Dealing with small sample volumes and analyte levels: the case of vitamin D metabolites in aqueous humor

Vitamin D is a very well known prohormone that plays a crucial role in the regulation of calcium and phosphate homeostasis, together with rickets and osteomalacia prevention. Recently a relationship between the levels of vitamin D in serum and the presence of primary open angle glaucoma has also been demonstrated (Goncalves et al. 2015). A major risk factor in this affection is the increase of the intraocular pressure due to aqueous humor draining blockage. However, the screening of the status of vitamin D metabolites in the aqueous humor has not been performed yet. As a consequence our objective was to developed a method for the determination of the main metabolites of vitamin D (including 24,25(OH)2D2/D3 and 25(OH)D2/D3) in aqueous humor by LC-MS/MS. The main challenges derived from this method development are related to two main drawbacks: the limited sample volume and the low levels of analytes expected. On the one hand, since the aqueous humor is extracted during the cataract surgery, only a few microliters can be drawn, and this can be performed only once. Besides, an initial screening of the normal levels in samples has shown very low levels in patient samples (from low ng/mL to pg/mL). This leads to the worst case scenario: an extremely low absolute amount of analite available to be measured within the system. In order to overcome these problems, the proposed sample preparation is based on a simple liquid-liquid extraction followed by derivatization with the commercially available reagent Amplifex®. This has permitted to reach these extremely low levels with an accurate sample preparation. The proposed methodology is under validation process