Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director – Aviv Analytical,
Joanne Yew, Temasek Life Sciences Laboratory and National University of Singapore, Department of Biological Sciences, Singapore.
Insects such as flies, ants and mosquitoes express species-specific blends of cuticular hydrocarbons (CHCs) on their surface that serve as pheromones and play an important role in their behavior and communication. In view of growing interest and importance of this research area recently several advanced tools were developed for improved CHCs analysis. However, one major limitation of these methods and instrumentations is that alkanes cannot be properly detected via some of the atmospheric pressure ionization ion sources while in standard electron ionization as used with GC-MS the molecular ion is often very weak or altogether missing thereby proper identification is hindered. Furthermore, standard GC-MS is limited to relatively small compounds up to about C40-C44. Thus, we explored the chemical structure of fly and mosquito waxes with the Aviv Analytical 5975-SMB GC-MS with Cold EI which significantly enhances the molecular ion abundances, amplifies and exposes isomer structural mass spectral effects and enables the elution of much larger hydrocarbons via the use of short columns with high column flow rates. In addition, it provides uniform response for accurate quantification of the waxes compounds which is superior to standard EI and in contrast to API ionization methods (APCI, DART MALDI etc). For further information on GC-MS with Cold EI please read the review article A. Amirav, A. Gordin, M. Poliak, T. Alon and A. B. Fialkov "Gas Chromatography Mass Spectrometry with Supersonic Molecular Beams" J. Mass. Spectrom. 43, 141-163 (2008)., which is available on request. In this post we report on the analysis of two fly samples. A) Male Drosophila melanogaster, and B) Male Drosophila mojavensis.
Keep reading how the 5975-SMB GC-MS with Cold EI drastically improves flies cuticular hydrocarbons analysis and bring novel tools for its characterization.
System: Aviv Analytical 5975-SMB GC-MS with Cold EI which is based on the combination of Agilent 5975 MSD with the Aviv Analytical Supersonic GC-MS technology (GC-MS with supersonic molecular beam interface and its unique fly-through Cold EI ion source).
Samples: The samples were received in empty vials that each contained around 10-20 micrograms of the fly waxes. It was diluted with 100 µL hexane and injected as is.
Injector: 250ºC injector temperatures with Pulsed Split or Pulsed Splitless injection at 50 PSIG (25 ml/min).
Column: 8.5 m 0.25 mm ID, 0.25µ film of DB-5MS UI
He column flow rate: 2 ml/min with flow program after 12 minutes to 12 ml/min at 4 ml/min.min.
Injection volume: 1.5 µl
Split ratio: Split 3
On column wax amount: estimated at the 50-100 ng range.
GC Oven: 80ºC followed by 20ºC/min to 330C and wait 5.5 min for total of 18 min.
Cold EI Ion Source: 12 mA emission, 700 mBar nozzle backing pressure, 60 ml/min total He flow rate (column plus make up at 250C transfer line temperature).
SMB transferline temperature: 250ºC with temperature program of 10ºC/min after 5 min to 330ºC.
Electron Energy: 70 eV
Mass Spectral range: 50-800 amu at 2 Hz scan rate.
Standard EI system: Shimadzu QP2010 Plus GC-MS with 30 m column with 1 ml/min helium column flow rate.
General Observations Summary and Advantages of Cold EI
- Dominant or highly abundant molecular ions were observed for all the flies hydrocarbon compounds, in sharp contrast to standard EI.
- Clear isomer mass spectral effects were observed via isomer related high mass fragments that enabled the elucidation of the isomers structures.
- Free acids were observed and identified with Cold EI in the two samples while they were missing in standard EI (of male D. Melanogaster), probably due to standard EI ion source tailing and acid reactions with the ion source metallic surface.
- The 5975-SMB GC-MS with Cold EI exhibited much lower limits of identification hence helped in the identification of minor compounds that could not be identified with standard EI. The significantly lower limits of identification emerged from three main reasons: a) the Cold EI TIC signal to noise ratio was superior to that of standard EI despite the fact that its injected amount was a third of the standard EI injection; b) at a given signal the availability of dominant or abundant molecular ions and high mass fragments significantly lowered the limits of identification. Please read more on the degree of molecular ion enhancement in Cold EI in our previous post; c) the use of short column with 2 ml/min column flow rate lowered the elution temperatures thereby reduced the column bleed interference. The significantly lower limit of identification enabled more than doubled number of fly wax compounds that could be identified in comparison with standard EI and also revealed a few errors in compounds identification via standard EI.
- The use of short column (8.5 m) ensured that all the wax sample compounds eluted from the column. The penalty was twice lower GC separation.
- The analysis took only 18 min, which is about twice faster than with the standard GC-MS analysis.
- The 5975-SMB GC-MS with Cold EI uniquely exhibits uniform response to all the wax compounds thereby as with GC-FID enables quantitative analysis of the wax composition. This is a distinct advantage over MALDI and all the atmospheric pressure ionization methods such as DART and DESI.
- The 5975-SMB GC-MS with Cold EI opens the door for a unique and highly effective isomer distribution analysis characterization of the waxes. This new procedure of data analysis with RSIM on the molecular ions is unique to Cold EI and can serve for species and cryptic species characterization.
- In the analysis of male Drosophila Melanogaster fly wax we found a reasonably large peak of vaccenyl acetate (the peak at 6.16 min in Figure 1 below) which acts as a pheromone in Drosophila. It showed abundant molecular ion m/z=310.3. In addition, at the high time shoulder of this peak we found another compound with molecular ion of m/z=308.3. This additional molecular ion is probably of C22H44 olefin isomer but possibly of a vaccenyl acetate related compound with two double bonds. The second option although less likely is interesting as it can be a compound with pheromone activity that colelutes with the vaccenyl acetate thus was not found up to now.
We believe that Cold EI is currently the most informative ion source available for the analysis of cuticular hydrocarbons, pheromones and lipids from living insects. Thus, it can serve as a step forward towards comprehensive hydrocarbon analysis and for advance entomology research. Further information is given in the figures and their captions below.
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