Salting-out assisted liquid-liquid extraction coupled with high performance liquid chromatography for the determination of vitamin D3 in milk samples

In this study, salting-out assisted liquid-liquid extraction (SALLE) as a simple and efficient extraction technique followed by high performance liquid chromatography was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate was used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250 x i.d 4.6 mm, 5 µm) high performance liquid chromatography column that was coupled with diode array detector (HPLC-DAD). Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 minutes. Good linearity (r2 > 0.993) was obtained within the range of 0.025 – 60.0 µg g-1 with the limit of detection (LOD) of 0.015 µg g-1 and limit of quantification (LOQ) of 0.025 µg g-1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 – 113.5 %.