Using the Routine Separation Dimension and Identification Criteria of ionKey/MS Ion Mobility to Enhance Specificity in Screening Complex Samples

Importance of the topic

Effective screening and authentication of flavonoid markers in complex botanical extracts are essential for ensuring the quality, safety, and regulatory compliance of functional foods and dietary supplements. Combining microflow chromatography with ion mobility mass spectrometry addresses challenges in specificity and sensitivity, enabling unambiguous identification of isomeric compounds at trace levels.

Objectives and overview of the study

This work evaluates the performance of the ionKey/MS System coupled to a SYNAPT G2-Si ion mobility mass spectrometer for the rapid profiling of flavonoid glycoside isomers in Passiflora species extracts. Key aims include demonstrating enhanced sensitivity over standard UPLC methods, validating linear response across femtogram concentrations, and achieving routine, collision cross section (CCS)-based identification of coeluting isomers.

Methodology

Microflow separations were performed on an ACQUITY UPLC M-Class System using 150 µm × 50 mm iKey BEH C18 devices in standard and post-column addition (PCA) formats. A water/acetonitrile gradient (0.1% formic acid) at 2 µL/min enabled both positive and negative electrospray without voltage changes. Passiflora edulis, alata, caerulea, and incarnata extracts were diluted up to 400:1, with calibration standards prepared from 0.1 to 10 pg/µL. Ion mobility separations used nitrogen-based travelling wave parameters, generating CCS values for marker flavonoids and supporting orthogonal discrimination of isomeric species.

Used Instrumentation

• ACQUITY UPLC M-Class System with microflow pump
• ionKey/MS System integrating iKey BEH C18 separation devices and microfluidic ESI emitter
• SYNAPT G2-Si high-resolution mass spectrometer with ion mobility (HDMSE) capability
• UNIFI Scientific Information System for CCS library management and data processing

Key results and discussion

Sensitivity tests for orientin and vitexin demonstrated linear responses (0.1–10 pg/µL) with correlation coefficients (r2>0.99) in both ion modes. MS-only methods achieved 100 fg/µL detection limits for marker flavonoids. Ion mobility resolved coeluting isomers (6-C and 8-C glycosides) by drift time differences and CCS measurements within 0.5% of library values. For the first time, coeluting vitexin and isovitexin were individually quantified using mobility-based precursor selection, overcoming limitations of mass accuracy alone.

Benefits and practical applications

• Enhanced selectivity through an added gas-phase separation dimension
• Reduced solvent and sample consumption via microflow operation
• Lower reliance on retention time by employing CCS as a robust identification parameter
• Capability to quantify isomeric analytes in complex matrices without additional standards

Future trends and applications

Expanding ion mobility CCS libraries and integrating machine learning for drift time prediction will further streamline compound identification. Broader adoption of microflow IM-MS may drive cost-effective quality control workflows in food, pharmaceutical, and environmental laboratories. Advances in gas-phase separations may also enable real-time reaction monitoring and high-throughput screening of natural products.

Conclusion

The combined ionKey/MS microflow platform with ion mobility MS offers a powerful approach for sensitive, specific, and reproducible profiling of flavonoid isomers in complex samples. The ability to resolve and quantify coeluting compounds using CCS enhances confidence in authenticity testing while reducing operational costs and reliance on high-purity reference standards.

References

• The use of collision cross section measurements (CCS) in food and environmental analysis. Waters Technical Note 720005374en, 2015.
• M. McCullagh et al. Using the routine separation dimension and identification criteria of UPLC ion mobility to enhance specificity in profiling complex samples. ASMS Poster PSTR134803078, 2014.