Biology Reference
In-Depth Information
sample.
24
An example of this type of separation is
shown in
Figure 2
.
Although specimens such as urine generally
present few problems for metabolite pro
peaks are only 3 to 5 seconds wide, requiring
similar speed of response from the detector
and thus a rapid duty cycle from the mass
spectrometer.
Although retention in RPLC is generally
controlled via changes in mobile phase composi-
tion, temperature can also be used as a variable
and, with the recent resurgence of interest in
high temperature (HT) LC, there have also
been some limited demonstrations of the poten-
tial usefulness of operating at higher tempera-
tures for pro
ling
by LC-based methods, with sample preparation
often limited to centrifugation and dilution,
protein-rich samples such as plasma/serum and
others are more problematic. Typically, the
proteins in such samples are removed by the
simple expedient of mixing the sample with an
excess (three volumes) of a solvent such as meth-
anol or acetonitrile, causing the proteins to
precipitate followed by their removal by centrifu-
gation. Solid-phase extraction (SPE) can also be
applied
27
to remove proteins from plasma and
an online method for protein removal,
28
ling samples containing mainly
polar metabolites, such as urine. Thus, operating
at high temperature decreases solvent viscosity,
meaning that higher
flow rates can be used if
desired, and also changes solvent properties
such that the need for organic modi
turbu-
ers can be
reduced or even eliminated. In one example of
the application of HT-UPLC to metabolite
pro
lent
ow chromatography (TFC), has been the
subject of a short investigation for plasma metab-
olome pro
ling. Plasma samples are
rst passed
ling, the temperature of the UPLC column
was maintained at 90
o
C
21
while a normal
RP-solvent gradient was used to elute the
urinary components. In another example, the
organic solvent was dispensed with and
a temperature gradient was performed to elute
urinary-excreted metabolites.
22
Lipids represent an important class of analytes
in their own right, which has led to the develop-
ment of the sub
though a
large particle-containing column
(25
e
50
m) in which small molecules are
retained and the proteins are eluted to waste as
a result of
m
“
turbulent
ow
”
caused by the combi-
nation of high
flow rates and large chromato-
graphic particles. Plasma can be directly
injected onto the LC system without compro-
mising chromatographic performance. The meth-
odology can be thought of as either online
extraction or a crude 2D-LC method, as the next
step is to elute the retained analytes from the
TFC column (using a reversed-phase solvent
gradient) on to a conventional HPLC column to
obtain the metabolic pro
eld of lipidomics. Gradient
RPLC has formed the basis of a number of meth-
ods
23
e
25
with gradient systems based on the use
of acetonitrile-aqueous ammonium formate (10
mM) 2:3 v/v versus acetonitrile-isopropanol 1:9
v/v (containing ammonium formate (10
mM)),
24
e
25
or 10 mM ammonium acetate versus
acetonitrile-isopropanol 5:2 v/v (containing
ammonium formate (10 mM).
26
Elution of lipids
was accomplished at 0.4 ml min and 55
o
Conan
Acquity
le. The preliminary
investigation was promising
28
but revealed inter-
esting differences between pro
les found by TFC
versus those of methanol-precipitated plasma
showing, for example, reduced amounts of phos-
pholipids (ca. tenfold reduction) for the former.
Finally, for samples such as blood (but also other
bio
HSS T3 column (2.1 mm i.d.
100
mm) via linear gradients rising
first from 100%
of the aqueous solvent to 40% of the organic
mobile phase over 3 min, then to 100% organic
solvent over the next 10 min, with this composi-
tion held for 2min before returning to the starting
conditions for 3 min prior to injection of the next
uids such as urine or bile), techniques such
as dried blood spots, in which the sample is
applied to a paper matrix and then subsequently
extracted with solvent, can be used to remove
protein. This type of blood spot technique has
been
brie
y
explored
for metabonomic
Search WWH ::
Custom Search