Analysis of Amprolium in Eggs Using Bond Elut Plexa SPE and LC/MS/MS

Importance of the Topic

The determination of veterinary drug residues in food products is critical for consumer safety and regulatory compliance. Amprolium, an antiprotozoal agent used in poultry production, must be monitored in eggs to ensure levels remain below maximum residue limits (MRLs) set by regulatory bodies.

Objectives and Study Overview

This study aimed to develop and validate a robust, sensitive analytical method for quantifying amprolium in eggs. The approach integrates solid-phase extraction (SPE) cleanup using Agilent Bond Elut Plexa cartridges with high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) on an Agilent 6470 system.

Methodology and Instrumentation

Sample Preparation:

• Homogenize 5 g of egg and extract with 10 mL of 5 % trichloroacetic acid.
• Shake, centrifuge, and collect 5 mL of supernatant.
• Condition Bond Elut Plexa SPE cartridges (200 mg, 3 mL) with methanol and water.
• Load sample, wash with water, dry under vacuum, and elute with 2 % formic acid in acetonitrile.
• Filter eluate and subject to LC/MS/MS analysis.

Used Instrumentation:

• Agilent 1260 Infinity UHPLC with Poroshell 120 HILIC-Z column (100 × 3.0 mm, 2.7 μm).
• Mobile phases: A) water with 0.1 % formic acid and 5 mM ammonium acetate; B) acetonitrile; gradient from 85 % B to 60 % B over 4 min at 0.4 mL/min.
• Agilent 6470 triple quadrupole LC/MS with Jet Stream ESI source; MRM transitions for amprolium: precursor 243.1 m/z, products 150.1 and 94.1 m/z.
• MS parameters: gas temp 250 °C, gas flow 7 L/min, sheath gas heater 325 °C, sheath gas flow 11 L/min, capillary 3500 V, nozzle 300 V.

Main Results and Discussion

• Calibration exhibited excellent linearity from 0 to 1000 ng/mL (R² = 0.9985).
• Method limit of quantitation (LOQ) was 10 ng/g and limit of detection (LOD) 1 ng/g.
• Matrix effect measured at approximately 40 %, indicating moderate ion suppression manageable via SPE cleanup.
• Recovery studies at spiking levels of 10, 20, and 100 ng/g yielded average recoveries of 70.8 %, 82.0 %, and 92.2 %, respectively, with relative standard deviations below 6 %.

Benefits and Practical Applications

The validated method delivers rapid, reliable quantitation of amprolium in eggs and meets MRL criteria of major regulatory agencies. The simplified SPE workflow reduces solvent use and processing time, making it suitable for routine food-safety monitoring laboratories.

Future Trends and Opportunities

Continued improvements in SPE materials and high-resolution MS may further enhance sensitivity and selectivity. Integration with automated sample handling could increase throughput for large-scale surveillance. Expanding the approach to multi-residue panels offers broader screening capabilities for food-control labs.

Conclusion

An efficient SPE–LC/MS/MS method using Agilent Bond Elut Plexa cartridges and an Agilent 6470 system was established for quantifying amprolium in eggs. The protocol demonstrates high linearity, low detection limits, good recoveries, and reproducibility, supporting its application in regulatory compliance testing.

References

No external references were cited in the original application brief.