Comparison of different extraction methods for the analysis of vitamin D and vitamin D metabolites in liver samples

Introduction: This study is focused on the development and comparison of different extraction methods for vitamin D (VD) analysis in liver samples. Background: VD deficiency is related to several diseases, so it is crucial to correctly detect and quantify its concentration in the human body. Despite the existing methodologies for VD analysis, the selectivity and the sensitivity of the methods for its analysis in biological samples should be improved. Also, to the best of our knowledge, there is no method that allows determining both types of VD metabolites – alcohols and esters – at the same time, especially there is no method in the literature that lets the esters to be detected. Aim(s): The aim of this study is to compare different extraction methods to find the most reachable and the greenest option for the analysis of VD and a group of VD metabolites in liver samples and to optimize the conditions of their extraction. Methods: Folch method, ultra-high pressure supercritical fluid extraction (UHPSFE) and dispersive liquid-liquid microextraction (DLLME) will be tested and optimized by using statistical design of experiments (DoE) prior to analysis by ultra-high performance supercritical fluid chromatography – triple quadrupole tandem mass spectrometry (UHPSFC-QqQ-MS/MS). The most suitable methodology will be validated. Results: Folch method and DLLME were optimized obtaining a non-valid model for the DLLME and a good one for Folch extraction, but with non-significant recovery values for ester metabolites and low ones for vitamin forms. UHPSFE appears to be the most suitable methodology, according to initial experiments, but further experiments should be carried out to confirm it . A solubility test using different solvents and gravimetric experiments showed that matrix effects are important and that classical Folch method and 2-propanol seem to be the best options for extracting the target analytes. Conclusion: Folch, DLLME and UHSFE have been combined with UHPSFC-QqQ-MS/MS for the evaluation of ten VD analogues in liver samples. Results for ester metabolites and vitamin D3 and D2 are still not satisfactory and should be improved. More experiments should be done to find the most suitable approach for the analysis of the VD compounds.