RADIOIMMUNOASSAY FOR THE DETERMINATION OF ALOSETRON IN HUMAN URINE AND SALIVA

The development of a radioimmunoassay (RIA) for the sub-ng ml(-1) dete rmination of alosetron, a potent and selective 5HT(3) receptor antagon ist, in human urine and saliva is described. The antiserum was raised in Soay sheep following primary and booster immunizations with an immu nogen prepared by conjugating alosetron-p-azobenzoic acid to bovine se rum albumin (BSA). The radioligand consisted of alosetron specifically 125-iodinated on the 2-position of the imidazole group. The mean (+/- standard deviation) theoretical sensitivity (minimum detectable dose corresponding to the imprecision of the zero standard) of the RIA is 3 .2 +/- 2.6 pg ml(-1) (n = 12) of alosetron in assay diluent (0.1% m/v gelatine-0.05% m/v sodium azide in 0.1 mol l(-1) phosphate buffer solu tion, pH 7.4). The working calibration range using 0.1 ml samples of s aliva and 20-fold diluted urine is 0.10-6.30 ng ml(-1) of alosetron. U rine samples were diluted prior to assay to overcome adverse matrix ef fects; consequently, the lower limit of quantification for undiluted u rine is 2.0 ng ml(-1) of alosetron. Inter- and intra-assay bias and im precision over the working calibration range were generally <+/-12% an d <13%, respectively, except at the 0.10 ng ml(-1) alosetron level, wh ere the corresponding values were <+/-17.3% and <20.2%. The antiserum was free from adverse cross-reactivity with either a synthetic precurs or of alosetron or with four major metabolites of the drug. The optimi zed RIA was applied to the determination of alosetron in serial sample s of saliva and urine taken from volunteers who had received a single oral dose (1 mg) of the drug. The salivary and urinary results from th is single-dose study are presented, together with associated serum con centrations (determined by means of HPLC with fluorescence detection), to demonstrate the applicability of the RIA method to saliva and urin e, and to confirm that the determination of the drug in these latter m atrices can indeed provide a useful, non-invasive indication of system ic exposure to alosetron.