Fast Analysis of Chiral and Structurally Related Isomers Using Supercritical Fluid Chromatography Mass Spectrometry

Significance of the Topic

High speed and high resolution separation of chiral and structurally related isomers is critical in pharmaceutical analysis and quality control. Applications spanning drug discovery, process optimization and impurity profiling require methods that combine high throughput with robust performance. Supercritical fluid chromatography coupled with mass spectrometry offers an alternative to traditional LC MS with enhanced selectivity and efficiency.

Objectives and Study Overview

This study evaluates the performance of an Agilent 1260 Infinity Analytical SFC system coupled to an Agilent 6130B single quadrupole mass spectrometer for rapid analysis of representative chiral and structural isomers. Key metrics include separation efficiency, analysis speed, reproducibility and quantitative capability.

Methodology

Samples of warfarin, metoprolol, atenolol impurity F, prednisolone, cortisone and ephedrine enantiomers were prepared in methanol. Chromatographic separation was optimized using a Lux Cellulose-1 column for chiral resolutions and a ZORBAX Rx SIL column for achiral separations. Mobile phase A was supercritical CO2, and mobile phase B was methanol modified with either 20 mM ammonium formate or 0.1 percent formic acid. Flow rates, modifier percentages and gradient schedules were tailored for each compound set.

Instrumentation

• Agilent 1260 Infinity Analytical SFC with binary pump, autosampler, column compartment and degasser
• Lux Cellulose-1 (4.6 × 150 mm, 3 µm) and ZORBAX Rx SIL (4.6 × 100 mm, 1.8 µm) columns
• Agilent 6130B single quadrupole mass spectrometer with electrospray ionization in positive mode
• Make-up flow and pre-MS heating to maintain CO2 supercritical state and prevent line freezing

Main Results and Discussion

SFC MS achieved baseline separation of all isomer pairs in under 3.5 minutes. Retention time RSD values were below 0.1 percent and peak area RSD values below 5 percent across six replicates. Modifier composition strongly influenced both chromatographic selectivity and MS signal intensity. Methanol containing 20 mM ammonium formate outperformed 0.1 percent formic acid in signal response and enantioselectivity. For atenolol impurity F, a linear calibration from 1 to 250 ng/mL was obtained with a lower limit of quantitation of 1 ng/mL and correlation coefficients exceeding 0.999.

Benefits and Practical Applications

• High throughput reduces cycle times and accelerates decision making in research and quality control
• Low organic solvent consumption and use of standard grade CO2 lower operational costs and environmental footprint
• Robust reproducibility supports reliable impurity profiling and enantiomeric purity assessments in pharmaceutical workflows

Future Trends and Applications

Future developments may integrate SFC with high-resolution and tandem mass spectrometry for comprehensive structural analysis. Exploration of novel chiral selectors and additive chemistries could expand selectivity across diverse compound classes. Automation and rapid screening platforms will streamline method development. Potential applications include metabolomics, natural products analysis and advanced pharmaceutical quality control.

Conclusion

The combination of Agilent 1260 Infinity Analytical SFC with single quadrupole MS delivers fast, sensitive and reproducible separations of chiral and structurally related isomers. Optimization of modifier composition is essential for optimal performance. This cost effective, green approach meets diverse analytical requirements in pharmaceutical development and beyond.

References

1. Dunkel M Vanhoenacker G David F Sandra P Vollmer M The Agilent 1260 Infinity SFC MS Solution Agilent Technologies Publication 5990 7972EN 2011
2. Zhang T Nguyen D Franco P Vollmer M Enantiomer Separation of Acidic Compounds Agilent Technologies Publication 5990 9594EN 2012