Bibian van der Voorn

96 CHAPTER 7 METHODS The Dutch Human Milk Bank (located at VUMC) provided milk from 30 mothers who gave birth to a term infant and donated their breast milk. Written informed consent was obtained from all donors prior to study enrollment. All donors who are donating to the Dutch Human Milk Bank are women who produce more breast milk than their own infant needs. They donate their spare milk voluntarily. Donors are not actively recruited. They register themselves, after which they receive more information. Prior to the first donation, donors were screened according to international guidelines, and milk was colle cted by standardized procedures (described at https://www. moedermelkbank.nl ). In short, donors collected breast milk at home in disposable bisphenol A free bott les (Sterifeed, Medicare Colgate Ltd, Devon, England), by use of a breast milk pump. They were asked to express all milk, including fore and hind milk, from one breast, completely. Immediately afterwards, the milk was stored at -20°C upon Holder pasteurization (30 minutes at 62.5°C) 1 . Samples that were used for this studywere randomly selected, spare samples of pooled milk that was used to feed infants at the NICU (n = 30). A pooled sample contained milk from one mother collected at different moments during several days. As part of standard care, one pre- and one post-pasteurization sample from each pooled sample was stored. When safe administration of the donor milk to an infant was assured, these spare samples could be used for our study. Pre- and post-pasteurisation sampleswere analysedwith the use of an isotope dilution LC–MS/MS method as described previously 11 , although with a slightly adjusted extraction method. In short, 0.5 mL of milk was used to assess cortisol and cortisone concentrations. Internal standards ( 13 C 3 -labeled cortisol and 13 C 3 -labeled cortisone) were added to the samples. Lipids were removed by washing the milk 3 times with 2 mL hexane. Samples were extracted using Isolute plates (Biotage, Uppsala, Sweden) and analyzed by LC–MS/MS (Acquity with Quattro Premier XE, Waters Corporation). The lower limit of quantitation was 0.5 nmol/L for cortisol and 0.3 nmol/L for cortisone. The intra-assay CVs were 4% and 5% at cortisol concentrations of 7 and 23 nmol/L, and 5% at cortisone concentrations of 8 and 33 nmol/L, respectively. The interassay CVs were <9% for cortisol and cortisone. The concentration found in the pre-pasteurization sample was set at 100% and plotted against the post-pasteurization concentration. Concentrations of cortisol and cortisone pre- and post-pasteurization were compared using Pearson correlation and