Calibration of the mass spectrometry system

Abstract

The present invention provides a system for analyzing a biological sample, the system comprising: a separation unit configured to separate components from the biological sample; an ionization unit configured to generate a plurality of ions from the components; an adjustable mass selection filter element; a detector configured to detect ions passing through the mass selection filter element; and a controller connected to the mass selection filter element and connected to the detector, wherein the controller is configured such that during operation of the system, the controller adjusts the mass selection filter element and activates the detector to measure at least three different ion signals corresponding to the plurality of ions, and determines a mass axis offset of the system based on the at least three different ion signals.

Description

Technical Field

[0001] The present disclosure relates to a mass spectrometry system. Background Art

[0002] Relative to some other analytical methods, mass spectrometry (MS) systems are widely used in the analysis of biological samples due to their high resolution and the ability to analyze relatively small sample volumes. As part of an analytical workflow, an MS system can be coupled to a liquid chromatography (LC) separation system. A complex sample such as a body fluid can be injected into the LC separation system and separated into sequentially eluted components, which are then analyzed on the MS system. The combination of LC separation and MS-based selective analysis enables quantitative analysis of different samples. Summary of the Invention

[0003] Multiple methods can be used to calibrate a mass spectrometry system. Before analyzing a sample, such systems typically undergo an initial calibration to ensure that the measured mass-to-charge ratio (m / z) is consistent with known values. If such a system is kept in use for a relatively long period of time, the initial calibration can drift due to factors such as temperature fluctuations. Continuing to use the system without recalibration can result in inaccurate ion m / z measurements. Since such measurements are typically used to identify analytes, it can lead to false or abnormal identifications.

[0004] Calibration of a mass spectrometry system can be performed by introducing a "standard" (or reference) sample and measuring the ion fragmentation pattern generated by the standard sample. However, for an MS system coupled to an LC column, introducing a standard sample can involve: disconnecting the column from the MS system to introduce the standard sample and putting the MS system into an "offline" mode. Depending on the nature of the standard sample, ionization parameters and other process parameters may also need to be adjusted. For an MS system that is used continuously or nearly continuously for analyzing samples, the downtime associated with modifying the instrument configuration and recalibrating the system results in reduced utilization, which has a negative impact on the overall sample measurement throughput.

[0005] The present disclosure features systems and methods for implementing an online calibration procedure that can be used for LC-MS systems. The mass spectrometer is not disconnected from the chromatography system or otherwise taken offline for calibration, so calibration can be performed quickly and accurately, and the calibrated system can resume use after a very short time interval. Since calibration does not involve disconnecting the LC and MS systems, a technician can easily perform the calibration, or even perform it in a fully automated manner, without significant mechanical intervention and instrument reconfiguration. In particular, for instruments operating in a clinical environment, performing calibration without such intervention can be highly desirable.

[0006] In one aspect, the present disclosure features a system for analyzing a biological sample, the system comprising: a separation unit configured to separate components from the biological sample; an ionization unit configured to generate a plurality of ions from the components; an adjustable mass selection filter element; a detector configured to detect ions passing through the mass selection filter element; and a controller connected to the mass selection filter element and the detector, wherein the controller is configured such that during operation of the system, the controller adjusts the mass selection filter element and activates the detector to measure at least three different ion signals corresponding to the plurality of ions, and determines a mass axis offset of the system based on the at least three different ion signals.

[0007] In another aspect, the present disclosure features a system for analyzing a biological sample, the system comprising: a separation unit configured to separate components from the biological sample; an ionization unit configured to generate a plurality of ions from the components; an adjustable mass selection filter element; a detector configured to detect ions passing through the mass selection filter element; and a controller connected to the mass selection filter element and the detector. The controller is configured such that during operation of the system, the controller is configured to: adjust the mass selection filter element to allow ions having a first mass-to-charge ratio q to pass through the mass selection filter element; activate the detector to measure a first ion signal corresponding to a common ion type among the plurality of ions; adjust the mass selection filter element to allow ions having a second mass-to-charge ratio q a <q to pass through the mass selection filter element; activate the detector to measure a second ion signal corresponding to the common ion type; adjust the mass selection filter element to allow ions having a third mass-to-charge ratio q b >q to pass through the mass selection filter element; activate the detector to measure a third ion signal corresponding to the common ion type; determine the intensity maxima of each of the first, second, and third ion signals, and fit the intensity maxima to a functional form that includes a local maximum within a mass-to-charge ratio range from q a to q b ; and determine a mass axis offset of the system based on an offset of the local maximum relative to the intensity maximum of the first ion signal, where (q - q a ) is 0.4 atomic mass units (amu) or less and (q b - q) is 0.4 atomic mass units (amu) or less.

[0008] Embodiments of any of these systems may include any one or more of the following features.

[0009] Each of the three different ion signals may correspond to a different mass-to-charge ratio of the mass selection filter element. The mass selection filter element may be configured such that ions corresponding to the mass-to-charge ratio pass through the mass selection filter element. The controller may be configured such that during operation of the system, the controller adjusts the mass selection filter element by adjusting one or more electric potentials applied to the electrodes of the mass selection filter element. The mass selection filter element may include a quadrupole electrode assembly.

[0010] Each of at least three different ion signals may correspond to a common ion type among a plurality of ions. The common ion type may have an associated mass-to-charge value q, and the controller may be configured to activate the detector to measure a first ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q - a) < q to pass through. The value a may be 0.4 atomic mass units (amu) or less (e.g., 0.2 amu or less). The controller may be configured such that during operation of the system, the controller activates the detector to measure a second ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q + b) > q to pass through. The value b may be 0.4 amu or less (e.g., 0.2 amu or less). The controller may be configured such that during operation of the system, the controller activates the detector to measure a third ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of q to pass through.

[0011] The controller may be configured such that during operation of the system, the controller determines a mass axis offset based on property values of the at least three different ion signals. The property may include the peak intensity of each of the at least three different ion signals and / or the area under each of the at least three different ion signals and / or the peak width of each of the at least three different ion signals and / or the magnitude of the derivative signal of each of the at least three different ion signals.

[0012] The controller may be configured such that during operation of the system, the controller fits a functional form to the property values, determines a local maximum of the functional form, and determines the mass axis offset based on the local maximum of the functional form. The functional form may correspond to a Gaussian function or a polynomial function.

[0013] The controller may be configured such that during operation of the system, the controller determines the mass axis offset by determining a mass offset associated with the local maximum of the functional form. The common ion type may have an associated mass-to-charge value q, and the controller may be configured such that during operation of the system, the controller determines the mass offset associated with the local maximum of the functional form relative to the mass-to-charge value q. The mass offset associated with the local maximum of the functional form may correspond to the mass axis offset.

[0014] The controller can be configured such that during operation of the system, the controller adjusts the mass axis calibration for the mass selection filter element based on the mass axis offset. At least three different ion signals can include five or more (e.g., seven or more) different ion signals.

