Sensitive Liquid Chromatographic Determination of Clarithromycin and 14-Hydroxy Clarithromycin in Human Plasma with Tandem Mass Spectrometry

Abstract

This paper describes a rapid, simple, sensitive, specific, and accurate analytical method for the determination of clarithromycin and 14-hydroxy clarithromycin (metabolite) in human plasma by high performance liquid chromatography with electrospray ionization tandem mass spectrometric detection (LC-MS/MS), using erythromycin as an internal standard (IS). The drug, metabolite, and internal standard were extracted by a precipitation method with the use of acetonitrile as a precipitant and separated on a C₁₈ analytical column (5 µ, 4.6 × 100 mm). The mobile phase consisted of acetonitrile: 10 mM ammonium formate buffer (90:10 v/v). Detection was carried out by positive electro spray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separations were obtained within 2.0 min and were linear in the concentration range of 36.5–5066.2 ng/mL for clarithromycin and 28.3–3934.1 ng/mL for 14-hydroxy clarithromycin. The limit of quantification for clarithromycin and 14-hydroxy clarithromycin were 36.5 and 28.3 ng/mL. The average recoveries for clarithromycin, 14-hydroxy clarithromycin, and the IS were 85.6%, 88.8%, and 85.0%, respectively. The results showed that the proposed method is rapid, sensitive, and reproducible to the quantitative determination of clarithromycin and 14-hydroxy clarithromycin in human plasma. Moreover, the proposed method was successfully applied for the bioequivalence study of clarithromycin.

Keywords:

• 14-Hydroxy clarithromycin
• Clarithromycin
• Liquid Chromatography
• Macrolide
• Precipitation
• Tandem mass spectrometry

Notes

^a Mean of six replicates.

^b Precision and Accuracy.

^c Mean of eighteen replicates.

∗2 times dilution

∗∗4 times dilution

∗Nominal concentration