Precision-cut liver slices as a model for assess hepatic cellular response of chitosan-glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol.

Zilpaterol and clenbuterol are two ß-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both ß-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

An Artemisia cina n-hexane extract reduces the Haemonchus contortus and Teladorsagia circumcincta fecal egg count in naturally infected periparturient goats.

The objective of this study was to evaluate an n-hexane extract of Artemisia cina (Acn-h) as a natural anthelmintic treatment for periparturient goats naturally infected with the nematodes Haemonchus contortus and Teladorsagia circumcincta. A total of 200 periparturient Alpine and Nubian goats were used. Deworming criteria were based on the following parameters: fecal egg account (epg), ocular mucosa color (OMC), and body condition (BC). A previous analysis using coprocultures of the flock revealed the presence of H. contortus (80%) and T. circumcincta (20%). The Acn-h contained two new compounds identified by mass spectrometry data as isoguaiacin and norisoguaiacin at 284.14 and 315 m/z. The lethal effects of Acn-h at 0.5, 1, 2, and 4 mg/mL on H. contortus adult stages were 31.6, 66.5, 81.3, and 86.9%, respectively (p < 0.05), showing similar efficacy at 2 and 4 mg/mL with albendazole (positive control group). Then, two experimental groups, with 100 goats in peripartum in each, were distributed randomly and treated at day 0 as follows: group 1 = 4 mg/kg of Acn-h as single oral dose, and group 2 = control group, treated with water (as a placebo). The epg, OMC, and BC parameters were recorded at 0 (periparturient period), 7 (birth period), and 23 (postpartum) days and analyzed using a completely randomized design with Duncan's test for comparison of means and analysis of variance. The following epg reductions were recorded in the Acn-h-treated group as follows: 20.1 ± 34.4 and 31.7 ± 38.2% at days 7 and 23 compared to the control group. During the whole experiment, no significant differences in OMC or BC were observed in relation to the control group, excepting at day 23 (p < 0.05) for BC in the group treated with A. cina. Thus, Acn-h can be a useful natural alternative tool for the control of the nematodes H. contortus and T. circumcincta in periparturient goat flocks.

3'-Demethoxy-6-O-Demethylisoguaiacin and Norisoguaiacin Nematocidal Lignans from Artemisia cina against Haemonchus contortus Infective Larvae.

Artemisia cina is a plant used in traditional Chinese medicine as a remedy for parasitic diseases. This study describes the isolation and chemical characterization of anthelmintic compounds of A. cina against Haemonchus contortus infective larvae (L3) through lethal testing. Previously, three extracts-n-hexane (HexAc), ethyl acetate (EtOAc) and methanol (MeOAc)-were evaluated at concentrations of 4 to 0.5 mg/mL, resulting in the HexAc extract with the greatest effect of 76.6% mortality of the larvae at 4 mg/mL. Then, this was chemically fractioned by polarity, obtaining seven fractions (C1F1-C1F7), and, when evaluated at concentrations from 2 to 0.25 mg/mL, the 2 mg/mL C1F5 fraction produced an effect against the nematode H. contortus of 100% mortality of the larvae. Thus, this fraction was fractionated again by column chromatography, obtaining twelve subfractions (C2F1-C2F12) which were evaluated from 1 to 0.125 mg/mL, with the C2F5 subfraction causing a nematicidal effect of 100% mortality. NMR analysis of one (1H, 13C and DEPT) and two dimensions (COSY, HSQC and HMBC) and mass spectrometry of this fraction allowed us to identify the mixture of 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin. Therefore, it can be assumed that the mixture of these compounds is responsible for the anthelmintic effect. These results indicate that A. cina containing anthelmintic compounds and might be used as an antiparasitic drug against H. contortus.

Determination of zilpaterol in a residue depletion study using LC-MS/MS in cattle plasma, muscle, liver and kidney.

Zilpaterol is a ß-agonist compound which promotes fat loss and muscle gain in cattle, providing economic benefits. However, zilpaterol residues in the animal might introduce a significant risk to humans after consumption. In the present manuscript, a highly specific, sensitive method using Selected Reaction Monitoring (SRM) in positive electrospray ionization (ESI +) mode by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS) for plasma, muscle, liver and kidney is presented. For method development, composition of the aqueous mobile phase, precipitation agent, and solid phase extraction (SPE) conditions were optimized. The method was fully validated showing a good linearity and recovery average greater than or equal to 97 % for all matrices. The method was applied to residue depletion studies in cattle after withdrawal of zilpaterol supplementation at 3, 4, 5 and 6 days showing that tissues can be consumed by humans after 4th day of zilpaterol withdrawal.

Development of a Novel Targeted Metabolomic LC-QqQ-MS Method in Allergic Inflammation.

The transition from mild to severe allergic phenotypes is still poorly understood and there is an urgent need of incorporating new therapies, accompanied by personalized diagnosis approaches. This work presents the development of a novel targeted metabolomic methodology for the analysis of 36 metabolites related to allergic inflammation, including mostly sphingolipids, lysophospholipids, amino acids, and those of energy metabolism previously identified in non-targeted studies. The methodology consisted of two complementary chromatography methods, HILIC and reversed-phase. These were developed using liquid chromatography, coupled to triple quadrupole mass spectrometry (LC-QqQ-MS) in dynamic multiple reaction monitoring (dMRM) acquisition mode and were validated using ICH guidelines. Serum samples from two clinical models of allergic asthma patients were used for method application, which were as follows: (1) corticosteroid-controlled (ICS, n = 6) versus uncontrolled (UC, n = 4) patients, and immunotherapy-controlled (IT, n = 23) versus biologicals-controlled (BIO, n = 12) patients. The results showed significant differences mainly in lysophospholipids using univariate analyses in both models. Multivariate analysis for model 1 was able to distinguish both groups, while for model 2, the results showed the correct classification of all BIO samples within their group. Thus, this methodology can be of great importance for further understanding the role of these metabolites in allergic diseases as potential biomarkers for disease severity and for predicting patient treatment response.