[0015] A common ion type can have an associated mass-to-charge value q, and the controller can be configured to activate the detector to measure n different ion signals out of at least three different ion signals, where each of the n different ion signals is measured using a mass selection filter element adjusted by the controller to allow ions with different mass-to-charge ratios such that (q - a n ) < q to pass through, where n is 2 or greater (e.g., where n is 3 or greater).

[0016] The controller can be configured to activate the detector to measure m different ion signals out of at least three different ion signals, where each of the m different ion signals is measured using a mass selection filter element adjusted by the controller to allow ions with different mass-to-charge ratios such that (q + b m ) > q to pass through, where m is 2 or greater (e.g., where m is 3 or greater). The values of n and m can be different.

[0017] The controller can be configured such that during operation of the system, the controller periodically determines a new mass axis offset value for the system and adjusts the mass axis calibration for the mass selection filter element based on the new mass axis offset value.

[0018] The system can include a temperature sensor configured to measure the temperature of components of the system or the temperature of the environment of the system, where the controller is configured such that during operation of the system, if the measured temperature is outside a selected temperature range, the controller determines a new mass axis offset value for the system and adjusts the mass axis calibration for the mass selection filter element based on the new mass axis offset value.

[0019] The controller can be configured such that during operation of the system, the controller determines the value of an attribute of at least one ion signal measured by the detector and corresponding to a biological sample, and if the attribute value is outside a selected range of values for the attribute, the controller determines a new mass axis offset value for the system and adjusts the mass axis calibration for the mass selection filter element based on the new mass axis offset value. The attribute can correspond to a member selected from the group consisting of the peak intensity of the ion signal, the width of the ion signal, the area under the ion signal, and a value obtained from the derivative signal of the ion signal.

[0020] The mass selection filter element can be a first mass selection filter element, at least three different ion signals can be a first set of at least three different ion signals, and the mass axis offset of the system can be associated with the first set of mass selection filter elements, and the system can include a second mass selection filter element located downstream of the first mass selection filter element. The controller can be connected to the second mass selection filter element and configured such that during operation of the system, the controller adjusts the second mass selection filter element and activates the detector to measure a second set of at least two different ion signals corresponding to a plurality of ions, and determines the mass axis offset of the system associated with the second mass selection filter element based on the second set of at least two different ion signals. The controller can be configured such that during operation of the system, the controller adjusts the mass axis calibration for the second mass selection filter element based on the mass axis offset associated with the second mass selection filter element.

[0021] The components of the sample can be a first component of a biological sample, the plurality of ions can be a first plurality of ions, and the mass axis offset is a first mass axis offset associated with the first component. The separation unit can be configured to separate a second component from the biological sample, the ionization unit can be configured to generate a second plurality of ions from the second component, and the controller can be configured such that during operation of the system, the controller adjusts the mass selection filter element and activates the detector to measure at least three different ion signals corresponding to the second plurality of ions, and determines a second mass axis offset of the system associated with the second component based on the at least three different ion signals corresponding to the second plurality of ions. The first and second components can be different. The controller can be configured such that during operation of the system, the controller determines the overall mass axis offset of the system based on the first and second mass axis offsets. The controller can be configured such that during operation of the system, the controller determines the overall mass axis offset of the system by averaging the first and second mass axis offsets.

[0022] The separation unit can include at least one chromatographic column. The separation unit can separate components from the biological sample by liquid chromatography.

[0023] Embodiments of the system can also include any other features described herein, and can include any combination of features connecting the same or different embodiments, unless otherwise explicitly stated.

[0024] In another aspect, the present disclosure features a method for determining a mass axis offset of a system for analyzing a biological sample, the method comprising separating components from the biological sample, generating a plurality of ions from the components, adjusting a mass selection filter element of the system, and measuring at least three different ion signals, where each ion signal corresponds to a common ion type among the plurality of ions and a different mass-to-charge ratio of ions passing through the mass selection filter element, and determining the mass axis offset of the system based on the at least three different ion signals, wherein the difference between any two mass-to-charge ratios corresponding to the ion signals is less than 0.5 atomic mass units (amu).

[0025] Embodiments of the method may include any one or more of the following features.

[0026] Each of the three different ion signals may correspond to a different mass-to-charge ratio of the mass selection filter element. The mass selection filter element may be configured such that ions corresponding to the mass-to-charge ratio pass through the mass selection filter element. The method may include adjusting the mass selection filter element by adjusting one or more electric potentials applied to electrodes of the mass selection filter element.

[0027] Each of the at least three different ion signals may correspond to a common ion type among the plurality of ions. The common ion type may have an associated mass-to-charge value q, and the method may include measuring a first ion signal among the at least three different ion signals, where the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q - a) < q to pass through. The value of a may be 0.4 atomic mass units (amu) or less (e.g., 0.2 amu or less).

[0028] The method may include measuring a second ion signal among the at least three different ion signals, where the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q + b) > q to pass through. The value of b may be 0.4 amu or less (e.g., 0.2 amu or less).

[0029] The method may include measuring a third ion signal among the at least three different ion signals, where the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of q to pass through.

[0030] The method may include determining the mass axis offset based on attribute values of the at least three different ion signals. The attribute may include the peak intensity of each of one or more of the at least three different ion signals; the area under each of the at least three different ion signals; the peak width of each of the at least three different ion signals; the magnitude of the derivative signal of each of the at least three different ion signals.

[0031] The method may include fitting a functional form to the attribute values, determining local maxima of the functional form, and determining a mass axis offset based on the local maxima of the functional form. The functional form may correspond to a Gaussian function. The functional form may correspond to a polynomial function.

[0032] The method may include determining a mass axis offset by determining a mass offset associated with a local maximum of the functional form. A common ion type may have an associated mass-to-charge value q, and the method may include determining a mass offset associated with the local maximum of the functional form relative to the mass-to-charge value q.

[0033] The mass offset associated with the local maximum of the functional form may correspond to the mass axis offset. The method may include adjusting the mass axis calibration for a mass selection filter element based on the mass axis offset.

[0034] At least three different ion signals may include five or more (e.g., seven or more) different ion signals.

[0035] A common ion type may have an associated mass-to-charge value q, and the method may include measuring n different ion signals out of at least three different ion signals, each of the n different ion signals being measured using an adjusted mass selection filter element, where the mass selection filter element is adjusted to pass ions having different mass-to-charge ratios such that (q - a n ) < q, where n is 2 or greater (e.g., 3 or greater). The method may include measuring m different ion signals out of at least three different ion signals, each of the m different ion signals being measured using a mass selection filter element adjusted by a controller, where the mass selection filter element is adjusted to pass ions having different mass-to-charge ratios such that (q + b m ) > q, where m is 2 or greater (e.g., 3 or greater). The values of n and m may be different.

[0036] The method may include periodically determining a new mass axis offset value and adjusting the mass axis calibration for a mass selection filter element based on the new mass axis offset value.

[0037] The method may include measuring the temperature of a component of the system or the temperature of the environment of the system, and if the measured temperature is outside a selected temperature range, determining a new mass axis offset value and adjusting the mass axis calibration for a mass selection filter element based on the new mass axis offset value.

