Benchmarking Lipid Quantitation from Skin Swab Sebum-Rich Samples to Establish Mass Spectrometry-Based Diagnostic...

Skin swab sampling provides an accessible, noninvasive method for collecting lipid-rich material from the skin surface, with growing potential for biomarker discovery and diagnostic testing. However, quantitative sebum analysis remains challenging because of variability in the amount collected and the complexity of the lipid matrix. This study evaluates the robustness of skin swab sampling for lipid analysis and the feasibility of quantifying skin-surface components using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple reaction monitoring was used to assess analytical linearity, matrix effects, and recovery following established protocols for swab collection, storage, and extraction. Robust linearity was observed for both spiked isotope-labeled internal standards (IS) and endogenous lipids across concentrations of 0.15-550 ng/mL (R² > 0.97). IS recovery varied between lipid classes, with consistent under-recovery. Although IS corrected for analytical variability during extraction and analysis, it could not fully account for differences in the amount of material collected. Endogenous lipids were, therefore, evaluated as normalization markers. Ratios between selected triacylglycerol (TG) species were highly conserved across samples, with coefficients of variation below 15% for TG 45:1/TG 47:1, TG 46:1/TG 48:1, and TG 47:1/TG 48:1. These ratios were stable across individuals and were independent of disease status. The reproducibility of TG recovery and stability of TG ratios suggest that endogenous lipid normalization can mitigate sampling and extraction variability. This approach provides a framework for improving quantitative reliability in skin swab lipidomics and represents a big step toward the development of reproducible mass spectrometry workflows for skin-swat-based biomarker studies.