Betasil Silica

Using the Tomtec Quadra 96 workstation, 0.1 mL of the ethyl acetate layer was transferred to a 96-well collection plate containing 0.4 mL of acetonitrile in each sample well. The solution was mixed 10 times by aspiration and dispersion on the Tomtec. The plate was then covered with a sealing mat and stored at 2 to 8°C until LC/MS/MS analysis. The HILIC-MS/MS system consisted of a Shimadzu 10ADVP HPLC system and Perkin Elmer Sciex API 3000 and 4000 tandem mass spectrometers operating in the positive ESI mode. The analytical column was Betasil silica (5 fim, 50 x 3 mm) and a mobile phase of acetonitrile water formic acid with a linear gradient elution from 95 5 0.1 to 73.5 26.5 0.1 was used for 2 min. The flow rate was 1.0 mL/min for the API 3000 and 1.5 mL/min for the API 4000 without any eluent split. The injection volume was 10 jjL and a run time of 2.75 min was employed. 

Song and Naidong [129] analyzed omeprazole and 5-hydroxyomepra-zole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry. Omeprazole and its metabolite 5-hydroxy omeprazole and the internal standard desoxyomeprazole were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 /il was injected onto a Betasil silica column (5 cm x 3 mm, 5 /rm) and detected by atmospheric pressure ionization 3000 and 4000 with positive electrospray ionization. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95 5 0.1 to 73.5 26.5 0.1 in 2 min). The flow-rate was 1.5 ml/min with total rim time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability, and recovery. 

Chromatographic Separation The LC system was a Shimadzu (Columbia, MD) series lOAD VP equipped with binary pumps, a degasser, and an SIL-HT autosampler. A Betasil silica column (50 x 3 mm, 5 pm) from Keystone Scientific (Bellefonte, PA) operated under HILIC mode was used. The isocratic mobile phase was composed of 5 95 (v/v) A B, where A was 0.05% trifluoroacetic acid (TFA) in water (v/v) and B was 0.05% TFA in acetonitrile (v/v). The flow rate was 0.6 mL/min and the injection volume was 10 pL. Separation was performed at ambient temperature. 

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