title: A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3.
abstract: Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D(3) [25(OH)D(3), the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D(3) in ESI-MS/MS and to separate 25(OH)D(3) from 3-epi-25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], a potent interfering metabolite. 25(OH)D(3) was extracted from two DBS punches (3  mm in diameter, equivalent to 5.3  μL of whole blood), purified using an Oasis HLB(®) cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D(4) was used as the internal standard. This method was reproducible (intra- and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0  ng/mL. The developed method enabled specific quantification of 25(OH)D(3) in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D(3).
authors.affiliation: School of Pharmaceutical Sciences and Global COE Program, University of Shizuoka, Shizuoka, Japan. higashi@u-shizuoka-ken.ac.jp
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Suzuki
authors.forename: Junji
authors.initials: J
authors.lastname: Hanai
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Inagaki
authors.forename: Jun Zhe
authors.initials: JZ
authors.lastname: Min
authors.forename: Kazutake
authors.initials: K
authors.lastname: Shimada
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D002112:Calcifediol / Q000031:analogs & derivatives / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D015997:Neonatal Screening / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014808:Vitamin D Deficiency / Q000097:blood* / Q000175:diagnosis
chemical_list: D002112:Calcifediol