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Quantification of physiological levels of vitamin D 3 and 25‐hydroxyvitamin D 3 in porcine fat and liver in subgram sample sizes
Author(s) -
Burild Anders,
Frandsen Henrik L.,
Poulsen Morten,
Jakobsen Jette
Publication year - 2014
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201400548
Subject(s) - chromatography , chemistry , vitamin , analyte , sample preparation , extraction (chemistry) , saponification , mass spectrometry , electrospray ionization , vitamin d and neurology , retinol , fat soluble vitamin , adipose tissue , biochemistry , endocrinology , medicine
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.