Lokesh Kumar Sugumar, Kishore K R Tetala
Vitamin D3 (VD3) and its metabolite 25-hydroxyvitamin D3 (25OHD3) are important biological molecules that play a significant role in human well-being. Conventional methods use highly hazardous solvents to extract them from their bound form, which is prior to their analysis in high-performance liquid chromatography (HPLC). Considering the World Health Organization's (WHO's) sustainable development goals (SDGs), there is a need for green solvents to extract valuable biological metabolites such as VD3 from human plasma. This study explored the potential of existing green solvents to liberate VD3 and 25OHD3 in human plasma samples. Five green solvents were shortlisted based on their physicochemical properties and low toxicity level. HPLC-UV chromatography conditions using a C8 column (BEH C8 1.7 µm, 2.1 × 150 mm2) were identified to achieve good peak-to-peak separation of 25OHD3 (2.74 min) and VD3 (4.5 min) within 5 min. The optimized method had an excellent limit of detection (LOD) for both VD3 (86 pg/mL) and 25OHD3 (89 pg/mL). Ethyl acetate has outperformed other green solvents and a solvent mixture of acetonitrile and methanol (in different ratios) in extracting both VD3 and 25(OH)D3 from human plasma samples (RSD < 2.2%). The stability of VD3 and 25OHD3 was tested with different plasma storage conditions (5°C, -20°C, -80°C, and lyophilized powder) for up to 60 days. Plasma stored at -20°C has significantly preserved the stability of VD3 and its metabolite 25OHD3, both of which were successfully extracted using ethyl acetate with minimal loss even on the 60th day of storage.