Quantitation of acetylcholinesterase inhibitors

Importance of the Topic

This application note addresses the quantitation of acetylcholinesterase inhibitors—neostigmine, pyridostigmine, and edrophonium—in human plasma. These compounds play a crucial role in treating neuromuscular disorders such as myasthenia gravis and are also of interest as potential nerve agent analogs. Achieving sensitive, reliable measurement at sub-nanogram levels is essential for clinical research, pharmacokinetics, and therapeutic monitoring.

Goals and Study Overview

The primary objectives of this study were to:

• Develop a UHPLC-MS/MS method with sensitivity from 0.050 to 50 ng/mL for target inhibitors.
• Employ a high-capacity SOLA WCX 30 mg SPE phase to minimize matrix effects and maximize analyte recovery.
• Demonstrate robust chromatographic separation and peak shape using the Accucore Polar Premium column.

Methodology and Used Instrumentation

Sample preparation relied on mixed-mode weak cation exchange SPE using Thermo Scientific™ SOLA™ WCX 30 mg cartridges. Plasma samples were diluted, conditioned, loaded, washed, and eluted under precisely controlled pH conditions to capture positively charged analytes and selectively remove matrix components.

Used Instrumentation:

• Vanquish™ Horizon UHPLC System with binary pump, split-sampler, heated column compartment, and active pre-heater.
• Accucore™ Polar Premium column (100 × 2.1 mm, 2.6 μm).
• TSQ Altis™ Triple Quadrupole Mass Spectrometer with H-ESI source.
• HyperSep™ 24-port glass vacuum manifold for SPE processing.

Chromatographic conditions included a 3.25-min gradient from aqueous ammonium formate/formic acid to methanol-based mobile phase, 30 °C column temperature, and 1.5 µL injections. MS/MS detection used SRM transitions for quantitation and confirmation ions with optimized collision energies.

Results and Discussion

The Accucore Polar Premium column delivered sharp peaks and baseline separation for all analytes. Calibration curves were linear (R2 ≥ 0.994) over four orders of magnitude with 1/response2 weighting. The method achieved a lower limit of quantitation of 50 pg/mL.

Precision and accuracy metrics across low, mid, and high QC levels met acceptance criteria (bias ±15%, CV <8.3%). Matrix effects were minimal (normalized factors ~1.05–1.09), demonstrating effective removal of endogenous interferences. Absolute recoveries ranged from 91% to 105% for all compounds, reflecting the high selectivity of the SOLA WCX phase.

Benefits and Practical Applications

This workflow offers several advantages:

• Exceptional sensitivity for trace-level quantitation in clinical and pharmacokinetic studies.
• High throughput with a 96-well SPE format and rapid 3.25-min chromatographic cycle.
• Reproducible extraction and minimal sample failure due to fritless SOLA cartridge architecture.
• Versatility for expanding to other cationic drug classes or biomarker panels.

Future Trends and Potential Applications

Advancements may include:

• Integration with automated SPE platforms to further increase throughput.
• Application to therapeutic drug monitoring in personalized medicine.
• Extension to metabolite profiling or toxicological screening of additional neuromodulators.
• Adoption of higher-load SPE formats or novel mixed-mode phases to accommodate larger sample volumes or more complex matrices.

Conclusion

By combining SOLA WCX mixed-mode SPE with UHPLC-MS/MS on an Accucore Polar Premium column, the method achieves sensitive, accurate, and reproducible quantitation of key acetylcholinesterase inhibitors in plasma. The streamlined workflow supports clinical research and QA/QC applications requiring trace-level detection and robust performance.