Routine and sensitive SPE-HPLC method for quantitative determination of pheophytin a and pyropheophytin a in olive oils

Abstract

A sensitive and specific HPLC method with tandem diode array-fluorescence detection (DAD-FL) has been developed and validated for the simultaneous determination of pheophytin a (phy a) and pyropheophytin a (pyrophy a) in olive oils. Pigments were extracted with reverse phase solid phase extraction (RP-SPE) and subsequently analysed by HPLC-DAD-FL. The chromatographic analysis was carried out isocratically on ODS2 RP column using methanol–acetone (1:1 v/v) at flow-rate of 2.0 ml min −1. Specificity of the method was assured by the simultaneous detection by UV–visible (410 nm) and FL ( λ Ex: 410 nm; λ Em: 672 nm). Both compounds could be baseline separated within 7 min. The method was validated and applied in olive oil samples recently extracted as well as stored during 12 months. The limit of detection (LOD) defined at a signal-to-noise ratio of about 3 was ∼21.6 ng g −1 for pyrophy a and ∼24.6 ng g −1 for phy a under FL detection, and ∼148.0 ng g −1 for both analytes under UV–visible detection. The calibration graphs were linear ( r 2 > 0.9999; p < 0.01) between 0.25–14.00 ng μl −1 for pyrophy a and 0.25–19.00 ng μl −1 for phy a, under both fluorescence and UV–visible detection conditions. Recoveries of phy a and pyrophy a were over 94% as estimated by the standard addition method. Relative standard deviation for the intra-day and inter-day determination of phy a and pyrophy a were lower than 3.7% and 8.0%, respectively.