Research Article Received: 28 May 2008, Revised: 1 July 2008, Accepted: 1 July 2008 Published online 17 September 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1118 Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2009; 23: 324–333 324 John Wiley & Sons, Ltd. Effect of chemically bonded stationary phases and mobile phase composition on b-blockers retention in RP-HPLC Boguslaw Buszewski, a * Tomasz Welerowicz a and Tomasz Kowalkowski a ABSTRACT: The effects of stationary and mobile phase on retention of 18 b-adrenolytic drugs ( b-blockers) have been studied. Four ‘deactivated surface’ stationary phases (polar-embedded or end-capped) were examined. Special attention was drawn to the cholesterolic (SG-CHOL) and alkylamide (SG-AP) stationary phases, and their application for analysis of the compounds. The retention of analyzed substances was also examined in terms of mobile phase composition. Sixteen different configurations of mobile phases were prepared, all based on methanol and acetonitrile with ammonium acetate and ammonium formate. The difference in retention between ammonium formate and acetate water solutions, and peak shape changes related to the addition of triethylamine (TEA), were investigated. Principal component analysis was used to find the similarities between station- ary phases. Polar-embedded phases synthesized on the same sorbent possess very similar properties. All phases based on silica gel compared with the monolithic column also showed similarities in retention of b-blockers. The addition of TEA to the mobile phase did not influence strongly the retention, and analysis of asymmetry factors showed only a little peak broadening for a few compounds on the monolithic column. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: polar-embedded stationary phases; monolithic stationary phase; β -blockers; bio-chromatography; chemometrics; principal component analysis Introduction The β-adrenergic receptor antagonists (β-blockers) are impor- tant substances of therapeutic value in the treatment of cardio- vascular disorders. The β-blockers can be used in the treatment of hypertension, angina pectoris, arrhythmia and congestive heart failure (Hardman and Limbird, 1996; Frishman, 1984; Fitzgerald, 1991; Brooks and Gillies, 1992). High-performance liquid chromato- graphy (HPLC) method is most frequently used for analyses of these drugs (Saarinen et al., 1995; Maguregui et al., 1995; Gonzalez et al., 1995). Basci et al. (1998) proposed the use of simplified mobile phase consisting of methanol:acetonitrile:phosphate buffer (10 mM, pH 3.0; 15:15:70, v:v:v). The method has been applied for separation of only five β-blockers, namely atenolol, practolol, metoprolol, oxprenolol and propranolol. Delamoye et al., 2004) applied gradient elution of acetonitrile–phosphate buffer pH 3.8 to analyze 13 β-blockers on C 18 column. The effect of chaotropic counter anion in buffered mobile phase and the role of organic modifier (methanol, acetonitrile) type on the retention behavior of some β-blockers has been also investigated (Hashem and Jira, 2006). Among classical reverse-phase (RP) system on octadecyl (C 18 ) stationary phases, an interesting method of micellar chromato- graphy was proposed by Rapado-Martinez et al. (1996, 1997). The ionized form of the compound with average pK a ≈ 9.5 is recommended to analyze the β-blockers and requires acidic conditions, where the phosphoric buffers with pH ~3.0 are commonly used. Recently, the use of ultra-performance liquid chromatography coupled with mass spectroscopy detection (UPLC-MS) has been adopted for the simultaneous identification of six β-blockers with superior separation performance (Wren and Tchelitcheff, 2006). Generally, the separation processes in high-performance liquid chromatography (HPLC) can be considered as a combination of specific and non-specific interactions between three main parts of the chromatographic arrangement: stationary phase, mobile phase and the solute (Vailaya and Horváth, 1998; Dorsey and Dill, 1989). Among the large-scale chromatographic packings, modified silica gel (SG) is still the most popular stationary phase. Chemically bonded stationary phases (CBSs) are prepared by the reaction of organo-silane and hydroxyl groups on the silica sur- face (silanols). The steric hindrance results in the fact that some of these hydroxyl groups are not blocked during the synthesis. This may cause ion-exchange interactions of basic compounds with these unblocked silanols. Such interactions appear as broaden- ing of the peak (‘tailing’) and affect the resolution and selectivity (Unger, 1990; Sander and Wise, 1987; Buszewski et al., 1998; Neue, 1997). There are a few ways to reduce such inconvenience during chromatographic analysis. The easiest is the addition of ‘small size’ basic substances to the mobile phase. Diethylamine (DEA) or tri- ethylamine (TEA) are commonly used for this purpose and, with phosphate buffers, can create an ion-paring environment. Station- ary phases with ‘deactivated surfaces’ are modified materials by * Correspondence to: B. Buszewski, Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St., PL- 87-100 Torun, Poland. E-mail: bbusz@chem.uni.torun.pl a Department of Environmental Chemistry and Bioanalytics, Faculty of Chem- istry, Nicolaus Copernicus University, 7 Gagarin St., PL- 87-100 Torun, Poland Abbreviations used: ACN, acetonitrile; CBS, chemically bonded stationary phases; DEA, diethylamine; EC, end-capped; PCA, principal component anal- ysis; SG, silica gel; TEA, triethylamine.