New Study Reviews Chromatography Methods for Flavonoid Analysis

A study published in the Journal of Chromatography Open, led by Ahmet Cetinkaya, a researcher of the Department of Analytical Chemistry at Ankara University, reviewed different chromatographic approaches for analyzing flavonoids in fruit (1).

Fresh fruits mixed.Tasty fruits background. | Image Credit: © travelbook - stock.adobe.com

In this review, the scientists discussed flavonoid analysis using various chromatographic methods, with details on studies in the literature for determining flavonoids in fruits. There is great diversity among flavonoid subclasses and their matrices, meaning there are no optimal sample pretreatment or analytical method for identifying them in their complex matrices. What pre-treatment is applied and what analytical methods are used depends on the type and quantity of target flavonoids, in addition to how complex a matrix is. Scientists are constantly developing, modifying, and applying novel sample pretreatments and analytical procedures to achieve simpler, faster, more environmentally friendly, and less expensive analysis methods. This has led to the creation of more rational and relevant methodologies. Further, selecting the appropriate method for chromatographically analyzing flavonoids depends on the purpose of the analysis, the desired level of sensitivity, and the type of fruit being studied.

Flavonoids are secondary metabolites responsible for plant properties such as color, aroma, and flavor. They typically consist of two benzene rings (A and B) and a pyrene ring (C) connecting these rings. They carry out important functions in plants, such as attracting pollinating insects; combating environmental stresses, such as microbial infection; and regulating cell growth (2). These substances have a wide range of biological activities, such as antioxidants, anti-inflammatory, anticancer, cardioprotective, and neuroprotective.

Due to these properties, flavonoids are widely used in the food, cosmetic, and pharmaceutical industries. Chromatographic analytical methods are the most commonly used methods for detecting, separating, and quantifying flavonoids in fruit species. Methods like high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), and liquid chromatography-tandem mass spectrometry (LC-MS) are crucial for analyzing flavonoids. Recently, hybrid systems combining MS and ultraviolet (UV) detectors have been developed to increase the techniques’ sensitivities. HPLC–MS techniques are among the preferred methods for determining flavonoids.

Flavonoid profiles between different fruit species were compared using these techniques. Though GC is less widely used, it uses derivatization steps to analyze non-volatile flavonoids. The accuracy and sensitivity may be limited compared to other methods. TLC, a simpler and more economical method, is still preferred for separating flavonoids. However, it is less widely used than HPLC due to its limited quantitative analysis capacity. There have been technological developments in chromatographic methods of analysis. Ultra-high-performance liquid chromatography (UHPLC) offers shorter analysis times and higher resolution, especially for separating complex flavonoid mixtures.

Research in these areas have significantly contributed to determining flavonoids and investigating their potential health benefits. Techniques like HPLC, GC, and TLC and their modern adaptations with MS and UV detectors have enabled researchers to achieve high levels of sensitivity and accuracy. With these methods, scientists have been able to identify and quantify flavonoids in different fruit species and obtain important information on their bioavailability and metabolic processes. Adding advanced tools, such as UHPLC, has enabled high-throughput analyses of flavonoid mixtures by increasing resolution and reducing analysis times. As such, chromatographic techniques remain an indispensable tool for flavonoid research.

As analytical techniques improve, deeper understandings of the bioactive compounds’ roles in human health will emerge. This will open up new avenues of application in areas like functional foods, dietary supplements, and therapeutic agents. According to the scientists, future perspectives should focus on developing sensitive and environmentally friendly analytical methods, in addition to the fastest and most selective extraction of target flavonoids from plants. 3D printer technology has been pitched as a new method for creating new sorbent supports, extraction sorbent containers, or interfaces that can help reduce the size of extraction devices and enable the coupling of selected analytical techniques, mainly flow analytical techniques.

References

(1) Cetinkaya, A.; Yayla, S.; Hurkul, M. M.; Ozkan, S. A. Comprehensive Review on Chromatographic Analysis of Flavonoids in Fruits. J. Chromatogr. Open 2025, 7, 100209. DOI: 10.1016/j.jcoa.2025.100209

(2) Flavonoids. Oregon State University 2016. https://lpi.oregonstate.edu/mic/dietary-factors/phytochemicals/flavonoids (accessed 2025-4-21)