阅读说明：本技术 Srm采集的可调停留时间 (Adjustable dwell time for SRM acquisition ) 是由 M·V·乌加罗夫 宋庆宇 于 2020-08-25 设计创作，主要内容包括：本发明公开一种分析样品的方法,其包含：设置多个跃迁的初始停留时间；在质谱分析期间监测所述跃迁；针对所述多个跃迁中的第一跃迁检测到信号强度高于第一阈值；响应于所述信号强度高于所述第一阈值,增加所述第一跃迁的停留时间；检测到所述第一跃迁的所述信号强度降至低于第二阈值；以及响应于所述信号强度降至低于所述第二阈值,减少所述第一跃迁的所述停留时间。(The present invention discloses a method of analyzing a sample, comprising: setting an initial dwell time for the plurality of transitions; monitoring the transition during mass spectrometry analysis; detecting a signal strength above a first threshold for a first transition of the plurality of transitions; increasing a dwell time of the first transition in response to the signal strength being above the first threshold; detecting that the signal strength of the first transition falls below a second threshold; and in response to said signal strength falling below said second threshold, reducing said dwell time of said first transition.)

1. A method of analyzing a sample, comprising:

setting an initial dwell time for the plurality of transitions;

monitoring the transition during mass spectrometry analysis;

detecting a signal strength above a first threshold for a first transition of the plurality of transitions;

increasing a dwell time of the first transition in response to the signal strength being above the first threshold;

detecting that the signal strength of the first transition falls below a second threshold;

reducing the dwell time of the first transition in response to the signal strength falling below the second threshold.

2. The method of claim 1, wherein increasing the first residence time results in decreasing the second residence time.

3. The method of claim 1, wherein decreasing the first residence time results in increasing a second residence time.

4. The method of claim 1, wherein the signal intensity is recalibrated relative to the residence time and the response is integrated over the peak duration to quantify the compound corresponding to the first transition.

5. The method of claim 1, wherein the initial dwell time is equal for each of the plurality of transitions.

6. The method of claim 1, wherein the initial dwell time is based on an expected intensity of the plurality of transitions.

7. The method of claim 1, wherein the initial residence time is based on a desired level of detection of a compound corresponding to the plurality of transitions.

8. The method of claim 1, further comprising reducing the dwell time of the first transition when the signal strength exceeds a third threshold.

9. A mass spectrometer, comprising:

an ion source configured to generate a stream of ions from a sample;

a quadrupole mass filter configured to:

selecting ions within a range of mass to charge ratios and rejecting ions outside the range of mass to charge ratios from the ion stream; and

repeating the cycling of a series of mass-to-charge ratios corresponding to a plurality of transitions, thereby pausing the dwell time corresponding to the transition over each range of mass-to-charge ratios;

a detector configured to generate a signal proportional to an intensity of an incoming ion stream;

a controller configured to:

setting an initial dwell time for the plurality of transitions;

monitoring a signal intensity of each transition of the plurality of transitions during mass spectrometry analysis;

detecting a signal strength exceeding a first threshold for a first transition of the plurality of transitions;

increasing a dwell time of the first transition in response to the signal strength being above the first threshold;

detecting that the signal strength of the first transition falls below a second threshold;

reducing the dwell time of the first transition in response to the signal strength falling below the second threshold.

10. The mass spectrometer of claim 9, wherein increasing the first residence time results in decreasing the second residence time.

11. The mass spectrometer of claim 9, wherein decreasing the first residence time results in increasing the second residence time.

12. The mass spectrometer of claim 9, wherein the controller is further configured to recalibrate the signal intensity relative to the residence time and integrate the response over a peak duration to quantify the compound corresponding to the first transition.

13. The mass spectrometer of claim 9, wherein said initial dwell time is equal for each of said plurality of transitions.

14. The mass spectrometer of claim 9, wherein said initial dwell time is based on expected intensities of said plurality of transitions.

15. The mass spectrometer of claim 9, wherein the initial residence time is based on a desired level of detection of a compound corresponding to the plurality of transitions.

