A quick approach for medetomidine enantiomer determination in dog plasma by chiral liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study.

In the present study, a rapid, sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of medetomidine enantiomers in dog plasma was developed and validated. The separation and individual quantification of chiral compounds can be a tricky task in LC. This is particularly true when target analytes have a relatively small mass, as is the case with medetomidine, a potent and highly specific α2-adrenoceptor agonist widely used in both human and veterinary medicine. The proposed approach is based on a quick liquid-liquid extraction with ethyl acetate and filtration prior to injection. The optimized mobile phase composition allowed to perfectly separate the two enantiomers of medetomidine in a short chromatographic run time, using a cellulose tris(4-methylbenzoate)-based chiral column. A lower limit of quantification of 0.1 ng/mL was reached for both analytes thanks to the high sensitivity and selectivity of MS/MS and the use of racemic medetomidine-d3 as internal standard prevented potential matrix effect. Linearity was satisfying (R2  > 0.99) over the range 0.1-25 ng/mL, as well as within- and between-session accuracy and precision, both always <15%. This method was also applied with success to a series of samples from a pharmacokinetic (PK) study aimed at comparing dex- and levomedetomidine behaviour after administration of the racemic mixture in dogs. The simple extraction procedure, which allows reduced solvent and time consumption without compromising analytical performances, makes this technique a useful tool for this kind of applications even when small animals are involved, due to the small amount of sample required.

Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (-)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases.

The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter-day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.

Investigation of the complexation between cyclodextrins and medetomidine enantiomers by capillary electrophoresis, NMR spectroscopy and molecular modeling.

The migration order of the enantiomers of medetomidine in the presence of cyclodextrins studied by capillary electrophoresis in phosphate buffer, pH 2.5, depended on the cavity size and the substitution pattern of the cyclodextrins. Opposite migration order was observed in the presence of ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) as well as randomly sulfated ß-CD (S-ß-CD) and heptakis(6-O-sulfo)-ß-CD (HS-ß-CD). This could be rationalized by the fact that dexmedetomidine formed more stable complexes with ß-CD and S-ß-CD, while levomedetomidine interacted stronger with γ-CD and HS-ß-CD. The structure of the complexes was derived from rotating frame nuclear Overhauser (ROESY) experiments for ß-CD, γ-CD and HS-ß-CD. In the case of the native CDs, the phenyl ring of medetomidine entered the cavity through the wider secondary rim of the CDs, whereas the protonated imidazole ring was positioned inside the CD cavity interacting with the sulfate groups of HS-ß-CD. Furthermore, molecular dynamics calculations also suggested opposite affinities of the medetomidine enantiomers toward ß-CD and γ-CD.

Exposure to the antifouling chemical medetomidine slows development, reduces body mass, and delays metamorphosis in wood frog (Lithobates sylvaticus) tadpoles.

Antifouling chemicals have a long history of causing toxicity to aquatic organisms. We measured growth and developmental timing in wood frog tadpoles exposed to the antifouling chemical medetomidine (10 nM-10 µM) starting at two different developmental stages in static renewal experiments. For tadpoles hatched from egg masses and exposed for 3 weeks to 100 nM and 1 µM, head width/total body length ratio was significantly shorter compared to control. For field-collected tadpoles at Gosner stage 24-25 and exposed for 2 weeks, 1 and 10 µM medetomidine significantly slowed development as measured by Gosner stage. Medetomidine (1 and 10 µM) significantly increased the time to metamorphosis by over 16 days on average, and at 100 nM and 1 µM, it significantly decreased mass at metamorphosis. We discuss the possible effects of antifouling chemicals containing medetomidine on globally threatened groups such as amphibians.

Affinity states of biocides determine bioavailability and release rates in marine paints.

A challenge for the next generation marine antifouling (AF) paints is to deliver minimum amounts of biocides to the environment. The candidate AF compound medetomidine is here shown to be released at very low concentrations, ie ng ml(-1) day(-1). Moreover, the release rate of medetomidine differs substantially depending on the formulation of the paint, while inhibition of barnacle settlement is independent of release to the ambient water, ie the paint with the lowest release rate was the most effective in impeding barnacle colonisation. This highlights the critical role of chemical interactions between biocide, paint carrier and the solid/aqueous interface for release rate and AF performance. The results are discussed in the light of differential affinity states of the biocide, predicting AF activity in terms of a high surface affinity and preserved bioavailability. This may offer a general framework for the design of low-release paint systems using biocides for protection against biofouling on marine surfaces.

Anion effect on the binary and ternary phase diagrams of chiral medetomidine salts and conglomerate crystal formation.

The binary phase diagrams of hydrogen halides salts of medetomidine (Med.HX, X:Br,I) and hydrogen oxalate salt of medetomidine (Med.Ox) were determined based on thermogravimetric/differential thermal analysis (TGA/DTA) and their crystal structure behavior was confirmed by comparison of the X-ray diffractometry and FT-IR spectroscopy of the racemate and pure enantiomer. All hydrogen halide salts presented racemic compound behavior. Heat of fusion of halides salt of (rac)-medetomidine decreased with ionic radius increase. Eutectic points for Med.HCl (previously reported), Med.HBr, and Med.HI rest were unchanged approximately. The solubility of different enantiomeric mixtures of Med.HBr and Med.HI were measured at 10, 20, and 30°C in 2-propanol showing a solubility increase with ionic radius. A binary phase diagram of Med.Ox shows a racemic conglomerate behavior. The solubility of enantiomeric mixtures of Med.Ox were measured at 10, 20, 30, and 40°C. The ternary phase diagram of Med.Ox in ethanol conforms to a conglomerate crystal forming system, favoring its enantiomeric purification by preferential crystallization.

