title: Determination of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum using liquid chromatography with tandem mass spectrometry.
abstract: Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH)2D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is considered to be the "gold standard" for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC-MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and 1α,25-dihydroxy vitamin D2 (1α,25(OH)2D2). The analytes were extracted from the matrix by liquid-liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH)2D. The assay was sensitive with a low limit of quantitation of 10.0pg/mL for both 1α,25(OH)2D3 and 1α,25(OH)2D2 using a 0.5mL sample aliquot. Linearity was obtained over the range of 10.0pg/mL to 500pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100±5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH)2D3 and 1α,25(OH)2D2 concentrations.
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Fang
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Songlin
authors.initials: S
authors.lastname: Yu
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Xinqi
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Xuzhen
authors.initials: X
authors.lastname: Qin
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China.
authors.forename: Liangyu
authors.initials: L
authors.lastname: Xia
authors.affiliation: Shanghai AB Sciex Analytical Instrument Trading Co., Ltd., Beijing, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, 100730, China. Electronic address: lingqiubj@aliyun.com.
authors.forename: Ling
authors.initials: L
authors.lastname: Qiu
mesh_terms: D002117:Calcitriol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004872:Ergocalciferols / Q000097:blood*; D006801:Humans; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction / Q000379:methods; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004872:Ergocalciferols; C003564:1,25-dihydroxyergocalciferol; D002117:Calcitriol
keywords: 1,25-Dihydroxy vitamin D2; 1,25-Dihydroxy vitamin D3; LC–MS/MS; Serum