Rapid Sample Preparation for Determination of PAHs in Wild-Caught Avian Eggs Utilizing QuEChERS Extraction and Ostro Pass-through 96-well Plate Cleanup Followed by UPLC-UV Analysis

Significance of the Topic

Polycyclic aromatic hydrocarbons (PAHs) are persistent environmental pollutants with known carcinogenic and mutagenic properties. Their lipophilic nature leads to bioaccumulation in avian eggs at concentrations far exceeding those in other tissues. Rapid, reliable determination of PAHs in egg samples is crucial for ecological risk assessment, wildlife conservation, and environmental monitoring following oil spills or chronic pollution.

Objectives and Study Overview

This study aimed to develop and validate a high-throughput sample preparation workflow for the quantification of 16 EPA priority PAHs in wild-caught avian eggs. By combining dispersive QuEChERS extraction with novel Ostro™ Pass-through 96-well plate cleanup and subsequent UPLC-UV analysis, the total preparation time was reduced from several days to approximately three hours.

Methodology and Instrumentation

Homogenized egg samples (≈1 g) were spiked with deuterated surrogates, extracted with 1% formic acid in acetonitrile, and treated with QuEChERS salts (magnesium sulfate/sodium acetate). The organic supernatant (0.5 mL) underwent protein and phospholipid removal via Ostro™ Pass-through plates under 10–15 psi. Elution was completed with an additional 0.25 mL acetonitrile rinse. Extracts were directly analyzed by UPLC-UV without pre-concentration.

Used Instrumentation

• Waters ACQUITY UPLC system with CSH C18 column (2.1×100 mm, 1.7 μm)
• ACQUITY UPLC PDA detector (200–450 nm scan, 1.2 nm resolution)
• Waters Ostro™ Pass-through 96-well cleanup plates
• MassLynx v4.1 and QuanLynx software for data acquisition and processing

Main Results and Discussion

Calibration curves for all 16 PAHs achieved correlation coefficients (R2) ≥0.9972 over three orders of magnitude (5–500 ng/mL). Method detection limits ranged from 1.7 to 8.7 ng/mL without additional concentration steps. Recoveries at 25 ng/mL spikes were 87.2–117.1% (≤2.8% RSD) and at 400 ng/mL spikes 89.3–105.5% (≤3.5% RSD). Analysis of 20 wild-caught eggs produced quantifiable PAH levels up to 4,412 ng/g. Ostro™ cleanup effectively removed matrix interferences, enabling clear chromatographic baselines and high sensitivity.

Benefits and Practical Applications

• Throughput increased by reducing sample prep time from days to hours.
• Minimal solvent usage and small sample mass enhance sustainability and cost-efficiency.
• Robust, reproducible cleanup ensures reliable data for environmental monitoring, toxicology studies, and regulatory compliance.

Future Trends and Potential Applications

Further extensions may include coupling this workflow with tandem MS or high-resolution mass spectrometry to improve selectivity and lower detection limits. Automation of the QuEChERS–Ostro protocol could facilitate large-scale monitoring. Adaptation to other complex matrices (e.g., tissues, sediments) and field-deployable kits represents promising directions.

Conclusion

The integration of QuEChERS extraction and Ostro™ Pass-through 96-well plate cleanup with UPLC-UV analysis offers a rapid, sensitive, and reproducible approach for PAH determination in avian eggs. This method outperforms traditional GPC and SPE workflows in speed, solvent consumption, and ease of use, making it an attractive alternative for environmental and wildlife research.

References

1. Yeudakimau AV, Provatas AA, Perkins CR, Stuart JD. Solid Phase Extraction and QuEChERS Sample Preparation Methods for Rapid Screening of Polycyclic Aromatic Hydrocarbons in Avian Blood and Egg Tissue by UPLC-UV. Analytical Letters. 46:999–1011 (2013).
2. Kumari R, Chaturvedi P. Optimization and Validation of an Extraction Method for the Analysis of Polycyclic Aromatic Hydrocarbons in Chocolate Candies. Journal of Food Science. 71:34–40 (2012).
3. Ma J, Xiao R, et al. Determination of 16 Polycyclic Aromatic Hydrocarbons in Environmental Water Samples by Solid-Phase Extraction Using Multi-Walled Carbon Nanotubes Coupled with GC-MS. Journal of Chromatography A. 1217:5462–5469 (2010).
4. Pereira MG, Walker AL, et al. Polycyclic Aromatic Hydrocarbons (PAHs) in Eggs from Gannets, Golden Eagles and Merlins. Organohalogen Compounds. 70:166–169 (2008).
5. Grova N, Salquebre G, et al. Determination of PAHs and OH-PAHs in Rat Brain by Gas Chromatography Tandem Mass Spectrometry. Chemical Research in Toxicology. 24:1653–1667 (2011).
6. Johnson YS. Determination of Polycyclic Aromatic Hydrocarbons in Edible Seafood by QuEChERS-Based Extraction and Gas Chromatography-Tandem Mass Spectrometry. Journal of Food Science. 77:131–137 (2012).
7. Gilgenast E, Boczkaj G, et al. Sample Preparation Procedure for the Determination of Polycyclic Aromatic Hydrocarbons in Petroleum Vacuum Residue and Bitumen. Analytical and Bioanalytical Chemistry. 401:1059–1069 (2011).
8. Wheaton JP, Chambers EE, Martin J, Fountain KJ. Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Clean-up Method. Waters Application Note 720004046en (2011).