Untargeted analysis and tentative identification of unknown substances in human tears by ultra-high performance liquid chromatography-high resolution mass spectrometry: Pilot study.

In this work, a new approach for the identification of unknown compounds in human tears has been developed and validated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) linked to an intelligent data acquisition mode (AcquireX DS-dd-MS) coupled to an automated data processing software (Compound Discoverer™ 3.2). As a pilot research study, four human tear samples from volunteers were analyzed. Data were acquired in both positive and negative ionization modes and exact mass, isotope pattern, and MS spectra match were used for the tentative identification. Following this approach, 58 substances were identified, 47 in positive mode and 11 in negative mode, with an estimated concentration ranging from 0.1 to 9000 ng mL. Most of them were amino acids, hormones, metabolites, and pharmaceuticals. In order to validate the proposed method, the system suitability was evaluated and 29 commercial analytical standards of the tentatively identified substances were analyzed, of which 28 were confirmed obtaining a high identification accuracy (96.6 %). These results confirm that the screening tool presented in this work can facilitate the discovery of new metabolites, novel potential biomarkers, and substances to which the person is exposed, such as pollutants.