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The final volume of methanol (solvent for clopidogrel) did not exceed 1.0% of the total incubation volume and was identical in all incubations including controls. Each reaction mixture was equilibrated for 4?min at 37��C in a shaking thermomixer. The reaction was initiated by adding the NADPH-regenerating system and the system incubated for the respective time at 37��C. Reactions were stopped by addition of 100??L of chilled acetonitrile (containing 6.5??M naproxen as internal standard) and cooled on ice for 10?min. Precipitated proteins were removed by centrifugation at 10?000?g http://www.selleckchem.com/products/pifithrin-alpha.html for 10?min and supernatants were analysed by HPLC as described below. The fraction of substrate metabolized was calculated as the difference between the measured and initial clopidogrel or clopidogrel carboxylate concentration expressed as a percentage of the initial concentration. Cytochrome P450 inhibition studies were performed in the presence of the respective inhibitors or substrates following the same incubation procedure as described for the kinetic experiments. Stock solutions http://en.wikipedia.org/wiki/Temsirolimus containing inhibitors (see figures) were prepared in methanol or in water. The final volume of methanol did not exceed 1.0% of the total incubation volume and was identical in all incubations including controls. The inhibitor concentrations are given in the figures. IC50 values were calculated by non-linear regression analysis using the software program GraphPad Prism version 4.00 (San Diego, CA, USA). Clopidogrel concentrations were determined using a LaChrom? high performance liquid chromatography (HPLC) system equipped with an UV detector operating at a wavelength of 235?nm, a column oven, a quaternary pump and an autosampler. The column temperature was maintained at 32��C and the injection volume was 30??L. Separation was performed on a Nucleosil 50-5-C18 column http://www.selleckchem.com/screening/pfizer-licensed-library.html equipped with a corresponding guard column using a gradient of solvent A (sodium phosphate 0.01?M, pH 3.0:acetonitrile; 50:50?v��v?1) and solvent B (sodium phosphate 0.01?M, pH 3.0:acetonitrile; 20:80?v��v?1). The gradient started at 80% A and 20% B for 2.5?min, changed to 100% B for 3.5?min and finally returned to the starting conditions for 4?min. The flow rate was 1?mL��min?1 and the total run time 10?min. The variability of the method was