## 4 Conclusions  
The method developed and validated in this work provides a simple and efficient way to analyze \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and \(\delta\)-tocopherol in different food matrices with high sensitivity and acceptable intra-day and inter-day precision and recovery using NP-HPLC with UV-Vis detection, which is one of the most common facilities available in laboratories conducting food analysis. Contrary to what is usually observed when using reversed-phase chromatographic columns, the use of a normal-phase column with an isocratic program allowed \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Food Matrix**} & **Extraction** & **Fortification Level** & \multicolumn{4}{c}{**Recovery (\%)**} \\ \cline{3-6}  & **Method** & **(mg/100 g fw)** & \multicolumn{2}{c}{\(\alpha\)**-T**} & \(\beta\)**-T** & \(\gamma\)**-T** & \multicolumn{1}{c}{**\(\delta\)-T**} \\ \hline High Oleic & - & 160 & 100.3 \(\pm\) 1.2 & 108.2 \(\pm\) 2.6 & 105.5 \(\pm\) 2.2 & 105.6 \(\pm\) 2.4 \\ Sunflower Oil & - & 320 & 100.3 \(\pm\) 1.4 & 106.2 \(\pm\) 2.5 & 105.8 \(\pm\) 2.3 & 106.3 \(\pm\) 1.7 \\ \hline \multirow{4}{*}{Mackerel} & \multirow{4}{*}{Folch} & 0.80 & 87.8 \(\pm\) 2.9 & 88.6 \(\pm\) 4.1 & 104.4 \(\pm\) 4.3 & 98.0 \(\pm\) 3.9 \\  & & 1.60 & 86.8 \(\pm\) 3.6 & 90.7 \(\pm\) 4.7 & 86.0 \(\pm\) 3.5 & 89.1 \(\pm\) 1.9 \\ \cline{1-1}  & \(n\)-hexane & 1.30 & 93.3 \(\pm\) 3.2 & 85.9 \(\pm\) 6.9 & 95.4 \(\pm\) 3.6 & 82.8 \(\pm\) 6.5 \\  & & 2.60 & 94.2 \(\pm\) 1.9 & 82.9 \(\pm\) 3.4 & 90.5 \(\pm\) 1.8 & 79.5 \(\pm\) 3.3 \\ \hline \multirow{4}{*}{Almond} & \multirow{4}{*}{Folch} & 10.0 & 81.9 \(\pm\) 2.6 & 77.8 \(\pm\) 3.4 & 82.9 \(\pm\) 0.9 & 80.3 \(\pm\) 4.5 \\  & & 20.0 & 85.8 \(\pm\) 0.7 & 83.9 \(\pm\) 2.2 & 81.3 \(\pm\) 2.5 & 88.8 \(\pm\) 1.2 \\ \cline{1-1}  & \(n\)-hexane & 10.0 & 89.1 \(\pm\) 2.0 & 78.0 \(\pm\) 2.1 & 84.5 \(\pm\) 2.4 & 86.2 \(\pm\) 0.9 \\ \hline \multirow{4}{*}{Spinach} & \multirow{4}{*}{Folch} & 0.16 & 81.8 \(\pm\) 2.4 & 89.2 \(\pm\) 2.3 & 86.6 \(\pm\) 5.7 & 88.4 \(\pm\) 1.6 \\  & & 0.32 & 87.0 \(\pm\) 1.0 & 85.1 \(\pm\) 1.4 & 87.7 \(\pm\) 3.4 & 86.2 \(\pm\) 2.9 \\ \cline{1-1}  & \(n\)-hexane & 0.16 & 85.7 \(\pm\) 1.9 & 89.2 \(\pm\) 3.8 & 84.9 \(\pm\) 2.4 & 80.6 \(\pm\) 3.3 \\ \cline{1-1}  & & 0.32 & 87.0 \(\pm\) 1.0 & 85.1 \(\pm\) 1.4 & 87.6 \(\pm\) 2.6 & 86.2 \(\pm\) 2.9 \\ \hline \multirow{4}{*}{Avocado} & \multirow{4}{*}{Folch} & 1.50 & 82.4 \(\pm\) 0.7 & 85.4 \(\pm\) 3.1 & 78.3 \(\pm\) 1.6 & 82.3 \(\pm\) 2.5 \\ \cline{1-1}  & & 3.00 & 81.3 \(\pm\) 0.5 & 84.1 \(\pm\) 1.5 & 80.4 \(\pm\) 0.4 & 88.7 \(\pm\) 3.4 \\ \cline{1-1}  & & 1.50 & 89.2 \(\pm\) 2.1 & 89.0 \(\pm\) 3.2 & 83.3 \(\pm\) 4.0 & 86.2 \(\pm\) 2.9 \\ \cline{1-1}  & \(n\)-hexane & 3.00 & 89.3 \(\pm\) 0.5 & 79.0 \(\pm\) 4.0 & 86.8 \(\pm\) 1.7 & 90.0 \(\pm\) 2.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Recovery values (%) of each tocopherol isoform in the five assayed foods after fortification with \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and \(\delta\)-tocopherols at two concentration levels. the complete separation of the four tocopherol isoforms in less than 10 min. The common problem of baseline drifting caused by differences in the UV absorbance of mobile phase solvents when working with a gradient method was avoided in this study by using an isocratic program throughout the analysis. In addition, two lipid extraction methods were compared to verify their suitability in terms of tocopherol quantification in different food matrices. Significantly higher amounts of \(\alpha\)- and \(\gamma\)-tocopherol were found in the avocado pulp when using the Folch method for lipid extraction instead of \(n\)-hexane. Conversely, in almonds, only the amount of \(\gamma\)-tocopherol was found to be significantly higher using Folch extraction compared to \(n\)-hexane. On the other hand, significantly higher levels of \(\alpha\)- and \(\gamma\)-tocopherol were found in mackerel fillets when \(n\)-hexane was used to extract the lipid fraction instead of the Folch method.