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 D-3 and 25-hydroxyvitamin D-3 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 D-3 and 25-hydroxyvitamin D-3 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 D-3 and 91-124% for 25-hydroxyvitamin D-3. 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 D-3 and 25-hydroxyvitamin D-3 in tissues from studies where sample sizes are limited.