Significance of Thermal Isomerisation on the Quantitation of Total Vitamin D 3 in Foods Brendon D. Gill 1 & Donald L. Gilliland 2 & Harvey E. Indyk 1 & Jackie E. Wood 1 & David C. Woollard 3 Received: 11 October 2018 /Accepted: 7 January 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Sample preparation techniques for the analysis of vitamin D 3 in food matrices typically utilise a saponification step, either at room temperature or at elevated temperatures. A calciferol (vitamin D 2 or isotope labelled vitamin D 3 ) is generally chosen as the internal standard to compensate for changes of previtamin D 3 –vitamin D 3 isomerisation during analysis, as well as to correct for analyte loss through complex sample preparation steps. Manufacturing practices and processing parameters contribute to previtamin D formation in food products. A significant proportion (5.6–8.3%) of the total vitamin D 3 in premixes was found as previtamin D 3 , indicating that it is likely, depending upon storage temperature and the time since manufacture, that a vitamin D 3 -fortified food product will contain a similar proportion of previtamin D 3 prior to analysis. Conversely, freshly prepared internal standard solutions have low previtamin D levels (< 1%). In lieu of direct measurement, this discrepancy in previtamin D content between the internal standard and analyte forms of vitamin D will lead to analytical bias. To mitigate this as a source of potential error, it is recommended that sample pretreatment steps are appropriately set and controlled. Based on this work, saponification times greater than 300, 120, or 60 min for temperatures of 60, 70, or 80 °C respectively should be employed and that saponification at room temperature be avoided. Keywords Vitamin D . Previtamin D . Saponification . Isomerisation Introduction Vitamin D, as ergocalciferol (vitamin D 2 ) and cholecalciferol (vitamin D 3 ), is a secosterol that plays a major role in bone health. Both forms are available through the diet, and vitamin D 3 is also formed in the skin via irradiation of 7-dehydrocholesterol to previtamin D 3 with subsequent thermal isomerisation (Fig. 1). The main biological function of vitamin D is controlling the absorption, transport and deposition of calcium and phosphorus as part of bone mineralisation. In addition to rickets in children and osteomalacia in adults, vitamin D deficiency has been asso- ciated with increased rates of cancer, diabetes, and cardiovascular disease (DeLuca 2004; Hewavitharana 2013; Higashi et al. 2010). The accurate analysis of vitamin D 3 in foods is challenging; aside from irreversible loss of vitamin D 3 through oxidative deg- radation, the reversible isomerisation of vitamin D 3 to previtamin D 3 is thermally induced, changing the relative proportion of each until equilibrium is reached (Mackay et al. 1979; Mulder et al. 1971). This isomerisation is not affected in vitro by solvent, pH or UV light (Keverling Buisman et al. 1968), although in vivo, biological macromolecules may play a catalytic role (Tian and Hollick 1995). An important consequence of this phenomenon for nutritional purposes is that the measurement of total vitamin D 3 (the sum of vitamin D 3 and previtamin D 3 ) is necessary to obtain complete, reliable and consistent results (de Vries et al. 1979). During analysis of vitamin D 3 in foods, alkaline hydrolysis is the preferred technique to remove triglycerides prior to or- ganic solvent extraction of the non-saponifiable fat-soluble vitamins. Such saponification procedures can be characterised as Bhigh temperature–short time^ or Blow temperature–long time^, with either strategy commonly used in numerous methods (Eitenmiller et al. 2008; Perales et al. 2005). The time/temperature for Bhot^ saponification is typically in range 60–80 °C for 30–60 min, whereas Bcold^ saponification is generally performed at room temperature for up to 24 h. Cold saponification has the advantage of limiting further * Brendon D. Gill brendon.gill@fonterra.com 1 Fonterra Co-operative Group Ltd., P.O. Box 7, Waitoa 3341, New Zealand 2 Abbott Nutrition, Nutritional Research Center, 3300 Stelzer Rd., Columbus, OH 43219, USA 3 Hill Laboratories, Private Bag 3205, Hamilton 3240, New Zealand Food Analytical Methods https://doi.org/10.1007/s12161-019-01434-6