title: Direct aqueous measurement of 25-hydroxyvitamin D levels in a cellular environment by LC-MS/MS using the novel chemical derivatization reagent MDBP.
abstract: Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D3(25(OH)D3) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri-n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D3in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D3directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D3into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. Graphical Abstract MDBP labelling of 25-hydroxyvitamin D in the extracellular space.
authors.affiliation: Institute of Bioanalytical Chemistry, Saarland University, Campus B2.2, 66123, Saarbrücken, Germany.
authors.forename: Miriam J
authors.initials: MJ
authors.lastname: Müller
authors.affiliation: Department of Internal Medicine 5 - Hematology/Oncology, University Hospital Erlangen, Ulmenweg 18, 91054, Erlangen, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Bruns
authors.affiliation: Institute of Bioanalytical Chemistry, Saarland University, Campus B2.2, 66123, Saarbrücken, Germany. Dietrich.Volmer@mx.uni-saarland.de.
authors.forename: Dietrich A
authors.initials: DA
authors.lastname: Volmer
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D052578:Ionic Liquids / Q000737:chemistry*; D008264:Macrophages / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry*; D013601:T-Lymphocytes / Q000378:metabolism; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000032:analysis; D014815:Vitamins / Q000032:analysis
chemical_list: D052578:Ionic Liquids; D010879:Piperazines; D014815:Vitamins; D014807:Vitamin D; C104450:25-hydroxyvitamin D
keywords: Cellular uptake; Chemical derivatization; LC-MS/MS; Vitamin D