Supplemental Materials
1. LC-MS/MS based quantitation
Samples obtained from in vitro incubations were analyzed by a LC-MS/MS system
consisting of a Shimadzu HPLC and an API4000 Qtrap mass spectrometer. The
Shimadzu HPLC was made of two LC-20AD pumps with a CBM-20A controller,
DGU-20A solvent degasser and a SIL-20A autosampler (Shimadzu, Columbia, MD,
USA). The mass spectrometer was equipped with a turbo-V ionization source
(Applied Biosystems, Ontario, Canada). Chromatographic separation was achieved
using an Agela Venusil XBP C18 column (50 × 2.1 mm; 5 μm particle size). Column
temperature was held at 40 oC. Gradient elution at a flow rate of 0.3 mL/min was
performed using the following mobile phase: A, acetonitrile:water with 0.1% formic
acid (5:95, v:v) and B, acetonitrile:water with 0.1% formic acid (95:5, v:v). The total
run time was 6 minute.
For MS/MS quantitation, the API4000 Qtrap mass spectrometer was operated under
the ESI positive mode (multiple reaction-monitoring, MRM mode) to detect
clopidogrel metabolites and the internal standard with a dwell time set to 100 ms. Ion
transitions monitored were: clopidogrel, 322.1 (M+H)  212.2 and 322.1 (M+H) 
184.1; 2-oxo-clopidogrel, 338.1 (M+H)  183.1 and 338.1 (M+H)  155.1;
clopidogrel carboxylic acid, 308.1 (M+H)  198.2, and diazepam (internal standard,
IS), 285.2 (M+H)  193.2. For sulfonylureas, ion transitions monitored were:
glipizide, 446.2 (M+H)  321.2 and 446.2 (M+H)  347.2; glimepiride, 491.3
(M+H)  352.2; gliclazide, 324.2 (M+H)  110.2. For sulfonylurea analysis, each
served as the other drug’s internal standard. After setting mass transition and collision
energy for each analyte and the internal standard, all other parameters were optimized
for the best sensitivity. Data were collected and processed using AB Sciex Analyst
1.5.2 data collection and integration software.
2. Fig. 1 Metabolic pathways of clopidogrel
3. Fig. 2. Depletion of gliclazide (GCZ), glimepiride (GMR) and glipizide (GPZ)
in human liver microsomes, human intestinal microsomes and recombinant
CYP2C9.
A. Human liver microsomes
B. Human intestinal microsomes
C. The recombinant human CYP2C9.