Novel sampling strategy for alive animal volatolome extraction combined with GC-MS based untargeted metabolomics: Identifying mouse pup pheromones.

In this study, we identify 11 mouse pup volatiles putatively involved in maternal care induction in adult females. For this purpose, we have adapted the dynamic headspace methodology to extract the volatolome of whole alive animals. Untargeted metabolomic methodology was used to compare the volatolome of neonatal (4-6 days) with elder pups until the age of weaning (21-23 days old). Pup volatolome was analyzed by gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) using automated thermal desorption for sample introduction. After data processing and multivariate statistical analysis, comparison with NIST spectral library allowed identifying compounds secreted preferentially by neonatal pups: di(propylen glycol) methyl ether, 4-nonenal, di(ethylene glycol) monobutyl ether, 2-phenoxyethanol, isomethyl ionone, tridecanal, 1,3-diethylbenzene, 1,2,4,5-tetramethylbenzene, 2-ethyl-p-xylene and tri(propylene glycol) methyl ether. Palmitic acid was enriched in the volatolome of fourth week youngsters compared to neonatal pups. The results demonstrated the great potential of the new sampling procedure combined with GC-MS based untargeted volatolomics to identify volatile pheromones in mammals.