an HPLC system consisting of a Perkin-Elmer Series 200 quaternary pump and
a Series 200 autosampler (Norwark, CT). A Turbo IonSpray probe is
employed for constant neutral loss scanning ofm/z129 in positive ion mode.
The instrument parameters are set as follows: declustering potential, 21;
focusing potential, 160; entrance potential, 10; collision energy, 40; collision
cell exit potential, 12; nebulizer gas, 6; curtain gas, 10; collision gas, 4; ionspray
voltage, 4500; temperature, 300C; step size, 0.1 Da; pause time, 5 ms; scan
rate, 2 s. The collision gas is nitrogen.
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 constant neutral loss scanning ofm/z129 or 27/29
in LC/MS/MS analysis may afford initial detection of GSH/NAc or cyano
adducts. Subsequent MS–MS analysis by CID of MH+species is required for
further structural elucidation of the adducts.
14.1.4 Protocol for Qualitative and Quantitative Analysis of Thiol Adducts
Using Dansyl Glutathione (dGSH)
14.1.4.1 Preparation of dGSH Dansyl glutathione (Fig. 14.1) is prepared by
derivatization of glutathione disulfide (GSSG) with dansyl chloride and
subsequent cleavage of the disulfide bond by dithiothreitol (Gan et al., 2005).
14.1.4.2 Incubation
(1) Prepare NADPH stock solution (20 mM) in water, test compound stock
solution (20 mM) in appropriate solvent,R-(+)-pulegone (Fig. 14.1)
stock solution (20 mM) in water:acetonitrile (1:1), dGSH stock solution
(20 mM, Note 4), potassium phosphate buffer (0.1 M, pH 7.4), and
chilled quenching solution (5 mM dithiothreitol in methanol). Human
liver microsomes (20 mg/mL) are thawed on ice.
(2) Dilute test compound stock solution to 1 mM with water (or20%
organic solvent if necessary) andR-(+)-pulegone stock solution to
1 mM with water.
(3) Prepare incubation mixture by adding 10mL each of stock solutions of
human liver microsomes, dGSH, and a test compound to 160mLof
0.1 M phosphate buffer in 1 mL deep well 96-well plate and preincubate
at 37C for 5 min. A blank incubation without addition of a test
compound is used to prepare a baseline chromatogram for identifica-
tion of adducts. A control incubation without dGSH is used for
identification of potential fluorescence interference from the testGLUTATHIONE,N-ACETYLCYSTEINE, AND POTASSIUM CYANIDE 457