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pro
led using a typical RP-gradient from 5 min
to 95% acetonitrile versus 0.1% aqueous formic
acid over 10 min at a
relatively low information content detectors such
as UV/vis (DAD) spectrophotometers and elec-
trochemical detectors up to MS and NMR spec-
trometers of much greater expense but
potentially of high information content. However,
as a result of its combination of sensitivity and
structure determination/identi
l/min.
Clearly, whatever type of separation and
mode of chromatography is employed, if meta-
bolic phenotypes are to be compared, the gener-
ation of repeatable pro
flow rate of 12
m
les from samples is of
central importance and therefore retention time
and peak shape must be stable over the whole
length of an analytical run (which may well
exceed 24 hrs on a routine basis). Although tech-
niques such as UPLC can demonstrate impres-
sive repeatability, researchers should be aware
that, depending upon the sample matrix, the
age of the column, and its previous exposure to
samples, it can take a number of injections before
the system is equilibrated, or conditioned, and
retention times stabilize.
10
e
16
In terms of the
chromatographic process, we assume that what
is happening during this
cation potential,
MS probably currently provides the best choice
for LC-metabolomic/metabonomic pro
ling.
MS is not without limitations, however, and it
should always be remembered that ionization
ef
c and may differ
widely even for structurally related compounds.
Currently, ESI, combined with time-of-
ciency is compound speci
ight
mass spectrometry (TOF-MS), is the method of
choice for LC-MS-based metabolomic/metabo-
nomic pro
ling, using both positive and nega-
tive modes to maximize the coverage of the
metabolome. Other ionization techniques, such
as atmospheric pressure chemical ionization
(APCI), are available and could potentially
have advantages for nonpolar metabolites, but
APCI has not yet been widely applied in metab-
olome pro
”
is that matrix components are interacting with
the column packing (or some other part of the
system, e.g., tubing or frits) in such a way that
active sites and silanols are masked. Condi-
tioning is matrix dependent, with samples such
as urine usually requiring fewer conditioning
injections than serum or plasma. Once condi-
tioned, we have found retention to be stable,
with the buildup of contamination in the ion
source of the mass spectrometer causing the
bulk of the analytical variability thereafter. It is
also our experience that in addition to condi-
tioning, column lifetime is matrix dependent.
Thus, analysis of urine generally provides longer
column lifetimes (several thousand samples)
than, for example, serum or plasma (up to
1,000 samples) before replacement is required.
“
conditioning phase
ling. Along with the use of TOF
instruments, metabolome pro
les have also
been determined using linear ion traps, as well
as hybrid spectrometers such as IT-TOF
48
or
13,48
although, where the highest
mass resolution is required, Fourier transform
ion cyclotron MS (FT-ICR) or Orbitrap MS
instruments can be used. The high-resolution
spectrometers provide excellent mass accuracy,
enabling more accurate determination of atomic
composition and so on to aid in metabolite char-
acterization and identi
the QTRAP
cation. One area of diffi-
-
culty that those performing this type of pro
ling
for biomarker research should be aware of,
though, is that different types of mass spectrom-
eter do not necessarily give the same responses.
Thus, although the dangers of trying to compare
the results of metabolite using different types of
chromatography are obvious, the problems of
comparing and correlating LC-MS data derived
from different MS instruments
d
even when the
same separation is used
d
are less obvious. That
DETECTION
Clearly, a range of LC detectors are available
that could, in principle, be used to interrogate
the separation and produce metabolite pro
les.
These include sensitive and inexpensive but
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