Clenbuterol in the horse: Confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration

Clenbuterol is a β2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 μg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of ∼150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of ∼500 pg/mL and then declined with a half-life of ∼7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 μg) was also administered intratracheally to five horses. Peak serum concentrations of ∼230 pg/mL were detected 10 min after administration, dropping to ∼50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach, offers considerable scientific and regulatory advantages over more traditional urine testing approaches

Production performance of pigs reared in different systems and fed increased energy content diets with or without green alfalfa

Abstract The objective of this study was to determine the effects of various feeding and housing systems on fattening performance, slaughter value and biochemical serum parameters in growing-finishing pigs. The experimental material comprised 90 growing-finishing pigs, divided into six groups of 15 animals that were diverse in terms of rearing (with or without free access to outdoor runs) and feeding systems (fed increased metabolizable energy (ME) content diets with or without green alfalfa). Different feeding regimes and rearing systems had no significant effects on most fattening results and the carcass traits of the pigs except for daily water and diet intake and feed/gain ratio. Daily water intake was lower in pigs fed complete diets plus green alfalfa forage. Low-density lipoprotein (LDL) cholesterol concentrations were statistically lower in the blood serum of Groups 3 and 6 than in Groups 2 and 4. High-density lipoprotein (HDL) cholesterol levels were significantly higher in the serum of Group 6, compared with other groups. Group 3 had significantly higher triglycerides levels compared with Groups 1, 2, 4, 5 and 6. Taking into consideration all these dependencies, it could be assumed that feeding regime and rearing with access to outdoor runs might improve not only blood lipid profile, but also the dietetic value of pork. It seems that feeding a complete diet with increased ME, plus green alfalfa forage and rearing indoors with free access to outdoor runs, gave the best results. _____________________________________________________________________________________

Review of Environmental Morphine Identifications: Worldwide Occurrences and Responses of Authorities

Opium poppies grow wild worldwide, and testing for morphine is now highly sensitive. Currently, many authorities worldwide do not pursue urinary morphine concentrations of less than 100 ng /ml. This is because such low urinary morphine concentrations are likely to be environmental morphine identifications (EMIs) and are also unlikely to be associated with pharmacological responses

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine

Amitraz (N′-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino] methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N′-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d6 deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intraveneous dose

Remifentanil in the Horse: Identification and Detection of its Major Urinary Metabolite

Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phyenylamino]-1- piperidinepropionic acid methyl ester) is a μ-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1- oxopropyl)phenylamino]-1-piperidinepropionic acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentanyl equivalents in these samples. Mass spectrometric analysis, using solid-phase extraction and trimethylsilyl (TMS) derivatization, showed the urine samples contained parent remifentanil in low concentrations, peaking at 1 h. More significantly, a major peak was identified as representing 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1- piperidinepropionic acid, arising from ester hydrolysis of remifentanil. This metabolite reached its maximal urinary concentrations at 1 h and was present at up to 10-fold greater concentrations than parent remifentanil. Base hydrolysis of remifentanil yielded a carboxylic acid with the same mass spectral characteristics as those of the equine metabolite. In summary, these data indicate that remifentanil administration results in the appearance of readily detectable amounts of 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]- 1-piperidinepropionic acid in urine. On this basis, screening and confirmation tests for this equine urinary metabolite should be optimized for forensic control of remifentanil

Detection and Confirmation of Ractopamine and Its Metabolites in Horse Urine after Paylean® Administration

