Analysis of Pharmaceutical Substances Using HPLC and UHPLC Methods

Význam tématu

In pharmaceutical quality control and development laboratories, maintaining continuity between legacy HPLC methods and advanced UHPLC platforms is critical for regulatory compliance, efficiency, and cost reduction.

Cíle a přehled studie

This study compares the performance of the Agilent 1290 Infinity LC and the Agilent 1290 Infinity II LC systems for analysis of antiepileptic drug standards, exploring method transfer from HPLC to UHPLC and optimization of run speed without sacrificing resolution or sensitivity.

Použitá instrumentace

The following modules and components were employed:

• Agilent 1290 Infinity II LC: High-Speed Pump G7120A, Multisampler G7167B with sample cooling, Multicolumn Thermostat G7116B, Diode Array Detector G7117B
• Agilent 1290 Infinity LC: Binary Pump G4220A with Jet Weaver mixer, Autosampler G4226A, Thermostat G1330B, Thermostatted Column Compartment G1316C, Diode Array Detector G4212A
• Columns: ZORBAX SB-C18 4.6 × 150 mm 5 µm and ZORBAX RRHD SB-C18 2.1 × 50 mm 1.8 µm
• Data system: Agilent OpenLAB CDS ChemStation Edition C.01.07

Použitá metodika

Three analytical workflows were evaluated:

1. HPLC on a 4.6 × 150 mm 5 µm column with 0.8 mL/min flow, 25 min runtime, gradient of water and acetonitrile.
2. UHPLC conversion on a 2.1 × 50 mm 1.8 µm column at 0.2 mL/min, simple transfer reducing cycle time to 13.5 min.
3. Speed-optimized UHPLC on the same 2.1 mm column at 1.5 mL/min and 80 °C, achieving 0.9 min separation and 2 min total cycle.

Hlavní výsledky a diskuse

All methods demonstrated excellent retention time precision (RSD below 0.05% for HPLC, improved on Infinity II) and peak area reproducibility (below 0.65% for HPLC, improved on Infinity II). Simple UHPLC transfer achieved over 90% runtime and 66% solvent savings while maintaining resolution. The speed-optimized UHPLC yielded a 20-fold analysis time reduction and over 95% solvent savings, with clear baseline separation of closely eluting antiepileptics at subminute scale. Linearity was >0.9999 in all cases and LOD/LOQ improved by more than tenfold after UHPLC conversion, remaining equivalent or better on the Infinity II system.

Přínosy a praktické využití metody

• Minimized revalidation and method transfer effort between legacy and modern LC platforms.
• Substantially increased throughput with faster cycles and reduced analysis costs.
• Lower solvent and sample consumption for cost and environmental benefits.
• High sensitivity and resolution ensure reliable quantification of pharmaceutical analytes.

Budoucí trendy a možnosti využití

Future developments may include automation of method conversion, further integration of high frequency detectors for enhanced MS coupling, and application of ultrafast UHPLC workflows in real-time process monitoring and high throughput screening.

Závěr

The Agilent 1290 Infinity II LC demonstrates equal or superior performance compared to the original Infinity system, enabling seamless conversion of validated HPLC methods to ultrafast UHPLC protocols with significant gains in speed, sensitivity, and efficiency.

Reference

• Gratzfeld-Huesgen A, Schneider S. Performance Characteristics of the Agilent 1290 Infinity II Multisampler. Agilent Technologies Technical Overview 2014.
• Schneider S. Performance Characteristics of the Agilent 1290 Infinity II Multicolumn Thermostat. Agilent Technologies Technical Overview 2015.
• Darwish HW et al. Stability-Indicating HPLC-DAD Method for Stiripentol Determination. Journal of Analytical Methods in Chemistry 2014.