RESUMO
Extraction efficiency was monitored, using 14C-neomycin and liquid scintillation counting, during development of a procedure to isolate neomycin from fortified kidney tissue. Chemiluminescence caused 119% false positive recovery at 0.16 ppm neomycin (1260 disintegrations/min (dpm) activity). However, the contribution of chemiluminescence to false positive recovery was negligible above 8 ppm neomycin (63,000 dpm activity). To reduce chemiluminescence, fortified kidney tissue was extracted with saline by homogenization, mixed with Protosol at 70 degrees C for 1 h, cooled 5 min at 4 degrees C and neutralized with acetic acid. The digest was mixed with Aquasol and incubated 1 h at 47 degrees C, followed by equilibration for 2 h in the counter. The procedure allows reliable monitoring of extraction efficiency down to about 407 dpm activity from 14C-neomycin (0.05 ppm) with maximum chemiluminescence of about 4%.
Assuntos
Rim/análise , Neomicina/isolamento & purificação , Animais , Radioisótopos de Carbono , Medições Luminescentes , Contagem de Cintilação/métodos , SolubilidadeRESUMO
A liquid chromatographic (LC) method is described for the determination of neomycin in animal tissues. Tissues are homogenized in 0.2M potassium phosphate buffer (pH 8.0); the homogenate is centrifuged, and the supernate is heated to precipitate the protein. The heat-deproteinated extract is acidified to pH 3.5-4 and directly analyzed by LC. The LC method consists of an ion-pairing mobile phase, a reverse phase ODS column, post-column derivatization with o-phthalaldehyde reagent, and fluorometric detection. The LC method uses paromomycin as an internal standard, and separates neomycin from streptomycin or dihydrostreptomycin because they have different retention times. The LC column separates neomycin in 25 min; the detection limit is about 3.5 ng neomycin. The overall recovery of neomycin from kidney tissues spiked at 1-30 ppm was 96% with a 9.0% coefficient of variation. The method was also applied to muscle tissue.
