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

Anthelmintic treatment is a common practice in horses. The presence of pharmaceutical residues in fields and pastures is well-documented affecting various organisms, like nematodes. This study describes the development and validation of a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method using multiple reaction monitoring (MRM) for extracting and determining the concentrations of four commonly used anthelmintics in horse manure. Manure was initially dried, then spiked with various concentrations (10–10,000 ng) of anthelmintics and extracted using an acetonitrile-water mixture containing 5 mM ammonium formate, with no need for further solid-phase extraction (SPE) cleaning. Recovery rates ranged from 82.3% for pyrantel to 120.8% for ivermectin, with standard deviations below 20%. Calibration curves were linear between 1 ng/mL and approximately 1.5 µg/mL for all four components. The lower limits of detection (LLODs) were 0.1 ng/mL for ivermectin, 0.2 ng/mL for moxidectin, 1.0 ng/mL for fenbendazole due to carryover, and 0.3 ng/mL for pyrantel, which formed two peaks. • Novel method for simultaneous extraction of anthelmintics from horse manure • Method allows efficient and reliable detection of the mainly used anthelmintics in horses • Basis for the extension of the method to other applications, including the analysis of anthelmintics in soil samples