Working solutions with different concentrations were prepared by dilution of stock with diluent (methanol and water; 50:50%, v/v). Preparation of calibration curve standards and quality control samples Calibration samples were prepared by spiking 950 ��L of control human plasma with the appropriate working solution of each analyte (25 ��L combined selleck chemicals Imatinib Mesylate dilution of losartan, losartan acid, and 25 ��L of amlodipine). Calibration samples were made at concentrations of 1000, 800, 600, 402, 201, 101, 10.5, 1.00, and 0.50 ng/mL for losartan and for losartan acid, and 10.10, 8.08, 6.06, 4.00, 2.00, 1.00, 0.50, 0.10, and 0.05 ng/mL for amlodipine. Quality control samples for losartan, losartan acid, and amlodipine were prepared at concentrations of 858, 854, 8.49 (higher quality control, HQC), 515, 512, 5.
09 (middle quality control 2, MQC2), 124, 123, 1.22 (middle quality control 1, MQC1), 1.55, 1.54, 0.15 (lower quality control, LQC) and 0.52, 0.51, 0.05 (lower limit quality control, LLOQ QC) ng/mL, respectively. Sample processing A 200-��L aliquot of human plasma sample was mixed with 25 ��L of the IS working solution (2000 ng/mL of irbesartan). To this, 200 ��L of extraction buffer (0.5% formic acid in water) was added after vortex mixing for 10 s. The sample mixture was loaded onto an Oasis HLB cartridge (30 mg/1 mL) that was pre-conditioned with 1.0 mL of methanol followed by 1.0 mL of extraction buffer. The extraction cartridge was washed with 1.0 mL of extraction buffer followed by 1.0 mL of water. Analytes and IS were eluted with 1.0 mL of 0.
5% ammonia in methanol and evaporated to dryness at 45��C under a stream of nitrogen. The dried extract was reconstituted in 1000 ��L of mobile phase and transferred into injector vials. From these, a 15-��L aliquot was injected into the chromatographic system. Pharmacokinetic study design A pharmacokinetic study on the drug was performed in healthy male subjects (n=6). The Ethics Committee approved the protocol, and the volunteers provided informed written consent. Blood samples were collected following oral administration of 50-mg tablet of losartan at pre-dose and 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.33, 2.67, 3, 3.33, 3.67, 4, 4.5, 5, 6, 8, 10, 12, 24, and 36 h, in EDTA Vacutainer collection tubes (BD, Franklin, NJ, USA). The tubes were centrifuged at 3200 rpm for 10 min and the plasma was collected.
The collected plasma samples were stored at �C70��C until their use. Method validation The validation of the above method was carried out as per US FDA guidelines.[24] The parameters determined were selectivity, specificity, matrix effect, linearity, precision, accuracy, recovery, stability, and dilution integrity. RESULTS AND Batimastat DISCUSSION Mass spectrometry Mass parameters were tuned in both, positive and negative ionization modes for the analytes. Good response was found in positive ionization mode.