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Quantification of physiological levels of vitamin D3 and 25‐hydroxyvitamin D3 in porcine fat and liver in subgram sample sizes
Authors:Anders Burild  Henrik L Frandsen  Morten Poulsen  Jette Jakobsen
Institution:1. Division of Food Chemistry, National Food Institute, Technical University of Denmark, , S?borg, Denmark;2. Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, , S?borg, Denmark
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.
Keywords:Derivatization  Method validation  Tissues  Vitamin D3
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