Abstract

DETERMINATION OF VITAMIN-D LEVEL IN FORTIFIED MILK BY ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY

Syed N. Alvi, Chafica El Tabache, and Muhammad M. Hammami*

ABSTRACT

A reliable ultra-performance liquid chromatography (UPLC) assay was developed for simultaneous quantification of vitamin D-2 (VD-2) and vitamin D-3 (VD-3) levels in VD-fortified milk, using laurophenone as an internal standard (IS). Milk samples were prepared by overnight incubation with a mixture of ethanol and 50% potassium hydroxide, extracted with hexane, and cleaned-up with ethanol and 5% potassium hydroxide. After evaporation, the residue was dissolved in methanol, centrifuged, and 5.0 μl of the clear solution was injected into Acquity UPLC BEH C18 column. The mobile phase, composed of methanol, acetonitrile, and water (80:10:10, v:v:v), was used at a flow rate of 0.35 ml/min and a run time of 15 minutes Eluents were monitored by photodiode array detector, with the wavelength set at 265 nm. The relationship between the concentration of VD-2 and VD-3 and their corresponding peak area ratios to the IS was linear over the range of 2.5 – 60 ng/ml. Mean extraction recovery for VD-2, VD-3, and IS was 93%, 94% and 70%, respectively. Stability of VD-2 and VD-3 before and after extraction was studied under various laboratory conditions. Further, the method was successfully used to measure vitamin D levels in milk samples from the local market.

Keywords: Laurophenone, VD-fortified milk.