SPE (Solid Phase Extraction)
olive oil spiked with a mixed sterol standard at 5 μg/g
Discovery DSC-Si, 1 g/6 mL (52656-U)
2 x 5 mL hexane
esters fraction: 2.5 mL of hexane:ethyl acetate (90:10); free sterols fraction; 5 mL of hexane:ethyl acetate (90:10) and 3 mL x 5 of ethanol:diethyl ether:hexane (50:25:25)
Evaporate to dryness and perform silylation as described in the Analysis Note.
SLB-5ms, 20 m x 0.20 mm I.D., 0.20 μm (28564-U)
125 °C (1 min), 10 °C/min to 325 °C (10 min)
MSD, rn/z 500 - 600
helium, 0.6 mL/min, constant
1 μL, splitless
4 mm I.D. single taper
Olive oil sample; fractionated, transesterified, and silylated
Weigh 0.25 gm oil into test tube. For spiked sample add 12.5 μL of 65-100 μg/mL mixed sterol std. Add 1 ml of hexane:ethyl acetate (90:10). After SPE fractionation, evaporate solvent from each fraction, add 1 ml of 30% methanolic sodium methoxide:MeOH:MTBE solution (13:27:60), mix and let sit at room temp for 30 min. Add 1 ml of water and 2 mL of n-heptane. Withdraw aqueous phase (bottom) and replace w/ 1 mL of 1% citric acid solution. Withdraw organic phase and dry over anhydrous sodium sulfate. Silylation: Evaporate organic phase to dryness. Add 125 μL of Sylon BFT and 125 μL of pyridine. Heat at 70°C for 20 min. The sample is then ready for GC-MS analysis.
Sterol content is one in a battery of tests used to analyze the composition and determine the grade and authenticity of olive oil. Test results are compared against standards such as those established by the International Olive Oil Council (IOC) and US Dept. of Agriculture. In vegetable oils, sterols exist in the free form, or esterified to fatty acids. In this application, sterols were analyzed in olive oil separately as free and esterified. This was accomplished by taking advantage of the polarity difference between these two groups to fractionate them using silica gel SPE. In place of saponification, the fractions were transesterified to liberate the esterified sterols. The fractions were then silylated, and analyzed by GC-MS. The silylated sterols all contained a significant molecular ion in their mass spectum, which made it possible to perform peak identification free of matrix interference. The sterols isolated in the free fraction are shown here.
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