Ion-pair high-performance liquid chromatographic procedure for the quantitative analysis of theophylline in serum...

An ion-pair chromatographic system is described for the separation of theophylline and related xanthines from serum samples. The mobile phase consisted of 0.02 M tetrabutylammonium ion and 0.015 M Tris buffer in water-acetonitrile-methanol (93:3.5:3.5, v/v/v) at a precisely controlled pH (7.50 +/- 0.02, adjusted with hydrochloric acid). The flow-rate was 1.2 ml/min through a 15 cm X 4.6 mm I.D., 5-micron reversed-phase column (Ultrasphere C18 ion pair). Xanthines were extracted from serum (100 microliter) with 1 ml of acidified chloroform-isopropanol (95:5, v/v). After reconstitution in 200 microliter of mobile phase, the extracted xanthines, including theophylline, caffeine, theobromine and 1.7-dimethylxanthine, were baseline-resolved in less than 15 min. The method correlates well with a common clinical immunoassay for theophylline (EMIT Syva, r2 = 0.999) and yields excellent recovery and precision (98-101% and better than 2% at therapeutic levels, respectively). In addition, the use of the ion-pair chromatography mode eliminates many of the interferences noted in the published literature for the common reversed-phase separations of theophylline.