Development of a high-performance thin-layer chromatography-based method for targeted glycerolipidome profiling of...

The conditionally essential very-long-chain polyunsaturated fatty acids (VLC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5 n-3), play a vital role in human nutrition. Their biological activity is thereby greatly influenced by the distinct glycerolipid molecule that they are esterified to. Here, microalgae differ from the conventional source, fish oil, both in quantity and distribution of VLC-PUFAs among the glycerolipidome. Therefore, the aim of this study was to develop a fast and reliable one-dimensional high-performance thin-layer chromatography (HPTLC)-based method that allows the separation and quantification of the main microalgal glycerolipid classes (e.g., monogalactosyldiacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG)), as well as the subsequent analysis of their respective fatty acid distribution via gas chromatography (GC) coupled to mass spectrometry (MS). Following optimization, method validation was carried out for 13 different lipid classes, based on the International Conference on Harmonization (ICH) guidelines. In HPTLC, linearity was effective between 100 and 2100 ng, with a limit of quantification between 62.99 and 90.09 ng depending on the glycerolipid class, with strong correlation coefficients (R² > 0.995). The recovery varied between 93.17 and 108.12%, while the inter-day precision measurements showed coefficients of variation of less than 8.85%, close to the limit of detection. Applying this method to crude lipid extracts of four EPA producing microalgae of commercial interest, the content of different glycerolipid classes was assessed together with the respective FA distribution subsequent to band elution. The results showed that the described precise and accurate HPTLC method offers the possibility to be used routinely to follow variations in the glycerolipid class levels throughout strain screening, cultivation, or bioprocessing. Thus, additional quantitative analytical information on the complex lipidome of microalgae can be obtained, especially for n-3 and n-6 enriched lipid fractions.