SEPARATION OF ISOBARIC AMINO ACIDS AND SMALL MOLECULE METABOLITES USING MULTIPASS ION MOBILITY ANALYSIS

Importance of the topic

Amino acids and small molecule metabolites often exist as isobaric or isomeric forms whose distinct biological activities demand analytical techniques capable of high-resolution separation. Traditional chromatography and mass spectrometry struggle to resolve such species, motivating the use of ion mobility spectrometry (IMS) for structural separation based on size, shape and charge.

Study objectives and overview

This work aimed to demonstrate enhanced separation of isobaric amino acids and small metabolites using a multipass cyclic IMS approach. By circulating ions through a racetrack geometry multiple times, the effective IMS resolution increases, enabling discrimination of challenging isomeric pairs.

Methodology and Instrumentation

Standard solutions and mixtures were directly infused into a Waters SELECT SERIES Cyclic IMS system. Both positive and negative ionization modes were screened, and various adducts (e.g., sodium acetate) were used to optimize ion mobility behavior. Separation conditions were tailored for each analyte set by varying the number of passes around the IMS racetrack.

Main results and discussion

• Leucine and isoleucine achieved baseline separation after 15 passes (resolution 252 Ω/ΔΩ), compared to 40 Ω/ΔΩ in a single-pass SYNAPT G2 system.
• D-Valine was resolved from 3-aminopentanoic acid after 23 passes (res. 312 Ω/ΔΩ).
• α-Aminoisobutyric acid and α-aminobutyric acid separated after 12 passes (res. 225 Ω/ΔΩ).
• Alanine and β-alanine showed partial separation in one pass, complete after 25 passes (res. 300 Ω/ΔΩ).
• Glucose-6-phosphate, glucose-1-phosphate and fructose-6-phosphate required 10 passes for full resolution (res. 205 Ω/ΔΩ).
• Lipid isomers GalCer and GlcCer separated after 20 passes (res. 290 Ω/ΔΩ).
• Adenosine protomers displayed two conformers resolved fully within three passes (res. 113 Ω/ΔΩ).

Benefits and practical applications of the method

The multipass cyclic IMS technique offers substantial resolution gains with minimal sample preparation, facilitating the identification and quantification of isomeric biomarkers in omics studies, pharmaceutical QA/QC and metabolomics workflows.

Future trends and potential applications

• Calculation of collision cross sections for species separated in multiple passes.
• Development of IMSn by selective peak slicing, reinjection and further separation to study interconversion phenomena.
• Integration with high-throughput omics platforms for comprehensive isomer profiling.

Conclusion

Multipass cyclic IMS provides a robust, high-resolution solution for separating isobaric and isomeric amino acids, metabolites and lipids. Its scalable resolution enhancement and compatibility with existing MS systems make it a powerful tool for advanced analytical challenges.

References

1. Campuzano I, Giles K, Neeson K, Richardson K. Waters Application Note 720003028 EN.
2. Isaac G, Olivos H, Plumb R. Waters Application Note 720007539 EN.