Journal of Chromatography B: Biomedical Sciences and Applications
Short communicationDetermination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection
Introduction
Macrolide antibiotics have been used for the treatment of bacterial infections caused by Gram-positive organisms. Although erythromycin (EM) have been widely used until 1980s, it is unstable to acid and its pharmacokinetics after oral administration are highly variable. Recently developed semisynthetic derivatives of EM such as clarithromycin (CAM), roxithromycin (RXM), and azithromycin (AZM) are stable to acid and more extensively distributed to tissues.
For pharmacokinetic studies of macrolide antibiotics, analytical methods using radioactivity, a microbiological assay [1], or high-performance liquid chromatographic (HPLC) with UV [2], fluorescent [3], or electrochemical detection [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15] have been employed. In recent years, HPLC methods with electrochemical detection have been widely used due to its high sensitivity and precision. In clinical settings, a more sensitive and simple method is required to determine the concentrations of several macrolide antibiotics in plasma. We have already reported a method for the determination of EM concentrations in rat plasma and liver by HPLC with electrochemical detection using a quite simple extraction procedure [14]. Kees et al. recently reported a highly sensitive method for the determination of EM, its ester, CAM, RXM, and AZM [15]. However, their method requires different mobile phases for these drugs, and changing of analytical conditions, such as mobile phases and columns, is laborious and requires additional stabilization times. In this study, therefore, we propose de novo HPLC method for the determination of EM, RXM, and AZM concentrations in rat plasma with amperometric detection, under a standardized, common analytical condition and extraction procedure. We also aimed to prove this method applicable for the determination of CAM by employing RXM as an internal standard.
Section snippets
Materials and animals
EM base, CAM, RXM and AZM were kindly provided by Dai Nippon Pharmaceutical (Osaka, Japan), Taisho Pharmaceutical (Tokyo, Japan), Hoechst Marion Roussel (Tokyo, Japan) and Pfizer Pharmaceuticals (Tokyo, Japan), respectively. Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Wako Pure Chemical Industries (Osaka, Japan), and tert.-butyl methyl ether (HPLC grade) from Aldrich (Milwaukee, USA). All other compounds used were of reagent grade.
Male Sprague-Dawley rats weighing
Chromatographic condition
The current–voltage relationships EM, CAM, RXM, and AZM were investigated. Peak height of each drug increased, as the applied potential increased from +400 to +1050 mV. The peak height became almost saturated at the potential of +950 mV. A detector cell potential of +850 to +1100 mV was reported to be required for the oxidation of macrolide antibiotics [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], being consistent with the result in this study. Although a higher peak was
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