Beta-agonists in Pork Using In-line SPE

Significance of the Topic

The illegal use of beta-agonists in livestock production and athletic doping raises significant food safety and public health concerns. Monitoring trace levels of compounds such as clenbuterol ensures regulatory compliance and protects consumers from toxic exposures.

Objectives and Study Overview

This work presents the development of an automated inline solid-phase extraction (SPE) coupled to reversed-phase high-performance liquid chromatography (RP-HPLC) for simultaneous determination of five beta-agonists (salbutamol, cimaterol, ritodrine, ractopamine and clenbuterol) in pork samples at low microgram-per-kilogram concentrations.

Methodology and Instrumentation

A mixed-mode cation-exchange SPE guard cartridge (Thermo Scientific Acclaim Mixed-Mode WCX-1, 3.0 × 10 mm) was integrated inline before an analytical column (Thermo Scientific Acclaim C30, 2.1 × 150 mm, 3 µm). An accelerated solvent extraction (ASE) system was used to extract 5 g of pork with acetonitrile:isopropanol (4:1). After chilling and fat precipitation, the extract was filtered and directly loaded onto the SPE cartridge via a 10-port auxiliary valve configured for reverse elution. Dual-pump gradient elution on a Thermo Scientific Dionex UltiMate 3000 RSLC system employed:

• Left pump: methanol and 5 mM ammonium formate (pH 7.5)
• Right pump: acetonitrile, water and 12.5 mM ammonium formate/50 mM formic acid (pH 3.1)

Flow rates were 0.40 mL/min per pump, injection volume 100 µL, column temperature 25 °C. Detection used a diode array detector at 210 nm (2 Hz, 0.5 s response time).

Main Results and Discussion

The inline SPE step provided effective removal of lipids and other interferences while concentrating target analytes. Chromatographic separation achieved baseline resolution of all five beta-agonists within a 15-minute run. Method performance demonstrated satisfactory recoveries and reproducibility at regulatory threshold levels.

Benefits and Practical Applications

• Automation of SPE minimizes manual handling, solvent evaporation and sample preparation time.
• Mixed-mode WCX cleanup enhances selectivity for cationic analytes in complex meat matrices.
• Rapid, high-throughput analysis supports routine screening in food safety and anti-doping laboratories.

Future Trends and Opportunities

Emerging mixed-mode and monolithic stationary phases may further improve extraction efficiency and speed. Coupling inline SPE with high-resolution mass spectrometry could expand detection capabilities to novel or emerging residues in food and biological samples.

Conclusion

The described inline SPE–RP-HPLC method provides a robust, sensitive and high-throughput platform for trace determination of beta-agonists in pork, meeting stringent regulatory and anti-doping requirements.