A validated qualitative method using oleuropein as a robust biomarker to confirm ground black pepper (Piper nigrum L.) adulteration with olive (Olea europaea L.) by-products

Olive stone and olive pomace were first identified as black pepper adulterants at the end of the 19th century. Nowadays, there is a worldwide boom in olive oil consumption. The olive oil and table olive industry generate tremendous volumes of olive wastes and this is presenting fraudsters with significant opportunities to use olive oil by-products as bulking agents in other foods such as black pepper. Despite the development of analytical methods for black pepper (Piper nigrum L.) authentication, it has been difficult to both detect and identify the specific fraudulent addition of Olea europaea L. waste by-products to this well-known spice. As such, there is a need for the development of a targeted method to address this issue. In this study, a secoiridoid phenolic biomarker of olive oil by-products, oleuropein, was identified as a target for the development of a novel analytical method, coupling ASE®) extraction and LC-MS/MS analysis for the identification of phenolic compounds as biomarkers of black pepper adulteration with olive wastes. The described operating conditions were used for the quantitation of oleuropein in olive oil by-products as well as the qualitative detection and identification of oleuropein in black pepper as a proof of the presence of olive waste addition as a bulking agent. Oleuropein was detected and quantified in all the olive oil by-product samples, even those subjected to processes such as olive-pomace extraction with hexane and sterilisation with steam heat-treatment. The method was validated for the qualitative detection of oleuropein in black pepper with selectivity and specificity values equal to 1 for 1.5% of olive pomace addition. The proposed method is simple and automated, requiring no purification step and can be applied as a rapid routine analysis to arm the industry against this potential fraud.