ratio of the target ion can lead to the formation of non-target product ions having m / z ratios that are the same as or close to the m / z ratios of the target product ions. This can give rise to a chemical noise background that is often observed in MRM signals.

[0006] The chemical noise spectrum is primarily due to solvent and analyte / solvent clusters and covers a wide m / z range with ions at nearly every m / z above about 50.Summary

[0007] In one aspect, a method of performing mass spectrometry is disclosed, which comprises introducing a plurality of ions, including one or more analyte ions and one or more chemical noise ions, into a first mass filter to select ions having m / z ratios within a specific range for passage through the first mass filter, accelerating the selected ions, introducing the accelerated ions into an ion trap containing a background gas for causing preferential collision induced dissociation (CID) of chemical noise ions passing through the first mass filter while substantially retaining the one or more analyte ions undissociated, allowing at least a portion of ions to exit the ion trap, and introducing at least a portion of the ions exiting the ion trap into a second mass filter configured to allow passage of the undissociated analyte ions.

[0008] The ions exiting the ion trap can include undissociated analyte ions, fragment ions generated via dissociation of the analyte ions, undissociated chemical noise ions, and fragment ions generated via dissociation of the chemical noise ions. The first mass filter is configured to block passage of chemical noise ions having m / z ratios that are separated from an m / z ratio of the analyte ion by a specific m / z value. By way of example, the specific m / z value can be in a range of about + / - 5 Da to about + / - 15 Da.

[0009] The analyte ions passing through the second mass filter can be dissociated to generate a plurality of analyte product ions. A mass spectrum of the analyte product ions can be determined, e.g., using any suitable mass analyzer, such as a time-of-flight (ToF) mass analyzer, or a linear ion trap.

[0010] In some embodiments, the first mass filter and the ion trap are disposed in a common first chamber. In such embodiments, the second mass filter can be disposed in a second chamber that is positioned downstream from the common chamber. In other embodiments, the first mass filter and the ion trap can be disposed in separate chambers, which can be, for example, maintained at different pressures. In various embodiments, the pressure within the ion trap can be maintained, e.g., in a range of about 1 mTorr to about 30 mTorr.

[0011] A DC offset voltage can be applied between the first mass filter and the ion trap for accelerating the ions exiting the first mass filter as they transition from the first mass filter to the ion trap. The DC offset voltage can be selected such that the accelerated ions acquire a kinetic energy at which the chemical noise ions are more likely to dissociate than the analyte ions. Byway of example, and without limitation, the DC offset voltage can be in a range of about 2 V to about 200 V, such as in a range of about 10 V to about 100 V.

[0012] In various embodiments, the ion trap can include a plurality of rods that are arranged in a multipole configuration, such as a quadrupole or a hexapole configuration. In such embodiments, a DC offset voltage can be applied between the rods of the ion trap and those of the first mass filter to establish an electric field for accelerating the ions exiting the first mass filter.

[0013] An RF voltage and a DC resolving voltage can be applied to the multipole rods of the first mass filter to generate a bandpass filter. In various embodiments, the bandpass of the bandpass filter can be in a range of about 10 Da to about 30 Da.

[0014] A DC gating voltage can be applied to the ion trap to establish a DC potential difference between the ion trap and a downstream component of the mass spectrometer, e.g., the second mass filter discussed above. The DC gating voltage can be modulated between a trapping voltage at which ions are trapped within the ion trap and a release voltage at which ions can exit the ion trap to enter the downstream component.

[0015] Segmented ion optic Q0, which includes four rods sets Q0A, Q0B, Q0C, and Q0D, is used in various embodiments as a mass filter. In various embodiments, after completion of an ion fill period during which a batch of ions is introduced into the Q0 and trapped within the Q0D segment, the introduction of the plurality of ions into the first mass filter can be temporarily stopped to allow the ions introduced into the Q0 to undergo cooling and preferential dissociation of the chemical noise ions. After the release of the ions from the Q0D ion trap, another batch of ions can be introduced into Q0 for processing. By way of example and without limitation, the time interval between successive ion filling periods can be, for example, in a range of about 12 ms to about 200 ms,

[0016] In various embodiments, the trapping voltage can be applied to the ion trap for a temporal duration in a range of about 3 ms to about 200 ms. Further, in some such embodiments, the release voltage can be applied to the ion trap to allow ions within the ion trap to exit for a temporal duration in a range of about 5 ms to about 20 ms.

[0017] In various embodiments, the DC offset voltage applied between the first mass filter andthe ion trap is configured to inhibit the return of ions introduced into the ion trap back to the first mass filter. By way of example, as noted above, the DC offset voltage can be in a range of about 2 V to about 200 V, e.g., in a range of about 10 V to about 100 V. More specifically, the ions entering the ion trap experience collisions with a background gas in the ion trap and hence lose some of their kinetic energy, which in turn allows the DC offset voltage to act like a barrier for inhibiting the return of the ions into the mass filter.

[0018] In a related aspect, a mass spectrometer is disclosed, which includes a first mass filter configured to receive a plurality of ions, where the plurality of ions includes one or more analyte ions and one or more chemical noise ions, said mass filter being configured to select ions having m / z ratios within a specific range for passage through the mass filter. An ion trap containing a background gas is positioned downstream of said first mass filter and is configured to receive ions passing through the first mass filter. By way of example, the ion trap can be maintained in a range of about 1 mTorr to about 30 mTorr.

[0019] At least one DC offset voltage source is configured to apply a DC offset voltage between the first mass filter and the ion trap for accelerating the ions passing through the first mass filter prior to entry thereof into the ion trap so as to cause preferential dissociation of the chemical noise ions while substantially retaining the analyte ions undissociated and a second mass filter is positioned downstream of the first mass filter and configured to allow passage of the undissociated analyte ions. By way of example, the DC offset voltage can be in a range of about 2 V to about 200 V, e.g., in a range of about 10 V to about 100 V.

[0020] In various embodiments, the mass spectrometer can further include one or more controllers for controlling operation of any of said first mass filter, said ion trap, said at least one DC offset voltage source and said second mass filter.

[0021] A DC gating voltage source operating under control of the controller(s) is configured to apply a DC gating voltage to the ion trap so as to establish a DC voltage offset between the ion trap and a downstream component of the mass spectrometer. The one or more controllers can send control signals to the DC gating voltage source to adjust the gating voltage applied to the ion trap between a trapping voltage that inhibits the exit of the ions from the ion trap and a release voltage that allows the ions within the ion trap to exit the trap. Further, the one or more control signals applied to the DC gating voltage source can instruct the DC gating voltage sourceto apply the trapping voltage for a temporal period in a range of about 3 ms to about 200 ms and to apply the release voltage for a temporal period in a range of about 5 ms to about 20 ms.

[0022] An ion lens can be positioned upstream of the first mass filter for directing the plurality of the ions into the first mass filter. A DC voltage source operating under control of the controller(s) can apply DC voltages to the ion lens to facilitate or inhibit entry of ions into the first mass filter. For example, the one or more controllers can apply control signals to the DC voltage source to temporally modulate the applied DC gating voltage so as to allow and inhibit transmission of the plurality of ions into the first mass filter during alternating temporal periods, respectively. By way of example, the time interval between the two successive time periods can be in a range of about 12 ms to about 200 ms.

[0023] Moreover, in various embodiments, the mass spectrometer can further include a dissociation cell that is positioned downstream of the second mass filter for causing dissociation of the analyte ions passing through the second mass filter. By way of example, and without limitation, the dissociation cell can be a collision cell in which the analyte ions can undergo collision induced dissociation.

[0024] In various embodiments, the first mass filter and the ion trap are disposed in a common first chamber while in some other embodiments, the first mass filter and the ion trap can be disposed in separate chambers, which may be maintained at different pressures. A second mass filter can be disposed in a second chamber positioned downstream of the chamber in which the ion trap is disposed, e.g., downstream of the common chamber in which both the first mass filter and the ion trap are disposed, to receive ions exiting the ion trap. The second mass filter can be configured to allow passage of undissociated analyte ions while blocking ions having m / z ratios that are separated from the m / z ratio of the undissociated analyte ions by a certain value. By way of example, in various embodiments, the second mass filter can include a plurality of rods arranged in a multipole configuration to which RF and DC resolving voltages can be applied to set the bandpass of the mass filter.

