Technical Note
The QSTAR® Elite and Mass Defect
Triggered IDA (MDt™ IDA): Improved
Ion Selection for Metabolism Studies
Overview
The difference between the exact mass and the nominal (nearest integer) mass of
a compound is known as the mass defect. In metabolism studies, closely related
molecules like the parent drug and its metabolites should have very similar mass
defects. We can take advantage of this fact during data acquisition to perform
MS/MS only on ions that fall within a user-defined mass defect window – effectively
performing MS/MS only on ions most likely to be drug or drug-metabolite related.
This has a large effect on minimizing the list of potential candidate ions and can be
used in an automated experiment and simplifies data interpretation post
acquisition.
Introduction
Automated LC/MS/MS workflows using Information Dependent Acquisition (IDA)
provide the framework for deriving maximum information from every LC
experiment. IDA lets you focus on specific ions of interest for increased
productivity. The benefits of IDA are the simultaneous collection of single MS and
MS/MS data to maximize the information in a single injection. The challenge in this
process is determining the ion(s) of interest, which are not necessarily the most
intense ion(s), all on an LC time scale.
Selection criteria tools available for IDA can help to select the ion of interest for
performing MS/MS studies while ignoring all the extraneous ions. The selection
criterion can include: upper and lower mass cut-offs, isotope pattern matching,
Inclusion and Exclusion lists, Dynamic Exclusion as well as Dynamic Background
Subtraction. While all of these tools in combination with one another can
significantly improve the ion selection process, there is another technique that can
help reduce the potential candidate list even further; triggering MS/MS based on an
ion’s mass defect.
The difference between the exact mass and the nominal (nearest integer) mass of
a compound is known as the mass defect. In metabolism studies, closely related
molecules like the parent drug and its metabolites should have very similar mass
defects. We can take advantage of this fact to select ions for MS/MS analysis
based on their mass defect, thereby concentrating only on those species that are
most likely related to the parent drug. This has a large effect on minimizing the list
of potential candidate ions and can significantly improve the number of drug and
drug-related compounds selected for MS/MS acquisition.
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Technical Note
The use of mass defect filtering post-acquisition was first described by Zhang, et
al., in 20031. The mass defect filter is a software-based post-acquisition filter that
takes advantage of high-resolution, high mass accuracy data and the predictable
mass defects of a parent drug and its corresponding metabolites. It allows the
user to filter the total ion chromatogram and/or mass spectrum to display only ions
that fall within the user-defined mass defect window (this option is still available on
all QSTAR® systems). Here, we apply this mass defect filtering process
dynamically as an IDA trigger criteria with the new QSTAR® Elite system.
The QSTAR® Elite system is the newest QqTOF instrument from Applied
Biosystems/MDS SCIEX with improved resolution and mass accuracy over other
QqTOF instruments. In addition to hardware improvements, the QSTAR® Elite
system contains a number of productivity enhancing software features including
dynamic mass defect filtering, which will be discussed in more detail below.
MDt™ IDA: The Concept
Mass Defect triggered IDA or MDt™ IDA is a new tool available to help filter the
MS/MS inclusion list when running IDA experiments based on the mass defect of
closely related species; like parent drug and drug-related metabolites. This is a
valuable tool in combination with other threshold criteria to help narrow down the
list of candidate ions for MS/MS acquisition and allow more time to be spent
acquiring data on more relevant species. The IDA process filters the candidate ion
list by mass defect, which is a user-defined value. Figure 1 shows part of the IDA
method used to create an IDA experiment incorporating the mass defect filtering
capabilities.
Figure 1. Mass defect filter option found
in IDA criteria tab of Acquisition method
within Analyst® QS 2.0 software. The
user inputs the chemical formula for the
parent drug and the corresponding mass
defect is calculated in mDa units. The
search window to select which ions
trigger MS/MS is user definable, with 4050 mDa typically a good starting point.
