Development of Analytical Methods for Identification of Small Molecules in Human Tears

The cornea is the eye's primary optical surface, which is constantly wetted by tears. Tears perform a role in the avascular tissue that is analogous to that of blood in the human body. The tear film is a complex biological fluid made up of three layers that are not as simple as they appear. Tears are created by the lacrimal glands' secretions, as well as some fluid seeping through the corneal and conjunctival tissue (1). Tears serve a variety of biological functions. The tear film's primary functions are to supply oxygen and nutrition to the cornea, hydrate and lubricate the ophthalmic surface, protect it from invading microorganisms, and create an optimal environment for ocular health and vision. A 'hydrated gel,' comprising soluble and insoluble mucins, proteins, and aqueous components coated by an upper lipid layer, has been described as part of the tear film (2). The composition of tears will be discussed in depth later in this work; briefly, it can be divided into 3 layers: the outer lipid, the middle aquas (the main layer), and the inner mucin layer. In this work, three projects are presented that seek to improve existing analytical methods and innovate new ones. Tears contain numerous potential biomarkers that may aid in early detection and monitoring of corneal conditions. The presence of small molecules such as free amino acids, neurotransmitters, hormones, and steroids hold promise of detecting biological conditions. The identification and detection of the potential biomarkers in this work involved various analytical instrumentation including high performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE); all three were used for separation. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) were used for ionization. Laser induced fluorescence (LIF) and mass spectrometry (MS) were used for detection. All sample pretreatments utilized different methods, of which several were optimized. The results obtained from the research described in this thesis offers new analytical tools for fast, cheap, and reliable tear analysis to track potential biomarkers in human tears. Firstly, amino acid content of tears was optimized using MALDI-TOF-MS analysis. A new method employing an amine labeling reaction was proven to be reliable for the determination of this class of potential biomarkers. In the second project, a comparison was made between sample collection methods with different commonly used analysis tools. The study employed the two absorptive sample collection methods and provided an opportunity to optimize sample handling in the pre-analytical stage. Additionally, chemical separation, conditions of ionization, and detection modes were studied for MS analysis of a number of potential tear biomarkers. The results include data to support accurate, sensitive, and selective methods for chemically diverse potential biomarkers. Finally, stress hormones and other neurotransmitters were detected and quantified in human tears and were benchmarked against saliva concentrations. This work in total is a significant contribution toward expanding and improving analytical tools for tears analysis for potential application in medical diagnostics.