Enantiomeric Separation of the Active Metabolite of Risperidone and its Application for Monitoring Metabolic Stability using UPC2/MS/MS

Importance of the topic

Enantiomeric resolution of drug metabolites is critical in pharmaceutical development to ensure safety, efficacy and accurate pharmacokinetic profiling. The active metabolite 9-hydroxyrisperidone exhibits chiral behavior that influences its biological activity and clearance rates. Applying a rapid and selective separation method aids in monitoring metabolic stability and supports decision making in lead optimization.

Objectives and overview of the study

This study aimed to develop and optimize a method combining ultra-performance convergence chromatography (UPC2) with tandem mass spectrometry (MS/MS) to separate and quantify the R and S enantiomers of 9-hydroxyrisperidone. The method was then applied to human liver microsomal incubations to track the formation rate of each enantiomer over a metabolic time course.

Methodology and instrumentation

Standards of racemic 9-hydroxyrisperidone were first analyzed to establish chromatographic conditions supporting baseline enantiomer separation. The optimized method employed a chiral Trefoil CEL2 column (3×150 mm, 2.5 µm) with a mobile phase of supercritical CO2 and methanol containing ammonium formate. Detection was performed using MS/MS in multiple reaction monitoring mode. Microsomal incubations of risperidone were quenched at 0, 15, 30, 60, 90 and 120 minutes to assess metabolic conversion.

Used instrumentation

• Waters UPC2 system
• Tandem quadrupole mass spectrometer
• Waters Trefoil CEL2 chiral column (3×150 mm, 2.5 µm)
• CO2 delivery module and automated backpressure regulator
• Data processing software for chromatographic integration

Main results and discussion

The optimized UPC2/MS/MS method achieved clear resolution of two major peaks at 3.92 min and 4.29 min, corresponding to the R and S enantiomers of 9-hydroxyrisperidone. Two additional peaks at 4.48 min and 4.76 min were attributed to minor 7-hydroxy risperidone metabolites. Time-course analysis showed that over 80 % of parent conversion occurred by 30 min. Based on literature precedent indicating preferential R-enantiomer formation, the earlier eluting peak was assigned as R-9-hydroxyrisperidone.

Benefits and practical application

This approach delivers high resolution, fast run times and MS compatibility, enabling reliable chiral metabolite profiling in drug metabolism studies. It supports metabolic stability assessments and helps identify enantiomer-specific clearance, guiding dosage form design and safety evaluations.

Future trends and potential applications

Advances may include coupling UPC2 with high-resolution mass spectrometry for comprehensive metabolite identification, adapting methods for high-throughput screening, and integrating green solvent strategies. Machine learning tools could enhance peak assignment and automate data interpretation in complex chiral analyses.

Conclusion

The study demonstrates that UPC2/MS/MS is a powerful tool for enantiomeric separation and quantitative monitoring of 9-hydroxyrisperidone in metabolic assays. The method’s speed, selectivity and compatibility with MS make it valuable for pharmaceutical research and development.

References

1. G. Mannens, M.L. Huang, W. Meuldermans, et al. Absorption, metabolism, and excretion of risperidone in humans. Drug Metab Dispos. 21:1134–1141, 1993.
2. N. Yasui-Furukori, M. Hidestrand, E. Spina, et al. Different enantioselective 9-hydroxylation of risperidone by human CYP2D6 and CYP3A4 enzymes. Drug Metab Dispos. 29:1263–1268, 2001.