Isocratic high-performance liquid chromatographic determination of thiacetazone by direct injection of plasma into an internal surface reversed-phase column.

This report describes the determination of thiacetazone in human and rat plasma by direct-injection high-performance liquid chromatography (HPLC). Plasma filtrate (50 microliters) was injected directly into the internal surface reversed-phase (ISRP) mixed-functional phenyl column (Capcell Pak, 50 x 4.6 mm, 5 microns) and eluted with an aqueous mobile phase containing 7.5% acetonitrile at a flow-rate of 1 ml/min. With UV detection at 322 nm, thiacetazone eluted at 11.0 min whereas endogenous interferences eluted before 5 min. The lower detection limit for a 50-microliter sample at a signal-to-noise ratio of 5 was 63 ng/ml, which was several hundred fold lower than its cytotoxic concentrations determined from in vitro cell line studies. At a concentration range of 0.17 to 2.7 micrograms/ml, the recovery of thiacetazone was 98.0 +/- 4.4% (mean +/- S.D.). The intra- and inter-day coefficients of variation were 3.0 +/- 1.4% and 4.2 +/- 2.1%, respectively. This method was successfully applied to study the pharmacokinetics of thiacetazone in rats. The direct injection method is simple, requires small sample volume and does not require sample extraction, internal standard, or gradient elution.