Liquid Chromatographic Methods Combined with Mass Spectrometry in Metabolomics - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 144

acid at 9.1, re-equilibrating for 0.9 min ready for
the next sample. For plasma extracts, protocols
for the RPLC-MS analysis of both serum/plasma
and urine have been reported. 10,11
For samples such as urine, minimal sample
preparation is required (often only centrifuga-
tion and dilution 11,14 ) and a typical UPLC sepa-
ration is shown in Figure 1 . With samples, such
as plasma or serum 9,10,15,16 or tissues 18 e 20 ,itis
essential to remove proteins prior to LC analysis
in order to protect the column. It is also the case,
as indicated previously, that relative contribu-
tions of polar and nonpolar metabolites (e.g.,
lipids) differ considerably between urine- and
blood-derived or tissue samples. As a result,
the gradient used for the analysis of such
samples often begins at a higher proportion of
the organic solvent than employed for urine.
Lipids are also more strongly retained on RPLC
columns and, as a result, their elution (and

column cleaning after the analysis) requires
more eluotropic solvent for elution and wash
steps. Generally, methanol (or other alcohols)
appears to be a better solvent for eluting lipidic
contaminants compared to, for example, acetoni-
trile and a typical (UPLC) analysis of such
a sample would use separation on a 1.7

m, 2.1

m

100 mm, Acuity BEH column 50 C eluted
with 0.1% aqueous formic acid (solvent A) and
methanol (also containing 0.1% formic acid,
solvent B) at 0.4 mL/min. The gradient program
employed a series of linear steps, beginning at
95% aqueous formic acid for 0.5 min., and
changing to 60% A at 2.5 min, then to 30% A at
4.5 min and finally rising to 100% B at 10 min.

This solvent composition was held for 2 min
before the column was returned to 95% aqueous
formic acid, where it was held for 2.5 min prior
to the next sample. The very high ef ciency of

UHPLC often means that chromatographic

FIGURE 1 UHPLC-TOFMS (+ESI) of rodent urine on an Acquity BEH C18 2.1 100 mm 1.7 m M column (maintained at
40 C) using a solvent gradient of 0.1% aqueous formic acid versus acetonitrile (containing 0.1% formic acid). The upper trace
shows the metabolites recovered from a

“ blood spot ” paper and the lower trace is of the unextracted urine. Details can be

found in reference 29.