Liquid Chromatographic Methods Combined with Mass Spectrometry in Metabolomics - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 150

Last, a further separation tool with potential
for the separation of highly polar metabolites
is aqueous normal phase chromatography
(ANPC). 43 This technique was used to pro

impressive, and high-resolution, separation

using a 50 m i.d. fused silica capillary, 200 cm

m

long and m porous C18-bonded

particles was applied to the separation of metab-
olites present in cell lysates of the microorganism
Shewanella oneidensis. 46 A reversed-phase
gradient was used, at a pressure of 20 Kpsi,
resulting in the detection of more than 5,000
metabolites over some 2,000 min.
Although long capillaries provide numerous
bene

filled with 3

m

le
metabolites in human urine and plant extracts
on a Cogent Diamond Hydride column

(100 m) via gradient elution using

15.9 mM ammonium formate or 13.0 mM ammo-
nium acetate and acetonitrile-water (containing
15.9 mM ammonium formate or 13.0 mM ammo-
nium acetate) 90:10 v/v at 0.4 mL/min.

2.1 mm, 4

m

ts, there are also some advantages to be
obtained from the use of shorter ones, so, for
example, metabolic pro

ling of the urine of
Zucker rats (a model for Type II diabetes) was
performed on a 320

Miniaturized LC Systems
In addition to the various different modes of
chromatography that can be used for sample
analysis, there is also a range of technical options
that can be useful for metabolome pro

m i.d. (10 cm) column

m

packed with a 3.5 m C18-bonded stationary

phase. 45 This analysis provided a more compre-
hensive pro

m

ling. So,
although not yet widely used for routine anal-
ysis, there are obvious bene

le than conventional HPLC-MS on
a column containing the same packing material,
and of the same length, with about twice as
many ions observed via the capillary method
(presumably as a result of reduced ion suppres-
sion). More recent developments have led to
capillary scale micro

ts to increasing
both sensitivity and resolution, as well as
providing minimal sample consumption, to be
derived from the use of miniaturized LC column
formats. Examples have been shown using micro
bore columns, with internal diameters from 0.5
to 1.0 mm, and capillary columns of varying
lengths in a number of metabolic pro

uidic systems such as the
Integrated Ceramic Micro . 47

uidic Devices In

this device, a 10 cm long, 300 m i.d. separations

m

ling stud-
ies. 44 e 46 For example, C18-bonded silica mono-
lithic columns (0.2 mm i.d., 30 e 90 cm) were
used to pro

channel, containing the 1.7 m Acquity BEH

stationary phase, was employed for the separa-
tion. An example of the type of separation that
can be achieved using this type of platform is
shown in Figure 4 . Here rat urine (1

m

le extracts of Arabidopsis thaliana 43
with MS detection.

In another example, an

l) was

m

FIGURE 4 A gradient RPLC-MS
separation of rat urine on a capillary
scale micro uidic device.