High-performance liquid chromatographic determination of azosemide in commercial animal feed

A high-performance liquid chromatographic (HPLC) method was developed for the assay of 2-chloro-5-(1H-tetrazol-5-yl)-4-[(2-thienylmethyl)amino]benzenesulfonamide (I) in animal feed diet mixtures. The sample is extracted with acetone, an aliquot of the extract is evaporated to dryness, and the residue is dissolved in chloroform. This solution can be subjected to either of two cleanup procedures. The first traps I on a sodium hydroxide-diatomaceuous earth column. Compound I is then recovered using an acetic acid-ethyl acetate-chloroform eluent, which is removed by evaporation, followed by dissolution of the residue in aqueous sodium hydroxide. The second procedure involves the direct extraction of I from the chloroform solution. A linear relationship exists between the HPLC UV detector response at 254 nm and concentration up to 170 microgram/ml. The sodium hydroxide extraction procedure resulted in an overall recovery (+/- SD) of 92.9 +/- 1.6 and 97.4 +/- 1.8% (n = 5) at levels of 200 and 6000 ppm, respectively. Overall recoveries (+/- SD) obtained by the diatomaceous earth column procedure for the 20- and 6000-ppm levels were 90.0 +/- 2.0 and 95.9 +/- 1.6% (n = 5), respectively.