Determination of Menbutone: Development and Validation of a Sensitive HPLC Assay according to the European Medicines Agency Guideline

Abstract

Menbutone is often used to stimulate the hepato-digestive activity in case of digestive disorders and hepatic insufficiency in different species (cattle, sheep, goats, pigs, horses, dogs) because it has choleretic and cholagogue effects and increases gastric and pancreatic juices. The objective was to develop and validate an HPLC method to quantify menbutone in sheep plasma using sparfloxacin as internal standard. The HPLC mobile phase consisted of acetonitrile:monopotassium phosphate solution. The method was validated according to EMA guideline (EMEA/CHMP/EWP/192217/2009). Mean retention times of menbutone and sparfloxacin were 4.5 and 2.2 min, respectively. The method met all specifications of the EMA guideline, being selective and linear in the range of 0.2–100 µg/mL (R2 ≥ 0.99). The within-run precision range was 0.19–8.21%, with an accuracy of 102.99–119.52% for the lower limit of quantitation (LLOQ). For the other values (LOW, MED, HIGH) the precision range was 0.01–4.77%, with an accuracy of 85.17–109.67%. The LLOQ was 0.2 µg/mL, and no interference from the biological matrix was found. Stability of menbutone in the biological matrix at different storage conditions was also demonstrated. Thus, the method can be used to determine menbutone concentrations in plasma sheep in different types of studies.

Pharmacokinetics of menbutone after intravenous and intramuscular administration to sheep

“…In fact, only few data are available for some pharmacokinetic parameters in the SPC of those medicinal products approved in the EU, as the holders of the marketing authorization are not obliged to make public those studies carried out to obtain this authorization. Several papers reported methods to determine menbutone residues in different tissues ( 7 , 13 , 14 ), and only one study has described in a preliminary way the distribution, metabolism and excretion of menbutone in rats ( 2 ).…”

“…Menbutone extraction from plasma samples was performed according to a method previously validated following the EMA guideline EMEA/CHMP/EWP/192217/2009 ( 7 , 8 ). Analysis was carried out by high-performance liquid chromatography (HPLC) with photodiode array detector (PDA model 2998) in a Waters Alliance e2695 system (Waters Corporation, Mildford, MA, USA).…”

“…Plasma samples were processed according to a previously published method ( 7 ). Solid-phase extraction (SPE) with Oasis HLB 1 cc 30 mg cartridges (Waters Corporation, Mildford, MA, USA) was used.…”

Development and Validation of a Sensitive HPLC Assay for Determination of Sparfloxacin According to the European Medicines Agency Guideline

“…According to a literature survey, there are few methodologies for determining SPAR in various liquids, plasma and dosage forms in humans [13][14][15][16]. In addition, there are few reports regarding pharmacokinetic analysis of SPAR in animals [2,3,8,17,18], and none of them have been carried out in sheep, although an HPLC method using it as an internal standard was validated in sheep plasma [19]. The numerous microbiological and pharmacokinetic advantages of SPAR compared to other members of its group could make it an excellent candidate to treat infections caused by susceptible pathogenic species in sheep.…”