Simultaneous determination of a novel antitrypanosomal compound (OSU-36) and its ester derivative (OSU-40) in plasma by HPLC: Application to first pharmacokinetic study in rats

Abstract

Purpose. To develop an HPLC-UV method for determination of a novel antitrypanosomal compound (OSU-36) and its ester prodrug (OSU-40) in rat plasma and to apply the method for pharmacokinetic evaluation of both compounds in rats. Methods. Since OSU-36 and OSU-40 were not stable in plasma resulting in highly non-linear calibration curves and poor sensitivity, the plasma samples were stabilized using paraoxon and ascorbic acid. The sample treatment included protein precipitation by acetonitrile; evaporation; reconstitution with acetonitrile and filtration. The chromatography conditions included Xterra RP18 3.5μm 4.6X100mm column and gradient mobile phase system of acetonitrile-water. Results. The limits of quantification (LOQ) using 200 μL of plasma sample were 50 ng/mL and 40 ng/mL for OSU-36 and OSU-40, respectively. The intra- and interday precision and accuracies were below 13% for low, medium and high concentration quality control samples for both compounds. While OSU-40 has been stable in all tested handling conditions, OSU-36 was unstable in plasma after 20 days storage at -80°C or at 4h 25°C storage. The developed method has been applied for a pharmacokinetic study in rats which revealed that an ester prodrug OSU-40 is rapidly converted to OSU-36 within the plasma compartment by plasma esterases. OSU-36, in turn, relatively quickly undergoes oxidative metabolism, including within the plasma compartment. Conclusions. A supplementation of rat plasma with an esterase inhibitor to prevent degradation of ester prodrug (OSU-40), and with antioxidant to prevent oxidation of OSU-36, is necessary for reliable determination of both compounds. Due to limited stability of OSU-36 even in stabilized rat plasma, long-term storage of samples or prolonged handling in room temperature conditions is not recommended.