16. The mass spectrometer of claim 9, wherein the controller is further configured to reduce the dwell time of the first transition when the signal strength exceeds a third threshold.

17. A method of analyzing a sample, comprising:

performing an initial mass spectrometry analysis on the sample to obtain signal intensities for a plurality of transitions;

setting a dwell time for the plurality of transitions based on the signal intensity determined by the initial mass spectrometric analysis of the sample;

performing a second mass spectrometry analysis on the sample using the residence time;

measuring ion intensities of the plurality of transitions as a function of time during the second mass spectrometry analysis; and

quantifying a compound corresponding to the plurality of transitions based on an integration of the ion intensity within at least one peak.

18. The method of claim 17, wherein the dwell time during the initial mass spectrometry analysis is equal for each of the plurality of transitions.

19. The method of claim 17, wherein the dwell time during the initial mass spectrometry analysis is based on expected intensities of the plurality of transitions.

20. The method of claim 17, wherein the residence time during the initial mass spectrometry analysis is based on a desired level of detection of a compound corresponding to the plurality of transitions.

Technical Field

The present disclosure relates generally to the field of mass spectrometry, including adjustable residence time for Single Reaction Monitoring (SRM) acquisition.

Background

Tandem mass spectrometry, known as MS/MS, is a popular and widely used analytical technique that subjects sample-derived precursor ions to fragmentation under controlled conditions to produce product ions. The product ion mass spectrum contains information that can be used for structural determination and for identifying sample components with high specificity. In a typical MS/MS experiment, a relatively small number of precursor ion species are selected for fragmentation, for example, those ion species with the greatest abundance or those ion species with mass-to-charge ratios (m/z) matching the values in the inclusion list.

Filter-type mass spectrometry systems can be used in a manner that simultaneously monitors multiple precursor-product pairs or transitions. Since only one type of ion can be separated in such a filter at any one time, the available analysis time must be split between all available transitions. Thus, while the instrument may sample a precursor close to 100% duty cycle, the overall sampling efficiency is about 0.1% (e.g., the separation window is 1Th for a mass range of 1000 Th). From the foregoing, it should be appreciated that there is a need for an improved method for scheduling transitions to maximize the value of the collected data.

Disclosure of Invention

In a first aspect, a method of analyzing a sample can comprise: setting an initial dwell time for the plurality of transitions; monitoring the transition during mass spectrometry analysis; detecting a signal strength above a first threshold for a first transition of the plurality of transitions; increasing a dwell time of the first transition in response to the signal strength being above the first threshold; detecting that the signal strength of the first transition falls below a second threshold; and in response to said signal strength falling below said second threshold, reducing said dwell time of said first transition.

In various embodiments of the first aspect, increasing the first dwell time may result in decreasing the second dwell time.

In various embodiments of the first aspect, decreasing the first dwell time may result in increasing the second dwell time.

In various embodiments of the first aspect, the signal intensity may be recalibrated with respect to the residence time, and the response may be integrated over the peak duration to quantify the compound corresponding to the first transition.

In various embodiments of the first aspect, the initial dwell time may be equal for each of the plurality of transitions.

In various embodiments of the first aspect, the initial dwell time may be based on an expected intensity of the plurality of transitions.

In various embodiments of the first aspect, the initial residence time may be based on a desired level of detection of the compound corresponding to the plurality of transitions.

In various embodiments of the first aspect, the method may further comprise reducing the dwell time of the first transition when the signal strength exceeds a third threshold.

In a second aspect, a mass spectrometer may include an ion source, a quadrupole mass filter, a detector, and a controller. The ion source may be configured to generate a stream of ions from a sample. The quadrupole mass filter may be configured to select ions within a range of mass to charge ratios and reject ions outside the range of mass to charge ratios from the ion stream; and repeating the cycling of a series of mass-to-charge ratios corresponding to the plurality of transitions, thereby pausing the dwell time corresponding to the transition over each range of mass-to-charge ratios. The detector may be configured to generate a signal proportional to the intensity of the incoming ion stream. The controller may be configured to: setting an initial dwell time for the plurality of transitions; monitoring a signal intensity of each transition of the plurality of transitions during mass spectrometry; detecting a signal strength exceeding a first threshold for a first transition of the plurality of transitions; increasing a dwell time of the first transition in response to the signal strength being above the first threshold; detecting that the signal strength of the first transition falls below a second threshold; and in response to said signal strength falling below said second threshold, reducing said dwell time of said first transition.

