Investigating the Chiral Metabolism of an Achiral Drug Using Agilent SFC‑MS/MS Technology

Significance of the Topic

Supercritical fluid chromatography coupled with tandem mass spectrometry offers a rapid and eco friendly platform for enantioselective separation and quantitation of drug metabolites which is essential for pharmacological profiling and safety assessment

Objectives and Overview

The study aimed to develop and validate a robust method for quantifying achiral risperidone and its chiral 7 hydroxy and 9 hydroxy metabolites in biological matrices using SFC MS MS The work includes method optimization specificity carryover assessment and in vitro metabolic application

Methodology and Instrumentation

Sample preparation included protein precipitation of incubated rat liver microsome and recombinant enzyme samples with methanol containing trazodone internal standard
Chromatography employed an Agilent 1260 Infinity SFC system with a CHIRALPAK AD 3 column at 45°C using supercritical CO2 and methanol with ammonium acetate and formic acid as mobile phases
Detection used an Agilent 6460 Triple Quadrupole mass spectrometer in positive ion MRM mode with optimized transitions for each analyte

Main Results and Discussion

• Baseline resolution of risperidone and all enantiomeric metabolites in under six minutes
• Lower limit of quantitation of 0.9 nM for risperidone and 9 hydroxy enantiomers and 0.5 nM for 7 hydroxy enantiomers
• Linearity over four orders of magnitude with correlation coefficients above 0.99 using weighted regression
• Accuracy within 90 to 110 percent and precision with RSD below 3 percent for area ratios and 0.7 percent for retention time
• Carryover below 0.008 percent and method robustness demonstrated over 300 consecutive injections
• In vitro study showed S enantiomer predominance in rat liver microsomes and CYP2D6 as the major enzyme responsible for hydroxy metabolite generation

Benefits and Practical Applications

This SFC MS MS approach delivers fast high sensitivity and green analytical workflow suitable for drug metabolism research enzyme phenotyping and quality control in pharmaceutical development

Future Trends and Potential Applications

• Extension to other chiral drug molecules and their metabolites
• Integration with high throughput platforms for screening of metabolic profiles
• Automation of sample preparation and data processing for regulatory bioanalysis
• Adoption in personalized medicine for monitoring enantiomeric pharmacokinetics
• Advancements in greener mobile phase additives and stationary phase chemistries

Conclusion

The validated SFC MS MS method meets bioanalytical performance criteria and enables rapid enantioselective quantitation of risperidone and its hydroxy metabolites with high sensitivity specificity and reproducibility making it a valuable tool for pharmaceutical research and development

Used Instrumentation

• Agilent 1260 Infinity Analytical SFC system with backpressure regulator
• CHIRALPAK AD 3 column 3 0 × 100 mm 3 µm
• Agilent 6460 Triple Quadrupole LC MS in positive ion MRM mode

References

1. Mannens G et al Absorption metabolism and excretion of risperidone in humans Drug Metabolism and Disposition 21 6 1134 1141
2. Meuldermans W et al The metabolism and excretion of risperidone after oral administration in rats and dogs Drug Metabolism and Disposition 22 1 129 138
3. Cabovska B Cox SL Vinks AA Determination of risperidone and enantiomers of 9 hydroxyrisperidone in plasma by LC MS MS Journal of Chromatography B Analyt Technol Biomed Life Sci 2009
4. De Meuldera M et al Validated LC MS MS methods for the determination of risperidone and the enantiomers of 9 hydroxyrisperidone in human plasma and urine Journal of Chromatography B 2008 870 8 16