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 cor responding pentobarbitone enantiomers which are usually present as met abolites of thiopentdne. Phenylbutazone was used as an internal standa rd: After acidification, the plasma samples were extracted with a mixt ure of ether and hexane (2:8). The solvent was evaporated to dryness a nd the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mmx4 mm I.D. Chiral AGP -CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate b uffer (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(-)-pentoba rbitone, R(+)-thiopentone, S(-)-thiopentone, and phenylbutazone, respe ctively. 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 an d 8 mu g/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 o f 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(-)-thiohentone. Linearity over the standard range, 0.01-40 mu g/ml, was shown by correlation coefficients > 0.998. This method h as proven suitable for pharmacokinetic studies of thiopentone enantiom ers after administration of mc-thiopentone in human plasma also and wo uld be suitable for pharmacokinetic studies of the pentobarbitone enan tiomers.