Proton NMR and HPLC Combine to Enhance Citrus Juice Authentication

In a recent study conducted by a team of researchers from SGF International and Geisenheim University in Germany, a potential strategy for assessing the quality of citrus juices was tested. This new approach, which involved using proton nuclear magnetic resonance (¹H NMR) spectroscopy and high-performance liquid chromatography with diode array detection (HPLC-DAD), was demonstrated to deliver more robust juice authentication protocols (1). This study was published in Food Chemistry and led by Markus Jungen and Christof B. Steingass (1).

Currently, the food and beverage analysis industry are seeking to find new ways to improve the quality control of citrus juices before they reach market (2,3). Citrus juices are popular consumer beverages, and they range from orange and grapefruit to lime and lemon. One of the biggest challenges is that these beverages are susceptible to mislabeling and adulteration (1).

Glasses of juice and citrus fruits | Image Credit: © Olena Rudo - stock.adobe.com

Traditional testing methods have focused on single techniques that may not capture the full scope of a juice’s chemical profile. In this study, the new dual-method approach allows for a comprehensive chemical characterization that supports both species-specific identification and the discovery of novel authenticity markers (1).

In their research, Jungen and Steingass, along with their team, applied targeted ¹H NMR spectroscopy to determine the concentrations of major juice constituents such as sugars, organic acids, and selected amino acids. These parameters were studied because they are crucial not only for identifying the type of juice, but they also assess freshness and processing quality (1). The resulting NMR data was benchmarked against existing industrial databases and reference guidelines, ensuring consistency and industry relevance.

NMR was not the only technique used in this study. The research team also used HPLC-DAD to measure minor but informative compounds, including phenolic substances, coumarins, and psoralens. These chemical groups, often overlooked in single-method analyses, play a significant role in juice differentiation (1). For instance, certain coumarins and psoralens can be species-specific, making them ideal candidates for verifying juice origins or identifying adulteration (1).

Combining ¹H NMR data with HPLC analysis of phenolic components was key to this study. It allowed the researchers to overcome limitations posed by considering singular data sets, especially in difficult cases such as differentiating lemon and lime juices (1).

The research team also employed both univariate and multivariate data analysis techniques, including principal component analysis (PCA), to explore the distinct chemical signatures of orange, grapefruit, lemon, and lime juices. Using PCA allowed the researchers to visualize how each juice variety clustered based on its compositional data, making it easier to spot inconsistencies or potential fraudulent blends (1).

The study not only confirmed several established authenticity markers, but it also proposed new minor compounds that could serve as future indicators. These findings are expected to improve the accuracy and reliability of juice differentiation, which is essential for protecting consumers and maintaining fair trade in the global juice market (1).

Future studies should include juices from other geographical origins, the researchers suggested in their conclusion. By doing so, researchers can compile a larger data set, which would allow their method to be adjusted further to improve it (1)

The study also proposes that future research could incorporate supervised multivariate classification methods, such as partial least squares discriminant analysis (PLS-DA), to refine marker selection and further validate the methodology (1).

Ultimately, this research underscores the value of integrative analytical techniques in modern food authentication. Because the research team was able to successfully integrate the strengths of ¹H NMR spectroscopy and HPLC-DAD together, Jungen and Steingass offer a new potential solution to properly and accurately evaluate citrus juice authenticity and quality (1).

References

1. Jungen, M.; Gilcher, C.; Patz, C.; et al. Phenolic Compounds and Quantitative ¹H NMR Spectroscopy for Authentication of Lemon, Lime, Orange, and Grapefruit Juices. Food Chem. 2025, ASAP. DOI: 10.1016/j.foodchem.2025.144379
2. Clark, C. J. Fast Determination by Fourier-transform Infrared Spectroscopy of Sugar–Acid Composition of Citrus Juices for Determination of Industry Maturity Standards. New Zealand J. Crop Hort. Sci. 2016, 44 (1), 69–82. DOI: 10.1080/01140671.2015.1131725
3. Brendel, R.; Schwolow, S.; Rohn, S.; Weller, P. Volatilomic Profiling of Citrus Juices by Dual-Detection HS-GC-MS-IMS and Machine Learning—An Alternative Authentication Approach. J. Agric. Food Chem. 2021, 69 (5), 1727–1738. DOI: 10.1021/acs.jafc.0c07447