ANALYSIS OF PHYTOCHEMICALS IN PROCESSED COCOA USING ION MOBILITY MASS SPECTROMETRY

Significance of the Topic

Cocoa beans are rich in bioactive phytochemicals associated with cardiovascular and anticancer benefits. Monitoring these compounds in processed cocoa products is essential for assessing nutritional value, ensuring quality control, and understanding the impact of manufacturing steps such as alkalization, roasting, and fermentation.

Objectives and Study Overview

• Apply non-targeted liquid chromatography–ion mobility–mass spectrometry (LC-IM-MS) to 67 commercial cocoa powders.
• Characterize processing effects on phytochemical composition through principal component analysis.
• Differentiate isomeric and conformational species using accurate mass, retention time, and collision cross section (CCS) values.

Methodology and Instrumentation

Samples were extracted with 70:30 ethanol–water, centrifuged, diluted, and analyzed in triplicate. Reverse-phase LC used a C18 column (1.7 µm, 2.1×100 mm) with a 17-minute gradient of 0.1% formic acid in water and acetonitrile at 0.75 mL/min and 45 °C. Electrospray ion mobility MS data (R~40) were acquired in positive and negative modes on a Waters Synapt G2-Si, recording TWCCSN2 fingerprints.

• Extraction: 5 min shake, centrifugation at 6 000 rpm, 1:10 dilution with methanol/0.1% formic acid.
• Chromatography: 10 µL injection, reverse-phase gradient.
• IM-MS: ESI+, ESI–, CCS measurement via travelling wave ion mobility.

Main Results and Discussion

Key methylxanthines (caffeine, theobromine, theophylline) matched reference TWCCSN2 values. Flavan-3-ol oligomers (procyanidin dimers, trimers, tetramers) and flavonoids (epicatechin, catechin, quercetin, vitexin) were identified by unique CCS fingerprints and retention times. Charge-bearing conformers (sodimers, potassimers) displayed distinct ATDs. Principal component analysis of CCS, retention time, and intensity data differentiated alkalized, organic, and other cocoa powder types, indicating significant processing-related shifts.

Benefits and Practical Applications

• High specificity for routine phytochemical profiling of cocoa powders.
• Rapid separation and identification of isomeric and conformational species without extensive MS/MS.
• Robust quality control and product authentication using CCS fingerprints.
• Evaluation of processing effects on nutritional and bioactive composition.

Future Trends and Applications

High-resolution cyclic ion mobility will enhance separation of unresolved conformers and complex oligomers. Expansion of CCS libraries in dual-polarity modes and integration with advanced chemometrics will support broader applications in food authentication, nutritional assessment, and industrial quality assurance across diverse botanicals.

Conclusion

Combining accurate mass, retention time, and CCS via LC-IM-MS provides a powerful, non-targeted approach for detailed profiling of cocoa phytochemicals. The method effectively resolves isomeric and conformational species, reveals processing influences, and advances analytical capabilities for food research and quality control.

Reference

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