QuEChERS and UHPLC–MS/MS Used to Analyze Vitamins in Yogurt

Scientists from the Korea Food Research Institute in Jeollabuk-do, Republic of Korea led a study where Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction was used alongside ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) to analyze vitamins D and K in yogurt. Their findings were published in the Journal of Chromatography A (1).

Bowl of sour cream or yogurt | Image Credit: © Mara Zemgaliete - stock.adobe.com

Yogurt is often viewed as a valuable source of macronutrients, such as proteins and micronutrients, including calcium, potassium, and phosphorus. When consumed in moderation, fermented foods like yogurt can deliver probiotic bacteria to the human gastrointestinal tract, adding health benefits beyond nutrition. However, despite these benefits, yogurt is often considered a relatively deficient source of fat-soluble vitamins, especially vitamins D and K.

These vitamins are central to maintaining calcium homeostasis and regulating bone metabolism. Low vitamin D can reduce intestinal calcium absorption, leading to osteoporosis and osteomalacia; further, this condition is a risk factor for different chronic diseases, such as diabetes, cancer, and cardiovascular diseases. Meanwhile, vitamin K helps the skeleton by acting as a cofactor in the post-translational carboxylation of several bone proteins. Various studies have examined these vitamins’ effect on bone metabolism, and when combined with calcium supplementation, vitamins D and K increased the bone mineral density and bone mineral content in humans. However, doses of these vitamins as presented in supplements cannot be obtained through natural dietary sources. As such, food fortification can be feasible for eradicating, or at least controlling, vitamin D and K deficiencies.

In this study, a novel approach was developed for simultaneously determining the contents of vitamins D₂, D₃, K₁, and K2 in yogurt fortified with nanoencapsulated vitamins D and K. This method combines QuEChERS extraction with UHPLC–MS/MS. QuEChERS effectively combines extraction and purification steps, greatly reducing solvent consumption and simplifying operations, while UHPLC–MS is a simple and accurate method. Moreover, this process’s flexibility allows for satisfactory results being guaranteed in a simple and inexpensive manner over a short time via parameter optimization.

In this study, QuEChERS optimization involved fine-tuning salt addition using response surface methodology based on the Box–Behnken design (an independent quadratic design in that it does not contain an embedded factorial or fractional factorial design) (2). Under optimized conditions, the developed method exhibited excellent linearity (R² > 0.999) across concentrations ranging from 0.5 to 500 µg/L. Limits of detection and quantification (LOD and LOQ) were found to range from 0.01–0.04 µg/L and 0.04–0.11 µg/L, respectively. Precision, accuracy, and recovery rates exceeded 94.88% and were accompanied by acceptable relative standard deviations (RSDs).

Comparative analysis with traditional methodologies showed the significant advantages of the proposed approach. Previous techniques, such as liquid-liquid extraction combined with saponification (a hydrolysis reaction where free hydroxide breaks the ester bonds between the fatty acids and glycerol of a triglyceride, resulting in free fatty acids and glycerol), are time-consuming and require high sample quantities (3). Further, dispersive liquid-liquid microextraction requires long analysis times and exhibits poor sensitivity, particularly with their LOD and LOQ values. In contrast, the sample preparation technique in this experiment proves more straightforward, efficient, and reliable than its counterparts. With these findings, QuEChERS seems feasible for stable vitamin quantification in yogurt, all while representing an innovative contribution to enhancing detection sensitivity and efficiency in food analysis.

References

(1) Kim, T-E.; Lee, M. H.; Kim, B-K.; Lee, J. H.; Chun, Y. G.; Jang, H. W. Optimization of the QuEChERS-UPLC-APCI-MS/MS Method for the Analysis of Vitamins D and K Nanoencapsulated in Yogurt. J. Chromatogr. A 2024, 1734, 465275. DOI: 10.1016/j.chroma.2024.465275

(2) Box-Behnken Designs. National Institute of Standards and Technology 2024. https://www.itl.nist.gov/di\v898/handbook/pri/section3/pri3362.htm (accessed 2024-8-26)

(3) Saponification. Elsevier B.V. 2024. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/saponification (accessed 2024-8-26)