GC-MS analysis of M. hyalina headspace
Headspace volatiles of a slant culture of M. hyalina grown on
potato dextrose agar (PDA) in a glass tube with stopper were collected
14 days after inoculation with a solid phase micro extraction (SPME)
fiber (Aldrich, red fiber, 100 µm PDMS) over 2 hours. As a control, the
headspace of the medium alone (PDA) was collected.
SPME fibers were desorbed in the injection port of a GC at 220 °C in
splitless mode and a helium flow of 1 mL/min through the chromatographic
column connected. The volatiles were separated chromatographically on a
ZB-5 ms column (30 m x 0.25 mm x 0.25 µm, Phenomenex) with an GC-oven
temperature program starting at 45 °C for 2 min, then heating up to 220
°C with a rate of 10°C/min, followed by a heating rate of 30°C/min to
280°C, and was maintained for 1.83 min. The column was connected to a
time-of-flight mass spectrometer (GCT, Micromass) via a transfer line
(280 °C). Ion source temperature was set to 250 °C and ionization energy
was 70 eV. For high resolution MS (HR-MS), heptacosa was continuously
streaming into the source and the calibrated HR-MS profile was locked at
m/z 218.9856.
A mixture of n-alkanes C8 – C20 inn -hexane (Aldrich) was measured before and after a sample
sequence under the same conditions except for the injector split ratio
(1:50). Retention times of the n -alkanes were used to calculate
the retention index (RI) for each peak in the GC-MS chromatogram
according to the method of
Vandendool
and Kratz (1963).
Compounds were identified based on their mass spectra (MS) in
combination with their individual RIs in comparison to MS and RI
database
(National
Institute of Standards and Technology, 2014) using MassFinder software
(Hochmuth,
2010) in combination with NIST MS Search. Authentic reference compounds
were used additionally for identification. For relative quantification,
identified peaks of the GC-MS total ion chromatogram (TIC) were
integrated.