[0025] Further understanding of various aspects of the present teachings can be found in the following detailed description in conjunction with the associated drawings, which are described briefly below.Brief Description of the Drawings

[0026] The drawings are not necessarily to scale or exhaustive. Instead, emphasis is generally placed upon illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated in this specification and constitute a part of it, illustrate several embodiments consistent with the disclosure. Together with the description, the drawings serve to explain the principles of the disclosure.

[0027] In the drawings:FIGS. 1A, IB, 1C, and ID demonstrate the effect of collision energy on reserpine ions and nearby chemical noise ions.FIG. 2 is a flowchart showing various steps of a process 200 according to some embodiments.FIGS. 3A and 3B, show an example of a mass spectrometer 300 according to some embodiments.FIG. 4A shows a portion of ion optics from a QJet ion guide to a QI mass filter.FIGS. 4B - 4E show the DC potentials applied to the ion optics at different stages in performing an MRM measurement.FIGS. 4F - 4J show DC voltages applied to the ion optics during various steps of performing an MRM analysis.FIGS. 5A and 5B show two Simion ion trajectory simulations.FIGS. 6A - 6D show examples of how ions having higher m / z ratios may fragment to produce product ions that can generate chemical noise.FIGS. 7A, 7B, and 7C provide schematic illustrations of a narrow bandpass filter required to mitigate chemical-noise producing fragment ions.FIGS. 8A, 8B, and 8C show examples of MS / MS spectra of a blank solution with the QI resolution set to transmit a 17 Da wide window centered at m / z 614.FIGS. 9A, 9B, and 9C show examples of MS / MS spectra of a blank solution with the QI resolution set to transmit a 17 Da wide window centered at m / z 655.FIGS. 10A, 1OB, 11A, 11B, 12A, 12B, 13A and 13B show the normalized cumulative intensity of the MRM 609 / 195 transition.FIG. 14 shows a plot of the integrated signal for the MRM transition 609 / 609 at CE = 10 eV.FIGS. 15A and 15B illustrate the number of ions transmitted into QOD with and without a 30 Da bandpass applied at the first mass filter.FIGS. 16 and 17 illustrate the estimated number of ions in QOD versus fill time.FIGS. 18A, 18B, 19A, 19B, 19C, 20A, 20B, 20C, 21A, 21B and 21C present the results of the fill time experiments at Q0 CE = 4 eV and 25 eV, andFIG. 22 is an illustration of the gain in signal to noise ratio compared to a normal nontrapping MRM experiment.Detailed Description

[0028] 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.

[0029] 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 means 10% greater or less than the value or range of values statedor 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 also refer 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.

[0030] 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

[0031] As used herein, the term “chemical noise ion,” and its plural, “chemical noise ions” refer to ions that are not target analyte ions of interest and their contribution to a mass spectrum of a sample can render the deconvolution of the mass spectrum more difficult, which can in turn typically lead to a better limit of quantification (i.e., a lower LLOQ).

[0032] The present disclosure relates to methods and systems for mass spectrometry and in particular to such methods and systems for reducing chemical noise in MRM mass spectra.

[0033] As noted above, chemical noise is primarily generated by solvent and analyte / solvent clusters. Generally, the intermolecular bonds of such clusters are weaker than the intramolecular bonds. For example, methanol-water molecules show infrared absorption bands in the 3570 cm'1to 3750 cm'1wavelength region, corresponding to 0.44 to 0.46 eV. Intramolecular bonds are typically stronger and can exhibit dissociation energies at 1 eV or higher.

[0034] As discussed in more detail below, in various embodiments, the amount of internal energy (e.g., collision energy) inputted into a plurality of ions, which can include both analyte ions and chemical noise ions, such as solvent or solvent / analyte clusters, can be controlled (tuned) so as to cause dissociation of a larger fraction of the chemical noise ions than that of the analyte ions. In other words, the input energy can be controlled to cause preferentially the dissociation of the chemical noise ions.

[0035] In various embodiments, the contribution of chemical noise ions having m / z ratios close to or the same as those of analyte ions of interest (e.g., within + / - 10 Da of an analyte of interest) can be reduced, and preferably eliminated. For example, in MRM analyses, the preferential dissociation of chemical noise ions at the same mass as the analyte ion removes those chemical noise ions that have a common product ion with the dissociated analyte ion, except the analyte ion remains largely undissociated. The chemical noise ions may be selectivelyfiltered relative to undissociated analyte ions, thereby reducing the contributions of the chemical noise ions to the resultant MRM transition.

[0036] Without being limited to any particular theory, and only by way of example, the rate of ion dissociation can be described by the so-called RRK expression below:where &Uni denotes the rate constant along the reaction coordinate in units of s’1, E* denotes the barrier along the reaction coordinate and S = 3N -1, where N denotes the number of atoms in the complex and S denotes the number of vibrational degrees of freedom. The RRK expression indicates that the larger the analyte, solvent or analyte / solvent complex, the more internal energy will be required to cause the complex to dissociate, e.g., a higher collision energy will be required. Conversely, smaller complexes will require a lower collision energy.

[0037] FIGS. 1A, IB, 1C, and ID demonstrate the effect of collision energy on reserpine ions and nearby chemical noise ions, illustrating that the degree of their fragmentation is in accordance with the RRK model. In particular, the intensity of m / z 609.3 at Q2 CE = 20 eV is 80% of the respective intensity at Q2 CE = 10 eV. In contrast, the intensity of the chemical noise peaks in the range of m / z 614 to m / z 617 at Q2 CE = 20 eV is ~ 25% of the intensity at Q2 CE = 10 eV. The greater reduction in the chemical noise intensity demonstrates that the bonds in ions that can lead to the generation of chemical noise are weaker than those in the analyte bonds.

[0038] Comparing the ratio of m / z 609.3 to m / z 616.3 at Q2 CE = 10 eV a ratio of 50: 1 is obtained whereas at Q2 CE = 20 eV the ratio climbs to 123: 1.

[0039] As shown in FIGS. 1C and ID, when the Q2 CE is increased to 25 eV the ratio climbs even higher to 268: 1. This demonstrates that tuning the collision energy can lead to an improved signal-to-noise ratio by reducing the amount of chemical noise that may be present at the same mass-to-charge ratios as those of the ions of interest.

[0040] FIG. 2 is a flowchart showing various steps of a process 200 according to some embodiments for performing mass spectrometry. At step 210, a plurality of ions is received by a first mass filter. The ions received by the first mass filter can include analyte ions as well as chemical noise ions. By way of example, the ions may be introduced in the first mass filter via asample preparation and ionization module. For instance, acoustic ejection of the sample from a sample plate may be used to eject the sample into an ionization source, which then ionizes the sample and produces ions that are received by the first mass filter. This process can result in the production of not only analyte ions but also chemical noise ions.

[0041] At step 220, the first mass filter is utilized to select ions having m / z ratios within the bandpass of the first mass filter for passage through the first mass filter. By way of example, the bandpass of the mass filter can be configured to allow passage of the target analyte ions and further block the passage of ions having m / z ratios that are separated from the m / z ratio of the target analyte ions by a value in a range of about + / - 5 Da to about + / - 15 Da.

[0042] At step 230, the ions passing through the first mass filter are accelerated and the accelerated ions are introduced into an ion trap, which contains a background gas. At step 240, the ions undergo cooling in the ion trap. Further, the energy of the accelerated ions imparts a kinetic energy to the ions that is more likely to cause collisional dissociation of the chemical noise ions while retaining the analyte ions in a substantially undissociated state, though some analyte ions may undergo dissociation, resulting in the generation of some analyte product ions.

