title: Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate.
abstract: 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 C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-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 microg/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.
authors.affiliation: School of Pharmacy and Institute of Pharmaceutical Innovation, School of Life Sciences, Bradford, UK.
authors.forename: M
authors.initials: M
authors.lastname: Mashat
authors.forename: H
authors.initials: H
authors.lastname: Chrystyn
authors.forename: B J
authors.initials: BJ
authors.lastname: Clark
authors.forename: K H
authors.initials: KH
authors.lastname: Assi
mesh_terms: D000280:Administration, Inhalation; D000328:Adult; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000302:isolation & purification / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry*; D008297:Male; D015203:Reproducibility of Results; D014031:Tobramycin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine*
chemical_list: D000900:Anti-Bacterial Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D016650:Fluorescein-5-isothiocyanate; D014031:Tobramycin