In contrast to a plethora of known insect pheromones, a paucity of mammalian pheromones has been identified, two of which have been in elephants (Albone, 1984; Brown and Macdonald, 1985; Wyatt, 2003; Burger, 2005). Elephants possess one of the world’s best chemosensory systems, due in no small measure to their prehensile trunk. The trunk is not only the gateway to smelling (primary olfaction), but also the means by which chemical signals are conveyed from their source to the openings of the vomeronasal organ ducts in the roof of the mouth (the flehmen response; secondary olfaction) (Rasmussen, 1999). The late L. E. L. “Bets” Rasmussen was a pioneer in the study of chemical signaling among elephants (Goodwin and Schulte, 2007). Among her many notable accomplishments are the identification of the urinary, preovulatory pheromone of the Asian elephant (Elephas maximus; Rasmussen et al., 1996a), and a chemical signal of musth in Asian male elephant temporal gland secretion (TGS; Rasmussen and Greenwood, 2003).

When prospecting for elephant pheromones, or those from any other mammal, four tasks must be accomplished:

(1) extraction of the volatile organic chemicals from the biological matrix;

(2) separation of the chemical components of the extraction mixture from each other;

(3) identification of the chemical compounds present in the mixture; and

(4) verification of bioactivity of the putative pheromones.

In our elephant research, and in general with similar investigations, the workhorse methodologies for separation and identification are gas chromatography (GC) and mass spectrometry (MS), respectively. Verification is achieved through behavioral bioassays, which in the case of elephants are quantified using flehmens and other distinctive trunk behaviors (Schulte et al., 2005; Schulte, 2006.) In this article we will discuss extraction procedures, primarily solventless ones that have been employed in the search for mammalian chemical signals.

For several years, the authors, an organic chemist (T.E.G.) and an animal behaviorist (B.A.S.), reaped the benefits of a productive collaboration and friendship with Bets Rasmussen (a biochemist). The main focus of our team was a study of chemical communication among African elephants (Loxodonta africana) (for example, see: Schulte et al., 2004; Bagley et al., 2006; Goodwin et al., 2006; Loizi et al., 2009). Our chemical analyses of volatile organic compounds in elephant excretions and secretions have involved solventless, and thus environmentally friendly (“green”), extraction methodologies. This report is not intended to be a comprehensive review of such procedures and mammalian applications thereof, but rather will focus on our work, along with selected examples of the techniques employed and discoveries made by other researchers in this area. (For more detailed comparisons and discussions of solventless extractions, see the following: Baltussen et al., 2002; Pillonel et al., 2002; Bicchi et al., 2004.)


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