2.3. Chemical Analysis
ALDRICH
Chemicals:Chloroform (HPLC grade) was from Baker (Deventer, The Netherlands). Methanol, acetonitrile and water (analytical grade) were from Fluka analytical/Sigma Aldrich (Munich, Germany). Standards (choline chloride (>99%) and betaine monohydrate (>99%)) were from Sigma-Aldrich (Munich, Germany). D4-choline chloride (choline-1,1,2,2-d4) as an internal standard was purchased from C/D/N Isotopes Inc. (Pointe-Claire, QC, Canada) and 1,2-diarachidoyl-sn-gylcero-3-phosphocholine as an internal standard was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The purity of the chemicals was checked by tandem mass spectrometry (see below). All other chemicals were of analytical grade and from various commercial sources.Extraction:Plasma and milk samples were extracted according to Bligh and Dyer [