The determined plasma concentrations of both amoxicillin and clavulanic acid

were in the range of the expected values upon the literature data for HPLC–UV and LC–MS methods. The working standard of amoxicillin trihydrate, amoxicillin d-4, clavulanate potassium and ampicillin trihydrate were obtained from Vivan life science (Mumbai, India). Ammonium acetate (GR grade), ortho phosphoric acid (GR grade), methanol (HPLC grade), acetonitrile (HPLC grade) were purchased from Merck Pvt. Ltd. Mumbai, India. Water was deionized and purified by a Milli-Q system from Millipore (Bedford, MA, USA) Oasis HLB 1 c.c, 30 mg solid phase extraction cartridges were procured from Waters (India) Pvt. Ltd. The blank human plasma with sodium heparin

anticoagulant was collected from Drs. Pathology Labs, Mumbai, Trametinib ic50 India. A Shimadzu HPLC system with a 5 μm HyPURITY advance C18 column (50 × 4.6 mm) was used for separation. The mobile phase was prepared by the addition of 2 mM ammonium acetate to acetonitrile (20:80 v/v). The flow rate was 0.8 mL/min. An API 4000 triple Quadrupole Mass Spectrometer (Applied Biosystem-SCIEX, Canada), equipped with a Turbo Ion spray source, was used for the LC–MS–MS analyses. The data processing was carried out using Analyst 1.5 software. The MS was operated in negative ion detection mode. Nitrogen was used as nebulizing turbo spray. The temperature of the vaporizer was set at 400 °C and the ESI needle voltage

was 4500 V. The declustering potential was set at 50 for AMX, AMX–D4, AMP and 30 for CLV. Collision energy for AMX, CLV, AMX-D4 and AMP see more was −25, −10, −15 and −15 V respectively. The mass spectrometer was operated at unit mass resolution with a dwell time of 200 ms per transition. Quantification was performed using multiple reactions monitoring (MRM) of the transition ions m/z 364.00 → m/z 223.00, m/z 198.00 → m/z 136.00, m/z 368.00 → 227.00 and m/z 347.90 → m/z 304.00 for AMX, CLV, AMX-D4 and AMP respectively. The stock solutions of AMX, CLV (1 mg/mL) and IS (1 mg/mL) were prepared, for calibration standards and quality control (QC) samples, by dissolving appropriate amounts of the compounds in methanol. unless Stock solutions of AMX and CLV were subsequently serially diluted with mobile phase to obtain working solutions which were then added to plasma to yield concentrations in the range 50.43–31500.68 and 25.28–6185.18 ng/mL. A working solution for each IS (60.0 μg/mL) was prepared in water. All solutions were stored at 2–8 °C. To 950 μL of drug free plasma, 50 μL of working solutions of AMX, CLV were added to yield final concentrations of 50.43, 100.81, 1047.84, 2526.68, 5250.16, 10500.47, 20600.49 and 31500.68 ng/mL for AMX and 25.28, 50.61, 201.75, 510.52, 1078.54, 2118.32, 4215.49 and 6185.18 ng/mL for CLV in plasma. QC samples of 50.55, 150.30, 9411.75 and 18823.24 ng/mL of AMX, 26.99, 76.98, 2368.62 and 4737.