Analyzing vitamin D in food is crucial for ensuring its nutritional content and adequacy in diets. We can make vitamin D through light exposure of the skin but many animals have this as an essential vitamin such as dogs because they produce too little. We’ve written many times about the nutritional value of vitamin D in relation to COVID, cancer, bone conditions etc. but analysis means we can truly measure what is available in the diet be it for people and pets.
Vitamin D is a fat-soluble vitamin. More accurately this vitamin is described structurally as belonging to the secosteroids. There are two major forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is the main form found in human and pet food and is also the form that is most often used in fortification. Vitamin D3 can be split further into 25-hydroxyvitamin D3 as well.
Vitamin D can thermally isomerize to previtamin D. The transformation is reversible and both forms are biologically active. It is thought that the relative content of previtamin D could be up to 22% of the total vitamin D at 80ºC. It is thought a good idea to individually measure both previtamin D and vitamin D contents.
The structure of vitamin D2 shows a double bond at the C22 position and a methyl group at the C24 position that vitamin D3 lacks.
Sources
Fish is a popular food high in natural vitamin D as is cod liver oil and some mushrooms (vitamin D2). It is extremely high in commercial pet foods. Vitamin D3 and 25-hydroxyvitamin D3 are compounds commonly found in foods.
Various methods are employed for this purpose, each with its advantages and limitations. The subject has been examined over many years (Parrish & Richter, 1979).
Vitamin D fortification is mandatory in Canada for fluid milk (110 IU/250 mL) and margarine (53 IU/10 g).
The current recommendation is that the AAFCO would like between 12.5 and 75 micrograms/kg of vitamin D in dog food. Pet foods contain about 0.1 to 0.5 IU per gram.
One of the main reasons for its measurement is that a deficiency or overdose causes sickness. A lack leads to rickets whilst an overdose can lead to death. Too little also means raised possibility of cancer and chronic kidney disease. Too much produces hypercalcaemia and calcification of soft tissues.
The most common methods used for vitamin D analysis in food are categorised below. Most common methods are based on chemical analysis, especially liquid chromatography coupled to mass or UV spectrometry. The main issue is the analysis of vitamin D with interferences from the sample matrix.
Sample Preparation
Sample preparation is a critical feature of analysis. In most cases samples are treated with KOH and then heated in a boiling water bath. The addition of strong alkali saponifies the sample and separates fat from the vitamin D. If the sample is too oily there can still be an oily residue as in the case of measuring the vitamin in fish. The sample is cooled and then extracted three times with petroleum ether or hexane. The samples are extracted and then dried with sodium sulphate. Samples are then concentrated by rotary evaporation.
In some cases protein precipitation is needed because vitamin D is sometimes bound to animal proteins with globulin characteristics. This means that when carrier protein is hydrolysed it releases vitamin D. Very often blood and milk contain major amounts of this vitamin D.
Extraction is needed to separate vitamins of interest from other substances. The main types of separation include liquid-liquid extraction (Tai et al., 2015), solid-phase extraction (SPE) (Higashi et al., 2011; Mena-Bravo et al., 2016) and supported liquid extraction (Geib et al., 2016).
Chemical Analysis
Chemical methods involve the extraction and quantification of vitamin D using chemical reagents. The Gold Standard method is liquid chromatography with mass spectroscopy (LC-MS/MS) as the detection method (Roth et al., 2008; Meunier et al., 2018). The LC-MS/MS is used to measure 25-hydroxyvitamin D (D2 and D3) in total plasma and serum as a standard clinical approach (Meunier et al., 2018).
One common technique is liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) (Reynolds & Judd, 1984). HPLC separates the components of a mixture based on their interactions with the stationary phase and mobile phase, allowing for the detection and quantification of vitamin D compounds. It is a standard method in the AOAC and EN.
The European Committee for Standarization accepted the AOAC Method 2002.05 which is titled ‘Cholecalciferol (Vitamin D3) in Selected Foods’. They also use Method EN12821 ‘Foodstuffs – Determination of vitamin D by high performance liquid chromatography – measurement of cholecalciferol (D3) and ergocalciferol (D2)’. The International Dairy Federation developed a method called IDF Standard 177: 1996 ‘Dried Skimmed Milk, Determination of Vitamin D Content’.
Methods from the AOAC include Method 2011.11: UHPLC/MS/MS Analysis of Vitamin D in Infant Formula on Titan™ C18 have been detailed by Merck and is an extremely common method. The procedure also details saponification and liquid-liquid extraction. The mobile phase is [A] 0.1% formic in methanol:water (20:80); [B] 0.1% formic acid in methanol. The gradient depending on flow-rate and column is 60 to 90% B in 0.4 min; to 100% B in 0.3 min; held at 100% B for 7.8 min; to 60% B in 0.1 min; held at 60% B for 1.5 min. This method is used for measurement of vitamin D in infant formula and other medical foods. It also describes methods using saponification followed by liquid-liquid extraction.
Specific methods tested include liquid chromatography coupled to mass spectrometry (Dimartino, 2007), ultra-performance liquid chromatography (UPLC)/MS/MS on infant formula and pet food (Huang & Winters, 2011), HPLC UV-DAD and LC-MS/MS (Bilodeau et al., 2011).
The paper by Reynolds & Judd (1984) discusses the methods that have been tested when it comes to preparing a sample containing vitamin D for analysis. The cleaning-up techniques include sterol precipitation (Panalaks, 1971), conventional column chromatography using alumina (Bell & Christie, 1974); and Sephadex, thin-layer chromatography and preparative HPLC (De Vines & Borsje, 1982). All work-up methods though add time to the analysis.
The OTSC (Office of the Texas State Chemist) (Li, 2021) Method discusses the work up of samples for vitamin D3 analysis in pet food prior to using LC-MS/MS. A suitable amount of sample is ground and then saponified by refluxing in a 3:1 ethanol to water mix with added sodium ascorbate and potassium hydroxide. The vitamin D is extracted with hexane and then evaporated with bubbling nitrogen. The concentrated sample is reconstituted in methanol/water with sonication before applied to a liquid chromatograph. Based on spiking levels of between 10 and 40 ng/g sample, the recovery percentage is above 100 with an RSD of 11.5% or lower as the level of spiking rises. The limit of detection is 1.27 ug/kg with an LOQ of 4.23 ug/kg or 0.17 KU/kg. The OTSC were quick to point out that both resolution and signal intensity dropped after 1000 injections. One consequence was that samples with a low vitamin D3 level would not be measured and that replicates would take longer to run.
Derivatization enhances ionization efficiency for specific compounds that results in increased sensitivity of the method and provides opportunities for low abundant metabolites to be measured. The downside is that derivatization increases sample preparation time and thus the total cost of analysis. There are two probable points for reaction: the cis diene moiety at C-5/6 and C-19 and the hydroxyl group.
A more refined method based on using PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) derivatization instead of reconstitution in methanol/water with sonication. A comparison of the two methods led to the conclusion that in the 1st method, it could be sued for regulatory sample analysis but the sensitivity and robustness of the technique dropped off over time unless there was reconstitution of the LC-MS/MS. The issues were resolved in the 2nd method using PTAD derivatization which had better sensitivity and less interference.
A method using trimethylsilyl derivatization and analysis by gas chromatography with tandem quadrupole detection, or GC/MS/MS. The use of gas chromatography means that instrumentation required is cheaper than those exploiting liquid chromatography. The tandem quadrapole GC/MS/MS is more expensive than a single quadrapole instrument but it is adaptable for the latter (Lehner et al., 2021).
The AOAC issued a Standard Method Performance Requirement (SMPRs) [AOAC SMPR 2015.016] to determine vitamin D in dietary supplements and other finished products.
Spectrophotometry
UV-visible spectrophotometry is another widely used method for quantifying vitamin D. This technique relies on the absorption of UV or visible light by vitamin D compounds. By measuring the absorbance of the sample at specific wavelengths, the concentration of vitamin D can be determined.
The use of Near Infra-Red spectroscopy has been tried but has not seen widely limited success (Jia et al., 2018). Near-Infrared Reflectance spectroscopy has been used for multiple vitamin mixes (Pires et al., 2001).
Immunoassays
Immunoassays, such as enzyme-linked immunosorbent assays (ELISA), use antibodies to specifically detect and quantify vitamin D compounds. The most common method of assay for 25-OH-D is the FDA approved version (Wyness et al., 2015).
ELISA kits are commercially available and offer a relatively simple and rapid method for analyzing vitamin D in human serum and plasma samples. There are circumstances where it has been applied to food samples but there are no companies that provide the test kits. It remains to be a method with potential. DL.D Diagnostica GmbH have published a paper describing their method exploiting this technology. Several automated immunoassays have been introduced for clinical use. Recently, Roche Diagnostics launched the Vitamin D (25‐OH) Total Assay (Roche, Basel, Switzerland) for clinical use. It’s worth considering products too from Abcam (Boston, Massachussets) and from Sigma-Aldrich.
Mass Spectrometry
Mass spectrometry (MS) is a highly sensitive technique capable of identifying and quantifying compounds based on their mass-to-charge ratio. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are commonly used for analyzing vitamin D in food samples. The methods for clinical detection have been well reviewed (Xu et al., 2024).
Vitamin D in pet food has been measured using liquid chromatography with tandem mass spectrometry (Huang et al., 2009). Because food matrices are extremely complex including pet foods and animal feeds, Ms/Ms is the first choice of instrumentation. If HPLC is optimised it is possible to seperately measure previtamin D and vitamin D. Pet foods have very low levels of vitamin D and to separate vitamin D from complex pet food matrices means using longer HPLC run times.
These methods offer high specificity and sensitivity but require specialized equipment and expertise. The main issue is if the vitamin D derivatives have the same molecular weight and roughly similar structure because ionization patterns are so similar (Zelzer et al., 2018). We have already referenced this method earlier in relation to liquid chromatography.
Bioassays
Bioassays are one of the oldest analytical methods especially when assessing vitamins (Baker & Wright, 1940). The AOAC method 936.14 (45.3.02) ‘Vitamin D in Milk, Vitamin Preparations, Feed Concentrates, rat Bioassay’ is still quoted as the main test. There is a version of chicks too. .
They involve measuring the biological activity of vitamin D compounds using cell cultures or animal models. While less commonly used for routine analysis, bioassays provide valuable information on the bioavailability and physiological effects of vitamin D in food.
Molecular Methods
Polymerase chain reaction (PCR) techniques can be used to detect and quantify the expression of genes involved in vitamin D metabolism in food samples. These molecular methods can provide insights into the vitamin D content of food at the genetic level.
Each method has its strengths and limitations in terms of sensitivity, specificity, cost, and complexity as we often like to say. At FoodWrite we’ve often had to consider a number of methods depending on the type of food. Pet food is a good example of understanding the nutritional value of the food having worked on various formulations for clients. Choosing the appropriate method depends on factors such as the type of food matrix, required sensitivity, and available resources. Combination approaches, such as using both chemical and immunoassay methods, may provide more comprehensive insights into the vitamin D content of food samples.
Health & Safety Issues
In the USA, the FDA in December 2018 recalled eight brands of dog food because they had toxic levels of vitamin D. Then in January 2019, Hill’s Pet Nutrition notified the FDA of a vitamin D issue in their dog food that led to a recall and by May that year had recalled 44 varieties of food.
Stability of Vitamin D in Food
Food fortified with vitamin D have been tested and generally the losses are minor if at all even on storage. Vitamin D, when heated to 150 °C in the presence of air is completely destroyed (Chen et al., 1965). Vitamin D in solution is unstable in the presence of calcium carbonate (Liebscher, 1938).
Dried crystalline vitamin D2 and D3 are both sensitive to atmospheric oxygen and will decompose within a few days at ambient temperature. The D3 form is more stable because it has one less double bond. Both vitamins can be destroyed by UV light but are more to irradiation than other vitamins. They are also less stable in acidic pH solutions.
Vitamin D is stable when contained in an oil carrier where oxygen is excluded (Fritz et al., 1942 a & b). It is however susceptible to air oxidation. It is better to store vitamin D in oil solutions and to use it in that format. Adding an antioxidant such as tocopherol helps greatly. Preparations for analysis are stored in brown glass vials with inert gas flushing.
Instability comes from the presence of the double bonds which makes them susceptible to isomerization. The isomerization rates of both vitamins (ergocalciferol and cholecalciferol) is similar. The previtamin form which occurs on heating is seen when cholecalciferol (D3) isomerises between ergocalciferol and precalciferol. There is an equilibrium formed and that ratio is temperature dependent (Ottaway, 2012).
Spray-dried milk, vitamin D fortified UHT-milk (chocolate milk as the example) and cheese are all foods where the vitamin content remains almost unchanged (Hanson & Metzger, 2010).
Jakobsen and Knuthsen (2014) investigated vitamin D in foodstuffs during cooking. More specifically, vitamin D compounds found in margarine and eggs when cooked in an oven for 40 minutes at a typical cooking temperature were the least stable. The retention was 39 to 45%. Frying on the other hand showed a retention of 82 to 84%. The retention of vitamin D in boiled eggs was 86% to 88%.
When fish is cooked in a microwave oven at a temperature between 172C and 200C for 20 minutes the loss of vitamin D3 is less than 10%. Fried salmon was worse than baked salmon with a loss of about 50%. Beef samples lose between 35 and 40% of their vitamin D content during cooking. Slow cooking is more destructive than a rapid cook. One study found an increase in cholecalciferol and 25(OH)-D3 when a longissimus steak was cooked at 71C because of moisture content changes in the meat (Schmid & Walther, 2013).
A great deal of pet food is extruded and then dried into kibble. Unlike other foods, the extruded pet food is wet and then must be extensively dried to stop mould growth. Whilst one of the issues is loss of lysine and of linoleic acid, there is some loss of vitamin D.
In fortified milk, light exposure causes little loss of vitamin D3. It is similarly surprising how little vitamin D is lost when milk passes through a considerable thermal process with spray drying.
References
Association Française de Normalisation (AFNOR) (2009). NF EN 12821, Foodstuffs—determination of vitamin D by high-performance liquid chromatography: measurement of cholecalciferol (D3) and ergocalciferol (D2). Saint Denis, France: AFNOR, May 2009
Baker, A. Z., & Wright, M. D. (1940). Biological assay of vitamin D 3. I. Assay methods at present in use, with particular reference to Olsson’s radiographic technique. Analyst, 65(771), pp. 326-335.
Bilodeau, L., Dufresne, G., Deeks, J., Clément, G., Bertrand, J., Turcotte, S., … & Fouquet, A. (2011). Determination of vitamin D3 and 25-hydroxyvitamin D3 in foodstuffs by HPLC UV-DAD and LC–MS/MS. Journal of Food Composition and Analysis, 24(3), pp. 441-448
Chen Jr, P. S., Terepka, A. R., Lane, K., & Marsh, A. (1965). Studies of the stability and extractability of vitamin D. Analytical Biochemistry, 10(3), pp. 421-434.
De Vines, E. J., and Borsje, B. (1982) J. Assoc. Off. Anal. Chem., 65, pp. 1228
Fritz, J. C., Archer, W. F., & Barker, D. K. (1942a). Observations on the stability of vitamin D. Poultry Science, 21(4), pp. 361-369.
Fritz, J. C, J. L. Halpin, J. H. Hooper, and E. H. Kramke, (1942b). Oxidative destruction of vitamin D. Ind. Eng. Chem. 34 pp. 979-982 .
Geib, T., Sleno, L., Hall RA, Stokes CS, Volmer DA. (2016) Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS / MS Analysis of 25-Hydroxyvitamin D in Human Serum. J Am Soc Mass Spectrom. 27(8) pp. 1404–1410. doi: 10.1007/s13361-016-1412-2
Hanson, A. L., & Metzger, L. E. (2010). Evaluation of increased vitamin D fortification in high-temperature, short-time–processed 2% milk, UHT-processed 2% fat chocolate milk, and low-fat strawberry yogurt. Journal of Dairy Science, 93(2), pp. 801-807
Higashi T, Suzuki M, Hanai J, Inagaki S, Min JZ, Shimada K, et al. A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3. J Sep Sci. 34(7) pp. 725–732. doi: 10.1002/jssc.201000911
Huang, M., LaLuzerne, P., Winters, D., & Sullivan, D. (2009). Measurement of vitamin D in foods and nutritional supplements by liquid chromatography/tandem mass spectrometry. Journal of AOAC International, 92(5), pp. 1327-1335 (Article).
Huang, M., & Winters, D. (2011). Application of ultra-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in foods and nutritional supplements. Journal of AOAC International, 94(1), pp. 211-223
Jackson, P. A., Shelton, C. J., and Frier, P. J. (1982) Analyst, 107, pp. 1363
Jakobsen, J., & Knuthsen, P. (2014). Stability of vitamin D in foodstuffs during cooking. Food Chemistry, 148, pp. 170-175.
Jia, L. P., Tian, S. L., Zheng, X. C., Jiao, P., & Jiang, X. P. (2018). Application of near-infrared spectroscopy in the detection of fat-soluble vitamins in premix feed. In Fourth Seminar on Novel Optoelectronic Detection Technology and Application (Vol. 10697, February pp. 457-464). SPIE.
Kritikos G, Weidner N, Atkinson JL, Bayle J, van Hoek I, Verbrugghe A. (2018) Quantification of vitamin D3 in commercial dog foods and comparison with Association of American Feed Control Officials recommendations and manufacturer-reported concentrations. J Am. Vet Med. Assoc. Jun 15; 252(12) pp. 1521-1526. doi: 10.2460/javma.252.12.1521. PMID: 29889635.
Lehner, A., Johnson, M., Zimmerman, A., Zyskowski, J., & Buchweitz, J. (2021). Vitamin D analyses in veterinary feeds by gas chromatography-tandem mass spectrometry. European Journal of Mass Spectrometry, 27(1), pp. 48-62.
Li, W. (2021) Analysis of Vitamin D3 in animal feed by LC-MS/MS. Presentation january 21, 2021 at the AAFCO Annual Virtual Mtg.
Liebscher, W., 1938. Stability of vitamin D in mixtures containing lime. Ztschr. Tierenahrung Futtermittelk 1 pp. 265-270
Mena-Bravo, A., Priego-Capote, F., & De Castro, M. L. (2016). Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3. Journal of Chromatography A, 1451, pp. 50-57.
Meunier C, Montérémal J, Faure P, et al. Four years of LC-MS/MS method for quantification of 25-hydroxyvitamin D (D2+D3) for clinical practice. J Chromatogr B Analyt Technol Biomed Life Sci 989: pp. 54–61
Ottaway, P.B. (2012) Vitamin D. Chapt. 9. The Technology of Vitamins in Food. Springer Science BV
Panalaks, T. (1971) J. Assoc. Of Anal. Chem., 54, pp. 1299
Parrish, D. B., & Richter, E. F. (1979). Determination of vitamin D in foods: a review. Critical Reviews in Food Science & Nutrition, 12(1), pp. 29-57
Reynolds, S. L., & Judd, H. J. (1984). Rapid procedure for the determination of vitamins A and D in fortified skimmed milk powder using high-performance liquid chromatography. Analyst, 109(4), pp. 489-492
Roth HJ, Schmidt-Gayk H, Weber H, et al. (2008) Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography-tandem mass spectrometry as a reference. Ann Clin Biochem 45: pp. 153–159
Tai, S.S.C., Nelson, M.A. (2015) Candidate Reference Measurement Procedure for the Determination of (24 R ),25-Dihydroxyvitamin D3 in Human Serum Using Isotope- Dilution Liquid Chromatography − Tandem Mass Spectrometry. Anal Chem. 87(15) pp. 7964–7970. doi: 10.1021/acs.analchem.5b01861
Wyness S.P., Straseski J.A. (2015) Performance characteristics of six automated 25-hydroxyvitamin D assays: mind your 3s and 2s. Clin. Biochem. 48 pp. 1089–1096. 2015/08/15
Xu, Z., Yu, K., Zhang, M., Ju, Y., He, J., Jiang, Y., … & Jiang, J. (2024). Accurate Clinical Detection of Vitamin D by Mass Spectrometry: A Review. Critical Reviews in Analytical Chemistry , pp. 1-25
Zelzer, S., Goessler W, Herrmann M. (2018) Measurement of vitamin D metabolites by mass spectrometry, an analytical challenge. J Lab Precis Med. 3: pp. 99–13
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