Performance of tiloronoxim and tilorone determination in human blood by HPLC–MS/MS: Method validation, uncertainty assessment and its application to a pharmacokinetic study

A highly sensitive and selective HPLC–MS/MS method is presented for the quantitative determination of tiloronoxim and its metabolite tilorone in human blood. An aliquot of 200 μl human blood was extracted with a mixture of chloroform/ethyl ether (1/2, v/v), using metoprolol as the internal standard (the IS). Separation was achieved on an Xterra MS C18 column (50 mm × 2.1 mm, 5 μm) with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). Detection was performed using positive MRM mode on a TurboIonSpray source. The mass transitions monitored were m/z 426.3 → 100.0, m/z 411.3 → 100.0 and m/z 268.3 → 116.1 for tiloronoxim, tilorone and the IS, respectively. The method was fully validated using total error theory, which is based on β-expectation tolerance intervals and include trueness and intermediate precision. The method was found to be accurate over a concentration range of 1–100 ng/ml for both compounds. The measurement uncertainty based on β-expectation tolerance intervals was assessed at each concentration level of the validation standards. This method was successively applied to a pharmacokinetic study of tiloronoxim in healthy volunteers.