Pressurized liquid extraction and dispersive liquid–liquid microextraction for determination of tocopherols and tocotrienols in plant foods by liquid chromatography with fluorescence and atmospheric pressure chemical ionization-mass spectrometry detection

Pressurized liquid extraction (PLE) and dispersive liquid–liquid microextraction (DLLME) were used to isolate and preconcentrate tocopherols and tocotrienols from plant foods. The Taguchi experimental method was used to optimize the six factors (three levels for each factor), affecting DLLME, namely: carbon tetrachloride volume, methanol volume, aqueous sample volume, pH of sample, sodium chloride concentration and time of the centrifugation step. The influencing parameters selected were 2 mL of methanol:isopropanol (1:1) (disperser solvent), 150 µL carbon tetrachloride (extraction solvent) and 10 mL aqueous solution. The organic phase was injected into reversed-phase liquid chromatography (LC) with an isocratic mobile phase composed of an 85:15 (v/v) methanol:water mixture and a pentafluorophenyl stationary phase. Detection was carried out using both fluorescence and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in negative ion mode. Quantification was carried out by the standard addition method. Detection limits were in the range 0.2–0.3 ng mL−1 for the vitamers with base-line resolution. The recoveries obtained using the optimized DLLME were in the 90–108% range, with RSDs lower than 6.7%. The APCI-MS spectra, in combination with fluorescence spectra, permitted the correct identification of compounds in the vegetable and fruit samples. The method was validated according to international guidelines and using two certified reference materials.