Stability-indicating assay for phenylbutazone: high-performance liquid chromatographic determination of hydrazobenzene and azobenzene in degraded aqueous phenylbutazone solutions.

A high-performance liquid chromatographic method was developed for the simultaneous determination of azobenzene, hydrazobenzene, and four other decomposition products in phenylbutazone injectable formulations. Separation was achieved on a C18 column, with 0.1 M Tris-citrate buffer (pH 5.25) and acetonitrile (52:48), at a flow rate of 2 mL/min and a detection wavelength of 237 nm. Diphenylamine was used as an internal standard. The limit of quantitation is 0.5% (with respect to phenylbutazone) of each degraded product. The detectability is 2.4 X 10(-3) micrograms for azobenzene and 1.5 X 10(-3) micrograms for hydrazobenzene. The limit of quantitation may be lowered to 0.1% (with respect to phenylbutazone) for azobenzene and hydrazobenzene in the presence of the two major decomposition products, which have been determined in commercially available injectable formulations. A higher sensitivity was obtained for azobenzene using the mobile phase 0.1 M Tris-citrate buffer (pH = 5.25) and acetonitrile (40:60) with detection at 314 nm. Under these conditions, 0.025% (with respect to phenylbutazone) of azobenzene is quantitated.