Exploring the Potential of Green Solvents to Extract Vitamin D 3 and the Metabolite 25OHD 3 From Human Plasma and the Influence of Plasma Storage Co
Lokesh Kumar Sugumar, Kishore K. R. TetalaABSTRACT
Vitamin D 3 (VD 3 ) and its metabolite 25‐hydroxyvitamin D 3 (25OHD 3 ) 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 VD 3 from human plasma. This study explored the potential of existing green solvents to liberate VD 3 and 25OHD 3 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 mm 2 ) were identified to achieve good peak‐to‐peak separation of 25OHD 3 (2.74 min) and VD 3 (4.5 min) within 5 min. The optimized method had an excellent limit of detection (LOD) for both VD 3 (86 pg/mL) and 25OHD 3 (89 pg/mL). Ethyl acetate has outperformed other green solvents and a solvent mixture of acetonitrile and methanol (in different ratios) in extracting both VD 3 and 25(OH)D 3 from human plasma samples (RSD < 2.2%). The stability of VD 3 and 25OHD 3 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 VD 3 and its metabolite 25OHD 3 , both of which were successfully extracted using ethyl acetate with minimal loss even on the 60 th day of storage.