LC-FLD analysis of 4 PAHs in olive oil samples using AZURA® GPC Cleanup System

Importance of Topic

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants known for their carcinogenic and mutagenic properties. Dietary intake is a major exposure route for non-smokers, and edible oils can accumulate PAHs during seed drying, extraction, and processing steps. Regulatory authorities in the EU have set maximum limits for benzo(a)pyrene (BaP) and the sum of four marker PAHs (PAH4) in oils and fats, driving the need for sensitive and reliable analytical cleanup and detection methods.

Objectives and Study Overview

This work aims to develop and validate a sample cleanup protocol for olive oil prior to high-performance liquid chromatography with fluorescence detection (HPLC-FLD), using the AZURA® GPC Cleanup System. The method targets quantification of BaP and three additional PAHs (benzo(a)anthracene, benzo(b)fluoranthene, chrysene) in accordance with Commission Regulation (EU) No 835/2011. Four commercial olive oil samples were processed and analyzed to assess method performance and compliance.

Methodology and Instrumentation

Sample Preparation and GPC Cleanup:

• Weighed 150 mg of olive oil, diluted to 2 mL with cyclohexane: dichloromethane (70:30, v/v).
• Ultrasonic mixing and filtration through a 0.45 μm PTFE syringe filter.
• AZURA® GPC Cleanup System procedure: loop rinsing with mobile phase, sample loading, fraction collection via automatic valve switching.
• Concentration under nitrogen, reconstitution in HPLC mobile phase.

Chromatographic Analysis:

• HPLC column: Nucleosil 100-5 C18 PAH, 150 × 4 mm, 5 μm, with precolumn.
• Mobile phase gradient of water (A) and acetonitrile (B), flow rate 1.2 mL/min, 30-minute runtime at 20 °C.
• Fluorescence detection with excitation/emission settings optimized for each analyte.

Použitá instrumentace

AZURA® GPC Cleanup System:

• Pump AZURA P 6.1L
• Autosampler AS 6.1L
• Fractionation valve

HPLC-FLD System:

• Pump AZURA P 6.1L
• Autosampler AZURA AS 6.1L
• CT 2.1 column thermostat
• Fluorescence Detector RF-20A
• Column: Nucleosil C18 PAH

Main Results and Discussion

The method exhibits high linearity (R² ≥ 0.9989) across calibration ranges (0.1–20 μg/L). Limits of detection (LODs) for individual PAHs ranged from 0.01 to 0.04 μg/L; limits of quantification (LOQs) from 0.04 to 0.13 μg/L, well below EU maximum levels (2.0 μg/kg for BaP, 10.0 μg/kg for PAH4). Intra- and inter-day precision (RSD) remained under 5%. Recoveries from spiked olive oil were between 60% and 100%, meeting regulatory criteria. Analysis of four commercial samples showed all PAH4 sums under 10 μg/kg and non-detectable BaP levels, confirming method suitability and compliance.

Benefits and Practical Applications

• Rapid and automated sample cleanup reduces manual handling and solvent use.
• High sensitivity and precision ensure compliance with strict regulatory limits.
• Applicable to routine quality control in food safety laboratories.
• Flexible configuration allows adaptation to other lipophilic contaminants.

Future Trends and Potential Applications

Advancements may include coupling GPC cleanup with mass spectrometry for broader analyte screening, miniaturization of cleanup columns to reduce solvent consumption, integration into fully automated workflows, and exploration of greener mobile phases. Real-time monitoring and on-site portable systems could further streamline food safety assessments.

Conclusion

The AZURA® GPC Cleanup System combined with HPLC-FLD provides a robust, high-throughput approach for quantifying PAH4 in olive oil. The method meets EU regulatory requirements, offering excellent sensitivity, precision, and recovery. Its implementation enhances laboratory efficiency and supports ongoing food safety monitoring.

Reference

[1] Vasudha Bansal, Ki-Hyun Kim. Environment International 84 (2015) 26–38.
[2] Moon HB, Kannan K, Lee SJ, Ok G. Arch. Environ. Contam. Toxicol. 51 (2006) 494–502.
[3] Commission Regulation (EC) No 1881/2006, 19 Dec. 2006.
[4] Commission Regulation (EU) No 835/2011, 19 Aug. 2011.
[5] Commission Regulation (EU) No 836/2011, 19 Aug. 2011.