quaternary pump and a Series 200 autosampler (Norwark, CT). The mass
spectrometer employs an electrospray ionization probe with positive ion
detection. A Turbo IonSpray probe is employed in positive ion mode. The
instrument parameters are optimized and set as follows: declustering potential,
36 (GS–AM), 46 (GSSG), 26 (g-Glu-Glu); focusing potential, 250 (GS–AM),
340 (GSSG), 230 (g-Glu-Glu); entrance potential, 10 (GS–AM, GSSG,
g-Glu-Glu); collision energy, 27; collision cell exit potential, 16; nebulizer
gas, 6; curtain gas, 10; collision gas, 4; ionspray voltage, 3000; temperature,
150 C. The collision gas is nitrogen. Samples (100mL) are loaded onto an
Agilent Zorbax SB-phenyl column (4.6 250 mm, 5mm, Wilmington, DE) at
the flow rate of 1 mL/min with a 1:5 split to the mass spectrometer ion source
and a waste container. The mobile phase consists of 5 mM ammonium acetate
in H 2 O–acetonitrile–trifluoroacetic acid (95:5:0.1, v/v/v). The HPLC run time
is 7 min. The quantitation is based on multiple reaction monitoring (MRM) of
the transitions ofm/z365.2 –>236.1 (GS–AM), 613.1 –>355.1 (GSSG), and
277.0 –>148.0 (g-Glu-Glu).
14.4 Perspectives
In addition to their propensity to covalent modification of proteins, some
reactive metabolites also can react with DNA molecules to form corresponding
adducts (Guengerich, 1992; Miller and Miller, 1981; Pereg et al., 2002; Wogan
et al., 2004). Therefore, screening of DNA adduct formation could provide
some guidance in lead optimization when certain structural modifications can
be made to eliminate the potential risk of genotoxicity of a suspected mutagen
based on structural information of DNA adducts. Some reactive intermediates
(e.g., epoxide, quinone, quinone methide, and carbocation) readily react with
nucleosides. Other reactive metabolites (e.g.,N-hydroxylamines of aromatic
amines), however, usually do not initially react with nucleosides, but require
additional bioactivation steps (e.g., O-acetylation, glucuronidation, or sulfa-
tion). Simple incubation systems containing liver fractions in the presence of
cofactors and nucleosides could be useful for generation of adducts, and LC/
MS/MS is the method of choice for characterization of adduct structures.
Because of the availability of several well-established platforms for the
prediction of mutagenicity (Muller et al., 1999), the widespread use of the
DNA adduct screening is not expected.
With significant advances of the LC/MS/MS and other bioanalytical techno-
logies, it becomes possible to qualitatively characterize and monitor covalent
drug–protein adducts (Yang et al., 2006). Elucidation of the protein target
would not only yield information leading to possible mechanism of toxicity but
also provide lead proteins to search for antibodies as a potential marker of
toxicity (hypersensitivity) in subjects (Boelsterli et al., 1995; Cohen et al., 1997;
Welch et al., 2005). In addition, the covalent modification of cytochrome P450
enzymes is often mechanistically intriguing for mechanism-based cytochrome
470 PROTOCOLS FOR ASSESSMENT