Determination of Main and Trace Enantiomers in One Run Using the Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System

Significance of Topic

Chiral analysis is critical for pharmaceutical products to ensure efficacy and safety by distinguishing enantiomers that may have different biological effects. High resolution detection of both main and trace enantiomers in a single run can streamline quality control and regulatory compliance.

Objectives and Overview

This study demonstrates the application of the Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer to quantify major and minor enantiomers of propranolol in one chromatographic run. The aim was to achieve a wide dynamic detection range, sensitive trace analysis, and accurate enantiomeric excess determination.

Used Instrumentation

• Agilent 1200 Infinity HDR-DAD Impurity Analyzer System with solution kit
• Agilent 1290 Infinity DAD modules with 60 mm and 3.7 mm flow cells
• Thermostatted column compartment and autosampler
• Binary pump and temperature-controlled sample compartment
• Agilent OpenLAB CDS ChemStation for acquisition and evaluation

Methodology

Chromatographic separation was carried out on a 250 × 4.6 mm YMC Chiral Cellulose C column using n-hexane/2-propanol/diethylamine (80/20/0.1) under isocratic conditions at 25 °C and 1 mL/min flow. Detection was performed at 230 nm with reference at 400 nm and 10 Hz acquisition. Sample preparation involved dissolving S-propranolol and R-propranolol in ethanol at concentrations designed to cover trace and main compound levels. Linearity, limit of detection, and precision were evaluated over a concentration range from ng to μg scale.

Main Results and Discussion

• Linearity for S-propranolol from 17 ng to 10 700 ng showed a correlation coefficient of 0.99996
• Limit of detection was determined as low as 2.34 ng for the R-enantiomer and 3.64 ng for the S-enantiomer (signal to noise = 3)
• Precision over five injections yielded retention time RSD ~0.1% and area RSD between 0.25% and 0.64%
• A single injection detected the main S-propranolol peak at ∼3 700 mAU and the trace R-propranolol at ∼14 mAU
• The method delivered an enantiomeric excess value of 99.87% for S-propranolol in one run without volume adjustments

Benefits and Practical Use

The HDR-DAD system’s broad linear dynamic range removes the need for separate injections or detector tuning for high and low concentration analytes. This capability simplifies workflow, reduces analysis time, and ensures reliable quantitation of both major and trace enantiomers in pharmaceutical quality control.

Future Trends and Opportunities

Enhanced diode array detectors with extended dynamic ranges are poised to support more complex chiral separations and multi-enantiomer assays. Integration with advanced data analysis software and coupling with mass spectrometry could further improve sensitivity, throughput, and specificity in enantiomeric impurity profiling.

Conclusion

The Agilent 1200 Infinity HDR-DAD Impurity Analyzer successfully quantifies main and trace enantiomers of propranolol in a single HPLC run. Its high sensitivity, wide linear range, and robust precision facilitate accurate determination of enantiomeric excess and streamline chiral analysis in pharmaceutical development and production.

References

1. US Food and Drug Administration. Development of New Stereoisomeric Drug Guidance; 1992.
2. IUPAC. Compendium of Chemical Terminology (Gold Book); Blackwell Scientific, 1997.
3. Huesgen AG. Comparison of Sensitivity and Linearity of the Agilent 1200 HDR-DAD; Agilent Application Note, 2013.
4. Sigma-Aldrich. Product Information HLD/RXR 12/02; 2014.