New LC-MS/MS Method Tracks Four Artificial Sweeteners in Dairy Foods with Unprecedented Precision

Artificial sweeteners have quietly become one of the most pervasive ingredients in the modern food supply, hiding in everything from diet yogurts to lactic acid bacteria drinks. Now, a team of researchers in South Korea has developed and rigorously validated an analytical method capable of simultaneously measuring four of the most widely used sugar substitutes in dairy products, and their findings offer both reassurance and a few surprises about what is actually inside the yogurt cups and milk cartons lining supermarket shelves.

The study, published in Food Science of Animal Resources, focused on acesulfame potassium, sodium saccharin, sucralose, and aspartame, four high-intensity sweeteners approved for use in Korea. Each is hundreds of times sweeter than table sugar, which means only tiny amounts are needed to deliver sweetness with little or no caloric load. But that very potency creates a regulatory challenge: because the effective doses are so small, food safety agencies need analytical tools sensitive enough to detect them at vanishingly low concentrations before they can verify that manufacturers are staying within legal limits.

The researchers turned to liquid chromatography coupled with tandem mass spectrometry, or LC-MS/MS, a technique that has become the gold standard for quantifying multiple compounds in complex food matrices. The method works by first separating the chemical components of a sample as they travel through a chromatography column, then fragmenting the molecules of interest and detecting them based on their characteristic mass-to-charge ratios. In this study, the team used a Thermo Scientific Vanquish UHPLC system coupled to a TSQ Quantis mass spectrometer, with chromatographic separation achieved on a C18 column using a mobile phase of ammonium acetate and acetic acid in water and methanol. Each analytical run took just 11 minutes, a significant improvement in throughput over earlier methods that sacrificed no accuracy in exchange for the speed.

One of the trickiest aspects of analyzing dairy products is that milk proteins and fats can interfere with the detection of target compounds, a phenomenon known as the matrix effect. To combat this, the researchers optimized a deproteinization step using two classic reagents: potassium ferrocyanide trihydrate and zinc sulfate heptahydrate, which together precipitate proteins out of solution. Through a systematic experiment involving ten different reagent volume combinations applied to a protein-rich thick fermented milk matrix spiked with known amounts of each sweetener, they identified the sweet spot: 0.6 milliliters of each reagent. Statistical analysis using one-way ANOVA followed by Duncan’s multiple range test confirmed that this combination maximized recovery rates for all four sweeteners simultaneously.

The validation results were striking. Calibration curves showed linearity with coefficients of determination of 0.9998 or better for all four sweeteners, far exceeding the US Food and Drug Administration’s minimum criterion of 0.995. Limits of detection ranged from just 0.002 milligrams per liter for acesulfame potassium to 0.103 milligrams per liter for sucralose, while limits of quantification spanned 0.007 to 0.312 milligrams per liter. Accuracy, measured through recovery experiments at low, medium, and high spike concentrations across all four dairy matrices, fell between 84.40 and 103.97 percent, and precision remained within a relative standard deviation of 6.67 percent. All of these figures comfortably satisfied the validation criteria set by both AOAC International and the International Council for Harmonisation.

Matrix effects, the perennial bogeyman of LC-based food analysis, proved remarkably benign in this method. Following European Commission SANTE guidelines, the team compared analyte responses in matrix-matched standards against solvent-only standards across fermented milk, processed milk, thick fermented milk, and lactic acid bacteria beverages. All four sweeteners showed matrix effects within plus or minus 10 percent, meaning the dairy matrix barely disturbed the signal at all. This allowed the researchers to use simple solvent-based calibration curves rather than more laborious matrix-matched calibration, streamlining routine analysis without compromising reliability.

To demonstrate that the method would hold up outside a single laboratory, the team conducted an inter-laboratory validation between two independent facilities. Thick fermented milk and lactic acid bacteria beverage samples were spiked with all four sweeteners at three concentration levels and analyzed in triplicate at both sites. Recovery rates ranged from 90.89 to 102.74 percent, and relative standard deviations stayed below 7.78 percent, both well within AOAC acceptance criteria for analytes in the 0.1 to 10 milligram per kilogram range. The researchers also calculated measurement uncertainty, accounting for contributions from stock solutions, sample preparation, calibration, and repeatability, and found expanded uncertainties between 2.74 and 10.97 percent across all matrices, comfortably below the Codex Alimentarius threshold of 22 percent.

With the method fully validated, the researchers applied it to 40 dairy products purchased from Korean retail stores, supermarkets, convenience stores, and online platforms between June and September 2024. The results painted a nuanced picture of sweetener use across the industry. Acesulfame potassium appeared exclusively in processed milk, detected in five of ten samples at concentrations between 120.82 and 161.76 milligrams per kilogram, well below the Korean Food Code limit of 0.50 grams per kilogram. Sucralose was the most ubiquitous, found in 88 percent of all samples across every category, with concentrations ranging from 14.53 to 172.96 milligrams per kilogram, all safely under the 0.40 grams per kilogram ceiling. Sodium saccharin, which is entirely prohibited in these products, was not detected in any sample, affirming regulatory compliance.

Aspartame told a more interesting story. It was found only in fermented milk, where five of ten samples tested positive at concentrations between 65.08 and 269.42 milligrams per kilogram. Because Korean regulations impose no specific numeric limit on aspartame, requiring only that it be used at the minimum level necessary to achieve its intended technical effect, these findings underscore the importance of continued surveillance. The researchers note that recent studies have raised questions about potential health implications of artificial sweetener consumption, including metabolic disturbances, alterations to gut microbiota, and possible links to cardiovascular disease, making reliable monitoring tools all the more essential for both regulatory compliance and health risk assessment.

The broader significance of this work lies in its completeness. Many analytical studies validate a method on paper but stop short of demonstrating real-world applicability or cross-laboratory reproducibility. By combining optimized sample preparation, thorough single-laboratory validation, formal uncertainty analysis, inter-laboratory confirmation, and a market survey of 40 commercial products, this study delivers a turnkey solution for food safety laboratories. As demand for reduced-sugar dairy products continues to grow globally, regulators and manufacturers alike now have a faster, more sensitive, and more robust tool for keeping the sweeteners in our yogurt and milk exactly where the law says they should be.

Subject of Research: Validation of an LC-MS/MS method for quantifying four artificial sweeteners in dairy-based foods and beverages

Article Title: Quantitative analysis, validation, and application of an LC-MS/MS method for four artificial sweeteners in dairy-based foods and beverages

Article References: Kim, K.-W., Kim, D., Kang, J., Yun, C.-I., & Kim, Y.-J. (2026). Quantitative analysis, validation, and application of an LC-MS/MS method for four artificial sweeteners in dairy-based foods and beverages. Food Science of Animal Resources, 46(1), Article 48. https://doi.org/10.1007/s44463-025-00010-3

Image Credits: AI Generated

DOI: 10.1007/s44463-025-00010-3

Keywords: artificial sweeteners, LC-MS/MS, dairy products, food safety, acesulfame potassium, sucralose, aspartame, sodium saccharin, method validation, matrix effects, Korean Food Code, mass spectrometry