2.3.10. Fatty Acids Determination Using GC-MS

Prior the analysis, the bee pollen was extracted for 48 h with n-hexane at room temperature [37]. The bee pollen oil (0.1 g) was mixed with 1 mL of n-hexane and 1 mL of 15% BF3 in methanol. The mixture was thermostated for 15 min at 60 ◦C in a water bath. The mixture was cooled to 20 ◦C and mixed with 5 mL of NaCl saturated solution, after mixing the solution was centrifuged for 5 min at 3000 rpm. The supernatant was filtered with a 0.45 μm nylon filter and kept at −20 ◦C prior analysis. The separation of the fatty acids methyl esters was carried out on SUPELCOWAX 10 column (60 m × 0.25 mm i.d., 0.25 μm film thickness; Supelco Inc., Bellefonte, PA, USA) using a Shimadzu GC-MS instrument (GC MS-QP 2010 Plus, Shimadzu, Japan) equipped with an AOC-01 auto-injector that was used to perform the gas chromatographic-mass spectrometric analyses. The initial oven temperature was 140 ◦C and was increased to 220 ◦C at a rate of 7 ◦C/min and then held at this temperature for 23 min. The flow rate of the carrier gas (He) and the split ratio were 0.8 mL/min and 1:24, respectively. Identification of FAMEs was done by comparing their retention times to those of known standards (37 component FAME Mix, Restek, Bellefonte, PA, USA, 35077) and the resulting mass spectra to the ones from our database (NIST MS

Search 2.0) [17]. The results are expressed as μg/g bee pollen; all the determinations were carried out in triplicate.
