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Phospholipids were extracted using chloroform and methanol and separated on C18 column. Repeatability, intermediate reproducibility, and recovery values were calculated and a large sample set of human milk analyzed. In human milk, phospholipid classes were quantified at concentrations of 0.6 mg\/100 g for phosphatidylinositol; 4.2 mg\/100 g for phosphatidylethanolamine, 0.4 mg\/100 g for phosphatidylserine, 2.8 mg\/100 g for phosphatidylcholine, and 4.6 mg\/100 g for sphingomyelin. Their relative standard deviation of repeatability and intermediate reproducibility values ranging between 0.8 and 13.4 % and between 2.4 and 25.7 %, respectively. The recovery values ranged between 67 and 112 %. Finally, the validated method was used to quantify phospholipid classes in human milk collected from 50 volunteers 4 weeks postpartum providing absolute content of these lipids in a relatively large cohort. 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