Human Tear Metabolomics Using Liquid Chromatography-Q Exactive-HF Mass Spectrometry

Aim: To putatively identify and characterise human tear metabolites in a normal subject on an untargeted platform of liquid chromatography-Q exactive-HF mass spectrometry. Methods: Four samples of unstimulated tears were collected from both eyes on four consecutive days between 1 and 2 pm using a microcapillary tube and pooled from both eyes each day. Untargeted analysis of the tears was performed by chromatographic separation of constituent metabolites in both CSH-C18RP (Charged Surface Hybrid - C18 Reversed Phase) and SeQuant ZIC-pHILIC (Zwitterionic - polymeric Hydrophilic Interaction Liquid Chromatography) columns, followed by heated electrospray ionization (HESI) and the acquisition of mass spectra using QExactive-HF mass spectrometry. Compound Discoverer software (v2.0) was used for data analysis. Result: Eighty-two metabolites were tentatively identified. Seventy compounds (85.4 %) were observed in all four samples with a coefficient of variation (CV) less than 25 %. Fifty-nine metabolites (71.9 %) were novel in healthy tears. Amino acids were the most frequently detected metabolites in the tears (28 %), followed by carbohydrates (12.2 %), carboxylic acids (8.5 %), carnitines (6.1 %) and glycerophospholipids (4.9 %), respectively. Conclusion: The current untargeted platform is capable of detecting a range of tear metabolites across several biological categories. This study provides a baseline for further ocular surface studies. Keywords: metabolites, tears, orbitrap, amino acids, QExactive-HF, carbohydrate, carnitine.