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Simple Fast Quantification of Cholecalciferol, 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in Adipose Tissue Using LC-HRMS/MS.


ABSTRACT: Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D3 (25(OH)D3), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid-liquid or solid-phase extraction followed by derivatization using Amplifex® reagents to improve metabolite stability and ionization efficiency. Here, the method is optimized by co-eluting stable isotope-labelled internal standards to calibrate each analogue and to spike biological samples. Intra-day and inter-day relative standard deviations were 0.8-6.0% and 2.0-14.4%, respectively for the three derivatized metabolites. The limits of quantification (LoQ) achieved with Amplifex® derivatization were 0.02 ng/mL, 0.19 ng/mL, and 0.78 ng/mL for 1,25(OH)2D3, 25(OH)D3 and cholecalciferol, respectively. Now, for the first time, 1,25(OH)2D3 can be co-quantified with cholecalciferol and 25(OH)D3 in mouse adipose tissue. This validated method is successfully applied to study the impact of obesity on vitamin D status in mice.

SUBMITTER: Bonnet L 

PROVIDER: S-EPMC6770531 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Simple Fast Quantification of Cholecalciferol, 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in Adipose Tissue Using LC-HRMS/MS.

Bonnet Laurianne L   Margier Marielle M   Svilar Ljubica L   Couturier Charlene C   Reboul Emmanuelle E   Martin Jean-Charles JC   Landrier Jean-François JF   Defoort Catherine C  

Nutrients 20190822 9


Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D<sub>3</sub> (25(OH)D<sub>3</sub>), and 1,25-dihydroxyvitamin D<sub>3</sub> (1,25(OH)<sub>2</sub>D<sub>3</sub>) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid-liquid or solid-phase extrac  ...[more]

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