Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

doi:10.1016/S0378-4347(99)00512-5

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 738, Issue 2, 11 February 2000, Pages 405-411

https://doi.org/10.1016/S0378-4347(99)00512-5 Get rights and content

Abstract

In this study, a high-performance liquid chromatographic method was developed for the quantitative determination of erythromycin (EM), roxithromycin (RXM), and azithromycin (AZM) in rat plasma with amperometric detection under a standardized common condition using clarithromycin (CAM) as an internal standard. This method was also proved to be applicable for the determination of CAM by employing RXM as an internal standard. Each drug was extracted from 150 μl of plasma sample spiked with internal standard under an alkaline condition with tert.-butyl methyl ether. The detector cell potential for the oxidation of the drugs was set at +950 mV. The linearity of the calibration curves were preserved over the concentration ranges of 0.1–10 μg/ml for EM and RXM, and 0.03–3.0 μg/ml for CAM and AZM. Coefficients of variation and relative error were less than 9% and ±7%, respectively. The analytical method presented here was proved to be useful for the investigation of the pharmacokinetic characteristics of EM, CAM, RXM, and AZM in rats.

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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Short communicationDetermination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

Abstract

Introduction

Section snippets

Materials and animals

Chromatographic condition

J. Pharm. Sci.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Short communication
Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection