In House Validated UHPLC Protocol for the Determination of the Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil Fit for the Purpose of the Health Claim Introduced by the EC Regulation 432/2012 for "Olive Oil Polyphenols"

Molecules. 2019 Mar 16;24(6):1044. doi: 10.3390/molecules24061044.


An ongoing challenge in olive oil analytics is the development of a reliable procedure that can draw the consensus of all interested parties regarding the quantification of concentrations above the required minimum value of 5 mg of bioactive "olive oil polyphenols" per 20 g of the oil, to fulfill the health claim introduced by the European Commission (EC) Regulation 432/2012. An in-house validated ultra-high performance liquid chromatography (UHPLC) protocol fit for this purpose is proposed. It relies on quantification of the total hydroxytyrsol (Htyr) and tyrosol (Tyr) content in the virgin olive oil (VOO) polar fraction (PF) before and after acidic hydrolysis of their bound forms. PF extraction and hydrolysis conditions were as previously reported. The chromatographic run lasts ~1/3 of the time needed under high performance liquid chromatography (HPLC) conditions, this was also examined. Eluent consumption for the same piece of information was 6-fold less. Apart from being cost effective, a larger number of samples can be analyzed daily with less environmental impact. Two external curves, detection at 280 nm and correction factors for molecular weight difference are proposed. The method, which is fit for purpose, is selective, robust with satisfactory precision (percentage relative standard deviation (%RSD) values < 11%) and recoveries higher than 87.6% for the target analytes (Htyr, Tyr). Standard operational procedures are easy to apply in the olive oil sector.

Keywords: European Commission Regulation 432/2012; UHPLC; diode array detection; health claim; hydroxytyrosol; olive oil phenols; tyrosol; validation; virgin olive oil.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Humans
  • Hydrolysis
  • Olive Oil / chemistry*
  • Phenylethyl Alcohol / analogs & derivatives*
  • Phenylethyl Alcohol / chemistry
  • Phenylethyl Alcohol / isolation & purification
  • Polyphenols / chemistry*
  • Polyphenols / isolation & purification


  • Olive Oil
  • Polyphenols
  • 3,4-dihydroxyphenylethanol
  • 4-hydroxyphenylethanol
  • Phenylethyl Alcohol