Separation of plasma from blood samples. The blood samples collected were centrifuged for 10 min at 2000 rpm (Remi Equipment, Mumbai), and the supernatant (i.e., plasma) was pipetted into clean and dry test tubes and immediately frozen at —20 °C until further study was conducted.

Extraction of plasma. The authors quantified rosiglitazone in plasma samples using high-performance liquid chromatography (HPLC) according to a validated method (18). The authors filled 0.9 mL of plasma separated from each blood sample into 10-mL centrifuge tubes. Next, 0.1 mL of the internal standard (pioglitazone 1000 ng/mL) was added to each of those tubes so that the concentration of pioglitazone was 100 ng/mL in all the tubes. The tubes were vortexed for 1 min, then 200 μL of disodium tetraborate was added before the tubes were vortexed for an additional minute. Then 5 mL of dichloromethane was added to the tubes, which then were capped and shaken horizontally for 10 min. The tubes were centrifuged at 3000 rpm for 10 min at room temperature. The organic layer was then removed and evaporated to dryness under a stream of nitrogen at ambient temperature (Zymark Turbo evaporator, Zymark Corporation, San Diego, CA). The authors added 5 mL of a solvent mixture [n-hexane-dichloromethane (80:20 v/v)] to the residue and vortexed the tubes for 30 s. Then, 350 μL of phosphate buffer solution, pH 2.3, was added to the tube, and the solution was vortexed for 2 min and centrifuged at 3000 rpm for 10 min. After phase separation by centrifugation and subsequent removal of the top organic layer, the aqueous phase containing the extracted analytes was analyzed by HPLC (Perkin Elmer, Knauer, Berlin) by injecting 100 μL of the samples in the chromatographic system.

Preparation of mobile phase and buffer. The mobile phase consisted of methanol and mixed-phosphate buffer in the ratio of 30:70 (v/v). Mixed-phosphate buffer (10 mM; pH 2.6) was prepared by dissolving 1.41 g of dibasic sodium phosphate and 1.56 g of monobasic sodium phosphate in 800 mL of water, adjusting the pH of 2.6 with ortho-phosphoric acid, and diluting the solution to 1000 mL with water.

Preparation of standard stock solution. Standard stock solutions were prepared by dissolving 13.24 mg of rosiglitazone maleate (equivalent to 10 mg of rosiglitazone as free base; molecular weights of rosiglitazone maleate and rosiglitazone as free base are 473.52 and 357.44 g/mol, respectively) in acetonitrile in a 100-mL volumetric flask to yield a primary solution with a concentration of 100 μg/mL. Secondary and working standard solutions were prepared by dilution with the buffer solution, pH 2.3.

Internal standard stock solution was prepared by dissolving 10 mg of pioglitazone in acetonitrile in a 100-mL volumetric flask. This solution was further diluted with the buffer solution, pH 2.3, to yield a concentration of 10 μg/mL.

Preparation of standard plasma. Six separate test tubes, each containing 0.8 mL of drug-free plasma, 0.1 mL of working stock solution of rosiglitazone, and 0.1 mL of internal standard solution (i.e., pioglitazone) were used, which provided a concentration of 100 ng/mL, to yield concentrations of 25, 100, 250, 500, 750, and 1000 ng/mL of rosiglitazone. The extraction of plasma was carried out as described above.

Instrumentation. The chromatographic analysis was carried out on a BDS Hypersil C18 column (5-μm particle size, 250 mm X 4. 6-mm internal diameter, Knauer, Berlin) maintained at 30 °C. The analytes were eluted using a mobile-phase composition of 10 mM mixed-phosphate buffer, pH 2.6, and methanol (70:30, v/v) at a flow rate of 1 mL/min. The mobile phase was premixed, filtered through a 0.45-μm membrane filter (Millipak, Millipore, Billerica, MA), and degassed before use. The peaks were determined using a UV detector (Genesys, Thermoelectocorporation) set at a wavelength of 318 nm.

Calibration curve in plasma. The calibration curve of rosiglitazone was calculated in plasma. Different concentrations such as 25, 100, 250, 500, 750, and 1000 ng/mL of the drug were made in blank plasma. The peak area of drug and internal standard were noted, and the peak area ratio was calculated with the following formula: