DETERMINATION OF JOSAMYCIN RESIDUES IN PORCINE TISSUES USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PRECOLUMN DERIVATIZATION AND SPECTROFLUOROMETRIC DETECTION

A simple, selective and sensitive high-performance liquid chromatograp hic (HPLC) method has been developed for the measurement of josamycin residues in four porcine tissues (i.e., muscle, liver, kidney and fat) . The sample preparation consisted of a homogenization step in an acet onitrile-10 mmol l(-1) phosphate buffer mixture, pH 6.0 (35 + 65), cen trifugation and a liquid-liquid extractive clean-up of the resulting s upernatant with isooctane. Pre-column derivatization of josamycin was performed using cyclohexa-1,3-dione in ammonium acetate buffer, pH 5.0 (90 degrees C for 2 h). The derivative was chromatographed in an isoc ratic reversed-phase HPLC system. A LiChrospher RP 18 end-capped (5 mu m) column was eluted with an acetonitrile-methanol-10 mmol l(-1) phos phate buffer mixture, pH 6.0 (45 + 5 + 50). The capacity factor of the josamycin derivative was 17.5. Detection was achieved using spectrofl uorimetry (lambda(ex) = 375 nm; lambda(em) = 450 nm). The structure of the derivative was assessed by using mass spectrometry. Full selectiv ity was obtained in the HPLC system versus other macrolide antibiotics (tylosin, spiramycin and erythromycin), aldehydes (formaldehyde, acet aldehyde and benzaldehyde) and endogenous compounds. Linearity and rep eatability were tested. Correlation coefficients, for calibration curv es in the range of 0.1-3.2 mu g g(-1), were greater than 0.999 for all tissues and the relative standard deviation (s(r)) was 4.9% (1.6 mu g g(-1); n = 6); recovery was higher than 88%.