## 4 Conclusions  
The present study aimed to perfect an extraction method of volatiles adaptable to small samples, valid in terms of repeatability, discriminative potency, and feasibility with minimal handling of the highly unstable human milk matrix. As the ultimate application of this extraction method was to identify molecular vectors bearing olfactory effect on newborn infants, the extraction procedure focused on the headspace developing over standing milk. Several extraction methods were designed after published procedures and the currently available sorbent polymers. Comparing the volatile profiles obtained by conventional HS-SPME-Car/PDMS and D-HS-Tenax extraction methods showed that D-HS-Tenax enables better detection and identification of a greater number of compounds in human milk headspace than HS-SPME-Car/PDMS. This result is primarily explained by the greater amount of sorbent used in the D-HS cartridge. However, the HS-SPME-Car/PDMS extraction appears more efficient in recovering short-chain fatty acids, commonly found in human milk (and amniotic fluid) and which are olfactorily active on newborn infants [56; 64; 65; 66]. The combination of the powdered polar sorbent Carboxen with apolar Tenax through the D-HS-trilayer method led to the ice-plug formation in the desorption system and was thus not workable. Therefore, considering the strong extraction potency of the D-HS polymer and the large range of polar compounds recovered by HS-SPME-Car/PDMS, innovative procedural variations of the latter headspace methods were assessed to analyze volatile compounds from human milk. The first variation (S-HS-MultiSPME), consisting of 10 Car/PDMS SPME fibers in a static extraction procedure, was conceived to increase the sensitivity of the HS-SPME. It led to a 6-fold amplification of the global signal compared to the use of a single fiber. The second variation (D-HS-MultiSPME), combining the same 10 SPME fibers but in a dynamic extraction procedure, led to a lower increase of the GC signal (4-fold increase) compared to S-HS-MultiSPME. However, it better-recovered aldehydes, terpen and alcohols relative to static extraction, resulting in extract profiles that resembled those obtained from conventional D-HS-Tenax extraction. Finally, considering the complementary sorbent potential of Car/PDMS fibers and the Tenax in terms of molecular polarity, a last procedural variation combined 10 Car/PDMS fibers with the Tenax sorbent in a unique dynamic extraction step (D-HS-MultiSPME/Tenax). This method widened the range of polarity of compounds recovered from human milk. Among the six extraction procedures tested in this study, it allowed the detection of compounds from the largest range of polarities and the highest concentrative yield with adequate repeatability. This association may constitute a suitable alternative to currently unavailable commercial solutions. D-HS-MultiSPME/Tenax method was demonstrated to be suitable for discriminating individual 5-mL human milk samples. Moreover, it identified more than 60 compounds in human milk samples belonging to the main chemical families reported in the literature: i.e., carboxylic acids, esters, alcohols, aldehydes, terpenes and ketones. In addition, this method recovered several compositionally minor compounds such as furans, lactones, pyrrole and pyrazine from human milk. Among them, pyrazine and the 2-methylpyrrole were, to our knowledge, identified for the first time in human milk. Finally, the D-HS-MultiSPME/Tenax extraction method constitutes a valuable tool to push forward the chemical exploration of the effluvium of human milk and colostrum. This new method can now be put into systematic use to delve deeper into the understanding of the compound(s) that convey the strongest informative value to newborn infants and are most active in eliciting their attention, attraction and appetence (e.g., [3; 67]).