Determination of Auranofin, a Chrysotherapy Agent, in Urine by HPLC with a Postcolumn Reaction and Visible Detection

Auranofin [(2, 3, 4, 6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S)(triethylphosphine)gold(I) : AF] is a unique orally active chrysotherapy agent. A HPLC method has been developed for determining AF in urine. The proposed method comprises initial chromatographic separation of AF followed by on-line decomposition by potassium iodide with a released mercapto group undergoing a color-developing reaction with 5, 5'-dithiobis(2-nitrobenzoic acid). An aliquot (100μl) of a urine sample was chromatographed on a YMC AM-302 octadecylsilica column (4.6mm i.d.×15cm, ambient) with a water-methanol (35 : 65) eluent delivered at a flow rate of 1ml/min. A reagent solution for a postcolumn reaction comprised of 50μM 5, 5'-dithiobis(2-nitrobenzoic acid), 0.3M potassium iodide and a 50mM phosphate buffer (pH 7.4), was delivered at a flow rate of 0.5ml/min. The postcolumn reactor consisted of a poly(tetrafluoroethylene) tube (0.5mm i.d.×5m) at 60°C. Detection wavelength was 412nm. The identity of the AF peak was confirmed by a 3-dimensional chromatogram as well as by atomic absorption spectrophotometric analysis of gold in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and AF concentration in the range 0.1 to 10μM, with a correlation coefficient of 0.999. The detection limit was 50nM (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.