Quantification and enantiomeric distribution of clenbuterol in several bovine tissues using UHPLC-tandem mass spectrometry: Evaluation of a risk factor associated with meat contamination.

Clenbuterol (Clb) (4-amino-α-[(tert-butylamine) methyl]-3,5-dichlorobenzyl alcohol) is a sympathomimetic agent that exhibits ß2-agonist activity. It is applied as a bronchodilatory, tocolytic, and mucolytic agent and is authorized for clinical management in both human and veterinary therapeutics as a racemic mixture. However, its use is strictly prohibited in animals destined for food production in countries in the European Union and in the United States and Mexico, among many others. The R-(-) enantiomer in clenbuterol stimulates ß2-receptors, whereas the S-(+) enantiomer blocks the effect of ß1-receptors. The aims of this study were to develop a method for detecting and quantifying Clb and its enantiomeric distribution in several bovine tissues. The UHPLC-MS/MS method developed to quantify the target compound at trace levels in these tissues combines high sensitivity with good selectivity and short chromatographic run time. The tissue samples tested were found to contain racemic Clb in concentrations of 5-447 pg g-1 . The enantiomeric analysis of Clb showed that R-(-)-Clb is present at higher concentrations in some tissues, whereas S-(+)-Clb was detected in a ratio of 55/45 in the liver and heart tissues.

Recent Developments and Applications of Nanosystems in the Preservation of Meat and Meat Products.

Due to their high water, lipid, and protein content, meat and meat products are highly perishable. The principal spoilage mechanisms involved are protein and lipid oxidation and deterioration caused by microbial growth. Therefore, efforts are ongoing to ensure food safety and increase shelf life. The development of low-cost, innovative, eco-friendly approaches, such as nanotechnology, using non-toxic, inexpensive, FDA-approved ingredients is reducing the incorporation of chemical additives while enhancing effectiveness and functionality. This review focuses on advances in the incorporation of natural additives that increase the shelf life of meat and meat products through the application of nanosystems. The main solvent-free preparation methods are reviewed, including those that involve mixing organic-inorganic or organic-organic compounds with such natural substances as essential oils and plant extracts. The performance of these additives is analyzed in terms of their antioxidant effect when applied directly to meat as edible coatings or marinades, and during manufacturing processes. The review concludes that nanotechnology represents an excellent option for the efficient design of new meat products with enhanced characteristics.

Detection of clenbuterol residues in beef sausages and its enantiomeric analysis using UHPLC-MS/MS: A risk of unintentional doping in sport field.

Clenbuterol (Clb) can be present in Mexico often but not all over the world in animal tissues and organs, therefore, potentially is derived from animal sources as well. The aims of this study were to develop and validate a method for detecting traces of clenbuterol in beef sausages. A calibration curve showed linearity in the range of 20-500 pg ml-1 . The limit of detection (LOD) and lower limit of quantification (LLOQ) were 5 and 10 pg g-1 , respectively. The analyte recovery was from 95.70% to 100.40% with an intraday relative standard deviation (RSD%) of 0.99%-2.10% and an interday RSD% of 0.54%-2.34%, R2 = 0.9998. The methodology developed was applied successfully in 15 samples of beef sausage, and 73.3% of the samples tested contained racemic clenbuterol in concentrations between 30 and 471 pg g-1 . The UHPLC-MS/MS method developed combines high sensitivity with good selectivity and short chromatographic run time. Additionally, the enantiomeric analysis of clenbuterol performed in beef sausages showed a 59% for R-(-)-Clb and 41% for S-(+)-Clb. The presence of clenbuterol in beef sausages could represent a risk of unintentional doping in sport field, because the clenbuterol is a banned substance included in the World Anti-Doping Agency's (WADA) list of prohibited substances.

Determination of clenbuterol at trace levels in raw gelatin powder and jellies using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry.

Clenbuterol is present in animal tissues and organs and, therefore, potentially present in gelatin derived from animal sources. The objective of this study was to develop a method for identify an quantify traces of clenbuterol in gelatin and jellies. The clenbuterol calibration curve showed linearity in the range of 20-1000 pg mL-1. The detection and quantification limits were 5 pg g-1 and 10 pg g-1, respectively. The recovery of the analyte ranged from 93.4 to 98.7% with an intra-day RSD% (n = 4) of 1.25%-3.25%, and an inter-day RSD% (n = 12) of 0.5%-2.25%, with good linearity (R2 = 0.99). The method developed and validated was successfully applied in 54 gelatin samples, 57.4% of which showed clenbuterol. This UHPLC-MS/MS method combines high sensitivity with good selectivity and short chromatographic run time.

Influence of Stabilizing and Encapsulating Polymers on Antioxidant Capacity, Stability, and Kinetic Release of Thyme Essential Oil Nanocapsules.

The release kinetics, stability, and antioxidant capacity of thyme essential oil polymeric nanocapsules as a function of encapsulating (poly-ε-caprolactone and ethylcellulose) and stabilizing (polyvinyl alcohol and Pluronic® F-127) polymers were established. Samples were evaluated in terms of particle size, zeta potential, release kinetics, calorimetry, infrared spectra, antioxidant capacity, and diffuse reflectance. The particle size obtained was below 500 nm in all cases, ensuring nanometric size. Zeta potential as a function of the stabilizing polymer. Encapsulation efficiency was higher in the samples that contained ethyl cellulose (around 70%), associated with its affinity for the molecules contained in the essential oil. Differential scanning calorimetry revealed a strong dependence on the encapsulating polymers as a function of the melting temperatures obtained. Infrared spectra confirmed that the polymeric nanocapsules had the typical bands of the aromatic groups of thyme essential oil. The antioxidant capacity evaluated is a function exclusively of the active content in the nucleolus of the nanocapsules. Nanoencapsulation was not a significant factor. Diffuse reflectance revealed high physical stability of the dispersions related directly to the particle size and zeta potential obtained (either by ionic or steric effect). These findings confirm favorable characteristics that allow proposing these systems for potential applications in food processing and preservation.

Quantification and Stereochemical Composition of R-(-) and S-(+)-Clenbuterol Enantiomers in Bovine Urine by Liquid Chromatography-Tandem Mass Spectrometry.

Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a ß2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10-8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10-10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P < 0.05) using the Wilcoxon test.

Development of in-house materials for the verification of specific gravity measurements: Homogeneity, stability, and proficiency studies.

We developed and evaluated the properties of in-house urine reference materials for the verification of laboratory refractometers, which are frequently used in clinical chemistry and doping testing laboratories. Urine was gathered from 26 healthy volunteers (16 male 30 ± 5 years old and 10 female 29 ± 4 years old), from which two urine batches were obtained: one with a low specific gravity (1.012± 0.003) and the other with a high specific gravity (1.027 ± 0.003). Homogeneity studies were conducted over 20 consecutive days. For short-term stability studies, aliquots of both urine batches were stored at -20 ± 2°C; 3 ± 2°C; 20 ± 2°C; 45 ± 2°C for 0, 2, 7, 14 and 35 days, under both light and dark conditions. Similarly, another study was conducted to measure the long-term stability of urine at -20 ± 2°C, over a 24-month evaluation period. Our data showed that the urine was homogeneous and stable at -20 ± 2°C, 3 ± 2°C, 20 ± 2°C, and 45 ± 2°C under both light and dark conditions. In all cases, the urine was evaluated by specific gravity and no statistically significant differences (p ≤ 0.05) were recorded. Additionally, a proficiency test was conducted in collaboration with 15 ISO/IEC 17025 accredited laboratories, and z-scores and performance factors were evaluated. These data indicate that this material could be used for the verification of refractometers.

A Novel Phospholipase A2 Isolated from Palythoa caribaeorum Possesses Neurotoxic Activity.

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum. This enzyme, which is Ca2+-dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA2´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA2 enzymes.

Banned substances and their incidence: A retrospective view of the national laboratory of prevention and doping control of Mexico. / Sustancias dopantes y su incidencia: una visión retrospectiva del laboratorio nacional de prevención y control del dopaje de México.

The use of banned substances to increase athletes' performance has been a scourge in international sport. In this sense, the World Anti-Doping Agency (WADA-AMA) has implemented a series of standards that harmonize the fight against doping. In particular, accredited WADA-AMA laboratories play an important role in the eradication of sports doping. This report shows the data obtained in the National Laboratory of Prevention and Control of Doping (LNPCD-CONADE) according to the incidence of Adverse Analytical Findings (AAF) in the 2009-2015 interval, which were obtained from the analysis of a total of 18,085 biological doping-control samples. The distribution of samples was analyzed as a function of gender, type of sport either in competition or out of competition, as well as the prevalence of AAF during the period of time analyzed and the relation regarding group of doping substance and type of sport. The data presented here were compared with those reported worldwide by the WADA-AMA and it was observed that in the cases of substances of the S1 group, the percentage reported by the LNPCD-CONADE is higher than the one reported worldwide. The opposite was observed for AAF presented by some substance from Groups S6 and S8. Likewise, a higher prevalence in the use of doping substances by male athletes (75%) is observed compared to that observed in female athletes (25%). The sports with the highest number of AAF detected in the laboratory were baseball, cycling, and athletics.

El uso de sustancias para incrementar el desarrollo deportivo de atletas ha sido un flagelo en el deporte internacional. En este sentido la Agencia Mundial Antidopaje (WADA-AMA) ha implementado una serie de estándares que permiten armonizar la lucha contra el dopaje desde diferentes aristas. Particularmente los laboratorios acreditados por la WADA-AMA forman parte importante en la erradicación de dopaje deportivo. En este informe se muestran los datos obtenidos en el Laboratorio Nacional de Prevención y Control del Dopaje  (LNPCD-CONADE) de acuerdo a la incidencia de Resultados Analíticos Adversos (RAA) en el periodo 2009-2015, los cuales fueron obtenidos del análisis de un total de 18,085 muestras biológicas de control antidopaje. Se hace un análisis de la distribución de muestras de acuerdo al género, tipo de deporte ya sea en competición o fuera de competición, así como de la prevalencia de RAA durante el periodo de tiempo analizado y la relación respecto al grupo de sustancia dopante y tipo de deporte. Los datos aquí presentados se compararon con los disponibles en la página electrónica de la WADA-AMA y se observó que en los casos de sustancias del grupo S1 es más alto el porcentaje que se reporta por el LNPCD-CONADE que el reportado a nivel mundial, caso contrario se determinó para RAA que presentaron alguna sustancia del grupo S6 y S8.  Así mismo se observa una mayor prevalencia en el uso de sustancias dopantes por atletas masculinos (75%) comparado con el 25 % observado en atletas femeninos. Los deportes con mayor número de RAA detectados en el laboratorio fueron béisbol, ciclismo y atletismo.

Resolution of R-(-) and S-(+)- enantiomers of clenbuterol in pharmaceutical preparations and black-market products using liquid chromatography-tandem mass spectrometry.

Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, and amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non-Olympic athletes that have adverse analytical findings (AAFs). In some cases, the presence of these substances could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R-(-) and S-(+)-enantiomers of clenbuterol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a chiral column in 15 min with good separation. The method developed can also be used for the analysis of other biological matrices such as urine, serum, and meat. The resolution between two peaks' (Rs ) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black-market products has not been reported previously. The information shown could be used by national anti-doping organizations and the athletes with AAFs attributed to clenbuterol. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of bis-1,4-dihydropyridine in the kidney of diabetic rats.

The in vivo effectiveness of 4-dihydropyridine (bis-1,4-DHP), a new calcium-channel blocker, as a nephroprotector in isolated perfused kidney was evaluated by determining its effects on parameters associated with renal injury in diabetic rats. Diabetes in male Wistar rats, control, diabetic, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP, was induced by a single administration of STZ (55 mg·kg(-1), i.p.). In the drug-treated groups, treatment with bis-1,4-DHP (10 mg·kg(-1)·day(-1)) started one week before diabetes induction; bis-1,4-DHP was dissolved in DMSO (0.3%) and suspended in drinking water with carboxymethyl cellulose (3%). Parameters evaluated were body weight, blood glucose, albuminuria, proteinuria, creatinine, urea excretion, kidney's weight / body weight ratio, and kidney perfusion pressure in all rat groups at different times of diabetes (2, 4, 6, and 10 weeks). Kidney weight of diabetic rats significantly increased vs. control, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP rats at different times of diabetes. The ratios % kidney weight / 100 g body weight were different between control, control + bis-1,4-DHP, and diabetic + bis-1,4-DHP rats vs. diabetic rats (P < 0.05). Kidney perfusion pressure was decreased by diabetes, while it was partially recovered by bis-1,4-DHP treatment in response to phenylephrine. Bis-1,4-DHP had a tendency to decrease hyperglycemia vs. diabetic rats, even though glycemia was too high as compared with controls, and it ameliorated albuminuria, creatinine, and urea excretion, suggesting a favorable effect on renal haemodynamics. Bis-1,4-DHP, by inhibiting Ca(2+) entrance, induced vasodilation in renal vascular bed and thus may have a nephroprotective effect against diabetes-induced renal dysfunction, but does not have significant impact on hyperglycemia.

Antimalarial activity of ultra-short peptides.

Ultra-short peptides 1-9 were designed and synthesized with phenylalanine, ornithine and proline amino acid residues and their effect on antimalarial activity was analyzed. On the basis of the IC(50) data for these compounds, the effects of nature, polarity, and amino acid sequence on Plasmodium berghei schizont cultures were analyzed too. Tetrapeptides Phe-Orn-Phe-Orn (4) and Lys-Phe-Phe-Orn (5) showed a very important activity with IC(50) values of 3.31 and 2.57 microM, respectively. These two tetrapeptides are candidates for subsequent in vivo assays and SARS investigations.