A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2006-01, Vol.830 (2), p.355-358
• Main Authors: Bahrami, Gholamreza, Mohammadi, Bahareh
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Azithromycin; Azithromycin - blood; Biological and medical sciences; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; FMOC-Cl; Fundamental and applied biological sciences. Psychology; General pharmacology; HPLC; Humans; Macrolide antibiotics; Medical sciences; On-line derivatization method; Pharmacology. Drug treatments; Reproducibility of Results; Reverse phase chromatography; Serum
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2005.10.044

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile–phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.