Pharmacokinetics of dexmedetomidine combined with methadone following oral-transmucosal and intramuscular administration in dogs

Oral-transmucosal (OTM) drug delivery refers to noninvasive and painless administration of medical preparations through any oral cavity membrane to achieve systemic effects (Sattar et al., 2014). Regarding sedative drugs, OTM administration is very attractive in veterinary medicine, especially for patients difficult to inject and restrain (Messenger et al., 2016). This study aims to compare the pharmacokinetics of dexmedetomidine after OTM and intramuscular (IM) administration combined with methadone. After obtaining Ethical Committee approval and owner’s written consent, eight dogs, were administered with dexmedetomidine (10 mg/kg) and methadone (0.4 mg/kg) by OTM and other 4 dogs by IM route. Blood samples were collected at prefixed times up to four hours. Dexmedetomidine was quantified by a validated HPLC-MS method. On dexmedetomidine concentrations, a pharmacokinetic analysis was carried out with a noncompartmental approach (Phoenix WinNonlin® 7.0, Pharsight, Cary, NC). Mean ± SD terminal half-lives of dexmedetomidine were 187.42 ± 109.66 and 94.78 ± 34.08 min after OTM and IM administration, respectively. Maximum serum (Cmax) concentrations were 0.83 ± 0.32 and 9.09 ± 2.46 ng/mL for OTM and IM administration, respectively. Time to maximum concentration (Tmax) were 44.38 ± 32.16 and 21.25±11.39 min by OTM and IM administration, respectively. Area under the curve from 0 to the last measured concentration (AUClast) were 103.75 ± 30.23 and 614.87 ± 77.15 min*ng/mL for OTM and IM administration, respectively. Cmax, Tmax and AUClast values by OTM route demonstrate a lower and delayed absorption of the drug compared to IM. To complete the study, the pharmacokinetic analysis of methadone is foreseen, so as a clinical trial to compare the clinical effects of the combination of dexmedetomidine and methadone by OTM and IM administration and to establish an effective dosage of oral-transumucosal route in dogs for this association

Integrated analytical approach in veal calvesadministered the anabolic androgenic steroidsboldenone and boldione: urine and plasma kineticprofile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-QMS/ MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone a- and b-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE,MALDI-TOF-MS and mLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasmasamples collected before treatment,was found upregulated even 36 h after hormone treatment.Extensivemassmapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed byWestern blot analysis confirmed a significant and time dependent increase of thisApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine

Transgenerational toxicity of flumequine over four generations of Daphnia magna

In this study, the effects of both continuous and alternate exposure to 2 mg L-1 of flumequine (FLU) on survival, growth and reproduction of Daphnia magna were evaluated over four generations. Mortality was the most evident effect, with an average mortality rate of 23\ub114 % across generations. Individuals destined to succumb were identifiable well in advance through their discolouration and lack of development, and limited or zero reproductive capacity. Inhibition of reproduction in surviving mothers varied across the four generations (14.3\ub117 %) without an apparent correlation with the duration of exposure over generations. Significant reproductive inhibition was observed in the generation that followed three non-exposed generations (the fourth generation), pointing to a transgenerational toxicity of FLU. In another experiment, in vitro exposure of 72 D. magna embryos to 2 mg L-1 FLU caused 14% mortality (versus 7% in the control). Among the 62 individuals that hatched alive, six showed birth defects and only one was able to survive the next few days. The other, apparently healthy newborns were randomly assigned to two groups and submitted to a reproduction test, either in the absence or in the presence of 2 mg L-1 FLU. A high mortality rate and/or strongly significantly inhibited reproduction were detected in both groups. As with previously run analogous tests with enrofloxacin, the multigenerational and embryonic tests showed a clear disruption to this crustacean population which would not be evidenced by the standard official acute and chronic tests. This indicates the necessity of taking a different and more comprehensive approach to the evaluation of substances having an inherent ability to interact with genetic material

Validation of a high liquid chromatography method (HPLC) for simultaneous quantification of skatole and indole in porcine fat

