Agilent Hybrid SFC/UHPLC-Triple Quadrupole System for Achiral and Chiral Metabolite Analysis

Importance of the topic

Metabolite profiling of drugs and their biotransformation products is essential for understanding pharmacokinetics, safety and efficacy. In particular, warfarin generates multiple stereo- and regio-isomeric hydroxylated metabolites that are difficult to separate and quantify by mass spectrometry alone due to similarity in fragmentation patterns. Combining achiral and chiral chromatographic techniques ensures unambiguous identification and accurate quantitation of these trace-level isomers in drug metabolism studies.

Objectives and Study Overview

This application note demonstrates a workflow for seamless switching between reversed-phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC) on a single Agilent 1260 Infinity Hybrid SFC/UHPLC system coupled to a 6460 Triple Quadrupole mass spectrometer. The goals were to develop and validate methods for both achiral and chiral separation of warfarin and its five hydroxylated metabolites, and to apply the stereospecific assay to quantify enantiomeric metabolites formed during rat hepatocyte incubation.

Methodology

• Sample preparation involved creating a racemic standard mix of warfarin and five hydroxylated metabolites, with calibration levels spanning sub-nanomolar to micromolar concentrations.
• Achiral RPLC was carried out on a phenyl-hexyl column (2.1×150 mm, 1.8 µm) at 25 °C using a water/methanol gradient with formic acid additive.
• Achiral SFC used a ZORBAX RX-SIL column (4.6×150 mm, 5 µm) with CO₂/methanol mobile phase at 60 °C and 140 bar backpressure.
• Chiral SFC separation employed a CHIRALPAK OD-3 column (3×100 mm, 3 µm) serially connected to the RX-SIL column to resolve enantiomers of all hydroxylated isomers in under 8 minutes.
• Mass spectrometry detection utilized positive-mode electrospray with multiple reaction monitoring (MRM) transitions optimized for parent and hydroxylated warfarin ions.

Instrumentation

• Agilent 1260 Infinity SFC Control Module (G4309A) with 2-position/10-port valve for automatic mode switching
• Agilent 1260 Infinity SFC Binary Pump and UHPLC Binary Pump
• Agilent 1290 Infinity Thermostatted Column Compartment with 2-position/6-port valve
• Agilent 1260 Infinity DAD and autosampler
• Agilent 6460 Triple Quadrupole LC/MS with Jet Stream electrospray source
• Make-up flow and flow splitter kits for optimal backpressure and sensitivity

Results and Discussion

• Achiral RPLC provided baseline resolution of warfarin and five hydroxylated metabolites, while achiral SFC offered complementary elution order and a faster analysis time.
• Serial coupling of the RX-SIL and CHIRALPAK OD-3 columns in SFC mode achieved baseline separation of all regio- and stereoisomeric hydroxyl warfarin enantiomers.
• Method validation demonstrated limits of detection below 0.15 nM and quantitation below 0.3 nM, linearity over four orders of magnitude (R² ≥ 0.99), retention time RSD < 0.2 %, and peak area precision ≤ 5 % (≤ 10 % at LOQ).
• Application to rat hepatocyte incubations showed significant formation of 4′-, 6- and 7-hydroxywarfarin enantiomers and lower levels of 8- and 10-hydroxy isomers after 24 hours.

Benefits and Practical Applications

• Automated switching between RPLC and SFC modes eliminates manual hardware reconfiguration and reduces downtime.
• Combined achiral/chiral analysis on a single platform provides orthogonal separation selectivity for confident isomer quantitation.
• High sensitivity and fast cycle times enable trace-level metabolite profiling in drug discovery and bioanalytical laboratories.

Future Trends and Applications

• Expansion of hybrid SFC/UHPLC workflows to a broader range of chiral drugs and complex metabolite matrices.
• Integration with high-resolution mass spectrometry for structure elucidation of unknown metabolites.
• Increased automation and data-driven method optimization using advanced software and machine learning.
• Adoption in regulated environments for routine chiral metabolite monitoring in pharmacokinetic and toxicology studies.

Conclusion

The Agilent 1260 Infinity Hybrid SFC/UHPLC system coupled to a 6460 Triple Quadrupole MS streamlines achiral and chiral metabolite analysis by enabling fast, sensitive and fully automated method switching. The validated assay for warfarin and its hydroxylated enantiomers delivers robust quantitation over a wide concentration range and is readily applied to in vitro biotransformation studies.

References

1. Jones DR, Miller GP. Assays and applications in warfarin metabolism: what we know, how we know it and what we need to know. Expert Opin Drug Metab Toxicol. 2011;7(7):857–874.
2. Kaminsky LS, Zhang ZY. Human P450 metabolism of warfarin. Pharmacol Ther. 1997;73:67–74.
3. Jones D, Moran JH, Miller GP. Warfarin and UDP-glucuronosyltransferases: writing a new chapter of metabolism. Drug Metab Rev. 2010;42:55–61.
4. Agilent Technologies. Agilent 1260 Infinity Analytical Hybrid SFC/UHPLC system user manual. 2015;G4309-90121.