Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study

[Image: see text] Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows. (31)P nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP)–mass spectrometry (MS) can use certified species-unspecific standards to validate shotgun or liquid chromatography (LC)-MS-based lipidomics approaches. We further introduce a novel lipid class quantification strategy based on lipid class separation and mass spectrometry using an all ion fragmentation (AIF) approach. Class-specific fragments, measured over a mass range typical for the lipid classes, are integrated to assess the lipid class concentration. The concept proved particularly interesting as low absolute limits of detection in the fmol range were achieved and LC-MS platforms are widely used in the field of lipidomics, while the accessibility of NMR and ICP-MS is limited. Using completely independent calibration strategies, the introduced validation scheme comprised the quantitative assessment of the complete phospholipid sub-ome, next to the individual lipid classes. Komagataella phaffii served as a prime example, showcasing mass balances and supporting the value of benchmarks for quantification at the lipid species level.