Development and applications of methods for assessing fat soluble micronutrients (FSMs) in dried blood spots and Plasma by HPLC and HPLC-MS/MS

Epidemiological studies have shown that deficiencies in vitamin A and vitamin E are very high in developing countries, whereas vitamin D deficiency is a global health concern. Deficiencies in fat soluble vitamins have been associated with negative health consequences, and are most severe when they occur during pregnancy, where they may lead to complications in both the mother and the offspring. In order to better understand the status of populations and associations with diseases, the establishment of analytical methods for determining fat soluble micronutrient (FSM) concentrations is essential. Therefore, the central aim of this thesis was to develop simple and reliable assays for FSMs in human blood and plasma, which would be suitable for application in large-scale clinical studies. The process of analytical method development in this thesis included: i) establishing and validating a dried blood spot (DBS) test for the measurement of retinol (biomarker for vitamin A) concentrations; ii) an assay to simultaneously determine all FSM (retinol, α-tocopherol, γ-tocopherol, β-tocopherol, 25OHD3, 25OHD2, lutein, zeaxanthin, lycopene and β-carotene) concentrations using a minimal volume of plasma; and iii) to validate these methods through the measurement of concentrations of FSMs in a sub-set of plasma samples in an ongoing clinical study (n = 103 women at two time points, one of which is first and the other the second trimester). This enabled me to evaluate the impact different anti-coagulants on FSM concentrations, and to obtain pilot data on the concentrations of FSMs in pregnant women in South Australia, and how they change across gestation. The DBS method that I established for measuring retinol showed good reliability and robustness when extracted using acidic conditions. Additionally, using this method, retinol in DBS was shown to be structurally stable at room temperature for 10 weeks, when stored in the dark and with desiccants. The measurement of all FSMs in plasma also showed reliable and sensitive results including high precision for all analytes (coefficient of variation 20 h before processing. The assessment of FSMs in the group of pregnant women in South Australia indicated vitamin A, E and carotenoid sufficiency, as well as low prevalence of vitamin D insufficiency (< 10%). Significant changes in concentrations of vitamin D, E and carotenoids between the first trimester and second trimester during pregnancy were also identified. In conclusion, this thesis reports the successful development of methods for measuring vitamin A in DBS and FSMs in plasma using HPLC, which are reliable, accurate and robust. The DBS method will be a valuable tool for clinical studies and studies in remote areas where sample collection and transportation are difficult.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2016