Identification of pheromones in mouse urine by head-space solid phase microextraction followed by gas chromatography–mass spectrometry

Given the key role of pheromones in animal communication and behaviour, there is need to identify the different classes of these molecules under varying physiological conditions. However, the highly volatile nature of pheromones and the fact that they occur at very low concentrations in urine makes this task all the more difficult. Herein, we present a method of detecting and identifying the five main pheromones known: 2-sec-butyl-4,5-dihydrothiazole, geraniol, indole, trans-beta farnesene and trans-alpha farnesene in individual urine microsamples taken from male mice. Urine volumes as small as 20 μl were subjected to solid phase microextraction (SPME) followed by gas chromatography–mass spectrometry (GC–MS). This selective analytical method permits the rapid detection of these pheromones free from cross-contaminants as a clearly distinguishable spectral signals. Highest recovery rates of natural pheromones were achieved by extraction on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre of 85 μm film thickness. This selective, sensitive and accurate method will help address the question of possible links between certain pheromone classes, and social and reproductive behaviour in mice.