Biology Reference
In-Depth Information
INTRODUCTION
UPLC) techniques to take advantage of the
improved chromatographic resolution provided
by these more advanced LC techniques.
8,9
A
more limited number of applications has also
been published for capillary and miniaturized
LC systems. Obviously, the operation of the
hyphenated technique of LC-MS, despite the enor-
mous technical advances made in the last two
decades,
The application of liquid chromatography
(LC) for metabolomic/metabonomic studies, in
which untargeted analytical methods are used
in an attempt to obtain metabolic pro
les of
samples of bio
uids or tissues that are as
comprehensive as possible in order to advance
biological understanding and discover novel
biomarkers of various conditions, forms an
increasingly important area of research.
1,2
Although in principle a wide range of analytical
methodologies could be used for such work, in
practice the major techniques employed on
a routine basis are gas chromatography (GC),
nuclear magnetic resonance (NMR, especially
1
HNMR) spectroscopy, capillary electrophoresis
(CE), and increasingly liquid chromatography
(LC). Indeed, the rise of LC-based methods
d
in
particular those using LC-MS
d
has been very
rapid, given that published applications only
began to appear early in this century (see, e.g.,
Plumb, Stumpf, et al. and Plumb, Granger,
et al.
3,4
). However, LC-based methods have
rapidly expanded in their application in this
type of holistic, or global, form of metabolic
pro
culty, and factors
such as sample-dependent ion suppression/
enhancement represent an ever-present problem
when the composition of the sample is both
unknown and variable. Such considerations
mean that although LC-MS can be used to rapidly
generate large volumes of data in the pursuit of
metabolic pro
is not without dif
ling, the value of that data depends
verymuch on the ability of the analyst to ensure its
quality. Clearly, there is always some level of
con
ict between the desire for the most compre-
hensive metabolite pro
le technically possible
and the urgent need for high sample throughput.
Indeed, in order to maximize throughput, many
researchers advocate eliminating the separation
step completely and infuse the samples directly
into the MS. However, our view is that except for
very simple matrices, the disadvantages of such
a
approach with its attendant
problems based on matrix interferences
d
in
particular, ion suppression or ion enhancement
and the dif
“
separation-free
”
ling because of their ready compatibility
with aqueous biological samples, which
d
like
urine or blood plasma
d
can usually be analyzed
with minimal sample preparation compared to
GC, for example. The use of LC-MS also poten-
tially affords high sensitivity (though this is
analyte-dependent) combined with spectro-
metric information that greatly aids analyte iden-
ti
culty of distinguishing between
isobaric species and structural isomers
d
outweigh any possible increase in throughput. In
particular, the requirement to obtain the most
comprehensive metabolite pro
les requires a
chromatographic separation. The need to reduce
the length of the chromatographic run to
a minimum has, however, been one of the driving
forces behind the adoption of UHPLC-MS and the
displacement of the lower resolution HPLC-MS
methods. Thus, the peaks obtained via UHPLC
are usually only 2 to 5 seconds wide at the base,
affording signi
cation. As a result of these favorable analytical
properties, there has been an enormous expansion
in the use of LC-MS-based techniques.
5
e
7
Initially,
the LC systems employed were based on conven-
tional high-performance liquid chromatography
(HPLC) with separations performed on 3 to 5
m
particles. More recently, however, there has been
a general move towards the use of separations
on sub-2
m
cantly greater peak capacity than
conventional HPLC methods. However, if the
need for high throughput is paramount, then it is
quite possible to use UPLC run times of
m particles and employing higher pres-
sures than conventional HPLC in ultra-high-
pressure LC (UHPLC, or ultra performance,
m
Search WWH ::
Custom Search