Analysis of Phospholipids in Natural Samples by Normal Phase HPLC and Corona Charged Aerosol Detection

Significance of the Topic

Phospholipids are vital amphiphilic molecules in cell membranes and functional ingredients in food, nutraceutical, and cosmetic products. Reliable quantitation of these non-volatile, non-chromophoric lipids ensures quality control, nutritional labeling accuracy, and supports research into lipid metabolism and formulation development.

Objectives and Study Overview

This study aimed to establish a robust normal-phase HPLC method combined with Corona™ Veo™ Charged Aerosol Detection (CAD) to measure six primary phospholipid classes—phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and lysophosphatidylcholine (LPC)—in natural samples such as egg yolk, lecithin, and krill oil. Key performance parameters including sensitivity, linear range, precision, and applicability to real matrices were evaluated.

Methodology and Instrumentation

The analytical workflow combined a Thermo Scientific™ Dionex™ UltiMate™ LPG3400-SD normal-phase HPLC system with a Hypersil GOLD™ Silica column (3 µm, 3 × 150 mm) at 50 °C and a Corona Veo SD CAD operated at 50 °C evaporator temperature. Mobile phases consisted of diethylamine-formate buffer (pH 3.0), 2-propanol, and iso-octane with a gradient flow of 0.2–0.8 mL/min. Sample preparation protocols involved solvent extraction with methanol/chloroform mixtures, protein precipitation, centrifugation, and filtration.

• HPLC system: LPG3400-SD pump, WPS3000 RS autosampler, TCC3000 RS column oven
• Column: Hypersil GOLD™ Silica, 3 µm, 3 × 150 mm
• Detector: Corona Veo SD Charged Aerosol Detector, 10 Hz data rate, 3.6 s filter, PowerFunction 1.40
• Mobile phases: A) 0.5% diethylamine-formate pH 3.0, B) 2-propanol, C) iso-octane

Main Results and Discussion

Calibration curves for PC, PE, PS, PI, and LPC showed excellent linearity (R2 ≥ 0.995) over four orders of magnitude (78–10 000 ng on column). Limits of detection ranged from 10 to 22 ng, with limits of quantitation between 31 and 67 ng. Analysis of egg yolk yielded a total phospholipid content of 86.6 mg/g, closely matching literature values obtained by 31P-NMR. Lecithin samples demonstrated 792 mg/g total phospholipids, while krill oil contained 44.1 mg/g, aligning within 15% of AOCS official method results. Chromatograms were free of significant interferences, and retention times allowed near-single peak resolution of target classes.

Benefits and Practical Applications

This NP-HPLC–CAD approach offers:

• High sensitivity and reproducibility for non-chromophoric lipids
• Wide dynamic range without compound-specific response calibration
• Compatibility with gradient elution and simple operation compared to mass spectrometry
• Direct analysis of complex food and feed samples with minimal cleanup

Future Trends and Opportunities

Advancements may include coupling CAD detection with high-resolution mass spectrometry for structural lipidomics, automation of sample preparation for high-throughput screening, and expansion to other polar lipid classes. Integration with chemometric data analysis could further improve profiling of complex biological matrices.

Conclusion

The developed normal-phase HPLC method combined with charged aerosol detection provides a sensitive, precise, and user-friendly platform for routine quantitation of major phospholipids in diverse natural samples. It delivers reliable compositional data that align well with established analytical techniques.

References

1. El Bagir NM, et al. Lipid Composition of Egg Yolk and Serum in Laying Hens Fed Diets Containing Black Cumin (Nigella sativa). Int J Poult Sci. 2006;5(6):574–578.
2. Walker LA, Wang T, Xin H, Dokle D. Supplementation of Laying-Hen Feed with Palm Tocos and Algae Astaxanthin for Egg Yolk Nutrient Enrichment. J Agric Food Chem. 2012;60:1989–1999.
3. AOCS Official Method Ja 7c-073. Analysis of Phospholipids in Krill Oil. 2014.