THE IMPACT of PPB MASS ACCURACY UPON BIOTRANSFORMATION PRODUCT IDENTIFICATION USING NEGATIVE ION NON -TARGETED URINARY SCREENING MULTI-REFLECTING TIME-OF-FLIGHT LCMS

Importance of Topic

High-resolution mass spectrometry is essential for non-targeted screening of drug metabolites in complex biological fluids. Achieving part-per-billion mass accuracy and high resolving power enables reliable identification of known and unknown biotransformation products. Such capability supports drug development, therapeutic monitoring, forensic toxicology and quality control by minimizing false positives and enhancing confidence in analyte assignment.

Study Objectives and Overview

This work evaluates the impact of routine ppb mass accuracy on the identification of urinary drug metabolites using negative-ion LC–MS non-targeted screening. A healthy volunteer received acetaminophen, carbamazepine and naproxen; urine was sampled over six hours. The goal was to demonstrate how mass accuracy and fine isotope structure (FIS) improve confidence in detecting phase I and II metabolites under a data-independent acquisition (DIA) MSE workflow.

Methodology

Sample Preparation and Chromatography

• Human urine diluted 1:10 in water
• Analysis time points: pre-dose, 2, 4 and 6 hours post-dose
• Reverse-phase separation: C18 column (100×2.1 mm, 1.8 µm) at 40 °C; 12 min gradient; 0.5 mL/min; 0.1% formic acid in water/acetonitrile
• Injection volume: 5 µL

Mass Spectrometry

• Multi-Reflecting Time-of-Flight (MRT) mass spectrometer operating in ES− mode
• System resolving power >200 000 FWHM across 10 Hz acquisition duty cycle
• DIA MSE acquisition acquiring precursor and fragment ions simultaneously over full mass range

Data Analysis

• MassLynx™ and waters_connect™ for data processing
• TIBCO Spotfire® for visualization and fine isotope structure assessment

Instrumention Used

SELECT SERIES™ hybrid quadrupole Multi-Reflecting Time-of-Flight mass spectrometer (Waters Corporation) with ES− electrospray interface. Software: MassLynx 3.1, waters_connect, Tibco Spotfire 6.0.

Main Results and Discussion

Chromatographic Fidelity and Resolution

• Analysis of carbamazepine-N-glucuronide showed 19 data points over a 3.6 s peak and mass resolution of 189 000 at m/z 411

Mass Accuracy Performance

• Observed RMS mass errors of ~754 ppb for precursor and fragment ions over 24 h
• Stable accuracy across major and minor metabolites

Metabolite Identification

• Detection of naproxen sulfate at tr 4.75 min with precursor error of 21 ppb and fragment ions at 143 and 171 m/z
• Identification of carbamazepine-O-sulfate with characteristic A+2 fine isotope structure and ppb-level errors for precursor (68 ppb) and fragments (216 and 68 ppb)
• Acetaminophen sulfate characterized by enhanced FIS in REM mode with system resolution >300 000 FWHM

Benefits and Practical Applications

• PPB mass accuracy and high resolution improve analyte selectivity in complex matrices
• Fine isotope structure adds confirmatory evidence for elemental composition
• Non-targeted DIA workflow retains chromatographic peak fidelity at 10 Hz
• Enhanced informatics processing reduces false detection rates and accelerates data review

Future Trends and Potential Applications

Integration of high-resolution screening with machine-learning algorithms can further automate metabolite annotation. Advancements in ion mobility and hybrid fragmentation techniques may enhance structural elucidation. Expansion to large-scale clinical and environmental studies will leverage improved mass accuracy to uncover low-abundance biomarkers.

Conclusion

Routine sub-ppm mass accuracy combined with high resolving power and fine isotope structure analysis significantly elevates confidence in non-targeted urinary screening of drug biotransformation products. The demonstrated workflow supports comprehensive metabolite coverage without sacrificing chromatographic performance, paving the way for more stringent analytical protocols.

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