Analysis of Flavonoids in Juices with the ACQUITY QDa Mass Detector

Importance of the Topic

Flavonoids are naturally occurring polyphenols prevalent in citrus fruits and valued for their sensory attributes and health benefits. Their diverse structural forms—flavanones and polymethoxylated flavones—serve as markers of product authenticity and quality in the food and beverage industry. Accurate, selective, and high‐throughput analysis of these compounds supports quality control, regulatory compliance, and ensures consistency across citrus‐derived products.

Objectives and Study Overview

This application note evaluates the performance of the Waters ACQUITY UPLC H-Class system coupled with the QDa Mass Detector for rapid quantitation of eight representative flavonoids in citrus juice. The study compares mass detection selectivity and sensitivity against traditional UV/Vis approaches, and demonstrates simplified sample preparation and accelerated chromatographic methods.

Methodology

A reversed‐phase UPLC method was developed using an ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm) at 35 °C. Mobile phases comprised 0.1% formic acid in water (A) and acetonitrile (B). A 13-minute gradient (98–2% A to 0–100% A) at 0.6 mL/min enabled baseline‐equivalent analysis times four times shorter than conventional methods. Sample injections of 2 µL were conducted at 4 °C. Flavonoid detection employed selective ion recording (SIR) in positive electrospray ionization mode (ESI+), targeting specific monoisotopic m/z values for each analyte.

Instrumentation

• ACQUITY UPLC H-Class System
• ACQUITY UPLC PDA Detector
• ACQUITY QDa Mass Detector (ESI+ SIR mode)
• MassLynx MS Software

Main Results and Discussion

SIR chromatograms demonstrated clear separation and selective detection of eight flavonoids—including narirutin, diosmin, hesperidin, didymin, sinensetin, nobiletin, tetramethoxyscutellarein, and tangeretin—even when chromatographic peaks partially overlapped. The QDa detector resolved coeluting nobiletin and tetramethoxyscutellarein peaks by monitoring m/z 403.2 and 343.1, respectively—a challenge for UV/PDA detection. Recovery studies (n=10) in citrus juice showed accuracy between 82% and 139% and repeatability RSD ≤ 8%, matching or exceeding existing QC methods.

Benefits and Practical Applications

• Improved selectivity over UV/Vis or PDA detection, reducing reliance on full baseline separation.
• Accelerated analysis cycles (13 minutes total runtime) enhance sample throughput.
• Simplified sample preparation using a single dilution ratio across concentration ranges.
• Cost-effective mass detection suitable for QC laboratories and manufacturing environments.

Future Trends and Opportunities

Advances in compact mass detectors like QDa will broaden routine adoption of MS-based assays in quality control. Integration with automated sample handling and data processing will further increase throughput. Emerging ion mobility and high-resolution MS technologies may supplement SIR to resolve isobaric flavonoids. Expanding target analyte libraries and method multiplexing can support comprehensive profiling of plant polyphenols and complex matrices.

Conclusion

The Waters ACQUITY UPLC H-Class system with QDa Mass Detector offers a robust, selective, and rapid platform for routine flavonoid analysis in citrus juice. By combining streamlined chromatographic conditions with targeted mass detection, the method achieves high accuracy and precision while substantially increasing laboratory productivity and cost efficiency.

References

1. Lee HS, Widmer BW. Phenolic compounds. In: Nollet LML, ed. Handbook of Food Analysis. Marcel Dekker; 1996:821–894.
2. Gattuso G, Barreca D, Gargiulli C, Leuzzi U, Caristi C. Flavonoid composition of citrus juices. Molecules. 2007;12:1641–1673.
3. AOAC Official Method 999.05. Naringin and Neohesperidin in Orange Juice. Official Methods of Analysis. AOAC International.
4. Widmer W. Determination of Naringin and Neohesperidin in Orange Juice by Liquid Chromatography with UV Detection to Detect the Presence of Grapefruit Juice: Collaborative Study. J AOAC Int. 2000;83(5):1155.