Development of an UHPLC Method for Azithromycin Tablets Using ChromSword Auto Software

Significance of the Topic

High-throughput and reliable impurity profiling in pharmaceutical QC demands rapid and reproducible chromatographic methods. Traditional HPLC method development relies heavily on trial-and-error and analyst expertise, often requiring long run times and extensive resource consumption. Automated UHPLC method development platforms can dramatically reduce development time, solvent use, and improve robustness for routine analysis of azithromycin tablets.

Objectives and Study Overview

This study aimed to develop a fast, robust UHPLC method for the analysis of azithromycin and its three specified impurities (M, E, A) in tablet formulations. Key goals included reducing run time compared to the USP procedure, achieving baseline separation of critical impurity pairs, and defining a scientifically sound design space for method robustness.

Methodology and Instrumentation

The workflow consisted of four stages:

• Screening: Agilent Method Scouting Wizard evaluated eight Poroshell stationary phases, two organic solvents, and eight pH buffers to identify promising column–mobile phase combinations.
• Rapid Optimization: ChromSword Auto algorithm performed iterative gradient adjustments with automatic peak tracking to refine separation on selected columns.
• Robustness Testing: ChromSword AutoRobust employed a three-level full factorial design to study deliberate variations in flow rate, column temperature, and gradient breakpoint at three pH values, generating two-dimensional contour plots to locate the robust region.
• Verification: Method performance was confirmed at center and edge conditions of the design space, assessing critical pair resolution, relative retention times, peak purity, and reproducibility over 250 injections.

Main Results and Discussion

Screening identified Agilent Poroshell HPH-C8 and Poroshell EC-C18 at pH 10.8 with acetonitrile as the strongest solvent for baseline separation. Rapid optimization on HPH-C8 with the USP buffer (pH 8.9 phosphate) achieved critical pair resolution >2.0 and tailing factor <2.0 within a 6-minute run. Robustness experiments established a design space over flow rate (±0.2 mL/min), temperature (±5 °C), and gradient breakpoint (±0.2 min) at pH 8.7–9.1. Contour plots guided a final gradient adjustment, guaranteeing resolution >2. Verification confirmed >99.9 % peak purity, RSD <0.05 % in retention time and <0.85 % in area over 250 injections, and stable column backpressure (~150 bar).

Benefits and Practical Applications

• Run time reduction from 93 min (USP method) to 6 min enhances sample throughput.
• Automated workflows minimize analyst intervention and method development costs.
• Defined design space supports regulatory flexibility and risk-based QC control.
• High reproducibility and peak purity ensure compliance with pharmaceutical standards.

Future Trends and Potential Applications

Automated UHPLC method development is poised to integrate machine learning models for predictive retention and selectivity. Future directions include coupling with mass spectrometry for impurity identification, real-time process analytical technology (PAT) integration, and expansion to complex biologics and chiral separations. Enhanced software intelligence will further reduce development cycles and foster green analytical chemistry.

Conclusion

The combination of Agilent 1200 Infinity Series LC Method Development System, Method Scouting Wizard, and ChromSword Auto software enabled a fully automated, robust UHPLC method for azithromycin tablets. The approach achieved dramatic reductions in run time and solvent consumption while delivering excellent precision, reproducibility, and chromatographic performance. A statistically defined design space underpins method reliability under routine QC conditions.

Used Instrumentation

• Agilent 1200 Infinity Series LC Method Development Solution
• Agilent 1290 Infinity Binary Pump, Valve Drive, Autosampler, Column Compartment
• ChromSword Auto 4.0 with Algorithm feature
• ChromSword AutoRobust 3.6 for DoE robustness testing
• Agilent Method Scouting Wizard in OpenLab CDS
• Agilent 1290 Infinity Diode Array Detector

Reference

1. Application of ChromSword Software for Automatic HPLC Method Development and Robustness Studies, March 2013, ChromSword.
2. Automated method development using Agilent 1100 Series HPLC systems, Agilent ChemStation and ChromSword Software, 2004.
3. Azithromycin monograph, USP-NF 37, Official from May 2014, pp 1886–1897.
4. Wikipedia, Azithromycin entry, accessed 2015.