Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma

Abstract

Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 °C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 °C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C 18 column at ∼6.9 and ∼8.1 min, respectively. The eluent ACN–water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 °C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at ∼35.2 and ∼37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 μl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-μl plasma volume.