Use of solid-phase microextraction to detect semiochemicals in synthetic and biological samples



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The objective of the following studies was to determine the effectiveness of solid-phase microextraction (SPME) at identifying semiochemicals (chemical messengers) in both synthetic and biological samples. Solid-phase microextraction, an alternative to traditional sample fractionation, employs specialized polymer fibers to detect molecules in a sample’s headspace. Solid-phase microextraction was previously used in the fields of environmental toxicology and food science to identify contaminants and was only recently applied to the fields of insect and mammalian semiochemicals. Solid-phase microextraction provides many benefits, namely the ability to detect unstable volatile molecules, making it optimal for use in the field of semiochemicals. The first study’s primary objective was to determine if a known semiochemical, 2-methyl-but-2-enal (2M2B), produced in commercial dog collars and behavioral adjustments sprays could be detected via SPME followed by gas chromatography-mass spectrometry (GC-MS) analysis. Collar pieces were placed in glass jars while the SPME fibers equilibrated to the surrounding airspace. Gas chromatography total ion chromatographs (TICs) identified the molecule in both the sprays and collars. A longitudinal study demonstrated that when the collar was left in the open air environment 2M2B continued to vaporize off the collar for about 50 days. A 30-day study comparing the collars worn on dogs determined that concentration of molecule remaining on the collar varied, possibly due to activity levels. The second study’s primary objective was to determine if SPME could be used to determine unique volatile molecules present in boar saliva. Components of boar saliva have been used in the past to check heat in sows for artificial insemination with weak results. Six saliva samples were collected, 3 from boars and 3 from sows for analysis with SPME followed by GC-MS. Total ion chromatographs were averaged for both sexes to determine molecules common to each sex. The averaged sow TIC was subtracted from its male equivalent to reveal 3 molecules unique to the boar: quinoline, androstenone, and androstenol. The molecules were verified in each of the individual boar saliva samples and were not present in any of the sow samples. The third study’s primary objective was to determine if SPME could be used to isolate and identify any molecules in ferret odor that elicited a defensive behavioral response in rats. A GC TIC identified 20 unique peaks/molecules. A literature review of the molecules narrowed behavioral testing down to 7 semiochemical candidates. In a simple behavioral paradigm,rats were conditioned to consume food pellets at the ends of a Y-maze until reaching a baseline amount. The animals underwent experiments consisting of a pre-exposure control trial followed by an individual semiochemical candidate exposure. Rats significantly decreased food consumption in response to two molecules, 2-methylbutyric acid and hexanal, but not to the same magnitude as ferret odor exposure. These data suggest that a combinational and concentrational calibration of these semiochemicals is required before a synthetic form of ferret odor can mimic its natural source. Based on the data of these studies SPME can be effective at detecting known semiochemicals in synthetic sources as the case with 2-methyl-but-2-enal and the synthetic dog collars. Problems arise when trying to create a semiochemical profile in biological samples due to the inherent noise in crude samples and the surrounding headspace. Further research would benefit from profiling multiple related samples, such as samples from multiple ferrets and narrowing down semiochemical candidates to molecules present in all samples. Solid-phase microextraction fibers are also prone to contaminate easily, and even after a fiber is conditioned large interfering peaks can occur in the GC TIC. An alternative method is proposed focusing on trapping sterile air blown directly over a sample. The sterile air can then equilibrate over a polymer surface on which the molecules can be extracted and injected into the GC-MS.



SPME, GC-MS, Pheromone, Semiochemical, Pig, Ferret, Rat, Dog