Analysis of Polyether Antibiotics in Animal Feeds by HPLC with Post-Column Derivatization

Polyether antibiotics are commonly used for preventing coccidiosis and other infections in poultry and for improving feed efficiency for beef cattle and swine. The use of polyether antibiotics is strictly regulated, with only specific ionophores approved for use in feeds intended for different animals.

Analysis of polyether antibiotics by HPLC with post-column derivatization and UV–vis detection has been proven to successfully identify and quantify monensin, narasin, and salinomycin in medicated feeds, supplements, and premixes as well as to determine trace contamination levels in non-medicated feeds (1,2).

Table I: Polyether antibiotics in certified medicated feeds

Post-column derivatization of polyether antibiotics is done using highly acidic vanillin or DMAB reagents. Pinnacle PCX derivatization system (Pickering Laboratories, Inc.) has an inert flow path and automated system wash capabilities that make it uniquely suitable for handling corrosive reagents. The two-pump system is recommended to extend reagent stability, but the single-pump system for this application is also available.

Table II: Spike recoveries for monensin

Adding a fluorescence detector to the instrumentation allows for using the same extraction procedure and HPLC conditions to also determine lasalocid which doesn't require post-column derivatization.

Method

Sample Preparation

To 25 g of finely ground feed sample, add 100 mL of extraction solution (90% methanol–10% water). Shake for 1 h at high speed using mechanical shaker. Let the solids settle and filter an aliquot of the extract for injection. Dilute with extraction solution if needed to fit the calibration curve. Use 2.5 g portion when testing premixes.

Figure 1: Standard mixture of monensin, salinomycin, and narasin.

Analytical conditions

Analytical Column: Polyether Column, C18, 4.6 × 250 mm, Catalog No. 2381750

Temperature: 40 °C

Flow rate: 0.7 mL/min

Mobile Phase: 90% methanol, 10% of 5% acetic acid solution in water, isocratic

Injection volume: 20 µL

Figure 2: Certified medicated beef feed sample containing 267 g/ton of monensin.

Post-Column Conditions

Post-column System: Pinnacle PCX

Reactor Volume: 1.4 mL

Reactor Temperature: 90 °C

Reagent 1: Concentrated sulfuric acid/methanol (4:96 v/v)

Reagent 2: 60 g of vanillin in 950 mL of methanol

Reagents Flow Rate: 0.3 mL/min

Detection: UV-vis 520 nm (for Lasalocid – FLD, Ex. 322 nm, Em. 370 nm)

Figure 3: Non-medicated bird feed sample spiked with monensin A (3.44 µg/g) and monensin B (0.16 µg/g).

Calibration

Monensin A: 0.1–50 ppm, R² = 0.999

Monensin B: 0.0035–0.7 ppm, R² = 0.999

Lasalocid acid: 0.25–50 ppm, R² = 0.999

Figure 4: Certified medicated milk replacer containing 72 g/ton of lasalocid.

Conclusion

Analysis of polyether antibiotics by HPLC with post-column derivatization is a robust and sensitive method that utilizes standard equipment and could easily be adopted by testing laboratories. It allows for testing of different ionophores at wide range of concentrations, including at trace levels. Using Pinnacle PCX post-column derivatization system, factory configured for the analysis, guarantees stable and reproducible results.

References

(1) H. Campbell and G. Nayeri, J. AOAC Int. 89, 1229–1242 (2006).

(2) AOAC Official Method 997.04. Monensin in Premix and Animal Feeds.

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