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Study of blood collection and sample preparation for analysis of vitamin D and its metabolites by liquid chromatography-tandem mass spectrometry

Summary The analysis of vitamin D status, with special emphasis on 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, is gaining interest in clinical studies due to the classical and non-classical effects attributed to this prohormone. In this research, the in?uence of the two steps preceding determination (viz. sample collection and preparation) on the quantitative analysis of vitamin D and its more important metabolites has been studied. Two preparation approaches, deproteination and solid- phase extraction (SPE), have been evaluated in terms of sensitivity to delimit their application, thus establishing that detection of 1,25-dihydroxyvitamin D cannot be addressed by protein precipitation. Concerning sample collection, serum and plasma reported high accuracy (above 83.3%) for vitamin D and metabolites, while precision, expressed as relative standard deviation, was below 12.9% for all analytes in both samples. Statistical analysis revealed that serum and plasma provided similar physiological levels for vitamin D3, 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3, while signi?cantly different levels were obtained for 1,25-dihydroxyvitamin D3, always higher in plasma than in serum. Sample collection and treatment have proved to be signi?cant in the analysis of vitamin D and its relevant metabolites.
Category 3. Symbiosis User Application
These are applications that have been made by Symbiosis customers. They are validated and have been published.
Matrix serum and plasma
Market Clinical
Cartridge HySphere C8
Detection Agilent 6410 triple quadrupole mass spectrometer
Compounds 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, Vitamin D2 and Vitamin D3. ,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D, 24,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D
Compound Group Vitamin D
Sample_volume 200µL
Contact A. Mena-Bravo, F. Priego-Capote, M.D. Luque de Castro
Account a Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba, Spain b Agroalimentary Excellence Campus, ceiA3, University of Córdoba, Córdoba, Spain c Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain
Published Analytica Chimica Acta 879 (2015) 69-76
Date 4-8-2015
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Remarks The developed research has allowed elucidation of key aspects on collection and preparation of blood for analysis of vitamin D and its metabolites. Thus, sample preparation based on SPE provides lower quantitation limits for all the target analytes than deproteination. The type of sample in?uences the sensitivity of the method since plasma is better for determination of the dihydroxymeta- bolites, while the two monohydroxymetabolites are determined with better sensitivity in serum. Statistical comparison of the use of plasma or serum as target sample has shown that the concentration of 1,25(OH)2D3 in plasma was higher than that in serum. This ?nding is important taking into account that quantitation of this dihydroxymetabolite is the most limiting aspect of methods for assessment of vitamin D status. The use of collection tubes with or without coating gel shows signi?cant statistical differences only for vitamin D3 in plasma, which yielded a higher concentration when the sample was collected in conventional plasma tubes as compared with plasma-gel tubes.

Status published