Isoxyl assays in plasma

Isoxyl is an effective drug to treat multi-drug resistant (MDR) tuberculosis but was abandoned due to failure in some clinical outcomes. The aim of this study was to develop and validate a reverse-phase high-performance liquid chromatographic (HPLC) method for determination of isoxyl concentrations in plasma, a prerequisite for understanding poor in vivo behavior of the drug. In the method, isoxyl was extracted from guinea pig plasma with acetonitrile and quantified by a Hewlett Packard 1100 series HPLC coupled with a Spherisorb 5 μm ODS2 (2 × 100 mm) column and UV detection at 270 nm. The mobile phase was 70% ACN in 20 mM ammonium acetate buffer. The isoxyl peak was eluted at 4.8 min with no interference with the peaks of impurities from plasma and internal standard. Recovery of isoxyl from guinea pig plasma was >68%, and LOQ (Limit of Quantification) was 0.25 μg/ml which was 8 times lower than the reported minimal inhibitory concentration (MIC, 2 μg/ml). The HPLC method was sensitive, reproducible, and accurate for quantification of isoxyl in guinea pig plasma according to FDA guidance for bioanalytical method validation. The method was utilized to quantify isoxyl plasma concentrations following oral administration of the drug to guinea pigs. The results suggest that the poor clinical outcomes of the drug may have been caused by the extremely low isoxyl plasma concentrations which were far below the MIC for action on Mycobacterium tuberculosis.