High Throughput\nLC-MS Platform for Large Scale Screening\nof Bioactive Polar Lipids in Human Plasma and Serum

Lipids play a key role in many biological processes,\nand their\naccurate measurement is critical to unraveling the biology of diseases\nand human health. A high throughput HILIC-based (LC-MS) method for\nthe semiquantitative screening of over 2000 lipids, based on over\n4000 MRM transitions, was devised to produce an accessible and robust\nlipidomic screen for phospholipids in human plasma/serum. This methodology\nintegrates many of the advantages of global lipid analysis with those\nof targeted approaches. Having used the method as an initial “wide\nclass” screen, it can then be easily adapted for a more targeted\nanalysis and quantification of key, dysregulated lipids. Robustness\nwas assessed using 1550 continuous injections of plasma extracts onto\na single column and via the evaluation of columns from 5 different\nbatches of stationary phase. Initial screens in positive (239 lipids,\n431 MRM transitions) and negative electrospray ionization (ESI) mode\n(232 lipids, 446 MRM transitions) were assessed for reproducibility,\nsensitivity, and dynamic range using analysis times of 8 min. The\ntotal number of lipids monitored using these screening methods was\n433 with an overlap of 38 lipids in both modes. A polarity switching\nmethod for accurate quantification, using the same LC conditions,\nwas assessed for intra- and interday reproducibility, accuracy, dynamic\nrange, stability, carryover, dilution integrity, and matrix interferences\nand found to be acceptable. This polarity switching method was then\napplied to lipids important in the stratification of human prostate\ncancer samples.