Acetonitrile

CHAPTER II

2. MATERIALS AND METHODOLOGY

2.1 Reagents HPLC-grade acetonitrile was purchased from J T backer (Phillipsburg, USA); formic acid was purchased from Fluka (St. Louis, MO 63178, USA), purified water collected through Milli-Q water purification system (Millipore, Bedford, MA, USA). Reference substances Methyl methanesulfonate and Ethyl methanesulfonate were purchased from Acros organics (500 American Road, Morris Plains, USA) and Emtricitabine, Lopinavir, Ritonavir and Fosamprenavir Calcium API samples were obtained from Ranbaxy laboratories Ltd (India).

2.2 Apparatus

The LC system used was an Agilent 1100 series LC system (Agilent Technologies, Waldbronn, Germany) consisting of a 1100 series pump with a degasser, a temperature controlled

In case of Ritonavir the standard concentration was prepared of concentration 0.125µg per ml for both MMS and EMS. Standard preparation was done by preparing stock solution of both the analytes in acetonitrile and then suitability diluting with diluent. Sample solution of 100 mg per ml concentration was prepared for Lopinavir and Ritonavir in diluent using sonication.

The LC system used was an Agilent 1100 series LC system. The analytical column was Atlantis T3 (150 x 4.6mm) 3.0µm. The mobile phase consisted of eluent A of Formic acid 0.1% (v/v in water) and eluent B of acetonitrile, at a flow rate of 1.0 ml per min. The gradient elution mode was used for analysis. In gradient program eluent B was kept 40% for 6min and in 7min eluent B was changed to 80%, followed by a hold time of 5 min, then in 1min eluent B was changed to 40% and a re-equilibration period of 7 min was given. Premixed and degassed solution of water and acetonitrile in the ratio of 30:70 was used as diluent. The run time for standard was kept as 6min and for sample and blank as 20 minutes. Column oven temperature was maintained at 30°C. Injection volume was 50 µL. The control of the HPLC system and data collection was by Empower software. All the solutions were filtered through 0.45µ nylon

Splitter was used to achieve a flow rate of 0.2 mL per min in to the mass spectrometer. The operating conditions were, Ion spray voltage was kept as -4500 V and source temperature 500°C. Curtain gas was applied at 20 psi and collision gas at high. The ion source gas1 and gas 2 were kept at 40 and 55 psi respectively. Venting was done using valco valve. Venting was given from 0.1 min. to 10.0 min. and from 22.1 min. to 32.0 min. The MRM transitions of m/z 202.0>137.80 and 202.0>108.20 were selected for quantification of Methyl, Ethyl and Isopropyl