EuroResidue: IMPROVED SPE FOR LC-MS/MS DETERMINATION OF RACTOPAMINE IN PORCINE LIVER

Importance of the Topic

The detection of ractopamine residues in porcine liver is critical for ensuring compliance with food safety regulations and protecting public health. Ractopamine, a β-agonist used as a growth promoter, is authorized in only a few countries with strict maximum residue limits and banned elsewhere under zero-tolerance policies. Reliable, sensitive analytical methods capable of quantifying trace levels of ractopamine in complex matrices like liver tissue are essential for regulatory enforcement and international trade.

Objectives and Study Overview

This study aimed to develop and validate an improved sample preparation workflow for the determination of ractopamine in porcine liver using LC–MS/MS. Key goals included:

• Increasing sample throughput and precision by transitioning from manual SPE cartridges to a 96-well plate format.
• Demonstrating enhanced sensitivity and repeatability at regulatory target levels (0.5 µg/kg and below).
• Comparing performance between a traditional vacuum manifold and the automated Otto SPEcialist positive pressure system.

Methodology and Instrumentation

Sample Preparation and SPE Clean-Up:

• Extraction: Modified AOAC 2011.23 procedure with methanolic extraction of homogenized porcine liver.
• SPE Clean-Up: Comparison of Oasis MCX cartridges on a vacuum manifold versus Oasis MCX 96-well plate on the Otto SPEcialist positive pressure platform.
• SPE Protocol: Conditioning with 1 mL MeOH, loading 1.62 mL supernatant, washing with 1 mL MeOH, eluting with 0.8 mL 5% NH₄OH in MeOH.

Instrumentation:

• LC System: ACQUITY UPLC I-Class Plus with BEH C18 column (2.1 × 100 mm, 1.7 µm) at 40 °C.
• Mobile Phase: A = 0.1% formic acid in water; B = methanol; injection volume 4 µL.
• MS System: Xevo TQ-XS with electrospray ionization in positive mode; MRM transitions m/z 302.2 > 164.1 (CE 14 eV) and 302.2 > 121.0 (CE 22 eV); cone voltage 35 V.

Results and Discussion

Chromatographic analysis of spiked liver samples at 0.10 and 0.01 µg/kg demonstrated clear detection of ractopamine with the 96-well plate workflow. Recovery and repeatability data from six replicates at 0.1 µg/kg showed:

• Comparable mean recoveries between cartridge and plate methods.
• Significantly improved precision (lower relative standard deviation) with the Otto SPEcialist positive pressure system.

The automated plate format reduced variability associated with manual handling and minimized the risk of cross-contamination.

Benefits and Practical Applications

The optimized SPE approach offers:

• Higher throughput through 96-well plate processing.
• Enhanced repeatability and sensitivity at trace levels.
• Reduced solvent consumption and elimination of filter and tube consumables, resulting in cost savings.
• Lower risk of cross-contamination and improved laboratory efficiency.

Future Trends and Possibilities

Advances likely to shape this field include:

• Further integration of SPE automation with LC–MS/MS workflows for fully robotic sample preparation.
• Development of novel mixed-mode sorbents for broader application to other β-agonists and veterinary drugs.
• Miniaturization and high-throughput microscale SPE formats to reduce sample and solvent volumes.
• Coupling with high-resolution mass spectrometry for non-targeted screening and multi-residue analysis.

Conclusion

The implementation of the Otto SPEcialist positive pressure system combined with Oasis MCX 96-well plates provides a robust, high-throughput, and cost-effective workflow for reliable LC–MS/MS quantification of ractopamine in porcine liver at sub-µg/kg levels. This enhanced method meets regulatory sensitivity requirements and offers significant improvements in precision and operational efficiency.