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Journal article
A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue
Analytical and bioanalytical chemistry, Vol.415(7), pp.1357-1369
2023
PMID: 36705732
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.
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Details
- Title
- A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue
- Authors/Creators
- Andrea Joy Stephenson - Curtin UniversityBenjamin Hunter - Curtin UniversityPaul Nicholas Shaw - The University of QueenslandNur Sofiah Abu Kassim - Cawangan Negeri Sembilan, Kampus Kuala Pilah, Kuala Pilah, MalaysiaRobert Trengove - Perth, WA Australia Perth, AustraliaRyu Takechi - Curtin UniversityVirginie Lam - Curtin UniversityJohn Mamo - Curtin University
- Publication Details
- Analytical and bioanalytical chemistry, Vol.415(7), pp.1357-1369
- Publisher
- Springer Berlin Heidelberg
- Grant note
- GNT1122734; GNT1135590; GNT2001090 /
- Identifiers
- 991005599565407891
- Copyright
- © 2023 The Author(s)
- Murdoch Affiliation
- Murdoch University
- Language
- English
- Resource Type
- Journal article
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- Citation topics
- 1 Clinical & Life Sciences
- 1.80 Bone Diseases
- 1.80.279 Vitamin D
- Web Of Science research areas
- Biochemical Research Methods
- Chemistry, Analytical
- ESI research areas
- Chemistry