Biology Reference
In-Depth Information
approximately 2 min (see, e.g., Plumb et al.
9
)and
analyze 20 to 30 urine samples/h while perhaps
achieving the same level of metabolome coverage
as a 10 to 15 min HPLC analysis. Currently,
however, chromatographic run times (for both
HPLC and UHPLC) are typically in the range of
10 to 20 min. This range allows for reasonable
sample throughput and good chromatographic
resolution and detailed protocols of this type for
bio
considered and the various modes and options
for chromatography described.
CHROMATOGRAPHIC METHODS
FO
R METABOLITE PROFILIN
G
Reversed-Phase LC Separations
HPLC-based separations in global metabolic
pro
uids such as serum/plasma and urine and
tissue extracts have been described.
12
e
16
When,
on the other hand, a more detailed investigation
of one or a few samples is the aim, then resolution,
rather than speed of analysis, is the main require-
ment, and the use of very high resolution systems
such as multidimensional or capillary LC may
represent the best strategy.
Irrespective of the desired analytical
outcome, there are a limited number of options
with respect to the mode of chromatography
and chromatographic phases that are avail-
able for use in metabolomics/metabonomics.
Currently, the most widely employed type of
separation is conventional gradient reversed-
phase (RP) chromatography. Such separations
need to be carefully optimized for the metabo-
lites likely to be present in a given matrix; for
example, a method optimized for urine would
be much less suitable for another matrix such
as plasma, as the latter is lipid rich and urine
is composed of much more
m particle size
stationary phases packed into 2.1 to 4.6 mm i.d.
columns 5 to 15 cm in length. Typical RP gradi-
ents for HPLC that we have found useful for
the analysis of samples such as urine used a
2.1 mm i.d.
ling studies employ 3 to 5
m
10 cm column packed with, e.g.,
m, C18-bonded Symmetry
packing held
at 40
C and with a solvent delivery
3.5
m
flow rate of
l/min.
17
For UHPLC (UPLC)-basedmetabo-
lite pro
600
m
m stationary phases
are employed, the columns used vary from 5 to
15 cm in length and are of aproximately 2 mm
i.d.
9,14
e
17
Apart from the higher operating pres-
sures used in UPLC compared to HPLC, the
mobile phases, gradient elution pro
ling, in which sub 2
m
les, and
flow rates used are similar. RPLC is currently
the most widely used separation mode for metab-
olomic/metabonomic pro
ling, stemming partly
from the fact that it is well suited to the analysis of
aqueous samples (e.g., urine, bile, and protein-
precipitated plasma/serum). Gradient elution
is usually over 10 to 30 min depending on the
type of analysis with 2 to 5 min at the end
of the runs for column washing and re-
equilibration. Thus, for urine a typical method
would use 0.1% aqueous formic acid and acetoni-
trile (also containing 0.1% formic acid) for chro-
matography with a gradient beginning at 100%
aqueous formic acid (held for 0.5 min) rising in
a linear gradient to 20% acetonitrile over 0.5 to
4 minutes and then continuing to 95% acetonitrile
at 8 min. The solvent composition would then be
held at 95% acetonitrile for a further minute to
wash strongly retained contaminants from the
column, then returning to 100% aqueous formic
metabolites.
Indeed, one of the limitations of RPLC is that the
analysis of polar/ionic metabolites represents
a considerable challenge because these are often
poorly retained, eluting at, or near, the solvent
front. RPLC is therefore most suitable for the
pro
“
polar
”
“
”
ling of
medium
to nonpolar metabolites.
The pro
ling of these unretained metabolites
demands a different strategy, and the use of
separations based on either hydrophilic interac-
tion (HILIC), ion-pair (IPLC), ion-exchange
(IEC), or aqueous normal phase chromatog-
raphy (ANPC).
In this chapter, the practice of LC-MS
for metabonomic/metabolomic
studies
is
Search WWH ::
Custom Search