Lipids can be found in all living organisms, and complex lipids are typically determined from biological samples and food products. Samples are usually prepared prior to analysis. Liquid-liquid extraction (LLE) is obviously the most often used technique to isolate lipids. Two fundamental protocols are Folch and Bligh & Dyer methods. In both methods, the extraction is based on lipid partitioning between chloroform and water-methanol phases. Methyl-tert-butyl ether offers an environmentally friendly alternative to chloroform. Total lipid fraction can be further separated by solid-phase extraction. Complex lipids are typically isolated from other lipid species with silica SPE cartridges.
Three main techniques used in quantitative determination of complex lipids are thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and direct infusion mass spectrometry (MS). Thin layer chromatography is a traditional technique, but its applicability is limited due to poor resolution and requirement of post-column derivatization. Instead, HPLC provides an efficient separation and it is easily coupled with several detectors. HPLC methods are the most commonly used in lipid analysis. Direct infusion mass spectrometry is the incoming technique. Lipid molecules can be precisely identified during the fast measurement. Other advantages are excellent selectivity and sensitivity.
New method for glycolipids was developed during the experimental period. Glycolipids were isolated from bio oil samples using solid phase extraction cartridges. Normal phase liquid chromatography was utilized to separate glycolipids, and detection was carried out with parallel tandem mass spectrometry (MS/MS) and evaporative light scattering detection (ELSD). Quantification was based on ELSD measurements, whereas MS/MS was adopted to confirm the identification. Developed method was validated and following parameters were determined: linearity, trueness, precision, measurement uncertainty, detection and quantification limits.
Precisions were successful and they were mainly between 5-15 %. Trueness results were however more undesired, because measured concentrations were typically higher than theoretical concentrations. Results were dependent on analyte, but generally they varied between 66 % and even 194 %. Validation pointed out that method needs further development. Mass spectrometric quantification can be considered, if appropriate internal standards would be available.
