Comparison of Single Phase and Biphasic Extraction Protocols for Lipidomic Studies Using Human Plasma

Lipidomic profiling of plasma is an emerging field, given the importance of lipids in major cellular pathways, and is dependent on efficient lipid extraction protocols. Recent attention has turned to plasma lipidomics as a means to identify potential diagnostic and prognostic biomarkers related to dementia, neuropsychiatric health and disease. Although several solvent-based lipid extraction protocols have been developed and are currently in use, novel and more efficient methods could greatly simplify lipid analysis in plasma and warrant investigation. Human plasma from normolipidemic adult volunteers was collected to evaluate three different solvent extraction protocols, including the classical Folch method, the methanol/tert-butyl methyl ether (MTBE) (Matyash) method, and a recent single-phase methanol/1-butanol (Alshehry) method. Extracted lipids were analyzed using liquid chromatography mass spectrometry (LC-MS) in positive and negative ion mode. Overall, more than 500 different lipids were identified in positive and negative ion mode combined. Our data show that the single phase Alshehry method was as effective as the Folch and Matyash methods in extracting most lipid classes and was more effective in extraction of polar lipids. Normalized peak areas of the Alshehry method were highly and positively correlated with both the Folch and Matyash methods (r(2) = 0.99 and 0.97, respectively). Within- and between- subject correlations were r = 0.99 and 0.96, respectively. Median intra-assay coefficient of variation (CV%) in positive mode was 14.1, 15.1, and 21.8 for the Alshehry, Folch and Matyash methods, respectively. Median Alshehry inter-assay CV (collected over 5 separate days) was 14.4%. In conclusion, the novel Alshehry method was at least as good as, if not better than the established biphasic extraction methods in detecting a wide range of lipid classes, using as little as 10 μL of plasma, and was highly reproducible, safer and more environmentally-friendly as it doesn't require chloroform.