[Simultaneous determination of monepantel and its metabolite residues in cow and sheep milk by solid phase extraction-high performance liquid chromatography-tandem mass spectrometry].

A method for the rapid and simultaneous determination of monepantel and its metabolite residues in cow and sheep milk has been developed using high performance liquid chromatography-tandem mass spectrometry. The samples were extracted with acetonitrile to precipitate the proteins and passed through a neutral alumina solid phase extraction (SPE) cartridge. The extracts were separated on an Inertsil C8-3 column (150 mm×4.6 mm, 5 µm) with a gradient elution program consisting of methanol and ammonium acetate. A triple stage quadrupole mass spectrometer equipped with an electrospray ionization source in negative ion mode was used to detect monepantel and its metabolite residues. The quantification of the analytes was performed by the external standard method. Good linear relationships between the peak areas and mass concentrations of the analytes were obtained in the range of 0.1-5.0 µg/L with correlation coefficients greater than 0.99 (n=6). The limits of quantification were 2.0 µg/kg. The recoveries of the analytes in cow and sheep milk at three spiked levels (2.0, 50, and 100 µg/kg) were in the range of 90.1%-103.3% with relative standard deviations between 2.0% and 6.2% (n=6). This method is fast, accurate, and highly sensitive, exhibits strong anti-interference capability with good recovery and repeatability, and can thus be used to simultaneously determine monepantel and its metabolite residues in cow and sheep milk.

Pharmacokinetic assessment of the monepantel plus oxfendazole combined administration in dairy cows.

Monepantel (MNP) is a novel anthelmintic compound launched into the veterinary pharmaceutical market. MNP is not licenced for use in dairy animals due to the prolonged elimination of its metabolite monepantel sulphone (MNPSO2 ) into milk. The goal of this study was to evaluate the presence of potential in vivo drug-drug interactions affecting the pattern of milk excretion after the coadministration of the anthelmintics MNP and oxfendazole (OFZ) to lactating dairy cows. The concentrations of both parent drugs and their metabolites were measured in plasma and milk samples by HPLC. MNPSO2 was the main metabolite recovered from plasma and milk after oral administration of MNP. A high distribution of MNPSO2 into milk was observed. The milk-to-plasma ratio (M/P ratio) for this metabolite was equal to 6.75. Conversely, the M/P ratio of OFZ was 1.26. Plasma concentration profiles of MNP and MNPSO2 were not modified in the presence of OFZ. The pattern of MNPSO2 excretion into milk was also unchanged in animals receiving MNP plus OFZ. The percentage of the total administered dose recovered from milk was 0.09 ± 0.04% (MNP) and 2.79 ± 1.54% (MNPSO2 ) after the administration of MNP alone and 0.06 ± 0.04% (MNP) and 2.34 ± 1.38% (MNPSO2 ) after the combined treatment. The presence of MNP did not alter the plasma and milk disposition kinetics of OFZ. The concentrations of the metabolite fenbendazole sulphone tended to be slightly higher in the coadministered group. Although from a pharmacodynamic point of view the coadministration of MNP and OFZ may be a useful tool, the presence of OFZ did not modify the in vivo pharmacokinetic behaviour of MNP and therefore did not result in reduced milk concentrations of MNPSO2 .

Comparison of biotransformation and efficacy of aminoacetonitrile anthelmintics in vitro.

The present in vitro study was designed to test and compare anthelmintic activity, hepatotoxicity, and biotransformation of four selected aminoacetonitrile derivatives (AADs): monepantel (MOP, anthelmintic approved for the treatment), AAD-970, AAD-1154, and AAD-1336. Micro-agar larval development test, MTT test of cytotoxicity, and biotransformation study coupled with Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique were used for this purpose. Larvae of two Haemonchus contortus strains (drug susceptible and multi-drug resistant) and primary cultures of rat and ovine hepatocytes served as model systems. All AADs (including MOP) exhibited significant larvicidal effect in H. contortus susceptible as well as multi-resistant strains, much higher than those of reference anthelmintics thiabendazole and flubendazole. AAD-1154 provides the best results for most tested parameters among all AADs in this study. The cytotoxicity test showed that all AADs can be considered as nontoxic for hepatocytes. In the biotransformation study, Phase I and Phase II metabolites of AADs were identified and schemes of possible metabolic pathways in ovine hepatocytes were proposed. Biotransformation of MOP was much more extensive than biotransformation of other AADs. Based on obtained results, AAD-1154 and AAD-1336 can be considered as promising candidates for further in vivo testing.

Accumulation of monepantel and its sulphone derivative in tissues of nematode location in sheep: pharmacokinetic support to its excellent nematodicidal activity.