In various embodiments of the second aspect, increasing the first dwell time may result in decreasing the second dwell time.

In various embodiments of the second aspect, decreasing the first dwell time may result in increasing the second dwell time.

In various embodiments of the second aspect, the controller may be further configured to recalibrate the signal intensity relative to the dwell time and integrate the response over the peak duration to quantify the compound corresponding to the first transition.

In various embodiments of the second aspect, the initial dwell time may be equal for each of the plurality of transitions.

In various embodiments of the second aspect, the initial dwell time may be based on an expected intensity of the plurality of transitions.

In various embodiments of the second aspect, the initial residence time may be based on a desired level of detection of the compound corresponding to the plurality of transitions.

In various embodiments of the second aspect, the controller may be further configured to decrease the dwell time of the first transition when the signal strength exceeds a third threshold.

In a third aspect, a method of analyzing a sample can comprise: performing an initial mass spectrometry analysis on the sample to obtain signal intensities for a plurality of transitions; setting a dwell time for the plurality of transitions based on the signal intensity determined by the initial mass spectrometric analysis of the sample; performing a second mass spectrometry analysis on the sample using the residence time; measuring ion intensities of the plurality of transitions as a function of time during the second mass spectrometry analysis; and quantifying a compound corresponding to the plurality of transitions based on an integration of the ion intensity within at least one peak.

In various embodiments of the third aspect, the dwell time during the initial mass spectrometry analysis may be equal for each of the plurality of transitions.

In various embodiments of the third aspect, the dwell time during the initial mass spectrometry analysis can be based on expected intensities of the plurality of transitions.

In various embodiments of the third aspect, the residence time during the initial mass spectrometry analysis can be based on a desired level of detection of a compound corresponding to the plurality of transitions.

Drawings

For a more complete understanding of the principles disclosed herein and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

fig. 1 is a block diagram of an example property spectrum system, in accordance with various embodiments.

Fig. 2, 3, and 4 are flow diagrams illustrating exemplary methods of determining dwell time in accordance with various embodiments.

Fig. 5 is a timing diagram illustrating dwell times during analysis in accordance with various embodiments.

Fig. 6 is a block diagram illustrating an exemplary data analysis system, in accordance with various embodiments.

It should be understood that the drawings are not necessarily drawn to scale, nor are the objects in the drawings necessarily drawn to scale relative to one another. The drawings are presented to provide a clear and easy to understand depiction of various embodiments of the apparatus, system, and method disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way.

Detailed Description

Embodiments of systems and methods for ion separation are described herein.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, it will be understood by those skilled in the art that the various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Moreover, those of skill in the art will readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that these sequences may be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong.

It should be understood that there is an implicit "about" preceding the temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprising", "containing" and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

As used herein, "a" or "an" may also mean "at least one" or "one or more". Moreover, the use of "or" is inclusive such that the phrase "a or B" is true when "a" is true, "B" is true, or both "a" and "B" are true. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

A "system" is intended to describe a set of real or abstract components, which system comprises an ensemble of components, wherein each component interacts or is related to at least one other component within the ensemble.

Mass spectrum platform

Various embodiments of mass spectrometry platform 100 can include components as shown in the block diagram of figure 1. In various embodiments, the elements of fig. 1 may be incorporated into mass spectrometry platform 100. According to various embodiments, the mass spectrometer 100 may include an ion source 102, a mass analyzer 104, an ion detector 106, and a controller 108.

