Milk Foodomics Analysis by GC-MS and NMR

Milk foodome was measured as previously described using GC–MS and ¹HNMR spectroscopy (16 (link)). Briefly, frozen milk samples were thawed at room temperature and vigorously vortexed until homogenization. After sonication, 200 μl milk was mixed with 300 μl of 80% methanol (containing 10 ppm internal standard, sorbitol) and 100 μl dichloromethane and vigorously vortexed followed by centrifugation at 13,572 g for 10 min at 4°C. Then 100 μl upper aqueous layer was transferred into a 200 μl glass insert and dried overnight using ScanVac (Labogene, Lynge, Denmark) at 40°C and 1,000 rpm. Immediately after drying, glass inserts were sealed with airtight magnetic lids into GC–MS vials, stored at 4°C,and analyzed by GC–MS within 24 h. For ¹H NMR analysis, frozen milk samples were thawed at room temperature, vigorously vortexed until homogenization and 1.8 ml of milk samples were centrifuged at 13,572 g for 30 min at room temperature. Then, 600 μl of an aliquot from the clear solution was mixed with 135 μl of phosphate buffer in deuterated water and transferred into NMR SampleJet tubes of L = 103.5 mm and O.D. = 5.0 mm, kept at 5°C and analyzed within 24 h. GC–MS and ¹H NMR datasets were subsequently processed using PARADASe (22 (link)) or SigMa (23 (link)) software, respectively, to convert the raw milk foodomics data into an informative metabolite table.