[0038] The method may include determining a value of an attribute corresponding to at least one ion signal of a biological sample, and if the attribute value is outside a selected range of attribute values, determining a new mass axis offset value and adjusting a mass axis calibration for a mass selection filter element based on the new mass axis offset value. The attribute may correspond to a member selected from the group consisting of peak intensity of an ion signal, width of an ion signal, area under an ion signal, and a value obtained from a derivative signal of an ion signal.

[0039] The mass selection filter element may be a first mass selection filter element, at least three different ion signals may be a first group of at least three different ion signals, and the mass axis offset may be associated with the first mass selection filter element, and the method may include adjusting a second mass selection filter element of the system located downstream of the first mass selection filter element, measuring a second group of at least two different ion signals corresponding to a plurality of ions, and determining a mass axis offset associated with the second mass selection filter element based on the second group of at least two different ion signals. The method may include adjusting a mass axis calibration for the second mass selection filter element based on the mass axis offset associated with the second mass selection filter element.

[0040] A component of the sample may be a first component of the biological sample, the plurality of ions may be a first plurality of ions, and the mass axis offset may be a first mass axis offset associated with the first component, and the method may include separating a second component from the biological sample, generating a second plurality of ions from the second component, adjusting the mass selection filter element and measuring at least three different ion signals corresponding to the second plurality of ions, and determining a second mass axis offset associated with the second component based on the at least three different ion signals corresponding to the second plurality of ions. The first and second components may be different.

[0041] The method may include determining an overall mass axis offset based on the first and second mass axis offsets. The method may include determining an overall mass axis offset by averaging the first and second mass axis offsets.

[0042] The method may include separating components from the biological sample by liquid chromatography.

[0043] Embodiments of the method may also include any other features described herein and may include any combination of features connecting the same or different embodiments, unless otherwise explicitly stated.

[0044] As used herein, the term "about" means "approximately" (e.g., indicating plus or minus 10% of a value).

[0045] References to "one embodiment", "an embodiment", "example embodiment", etc. in the specification mean that the described embodiment may include specific aspects, features, structures, or characteristics, but each embodiment does not necessarily include the specific aspects, features, structures, or characteristics. Additionally, such phrases may, but do not necessarily, refer to the same embodiment mentioned elsewhere in the specification. Further, when connecting embodiments to describe specific aspects, features, structures, or characteristics, it should be noted that whether explicitly stated or not, such aspects, features, structures, or characteristics are within the knowledge of those skilled in the art and may be related or connected to other embodiments.

[0046] Unless otherwise explicitly specified, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "mass shift" includes a plurality of such mass shifts.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or current to those described herein may be used in the practice or testing of the present invention, suitable methods and systems are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a schematic diagram showing an example of a liquid chromatography - mass spectrometry system.

[0049] Figure 2 is a schematic diagram showing the use of Figure 1 of the system for an example of a sample analysis workflow.

[0050] Figure 3 is a schematic diagram showing measured ion peaks and detection windows.

[0051] Figure 4 is a schematic diagram showing ion peaks corresponding to three different mass shift values.

[0052] Figure 5 is a schematic diagram showing fitting the measured ion peak intensities to a functional form and the mass axis shift relative to the nominal zero mass shift.

[0053] Like reference symbols denote like elements. DETAILED DESCRIPTION

[0054] Introduction

[0055] A liquid chromatography - mass spectrometry (LC - MS) system is used to analyze various biological samples. Such systems implement an end - to - end workflow in which a sample (e.g., a body fluid such as blood, urine, etc.) is injected into the inlet of a liquid chromatography column, the sample is separated into components on the column, and the individual components are eluted from the column. The eluted components are directed into a mass spectrometer where they are ionized and analyzed. The mass spectrometer measures the ion fragmentation patterns associated with each component. Each ion fragmentation pattern consists of one or more peaks that correspond to ion fragments with specific m / z ratios. The peak pattern (e.g., the m / z ratios and intensities of the peaks) of a particular analyte effectively serves as a “fingerprint” of the analyte.

[0056] Due to the complexity of the fragmentation patterns, a wide variety of components can be identified and quantified based on these measurements. Typically, identification is performed by comparing the measured ion fragmentation patterns with reference information (e.g., previous measurements of known components or simulated ion fragmentation patterns). Identification of specific components can also be performed based on the time interval between the initial introduction of the sample (e.g., injection into the inlet of the LC - MS system) and the elution of the component from the LC column, or the time interval between the initial introduction of the sample and the measurement of the ion fragmentation pattern of the component in the mass spectrometer. Certain components migrate through the LC column at specific rates, and the elapsed time interval can be used as an indicator of the component's identity. Similar to the ion fragmentation patterns described above, the elapsed time interval can be compared with reference information (e.g., previous measurements of the migration and / or measurement times of known components) to determine the identity of the component.

[0057] To ensure accurate component identification and that the component population can be quantitatively measured, LC - MS systems are typically calibrated before use. Additionally, when such systems are used continuously or nearly continuously, such as in a clinical or laboratory setting, the system can be periodically recalibrated, and / or when drift in the system calibration is detected or suspected. Conventional recalibration procedures involve taking the systems offline so that they are no longer analyzing biological samples. Additionally, for many LC - MS systems, conventional calibration procedures can involve disconnecting the column from the mass spectrometer to introduce a reference sample, and in some cases, changing the configuration of the mass spectrometer to analyze the reference sample (e.g., changing the configuration from LC - MS to direct infusion). In other words, such procedures can involve reconfiguring a large number of intervening instruments, and this procedure is either performed by the user or deferred until a properly trained technician is available to perform the work. After calibration, the liquid chromatography column is reconnected to the mass spectrometer, and if necessary, the analytical configuration of the LC - MS system is adjusted to analyze biological samples.

[0058] For LC-MS systems deployed in clinical and laboratory settings, the level of user intervention in the aforementioned conventional calibration procedures can be highly disadvantageous. System users in these settings may have little training or experience with chromatography and / or mass spectrometry hardware and system configuration. Additionally, the time for offline calibration of the LC-MS system represents downtime - during which no samples are analyzed - and this downtime reduces the effective duty cycle and utilization of the system. In high-throughput environments where hundreds or thousands of samples are analyzed per day, such downtime can be a significant drawback.

[0059] The present disclosure features systems and methods for implementing an online calibration procedure during which the LC-MS system does not go offline. That is, the chromatographic column is not disconnected from the mass spectrometer. As a result, the calibration procedure can be performed more quickly than some conventional calibration procedures and with significantly less user intervention. In fact, some implementations may involve no user intervention at all and can be performed in a fully automated manner by the LC-MS system.

[0060] The systems and methods described herein can implement a calibration procedure that adjusts (e.g., optimizes) multiple calibration parameters in a single calibration procedure. Thus, for example, when the LC-MS system includes multiple ion filtering stages, each stage can be independently calibrated and adjusted in a single calibration procedure such that the system is fully calibrated at the end of the procedure. After calibration, the system can immediately return to the analysis of biological samples.