Development and Validation of a Stability-Indicating HPLC Method for the Simultaneous Determination of trans-Resveratrol and cis-Resveratrol in an Injectable Solution.

trans-Resveratrol, a phytochemical compound with antioxidant power and various therapeutic effects, such as cardioprotective, chemopreventive, and neuroprotective, among others, has disadvantages of poor solubility and limited stability, creating difficulties for the development of new strategies for its quantification. This study developed and validated an analytical stability method for trans-resveratrol by high-pressure liquid chromatography with photodiode-array detection (HPLC-PDA), which allowed its quantification in the presence of its degradation products. The quantification of trans-resveratrol occurred at a retention time of 2.6 min, with ammonium formate (10 mM, pH = 4)/acetonitrile, 70/30 v/v, as mobile phase. The validation met the ICH Q2 criteria of specificity, method linearity (2.8-4.2 µg/ml), precision and accuracy, robustness, quantification limit (0.176 µg/ml), and detection (0.058 µg/ml). As degradation products, cis-resveratrol was observed at 3.9 min, which could be resveratrone in 3.2 min and five unidentified products in 0.7, 1.0, 1.4, 1.8, and 5 min. Some solutions subjected to temperature stress of 40 and 60°C, UV light, and acidic and basic hydrolysis exhibited colour changes. An analytical method was obtained by HPLC-PDA, which allowed quantifying the stability of trans-resveratrol in a fast and specific manner in the presence of its degradation products.

Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC-MS/MS.

Zilpaterol and Clenbuterol are ß-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these ß-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 µg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 µg/mL and 2.483 ± 0.36 µg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 µg/mL and 0.822 ± 0.11 µg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.

In Vitro Nematocidal Effect and Anthelmintic Activity of Artemisia cina Against Haemonchus contortus in Gerbils and Relative Expression of Hc29 Gene in Transitional Larvae (L3-L4).

PURPOSE: (1) To assess the in vitro activity of Artemisia cina against Haemonchus contortus L3 (HcL3) and in transitional (L3-L4) larvae (HcTrL3-L4); (2) to quantify the relative expression of the Hc29 gene in HcTrL3-L4 exposed to the A. cina n-hexane extract; and (3) to assess the anthelmintic activity (AA) of the A. cina organic extracts in gerbils artificially infected with H. contortus (HcArt/inf/gerbs). METHODS: The in vitro assay was carried out in 96-well microtitration plates. The following A. cina extracts: ethyl acetate (Ac-EtOAcEx), n-hexane (Ac-n-HexEx), and methanol (Ac-MethEx) were assessed at 1 and 2 mg/mL against HcL3 and HcTrL3-L4 at 24 h exposure. Relative expression of the Hc29 gene in HcTrL3-L4 was obtained by RT-PCR. For assessing the AA, six groups of five HcArt/inf/gerbs were used. Groups were treated orally with 4 mg/kg BW of A. cina extracts. Five days after treatment, the gerbils were necropsied and nematodes counted. RESULTS: The highest in vitro activities (75 and 82.6%) were shown by Ac-n-HexEx at 1 and 2 mg/mL, respectively. For HcTrL3-L4 the highest in vitro activities (69 and 23%) were shown by Ac-n-HexEx and isoguaiacine at 0.625 mg/mL, respectively. Also, upregulation of H. contortus Hc29 gene by 13- and 80-fold (p < 0.01) was observed on the HcTrL3-L4 stage after exposure to Ac-n-HexEx extract and isoguaiacine at 0.078 mg/mL, respectively. Reduction percentage was 100% in HcArt/inf/gerbs treated with Ac-n-HexEx. CONCLUSIONS: We conclude that the Ac-n-HexEx and isoguaiacine compound had anthelmintic efficacy against H. contortus and L3 and HcTrL3-L4.

Evaluation of R- (-) and S- (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC-TQ-MS.

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol-fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S- (+) and R- (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC-TQ-MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5-800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.

Troubleshooting in Large-Scale LC-ToF-MS Metabolomics Analysis: Solving Complex Issues in Big Cohorts.

Metabolomics, understood as the science that manages the study of compounds from the metabolism, is an essential tool for deciphering metabolic changes in disease. The experiments rely on the use of high-throughput analytical techniques such as liquid chromatography coupled to mass spectrometry (LC-ToF MS). This hyphenation has brought positive aspects such as higher sensitivity, specificity and the extension of the metabolome coverage in a single run. The analysis of a high number of samples in a single batch is currently not always feasible due to technical and practical issues (i.e., a drop of the MS signal) which result in the MS stopping during the experiment obtaining more than a single sample batch. In this situation, careful data treatment is required to enable an accurate joint analysis of multi-batch data sets. This paper summarizes the analytical strategies in large-scale metabolomic experiments; special attention has been given to QC preparation troubleshooting and data treatment. Moreover, labeled internal standards analysis and their aim in data treatment, and data normalization procedures (intra- and inter-batch) are described. These concepts are exemplified using a cohort of 165 patients from a study in asthma.