Quantification of vitamin D and 25-hydroxyvitamin D in soft tissues by liquid chromatography–tandem mass spectrometry

Abstract

Inadequate data on tissue distribution of vitamin D and its metabolites remains a barrier to defining health outcomes of vitamin D intake and 25-hydroxyvitamin D (25(OH)D) status. The purpose of this study was to develop a method for the analysis of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), 25(OH)D2, and 25(OH)D3 in soft tissues, and determine distribution in select tissues from a dose-response study of vitamin D2 and vitamin D3 in rats. Liver, gastrocnemius muscle, and epididymal fat homogenates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization following liquid-liquid extraction, solid-phase extraction, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). A dose-response was observed in most tissues for vitamin D and 25(OH)D from both vitamers. Vitamin D concentration was greater in epididymal fat than gastrocnemius muscle and liver, but 25(OH)D concentration was not significantly different between tissues. Soft tissues of rats fed crystalline vitamin D3 had higher concentrations of total vitamin D than those of rats fed yeast-derived vitamin D2, while total 25(OH)D concentrations were similar between vitamin D sources. This method is well suited to more complete studies of vitamin D bioavailability and metabolite tissue distribution.

Chromatographic separation of PTAD-derivatized 25-hydroxyvitamin D3 and its C-3 epimer from human serum and murine skin

“…These proportions are in close agreement with previously published results for both adults [5,6] and infants [4]. This is the first report of skin levels of 25OHD 3 and C3epi in mice, and the levels of 25OHD 3 are comparable to those in fat, muscle, and liver of rats, as reported by Lipkie and colleagues [13]. If left unresolved, C3epi would have represented a range between 3.7% and 22.1% of the total 25OHD 3 measurement in skin.…”

“…Some of these were limited by sample size or analyte concentration, and thus utilized PTAD derivatization to enhance MS/MS signal [13,26,27]. The present study is the first account of near-complete chromatographic resolution of PTAD-derived C3epi and 25OHD 3 , thus allowing separate detection of these compounds in samples with low abundance of analytes.…”

“…Dried extracts and standards were derivatized with PTAD according to Lipkie et al [13]. Briefly, 100 L of a 2 mg/mL PTAD solution was added to the extracts, which were then vortexed for 10 min.…”

Synthesis of Low Abundant Vitamin D Metabolites and Assaying Their Distribution in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) as a New Tool for Diagnosis and Risk Prediction of Vitamin DRelated Diseases

“…Other biological matrices have rarely been used, although vitamin D metabolites have successfully been quantified in saliva [48] and various soft tissues [49]. In the following sections, we discuss the requirements and characteristics of LC-MS/MS of vitamin D metabolites; that is, chromatographic separation, ionization, m/z analysis, interferences and accuracy issues, multimetabolite screening applications, and the clinical role of vitamin D fingerprinting methods.…”

“…Several promising PTAD assays have been described in the literature [39,49,[62][63][64]. The advantage of using the cis-diene moiety is that all vitamin D metabolites contained in the sample are converted when the reagent is present at sufficient concentrations (i.e., in large excess quantities) as all relevant vitamin D metabolites possess this structural motif.…”