[0043] Subsequently, at step 250, the ions within the ion trap are allowed to exit the trap and the exiting ions are introduced into a second mass filter that is positioned downstream of the first mass filter. The second mass filter is configured to allow passage of undissociated analyte ions. By way of example, in embodiments in which the second mass filter includes a plurality of rods that are arranged in a multipole configuration, an RF voltage and a DC resolving voltage applied to those rods can be selected to allow passage of the undissociated analyte ions through the mass filter while blocking ions outside the bandpass of the mass filter.

[0044] At step 260, the ions passing through the second mass filter can be dissociated, e.g., via collision-induced fragmentation, to generate a plurality of product ions associated with the analyte precursor. At step 270, a mass spectrum of these product ions can be determined. The third mass filter can be scanned to allow passage of different product ions during consecutive time intervals and a downstream ion detector can detect the product ions passing through the third mass filter to generate ion detection signals indicative of the intensity of the product ions. In various embodiments, the ion detection signals can be collected and analyzed to generate a mass spectrum of the product ions.

[0045] The present teachings can be implemented using a variety of mass spectrometers. By way of example, in various embodiments, the mass spectrometer may be a quadrupole mass spectrometer, a time-of-flight (ToF) mass spectrometer, an ion trap mass spectrometer, a triple quadrupole mass spectrometer, a hybrid instrument including QToF or trapping instruments, such as a linear ion trap, a 3D ion trap, an Orbitrap, an electrostatic ion trap, or a combination of these with each other or other types of mass spectrometers.

[0046] In some embodiments, the mass spectrometer may be an MS / MS mass spectrometer configured to perform MS / MS analysis of the sample ions that it receives. In such embodiments, the mass spectrometer may include a mass filter that is configured to select one or more precursor ions, and a collision cell to fragment the selected precursor ions. These fragments are then detected by an ion detector of the mass spectrometer to generate ion detection signals that may then be processed to generate a mass spectrum of the fragment ions.

[0047] For example, with reference to FIGS. 3 A and 3B, an example of a mass spectrometer 300 according to an embodiment includes an ion source 302 that generates a plurality of ions. A variety of ion sources can be employed in the practice of the present teachings. 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, 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.

[0048] The ions generated by the ion source 302 pass through an orifice 304a of a curtain plate 304 and an orifice 306a of an orifice plate 306, which is positioned downstream of the curtain plate 304 and is separated from the curtain plate 304 such that a gas curtain chamber is formed between the orifice plate 306 and the curtain plate 304. A curtain gas supply (not shown) can provide a curtain gas flow (e.g., of nitrogen) between the curtain plate 304 and the orifice plate 306 to help keep the downstream sections of the mass spectrometer clean by declustering and evacuating neutral particles. The curtain chamber can be maintained at an elevated pressure (e.g., a pressure greater than the atmospheric pressure) while the downstream sections of the mass spectrometer can be maintained at one or more selected pressures via evacuation throughone or more vacuum pumps (not shown).

[0049] The ions passing through the orifice plate 306 are received by an ion guide QJet® that focuses the received ions into an ion beam that is transmitted into a segmented ion optic Q0, which includes four rods sets QOA, QOB, QOC, and QOD

[0050] The ion guide QJet® includes four rods 330 (two of which are visible in FIGS. 3A and 3B) that are arranged according to a quadrupole configuration. An RF source 332 applies RF voltages to the four rods 330 in a manner known in the art to provide radial focusing of the ions passing through the ion guide QJet. An ion lens IQ0 is disposed between the ion guide QJet® and Q0 to allow differential pumping of the QJet® and Q0 chambers and can provide enhanced transmission and focusing of the ions. In some embodiments, the Q0 chamber can be maintained in a range of about 3 mTorr to about 10 mTorr.

[0051] In this embodiment, each of the four sets of rods QOA, QOB, QOC, and QOD, which are positioned in series relative to one another, includes four rods that are arranged in a quadrupole configuration to provide a passageway through which ions received via an inlet 334 of Q0 can propagate to its outlet 336 through which the ions exit Q0. In this embodiment, the RF voltage source 332 (or a separate RF voltage source) and a DC voltage source 340a apply RF and DC voltages to the rods of the Q0 so as to provide radial focusing of the ions as well as establish a bandpass window (i.e., a transmission window) in the QOC segment for passage of target ions of interest through that segment. The DC voltage source 340a, or another voltage source, can further provide DC offset voltages between each of the Q0 segments and a downstream segment to facilitate the passage of the ions through Q0.

[0052] More specifically, the DC voltage source 340a can apply a resolving DC voltage to the rods of the QOC segment such that the combination of the applied RF voltage and the DC resolving voltage results in the QOC segment functioning as a bandpass filter. In other words, ions having m / z ratios that fall within the bandpass window of the mass filter QOC can pass through the mass filter QOC while transmission of ions having m / z ratios that fall outside the bandpass window is substantially reduced, and preferably inhibited. The bandpass of the QOC mass filter can be selected such that analyte ions of interest will pass through QOC, but ions, including chemical noise ions having m / z ratios separated from the m / z ratio of the analyte ions of interest by more than a specific value, e.g., by a value in a range of about + / -5 Da to about + / -10 Da, are blocked from passing through the mass filter.

[0053] In other words, in this embodiment, RF voltages are applied to the first, second, and fourth sets of rods QOA, QOB, and QOD, while an RF voltage as well as a resolving DC voltage (for setting the bandpass window of the mass filter QOC) are applied to the third set of rods QOC. Further, the RF voltages applied to the QOA and QOB rod sets have a lower amplitude than the amplitude of the RF voltages applied to QOC rod set because in this embodiment the RF voltages applied to QOA and QOB rod sets are obtained, via capacitive coupling, from the RF voltage supply that applies RF voltages to the QOC rod set. Further, in this embodiment, the amplitude of the RF voltages applied to the QOB rod set is slightly lower than the amplitude of RF voltages applied to the QOA rod set. By way of example, this can be achieved by lowering the coupling capacitance, e.g., by about 10%, such as from 220 pF to about 200 pF. This ensures that the RF amplitude applied to QOB remains lower than the respective RF amplitude applied to QOA, which leads to a higher effective potential at QOA than QOB.

[0054] By way of example, such a variation in the amplitude of the applied RF voltage may be implemented to account for the tolerances of the coupling capacitors, e.g., a typical tolerance of + / - 5%. This can reduce the probability of creating an effective potential that could generate an off-axis field pointing toward QOA rather than toward QOB. Consequently, the ions at the exit of the QOB rod set exhibit a higher value for the Mathieu q parameter than those at the entrance to the QOC segment.

[0055] A description of why the effective potential for QOB is greater than at QOC is in order. As shown in Fig. 4A, the Mathieu q value decreases from QOA to QOD. QOC has the highest RF amplitude which means that, in order for Q0A / Q0B to have a higher q value, the field radius of the rods had to be reduced. By way of example, in an embodiment in which the Q0A / Q0B field radius is 3.88 mm, the Q0C / Q0D field radius may be 4.17 mm. To make the smaller field radius for Q0A / Q0B the rods may be made larger in diameter than the Q0C / Q0D rods. In various embodiments, the rods for each segment can have the same center, which makes mechanical assembly and alignment easier. The Mathieu q value for each segment is shown in Fig. 4A. The smaller field radius increases the q value. QOD has the same field radius as QOC because its RF amplitude is lower than that of QOC due to the capacitive coupling from QOC. The higher q value at the exit of the QOB rod set results in a higher effective potential at the exit of the QOBrod set than at the entrance of the QOC rod set, thereby reducing ion reflections at the QOB / QOC boundary.

[0056] The voltage source 340a can also apply DC voltage offsets between each of the QOA, QOB, QOC, and QOD segments and a neighboring segment (the voltages depicted in FIG. 3B are for illustration purposes only and other voltages may also be employed). The DC voltage offset between the QOC and QOD can accelerate the ions passing through QOC so as to impart a sufficient kinetic energy to those ions for causing preferential dissociation of the chemical noise ions.

