Paired-ion liquid chromatographic method for the analysis of a phenanthrenemethanol antimalarial in whole blood

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of the candidate antimalarial (+/-)-(1,3-dichloro-6-trifluoromethyl-9-phenanthryl)-3-di-(n-butyl )aminopropanol hydrochloride in whole blood. A reversed-phase, paired-ion (lauryl sulfate) system achieved separation of the antimalarial and internal standard from interfering constituents with a sensitivity limit of 10 ng/mL by UV detection (254 nm). Chromatographic variables (counterion concentration, pH, and column temperature) were examined to determine their effect on assay characteristics (retention, efficiency, and relative response) in clinical analysis. The antimalarial was isolated from 2.0 mL of whole blood using overnight extraction with 30% ethyl acetate in hexane followed by an acid/base partition sequence to remove major interferences. Overall recovery for the antimalarial was 84% with a CV of 5.0%, and the recovery of the internal standard was 81% (CV = 3.6%). The assay was validated by analysis of both intra- and interlaboratory samples. The assay was applied to the analysis of whole blood samples taken from a 30-year-old healthy human male who had received a single 14.1-mg/kg oral dose. The stability of the antimalarial in whole blood for up to 4 months and in sample extracts for up to 34 d at -17 degrees C was also demonstrated.