ionkey/MS - APPLICATION COMPENDIUM

Importance of the Topic

Steviol glycosides from Stevia rebaudiana are widely used natural sweeteners. Their purity, isomer composition, and potential co-extracted impurities directly affect flavor and safety. Conventional analytical methods often lack the specificity to resolve co-eluting isomers at trace levels in complex food matrices.

Objectives and Study Overview

This work presents a novel screening and profiling strategy for steviol glycosides in food. The goal was to detect and differentiate all major steviol glycoside isomers—including pairs of structural isomers—at low picogram levels in processed food products, using the combined selectivity of microflow LC separation, ion mobility CCS measurements, and high-resolution mass spectrometry.

Methodology and Instrumentation

• Sample cleanup involved fat removal by liquid-liquid extraction and C18 SPE to produce extracts at 10 mg/mL of chocolate spread.
• Separation was performed on an ionKey™ BEH C18 PCA iKey Separation Device (150 μm × 50 mm, 1.7 μm) integrated into the ionKey/MS™ System.
• The SYNAPT G2-Si HDMS™ was operated in ESI-negative mode with traveling-wave IMS, acquiring CCS N₂ values.
• Data were processed in UNIFI® Scientific Information System, utilizing a custom CCS library of steviol glycosides for screening and confirmation.

Main Results and Discussion

• TW CCS N₂ values were determined for eleven steviol compounds (e.g., stevioside 269.64 Ų vs. rebaudioside B 261.19 Ų) at concentrations down to 100 pg/μL.
• Detection of stevioside isomer pairs (rubusoside/steviolbioside; rebaudioside B/stevioside; rebaudioside A/E; rebaudioside C/D) was unambiguous via CCS differentiation, even when co-eluting chromatographically.
• Mass measurement accuracy was maintained within 2 ppm RMS; CCS errors were <0.4% across all levels down to 1 pg/μL.
• At ≤1 pg/μL, rubusoside was detected with a signal-to-noise sufficient for confident assignment (680 fg on-column), where standard mass-only screening would risk false negatives.

Benefits and Practical Applications

• The ionKey/MS System with IMS provides high-throughput, sensitive screening of steviol glycosides at trace levels in complex foods.
• CCS filtering dramatically reduces false positives from in-source fragments and matrix interferences.
• Microflow operation reduces solvent usage by >150-fold compared to 2.1 mm methods.
• Rapid authentication of natural vs. semi-synthetic or adulterated sweeteners, supporting regulatory compliance with JECFA purity standards.

Future Trends and Potential Uses

• Expansion of CCS libraries to cover additional glycosides and plant metabolites for comprehensive natural product profiling.
• Integration with untargeted HRMS workflows for discovery of novel sweetener compounds or adulterants.
• Application in other food authenticity and contamination screening assays where high specificity is required.

Conclusion

The combination of integrated microflow LC separation, ion mobility CCS measurements, and high-resolution MS on the ionKey/MS System enables selective, sensitive profiling of steviol glycoside isomers at sub-pg levels in complex food matrices. This approach offers a robust, high-throughput solution for food authenticity, purity assessment, and regulatory compliance.

Reference

• World Health Organization. Joint FAO/WHO Expert Committee on Food Additives (JECFA). Evaluation of Steviol Glycosides. WHO Technical Report Series; 2010.
• Geuns JMC, Cornelis BO, Devos L, Augustijns P, Salaün JP. Phytochemistry. 2003;62(3):467–479.
• McCullagh M, Douce D, Rao R, Goscinny S. A Novel Strategy to Screen and Profile Steviol Glycosides of Natural Sweeteners in Food Using ionKey/MS Ion Mobility Mass Spectrometry. Waters Application Note; 2015.720005211EN.