[0061] Conventional calibration procedures typically rely on dedicated calibration samples, such as materials containing polypropylene glycol polymers. Such materials can generate ion peaks at m / z values that are relatively close to the expected ion peaks from specific target sample components. However, in some cases, the ion peaks generated from the dedicated calibration samples may be relatively far from the expected ion peaks corresponding to the target sample components. In such cases, calibrating the system based on the dedicated calibration samples may result in a system where the calibration within the m / z window of interest remains questionable.

[0062] In contrast, the systems and methods described herein can be used with a variety of reference samples for system calibration, ensuring that calibration can always be performed within the m / z region corresponding to the sample components to be measured. In some embodiments, the reference samples for calibration are isotopically enriched or isotopically labeled versions of specific sample components. Examples of such reference samples include, but are not limited to, testosterone, gabapentin, and cyclosporine. More generally, any reference sample can be used, and the choice of reference sample can depend on the nature of the target sample components.

[0063] Isotopically labeled reference samples typically include isotopic substitution at one or more sites within the molecular structure of the sample molecule. Isotopes that can be used as labels (instead of their more common bulk counterparts) include, but are not limited to, carbon-13, deuterium, tritium, oxygen-18, and phosphorus, fluorine, chlorine, bromine, iodine, sulfur, and nitrogen. Isotopically labeled reference samples can typically substitute the molecular structure of the reference sample at one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, or even more) sites. In some embodiments, each site within the molecular structure of the reference sample corresponding to a certain type of atom can be isotopically substituted (e.g., each C atom can be substituted by 13 C, or each H atom can be substituted by 2 H or 3 H atoms).

[0064] The systems and methods described herein can be used with systems for measuring a variety of biological samples. Examples of such samples include, but are not limited to, blood, plasma, urine, saliva, lymphatic fluid, interstitial fluid, and cerebrospinal fluid.

[0065] Liquid Chromatography-Mass Spectrometry System

[0066] Figure 1 FIG. is a schematic diagram showing an example of a liquid chromatography-mass spectrometry (LC-MS) system 100. The system 100 includes an inlet 102 connected to a liquid chromatography column 104. The column 104 is coupled to a mass spectrometer 106 through an optional valve 122 connected to an optional waste container 124. The mass spectrometer 106 includes an ionizer 108, a skimmer 110, quadrupoles 112, 114, and 116, and a detector 118. Each component can optionally be connected to a controller 120, which typically includes at least one electronic processor, at least one storage unit, at least one display device, and at least one interface for receiving instructions and data from a user of the system 100.

[0067] During operation of the system 100, a sample is introduced into the inlet 102, e.g., by direct injection. After introduction, the sample enters the column 104 and deposits on the column material (e.g., resin material). As one or more solvents flow through the column material, the sample migrates through the column material. As the sample migrates collectively, different components of the sample migrate at different rates and thus reach the end of the column at different times. As described above, the elution time of a certain sample component can be specific to that component and can be used to identify the component (e.g., by comparing the elution time of the component with reference information including the elution times of known sample components).

[0068] The column 104 may optionally be connected to the valve 122 (as described above), which in turn may optionally be connected to the waste container 124. During operation of the system 100, the valve 122 may optionally be activated by the controller 120 to direct the eluent from the column 104 to the waste container 124, or to the mass spectrometer 106. Selectively directing only a portion of the eluent to the mass spectrometer 106 can ensure that only the target components in the sample are measured.

[0069] In some embodiments, to facilitate the selective direction of a portion of the eluent from the column 104 to the waste receiver 124 or to the mass spectrometer 106, the valve 122 may include a detector connected to the controller 120 that generates an electrical signal when a component of the sample elutes from the column 104 and reaches the detector. The controller 120 receives the electrical signal and may determine whether to direct the eluent into the waste container 124 or into the mass spectrometer 106. In certain embodiments, the controller 120 determines the direction of the eluent based on the time interval between the introduction of the sample at the inlet 102 and the detection of the component emerging from the downstream end of the column 104. In some embodiments, the elapsed time may be compared to reference information including the elution times of known sample components to at least preliminarily identify the generated component. Based on this preliminary identification, the controller 120 may determine whether the component is a target component (and thus directed to the mass spectrometer 106), or whether the component is a non-target component (and directed to the waste container 124). When sample components do not elute from the column 104 (e.g., during a time interval when only eluent solvent flows out of the column 104), the eluent may also optionally be directed to the waste container 124 instead of the mass spectrometer 106.

[0070] Various detectors may be integrated into the valve 122, or more generally, be positioned between the column 104 and the mass spectrometer 106 to facilitate component detection when sample components elute from the column 104. Examples of suitable detectors include, but are not limited to, optical detectors such as photodiodes, photocells, spectroscopic detectors, and CCDs, and electrical detectors such as conductivity sensors and resistivity sensors.

[0071] The sample components entering the mass spectrometer 106 are received in the ionizer 108, where they are ionized to form an ion swarm. The ionizer 108 may be implemented as any one of a variety of different types of ionizers. Examples of suitable ionizers include, but are not limited to, electrospray ionizers, electron impact ionizers, atmospheric pressure chemical ionizers, thermospray ionizers, inductively coupled plasma ionizers, glow discharge ionizers, and photoionizers.

[0072] The ion swarm generated in the ionizer 108 passes through the extractor 110, which typically includes apertures of reduced size (relative to the exit aperture of the ionizer 108), and the extractor 110 reduces the ion swarm that is directed to the quadrupoles of the mass spectrometer 106. After passing through the extractor 110, the ions are separated and detected in the remainder of the mass spectrometer 106.

[0073] A variety of different mass spectrometer configurations can be used to separate, detect, and analyze ions generated from sample components. The mass spectrometer 106 is one example of such a configuration and will be discussed in detail below for illustrative purposes. However, it should be understood that the calibration methods described herein can be used with many different configurations of the mass spectrometer 106 and are in no way limited to Figure 1 the configuration shown.

[0074] In Figure 1 this example, the mass spectrometer 106 is implemented as a tandem mass spectrometer (e.g., tandem MS / MS) having three quadrupoles 112, 114, and 116. In the first quadrupole 112 (also referred to herein as "Q1"), the ions passing through the extractor 110 are filtered to select ions falling within a specific m / z value range for further analysis. Ions falling outside of this m / z value range are blocked and do not pass through the quadrupole 112. Instead, ions having an m / z within the desired range pass through the quadrupole 112 and enter the second quadrupole 114.

[0075] Typically, the quadrupole 112 includes four electrodes arranged symmetrically about a central axis. To selectively direct only ions having an m / z value within the desired range to the second quadrupole 114, the controller 120 adjusts the electric potential applied to the four electrodes. With the appropriate electric potential applied, the four quadrupole electrodes generate an oscillating radio frequency (RF) field that serves to direct the ions along the quadrupole 112 from one end to the other. For a specific RF field, ions within a specific m / z value range are directed out of the exit aperture of the quadrupole 112, and ions having an m / z outside of this range are rejected (e.g., blocked) within the quadrupole 112.

