Abstract
We have developed a rapid, simple, specific and quantitative assay for plasma theophylline by high performance liquid chromatography. For reagents of deproteinization, various water-miscible organic solvents such as methanol, ethanol, I-propanol, acetone, acetonitrile and tetrahydrofuran were studied. Methanol gives best result. 50μl of plasma is mixed with 250μl of methanol solution containing 7-(2-hydroxyethyl) theophylline (4μg/ml in methanol) as an internal standard. Under these conditions, plasma proteins are precipitated and total theophylline is dissolved into the methanol solution. After the centrifugation, 20μl of the supernatant is injected into the high performance liquid chromatograph equipped with a Waters U6K injector, a 4mm×30cm μBondapak C18 column, a Model 440UV detector set at 0.01 absorbance units (280nm) and a 6000A solvent delivery system. The flow rate of the eluent of 0.01M sodium acetate (pH 4.0): acetonitrile (10:1, v/v) is 1ml/min. Elution of theophylline occurs at 9 minutes and that of internal standard at 12 minutes. The amount of theophylline is calculated from the peak height ratio of theophylline to internal standard. Recoveries are 98-102% and the standard curve obtained from theophylline spiked plasma is linear in range of 0μg/ml to 30μg/ml. Within-run and day-to-day reproducibility date for 5μg/ml are±1.0% and±1.5%, respectively. The assay can be accomplished in less than 15 minutes.