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Technical Note
MDt™ IDA in Practice:
The ability to identify unknown species in complex matrices is typically very
difficult; generally the ion of interest is buried in a sea of other irrelevant ions. On
instruments such as triple quadrupoles, specific scan functions such as precursor
or neutral loss scanning can be used to help filter out those irrelevant ions. On
instruments such as the QSTAR® Elite, automated experiments like IDA are used
to target key ions for gathering MS/MS data for confirmation and structural
elucidation. Just as precursor scanning and neutral loss scanning aren’t always
perfect, some relevant ions are missed and some irrelevant ions are selected, IDA
or any type of automated MS/MS acquisition method can suffer from the same
problems. For metabolism studies especially, finding those unexpected or low
abundance metabolites and ignoring irrelevant ions can be challenging.
With a high resolution, high mass accuracy system like the QSTAR® Elite, one can
take advantage of the fact that metabolites and the corresponding parent drug are
closely related with respect to mass defect. Small structural changes that occur as
the result of drug metabolism don’t significantly alter the mass defect of the
compounds. This means one can target a very narrow mass defect window to filter
out those ions that aren’t drug-related. While it is possible to apply a mass defect
filter post-acquisition on most accurate mass instruments, applying this in real-time
is significantly more challenging. It requires that the instrument is capable of
acquiring accurate mass data on all ions being generated, on an LC time-scale, as
well as fast electronics and software processing to filter data on-the-fly and target
selected ions for MS/MS acquisition. This is the benefit of MDt™ IDA.
Figure 2 shows the comparison of the survey scan with and without mass defect
filtering. In Figure 2a, the original TIC doesn’t show any clear metabolite
presence. In Figure 2b, with mass defect filtering turned on, a much cleaner,
more well-defined picture emerges.
A)
Original TIC
B)
Mass Defect Filtered
Figure 2. Metabolite
profile of erythromycin.
A) original TIC. B) mass
defect filtered TIC. The
mass defect filtered TIC
shows
significant
definition in the profile,
clearly indicating the
presence of potential
ions
of
interest.
Because the data is
filtered dynamically as
the LC run proceeds,
MS/MS data is collected
for identification in the
same run.
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Technical Note
The mass defect filtering process cleans up the TIC and, more importantly, the
underlying the mass spectra. Figure 3 shows the impact on the mass spectrum.
MD Filtered TIC
Original Mass spectrum
MD Filtered Mass spectrum
Figure 3. Comparison of the unfiltered and filtered mass spectra for the erythromycin metabolism
study. A) mass defect filtered TIC. The highlighted region between 5.7 and 5.9 minutes compares
B) the original mass spectrum, and C) the mass defect filtered mass spectrum. For the automated
experiment, the dominant ion is drug related and chosen for MS/MS data collection from the filtered
spectrum, all in the same analysis.
The use of MDt™ IDA has a significant impact on the efficiency of the IDA selection
process, identifying more metabolites than standard IDA experiments while
collecting less irrelevant data.
# of ions selected
# of Metabolites ID'd
Standard IDA
912
10
with DBS
496
27
with DBS & MDt IDA
288
27
Table 1. The comparison of standard IDA with the addition of DBS and MDt™ IDA on the number
of metabolites identified for propanolol. MDt™ IDA shows significant improvement in overall
efficiency of the automated workflow, by significantly narrowing the search. A factor of 3 fewer ions
were selected for MS/MS collection while metabolite coverage improved from 32% to 87%. The
efficiency leads to higher quality, more relevant data and more time available for additional
experiments.
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Technical Note
Summary
Mass Defect triggered IDA (MDt™ IDA) is an important tool in combination with the
high resolution, high mass accuracy of the new QSTAR® Elite system for finding
drug related metabolites in complex mixtures.
Simple IDA implementation, yet powerful in helping to identify metabolites
whose mass defects are similar to the parent ion
A solid tool in combination with Dynamic Background Subtract (DBS)
Takes advantage of the mass accuracy and fast scanning capability of the
new QSTAR Elite system
Increases the number of metabolites discovered while decreasing the
amount of irrelevant data acquired
1
J. Mass Spectrom. 2003; 38: 1110-1112
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