Setup and transfer of a gradient HILIC method for the impurity analysis of temozolomide

Importance of the Topic

This application addresses the rapid and reliable analysis of impurities in temozolomide, a polar oncology drug. Traditional reversed-phase methods often struggle to retain such hydrophilic compounds, leading to lengthy run times, extensive column equilibration, and reliance on ion-pairing reagents. Implementing a gradient HILIC approach streamlines impurity profiling, reducing analysis time and solvent consumption while meeting pharmacopeial requirements.

Objectives and Study Overview

The primary goal was to develop a fast gradient HILIC method for temozolomide impurity analysis on an Agilent 1260 Infinity LC system and demonstrate its straightforward transfer to a Thermo Scientific Vanquish Core HPLC system. Key evaluation metrics included retention time reproducibility, peak resolution, and method robustness across instruments.

Methodology and Instrumentation

A Syncronis HILIC column (4.6 × 100 mm, 3 µm, 175 Å) was employed with mobile phases of 20 mM ammonium acetate at pH 5.1 in water (A) and in 10:90 water:acetonitrile (B). A gradient from 0 to 44% A over 5 min followed by re-equilibration yielded a total run time of 12 min. Column temperature was set to 30 °C, and autosampler temperature at 10 °C. Detection was at 266 nm with response times adjusted for each detector.

Used Instrumentation

• Agilent 1260 Infinity LC with quaternary pump G1311B, autosampler G1367E, column oven G1316A, VWD G1314F.
• Thermo Scientific Vanquish Core Quaternary HPLC with Pump C, Split Sampler CT, Column Compartment C, Variable Wavelength Detector C.

Results and Discussion

The HILIC method delivered baseline separation of temozolomide and its four related impurities (A, B, D, E) with only three to four column volumes of equilibration. Forced degradation samples confirmed impurity transformations. Upon transfer, both systems produced equivalent chromatograms: retention time deviations were under 0.1 min, with RSD < 0.08% for retention and < 0.4% for peak area. The Vanquish Core system exhibited slightly narrower peaks and improved resolution due to lower extra-column volume.

Benefits and Practical Applications

• Shortened analysis time (12 min vs. ~30 min in reversed phase).
• Minimal column equilibration under HILIC conditions.
• Smooth transfer between legacy and modern LC platforms without method revalidation.
• Reliable impurity identification supporting compliance with Ph. Eur. and USP guidelines.

Future Trends and Opportunities

Emerging HILIC research focuses on further reducing equilibration demands through dynamic column preconditioning, integrating high-throughput sample preparation, and exploring novel zwitterionic stationary phases for enhanced selectivity. Additionally, automated volumetric matching tools and cloud-based CDS protocols will simplify method transfers across global laboratories.

Conclusion

A fast gradient HILIC method for temozolomide impurity analysis was successfully established and transferred between two HPLC platforms. Equivalent chromatographic performance, combined with reduced run and equilibration times, illustrates the viability of HILIC for polar drug analysis and highlights the Vanquish Core system’s suitability for robust method implementation.

References

• European Pharmacopoeia Monograph 2780, Temozolomide.
• USP43–NF38, Temozolomide Monograph.
• Paul, C.; Grübner, M. Thermo Scientific WP-72711, 2018.
• McCalley, D.V. J. Chromatogr. A, 2018, 1554, 61–70.
• Müllner, T.; Franz, H. Thermo Scientific TN-73371, 2020.