# Extraction of Galactolipids from Waste By-Products:
## 2 Materials and Methods
### Analysis of Galactolipid Content  
The method presented by [PERSON] and co-workers was used for the qualitative and quantitative analysis of galactolipids [9]. The ground plant sample (6 g) was placed in a flask, 15 mL of isopropanol was added, and the mixture was kept at a temperature of 75 \({}^{\circ}\)C for 15 min. Subsequently, 7.5 mL of chloroform and 3 mL of water were added to the flask and then shaken for 1 h at 300 min\({}^{-1}\). The extract was collected and a sample was reextracted with 10 mL of chloroform-methanol mixture (2:1) three times. The extracts were collected and washed with 20 mL of 1M potassium chloride. The organic phase was dehydrated with 5 g of anhydrous magnesium sulfate, filtered through cotton wool, and concentrated on a rotary evaporator (R-300 Rotavapor, Buchi, Flawil, Switzerland) to approximately 20% of the initial volume. The extracts obtained using SFE and UAE methods were only subjected to the last part of the aforementioned protocol (from dehydration with MgSO\({}_{4}\)). Semi-preparative thin-layer chromatography was used to isolate the main groups of galactolipids. The extract was transferred to a 10 \(\times\) 20 cm TLC silica gel 60G (0.2 mm) plate (Merck, Darmstadt, Germany) and eluted with a mixture of chloroform-methanol-water (80:18:2). The zones corresponding to MGDGs and DGDGs typically had retention factors of 0.7 and 0.4, respectively. Nevertheless, standards were also placed on the plate and visualized under UV light after spraying with a primuline solution (0.05% in methanol-water (8:2) mixture). The zones corresponding to galactolipid groups were scraped, extracted with a mixture of chloroform-methanol (2:1), and filtered through cotton glass. Liquid chromatography analyses were performed on an H-class liquid chromatograph coupled to a mass spectrometer with a time-of-flight analyzer (UPLC-TOF-HRMS; Waters, Milford, MA, USA). The samples (5 \(\upmu\)L) were separated using a UPLC C18 Cortecs (2.1 \(\times\) 100 mm, 1.6 \(\upmu\)m) column (Waters). Despite using the same equipment, separate gradients were employed for the galactolipid groups--the details are presented below in Table 1. The mass spectrometer operated in the positive electrospray ionization mode. The ion source temperature was 150 \({}^{\circ}\)C, while the desolvation temperature was 350 \({}^{\circ}\)C. The nebulizing gas (N\({}_{2}\)) flow rate was 750 L min\({}^{-1}\), the cone gas flow rate was 40 L min\({}^{-1}\), and the capillary bias was 3.2 kV. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Time (min)**} & \multicolumn{2}{c}{**MGDGs**} & \multirow{2}{*}{**Methanol**} \\  & **Water** & & \\ \hline
0 & 25\% & & 75\% \\
80 & 0\% & & 100\% \\
90 & 0\% & & 100\% \\
95 & 25\% & & 75\% \\ \hline \hline \end{tabular}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Time (min)**} & \multirow{2}{*}{**Water**} & \multirow{2}{*}{**OGDGs**} & \multirow{2}{*}{**Methanol**} \\  & & & 65\% \\
0 & 0\% & & 80\% \\
50 & 0\% & & 0\% \\
60 & 0\% & & 100\% \\
80 & 0\% & & 0\% \\
85 & 35\% & & 65\% \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Gradients used for the LC-MS analysis of the galactolipids. The method implemented allowed the separation of galactolipids and their partial identification. As reported by the authors of the method, galactolipids in plant samples are present as a mixture of compounds with various fatty acids attached to the glycerol scaffold (Figure 1) [9]. The implementation of MS detection made it possible to distinguish between the compounds based on their \(m/z\) ratio, as the elongation of the carbon chain or saturation of the double bond can be easily identified. GOPO, the compound that, according to the literature, has the highest pharmacological potential is MGDG, with two \(\alpha\)-linoleic acids (18:3) attached; due to its structure, the identification is certain as no lipids of the same molecular mass could occur.