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

Significance of the topic

Plant steryl and stanyl esters are functional lipid ingredients known for their cholesterol-lowering effects in foods such as margarines and yogurts.
Accurate measurement of these esters is critical for product quality, nutritional labeling, and regulatory compliance.
Conventional offline cleanup methods (TLC, SPE) are labor-intensive and risk sample loss or contamination.
Integrating liquid chromatography with gas chromatography in an on-line setup enables seamless fractionation and analysis, improving throughput and reproducibility.

Objectives and study overview

The study aimed to develop and validate an on-line LC-GC/FID method for direct determination of intact plant stanyl fatty acid esters in enriched margarine.
Key goals included assessing calibration linearity, repeatability, robustness over multiple injections, and comparing performance against loop-type interfaces.

Methodology

Margarine samples (20–40 mg) were spiked with cholesteryl palmitate internal standard, extracted with n-hexane/MTBE (3:2 v/v), sonicated, filtered (0.45 µm) and diluted 1:5.
The Agilent 1220 Infinity LC (silica column, 250 × 2 mm, 5 µm; 27 °C; n-hexane/tert-butyl methyl ether 96:4; 0.2 mL/min) was coupled on-line to an Agilent 7890A GC via a 2-position/6-port valve with a 200 µL loop.
Transfer of the stanyl ester-containing fraction occurred at 4.25 min using the multimode inlet in solvent-vent mode.
GC separation employed a Restek Rtx-200MS column (30 m × 0.25 mm, 0.1 µm) with hydrogen carrier and a temperature program up to 340 °C; detection by FID.

Instrumentation

• Agilent 1220 Infinity LC with UV detector (205 nm)
• Agilent 7890A GC with Multimode Inlet (solvent vent mode)
• Agilent 2-position/6-port switching valve (200 µL loop)
• Eurospher-100Si LC column (250 × 2 mm, 5 µm)
• Restek Rtx-200MS GC column (30 m × 0.25 mm, 0.1 µm)

Main results and discussion

Nine individual plant stanyl esters were baseline-resolved by carbon chain length and unsaturation level.
Calibration over 0.2–1.0 µg injected yielded correlation coefficients (R²) between 0.995 and 0.999, demonstrating excellent linearity.
Repeatability tests (10 consecutive injections) showed relative standard deviations below 9% for all analytes.
No loss of chromatographic resolution was observed after over 600 on-line transfers.
Quantification in commercial enriched margarine indicated total plant stanyl ester content of 13.3 ± 0.5 g per 100 g sample, consistent with the labeled formulation.

Benefits and practical applications

• Eliminates offline cleanup steps and associated sample handling errors
• Reduces risk of analyte loss and contamination in a closed-system workflow
• Combines fractionation and analysis in a single automated run for high throughput
• Delivers robust, reproducible results suitable for routine quality control in food and nutrition laboratories

Future trends and potential applications

On-line LC-GC techniques may be extended to other complex lipid classes, such as tocopherol or phospholipid esters.
Coupling with high-resolution mass spectrometry could further enhance selectivity and structural elucidation.
Advances in microfluidics and miniaturized interfaces may improve solvent consumption and increase lab automation.

Conclusion

The Agilent on-line LC-GC/FID system provides a streamlined, accurate, and repeatable method for direct analysis of plant stanyl fatty acid esters in enriched margarine.
This approach simplifies sample preparation, maintains chromatographic performance over many injections, and supports routine quality assurance in the functional foods industry.

References

• A. Barnsteiner, T. Lubinus, A. di Gianvito, W. Schmid, K.–H. Engel, J. Agric. Food Chem. 2011, 59 (10), 5204–5214.
• P. Dugo, G. Dugo, L. Mondello, LC-GC Eur. 2003, 16 (12a), 35–43.
• T. Hyoetylaeinen, M.–L. Riekkola, J. Chromatogr. A 2003, 1000 (1–2), 357–384.
• R. Esche, A. Barnsteiner, K.–H. Engel, W. Kohlert, S. Fenzel, Agilent Technical Overview 5990-8025EN, 2011.