Evaluation of short-term storage prior to analysis of vitamin D3 and metabolites in human serum by liquid chromatography coupled to tandem mass spectrometry.

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 D3 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 D3 and its metabolites, being vitamin D3 and 1,25-dihydroxyvitamin D3 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 D3 and its metabolites were affected by this preprocessing step. The results revealed that only vitamin D3 experienced a decrease in serum concentration after two months, which does not constitute a real problem as vitamin D3 is not currently a crucial parameter to be determined in clinical trials due to its scant biological activity.