mass spectrometry
MMDF Example
Multiple Mass Defect Filter
(MMDF)
This figure shows the
excellent capability of
MMDF to capture both
Phase I and II metabolites
specifically and concurrently.
LC peaks of metabolites from
Phase I and Phase II are
visibly separated from the
background matrix by the
corresponding MDF(s). The
resulting chromatogram from
MMDF (bottom chromatogram)
is much cleaner and specific
than the original (top chromatogram), and combines the information from
both chromatograms filtered by separate MDF for Phase I and Phase II
(middle chromatograms).
Applying Multiple Mass Defect Filter (MMDF)
to Accurate Mass Data for Fast, Sensitive,
and Accurate Identification of Metabolites
What is MMDF?
MMDF is an acronym for Multiple
Mass Defect Filter. A new feature in
the latest version of Thermo Scientific
MetWorks™ Software, this post-acquisition data processing tool allows the
user to combine the results from up to
six different Mass Defect Filters (MDF).
In order to explain MMDF, one needs to
first understand the terms mass defect
and mass defect filter.
The term “mass defect” originates
from the fact that only the mono isotopic
element 12C, has an integer value for atomic
weight, i.e. 12.000000. Mass defect refers
to the difference between the exact mass of
an element (or a compound) and its closest
integer value. It may be positive (larger than
the nominal mass) or negative (smaller than
the nominal mass).
Mass Defect Filter (MDF) refers to a
post-acquisition data filtering technique
based on the mass defect of the parent
drug and its metabolites. In order to use
MDF, it is critically important to use high
mass accuracy data from high resolution
analyses, i.e. LTQ Orbitrap™ hybrid mass
spectrometer.
Element
Nuclide
Nominal Mass
Exact Mass
Mass Defect
Hydrogen
H
1
1.0078
0.0078
Carbon
12
C
12
12.0000
0.0000
Nitrogen
14
N
14
14.0031
0.0031
Oxygen
16
16
15.9949
-0.0051
Fluorine
19
F
19
18.9984
-0.0016
Sulfur
32
S
32
31.9721
-0.0279
Chlorine
35
CI
35
34.9689
-0.0311
O
Exact Mass and Mass Defect of Common Elements
Part of Thermo Fisher Scientific
How is MDF used in drug
metabolism?
Each parent compound has a mass
defect, which will be associated with its
metabolites because a large portion of the
parent compound structure usually remains
unchanged during biotransformation. In
other words, the mass defect of metabolites
will lie within a relatively narrow range.
Based on the molecular weight of the
parent compound, estimation can also be
made regarding the range of molecular
weight (MW) in which these metabolites
will occur. Mass Defect Filter (MDF) can
then be applied to filter out all ions that fall
outside of the expected MW range, as well
as those ions that are within the expected
MW range but exceed the expected mass
defect range. This data reduction technique
allows users to focus on the analysis of
species that are potential drug metabolite
candidates.
Why is MMDF more useful
than MDF?
Description of the Biotransformation
Formula Change
Nominal Mass Change (Da)
Accurate Mass Change (Da)
Oxidative debromination
[M+OH-Br]
-62
-61.9183
81.7
De-ethlylation (O, N, S)
[M-CH3CH]
-28
-28.0313
-31.3
33.9
Oxidative dechlorination
[M+OH-Cl]
-18
-17.9661
Phosphothionate oxidation
[M-S+O]
-16
15.9772
22.8
De-methylation (O, N, S)
[M-CH2]
-14
-14.0157
-15.7
Dehydrogenation
[M-2H]
-2
-2.0156
-15.6
Oxidation (Alcohol Aldehyde)
[M-2H]
-2
-2.0156
-15.6
Oxidative deanimation
[M+O-NH3]
-1
-1.0316
-31.6
Reduction
[M+2H]
2
2.0156
15.6
Hydroxylation
[M+O]
16
15.9949
-5.1
Epoxidation
[M+O]
16
15.9949
-5.1
Oxidation (N, S)
[M+O]
16
15.9949
-5.1
Oxidation (Aldehyde Carboxylic Acid)
[M+O]
16
15.9949
-5.1
Di-hydroxylation
[M+O2]
32
31.9898
-10.2
Tri hydroxylation
[M+O3]
48
47.9847
-15.3
Phase I Metabolic Modifications
Description of the Biotransformation
Formula Change
Nominal Mass Change (Da)
Accurate Mass Change (Da)
Mass Defect (mDa)
Methylation
[M+CH2]
14
14.0157
15.7
Acetylation
[M+COCH2]
42
42.0101
10.1
Glycine Conjugation (Carboxylic acids)
[M-OH+C2H4NO2]
57
57.0215
21.5
Sulfation
[M+SO3]
80
79.9568
-43.2
Glucuronidation
[M+C6H8O6]
176
176.0321
32.1
Glutathione Conjugation
[M+C10H15N3O6S]
305
305.0682
68.2
Phase II Metabolic Modifications
The ability to apply MMDF allows users
to capture metabolites from Phase I and
Phase II specifically and concurrently. It also
can, at the same time, capture metabolites
from hydrolysis or N-dealkylation, even
when the products from such processes
have mass defects that are significantly
different from the parent. These capabilities
are not possible with the use of a single
MDF. The results from MMDF are much
more specific, and the analysis provides
faster results.
What is the significance of MMDF
in drug metabolism?
MMDF provides speed, sensitivity, and
accuracy to researchers in drug metabolism.
The use of MMDF dramatically reduces the
data processing time in metabolite identification experiments by filtering out the vast
majority of matrix-related background ions,
which may share the same nominal mass as
potential metabolites. This allows users to
use low threshold values during data
MetWorks Software is compatible
with all Thermo Scientific mass
spectrometers capable of performing
MS/MS or greater fragmentation and
takes advantage of the high mass
accuracy and reproducible MSn
fragmentation data generated by
these products.
New MMDF Feature in MetWorks Software
processing so that metabolites at very low
levels can be easily identified. The resulting
chromatogram is very accurate and specific
because it is based on exact mass and mass
deficiencies, which are highly specific to
each parent drug compound of interest. It is
a very powerful tool that is simple to use.
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