title: Development and comparison of three liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry methods for determining vitamin D metabolites in human serum.
abstract: Liquid chromatographic methods with atmospheric pressure chemical ionization mass spectrometry were developed for the determination of the vitamin D metabolites 25-hydroxyvitamin D₂ (25(OH)D₂), 25-hydroxyvitamin D₃ (25(OH)D₃), and 3-epi-25-hydroxyvitamin-D₃ (3-epi-25(OH)D₃) in the four Levels of SRM 972, Vitamin D in Human Serum. One method utilized a C18 column, which separates 25(OH)D₂ and 25(OH)D₃, and one method utilized a CN column that also resolves the diastereomers 25(OH)D₃ and 3-epi-25(OH)D₃. Both methods utilized stable isotope labeled internal standards for quantitation of 25(OH)D₂ and 25(OH)D₃. These methods were subsequently used to evaluate SRM 909c Human Serum, and 25(OH)D₃ was the only vitamin D metabolite detected in this material. However, SRM 909c samples contained matrix peaks that interfered with the determination of the [²H₆]-25(OH)D₃ peak area. The chromatographic conditions for the C18 column were modified to remove this interference, but conditions that separated the matrix peaks from [²H₆]-25(OH)D₃ on the CN column could not be identified. The alternate internal standard [²H₃]-25(OH)D₃ did not suffer from matrix interferences and was used for quantitation of 25(OH)D₃ in SRM 909c. During the evaluation of SRM 909c samples, a third method was developed using a pentafluorophenylpropyl column that also separates the diastereomers 25(OH)D₃ and 3-epi-25(OH)D₃. The 25(OH)D₃ was measured in SRM 909c using all three methods, and the results were compared.
authors.affiliation: National Institute of Standards and Technology, Analytical Chemistry Division, Gaithersburg, MD 20899, USA. mary.bedner@nist.gov
authors.forename: Mary
authors.initials: M
authors.lastname: Bedner
authors.forename: Karen W
authors.initials: KW
authors.lastname: Phinney
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol