A Novel Strategy to Screen and Profile Steviol Glycosides of Natural Sweeteners in Food Using the ionKey/MS System’s Ion Mobility Mass Spectrometry

Importance of the Topic

Natural steviol glycosides derived from Stevia rebaudiana serve as high-potency, calorie-free sweeteners in foods and beverages worldwide. Their structural diversity, regulatory purity requirements (≥95% of seven defined glycosides), and low dosage levels in complex matrices underscore the need for highly selective and sensitive analytical methods to authenticate origin, detect isomeric variants, and ensure product safety and quality.

Objectives and Study Overview

The study presents a novel screening strategy combining microfluidic chromatography and ion mobility mass spectrometry to profile eleven steviol glycosides and aglycone in a challenging food matrix (chocolate spread). Key goals included:

• Rapid, low-level detection of target glycosides in complex extracts.
• Resolution of coeluting isomeric pairs (e.g. stevioside vs. rebaudioside B; rebaudioside A vs. E; rubusoside vs. steviolbioside).
• Establishment of collision cross section (CCS) values as an additional identification dimension.

Methodology and Instrumentation

Sample Preparation:

• Fat removal by liquid–liquid extraction followed by C18 SPE cleanup.
• Final extract dissolved in acetonitrile at 0.1 g/mL matrix load.
• Post-cleanup spiking at levels down to 1 pg/µL for fortification studies.

Chromatography and Ion Mobility:

• ACQUITY UPLC M-Class System with iKey BEH C18 PCA Separation Device (150 µm×50 mm, 1.7 µm) at 40 °C.
• Flow rate 2 µL/min, water/acetonitrile with 0.1% formic acid gradient.
• ionKey/MS System microfluidic ESI source coupled to SYNAPT G2-Si Q-ToF with Traveling Wave IMS using nitrogen drift gas.

Data Acquisition and Processing:

• Full-scan MS (50–1200 m/z) at 20,000 FWHM, lock-mass correction with leucine enkephalin.
• Collision energy ramp 30–70 eV for fragmentation.
• UNIFI Scientific Information System for component screening using accurate mass, retention time, fragmentation, and experimentally derived TW CCS_N2 library.

Main Results and Discussion

TW CCS_N2 values were determined for all analytes (e.g. rebaudioside A 298.9 Ų, stevioside 269.6 Ų, rebaudioside D 321.8 Ų). Mass accuracy at ≤100 pg on-column achieved RMS error <2 ppm and CCS error <0.4%. At trace levels (≤1 pg/µL), all targets were screened with a mass tolerance of 10 ppm and CCS tolerance of 2%, enabling confident identification down to ~680 fg despite matrix interferences.

Ion mobility resolved coeluting isobaric species such as stevioside and rebaudioside B at m/z 803.37, delivering drift-time aligned precursor and fragment spectra. This spectral cleanup minimized false positives and enhanced specificity in complex food extracts.

Benefits and Practical Applications

Key advantages of the ionKey/MS-IMS approach include:

• Enhanced sensitivity via reduced dead volume microfluidics.
• Additional separation dimension from CCS to distinguish isomers and source fragments.
• Lower solvent and standard consumption through microflow operation.
• Streamlined routine screening with integrated UNIFI workflows.

Applications extend to authenticity testing, purity verification, and regulatory compliance in functional foods, beverages, and nutraceutical quality control.

Future Trends and Possible Applications

Advances may include expanded CCS libraries for broader natural product classes, real-time multivariate screening combining CCS and MS/MS libraries, and integration into high-throughput quality assurance pipelines. Miniaturized interfaces and emerging ion mobility technologies promise further sensitivity gains and reduced analysis cost.

Conclusion

The combination of microfluidic LC, high-resolution MS, and ion mobility collision cross section measurement provides a robust, sensitive, and selective platform for screening steviol glycosides in complex food matrices. This strategy effectively resolves isomeric pairs at trace levels, reduces false positives, and supports regulatory and quality control demands while minimizing resource usage.

References

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