A Dispersive Liquid–Liquid Micro–Extraction Technique for the Pre–concentration and Quantification of Vitamin D₃ in Milk and Yogurt Samples Using a Non-Aqueous HPLC Method

Abstract

In present study, a DLLME-HPLC-UV method was developed and validated for the extraction, pre–concentration, and subsequently quantification of vitamin D3 (Vit D3) in milk and yogurt samples. In order to be able to extract Vit D3 from studied samples efficiently, the DLLME procedure was optimized with respect to the parameters affecting the extraction efficacy, where acetonitrile (2 mL as disperser solvent) resulting from the protein precipitation procedure was mixed with 80 µL carbon tetrachloride (as an extraction solvent) respectively. The extracted samples were quantitatively analyzed with a HPLC technique using a C8 column (250 mm × 4.6 mm, 5 μm) at room temperature (25 °C), mobile phase of acetonitrile/methanol (90:10% v/v) in isocratic elution mode at a flow rate of 1.2 mL/min and UV detection at 265 nm. The method validation results revealed that the method was linear in the concentration range of 2 to 60 ng/mL (r = 0.9997) with a LOD of 0.9 ng/mL and LLOQ of 2 ng/mL; the method was accurate (-2.1% ≤ RE% ≤ +0.6%) and precise (1.2% ≤RSD% ≤ 11.3%) and its recovery was in the range of 86.6 to 113.3%. The obtained results indicated that the method could be utilized as an easy to use technique for the monitoring Vit D3 in dairy products, especially milk and yogurt samples.