The amino-acetonitrile derivatives (AADs) are a new class of anthelmintic molecules active against a wide range of sheep gastrointestinal (GI) nematodes including those that are resistant to other anthelmintic families. The plasma disposition of monepantel (MNP) has been previously characterized in sheep. However, information on drug concentration profiles attained at tissues of parasite location is necessary to fully understand the pharmacological action of this novel compound. The current work aimed to study the relationship between the concentrations of MNP parent drug and its main metabolite monepantel sulphone (MNPSO2), measured in the bloodstream and in different GI tissues of parasite location in sheep. Twenty two (22) uninfected healthy Romney Marsh lambs received MNP (Zolvix, Novartis Animal Health) orally administered at 2.5 mg/kg. Blood samples were collected from six animals between 0 and 14 days post-treatment to characterize the drug/metabolite plasma disposition kinetics. Additionally, 16 lambs were sacrificed at 8, 24, 48 and 96 h post-administration to assess the drug concentrations in the GI fluid contents and tissues. MNP and MNPSO2 concentrations were determined by HPLC. MNP parent compound was rapidly oxidized into MNPSO2. MNP systemic availability was significantly lower than that observed for MNPSO2. The peak plasma concentrations were 15.1 (MNP) and 61.4 ng/ml (MNPSO2). The MNPSO2 to MNP plasma concentration profile ratio (values expressed in AUC) reached a value of 12. Markedly higher concentrations of MNP and MNPSO2 were measured in both abomasal and duodenal fluid contents, and mucosal tissues compared to those recovered from the bloodstream. A great MNP availability was measured in the abomasal content with concentration values ranging between 2000 and 4000 ng/g during the first 48 h post-treatment. Interestingly, the metabolite MNPSO2 was also recovered in abomasal content but its concentrations were significantly lower compared to MNP. The parent drug and its sulphone metabolite were detected in the different segments of the sheep intestine. MNPSO2 concentrations in the different intestine sections sampled were significantly higher compared to those measured in the abomasum. Although MNP is metabolized to MNPSO2 in the liver, the large concentrations of both anthelmintically active molecules recovered during the first 48 h post-treatment from the abomasum and small intestine may greatly contribute to the well-established pharmacological activity of MNP against GI nematodes.

Metabolic pathways of anthelmintic drug monepantel in sheep and in its parasite (Haemonchus contortus).

Monepantel (MOP) is a new anthelmintic drug intended for the treatment and control of gastrointestinal roundworms (nematodes) infection and associated disease in sheep. The aim of our study was to find out metabolic pathways of MOP in sheep in vivo and in its parasite Haemonchus contortus ex vivo. MOP biotransformation in two H. contortus strains with different sensitivity to anthelmintics was also compared. Ultra high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) technique is used for the identification of MOP metabolites in ovine urine, faeces, and nematodes. MOP biotransformation study in sheep in vivo led to the identification of 13 MOP metabolites; 7 of them have not been described previously in in vitro study. The study of MOP biotransformation in H. contorus ex vivo reveals four MOP metabolites. The nitrile hydrolysis as a new biotransformation pathway in helminths ex vivo is reported here for the first time. Unlike sheep, H. contorus nematodes are not able to metabolize MOP via phase II biotransformation. Nematodes of resistant White river (WR) strain form more types of MOP metabolites than nematodes of sensitive inbred susceptible Edinburgh (ISE) strain. Based on obtained results, schemes of metabolic pathways of MOP in sheep and nematodes are proposed.

Determination of the new anthelmintic monepantel and its sulfone metabolite in milk and muscle using a UHPLC-MS/MS and QuEChERS method.

This is the first paper to report a method for the detection of the new anthelmintic monepantel and its sulfone metabolite in goat's milk and ovine muscle. Samples were extracted and purified using a modified QuEChERS method. A concentration step was included when analyzing in the low µg kg(-1) range. Analysis was carried out by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in a 13min run time using atmospheric pressure electrospray ionisation in the negative mode (ESI(-)) and multiple reaction monitoring (MRM) scanning. Monepantel (m/z 472) and monepantel-sulfone (m/z 504) both had product ions at m/z 186 and m/z 166. The method has been single-laboratory validated according to the 2002/657/EC guidelines. The mean recovery in milk was 108 and 106% for monepantel and monepantel-sulfone, respectively. The mean recovery in muscle was 109 and 108% for monepantel and monepantel-sulfone, respectively. The coefficients of variation for the within laboratory repeatability and reproducibility were ≤6.4% in milk and ≤14.2% in muscle. The decision limits (CCα) in milk were 2.20 and 2.08 µg kg(-1) for monepantel and monepantel-sulfone, respectively. The decision limits (CCα) in muscle were 771 and 746 µg kg(-1) for monepantel and monepantel-sulfone, respectively.

Assessment of risk of monepantel faecal residues to dung fauna.

OBJECTIVE: To evaluate the safety of monepantel and its major metabolite for dung fauna. Monepantel is a new oral anthelmintic drug for use in sheep at a dose of 2.5 mg active ingredient/kg body weight. Hazard (toxicity) is related to the expected exposure. DESIGN: The methodology was based on the Organisation for Economic Cooperation and Development (OECD) draft guideline for testing of chemicals, so dung from cattle, not sheep, and eggs of the dung fly, Scathophaga stercoraria and larvae of the dung beetle Aphodius constans were used. PROCEDURES: Monepantel or its sulfone metabolite were mixed into bovine faeces in which either dung fly eggs or dung beetle larvae were placed and their development observed. The primary endpoint was survival. Real exposure data (faecal concentrations) for comparison with the generated laboratory data were taken from a kinetics study in sheep dung. RESULTS: The no-observed effect concentration (NOEC) for monepantel was >1000 mg/kg substrate for dung flies and 250 mg/kg for dung beetles. The sulfone metabolite was slightly more toxic, with a NOEC of 500 mg/kg for dung flies and 125 mg/kg for dung beetles. CONCLUSION: A comparison of the results to the maximum concentrations of 15 mg monepantel and 4.5 mg sulfone per kg dung observed under an exaggerated dosing regime in sheep indicates that monepantel poses no risk to insect dung fauna when used as recommended. The study is considered valid because representatives of both genera were able to develop in bovine or ovine dung.