Complexation chemistry for tuning release from polymer coatings.

The strategy of metal ion complexation is employed to design a delivery system for an antifouling agent (AFA) in marine paints. A poly(1-vinylimidazole-co-methyl methacrylate) copolymer (PVM), together with Cu2+ or Zn2+ formed a PVM-M2+ complex. The AFA, Medetomidine, was then coordinated into the complex. The coordination strength was investigated in solution by 1H NMR and on solid surfaces by using the Quartz Crystal Microbalance with Dissipation monitoring technique (QCM-D) and Surface Plasmon Resonance (SPR). From the 1H NMR experiments strong interactions were observed between Cu2+ and the PVM-polymer and between Medetomidine and the PVM-Cu2+ complex. From the QCM-D and SPR measurements it was shown that Cu2+, compared to Zn2+, exhibited a larger affinity for the PVM-copolymer surface that resulted in higher degree of swelling of the polymer film. Large amounts of Medetomidine were adsorbed to the PVM-Cu2+ complex resulting in low desorption rates. However, the adsorbed amount of Medetomidine was lower to the Zn2+ doped polymer and a higher desorption rate was observed. These results indicate the possibility of tuning the release of Medetomidine by altering the coordinating metal ion, which may prove to be favorable in a paint formulation.

Impact of polymer surface affinity of novel antifouling agents.

In a previous study we found two agents, the alpha(2)-agonist medetomidine ((+/-)-4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) and the alpha(2)-agonist clonidine (2-(2,6-dichloroanilino)-2-imidazoline), that specifically and efficiently impede settlement of the barnacle Balanus improvisus, one of the most serious biofouling organisms in Swedish waters. Medetomidine, but not clonidine, is known to adsorb to solid polystyrene (PS) surfaces in the presence of salt, a feature that is of particular interest in attempts to develop an efficient antifouling surface. We show that medetomidine, but not clonidine, has a significant ability to adsorb to untreated (hydrophobic) PS in two different incubation media: filtered seawater (FSW) and deionized water (mQ). At negatively charged (hydrophilic) PS, medetomidine displays a strong interaction with the surface in both incubation media. At the hydrophilic PS, clonidine also displays a significant interaction with the surface when incubated in mQ and a weaker, but not significant, interaction when incubated in FSW. By studying the effects of time, incubation media, and pH on the adsorption of medetomidine and clonidine, we suggest that medetomidine is associated to hydrophobic PS by means of hydrophobic interactions, while the adsorption of medetomidine and clonidine to hydrophilic PS contains elements of electrostatic interaction. Using time-of-flight secondary ion mass spectroscopy (TOF-SIMS) we detected only weak signals from medetomidine on the hydrophobic PS surfaces, while strong medetomidine signals were observed on hydrophilic PS. This suggests that the adsorbed medetomidine, to a greater extent, desorbed from the hydrophobic rather than from the hydrophilic PS surfaces during exposure to vacuum. The strong surface affinity of medetomidine on both types of surfaces and the preserved antifouling activity are valuable features in designing a marine coating.

Medetomidine analogs as selective agonists for the human alpha2-adrenoceptors.

alpha(2)-Adrenoceptor (AR) agonists have therapeutic applications in a variety of diseases. Medetomidine, an alpha(2)-AR agonist, belongs to 4-substituted imidazole class of compounds and is highly selective for the alpha(2)-AR. The benzylic methyl group of medetomidine and naphthalene imidazole was proposed to interact with rat brain alpha(2)-ARs via a methyl binding pocket in a manner analogous to its presence in alpha-methyl norepinephrine. A series of derivatives containing hydrophilic and hydrophobic substituents, as well as chiral and conformationally rigid analogs were used. In current binding and functional studies using human alpha(2)-AR subtypes expressed in Chinese hamster ovary cells, optimal interactions were observed with the presence of the methyl group on the benzylic carbon atom of naphthyl imidazole. Data obtained with various analogs have demonstrated that size, electronegativity, lipophilicity, chirality and conformational flexibility of the substituents at the carbon bridge of naphthyl imidazole are important factors for interaction of the imidazole class of ligands with these alpha(2)-AR subtypes. Taken collectively, the results obtained support the existence of the methyl binding pocket for optimal ligand receptor binding interactions in human alpha(2)-AR subtypes. Further, the results also suggest that, additional modifications of medetomidine and naphthyl methyl imidazole at the benzylic carbon atom, and/or on the aromatic and imidazole ring systems could provide insights into the chemical requirements for optimizing alpha(2)-AR subtype selectivity. This could eventually lead to the discovery of promising compounds for the evaluation of the physiological importance of the three alpha(2)-AR subtypes.