Simple and efficient HPLC-MS/MS method for simultaneous extraction and quantification of anthelmintics in equine manure.

Overview

• This study presents a simple, fast, and reliable HPLC-MS/MS method to simultaneously extract and measure key anthelmintic drug residues in horse manure.
• The method enables accurate quantification of four commonly used anthelmintics with high recovery and sensitivity without requiring complex sample cleanup.

Introduction and Background

• Anthelmintics are drugs administered to horses to control parasitic worms, a routine aspect of equine health management.
• Post-treatment, residues of these pharmaceuticals are excreted and can accumulate in manure, which is often spread on fields and pastures.
• The presence of anthelmintic residues in the environment may impact non-target organisms such as beneficial nematodes, potentially affecting soil health and biodiversity.
• Therefore, sensitive and efficient analytical methods to monitor these residues in equine manure are important for environmental and veterinary safety assessments.

Objective

• The study aimed to develop and validate a high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method.
• The method targets simultaneous extraction and quantification of four widely used anthelmintic compounds in horse manure.

Method Development

• Sample Preparation:
  • Horse manure samples were initially dried to standardize moisture content and facilitate extraction.
  • Samples were spiked with varying concentrations of the target drugs ranging from 10 to 10,000 ng to assess recovery and calibration.
• Extraction:
  • The extraction solvent was an acetonitrile-water mixture with 5 mM ammonium formate, which helped in efficient extraction of the anthelmintics.
  • No complex solid-phase extraction (SPE) cleanup was necessary, simplifying the procedure.
• Detection and Quantification:
  • HPLC-MS/MS was used with multiple reaction monitoring (MRM) to specifically detect the chosen anthelmintics.
  • This provided high sensitivity and selectivity for simultaneous quantification of the drugs in one run.

Validation and Results

• Recovery:
  • Recovery rates ranged from 82.3% (pyrantel) up to 120.8% (ivermectin), indicating good extraction efficiency.
  • Standard deviations were below 20%, showing consistency and reproducibility.
• Calibration:
  • Calibration curves were linear between 1 ng/mL and about 1.5 µg/mL for all four drugs, enabling accurate quantification over a broad concentration range.
• Limits of Detection:
  • Lower limits of detection (LLODs) were very sensitive:
    • Ivermectin: 0.1 ng/mL
    • Moxidectin: 0.2 ng/mL
    • Fenbendazole: 1.0 ng/mL (affected by carryover)
    • Pyrantel: 0.3 ng/mL (noted to show two chromatographic peaks)

Significance and Applications

• The described method is novel in that it allows simultaneous extraction and quantification of multiple anthelmintics directly in horse manure without complex sample cleanup.
• Its efficiency and reliability make it a valuable analytical tool for monitoring pharmaceutical residues in equine waste.
• Given its adaptability, the method lays the groundwork for extensions to other sample types such as soil, enabling environmental monitoring of anthelmintic contamination beyond just manure.

Conclusion

• This research provides a streamlined, effective HPLC-MS/MS protocol to detect and measure principal anthelmintics in horse manure, facilitating improved environmental risk assessments.
• Its implementation can support better management of pharmaceutical residues from equine treatment practices, helping to protect soil ecosystems and non-target organisms.