title: Enhancing analysis throughput, sensitivity and specificity in LC/ESI-MS/MS assay of plasma 25-hydroxyvitamin D3by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) isotopologues.
abstract: The plasma/serum concentration of 25-hydroxyvitamin D3[25(OH)D3] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) assay of the serum/plasma 25(OH)D3for enhancing the sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3[3-epi-25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI-MS/MS assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D3by LC/ESI-MS/MS combined with the derivatization using the triplex (2H0-,2H3- and2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI-MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D3in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0ng/mL when a 5μL-plasma was used), precision (intra-assay RSD≤5.9% and inter-assay RSD≤5.5%), accuracy (98.7-102.2%), matrix effects, and capability of separating from an interfering metabolite, 3-epi-25(OH)D3. The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability.
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: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kittaka
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.
authors.forename: Akiho
authors.initials: A
authors.lastname: Nakata
authors.affiliation: Shizuoka Saiseikai General Hospital, 1-1-1 Oshika, Suruga-ku, Shizuoka 422-8527, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Shizuoka Saiseikai General Hospital, 1-1-1 Oshika, Suruga-ku, Shizuoka 422-8527, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sugiura
authors.affiliation: Divisions of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.affiliation: Divisions of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. Electronic address: higashi@rs.tus.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
mesh_terms: D000328:Adult; D000814:Aniline Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D002112:Calcifediol / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D007223:Infant; D057230:Limit of Detection; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014230:Triazoles / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: C000620455:4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione; D000814:Aniline Compounds; D014230:Triazoles; D002112:Calcifediol
keywords: Analysis throughput; Derivatization; Isotopologue; LC/ESI–MS/MS; Vitamin D