Analysis of plant stanyl fatty acid esters in enriched margarine using an on-line coupled Agilent 1220 Infinity LC-7890 GC system

Importance of the topic

Plant steryl and stanyl fatty acid esters are widely incorporated into functional foods such as enriched margarines and yogurts for their cholesterol‐lowering effects. Reliable quantification of these esters in complex lipid matrices is essential for quality control, nutritional labeling and regulatory compliance. Traditional off‐line fractionation techniques can be laborious and prone to sample loss or contamination. The development of an on‐line coupled liquid chromatography–gas chromatography (LC–GC) approach streamlines analysis and improves repeatability.

Objectives and study overview

This study aimed to establish and validate a fully on‐line LC–GC/FID method for direct quantification of intact plant stanyl fatty acid esters in commercial margarine without prior purification. Key goals included demonstrating linearity and precision of the method, comparing solvent vent vs loop‐type interfaces, and evaluating method robustness over multiple transfers.

Methodology and instrumentation used

Sample preparation involved weighing 20–40 mg of enriched margarine, spiking with cholesteryl palmitate internal standard, extracting lipids with n-hexane/MTBE, and filtering. A 2 µL aliquot was injected onto a silica LC column (Eurospher-100Si, 250×2 mm, 5 µm) with isocratic elution (n-hexane/MTBE 96:4, 0.2 mL/min at 27 °C). After 4.25 min separation, the stanyl ester‐rich fraction was transferred on‐line into the GC via a 2-position/6-port valve and a multimode inlet in solvent vent mode for solvent evaporation.

Used instrumentation

• Agilent 1220 Infinity LC system
• Agilent 7890A GC system with PTV multimode inlet
• 2-position/6-port switching valve with 200 µL loop
• LC column: Eurospher-100Si (250×2 mm, 5 µm)
• GC column: Restek Rtx-200MS (30 m×0.25 mm, 0.1 µm)
• FID detector

Main results and discussion

The on‐line LC–GC method exhibited excellent linearity (R² 0.995–0.999) across five calibration levels (0.2–1.0 µg total stanyl esters). Repeatability, assessed by ten consecutive injections, yielded coefficients of variation below 9% for all nine individual esters. The solvent vent interface maintained chromatographic resolution even after 600 transfers, outperforming loop‐type coupling by reducing solvent load in the GC inlet.

Benefits and practical applications

By integrating fractionation and separation in a single automated workflow, the method minimizes sample handling, lowers risk of contamination and improves throughput. Its robust performance makes it suitable for routine quality assurance of functional foods, nutritional studies and compliance testing of sterol‐enriched products.

Future trends and applications

Advancements may include coupling high‐resolution mass spectrometry for structural confirmation, miniaturizing interfaces for reduced solvent consumption, and extending the approach to other lipid ester classes. Integration with laboratory informatics could enable fully automated reporting and data management.

Conclusion

The on‐line coupled Agilent 1220 LC–7890 GC system offers a reliable, precise and efficient solution for quantifying plant stanyl fatty acid esters in enriched margarine. Its high linearity, repeatability and reduced solvent load support its adoption in routine analytical settings.

References

• A. Barnsteiner et al., J. Agric. Food Chem. 2011, 59, 5204–5214
• P. Dugo et al., LC–GC Eur. 2003, 16, 35–43
• T. Hyoetylaeinen, M.-L. Riekkola, J. Chromatogr. A 2003, 1000, 357–384
• R. Esche et al., Agilent Technical Overview 2011, publication 5990-8025EN