Headspace volatile analysis
Solid phase micro-extraction (SPME) combined with GC-MS was used to
analyze headspace volatiles using a Thermo-Fisher ISQ GC-MS
(Thermo-Fisher, Waltham, MA) with a Combi-Pal (CTC USA, MN) autosampler
equipped with a shaking incubator, fiber cleaning station, and SPME
holder, SPME injector insert, and Thermo-Fisher TG-WAXMS column (30 m x
0.25 mm x 0.25 μM). The SPME fiber was 1 cm, 50/30 μm
divinlybenzene/carboxen® on polydimethylsiloxane on a StableFlex fiber
(Millipore-Sigma, St. Louis, MO). An internal standard (IS) solution was
prepared by dissolving 4-methyl-1-pentanol in stripped soybean oil to a
concentration of 0.5 mg/g. Oil samples (0.1000 ± 0.0020 g) and IS
(0.0100 ± 0.0012 g) were weighed in 2 ml amber headspace vials. Using
the automated SPME headspace analyzer, samples were transferred to the
incubator and held for 5 min at 60 °C with shaking (250 rpm). The SPME
fiber was injected into the sample headspace and held for 30 min, then
volatiles were desorbed by injecting the fiber into the GC-MS injector
(250 °C, splitless mode) for 5 min at constant helium flow of 1.5
ml/min. The oven was held at 40 °C for 1 min, then heated to 240 °C at
10 °C/min where it was held for 2 min. The MS was operated in electron
ionization mode at 70 eV and 250 °C and scanning mass/charge m/z 35 –
350. Volatiles were identified by comparison of retention time and mass
spectra to commercial standards. Standard curves using the internal
standard method were developed using 5 μg IS and mixtures (0.005 to 10
μg/g oil) of commercial standards, and analyzing the standard mixtures
using the same procedure described above. Three experimental replicates
were analyzed in duplicate.