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

Importance of the topic

Saturated fatty acids (FAs) such as lauric, myristic, palmitic, and stearic acids can arise as impurities or degradation products in pharmaceutical and biopharmaceutical formulations containing polysorbates. Their presence may lead to particle formation, compromising drug stability, efficacy, and patient safety. Charged aerosol detection (CAD) coupled with high-performance liquid chromatography (HPLC) offers a universal, sensitive, and uniform response for non-volatile and many semi-volatile analytes, making it an attractive tool for routine FA analysis without derivatization.

Objectives and study overview

This work evaluates the enhanced evaporation temperature control and the new Temperature Coupling Mode of the Thermo Scientific™ Vanquish™ Charged Aerosol Detector P series. The study aims to determine optimal evaporation tube temperature (EvapT) and assess the impact of coupling EvapT and charging detection module (CDM) temperature on signal-to-noise (S/N) performance for four common saturated FAs under gradient HPLC conditions, and to verify method robustness over a 24-hour period.

Methodology

An existing gradient UHPLC method was adapted: mobile phase A was 0.05% formic acid in water, B was 0.05% formic acid in acetonitrile; a Hypersil GOLD C18 column (50×2.1 mm, 1.9 µm) was operated at 25 °C with a 5-minute gradient (75–85% B). A mixed standard (50 µg/mL each FA) was injected (10 µL). EvapT was varied between 25 °C and 40 °C to evaluate noise and S/N, and signal stability was monitored over 24 h (n=18).

Instrumentation

• Vanquish Flex Quaternary UHPLC system
• Vanquish Charged Aerosol Detector HP with adjustable EvapT and Temperature Coupling Mode
• Chromeleon™ CDS 7.3.2 for data acquisition and processing

Main results and discussion

• Noise levels remained essentially constant across EvapT settings from 25 °C to 40 °C.
• S/N decreased as EvapT increased; optimal S/N for all four FAs was achieved at 25 °C EvapT.
• Over 24 h, relative standard deviations of S/N were below 10%, demonstrating reliable method stability.
• Activating Temperature Coupling Mode (CDM temperature set 5 °C above EvapT) at 25 °C EvapT improved S/N by 46.8% for lauric acid, 35.7% for myristic acid, 20.3% for palmitic acid, and 9.4% for stearic acid, highlighting enhanced detection of more semi-volatile species.

Benefits and practical applications

• Improved detection sensitivity for semi-volatile fatty acids without complex derivatization.
• Robust, reproducible analysis over extended run times suitable for QA/QC laboratories.
• Optimized instrument parameter settings simplify method development for non-chromophoric analytes.

Future trends and applications

Emerging CAD enhancements may include further automation of temperature control, expansion to a wider range of semi-volatile compounds, and integrated workflows combining CAD with mass spectrometry for comprehensive impurity profiling. These developments could streamline quality control in pharmaceutical, biopharmaceutical, and food industries, supporting regulatory compliance and product safety.

Conclusion

The improved evaporation temperature control and Temperature Coupling Mode of the Vanquish CAD P series significantly enhance HPLC-CAD performance for saturated fatty acid analysis. Operating at 25 °C EvapT with coupled CDM temperature yields optimal S/N and demonstrates robust long-term stability, facilitating reliable FA quantitation in complex formulations.

References

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