proteins, lipids, and other endogenous material interfere with the detection of
drug-derived material. Mass spectrometry (MS) has become the analytical tool
of choice for the detection and identification of metabolites due to its ability to
easily couple to liquid chromatography (LC) systems through atmospheric
pressure ionization (API) interfaces. The high sensitivity and selectivity of liquid
chromatography/mass spectrometry (LC/MS) and its ability to separate, detect,
and identify many metabolites in the presence of endogenous material, makes it
well-suited for drug metabolism studies. In a previous article (Ma et al., 2006),
we discussed recent advances in the LC/MS technologies and current practices
for profiling, characterization, and identification of drug metabolites in drug
discovery and development settings. This chapter represents an extension of
the previous article with the incorporation of new developments in metabolite
identification and LC/MS instrumentation with particular emphasis on the
detection and identification of reactive metabolites and intermediates.
11.2 LC/MS Instrumentation
11.2.1 High Performance Liquid Chromatography (HPLC)
HPLC is now one of the most widely used analytical tools to separate complex
mixtures. Liquid chromatography uses differential analyte interactions
between a stationary phase and a liquid mobile phase to effect separation of
components in a mixture. The degree of separation depends on the extent of
interaction between the solute components and the stationary phase. The
interaction of the solute with mobile and stationary phases can be manipulated
through choices of solvents and stationary phases. The choice of chromato-
graphy conditions for metabolite identification studies can be critical. It is
important to separate the metabolites from matrix ions and from each other. A
good separation can facilitate the ionization and characterization of the
compounds of interest. Traditional metabolite characterization experiments
are conducted using reverse phase LC with a slow gradient and long column to
ensure adequate separation of drug metabolites in biological matrices.
Further advances in instrumentation and column technology are needed to
achieve significant increases in resolution, speed, and sensitivity in liquid
chromatography. Better resolution and reduced analysis time can be attained by
reducing the particle size. In 2004, columns with smaller particle sizes (1.7mm)
and instrumentation designed to work at 15,000 psi were developed and
commercialized by Waters Corporation to achieve a new level of performance.
This new technology is called ultra performance liquid chromatography
(UPLCTM). UPLC is rapidly gaining acceptance as an ideal separation tool for
complex mixture analysis in drug metabolism studies due to its superior
chromatographic resolution, sensitivity, and speed of analysis. The typical peak
widths generated by the UPLC system are in the order of 1–2 s. The reduction in
peak width increases the sensitivity by three to five fold. The increased efficiency
320 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY