The Analysis of Tetracycline and Sulfonamide Antibiotics in Shrimp Tissue using Liquid Chromatography Tandem Quadrupole Mass Spectrometry

Importance of the topic

Antibiotics such as tetracyclines and sulfonamides are extensively used in aquaculture to maintain animal health and boost yields, raising concerns over residue accumulation in seafood. Uncontrolled residues can pose health risks to consumers and promote antibiotic resistance. Reliable, sensitive detection of multiple drug classes in shrimp tissue is therefore critical for food safety and regulatory compliance.

Objectives and overview of the study

This application note presents a validated method for simultaneous analysis of a broad spectrum of veterinary antibiotics (tetracyclines, sulfonamides, trimethoprim, ormetoprim, dapsone) in shrimp tissue using LC-MS/MS. The study aims to demonstrate robust cleanup, sensitive quantitation, and adherence to EU regulatory requirements (MRLs, CCα, CCβ).

Methodology and extraction procedure

The sample preparation combines chelation, liquid–liquid defatting, and solid-phase extraction (SPE) to efficiently isolate analytes from homogenized shrimp tissue:

• Homogenized tissue (5 g) spiked with isotopically labeled internal standards.
• Extraction with succinate buffer and EDTA, hexane defatting, centrifugation.
• SPE cleanup on Oasis HLB cartridges, washing and methanolic elution.
• Drying and reconstitution in water/methanol (70/30, v/v).

Instrumentation

• Waters ACQUITY UPLC I-Class PLUS with Flow-Through-Needle sample manager.
• ACQUITY HSS C18 column (2.1 × 100 mm, 1.8 µm).
• Xevo TQ-S micro triple quadrupole mass spectrometer with ESI+.
• MassLynx and TargetLynx XS software for data acquisition and processing.

Chromatographic and MS conditions

Gradient elution with water/methanol (+0.1% formic acid) achieves separation of parent and epimeric tetracyclines without ion-pairing agents. Two MRM transitions per compound ensure identification (>3 identification points), with automatic dwell times to secure ≥12 data points per peak.

Main results and discussion

The method delivered precise separation of tetracycline isomers and high sensitivity across all analytes. Calibration curves (1/x weighting) were linear (R² > 0.99) with residuals < 20%. Recoveries ranged 88.6–106%, RSDr < 10.2%, RSDRL < 9.8%. Decision limits (CCα) and detection capabilities (CCβ) fell below established MRLs. No significant matrix interferences were observed in blank shrimp extracts.

Benefits and practical applications of the method

• Comprehensive multi-residue assay reduces analysis time and cost.
• Sensitivity well below regulatory limits enables reliable compliance testing.
• Robust cleanup and quantitation allow routine implementation in food safety and quality control laboratories.
• Potential for further dilution and high-throughput screening.

Future trends and potential applications

Advances may include extending the method to other seafood and tissue matrices after validation, integration with high-resolution mass spectrometry for non-target screening, automation of sample preparation, and development of portable platforms for on-site monitoring.

Conclusion

The validated LC-MS/MS method offers a fast, reliable, and sensitive solution for simultaneous determination of multiple antibiotic residues in shrimp. It meets EU regulatory criteria and supports effective monitoring of food safety in aquaculture products.

References

• Chen J., et al. (2019) Antibiotic Residues in Food: Extraction, Analysis, and Human Health Concerns. Journal of Agricultural and Food Chemistry, 67, 7569–7586.
• European Commission (2010) Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and MRLs in foodstuffs of animal origin. OJ L15, 1–72.
• CRL Guidance Paper (2007) State of the Art Analytical Methods for National Residue Control Plans. Community Reference Laboratories for Residues, Council Directive 96/23/EC.
• European Commission (2002) Decision 2002/657/EC implementing Directive 96/23/EC on analytical methods and result interpretation. OJ L221, 8–36.
• Young M.S.; Van Tran K.H. (2013) HPLC/UV Determination of Tetracyclines in Milk Using Mixed-Mode SPE and XP 2.5 µm Columns. Waters Application Note 720004582EN.