Exploring possibilities of calcium isotopic analysis in aqueous humor using a fast and miniaturized calcium isolation procedure

The potential of using isotope ratios to understand biochemical processes in the body has been investigated in the past few years. In addition, to carry out a more comprehensive study on the etiology and evolution of diseases, analysis of microsamples has gained importance. The isotopic study of aqueous humor could contribute to elucidating the pathophysiological mechanisms that occur inside the eye leading to the development of glaucoma. Using this method, it is known that proteins and essential elements such as calcium play important roles in aqueous humor outflow dynamics and/or glaucoma disease due to their behavior as biological regulators. Thus, aiming at exploring the possibilities of calcium isotopic analysis by multicollector ICP-MS in aqueous humor within the context of eye diseases, and more specifically glaucoma, an optimized Ca isolation purification procedure was developed. As aqueous humor volumes available are usually at the μL level, and considering that most of the procedures discussed in the literature require high volumes of eluents, we propose a fast and miniaturized procedure applicable to low sample volumes, combining a reduced amount of resin and low eluent consumption. In the selected protocol, about 0.25 mL of sample were purified, using 0.5 mL of cation exchange resin and requiring just 21 mL of 1 M HCl as an eluent. The results showed the capabilities of the proposed approach for the analysis of Ca isotopic ratios in human aqueous humor. Further studies will be addressed using this novel procedure in larger cohorts of both glaucoma patients and control subjects.