Extraction and Analysis of Lipids from Human Milk and Infant Formula

Total lipids were extracted from HM and IFs according to the method of Zhang et al. [19 (link)]. HM samples were mixed with CHCl₃/CH₃OH (2:1, v/v) at a ratio of 1:3, followed by 30 min of ultrasonic treatment at 30 °C. After 10 min of centrifugation at 16,994× g and 4 °C, the organic phase was transferred to a new vial, and the same extraction process was performed on the remaining water phase once more. The mixed organic phase was evaporated to about 5 mL under vacuum, and further dried under a stream of nitrogen. IFs were dissolved in ultrapure water at a ratio of 1:10 before mixing with CHCl₃/CH₃OH (2:1, v/v). The obtained dry lipid was stored at −20 °C for further gas chromatography (GC) analysis.
The process for lipid extraction was performed following the method of Li et al. [12 (link)], with slight modifications. Briefly, 60 μL milk sample and 340 μL ultrapure water were collected and placed in an EP tube (A). Then, 960 μL extraction liquid (MTBE:CH₃OH = 5:1, v/v) and 70 μL internal standard mixture ((d5-17:0/17:1/17:0) TG, 15:0 lyso PC, (19:0/19:0) PE, 13:0 lyso PE, d18:1/6:0 SM, (17:0/17:0) PS, and (17:0/17:0) PG were mixed in equal quantities in the concentration of 250 μg/mL) were added to each sample. The sample was mixed to homogeneity, vortexed, sonicated for 10 min, and then centrifuged for 10 min at 16,994× g and 4 °C. The supernatant was transferred to a new EP tube (B), and the remaining aqueous phase was extracted again with extraction liquid. The organic phases were combined and dried in a vacuum concentrator at 30 °C before 100 μL isopropyl alcohol was added for reconstitution. The samples were then transferred to a fresh glass vial for ultra-high-performance liquid chromatography quadruple time-of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis.