Determination of a chemopreventive agent, Oltipraz, in rat plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.

Stability, blood partition and protein binding of an antifibrotic agent, oltipraz.

The stability, blood partition and factors influencing the binding of oltipraz to 4% human serum albumin (HSA) were evaluated. Oltipraz was relatively stable in various pH (1-12) solutions for up to 48-h incubation, however, it was unstable in pH 13 solution and rat plasma and urine. Oltipraz reached an equilibrium fast (within 30 s mixing manually) between plasma and blood cells o f rabbit blood and the plasma-to-blood cells concentration ratios were independent of initial blood concentrations of oltipraz, 1 and 5 microg/ml; the ratios ranged from 0.908 to 1.004. The binding of oltipraz to 4% HSA was independent of oltipraz concentrations ranging from 1 to 100 microg/ml using an equilibrium dialysis technique: the mean value was 95.0%. However, the binding of oltipraz was dependent on HSA concentrations (the low concentrations, 3, 2, 1 and 0.5% of HSA caused an increase in the unbound fraction of oltipraz by 1.32, 1.98, 3.44 and 5.31 times, respectively, compared with the mean value from 4-6% HSA), incubation temperature (the unbound fractions at 37 degrees C and 22 degrees C increased 1.89 and 1.73 times, respectively, than that at 4 degrees C) and the buffer pHs (the unbound fractions were 6.36, 6.51, 5.60 and 4.63% for buffer pHs of 5.8, 6.4, 7.4 and 8.0, respectively).