Analysis of Macrolide Antibiotics in Bovine Muscle Tissue Using Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

Significance of the Topic

The widespread use of macrolide and lincosamide antibiotics in veterinary practice requires reliable monitoring of residues in meat products to protect consumer health. Regulatory agencies establish maximum residue limits (MRLs) to prevent allergic reactions and antibiotic resistance. A robust, high-throughput analytical approach is essential for compliance testing and public safety.

Objectives and Study Overview

This study aimed to develop and validate a single-run liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of 18 macrolide veterinary drugs in bovine muscle. Validation followed European Commission Decision 2002/657/EC, demonstrating suitability for routine screening and potential extension to confirmatory analysis.

Methodology and Instrumentation

Sample preparation combined buffer extraction, protein precipitation, and solid-phase extraction (SPE) using Oasis PRiME HLB cartridges. Matrix-matched calibration levels ranged from half to 1.5× MRLs. Chromatographic separation employed an ACQUITY UPLC I-Class PLUS system with FTN sample manager and an ACQUITY HSS T3 column (2.1×100 mm, 1.8 µm). Mobile phases consisted of water and acetonitrile with 0.1% formic acid under a stepped gradient. Detection used a Xevo TQ-S micro triple quadrupole MS in positive electrospray ionization mode. Two MRM transitions per analyte with automatic dwell-time adjustment ensured at least 12 data points per peak. Data processing utilized MassLynx and TargetLynx XS.

Main Results and Discussion

Chromatographic run time was 11 minutes with analytes eluting between 1.9 and 7.3 minutes. No significant matrix interferences were observed in blank extracts. Calibration curves in matrix achieved R² > 0.99 (one at 0.98) using quadratic 1/x weighting; residuals remained < 20%. Mean recoveries ranged from 81 to 111% (CP-60,300 marginally above target), repeatability (RSDr) and reproducibility (RSDRL) values were < 12% for all compounds. Decision limits (CCα) and detection capabilities (CCβ) fell at or below regulatory thresholds, demonstrating sensitivity well below MRLs.

Benefits and Practical Applications

• Simultaneous quantitation of multiple macrolides in a single run
• Fast cycle time and minimal injection volume
• Low detection limits enabling dilution to mitigate matrix effects
• Compliance with EU regulatory guidelines for screening purposes

Future Trends and Potential Applications

Advancements may include adaptation to other tissue types and species, integration of hydrolysis steps for confirmatory analysis of tulathromycin, further miniaturization and automation of sample cleanup, and deployment of high-resolution mass spectrometry for broader screening of emerging veterinary drug residues. Integration with antibiotic-resistance surveillance programs will enhance food safety monitoring.

Conclusion

The validated UPLC-MS/MS method delivers rapid, sensitive, and robust multi-residue analysis of macrolide antibiotics in bovine muscle, meeting stringent regulatory criteria. Its high throughput and reliable performance make it suitable for routine residue monitoring and quality control in food safety laboratories.

Applied Instrumentation

• Waters ACQUITY UPLC I-Class PLUS with FTN Sample Manager
• ACQUITY HSS T3 column (2.1×100 mm, 1.8 µm)
• Waters Xevo TQ-S micro triple quadrupole mass spectrometer
• MassLynx Software and TargetLynx XS Application Manager
• Oasis PRiME HLB SPE cartridges

References

1. Regulation (EU) 2019/6 of the European Parliament and of the Council on veterinary medicinal products. Official Journal of the European Union, L4, 7 Jan 2019, pp. 43–167.
2. Commission Regulation (EU) No 37/2010 on pharmacologically active substances and MRL classification in food of animal origin. Official Journal of the European Union, L15, 20 Jan 2010, pp. 1–72.
3. 21 C.F.R. § 556.292 (2018). Code of Federal Regulations, Title 21 – Food and Drugs.
4. World Health Organization. WHO Guidelines on Use of Medically Important Antimicrobials in Food-Producing Animals. Geneva: WHO; 2017.
5. Commission Decision 2002/657/EC implementing Directive 96/23/EC on analytical method performance and result interpretation. Official Journal of the European Union, L221, 17 Aug 2002, pp. 8–36.