GC Analysis of Sterols (Silylated Derivatives) in Olive Oil (Free Sterol Fraction) on SLB®-5ms after SPE using Discovery DSC-Si
Materials
analytical column
CONDITIONS
sample preparation
SPE (Solid Phase Extraction)
sample/matrix
olive oil spiked with a mixed sterol standard at 5 μg/g
SPE tube/cartridge
Discovery DSC-Si, 1 g/6 mL (52656-U)
condition
2 x 5 mL hexane
sample addition
1 mL
elution
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)
eluate post-treatment
Evaporate to dryness and perform silylation as described in the Analysis Note.
column
SLB-5ms, 20 m x 0.20 mm I.D., 0.20 μm (28564-U)
oven
125 °C (1 min), 10 °C/min to 325 °C (10 min)
inj. temp.
250 °C
detector
MSD, rn/z 500 - 600
MSD interface
325 °C
carrier gas
helium, 0.6 mL/min, constant
injection
1 μL, splitless
liner
4 mm I.D. single taper
sample
Olive oil sample; fractionated, transesterified, and silylated
Description
Analysis Note
Sample pre-treatment
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.
Legal Information
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