Liquid chromatography–tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration

We describe a liquid chromatography–electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid–liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water–acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5–1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5–1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.