beta-blockers are generally determined using high-performance liquid chromatography (HPLC). Previous HPLC separations of beta-blockers have often required a mobile phase containing three components; acetonitrile or methanol to control the retention; buffer to control the ionic strength and pH of the mobile phase; ion-pairing reagent to provide adequate retention of beta-blockers or organic amines as masking agent to reduce peak tailing. Due to the complexity of the mobile phases employed, development od these assays can be a laborious process. Additionally, alkyl sulphonates and organic amines dramatically reduces the life-time reduction of silica based C-18 columns. The results of this study demonstrated that the addition of tested alkyl sulphonates and organic amines is not essential for an adequate separation of beta-blockers. In this study, we developed a simple HPLC method for the simultaneous separation of model beta-blockers, atenolol, practolol, metoprolol, oxprenolol and propranolol. Atenolol, practolol, metoprolol, oxprenolol and propranolol adequately separated with high peak symmetries using a mobile phase consisted of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (15:15:70, v/v/v). By altering only the fraction of methanol with respect to acetonitrile, method development becomes a more efficient separation. Furthermore, atenolol, practolol, metoprolol, oxprenolol and propranolol can be detected up to 0.25, 5, 10, 50 and 10 ng ml(-1). In this publication, we present the simultaneous separation of beta-blockers having a wide range of polarity. It is proposed that this new mobile phase, consisting only acetonitrile, methanol and phosphate buffer can be used for the analysis of the several beta-blockers presently in doping control analysis as well as others. (C) 1998 Elsevier Science B.V. All rights reserved.