The analysis of triacylglycerols and phospholipids - phosphatidylcholines allowed the use of shotgun lipidomics to identify very long-chain fatty acids and very long-chain polyunsaturated fatty acids in microalgae. These fatty acids were determined in triacylglycerols by positive electrospray ionization of neutral loss scans of different fatty acids, e.g. 24:0, 24:1ω9, 24:6ω3, 26:0, 26:1ω9, 28:0, 28:1ω9, 28:2ω6, and 28:8ω3. Likewise, very long-chain fatty acids in phosphatidylcholines were identified by negative electrospray ionization mass spectrometry in the selected ion-monitoring of the two most important ions (R1COO- and R2COO-). The limit of detection was determined at 10 nmol/L (∼11 pg/μL) in triacylglycerols and 8.6 nmoles/L (∼8 pg/μL) in phosphatidylcholines. The use of liquid chromatography-mass spectrometry is suitable for very long-chain polyunsaturated fatty acids with up to 8 double bonds due to the time of analysis as well as for reasons of lower thermal stability of polyunsaturated fatty acids towards saturated fatty acids, but gas chromatography-mass spectrometry is better suited for the analysis of saturated very long-chain fatty acids.

Department of Biotechnology Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Ecology Faculty of Science Charles University Viničná 7 128 44 Prague Czech Republic

Institute of Botany The Academy of Sciences of the Czech Republic Dukelská 135 379 82 Třeboň Czech Republic

Institute of Microbiology The Academy of Sciences of the Czech Republic Centre Algatech Laboratory of Cell Cycles of Algae Opatovický mlýn 237 379 81 Třeboň Czech Republic

Institute of Microbiology The Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Prague Czech Republic

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Characterization of Archaea membrane lipids in radioactive springs using shotgun lipidomics