HPLC Method with UV Detection for the Determination of trans‐Resveratrol in Plasma

Abstract

The main representative of phytoalexins (biologically active substances produced by the plant as an immediate immunological response to infections or abiotic stresses) in wine grapes (Vitis vinifera) is the stilbene trans‐resveratrol. The proposed method for the determination of trans‐resveratrol in blood plasma is based on reversed phase HPLC utilizing UV detection (310 nm), under isocratic conditions (1.0 mL/min), with mobile phase consisting of acetonitrile‐phosphate buffer pH 4.8 (30 mM) 25∶75 v/v, a C₁₈ Novapack 150×4.0 mm column (4 µm particle size), and carbamazepine as internal standard (5 µg/mL). Plasma samples (1 mL) are buffered with phosphate buffer (0.4 mL) pH 6.0 and extracted with 3×3 mL of ethyl acetate. trans‐resveratrol is eluted at around 4.6 min, whereas carbamazepine at approximately 9.6 min, yielding a resolution of 4.4. The method appears to be linear within a range of 0.15–4.0 µg/mL (r=0.9998), with good repeatability (%RSD=0.86) and reproducibility (%RSD=2.2). Detection and quantification limits were found equal to 0.10 and 0.33 µg/mL, respectively. Accuracy, expressed as recovery determined at two concentrations of 0.3 and 3.0 µg/mL (6‐replicates) were found to be 88.3±7.5% and 100.7±0.7%, respectively. The reported method is simple, rapid, accurate, and intended for use to further bioavailability studies, aiming to the development of pharmaceutical dosage formulations of resveratrol for oral administration.

Keywords:

• Trans‐resveratrol
• HPLC
• Plasma
• Carbamazepine

Acknowledgments

We are grateful to the General Secretary of Research and Technology of the Ministry of Research and Technology, to the Greek pharmaceutical company Lavipharm, and the Greek Ministry of Education (EPEAEK program) for financial support.