title: Quantification of physiological levels of vitamin D₃ and 25-hydroxyvitamin D₃ in porcine fat and liver in subgram sample sizes.
abstract: Most methods for the quantification of physiological levels of vitamin D3 and 25-hydroxyvitamin D3 are developed for food analysis where the sample size is not usually a critical parameter. In contrast, in life science studies sample sizes are often limited. A very sensitive liquid chromatography with tandem mass spectrometry method was developed to quantify vitamin D3 and 25-hydroxyvitamin D3 simultaneously in porcine tissues. A sample of 0.2-1 g was saponified followed by liquid-liquid extraction and normal-phase solid-phase extraction. The analytes were derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to improve the ionization efficiency by electrospray ionization. The method was validated in porcine liver and adipose tissue, and the accuracy was determined to be 72-97% for vitamin D3 and 91-124% for 25-hydroxyvitamin D3 . The limit of quantification was <0.1 ng/g, and the precision varied between 1.4 and 16% depending on the level of spiking. The small sample size required for the described method enables quantification of vitamin D3 and 25-hydroxyvitamin D3 in tissues from studies where sample sizes are limited.
authors.affiliation: Division of Food Chemistry, National Food Institute, Technical University of Denmark, Søborg, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Burild
authors.forename: Henrik L
authors.initials: HL
authors.lastname: Frandsen
authors.forename: Morten
authors.initials: M
authors.lastname: Poulsen
authors.forename: Jette
authors.initials: J
authors.lastname: Jakobsen
mesh_terms: D000818:Animals; D002112:Calcifediol / Q000032:analysis*; D002762:Cholecalciferol / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005223:Fats / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D013552:Swine; D053719:Tandem Mass Spectrometry
chemical_list: D005223:Fats; D002762:Cholecalciferol; D002112:Calcifediol
keywords: Derivatization; Method validation; Tissues; Vitamin D3