Simultaneous Determination of Oleuropein and Tyrosol in Plasma Using High Performance Liquid Chromatography with UV Detection

Abstract

Oleuropein (O) and Tyrosol (T) are polyphenolic compounds with potent biological activities including, but not limited to, an antioxidant activity. An increased interest by many pharmaceutical companies has been shown to develop a formulation of O and/or T. In this research effort, a fast, simple, and reliable analytical method for the determination of O and T in plasma is required in order to carry out bioavailability studies. In this work, a reversed‐phase isocratic high‐performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of O and T in plasma. The isolation of the polyphenolic analytes from plasma was carried out by liquid extraction with ethyl acetate after the addition of Na2SO4 to the sample (salting out effect), followed by evaporation to dryness at 50°C, and reconstitution with mobile phase (4 × preconcentration). Chromatographic analysis was performed using a C8 column with MeOH/CH3COOH 2% in water, 45:55 v/v as mobile phase, and UV detection at 280 nm. Vanillin (V) was used as internal standard. The recovery of the isolation procedure was 80% for O (CV = 6–18%) and 98% for T (CV = 3–8%). Retention times (min) were 4.6 for T, 6.2 for internal standard, and 8.9 for O, while endogenous plasma components were eluted before 4.3 min. Calibration curves of O and T in plasma were linear from 1–20 and 0.1–2.0 μg/mL at least, respectively (r > 0.9996 and 0.999, correspondingly). The detection limits of the method were 0.36 µg/mL for O and 0.09 µg/mL for T (4 × preconcentration). Precision and accuracy were determined from spiked plasma samples and were, for O CV = 5.7–8.3% and Er = 1.3–6.1% and for T CV = 6.1 − 0% and Er = 1.5–5%. The analytical methodology developed in this report is simple, rapid, accurate, and sensitive enough to be used in bioavailability studies.