A Sensitive Liquid Chromatographic Method for the Analysis of Clarithromycin with Pre-Column Derivatization: Application to a Bioequivalence Study

Document Type: Original Article

Authors

1 School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran

2 Young Researchers Club, Islamic Azad University, Kermanshah branch, Iran

3 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Abstract

Objective(s)
A sensitive liquid chromatographic method for the analysis of clarithromycin- a macrolide antibiotic- in human serum, using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described.
Materials and Methods
The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 ml/l; pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm.
Results
The analytical method was linear over the concentration range of 0.025-10 pg/ml of clarithromycin in human serum with a limit of quantification of 0.025 pg/ml. The assay is sensitive enough to measure drug levels obtained in human single dose studies.
Conclusion
In the present method, sensitivity and the running time of analysis have been improved and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.

Keywords


1.Chambers HF. Chloramphenicol, Tetracyclines, Macrolides, Clindamycin, and Streptogramins. In: Katzung BG. editor. Basic and clinical pharmacology. New York, NY: McGraw-Hill; 2004.p.760.

2.Chu SY, Sennello LT, Sonders RC. Simultaneous determination of clarithromycin and 14(R)- hydroxyclarithromycin in plasma and urine using high-performance liquid chromatography with electrochemical detection. J Chromatogr 1991; 57:199.

3.Hedenmo M, Eriksson BM. Liquid chromatographic determination of the macrolide antibiotics roxithromycin and clarithromycin in plasma by automated solid-phase extraction and electrochemical detection. J Chromatogr A 1995; 692:161. 

4.Taninaka PC, Ohtani H, Hanada E, Kotaki H, Sato H, Iga T. Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection. J Chromatogr B 2000; 738:405.

5.Niopas I, Daftsios AC. Determination of clarithromycin in human plasma by HPLC with electrochemical detection: validation and application in pharmacokinetic study. Biomed Chromatogr 2001; 15:507.

6. Choi SJ, Kim SB, Lee H-Y, Na DH, Soon YS, Lee SS, et al. Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection. Talanta 2001; 54:377.

7.Pappa-Louisi A, Papageorgiou A, Zitrou A, Sotiropoulos S, Georgarakis E, Zougrou F. Study on the electrochemical detection of the macrolide antibiotics clarithromycin and roxithromycin in reversed-phase high- performance liquid chromatography. J Chromatogr B 2001; 755: 57.

8.Kees F, Spangler S, Wellnhofer M. Determination of macrolides in biological matrices by high-performance liquid chromatography with electrochemical detection J Chromatogr A 1998;812:287.

9.Wibawa JID, Shaw PN, Barrett DA. Quantification of clarithromycin, its 14-hydroxy and decladinose metabolites in rat plasma, gastric juice and gastric tissue using high-performance liquid chromatography with electrochemical detection. J Chromatogr B 2003; 783:359.

10.Van Rooyen GF, Smit MJ, De Jager AD, Hundt HKL, Swart KJ, Hundt AF. Sensitive liquid chromatography- tandem mass spectrometry method for the determination of clarithromycin in human plasma. J Chromatogr B 2002; 768:223.

11.Amini H, Ahmadiani A. Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection. J Chromatogr B 2005; 817:193.

12.Torano JS, Guchelaar HJ. Quantitative determination of the macrolide antibiotics erythromycin, roxithromycin, azithromycin and clarithromycin in human serum by high-performance liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl chloride and fluorescence detection. J Chromatogr B 1998; 720: 89.

13.Bahrami Gh, Mirzaeei Sh, Kiani A. High performance liquid chromatographic determination of azithromycin in serum using fluorescence detection and its application in human pharmacokinetic studies. J Chromatogr B 2005; 820:277.

14.Bahrami Gh, Mohammadi B. A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum. J Chromatogr B 2006; 830:355.

15.Hori T, Hashimoto H, Konishi M. Determination of erythromycin A in rat plasma and urine by high- performance liquid chromatography with chemiluminescence detection using Tris (2,2- bipyridine) ruthenium (II). Biomed Chromatogr 2006; 20:17.