resulting in covalent protein binding in vivo may not be detected in
incubations with common trapping agentsin vitro(Zhang et al., 2003). In
such cases, false negative results might be obtained fromin vitrotrapping
experiments.
14.1.2 Detection of Glutathione/N-Acetylcysteine or Cyano Adducts
Using Ion-Trap Mass Spectrometer
14.1.2.1 Incubation Human or rat liver microsomes (1 mg protein/mL) are
suspended in phosphate buffer (100 mM, pH 7.4) containing EDTA (1 mM),
MgCl 2 (0.1 mM), and GSH (5 mM, Note 1) or NAc (5 mM) orKCN=K^13 C^15 N
(1 mM) in a total volume of 1 mL. A test compound in methanol is added to a
final concentration of 50mM, such that the concentration of methanol in the
incubation mixture does not exceed 0.2% (v/v). Incubations are performed in
the presence of NADPH (1.2 mM) at 37C for 60 min. Control experiments
contain liver microsomes and a test compound in the absence of either
NADPH or trapping agents (Note 2). The reaction is quenched by adding
2 mL of acetonitrile. The suspension is then sonicated for 5 min and
centrifuged at 20,800 gfor 10 min. The supernatants are removed and the
pellets are extracted twice with 1 mL of methanol–water (3:1, v/v). The extracts
are combined with the above supernatants and are evaporated to dryness under
nitrogen at room temperature. The residues are dissolved in 300mLof
methanol—water (3:1, v/v), and an aliquot (75mL) is loaded onto an HPLC
column for LC/MS/MS analysis (Zhang et al., 2005).
14.1.2.2 Instrumentation LC/MS/MS is carried out on a Finnigan LCQ
Deca XP Plus ion-trap mass spectrometer (San Jose, CA) interfaced to a HPLC
system consisting of two Shimadzu LC–10AD pumps (Kyoto, Japan),
a Shimadzu SIL–10AD autoinjector and a Finnigan UV6000LP photodiode
array detector (San Jose, CA). The electrospray ionization is employed in
the positive ion mode. The heated capillary temperature is set at 275C, the
normalized collision energy is 42%, the sheath gas flow rate is 60 units, and the
auxiliary gas flow rate is 20 units. The ion spray voltage, the capillary voltage,
and the tube lens offset are adjusted to achieve maximum sensitivity using the
test compound. The collision gas is helium.
14.1.2.3 LC/MS/MS Analysis The samples (75mL) are loaded onto an
Agilent Zorbax RX-C8 column (4.6 mm 250 mm, 5mm, Wilmington, DE).
The flow rate is set at 1 mL/min with a 1:5 split to the mass spectrometer ion
source and a waste container, respectively. The mobile phase consists of solvent
A (5 mM ammonium acetate in water–acetonitrile–acetic acid, 95:5:0.05, v/v/v)
and solvent B (5 mM ammonium acetate in acetonitrile–water–acetic acid,
95:5:0.05, v/v/v). The HPLC runs are programmed by a linear increase from
0% to 80% of solvent B during a 30 min period. The mass spectra are recorded
in full scan and data-dependent scans of MSn(n= 2, 3, or 4). The MSnspectra
452 PROTOCOLS FOR ASSESSMENT