Sulfonamide Antibiotic Residues in Milk on a Thermo Scientific™ Acclaim ™ 120 C18 Column

Importance of the Topic

The monitoring of sulfonamide antibiotic residues in milk is essential to protect consumer health and comply with regulatory maximum residue limits. Sulfonamides are widely used in veterinary medicine but can persist in dairy products if withdrawal times are not strictly observed. Reliable analytical methods are therefore required for routine screening and quantification of these compounds in complex matrices such as milk.

Objectives and Study Overview

This application note demonstrates an optimized high-performance liquid chromatography (HPLC) method that resolves eight sulfonamide antibiotics in milk using a single injection and gradient elution on a Thermo Scientific™ Acclaim™ 120 C18 column. The goal is to simplify the conventional two-injection approach while achieving baseline separation of all target analytes in one run.

Methodology and Instrumentation

The method employs the following instrumentation and conditions:

• Column: Thermo Scientific™ Acclaim™ 120 C18, 5 µm, 4.6 × 250 mm
• Pump: Thermo Scientific™ Dionex™ Summit P580 HPG/4
• Autosampler: ASI-100, injection volume 100 µL
• Mobile Phase A: 30 mM acetic acid adjusted to pH 4.2 with NaOH
• Mobile Phase B: Methanol
• Gradient Program: 0–20 min, A from 88% to 60%; 20–25 min, hold at 60% A
• Flow Rate: 1.5 mL/min
• Column Temperature: 35 °C
• Detector: UVD 340U UV at 265 nm with spectral confirmation
• Sample Preparation: AOAC 993.32 protocol for skim milk extracts

Main Results and Discussion

The optimized gradient achieved clear resolution of eight sulfonamide compounds—sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline—in under 25 minutes. Theobromine, an endogenous interferent, eluted separately without compromising analyte peaks. Signal responses were consistent for spiked milk extracts at 1.0 µg/g and external standards at 0.20 µg/mL, demonstrating the method’s linearity and sensitivity. Organic acid interferences were effectively displaced by pH adjustment of the mobile phase.

Benefits and Practical Applications

• Single-injection workflow reduces analysis time and solvent consumption.
• Gradient elution on a high-resolution C18 column ensures baseline separation of closely eluting sulfonamides.
• UV detection with spectral confirmation enhances specificity in routine QA/QC laboratories.
• Adaptable to regulatory monitoring programs for veterinary drug residues in dairy.

Future Trends and Potential Uses

Advances in column chemistry and detectors may further increase throughput and sensitivity. Coupling with mass spectrometry could enable multi-residue screening of additional antibiotic classes in a single run. Automation of sample preparation and integration within laboratory information management systems will streamline compliance testing and data reporting.

Conclusion

The described HPLC method using a Thermo Scientific™ Acclaim™ 120 C18 column offers a robust, efficient solution for the analysis of sulfonamide residues in milk. Its single-injection gradient approach simplifies routine testing while meeting performance requirements for regulatory compliance.

Reference

Thermo Fisher Scientific Inc. Sulfonamide Antibiotic Residues in Milk on a Thermo Scientific™ Acclaim™ 120 C18 Column. 2009.