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.
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