[0057] As discussed in more detail below, the QOD segment receives the ions passing through the mass filter QOC and functions as an ion trap in which the received ions are trapped. More specifically, in order to trap the ions within the ion trap QOD, the DC voltage source 340b, or another DC voltage source, can apply a DC voltage offset between an ion lens IQ1 positioned between the outlet of the ion trap and an inlet of a downstream mass filter QI to prevent the exit of the ions from the ion trap. Subsequent to collisional cooling and preferential dissociation of the chemical noise ions within the ion trap, the DC voltage offset between the mass filter QI and the lens IQ1 can be lowered so as to allow the ions within the ion trap to exit the trap and enter a downstream mass analyzer QI via passage through an RF ion lens STI.

[0058] The RF lens STI includes four rods arranged in a quadrupole arrangement to which RF voltages are applied for generating an electromagnetic field, which can focus the ions into the downstream mass filter QI. The mass filter QI includes four rods that are arranged in a quadrupole configuration. In this embodiment, the quadrupole rod set of the mass filter QI can be operated as a transmission RF / DC quadrupole mass filter for selecting the target analyte ions of interest. By way of example, the quadrupole rod set of the mass filter QI can be provided with RF / DC voltages suitable for operation in a mass-resolving mode. For example, parameters of applied RF and DC voltages can be selected so that the mass filter QI establishes a transmission window of a chosen m / z ratio corresponding to the undissociated target analyte ions such that these ions can traverse the mass filter QI largely unperturbed. Ions having m / z ratios falling outside the window, however, do not attain stable trajectories within the quadrupole and can be prevented from traversing the quadrupole rod set of the mass filter QI. It should be appreciated that this mode of operation is but one possible mode of operation for the mass filterQI. By way of example, and without limitation, the bandpass of the QI mass filter can be in a range of about 0.1 Da to about 100 Da.

[0059] The ions passing through the mass filter are received by a collision cell Q2 via a stubby lens ST2 and an ion lens IQ2 The collision cell Q2 contains a gas, such as nitrogen, to cause collisional fragmentation of at least a portion of the received ions to generate a plurality of analyte product ions.

[0060] In this embodiment, the collision cell Q2 includes four rods that are arranged in a quadrupole configuration and to which RF voltages can be applied to provide radial confinement of the ions received by the collision cell Q2.

[0061] The product ions generated by the collision cell Q2 are received by a downstream quadrupole mass analyzer Q3 via an ion lens IQ3 and a stubby lens ST3, which function to focus the product ions into the quadrupole mass analyzer Q3. The quadrupole mass analyzer Q3 includes four rods 314 that are arranged relative to one another in a quadrupole configuration and to which RF and DC voltages can be applied in a manner known in the art to provide mass analysis of the product ions. The ions passing through the mass analyzer Q3 are detected by a downstream detector 322 that generates ion detection signals in response to the incident ions. A pair of lenses 316 and 318 help focus the ions onto the detector.

[0062] In this embodiment, the lens 316 is implemented as a 90% transmission mesh that defines the end of the trapping region. The lens 318 in turn provides shielding for the trap region which reduces the field penetration from the float potential applied to the detector (e.g., -6 kV in positive ion mode, +4 kV in negative ion mode for the 4822D channeltron detection system, or, from the -15 kV applied to the HED and +5.5 kV detector float potential used in the high dynamic range detection system, such as those described in US10074529 and US9991104, which are herein incorporated by reference in their entirety).

[0063] An analyzer 324 in communication with the detector 322 receives the ion detection signals and processes the ion detection signals to generate a mass spectrum of the product ions, thereby allowing monitoring MRM transitions corresponding to the precursor ions. As is known in the art, the analyzer 324 can be implemented in hardware / firmware and / or software using techniques known in the art as informed by the present teachings. For example, the analyzer 324 can include a processor, one or more random access memory (RAM) modules, one or morepermanent memory modules and at least one communication bus for allowing communication among these and other components.

[0064] In use, during an ion fill step, the ions are introduced into the Q0 via passage through the IQO lens while the voltage applied to IQ1 is adjusted to ensure that the ions entering the Q0 are trapped within the QOD segment. In various embodiments, the fill time can be, e.g., in a range of about 1 ms up to about 200 ms. In various embodiments, the minimum fill time can be determined based on the desired signal-to-noise ratio from counting statistics for the ion of interest. By way of example, the maximum fill time can be set by the maximum number of ions that the ion trap can hold before space charge effects become evident. An illustration of potential space charge effects is seen in FIG. 14 as described above. In some embodiments, in an MRM analysis, the fill time will be determined by how many MRM transitions per second are required. As seen in figure 14 the integrated signal should be linear with fill time up until the point where the effect of space charge sets in. Accordingly, an indicator for the onset of space charge is the onset of the non-linearity in the curve. When no Q0 bandpass is applied, then even more ions enter the QOD trap, and the non-linear effect is seen at even lower fill times.

[0065] In various embodiments, for a high duty cycle, the fill time should be set low enough to get good counting statistics. Since the goal is MRM analysis, then the fill time will be determined by how many MRM transitions per second are required in these embodiments. Based on the timing numbers a 5 ms fill plus a 5 ms cool / fragment, a 10 ms drain / detect and 3 ms for emptying Q2 and resetting, will amount to a total of 23 ms in exemplary embodiments. This gives about 43 MRMs per second accordingly. Fast instruments run may be a 3 ms pause plus 2 ms dwell time for a total of 5 ms per MRM transition or 200 MRMs per second. The trapping inherently slows down the process, however, the signal-to-noise gains make up for the decreased duty cycle in such embodiments. Accordingly, the MRM trapping technique would be activated when a lower LLOQ is required.

[0066] After the completion of the fill step, during a period, e.g., in a range of about 5 ms to about 20 ms, in which ion cooling and preferential fragmentation of the chemical noise ions is achieved, the voltage applied to the IQO is adjusted to inhibit entry of additional ions into Q0.

[0067] Subsequent to completion of the ion cooling and fragmentation step, the voltage applied to the IQ1 lens is lowered to allow the ions within the QOD ion trap to exit the ion trap,that is, the ion trap is drained, and the exiting ions are received by the downstream mass filter QI, which is configured to allow the undissociated analyte ions to pass through while blocking ions having m / z ratios outside its bandpass.

[0068] Referring again to FIGS. 3A and 3B, in some embodiments, the QOB segment, rather than the QOC segment, can be configured as a mass filter and the ions can be accelerated to impart sufficient kinetic energy thereto at the boundary of the QOB and QOC segment, i.e., the ions exiting the QOB mass filter can be accelerated as they transition into the QOC segment. Similar to the previous embodiment, the ions can still be trapped within the QOD segment. Alternatively, in some such embodiments, the QOD segment may be implemented as a flow- through segment without ion trapping. In such a case, the QOD segment should be sufficiently long to allow for collisional cooling of the ions. If the collisional cooling were inadequate, the ion beam could potentially charge up the surface of the IQ1 lens. On the other hand, in such an implementation, if the length of the QOD segment were too long, the ions may slow down to a degree that they would require the assistance of space charge to move through the last segment. By way of example, and without limitation, in various embodiments, the length of the QOD segment can be in a range of about 1 cm to about 19 cm. According to the US patent US04963736, incorporated here by reference, a pressure times distance (pd) of 2.25e-2 Torr cm is recommended as a minimum for collisional cooling for ions with between 1 to 30 eV axial kinetic energy.

[0069] In such embodiments in which the QOB segment is configured as a mass filter, the length of the Q0A segment is selected to be longer than its respective length in the previous embodiment to ensure that ions undergo sufficient cooling in the Q0A segment prior to their entry into the QOB segment. By way of example and without limitation, the length of the Q0A segment can be in a range of about 2 cm to about 23 cm.

