Macrolide Analysis in Pork Using Bond Elut QuEChERS dSPE EMR—Lipid and Poroshell 120

Importance of topic

Macrolide antibiotics such as spiramycin, tilmicosin, oleandomycin, erythromycin, tylosin, roxithromycin, and josamycin are widely used in livestock to treat bacterial infections. Residues of these compounds in pork pose health risks and are regulated at low levels by national and international agencies. A rapid and reliable analytical approach is essential to enforce regulatory limits, protect consumers, and ensure food safety.

Study objectives and overview

The primary goal was to develop a multiresidue method for routine monitoring of seven macrolides in pork. The approach combines a QuEChERS dSPE cleanup step using Enhanced Matrix Removal—Lipid sorbent with high‐performance liquid chromatography on a Poroshell 120 EC-C18 column. Detection and quantification are performed by electrospray tandem mass spectrometry in positive multiple‐reaction‐monitoring mode. The method aims for simplicity, speed, low detection limits, and acceptable recoveries in a fatty matrix.

Sample preparation and methodology

The optimized procedure involves:

• Weighing 2.5 g homogenized pork into a 50 mL tube.
• Adding 8 mL water and vortexing for 1 min.
• Adding 10 mL acetonitrile and QuEChERS EN extraction salts, then shaking for 1 min.
• Centrifuging at 4 000 rpm for 5 min and transferring 5 mL supernatant to an EMR—Lipid dSPE tube.
• Vortexing for lipid removal and then centrifuging again.
• Polishing the extract with a final dSPE sorbent and salts, followed by centrifugation.
• Diluting 200 µL of the cleaned acetonitrile layer with 800 µL water before injection.

Used instrumentation

The analytical setup comprised:

• Agilent 1290 Infinity LC system with Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 µm).
• Mobile phase A: 10 mM ammonium acetate with 0.1% formic acid in water; phase B: acetonitrile; flow rate 0.5 mL/min; injection volume 2 µL; column temperature 30 °C; gradient 20–65% B in 5 min, hold, then re-equilibrate.
• Agilent 6460 Triple Quadrupole LC/MS with electrospray ionization in positive MRM mode; gas temp 300 °C; sheath gas temp 400 °C; capillary voltage 4 000 V.

Main results and discussion

Calibration curves constructed in matrix-matched samples were linear over 5–250 µg/kg with R2 > 0.9994. Limits of detection were below 0.1 µg/kg for all analytes. Recoveries at spike levels of 10, 20, and 100 µg/kg ranged from 63.9 to 98.4%, with relative standard deviations between 3.8 and 10.3%. Enhanced Matrix Removal—Lipid effectively eliminated triglycerides and phospholipids, reducing matrix effects and preserving column performance. Typical chromatograms showed well-resolved peaks within a 5 min run time.

Benefits and practical applications

This method offers a streamlined workflow for regulatory testing laboratories and quality-control settings:

• Fast sample turnaround with minimal hands-on time.
• Robust lipid removal extends column lifetime and enhances sensitivity.
• Low detection limits and broad dynamic range meet regulatory requirements.
• High reproducibility supports routine monitoring of veterinary drug residues.

Future trends and potential uses

Emerging directions include integrating high-resolution mass spectrometry for non-targeted screening, automating QuEChERS workflows for higher throughput, extending the method to other animal matrices such as beef and poultry, and developing greener solvent systems. Advances in microextraction and online cleanup may further reduce solvent consumption and sample preparation time.

Conclusion

The presented QuEChERS dSPE EMR—Lipid cleanup combined with Poroshell 120 EC-C18 LC/MS/MS provides a rapid, sensitive, and reliable tool for multiresidue analysis of macrolide antibiotics in pork. The protocol meets stringent regulatory limits and delivers consistent recoveries in a high-fat matrix, making it suitable for routine enforcement and quality assurance.

Reference

1. Anon. GB/T 23408-2009. Determination of macrolides residues in honey – LC-MS/MS method. General Administration of Quality Supervision, Inspection and Quarantine, Beijing, China.
2. SN/T 1777.2-2007. Determination of macrolide antibiotic residues in animal-derived food – Part 2: LC-MS/MS method. General Administration of Quality Supervision, Inspection and Quarantine, Beijing, China.
3. Zhai CH, Fu RJ. Macrolides in Honey Using Agilent Bond Elut Plexa SPE, Poroshell 120, and LC/MS/MS. Agilent Technologies Application Note 5991-3190EN, 2013.