Exploring the Impact of Part Per Billion Mass Accuracy for Metabolite Identification using Multi Reflecting Time of Flight MS with UPLC Part B

Significance of the Topic

High accuracy mass measurements in the low part-per-billion range combined with ultrahigh resolution mass spectrometry are critical for unambiguous identification of trace metabolites in complex biological matrices. This capability supports drug metabolism studies, environmental monitoring, and omics research by reducing false positives and increasing confidence in small molecule characterization.

Study Objectives and Overview

This study evaluates the impact of routine part-per-billion mass accuracy on non-targeted metabolite identification in human urine samples over a 24-hour period. Using negative electrospray ionization with a SELECT SERIES Multi Reflecting Time-of-Flight mass spectrometer coupled to UPLC, the authors aimed to demonstrate improved detection of pharmaceutical drugs and their metabolites including uncommon biotransformation products.

Methodology and Instrumentation

Urine samples from a healthy volunteer collected at 0, 2, 4, and 6 hours post-dose of acetaminophen, carbamazepine, and naproxen were diluted 1:10 and injected (5 µL) onto an ACQUITY UPLC HSS T3 C18 column at 40 °C. A Waters ACQUITY UPLC I-Class Premier system delivered a 10 Hz UPLC-MSE data independent acquisition. The SELECT SERIES MRT performed ES- detection across m/z 50–2400 with resolving power greater than 200 000 FWHM and routine ppb mass accuracy monitored over 24 hours.

Used Instrumentation

• Waters ACQUITY UPLC I-Class Premier
• ACQUITY UPLC HSS T3 C18 Column (100 mm × 2.1 mm, 1.8 µm)
• SELECT SERIES Multi Reflecting Time-of-Flight Mass Spectrometer
• MassLynx v4.2 and waters_connect 3.1.0 software

Main Results and Discussion

An overall root mean square mass error of 761 ppb was achieved for 1813 metabolite detections. Fine isotope structure and high resolution (> 200 000 FWHM) enabled differentiation of isomeric O-glucuronides and detection of rare conjugates such as desmethyl naproxen sulfate and carbamazepine hydroxy sulfate. MSE fragmentation (15–45 eV) yielded sub-ppm accuracy for precursors and fragments, improving structural elucidation and reducing the number of candidate elemental compositions.

Benefits and Practical Applications

• High confidence identification of small molecules with low false positive rates
• Detection of coeluting isomeric species in complex biological matrices
• Routine ppb mass accuracy at high duty cycle preserves chromatographic fidelity
• Additional specificity through fine isotope profile analysis

Future Trends and Opportunities

Integration of ultrahigh resolution mass spectrometry with advanced informatics and machine learning will further accelerate non-targeted studies. Expanded ionization modes, faster acquisition rates, and multi-omics platforms are expected to improve coverage of low-abundance metabolites and novel biotransformation products.

Conclusion

Routine part-per-billion mass accuracy combined with > 200 000 FWHM resolving power at 10 Hz acquisition provides a robust platform for confident non-targeted metabolite identification. This approach reduces ambiguity in elemental composition assignments and supports comprehensive profiling of small molecules in complex samples.

References

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