Electron Impact and Chemical Ionization High Resolution Time-of-Flight Mass Spectrometry Analyses of Blood Plasma Samples

Importance of the Topic

Metabolomics plays a central role in modern biomedical research and clinical diagnostics by profiling small molecules that reflect physiological and pathological processes. High resolution time-of-flight mass spectrometry with complementary ionization techniques enhances metabolite coverage and structural confidence in complex biofluids such as blood plasma.

Objectives and Study Overview

This study aimed to develop a rapid, comprehensive profiling workflow for lean, fatty, and obese Zucker rat plasma samples. Over 700 derivatized metabolites were analyzed using both electron impact and chemical ionization high resolution TOF mass spectrometry to evaluate throughput, identification accuracy, and biological trends.

Methodology and Instrumentation

• Sample Preparation: Protein precipitation with methanol, centrifugation, lyophilization, followed by derivatization with MSTFA and addition of fatty acid methyl ester standards.
• Gas Chromatography: Agilent 7890 GC system with Restek Rxi-5Sil MS capillary column; temperature program from 70°C to 300°C; helium carrier gas at constant flow.
• Mass Spectrometry: LECO Pegasus GC-HRT operated in high resolution mode (R 25 000) with EI source at 250°C and CI source at 200°C; spectral acquisition at 6 spectra per second; m/z ranges 60–520 for EI and 180–1400 for CI; internal calibration with PFTBA.
• Data Processing: ChromaTOF-HRT for deconvolution, alignment, and library matching; exported to third-party software for statistical analysis.

Main Results and Discussion

• EI-HRT chromatograms revealed diverse metabolite classes including amino acids, organic acids, sugars, fatty acids, and sterols with high quality deconvoluted spectra.
• Mass accuracy averaged better than 1 ppm, enabling robust elemental formula assignments for molecular and fragment ions.
• CI-HRT provided molecular ion information for compounds lacking EI molecular ions, such as serine, galactose, glucose, and inositol, and corrected misassignments from library searches.
• The combined workflow allowed repeated interrogation of archived data sets and efficient handling of coeluting peaks.
• Comparative trends across lean, fatty, and obese rat plasma highlighted metabolic shifts, for example a decrease in 2-hydroxybutanoic acid and an increase in octadecadienoic acid derivatives.

Benefits and Practical Applications

• Single-platform analysis with dual ionization enhances metabolome coverage and confidence in metabolite identification.
• Fast GC method and automated deconvolution support high throughput sample processing.
• Accurate mass data allow discrimination of isobaric and coeluting compounds.
• Seamless data export enables follow-up statistical and bioinformatics analyses.

Future Trends and Potential Applications

Integration with advanced bioinformatics and multivariate statistics will expand the interpretive power of metabolomic profiles. Ongoing work may include refinement of derivatization strategies, integration with liquid chromatography high resolution MS, and application to translational biomarker discovery and real-time metabolic flux studies.

Conclusion

The combined electron impact and chemical ionization high resolution TOF MS workflow provides a robust and high throughput strategy for comprehensive plasma metabolome profiling. Accurate mass measurements, effective deconvolution, and complementary ionization significantly enhance confidence in metabolite identification and enable detailed comparative studies of physiological states.