The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D3) and ergocalciferol (D2). The main source of vitamin D3 is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D3, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSn) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D3 in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSn and LC–MS/MS, respectively. Recoveries of vitamin D3 added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D3 was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).