Quantitation of the Aminoglycosides Group of Veterinary Drugs in Milk Samples

Significance of the topic

The reliable quantitation of veterinary aminoglycoside antibiotics in milk is essential for food safety, regulatory compliance, and mitigation of antimicrobial resistance. Aminoglycosides are polar, metal‑binding compounds that present analytical challenges: poor retention on conventional reversed‑phase columns, strong matrix effects in complex food matrices, and sensitivity loss when ion‑pairing reagents (e.g., HFBA) are used. A robust, MS‑compatible workflow that minimizes system contamination while achieving low limits of quantitation supports routine monitoring in dairy testing laboratories and high‑throughput quality control environments.

Objectives and study overview

The study aimed to develop and validate an LC–MS/MS method for quantifying nine veterinary aminoglycoside and related antibiotics in milk without using heptafluorobutyric acid (HFBA). Key objectives were to: ensure sufficient chromatographic retention and separation using an MS‑friendly mobile phase and polar‑compatible reversed‑phase column; implement a simple, reproducible sample extraction that addresses metal chelation and protein precipitation; achieve low limits of quantification (LOQs) appropriate for regulatory screening; and demonstrate method robustness across calibration levels using MRM ion ratios and matrix‑based calibration.

Methodology

Analytical scope: apramycin, erythromycin, kanamycin, spectinomycin, lincomycin, neomycin, spiramycin, tylosin, and virginiamycin M1 analyzed in milk.

Chemicals and mobile phases:

• Mobile phase A: 4.5 mM ammonium formate, 0.5 mM ammonium fluoride, 0.1% formic acid in water.
• Mobile phase B: acetonitrile with 0.1% formic acid.

Chromatography conditions (high‑level): flow 0.5 mL/min, injection 20 µL, column temperature 30 °C, acquisition time 12 min plus 2 min post time. Column: Agilent InfinityLab Poroshell SB‑AQ, 4.6 × 150 mm, 2.7 µm. Gradient program moved from aqueous‑rich to higher organic to elute the polar analytes while maintaining separation.

Sample preparation (workflow):

1. Spike 1 mL milk with standards for matrix‑based calibration.
2. Add 0.1 mL of 0.1 M EDTA solution and let stand 10 min to chelate divalent metals.
3. Extract with 1 mL of 5% trichloroacetic acid in 50:50 acetonitrile/water.
4. Vortex 1 min for protein precipitation.
5. Centrifuge at 9,000 rpm for 10 min.
6. Filter 1 mL supernatant through a nylon filter and transfer to HPLC vials.

Calibration and quantitation:

• Pre‑extraction spiked matrix calibration points used to compensate for matrix effects.
• Linearity: for six analytes 2.5–100 ng/mL; for kanamycin, spectinomycin, apramycin 25–300 ng/mL. Linear regression with 1/x weighting applied.
• MRM in positive ESI mode with compound‑specific transitions; ion ratios monitored to confirm identity and consistency (acceptance within ±30%).

Instrumentation used

The method was implemented on an Agilent platform: 1290 Infinity II LC (multisampler, high‑speed pump, multicolumn thermostat, diode array detector) coupled to an Agilent 6475 triple quadrupole LC/MS with Agilent Jet Stream (AJS) ion source. Key source parameters included gas temperature ~340 °C, gas flow 10 L/min, nebulizer 45 psi, capillary voltage ~3,700 V, sheath gas temperature ~390 °C. The column used was Agilent InfinityLab Poroshell SB‑AQ (4.6 × 150 mm, 2.7 µm).

Main results and discussion

Chromatography and sensitivity:

• The HFBA‑free mobile phase in combination with the SB‑AQ column provided adequate retention and separation of highly polar aminoglycosides without the drawbacks of ion‑pairing reagents (reduced ion suppression and less instrument contamination).
• Representative extracted ion chromatograms at 100 ng/mL demonstrated clear analyte peaks and retention‑time reproducibility across the panel.

Calibration, recoveries, and LOQs:

• Calibration accuracies for all calibration points were within ±20% of nominal concentrations.
• Recoveries in milk ranged approximately from 80% to 120% across analytes when using pre‑extraction spiked calibration, indicating acceptable extraction efficiency and compensation for matrix effects.
• LOQs achieved: 2.5 ppb for neomycin, erythromycin, lincomycin, tylosin, spiramycin, and virginiamycin M1; 25 ppb for kanamycin, spectinomycin, and apramycin.

Matrix effects and handling notes:

• Significant matrix effects were observed for several analytes, necessitating matrix‑matched calibration prepared by spiking prior to extraction.
• EDTA significantly improved recoveries by chelating calcium and other divalent cations that bind aminoglycosides, reducing analyte loss due to metal complexation.
• Certain standards (apramycin and neomycin) are prone to adsorption losses on glass and are recommended to be dissolved in water and stored in plastic vials.

Method robustness:

• MRM ion ratios remained within ±30% across calibration levels, supporting reliable compound identification in the matrix.
• Elimination of HFBA reduced the risk of long‑term contamination and extended equilibration times, improving instrument uptime for routine labs.

Benefits and practical applications

This workflow delivers several practical advantages for routine monitoring of antibiotic residues in dairy matrices:

• MS‑friendly method avoids HFBA, decreasing ion suppression and instrument maintenance burden.
• Simple, fast sample preparation suitable for moderate‑to‑high throughput testing with standard lab consumables.
• Matrix‑matched calibration and EDTA use yield reliable recoveries and accurate quantitation in a complex food matrix.
• LOQs align with many regulatory thresholds for aminoglycosides, enabling compliance testing and surveillance programs.

Future trends and potential uses

Anticipated developments and broader applications include:

• Adoption of alternative polar‑retentive stationary phases and smaller particle technologies to further improve chromatographic efficiency for highly polar antibiotics.
• Integration with automated sample preparation and online cleanup (SPE) to increase throughput and reproducibility for large surveillance studies.
• Expansion of the panel to include metabolite markers and other antibiotic classes, paired with non‑target screening strategies using high‑resolution MS for comprehensive residue surveillance.
• Further work on standardized matrix effect mitigation (stable isotope standards, automated matrix calibration procedures) to simplify inter‑laboratory transfer and regulatory validation.

Conclusion

An HFBA‑free LC–MS/MS method using an Agilent 1290/6475 platform and an SB‑AQ column was demonstrated to quantitate nine aminoglycoside and related veterinary antibiotics in milk with good sensitivity, reproducibility, and robustness. The protocol combines EDTA‑assisted extraction, simple protein precipitation, matrix‑matched calibration, and MRM monitoring to overcome matrix effects and metal binding challenges. The approach is practical for routine residue monitoring, minimizing MS contamination while delivering LOQs suitable for many regulatory needs.

Reference

1. Zhai A. Aminoglycosides in Milk using Agilent Bond Elute Plexa SPE, Agilent Poroshell 120, and LC/Tandem MS. Agilent Technologies Application Note, publication number 5991-1758 EN, 2013.