Determination of α-naphthylisothiocyanate and metabolites α-naphthylamine and α-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of α-naphthylisothiocyanate (1-NITC) and two metabolites α-naphthylamine (1-NA) and α-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C₁₈ 5-μm column, a mobile phase of acetonitrile-water (ACN-H₂O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80°C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4°C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.