Lipidomics has great promise in various applications; however, a major bottleneck in lipidomics is the accurate and comprehensive annotation of high-resolution tandem mass spectral data. While the number of available lipidomics software has drastically increased over the past five years, the reduction of false positives and the realization of obtaining structurally accurate annotations remains a significant challenge. We introduce Lipid Annotator, which is a user-friendly software for lipidomic analysis of data collected by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). We validate annotation accuracy against lipid standards and other lipidomics software. Lipid Annotator was integrated into a workflow applying an iterative exclusion MS/MS acquisition strategy to National Institute of Standards and Technology (NIST) SRM 1950 Metabolites in Frozen Human Plasma using reverse phase LC-HRMS/MS. Lipid Annotator, LipidMatch, and MS-DIAL produced consensus annotations at the level of lipid class for 98% and 96% of features detected in positive and negative mode, respectively. Lipid Annotator provides percentages of fatty acyl constituent species and employs scoring algorithms based on probability theory, which is less subjective than the tolerance and weighted match scores commonly used by available software. Lipid Annotator enables analysis of large sample cohorts and improves data-processing throughput as compared to previous lipidomics software.

Lipidomic approaches are widely used to investigate the relationship between lipids, human health, and disease. Conventional sample preparation techniques for the extraction of lipids from biological matrices like human plasma are based on liquid-liquid extraction (LLE). However, these methods are labor-intensive, time-consuming, and can show poor reproducibility and selectivity on lipid extraction. A novel, solid-phase extraction (SPE) approach was demonstrated to extract lipids from human plasma using a lipid extraction SPE in both cartridge and 96-well-plate formats, followed by analysis using a combination of targeted and untargeted liquid chromatography/mass spectrometry. The Lipid Extraction SPE method was compared to traditional LLE methods for lipid class recovery, lipidome coverage, and reproducibility. The novel SPE method used a simplified protocol with significant time and labor savings and provided equivalent or better qualitative and quantitative results than traditional LLE methods with respect to several critical performance metrics; recovery, reproducibility, and lipidome coverage.