A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum.
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.
Bahrami
Gholamreza
G
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran. gbahrami@kums.ac.ir
Mohammadi
Bahareh
B
D017963:Azithromycin / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results
D017963:Azithromycin