Accurate Mass Analysis of Flavonoids by LCMS-IT-TOF

Significance of the Topic

Flavonoids are widespread polyphenolic compounds in fruits, vegetables, grains and seeds, valued for their antioxidative, hormone-modulating and pigment properties. Precise structural elucidation of these minor bioactive molecules is essential for developing functional foods, nutraceuticals and pharmaceuticals, yet traditional isolation and purification demand substantial time and resources.

Study Objectives and Overview

This work demonstrates the use of a hybrid ion-trap time-of-flight mass spectrometer (LCMS-IT-TOF) for accurate mass and multistage MSn analysis of flavonoids in a mandarin orange methanol extract. The goals were to identify flavonoid glycosides and aglycones directly in a complex matrix, assess mass accuracy stability with external calibration, and predict structural details without full isolation.

Methodology and Instrumentation

A methanol extract of mandarin orange peel was analyzed using Shimadzu LCMS-IT-TOF equipped with Ballistic Ion Extraction and an internal temperature adjustment mechanism to ensure long-term mass accuracy.

• Column: Phenomenex Gemini C18 (2.0 × 150 mm, 5 µm)
• Mobile phase: 0.1% acetic acid in water (A) and acetonitrile (B), gradient from 0% to 100% B over 90 min
• Flow rate: 0.2 mL/min, injection volume: 0.2 µL, column temperature: 40 °C
• Ionization: ESI positive mode, probe voltage +4.5 kV, CDL and block heater at 200 °C
• Calibration: External standard method achieving mass errors within ±5 ppm

Main Results and Discussion

A total of eleven flavonoid peaks were resolved by HPLC–MS, with each compound assigned based on exact mass and MSn fragmentation patterns. Table 1 summarizes retention times, molecular formulas and mass accuracy for all identified flavonoids.

• Peak 6 (hesperidin): [M+H]+ at m/z 611.1945 (–5.1 ppm) yielding fragment ions at m/z 449 and 303 corresponding to sequential loss of rutinoside and glucose moieties.
• Peak 8 (dehydroxyhesperidin): m/z 595.2024 (–0.5 ppm) reflected loss of one hydroxyl group; MS/MS confirmed a C16H13O5 aglycone fragment.
• Peak 4 (rutin): although nominally isobaric with hesperidin, the [M+H]+ at m/z 611.1603 (–1.5 ppm) and unique fragmentation fingerprint supported its identification as quercetin‐3‐O‐rutinoside.

These examples illustrate how mass shifts of 16 Da reveal dehydroxylation, while MS3 spectra permit assignment of monosaccharide versus disaccharide components.

Benefits and Practical Applications of the Method

The LCMS-IT-TOF approach offers:

• Rapid, high-confidence flavonoid profiling in complex samples without extensive purification.
• Sub-5 ppm mass accuracy enabling unambiguous molecular formula assignments.
• Multistage MSn for detailed glycoside linkage and aglycone structure determination.

Future Trends and Potential Applications

Ongoing developments may include:

• Application to other plant metabolites, alkaloids and phenolic classes in food and environmental matrices.
• Coupling with data-driven workflows and spectral libraries for automated high-throughput screening.
• Advances in ion-transport and thermal management to further improve mass stability and sensitivity.

Conclusion

The Shimadzu LCMS-IT-TOF system enables comprehensive, accurate mass and MSn analysis of flavonoids directly in crude extracts. By combining external calibration stability with automated multistage fragmentation, this platform streamlines structural characterization of glycosides and aglycones, accelerating research and quality control in food, pharmaceutical and natural product chemistry.

References

• Shimadzu Corporation Application News, “Accurate Mass Analysis of Flavonoids by LCMS-IT-TOF,” No. C51