In various embodiments, the ion source 102 generates a plurality of ions from a sample. The ion source may include, but is not limited to: matrix-assisted laser desorption/ionization (MALDI) sources, electrospray ionization (ESI) sources, Atmospheric Pressure Chemical Ionization (APCI) sources, atmospheric pressure photoionization sources (APPI), Inductively Coupled Plasma (ICP) sources, electron ionization sources, chemical ionization sources, photoionization sources, glow discharge ionization sources, thermal spray ionization sources, and the like.

In various embodiments, the mass analyzer 104 may separate ions based on their mass-to-charge ratios. For example, the mass analyzer 104 can include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., an orbital ion trap) mass analyzer, a fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, and the like. In various embodiments, the mass analyzer 104 may also be configured to fragment ions using Collision Induced Dissociation (CID), Electron Transfer Dissociation (ETD), Electron Capture Dissociation (ECD), light induced dissociation (PID), Surface Induced Dissociation (SID), and the like, and further separate fragment ions based on mass-to-charge ratios.

In various embodiments, the ion detector 106 may detect ions. For example, the ion detector 106 may include an electron multiplier, a Faraday cup (Faraday cup), or the like. Ions exiting the mass analyzer may be detected by an ion detector. In various embodiments, the ion detector may be quantitative such that an accurate count of ions may be determined.

In various embodiments, the controller 108 may be in communication with the ion source 102, the mass analyzer 104, and the ion detector 106. For example, the controller 108 may configure the ion source or enable/disable the ion source. In addition, the controller 108 may configure the mass analyzer 104 to select a particular mass range to detect. Further, the controller 108 may adjust the sensitivity of the ion detector 106, for example, by adjusting the gain. In addition, the controller 108 may adjust the polarity of the ion detector 106 based on the polarity of the detected ions. For example, the ion detector 106 may be configured to detect positive ions or configured to detect negative ions.

Residence time

Filter-type mass spectrometry systems are used in a manner that simultaneously monitors multiple precursor-product pairs or transitions. Since only one type of ion can be separated in such a filter at any one time, the available analysis time must be split between all available transitions.

Disadvantageously, different compounds often exhibit different signal levels. In addition, the target quantification level may vary over a wide range. Thus, applying similar residence times for all compounds does not achieve an optimal distribution of available acquisition time and puts the "tricky" goal at a disadvantage.

Alternative methods are known in the art to address the uneven distribution of LC efflux peaks along the length of the chromatogram. For example, analysis of a particular subset of transitions may be scheduled to occur only during a predetermined time window, thereby reducing the number of transitions running in parallel and increasing the corresponding dwell time. However, the method does not distinguish between these transitions and applies the same dwell for all transitions.

In another approach, different residence times may be manually specified for all compounds. While this may achieve the goal, the task can be arduous and quite impractical with hundreds of compounds. In addition, since the level of analyte is typically unknown prior to LC/MS runs, such fixed value methods may not work in most cases.

Heretofore, several methods have been proposed to improve time utilization by dynamically changing the acquisition timing based on the acquired data. For example, Perst et al (US 7482580B 2) suggest that the time period for ion detection may be terminated based on the results of the detector output real-time monitoring to reduce the overall cycle time. This method may be applicable to both very strong and very weak transitions during collection where the quantitative results are statistically satisfactory. However, this method is not suitable for typical LCMS experiments where the peak shape and duration vary and reliable quantitative analysis requires full sampling of the ion efflux curve.

In another prior art example of Green (US 9881781B 2), it is taught to interrupt monitoring or reduce monitoring residence time in the presence of ions. This makes it possible to improve the analysis duty cycle. However, the success of this approach depends on whether the observed ion signal is reliably identified as a chromatographic peak, which may not always be readily identified for weaker signals and less than ideal peak shapes. This limits the usefulness of this approach and can even result in the loss of the actual peak if monitoring of the ion signal is terminated prematurely.

Further, it is critical that none of the above patents teach adjusting the residence time in real time based on the onset of ion chromatographic flux by detecting the rising edge of the chromatographic peak.