[0076] A subset of the ions entering the first quadrupole 112 passes through the stage 112 and enters the second quadrupole 114 (also referred to herein as "Q2"). The second quadrupole 114 is implemented as a collision cell in which the ions entering the stage 114 are fragmented to form a distribution of ions of relatively lower molecular mass. This distribution of ions of lower mass (derived from the relatively larger mass ions typically entering the stage 114 from the stage 112) passes through the stage 114 and enters the third quadrupole 116.

[0077] Within the second quadrupole stage 114, the controller 120 applies an electric potential to one or more electrodes to generate one or more electric fields, establishing a field gradient between the inlet and outlet apertures of stage 114. Ions entering from stage 112 are typically accelerated by the field gradient. Atoms or molecules of the neutral gas are introduced into stage 114 and collide with the accelerated ions entering from stage 112, generating (by collision) ion fragments that pass through stage 116. A variety of gases can be used for the fragmentation process, including but not limited to hydrogen, nitrogen, and inert gases such as argon.

[0078] After the distribution of ions of smaller mass (referred to herein as "fragment ions") enters the third quadrupole stage 116 (also referred to herein as "Q3"), the fragment ions are filtered in a manner similar to the filtering that occurs in stage 112. Specifically, stage 116 includes four electrodes arranged around a central symmetry axis, and the controller 120 adjusts one or more electric potentials applied to the four electrodes to generate an oscillating RF field within stage 116. The generated field guides a subset of the ion fragments (each ion fragment having an m / z that falls within a specific range) from one end of stage 116 to the other end and into the detector 118. Ion fragments having an m / z value outside of this range are rejected (e.g., blocked) within the quadrupole stage 116.

[0079] After the subset of ion fragments guided out of the third quadrupole stage 116 enters the detector 118, the detector measures the m / z value of the fragments. Specifically, the detector 118 generates a measurement signal corresponding to the fragments and transmits it to the controller 120, which determines the m / z value of the fragments based on the measurement signal.

[0080] The detector 118 can incorporate a variety of different detection techniques. In certain embodiments, the detector 118 corresponds to an electron multiplier, a Faraday cup, or a microchannel plate detector. In some embodiments, the detector 118 is an orbitrap-based detector. More generally, the detector 118 can implement any one or more known ion detection techniques.

[0081] In Figure 2 is schematically shown the overall workflow implemented by the system 100. Two components of the sample, testosterone and 13 C-labeled testosterone (the internal reference component) elute from the column 104 at the same time. The components are ionized within the ionizer 108 to generate molecular ions of each component. The molecular ions are selectively filtered in the Q1 stage and transferred to the Q2 stage, where they undergo fragmentation to form ion fragments having a molecular weight less than that corresponding to the molecular ions. The fragment ions are filtered in the Q3 stage and transferred to the detector 118, where they generate a detection signal having a specific m / z value.

[0082] Mass Axis Calibration

[0083] Calibration system 100 enables the ion signals generated in detector 118 to be attributed to ions having a specific m / z value. This is referred to as "mass axis calibration" of system 100. Generally, the mass axis calibration of the system corresponds to the relationship between the physical configuration of the mass selection filter element in system 100 and the actual m / z values corresponding to different configuration settings. Thus, for example, for a mass selection filter element, one or more electric potentials are applied (e.g., by controller 120) to the electrodes of the element to selectively filter ions having a specific m / z value (or within an m / z range), and the relationship between the applied electrode potential and the filtered m / z value corresponding to the applied potential is the mass axis calibration.

[0084] As used herein, the term "mass selection filter element" refers to a component of a mass spectrometry system that allows only charged particles having a specific mass value or range of mass-to-charge ratios to pass through the element. Such an element is typically configurable, but not always, and thus the range of mass values or mass-to-charge ratios allowed to pass is adjustable. It should be noted that "pass through" refers to the fact that the mass selection filter element effectively acts as a "gate" or "barrier" for the flow of charged particles. A mass selection filter element can generally be implemented in a variety of forms, including configurations where charged particles enter through an input port and exit through an output (i.e., pass through the element), and configurations where charged particles enter and exit through a common port. A mass selection filter element can also be implemented in such a configuration where the element deflects charged particles having a mass value or mass-to-charge ratio within or outside a selected range of values, or more generally, uses any mechanism to restrict charged particles from reaching a specific location in the system to only those having a mass value or mass-to-charge ratio falling within a specific range of values.

[0085] In Figure 1 In the illustrated system 100, quadrupoles Q1 and Q3 are both mass selection filter elements. Since Q1 and Q3 each act as an m / z filter for ions, each pole affects the measurement signal generated in detector 118. That is, the mass axis calibration in system 100 is more complex than the relationship between a set of electric potentials or other configuration settings of a single mass selection filter element and the set of m / z values corresponding to the potentials or settings. Instead, the mass axis calibration for system 100 corresponds to the electric potentials or configuration settings of poles Q1 and Q3, and their corresponding m / z values.

[0086] Typically, before performing measurements using system 100, the system is calibrated to establish a relationship between the potential applied to the electrodes of poles Q1 and Q3 and the m / z values of the ions filtered by each pole. After calibration, each pole can be independently configured by controller 120 to filter (i.e., allow passage of) only ions having a specific m / z value by applying an appropriate potential to the electrodes of each pole according to the calibration relationship of the pole. However, after extended use of system 100 and / or with changes in environmental conditions, it has been observed that the mass axis calibration of system 100 can drift such that the m / z values determined for the ions measured by detector 118 no longer correspond to the actual m / z values of the ions.

[0087] Drift in the mass axis calibration for system 100 can have several important consequences. In some embodiments, if the drift is large enough, ions can be misidentified from the mass spectrometry information measured by system 100. In certain embodiments, drift in the mass axis calibration causes system 100 to measure ion peaks with increased widths. The increased peak width results in a decrease in resolution and can increase isotope interference in samples having components that are structurally similar but differ only in one or more isotope-labeled positions. The increased peak width can also result in a decrease in the measured ion peak intensity value, which can reduce the sensitivity of system 100, as well as incorrect peak area measurements, which can result in incorrect peak area ratio calculations when comparing target sample components to internal reference components.

[0088] In Figure 3 the chart schematically shown is the effect of mass axis calibration drift. In Figure 3 it, the measured intensity of ion peak 300 corresponding to a sample component is shown as a function of the mass offset along the mass axis relative to the nominal value of 0. The mass axis 0 value represents the center of the effective measurement “window” 302 of ion peak 300. In other words, ion peak 300 is measured by the system within window 302 (which corresponds to a very narrow m / z value range). Because the mass axis calibration of the system is not aligned with the intensity maximum of ion peak 300, ion peak 300 will be detected with the measurement window 302 shifted relative to ion peak 300 (i.e., centered at an m / z value different from that of ion peak 300), such that the integrated peak signal is significantly less than the peak signal that would be measured if the measurement window 302 were aligned with ion peak 300. Thus, quantitative measurements that rely on accurate peak intensity measurements can be affected.

