deriving detailed structural information once preliminary data on the nature of
the metabolic change is available from LC/MS. Static NMR analysis is often
performed on a metabolite at high purity and thus requires time-consuming
isolation and purification steps to generate a suitable sample fromin vivoorin
vitroexperiments. The direct coupling of LC with NMR eliminates the need for
extensive sample purification steps and, therefore, increases its capability of
solving structural problems in complex mixtures. The simultaneous coupling of
LC with NMR and MS, enables the acquisition of both NMR and MS data
from a single chromatographic analysis and provides comprehensive data for
unambiguous structural determination (Dear et al., 2000; Sidelmann et al.,
2001). The application of this technique in pharmaceutical research
and development was recently reviewed in the literature (Lindon et al., 2000;
Yang, 2006).
11.6.2 LC/ICPMS
In the absence of radiolabeled drug or reference standards, the detection and
quantification of drug-related material in a biological sample is problematic.
Liquid chromatography combined with inductively coupled plasma MS
(ICPMS) could, in principle, offer an analytical solution (Marshall et al.,
2004; Montes-Bayon et al., 2003). The temperature in the ICP source is
approximately 5000 K, resulting in the production of singly charged elemental
ions with high efficiency. LC/ICPMS provides a sensitive and highly selective
platform for the detection and quantitation of drug metabolites containing
atoms such as bromine, chlorine, sulfur, and selenium, which are not
commonly found in endogenous material (Corcoran et al., 2000; Jensen
et al., 2005; Nicholson et al., 2001). Unfortunately, ICPMS does not provide
structural information and if one of the isotopes mentioned is not present in
the drug, the technique has limited value for metabolite profiling.
11.7 Characterization of Unstable Metabolites
Identification and quantification of unstable metabolites pose a serious challenge
for mass spectrometric investigations. The unstable metabolites may undergo
decomposition, rearrangement, and/or adduct formation during sample proces-
sing and mass spectrometric analysis. This may thereby lead to incorrect
determination of metabolite quantification and structural characterization. In
these cases, extra precautions are needed during sample preparation, while
conducting the LC/MS experiment, and when interpreting LC/MS results.
11.7.1 Glucuronides
Glucuronides are often thermally unstable in the ion source of mass
spectrometers and can break down to the aglycone via thermally induced
CHARACTERIZATION OF UNSTABLE METABOLITES 349