Determination of avoparcin in animal formulations by capillary electrophoresis

The separation of two closely related glycopeptides, alpha- and beta-avoparcin, which differ by the presence or absence of a Cl atom, is achieved by capillary zone electrophoresis (CZE) using 20 mM borate (pH 9.2) in bare, fused-silica capillaries. The pKa values of these two glycopeptides are determined from spectrophotometric titrations of the mixture. Absorption spectra recorded between pH 3 and 12 produced a maximum pH transition at 258 nm. Titration data fitted to two sigmoidal functions reveal pKas of 7.99 for beta-avoparcin and 10.3 for alpha-avoparcin. The differing pH dependence of the effective electrophoretic mobilities of alpha- and beta-avoparcin resulted in an optimum pH of 9.2 (roughly the average of the pKa values) for their separation. Borate is shown to be necessary for the optimal CZE separation. A linear dynamic range of 0.5-50 ppm is achieved with regression coefficients of 0.9999. The limit of detection ranges from 0.2 to 0.5 ppm using a pressure injection of 15 sec in a 50-cm (effective length) fused-silica capillary with UV detection. Finally, the effects of sodium dodecyl sulfate (SDS) concentration in the migration buffer on apparent mobility, efficiency, migration times, and resolution are discussed. The addition of 75 mM SDS to the running buffer allows baseline resolution of alpha- and beta-avoparcin, including its minor components. Both the CZE and micellar electrokinetic chromatography (MEKC) separations of alpha- and beta-avoparcin are highly reproducible and can readily be applied to the analysis of avoparcin bulk drug and formulated products.