Interferences in a high pressure liquid chromatographic assay of theophylline.

Interference by salicylic acid was noted in a high pressure liquid chromatographic assay of theophylline in serum. The acid eluted very close to theophylline in a mobile phase consisting of 0.01 M acetate buffer, pH 4.0, containing 28% methanol on a C-18 reverse-phase column. The two peaks could be resolved by switching to a mobile phase containing 18% methanol, 1.6% acetonitrile, and 1.6% acetic acid in water. However, in this mobile phase traces of the extracting solvent, ethyl acetate, caused a sharpening of the theophylline peak leading to spuriously high results. The problem was overcome by using chloroform to extract the theophylline from serum.