Journal of Bioequivalence & Bioavailability

Development and validation of a HPLC-FL method for fluorescein quantification in buccal permeability studies

6^th World Congress on Bioavailability & Bioequivalence: BA/BE Studies Summit

August 17-19, 2015 Chicago, USA

Monica Maria Coquemala da Silva, Thaisa Marinho Dezani, André Bersani Dezani and Cristina Helena Dos Reis Serra

Posters-Accepted Abstracts: J Bioequiv Availab

Abstract:

Permeability is an extremely important factor for absorption of drugs. However, the investigation of permeability using buccal
transmucosal route has grown because few studies are known about the mechanisms that influence. Drugs administered by the
buccal transmucosal route do not suffer the first pass effect and thus may increase the bioavailability. The aim of this study was to
develop and validate a chromatographic method (HPLC-FL) with fluorescent detection for quantification of fluorescein in ex-vivo
permeability studies using buccal mucosa of pigs. This method was developed and validated according to ICH guideline considering
parameters as selectivity/specificity, linearity, precision, accuracy, limit of detection and limit of quantification. The chromatography
conditions were: Gemini Phenomenex® column C18 (250 mm x 4.6 mm i.d., 5 μm particle size), isocratic mobile phase consisting of
a mixture of acetonitrile 20 mM and monobasic sodium phosphate buffer (40:60 v/v) and 1.0% triethylamine, pH 4.5 adjusted with
orthophosphoric acid. The flow rate was 1.0 mL/min, oven temperature set at 35ºC and volume injection of 30 μL. The excitation
wavelength of FL detector was set at 485 nm and emission wavelength of 515 nm. The method presented linearity in a range of 10- 600
ng/mL (r²= 0.9983). Precision and accuracy was also adequate with intraday and interday variations at maximum of 10%. Selectivity
and specificity of method was adequate since no interferences were noticed in chromatograms. The chromatographic method was
developed and validated for fluorescein quantification in samples in ex-vivo permeability studies using buccal mucosa of pigs.