Triclabendazole is one of the main drugs used to treat liver fluke in livestock. A rapid LC-MS/MS method was developed and validated to determine ovine plasma levels of triclabendazole sulfoxide. A Gemini NX-C18 column was used to achieve analytical separation, with gradient elution of a mobile phase composed of 0.1% formic acid in acetonitril and 0.1% formic acid in water at flow rate of 0.6 mL/min. MRM with positive ESI ionization was used for the detection of triclabendazole sulfoxide (m/z 360.10 from m/z 376.97). Fenbendazole was used as internal standard. Plasma protein precipitation with acetonitrile was used for sample processing. The method was validated with regards to selectivity, linearity (r > 0.9939), within run and between run precision (CV < 8.9%) and accuracy (bias < 8.9%) over the concentration range 1–100 µg/mL plasma. The method developed is simple, selective and can be applied in bioequivalence and bioavailability studies.
The experiment was conducted at the Vilniaus irgynas horse breeding farm in Lithuania, where horse strongyles resistance to fenbendazole (FBZ) was detected earlier by an FECR test. The experiment included 4 naturally-infected horses. The following anthelmintics were administered to three experimental horses: first FBZ @ 7.5 mg/kg BW, and subsequently ivermectin (IVM) @ 0.2 mg/kg BW 13-14 days later. One control horse was not treated. The elimination of strongyles after treatment with FBZ lasted 35-95 hours, and 36-38 hours after treatment with IVM. In total, 87422 small strongyles (cyathostomes) were expelled in the faeces of horses treated with FBZ and IVM. The treatment with FBZ in each experimental horse reduced the strongyle faecal egg count (FEC) by 94.5%, 86.3%, and 83.1% (average 88.0%), and strongyle worm burdens by 81.4%, 67.1%, 48.1% (average 65.5%), respectively. A total of 5491 cyathostomes were collected and thirteen species of cyathostomes were found (Cylicocyclus nassatus, Cyathostomum catinatum, Cylicocyclus leptostomus, Cylicostephanus longibursatus, Cylicocyclus ashworthi, Cylicostephanus goldi, Cyathostomum pateratum, Coronocyclus labiatus, Cylicostephanus minutus, Coronocyclus labratus, Cylicocyclus insigne, Cylicostephanus calicatu, Coronocyclus coronatus). It was determined that Cylicocyclus ashworthi, Cylicocyclus nassatus, and Cylicostephanus goldi had developed strong resistance to FBZ (54.69%, 53.02%, and 34.10%, respectively); Cyathostomum catinatum was less resistant (16.83%). Cylicostephanus longibursatus and Cyathostomum pateratum were considered to be weakly resistant, whereas Cylicocyclus leptostomus exhibited no resistance at all. The results of this study indicate that the use of the two anthelmintics have a potential value for detecting anthelmintic-resistance in horse strongyles and could be used as an alternative to necropsy. However, this recommendation requires further investigation.