Automated UHPLC method development for mebendazole and related impurities, from method scouting to robustness testing

Significance of the Topic

Monitoring mebendazole and its impurities is critical for ensuring the safety and efficacy of anthelmintic treatments. Automated UHPLC method development reduces time and resource demands while complying with ICH guidelines for impurity profiling.

Objectives and Study Overview

This study aimed to establish a fast and robust UHPLC method for mebendazole and related impurities using a fully automated workflow. The process included method scouting, rapid and fine optimization, and robustness testing.

Methodology and Instrumentation

Method development was performed on a Thermo Scientific Vanquish Quaternary Flex UHPLC system controlled by ChromSword Chromeleon Connect software. The workflow comprised three main steps: scouting of columns and mobile phases, optimization of gradient conditions, and design space exploration for robustness.

Used Instrumentation

• Thermo Scientific Vanquish Quaternary Flex System with Vanquish Horizon base
• Vanquish Quaternary Pump F
• Split Sampler FT and Diode Array Detector FG
• Automated Viper method scouting kit and 6-position/7-port switching valves
• ChromSword Chromeleon Connect modules Scout, Developer, AutoRobust, ReportViewer integrated with Chromeleon 7.3 CDS

Main Results and Discussion

Method scouting screened 32 conditions (4 columns x 4 aqueous phases x 2 solvents) in 51 h of instrument time, identifying Hypersil GOLD with pH 4.7 ammonium acetate buffer and acetonitrile as optimal. Rapid optimization yielded full separation in 13 min with minimal analyst time. Fine optimization refined a simple linear gradient yielding baseline resolution for all impurities within 11 min. Robustness testing via a Plackett-Burman design (17 runs) defined a design space where critical resolution remained above 2.4 across ±5% organic, ±5 °C, ±0.25 min gradient break, and ±0.2 pH.

Benefits and Practical Applications

Automated method development significantly reduced total instrument time (70.6 h) and analyst effort (3.75 h). The approach streamlines impurity profiling workflows, supports regulatory compliance, and facilitates method transfer among laboratories.

Future Trends and Potential Uses

Integration of machine learning and advanced chemometric tools may further accelerate LC method development. Expanded use of automated platforms can promote green chromatography by optimizing solvent use and energy efficiency.

Conclusion

The fully automated Vanquish and ChromSword workflow delivered a rapid, robust UHPLC method for mebendazole impurity analysis. This strategy enhances productivity, ensures high-quality method development, and supports regulatory requirements efficiently.

Reference

1. ICH Q2(R1) Validation of Analytical Procedures Text and Methodology 1995
2. ICH Q3A(R2) Impurities in New Drug Substances 2006
3. Swartz ME Krull IL Chromatogr North Am 2006 24 480–490
4. European Pharmacopoeia 7th Edition
5. Thermo Scientific Application Note 72939 Transfer of EP Method for Mebendazole to Vanquish UHPLC