Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba, Spain; University of Córdoba Agroalimentary Excellence Campus, ceiA3, Córdoba, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain; CIBER Fragilidad y Envejecimiento Saludable (CIBERfes), Instituto de Salud Carlos III, Spain.
Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba, Spain; University of Córdoba Agroalimentary Excellence Campus, ceiA3, Córdoba, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain; CIBER Fragilidad y Envejecimiento Saludable (CIBERfes), Instituto de Salud Carlos III, Spain.
Talanta. 2019 Jun 1;198:344-349. doi: 10.1016/j.talanta.2019.02.030. Epub 2019 Feb 6.
Vitamin D has been widely determined in clinical trials to elucidate its biochemical involvement in a great number of pathologies. The analysis of vitamin D and its hydroxymetabolites in biofluids such as serum or plasma is a challenging task due to limitations associated to the low concentrations of some metabolites (typically, dihydroxymetabolites), methodological interferences, and the low stability of the compounds. Among these limitations, efforts have been targeted at optimizing instrumental improvements to develop more sensitive and selective methods, while the stability of vitamin D and metabolites has not been exhaustively evaluated. In this research, several aspects regarding the short-term storage conditions of human serum have been studied to evaluate their influence on the determination of vitamin D and metabolites. An experimental plan has been applied to assess the influence of two relevant parameters: the storage temperature for a period of two months and the number of freeze-thaw cycles. The storage temperature affected in a different manner to vitamin D and its metabolites, being vitamin D and 1,25-dihydroxyvitamin D the two analytes more affected by this parameter. Concerning the freeze-thaw cycles, this variable must be limited to two cycles owing to its significant influence on the variability for quantitation of dihydroxymetabolites in human serum. Finally, lyophilization was also tested to check if serum concentrations of vitamin D and its metabolites were affected by this preprocessing step. The results revealed that only vitamin D experienced a decrease in serum concentration after two months, which does not constitute a real problem as vitamin D is not currently a crucial parameter to be determined in clinical trials due to its scant biological activity.
维生素 D 在临床试验中被广泛研究，以阐明其在许多病理中的生化作用。由于一些代谢物（通常是二羟代谢物）浓度低、方法学干扰以及化合物稳定性低等限制，分析生物体液（如血清或血浆）中的维生素 D 及其羟代谢物是一项具有挑战性的任务。在这些限制中，人们努力优化仪器改进，以开发更敏感和选择性的方法，而维生素 D 和代谢物的稳定性尚未得到充分评估。在这项研究中，研究了人类血清的短期储存条件的几个方面，以评估它们对维生素 D 和代谢物测定的影响。应用实验方案评估了两个相关参数的影响：两个月的储存温度和冻融循环的次数。储存温度以不同的方式影响维生素 D 和其代谢物，维生素 D 和 1,25-二羟维生素 D 是受该参数影响最大的两种分析物。关于冻融循环，由于其对二羟代谢物在人血清中定量的变异性有显著影响，这个变量必须限制在两个循环以内。最后，还测试了冻干以检查维生素 D 和其代谢物的血清浓度是否受到此预处理步骤的影响。结果表明，只有维生素 D 在两个月后血清浓度下降，这并不是一个真正的问题，因为由于其生物活性很少，维生素 D 目前不是临床试验中需要确定的关键参数。
