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-beta-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 (100 microliters) of a urine sample was chromatographed on a YMC AM-302 octadecylsilica column (4.6 mm i.d. x 15 cm, ambient) with a water-methanol (35:65) eluent delivered at a flow rate of 1 ml/min. A reagent solution for a postcolumn reaction comprised of 50 microM 5,5'-dithiobis(2-nitrobenzoic acid), 0.3 M potassium iodide and a 50 mM phosphate buffer (pH 7.4), was delivered at a flow rate of 0.5 ml/min. The postcolumn reactor 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 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 10 microM, 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.