A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue

Abstract

Despite its critical role in neurodevelopment and brain function, vitamin D (vit-D) homeostasis, metabolism, and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive, and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely 1,25(OH)₂D₃, 3-epi-1,25(OH)₂D₃, 1,25(OH)₂D₂, 25(OH)D₃, 25(OH)D₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂. Chromatographic separation was achieved on a pentaflurophenyl column with 3 mM ammonium formate water/methanol [A] and 3 mM ammonium formate methanol/isopropanol [B] mobile phase components. Detection was by positive ion electrospray tandem mass spectrometry with the EVOQ elite triple quadrupole mass spectrometer with an Advance ultra-high-performance liquid chromatograph and online extraction system. Calibration standards of each metabolite prepared in brain matrices were used to validate the detection range, precision, accuracy, and recovery. Isotopically labelled analogues, 1,25(OH)₂D₃-d₃, 25(OH)D₃-c₅, and 24,25(OH)₂D₃-d₆, served as the internal standards for the closest molecular-related metabolite in all measurements. Standards between 1 fg/mL and 10 ng/mL were injected with a resulting linear range between 0.001 and 1 ng, with an LLOD and LLOQ of 1 pg/mL and 12.5 pg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12-11.53% and 0.28-9.11%, respectively. Recovery in acetonitrile ranged between 99.09 and 106.92% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D₂, 1,25(OH)D₂, and 24,25(OH)₂D₂, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological and neuropsychiatric disorders, and neurodegeneration.

Keywords: 1,25(OH)D; 24,25(OH)D; 25(OH)D; 3-Epi-1,25(OH)D; Brain; Liquid chromatography tandem mass spectrometry; Vitamin D.

Fig. 1

OLE configuration

Fig. 2

Precursor/product ion spectrum of six vit-D metabolites (A) and associated isotopically labelled internal standards (B) in mouse brain

Fig. 3

Chromatograms of the quantitative transition signal for the six vit-D metabolite standards (A) and the chromatograms of the associated isotopically labelled internal standards (B) in mouse brain at a concentration of 100 ng/mL, with a mobile phase flow rate of 300 µL/min

Fig. 4

An unknown 7.5-min peak was observed with the 1,25(OH)₂D₃ transitions (399.3 > 135.2) for retention time 7.02 min later validated as 3-epi-1,25(OH)₂D₃