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

Abstract

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.

Popular Abstract

Comparison of different extraction methods for the analysis of vitamin D analogues in liver samples.
Vitamin D (VD) deficiency is related to several diseases, so it is crucial to correctly detect and quantify its concentration in the human body. One of the most challenging parts in the analysis of VD in biological samples is to extract the compounds, due to the complexity of these kinds of samples, as well as the low levels of concentration at which VD analogues are present in it, that required the application of very sensitive and selective techniques. Despite the existing methodologies for VD analysis, the existing methods for its analysis in biological samples should be improved.
The aim of this study is to compare different extraction methods to find the most suitable and the greenest option for the analysis of VD and its metabolites in liver samples. Finally, the parameters that can affect the extraction efficiency will be determined, studied and optimized.
Different methods were tested and the expected results were not obtained, especially because of the low extraction yield achieves for some of evaluated compounds including the ester metabolites and VD2 and VD3. On account of that, a backward step was done and some experiments were performed to study the viability of VD compounds extraction by using
different solvents. Classical Folch (i.e., a liquid-liquid extraction method using a mixture of chloroform with methanol and water), 2-propanol, acetone and acetonitrile were found as the most suitable options for extracting the target substances. Moreover, some gravimetric
experiments were done in order to study how much fat was extracted from the liver alongside the VD compounds depending on the solvent used. This study was carried out taking into account the hypothesis that the more fat is extracted, the more analytes akin to fat would be
extracted (these kinds of analytes were the ones that showed the worst results when extracting VD from liver samples). These experiments indicated that mixture of chloroform and methanol and 2-propanol are the best options for analyzing the desired compounds, as with these two
solvents the highest amount of fat was extracted.
More experiments should be done in order to find the most reachable approach to extract these compounds from the liver samples. However, this study constitutes the first step to recognize those parameters that have a remarkable effect on the extraction of vitamin analogues from such complex matrices.

• author: Mestres, Pol
• supervisor: Charlotta Turner; Margareta Sandahl; Barbara Socas Rodriguez; Daniel Molins Delgado
• organization: Department of Chemistry
• course: KEMP30 20201
• year: 2020
• type: L3 - Miscellaneous, Projetcs etc.
• subject: Chemistry
• keywords: analytical chemistry, green extraction techniques, liver, mass spectroscopy, supercritical fluid chromatography, vitamin D, analytisk kemi
• language: English
• id: 9019067
• date added to LUP: 2020-06-18 17:20:40
• date last changed: 2020-06-18 17:21:05