Fig. 2 illustrates an exemplary method 200 for determining dwell time. At 202, a weight can be assigned to the transition. The transition represents a particular fragment ion of the parent ion. The signal strength for a given transition may depend on the parent ion abundance and the rate of generation of a particular fragment ion. The parent ion abundance may depend on the concentration of the compound that generates the parent ion and the ion ionization efficiency of the compound that generates the parent ion. Lower intensity transitions can be given higher weight compared to higher abundance compounds, which can result in longer residence time for low abundance compounds.

In an exemplary embodiment, weights can be assigned based on expected intensities, e.g., based on known relative responses of analytes and the "liveness" of particular transitions. Transitions with higher expected intensities may be given lower weight than transitions with lower intensities. In other embodiments, weights may be assigned based on target regulatory limits. Compounds with lower regulatory limits may be given higher weight to ensure accurate detection of compounds at or near the regulatory limits.

At 204, a weight-building analysis method may be used to calculate the residence time. In an exemplary embodiment, the dwell time may be calculated according to equation 1. If N transitions are present simultaneously, DT_iIs the dwell time of the transition i, T_sIs the cycle time (analysis time available for all transitions), and W_iIs the weight of transition i.

Equation 1

A method can be established in which the respective dwell times are determined to maximize the acquisition time for transitions with the expected lower signal level. Additionally, acquisition "windows" or "segments" may also be scheduled to further optimize temporal usage. Also, the transition sequence can be optimized to reduce the intermediate scan (setup) time. Factors such as the m/z difference between the parent and product ions may be considered in the process.

At 206, a method can be performed and the intensity of the transition can be monitored according to the schedule determined at 204, and at 208, the compound can be quantified according to the measured intensity.

FIG. 3 illustrates another exemplary method 300 for determining dwell time. At 302, a first injection analysis of the sample may be performed. In various embodiments, all target transitions may be monitored during the initial analysis. The dwell times may be equal or assigned weights, for example, according to the method 200. Additionally, transitions can be scheduled and ordered during the acquisition window to reduce inter-scan delays.

At 304, a transition with a low signal can be identified from the initial analysis. Transitions where sufficient data is collected to adequately quantify the compound can be excluded from subsequent analysis of the same sample. By excluding transitions with sufficient data from the initial analysis, more time can be devoted to the analysis of low signal transitions.

At 306, the residence time may be empirically determined based on the quantitative results from the first run. The optimal dwell time can be calculated to assign a longer dwell time for weaker transitions. Furthermore, the retention time window of the transition can be reallocated if necessary.

At 308, a second injection is performed and a new quantitative analysis is performed.

This method may require splitting the available samples, or preparing double the amount. Moreover, this method requires doubling the analysis time, which can be considered as a disadvantage. However, optimal sensitivity and quantitative gain limitations should in many cases compensate for this potential disadvantage and enable experiments that cannot be performed due to the low sensitivity of some compounds.

Sensitivity can be further improved if the first injection run is not only used to estimate the intensity optimized for the second run, but is also used as a quantitative run at least for stronger ion signals. The second run can then be limited to those "weaker transitions" that are more likely to improve signal quality.

FIG. 4 illustrates another exemplary method 400 for dynamically determining dwell time. At 402, a set of initial dwell times may be determined. In various embodiments, transitions assigned time windows (segments) may be given equal dwell times. In various embodiments, the dwell time may be specified or determined according to the method 200. In further embodiments, an initial scan may be used to determine an initial dwell time as in method 300.

At 404, the transition can be monitored during the analysis. At 406, an increase in signal strength of the transition above a first threshold can be detected, indicating the beginning of the associated chromatographic peak.

At 408, in response to detecting increasing signal strength, a change can be triggered to increase the residence time of this particular compound at the expense of other transitions that are currently inactive. Subsequently, if a peak corresponding to the efflux of other compounds occurs in the mass spectrum, its corresponding residence time is increased to provide more acquisition time.

At 410, when the data acquisition module detects the falling edge of the peak and the intensity falls below a predetermined level, the transition is removed from the list of "active" transitions, thus resulting in a minimum (or zero) residence time during the chromatography run, as shown at 412.

