Steviol glycosides are intense natural sweeteners used in foods and beverages. Their acceptable daily
intake, expressed as steviol equivalents, is set at 0–4 mg/kg body weight. We report the development
and validation of a RP-HPLC method with fluorometric detection of derivatized isosteviol, formed by acid
hydrolysis of steviol glycosides. Dihydroisosteviol was used as an internal standard. Using this method,
the amount of steviol equivalents in commercial steviol glycoside mixtures and different foods can be
directly quantified. The method was successfully tested on strawberry jam, low-fat milk, soft drink,
yogurt and a commercial mixture of steviol glycosides. Calibration curves were linear between 0.01
and 1.61 mM steviol equivalents, with a quantification limit of 0.2 nmol. The % RSD of intra-day precision
varied between 0.4% and 4%, whereas inter-day precision varied between 0.4% and 5%, for high and medium
concentrations, and between 3% and 8% for low concentrations. Accuracy of the analysis varied
between 99% and 115%.