I got the HPLC method from a published paper and is the same method that Sirtris pharma uses.
Would you mind sharing the name/authors of this paper?
Is is for the blood plasma analysis..... Or go to pubmed and the keywords I have used are "HPLC Resveratrol plasma"
http://cebp.aacrjour.../full/16/6/1246Sample Preparation
After defrosting, plasma and urine samples were extracted with acidified methanol [5% methanol with 2% acetic acid (pH 4), equal volume for plasma, twice the volume for urine]. The extractant was centrifuged (9, 000 x g, 15 min) and the supernatant was analyzed. Fecal matter was homogenized (5-mL water containing 2% phosphoric acid) and the homogenate was centrifuged (4°C, 2,900 x g, 15 min). Analyte was isolated from the supernatant using solid-phase extraction (Oasis HLB, Waters; volume, 3 mL; packing weight, 30 mg). Solid-phase cartridges were primed (1-mL acidified methanol followed by 1-mL water), washed (1 -mL acidified methanol), and analyte was eluted with methanol. While urine extractant and column eluents obtained from fecal extracts were injected onto the high-performance liquid chromatography column directly, plasma extractant was dried under nitrogen and reconstituted in mobile phase before high-performance liquid chromatography analysis. Resveratrol content in stool was related to dry feces weight, which was obtained by drying (100°C) stool to weight constancy (24 h).
High-Performance Liquid Chromatography Analysis
Resveratrol and its metabolites were extracted and separated using a gradient UV-high-performance liquid chromatography system (Waters Breeze) as described before (29). Separation was achieved on a Waters Atlantis C18 column (4.6 x 150 mm, 3 µm; Waters) in combination with a Waters Atlantic C18 guard column (4.6 x 20 mm, 5 µm). Column oven temperature was 35°C (flow rate, 1 mL/min). The gradient elution system (A: 5 mmol/L ammonium acetate; B: 98% methanol, both with 2% propan-2-ol) was as follows with respect to B: 0 to 7 min 20%, 7 to 16 min 50%, 16 to 18 min 55%, 18 to 23 min 95%, then reequilibration to 0% B for 6 min before the next injection. Injection volume was 100 µL. The retention time of resveratrol was 18.6 min; its lower limit of detection was 5 ng/mL. This method has been validated for resveratrol in terms of interday and intraday variability, recovery, accuracy, and precision (29). As resveratrol metabolites were not available in sufficient quantities for method development, their quantities were calculated based on the assumption that recovery characteristics and relationship between peak area ratios and concentrations were the same as those for resveratrol. Metabolite concentrations are therefore described as "resveratrol equivalents." Authentic resveratrol 3-sulfate was provided by Royalmount Pharma. The identity of resveratrol and its metabolites was confirmed using an Agilent 1100 series high-performance liquid chromatography with in-line Applied Biosystems/MDS SCIEX API 2000 ion spray triple quadrupole mass spectrometer (Applied Biosystems) using the same chromatographic conditions as described above.
