Degradation studies of cholecalciferol (vitamin D3)

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Structures and properties of vitamin D and its isomerization products

Vitamin D

Vitamin D is a group of fat-soluble seco-steroid compounds with relatively low polarity. Vitamin D, including cholecalciferol (D3), (5Z,7E)-(3S)-9,10-seco-5,7,10(19)-cholestatrien-3-ol, and ergocalciferol (D2), (5Z,7E,22E)-(3S)-9,10-seco-5,7,10(19),22-ergostatetraen-3-ol, are originated from their precursors 7-dehydrocholesterol and ergosterol in animals and fungi, respectively. Whereas vitamin D3 is produced naturally in the skin by ultraviolet (UV) irradiation of  7-dehydrocholesterol, vitamin D2 is produced in fungi and yeast by a UVB-exposure of ergosterol (provitamin D2) (DeLuca & Schnoes, 1976; Japelt & Jakobsen, 2013). The cleavage of B-ring between C9 and C10 of vitamin D is central to the creation of the  5, 6, 7 cis-triene structure of previtamin D. These compounds have an open B-ring and  a conjugated triene system which plays an important role not only in the biological activity of vitamin D but also have an interestingly parallel synthesis from the chemistry point of view. Both vitamin D2 and D3 have a methylene group at C10 and the 5, 6 double bond is considered to be cis, and vitamin D2 has an extra methyl group at C24 and an additional double bond in its side chain at C22 (Koshy & Beyer, 1984; Martıneź -Núñez, Cabaleiro-Lago, FernándezRamos, Hermida-Ramón, & Peña-Gallego, 1999).

Previtamin D3

Previtamin D3, (6Z)-(3S)-9,10-seco-5(10),6,8-cholestatrien-3-ol, is an intermediate in the production of vitamin D. In terms of vitamin D formation, the first step is the photolytic opening of the B ring to form previtamin D, followed by the second step, which is thermally favoured transformation of previtamin D into vitamin D. It is well known that vitamin D in solution exists in an equilibrium between vitamin D and previtamin D (Mulder, Vries, & Borsje, 1971). In this regard, Johnsson & Hessel (1987) reported partial isomerization of vitamin D into its precursor previtamin D during the saponification process employed for its extraction. The conversion of vitamin D to previtamin D is a thermal equilibrium and it has been shown that the equilibrium rate constant is independent of the nature of the solvent. The percentage of vitamin D3 in equilibrium with previtamin D3 ranges from 98% at -20°C to 78% at 80°C. Thus, temperature is an important factor in the equilibrium of vitamin D3 to previtamin D3 (Koshy & Beyer, 1984; Naidoo, 2011). The UV absorption maxima in the spectrum of cis-vitamin D3 after heat treatment changes from 265 nm to 261 nm as a result of the contribution of the UV absorption spectra of previtamin D3 (Figure 2.3) (Verloop at al., 1959). The extinction coefficient also changes for the conversion of vitamin D to previtamin D (Hanewald, Rappoldt, & Roborgh, 1961; Hanewald, Mulder, & Keuning, 1968)

5,6-trans-vitamin D3

The cis configuration of vitamin D is important for its biological activity but the trans from has very low activity. Vitamin D could be rearranged by exposure to ultraviolet radiation to form  5,6 trans isomer, (3S,5E,7E)-9,10-Secocholesta-5,7,10-trien-3-ol, (Figure 2.4). Moreover, the trans isomer can irreversibly undergo further isomerization mostly to isotachysterol through exposure to heat or acids (Havinga, 1973; Jin et al., 2004). The cis and the trans forms of vitamin D3 display different UV spectra. While the cis form has a UV maxima at 265 nm (Figure 2.3 a), the trans form shows a UV maxima at 273 nm (Holick, Garabedian, & DeLuca, 1972; Verloop, Koevoet, & Havinga, 1955)


Vitamin D3 can be isomerized to corresponding isotachystero13 derivatives, (6E)-(3S)-9,10-seco5(10),6,8(14)-cholestatrien-3-ol, (Figure 2.6 a). In this reaction, the double bond system of the secosteroid is rearranged and the A-ring rotated to expose the hydroxyl group of C1. The spectral properties and the polarity of the molecule are thus altered (Trafford, Seamark, Turnbull, & Makin, 1981). Low pH is known to isomerize vitamin D3 to isotachysterol which exists as a pale yellow oily liquid (Jin et al., 2004). It was reported that vitamin D, previtamin D and tachysterol can be converted to isotachysterol in various acidic conditions such as HCl, BF3 and H3PO3, but the HCl procedure is the most efficient (Seamark, Trafford, & Makin, 1980; Verloop, Corts, & Havinga, 1960; Zhang et al., 2006). It was also reported that in the preparation of isotachysterol, the isomerization yield is affected by the temperature and volume of the reaction mixture and the duration of the reaction (Agarwal, 1990). Zhang et al. (2006) suggested a reaction model by which the acid induced formation of isotachysterol from vitamin D may sequentially proceed through the intermediates of previtamin D and tachysterol. Agarwal, 1990 reported that isotachysterol has absorption maxima at 278, 288 and 301 nm with its molar absorption being more than twice that of vitamin D at 265 nm (Kobayashi, 1965)

