DETERMINATION OF AURANOFIN, A CHRYSOTHERAPY AGENT, IN URINE BY HPLC WITH A POSTCOLUMN REACTION AND VISIBLE DETECTION

Auranofin ta-D-glucopyranosato-S)(triethylphosphine)gold(1): AF] is a unique orally active chrysotherapy agent. A HPLC method has been devel oped for determining AF in urine. The proposed method comprises initia l chromatographic separation of AF followed by on-line decomposition b y potassium iodide with a released mercapto group undergoing a color-d eveloping reaction with 5,5'-dithiobis(2-nitrobenzoic acid). An aliquo t (100 mul) of a urine sample was chromatographed on a YMC AM-302 octa decylsilica column (4.6mm i.d. x 15 cm, ambient) with a water-methanol (35: 65) eluent delivered at a flow rate of 1 ml/min. A reagent solut ion for a postcolumn reaction comprised of 50 muM 5,5'-dithiobis(2-nit robenzoic acid), 0.3 m potassium iodide and a 50 mm phosphate buffer ( pH 7.4), was delivered at a flow rate of 0.5ml/min. The postcolumn rea ctor consisted of a poly(tetrafluoroethylene) tube (0.5 mm i.d. x 5 m) at 60-degrees-C. Detection wavelength was 412 nm. The identity of the AF peak was confirmed by a 3-dimensional chromatogram as well as by a tomic absorption spectrophotometric analysis of gold in the column eff luent. Under the conditions described above, a linear relationship was obtained between peak height and AF concentration in the range 0.1 to 10 muM, with a correlation coefficient of 0.999. The detection limit was 50 nm (S/N= 3 at 0.005 AUFS) and the reproducibility was within 4% for 5 determinations. The AF concentrations in the urine of a rabbit given AF intraperitoneally were determined.