compound and its metabolites.R-(+)-Pulegone is used as a positive
control.
(4) Add 10mL of the NADPH stock solution to start the reactions. After
30 min of incubation, 400mL of 5 mM dithiothreitol in methanol is
added into each well to stop the reactions (Note 5).
(5) After vortexing and centrifugation, 60mL of supernatants is injected
into a HPLC column for analyses.14.1.4.3 Instrumentation LC/MS/MS is carried out on a Finnigan LCQ ion-
trap mass spectrometer interfaced to a Shimadzu LC-10Avp HPLC system
consisting of two pumps, a refrigerated autoinjector and a fluorescence detector.
The electrospray ionization is employed in negative ion mode. Full scan mass
monitoring is employed at mass range of 500–1100 Da. The heated capillary
temperature is set at 270C, the sheath gas flow rate is 70 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 dGSH. When MSn
(n= 2, 3, or 4) spectra of dGSH adducts are collected, the normalized collision
energy is 20–30%. The collision gas is helium.
14.1.4.4 LC/MS/MS Analysis The samples (60mL) are loaded onto a
Phenomenex Prodigy ODS-2 column (4.6 mm 150 mm). The flow rate is set
at 1 mL/min. Fluorescence signal is recorded at excitation wavelength of 340 nm
and emission wavelength of 525 nm. The HPLC eluent after fluorescence
detector is split to allow a flow of 200mL/mL into the electrospray source of the
ion-trap mass spectrometer. The mobile phase consists of solvent A (0.1% formic
acid in water) and solvent B (0.1% formic acid in acetonitrile). The HPLC runs
are programmed at 20% of B for 3 min, followed by a linear gradient to 50% B at
23 min, a linear gradient to 90% B at 40 min, 90% B at 43 min, 20% B at 43 min,
and 20% B at 50 min. Total run time is 50 min.
14.1.4.5 Data Analysis A calibration curve of the fluorescence detector
should be obtained upon installation and once every month using a
concentration range of dGSH in water:5 mM dithiothreitol in methanol
(1:2). QC standards (preferable with a concentration of 1mM) should be
included in each batch of analysis to ensure accuracy of calibration. The
fluorescence response factor is usually very consistent. Thus, daily calibration
is not recommended.
Fluorescence chromatograms from the blank, test compound control, and
sample incubations are superimposed. Additional peaks present only in the
sample chromatogram are considered adducts. Mass spectra (full scan and
MSn) of the peak provide additional confirmation. Quantitation is based on
comparison of fluorescence peak area of adduct versus that of dGSH standard
(external calibration). Quantitative analysis should not be performed if the test
compound or its metabolites have fluorescence interference at the wavelength
458 PROTOCOLS FOR ASSESSMENT