Improving peak results using a custom injection program to reduce solvent strength prior to sample injection

Importance of the Topic

Pharmacopoeia monographs mandate strict system suitability criteria in HPLC analyses. When samples are dissolved in strong solvents like DMF, early-eluting peaks can distort, compromising asymmetry, resolution, and efficiency. Custom injection programs reduce solvent strength immediately before injection, offering a practical route to improve peak performance without modifying validated chromatographic methods.

Study Objectives and Overview

The goal of this study was to evaluate how user-defined injection routines affect chromatographic parameters—peak asymmetry, resolution, and plate number—in a European Pharmacopoeia mebendazole method. A Thermo Scientific Vanquish Core HPLC system was employed to compare normal injection with two custom injection sequences (A and B) on a mebendazole system suitability test (SST) reference standard containing the API and seven impurities.

Methodology and Instrumentation

The EP gradient method used a Hypersil GOLD column (100×4.6 mm, 3 µm) and mobile phases of 7.5 g/L ammonium acetate buffer in water (A) and acetonitrile (B). The gradient ran from 20% B to 90% B over 25 min. Sample injections (10 µL) comprised 1 mg/mL mebendazole SST in DMF.

• Custom injection A: draw 10 µL sample then 90 µL initial mobile phase (20:80 B:A).
• Custom injection B: sandwich 10 µL sample between two 45 µL buffer (A) aliquots.

Instrumentation included:

• Vanquish Core Quaternary HPLC system
• Quaternary Pump C and Diode Array Detector CG
• Vanquish Sampler CT autosampler
• Vanquish Column Compartment C

Data acquisition and processing used Chromeleon 7.3 CDS.

Main Results and Discussion

Normal injection produced fronting for early impurities A–C (asymmetry 0.76–0.83). Custom injection A improved asymmetry to 0.95–0.98; custom B yielded 0.87–0.89. Resolution and plate numbers increased two- to fourfold with custom routines. Retention time precision remained excellent (<0.1% RSD) across all methods. Peak area precision improved (<0.5% RSD) for most impurities, except impurity G, which showed higher variability (up to 3.3%) due to its hydrophobicity and ionization behavior.

Benefits and Practical Application

• Improves peak symmetry and narrows peaks, enhancing resolution and efficiency.
• Avoids modification of official gradient or pH, maintaining method compliance.
• Automates dilution and solvent strength adjustment for routine QC workflows.

Future Trends and Potential Applications

Custom injection protocols can be extended to automated sample preparation tasks—derivatization, calibration standard generation, and internal standard addition—within the Vanquish platform. This flexibility supports complex analytical workflows in pharmaceutical QC and industrial laboratories.

Conclusion

Custom injection programs on the Vanquish autosampler effectively mitigate solvent mismatch effects in EP methods, delivering superior system suitability metrics without altering chromatographic conditions. This strategy streamlines regulatory compliance and enhances data quality in routine HPLC analyses.

Reference

1. European Pharmacopoeia 9.2, Chapter 2.2.46: Chromatographic separation techniques, system suitability.
2. McMath A. The European Pharmacopoeia and certificates of suitability (CEP), EMA presentation, 2019.
3. Alsehli J.; Dolan J.W. The Role of the Injection Solvent, LCGC Asia Pacific, 2012, 15(4), 19–21.
4. European Pharmacopoeia, 7th Edition overview.
5. European Pharmacopoeia 9.5, Monograph 0845: Mebendazole.
6. Eluotropic strength of common solvents, sanderkok.com, accessed July 2019.