NITISINONE IN HUMAN PLASMA: UPLC-MS/MS ASSAY VALIDATION AND STABILITY STUDIES

A simple, precise, and rapid ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS method for determination of nitisinone in 20 µl human plasma was developed and validated. Nitisinone 6C13 was used as an internal standard (IS). Chromatographic analysis was performed on an Atlantis dC18 column (2.1 x 100 mm, 3 µm) using a mobile phase consisting of methanol and 10 mM ammonium-acetate (90:10, v;v) that was delivered at a flow rate of 0.25 ml/min. The eluents were monitored using electrospray ionization in the positive ion mode set at transition set of mass-to-charge (m/z): 330 → 217.92 and 336 → 217.91 for nitisinone and nitisinone-13C6 (internal standard, IS), respectively. The retention time of nitisinone and IS both were about 0.88 minutes, respectively. Relationship between nitisinone concentration and peak area ratio of nitisinone to the IS was linear (R² ≥ 0.9991) in the range of 2–100 µg/ml and the intra- and inter-day coefficient of variations were 1.9% to 4.5% and 3.2% to 6.2%, respectively. Extraction recoveries for nitisinone and the IS were 93%, and 98%, respectively. Nitisinone stability was evaluated in processed samples (stored at room-temperature for 24 hours) and unprocessed sample (stored at room-temperature for 24 hours or at -20°C for 8 weeks) and after 3 freeze-thaw cycles was ≥ 91%.