Microassay for the simultaneous determination of theophylline and dyphylline in serum by high-performance liquid chromatography.

An improved and simplified high-performance liquid chromatographic method is presented for the simultaneous determination of theophylline and dyphylline in serum. The internal standard, beta-hydroxyethyltheophylline (20 microliters of a 100 micrograms/ml solution) is added to 50 microliters of serum. Serum proteins are precipitated by the addition of 30 microliters of 40% trichloroacetic acid solution. The mobile phase consists of a sodium acetate buffer with 6% acetonitrile. Chromatogram run time is 6 min. The sensitivity limit for both compounds is 0.25 micrograms/ml. This method is interference-free from the major metabolites of theophylline and other drugs commonly coadministered with theophylline.