Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(−)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid–liquid extraction of liver microsomal samples with methyl tert -butyl ether, EPH and PEPH were separated on a C 18 reversed-phase column (200 mm × 4.6 mm, 5 μm) with methanol–0.5% sodium dodecyl sulfate–phosphoric acid–triethylamine 60:40:1.25:1 ( v / v ) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5–60.0 μg mL −1 , the limit of quantification was 1.5 μg mL −1 , and intra-day and inter-day variability was <10.0%. Average extraction recovery of the two analytes was >73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and β-naphthoflavone.