DATA INDEPENDENT ANALYSIS EVOLUTION: EXPLORING THE USE OF HIGH RESOLVING POWER MULTI- REFLECTING TIME-of-FLIGHT MASS SPECTROMETRY FOR METABOLITE IDENTIFICATION

Significance of the Topic

The identification of small molecule metabolites in complex biological matrices is critical for drug development and safety assessment. Data independent acquisition combined with high resolving power mass spectrometry enhances analyte selectivity and confidence in identification.

Study Objectives and Overview

The study aimed to evaluate the performance of a high resolving power hybrid quadrupole Multi Reflecting Time-of-Flight mass spectrometer operating at over 200000 FWHM for non-targeted urinary screening of therapeutic drugs and metabolites. Time course samples were collected at 0, 2, 4 and 6 hours post dose to assess mass accuracy stability and identification capability.

Methodology

The analysis employed a data independent acquisition workflow (LCMSE ES positive) at 10 Hz frequency. Urine samples diluted 1 to 10 were separated by reverse phase liquid chromatography on a C18 column using a 12 minute gradient with 0.1 percent formic acid in water and acetonitrile at 0.5 mL per minute. A 5 microliter injection volume was used. Data processing and visualization were performed with MassLynx, waters_connect and Tibco Spotfire software.

Instrumentation

• Hybrid quadrupole Multi Reflecting Time-of-Flight mass spectrometer operating above 200000 FWHM
• Resolution Enhanced Mode achieving over 300000 FWHM for fine isotope structure analysis
• Reverse phase UPLC system with C18 column (100 mm x 2.1 mm, 1.8 micron)

Results and Discussion

Chromatographic peak fidelity was maintained at high acquisition speed allowing accurate profiling of parent drugs and conjugated metabolites. Mass accuracy remained at 549 ppb RMS over 24 hours facilitating confident identification. Seventy five percent of precursor measurements showed errors below 500 ppb and ninety five percent were within 1 ppm. Application of combined precursor and fragment mass tolerance filters reduced false detection rates in metabolite workflows. Fine isotope structure provided an additional layer of confirmation for known and unknown conjugates.

Benefits and Practical Applications

• Routine achievement of part per billion mass accuracy enhances non-targeted screening workflows
• Improved specificity reduces false positives and streamlines retrospective analysis
• Fine isotope structure information aids structural confirmation of metabolites

Future Trends and Opportunities

Advances in multi reflecting time-of-flight design and operation at higher resolving power may enable sub-part per billion accuracy. Integration of fine isotope structure data into automated informatics pipelines will broaden applications across metabolomics and pharmacokinetics. Retrospective analysis capabilities can be expanded by leveraging archived high resolution datasets.

Conclusion

High resolving power multi reflecting time-of-flight mass spectrometry with data independent acquisition delivers stable part per billion mass accuracy over extended periods, enabling confident and efficient metabolite identification in complex matrices. Fine isotope structure and enhanced resolution modes further strengthen structural elucidation and reduce false detection rates.

Reference

1 Cooper Shepherd D Wildgoose J Kozlov B Johnson WJ Tyldesley Worster R Palmer M Hoyes J McCullagh M Jones E Tonge R Marsden Edwards E Nixon P Verenchikov A Langridge J Novel Hybrid Quadrupole Multireflecting Time of Flight Mass Spectrometry System Journal of the American Society for Mass Spectrometry 2023 34 2 264 272
2 McCullagh M Palmer M Eatough D Marsden Edwards E Langridge J Cooper Shepherd D Exploring the Impact of Part Per Billion Mass Accuracy for Metabolite Identification using Multi Reflecting Time of Flight MS with UPLC Part A 720007896 April 2023