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Previtamin D3 can be isomerized by ultraviolet (UV) radiation to tachysterol3, (6E)-(3S)-9,10seco-5(10),6,8-cholestatrien-3-ol, (Figure 2.7). Tachysterol, which is a 7,8 cis-isomer of isotachysterol and a trans-isomer of previtamin D, could also be generated by chemical isomerization (Verloop et al., 1960). The irreversible isomerisation to inactive products has a major impact on formulation of vitamin D so that all aspects of isomerisation need to be considered. The UV absorption of tachysterol was considered to agree more with a trans structure with an absorption peak at 281 nm and shoulders at 272 and 289 nm (Figure 2.3 c) (Lugtenburg & Havinga, 1969; Verloop et al., 1959)

Food fortification


Food fortification, defined by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United Nations (Allen et al., 2006), refers to  “the practice of deliberately increasing the content of an essential micronutrient, ie. vitamins and minerals (including trace elements) in a food irrespective of whether the nutrients were originally in the food before processing or not, so as to improve the nutritional quality of the food supply and to provide a public health benefit with minimal risk to health”. WHO and FAO (2006) have identified food fortification as the second strategy of four, in decreasing the incidence of nutrient deficiencies at the global level. As micronutrient malnutrition (MNM) has many adverse effects on human health, food fortification has had a long history of controlling the deficiencies of different micronutrients such as vitamin D, A, B, iodine and iron.  The foods that are most commonly fortified include milk and milk products, infant formulas, flour, salt, and oils and fats. There are several forms of food fortification: foods that are widely consumed by general population, foods fortified for specific populations, and foods which are fortified by food manufacturers. Food fortification carries a number of advantages and limitations over supplementation as follows (Allen et al., 2006):
– Consuming fortified foods regularly maintains nutrients more efficiently and effectively in the body. However, it has a less immediate impact but is usually the shortest way to treat deficiencies compared to supplementation.
– One of the most important advantages of food fortification is a prolonged impact for a population who needs a continual supply of micronutrients for growth and development.  On the other hand, supplements are needed to satisfy the requirements of selected population groups such as infants and pregnant women who need adequate amounts of some micronutrients.
– In contrast to supplements, which are consumed by individuals who are willing to take them and can afford to buy them, fortified foods are widely distributed and consumed. Thus, a large proportion of the population can be accessed by food fortification.
– Since broad access to fortified foods may cause increase levels of micronutrients for all population groups, it could be harmful for those who do not suffer from malnutrition.
– Since foods are complex mixtures, some technological issues relating to fortification including appropriate levels of nutrients, homogeneous distribution of the nutrients within the food matrix, stability of fortificants, nutrient interactions, as well as accessibility by consumers have to be resolved. Additionally, due to the likelihood interactions between the added micronutrients and the food vehicles, and with other nutrients (either added or naturally present), the fate of fortificants should be investigated in fortified foods.
Although multiple micronutrients fortification is technically possible, fortified foods alone may not be sufficient to supply all micronutrients, but, supplement intake might be able to resolve this issue. While fortification carries a minimal risk of chronic toxicity, it still can cause a risk of excess. Supplement intake, however, is likely to be better regulated by the individual. However, there is a low compliance for using supplements and those who disregard the instructions may have a high risk of, for example, vitamin D deficiency (Holick, 2006). Therefore, fortification of regularly consumed foods may be more appropriate since they do not require individual compliance or changes in purchasing patterns. Food fortification is a complex task and there are several factors to be considered in this matter. Nature of the fortificant: The micronutrient, which is used for fortification, should be safe, stable, and well absorbed. The fortificant should resist processing and storage conditions without significant degradation. Nature of the food vehicle: An appropriate food vehicle that protects the fortificant from degradation during processing and storage should be chosen. The food vehicle containing components that could compromise the stability and/or absorption of the fortificant should be avoided. Consumer satisfaction:
– The fortificant should not adversely affect the quality of final product such as chemical composition, cooking properties or sensory qualities (e.g. colour, flavours or texture).
– Non-compliance of fortification: The amount of micronutrient should be safe and sufficient to prevent deficiency problem. Vitamins A and D are considered toxic in excessive levels of consumption.
– These fat-soluble vitamins accumulate in tissue over time and are not easily eliminated from the body.
– Excessive intake of vitamin D causes serious adverse effects such as hypercalcemia, dehydration, heart damage, kidney damage and soft tissue calcification (Rubin et al., 2005; Viswanath, 2013)


