Extraction of Sulfonamides from Chicken Liver using Supercritical Fluoroform (CHF3)

Importance of the Topic

Veterinary sulfonamides are widely used antimicrobial agents that can accumulate in animal tissues, including chicken liver. Their presence at trace levels poses food safety and regulatory concerns. Efficient extraction and cleanup methods are essential for reliable quantification and monitoring of these residues in complex biological matrices.

Objectives and Study Overview

This study aimed to develop and validate a supercritical fluid extraction approach using fluoroform (CHF3) modified with methanol to isolate three sulfonamides—sulfamethazine (SMZ), sulfadimethoxine (SDM) and sulfaquinoxaline (SQA)—from fortified chicken liver. The method was evaluated for recovery, reproducibility and cleanup efficiency compared to conventional procedures.

Methodology

• Sample Preparation: One gram of homogenized chicken liver was spiked with a known mixture of SMZ, SDM and SQA, then blended with 2 g of Spe-ed Matrix adsorbent.
• Extraction Vessel Assembly: Spe-ed Wool plugs were placed above and below the sample bed within a 10 mL extraction vessel; remaining void volume filled with additional adsorbent and compressed.
• Supercritical Extraction: Conducted at 6500 psi and 40 °C with a CHF3 flow of 2 L/min and methanol modifier at 200 µL/min for 30 minutes.
• Collection and Cleanup: Extracts were captured on C18 SPE cartridges and eluted with 5 mL of 50:50 phosphate buffer (0.5 M, pH 7.1) and methanol.
• Analysis: Eluates were analyzed by HPLC using a C18 column (250 × 4.6 mm, 5 µm) with a 60:40 phosphate buffer/methanol mobile phase at 1 mL/min and UV detection at 265 nm.

Used Instrumentation

• Spe-ed SFE-2 or Helix Supercritical Fluid Extraction System (Applied Separations)
• Methanol modifier pump
• Waters 6000 HPLC system
• C18 analytical column (250 × 4.6 mm, 5 µm)
• C18 SPE cartridges (Applied Separations) and Spe-ed Polypropylene Wool

Main Results and Discussion

Recovery for SMZ approached quantitative levels at 99.8 % with an RSD of 4 %. SDM exhibited 85.8 % recovery (RSD 12 %), while SQA yielded lower recovery at 31.2 % (RSD 14 %). The supercritical CHF3 extraction provided cleaner extracts with significantly reduced co-extracted lipids, simplifying downstream cleanup. The variability observed for SQA suggests further optimization may be needed for more polar or strongly protein-bound analytes.

Benefits and Practical Applications

• Accelerated extraction compared to traditional solvent methods, reducing analysis time.
• Minimized solvent usage and improved environmental profile through supercritical fluid technology.
• Enhanced selectivity and cleanliness of extracts, lowering interference in HPLC analysis.
• Adaptable to routine monitoring in food safety laboratories and regulatory compliance testing.

Future Trends and Applications

Ongoing developments in supercritical fluid modifiers and stationary phase chemistries may further improve extraction efficiency for a broader range of analytes. Integration with online coupling to HPLC or mass spectrometry could enable fully automated workflows. Exploration of alternative green solvents and mixed supercritical systems may enhance sustainability and performance in complex matrices.

Conclusion

The methanol-modified supercritical fluoroform extraction method offers a robust, efficient and environmentally friendly alternative for isolating veterinary sulfonamides from chicken liver. High recoveries, reduced lipid co-extraction and straightforward SPE cleanup make this approach well suited for routine analytical laboratories focused on food safety and residue monitoring.

Reference

• Ashraf-Khorassani M., Taylor L. Comparison of Supercritical CHF3 and CO2 and Methanol-modified CHF3 and CO2 for Extraction of Sulfonamides from Chicken Liver. Journal of AOAC International. 1996;79(5):1043–1049.