LC-MS/MS Analysis of Edrophonium, Neostigmine, and Pyridostigmine in Plasma Using HILIC Chromatography and Weak Cation-Exchange SPE

Importance of the Topic

Rapid and reliable quantification of acetylcholinesterase inhibitors in plasma is critical for therapeutic drug monitoring in conditions such as myasthenia gravis.

Study Objectives and Overview

This work aims to develop a fast, sensitive LC-MS/MS assay to quantify edrophonium, neostigmine, and pyridostigmine in human plasma using mixed-mode SPE and HILIC separation. The focus is on achieving robust extraction, minimal matrix effects, and high throughput with cycle times under 3 minutes.

Methodology and Instrumentation

Sample preparation utilized Thermo Scientific SOLA WCX weak cation-exchange solid phase extraction to selectively retain quaternary ammonium compounds. Extraction steps included conditioning with acidified methanol, sample loading, washing with phosphate buffer and methanol, and elution with formic acid in methanol. Extracts were dried and reconstituted in acetonitrile/ammonium formate (80:20 v/v, pH 3.3).
Separation was performed on a Syncronis HILIC UHPLC column (1.7 μm, 100×2.1 mm) at 25 °C using acetonitrile/ammonium formate (90:10 v/v, pH 3.3) at 0.5 mL/min.
Detection employed a TSQ Vantage triple quadrupole MS with HESI source in positive mode and fast SRM transitions optimized for each analyte.

Main Results and Discussion

The method achieved baseline separation of all analytes and the internal standard in under 3 minutes, supporting high throughput. Calibration was linear from 0.1 to 100 ng/mL (r2 ≥0.9968). Accuracy deviations were within ±9% across six calibration levels.
Precision (n=20 at 0.3 ng/mL) showed CVs below 6% with internal standard correction.
Recovery values exceeded 74% (range 74.9–90.9%), and matrix suppression was minimal (<8%), demonstrating effective SPE clean-up. The lower limit of quantification was established at 0.1 ng/mL.

Practical Benefits and Applications

• The mixed-mode SOLA WCX SPE simplifies extraction of permanently charged analytes without high salt elution, reducing sample handling and solvent use.
• Rapid HILIC separation with a sub-3-minute run time enhances laboratory throughput for bioanalytical and clinical studies.
• Robust performance with low matrix effects and high reproducibility supports reliable therapeutic drug monitoring and pharmacokinetic profiling.

Future Trends and Opportunities

• Integration of automated SPE and UHPLC-MS workflows to further increase throughput and reduce manual steps.
• Expansion to multiplexed assays for broad panels of polar, ionizable drugs and metabolites using HILIC and mixed-mode SPE strategies.
• Application of high-resolution MS and advanced data processing for improved specificity in complex biological matrices.

Conclusion

The described LC-MS/MS method delivers fast, sensitive, and reproducible quantification of edrophonium, neostigmine, and pyridostigmine in plasma. The combination of SOLA WCX SPE and Syncronis HILIC UHPLC ensures low matrix interference, high recovery, and sub-3-minute analysis, meeting the demands of high-throughput bioanalysis.