Analysis of Doxorubicin Hydrochloride with the Agilent 1260 Infinity II Prime LC as per USP Monograph Method

Significance of the Topic

Doxorubicin hydrochloride is a widely used chemotherapeutic agent, necessitating robust and efficient analytical methods for quality control. The recent USP monograph modernization emphasizes faster, reproducible assays compatible with both HPLC and UHPLC platforms. Upgrading to sub-2 µm column methods enhances laboratory throughput and accuracy in routine and regulatory environments.

Study Objectives and Overview

This study evaluates the performance of the Agilent 1260 Infinity II Prime LC system for the USP monograph method of doxorubicin hydrochloride analysis. Key aims include method transfer from conventional to UHPLC conditions, validation of assay precision, and verification of related substances detection on a sub-2 µm column.

Methodology

• Sample Preparation:
  
  • Diluent: 1:1 mix of mobile phase A and B (protected from light).
  • System suitability solution: 0.1 mg/mL each of doxorubicin and epirubicin standards.
  • Standard and sample solutions: 0.1 mg/mL doxorubicin hydrochloride in diluent.
  • Related substances mix: 0.002 mg/mL of doxorubicin, doxorubicinone, daunorubicin, daunorubicinone.
• Chromatographic Conditions:
  
  • Column: 2.1 × 100 mm, 1.7 µm L1 packing.
  • Mobile Phase A: 0.1% TFA in water; B: 80% acetonitrile, 20% methanol, 0.1% TFA.
  • Gradient: 10% B to 75% B over 15 min, hold, then re-equilibration.
  • Flow Rate: 0.5 mL/min; Column Temp: 35 °C; Injection: 2 µL.
  • Detection: UV at 254 nm.

Used Instrumentation

• Agilent 1260 Infinity II Flexible Pump (G7104C)
• Agilent 1260 Infinity II Multisampler (G7167A)
• Agilent 1260 Infinity II Multicolumn Thermostat (G7116A)
• Agilent 1260 Infinity II Diode Array Detector HS (G7117C)
• Software: Agilent OpenLab CDS Workstation v2.3.0 (M8413AA)
• Solvents: LC-grade, TFA LC/MS-grade, Milli-Q ultrapure water

Main Results and Discussion

System suitability tests met USP <621> criteria: relative retention time for epirubicin was 1.05 min; resolution >2.9; %RSD for area and retention time well below 0.73%.
Repeatability over 25 injections showed area RSD of 0.07% and retention time RSD of 0.13%.
Related substances assay achieved RSD <1% for all impurities and clear separation of doxorubicinone, daunorubicin, and daunorubicinone with resolution >4.7.

Benefits and Practical Applications

• Seamless method transfer from conventional HPLC to UHPLC conditions using ISET.
• High precision and reproducibility for routine QC laboratories.
• Compatibility with compendial specifications up to 800 bar backpressure.
• Flexibility to run a broad range of assays on a single platform.

Future Trends and Opportunities

• Integration with mass spectrometry detectors for extended impurity profiling.
• Further compendial modernization of anticancer drug monographs.
• Automation and data analytics integration for enhanced laboratory efficiency.
• Development of green solvent methods to reduce environmental impact.

Conclusion

The Agilent 1260 Infinity II Prime LC demonstrates robust performance and compliance with the USP monograph for doxorubicin hydrochloride, offering reliable, high-throughput analysis on sub-2 µm columns. Its dual HPLC/UHPLC capability and intelligent system emulation facilitate method standardization and lab productivity.

References

1. United States Pharmacopeia 39, Doxorubicin Hydrochloride Monograph 3602.
2. USP General Chapter <621> Chromatography, USP 38.