[0070] In some embodiments, the QOC and the QOD segments may be combined into a single segment or the QOC segment may be removed if the objective was to only perform trapping. In these embodiments, the bandpass can be created at the QOB segment. The Q0A segment would have to be of sufficient length to accomplish enough collisional cooling before the ions enter the QOB segment. Ions are then accelerated into QOC by having a DC offset between QOB and QOC. Ions are then trapped in QOC. There is no QOD segment in such embodiments. This isY1simply a three segment QO with the bandpass placed on the middle, QOB, segment.

[0071] In some embodiments, the use of four segments can allow, a) for increased functionality for other scan modes, and b) it can ensure the pd is high enough to provide good collisional cooling, e.g., under circumstances such as a low QO pressure caused by the ion source running at a high temperature.

[0072] In another embodiment, three mass filters and two collision cells positioned in series are employed. For example, the first mass filter can be used to transmit a wide mass range, e.g., the first mass filter can have a bandpass of about 10 Da. As such, the first mass filter can remove chemical noise ions from the low and the high mass regions but it does not affect the transmission of the analyte of interest. The mass regions comprise what is not included in the bandpass. For example, if the analyte ion is at m / z 609 and a 10 Da bandpass centered on m / z 609 is used, then ions are transmitted from m / z 604 to m / z 614. The low mass region includes ions that are < m / z 604 and the high mass region includes ions that are > m / z 614. The remaining chemical noise ions within the bandpass of the first mass filter, which pass through the first mass filter, can be removed through CID in the first collision cell that is positioned downstream of the first mass filter. The ions exiting the first collision cell enter the second mass filter, which can operate at unit resolution. The second collision cell positioned downstream of the second mass filter is used to cause fragmentation of at least a portion of ions passing through the second mass filter. The resulting product ions are analyzed by the third mass filter. These embodiments represent a penta-quad example as shown in JASMS v7 pl 126 to 1137 1996, incorporated here by reference. In an exemplary implementation, the first mass filter is operated at a pressure of 1 e- 5 Torr with a wide bandpass, 10 Da, which ensures 100% transmission of the analyte ion because of the low resolution. Reducing the chemical noise occurs in the first collision cell and the ions that exit the first collision cell have m / z ratios within the 10 Da bandpass centered on the analyte ion with preferentially reduced chemical noise. This accomplishes the reduction of the chemical noise except that no trapping has occurred. At this point an analyte ion is selected in the second mass filter which is operating at unit resolution. The analyte ion is then passed into the second collision cell for fragmentation followed by mass selection of the fragment ions in the third mass filter. The following examples are provided for further elucidation of various aspects of the present teachings and are not intended to indicate necessarily the optimal ways of practicing the invention and / or optimal results that may be obtained.

[0073] Examples

[0074] Experiments were carried out on a modified Sciex QTrap 6500+mass spectrometer. The modifications included providing a four segment Q0 ion optic and ST1 / ST3 pre-filters that have a reduced field radius and are rotated to prevent the trapping of ions in the pre-filter ion optics. The rotated pre-filters are described in US20220028677, Effective Potential Matching at Boundaries of Segmented Quadrupoles in a Mass Spectrometer, which is herein incorporated by reference in its entirety. The ability to turn the ion beam off using the IQO lens is used in the technique described below. Using the IQO lens to turn the ion beam off is described in US20220254619 Increased Dynamic Range for the Attenuation of an Ion Beam, which is also incorporated herein by reference in its entirety.

[0075] Dilutions of a reserpine solution, 0.017 pmol / pl, 0.0017 pmol / pl and 0.00017 pmol / pl were used for characterizing the trap and in particular the ion release capabilities of the Q0D trap. A set of chemical noise background experiments were carried out using the same solution to dilute the reserpine samples, 50 / 50 water / acetonitrile with 0.1 % formic acid.

[0076] FIG. 4A shows a portion of the ion optics from the QJet ion guide to the QI mass filter. The Q0 segments are labelled with the values of Mathieu stability parameter q. The q values are decreasing from the Q0A to the Q0D segment. This is done to keep the effective potentials decreasing as the ions move from one segment to the next towards the IQ1 lens. This keeps the off-axis axial fields pointing downstream as the ions move through the Q0 ion optic, which can in turn prevent reflections of the ions at the boundaries and allows the ions to travel quickly through the segmented Q0 ion guide (< 1 ms for m / z 609 at the DC potential offsets shown in the figure).

[0077] In various embodiments, the purpose of the first mass filter is to eliminate or remove the chemical noise ions at other masses, mostly greater in mass than the analyte ion, that may dissociate into chemical noise ions at the same mass as the analyte ion when accelerated into the ion trap.

[0078] For example, if the analyte mass is m / z 500, the first mass filter may allow ions in the range m / z 485 to m / z 515 to be accelerated into the trap. An ion at m / z 550 is not allowed into the trap. If m / z 550 was allowed into the trap and dissociated to produce m / z 500, the samemass as the analyte ion, then one would have removed the original chemical noise at m / z 500 and replaced it with new chemical noise from the dissociation of m / z 550. In various embodiments, the first mass filter prevents this process from occurring.

[0079] FIGS. 4B - 4E show the DC potentials applied to the ion optics at different stages in performing an MRM measurement. In the first step, the DC offsets are set to allow the transmission of the ion beam into the Q0 ion optic. This step was 0.5 ms in duration. The kinetic energy of the ion beam was set by the potential difference between Q0C / Q0D and is referred to as Q0 CE. In this example, this voltage difference was set to 4 V.

[0080] The Q0C was configured as a mass filter with a 30 Da bandpass. The second step is to raise the DC voltage applied to IQ1 to 30 V to trap ions in Q0D. The duration of the second step corresponds to the ion trap fill time. In the experiments described below, the ion trap fill time was varied from 1 ms to 200 ms.

[0081] The range of the bandpass, when implemented in the Q0C segment, may be from 10 Da up to about 30 Da centered on the analyte mass. In some embodiments, below about 30 Da, the analyte intensity will drop for singly charged ions. However, multiply charged ions may maintain their intensity at a smaller bandpass, a phenomenon which has been observed experimentally. In some embodiments, the minimum ranges are determined by the quality of the mass filter and the operating pressure. If the first mass filter were separated from the ion trap or collision cell, then it may be operated at a lower pressure and the bandpass may be made smaller. Accordingly, in various embodiments, the range depends upon the operating conditions of the first mass filter.

[0082] In various embodiments, the collision energy profiles of the chemical noise and the analyte ions are different in order to allow preferential dissociation of the chemical noise ions relative to the analyte ions. In many cases the CE profiles for causing dissociation of the chemical noise ions and the target analyte ions will likely overlap. Accordingly, a fraction of both the chemical noise and analyte ions may dissociate at the same collision energy.

[0083] In the third step, the DC voltage offset applied to the IQ0 lens is dropped to -35 V, which turns off the ion beam. The duration of the third step, which corresponds to a cooling step, is set at 10 ms. During this time period, ions can undergo fragmentation in Q0D and cool both translationally and internally. The choice of 10 ms was based on the fact that ions will coolrapidly at the 5.5 mTorr pressure in the QO region.

[0084] The fourth step is the drain step in which the DC voltage offset applied to the IQ1 lens is dropped to its normal transmitting value and ions are allowed to drain out of QOD. The choice of a 20 ms drain period was determined from a series of experiments in which the ion beam intensity was measured at 2.5 ms intervals. The ion beam is still turned off at IQO during this step.

[0085] In some embodiments, the DC voltage offset may be as low as 5 V or less for low mass chemical noise ions and fragile analyte ions. It may be higher for high mass chemical noise ions and / or analyte ions requiring a higher collision energy to fragment. In some embodiments, an energy in the range from 2 eV to 50 eV (DC offset from 2 V to 50 V) is accordingly adopted. In other embodiments, even higher values, up to 200 V, can be adopted for heavier ions.

