## 2 Results and Discussion
### Comparison of Headspace Extraction Methods
#### 2.1.2 Dynamic Headspace with Tenax (D-HS-Tenax)
Similar observations were made when DVB/Car/PDMS SPME fiber and D-HS-Tenax were compared for the volatile fraction extraction of dairy products [44]. Indeed, the polar Carboxen sorbent makes these SPME fibers more efficient for extracting polar molecules such as short-chain fatty acids compared to the apolar Tenax. This limitation of Tenax for extracting polar compounds has previously been underlined [41]. Finally, the 6 following ketones were detected in the D-HS extract: 3-octanone (LRI: 1264), 6-methyl-5-hepten-2-one (LRI: 1351), 2-nonanone (LRI: 1397), 3-octen-2-one (LRI: 1423), 3, 5-octalation-2-one (LRI: 1525), 2-undecanone (LRI: 1611). However, the 2-propanone, which was strongly represented in the HS-SPME extract, is not detected in the present D-HS extract. As an intermediary outcome, the D-HS-Tenax extraction enables more compounds to be detected and, to a greater extent, than the HS-SPME extraction with a Car/PDMS fiber. This result can be partly explained by the greater quantity of sorbent used in the D-HS cartridge (200 mg) compared to an SPME fiber (0.5 \(\upmu\)g). However, the D-HS-Tenax method displays a lack of short-chain acid recovery. This chemical family is characteristic of milk. Some of its representatives, namely butanoic and nonanoic acids, were previously detected by newborn infants when presented as pure compounds [55; 56]. Moreover, newborns appear to be more sensitive than adults to short-chain carboxylic acids found in sweat [57]. Hence, carboxylic acids are compounds not to overlook to choose an extraction method suitable for analyzing human milk's volatile fraction. Therefore, the interest in D-HS-Tenax is mitigated regarding the objective of a non-targeted exploration of the volatile fraction of human milk. Conversely, the presence of the polar Carboxen in Car/PDMS SPME fibers enables short-chain acids to be recovered. 1.3 Dynamic Headspace Extraction with a Trilayer Cartridge Combining Tenax TA, Carbograph, and Carboxen (D-HS-Trilayer)  
To extend the range of compounds extracted by the dynamic headspace procedure to more polar compounds, a custom-assembled cartridge built in associating Tenax and the more polar Carboxen 1003 in powder form was evaluated. The composite cartridge thus obtained was completed by the third layer of Carbograph 1 TD sorbent, ensuring the extraction of compounds from the range C5-C7 covered neither by Carboxen 1003 (C2-C5) nor by Tenax (C7-C30). Hence, this trilayer cartridge was tested in a dynamic extraction mode according to the extraction and desorption conditions established for the D-HS-Tenax extraction. Despite optimization of the extraction procedure aiming to limit water trapping, the highly polar Carboxen in the trilayer tube have excessively strong water vapor retention leading to the ice-plug formation in the cryotrap desorption system leading, thus to un-reproducible chromatograms. Such water issues were reported in a previous study using combinations of carbon molecular sieves to extract volatile compounds from cow's milk [42]. Hence, the association of apolar Tenax with the commercial Carboxen in a powdered form in the D-HS-trilayer method does not lead to exploitable results. Therefore, three innovative variations of HS-SPME and D-HS methods were designed to combine the high concrutable power of the D-HS-Tenax method and the large polar range recovery of the HS-SPME-Car/PDMS.