High-performance liquid chromatographic separation and nanogram quantitation of bupivacaine enantiomers in blood.

Chiral separation of rac-bupivacaine extracted from blood was achieved with similar limits of detection but using a much simpler sample preparation than reported previously. The simple one-step sample preparation devised was highly robust and efficient and allowed a very high throughput of samples. The high-performance liquid chromatography (HPLC) conditions used gave baseline separation of the enantiomers with high sensitivity. R-(+)-bupivacaine and S-(-)-bupivacaine blood concentrations were determined using a chiral stationary phase (AGP, ChromTech) with diode array detection at 220 nm; this wavelength produced a stable baseline allowing semi-automated analysis. Sample preparation involved addition of internal standard (diphenhydramine), basification of blood, extraction with n-hexane, concentration of the extract to dryness and reconstitution in 0.002 M phosphoric acid. At rac-bupivacaine concentrations of 0.5, 5 and 50 microg/ml in blood, assay accuracy as estimated by coefficients of variation (C.V.s), were 3.3, 1.4, and 1.6%, respectively, for R-(+)-bupivacaine and 3.7, 2.0 and 1.5%, respectively, for S-(-)-bupivacaine. Using 0.6-ml samples, the estimated limits of detection for R-(+)-bupivacaine and S-(-)-bupivacaine were both 15 ng/ml of blood. Calibration curves (n=188) were linear from 0.1 to 50 microg/ml with all correlation coefficients being greater than 0.99. This semi-automated method was applied to studies involving whole body pharmacokinetics with intravenous doses ranging from 12.5 to 350 mg and regional myocardial pharmacokinetics with coronary arterial doses ranging from 2.5 to 12.5 mg. These studies generated approximately 12000 blood samples.