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coverage and the best results are obtained if both
types of separation are used. Although they can
be used separately, it is also technically feasible
to combine them into a single 2D system and
indeed such a combined HILIC and RP separa-
tion.
39
The resulting separation method was
successfully used to provide metabolic pro
recognized that this methodology has long-term
consequences for MS-based applications as
a result of the contamination of the instrument
by the ion pair reagent. This contamination can
prove extremely resistant to cleaning, and it
may therefore be necessary to dedicate a mass
spectrometer solely to this type of analysis.
However, where the pro
les of
urine samples obtained from lung cancer patients.
Here polar compounds were separated on a TSK
gel amide-80 column (2.0
ling of polar acidic
metabolites is required, the disadvantages of
IPLC may be compensated for by the utility of
the method. Though many examples of the use
of the IPLC technique involve targeted analysis,
an example of this type of method, with detection
via an Orbitrap
50 mm, 3
m
m) and
the less polar ones using a Hypersil
ODS2, C18
column (4.6
m) and RPLC, and
was found to be reproducible and robust.
An example of the use of UPLC for both
HILIC and RPLC analysis of urine from Zucker
rats
32
is shown in
Figure 3
.
150 mm, 5
m
mass spectrometer that was
used for the sensitive and speci
c detection of
both unknown and known metabolites (5 ng/ml
for 80 metabolite standards) used of RP-IPLC
on a Synergi
Hydro-RP 2.5
m C18 column
m
Other Approaches to the Pro
ling
(100 mm
2 mm i.d.) employing a
flow rate of
of Polar and Ionic Metabolites
Although HILIC has been widely used for
polar compounds as discussed previously, it
does not yet provide a complete answer to the
analysis of polar, ionic compounds and it is argu-
able that techniques such ion exchange chroma-
tography (IEC) should provide another
approach to the separation of such metabolites
present in samples. Preliminary studies using
IEC for pro
L/min.
40
In a study on a strain of yeast engi-
neered such that a gene of unknown function
(YKL215C) was knocked out, the accumulation
of oxoproline was observed, allowing the gene
to be identi
200
m
ed as oxoprolinase. The IP reagent
was tributylamine (10 mM) (and 15 mM acetic
acid) with a water-methanol gradient. The start-
ing conditions for the gradient were 97:3 water/
methanol for 2.5 min, increasing the methanol
content to 20% methanol at 5 min, then, from
7.5 min, increasing to 55% methanol at 13 min,
then, at 15.5 min, rising to 95% methanol at
18.5 min. This solvent composition was main-
tained for a further 0.5 min before returning
to the starting conditions. Tributylamine was
also used for the analysis of cell extracts of Meth-
ylobacterium extorquens with a nanoscale LC-MS
system
41
and a hexylamine-based IP system
42
has been used to analyze extracts of Lactobacillus
plantarum, E. coli, and B. subtilis by LC-MS.
IPLC, despite the manifest disadvantage of the
need to essentially dedicate an instrument to
the technique, provides a robust and reliable
method for polar acidic metabolites and may
well become
ling rat urine samples from (fa/fa)
and (
e
/
e
) Zucker rats, representing obese and
lean phenotypes, were promising, demonstrating
separation between the two classes on multivar-
iate statistical analysis by principal component
analysis (PCA). In this study, samples were
analyzed on an IonPac
250 mm
column. Separations were performed using
gradient elution with the starting conditions
being 5 mM NaOH for 1 min, changing with an
exponential curve to 100 mM NaOH at 12 min
at a
AS18 2
l/min. These conditions
weremaintained for a further 3min before return-
ing to the starting conditions for the next 8min (to
re-equilibrate the column) prior to the next injec-
tion. Another alternative to HILIC is ion-pair
liquid chromatography (IPLC), but it has to be
flow rate of 300
m
firmly established in this role in the
future.
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