Comparison between ammonium formate and ammonium fluoride in the analysis of stratum corneum lipids by reversed phase chromatography coupled with high resolution mass spectrometry

Abstract

Lipids are key constituents of the barrier function in the human stratum corneum (SC), which is the outermost layer of the epidermis and amenable to non-invasive sampling by tape stripping. The three major lipid classes in the SC, i.e., ceramides, fatty acids, and cholesterol, present equimolar concentration. Liquid chromatography coupled with mass spectrometry (LCMS) is elective in profiling lipids in the SC in both positive and negative ion modes. Nevertheless, the latter one allows for the simultaneous detection of the three major epidermal components of the SC. Determination of ceramides in the SC poses analytical challenges due to their wide range of structures and concentrations especially in the case of limited sample amounts. Ammonium formate is a commonly used modifier added to the mobile phase to assist ionization. However, it introduces uncertainty in the identification of ceramides when operating in negative ion mode, even with high resolution MS. We tested the advantages of using fluoride in the lipid profiling of SC and unambiguous identification of ceramides subclasses. The use of fluoride enhanced the ionization of ceramides, regardless the specific substructure, solved misidentification issues, and was successfully applied to the simultaneous detection of all three lipid classes in the human SC.

JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines

“…The maximum operating pressure was 600 bar/9000 psi. Cell extracts were eluted with a binary gradient of (A) 0.2 mM NH 4 F in water (18.2 Ω) and (B) 0.2 mM NH 4 F in methanol/isopropanol 80/20 [25]. Following a hold time of 2 min in 40% B, the gradient 40-99% occurred between 2.0 and 36.0 min; 99% B was held from 36.0 to 46.0 min.…”