GC-MS methods for the analysis of pollen/anther odors

Pollen sampling — freshly collected mature anthers are used for analysis. The freshly collected anthers (1-3 per analysis depending on the fresh mass) from single flowers are  filled into quartz microvials for direct analysis via thermal desorption and coupled gas chromatography and mass spectrometry (GC-MS). 

Gas chromatography (GC)/mass spectrometry (MS) — The samples are analyzed on a Varian Saturn 2000 System using a 1079 injector that had been fitted with the ChromatoProbe kit. This kit allows the thermal desorption of small amounts of solids or liquids contained in quartz microvials (Amirav and Dagan, 1997; Wilkinson and Ladd, Undated). The ChromatoProbe microvial is loaded into the probe, which was then inserted into the modified GC injector. The injector split vent is opened (1/20) and the injector heated to 40° C to flush any air from the system. The split vent is closed after 2 min and the injector is heated at 200°C/min, then held at 150°C for 2 min, after which the split vent is opened (1/20) and the injector cools down.

A ZB-5 column (5% phenyl polysiloxane) is used for the analyses (60 m long, inner diameter 0.25 mm, film thickness 0.25 µm, Phenomenex). Electronic flow control (EFC) is used to maintain a constant helium carrier gas flow of 1.8 mL/min. The GC oven temperature is held for 4.6 min at 40°C, then increases by 6°C per min to 260°C and held for 1 min. After each run the column is cleaned by heating at 100°C/min to 300°C. The MS interface was 260°C and the ion trap works at 175°C. The mass spectra are taken at 70eV (in EI mode) with a scanning speed of 1 scan/sec from m/z 40 to 650. The GC-MS data is processed using the Saturn Software package 5.2.1. Component identification is carried out using the NIST 2000 mass spectral data base (NIST algorithm) and confirmed by comparison of retention times with published data (Davies, 1990; Adams, 1995). Identification of individual components is confirmed by comparison of both mass spectrum and GC retention data (Kovats Index) with those of authentic standards.

ADAMS, R. P. 1995. Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, Carol Stream, Illinois, USA.

AMIRAV, A., AND S. DAGAN. 1997. A direct sample introduction device for mass spectrometry studies and GC-MS analysis. European Journal of Mass Spectrometry 3: 105-111.

DAVIES, N. W. 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. Journal of Chromatography 503: 1-24.

WILKINSON, S., AND P. LADD. Undated. The use of a solids inlet system to identify essential oils in anthers and leaves of flowering plants. Varian GC/MS Application note number 65. http://www.varianinc.com/image/vimage/docs/products/chrom/apps/gcms65.pdf