Quantification of physiological levels of vitamin D₃ and 25-hydroxyvitamin D₃ in porcine fat and liver in subgram sample sizes

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