High-performance liquid chromatographic determination of thiopentone enantiomers in sheep plasma

An HPLC method was developed to determine the plasma concentrations of R(+)- and S(-)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma sample were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm x 4 mm I.D. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k' values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(-)-pentobarbitone, R(+)-thiopentone, S(-)-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S(-)-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 micrograms/ml were, respectively, 4.8, 4.4 and 3.5% for R(+)-thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(-)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentone and 8.8, 7.4 and 9.6% for S(-)-thiopentone. Linearity over the standard range, 0.01-40 micrograms/ml, was shown by correlation coefficients > 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac-thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone enantiomers.