Simple Fast Quantification of Cholecalciferol, 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in Adipose Tissue Using LC-HRMS/MS.
Laurianne BonnetMarielle MargierLjubica SvilarCharlene CouturierEmmanuelle ReboulJean-Charles MartinJean-François LandrierCatherine DefoortPublished in: Nutrients (2019)
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.
Keyphrases
- adipose tissue
- insulin resistance
- ms ms
- solid phase extraction
- simultaneous determination
- liquid chromatography
- mass spectrometry
- liquid chromatography tandem mass spectrometry
- high performance liquid chromatography
- high fat diet induced
- gas chromatography
- tandem mass spectrometry
- high resolution mass spectrometry
- high fat diet
- gas chromatography mass spectrometry
- ultra high performance liquid chromatography
- metabolic syndrome
- type diabetes
- molecularly imprinted
- weight loss
- multiple sclerosis
- skeletal muscle
- physical activity