759 (2001) 209–218 Journal of Chromatography B, www.elsevier.com / locate / chromb Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin * Kalliopi A. Georga, Victoria F. Samanidou, Ioannis N. Papadoyannis Laboratory of Analytical Chemistry Department of Chemistry, Aristotle University of Thessaloniki, GR-54006 Thessaloniki, Greece Received 16 November 2000; accepted 13 April 2001 Abstract An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7- dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C , 25034 mm, 5 mm, was 4 2 operated at ambient temperature with back pressure values of 80–110 kg / cm . The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng / ml. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-ml injection, while linearity held up to 8 ng/ ml for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng / ml and for caffeine for which it was 20 ng/ ml. The statistical evaluation of the method was examined performing intra-day ( n56) and inter-day calibration (n57) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v / v) as eluent, requiring small volumes, 40 ml of blood serum and 100 ml of urine.  2001 Elsevier Science B.V. All rights reserved. Keywords: Caffeine; Methylxanthine; Methyluric acid 1. Introduction which are pharmacologically active possessing a wide range of therapeutic activity. Theophylline As extensively reported in literature caffeine (CA) (TP), acts as a powerful bronchodilator, within a is ubiquitous in the human diet. Major caffeine narrow serum therapeutic range. Similarly to theo- metabolites include N-demethylated derivatives phylline, caffeine and theobromine (TB) stimulate the central nervous system (CNS), gastric acid secretion and cardiac muscle, relax smooth bronchial *Corresponding author. Tel.: 130-31-997-793; fax: 130-31- muscle and enhance diuresis. Theophylline stimula- 997-719. E-mail address: papadoya@chem.auth.gr (I.N. Papadoyannis). tion of CNS is more profound and more dangerous 0378-4347 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0378-4347(01)00251-1