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studies
29,30
and an example of the UPLC pro
et al.
33
). Examples using the format HPLC
include separations performed with ZIC-HILIC
(100
le
of urine obtained following this type of dried
blood spotting compared to direct analysis of
the sample is shown in
Figure 1
. All of these tech-
niques render samples suitable for RPLC but
each method gives slightly different metabolite
pro
m)
34
or the Aphera NH2
polymer column (150
4.6 mm, 3.5
m
m)
35
and
UHPLC applications of HILIC include examples
based on the Acquity BEH HILIC (2.1
2 mm, 5
m
50 mm,
ling
32,36
in, for example,
animal disease model metabotyping,
32
toxico-
logical investigations, or for cancer biomarker
discovery.
37
les.
1.7
m) material for pro
m
Hydrophilic Interaction Liquid
Chromatography (HILIC)
As indicated earlier, polar/ionizable, metabo-
lites (sugars, amino acids, organic acids, etc.)
pose a problem for RPLC methods due to poor
retention. For the analysis of such analytes, the
current standard approach in metabolomics is
to use HILIC. Such separations employ solvents
containing a very high proportion of organic
modi
In the
latter
application,
the
pro
ling of metabolites present in the urine of
rats exposed to galactosamine (a model hepato-
toxin) was performed by UPLC on an Acquity
BEH HILIC column (2.1
100 mm, 1.7
m) at
m
a
flow rate of 0.4 mL/min and a column temper-
ature of 40
C. A solvent gradient separation was
used with 0.1% (v/v) formic acid and 10 mM
ammonium acetate in acetonitrile-water 95:5
v/v as the initial organic and 0.1% aqueous
formic acid and 10 mM ammonium acetate in
acetonitrile-water 50:50 as aqueous solvent.
The initial solvent composition employed was
99.0% organic and 1% aqueous solvents for
1.0 min, followed by a linear gradient of the
aqueous solvent over the next 11.0 min. On
completion of the gradient, the solvent composi-
tion was returned to the starting conditions
(0.1 min) and allowed to re-equilibrate for
4 min before injection of the next sample.
Although HILIC has been used for polar
metabolites as described earlier, it has also
been applied as a complementary method for
polar lipid separations on a Diol column to
complement an RPLC separation in a 2D separa-
tion. Here the eluent from the HILIC column was
collected manually, desalted, and concentrated
before reanalysis by RPLC.
37
The isocratic sepa-
ration used a 5
er, and indeed the water content should
be limited to no more than 50%; as a result, MS
sensitivity in HILIC is often better than for
RP methods because ionization ef
ciency is
improved due to ef
cient generation of spray
conditions. The value of HILIC for untargeted
metabolite pro
ling has now been demonstrated
in a wide range of applications
31
e
34
and, when
combined with RPLC on the same samples,
probably provides the most comprehensive
metabolome coverage currently available by
LC using
methodology. HILIC can
either be used directly on samples such as urine
or samples can be extracted using, for example,
SPE
“
routine
”
first to separate polar from nonpolar metab-
olites. This approach was employed for the anal-
ysis of rat urine samples
31
in which the
unretained portion of the sample following SPE
was analyzed via HILIC/electrospray ionization
(ESI)-MS (and the retained portion by RP-LC/
ESI-MS). The polar metabolites obtained in this
way were pro
MNucleosil
100-5OH packing
m
contained in a 10 mm i.d.
250 mm column,
with a mobile phase consisting of a mixture of
cyclohexane and isopropanol-water-acetic acid-
28% aqueous ammonia 86:13:1:0.12 (v/v)
led using HPLC on a ZIC-HILIC
column (100
2.1 mm, 3.5 mm). Although still
not as widely employed as RPLC, the use of
HILIC has continued to increase with examples
of both HPLC and UHPLC-based metabolo-
mic/metabonomic studies (reviewed in Spagou
in
flow rate of 1 ml min.
38
It is clear that RP and HILIC, if used alone,
will not provide comprehensive metabolome
a ratio of 1:9 v/v at a
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