[0089] It has been determined that a unit resolution of 0.7 is appropriate for the adequate separation of the isotope-substituted components, and a mass axis accuracy of ±0.1 amu should be maintained during operation of the system 100. To maintain these operating conditions, particularly when the system 100 is operated for long periods of continuous or near-continuous use and / or is subject to environmental conditions that may fluctuate (whether or not during operation of the system 100), the mass axis calibration should be corrected to account for drift in the system calibration.

[0090] The systems described herein are configured to monitor the mass axis calibration and correct the mass axis calibration as needed to ensure that the system produces accurate and reproducible mass spectral information of the sample components. In some embodiments, the system 100 verifies the mass axis calibration at regular time intervals and / or upon receipt of an instruction from a user of the system 100. In certain embodiments, the system 100 may include one or more sensors that measure environmental conditions, and the controller 120 initiates verification of the mass axis calibration based on the sensor measurements. For example, referring Figure 1 , the system 100 may optionally include a temperature sensor 126 connected to the controller 120. The temperature sensor 126 may be positioned to measure the ambient temperature of the environment surrounding the system 100. Alternatively, the temperature sensor 126 may be positioned to measure the temperature of one or more components of the system 100. If the temperature measured by the sensor 126 is outside a predetermined temperature range, the controller 120 may initiate verification of the mass axis calibration for the system 100.

[0091] In some embodiments, the system 100 verifies the mass axis calibration based on a comparison between parameters associated with the measured mass spectral information. For example, the controller 120 may determine the peak width associated with one or more sample components and compare the determined peak width to the peak width determined from similar sample components at different times. As an example, if the determined peak width has increased or decreased significantly (e.g., as determined by calculating a peak width ratio) in subsequent measurements, the controller 120 may initiate verification of the mass axis calibration of the system 100.

[0092] To verify the mass axis calibration of system 100, for each mass selection filter element of system 100, controller 120 measures the signal corresponding to an ion peak of a known m / z, where there are three different mass offsets for the mass selection filter element: a negative mass offset, a zero mass offset, and a positive mass offset. The mass offsets are respectively for the known m / z values of the measured ion peaks. Thus, for example, in the measurement of an ion peak with a known m / z value q, the ion peak at the negative mass offset is measured by adjusting the configuration of the mass selection filter element to allow ions with an m / z value of (q - a) to pass through, where a is the negative mass offset. To measure the ion peak at the zero mass offset, the configuration of the mass selection filter element is adjusted to allow ions with an m / z value of q to pass through. To measure the ion peak at the positive mass offset, the configuration of the mass selection filter element is adjusted to allow ions with an m / z value of (q + b) to pass through, where b is the positive mass offset.

[0093] Figure 4 is a schematic diagram showing the measured ion peaks corresponding to the negative mass offset -a (peak 402), the zero mass offset (peak 404), and the positive mass offset +b (peak 406). The peaks respectively have intensity maxima 402a, 404a, and 406a. After determining the intensity maxima, the intensity maximum values are fitted into a functional form having a local maximum within the mass offset range (-a, +b). The local maximum represents the mass axis offset, which is the deviation of the mass axis calibration from the actual m / z value of the measured ions.

[0094] Figure 5 is a schematic diagram showing the measured intensity maxima 402a, 404a, and 406a, which are plotted as a function of the mass offset. The intensity maximum values have been fitted into a functional form 502 having a local maximum 504 within the mass offset range defined by the maxima 402a, 404a, and 406b. The intensity maximum 404a corresponds to the measurement of the ion peak at the zero mass offset value. If system 100 is perfectly calibrated, the local maximum 504 of the functional form 502 will be the same as the maximum 404a. However, due to drift in the calibration of system 100, the local maximum 504 is no longer aligned with the maximum 404a, indicating that the mass axis calibration of system 100 should be adjusted to match the known m / z value of the ion peak.

[0095] Mass axis offset - the mass axis calibration should be adjusted to compensate for the amount of drift - is represented by the difference between the local maximum 504 and the mass axis offset of the maximum 404a. The controller 120 determines this value in the system 100 and then applies a correction to the calibration of the mass selection filter element of the system 100. For example, if the calibration information of the system 100 corresponds to a functional relationship (e.g., a calibration curve) between one or more applied potentials (or other configuration settings) and the corresponding filtered m / z values of the mass selection filter element, the controller 120 applies an appropriate shift to the functional relationship to account for the drift. In some embodiments, the controller 120 applies the correction to the m / z values initially used to determine the functional relationship and recalculates the functional relationship between one or more applied potentials (or other configuration settings) and the corresponding filtered m / z values.

[0096] In some embodiments, the magnitudes of the negative mass offset a and the positive mass offset b are the same. In certain embodiments, the magnitudes of a and b are different. For example, when the mass axis offset can occur in a biased manner, different offsets can be used. By selecting negative and positive mass offsets of different magnitudes, the local maximum 504 of the functional form 502 can be resolved more accurately, resulting in a more accurate measurement of the mass axis offset 506.

[0097] The magnitudes of the mass offsets a and b can generally be selected to ensure sufficient sampling of both negative and positive mass offsets. Thus, the magnitude of a and / or the magnitude of b can be 0.01 atomic mass units (amu) or greater (e.g., 0.03 amu or greater, 0.05 amu or greater, 0.07 amu or greater, 0.1 amu or greater, 0.12 amu or greater, 0.15 amu or greater, 0.2 amu or greater, 0.25 amu or greater, 0.3 amu or greater, 0.35 amu or greater, 0.4 amu or greater).

[0098] The detected ion peaks corresponding to a particular sample component and used to measure peak intensity are as Figure 4 and 5 shown, and as described above, generally, the ion peaks associated with any sample component can be used to measure peak intensity. However, it has also been found that in order to avoid detecting ion peaks corresponding to isotopically substituted counterparts of sample components, it can be advantageous if the magnitude of a and / or b is 0.2 amu or less (e.g., between 0.2 amu and 0.01 amu, between 0.15 amu and 0.01 amu, between 0.1 amu and 0.01 amu, between 0.05 amu and 0.01 amu).

[0099] In some embodiments, as Figure 4 and 5As described above, the ion peak intensity measurements are performed at three different mass offsets: -a, 0, and +b. However, more generally, to improve the accuracy of calculating the mass axis offset 506, the ion peak intensity measurements may be performed at more than three different mass offsets. For example, in some embodiments, the ion peak intensity measurements may be performed at a plurality of negative mass offsets, represented by the values (-a1)...(-a n )), where n is the number of negative mass offset values. Typically, n can be 1 or greater (e.g., 2 or greater, 3 or greater, 4 or greater, 5 or greater, 7 or greater, 10 or greater, or even greater).

