Analysis of Anti-epileptic Drugs with the Agilent 1290 Infinity II LC

Importance of the Topic

Qualitative and quantitative analysis of anti-epileptic pharmaceuticals is critical to ensure drug purity, stability, and patient safety under various environmental conditions. High performance liquid chromatography (HPLC) remains a standard analytical technique in pharmaceutical laboratories. However, the demand for higher sample throughput, reduced solvent consumption, and lower operational costs has driven the transition to ultrahigh performance liquid chromatography (UHPLC) methods. This shift supports more efficient workflows in quality control, research, and regulatory compliance.

Objectives and Study Overview

• Transfer an existing HPLC method for seven anti-epileptic compounds from a conventional 4.6×150 mm, 5 µm column to UHPLC using sub-2 µm stationary phases.
• Optimize UHPLC separations for both moderate and ultrafast analyses on 4.6×50 mm and 2.1×50 mm, 1.8 µm columns, respectively.
• Evaluate performance in terms of precision, resolution, linearity, detection limits, cycle time, and solvent/sample savings.

Used Instrumentation

• Agilent 1290 Infinity II LC system comprising:
• High-speed Pump (G7120A)
• Multisampler (G7167B)
• Multicolumn Thermostat (G7116B)
• Diode Array Detector with Max-Light cell (G7117B)
• ZORBAX SB-C18 columns:
• 4.6×150 mm, 5 µm
• 4.6×50 mm, 1.8 µm
• 2.1×50 mm, 1.8 µm

Methodology

The sample comprised seven anti-epileptic drugs (2-ethyl-2-phenylmalonamide, ethosuximide, primidone, carbamazepine, phenytoin, carbamazepine-10,11-epoxide, stiripentol) each at 25 ng/µL, with caffeine as an internal reference. LC-grade water and acetonitrile formed the mobile phases under gradient elution. Chromatographic conditions were adapted to each column dimension, with detection at 204 nm (4 nm bandwidth) and reference at 360 nm (80 nm bandwidth). Data acquisition rates and injection volumes were adjusted to the column geometry and analysis time.

Main Results and Discussion

• Precision and Resolution:
  • HPLC on 4.6×150 mm, 5 µm column achieved retention time RSD <0.042 % and area RSD <0.15 %.
  • UHPLC on 4.6×50 mm, 1.8 µm column reduced cycle time by >66 % with RT RSD <0.085 % and area RSD <0.42 %.
  • Ultrafast UHPLC on 2.1×50 mm, 1.8 µm column completed separation within 0.9 min, with RT RSD <0.19 % and area RSD <0.5 %, maintaining baseline resolution for all analytes.
• Linearity:
  • All methods displayed excellent linearity (R >0.9995) across ten concentration levels from 0.195 to 50 µg/mL.
• Sensitivity:
  • UHPLC on the 4.6×50 mm column improved limits of detection by >100× and on the 2.1 mm column by >10× compared to HPLC.
• Efficiency Gains:
  • UHPLC methods achieved up to 95 % solvent savings and a 75 % reduction in sample volume on the 2.1 mm column.

Benefits and Practical Applications

The optimized UHPLC workflows deliver significant time savings and cost reductions while preserving analytical performance. The approach suits high-throughput pharmaceutical quality control, stability testing, and routine monitoring of drug purity. Reduced solvent usage and lower sample consumption support green chemistry initiatives and minimize laboratory waste.

Future Trends and Applications

• Integration of UHPLC with mass spectrometry for enhanced selectivity and structural identification.
• Development of automated, high-throughput LC platforms for complex drug panels.
• Implementation of miniaturized and portable UHPLC systems for on-site and real-time monitoring.

Conclusion

Transferring a conventional HPLC method for anti-epileptic drugs to UHPLC using the Agilent 1290 Infinity II LC system achieves dramatic improvements in analysis speed, solvent and sample economy, sensitivity, and throughput, without compromising reproducibility or resolution.

Reference

• Darwish H. W. et al. Stability-Indicating HPLC-DAD Method for Determination of Stiripentol. Journal of Analytical Methods in Chemistry. 2014; Article ID 638951.