Quantitation of Aminoglycosides group of veterinary drugs in milk samples usingLC-MS/MS without ion pairing agents in the mobile phase

Importance of the topic

The presence of aminoglycoside antibiotics in milk is a food-safety concern due to their therapeutic use in livestock and potential risks to human health. Reliable, routine residue monitoring is necessary to ensure compliance with regulatory limits and to protect consumers. Developing LC–MS/MS methods that are sensitive, robust, and MS-friendly—avoiding problematic reagents that cause ion suppression or instrument contamination—improves throughput and long-term laboratory performance.

Objectives and study overview

This poster describes development and validation of a selective LC–MS/MS workflow for quantifying multiple aminoglycoside and related veterinary antibiotics in milk without using ion-pairing reagents (e.g., HFBA). The goals were to: (1) establish chromatographic conditions that provide adequate retention and separation while remaining MS-compatible, (2) optimize a simple extraction compatible with high throughput, and (3) demonstrate linearity, recovery, and reproducibility across relevant concentration ranges for regulatory monitoring.

Methodology

Sample preparation:

• Start with 1 mL milk sample; add 1 mL 0.1 M EDTA and allow to stand for 10 minutes to chelate divalent metals.
• Extract analytes with 1 mL acetonitrile:water (1:1 v/v) and vortex 1 minute to precipitate proteins.
• Centrifuge at 9000 rpm and filter supernatant through a nylon filter before analysis.

Chromatography and mobile phases:

• Mobile phase A: 4.5 mM ammonium formate, 0.5 mM ammonium fluoride and 0.1% formic acid in water.
• Mobile phase B: acetonitrile with 0.1% formic acid.
• Analytical column: Poroshell SB AQ, 150 x 4.6 mm, 2.7 μm.
• Gradient: low organic (~10% B) at injection, ramping to high organic (≈90% B) to elute retained analytes and clean the column; the run supports efficient separation without HFBA.

Mass spectrometry:

• Triple-quadrupole MS used in electrospray ionization, positive mode, with multiple reaction monitoring (MRM) transitions optimized per analyte.

Instrumentation used

The method was implemented on an Agilent 1290 Infinity III UHPLC coupled to an Agilent 6475A LC/TQ (triple quadrupole) instrument. Electrospray ionization in positive polarity and MRM acquisition were employed for selective quantitation.

Main results and discussion

Use of EDTA in the extraction protocol markedly improved recoveries by preventing formation of aminoglycoside–metal complexes (calcium and other divalent ions), which otherwise reduce extraction efficiency. Improvements were most prominent for analytes with high metal affinity.

Calibration and quantitation:

• Matrix-based calibration (pre-spike approach) was necessary due to pronounced matrix effects observed for several analytes; this approach compensated for ion suppression/enhancement from milk components.
• For seven target analytes, linearity was achieved over 2–100 ng/mL (0.002–0.1 μg/mL) with appropriate weighting, yielding recoveries in the acceptable 80–120% range.
• Kanamycin, spectinomycin and apramycin required higher calibration ranges (25–300 ng/mL) to cover expected concentrations and maintain linear response.

Method robustness:

• MRM ion-ratio consistency confirmed selective detection and supported method specificity.
• Handling notes: apramycin and neomycin exhibit adsorption losses on glass; the study recommends dissolving these standards in water and storing in plastic vials to preserve standard integrity.

Key performance takeaways include reliable retention and separation without ion-pairing agents, maintained MS compatibility, reproducible batch-to-batch performance, and suitability for high-throughput screening workflows. The method covers typical regulatory action levels reported for aminoglycosides (approximately 10–200 ppb), depending on the analyte and jurisdiction.

Benefits and practical applications

• MS-friendly mobile phases (ammonium formate + ammonium fluoride + formic acid) avoid long-term contamination and ion suppression associated with HFBA, reducing maintenance and improving sensitivity.
• Simple, rapid sample preparation with EDTA-assisted extraction supports higher sample throughput and consistent recoveries from milk matrices.
• Matrix-based calibration ensures quantitative accuracy in complex food matrices where matrix effects are significant.
• The workflow is applicable for routine monitoring of veterinary drug residues in dairy testing laboratories, regulatory control, and quality assurance/quality control programs.

Future trends and potential applications

• Further miniaturization and automation of the sample-preparation step (e.g., automated liquid handling, 96-well formats) can increase throughput for large surveillance programs.
• Expanded analyte panels combining aminoglycosides with other antibiotic classes in a single method would improve laboratory efficiency and broader residue surveillance capability.
• Integration of high-resolution MS or hybrid workflows could provide enhanced screening for unexpected metabolites or analogues while maintaining targeted MRM for quantitation.
• Refinement of mobile-phase additives and column chemistries that maximize retention of polar, multi-ionic analytes without ion-pairing reagents remains an active area to improve chromatographic performance.

Conclusion

The presented LC–MS/MS method delivers a practical, MS-compatible approach for quantifying aminoglycoside antibiotics in milk without ion-pairing reagents. Key strengths are the EDTA-enhanced extraction, matrix-based calibration to address matrix effects, and robust chromatographic conditions that provide good retention and reproducibility. The method is well suited for routine screening and compliance testing across typical regulatory concentration ranges.

Reference

Agilent Technologies. Aminoglycosides in Milk using Agilent Bond Elute Plexa SPE, Agilent Poroshell 120, and LC/Tandem MS. Application Note 5991-1758 EN.