Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

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• 作者：M. Mashat ; H. Chrystyn ; B.J. Clark ; K.H. Assi
• 关键词：Tobramycin ; Fluorescein isothiocyanate (FITC) ; HPLC ; Assay ; Validation ; Urinary pharmacokinetic
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2008
• 出版时间：15 June 2008
• 年：2008
• 卷：869
• 期：1-2
• 页码：59-66
• 全文大小：718 K

文摘

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C₁₈ column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile 13;methanol 13;glacial acetic acid 13;water (420:60:5:515, v/v/v/v). The tobramycin 13;FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 μg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99 % (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2 % , and intra-day and inter-day precision (in term of % coefficient of variation) were <4.89 % and 8.25 % , respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI^®) by different nebuliser systems.