Bibian van der Voorn

68 CHAPTER 5 Human mother’s milk is recommended as the standard nutrition for neonates, due to its widely acknowledged benefits. Besides being a source of nutrients, milk contains a variety of non-nutritive bioactive and immunomodulatory components 1 . Glucocorticoids are found in mother’s milk, bound mainly to CBG and albumin 2 . Glucocorticoids are suggested to induce proliferation and differentiation of glandular cells in the mammary gland, and also to influence the neonate via the mother’s milk 2,3 . Despite efforts to optimize the contents of formula feeding, human milk is still recommended for its widely acknowledged benefits. Maternal glucocorticoids might be one of the factors contributing to the advantages of breast milk over formula feeding and are therefore worthwhile to investigate. In order to be able to measure glucocorticoids in human milk we developed a reliable LC-MS/MS method to determine cortisol and cortisone in mother’s milk. In this letter, we describe this method and the results of the validation of our assay, including experiments that investigated the stability of cortisol and cortisone in human milk. Donor mother’s milk of 13 healthy mothers, donated 8 to 28 wks postpartum to the Dutch Human Milk Bank of the VUMC, Amsterdam was used. All samples were stored in polypropylene vials at −20°C. Preparation of the samples was initiated by the addition of 2 H 4 labeled cortisol (Cambridge Isotope Laboratories) and 2 H 8 labeled cortisone (CDN Isotopes Inc.), both serving as internal standards, to 200 µL thawed milk and thorough mixing. To remove undesired lipids, the milk was washed by the addition of 2 mL hexane 2 . Capped tubes were mixed for 2 minutes in a multivortexer and centrifuged for 2 minutes, at 19°C, 1,900 g, resulting in the separation of the lipid layer from the aqueous layer. Thereafter the sample was frozen in a −60°C CO 2 ice bath, which enabled the liquid hexane to be decanted from the frozen milk. This washing procedure was completed three times, in total. Forty µL of the washed milk was injected onto a Symbiosis online solid phase extraction (SPE) system (Spark Holland, Emmen, The Netherlands). Online SPE with C8 cartridges (Spark Holland) was performed for further purification of the samples. The analyte was eluted from the cartridge with methanol-water and focused on the Synergi Hydro RP column (Phenomenex, Utrecht, The Netherlands), which was equipped with a C18 guard column (Phenomenex). A linear binary gradient from 55 to 61%methanol containing 0.1% formic acid and 2 mmol/L ammonium acetate was applied, after which the methanol content was increased to 100%. Cortisol, cortisone and their internal standards eluted at retention times of 5.2 and 4.8 minutes, respectively. The total run time was 7.5 minutes. A Quattro Premier XE tandem mass spectrometer (Waters Corp., Milford, MA), which operated in the electrospray positive ionization mode, served as detection instrument which operated in the electrospray