Micro-scale method for theophylline in body fluids by reversed-phase, high-pressure liquid chromatography

We describe a micro-scale method for determining serum theophylline. The chromatography system includes a muBondapack C18 column and acetonitrile, 70 ml/liter of sodium acetate buffer (10 mmol/liter, ph 4.0) as the mobile phase. Test serum or plasma, 30 mul, is mixed with an equal quantity of a solution containing the internal standard, beta-hydroxyethyltheophylline in acetonitrile/sodium acetate buffer (20 mmol/liter, pH 4.0), 7/43 by vol. After the precipitate is removed by centrifugation, the mixture is chromatographed and the amount of theophylline calculated from the ratio between peak heights for theophylline and the internal standard. Advantages include easy sample preparation, involving only addition of internal standard and centrifugation before injection, long column life, and the suitability of the internal standard, which is adjusted to a peak height equivalent to 20 mg of theophylline per liter for easy computation of results.