Boar taint is a meat quality defect that may lead to an unpleasant experience during the consumption of meat and meat products with negative economic repercussions for food industry. It can occur when meat or fat from entire male pigs is heated, due to excessive accumulation of skatole, indole and androstenone (1). For a fast identification and quantification of skatole and indole, an high-pressure liquid chromatography analytical method, coupled with a fluorimetric detector (HPLC-FD), has been set up and validated. The extraction of the target analytes (skatole, indole and internal standard, 2-methylindole) from lipid matrix (approximately 0.5 ± 0.05 g of subcutaneous fat taken from female pigs at slaughterhouse) was performed by sample liquefaction, addition of pure methanol and separation of the organic solvent was obtained by cooling and centrifugation steps (2). Chromatography separation was achieved using a simple isocratic HPLC method (3). In addition to the limit of detection (LOD) and quantification (LOQ), linearity, recovery, accuracy and sensitivity were evaluated for the validation of the method. A good linearity was confirmed by the correlation coefficient (R2) of the standards calibration curves always ≥ 0,99, together with the recovery from matrix for both analytes, always ≥ 96%. The precision of the method, intra-and inter-assay, expressed as relative standard deviation (RDS%) was good for each analyte, always below the limit of 10%. Good results were obtained also for LOD and LOQ, amounting to 1,562 ng/g and 2,312 ng/g for both compounds, concentrations far lower than those perceived by human smell. The proposed method is suited for rapid routine analyses because of its high sensitivity and precision, required by small sample size and was always reliable to identify and quantify target compounds in all samples analyzed. The follow application of the method to samples (28) taken at slaughterhouse by different subjects (sows, entire and castrated male), has allowed always to identify and quantify skatole and indole, confirming the robustness of the method. The small sample size of sample used, allow the application of the method also to samples of fatty tissue taken from pigs by subcutaneous biopsy

Total aflatoxins in corn: analytical performance of two elisa kit

A careful and accurate control of corn for the presence of Aflatoxins (AFs) is crucial since they represent a risk both for agriculture and for human and animal health, in particular AF B1 is recognized as the most potent hepatocarcinogenic of natural origin for human (1). For these reasons accurate, sensitive, reproducible and rapid methods are required to guarantee food safety. The aim of this work was to evaluate two commercial ELISA kits (A and B) in terms of reliability and accuracy to establish the level of contamination in corn samples by AFs. Both kits used had the same analytical procedure (direct and competitive) and also the same range of determination (1-20ppb). To evaluate the reliability in terms of analytical response, the results obtained from kit analysis, were compared with those obtained by liquid chromatographic analysis (HPLC) of corn samples fortified with AFs in the concentration range of 2.16 to 18.49 ppb (4 points, 4 replicates each). The average of the values of kit B deviated from the statistical point of view, significantly, from values obtained after HPLC analysis, while the curve of the results obtained from analysis by kit A, was comparable to that obtained with chromatography. The recovery and repeatability of ELISA kits were evaluated taking into account the performance criteria for methods for determining AFs according to European Regulation 401/2006/EC. Kit A showed medium recovery always greater than kit B, characterized by recovery rates next to the minimum performance values required. Kit B therefore underestimates the AFs concentration in corn samples, especially for the lower levels of contamination, confirming his lowest reliability and accuracy. Regarding repeatability (RDS%), an index of accuracy, was acceptable at all concentration levels for both kits, respecting the criteria imposed by European Regulation 401/2006/EC (2). Finally, we evaluated the analytical performance over time of ELISA kits in three different sessions of work, after 10 days apart (T0, T10, T20). Both kits maintained unchanged analytical performance up to 20 days after the opening, if properly stored

Pyrrolizidine alkaloids and relative N-oxides evaluation in Italian honey from the Veneto region (Italy) by LC-MS/MS.

