Cost effective, robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: application to insects.

The quantification of phospholipid classes and the determination of their molecular structures are crucial in physiological and medical studies. This paper's target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation processes of poikilothermic organisms. We introduce a set of simple and cost-effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid-phase extraction and high-performance thin-layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chromatography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high-performance liquid chromatography with mass spectrometry, which gives information about the headgroup moiety and attached fatty acids. The total content of each phospholipids class is assessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high-performance liquid chromatography analysis.