[0086] The fifth and the sixth steps are the same as the first step for the Q0 ion optics with a difference in the fifth step, where the Q3 mass is dropped to 5 Da for a period of 2 ms to empty the Q2 collision cell which eliminates MRM cross talk. In the sixth step the Q3 mass is returned to the fragment ion mass.

[0087] In the experiments discussed herein, to increase Q0 CE, the potential offsets for the QJet / IQO / QOA / QOB and Q0C are all increased. By way of example, FIGS. 4FA - 4 J show DC voltages applied to the ion optics during various steps of performing an MRM analysis, where the potential offsets are set such that Q0 CE = 25 eV. The steps are the same as those discussed above in connection with FIGS. 4A - 4E.

[0088] QOD filling simulation

[0089] One key aspect of the present teachings is the ability to fill QOD ion optic while the bandpass is applied to Q0C ion optic without any loss of ions. Simulations show that applying a bandpass to the Q0C ion optic does not cause any loss of ions during the fill step.

[0090] FIGS. 5A and 5B show two Simion ion trajectory simulations in which Q0 CE was set at 4 and 25 eV, respectively. The parameters of the simulation were 5 mTorr nitrogen, and collision cross section G = 280 A2, 10 ms duration, Q0C = -6 V and 15 V, QOD = -10 V, IQ1 = 30 V, m / z 609, Mathieu parameters a, q = 0.22, 0.706 for Q0C and the initial KE = 1 eV withthe ion started in the QOC segment.

[0091] In some embodiments, the pressure in the ion trap may range from 1 mTorr to 30 mTorr. The lower the pressure the fewer collisions and the longer the time required for the chemical noise ions to build up enough internal energy from the collisions to dissociate. In some embodiments in which the pressure is set high, the DC offset used to accelerate the ions will have to be higher to overcome the collisions that can thermalize the chemical noise ions and reduce the dissociation rate. That is, there are two competing processes, namely, dissociation and cooling.

[0092] In some embodiments, with the segmented Q0, the pressure is set to be between about 3 mTorr to 10 mTorr. At 3 mTorr, the pressure is barely able to maintain the amount of collisional cooling required for improved ion transmission while at 10 mTorr, a pump used to apply a negative pressure to Q0, such as a turbopump, may begin to lose its pumping efficiency. In various embodiments, a practical range for the segmented Q0 can be in a range of about 3 to 10 mTorr, e.g., of nitrogen. In some embodiments, the local pressure can be increased by installing a sleeve around the ion guide.

[0093] The model shows that the ions can be trapped without ion losses at the Q0C / Q0D boundary. The only requirement is the presence of a collision gas to reduce the ion’s axial kinetic energy so that the ions cannot overcome the potential barrier at the Q0C / Q0D boundary after reflecting back from the IQ1 lens. The squares mark the collision events along the path of the ion trajectory. Only one trajectory is shown in each case for clarity. Running a large number of ion trajectories resulted in all ions becoming trapped in the Q0D segment.

[0094] The simulation data shows that a bandpass can be applied to the QOC ion optic while Q0D is being filled without any loss of ions.

[0095] Fragmentation of ions generating chemical noise

[0096] FIGS. 6A - 6D show examples of how ions having higher m / z ratios can fragment to produce product ions that can generate chemical noise in a region previously cleared of chemical noise. FIGS. 6A and 6B show mass spectra acquired, respectively, with and without dipole excitation applied to remove ions in the region centered at m / z 395 with a DC potential drop of 4 V between Q0A and Q0B. The dipole excitation was applied to Q0A.

[0097] FIGS. 6C and 6D show mass spectra acquired, respectively, with and without dipole excitation applied to remove ions in the region centered at m / z 395 with a potential drop of 41 V between QOA and QOB. The overall intensity of the mass spectra was reduced by a factor of two but, the hole centered at m / z 395 has been repopulated from fragments of higher mass ions, which generate chemical noise. This demonstrates the need to remove chemical noise ions from outside the region of interest using a narrow bandpass filter as disclosed herein.

[0098] FIGS. 7A, 7B, and 7C provide schematic illustrations of why a narrow bandpass filter is required to remove ions that can dissociate to generate chemical-noise producing fragment ions prior to subjecting precursor ions to collision induced dissociation. For example, the application of a 30 Da bandpass to the Q0C ion optic can remove ions with high m / z ratios that can dissociate to produce ions at the mass of an analyte of interest (thick line) when high mass ions are dissociated by a high Q0 CE to generate product ions that cause chemical noise. It is also possible that multiply charged low mass ions could lose a charge and dissociate to a mass at or close to analyte mass of interest. Applying a narrow bandpass prior to the CID event in the Q0 ion optic removes the low and high mass ions that could contribute to chemical noise at the mass of interest.

[0099] FIGS. 8A, 8B, and 8C show examples of MS / MS spectra of a blank solution (50 / 50 water / acetonitrile, 0.1% formic acid) with the QI resolution set to transmit a 17 Da wide window centered at m / z 614. More specifically, FIGS. 8A and 8B show the spectra acquired with the ion collision energy (CE) set to 10 eV and 30 eV, respectively. FIG. 8C shows a portion of each spectrum in an m / z region in which ions generating chemical noise can be present.

[0100] The spectra shown in FIGS. 8A - 8C illustrate that at CE = 10 eV, the ion signals associated with chemical noise are present at almost every mass. At CE = 30 eV, the chemical noise spectrum exhibits more structure, but it is still present at every mass. A comparison of the spectra acquired at CE = 10 eV and CE = 30 eV shows that the chemical noise has been reduced to a few percent at the higher collision energy.

[0101] FIGS. 9A, 9B, and 9C show examples of MS / MS spectra of a blank solution (50 / 50 water / acetonitrile, 0.1% formic acid) with the QI resolution set to transmit a 17 Da wide window centered at m / z 655. More specifically, FIGS. 9A and 9B show the spectra acquired with the ioncollision energy (CE) set to 10 eV and 30 eV, respectively. FIG. 9C shows a portion of each spectrum in an m / z region in which ions generating chemical noise can be present. In this example, the chemical noise peak at m / z 663.5 appears to create ion fragments corresponding to successive losses of 56 Da from the parent ion to create m / z 607.4, m / z 551.4 and m / z 495.3. The m / z 663.5 signal has been reduced by a factor of eight at CE = 30 eV compared to CE = 10 eV. The m / z 663.5 chemical noise ion is an example of a relatively stable solvent complex. If an analyte ion with m / z 663.5 dissociates to either m / z 607.4, m / z 551.4 or m / z 495.3 then the chemical noise background in the measurement would be high leading to a high lower limit of quantitation (LLOQ). In such a case, removing the chemical noise prior to mass analysis in QI would lead to a significantly lower LLOQ.

[0102] Drain time

[0103] FIGS. 10A, 10B, 11 A, 11B, 12A, 12B, and 13A and 13B show the cumulative intensity of the MRM 609 / 195 transition as a function of the Q0D drain time and the fill time for the blank sample and samples having different concentrations of reserpine. A 30 Da bandpass was employed during these experiments. The intensity of the MRM 609 / 195 transition was measured every 2.5 ms for 20 ms with a 0.5 ms pause used between measurements as the ions were draining out of Q0D for detection. The signal was added for each measurement and plotted as a function of the drain time. The total signal changed very little beyond a drain time of 20 ms so this time was chosen as the optimal drain time.

[0104] One characteristic that is evident is the role that space charge plays in how fast Q0D is drained. In each plot, the time required to drain Q0D decreased with increasing fill time, which corresponds to increasing space charge. With no space charge present in Q0D the drain time was determined by the thermal velocities of the ions. It should be noted that there is no delay or indication of any issue with the ions draining out of Q0D which is believed to be due to the use of the rotated STI ion optic. As noted above, the rotated STI was employed to prevent ion trapping in the STI ion optic.

[0105] 30 Da Bandpass vs No Bandpass

[0106] In these experiments, the Q0D segment was 55 mm long with a field radius of 4.17 mm and operated at a drive frequency of 1MHz and an RF level corresponding to Mathieu parameter q = 0.64. An estimate of the trapping capacity of the Q0D segment can be obtainedfrom knowledge of the number of ions that are introduced into the QOD trap.

