title: Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2 , vitamin D3 , 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.
abstract: Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.
authors.affiliation: Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA. jadamec2@unl.edu
authors.forename: Jiri
authors.initials: J
authors.lastname: Adamec
authors.forename: Amber
authors.initials: A
authors.lastname: Jannasch
authors.forename: Jianjie
authors.initials: J
authors.lastname: Huang
authors.forename: Emily
authors.initials: E
authors.lastname: Hohman
authors.forename: James C
authors.initials: JC
authors.lastname: Fleet
authors.forename: Munro
authors.initials: M
authors.lastname: Peacock
authors.forename: Mario G
authors.initials: MG
authors.lastname: Ferruzzi
authors.forename: Berdine
authors.initials: B
authors.lastname: Martin
authors.forename: Connie M
authors.initials: CM
authors.lastname: Weaver
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000032:analysis*; D002112:Calcifediol / Q000032:analysis*; D002762:Cholecalciferol / Q000032:analysis*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D004872:Ergocalciferols / Q000032:analysis*; D006801:Humans; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D004872:Ergocalciferols; D002762:Cholecalciferol; D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol