Measuring Vitamin D3 in Hen's Egg Yolk with HPLC

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Researchers have developed a method employing high performance liquid chromatography with ultraviolet detection (HPLC-UV) to determine vitamin D3 in food (even in the presence of vitamin D2, and with a specific focus on egg yolk) in a cost-effective and quantitative manner.

A team of researchers from the University of Perugia and the University of Bologna (both in Italy) developed a high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the quantitative analysis of vitamin D3 in egg yolk samples, even with the presence of vitamin D2 derived from the animal's diet. A paper based on this research was published in the Journal of Separation Science (1).

A key advantage of this method is its ability to clearly resolve vitamin D2 from vitamin D3, the scientists wrote, ensuring a satisfactory chromatographic peak separation. Designed to enable the facile quantification of both vitamin D2 and vitamin D3 after the saponification and extraction steps (while avoiding preliminary chromatography-based purification procedures), the approach not only streamlines the analytical workflow but also improves the reliability of the quantification process.

Vitamin D is primarily recognized for its role in maintaining calcium and phosphorus homeostasis (2). However, it is also connected to other health benefits such as preventing cardiovascular disease, rheumatoid arthritis, and diabetes (3-7). While vitamin D can be synthesized internally, dietary intake is critical to sustain suitable levels, thus highlighting its important role in human physiology (1). Vitamin D occurs in two principal forms in nature—ergocalciferol (commonly referred to as vitamin D2, which comes from plant-sourced foods) and cholecalciferol (commonly referred to as vitamin D3, which comes from animal-sourced foods) (8).

The analytical detection and quantification of vitamin D2 and vitamin D3 in hen eggs present significant challenges. For example, the very low concentrations of these compounds (a few micrograms per 100 g of food matrix) require a sensitive method of detection to navigate the presence of other abundant compounds, including fats, proteins, and other fat-soluble vitamins. Coupling of high-performance liquid chromatography (HPLC) to tandem mass spectrometry (HPLC-MS/MS) is frequently reported in the literature and is considered the most appropriate technique for accurately quantifying vitamin D in complex food matrices and is frequently reported in previous analysis (9,10). However, the high costs associated with HPLC-MS/MS systems, as well as the significant technical skill necessary, continue to obstruct routine application of that method in laboratories that perform analysis related to food control (1).

The authors of the study report that their method was validated following a research-based protocol, demonstrating satisfactory linearity, precision, accuracy, LOQ, and robustness. The method was then successfully applied to the analysis of real egg yolk samples. Moreover, the authors state that this method can easily be adapted for use with HPLC-MS systems, which allows for additional sensitivity enhancements, making the method suitable for a wide range of laboratory settings, and providing a robust and efficient analytical tool for assessing vitamin D levels in food matrices, particularly in egg yolks (1).

Broken egg with yolk in the shell. © TATIANA - stock.adobe.com

Broken egg with yolk in the shell. © TATIANA - stock.adobe.com

References

1. Varfaj, I.; Mancinelli, A. C.; Migni, A.; Mercolini, L.; Castellini, C.; Galli, F.; Bartolini, D.; Sardella, R. A Cost-Effective Nonaqueous Reversed-Phase High-Performance Liquid Chromatography Method to Measure Vitamin D3 in Hen's Egg Yolk. J. Sep. Sci. 2025, 48 (1), e70087. DOI: 10.1002/jssc.70087

2. Balachandar, R.; Pullakhandam, R.; Kulkarni, B.; Sachdev, H. S. Relative Efficacy of Vitamin D2 and Vitamin D3 in Improving Vitamin D Status: Systematic Review and Meta-Analysis. Nutrients 2021, 13 (10), 3328. DOI: 10.3390/nu13103328

3. Wang, H.; Chen, W.; Li, D.; Yin, X.; Zhang, X.; Olsen,N.; Zheng, S. G. Vitamin D and Chronic Diseases. Aging Dis. 2017, 8 (3), 346-353. DOI: 10.14336/AD.2016.1021

4. Athanassiou, L.; Mavragani, C. P.; Koutsilieris, M. The Immunomodulatory Properties of Vitamin D. Mediterr. J. Rheumatol. 2022, 33 (1), 7-13. DOI: 10.31138/mjr.33.1.7

5. Jeon, S. M.; Shin, E. A. (2018). Exploring Vitamin D Metabolism and Function in Cancer. Exp. Mol. Med. 2018, 50 (4), 1-14. DOI: 10.1038/s12276-018-0038-9

6. Bikle, D. D. Extraskeletal Actions of Vitamin D. Ann N Y Acad Sci. 2016, 1376 (1), 29-52. DOI: 10.1111/nyas.13219

7. Holick M. F. Sunlight and Vitamin D for Bone Health and Prevention of Autoimmune Diseases, Cancers, and Cardiovascular Disease. Am. J. Clin. Nutr. 2004, 80 (6s), 1678S-1688S. DOI: 10.1093/ajcn/80.6.1678S

8. Vitamin D2 vs. D3: What’s the Difference? Healthline website.https://www.healthline.com/nutrition/vitamin-d2-vs-d3 (accessed 2024-01-24)

9. Usoltseva, L.; Ioutsi, V.; Panov, Y.; Antsupova, M.; Rozhinskaya, L.; Melnichenko, G.; Mokrysheva, N. Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization. Int. J. Mol. Sci. 2023, 24 (9), 8111. DOI: 10.3390/ijms24098111

10. Yin, S.; Yang, Y.; Wu, L.; Li, Y.; Sun, C. Recent Advances in Sample Preparation and Analysis Methods for Vitamin D and its Analogues in Different Matrices. TrAC, Trends Anal. Chem. 2019, 110, 204-220. DOI: 10.1016/j.trac.2018.11.008

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