[0100] Similarly, to improve the accuracy of calculating the mass axis offset 506, the ion peak intensity measurements may be performed at a plurality of positive mass offsets, represented by the values (+b1)…(+b m ), where m is the number of positive mass offset values. Typically, m can be 1 or greater (e.g., 2 or greater, 3 or greater, 4 or greater, 5 or greater, 7 or greater, 10 or greater, or even greater).

[0101] In some embodiments, the number of negative mass offset values n and the number of positive mass offset values m are the same. In certain embodiments, n and m are different. For example, if there is a bias in the mass axis offset, it may be advantageous for either n or m to be larger, depending on the bias direction. In other words, for a positive mass axis offset, it may be advantageous for m to be greater than n. Conversely, for a negative mass axis offset, it may be advantageous for n to be greater than m.

[0102] In certain embodiments, the total number of mass offset values at which peak intensity measurements are made and fitted to the functional form 502 is 3 or more (e.g., 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more, 12 or more, 15 or more, or even more). The total number of mass offset values can typically be even or odd.

[0103] In some embodiments, the peak intensity value corresponding to zero mass offset is not fitted to the functional form 502. In other words, Figure 4 the peak 404a in is not fitted to the functional form 502. However, the local maximum 504 of the functional form 502 is still determined in the same way, and the mass axis offset 506 can still be calculated from the difference in the mass offset of the local maximum 506 and the peak intensity 404a as described above.

[0104] In some embodiments, the functional form 502 is a Gaussian functional form. It has been found that by using a Gaussian functional form to represent the correlation between measured peak intensity and mass shift, a particularly accurate mass axis shift can be calculated based on the local maximum 504 of the Gaussian functional form. In certain embodiments, other functional forms having a local maximum within the mass shift interval (-a, +b) can be used. For example, a parabolic functional form, a polynomial functional form, and an exponential functional form can be used. More complex functional forms can also be used, including combinations of any of the foregoing functional forms and / or other functional forms.

[0105] In the previous discussion, the peak intensities 402a, 404a, and 406a were fit to the functional form 502 to determine the local maximum 504. However, quantities other than peak intensity can be fit to the functional form 502 and used to determine the mass axis shift. For example, in some embodiments, the integral area under each of the peaks 402, 404, and 406 in Figure 4 can be calculated and fit to the functional form 502 to determine the mass axis shift. In certain embodiments, the widths (e.g., full width at half maximum) of the peaks 402, 404, and 406 in Figure 4 can be calculated and fit to the functional form 502 to determine the mass axis shift. In certain embodiments, another parameter associated with the peaks 402, 404, and 406, such as the first derivative value at one or more points on each peak, or the second derivative value at one or more points on each peak, can be calculated and fit to the functional form 502 to determine the mass axis shift. Combinations of any of the foregoing quantities (and other quantities) can also be fit to the functional form 502, particularly when it is determined that such combinations result in a more accurately measured mass axis shift 506.

[0106] In some embodiments, multiple mass axis shifts can be determined for the system 100 based on different criteria, and then a final mass axis shift 506 can be determined by the controller 120 based on the set of mass axis shifts. For example, a first mass axis shift can be determined by fitting the measured values associated with the first property of the peaks 402, 404, and 406 in Figure 4 to a first functional form, thereby determining the first mass axis shift as described above. Then, the measured values associated with the second property (different from the first property) of the peaks 402, 404, and 406 can be fit to a second functional form, thereby determining the second mass axis shift as described above.

[0107] Typically, a set of peaks used to determine the first and second mass axis offsets can be the same or different (i.e., these peaks can correspond to the same set of mass offsets, or different sets of mass offsets). Additionally, the set of peaks can correspond to ions associated with a common sample component, or a set of peaks used to determine the first mass axis offset can be associated with a first sample component, and a set of peaks used to determine the second mass axis offset can be associated with a second sample component. Further, the set of peaks used to determine the first mass axis offset can be associated with a first ion derived from a sample component, and the set of peaks used to determine the second mass axis offset can be associated with a second ion, different from the first ion, but also derived from a sample component.

[0108] The number of peaks used to determine the first mass axis offset and the number of peaks used to determine the second mass axis offset can be the same or different. Additionally, the first and second functional forms can be the same or different, depending on criteria such as the nature of the peak attributes being fit, and the accuracy of different functional forms for determining the mass axis offset based on the fit peak attributes.

[0109] Although the foregoing examples refer to two different mass axis offsets, more generally it should be understood that the systems and methods described herein can measure any number of different mass axis offset values before determining a final mass axis offset value. The final mass axis offset value can be determined in various ways. In some embodiments, for example, the members of a group of mass axis offset values are averaged to determine the final mass axis offset value. In certain embodiments, the final mass axis offset value is determined to be the most common mass axis offset value in the group of values. In some embodiments, the final mass axis offset value is determined to be the median of the group of mass axis offset values. Other methods for determining the final mass axis offset value from the group of mass axis offset values can also be used.

[0110] The foregoing discussion has focused on determining mass axis offset values to correct the mass axis calibration associated with a single mass selection filter element in system 100. However, referring Figure 1 , system 100 includes two mass selection filter elements: quadrupoles Q1 and Q3. The foregoing method is applicable to determining mass axis offset values and correcting mass axis calibration for each mass selection filter element in system 100. More generally, for a system 100 that includes M mass selection filter elements, the foregoing method can be used to determine M independent mass offset values, and thus determine M independent corrections to the mass axis calibration, one for each of the M mass selections of mass selection filter elements.

[0111] To evaluate the effectiveness of the foregoing method for determining the mass axis offsets and associated mass axis calibration adjustments for Q1 and Q3 poles in system 100, a sample containing testosterone, gabapentin, cyclosporine, and 13 C-labeled internal reference compound testosterone- 13 C3, gabapentin-13 Samples of serum of C3 and cyclosporine-d10 were introduced into system 100. For each of these components, five different ion peaks were measured, corresponding to the mass offsets of the Q1 and Q3 poles shown in Table 1.

[0112]

[0113] Table 1

[0114] The measurements were repeated 10 times, and the measured ion peaks were integrated. Based on the integrated peak area values, the mass axis offsets were determined for each of the Q1 and Q3 poles of each unlabeled spiked component of the sample. For each of the Q1 and Q3 poles, the final mass axis offset was calculated as the median of a set of mass axis offsets determined for that pole from each unlabeled spiked component of the sample. Then, the Q1 and Q3 poles of system 100 were corrected such that their mass axis calibrations reflected the corresponding final mass axis offsets determined for each pole.

[0115] It should be noted that in the above investigation, both the analyte and its isotopically labeled counterpart were present in the analyzed sample. Typically, an isotopically labeled counterpart of the target analyte can be introduced for purposes such as compensating for irregularities in the sample preparation process. Since they are typically introduced at known concentrations, they provide an internal reference standard for each analyte of interest.

[0116] The measured values associated with the unlabeled analyte, the isotopically labeled reference compound, or both can be used to determine the mass axis offset in the methods described herein. In some embodiments, due to their known concentrations in the sample, the counterparts of the isotopically labeled target analyte are used to determine the mass axis offset, particularly when the concentration of the target analyte is unknown and may be too low to reliably provide a suitable measurement signal for determining the mass axis offset. In certain embodiments, both the unlabeled analyte and the labeled counterpart are used to determine the mass axis offset.

