Rapid and sensitive profiling of tear wax ester species using high performance liquid chromatography coupled with tandem mass spectrometry

• A rapid and sensitive HPLC-MRM method for quantitation of wax esters in human tears.
• 13C-labeled oleic acid-containing wax esters synthesized in-house as standards.
• Total of 141 individual waxes with various combinations of acids and alcohols found.
• Method is transferrable for analyzing wax esters in other biological fluids.

A rapid and sensitive method was developed for quantitative profiling of wax esters (WEs) in human tear lipidome. Individual WE species was separated by liquid chromatography and detected by electrospray ionisation mass spectrometry using specific multiple reaction monitoring (MRM) scanning. Palmitoyl palmitate and in-house synthesized wax esters 13C18:1(oleic acid-1,2,3,7,8,9,10-13C7)C26:0 were used as internal standards for quantitation of WEs containing saturated and unsaturated fatty acids (FA), respectively. The limit of detection was approximately 70 nmol/L. The linearity range of the liquid chromatography (LC)-MRM detection for WEs was about three orders of magnitude. Quantitative analyses of 141 individual WE in the human tear lipidome demonstrated that species comprising FA18:1 and FA16:1 each accounted for 47.7% and 24.0% (molar%) of total WE, while fatty alcohols in WEs of human tears ranged from 17 carbons to 32 carbons with predominant species represented by C24, C25 and C26.