Hilic-Ms: from targeted to untargeted
Narduzzi Luca, Arapitsas Panagiotis, Della Corte Anna, Angeli Andrea, Mattivi Fulvio.
Research and Innovation Center, Fondazione Edmund Mach, Via E. Mach 1, San Michele all’Adige 38100 (TN) Italy.
Introduction
The development of a new holystic chromathographic method is one of the most challenging part of a metabolomics experiment; untargeted methods do require, not only a
good detection for how many metabolites as possible, but also high stability and repeatability during the analysis, in order to use statistics software to determine alignment, chemometrics
and to define a metabolic space.
To design an untargeted method, a good strategy is to start from a targeted one, already validated for as many compounds as possible, and try to adapt it to untargeted values,
modifying the required parameters in order to introduce the due stability and repeatability in very long batches of analysis. In this work, using an already validated targeted method by
HILIC-MS/MS (Gika et al. 2012, JCA), we developed a new method for untargeted analysis of polar compounds in grape, using 4 main developing steps:
A) chromathographic
modifications;
B) sample dilution;
C) column stabilization; D) instrumental stability.
D)
instrumental stability: The number of
the features was decreasing during the
on-going injections (Figure 5B), as peak area was
dramatically dropping (such as adenosine)
after a certain number of analysis (Figure 6).
Taking
in
consideration
the
following
parameters: Leu-enk accuracy and intensity,
system pressure, peak accuracy and intensity
in STDmixes and QCs samples, number of
features and PCA overview, allowed us to
restore the best instrumental conditions.
Fig. 2: A & B show the number of features (obtained with XCMS peak picking and alignment software) during QC injections between different dilution values both wine and grape. C & D show peak area of some common grape compounds at different dilution levels (measured with Waters targetlynx software). Figure E & F show the peak shape of some metabolites at different ACN concentration. 1:2 ratio showed best sensitivity, peak shape and number of features.
The method:
1. Grape samples were extracted as
reported in the paper Theodoridis et al.
(2011). Briefly the samples were
extracted with CH3OH/H2O/CHCl3
2/1/1, the acqueous fraction was collect
and diluted with ACN in order to
eliminate
contaminants
(check
paragraph B). The filtered was
analyzed by UPLC HILIC AMIDE (BEH
Amide Waters) coupled to a Synapt
HDMS
(Waters)
with
the
chromathographic condition described
in Figure 1.
A)
chromathographic
modifications:
A clean-up
step was added in the
chromathographic run at
30-35 minutes with 80% of
eluent, to avoid accumulation
effects in the column (Figure
1). Concentration of the
ammonium formiate salt was
set equal for both eluents (20
mM) to stabilize pH, while, a
weekly preparation of the
eluents was done using salt
stock solution
B)
sample dilution: A dilution
step was added to the sample
extraction, adding ACN followed by
a 1 hour storage at 4°C. The best
dilution ratio was selected testing
many different concentration, as
showed in figure 2. Best results
were obtained with dilution ranging
from 1:2 to 1:3. To achieve higher
sensitivity, we choose 1:2 dilution.
After 4° C storage, samples showed
a white precipitate that we avoided
to inject.
C)
column stabilization:
Sample analysis in new HILIC
AMIDE columns showed a
trend in delta pressure
(Figure 4) and PCA analysis
(Figure 3a). To avoid trends,
we perfomed a so called
column stabilization with 100
QC
injections.
In
our
experiment, after 40 to 50
injections, columns looks like
to achieve better stability
(Figure 3B).
blanks
samples
Fig n. 3: figure 3A shows a PCA of the equilibration step; in the red spot is possible to see how after 40 injections on a new column, the signal looks more repeatable between different injections. Figure 3B: shows equilibration step in a new column plus 4 different batch analysis (around 180 grape extract injections each one). The difference given by Principal Component 1 (the main one) decreases during the analysis, reaching a bottom in batches B, C and D, indicating a rise in stability.
Conclusions
The aim of this work was to obtain a
chromatographic method capable to improve the
stability and repeatability of long sequences. The
method adjustements (A&B), the choice of a
stabilization protocol of new columns (C) and the
control of several LC-MS parameters (D) during the
sequence, allowed to define a set of conditions
useful to reduce the systematic error which can be
critical on HILIC columns. This is a pre-requisite for
the succesful use of multivariate statistical analysis
in untargeted metabolomics.
Fig n.4: a comparison between range pressure (delta between maximum and minimum pressure) between equilibration steps, and the first batch of analysis.
Acknownledgements
We would like to thank Domenico Masuero and Cesare Lotti for
technical help. We are also grateful to the FEM bio-statistics and
data management group for statistical support.
cleaning
Fig n.6: shows peak area of few compounds during the analysis. System clean-up restores the initial condition.
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Fig n.5: Fig 5A shows the number of features against the number of injections, better visualized in the zooming present (figure 5B ).
References
1: Georgios Theodoridis, Helen Gika, Pietro Franceschi, Lorenzo Caputi, Panagiotis Arapitsas, Mattias Scholz, Domenico Masuero, Ron Wehrens, Urska Vrhovsek, Fulvio Mattivi
LC-MS based global metabolite profiling of grapes: solvent extraction protocol optimisation
Metabolomics (2012) 8:175–185 DOI 10.1007/s11306-011-0298-z
2: Helen Gika, Georgios Theodoridis, Urska Vrhovsek, Fulvio Mattivi
Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometry
Journal of Chromatography A, 2012), 1259 :( 121– 127)
Fig. 1: the figure shows the main differences between the previous chromathographic targeted method (Gika et al. 2012), and the new untargeted one. The method consistes in 3.5 of 100 A, up to 28% B at 25 min, 60% B at 30 minutes and then a 5 minutes clean up step with 80% of B and final 10 minutes ri-equilibration to 100% A.
