Unravelling the polyphenolic content of berry juices using focusing-modulated comprehensive two-dimensional liquid chromatography

Significance of the topic

Polyphenols in berries are recognized as potent antioxidants and bioactive agents with health‐promoting effects, including anti‐inflammatory, anti‐carcinogenic, and neuroprotective properties. Reliable profiling of these compounds in commercial juices supports quality control, authenticity verification, and optimization of functional food and nutraceutical products.

Aims and study overview

This study developed and applied a novel two‐dimensional liquid chromatography platform combining hydrophilic interaction (HILIC) in the first dimension and reversed‐phase (C18) chromatography in the second dimension, with focusing modulation, to comprehensively profile polyphenolic compounds in seven commercially available berry juices (bilberry, blackcurrant, blueberry, chokeberry, elderberry, honeyberry, and raspberry). A total of 104 polyphenols across different classes were characterized and quantified.

Methodology and instrumentation

The method employed HILIC (150×1.0 mm) as 1D and core‐shell C18 (50×4.6 mm) as 2D columns, connected via high‐pressure switching valves and trapping columns for peak focusing. A Shimadzu Nexera X2 LC×LC system with PDA detection (190–550 nm) and an LCMS-8050 ESI‐MS provided spectral identification and quantification. Mobile phases included 0.1% formic acid in acetonitrile and water, operated under segmented‐in‐fraction gradients to optimize orthogonality and peak capacity.

Main results and discussion

The optimized HILIC×RP‐LC method achieved corrected peak capacities up to 1372 and orthogonality values around 0.8. Validation metrics showed linearity (R2>0.993), low LODs (0.02–0.90 mg/L), LOQs below 1 mg/L (except quercetin), precision (intra‐ and inter‐day RSD<1.3%), and recoveries of 84–119%. Elderberry juice exhibited the highest total polyphenolic content (917.8 mg/100 mL), followed by chokeberry (515.7 mg/100 mL) and honeyberry (439.5 mg/100 mL); raspberry had the lowest (103.6 mg/100 mL). Flavonol glycosides dominated in elderberry and honeyberry, while hydroxycinnamic acids were most abundant in blueberry and chokeberry.

Benefits and practical applications

By providing extensive separation and reliable quantification, the method serves multiple applications:

• Quality control and standardization of berry‐based products in pharmaceutical and food industries
• Authentication and detection of adulteration
• Support for nutritional labeling and regulatory compliance
• Data for clinical and nutritional studies on health benefits of polyphenols

Future trends and applications

Advancements may include integration with high‐resolution mass spectrometry for deeper structural elucidation, extension to other complex food matrices, and miniaturized or multiplexed 2D LC configurations. Emerging stationary phases and automated data processing will further increase throughput and data richness for metabolomic and quality assurance workflows.

Conclusion

The presented HILIC×RP‐LC‐PDA‐ESI‐MS approach delivers high separation efficiency, broad polyphenolic coverage, and robust quantitative performance for commercial berry juices. This method can be advantageously applied to ensure product quality, study bioactive profiles, and guide the development of functional food and nutraceutical formulations.

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