An Improved Global Method for the Quantitation and Characterization of Lipids by High Performance Liquid Chromatography and Corona Charged Aerosol Detection

Importance of the Topic

Analysis of lipid mixtures spanning paraffins, free fatty acids, fatty alcohols, esters and acylglycerols is critical across biotechnology, food science, pharmaceuticals and industrial QA/QC. Conventional gas chromatography methods often require laborious derivatization while standard HPLC with UV detection lacks sensitivity for non-chromophoric lipids. A universal high-performance liquid chromatography (HPLC) method paired with charged aerosol detection (CAD) offers broad applicability, high sensitivity and a wide dynamic range for seamless, derivatization-free lipid profiling in complex matrices.

Study Objectives and Overview

This work aimed to develop and validate a single, global HPLC-CAD protocol for high-resolution separation and quantitation of multiple lipid classes in one analysis. Using a Thermo Scientific Accucore C18 column and the Corona ultra RS detector, the study assessed selectivity, calibration linearity, sensitivity and precision across five lipid families. The method was applied to two complex oil samples—algal oil and certified emu oil—to demonstrate its real-world performance.

Methodology and Instrumentation

Standard and sample preparation:

• Stock standards dissolved at 10 mg/mL in methanol/chloroform (1:1), adjusting to 1:3 for highly hydrophobic paraffins.
• Samples dissolved at 10 mg/mL in methanol/chloroform (1:1) or methanol/tetrahydrofuran (1:1), then centrifuged at 10 000 g for 3 min.

Instrument setup:

• HPLC system: Thermo Scientific Dionex UltiMate 3000 DGP-3600RS pump, WPS-3000RS autosampler, TCC-3000RS column oven at 40 °C.
• Column: Accucore C18, 2.6 µm, 3.0×150 mm.
• Mobile phases: A = MeOH/H₂O/Acetic acid (600:400:4), B = THF/ACN (50:950), C = Acetone/ACN (900:100).
• Gradient: 90% A initial, ramping to mixtures of B and C over 65 min; flow rate 1.0–1.5 mL/min; injection 2–10 µL.
• Detector: Corona ultra RS charged aerosol; nebulizer 15 °C; filter 3; data rate 10 Hz; power function 1.00.
• Data processing: Thermo Scientific Chromeleon 7.1 SR1.

Main Results and Discussion

Separation and selectivity:
All analytes within each lipid class and between classes were baseline resolved using the ternary gradient. Five classes—paraffins, fatty alcohols, fatty acids, esters and acylglycerols—were eluted in a single 65 min run.

Calibration and sensitivity:
Quadratic calibration curves covering 10–10 000 ng on-column were generated by fitting inverted axis detector response, with correlation coefficients (r²) > 0.996. Limits of quantitation ranged from 2 to 20 ng for most compounds. Application of class-specific power function values enabled linearization when required.

Sample applications:

• Algal oil: Complex profile resolved across paraffins to high-molecular-weight triglycerides.
• Emu oil: TAG distribution grouped by equivalent carbon number (ECN) matched certified specifications; major ECN-44 to ECN-48 triglycerides recovered at 86–103%, demonstrating quantitative capability.

Benefits and Practical Applications

• Universal profiling of diverse lipid classes in one method without derivatization.
• High sensitivity to single-digit nanogram levels and uniform detector response across chemical classes.
• Wide dynamic range supporting quantitative assays in food, biofuel, cosmetic and clinical laboratories.
• Method and mobile phases compatible with mass spectrometry for peak identification and lipidomics workflows.

Future Trends and Potential Applications

Integration with high-resolution MS will enable detailed lipid structure elucidation and isomer discrimination. Miniaturized and fast-gradient versions could increase throughput in screening laboratories. Advanced data processing and automation will further streamline lipidomics and QC workflows in regulated and research settings.

Conclusion

This improved global HPLC-CAD method using a core-shell C18 column and charged aerosol detection delivers sensitive, uniform and high-resolution quantitation of multiple lipid classes in a single analysis. It offers robust performance for complex samples, broad dynamic range, and compatibility with MS, making it a versatile tool in analytical chemistry and industry applications.

References

• Thermo Fisher Scientific. An Improved Global Method for the Quantitation and Characterization of Lipids by HPLC and Corona Charged Aerosol Detection. Poster PN70533, 2013.
• Emushop.com. Specifications of triacylglycerol content of emu oil by AOCS HPLC method Ce 5b-89. Accessed February 12, 2013.