Quantitative analysis of multi-class antibiotic residues in milk using LC/MS/MS

Importance of the Topic

Monitoring antibiotic residues in milk is critical to protect public health, prevent allergic reactions, and limit the development of antibiotic-resistant bacteria. A single, sensitive method capable of detecting multiple classes of antibiotics in complex food matrices streamlines quality control and ensures regulatory compliance.

Study Objectives and Overview

This study aimed to develop and validate a unified LC-MS/MS method for simultaneous quantitation of ten antibiotics across six classes (β-lactams, sulfonamides, tetracyclines, macrolides, amphenicols, and dihydrofolate reductase inhibitors) in raw milk. The key goals were high sensitivity (LOQ ≤1 ppb), excellent linearity, and minimal analytical cross-talk.

Methodology

Sample Preparation

• 2 mL raw milk + 8 mL acetonitrile; ultrasonic extraction for 5 min, centrifugation.
• Evaporation of 8 mL supernatant under nitrogen; reconstitution in 8 mL water, 0.22 µm filtration.
• Matrix-matched calibration standards prepared at 0.5–20 ppb to compensate for ion suppression/enhancement.

Chromatographic and MS Conditions

• UHPLC Nexera with Shim-pack XR-ODS column (50 × 3 mm, 2.2 µm), flow 0.3 mL/min, 40 °C.
• Mobile phases: A = water, B = 0.1 % formic acid in acetonitrile; gradient from 10 to 100 % B in 2.7 min.
• Injection: 15 µL; total run time 6 min.
• Triple quadrupole LCMS-8040 with ultrafast polarity switching (15 ms), UFsweeper II, dwell time 0.8 ms, pause time 1 ms.
• ESI in positive/negative modes; nebulizing gas 2 L/min, drying gas 10 L/min, DL temp 250 °C, heat block 350 °C.

Instrumentation used

• UHPLC Nexera system (Shimadzu)
• LCMS-8040 triple quadrupole mass spectrometer with UFsweeper II technology
• Shim-pack XR-ODS analytical column
• Nitrogen evaporator and ultrasonic bath for sample prep

Results and Discussion

Ten antibiotics were separated in a single 6 min run with retention times ranging from 1.17 to 3.38 min. Calibration curves showed excellent linearity (r2 ≥0.9926) over 0.5–20 ppb. LOQs met criteria of S/N >10, accuracy 80–120 %, and RSD <16 % (n=6). Representative MRM chromatograms confirmed baseline separation and minimal cross-talk, demonstrating method robustness in a milk matrix.

Benefits and Practical Applications

• Consolidates multiple analytical workflows into a single rapid assay.
• High throughput and low sample volume requirements aid routine quality control in dairy laboratories.
• Matrix-matched calibration ensures reliable quantitation in complex food samples.

Future Trends and Potential Applications

Advancements may include automated sample preparation robotics, expansion to other food and environmental matrices, integration with high-resolution mass spectrometry for non-target screening, and AI-driven data interpretation to further enhance sensitivity, throughput, and traceability.

Conclusion

The developed UHPLC-MS/MS method on the Shimadzu LCMS-8040 platform enables simultaneous, sensitive, and selective quantitation of ten antibiotics in milk. Ultrafast polarity switching and advanced ion-sweep technology ensure minimal cross-talk and high reproducibility, significantly simplifying dairy antibiotic residue analysis.

Reference

• Helio A. Martins-Junior, Tereza A. Kussumi et al. A rapid method to determine antibiotic residues in milk using liquid chromatography coupled to electrospray tandem mass spectrometry. J. Braz. Chem. Soc. 18(2):1781–1788 (2007).
• Kwon H., Lehotay S. J., Geis-Asteggiante L. Variability of matrix effects in LC-MS and GC-MS analysis of pesticide residues after QuEChERS sample preparation. J. Chromatogr. A 1270:235–245 (2012).