[0107] FIG. 14 shows a plot of the integrated signal for the MRM transition 609 / 609 at CE = 10 eV. With a 30 Da bandpass, the signal begins to deviate from linearity at a fill time ~ 100 ms, whereas with no bandpass the signal deviates from linearity at ~ 50 ms. This difference is believed to be due to the presence of space charge. An estimate of the total number of ions in QOD was obtained from the peak intensities of the ions in the QI scans with and without the bandpass applied while the RF amplitude was held constant on the Q0 ion optic at the level required for the bandpass (FIGS. 15A and 15B). With the 30 D bandpass, QOD is filled with ions in a narrow mass range while with no bandpass the mass range can span several hundred Da.

[0108] The estimated number of ions versus fill time is shown in FIGS. 16 and 17. With the 30Da bandpass the number of ions in QOD at a 100 ms fill is ~ 2 *106ions (0.017 pmol / pl reserpine) while with no bandpass the number of ions is calculated to be ~ 8 106ions at a 50 ms fill time. The data indicates that the number of ions that the QOD trap can hold is on the order of 106ions. There will be some variation of this number as the trapping parameters will vary with the mass to be trapped.

[0109] Fill Time Linearity and Signal to Noise Gains

[0110] Experiments were conducted to examine the fill linearity of various embodiments of the present teachings by monitoring the MRM 609 / 195 transition. The signal-to-noise ratio was also measured. A drain time of 20 ms was used together with Q2 CE = 43 eV. The linearity was maintained across the entire fill range for the blank sample as well as samples having concentrations of 0.00017 pmol / pl, and 0.0017 pmol / pl. A slight deviation from linearity was observed for the sample having a concentration of 0.017 pmol / pl at the highest fill time. The results of the fill time experiments at Q0 CE = 4 eV and 25 eV are presented in FIGS. 18A, 18B, 19A, 19B, 19C, 20A, 20B, 20C, 21A, 21B and 21C. More specifically, FIG. 18A shows the ion intensity of a blank sample as a function of fill time for Q0 CE = 4 eV and Q0 CE = 25 eV, and FIG. 18B presents a portion of the data depicted in FIG. 18A in a fill time range of 0 to 30 ms.

[0111] FIG. 19A shows the ion intensity as a function of fill time for a reserpine sample with a concentration of 0.00017 pmol / pl and FIG. 19B depicts a portion of the data presented in FIG. 19A in a fill time region extending from 0 to 30 ms. FIG. 19C presents the signal-to-noise ratioof the reserpine sample at the concentration of 0.00017 pmol / pl as a function of the fill time for Q0 CE = 4 eV and Q0 CE = 25 eV. By way of further illustration, FIG. 20A shows the ion intensity as a function of fill time for a reserpine sample with a concentration of 0.0017 pmol / pl as a function of fill time and FIG. 20B depicts a portion of the data presented in FIG. 20A in a fill time region extending from 0 to 30 ms. FIG. 20C presents the signal-to-noise ratio of the reserpine sample at the concentration of 0.0017 pmol / pl as a function of the fill time for Q0 CE = 4 eV and Q0 CE = 25 eV. Finally, FIG. 21A shows the ion intensity as a function of fill time for a reserpine sample at a concentration of 0.017 pmol / pl and FIG. 21B depicts a portion of the data presented in FIG. 21A in a fill time range extending from 0 to 30 ms, and FIG. 21C presents signal-to-noise of the reserpine sample as a function of fill time at the concentration of 0.017 pmol / pl.

[0112] The signal-to-noise ratio plots show that the signal-to-noise ratio is relatively constant as a function of fill time. For a Q0 CE = 25 eV relative to a Q0 CE = 4 eV, an improvement of about a factor of 1.4 was observed. The signal-to-noise ratio plots also include data corresponding to a conventional MRM experiment performed at a Q0 CE = 4 eV with a 30-Da bandpass on Q0C, which shows that trapping of the ions has led to an improvement of the signal-to-noise ratio by allowing more time for the chemical noise ions to dissociate than would be available in the normal flow through operational mode. As seen in FIG. 22, this improvement is about a factor of two. Without being limited to a particular theory, it is believed that the modest improvement in the signal-to-noise ratios relative to the conventional MRM experiment and at a higher Q0 CE is due to a low concentration of chemical noise ions in close proximity to the monitored transition.

[0113] Quantitation

[0114] The linearity of the ion intensity as a function of the fill time, which can be observed in the data presented in FIGS. 18A, 18B, 19A, 19B, 20A, 20B, as well as 21A and 21B, suggests that the Q0D trap can be filled quantitatively. As a check, the ratio of the intensity of the normal MRM (i.e., MRM observed in a flow-through mode) at a 20 ms dwell time to the intensity of the Q0 trapping (i.e., via the Q0C trap in these experiments) was calculated and is presented in Table 1 below.Table 1

[0115] FIG. 22 presents data that provides a comparison of the signal-to-noise ratio for samples of reserpine at different concentrations relative to a blank sample. The data shows that a similar gain in signal-to-noise ratio is achieved at different concentrations of reserpine.

[0116] It should be noted that the collision energy (CE) used for normal MRM was 38 eV and the CE used for trapping MRM was 43 eV. The choice of a lower collision energy for the normal MRM was because of the fast transit time of ions through the Q0 ion optic causes some heating of the ions, whereas the ions trapped in the QOD ion trap are cooler internally and hence would require a higher collision energy to achieve an optimal collision induced fragmentation. The last column of the above table shows that the ratio of the normal MRM intensity to Q0 trapping MRM intensity is about 0.9 for all three sample concentrations, which is close to one and hence can be interpreted that the QOD trap was filled with about the same number of ions that were observed with the normal MRM at a dwell time of 20 ms. Further, the consistency across the different concentrations indicates that there were no losses due to trapping when the number of ions was varied over the concentration range. In other words, the data shows that the present teachings can be implemented in a quantitative manner.

[0117] 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 asAlthough some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent description of a corresponding block or item or feature of a corresponding apparatus. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus.

[0118] Depending on certain implementation requirements, embodiments of the invention can be implemented in hardware and / or in software. The implementation can be performed using a non-transitory storage medium such as a digital storage medium, for example a floppy disc, 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.

[0119] 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.

[0120] 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.

[0121] 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.

Claims

Claims1. A method of performing mass spectrometry, the method comprising: introducing a plurality of ions, including one or more analyte ions and one or more chemical noise ions, into a first mass filter to select ions having m / z ratios within a specific range for passage through the first mass filter; accelerating the selected ions; introducing the accelerated ions into an ion trap containing a background gas for causing preferential collision induced dissociation (CID) of chemical noise ions passing through the first mass filter while substantially retaining the one or more analyte ions undissociated; allowing at least a portion of ions to exit the ion trap; and introducing at least a portion of the ions exiting the ion trap into a second mass filter configured to allow passage of the undissociated analyte ions.

2. The method of Claim 1 , wherein the ions exiting the ion trap comprise undissociated analyte ions, fragment ions generated via dissociation of the analyte ions, undissociated chemical noise ions, and fragment ions generated via dissociation of the chemical noise ions.

3. The method of Claim 1 or Claim 2, wherein said first mass filter is configured to block passage of chemical noise ions having m / z ratios that are separated from an m / z ratio of the analyte ion by at least a specific m / z value.

4. The method of Claim 3, wherein the specific m / z value is in a range of about + / -5 Da to about + / - 15 Da.

5. The method of any one of Claims 1-2 and 4, further comprising causing dissociation of the analyte ions passing through the second mass filter to generate a plurality of analyte product ions.

6. The method of Claim 5, further comprising determining a mass spectrum of said analyte product ions by introducing said analyte product ions to a third mass filter and utilizing a detector downstream of the third mass filter to monitor an intensity of product ions passing through the third mass filter.

