A Routine Separation Approach to Enhance Specificity and Identification in Authentication Profiling of Natural Food Products Using UPLC-IM-MS

Significance of the Topic

The authentication and profiling of flavonoid isomers in natural food products is critical for ensuring quality, safety and consistent efficacy in phytomedicines and functional foods. Flavonoid glycosides such as vitexin, isovitexin, orientin and isoorientin occur as structural isomers in complex botanical extracts. Conventional chromatographic and mass spectrometric methods often struggle to resolve these isomers, leading to ambiguous identification and potential substitution of active species. The integration of UPLC with ion mobility mass spectrometry (IM-MS) and collision cross section (CCS) measurements presents a powerful approach to overcome these challenges and enhance specificity in routine authentication workflows.

Objectives and Study Overview

This study aimed to develop a robust, non‐targeted screening workflow using UPLC‐IM‐MS and travelling wave CCS (TW CCS N2) to:

• Uniquely differentiate and identify 6‐C and 8‐C flavonoid glycoside isomers in Passiflora species extracts.
• Create a reference library of accurate CCS values for marker flavonoids.
• Demonstrate routine screening of complex herbal extracts (P. incarnata, P. edulis, P. caerulea, P. alata) for authentication profiling.

Methodology and Instrumentation

Sample preparation involved hydroethanolic extraction (2:1 v/v), SPE cleanup on RP‐18 cartridges and dilution to appropriate concentrations. UPLC‐IM‐MS conditions included:

• ACQUITY UPLC I‐Class with BEH C18 column (100×2.1 mm, 1.7 µm) at 45 °C.
• Gradient elution with 0.1% formic acid in water and acetonitrile, flow 0.75 mL/min, 2 µL injection.
• SYNAPT G2‐Si with ESI in negative mode, capillary 2.3 kV, desolvation 600 °C.
• Ion mobility parameters: T‐Wave velocity 1000→300 m/s, pulse height 40 V, nitrogen gas 90 mL/min.
• Data acquired in HDMSE mode (50–1200 m/z, 10 spectra/s, collision energy ramp 35–75 eV).

Main Results and Discussion

TW CCS N2 values measured for marker isomers matched library standards with errors <0.5%. Key findings included:

1. Distinct CCS values for 6‐C vs. 8‐C flavonoid glycosides: orientin (187.64 Å2) vs. isoorientin (197.68 Å2), vitexin (188.47 Å2) vs. isovitexin (195.03 Å2).
2. Reliable mass accuracy <5 ppm across four Passiflora species.
3. Ion mobility separation resolved coeluting isomers and isobaric background compounds, enabling acquisition of ‘clean’ collision‐induced product ion spectra.
4. Unique fragment ion ratios in HDMSE allowed unambiguous differentiation of isomer pairs (e.g. m/z 284/285 and 297–299 ratios for orientin/isoorientin; m/z 281–284 for vitexin/isovitexin).
5. TW CCS screening identified flavonoid markers in >10 000 analytes per acquisition.

Benefits and Practical Applications

The combined UPLC‐IM‐MS approach delivers:

• High specificity in isomer identification regardless of retention time shifts.
• Increased peak capacity through orthogonal separation dimensions.
• Reduced spectral complexity and improved confidence in marker detection.
• Capability for routine QA/QC and authentication of botanical and food products.

Future Trends and Potential Applications

Advancements expected in the field include:

• Expansion of global CCS databases and integration with informatics platforms.
• Coupling of ion mobility with novel separation techniques (e.g. multidimensional LC).
• Real‐time process monitoring and inline authentication in industrial workflows.
• Application of machine learning to predict CCS and accelerate compound annotation.

Conclusion

The use of UPLC‐IM‐MS with TW CCS measurements and HDMSE fragmentation provides a robust, high‐throughput strategy for the unambiguous identification of flavonoid isomers in complex natural extracts. This workflow enhances specificity beyond conventional LC‐MS approaches and supports rigorous authentication profiling in food, pharmaceutical and botanical industries.

References

• McCullagh M, Douce D, Pereira CAM, Yariwake JH. Routine UPLC‐IM‐MS screening for flavonoid glycoside isomers in Passiflora. Waters Application Note; 2015.
• Zucolotto SM, Fagundes C, Reginatto FH, et al. Analysis of C‐glycosyl flavonoids from South American Passiflora by HPLC‐DAD and HPLC‐MS. Phytochem Anal. 2012;23:232–239.
• Pereira CAM, Yariwake JH, McCullagh M. Distinction of C‐glycosylflavone isomers using LC‐MS exact mass and in-source CID. Phytochem Anal. 2005;16:295–301.
• March RE, Lewars EG, Stadey CJ, Miao X, Zhao X, Metcalfe CD. Comparison of flavonoid glycosides by electrospray tandem MS. Int J Mass Spectrom. 2006;248:61–85.