###### Abstract  
This study developed, validated, and tested a simple method for tocopherol analysis on five different food matrices (sunflower oil, mackerel fillets, almonds, spinach, and avocado pulp). Tocopherol extraction from foods was carried out by the Folch method and with \(n\)-hexane, and the identification and quantification oftocopherol isoforms (\(\alpha\), \(\beta\), \(\gamma\), and \(\delta\)) was performed using normal-phase liquid chromatography with ultraviolet-visible detection (NP-HPLC-UV-Vis). The normal-phase column fully separated the four tocopherol isoforms in less than ten minutes. Linearity was shown to be excellent for the four isoforms in the assayed range (10-375 ppm, R\({}^{2}\) > 0.99). Furthermore, the limits of detection (LOD) and quantification (LOQ) ranged from 0.32 to 0.63 ppm, and from 1.08 to 2.11 ppm, respectively. The intra-day and inter-day precision were assessed at different concentrations (10, 100, and 250 ppm) for each tocopherol isoform and they were within the range of acceptable values. Recovery rates were above 80% in most cases for all of the assayed food matrices, regardless of the extraction method (Folch solvents or \(n\)-hexane). \(\alpha\)-Tocopherol was the main isoform found in all tested foods, and sunflower oil was the sample with the highest content, followed by almond, avocado pulp, mackerel fillet, and spinach. This method provides a convenient alternative for obtaining a complete profile of the four tocopherol isoforms in a variety of food matrices and for tracking the potential degradation kinetics of fortified foods during their processing and storage. Keywords:vitamin E; tocopherols; validation; HPLC; foods +
Footnote †: journal:Additionally, tocopherols help to increase the shelf life and stability of foods due to their antioxidant properties and interaction with polyunsaturated fatty acids in membrane lipids, protecting them against lipid peroxidation and scavenging reactive oxygen species (ROS) [2,9]. According to the Institute of Medicine of the US National Academies, the Dietary Reference Intakes (DRI) for \(\alpha\)-tocopherol as the active form of vitamin E in humans are 5 mg (7.5 IU) for children between 1 and 3 years old, 6 mg (9.0 IU) for children between 4 and 10 years old, 9 mg (13.4 IU) for children between 11 and 14 years old, 12 mg (17.9 IU) for those older than 14 years, and 16 mg (23.9 IU) for breastfeeding women [10]. Vitamin E is found in several food groups. In general, nuts, seeds, vegetable oils, grains, and green leafy vegetables are considered good sources of vitamin E [2,9,11]. \(\alpha\)-Tocopherol is found in high levels in green leafy vegetables, wheat germ and its oil, sunflower seeds and oil, rice bran, almonds, hazelnuts, grape seeds, and olive oil, while \(\gamma\)-tocopherol is found primarily in the seeds and oils of corn, rapeseed, canola, soybeans, and peanuts. Soybeans, sunflower seeds, and raspberries are also sources of \(\delta\)-tocopherol [2,11]. Tocopherols are identified and quantified in foods by chromatographic methods, including gas chromatography, capillary electrochromatography, supercritical fluid chromatography, thin-layer chromatography, and normal- or reversed-phase high-performance liquid chromatography (NP/RP-HPLC) [12,13]. HPLC is a suitable and versatile tool fortocopherol analysis [11]. The combined use of different stationary phases and detection methods, such as light absorption (diode array or UV-Visible detectors), fluorescence, or mass spectrometry, allows a suitable separation of tocopherol isoforms with good reproducibility and precise identification and quantification [14, 15, 16, 17]. This work aimed to validate a method for the identification and quantification of tocopherol isoforms (\(\alpha\), \(\beta\), \(\gamma\), and \(\delta\)) in different foods (sunflower oil, mackerel fillet, almonds, avocado, and spinach) by HPLC-UV-Vis.