7. The method of Claim 6, wherein the utilized detector is selected from the group consisting of a channeltron ion detector, a discrete dynode detector, a multi-channel plate detector, and an optical detector.

8. The method of any one of Claims 1-2, 4, and 6-7, wherein said first mass filter and said ion trap are disposed in a common first chamber.

9. The method of Claim 8, wherein said second mass filter is disposed in a second chamber positioned downstream from said common first chamber.

10. The method of Claim 1, further comprising applying a DC offset voltage between said first mass filter and said ion trap for accelerating the selected ions.

11. The method of Claim 10, wherein said DC offset voltage is selected such that said accelerated ions acquire a kinetic energy at which the chemical noise ions are more likely to dissociate than the analyte ions.

12. The method of Claim 10, wherein said first mass filter comprises a first plurality of rods arranged in a multipole configuration.

13. The method of Claim 12, wherein said ion trap comprises a second plurality of rods arranged in a multipole configuration.

14. The method of Claim 13, wherein said DC offset voltage is applied between at least one of said first plurality of rods and at least one of said second plurality of rods.

15. The method of Claim 14, wherein said DC offset voltage is in a range of about 2 eV to about 200 eV .

16. The method of any one of Claims 12-15, further comprising applying an RF voltage and a DC resolving voltage to said first plurality of multipole rods of the first mass filter togenerate a bandpass filter.

17. The method of Claim 16, wherein said RF voltage and said DC resolving voltage are selected such that the bandpass filter exhibits a bandpass in a range of about 10 Da to about 30 Da.

18. The method of any one of Claims 1-2, 4, 6-7, and 9-15, further comprising maintaining a pressure within said ion trap in the range of about 1 mTorr to about 30 mTorr.

19. The method of Claim 1-2, 4, 6-7, and 9-15, further comprising applying a DC gating voltage between the ion trap and a downstream component of the mass spectrometer.

20. The method of Claim 19, further comprising modulating the DC gating voltage between a trapping voltage at which ions are trapped within the ion trap and a release voltage at which ions can exit the ion trap to enter the downstream component.

21. The method of Claim 20, further comprising temporarily ceasing for a specific time interval the step of introducing the plurality of ions into the first mass filter after completion of introduction of a batch of ions into the first mass filter.

22. The method of Claim 21, wherein said specific time interval is selected so as to allow at least a portion of the batch of ions introduced in to the first mass filter to undergo cooling and dissociation.

23. The method of Claim 21, wherein said specific time interval is in a range of about 5 ms to about 30 ms.

24. The method of Claim 20, further comprising applying the trapping voltage for a temporal duration in a range of about 3 ms to about 200 ms.

25. The method of Claim 24, further comprising applying the release voltage for a temporal duration in a range of about 5 ms to about 20 ms.

26. The method of Claim 20, wherein said DC offset voltage is configured to inhibit return of ions from the ion trap into the first mass filter.

27. The method of ant one of Claims 1-2, 4, 6-7, and 9-15, further comprising maintaining apressure within said ion trap in the range of about 1 mTorr to about 30 mTorr.

28. A mass spectrometer, comprising: a first mass filter configured to receive a plurality of ions, wherein said plurality of ions include one or more analyte ions and one or more chemical noise ions, said mass filter being configured to select ions having m / z ratios within a specific range for passage through the mass filter; an ion trap positioned downstream of said first mass filter and configured to receive ions passing through the first mass filter, said ion trap containing a background gas; at least one DC offset voltage source configured to apply a DC offset voltage between the first mass filter and the ion trap for accelerating the ions passing through the first mass filter prior to entry thereof into the ion trap so as to cause preferential dissociation of said chemical noise ions while substantially retaining the analyte ions undissociated; and a second mass filter positioned downstream of the first mass filter and configured to allow passage of the undissociated analyte ions.

29. The mass spectrometer of Claim 28, further comprising one or more controllers for controlling operation of any of said first mass filter, said ion trap, said at least one DC offset voltage source, and said second mass filter.

30. The mass spectrometer of Claim 28, wherein said first mass filter is configured to block passage of chemical noise ions having m / z ratios that are separated from an m / z ratio of the analyte ion by at least a specific m / z value.

31. The mass spectrometer of claim 30, wherein the specific m / z value is in a range of about + / - 5 Da to about + / - 15 Da.

32. The mass spectrometer of any one of Claims 28-31, further comprising a dissociation cell positioned downstream of the second mass filter for causing dissociation of the analyte ions passing through the second mass filter.

33. The mass spectrometer of Claim 32, wherein said dissociation cell comprises a collision cell in which at least a portion of the analyte ions undergo collision induced dissociation.

34. The mass spectrometer of any one of Claims 28-31, wherein said first mass filter and said ion trap are disposed in a common first chamber.

35. The mass spectrometer of any one of Claims 28-31, wherein said first mass filter and said ion trap are disposed in two separate chambers.

36. The mass spectrometer of Claim 35, wherein said two separate chambers are maintained at different pressures.

37. The mass spectrometer of Claim 34, wherein said second mass filter is disposed in a second chamber positioned downstream from said common first chamber.

38. The mass spectrometer of any one of Claims 28-31, wherein said first mass filter comprises a first plurality of rods arranged in a multipole configuration.

39. The mass spectrometer of Claim 38, wherein said ion trap comprises a second plurality of rods arranged in a multipole configuration.

40. The mass spectrometer of Claim 39, wherein said DC offset voltage is applied between at least one of said first plurality of rods of the first mass filter and at least one of said second plurality of rods of the ion trap.

41. The mass spectrometer of Claim 40, wherein said DC offset voltage is in a range of about 2eV to about 200 eV.

42. The mass spectrometer of Claim 41, further comprising an RF voltage source and a DC voltage source operating under control of said one or more controllers and configured to apply, respectively, an RF voltage and a DC resolving voltage to said first plurality of multipole rods of the first mass filter to generate a bandpass filter.

43. The mass spectrometer of Claim 42, wherein said bandpass filter provides a bandpass in a range of about 10 Da to about 30 Da.

44. The mass spectrometer of any one of Claims 28-31, wherein said ion trap is maintained ata pressure in the range of about 1 mTorr to about 30 mTorr.

45. The mass spectrometer of Claim 29, further comprising a DC gating voltage source operating under control of said one or more controllers and configured to apply a DC gating voltage between the ion trap and a downstream component of the mass spectrometer.

46. The mass spectrometer of Claim 45, wherein said one or more controllers are configured to apply one or more control signals to said DC gating voltage source for modulating said DC gating voltage between an ion trapping voltage at which ions remain trapped within the ion trap and an ion release voltage at which ions within the ion trap can exit the ion trap.

47. The mass spectrometer of Claim 46, wherein said one or more control signals applied to the DC gating voltage source instruct the DC gating voltage source to apply said gating voltage for a temporal period in a range of about 3 ms to about 200 ms.

48. The mass spectrometer of Claim 47, wherein said one or more control signals applied to the DC gating voltage source instruct the DC gating voltage source to apply said release voltage for a temporal period in a range of about 5 ms to about 20 ms.

49. The mass spectrometer of any one of Claims 29-31 and 45-48, further comprising an ion lens positioned upstream of said first mass filter for directing the plurality of the ions into the first mass filter.

50. The mass spectrometer of Claim 49, further comprising a DC voltage source for applying a DC voltage to the ion lens.

51. The mass spectrometer of Claim 50, wherein said one or more controllers apply control signals to said DC voltage source to temporally modulate said applied DC voltage so as to allow and inhibit transmission of the plurality of ions into the first mass filter during alternating temporal periods, respectively.

52. The mass spectrometer of Claim 51, wherein a time interval between two successive temporal periods is in a range of about 5 ms to about 30 ms.

3. The mass spectrometer of any one of Claims 28-31 and 45-48, wherein said DC offset voltage is configured to inhibit return of the ions within the ion trap back to the first mass filter.

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