###### Abstract  
The odor of human milk induces search-like movements and oral activation in newborns, which increases their chances of taking advantage of milk intake and benefits. However, the underlying volatile fraction of human milk remains understudied. This study aimed to devise a simple method to extract a wide range of volatile compounds from small-volume human milk samples. Headspace solid phase micro-extraction (HS-SPME) with a Car/PDMS fiber and dynamic headspace extraction (D-HS) with a Tenax or a trilayer sorbent were tested because of their selective affinity for volatiles. Then, innovative variations of these methods were developed to combine their respective advantages in a one-step extraction: Static headspace with multiple SPME fibers (S-HS-MultiSPME), Dynamic headspace with multiple SPME fibers (D-HS-MultiSPME) and dynamic headspace with multiple SPME fibers and Tenax (D-HS-MultiSPME/Tenax). The extracts were analyzed by gas chromatography coupled with mass spectrometric and flame ionization detection. The relative performances of these methods were compared based on qualitative and semi-quantitative analyses of the chromatograms. The D-HS technique showed good sensitivity for most compounds, whereas HS-SPME favored the extraction of acids. The D-HS-MultiSPME/Tenax identified more than 60 compounds from human milk (some for the first time) and evidence of individual singularities. This method that can be applied to volatilome analysis of any biological fluid should further our understanding of human milk odor. headspace extraction; solid phase micro-extraction; dynamic headspace analysis; human milk; volatilome; olfacto; neonate +
Footnote †: journal: _m molecules_  
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Thus far, the volatiles of human milk were mostly traced in the context of environmental contamination of the first food [12; 13; 14; 15], including volatile organic compounds (VOCs; e.g., chloroform, benzene and toluene) or semi-volatile organic pollutants [POPs; e.g., polychlorinated biphenyls (PCBs), polychlorinated dibenzo-\(p\)-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)). Tobacco smoke also leads to the transfer of pollutants into breast-milk, especially nicotine [16; 17]. The presence of nitro-musk's was related to their use as fragrances in cosmetics, soaps or laundry products [18]. Besides, some flavor compounds (e.g., d-carvone, l-menthol or trans-anethole) from the mother's diet were shown to pass into milk [19]. Finally, the profile of VOCs in ejected breast milk is easily modified by heating processes and storage conditions, leading to a Maillard reaction and lipid oxidation [20; 21; 22; 23; 24]. Among VOCs, carboxylic acids were estimated to represent up to 80% of the total VOC content of human milk [25]. The presence of aldehydes, ketones, alcohols, furans, lactones, nitrogen-containing compounds, esters and terpenes reveals the considerable chemodiversity of human milk effluvium [11; 25; 26; 27; 28]. Most of these volatiles occur in low, even traces concentrations. Subsequent difficulties of chemical identification are increased by the generally rather small volumes available of fresh human milk samples. Thus, before making assumptions about their behavioral activity in neonates, VOCs' composition in human milk needs more investigation. Different extraction methods were previously used to trap volatiles from human milk before GC analysis, such as solvent extraction [29], solvent-assisted flavor evaporation (SAFE) [23; 24; 30; 31; 32], or simultaneous distillation-extraction (SDE) [25]. These methods led to identifying compounds from the main chemical classes in human milk, but the elimination of the solvent and the temperatures generally applied (>50 \({}^{\circ}\)C) may have induced a loss of VOCs [33]. In addition, the amount of sample required was often large, reaching up to one liter [25].