Determination of Beta Agonists in Animal Tissues and Urine Using Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

Importance of the Topic

Beta-adrenergic receptor agonists are synthetic compounds used illicitly to promote growth and alter body composition in livestock. Regulatory agencies worldwide impose strict limits or bans on these residues due to potential human health risks. Reliable analytical methods are essential for official controls, trade compliance, and food business operators’ due diligence.

Objectives and Overview of the Study

This work evaluates a targeted liquid chromatography–tandem quadrupole mass spectrometry method for simultaneous determination of 16 β-agonists in animal tissues and urine. The aim is to demonstrate the performance of the Waters ACQUITY UPLC I-Class System coupled with a Xevo TQ-S micro instrument for both screening and confirmatory analysis across diverse matrices, meeting international regulatory requirements and challenging pre-export action levels.

Methodology and Instrumentation

• Sample Preparation: Meat and fish samples were blended with aqueous buffer/methanol, subjected to enzyme hydrolysis to cleave conjugates, then cleaned by mixed-mode SPE. Urine samples underwent enzyme hydrolysis followed by SPE. Stable isotope-labeled analogues were spiked prior to extraction for quality control.
• Chromatography: ACQUITY UPLC I-Class with FTN autosampler and CORTECS UPLC C18+ column (2.1×100 mm, 1.6 µm). Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B). Gradient elution over 11 min at 0.6 mL/min, column at 40 °C, sample tray at 10 °C, 5 µL injection.
• Mass Spectrometry: Xevo TQ-S micro triple quadrupole, ESI positive mode, capillary 1.0 kV, desolvation 600 °C, gas flow 1000 L/hr. MRM acquisition with optimized transitions and collision energies. Data processed with MassLynx 4.1 and TargetLynx XS.

Used Instrumentation

• ACQUITY UPLC I-Class System with FTN Autosampler
• CORTECS UPLC C18+ Column, 2.1×100 mm, 1.6 µm
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• MassLynx 4.1 and TargetLynx XS Application Manager Software

Main Results and Discussion

• Sensitivity and Selectivity: Achieved standard screening target concentrations from 0.05 to 5 µg/kg, sufficient to meet Codex, EU, US, and stringent Russian action levels. No significant interferences observed except a minor isobaric overlap for clenproperol in liver.
• Precision and Carryover: Replicate injections (n=25) of matrix-extracted standards yielded RSDs <5% for most analytes, improved to <4% with internal standards. Carryover was <1% except 1.3% for salmeterol, consistent with background signals.
• Linearity and Accuracy: Calibration curves in bovine liver matrix exhibited r²>0.99 for clenbuterol (0.05–1.0 µg/kg) and ractopamine (0.25–5.0 µg/kg). Recoveries in proficiency test materials and spiked blanks averaged 100–101% (RSD<3.7%).
• Identification Criteria: Ion ratio deviations and retention time shifts fell within EU Decision 2002/657/EC tolerances (±20% ion ratio; ±2.5% retention time).

Benefits and Practical Applications

• Comprehensive screening and confirmation workflow for official control laboratories and food producers.
• Meets multiple regulatory frameworks and stringent pre-export testing specifications.
• Applicable to a wide variety of matrices including muscle, offal, fish, and urine.

Future Trends and Applications

Anticipated developments include automation of sample processing, expansion to emerging β-agonist analogues, coupling with high-resolution mass spectrometry for enhanced confirmatory power, and adaptation of the workflow for processed foods and complex matrices.

Conclusion

The validated UPLC-MS/MS method employing stable isotope internal standards provides high sensitivity, selectivity, and precision for the determination of 16 β-agonists in diverse animal matrices. It supports both rapid screening and confirmed quantification to ensure compliance with global regulatory and trade requirements.

Reference

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