Characterization of four saturated fatty acids using gradient HPLC-CAD highlighting optimized evaporation temperature control features

Importance of the Topic

Accurate quantification of free fatty acids (FAs) is critical in pharmaceutical and biopharmaceutical quality control, especially for monitoring polysorbate degradation and impurity levels in formulations. Charged aerosol detection (CAD) offers a universal, sensitive approach for non-volatile and semi-volatile compounds without derivatization, providing uniform response across analytes.

Objectives and Study Overview

This study evaluates the impact of enhanced evaporation temperature control and a novel Temperature Coupling Mode in the Thermo Scientific Vanquish CAD HP system on the analysis of four common saturated FAs (lauric, myristic, palmitic, stearic acids). Key goals include identifying optimal evaporation tube temperatures, assessing signal-to-noise (S/N) performance, and demonstrating method robustness over 24 hours.

Methodology and Instrumentation

A reversed-phase gradient HPLC method was employed using a Thermo Scientific Hypersil GOLD C18 column (50 × 2.1 mm, 1.9 µm). Mobile phases were 0.05% formic acid in water (A) and acetonitrile (B). A gradient from 75% B to 85% B and back over a 5 min cycle at 0.75 mL/min was used. Sample preparation involved individual FA stock solutions (0.25 mg/mL) combined into a 50 µg/mL working solution.

Key instrument parameters:

• Detector: Thermo Scientific Vanquish Detector CAD HP with active evaporation tube temperature control
• Temperature Coupling Mode: on/off comparisons at EvapT = 25 °C and 50 °C
• Charging Detection Module (CDM) stabilized at fixed offsets to EvapT
• Autosampler at 6 °C; injection volume 10 µL; data rate 10 Hz; filter 5 s
• Data analysis: Thermo Scientific Chromeleon CDS 7.3.2

Main Results and Discussion

Noise analysis across EvapT settings (25–40 °C) showed minimal variation. S/N ratios declined as EvapT increased, reflecting greater analyte volatility. The optimum EvapT of 25 °C delivered the highest average S/N for all four FAs over 24 h (RSD <10%). Activating Temperature Coupling Mode at 25 °C further enhanced S/N by 9.4% (stearic acid) up to 46.8% (lauric acid), attributed to reduced CDM temperature and minimized semi-volatile evaporation losses. The method exhibited consistent performance in continuous runs, validating robustness for routine analysis.

Benefits and Practical Applications

• Improved sensitivity and reproducibility for semi-volatile and non-volatile FAs without derivatization
• Rapid, gradient HPLC-CAD workflow suitable for raw material and formulation QC
• Enhanced control over evaporation and charging conditions simplifies method transfer and automation

Used Instrumentation

• Thermo Scientific Vanquish Detector CAD HP
• Thermo Scientific Hypersil GOLD C18 column (50 × 2.1 mm, 1.9 µm)
• Thermo Scientific Vanquish UHPLC system
• Chromeleon Chromatography Data System 7.3.2

Future Trends and Applications

Continued innovations in temperature management and coupling strategies can extend CAD applicability to a broader range of semi-volatile analytes, including complex lipids and surfactant degradation products. Integration with high-resolution mass spectrometry, real-time process monitoring, and advanced data analytics will further elevate QA/QC capabilities in pharmaceutical development.

Conclusion

The enhanced evaporation temperature control and Temperature Coupling Mode in the Vanquish CAD HP system deliver significant S/N improvements for saturated FAs, with an optimal EvapT of 25 °C and coupling activated. The method is robust over extended runs and supports reliable FA quantitation in pharmaceutical contexts.

References

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