Thus, an "active" transition at any time during the run can increase the acquisition time compared to a conventional run with a uniform dwell.

Even though the dwell time is dynamically set and can be adjusted during the actual peak outflow, repeatable quantitation can be achieved because the data processor can continually recalibrate the signal based on the current dwell. The total response is then calculated as the integral of the entire peak duration, as shown at 414.

In various embodiments, the dwell time may be reduced after step 408, while the signal strength exceeds a second threshold indicative of a strong transition before step 410.

Fig. 5 provides an illustration of an exemplary analysis. During cycle 1, both transition x and transition y are below the threshold.

During cycle 2, a transition x above the threshold is detected at 502, while a transition y is still below the threshold at 504.

For cycle 3, the dwell time for the transition x increases significantly as the other dwell times, including the dwell time for the transition y, decrease. However, at 506, the intensity of the transition y is measured to be above the threshold.

For cycles 4 and 5, both transition x and transition y are in an active state and the dwell time of transition y increases. When transition x is the only active transition, transition x is not reduced to the extent of other inactive transitions, although the dwell time of transition x is reduced relative to cycle 3.

During cycle 6, the strength of the transition x drops below the threshold, removing the transition x from the active list. Thus, in cycle 7, the dwell time of the transition x is reduced to 0, thereby further increasing the dwell time of the transition y, since the transition y is the only active transition during cycles 7 and 8. During cycle 8 at 510, the transition y falls below the threshold and can be removed from the active transition list.

Computer implemented system

Figure 6 is a block diagram illustrating a computer system 600 upon which embodiments of the present teachings may be implemented that may incorporate or communicate with a system controller (e.g., controller 110 shown in figure 1) such that the operation of components of an associated mass spectrometer may be adjusted according to calculations or determinations made by computer system 600. In various embodiments, computer system 600 may include a bus 602 or other communication mechanism for communicating information, and a processor 604 coupled with bus 602 for processing information. In various embodiments, computer system 600 may also include a memory 606, which may be a Random Access Memory (RAM) or other dynamic storage device, coupled to bus 602 and instructions to be executed by processor 604. Memory 606 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 604. In various embodiments, computer system 600 may further include a Read Only Memory (ROM)608 or other static storage device coupled to bus 602 for storing static information and instructions for processor 604. A storage device 610, such as a magnetic disk or optical disk, may be provided and coupled to bus 602 for storing information and instructions.

In various embodiments, computer system 600 may be coupled via bus 602 to a display 612, such as a Cathode Ray Tube (CRT) or Liquid Crystal Display (LCD), for displaying information to a computer user. An input device 614, including alphanumeric and other keys, may be coupled to bus 602 for communicating information and command selections to processor 604. Another type of user input device is cursor control 616, such as a mouse, a trackball, or cursor direction keys for communicating direction information and command selections to processor 604 and for controlling cursor movement on display 612. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), which allows the device to specify positions in a plane.

Computer system 600 may perform the teachings of the present invention. Consistent with certain embodiments of the present teachings, results may be provided by computer system 600 in response to processor 604 executing one or more sequences of one or more instructions contained in memory 606. Such instructions may be read into memory 606 from another computer-readable medium, such as storage device 610. Execution of the sequences of instructions contained in memory 606 may cause processor 604 to perform processes described herein. In various embodiments, the instructions in the memory may order the use of various combinations of logic gates available within the processor to perform the processes described herein. Alternatively, hardwired circuitry may be used in place of or in combination with software instructions to implement the teachings of the present invention. In various embodiments, the hardwired circuitry may include necessary logic gates that operate in the necessary sequence to perform the processes described herein. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

While the present teachings are described in conjunction with various embodiments, there is no intent to limit the present teachings to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

Further, in describing various embodiments, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that a method or process does not rely on the particular sequence of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. Other sequences of steps are possible, as will be appreciated by those of ordinary skill in the art. Accordingly, the particular sequence of steps set forth in this specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.