Pyrrolizidine alkaloids (PAs) are the most common class of natural toxins (approximately 350 types), synthesised by a wide variety plant species and the presence of PAs in food and feed clearly represents a potential risk to human and animals. In this study, an analytical method by liquid chromatography coupled to mass spectrometry (LC-MS/MS) was applied to evaluate and characterise PA and their N-oxides (PANOs) content in Italian honey. Echimidine, heliotrine, lycopsamine, senecionine, seneciphylline, retrorsine and the corresponding PANOs were considered. For the matrix clean-up and the extraction of PAs/PANOs, the solid phase extraction with non-polar/cation-exchange polymeric cartridges, PLEXA 200 mg/6 mL, (Agilent Technologies, USA) were adopted. Analyses were performed by a LTQ XL ion trap, equipped with a heated electrospray ionisation probe, operating in positive ion mode (Thermo Fischer Scientific, San Jose, CA, USA). The linearity of the calibration curves was evaluated in the range of 0.25-50 ug kg-1, the mean recovery ranged from 82.70 % to 104.16 %; LOQ was 0.25 ug kg-1 for all PAs/PANOS, fully satisfying the limit stated in the document \u201cSummary report of the standing committee on plants, animals, food and feed held in Brussels on 1 July 2014\u201d. 147 honey samples from Veneto region were analysed, according to their geographical origin. Analyses showed that 69% of the honey samples were negative for all PAs/PANOs monitored, 31% were positive. In all positive samples the contribute from target PANOs was negligible (range 0.39-1.52 \ub5g kg-1). The total content of PAs/PANOs monitored in Veneto honey ranged from 0.25 to 64.44 \ub5g kg-1. These results are not consistent with previous studies about honey samples from other European countries. Taking into account the indication from CONTAM Panel (EFSA 2011), based on the Margin of Exposure (MOE) approach, a honey sample must not exceed a PAs/PANOs total content of 21 \ub5g kg-1; only 9% of our samples exceed this limit. Moreover, preliminary results showed an influence of two important factor as season (summer > spring) and territory (hills > lowland) on the total content of PAs/PANOs in honey. Honey produced from local beekeepers of Veneto region is not a real threat to health, while a monitoring program of PAs/PANOs in honey is highly desirable to risk assessment

In vitro hydroxylation of testosterone in rabbits: influence of age and sex*

Introduction Hydroxylated metabolites of testosterone (T) have been proposed as useful markers of specific isoform P450 activity in rats (1). Only few studies have been devoted to characterisation of P450 biotransformation activity in food producing species thus the aim of this study was to define the influence of sex and age in rabbits on the metabolic profile of hydroxylated testosterone (OHT) metabolites using an HPLC method able to separate nine metabolites. The role played by CYP3A was also studied in Rifampicin (RIF) induced rabbits. Material and methods Liver microsomes were obtained (2) from rabbits. five male (3\ub10.4 kg), five female (3\ub10.2 kg), five young male (1,5\ub10,3 kg), and two male induced with RIF (50 mg/kg, 4 days, ip). Microsomes (0.2 mg/ml) were incubated for 10 min, at 37\ub0C with 250 \ub5M T. Hydroxylated metabolites were extracted with methylene chloride and separated according to Purdon (3) with HPLC using a C18column and ternary phase gradient elution. Elution times were compared with those of pure standards (Steraloids). Discussion Main differences are: the absence in males of 16alfa-OHT; the greater production (*P<0,001) of 6beta-OHT, 11alfa-OHT, 16beta-OHT and 2beta-OHT compared to females; the decreased production (#P<0.001) of 6beta-OHT and 16alfa-OHT in young rabbits versus adult ones. Based on data reported for other species (1, 4) apparently, the isoforms involved in rabbits are CYP3A for 6beta-OHT, and 2beta-OHT; and CYP2B for 16alfa-OHT, and 16beta-OHT. RIF induced microsomes gave a significantly (\ub0P<0,001) greater quantities of 6beta-OHT, 2beta-OHT supporting, also in rabbits, the CYP3A role played in the production of these hydroxyl-derivatives

Valutazione, con Daphnia magna, della tossicità acuta di quattro antibatterici

Oxytetracycline (OTC), tylosin (TYL), sulfadimethoxine (SDM) and sulfamethazine (SMZ) are largely employed for mass treatments in food animals and so prone to contaminate the environment. According to the EMEA guidance document on Environmental Impact Assessment for veterinary drugs, a Predicted No Effect Concentration in water should be derived from the acute toxicity test in Daphnia magna. Using Daphtoxkit® (compliant with OECD Guideline 202) their toxicity on Daphnia magna was evaluated. Results shows a low sensitivity to OTC and SDM, confirms a moderate sensitivity to TYL (EC50=1012 mg/L) and highlights the particular sensitivity to SMZ (EC50=112 mg/L). This is of interest because SMZ is one of the most widely used sulfonamide and has been detected in soil and in wells