Novel fatty-acid eutectics for solidified floating-DES drop extraction of vitamin D3 from horse milk followed by HPLC determination

Due to the nutritional importance of vitamin D₃, the development of new extraction methods for this vitamin is highly desirable. Herein, we designed hydrophobic deep eutectic solvents (HDESs) based on stearic acid (SA), oleic acid (OA), and choline chloride (ChCl) for the vitamin D₃ extraction from horse milk. Accordingly, the extracted complex of vitamin D₃/HDES was solidified, back-extracted, and the vitamin D₃ amount was determined by HPLC analysis. Among the examined hydrophilic and hydrophobic DESs, ChCl:SA (1:2) (HDES-3) and sodium stearate (NaSt):OA (1:4) (HDES-11) demonstrated higher extraction yields 1000.1 µg/L for HDES-3 than 800 µg/L for HDES-11. Either the higher regularity in non-bent layers of SA or the smaller hydrophilic core of HDES-3 are accountable for its interlayer hydrophobic interactions with fat-soluble vitamin D₃. The intra/inter-day repeatability (4.86 %-8.71 % RSD, linearity 0.5–10 mg/L, R² coefficient 0.9983, LOD 0.17 mg/L, and LOQ 0.5 mg/L are analytical concerns of this method. Results suggest vitamin D₃ enrichment of horse milk and proficiency of the fatty acid-based HDESs for improving solidified floating-DES drop extraction. This method offers a suitable, bio-degradable, and non-toxic alternative for extracting oil-soluble food compositions and natural compounds.