results in the reduction of analytical run time by a factor of 5, while still
maintaining the chromatographic resolution (Johnson and Plumb, 2005; Plumb
et al., 2004). The overall enhancement in the chromatographic resolution and
peak capacity is translated into a reduction in the number of coeluting species.
Castro-Perez et al. reported the detection of additional prochlorperazine
metabolites from a rat liver microsomal incubation using UPLC coupled to a
Q-TOF mass spectrometer, as shown in Fig. 11.1 (Castro-Perez et al., 2005a). In
the HPLC separation, just three of the doubly hydroxylated metabolites were
detected; however, with the UPLC analysis, all eight metabolites were detected
within 4.5 min run time. UPLC systems with similar technology are now
available from other manufacturers (Frank, 2006; Pereira et al., 2006).
11.2.2 Atmospheric Pressure Ionization Methods
A prerequisite for detection, identification, and quantification of any species by
MS is that all analytes must be converted into gas-phase ions before they enter
the mass analyzer. API techniques are most widely used for metabolite
identification, mainly due to their ability to couple to liquid chromatography
and generate intact gas-phase molecular ions at very high sensitivity (Rossi,
2002; Voyksner, 1997).
11.2.2.1 Electrospray Ionization (ESI) Much of the current importance of
the electrospray technique derives from the pioneering work of Dole et al.
(1968) who first recognized the possibility of generating gas-phase ions by
FIGURE 11.1 Comparison of the extracted ion chromatograms for doubly hydro-
xylated metabolites (m/z 406) of prochlorperazine from incubations with rat liver
microsomes obtained from HPLC/MS (a) and UPLC/MS (b). Reprinted from Castro-
Perez et al. (2005a) with permission of John Wiley and Sons Limited.
LC/MS INSTRUMENTATION 321