[0117] It should also be noted that the reference compound added to the sample and used to determine the mass axis offset does not need to be isotopically labeled. Typically, any reference compound can be added to the sample and used in the methods described herein to determine the mass axis offset and correct the mass axis calibration.

[0118] After correcting the mass axis calibration, the above measurements for each unlabeled spiked component in the sample were repeated, and the mass axis offsets of Q1 and Q3 for each component were calculated. Table 2 shows the mass axis drifts calculated for the components of the sample before and after correcting the mass axis calibrations of the Q1 and Q3 quadrupole poles of system 100.

[0119]

[0120] Table 2

[0121] As is clearly evident from the data shown in Table 2, the mass axis offset measured after calibrating the mass axis of poles Q1 and Q3 according to the method described herein is, in some cases, more than an order of magnitude smaller than the initial mass axis offset. This significant reduction in the mass axis offset strongly indicates that the method described herein is highly effective for recalibrating a mass spectrometry-based analysis system to compensate for mass axis offset caused by calibration drift.

[0122] Hardware and Software Components

[0123] Controller 120 can be implemented using a variety of different hardware and software components and combinations thereof. In some embodiments, controller 120 includes at least one electronic processor capable of executing software-based instructions to perform any of the functions described herein. In certain embodiments, controller 120 includes one or more dedicated electronic circuits, such as an application specific integrated circuit (ASIC) capable of performing any of the functions described herein.

[0124] Controller 120 may optionally include at least one memory unit. The memory unit may include, for example, random access memory (RAM), read only memory (ROM), and / or any other type of volatile or non-volatile storage medium for software instructions.

[0125] Controller 120 may optionally include at least one storage unit. The storage unit may include any type of medium for storing information readable by the controller (e.g., one or more electronic processors of the controller), including software instructions, calibration settings and information (including mass axis calibration settings and information, such as one or more calibration curves / relationships and corresponding mass spectrometry information for determining the calibration curves / relationships), measurement information (e.g., mass spectrometry information measured by detector 118 and transmitted to controller 120), and data values and other information determined by controller 120 from the measurement information. The at least one storage unit may include various types of tangible storage media, including magnetic storage devices such as hard disk drives, persistent solid state storage devices; rewritable and non-rewritable optical storage media such as CDs and DVDs; programmable circuit elements such as FPGAs, and other types of writable and non-writable storage media.

[0126] The controller 120 may optionally include at least one interface to allow the system 100 to transmit information and / or receive information. The interface may include, for example, a display unit for displaying information to a user of the system 100. The interface may include a transmitter to allow the system 100 to transmit information to a remote device via one or more networks, including a dedicated peer-to-peer network, a wireless network, and a distributed network such as the Internet. The interface may include a human-machine interface device that includes one or more components, such as a keyboard, a mouse, a touch screen, a keypad, a remote control, and any other similar components that allow a user to issue instructions to the system 100. The interface may also include a receiver for receiving information from a remote device via any of the above networks.

[0127] The system 100 may include software instructions that, when executed by the controller 120, cause the controller 120 to perform any of the functions described herein. The software instructions may be encoded in any of the above storage media, embodied in any of the above memory units, encoded in the circuitry of any processor or ASICs of the controller 120, or may be received by the controller 120 from a remote device via the receiver, installed in a memory unit or storage unit of the controller 120, and executed by one or more processors.

[0128] The software instructions may be implemented in a computer program using standard programming techniques. Each such computer program may be implemented in a high-level procedural or object-oriented programming language, or in assembly or machine language. The language may be a compiled or interpreted language, and the specific operations or steps to be performed by one or more processors and / or electronic circuits of the controller 120 may optionally be generated dynamically by executing the computer program.

[0129] In other embodiments

[0130] It should be understood that the above description is intended to illustrate and not limit the scope of the invention, and embodiments other than those explicitly described are within the scope of the invention.

Claims

1. A system for analyzing a biological sample, the system comprising: a separation unit configured to separate components from the biological sample; an ionization unit configured to generate a plurality of ions from the components; an adjustable mass selection filter element; a detector configured to detect ions passing through the mass selection filter element; and a controller connected to the mass selection filter element and connected to the detector, wherein the controller is configured such that during operation of the system, the controller: adjusts the mass selection filter element and activates the detector to measure at least three different ion signals corresponding to the plurality of ions; and determines a mass axis offset of the system based on the at least three different ion signals; wherein: the ionization unit generates the plurality of ions from the components of the biological sample, and the detector measures the at least three different ion signals corresponding to the plurality of ions.

2. The system according to claim 1, wherein each of the three different ion signals corresponds to a different mass-to-charge ratio for the mass selection filter element.

3. The system according to claim 1, wherein the mass selection filter element comprises a quadrupole electrode assembly.

4. The system according to claim 1, wherein each of the at least three different ion signals corresponds to a common ion type among the plurality of ions.

5. The system according to claim 4, wherein the common ion type has an associated mass-to-charge value q, and wherein the controller is configured to activate the detector to measure a first ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q - a) < q to pass through.

6. The system according to claim 5, wherein a is 0.4 atomic mass units (amu) or less.

7. The system according to claim 5, wherein the controller is configured such that during operation of the system, the controller activates the detector to measure a second ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of (q + b) > q to pass through.

8. The system according to claim 7, wherein b is 0.4 amu or less.

9. The system according to claim 7, wherein the controller is configured such that during operation of the system, the controller activates the detector to measure a third ion signal among the at least three different ion signals, wherein the mass selection filter element is adjusted to allow ions having a mass-to-charge ratio of q to pass through.

10. The system according to claim 4, wherein the controller is configured such that during operation of the system, the controller determines the mass axis offset based on attribute values of the at least three different ion signals.

11. The system according to claim 10, wherein the controller is configured such that during operation of the system, the controller fits a functional form to the property value, determines a local maximum of the functional form, and determines the mass axis offset based on the local maximum of the functional form.

12. The system according to claim 11, wherein the functional form corresponds to at least one member selected from the group consisting of: a Gaussian function or a polynomial function.

13. The system according to claim 11, wherein the common ion type has an associated mass-to-charge value q, and wherein the controller is configured such that during operation of the system, the controller determines the mass axis offset by determining a mass offset associated with the local maximum of the functional form relative to the mass-to-charge value q.

14. The system according to claim 1, wherein the controller is configured such that during operation of the system, the controller adjusts the mass axis calibration for the mass selection filter element based on the mass axis offset.

15. The system according to claim 14, wherein the controller is configured such that during operation of the system, the controller: determines a value of an attribute of at least one ion signal measured by the detector and corresponding to the biological sample; and if the attribute value is outside a selected range of values for the attribute, determines a new mass axis offset value for the system and adjusts the mass axis calibration for the mass selection filter element based on the new mass axis offset value.

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