Determination of Sterols in Olive Oil using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID

Significance of the Topic

Olive oil, prized for its nutritional and health benefits, faces widespread fraud and adulteration due to its high market value. Accurate profiling of sterols and triterpene diols in extra virgin olive oil (EVOO) is essential for verifying authenticity, ensuring safety, and protecting both consumers and reputable producers.

Objectives and Study Overview

The main goal of this work was to evaluate an improved analytical protocol for quantifying sterols, erythrodiol, and uvaol in olive oil. The study replaced traditional liquid–liquid extraction and thin-layer chromatography with supported liquid extraction (SLE) on diatomaceous earth and solid-phase extraction (SPE) cleanup, followed by gas chromatography–flame ionization detection (GC-FID). Parallel analysis of pure EVOO and a 50:50 EVOO–canola blend demonstrated method performance and the ability to detect adulteration.

Methodology and Instrumentation

The procedure consisted of:

• Saponification of oil (200 mg) with 2 M KOH in 95 % ethanol and internal standard addition (cholestanol).
• Supported liquid extraction on a Strata DE SLE cartridge to isolate the unsaponifiable fraction using diethyl ether.
• SPE cleanup on a Strata Si-1 silica cartridge, with activation by ethanolic KOH, washing with hexane/ether mixtures, and elution of target analytes.
• Derivatization with BSTFA/pyridine and analysis by isothermal GC-FID on a Zebron ZB-5PLUS column (30 m × 0.25 mm, 0.25 µm) at 260 °C for 70 min.
  

Instrumentation Used

• Strata DE SLE cartridge (60 cc, 20 mL load capacity) with anhydrous sodium sulfate drying tube.
• Strata Si-1 SPE cartridge (1 g, 6 mL) activated with potassium hydroxide in ethanol.
• GC-FID system fitted with Zebron ZB-5PLUS column and single taper Z-liner.

Main Results and Discussion

Chromatograms of standard mixtures and real EVOO samples showed full resolution of nine analytes, with a clear distinction between pure EVOO and a 50:50 EVOO–canola adulterated blend. Method accuracy, assessed through fortified EVOO samples, yielded recoveries between 75 % and 134 % for key sterols and diols, comparable or superior to IOC reference procedures. According to IOC criteria, the pure EVOO met all eight sterol standards, while the adulterated sample failed four criteria (including brassicasterol and campesterol limits), confirming robust detection of adulteration. Throughput improved markedly, allowing up to 16 samples processed in parallel over two days without laborious glassware handling.

Benefits and Practical Applications

• Reduced solvent use and elimination of emulsions and TLC steps.
• Higher sample throughput with parallel SLE extractions.
• Improved reproducibility and ease of cleanup without separatory funnels.
• Sensitive and quantitative discrimination of olive oil authenticity.

Future Trends and Potential Applications

Further integration with automated sample-prep platforms, coupling to mass spectrometry for enhanced selectivity, and miniaturized cartridges for field screening may advance rapid quality control. Expansion of the method to detect geographical origin markers and additional adulterants is also anticipated.

Conclusion

The combined SLE–SPE–GC-FID workflow offers a reliable, high-throughput alternative to traditional IOC methods for sterol analysis in olive oil. It delivers accurate quantitation, effective adulteration detection, and streamlined laboratory operations.

References

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2. International Olive Council. Determination of the Composition and Content of Sterols and Triterpene Dialcohols by Capillary Column Gas Chromatography; COI/T.20/Doc. 30/Rev. 1, 2013.
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4. Azadmard-Damirchi S, Dutta PC. Novel solid-phase extraction method to separate 4-desmethyl-, 4-monomethyl-, and 4,4′-dimethylsterols in vegetable oils. J Chromatogr A. 2006;1008:183–187.
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