Vitamin D fortification

Vitamin D is a regulator of calcium metabolism and is involved in the absorption of calcium in the intestines. Moreover, vitamin D is required for bone growth in the mineralization process. Chronic severe vitamin D deficiency in children causes bone deformation due to poor mineralization, which is known as rickets. In adults, vitamin D deficiencies can result in muscle weakness, bone pain, osteomalacia and rapid development of osteoporosis. Apart from the effects of vitamin D on skeletal health, it may play a role for regulating cell growth, the immune system, and other physiological processes (Bischoff-Ferrari et al., 2004; Bischoff-Ferrari, Giovannucci, Willett, Dietrich, & Dawson-Hughes, 2006; Giovannucci, 2005; Lappe, Travers-Gustafson, Davies, Recker, & Heaney, 2007). Vitamin D3 is mostly produced via the exposure of the skin to the sun. Vitamin D intake from sunlight depends on latitude, season, cloud cover, ozone level, surface reflection, altitude, outdoor practices, skin type, obesity, age and clothing (Engelsen, 2010; Lips, 2010; Rockell et al., 2005; Webb, Kline, & Holick, 1988). Although the human body is able to generate vitamin D through exposure to the sun, individuals who do not receive sun exposure, people with dark pigmented skin and older people are high-risk groups of vitamin D deficiency (Kiely & Black, 2012; Whiting, Green, & Calvo, 2007). Only a limited number of natural foods contain vitamin D. Vitamin D3 is present in animal foods such as eggs, fish species (salmon, herring and mackerel) and liver. Vitamin D2 also has been found in some wild mushrooms, where it appears to be formed from the action of UV on the provitamin, ergosterol (Teichmann et al., 2007). Other plant sources used as food may contain ergosterol that is not converted to vitamin D2 (Lamberg-Allardt, 2006). Vitamin D malnutrition is likely to be recognized as a significant public health problem in many countries. The importance of vitamin D in human diet has been recognized since the early 1900’s (McCollum, Simmonds, Becker, & Shipley, 1922). Few countries fortify foods with vitamin D. Vitamin D3 is now the permitted form for addition to foods in Canada, while the USA and European Union (EU) permits use of either vitamin D2 or vitamin D3. In these countries, foods usually fortified with vitamin D are milk products, margarines and breakfast cereals (Lamberg-Allardt, 2006). In Australia, it is mandatory for margarines to contain no less than 55 μg/kg of vitamin D. This mandatory requirement does not apply to these foods for sale in New Zealand (Food Standards Australia New Zealand, 2016). Vitamin D also may be sourced from dietary supplements (usually in pill, capsule, tablet or other controlled dosage form). The vitamin D present in supplements can be in the form of both vitamin D2 and vitamin D3. However, vitamin D2 is rarely used as the fortificant in supplements (Rockell, Skeaff, Logan, & Green, 2008)

1. Introduction and Research Objectives
1.1. Research background
1.2. Research objectives
1.3.Thesis framework
2. Literature Review
2.1. Structures and properties of vitamin D and its isomerization products
2.2. Food fortification
2.3. Vitamin D stability
2.4. Lipids
2.5. Analysis of vitamin D and its related products
2.6. Conclusions
3. Degradation studies of cholecalciferol (vitamin D3) using  HPLC-DAD, UHPLC-MS/MS and chemical derivatization
3.1. Introduction
3.2. Material and methods
3.3. Results and discussions
3.4. Chemical derivatization
3.5. Conclusions
4. Lipid oxidation and vitamin D3 degradation in simulated  whole milk powder as influenced by processing and storage
4.1. Introduction
4.2. Materials and methods
4.3. Results and discussions
4.4. Conclusions
5. Identification of vitamin D3 oxidation products using high-resolution and tandem mass spectrometry
5.1. Introduction
5.2. Material and methods
5.3. Results and discussion
5.4. Conclusions
6. Conclusions and Future work
6.1. General conclusions
6.2. Future work

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