Vitamin D includes a group of steroids molecules which is responsible for numerous cellular processes including calcium and phosphorous homeostasis (Chen et al., 2016; Japelt & Jakobsen, 2013), glucose metabolism (H, O, Da, O, & Ng, 2013; Jamka et al., 2015), endocrine signalling (Al-Hendy, Diamond, Boyer, & Halder, 2016; Santos et al., 2017), cellular proliferation, differentiation and apoptosis process (Arboleda Alzate, Rodenhuis-Zybert, Hernandez, Smit, & Urcuqui-Inchima, 2017; Bartels et al., 2013; Bosetti et al., 2016; Hu & Zuckerman, 2014; Hunten & Hermeking, 2015), and development of immune response (Al-Jaderi & Maghazachi, 2013; Alhassan Mohammed et al., 2017; Boontanrart, Hall, Spanier, Hayes, & Olson, 2016). Even though the relationship of vitamin D deficiency correlates with various diseases, its deficiency or insufficiency is widespread among the children and adults across the world (Holick, 2002; Lopez-Sobaler et al., 2017; Malabanan, Veronikis, & Holick, 1998; Ovesen, Andersen, & Jakobsen, 2003). Due to its immense physiological importance, vitamin D is currently included in various foods and beverages worldwide. A wide variety of methodologies have been developed for the quantification of vitamin D in food feed environmental, clinical and pharmaceutical samples. The analysis of vitamin D compounds is further complicated by their compound structural similarity of metabolites, the predominantly hydrophobic/ lipophilic nature of vitamin D compounds and the hydrophilic nature of some of the metabolites, and their instability in the presence of heat or UV light. Accordingly, analysis requires selective and rapid methods and among these, there are two main types of methods which are used routinely, namely: competitive immunoassays and methods based on chromatographic separation followed by non-immunological direct detection (HPLC-UV and LC–MS/MS). Immunoassays are frequently used as commercial kits that include RIA, ELISA, CLIA, and ECL methods. Immunoassays are readily automated, suitable for high throughput and do not require high level technical skills to obtain satisfactory results. However, the major disadvantage is that of specificity as immunoassays are not able to detect D2, thus making them unsuitable for monitoring any food, feed, water and environment samples. In recent years, LC- MS/MS has been using for vitamin D analysis because it conveys both high specificity and sensitivity (Qi et al., 2015). LC-MS methods allow the separation of compounds based on their polarities, ionization behaviors, and mass to-charge (m/z) ratios and can offer very low limits of quantitation. However, these LC-MS methods also have challenges include, abundant isobaric and isomeric interferences (Volmer, 2015), low ionization efficiencies for mass spectrometric analyses (Hewavitharana et al., 2014) and matrix effects (Gomes et al., 2013). Therefore, increasing the selectivity and sensitivity for determination of vitamin D metabolites has been the aim of various methods and has focused on sample clean-up/extraction and analyte concentration prior to determination.
Establishing an appropriate extraction method for vitamin D is crucial as it cannot be assessed by the validation process (Heijboer et al., 2012). Therefore, the release of the vitamin from the sample matrix is a crucial step that affects the sensitivity and reproducibility of the analytical process. Ideally, the extraction method must be capable of dislodging the entire vitamin content from the matrix before analysis.
A variety of extraction methods was developed in recent year and compared and the results showed high variability illustrating inconsistency in the extent of vitamin D release from the matrix (Heijboer et al., 2012). On the contrary, traditionally, saponification has been used for the extraction of vitamin D from foodstuffs including milk and LLE and SPE also have been used as extraction/sample clean-up procedures in vitamin D assays analyses after the release from matrix components such as protein and fat. Traditionally, the process of alkaline saponification for the extraction of vitamins and step-wise HPLC analyses has been widely used for analysis of lipophilic vitamins in animal feeds and the most common procedure by which to extract vitamin D compounds from foodstuff (Berg et al., 1986). The hydrolysis reaction attacks ester bonds and releases the fatty acids from the glycerol of glycerides and phospholipids, as well as from esterified sterols and carotenoids (Thompson et al., 1982). This reaction also frees vitamin D from any binding matrix that may exist in the sample matrix. Given the lack of stability of vitamin D, it is common to use antioxidants such as butylated-hydroxytoluene (2,6-di-tert-butyl-4-methylphenol) and ascorbic acid in the saponification process (Demchenko et al., 2011; Japelt et al., 2011; Kienen et al., 2008; Trenerry et al., 2011; Perales et al., 2005), combined with potassium hydroxide in ethanol or water solutions. The importance of potassium concentration in ethanol or methanol in saponification to obtain vitamin D in milk matrix has been reported in various studies. However, there was no significant difference when either methanol or ethanol were used (Paixao & Stamford, 2002). Ethanolic KOH prevents the formation of emulsions and mixes well with fat, but it requires daily preparation (Perales et al., 2005). In contrast, aqueous KOH does not mix well with fat, but is more stable – this probably being the reason why it is more often used (Perales et al., 2005).
Hot saponification consists of treating the sample with ethanolic or aqueous KOH at temperatures between 60–100°C and times range of 20–45 min while cold saponification consists of treating the sample overnight with ethanolic or aqueous KOH at room temperature, under slow constant stirring (Perales et al., 2005). Thermal isomerisation of vitamin D to pre-vitamin D may be avoided in a cold saponification procedure (isomerisation losses of less than 5% under cold conditions versus about 10–20% under hot conditions). Furthermore, this method provides satisfactory extraction and recovery and is simpler to operate with less operator attention (Thompson et al., 1982; Thompson et al., 1977).
Once saponification has been completed, the non-saponifiable fraction is extracted with organic solvents that are not miscible in water. Preferably with hexane instead of di-ethyl ether because di-ethyl ether is more inflammable and unstable than hexane and the latter can be simply removed at low pressure at a temperature below 50°C) (Thompson et al., 1982). In addition, there are various other conditional factors such as sample particle size (mesh), ratio of sample to reagent, extraction time, extraction equipment and pre-purification that can affect extraction efficiency (Qian & Sheng, 1998). Subsequently, evaporation is used to remove the organic solvents. These extraction methods were further simplified in this study to reduce the complexity involved in the process. Since efficient extraction of vitamin D and purification was the main challenge in all the analytical technique, therefore, we use this extraction method and UV-visible spectrophotometer instead of HPLC, LC-MS/MS to quantify vitamin D in food feed, environmental and pharmaceutical samples.