We have investigated the detection, confirmation, and metabolism of the beta-adrenergic agonist ractopamine administered as Paylean to the horse. A Testing Components Corporation enzyme-linked imunosorbent assay (ELISA) kit for ractopamine displayed linear response between 1.0 and 100 ng/ml, with an 1-50 of 10 ng/ml, and an effective screening limit of detection of 50 ng/mL. The kit was readily able to detect ractopamine equivalents in unhydrolyzed urine up to 24 h following a 300-mg oral dose. Gas chromatography-mass spectrometry (GC-MS) confirmation comprised glucuronidase treatment, solid-phase extraction, and trimethylsilyl derivatization, with selected-ion monitoring of ractopamine-tris(trimethylsilane) (TMS) m/z 267, 250, 179, and 502 ions. Quantitation was elaborated in comparison to a 445 Mw isoxsuprine-bis(TMS) internal standard monitored simultaneously. The instrumental limit of detection, defined as that number of ng on column for which signal-to-noise ratios for one or more diagnostic ions fell below a value of three, was 0.1 ng, corresponding to roughly 5 ng/mL in matrix. Based on the quantitation ions for ractopamine standards extracted from urine, standard curves showed a linear response for ractopamine concentrations between 10 and 100 ng/mL with a correlation coefficient r \u3e 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r \u3e 0.99. The lower limit of detection for ractopamine in urine, defined as the lowest concentration at which the identity of ractopamine could be confirmed by comparison of diagnostic MS ion ratios, ranged between 25 and 50 ng/mL. Urine concentration of parent ractopamine 24 h post-dose was measured at 360 ng/mL by GC-MS after oral administration of 300 mg. Urinary metabolites were identified by electrospray ionization (+) tandem quadrupole mass spectrometry and were shown to include glucuronide, methyl, and mixed methyl-glucuronide conjugates. We also considered the possibility that an unusual conjugate added 113 amu to give an observed m/z 415 [M+H] species or two times 113 amu to give an m/z 528 [M+H] species with a daughter ion mass spectrum related to the previous one. Sulfate and mixed methyl-sulfate conjugates were revealed following glucuronidase treatment, suggesting that sulfation occurs in combination with glucuronidation. We noted a paired chromatographic peak phenomenon of apparent ractopamine metabolites appearing as doublets of equivalent intensity with nearly identical mass spectra on GC-MS and concluded that this phenomenon is consistent with Paylean being a mixture of RR, RS, SR, and SS diastereomers of ractopamine. The results suggest that ELISA-based screening followed by glucuronide hydrolysis, parent drug recovery, and TMS derivatization provide an effective pathway for detection and GC-MS confirmation of ractopamine in equine urine

Effect of rearing system conditions of fatteners on meat quality and profile of fatty acids in m. Longissimus dorsi

Badano jakość mięsa tuczników utrzymywanych alkierzowo lub na wolnym wybiegu, w kojcach ściołowych lub bezściołowych i żywionych w okresie tuczu mieszanką pełnoporcjową lub mieszanką pełnoporcjową i, dodatkowo, zielonką z lucerny. Tuczniki mieszańce [♀(♀polska biała zwisłoucha × ♂ wielka biała polska) × ♂ (♀ pietrain × ♂ duroc)] podzielono na 6 grup doświadczalnych po 15 sztuk w każdej. Z tusz tuczników pobrano próbki mięśnia najdłuższego grzbietu (m. Longissimus dorsi), w których oznaczono podstawowy skład chemiczny, właściwości fizykochemiczne, cechy sensoryczne i profil kwasów tłuszczowych. Najmniejszą zawartość tłuszczu surowego stwierdzono w mięsie pozyskanym z tuczników utrzymywanych na ściółce i żywionych paszą z dodatkiem zielonki z lucerny. Najwyższy poziom kwasów tłuszczowych jednonienasyconych (MUFA) stwierdzono w próbach mięsa pochodzących z tuczników utrzymywanych bezściołowo i z dostępem do wybiegu, niezależnie od zastosowanego żywienia. Najmniej pożądanym zapachem i największą soczystością charakteryzowało się mięso pozyskane ze świń utrzymywanych na ściółce, żywionych mieszanką pełnoporcjową i dodatkowo zielonką. W warunkach przeprowadzonego i opisanego doświadczenia nie można jednoznacznie stwierdzić, który sposób żywienia i utrzymania tuczników wpłynął na istotną poprawę jakości pochodzącego z nich mięsa.Analyzed was the meat quality of fatteners, which were reared indoor or outdoor, in bedded or nonbedded pens, and fed a complete feed mixture or a complete feed mixture and, additionally, a green forage of alfalfa. The crossbred fatteners [♀(♀ PL × ♂ PLW) × ♂ (♀ Pietrain × ♂ Duroc)] were divided into six experimental groups, 15 pigs in each group. Samples of m. Longissimus dorsi were collected from the fattener carcasses and in the samples determined were the basic chemical composition, physicochemical and sensory properties, and fatty acid profile. The lowest level of crude fat was found in the meat samples derived from the fatteners reared indoor in bedded pens and fed a fodder with green forage of alfalfa added. The highest level of monounsaturated fatty acids (MUFA) was obtained in the meat samples derived from the fatteners kept outdoor, without bedding, regardless of the feeding type applied. The meat derived from the fatteners kept in bedded pens, fed a complete feed mixture and, additionally, green forage of alfalfa was characterized by the least desirable aroma and the best juiciness. Under the particular conditions of the experiment accomplished, it was impossible to clearly and precisely determine what methods of feeding and rearing the fatteners impacted the significant improvement of the quality of meat thereof