title: Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications.
abstract: The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC-MS/MS would allow more accurate determination. Furthermore, LC-MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC-MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC-MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC-MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze-thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC-MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3required as little as 20 μL serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D3and 25(OH)D2.
authors.affiliation: Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ishige
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan.
authors.forename: Shoujiro
authors.initials: S
authors.lastname: Ogawa
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.|Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.
authors.forename: Motoi
authors.initials: M
authors.lastname: Nishimura
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.|Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsushita
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.affiliation: Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan. fnomura@faculty.chiba-u.jp.
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000097:blood*; D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D000814:Aniline Compounds / Q000737:chemistry; D002112:Calcifediol / Q000097:blood*; D002117:Calcitriol / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014230:Triazoles / Q000737:chemistry
chemical_list: C000620455:4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione; D000814:Aniline Compounds; D014230:Triazoles; D015652:25-Hydroxyvitamin D 2; D015650:24,25-Dihydroxyvitamin D 3; D002117:Calcitriol; D002112:Calcifediol
keywords: 24,25(OH)2D3; 25(OH)D2; 25(OH)D3; 3-epi-25(OH)D3; 4-(4′-Dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD); Clinical laboratory