mass resolution of 10^4 –10^5 with a mass accuracy better than 2 ppm. During the
past decade, the application of the sector instruments has declined owing to the
rapid advancement of TOF and Fourier transform (FT) mass spectrometry,
which are capable of high resolution but more importantly, are more
compatible with atmospheric pressure ionization sources.
Liquid chromatography coupled with hybrid quadrupole time-of-flight mass
spectrometers has emerged as a powerful tool in accurate mass measurement
for metabolite identification, owing to its ease of use, fast acquisition speed,
high sensitivity, and high mass accuracy in both TOF–MS and MS/MS mode
(Chernushevich et al., 2001). Eckers and co-workers reported a mass accuracy
of within a fewmTh of the theoretical value for protonated and deprotonated
molecule ions as well as product ions using a Q-TOF instrument (Eckers et al.,
2000; Wolff et al., 2001). The hybrid LTQ–FTICR and LTQ-FT-Orbitrap
mass spectrometers integrate ease of operation, standard HPLC flow rate
compatibility and automatically triggered data-dependent MSnexperiments
with high mass resolving power and mass accuracy. These high resolution
instruments have demonstrated great value for rapid confirmation of expected
metabolites or structural elucidation of unusual metabolites (Gratz et al., 2006;
Peterman et al., 2006; Sanders et al., 2006).
11.4.5 Nanospray Ionization (NSI) MS for Metabolite Identification
Nanospray ionization is a variation of regular ESI in that the typical flow rate
is reduced to between 30 and 200 nL/min. Because of the very low flow rate at
which samples are consumed, this technique is rapidly being integrated in many
analytical applications including proteomics, metabolite characterization, and
pharmaceutical analysis. An approach of combining fraction collection with
automated chip-based NSI MS was recently introduced for metabolite
identification (Hop, 2006; Staack et al., 2005). LC effluent was collected into
a 96-well plate and the fractions of interest were infused using an automated
chip-based nanospray system for structure elucidation.
Compared to conventional LC/MS analysis, the NSI technique has some
distinct advantages. For example, a complete mass spectral analysis of a
sample using online LC/MS usually requires multiple injections to acquire the
necessary data, while NSI typically requires only a single infusion of selected
chromatographic fractions. Due to the low flow rates involved, mass spectral
acquisition on any given peak can be extended for prolonged periods. This
allows many different types of mass spectrometric experiments to be performed
including, constant neutral loss scan, precursor ion scan, product ion scan,
MSn, and even polarity switching. In addition, online LC/MS typically requires
nanogram quantities of drug metabolites to acquire high quality spectra
suitable for structural characterization, while NSI, due to the ability to signal
average for a long period of time, consumes picogram quantities to generate
the same high quality MS/MS spectra (Borts et al., 2004). NSI also exhibits
more uniform ionization efficiencies between metabolites and parent drug. This
TECHNIQUES FOR IMPROVING METABOLITE DETECTION 343