Plozza, Timothy Edward (2017) The analysis of fat-soluble vitamins in dairy cow milk by high performance liquid chromatography-ion trap mass spectrometry. Research Master thesis, Victoria University.

Abstract

Fat-soluble vitamins A, D, E, and vitamin A precursor β-carotene are essential nutrients for the human body. The analysis of these compounds is difficult due to low levels (ppt - ppm), and physical and chemical similarity to other compounds present in foods. Traditionally, the determinative step uses high performance liquid chromatography (HPLC) coupled to either ultraviolet-visible (A, D, E and β-carotene) or fluorescence (A and E) detection. The work described in this thesis demonstrates that HPLC coupled with mass spectrometry (MS) is a viable alternative due to its superior selectivity and sensitivity, reducing the need for time-consuming sample preparation, and enabling the analysis of several vitamins in a single analytical method. High performance liquid chromatography-ion trap mass spectrometry (HPLC-MSn) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were developed to measure the levels of vitamin D3 in fortified commercial cow’s milk (2 μg/100 ml), unfortified commercial and fresh cow’s milk (0.01-0.05 μg/100 ml). An additional solid phase extraction step enabled the lower levels (<0.1 μg/100 ml) to be measured. 25-hydroxyvitamin D3 was also measured, although the extraction procedure was not fully optimised for this compound. A single HPLC-MSn method suitable for the analysis of all trans-retinol (vitamin A), α- tocopherol (vitamin E) and β-carotene in cow’s milk was also developed. This method was used to measure the effects of different supplementary feeding regimes on the fatsoluble vitamin content of milk from pasture-fed dairy cows. Typical levels were all trans-retinol, 45 μg/100 ml; α-tocopherol, 150 μg/100 ml; and β-carotene, 12 μg/100 ml. The results from the feeding experiment showed reductions in the levels of β-carotene and α-tocopherol in milk due to the feeding of Partial Mixed Rations, most likely due to reduced intake of pasture, which was the major source of these compounds in the cows’ diet.

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