To create a technique suited for analyzing proanthocyanidins and their properties, scientists combined high-speed countercurrent chromatography (HSCCC) and electrospray ionization–tandem mass spectrometry (ESI–MS/MS).
Researchers from Leibniz University, Hannover (Hannover, Germany) and Technische Universität Braunschweig (Braunschweig, Germany) developed a new means of profiling dimeric procyanidins (PCs). Their findings were published in the Journal of Chromatography A (1).
Proanthocyanidins are polyphenolic compounds that are known in the nutraceutical field due to potential health benefits (2). These compounds are also present in agro-industrial waste due to their occurrence in the fruit pods, seeds, seed shells, and bark. In 2022, approximately 1.05 billion tons of food waste was discarded across the world, from which bioactive compounds could be extracted. A-type proanthocyanidins are produced by oxidative conversion of B‑type procyanidins, such as B1 and B2, induced by oxidative processes during food processing, or by direct transformation via enzymes or radical agents, such as 2,2‑diphenyl‑1‑picryl-hydrazyl (DPPH) radicals.
In this study, the researchers investigated reaction by‑products using high‑speed countercurrent chromatography (HSCCC), followed by sequential off-line flow‑injection electrospray ionization–tandem mass spectrometry (ESI–MS/MS) profiling. The study applied selected single ion-traces to monitor targeted PCs by off‑line injection profiling using ESI‑MS/MS with recovered HSCCC fractions. This approach enabled the rapid identification of main structures, clear detection of co‑elution effects, and identification of 19 metabolites. Specifically, off‑line flow injection ESI‑MS/MS profiling and the coupling of HSCCC with LC-ESI‑MS/MS (HSCCC x LC‑ESI‑MS) were applied to fractionate metabolites from a semi‑synthetic approach using agro‑industrial waste.
High-speed countercurrent chromatography (HSCCC) is an all-liquid separation technique that involves a system of immiscible solvents without using a solid chromatographic phase. This technique can offer various advantages, including low solvent consumption, high repeatability, high recovery, and high solute loading capability (3). This technique has been used for separating ingredients from natural products and other substances, especially for obtaining phenolic fractions or individual phenolics.
In this experiment, the scientists demonstrated the DPPH radical oxidation of a mixture of dimeric A-type procyanidins B2 and B4. These substances are obtained through semi-synthetic acid‑catalyzed depolymerization of Salix alba polymer after (–)‑epicatechin addition. Under reaction conditions of 50.0 °C for 185 min and a molar ratio of 9/31.25 (n/n) of B‑type to DPPH radicals, various reaction products, such as A‑type PCs, spirocyclisation products (m/z 575 [M−H]−), (bis)methylated B‑types and oxidised PCs (m/z 591, 581 and 605 [M−H]−), as well as further oxidized products (m/z 573 [M−H]−) were detected.
HSCCC x LC‑PDA monitoring at λ= 280 nm revealed oligomeric and polymeric compounds being present, formed through intermolecular nucleophilic additions. The HSCCC separation in combination with an off‑line ESI‑MS/MS profiling and a 2D‑plot of HSCCC fractions versus LC‑ESI‑MS/LC‑PDA was successfully applied for the semi‑synthetic reaction approach of radical‑induced oxidation of dimeric B‑type PCs. This optimized solvent system can be applied for further scale-up processes in pre-purification studies of oxidized PCs. Overall, this study demonstrated a new method for isolating the poorly investigated oxidation products of B-type PCs from a complex reaction mixture; this approach could be used for future biological studies investigating the health‑promoting properties of this substance class.
(1) Fischer, A.; Gök, R.; Jerz, G.; Esatbeyoglu, T. Profiling of B-type Procyanidins Oxidation Products by Off-Line High Speed Countercurrent Chromatography x Electrospray Ionisation Mass Spectrometry Flow-Injection. J. Chromatogr. A 2025, 1757, 466111. DOI:
(2) Mannino, G.; Chinigò, G.; Serio, G.; et al. Proanthocyanidins and Where to Find Them: A Meta-Analytic Approach to Investigate Their Chemistry, Biosynthesis, Distribution, and Effect on Human Health. Antioxidants 2021, 10 (8), 1229. DOI:
(3) Li, L.; Zhao, J.; Yang, T.; Sun, B. High-Speed Countercurrent Chromatography as an Efficient Technique for Large Separation of Plant Polyphenols: A Review. Food Res. Int. 2022, 153, 110956. DOI:
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