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L. T. Davis, R. D. Glickman, N. Kumar, L. M. Nijm, E. Y. Tu, L. J. Ulanski, II, R. Fiscella, Jr.; Development of an Adaptable HPLC Method for The Analysis of Frequently Used Antibiotics in Ocular Samples. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1818. doi: https://doi.org/.
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To implement a high performance liquid chromatography (HPLC) method for measuring the concentrations of four antibiotics, specifically moxifloxacin, gatifloxacin, linezolid, and cefuroxime, in ocular tissue. A number of different methods have been published describing measurement of ophthalmic drugs; however, we have developed an adaptable and sensitive chromatographic method with wide application.
HPLC analyses were performed at room temperature using a reversed-phase LiChrospher RP-18, 125 x 4 mm, 5 µm pore column (Phenomenex), plus a guard column. The LC system consisted of an HPLC pump operated in isocratic mode at 0.5 ml/min and an autosampler. A common mobile phase and detection method was used for linezolid and cefuroxime. The mobile phase consisted of 25% acetonitrile, 25 mM sodium acetate buffer, pH 5.0, with detection at 255 nm (linezolid) and 274 nm (cefuroxime) using a photodiode array. A common mobile phase and detection method was also employed for gatifloxacin and moxifloxacin. This mobile phase consisted of 20% acetonitrile, 0.03 M tetrabutylammonium Cl (Sigma/Fluka), and 0.1% trifluoroacetic acid, pH 3.0. Although UV detection (295 nm) can be used with the fluoroquinolones, greater sensitivity is obtained by fluorescence detection with excitation at 345 nm and emission at 470 nm.
For each antibiotic, the drug was successfully detected in aqueous and vitreous humor samples from 11 rabbit eyes, and the concentrations were measured reliably. The sensitivity (LOQ) for linezolid was approximately 0.05 µg/ml for both aqueous and vitreous samples. The LOQ for cefuroxime was approximately 0.05 µg/ml for aqueous and 0.5 µg/ml for vitreous. For gatifloxacin, the LOQ was about 0.01 µg/ml for both aqueous and vitreous samples. For moxifloxacin, the LOQ was about 0.05 µg/ml for aqueous and 0.1 µg/ml for vitreous samples.
A fairly straightforward HPLC method was developed that was easily adapted to each antibiotic, by adjusting the composition of the mobile phase and changing the detection method. Pharmacokinetic results obtained with this HPLC analytical methodology will be presented separately by Nijm et al.
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